RU2833879C1 - Cystathionine beta-synthase enzyme therapy for treating diseases caused by high homocysteine levels - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method of treating CBS deficiency in an individual, comprising determining a metabolic rate of severity of disease or disease progression in an individual, where the metabolic indicator of disease severity or disease progression is total homocysteine (tHcy), and administering to an individual a therapeutically effective amount of a pharmaceutical composition containing a drug substance containing an isolated cystathionine protein-β-synthase (CBS), and where the individual is a patient with a genetically non-heritable disease associated with high levels of tHcy, or a patient with a non-heritable disease associated with CBS deficiency.

EFFECT: invention provides a method of treating individuals with CBS deficiency and/or high tHcy levels, in which HCU has not been diagnosed or its genetic predisposition has not been detected.

2 cl, 4 dwg, 10 tbl, 8 ex

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/895,230, filed September 3, 2019, and U.S. Provisional Patent Application No. 62/983,862, filed March 2, 2020, the contents of each of which are incorporated herein by reference in their entirety.

Link to the list of sequences

[0002] This application is filed together with a sequence listing provided in electronic format. The sequence listing file named 2089_1006PCT_SL.txt was created on September 3, 2020 and has a size of 18,341 bytes. The information provided in the Sequence Listing in electronic format is incorporated herein by reference in its entirety.

Field of invention

[0003] The present invention relates to compositions and methods of enzyme therapy for the treatment of homocystinuria and for the treatment of conditions associated with elevated homocysteine levels using the medicament described herein.

Prerequisites for the creation of an invention

[0004] Classic homocystinuria, referred to herein as HCU or HCU type 1, and also known as homocystinuria with cystathionine β-synthase deficiency (CBSDH), is a rare disorder that affects both children and adults. HCU is a rare autosomal recessive metabolic disorder characterized by excess of the compound homocysteine (Hcy) in urine, tissue, and plasma due to decreased or absent activity of the enzyme cystathionine β-synthase (CBS) (see Kraus et al., In: Carmel R, Jacobsen DW, eds. Homocysteine in Health and Disease. Cambridge, United Kingdom: Cambridge University Press; 2001:223–243; Sacharow et al., Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mefford HC, et al, eds. GeneReviewsTM [Internet]. Seattle (WA): University of Washington, Seattle, 2017; each of which is incorporated by reference in its entirety).

[0005] The diagnosis of HCU can be confirmed by molecular genetic testing of the CBS gene as described by Sacharow et al. CBS is an enzyme involved in the metabolism of the sulfur-containing amino acid methionine (Met), which is present in food proteins (see Maclean et al. J Biol Chem. 2012; 287(38):31994-32005, which is incorporated herein by reference in its entirety).

[0006] HCU may be suspected based on 1) clinical features including ectopia lentis (displacement of the lens of the eye) and/or severe myopia, asthenic frame (tall and thin), skeletal abnormalities, early-onset osteoporosis and/or thromboembolism, unexplained developmental delay/mental retardation; 2) neonatal testing for hypermethioninemia or, in particular, a positive family history of CBS deficiency, which may identify the patient before symptoms develop; and 3) family history. All of these clinical features and the age at which symptoms appear in patients vary considerably. Current testing approaches generally fail to identify infants with less severe CBS deficiency and identify only a small proportion of patients with more severe HCU (see Huemer et al. J Inherit Metab Dis. 2015 Nov; 38(6):1007–19; Yap, Orphanet Encyclopedia [online serial]. 2005, pages 1–13; Schiff et al. Neuropediatrics. 2012 Dec; 43(6):295–304, each of which is incorporated by reference in its entirety).

[0007] In addition to homocystinuria (i.e., diagnosed or genetically inherited HCU), elevated plasma total homocysteine levels have been associated with an increased risk of osteoporosis and/or bone fracture (see van Meurs et al. N Engl J Med 2004; 350:2033-2041). In addition, elevated tHcy levels are “a modifiable risk factor for cognitive decline, dementia, and Alzheimer’s disease” (Smith, A. David, et al. “Homocysteine and dementia: an international consensus statement” Journal of Alzheimer's Disease 62.2 (2018): 561-570).

[0008] Thus, there is currently a need to develop methods to identify, stratify, and treat individuals with CBS deficiency and/or elevated tHcy levels who have not been diagnosed with HCU or have no identified genetic predisposition to it.

The essence of the invention

[0009] The present invention relates to methods for improving cognitive function in an individual having an elevated plasma total homocysteine (tHcy) level, wherein said methods comprise administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising: a drug substance comprising an isolated cystathionine β-synthase (CBS) protein comprising SEQ ID NO: 1; a PEG molecule covalently linked to the CBS protein; and a pharmaceutically acceptable excipient, diluent, or adjuvant.

[0010] In some embodiments of the invention, methods for improving cognitive function in an individual with elevated plasma total homocysteine (tHcy) levels further comprise treating cognitive or behavioral disorders.

[0011] In some embodiments of the invention, the treatment of cognitive or behavioral disorders includes parent behavior training (BPT) or behavioral classroom management (BCM).

[0012] The present invention also relates to methods for reducing skeletal frailty in an individual with elevated plasma total homocysteine (tHcy) levels, said method comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising: a drug substance comprising an isolated cystathionine β-synthase (CBS) protein comprising SEQ ID NO: 1; a PEG molecule covalently linked to the CBS protein; and a pharmaceutically acceptable excipient, diluent, or adjuvant.

[0013] In some embodiments of the invention, the skeletal fragility of an individual is assessed by bone mineral density.

[0014] In some embodiments, the PEG molecule covalently linked to the CBS protein is ME-200GS.

[0015] In some embodiments, the therapeutically effective amount comprises a dose of about 0.25 mg/kg to about 10 mg/kg of the drug. In some embodiments, the therapeutically effective amount comprises a dose of about 0.25 mg/kg to about 10 mg/kg of the drug, administered twice daily.

[0016] In some embodiments, the dose is about 0.33 mg/kg of drug. In some embodiments, the dose is about 0.33 mg/kg of drug administered twice daily.

[0017] In some embodiments, the dose is about 0.66 mg/kg of drug. In some embodiments, the dose is about 0.66 mg/kg of drug administered twice daily.

[0018] In some embodiments, the dose is about 1.0 mg/kg of drug. In some embodiments, the dose is about 1.0 mg/kg of drug administered twice daily.

[0019] In some embodiments, the dose is about 1.5 mg/kg of drug. In some embodiments, the dose is about 1.5 mg/kg of drug administered twice daily.

[0020] In some embodiments of the invention, the dose is administered by the patient himself.

[0021] In some embodiments of the invention, the methods further comprise administering to the individual one or more of vitamin B6 and betaine.

[0022] In some embodiments of the invention, the individual is on a methionine (Met) restricted diet.

[0023] In some embodiments, the methods further comprise discontinuing or restricting the consumption of a methionine-restricted diet. For example, after administration of a pharmaceutically effective amount of a medicament described herein, the individual may be discontinued from a methionine-restricted diet or may be placed on a milder methionine-restricted diet.

[0024] In some embodiments of the invention, the methods further comprise administering an antiplatelet agent.

[0025] In some embodiments, the antiplatelet agent is a warfarin-lowering agent or an anticoagulant.

[0026] In some embodiments of the invention, administration of a therapeutically effective amount of the pharmaceutical composition is carried out approximately once every 3 days.

[0027] In some embodiments, the administration of a therapeutically effective amount of the pharmaceutical composition is performed about once every 2 days. In some embodiments, the administration of a therapeutically effective amount of the pharmaceutical composition is performed about once a day.

[0028] In some embodiments of the invention, the administration of a therapeutically effective amount of the pharmaceutical composition is carried out approximately twice a day.

[0029] In some embodiments of the invention, administration of a therapeutically effective amount of the pharmaceutical composition is carried out approximately once per week.

[0030] In some embodiments of the invention, administration of a therapeutically effective amount of the pharmaceutical composition is carried out approximately twice a week.

[0031] In some embodiments, administration of a therapeutically effective amount of the pharmaceutical composition is repeated for about 6 weeks.

[0032] In some embodiments, administration of a therapeutically effective amount of the pharmaceutical composition is repeated for about 3 months.

[0033] In some embodiments, administration of a therapeutically effective amount of the pharmaceutical composition is repeated for about 6 months.

[0034] In some embodiments of the invention, administration of a therapeutically effective amount of the pharmaceutical composition is repeated for more than 6 months.

[0035] In some embodiments of the invention, administration of a therapeutically effective amount of the pharmaceutical composition is repeated throughout the life of the individual.

[0036] In some embodiments of the invention, elevated plasma total homocysteine (tHcy) levels in an individual include tHcy levels greater than about 5 μmol/L.

[0037] In some embodiments of the invention, elevated plasma total homocysteine (tHcy) levels in an individual include tHcy levels greater than about 10 μmol/L.

[0038] In some embodiments of the invention, elevated plasma total homocysteine (tHcy) levels in an individual include tHcy levels greater than about 15 μmol/L.

[0039] In some embodiments of the invention, the individual with elevated plasma total homocysteine (tHcy) levels is a patient with genetically inherited HCU.

[0040] In some embodiments of the invention, an individual with elevated plasma total homocysteine (tHcy) levels is a patient with a non-genetically inherited disease associated with elevated tHcy levels or CBS deficiency.

[0041] The present invention also relates to methods of treating CBS deficiency in an individual, comprising: determining a level of a metabolic indicator of disease severity or disease progression in the individual and administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising: (i) a drug substance comprising an isolated cystathionine β-synthase (CBS) protein comprising SEQ ID NO: 1; (ii) a PEG molecule covalently linked to the CBS protein; and (iii) a pharmaceutically acceptable excipient, diluent, or adjuvant, wherein the individual is a patient with genetically inherited HCU or with a genetically non-heritable disease associated with elevated tHcy levels or CBS deficiency, and wherein the therapeutically effective amount of the pharmaceutical composition comprises a dose of the drug substance adjusted in accordance with the level of a metabolic indicator of disease severity or disease progression in the individual.

[0042] In some embodiments of the invention, determining the level of a metabolic indicator of disease severity or disease progression in an individual comprises collecting a blood or plasma sample from the individual, assessing the level of one or more metabolic indicators of disease severity or disease progression in the sample, and comparing the level of one or more metabolic indicators of disease severity or disease progression to the level of the metabolic indicator in a control sample collected from a healthy individual.

[0043] In some embodiments, the dose of the drug adjusted according to the level of the metabolic indicator of disease severity or disease progression in the individual comprises a low dose, a medium dose, or a high dose of 20NHS PEG-CBS.

[0044] In some embodiments, the low dose of 20NHS PEG-CBS is from about 0.25 mg/kg to about 1.0 mg/kg 20NHS PEG-CBS and is administered once or twice daily.

[0045] In some embodiments, the average dose of 20NHS PEG-CBS is from about 0.5 mg/kg to about 1.5 mg/kg 20NHS PEG-CBS and is administered once or twice daily.

[0046] In some embodiments, the high dose of 20NHS PEG-CBS is from about 1 mg/kg to about 2 mg/kg 20NHS PEG-CBS and is administered once or twice daily.

[0047] In some embodiments, the high dose of 20NHS PEG-CBS is from about 2 mg/kg to about 10 mg/kg 20NHS PEG-CBS and is administered once or twice daily.

[0048] In some embodiments, the metabolic indicator of disease severity or progression is total homocysteine (tHcy), methionine, creatinine, C-reactive protein, dimethylglycine, alanine aminotransferase, protein C, aspartate aminotransferase (AST), antithrombin III, and/or apolipoprotein A.

[0049] In some embodiments, the metabolic indicator of disease severity or progression is tHcy, and a dose of 20NHS PEG-CBS is administered to an individual having tHcy at a level slightly above low, moderate, or high.

[0050] The present invention also relates to methods of treating an individual suffering from homocystinuria or suspected of having homocystinuria, wherein said methods comprise: assessing a level of one or more of creatinine, high-sensitivity C-reactive protein, fibrinogen, or a protein C activity level in the individual; comparing the one or more levels to a known range of values for one or more levels in a group of individuals who are known not to suffer from homocystinuria or in a group of individuals who suffer from homocystinuria, or in both; assessing disease progression or disease severity in the individual according to the one or more levels assessed in the individual; adjusting a dose of enzyme therapy for the individual according to the disease progression or disease severity; and administering enzyme therapy to the individual. In these methods, performing enzyme therapy comprises administering a medicinal substance comprising an isolated cystathionine β-synthase (CBS) protein comprising or consisting of SEQ ID NO: 1, wherein said CBS protein has a PEG molecule covalently linked to the CBS protein.

Brief description of the drawings

[0051] The above and other objects, features and advantages of the present invention will become apparent from the following description of specific embodiments thereof as shown in the accompanying drawings. The drawings are not necessarily to scale, and emphasis is instead placed on illustrating the principles of the various embodiments of the invention.

[0052] Figure 1 is a graph showing the number of patients with high, normal, or low laboratory values for certain biological markers tested in a pathogenesis study in patients with HCU. In Figure 1, ALT-SGPT=alanine aminotransferase-serum glutamic acid-pyruvic acid transaminase; AST-SGOT=aspartate aminotransferase-serum glutamic acid-pyruvic acid transaminase; DMG=dimethylglycine; hsCRP=high-sensitivity C-reactive protein; P1NP=procollagen type 1 N-terminal propeptide.

[0053] Figure 2 shows correlations of plasma tHcy levels ≤100 μM and >100 μM in the case of skeletal fragility.

[0054] Figure 3 shows the median and quartile NIH Toolbox scores for the cognitive functions tested. The boxes in the Toolbox plots span the data from the 25th to the 75th percentile, and the median is represented as a horizontal line in the box. The "fringes" represent the minimum and maximum values. Composite analysis of "crystallized intelligence" = a domain of cognition reflecting past cognitive ability and knowledge. Composite analysis of "fluid intelligence" = a domain of cognition reflecting the ability to learn, solve new problems, and remember.

[0055] Fig. 4 shows the effect of tHcy levels on cognitive function.

Detailed description of the invention

I. Introduction

[0056] Homocystinuria with cystathionine beta-synthase deficiency (HCU) is characterized by elevated plasma homocysteine (Hcy) levels along with high Met levels and decreased cysteine (Cys) concentrations (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Morris et al. J Inherit Metab Dis 2017;40:49-74; NORD, Kraus JP. Homocystinuria due to cystathionine beta-synthase deficiency. NORD [serial online] 2017; each of which is incorporated by reference in its entirety). To date, over 180 different mutations in the CBS gene associated with HCU have been identified (see Human Genome Mutation Database. 2017. Document Type: Web source. Available from hgmd.cf.ac.uk/ac/index.php, which is hereby incorporated by reference in its entirety). Homocysteine (Hcy) is a naturally occurring amino acid that, together with serine, serves as a substrate for the CBS enzyme. CBS regulates the unidirectional flow of sulfur from methionine (Met) to cysteine (Cys), acting at the crossroads of transmethylation, transsulfuration, and remethylation (see Maclean et al. J Biol Chem. 2012;287(38):31994–32005, which is hereby incorporated by reference in its entirety). Native CBS is activated by binding the allosteric activator S-adenosylmethionine (SAM) and catalyzes a β-substitution reaction in which serine is condensed with Hcy via a pyridoxal 5'-phosphate (pyridoxine or vitamin B6)-dependent mechanism to form cystathionine (Cth). Cystathionine γ-lyase (CGL), acting downstream of CBS, uses Cth as a substrate to form Cys. Thus, proper CBS function is essential for the regulation of Hcy, Met, and Cys metabolism.

[0057] Normal total homocysteine (tHcy) levels vary with age, sex, and diet, but are typically in the range of 4.5 to 11 μmol/L (see Quest Diagnostics Reference Range; questdiagnostics.com, which is incorporated herein by reference in its entirety). Men typically have slightly (1 to 2 μmol/L) higher tHcy levels than women, and approximately double the mean values are observed in patients from childhood to age 80 (see Refsum et al. Clin Chem 2004; 50:3-32, which is incorporated herein by reference in its entirety). In folate-supplemented groups, the upper limit (97.5%) of tHcy levels is approximately 12 μmol/L in adults <65 years and 16 μmol/L in adults >65 years. Many patients with HCU have severe hyperhomocysteinemia with total tHcy levels greater than 100 μmol/L, while others have elevations ranging from mild to several times normal (see Morris et al. J Inherit Metab Dis 2017;40:49-74, which is incorporated herein by reference in its entirety). tHcy levels have been found to correlate highly with disease severity (see Yap et al. J Inherit Metab Dis 1998;21:738-747, which is incorporated herein by reference in its entirety).

[0058] The severity and presentation of the signs and symptoms of HCU vary widely among patients (see Karaca et al. Gene 2014; 534:197-203; Trondle et al. Acta Med Austriaca 2001; 28:145-151; Kluijtmans et al. Am J Hum. Genet 1999; 65:59-67; each of which is incorporated by reference in its entirety). Many patients have severe hyperhomocysteinemia with total homocysteine (tHcy) levels greater than 100 μmol/L, while others have tHcy levels ranging from low to several times normal (see Morris et al. J Inherit Metab Dis 2017; 40:49-74), which is incorporated by reference in its entirety). Significantly elevated tHcy levels generally correlate with more severe disease, whereas lower levels generally correlate with milder disease.

[0059] HCU is typically classified based on the individual's response to treatment aimed at lowering total homocysteine (tHcy) levels using pyridoxine (vitamin B6), a cofactor for the CBS enzyme necessary for normal CBS function (see Mudd et al., Am J Hum Genet 1985;37:1-31; Abbott et al., Am J Med Genet 1987;26:959-969, which are each incorporated by reference in their entireties). In general, patients who respond to pyridoxine have lower tHcy levels, resulting in a milder form of the disorder. These patients may develop only one or a few symptoms of HCU later in life, and many remain undiagnosed. Consequently, patients who respond well to pyridoxine are believed to be underrepresented in most studies.

[0060] Retrospective studies have shown that patients with the highest tHcy levels (treated or untreated) have more severe symptoms and at an earlier age (see Yap et al. J Inherit Metab Dis 1998; 21:738-747; Mudd et al. Am J Hum Genet. 1985; 37:1-31, which are incorporated herein by reference in their entireties). Untreated individuals with elevated tHcy levels typically have developmental delay, thromboembolism, severe myopia with subsequent lens dislocation, osteoporotic fractures, Marfan-like habitus (particularly elongated long bones), and psychiatric disorders including learning disabilities (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Morris et al. J Inherit Metab Dis 2017;40:49-74; NORD, Kraus JP. Homocystinuria due to cystathionine beta-synthase deficiency. NORD [serial online] 2017; each of which is incorporated by reference in its entirety). Some patients with elevated tHcy levels have severe multisystem disease with childhood-onset. Without treatment, life expectancy in more severe patients is markedly reduced (see Morris et al. J Inherit Metab Dis 2017;40:49-74, which is incorporated herein by reference in its entirety).

[0061] Hcy-derived compounds, referred to as tHcy, consist of free thiol-homocysteine (Hcy-SH or fHcy), disulfides (such as homocysteine-cysteine and homocysteine), and protein-bound homocysteine (see publications by Ueland; Nord Med 1989; 104:293-298); Mudd et al. N Engl J Med 1995; 333:325; Mudd et al. Arterioscler Thromb Vasc Biol 2000; 20:1704-1706, each of which is hereby incorporated by reference in its entirety). The distinction between the sulfhydryl form (homocysteine; Hcy) and the disulfide form (homocysteine) (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; which is incorporated herein by reference in its entirety) is important because many pathophysiological effects depend on the presence of the sulfhydryl group in Hcy (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Ueland et al. Nord Med 1989; 104: 293-298; Mudd et al., N Engl J Med 1995; 333:325, each of which is incorporated herein by reference in its entirety).

[0062] CBS is primarily expressed in the liver, pancreas, kidney, and brain (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is hereby incorporated by reference in its entirety). The catalytic domain binds to pyridoxal 5'-phosphate (a cofactor, also known as pyridoxine or vitamin B6), and the regulatory domain binds to SAM (an allosteric activator).

[0063] Insufficient levels of CBS enzymatic activity block the first step transsulfuration pathway, resulting in accumulation of Hcy, elevated SAH and Met levels, and decreased Cth and Cys levels. Clinical data on Met metabolite dysregulation reviewed herein have demonstrated that elevated Hcy (most commonly measured clinically as plasma tHcy) is the most important contributor to the pathophysiology of HCU.

[0064] Higher than normal levels of Hcy modify the sulfhydryl groups of proteins, preventing proper protein cross-linking and leading to structural abnormalities in many body systems. Elevated Hcy levels also disrupt intracellular signaling, leading to endothelial dysfunction and ultimately thromboembolism and vascular disease. In HCU, accumulation of Hcy leads to the development of ocular, skeletal, vascular, and psychiatric diseases.

[0065] The diagnosis of HCU is sometimes confirmed by molecular genetic testing of the CBS gene (see Sacharow et al. Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mefford HC, et al, editors. GeneReviewsTM [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2017, which are incorporated herein by reference in their entireties). Current screening approaches generally fail to identify infants with less severe CBS deficiency and identify only a small proportion of patients with more severe HCU (see Huemer et al. J Inherit Metab Dis. 2015 Nov;38(6):1007-19; Yap, Orphanet Encyclopedia [online serial]. 2005, pages 1-13; Schiff et al. Neuropediatrics. 2012 Dec;43(6):295-304).

[0066] The criterion of choice for determining Hcy levels in clinical specimens is tHcy, which includes free Hcy as well as Hcy bound to protein or present in the form of disulfides. Normal tHcy levels vary with age, gender, and diet, but are typically between 4.5 and 11 μM (QUEST DIAGNOSTICS™ reference range). Many patients with HCU have severe hyperhomocystinuria with total tHcy levels greater than 100 μM, while others have levels that range from mild to several times higher than normal (see Morris et al. J Inherit Metab Dis. 2017 Jan;40(1):49-74, which is incorporated herein by reference in its entirety). tHcy levels are highly correlated with disease severity (see Yap et al. J Inherit Metab Dis 1998; 21: 738-47).

[0067] Studies have shown that decreased Hcy levels in patients with HCU correlate with less severe clinical symptoms (see Yap et al. J Inherit Metab Dis 1998; 21: 738-47; Yap et al. Arterioscler Thromb Vasc Biol. 2001). Dec; 21(12):2080-5; which are incorporated herein by reference in their entireties). The pathways by which homocysteine levels cause damage to these systems have been well described in the literature (see Ajith et al. Clin Chim Acta 2015;450:316-321; Behera et al. J Cell Physiol 99999:1-6, 2016; Saha et al. FASEB J 2016;30:441-456; which are each incorporated by reference in their entireties) and have initiated research into the role of Hcy in a large group of individuals that has identified a significant pathogenic role for Hcy in disease.

[0068] One of the goals of treatment using the medicament described herein is to increase the activity of the CBS enzyme in the bloodstream, which leads to improved metabolic control, and thereby to a reduction in the clinical manifestations of diseases and a slowing down or prevention of their further progression. High molecular weight compounds such as enzymes have a limited ability to penetrate tissues and are therefore mainly present in the plasma. These proteins usually remain in the bloodstream for a short period of time, since they are removed from the bloodstream by several mechanisms (see Vugmeyster et al. World J Biol Chem. 2012;3(4):73-92, which is incorporated herein by reference in its entirety). Ideally, the administered CBS should maintain high activity in the plasma for a period of time sufficient to achieve a sustained effect on the metabolism of sulfur-containing amino acids. This goal can be achieved through PEGylation, i.e., the attachment of PEG moieties to the surface of the protein. PEGylation of proteins is a widely used strategy that has been shown to minimize proteolysis, immune response, and antigenicity, while increasing protein stability and size and decreasing renal excretion (see Kang et al. 2009;14(2):363-380, which are incorporated by reference in their entireties). The drug product described herein is a PEGylated htCBS C15S enzyme prepared for administration to an individual and intended for long-term systemic exposure.

A. Clinical manifestations of homocystinuria

[0069] There is compelling evidence indicating a causal effect of elevated tHcy levels on adverse clinical outcomes in the four systems commonly affected in patients with HCU (i.e., the eye, skeleton, cardiovascular system, and nervous system). These data are further supported by studies in a large group of individuals demonstrating a significant association between moderately elevated tHcy levels and adverse outcome.

1. Eyes

[0070] Abnormalities affecting the eyes may be an early clinical feature of HCU. Many individuals develop a displacement of the lens of the eye from the center of the eyeball (ectopia lentis). Affected individuals also typically develop severe myopia (nearsightedness or nearsightedness) and iridodonesis (trembling of the iris). Ectopia lentis and myopia typically develop after the first year of life and before age 10, usually in untreated individuals (see Mudd et al. Am J Hum Genet 1985; 37: 1-31, which is hereby incorporated by reference in its entirety). Other less common ocular abnormalities include cataracts, optic nerve degeneration, and glaucoma. Some people may have retinal detachment, which can cause blurred vision or "spots" in the visual field (see Burke et al. Br J Ophthalmol, 1989; 73(6): 427-31, which is incorporated herein by reference in its entirety).

[0071] Elevated Hcy levels are an important and independent risk factor for ocular complications, particularly lens dislocation, in patients with HCU and in the general population (see Mudd et al. Am J Hum Genet 1985; 37: 1-31; Ajith et al. Clin Chim Acta 2015; 450:316-321; Mulvihill et al. J AAPOS 2001; 5:311-315, which are each incorporated by reference in their entireties). Even with pharmacologic and dietary interventions, most patients with HCU eventually develop ocular complications. Reduction in Hcy levels has been observed to delay and possibly prevent lens dislocation in patients with HCU (see Yap et al. J Inherit Metab Dis 1998; 21: 738-47, which is incorporated herein by reference in its entirety).

2. Central nervous system

[0072] Developmental delay and learning problems, such as cognitive symptoms, may also be early signs of HCU, occurring between one and three years of age (see Screening, Technology and Research in Genetics (STAR-G) Project. 2016. Homocystinuria. Available at newbornscreening.info; National Institutes of Health (NIH), US National Library of Medicine, Genetics Home Reference. Homocystinuria. 2016. ghr.nlm.nih.gov; which are incorporated herein by reference in their entireties). Intelligence quotient (IQ) in people with HCU has been reported to range from 10 to 138. Patients with the highest tHcy levels are more likely to have a lower IQ (with a mean IQ of 57 in the absence of treatment) compared with less severe patients (with a mean IQ of 79) (see Sacharow et al., Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mefford HC, et al, editors. GeneReviewsTM [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2017, which is incorporated herein by reference in its entirety).

[0073] Seizures occur in approximately 20% of untreated patients with HCU (see Mudd et al. Am J Hum Genet 1985; 37: 1-31, which is hereby incorporated by reference in its entirety). Many individuals suffer from psychiatric disorders, including personality disorder, anxiety, depression, obsessive-compulsive disorder, and psychotic episodes (see Sacharow et al. 2017). Extrapyramidal features such as dystonia may also occur (see Screening, Technology and Research in Genetics (STAR-G) Project. 2016. Homocystinuria. Available at newbornscreening.info, which is hereby incorporated by reference in its entirety).

[0074] Studies have shown that early reduction of Hcy levels induced by a low-Met diet, folate/vitamin B supplementation, and/or pyridoxine and betaine therapy can delay and sometimes prevent or reverse the progression of various neurodevelopmental disorders and achieve normal IQ in patients with HCU (see El Bashir et al. JIMD Rep 2015;21:89-95; Yap et al. J Inherit Metab Dis 2001;24:437-447; which are incorporated herein by reference in their entireties). The association between elevated Hcy levels and central nervous system (CNS) symptoms, including cognitive symptoms, neurodegenerative diseases, seizures, dystonia, psychosis, cognitive impairment, dementia, and depression, has been well documented in patients with HCU and in the general population (see Abbott et al. Am J Med Genet 1987;26:959-969; Shimke et al. JAMA 1965;193:711-719; Herrmann et al. Clin Chem Lab Med 2011;49:435-441; which are each incorporated by reference in their entireties).

3. Skeletal system

[0075] People with HCU often develop a variety of skeletal abnormalities. People with these abnormalities are often tall and thin with a "Marfan-like" body habitus, which is characterized by thinning and elongation of the long bones (dolichostenomelia) and in which the knees are bent inward so that they touch when the legs are straightened ("knees that turn out" or genu valgum), the feet are very arched (pes cavus), there is an abnormal lateral curvature of the spine (scoliosis), an abnormally protruding rib cage (pectus carinatum), or an abnormally sunken rib cage (pectus excavatum). By adolescence, 50% of people show signs of osteoporosis (see Screening, Technology and Research in Genetics (STAR-G) Project. 2016. Homocystinuria, available at newbornscreening.info, which is incorporated herein by reference in its entirety). HCU is associated with an increased risk of osteoporotic fractures, which may be partly related to low bone mineral density (see Mudd et al. Am J Hum Genet 1985; 37: 1–31; Weber et al. Mol Genet Metab 2016; 117:351–354; which are incorporated herein by reference in their entireties).

[0076] In a study of 25 Irish patients with HCU followed for 25 years, the risk of skeletal abnormalities was found to be significantly lower in patients with adequate compliance with Hcy-lowering treatment compared to patients who were non-compliant (see Yap et al. J Inherit Metab Dis 1998; 21:738-47, which is incorporated herein by reference in its entirety).

4. Cardiovascular system

[0077] The association between HCU and vascular disease was first demonstrated in 1985 in an epidemiologic study of patients with moderate to severely elevated Hcy levels due to homozygous HCU (see Mudd et al. Am J Hum Genet 1985; 37: 1-31, which is hereby incorporated by reference in its entirety). Thromboembolism is the most serious, often life-threatening complication of HCU and can involve any vessel. It is a major cause of morbidity and early death in patients with HCU (see Yap et al. Arterioscler Thromb Vasc Biol. 2001 Dec; 21(12):2080-5, which is hereby incorporated by reference in its entirety).

[0078] The risk of thromboembolic complications was approximately 25% at age 16 years and 50% at age 29 years. Several reports suggest that tHcy-lowering treatment significantly reduces the incidence of vascular complications, a major cause of morbidity in patients with HCU (see Wilcken et al. J Inherit Metab Dis 1997; 20:295-300; Yap et al. Arterioscler Thromb Vasc. Biol. 2001 Dec; 21(12):2080-5, which are incorporated herein by reference in their entireties). Since then, a number of other studies have demonstrated an increased risk of vascular complications, particularly venous thrombosis, in patients with HCU (see Kelly et al. Neurology 2003;60:275-279; Magner et al. J Inherit Metab Dis 2011;34:33-37; which are incorporated herein by reference in their entireties).

5. Additional symptoms

[0079] Several additional, although rare, features have been reported in patients with HCU, including very thin, fragile skin, brittle hair, skin color changes (hypopigmentation), and a rash on the cheeks (malarials). Some individuals may develop fatty changes in the liver, protrusion of part of the intestine through a tear in the abdominal wall (inguinal hernia), or inflammation of the pancreas. Abnormal forward-backward curvature of the spine (kyphosis) and collapse of the lung (spontaneous pneumothorax) have also been reported in patients with HCU (see Yap; Orphanet Encyclopedia [online serial]. 2005, pages 1-13, which is incorporated by reference in its entirety).

B. Elevated homocysteine levels in groups of patients with diseases that are not genetically inherited

[0080] Normal tHcy levels vary with age, sex, and diet, but typically range from 4.5 to 11 μmol/L (see Quest Diagnostics Reference Range; questdiagnostics.com). Men tend to have slightly (1 to 2 μmol/L) higher tHcy levels than women, and there is an approximate doubling of the mean values in patients from childhood to age 80 (see Refsum et al. Clin Chem 2004; 50:3-32, which is incorporated by reference in its entirety). In folate-supplemented groups, the upper limit (97.5%) of tHcy levels is approximately 12 μmol/L in adults <65 years and 16 μmol/L in adults >65 years.

[0081] Individuals with higher than normal tHcy levels are at higher risk of developing complications associated with elevated tHcy levels. For example, elevated plasma total homocysteine levels have been associated with an increased risk of osteoporosis and/or bone fracture (see van Meurs et al. N Engl J Med 2004; 350:2033-2041; which is incorporated herein by reference in its entirety). Additionally, elevated homocysteine levels are a known risk factor for the development of cognitive symptoms, including dementia and Alzheimer's disease (see Smith, A. David, et al. "Homocysteine and dementia: an international consensus statement." Journal of Alzheimer's Disease 62.2 (2018): 561-570; which is incorporated herein by reference in its entirety).

[0082] Thus, patients with elevated tHcy levels, i.e., above the normal range of 4.5 to 11 μmol/L, can be treated with the compounds and methods described herein to alleviate skeletal, cardiovascular, and/or cognitive symptoms caused by elevated tHcy levels, regardless of genetically inherited cystathionine β-synthase deficiency.

II. Compositions

A. Native human CBS enzyme

[0083] The full-length native CBS enzyme is a tetramer with four identical monomers, where each monomer (63 kDa in size) consists of three functional domains. The first domain is the N-terminal region of approximately 70 amino acids that binds to heme and is thought to be involved in the redox reaction and/or enzyme folding. The second domain is the central domain, which contains the catalytic core and mediates folding of PLP-dependent type II enzymes. The PLP coenzyme is deeply buried in the cleft between the N- and C-terminal domains. The third region is the C-terminal regulatory domain, consisting of a tandem pair of CBS motifs that, when bound to S-adenosylmethionine (SAM), activate the enzyme. Removal of the regulatory region results in a constitutively active enzyme (see Miles et al. J Biol Chem. 2004 Jul 16; 279(29):29871-4, which is incorporated herein by reference in its entirety).

[0084] The pyridoxal-5'-phosphate (PLP)-dependent enzyme fold contains a heme group. This group catalyzes the PLP-dependent beta-substitution reaction in which it condenses L-homocysteine with L-serine to form L-cystathionine. This group is allosterically regulated by the binding of S-adenosyl-L-methionine (Ado-Met) to the C-terminal regulatory domains, resulting in a conformational rearrangement of these domains and the release of the autoinhibitory block. Activation of CBS can also be achieved by complete removal of the C-terminal regulatory domains, yielding a constitutively active dimeric form of the enzyme (see Miles et al. J Biol Chem. 2004 Jul 16; 279(29):29871-4; Ereno-Orbea et al. Proc Natl Acad Sci USA 111(37), E3845-3852 (2014), each of which is incorporated herein by reference in its entirety).

[0085] The active substance in the drug product described in the present application is a recombinant truncated human CBS protein with a cysteine to serine substitution at amino acid position 15 of the protein (htCBS C15S) compared to the amino acid sequence of SEQ ID NO: 2 in the sequence listing, which is the native CBS protein modified by the addition of polyethylene glycol (PEG). This enzyme is also known as htCBS C15S. In some embodiments, the htCBS C15S drug substance has the amino acid sequence of SEQ ID NO: 1.

[0086] This form of the enzyme has a high propensity to aggregate, which poses serious limitations for the production and recovery of human CBS (hCBS). PEGylated htCBS C15S (including "20NHS PEG-CBS" as defined herein) was engineered to form dimers rather than tetramers, which are less prone to aggregation. High molecular weight compounds such as enzymes are removed from the circulation by degradation through proteolysis and various clearance mechanisms (see Vugmeyster et al. World J Biol Chem. 2012;3(4):73-92, which is incorporated herein by reference in its entirety). PEGylation is known to minimize proteolysis and immunogenicity while increasing protein stability and decreasing renal excretion (see Kang et al. 2009;14(2):363-380, which is incorporated herein by reference in its entirety). These structural modifications make the drug product comprising PEGylated htCBS C15S described herein a more suitable candidate than native hCBS as an enzyme therapy (ET) for the treatment of HCU.

[0087] Native CBS is an intracellular enzyme, but the mechanism by which the enzyme is uptaken from the extracellular environment to its primary intracellular site of action is unknown, whereas PEGylated htCBS C15S acts extracellularly. Unlike native endogenous CBS, PEGylated htCBS C15S corrects metabolic disorders by acting directly in the bloodstream and indirectly in tissues, and does not require SAM for activation. Native hCBS is activated in cells by binding of S-adenosylmethionine (SAM) to its C-terminal regulatory domain. However, circulating levels of SAM in both patients and healthy individuals are far below the levels required to activate CBS (see Stabler et al. Metabolism, 2002. 51(8): p.981-8, which is incorporated herein by reference in its entirety). Therefore, introducing native CBS into the circulation would be ineffective because CBS would not be activated. PEGylated htCBS C15S, although it remains in the circulation and does not enter cells, was engineered to bypass the requirement for SAM activation by removing the C-terminal regulatory domain of CBS that renders the enzyme constitutively active.

B. Enzyme therapy (ET)

[0088] PEGylated htCBS C15S is a PEGylated truncated hCBS with a cysteine to serine substitution at position 15 for ET and can be used to treat HCU and/or to treat a disease caused by elevated plasma total homocysteine levels in patients in whom the disease is not genetically inherited. This modification optimizes the enzyme to form dimers rather than tetramers and is constitutively active.

[0089] PEGylated htCBS C15S complements the deficiency in CBS activity, thereby reducing plasma homocysteine (Hcy) and methionine (Met) levels, increasing cystathionine (Cth) levels, and normalizing cysteine (Cys) levels in HCU models. Reduction in total Hcy (tHcy) levels is a major treatment goal (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74, which is hereby incorporated by reference in its entirety) and significantly correlates with improvement in clinical symptoms (ocular, skeletal, vascular, and neurologic) (see Yap; Orphanet Encyclopedia [online serial]. 2005, pages 1-13, which is hereby incorporated by reference in its entirety).

[0090] PEGylated htCBS C15S reduces plasma homocysteine (Hcy) levels in individuals with elevated Hcy levels, including individuals with a non-genetically inherited increase in tHcy levels. PEGylated htCBS C15S is a recombinant form of the human native CBS enzyme produced by E. coli bacteria. The DNA sequence of native human CBS (SEQ ID NO: 3 in the sequence listing) has been genetically modified to remove the C-terminal regulatory region (amino acids 414-551) (SEQ ID NO: 4) to produce truncated human CBS. The DNA sequence of truncated human CBS was further modified to introduce a T→A point mutation at position 43 of the coding region of DNA (corresponding to SEQ ID NO: 3), resulting in a cysteine to serine substitution at position 15 of the translated protein, generating human truncated CBS C15S (htCBS C15S) (SEQ ID NO: 5). This change reduces the degree of aggregation and ensures consistency between batches compared to native hCBS.

[0091] The enzyme was further modified in E. coli bacteria during expression, resulting in the removal of the first Met from the protein, as shown in SEQ ID NO: 1. Following purification, the htCBS C15S enzyme was further modified by PEGylation using 20 kDa PEG molecules functionalized with N-hydroxylsuccinimide ester, which react with primary amines on the surface of the protein. On average, approximately 5.1 PEG molecules are attached to each monomer unit of the enzyme, forming a heterogeneous dimeric product with an average molecular weight of 290 kDa.

C. PEGylation of htCBS C15S to 20NHS-PEG-CBS

[0092] ME-200GS (also called methoxy-PEG-CO(CH ₂ ) ₃ COO-NHS) is used here for PEGylation of htCBS C15S:

ME-200GS has a molecular weight of 20 kDa and the chemical name α-succinimidyloxyglutaryl-ω-methoxypolyoxyethylene. ME-200GS targets free amines on the surface of htCBS C15S. An amide bond is formed between the PEG and a lysine residue on htCBS C15S. The resulting molecule is referred to herein as "20NHS PEG-CBS" and is a PEGylated human cystathionine beta synthase molecule that is truncated and has the C15S mutation as shown in SEQ ID NO: 1.

D. Post-translational modifications

[0093] Post-translational modifications may require additional biotechnological steps to separate modified and unmodified polypeptides, which increases costs and reduces the efficiency of biologics production. Accordingly, in some embodiments, the production of a polypeptide agent in a cell is enhanced by modulating the expression of a target gene encoding a protein that affects a post-translational modification. In additional embodiments, the production of biologics is increased by modulating the expression of a first target gene encoding a protein that affects a first post-translational modification and modulating the expression of a second target gene encoding a protein that affects a second post-translational modification.

[0094] In addition, proteins expressed in prokaryotic or eukaryotic cells may undergo several post-translational modifications that may adversely affect production and/or the structure, biological activity, stability, homogeneity, and/or other properties of the biological product. Many of these modifications occur spontaneously during cell growth and expression of the polypeptide and may occur at several sites, including the peptide backbone, amino acid side chains, and the amino and/or carboxyl termini of the polypeptide. In addition, a polypeptide may contain several different types of modifications. For example, proteins expressed in bacterial cells such as E. coli can undergo acetylation, histone capture, carboxylation, and/or deamidation (see Yang et al., PNAS 111 (52) E5633-E5642 (2014), which is hereby incorporated by reference in its entirety). For example, proteins expressed in avian and mammalian cells such as Chinese hamster ovary (CHO) cells can undergo acetylation, carboxylation, gamma-carboxylation, histone capture, deamidation, N-terminal cyclization, and glutamine deamidation, as well as asparagine deamidation.

[0095] In some embodiments, protein production is enhanced by modulating the expression of a target gene that encodes a protein involved in protein deamidation. Proteins can be deamidated in several ways, including cyclization and deamidation of N-terminal glutamine and deamidation of asparagine. Thus, in one embodiment, the protein involved in protein deamidation is N-terminal asparagine amidohydrolase. Deamination of a protein may result in altered structural properties, decreased activity, decreased biological activity, decreased potency, increased immunogenicity, and/or other undesirable properties and may be assessed by several methods including, but not limited to, charge-based separation of proteins such as by ion exchange chromatography, HPLC, isoelectric focusing, capillary electrophoresis, native gel electrophoresis, reversed phase chromatography, hydrophobic interaction chromatography, affinity chromatography, mass spectrometry, or the use of L-isoaspartyl methyltransferase.

[0096] In some embodiments, the protein that influences protein secretion is a molecular chaperone selected from the group consisting of: Hsp40, HSP47 (also called serpin peptidase inhibitor cladotype H; heat shock protein 47), HSP60, Hsp70, HSP90, HSP100, protein disulfide isomerase, peptidylprolyl isomerase, calnexin, Erp57 (protein disulfide isomerase family A member 3), and BAG 1. In some embodiments, the protein that influences protein secretion is selected from the group consisting of γ-secretase, p115, signal recognition particle (SRP) protein, secretin, and a kinase (e.g., MEK).

[0097] It is contemplated that further optimization may be achieved by systematically adding or removing nucleotides to create longer or shorter sequences and testing the sequences obtained by "window-skipping" the longer or shorter length up or down the target RNA starting at a specified point. Combining this approach to generate new candidate targets with testing the efficacy of effector RNA molecules based on these target sequences in an inhibition assay, as known in the art or as described herein, may result in further improvements in inhibition efficiency. Moreover, such optimized sequences can be adjusted, for example, by introducing modified nucleotides as described herein or as known to those skilled in the art, adding or modifying overhangs, or by introducing other modifications known to those skilled in the art and/or discussed herein, to further optimize the molecule (e.g., increasing serum stability or circulating half-life; increasing thermal stability; enhancing transmembrane delivery; targeting to a specific site or cell type; enhancing interaction with silencing pathway enzymes; enhancing release from endosomes; etc.) used as an expression inhibitor.

E. Stability

[0098] The drug substance or drug product is stable at various temperatures and under various storage conditions. In some embodiments, the drug substance or drug product is stable when stored at -65°C and -20°C. Alternatively, the drug substance or drug product may be stable when stored at a temperature in the range of from about 2°C to about 8°C. Alternatively, the drug substance or drug product may be stable when stored at a temperature within the range of 25°C±2°C. For example, the drug substance or drug product remains stable at a temperature of from 20°C to 25°C. In some embodiments, the drug substance or drug product is stable under reducing conditions. In some embodiments, the drug substance or drug product is stable under non-reducing conditions. In some embodiments, the drug substance or drug product remains stable for at least 2 days, at least 7 days, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or at least 12 months. For example, the drug substance or drug product remains stable upon storage for about 2 days. For example, the drug substance or drug product remains stable upon storage for about 7 days. For example, the drug substance or drug product remains stable upon storage for about 1 month. For example, the drug substance or drug product remains stable upon storage for about 2 months. For example, the drug substance or drug product remains stable upon storage for about 3 months. For example, the drug substance or drug product remains stable upon storage for about 6 months. For example, the drug substance or drug product remains stable upon storage for about 12 months. For example, a medicinal substance or medicinal product remains stable upon storage for approximately 18 months.

[0099] In some embodiments, the drug substance or drug product is stable when stored at -65°C for up to 18 months. In some embodiments, the drug substance or drug product is stable when stored at a temperature of about 2°C to about 8°C for up to 3 months. In some embodiments, the drug substance or drug product is stable when stored at 25°C±2°C for up to 1 month.

[0100] In some embodiments, the drug substance or drug product remains stable for at least 3 freeze-thaw cycles. In some embodiments, the drug substance or drug product remains stable for up to 6 freeze-thaw cycles. For example, the drug substance or drug product remains stable for 5 freeze-thaw cycles. In some embodiments, the drug product remains stable after injection from a syringe.

III. Pharmaceutical compositions

[0101] The drug product described herein, comprising PEGylated htCBS C15S, is intended to restore metabolic control and attenuate clinical manifestations of disease by reducing homocysteine levels and normalizing cysteine levels in patients with HCU. htCBS C15S is produced by recombinant technology using E. coli BL21 (DE3) and is formulated as a sterile drug product in 15 mM potassium phosphate, 8% (w/v) trehalose, pH 7.5. The drug product is intended for administration by subcutaneous (SC) injection.

[0102] The activity of PEGylated htCBS C15S in the bloodstream also improves or even completely normalizes metabolite profiles in tissues (see WO 2017/083327, which is incorporated herein by reference in its entirety). Therefore, the drug product does not necessarily have to be delivered into its native intracellular environment.

[0103] The drug product reduces the accumulation of toxic Hcy in patients with HCU; normalizes circulating Cys levels; increases circulating Cth levels; and/or prevents, delays the onset, and/or eliminates the development of HCU symptoms. The drug product provides at least one of these benefits, allowing patients to enjoy a normal diet. In fact, an increase in Cth activity even with regular feeding (e.g., 4.0 g/kg MET) indicates increased drug activity and/or decreased renal clearance.

[0104] The molecular mass of the drug substance, calculated from isotope-averaged molecular mass using SEC/UV/MS, is 45.290 kDa for the monomer and 90.58 kDa for the dimer.

[0105] All lots are clear liquid, substantially free of visible particles, and dark red in color. In addition, SDS-PAGE, both under reducing and non-reducing conditions, and Western blot analysis were consistent for each lot. Each of these methods demonstrated a characteristic, uniform, and consistent pattern of PEGylation variant. Co-medications, including anticoagulants, vitamin and mineral supplements, betaine, antidepressants, may also be administered in combination with the drug product described herein to enhance the efficacy of the pharmaceutical composition.

IV. Preparations

[0106] For the above therapeutic uses, the dose administered will vary depending on the compound used, the route of administration, the desired treatment, and the disorder indicated. For example, a daily dose of a compound of the invention by inhalation may be in the range of 0.05 micrograms per kilogram of body weight (mcg/kg) to 100 micrograms per kilogram of body weight (mcg/kg). Alternatively, if the compound is administered orally, the daily dose of a compound of the invention may be in the range of 0.01 micrograms per kilogram of body weight (mcg/kg) to 100 milligrams per kilogram of body weight (mg/kg).

[0107] The protein with the amino acid sequence of SEQ ID NO: 1, which has been PEGylated to obtain the drug substance described in the present application, can be used alone, but is typically administered in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable adjuvant, diluent or carrier. Thus, the present invention also relates to a pharmaceutical composition comprising the drug substance described in the present application in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0108] Pharmaceutically acceptable adjuvants, diluents or carriers that can be used in the pharmaceutical compositions of the invention are those adjuvants, diluents or carriers that are commonly used in pharmaceuticals and include, but are not limited to, sugars, sugar alcohols, starches, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride; zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, block copolymers of polyethylene and polyoxypropylene, polyethylene glycol and wool fat (lanolin).

[0109] The pharmaceutical compositions of the invention may be administered orally, parenterally, by inhalation, rectally, intranasally, buccally, vaginally, or via an implanted reservoir. In one embodiment, the pharmaceutical composition may be administered orally. In one embodiment, the pharmaceutical composition may be administered subcutaneously. The pharmaceutical compositions of the invention may contain any standard non-toxic pharmaceutically acceptable adjuvants, diluents, or carriers. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal injection or infusion, and intralesional and intracranial injection or infusion.

[0110] The pharmaceutical compositions can be prepared in the form of a sterile injectable preparation, for example, as a sterile aqueous or oleaginous injection suspension. The suspension can be prepared by standard techniques known to those skilled in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injection solution or suspension in a non-toxic parenterally acceptable adjuvant, diluent or carrier, for example, as a solution in 1,3-butanediol. Among the acceptable adjuvants, diluents and carriers that can be used are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as a solvent or suspending medium. Any bland fatty oil may be used for this purpose, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are suitable for the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersing agent.

[0111] The pharmaceutical compositions of the invention can be administered orally in any suitable dosage form, including, but not limited to, capsules, tablets, powders, granules, and aqueous suspensions and solutions. These dosage forms are prepared according to methods well known to those skilled in the art of pharmaceutical preparation. In the case of tablets for oral use, carriers such as lactose and corn starch are commonly used. Lubricants such as magnesium stearate are also commonly added. For oral administration in the form of capsules, suitable diluents such as lactose and dried corn starch are used. For oral administration of aqueous suspensions, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents can be added.

[0112] The pharmaceutical compositions of the invention may also be prepared as suppositories for rectal administration. These compositions may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

[0113] The pharmaceutical compositions of the invention can be administered by intranasal aerosol or inhalation. Such compositions are prepared by methods well known to those skilled in the art of pharmaceutical preparation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons and/or other known solubilizing or dispersing agents.

[0114] The pharmaceutical compositions described herein can be prepared in a form suitable for administration through the circulatory system as described in WO 2015/153102, WO 2016/183482, and WO 2018/009838, each of which is incorporated herein by reference in its entirety. The CBS protein can be encoded by a recombinant nucleic acid expressed by nucleated hematopoietic cells (EHC), including erythroid or thromboid cells. For example, erythroid cells are red blood cells, erythrocytes, or reticulocytes. For example, thromboid cells are platelets. In some embodiments, the encoded CBS protein is associated with a translated membrane-anchored polypeptide. In some embodiments, the CBS protein is localized on the surface of the EHC. The CBS protein may be cleaved to activate the enzyme in the extracellular space. Alternatively, the internally localized CBS protein may be released into the extracellular space by lysis of the EHC. Alternatively, the enzymatic target of the CBS protein may enter the EHC and then exit through the membrane after modification. In some embodiments, the CBS protein has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 in the Sequence Listing provided herein.

[0115] Depending on the route of administration, the pharmaceutical composition will contain from 0.05 to 99% by weight (percent by weight), more particularly from 0.05 to 80% by weight, even more particularly from 0.10 to 70% by weight, and still more particularly from 0.10 to 50% by weight of the active ingredient, where all percentages by weight are based on the total composition.

[0116] Conventional procedures for selecting and preparing suitable pharmaceutical compositions are described, for example, in Pharmaceuticals - The Science of Dosage Form Design, M.E. Aulton, Churchill Livingstone, 1988, which is incorporated herein by reference in its entirety. In some embodiments, the drug product is prepared for administration to an individual at a dose of about 50 mU/μL. A lyophilized preparation can be used for administration to a human after reconstitution.

A. Lyophilization

[0117] The pharmaceutical compositions can be prepared as a lyophilized preparation. In some embodiments, the lyophilized preparation comprises a drug substance, a buffer, and an excipient. In some embodiments, after reconstitution of the lyophilized preparation in a suitable dilution buffer, water, or any other pharmaceutically acceptable adjuvant, diluent, or carrier, the concentration of the drug substance is about 20-30 mg/mL. In some embodiments, the concentration of the drug substance is about 20 mg/mL, about 21 mg/mL, about 22 mg/mL, about 23 mg/mL, about 24 mg/mL, about 25 mg/mL, about 26 mg/mL, about 27 mg/mL, about 28 mg/mL, about 29 mg/mL, or about 30 mg/mL. In some embodiments, the concentration of the drug substance is about 25.4 mg/mL. In some embodiments, after reconstitution of the lyophilized preparation in a suitable dilution buffer, water, or any other pharmaceutically acceptable adjuvant, diluent, or carrier, the concentration of the potassium phosphate buffer is 15 mM. In some embodiments, the excipient is trehalose at a concentration of 8% (w/v). In some embodiments, the preparation comprises sucrose such that after reconstitution of the lyophilized preparation in a suitable dilution buffer, water, or any other pharmaceutically acceptable adjuvant, diluent, or carrier, the concentration of sucrose is 5%. In some embodiments, the temperature at the onset of degradation (Tc,on), as determined by freeze-drying microscopy, is -21°C. In some embodiments, the preparation has a pH of 7.5.

[0118] In some embodiments, the lyophilization process can be accomplished in 48 hours or less without melting the crystalline cake structure. The lyophilization process can be optimized to adjust one or more of the following parameters or properties, such as, but not limited to, (i) reduced reconstitution time of the lyophilized drug (e.g., less than 1 minute), (ii) reduced viscosity, allowing for a more concentrated drug product, (iii) inclusion of an isotonic buffer to minimize pain in patients, and/or (iv) a decrease in the degree of de-PEGylation.

[0119] A lyophilized preparation can be prepared according to the following protocol. Three days before preparation, the drug substance (stored at -80°C) at a concentration of 20-30 mg/mL or approximately 25 mg/mL is thawed for 72 hours at 2-8°C in a refrigerator. After thawing, the drug substance is homogenized by gentle shaking. Dialysis is carried out under controlled conditions at 2-8°C for 24 hours. In this case, dialysis cassettes with a cutoff of 20 kDa are used, and the buffer is changed three times at a volume ratio greater than or equal to 1:50 each time. The buffer is changed at 3 and 6 hours throughout the dialysis time. The last dialysis step is carried out overnight. After dialysis, the drug is removed from the dialysis cassettes and filtered using a polyvinylidene difluoride (PVDF) filter with a pore size of 0.22 μm. After filtration, vials are filled with a volume of 1.0 ml under laminar air flow conditions.

[0120] Lyophilization is carried out in an experimental freeze dryer Epsilon 2-12D (Martin Christ, Osterode, Germany). The pressure in the chamber is regulated by a capacitive sensor and a vacuum pump and a controlled dosage of nitrogen.

[0121] After equilibration of the vials to 5°C, the vials are frozen to -45°C and equilibrated for an additional 5 hours at -45°C. The storage temperature is set to -15°C for 31 hours during primary drying. Secondary drying is carried out at a storage temperature of 40°C for 2.5 hours. At the end of the lyophilization process, the chamber is aerated with nitrogen to 800 mbar and the vials are sealed and then lifted onto storage stands. After sealing, the chamber is aerated with nitrogen until atmospheric pressure is reached.

[0122] During the lyophilization process, the product temperature, storage temperature, refrigerator temperature and chamber pressure (using a capacitive sensor and a Pirani sensor) are monitored. The product temperature is controlled by Pt100 sensors (OMEGA™).

V. Treatment of diseases, disorders or conditions

[0123] Individuals with HCUk are usually asymptomatic at birth, and if left untreated, symptoms develop over time, some in infancy and many in childhood, and because the disease has a broad spectrum of symptoms, some patients do not develop symptoms until adulthood (see Yap, 2005; Mudd et al. Am J Hum Genet 1985; 37:1–31; Morris et al. Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J Inherit Metab Dis 2017; 40:49–74; Mudd et al. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7 ed. New York: McGraw Hill; 2001;1279-1327, each of which is hereby incorporated by reference in their entireties). Four major organ systems are typically affected: the eye, skeletal and vascular system, and the central nervous system. However, other organs such as the liver, pancreas, gastrointestinal tract, and skin, including hair follicles, may also be affected (see Mudd et al. Am J Hum Genet 1985;37:1-31; Morris et al. J Inherit Metab Dis 2017;40:49-74; Muacevic-Katanec et al. Coll Antropol 2011;35:181-185; Suri et al. J Neurol Sci 2014;347:305-309, each of which is hereby incorporated by reference in their entireties).

[0124] The obtained data showed that the reduction of Hcy levels may serve as an indicator of the successful use of enzyme therapy (ET) in the treatment of HCU. This is consistent with the definition by the NIH-FDA Biomarker Working Group (see FDA-NIH Biomarker Working Group.BEST (Biomarkers, EndpointS, and other Tools) Resource [Internet]. Silver Spring (MD): Food and Drug Administration (US); 2016-. Reasonably Likely Surrogate Endpoint. 2017 Sep 25. Co-published by National Institutes of Health (US), Bethesda (MD). ncbi.nlm.nih.gov/books/NBK326791/, which are incorporated herein by reference in their entireties), namely, “pharmacodynamic level/response biomarkers that change in response to exposure to a drug product” and that are further closely related to the marker, and in the case of ET for HCU, in response to the mechanism of action of the drug. Thus, blood or plasma Hcy levels are not only a valuable marker for pharmacodynamic studies, but have also previously been recognized as a “reasonably plausible surrogate endpoint” for homocystinuria (HCU).

[0125] In infants and children with HCU, the priority is to prevent complications associated with HCU and to ensure adequate growth and normal intellectual development (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is incorporated herein by reference in its entirety). In patients diagnosed later in life, the goals of treatment should be to prevent life-threatening thromboembolism and to minimize the progression of complications already identified. To achieve these goals, the biochemical abnormalities associated with HCU should be reduced and, if possible, normalized (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is incorporated herein by reference in its entirety).

[0126] According to the 2016 HCU Diagnosis and Treatment Guidelines, Hcy levels should be maintained as close to normal as possible. This is generally not possible in patients with HCU receiving currently available treatments, and therefore target doses below 50 μmol/L in pyridoxine-responsive HCU patients and below 100 μmol/L in pyridoxine-non-responsive patients have been suggested (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is incorporated by reference in its entirety). As noted previously, pyridoxine-non-responsive patients typically have higher Hcy levels than pyridoxine-responsive patients. Although two treatment options are recommended for patients with the same disease, they have been designed to be achievable rather than optimized and to minimize complications.

[0127] Overall, the long-term treatment needed to treat HCU, especially since it most often relies on dietary restrictions and supplements, will not be effective if compliance is poor or inconsistent over a lifetime. ET for the treatment of HCU avoids many of these pitfalls. By compensating for the metabolic defect in HCU through a mechanism that does not require severe Met restriction or Cys supplementation, ET can be expected to result in more consistent reductions in Hcy and non-harmful increases in Met levels, as well as improved well-being or normalization of diet.

[0128] There are currently no cures for HCU that address the underlying genetic causes of the condition, but the primary therapeutic goal is to reduce tHcy levels as much as possible (see Morris et al. J Inherit Metab Dis. 2017 Jan;40(1):49-74, which is incorporated by reference in its entirety). Therefore, current therapy is aimed at correcting the biochemical abnormalities, thereby reducing the risk of adverse clinical manifestations of the disease. Hcy levels are rarely completely normalized with the treatments currently available for patients with HCU.

[0129] A combination of strategies is required to achieve treatment goals in most patients. These treatment strategies include: 1) increasing residual CBS activity by administering pharmacologic doses of pyridoxine (vitamin B6, a cofactor for CBS, along with folate) in pyridoxine-sensitive patients (see Yap et al. Arterioscler Thromb Vasc Biol. 2001 Dec; 21(12):2080-5, which is incorporated herein by reference in its entirety); 2) reducing methionine load through severe dietary/protein restriction while supplementing the diet with foods not in the metabolic block; and 3) enhancing alternative metabolic pathways to reverse the effects of CBS deficiency, such as administering betaine (a methyl donor) to enhance remethylation of Hcy to Met. Some embodiments of the invention include administration of folate supplements and (if necessary) vitamin B12 (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74, which is incorporated herein by reference in its entirety).

A. Current treatment methods for HCU

[0130] In some embodiments, patients with HCU must receive adequate folate supplementation and (if needed) vitamin B12 supplementation (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is incorporated herein by reference in its entirety). In addition, patients must receive pyridoxine therapy (if a positive response), Met-restricted dietary therapy with Cys supplementation, and/or betaine therapy. Most patients require a combination of strategies to achieve treatment goals.

[0131] The most commonly prescribed treatment is a combination of diet and betaine administration, followed by betaine administration alone and diet alone (see Adam et al. Mol Genet Metab 2013; 110:454-459, which is incorporated herein by reference in its entirety). When patients are over 16 years of age, they are most often prescribed betaine without diet, due to poor compliance with a Met-restricted diet in adults. However, compliance with betaine in adults is also poor. Median protein intake varies widely among patients and increases sharply with age.

[0132] Consistent with these findings, a recent report found that only four adult patients out of 24 patients prescribed a low-protein diet with specific amino acid supplements appropriate for the treatment of HCU were compliant with the treatment regimen (see Lorenzini et al. J Inherit Metab Dis. 2017 Oct 4, which is incorporated by reference in its entirety). Several HCU experts have described similarly wide variability in patients in the United States (Rare Disease Research Advisory Board, comprised of physicians who are experts in HCU in the United States). A study comparing tHcy values in untreated and treated patients (25 and 93 patients, respectively) found no significant differences between the two groups (tHcy ranged from 15.7 to 281.4 and 4.8 to 312 μmol/L, respectively, and median values ranged from 125.0 to 119.0 μmol/L, respectively), although this study did not provide any details on the patients' treatment regimens (see Stabler et al. JIMD Rep 2013; 11:149-163, which is incorporated by reference in its entirety). These results lead to the following conclusion or set of conclusions: patients either had a heterogeneous disease presentation; standard treatment was ineffective; and/or treatment adherence was poor.

1. Pyridoxine

[0133] Following diagnosis, patients are tested for sensitivity to pyridoxine, a cofactor of CBS. Administration of pyridoxine (vitamin B6) at pharmacological doses increases residual CBS activity in individuals who have been shown to be sensitive to pyridoxine. The definition of pyridoxine sensitivity varies widely among study centers, although according to the 2016 Guidelines presented as part of the European Homocystinuria and Methylation Defects (EHOD) Network and Registry, pyridoxine sensitivity is defined as a 20% decrease in tHcy levels within 6 weeks of initiating pyridoxine treatment. Patients with severely elevated tHcy and those with mildly to moderately elevated tHcy may be defined as sensitive, despite having very different tHcy levels. In addition, different treatment centers define pyridoxine sensitivity differently, and therefore classifying patients by tHcy levels rather than by their pyridoxine sensitivity is more appropriate and rigorous. In general, patients who respond to pyridoxine have some residual CBS activity and therefore lower tHcy levels, resulting in less severe disease symptoms.

[0134] Pyridoxine is generally considered safe for patients with HCU (see Yap; Orphanet Encyclopedia [online serial], 2005, pages 1-13, which is incorporated herein by reference in its entirety). Its most commonly reported adverse effects include peripheral neuropathy in patients receiving high doses, defined as greater than 900 mg/day (see Schaumburg et al. N Engl J Med 1983; 309:445-448; Ludolph et al. Eur J Pediatr 1993; 152:271; which are hereby incorporated by reference in their entirety), apnea and unresponsiveness in neonates receiving 500 mg/day pyridoxine (see Mudd et al. Am J Hum Genet 1985; 37: 1-31, which is hereby incorporated by reference in its entirety), and rhabdomyolysis (see Shoji et al. J Inherit Metab Dis 1998; 21:439-440, which is hereby incorporated by reference in its entirety).

[0135] Although pyridoxine treatment is now widely used, it only produces modest reductions in tHcy levels, and most patients identified as responsive to it fail to have their tHcy levels significantly reduced, let alone normalized, after pyridoxine alone, as their initial levels are many times higher than normal (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74, which is incorporated herein by reference in its entirety). In addition to long-term pyridoxine treatment, folate and, if needed, vitamin B12 supplementation are recommended for patients with pyridoxine-responsive HCU.

2. Dietary restrictions

[0136] Lifelong dietary restrictions have previously been recommended for all patients with HCU (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74; Walter et al. Eur J Pediatr. 1998 Apr;157 Suppl 2: S71-6; which are incorporated by reference in their entirety). The recommended diet was extremely restrictive and aimed at reducing Met intake by restricting protein content.

[0137] The mainstay of current therapy for patients with HCU is a lifelong low-protein diet containing only 5 g of natural protein per day (www.hcunetworkamerica.org) supplemented with Met-free L-amino acids and, in many cases, Cys supplementation in the diet (see Yap et al. J Inherit Metab Dis. 1998; 21: 738-47; Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74; which are each incorporated by reference in their entireties). The severely restricted diet consists of low-methionine cereal-based foods; low-methionine fruits and vegetables; low-methionine medicinal foods; oils; and sugars. Foods that should be avoided include meat, chicken, fish, eggs, milk, yogurt, cheese, soy products, nuts, legumes, and many fruits and vegetables, which contain moderate amounts of Met. Since native CBS is a key enzyme in Met metabolism, Met supplementation, an essential amino acid found in many foods, results in increased plasma tHcy concentrations and decreased concentrations of the downstream metabolites Cth and Cys.

[0138] Processed foods, baked goods, and ready-to-eat foods should be strictly limited, as they often contain milk, eggs, or flour. The amounts of protein required and ingested by each patient with HCU vary and are likely to change over time. This amount is adjusted according to tHcy levels, which are monitored by frequent blood tests (according to the ASIEM Guidelines for Protein Testing in Homocystinuria). Most patients on dietary therapy also require a daily intake of an unpalatable Met-free synthetic amino acid mixture to prevent secondary malnutrition, for proper growth in children, and for proper digestion in adults (see Morris et al. J Inherit Metab. Dis. 2017 Jan; 40(1):49-74, which is incorporated herein by reference in its entirety).

[0139] Although dietary modifications with a combination of vitamin supplements can reduce tHcy levels to some extent in patients who fully adhere to a strictly restrictive diet, tHcy levels in most patients with HCU remain several times or even orders of magnitude higher than normal. Most people have great difficulty fully adhering to such an altered diet throughout their life, and the resulting periods of poor metabolic control have generally negative consequences (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40 (1): 49-74, which is incorporated herein by reference in its entirety). Dietary compliance is often poor and typically worsens during adolescence and adulthood (see Walter et al. Eur J Pediatr. 1998 Apr;157 Suppl 2:S71-6; Schiff et al. Neuropediatrics. 2012 Dec;43(6):295-304; Garland et al., Paediatr Child Health, 1999 Nov;4(8):557-62, which are each incorporated by reference in their entireties). Furthermore, eating foods high in Met does not cause an immediate adverse physical response, further exacerbating the difficulty in dietary compliance (www.hcunetworkamerica.org). Limiting Met intake is even more challenging in children because of the need to ensure sufficient Met is available to stimulate growth and development. Most patients who have been recommended dietary therapy also require Cys-rich, Met-free L-amino acid supplementation for proper growth in children and proper nutrition in adults (see Morris et al. J Inherit Metab Dis 2017;40:49-74, which is incorporated herein by reference in its entirety).

[0140] A recent report reported that only four adult patients out of 24 patients prescribed a low-protein diet with specific amino acid supplements appropriate for the treatment of HCU were compliant with the regimen (see Lorenzini et al. J Inherit Metab Dis. (2018) 41:109–115, which is incorporated herein by reference in its entirety). Several HCU experts have described similar wide variability in patients in the United States

3. Betaine Supplement

[0141] Problems associated with diet-based therapy alone have called for other approaches to lowering Hcy levels, most notably betaine (N,N,N-trimethylglycine, marketed as CYSTADANE™) supplementation at least twice daily. Betaine is rarely effective as monotherapy (see Sakamoto et al. Pediatr Int 2003; 45:333-338, which is incorporated herein by reference in its entirety) and is typically used as an adjunct to pyridoxine and/or in a Met-restricted diet (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74; which is incorporated herein by reference in its entirety).

[0142] Betaine does not reverse the underlying CBS deficiency, but rather induces an alternative pathway that results in the remethylation of Hcy back to Met and correction of the partial misfolding of CBS mutants (see Kopecka et al. J Inherit Metab Dis 2011; 34: 39-48, which is hereby incorporated by reference in its entirety). In the presence of betaine, the enzyme betaine homocysteine methyltransferase (BHMT) remethylates Hcy to Met (see Singh et al. Genet Med 2004; 6:90-95, which is hereby incorporated by reference in its entirety), resulting in a partial reduction in Hcy levels and an increase in already highly elevated Met levels. Metabolites acting downstream of CBS are not effective upon betaine administration, and Cys supplementation may be required. Furthermore, betaine treatment has been associated with white matter abnormalities, a marker of cerebral vascular injury (see Prins et al. Nat Rev Neurol 2015; 11:157–165, which is hereby incorporated by reference in its entirety) in patients with and without acute cerebral edema (see Devlin et al. J Pediatr 2004, 144:545–548; Yaghmai et al. Am J Med Genet 2002; 108:57–63; which is hereby incorporated by reference in its entirety) and without such edema (see Vatanavicharn et al. J Inherit Metab Dis 2008; 31 Suppl 3:477–481; Brenton et al. J Child Neurol 2014; 29:88–92; Sasai et al. Tohoku J Exp Med 2015; 237:323-327; which is hereby incorporated by reference in its entirety). Although betaine treatment is known to exacerbate methionine accumulation, Schwann et al. showed that methionine is the main agent causing cerebral edema (see Schwahn, Bernd C., et al. JIMD reports 52.1 (2020): 3-10, which is hereby incorporated by reference in its entirety).

[0143] Betaine can be unpleasant to the taste (see Walter et al. Eur J Pediatr. 1998 Apr;157 Suppl 2:S71-6, which is hereby incorporated by reference in its entirety) and can cause an unpleasant fishy body and/or breath odor (see Manning et al., JIMD Rep 2012;5:71-75, which is hereby incorporated by reference in its entirety). Both effects are potentially exacerbated by the need for high doses (greater than 6 g/day in adults and children). Consequently, compliance with treatment is generally poor (see Adam et al. Mol Genet Metab. 2013 Dec;110(4):454-9; Walter et al. Eur J Pediatr. 1998 Apr;157 Suppl 2:S71-6; Sakamoto et al., Pediatr Int 2003;45:333-338, which are incorporated herein by reference in their entireties).

[0144] The betaine-based pharmaceutical, CYSTADANE™, was approved by the FDA in 2006 and is indicated for the reduction of elevated blood Hcy levels in homocystinuria, including CBS deficiency, 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency, and cobalamin cofactor metabolism (CBL) defects. (See Recordati. CYSTADANE™ Product Information (PI). 2017. Ref Type: Online Source, which is incorporated herein by reference in its entirety.)

[0145] The largest study to date of dietary treatment in patients with pyridoxine-unresponsive HCU has shown that betaine is a common treatment choice, particularly in late-diagnosed patients, adolescents, and adults. The use of betaine as primary therapy in the 34% of patients without diet is thought to be due to diet non-compliance, as there have been no controlled studies examining the long-term efficacy of betaine when administered without diet (see Adam et al. Mol Genet Metab 2013;110:454-459, which is incorporated herein by reference in its entirety). Indeed, a study in a mouse model of HCU showed that the effect of betaine treatment, which significantly lowers tHcy levels, diminished over time (see Maclean KN. Betaine treatment of cystathionine b-synthase-deficient homocystinuria; does it work and can it be improved? Dove press 2012; 2:23-33, which is incorporated herein by reference in its entirety).

4. Antiplatelet therapy

[0146] In addition to Hcy-lowering therapy, patients with poorly controlled Hcy levels and/or patients with additional risk factors for thrombosis (e.g., patients with factor V Leiden, a history of thrombosis, and pregnant patients) may be given antiplatelet agents (e.g., aspirin, dipyridamole, or clopidogrel) (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74; which is incorporated by reference in its entirety). COUMADIN™ blood thinners may also be given to patients with a history of venous thrombosis. However, anticoagulants are associated with an increased risk of cerebral hemorrhage, and the use of such anticoagulants should be determined on an individual patient basis (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74, which is incorporated herein by reference in its entirety).

5. Clinical results of modern treatment methods

[0147] The manifestations of HCU continue to progress with classic clinical symptoms, leading to varying degrees of disability and impacting the quality of life of patients. Regardless of the age at onset of the disease, loss of biochemical control at any age is associated with the development of serious complications that may be life-threatening (see Walter et al. Eur J Pediatr. 1998 Apr;157 Suppl 2:S71-6, which is incorporated herein by reference in its entirety).

[0148] Treatment should be continued throughout life, as periods of poor metabolic control have cumulative harmful consequences that can lead to severe complications and premature death (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74, which is incorporated herein by reference in its entirety).

[0149] Without treatment, the prognosis for pyridoxine-resistant HCU is poor, and the life expectancy of patients is markedly reduced. Since the disease was first described in 1962, no randomized controlled trials of dietary or other therapy for HCU have been conducted (see Carson et al. Arch Dis Child. 1969 Jun; 44(235):387-92; Gerritsen et al. Biochem Biophys. Res Commun. 1962 Dec 19;9:493-6, which is incorporated herein by reference in its entirety). However, some experimental studies have been published.

[0150] An international study documenting the pathogenesis of 629 untreated CBS patients showed that the risk of complications increases with age (see Mudd et al. Am J Hum Genet 1985; 37: 1-31, which is incorporated by reference in its entirety). Treatment (pyridoxine, Met-restricted diet) reduced plasma tHcy levels and markedly reduced the risk of thromboembolic complications and lens dislocation, even in the presence of poor dietary compliance.

[0151] Yap et al. (Arterioscler Thromb Vasc Biol. 2001 Dec;21(12):2080-5) conducted an international multicenter study of 158 treated patients. The incidence of cardiovascular disease was markedly reduced in this treatment group compared with historical controls (see Mudd et al. Am J Hum Genet 1985;37:1-31, which is incorporated by reference in its entirety). This apparent benefit correlated with lower (but not normalized) plasma tHcy levels in treated patients. It was noted that consistent adherence to the required regimen was very difficult to achieve.

[0152] Overall, although several treatment strategies for HCU are available, they fail to restore tHcy levels to near normal levels in most patients. Moreover, their long-term effectiveness is compromised by poor or inconsistent adherence to treatment throughout life. Thus, consistent Hcy reduction is difficult to maintain in patients with HCU. Treatment with the drug described herein aims to avoid many of these pitfalls. By compensating for the metabolic defect in HCU through a mechanism that does not require severe Met restriction or Cys supplementation, enzyme therapy (ET) is expected to result in more consistent Hcy reduction and a benign increase in Met levels.

B. Ways to improve cognitive function

[0153] The present patent application relates to methods for improving cognitive function in an individual suffering from or suspected of having homocystinuria. These methods may comprise administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising: a drug substance comprising an isolated cystathionine β-synthase (CBS) protein (SEQ ID NO: 1); a PEG molecule covalently linked to the CBS protein; and a pharmaceutically acceptable excipient, diluent, or adjuvant. In some aspects, combining an enzyme therapy comprising administering a pharmaceutical composition comprising the htCBS C15S protein as described herein with one or more therapies for improving cognitive or behavioral function may provide additional improvement in cognitive function or behavioral function in an individual suffering from or suspected of having homocystinuria. In some embodiments, treatment or therapy for neurobehavioral or cognitive disorders includes supports similar to those used for other executive function problems such as ADHD, including, for example, behavioral therapies such as behavioral parent training (BPT) and behavioral classroom management (BCM) (see, for example, Pelham Jr, William E., and Gregory A. Fabiano. “Evidence-based psychosocial treatments for attention-deficit/hyperactivity disorder.” Journal of Clinical Child & Adolescent Psychology 37.1 (2008): 184-214; and Pfiffner, Linda J., and Lauren M. Haack. “Behavior management for school-aged children with ADHD.” Child and Adolescent Psychiatric Clinics 23.4 (2014): 731-746, the contents of each of which are incorporated herein by reference in their entireties). Thus, the present invention relates to a combination therapy comprising the enzyme therapy described herein in combination with neurobehavioral or cognitive therapy to further improve cognitive function in individuals suffering from or suspected of having homocystinuria. It is expected that such a combination therapy will be effective in improving the patient's cognitive function, including cognitive development in children, to a greater extent than enzyme therapy or neurobehavioral/cognitive therapy alone.

[0154] Pharmaceutical preparations for use in methods of enhancing cognitive function may include ME-200GS, which may be administered as a PEG molecule. The pharmaceutical preparations may comprise the drug substance at a concentration of about 25.4 mg/mL, optionally in 5% sucrose, about 15 mM potassium phosphate; and about 8% (w/v) trehalose. The pharmaceutical preparations may be lyophilized.

C. Treatment options based on metabolic parameters

[0155] Also described herein are methods of treating an individual suffering from or suspected of having homocystinuria, the methods comprising: determining a level of one or more of creatinine, high-sensitivity C-reactive protein, fibrinogen, or protein C activity in the individual; comparing the one or more levels to a known range of values for one or more levels in a group of individuals known not to have homocystinuria or in a group of individuals known to have homocystinuria, or both; assessing disease progression or disease severity in the individual based on the one or more levels assessed in the individual; adjusting a dose of enzyme therapy for the individual based on the disease progression or disease severity; and administering the enzyme therapy to the individual. In these methods, performing enzyme therapy includes administering a medicament comprising an isolated cystathionine β-synthase (CBS) protein comprising or consisting of SEQ ID NO: 1, wherein the CBS protein has a PEG molecule covalently linked to the CBS protein.

[0156] In the methods described herein, methionine levels ≥600 μM can be used to identify and stratify individuals based on response to a methionine-restricted diet and/or betaine and/or vitamin supplementation in pediatric and adult patients with HCU. In some embodiments, methionine levels >1000 μM can be used to identify and stratify individuals based on response to a methionine-restricted diet and/or betaine and/or vitamin supplementation in pediatric and adult individuals with HCU.

[0157] Dimethylglycine (DMG) levels above the upper limit of normal (ULN) can be used to identify and stratify individuals based on response to betaine in pediatric and adult patients with HCU. Creatinine levels below the lower limit of normal (LLN) can be used to identify and stratify individuals who may benefit from enzyme therapy administered in accordance with the methods described herein. In some embodiments, high-sensitivity C-reactive protein (hsCRP) levels above the ULN can be used to identify and stratify individuals who may benefit from enzyme therapy administered in accordance with the methods described herein. In some embodiments, low levels of protein C activity and/or low levels of fibrinogen can be used to identify and stratify individuals who may benefit from enzyme therapy administered in accordance with the methods described herein.

[0158] The methods described herein may further include molecular genetic testing of the CBS gene in an individual, wherein identification of a mutation in the CBS gene indicates the severity of the disease or confirms a diagnosis of homocystinuria. Non-limiting examples of mutations of the CBS gene are provided in Table 3.

D. Methods to reduce skeletal fragility

[0159] In some embodiments, the present invention relates to methods for reducing skeletal fragility in an individual suffering from or suspected of having homocystinuria. The methods may comprise administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising: a drug substance comprising an isolated cystathionine β-synthase (CBS) protein (SEQ ID NO: 1); a PEG molecule covalently linked to the CBS protein; and a pharmaceutically acceptable excipient, diluent, or adjuvant.

[0160] Pharmaceutical compositions for use in methods for reducing skeletal frailty may comprise ME-200GS as a PEG molecule. The administered pharmaceutical compositions may comprise the drug substance at a concentration of about 25.4 mg/mL, optionally in 5% sucrose, about 15 mM potassium phosphate; and about 8% (w/v) trehalose. The pharmaceutical compositions may be lyophilized.

E. Ways to relieve cardiovascular symptoms

[0161] In some embodiments, the invention provides methods for reducing cardiovascular symptoms and vascular complications as described herein in an individual with or suspected of having homocystinuria (see van Meurs et al. N Engl J Med 2004; 350:2033-2041; Hankey et al., Lancet, 1999, 354:407-413, which are each incorporated by reference in their entireties). The methods may comprise administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising: a drug substance comprising an isolated cystathionine β-synthase (CBS) protein (SEQ ID NO: 1); a PEG moiety covalently linked to the CBS protein; and a pharmaceutically acceptable excipient, diluent, or adjuvant.

[0162] Pharmaceutical compositions for use in methods of reducing cardiovascular symptoms and vascular complications may comprise ME-200GS as a PEG molecule. The administered pharmaceutical compositions may include the drug substance at a concentration of about 25.4 mg/mL, optionally in 5% sucrose, about 15 mM potassium phosphate; and about 8% (w/v) trehalose. The pharmaceutical compositions may be lyophilized.

F. Methods for reducing elevated tHcy levels in a group of patients with a non-genetically inherited disease

[0163] The methods described herein involve treating a population of patients with a genetically non-heritable disorder associated with elevated tHcy levels. The terms "patient with a genetically non-heritable disorder" or "individual with a genetically non-heritable disorder" refer to one or more individuals who do not have or have not been diagnosed with a genetically inheritable deficiency of cystathionine β-synthase (e.g., who lack a missense mutation or a loss-of-function mutation in one or more alleles of the CBS gene).

[0164] Individuals with a non-genetically inherited disorder associated with elevated tHcy levels may have skeletal abnormalities or be at increased risk of developing skeletal abnormalities, such as increased incidence of bone fractures, decreased bone mineral density, and/or osteoporosis (see, e.g., Filip, Alexandru, et al. "The Relationship between Homocysteine and Fragility Fractures-A Systematic Review." Annual Research & Review in Biology (2017): 1-8; van Meurs et al. N Engl J Med 2004; 350:2033-2041; which are each incorporated by reference in their entireties). Thus, some embodiments of the methods described herein include treating or ameliorating skeletal symptoms associated with elevated tHcy levels by administering to the individual the pharmaceutical compositions or pharmaceutical preparations described herein. In some embodiments, administering the disclosed pharmaceutical compositions or pharmaceutical preparations to individuals with a non-genetically inherited disease associated with elevated tHcy levels reduces tHcy levels in the individuals. In some embodiments, administering the disclosed pharmaceutical compositions or pharmaceutical preparations to individuals with a non-genetically inherited disease associated with elevated tHcy levels reduces skeletal symptoms associated with elevated tHcy levels in the individual, including risk of bone fracture, decreased bone mineral density, and/or osteoporosis.

[0165] Individuals with a non-genetically inherited disorder associated with elevated tHcy levels may have cognitive symptoms or cognitive impairment or be at increased risk of developing cognitive symptoms or cognitive impairment, such as increased prevalence of cognitive decline, dementia, and/or Alzheimer's disease (see, e.g., Smith, A. David, et al. "Homocysteine and dementia: an international consensus statement." Journal of Alzheimer's Disease 62.2 (2018): 561–570; Smith, A. David, and Helga Refsum. "Homocysteine, B vitamins, and cognitive impairment." Annual review of nutrition 36 (2016): 211–239; Setién-Suero, Esther, et al. "Homocysteine and cognition: a systematic review of 111 studies." Neuroscience & Biobehavioral Reviews 69 (2016): 280-298; which are incorporated herein by reference in their entireties). Thus, some embodiments of the methods described herein involve treating or ameliorating cognitive symptoms associated with elevated tHcy levels by administering to an individual the pharmaceutical compositions or pharmaceutical formulations described herein. In some embodiments, the methods described herein involve treating or preventing or slowing the progression of neurodegenerative disorders associated with elevated tHcy levels, including Alzheimer's disease (see Farina, Nicolas, et al. "Homocysteine concentrations in the cognitive progression of Alzheimer's disease." Experimental Gerontology 99 (2017): 146-150, which is incorporated herein by reference in its entirety). In some embodiments, administering the disclosed pharmaceutical compositions or pharmaceutical preparations to individuals with a non-genetically inherited disease associated with elevated tHcy levels results in a decrease in tHcy levels in the individuals. In some embodiments, administering the disclosed pharmaceutical compositions or pharmaceutical preparations to individuals with a non-genetically inherited disease associated with elevated tHcy levels results in a decrease in cognitive symptoms associated with elevated tHcy levels in the individual, including cognitive decline, dementia, or Alzheimer's disease.

[0166] Individuals with a non-genetically inherited disease associated with elevated tHcy levels may have vascular disease or an increased risk of developing vascular disease (see, e.g., Kang, Soo-Sang, and Robert S. Rosenson. "Analytic approaches for the treatment of hyperhomocysteinemia and its impact on vascular disease." Cardiovascular drugs and therapy 32.2 (2018): 233-240, the contents of which are incorporated herein by reference in their entirety). Thus, some embodiments of the methods described herein involve treating, ameliorating, or preventing vascular disease.

[0167] Individuals with a non-genetically inherited disorder associated with elevated tHcy levels may have stroke or be at increased risk of developing stroke (see, e.g., Spence, J. David. "Homocysteine lowering for stroke prevention: unravelling the complexity of the evidence." International Journal of Stroke 11.7 (2016): 744-747, the entire contents of which are incorporated herein by reference). Thus, some embodiments of the methods described herein include treating, ameliorating, or preventing stroke.

[0168] Individuals with a non-genetically inherited disease associated with elevated tHcy levels may have eye diseases or an increased risk of developing eye diseases (see, for example, Ajith, Thekkuttuparambil Ananthanarayanan. "Homocysteine in ocular Diseases." Clinica Chimica Acta 450 (2015): 316-321, the entire contents of which are incorporated herein by reference). Thus, some embodiments of the methods described herein include treating, ameliorating, or preventing eye diseases.

[0169] Elevated tHcy levels can affect a wide range of metabolic and physiological systems. Accordingly, individuals with a non-genetically inherited disease may have a variety of phenotypic symptoms or have an increased risk of developing such symptoms, including, but not limited to, skeletal symptoms, cognitive symptoms, ocular disease, cardiovascular symptoms, and reproductive symptoms.

[0170] For example, women with a non-genetically inherited disorder associated with elevated tHcy levels may have a miscarriage or an increased risk of miscarriage (see Cavallé-Busquets, Pere, et al. "Moderately elevated first trimester fasting plasma total homocysteine is associated with increased probability of miscarriage. The Reus-Tarragona Birth Cohort Study." Biochimie (2020), the contents of which are incorporated herein by reference in their entirety). Accordingly, the methods described herein may avoid miscarriage in women with moderately elevated tHcy levels in the first trimester.

[0171] Individuals, including children and adolescents, with a non-genetically inherited disorder associated with elevated tHcy levels may have or be at increased risk for developing anxiety and depression (see Folstein, Marshal, et al. "The homocysteine hypothesis of depression." American Journal of Psychiatry 164.6 (2007): 861–867; Chung, Kuo-Hsuan, Hung-Yi Chiou, and Yi-Hua Chen. "Associations between serum homocysteine levels and anxiety and depression among children and adolescents in Taiwan." Scientific reports 7.1 (2017): 1–7; see also Mech, Arnold W., and Andrew Farah. "Correlation of clinical response with homocysteine reduction during therapy with reduced B vitamins in patients with MDD who are positive for MTHFR C677T or A1298C polymorphism: a randomized, double-blind, placebo-controlled study." The Journal of Clinical Psychiatry 77.5 (2016): 668-671; the contents of which are incorporated herein by reference in their entirety). Accordingly, the methods described herein may prevent the development of anxiety or depression in children and adolescents with elevated tHcy levels.

[0172] Individuals with a non-genetically inherited disorder associated with elevated tHcy levels may have or be at increased risk for renal complications and/or cardiovascular disease and related conditions, including coronary artery disease and stroke (see Guldener, Coen. "Why is homocysteine elevated in renal failure and what can be expected from homocysteine-lowering?." Nephrology Dialysis Transplantation 21.5 (2006): 1161–1166; Clarke, Robert, Sarah Lewington, and Martin Landray. "Homocysteine, renal function, and risk of cardiovascular disease." Kidney international 63 (2003): S131–S133; Yang, Hui-Fang, et al. "Does serum homocysteine explain the connection between sexual frequency and cardiovascular risk?" The Journal of Sexual Medicine 14.7 (2017): 1–1. 910-917; the contents of which are hereby incorporated by reference in their entirety). Accordingly, the methods described herein provide for the treatment or prevention of renal complications, including, for example, renal failure and chronic kidney disease, as well as cardiovascular diseases in individuals with elevated tHcy levels.

VI. Dosage and administration

[0173] In some embodiments, the drug product may be administered to the individual by subcutaneous (SC), intravenous (IV), or intraperitoneal (IP) injection. In one embodiment, the drug product may be administered to the individual 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In another embodiment, the drug product is administered more than 20 times. In another embodiment, the drug product is administered more than 100 times. Alternatively, the drug product may be administered throughout the life of the individual.

[0174] In some embodiments, the drug product may be administered repeatedly every 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, daily, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, and monthly. In some embodiments, the drug product is administered once every 3 days, once every 2 days, or once daily.

[0175] In some embodiments, the administration of the drug product may be a series of doses that may be administered at intervals of minutes, hours, days, or weeks. For example, the number of doses in the series may be 1, 2, 3, 4, 5, or 6. As a non-limiting example, the individual is administered 3 doses at intervals of 24 hours. As another non-limiting example, the individual is administered 5 doses at intervals of 12 hours. The individual may be a human.

[0176] In some embodiments of the invention, the administration of the medicinal product may be carried out according to a schedule of administration of a series of doses with a break between the administration of the first series and the second series of doses. The interval between doses may be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months. The number of doses in a series may be 2, 3, 4, 5, or 6. As a non-limiting example, an individual may be administered a first series of 5 doses 12 hours apart, and then 14 days after the first dose, the individual may be administered a second series of 5 doses 12 hours apart. As another non-limiting example, an individual is administered two series of doses over 8 weeks, where the first series is a dose administered twice a week for two weeks, and the second series is a dose administered three times a week for 6 weeks.

[0177] In some embodiments, the drug product may be administered at least once after the betaine has been administered to the individual. The time between the administration of betaine and the administration of the drug can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months or 18 months. As a non-limiting example, the medicinal product may be administered 14 days after the individual has been administered betaine. As another non-limiting example, the medicinal product may be administered to the individual in two doses after the individual has been administered betaine. As another non-limiting example, the medicinal product may be administered 14 or 15 days after the administration of betaine.

[0178] In some embodiments, a drug product may be administered to an individual in combination with betaine. In some embodiments, administering betaine involves self-administering an over-the-counter supplement. In some embodiments, the individual is prescribed betaine, such as cystadan. Administering betaine may involve administering various supplements or drug formulations, which may include betaine HCl, trimethylglycine, betaine anhydrous, or other forms. The combination of betaine and a drug formulation described herein may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 times. In some embodiments, the drug formulation may be administered in combination with betaine more than 15 times. Additional combination therapies that may be prescribed to the patient include administration of the drug product and at least one course of treatment to lower tHcy levels, such as a very low protein/Met diet and/or vitamins/supplements.

[0179] In some embodiments, the dosage of a drug administered to an individual is determined based on characteristics of the individual, such as age, gender, and body weight. For example, the drug may be prepared in a dosage form, such as a vial, wherein the amount of the drug in the dosage form is predetermined based on the age of the individual, the weight of the individual, and/or the gender of the individual. The drug may be administered in different dosage forms over a set period of time depending on the age, weight, gender, and/or other characteristics of the individual to adjust the effect of the drug on the individual over time. The dosage form of the drug may be provided in any therapeutically effective amount.

[0180] In some embodiments, the dosage of the drug administered to the individual can be from about 0.25 mg/kg to about 10 mg/kg. For example, the dosage is about 0.33 mg/kg, about 0.66 mg/kg, 1.0 mg/kg, or 1.5 mg/kg. Alternatively, the dosage is about 2 mg/kg, about 7 mg/kg, and about 10 mg/kg. For example, the dosage can be about 0.5 mg/kg. Alternatively, the therapeutically effective amount is a dose selected from the range of about 5.0 mg/kg to about 50 mg/kg and about 10.0 mg/kg to about 25 mg/kg. For example, the dosage is selected from the group consisting of: about 0.25 mg/kg, about 0.33 mg/kg, about 0.66 mg/kg, about 1.00 mg/kg, about 1.10 mg/kg, approximately 1.20 mg/kg, approximately 1.30 mg/kg, approximately 1.40 mg/kg, approximately 1.50 mg/kg, approximately 1.60 mg/kg, approximately 1.70 mg/kg, approximately 1.80 mg/kg, approximately 1.90 mg/kg, approximately 2.00 mg/kg, approximately 3.00 mg/kg, approximately 4.00 mg/kg, approximately 5.00 mg/kg, approximately 6.00 mg/kg, approximately 7.00 mg/kg, approximately 8.00 mg/kg, approximately 9.00 mg/kg, approximately 10.0 mg/kg, approximately 11.0 mg/kg, approximately 12.0 mg/kg, approximately 13.0 mg/kg, approximately 14.0 mg/kg, approximately 15.0 mg/kg, approximately 16.0 mg/kg, approximately 17.0 mg/kg, approximately 18.0 mg/kg, approximately 19.0 mg/kg, approximately 20.0 mg/kg, approximately 21.0 mg/kg, approximately 22.0 mg/kg, approximately 23.0 mg/kg, approximately 24.0 mg/kg, approximately 25.0 mg/kg, approximately 26.0 mg/kg, approximately 27.0 mg/kg, approximately 28.0 mg/kg, approximately 29.0 mg/kg, approximately 30.0 mg/kg, approximately 31.0 mg/kg, approximately 32.0 mg/kg, approximately 33.0 mg/kg, approximately 34.0 mg/kg, approximately 35.0 mg/kg, approximately 36.0 mg/kg, approximately 37.0 mg/kg, approximately 38.0 mg/kg, approximately 39.0 mg/kg, approximately 40.0 mg/kg, approximately 41.0 mg/kg, approximately 42.0 mg/kg, about 43.0 mg/kg, about 44.0 mg/kg, about 45.0 mg/kg, about 46.0 mg/kg, about 47.0 mg/kg, about 48.0 mg/kg, about 49.0 mg/kg, and about 50.0 mg/kg. The doses described herein may be administered once a day or, in some embodiments, twice a day.

[0181] In some embodiments, the drug is administered to an individual on a methionine-restricted diet. Alternatively, the drug is administered to an individual who is not on a methionine-restricted diet. In some embodiments, the drug is administered to an individual with elevated tHcy levels. In some embodiments, the individual is a patient with genetically non-heritable HCU.

[0182] In some embodiments, the drug may be administered together with another therapeutic agent for the treatment of HCU. As used herein, the term "co-administration" means the administration of two or more components. These co-administration components include, but are not limited to, betaine or vitamin B6. Co-administration means administration of two or more components simultaneously or with an interval of time between administrations, such as 1 second, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 1.5 days, 2 days or 3 days. In some embodiments of the invention, the time interval between administration of two or more components exceeds 3 days.

[0183] In some embodiments, the drug may be used as a parenteral agent for chronic administration to patients via subcutaneous (SC) injection with an initial dosing interval of once per week. For example, 6 doses of the drug are administered weekly. In some embodiments, the individual may be between 18 and 65 years of age. In some embodiments, the same treatment may be administered to an individual at least 16 years of age.

[0184] In some embodiments, the administration is for 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days. In some embodiments, the administration is for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks. 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks or 52 weeks.

[0185] In some embodiments, individuals with CBS deficiency, including patients with genetically inherited HCU or patients with genetically non-heritable HCU with elevated tHcy levels or with CBS deficiency, are administered various doses for treatment as described herein based on the level(s) of one or more metabolic indicators or other indicators or based on the severity or progression of the disease. For example, an individual may be administered a dose of 20NHS PEG-CBS within a proposed therapeutic dose range (e.g., 0.25 to 10 mg/kg twice daily) based on a determination of the individual's tHcy level or other metabolic indicator level. Thus, individuals may be stratified according to the metabolic indices of disease severity or progression described herein and may be administered a dose of 20NHS PEG-CBS depending on the severity or progression of the disease.

[0186] As a non-limiting example, the tHcy level in an individual with CBS deficiency may be determined. The estimated tHcy level may be stratified as slightly above low, moderate, or high.

[0187] In some embodiments, tHcy levels slightly above low are in the range of about 10 μmol/L to about 50 μmol/L, i.e., about 10 μmol/L, about 15 μmol/L, about 20 μmol/L, about 25 μmol/L, about 30 μmol/L, about 35 μmol/L, about 40 μmol/L, about 45 μmol/L, or about 50 μmol/L.

[0188] In some embodiments, the slightly above average tHcy levels are in the range of about 50 μmol/L to about 100 μmol/L, i.e., about 50 μmol/L, about 55 μmol/L, about 60 μmol/L, about 65 μmol/L, about 70 μmol/L, about 75 μmol/L, about 80 μmol/L, about 85 μmol/L, about 90 μmol/L, about 95 μmol/L, or about 100 μmol/L.

[0189] In some embodiments, tHcy levels slightly above high are in the range of about 100 μmol/L or higher, such as about 100 μmol/L to about 1000 μmol/L or about 100 μmol/L to about 500 μmol/L; that is, about 100 μmol/L, about 110 μmol/L, about 120 μmol/L, about 130 μmol/L, about 140 μmol/L, about 150 μmol/L, about 160 μmol/L, about 170 μmol/L, about 180 μmol/L, about 190 μmol/L, about 200 μmol/L, about 210 μmol/L, about 220 μmol/L, about 230 μmol/L, about 240 μmol/L, about 250 μmol/L, about 260 μmol/L, about 270 μmol/L, about 280 μmol/L, about 290 μmol/L, about 300 μmol/L, about 310 μmol/L, about 320 μmol/L, about 330 μmol/L, approximately 340 μmol/L, approximately 350 μmol/L, approximately 360 μmol/L, approximately 370 μmol/L, approximately 380 μmol/L, approximately 390 μmol/L, approximately 400 μmol/L, approximately 410 μmol/L, approximately 420 μmol/L, approximately 430 μmol/L, approximately 440 μmol/L, approximately 450 μmol/L, approximately 460 μmol/L, approximately 470 μmol/L, approximately 480 μmol/L, approximately 490 μmol/L or approximately 500 μmol/L.

[0190] In some embodiments, a dose of 20NHS PEG-CBS within the expected therapeutic dose range (0.25 to 10 mg/kg) is administered to the individual based on the determination of the individual's tHcy level as slightly above a low, medium, or high level. For example, the individual may be administered a low dose, a medium dose, or a high dose, respectively. In some embodiments, the low dose of 20NHS PEG-CBS is from about 0.25 mg/kg to about 1.0 mg/kg, such as about 0.25 mg/kg, about 0.50 mg/kg, about 0.75 mg/kg, or about 1 mg/kg. In some embodiments, the mid dose of 20NHS PEG-CBS is from about 0.5 mg/kg to about 1.5 mg/kg, such as about 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/kg, or about 1.5 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS is from about 1 mg/kg to about 2 mg/kg, such as about 1 mg/kg, about 1.25 mg/kg, about 1.50 mg/kg, about 1.75 mg/kg, or about 2 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS comprises a dose greater than about 2 mg/kg, such as about 2.25 mg/kg, about 2.50 mg/kg, about 2.75 mg/kg, about 3 mg/kg, about 3.25 mg/kg, about 3.50 mg/kg, about 3.75 mg/kg, about 4 mg/kg, about 4.25 mg/kg, about 4.50 mg/kg, about 4.75 mg/kg, or about 5 mg/kg or higher.

[0191] In some embodiments, the drug is administered as a combination therapy with pyridoxine (also referred to as vitamin B6) and/or antiplatelet therapy. In some embodiments, vitamin B12 is administered as a combination therapy with pyridoxine and/or antiplatelet therapy. In some embodiments, folate/folic acid is administered as a combination therapy with pyridoxine and/or antiplatelet therapy. Accordingly, some embodiments of the methods of treatment described herein comprise administering the drug described herein together with one or more of pyridoxine, antiplatelet agents, vitamin B12, and/or folate/folic acid.

VII. Patient stratification

[0192] As used herein, the term "patient stratification" refers to the use of clinical, biochemical, molecular, behavioral, cognitive, or other indicators of disease progression or disease severity in patients with or suspected of having HCU. In some embodiments, stratifying patients according to clinical, biochemical, molecular, behavioral, cognitive, or other indicators of disease progression or disease severity comprises quantitatively or qualitatively recording one or more clinical, biochemical, molecular, behavioral, cognitive, or other indicators of disease progression or disease severity in a patient or a group of patients and ranking the patient or group of patients on a disease progression or severity scale according to the recorded qualitative or quantitative indicators for the patient or group of patients compared to corresponding quantitative or qualitative data or observations from a group of healthy patients (i.e., a group of patients known not to have HCU) and/or a control group of patients known to have HCU. Stratification of patients according to the methods described herein may facilitate clinical diagnosis, research and/or treatment of CSBDH in various aspects. For example, stratification of patients based on clinical, biochemical, molecular, behavioral, cognitive or other indicators of disease progression or disease severity described herein may serve as criteria for inclusion of individuals in a clinical trial program, determination of therapeutic doses, determination of a treatment regimen and/or information on additional treatments or therapies that may be administered in combination with the enzyme therapy methods described herein to alleviate a patient's symptoms and/or improve a patient's quality of life. In some embodiments, individuals suitable for inclusion in an effective enzyme therapy program using a medicament described herein are patients diagnosed with HCU based on confirmation of genetic CBS-deficiency homocystinuria using a CBS gene mutation assay and a plasma tHcy level greater than or equal to 80 μM. In some embodiments of the invention, individuals suitable for inclusion in a clinical trial program for enzyme therapy using a medicament described herein are patients diagnosed with HCU based on confirmation of genetic CBS-deficient homocystinuria by CBS gene mutation testing and a plasma tHcy level greater than or equal to 80 μM.

[0193] In some embodiments, individuals with CBS deficiency are administered various therapeutic doses, as described herein, based on the level(s) of one or more metabolic indicators. For example, an individual may be administered a dose of 20NHS PEG-CBS within a proposed therapeutic dose range (e.g., 0.25 to 10 mg/kg twice daily) based on an assessment of the individual's tHcy level or another metabolic indicator. Thus, individuals may be stratified based on the metabolic indicators of disease severity or progression described herein and may be administered a dose of 20NHS PEG-CBS based on the severity or progression of the disease.

[0194] As a non-limiting example, the tHcy level in an individual with CBS deficiency may be determined. The estimated tHcy level may be stratified as slightly above low, moderate, or high.

[0195] In some embodiments, tHcy levels slightly above low are in the range of about 10 μmol/L to about 50 μmol/L, such as about 10 μmol/L, about 15 μmol/L, about 20 μmol/L, about 25 μmol/L, about 30 μmol/L, about 35 μmol/L, about 40 μmol/L, about 45 μmol/L, or about 50 μmol/L.

[0196] In some embodiments, the slightly above average tHcy levels are in the range of about 50 μmol/L to about 100 μmol/L, i.e., about 50 μmol/L, about 55 μmol/L, about 60 μmol/L, about 65 μmol/L, about 70 μmol/L, about 75 μmol/L, about 80 μmol/L, about 85 μmol/L, about 90 μmol/L, about 95 μmol/L, or about 100 μmol/L.

[0197] In some embodiments, tHcy levels slightly above high are in the range of about 100 μmol/L or higher, such as about 100 μmol/L to about 1000 μmol/L or about 100 μmol/L to about 500 μmol/L; that is, about 100 μmol/L, about 110 μmol/L, about 120 μmol/L, about 130 μmol/L, about 140 μmol/L, about 150 μmol/L, about 160 μmol/L, about 170 μmol/L, about 180 μmol/L, about 190 μmol/L, about 200 μmol/L, about 210 μmol/L, about 220 μmol/L, about 230 μmol/L, about 240 μmol/L, about 250 μmol/L, about 260 μmol/L, about 270 μmol/L, about 280 μmol/L, about 290 μmol/L, about 300 μmol/L, about 310 μmol/L, about 320 μmol/L, about 330 μmol/L, approximately 340 μmol/L, approximately 350 μmol/L, approximately 360 μmol/L, approximately 370 μmol/L, approximately 380 μmol/L, approximately 390 μmol/L, approximately 400 μmol/L, approximately 410 μmol/L, approximately 420 μmol/L, approximately 430 μmol/L, approximately 440 μmol/L, approximately 450 μmol/L, approximately 460 μmol/L, approximately 470 μmol/L, approximately 480 μmol/L, approximately 490 μmol/L or approximately 500 μmol/L.

[0198] In some embodiments, the individual is administered a dose of 20NHS PEG-CBS within the intended therapeutic dose range (e.g., 0.25 to 10 mg/kg twice daily) based on the individual's estimated tHcy level being slightly above low, medium, or high. For example, the individual may be administered a low dose, medium dose, or high dose, respectively. In some embodiments, the low dose of 20NHS PEG-CBS is from about 0.25 mg/kg to about 1.0 mg/kg, such as about 0.25 mg/kg, about 0.50 mg/kg, about 0.75 mg/kg, or about 1 mg/kg. In some embodiments, the mid dose of 20NHS PEG-CBS is from about 0.5 mg/kg to about 1.5 mg/kg, such as about 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/kg, or about 1.5 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS is from about 1 mg/kg to about 2 mg/kg, such as about 1 mg/kg, about 1.25 mg/kg, about 1.50 mg/kg, about 1.75 mg/kg, or about 2 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS comprises a dose greater than about 2 mg/kg, such as about 2.25 mg/kg, about 2.50 mg/kg, about 2.75 mg/kg, about 3 mg/kg, about 3.25 mg/kg, about 3.50 mg/kg, about 3.75 mg/kg, about 4 mg/kg, about 4.25 mg/kg, about 4.50 mg/kg, about 4.75 mg/kg, or about 5 mg/kg or higher.

A. Clinical picture

[0199] According to the Guidelines for the Diagnosis and Management of HCU, the disease should be suspected in children with severe or rapidly progressive myopathy, lens dislocation, and/or developmental delay (see Morris et al. Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J Inherit Metab Dis 2017;40:49-74, which is incorporated herein by reference in its entirety). Tests should also be performed in adults with thromboembolism and/or lens dislocation but no other symptoms, and in patients with multisystem disease including ocular disease, connective tissue disease, neuropsychiatric disease, and vascular complications (see Mudd et al. Am J Hum Genet 1985; 37:1–31; Morris et al. Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J Inherit Metab Dis 2017; 40:49–74; Kelly et al. Neurology 2003; 60:275–279, each of which is incorporated by reference in its entirety).

B. Biochemical analysis

1. Total homocysteine levels

[0200] Plasma tHcy levels are determined by the sum of all free and bound homocysteine species after plasma treatment with a reducing agent. In healthy individuals with stable dietary habits, tHcy levels remain relatively constant over time (see Refsum et al. Clin Chem 2004; 50:3-32; McKinley et al. Clin Chem 2001; 47:1430-1436; each of which is incorporated herein by reference in its entirety). However, consumption of a protein-rich meal can increase tHcy levels by approximately 10% for several hours (see Verhoef et al. Am J Clin Nutr 2005; 82:553-558, which is incorporated herein by reference in its entirety). A study in individuals with hyperhomocysteinemia (tHcy>40 μmol/L) showed that individual tHcy levels varied by up to 25% over a period of 4 to 8 months. However, no information was provided on the variation in diets, assay methods, or sampling times, making interpretation of the data difficult (see Refsum H, Smith AD, Ueland PM et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin Chem 2004; 50:3-32, which is incorporated by reference in its entirety).

[0201] In patient groups not receiving folate supplementation, the corresponding upper baseline limits are approximately 15 and 20 μmol/L, respectively. To confirm the diagnosis of CBS in neonates, the expected plasma tHcy level should be between 50 and greater than 100 μmol/L and the plasma Met level should be between 200 and 1500 μmol/L (i.e., 3-23 mg/dL) (see Sacharow et al. Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. GeneReviews 2017, which is incorporated herein by reference in its entirety). To confirm the diagnosis of CBS in untreated older adults, the expected plasma tHcy level is greater than 100 μmol/L and the plasma Met level is greater than 50 μmol/L (i.e., greater than 0.7 mg/dL). In a control neonate or older adult, the expected plasma tHcy level is less than 15 μmol/L and the Met level is between 10 and 40 μmol/L (0.2 and 0.6 mg/dL).

[0202] In some embodiments, individuals with elevated tHcy and/or CBS deficiency are administered various therapeutic doses as described herein based on the tHcy level(s). For example, an individual may be administered a dose of 20NHS PEG-CBS within a predicted therapeutic dose range (e.g., 0.25 to 10 mg/kg) based on an assessment of the individual's tHcy level. Thus, individuals may be stratified according to the metabolic indices of disease severity or progression described herein and may be administered a dose of 20NHS PEG-CBS based on the severity or progression of the disease.

[0203] As a non-limiting example, the tHcy level in an individual with CBS deficiency may be assessed. The assessed tHcy level may be stratified as slightly above low, moderate, or high.

[0204] In some embodiments, the slightly above low tHcy levels are in the range of about 10 μmol/L to about 50 μmol/L, such as about 10 μmol/L, about 15 μmol/L, about 20 μmol/L, about 25 μmol/L, about 30 μmol/L, about 35 μmol/L, about 40 μmol/L, about 45 μmol/L, or about 50 μmol/L.

[0205] In some embodiments, the slightly above average tHcy levels are in the range of about 50 μmol/L to about 100 μmol/L, i.e., about 50 μmol/L, about 55 μmol/L, about 60 μmol/L, about 65 μmol/L, about 70 μmol/L, about 75 μmol/L, about 80 μmol/L, about 85 μmol/L, about 90 μmol/L, about 95 μmol/L, or about 100 μmol/L.

[0206] In some embodiments, tHcy levels slightly above high are in the range of about 100 μmol/L or higher, such as about 100 μmol/L to about 1000 μmol/L or about 100 μmol/L to about 500 μmol/L; that is, about 100 μmol/L, about 110 μmol/L, about 120 μmol/L, about 130 μmol/L, about 140 μmol/L, about 150 μmol/L, about 160 μmol/L, about 170 μmol/L, about 180 μmol/L, about 190 μmol/L, about 200 μmol/L, about 210 μmol/L, about 220 μmol/L, about 230 μmol/L, about 240 μmol/L, about 250 μmol/L, about 260 μmol/L, about 270 μmol/L, about 280 μmol/L, about 290 μmol/L, about 300 μmol/L, about 310 μmol/L, about 320 μmol/L, about 330 μmol/L, approximately 340 μmol/L, approximately 350 μmol/L, approximately 360 μmol/L, approximately 370 μmol/L, approximately 380 μmol/L, approximately 390 μmol/L, approximately 400 μmol/L, approximately 410 μmol/L, approximately 420 μmol/L, approximately 430 μmol/L, approximately 440 μmol/L, approximately 450 μmol/L, approximately 460 μmol/L, approximately 470 μmol/L, approximately 480 μmol/L, approximately 490 μmol/L or approximately 500 μmol/L.

[0207] In some embodiments, a dose of 20NHS PEG-CBS within the expected therapeutic dose range (0.25 to 10 mg/kg) is administered to the individual based on an assessment of the individual's tHcy level as being slightly above low, medium, or high. For example, the individual may be administered a low dose, medium dose, or high dose, respectively. In some embodiments, the low dose of 20NHS PEG-CBS is from about 0.25 mg/kg to about 1.0 mg/kg, such as about 0.25 mg/kg, about 0.50 mg/kg, about 0.75 mg/kg, or about 1 mg/kg. In some embodiments, the mid dose of 20NHS PEG-CBS is from about 0.5 mg/kg to about 1.5 mg/kg, such as about 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/kg, or about 1.5 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS is from about 1 mg/kg to about 2 mg/kg, such as about 1 mg/kg, about 1.25 mg/kg, about 1.50 mg/kg, about 1.75 mg/kg, or about 2 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS comprises a dose greater than about 2 mg/kg, such as about 2.25 mg/kg, about 2.50 mg/kg, about 2.75 mg/kg, about 3 mg/kg, about 3.25 mg/kg, about 3.50 mg/kg, about 3.75 mg/kg, about 4 mg/kg, about 4.25 mg/kg, about 4.50 mg/kg, about 4.75 mg/kg, or about 5 mg/kg or higher.

2. Methionine levels

[0208] High to above-normal Met levels (reference ranges typically 40 to 45 and 12 to 15 μmol/L, respectively) in combination with low to below-normal Cth levels (reference ranges 0.05 to 0.08 and 0.35 to 0.5 μmol/L, respectively) may be useful in distinguishing HCU from HCU caused by genetic and dietary defects in Hcy remethylation (see Morris et al. J Inherit Metab Dis 2017; 40:49-74; Stabler et al. JIMD Rep 2013; 11:149-163; Bartl et al., Clin Chim Acta 2014; 437:211-217, each of which is hereby incorporated by reference in its entirety). Another suitable assay measures the production of Cth from Hcy and serine in cultured fibroblasts using radioactive or deuterium-labeled substrates (see Morris et al. J Inherit Metab Dis 2017; 40:49-74; Kraus JP. Methods Enzymol 1987; 143:388-394; Smith et al., J Chromatogr B Analyt Technol Biomed Life Sci 2012; 911:186-191, each of which is incorporated by reference in its entirety). However, enzyme assays do not always distinguish between pyridoxine-sensitive and -resistant individuals, and in mild cases, enzyme activity may be normal (Alcaide et al. Clin Chim Acta 2015; 438:261-265, which is incorporated by reference in its entirety). More recently, rapid stable isotope assays that measure the activity of CBS released from organs into plasma have shown 100% sensitivity in pyridoxine-non-responders and only 86% sensitivity in pyridoxine-responsive patients (Alcaide et al. Clin Chim Acta 2015; 438:261). 265; Krijt et al., J Inherit Metab Dis 2011;34:49-55, which are hereby incorporated by reference in their entirety).

[0209] Methionine levels ≥600 μM, and in some cases greater than ≥1000 μM, and dimethylglycine (DMG) levels above the upper limit of normal (ULN) are observed in significant amounts even in patients treated with a natural protein-restricted diet and/or a Met-free L-amino acid mixture and supplements including betaine, as described in Examples 4-5 and shown in Table 5 and Fig. 1. These data indicate that current diet and medical treatment have little effect. In particular, contrary to popular belief, betaine alone is not an adequate treatment for HCU. Further studies are needed to understand the relationship between patient response to standard treatment and elevated methionine, DMG, and other biochemical parameters. In some embodiments of the methods described herein, assessing and monitoring levels of various biochemical markers may be useful for identifying and stratifying individuals with HCU in terms of disease severity and patient response to treatment.

[0210] In some embodiments, individuals with CBS deficiency are administered various therapeutic doses as described herein based on their methionine level(s). For example, an individual may be administered a dose of 20NHS PEG-CBS within the expected therapeutic dose range (0.25 to 10 mg/kg) based on an assessment of the individual's methionine level. Thus, individuals may be stratified based on the metabolic indices of disease severity or progression described herein and may be administered a dose of 20NHS PEG-CBS based on the severity or progression of the disease.

[0211] As a non-limiting example, the tHcy level in an individual with CBS deficiency can be assessed. The assessed tHcy level can be stratified as slightly above low or high. In some embodiments, the slightly above low methionine level is from about 10 μmol/L to about 100 μmol/L, and the slightly above high methionine level is from about 100 μmol/L to about 500 μmol/L or above about 150 μmol/L.

3. Creatinine levels

[0212] Creatinine levels below the lower limit of normal (LLN) can be used to identify and stratify HCU in pediatric and adult patients, including stratification based on the patient's response to a methionine-restricted diet and/or to betaine and/or vitamin supplementation. Decreased creatinine levels may be associated with low muscle mass following protein restriction in individuals on a protein-restricted diet. Thus, creatinine levels below the LLN can be used to monitor and stratify individuals who have been placed on a restricted diet and/or individuals who are on such a diet.

4. C-reactive protein levels

[0213] High-sensitivity C-reactive protein (hsCRP) levels above the ULN can be used to identify and stratify HCU in patients, including stratification based on the patient's response to a methionine-restricted diet and/or betaine and/or vitamin supplementation.

[0214] Low levels of protein C activity and/or low fibrinogen levels can be used to identify and stratify HCU in patients, including stratification based on the patient's response to a methionine-restricted diet and/or betaine and/or vitamin supplementation.

5. Other metabolic indicators

[0215] More precise biochemical analysis using a number of biochemical markers, individually or in combination, can be performed to stratify patients for the diagnosis or treatment of HCU (see Example 5, Table 5, Fig. 1). Alternatively, biochemical markers can be used to monitor a patient's response to traditional non-ET therapies. The ET treatment methods described herein can, in some cases, correct increases or decreases in these biochemical markers outside of normal ranges in individuals on a methionine-restricted diet or in individuals taking vitamin or betaine supplements. In addition to elevated tHcy and Met levels and low cystathionine and total cysteine levels, the following biochemical markers can help identify and stratify HCU in individuals, such as based on a patient's response to a restricted diet and/or betaine and vitamin supplement treatment methods.

[0216] In some embodiments, methionine levels, DMG levels, ALT levels, creatinine levels, hsCRP levels, and/or protein C activity levels can be used individually or in combination to identify and stratify HCU in patients, including stratification based on the patient's response to a methionine-restricted diet and/or betaine and/or vitamin supplementation. Using multiple of these biochemical indices can improve the sensitivity and accuracy of diagnosis and stratification. These biochemical indices can be further combined with biochemical indices that are known to be within normal limits in patients with HCU. For example, normal levels of aspartate aminotransferase (AST), antithrombin III, and apolipoprotein A can be used to further identify and stratify patients with HCU.

6. Correction of CBS deficiency treatment according to metabolic parameters

[0217] In some embodiments, the treatment of CBS deficiency as described herein may be dose adjusted based on levels of one or more metabolic indicators of disease severity or progression provided herein. For example, if the assessment or determination of one or more metabolic indicators of disease progression or severity indicates severe or marked progression of CBS deficiency, then in the treatment of CBS deficiency, the 20NHS PEG-CBS preparation may be administered at a low dose, at a medium dose, or at a high dose. In some embodiments, the low dose of 20NHS PEG-CBS is from about 0.25 mg/kg to about 1.0 mg/kg, such as about 0.25 mg/kg, about 0.50 mg/kg, about 0.75 mg/kg, or about 1 mg/kg. In some embodiments, the mid dose of 20NHS PEG-CBS is from about 0.5 mg/kg to about 1.5 mg/kg, such as about 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/kg, or about 1.5 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS is from about 1 mg/kg to about 2 mg/kg, such as about 1 mg/kg, about 1.25 mg/kg, about 1.50 mg/kg, about 1.75 mg/kg, or about 2 mg/kg. In some embodiments, the high dose of 20NHS PEG-CBS comprises a dose greater than about 2 mg/kg, such as about 2.25 mg/kg, about 2.50 mg/kg, about 2.75 mg/kg, about 3 mg/kg, about 3.25 mg/kg, about 3.50 mg/kg, about 3.75 mg/kg, about 4 mg/kg, about 4.25 mg/kg, about 4.50 mg/kg, about 4.75 mg/kg, or about 5 mg/kg or higher.

C. Molecular diagnostics

[0218] Molecular genetic testing can be accomplished by testing either a single gene or a panel of multiple genes (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Sacharow SJ, Picker JD, Levy HL. Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. GeneReviews 2017; Morris et al. J Inherit Metab Dis 2017; 40:49-74; Katsanis et al. Nat Rev Genet 2013; 14:415-426; each of which is incorporated herein by reference in its entirety). Individuals at high risk for having a particular CBS mutation should be screened with targeted single gene testing. However, this is only useful for select groups with a common CBS mutation (e.g., 93% of individuals with HCU from Qatar have the p.Arg336Cys; c.1006C>T mutation) and for individuals from families with a known pathogenic variant. In other patients, the CBS gene can be sequenced and target gene deletion/duplication testing can be performed only if one or no pathogenic variant is detected. Table 3 provides a non-limiting list of genetic mutations that can be used to identify and stratify HCU in patients.

[0219] Alternatively, simultaneous molecular testing of multiple genes can be accomplished using a multigene panel. Methods used may include sequence analysis, deletion/duplication analysis, and other non-sequencing-based tests (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is hereby incorporated by reference in its entirety). Typically, molecular genetic testing is reserved for high-risk groups with a limited number of common mutations (see Morris et al. J Inherit Metab Dis 2017; 40:49-74; Huemer et al. J Inherit Metab Dis 2015; 38:1007-101, each of which is hereby incorporated by reference in its entirety).

D. Pyridoxine sensitivity tests

[0220] In the clinic, pyridoxine sensitivity testing is performed to determine whether pyridoxine supplementation should be given to patients with HCU. Because different treatment centers define pyridoxine sensitivity differently (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is incorporated by reference in its entirety), classifying patients by tHcy levels rather than by their pyridoxine sensitivity is more stringent. Pyridoxine response is not a measure of metabolic control, but rather indicates that some residual CBS activity remains.

E. Newborn screening (NBS)

[0221] Typically, NBS tests for HCU deficiency are performed by analyzing dried blood spots to determine Met levels. Alternatively, assessment of tHcy, rather than Met, levels in dried blood spots for NBS is available at several Centers worldwide. This test is used as a second-tier test to reduce the rate of false-positive NBS results in individuals with high Met levels (see Turgeon et al. Clin Chem 2010; 56:1686-1695, which is incorporated herein by reference in its entirety) and is not used to improve sensitivity or reduce the rate of false-negative results.

F. Cognitive functions

[0222] Patients with lower plasma tHcy levels have better performance on executive function. The NIH Instrumentation Suite or other neurobehavioral or cognitive assessment methods can be used to assess and stratify patients according to their cognitive performance over time, including monitoring treatment response. Correlations between cognitive performance and tHcy levels can also be used to stratify patients (see, for example, Table 9, Fig. 3, and Fig. 4). Reduced tHcy levels in an individual following ET as described herein can be used as an indicator that the individual will have an improved outcome from treatment for a neurobehavioral or cognitive disorder. On the other hand, elevated tHcy levels may indicate that the individual needs or may benefit from treatment for a neurobehavioral or cognitive disorder in combination with ET as described herein. For example, tHcy levels >50 μM, >60 μM, >70 μM, >80 μM, >90 μM, or >100 μM may be used as a threshold for assessing and/or monitoring neurobehavioral and cognitive function and/or as an indicator of patient responsiveness or need for treatment of neurobehavioral or cognitive disorders or in the treatment of patients with or suspected of having HCU. In some embodiments, treatment of neurobehavioral or cognitive disorders includes supports similar to those used for other executive function problems such as ADHD, including, for example, behavioral therapies such as parent behavior training (BPT) and behavior management in the classroom (BCM) (see, for example, Pelham Jr, William E., and Gregory A. Fabiano. “Evidence-based psychosocial treatments for attention-deficit/hyperactivity disorder.” Journal of Clinical Child & Adolescent Psychology 37.1 (2008): 184-214; and Pfiffner, Linda J., and Lauren M. Haack. “Behavior management for school-aged children with ADHD.” Child and Adolescent Psychiatric Clinics 23.4 (2014): 731-746, the contents of each of which are hereby incorporated by reference in their entireties).

G. Stratification of patients with a non-genetically inherited disease

[0223] The compositions and methods described herein can be used to treat or ameliorate complications or conditions associated with elevated tHcy levels, including in individuals or groups of individuals with non-genetically inherited diseases associated with elevated tHcy levels.

[0224] Using the Broad Institute GnomAD database for conservative screening of CBS mutation frequencies, it was estimated that thousands of patients with loss-of-function and/or missense CBS variants have been identified in the United States. Some estimates suggest that there are over 4,000 individuals with loss-of-function and/or missense CBS variants, while other estimates suggest that there are over 14,000 individuals with loss-of-function and/or missense CBS variants.

[0225] tHcy testing is common and inexpensive. An estimated 5 million patients are tested for tHcy each year. Between 31,000 and 35,000 patients have tHcy levels greater than 2 standard deviations above the mean. Many patients with elevated tHcy have a history of diagnoses that are also associated with classic HCU, but less than 10% have a documented diagnosis of HCU or sulfur amino acid metabolism disorders. Many of these patients may have non-genetically inherited elevated tHcy levels.

[0226] As used herein, the term "elevated tHcy levels" refers to tHcy levels (i.e., plasma total homocysteine levels) that are above the normal mean value expected for an individual based on age, gender, diet, or other factors. In some embodiments, in individuals with elevated tHcy levels, such levels are above about 4 μmol/L, above about 5 μmol/L, above about 6 μmol/L, above about 7 μmol/L, above about 8 μmol/L, above about 9 μmol/L, above about 10 μmol/L, above about 11 μmol/L, above about 12 μmol/L, above about 13 μmol/L, above about 14 μmol/L, above about 15 μmol/L, above about 16 μmol/L, above about 17 μmol/L, above about 18 μmol/L, above about 19 μmol/L, above about 20 μmol/L, above about 21 μmol/L, above about 22 μmol/L, above about 23 μmol/L, above about 24 μmol/L, above about 25 μmol/L, above about 26 μmol/L, above about 27 μmol/L, above about 28 μmol/L, above about 29 μmol/L, above about 30 μmol/L. In some embodiments, the elevated tHcy levels can be as high as 100 μmol/L. In some embodiments, the elevated tHcy levels can be above 100 μmol/L. Thus, some embodiments of the present invention provide for elevated tHcy levels in an individual in the range of about 4 μmol/L to about 100 μmol/L or higher.

[0227] Accordingly, some embodiments of the disclosed methods and compositions include stratifying a patient population based on elevated tHcy levels and selecting individuals from the population for treatment of a disease associated with elevated tHcy levels, including treating and ameliorating conditions and complications associated with elevated tHcy levels. In some embodiments, treating individuals with elevated tHcy levels allows for amelioration of associated conditions and complications, such as cognitive or skeletal pathologies, independent of any underlying pathology, such as CBS deficiency or HCU.

VIII. Phenotypic results

[0228] Retrospective studies have shown a proportional relationship between tHcy levels and treatment outcome. Patients with the highest tHcy levels (treated or untreated) have more severe symptoms at an earlier age, whereas patients with lower tHcy levels have fewer symptoms and those symptoms progress less rapidly (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Mudd et al. Am J Hum Genet 1985; 37:1-31, each of which is incorporated by reference in its entirety). Individuals with elevated tHcy levels typically have developmental delay, thromboembolism, severe myopia with subsequent lens dislocation, osteoporotic fractures, Marfan-like habitus (particularly elongated long bones), and/or mental retardation such as learning disabilities (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Morris et al. J Inherit Metab Dis 2017; 40:49-74; NORD, Kraus JP. Homocystinuria due to cystathionine beta-synthase deficiency. NORD [serial online] 2017, each of which is incorporated by reference in its entirety). Regarding the spectrum of CBS deficiency, some patients have severe multisystem disease beginning in childhood, whereas patients with less severe Hcy elevations may remain undiagnosed into adulthood (see Morris et al. J Inherit Metab Dis 2017;40:49-74, which is incorporated by reference in its entirety). Life expectancy is markedly reduced in patients with severely elevated Hcy, although even patients with moderately elevated tHcy suffer multiple negative clinical outcomes.

[0229] Substantial evidence has been obtained indicating a causal effect of elevated tHcy levels and adverse clinical outcomes in four systems commonly affected in patients with HCU (eyes, skeleton, cardiovascular system, and nervous system). In the eye, commonly observed phenotypic features include: ectopia lentis, iridodonesis, myopia, and less commonly observed phenotypic features include: glaucoma, optic atrophy, retinal degeneration, retinal detachment, cataract, and corneal pathology. In the skeletal system, the commonly observed phenotypic features are: osteoporosis, biconcave vertebrae, scoliosis, increased length of long bones, uneven widening of the metaphysis, metaphyseal spicules, abnormal size/abnormal shape of epiphyses, line of growth arrest, pes cavus and high-arched palate, while the less commonly observed phenotypic features are: arachnodactyly, enlarged carpal bones, age-related bone pathology, pectus carinatum/hollow chest, genu valgus, kyphosis and short fourth metacarpal. In the vascular system, the commonly observed phenotypic features are: vascular occlusion, erythema malarum and "marbled reticular skin". In the central nervous system, frequently observed phenotypic features are: cognitive symptoms, psychiatric disorders and extrapyramidal features, and less frequently observed phenotypic features are: epileptic seizures and abnormal electroencephalogram. In other body systems, frequently observed phenotypic features are: light brittle hair, thin skin, fatty changes in the liver, inguinal hernia, myopathy, endocrine pathologies, decreased levels of coagulation factors and spontaneous intestinal perforation.

[0230] There was a significant association between moderately elevated tHcy levels and adverse outcomes, but the data also showed that lowering tHcy levels had a positive effect on clinical symptoms. The literature on HCU is sparse due to the rarity of the disease and the paucity of studies in this area, but it provides valuable insights into improving clinical outcomes in a group of individuals with highly elevated Hcy levels. Conversely, studies in a broader group of individuals benefit from larger sample sizes but smaller elevations in tHcy levels. Overall, these studies consistently demonstrate that elevated tHcy levels are a good predictor of adverse clinical outcomes and that pharmacologic treatment aimed at lowering these levels is effective.

[0231] The potential outcome of treating HCU with the medicinal product described herein is to reduce plasma tHcy concentrations to the lowest possible levels while maintaining a more restrictive diet, including higher Met concentrations than other HCU treatments and treatments using other essential amino acids. In infants and children with HCU, the priority is to prevent complications associated with HCU and to ensure adequate growth and normal intellectual development (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74, which is incorporated by reference in its entirety). In patients diagnosed later in life, the priority may be to prevent life-threatening thromboembolism and to minimize the progression of complications already identified. To achieve these goals, the biochemical abnormalities associated with HCU can be reversed and, if possible, normalized (see Morris et al. J Inherit Metab Dis. 2017 Jan; 40(1):49-74, which is incorporated herein by reference in its entirety). A survey comparing dietary treatments for patients with HCU across 29 centers in 8 European countries found that there was no consensus among treatment centers regarding target ranges for plasma tHcy levels, with the median recommended target being less than 55 μM and ranging from less than 20 to 100 μM for the 29 centers (see Adam et al. Mol Genet Metab. 2013 Dec; 110(4):454-9, which is incorporated herein by reference in its entirety).

[0232] A cutoff value of greater than or equal to 80 μM for tHcy levels acceptable for inclusion in the treatment program was chosen here to avoid excluding patients with prior plasma tHcy levels of approximately 100 μM, given the 25% inter-individual variability in plasma tHcy levels tested several months apart (see Refsum et al. Clin Chem 2004; 50:3-32; Guttormsen et al., J Clin Invest. 1996, 98(9):2174-83; each of which is incorporated herein by reference in its entirety) to provide levels high enough to detect a clinically significant decrease in a small number of patients. The 25% interindividual variability in plasma tHcy levels tested several months apart (see Refsum et al. Clin Chem 2004; 50:3-32; Guttormsen et al., J Clin Invest. 1996, 98(9):2174-83; each of which is incorporated by reference in its entirety) may be due in part to changes in diet, medication, or nutritional supplement use in HCU patients over time.

[0233] Without treatment, the prognosis for patients with HCU who do not respond to pyridoxine is poor (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is incorporated herein by reference in its entirety). In 1985, an international retrospective study documenting the pathogenesis of HCU in 629 patients using pretreatment analysis found that 70% of patients had lens dislocation by 10 years of age and 85% of patients had symptoms by 12 years of age (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Mudd et al., Skovby F. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases, 7 ed., New York: McGraw Hill, 2001, 1279-1327, which are incorporated by reference in their entireties). Overall, 50% of patients had radiographic evidence of spinal osteoporosis by age 15 years, and 23% of pyridoxine non-responders (4% of responders) had died by age 30 years (see Mudd et al. Am J Hum. Genet 1985; 37:1-31, which is incorporated herein by reference in its entirety).

[0234] Overall, the available data indicate that current approaches to treating HCU, including restrictive diet and nutritional supplements, are ineffective in halting disease progression in most patients. Consequently, there is a largely unmet medical need to develop well-tolerated therapies that can reverse or normalize the metabolic abnormalities in HCU and slow or halt the progression of clinical disease manifestations.

[0235] A causal relationship has been found between elevated Hcy levels and key clinical outcomes associated with HCU, including ocular diseases (particularly lens luxation), skeletal pathologies (particularly osteoporosis), vascular pathologies (particularly stroke and small vessel disease), and various CNS lesions (particularly cognitive function). The relationship between elevated tHcy levels and negative clinical outcomes, and conversely, lowering Hcy levels and improving clinical outcomes, is further supported by numerous studies in general populations. Overall, these clinical data highlight the need for early diagnosis of HCU and prompt treatment to bring Hcy levels as close to normal as possible.

[0236] Although no studies have been published assessing the quality of life (QoL) of patients with HCU, unpublished reports indicate that patients and their caregivers suffer from the psychosocial consequences of following a severely restricted and socially isolating diet and are extremely concerned about the long-term clinical complications of the disease. Not surprisingly, patients seek to moderate their diet whenever possible without compromising their long-term outlook.

[0237] The close relationship between tHcy levels and key clinical outcomes in patients with HCU indicates that changes in tHcy levels are a reliable surrogate marker for a combination of clinical endpoints in HCU. Thus, changes in tHcy levels are useful for (i) monitoring patient disease progression in the clinic, (ii) predicting the clinical benefit of new treatments in clinical trials, and (iii) predicting therapy efficacy.

[0238] tHcy levels also provide a reliable surrogate marker for selecting individuals with non-genetically inherited CBS deficiency. According to the methods described herein, individuals with elevated tHcy levels and/or CBS deficiency can be treated with the PEGylated htCBS C15S drug product described herein to treat or ameliorate symptoms associated with elevated tHcy levels, including ocular, skeletal, cardiovascular, and neurological symptoms caused by elevated tHcy levels.

[0239] For example, the drug normalizes or increases the flexibility of the femoral artery in an individual compared to the situation that was observed before the drug was administered to the individual. For example, I278T mice have significantly lower femoral artery flexibility compared to wild-type mice. In fact, a Met-restricted diet can result in a decrease in femoral artery diameter in I278T mice compared to a normal diet in both drug-treated and drug-untreated mice.

[0240] Previous studies in 3 mouse models of the disease have demonstrated that htCBS C15S is effective after systemic administration as described in WO 2017/083327, which is incorporated herein by reference in its entirety. These studies have shown a reduction in extracellular Hcy levels in plasma and intracellular Hcy levels in tissues such as the brain by up to 90%. It has been observed that drug administration results in a concentration gradient with a flux of Hcy from higher concentrations in the intracellular space to lower concentrations in the extracellular space where the drug can further process it. Extracellular PEGylated htCBS C15S serves as a "sink" for Hcy. Thus, the drug restores control of the Met pathway in the animal model of HCU.

[0241] These studies also showed that subcutaneous administration of PEGylated htCBS C15S to mice with the HCU model corrected metabolite levels, including an increase in Cth levels and normalization of Cys levels. In addition, PEGylated htCBS C15S positively affected the phenotypic manifestation of the disease in mice, including facial alopecia, poor liver histology, osteoporosis, abnormal body habitus, diabetic retinopathy (possibly caused by renal disease), and macular and optic nerve atrophy due to retinal vascular occlusion or non-arteritic ischemic optic neuropathy, and increased cytokine and lipid levels. PEGylated htCBS C15S also rescued CBS knockout (KO) mice from early death (Looker et al. Diabetologia 2003; 46:766–772; Pusparajah et al. Front Physiol 2016; 7:200; Gerth et al. J AAPOS 2008; 12:591–596; Stanger et al. Clin Chem Lab Med 2005; 43:1020–1025; Cahill et al. Am J Ophthalmol 2003; 136:1136–1150; Minniti et al. Eur J Ophthalmol 2014; 24:735–743; each of which is hereby incorporated by reference in its entirety). It was also observed that PEGylated htCBS C15S was well tolerated without any toxicological effects observed with chronic dosing in animal models of the disease.

[0242] PEGylated htCBS C15S acts in the extracellular space and is expected to reduce plasma tHcy concentrations regardless of patient genetics, concomitant therapy, or baseline tHcy levels. Thus, an appropriate study population should include both pyridoxine-responsive and non-responsive patients.

[0243] In healthy humans, tHcy levels range from approximately 5 to 15 µM (see OECD Environmental Health and Safety Executive's publication, Standard Laboratory Practice and Compliance Monitoring Series No. 1 ENV/MC/CHEM(98)17, "Principles of Standard Laboratory Practice (Revised 1997), which is incorporated herein by reference in its entirety), and 98% of tHcy is present in the disulfide or protein-bound form. Only 2% of tHcy exists as unbound, free, reduced aminothiol, which can serve as a substrate for the enzyme (see EMA: Guidance on bioanalytical method validation, EMEA/CHMP/EWP/192217/2009, version 1, 21 July 2011; ATL-15-1419 as per the Atlanbio study report entitled “LC-MS/MS determination of cystathionine D4 as a drug with cystathionine beta synthase activity in monkey plasma samples collected during study 529736”, which is incorporated herein by reference in its entirety). On the other hand, patients with HCU not only have plasma tHcy levels that can reach 400 μM or more, but also exhibit a dramatically altered balance, with free homocysteine levels reaching 10-25% of baseline tHcy values.

[0244] In mouse models, administration of PEGylated htCBS C15S resulted in up to 90% reduction in tHcy levels. Thus, the initial levels of free homocysteine available to the enzyme (10-25% of the total) cannot be the sole cause of the significant reduction in reported tHcy levels, and additional pools must be available to the enzyme. For example, if free Hcy becomes deficient as a result of PEG-htCBS activity, then the balance between free Hcy and Hcy adducts (in the form of protein-bound Hcy or disulfides) in plasma is altered in favor of the formation of free Hcy, which can then be utilized by the enzyme.

A. Eye pathologies

[0245] Elevated Hcy levels are a significant and independent risk factor for ocular pathologies, particularly lens dislocation, in patients with HCU and in the general population. Even with pharmacological and dietary interventions, most patients with HCU eventually develop ocular pathologies. Reduction of Hcy levels has been shown to delay and possibly prevent lens dislocation in patients with HCU (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Mudd et al. Am J Hum Genet 1985; 37:1–31; Martinez-Gutierrez et al. Int Ophthalmol (2011) 31:227–232; Ajith et al. Clin Chim Acta 2015; 450:316–321; Mulvihill et al. J AAPOS 2001; 5:311–315; Marti-Carvajal et al. Cochrane Database Syst Rev 2015; 1:CD006612; Sweetser et al. N Engl J Med 2016, 375:1879-1890; Sadiq et al. Semin Ophthalmol 2013; 28:313-320; Wright et al. Homocysteine, folates, and the eye. Eye (Lond) 2008; 22:989-993; Lieberman et al. Am J Ophthalmol 1966, 61:252-255; Harrison et al. Ophthalmology 1998, 105:1886-1890; Ramsey et al. Am J Ophthalmol 1972; 74:377-385; Couser et al. Ophthalmic Genet 2017, 38:91-94; Ghorbanihaghjo et al. Mol Vis 2008, 14:1692-1697; Javadzadeh et al. Mol Vis 2010; 16:2578-2584; Seddon et al. Am J Ophthalmol 2006; 141:201-203; Coral et al. Eye (Lond) 2006; 20:203-207; Axer-Siegel et al. Am J Ophthalmol 2004, 137:84-89; Heuberger et al. Am J Clin Nutr 2002; 76:897-902; Huang et al. Sci Rep 2015; 5:10585; Sen et al. Indian J Clin Biochem 2008; 23:255-257; Yousefi et al. Protein Pept Lett 2013; 20:932-941; Gerth et al. J AAPOS 2008; 12:591-596; Stanger et al. Clin Chem Lab Med 2005, 43:1020-1025; Cahill et al. Am J Ophthalmol 2003, 136:1136-1150; Minniti et al. Eur J Ophthalmol 2014, 24:735-743; Turkcu et al. Medicina (Kaunas) 2013,49:214-218; Vessani et al. Am J Ophthalmol 2003; 136:41-46; Leibovitch et al. J Glaucoma 2003, 12:36-39; Leibovitz et al. Medicine (Baltimore) 2016;95:e4858; Michael et al. Mol Vis 2009; 15:2268–2278; Clement et al. J Glaucoma 2009; 18:73–78; Cumurcu et al. BMC Ophthalmol 2006; 6:6; Bleich et al. J Neural Transm (Vienna) 2002;109:1499–1504; Lee et al. Curr Eye Res 2017;1–6; Wang et al. Am J Ophthalmol 2004; 137:401–406; Ganapathy et al. Invest Ophthalmol Vis Sci 2009; 50:4460–4470, each of which is incorporated herein by reference in its entirety). Even with pharmacologic and dietary interventions, most patients with HCU eventually develop ocular complications. Reduction of Hcy levels has been shown to delay and possibly prevent lens dislocation in patients with HCU (see Yap et al. J Inherit Metab Dis 1998; 21:738-747, which is incorporated herein by reference in its entirety).

[0246] One of the most common and earliest manifestations of HCU is ectopia lentis (dislocation of the lens) (see Mulvihill et al. J AAPOS 2001; 5:311-315, which is incorporated herein by reference in its entirety). This typically occurs after two years of age and is seen in approximately 50% of untreated, pyridoxine-unresponsive patients by age six and in 50% of untreated, pyridoxine-responsive patients by age 10 (see Mudd et al. Am J Hum Genet 1985; 37:1-31, which is incorporated herein by reference in its entirety). The dislocation may be partial (subluxation) or complete, and although it may occur inferiorly or nasally, it is usually bilateral (see Mulvihill et al. J AAPOS 2001; 5:311-315; Sweetser et al. N Engl J Med 2016; 375:1879-1890, which are incorporated herein by reference in their entireties).

[0247] Lens dislocation often follows a period of rapidly progressive myopia, which can result in marked astigmatism, monocular diplopia, and decreased visual acuity (see Sadiq et al. Semin Ophthalmol 2013;28:313-320, which is incorporated herein by reference in its entirety). Overall, myopia (greater than 1 diopter [D]) is thought to occur in approximately 85% of patients with HCU, with very high myopia (greater than 5D) affecting 50% to 76% of patients. Iridodonesis (trembling of the iris following eye movement) occurs in approximately 56% of patients, and spherophakia (a small spherical lens prone to subluxation) affects 50% of patients (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Mulvihill et al. J AAPOS 2001; 5:311–315, each of which is incorporated by reference in its entirety). Other complications associated with HCU include cataract formation, chronic vitritis (inflammation of the vitreous) and chorioretinal inflammation, pupillary block with acute and/or chronic angle-closure glaucoma, and (in children) amblyopia (lazy eye) (see Sadiq et al. Semin Ophthalmol 2013; 28:313-320, which is incorporated herein by reference in its entirety).

[0248] Data from a long-term retrospective study of 25 patients with HCU up to the age of 24 years suggest that lens dislocation can be prevented or at least significantly reduced and delayed in patients whose tHcy levels are persistently reduced from an early age (see Yap et al. J Inherit Metab Dis 1998;21:738-747, which is incorporated by reference in its entirety). Early Hcy-lowering treatment is also associated with a reduced risk of common ocular complications, including myopia progression. Supporting evidence comes from a case-control study of 32 HCU patients and 25 control siblings, in whom early Hcy-lowering treatment was associated with a significant reduction in ocular complications compared with patients who were treated later in life or who were not fully compliant with treatment recommendations (see El Bashir et al. JIMD Rep 2015;21:89-95, which is incorporated herein by reference in its entirety).

[0249] The largest and longest-running study to date of eye disease in 25 patients with cobalamin C deficiency, which is also characterized by elevated tHcy levels, found that macular degeneration, optic nerve pallor, nystagmus, strabismus, and vascular changes were present in the majority of patients.

[0250] Numerous studies in HCU patients and the general population have demonstrated an association between elevated Hcy levels and a variety of ocular diseases (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Ajith, Clin Chim Acta 2015; 450:316-321; Mulvihill et al. J AAPOS 2001; 5:311-315; Wright et al. Eye (Lond) 2008;22:989-993, each of which is hereby incorporated by reference in its entirety), including myopia and lens luxation (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Mudd et al. Am J Hum Genet 1985; 37:1-31; Martinez-Gutierrez et al. Int Ophthalmol 2011; 31:227-232; Suri et al. J Neurol Sci 2014; 347:305-309; Mulvihill et al. J AAPOS 2001; 5:311-315; Lieberman et al. Am J Ophthalmol 1966; 61:252-255; Harrison et al. Ophthalmology 1998; 105:1886-1890; Ramsey et al. Am J Ophthalmol 1972; 74:377-385; Couser et al. Ophthalmic Genet 2017;38:91-94; each of which is hereby incorporated by reference in its entirety), iridodonesis (see Mulvihill et al. J AAPOS 2001; 5:311-315, which are each incorporated by reference in their entireties), retinal arteriosclerosis (see Ghorbanihaghjo et al. Mol Vis 2008;14:1692-1697, which are each incorporated by reference in their entireties), age-related macular degeneration (see Javadzadeh et al. Mol Vis 2010; 16:2578-2584; Seddon et al. Am J Ophthalmol 2006; 141:201-203; Coral et al. Eye (Lond) 2006; 20:203-207; Axer-Siegel et al. Am J Ophthalmol 2004;137:84-89, each of which is incorporated by reference in their entireties), age-related maculopathy (AMD) (See Heuberger et al. Am J Clin Nutr 2002; 76:897-902; Huang et al. Sci Rep 2015; 5:10585, which are each incorporated by reference in their entireties), cataracts (See Sen et al. Indian J Clin Biochem 2008; 23:255-257; Yousefi et al. Protein Pept Lett 2013; 20:932-941, which are each incorporated by reference in their entireties), diabetic retinopathy (possibly due to kidney disease) (See Looker et al. Diabetologia 2003; 46:766-772; Pusparajah et al. Front Physiol 2016; 7:200, which are each incorporated by reference in their entireties) and macular and optic nerve atrophy due to retinal vascular occlusion or non-arteritic ischemic optic neuropathy (see Gerth et al. J AAPOS 2008; 12:591-596; Stanger et al. Clin Chem Lab Med 2005; 43:1020-1025; Cahill et al. Am J Ophthalmol 2003; 136:1136-1150; Minniti et al. Eur J Ophthalmol 2014; 24:735-743, each of which is incorporated by reference in their entireties).

[0251] A retrospective study of 629 patients with HCU showed that lens dislocation typically occurs after the age of two years and occurs in approximately 50% of untreated, pyridoxine-unresponsive patients by the age of six years and in 50% of untreated, pyridoxine-responsive patients by the age of 10 years (see Mudd et al. Am J Hum Genet 1985; 37:1-31, which is incorporated herein by reference in its entirety). The largest and longest-running study to date of ocular disease in 25 patients with cobalamin C deficiency, which is also characterized by elevated tHcy levels, found that macular degeneration, optic nerve pallor, nystagmus, strabismus, and vascular changes were present in the majority of patients (see Brooks et al. Ophthalmology. 2016 Mar; 123(3):571-82, which is incorporated herein by reference in its entirety).

[0252] Numerous studies in HCU patients and the general population have demonstrated an association between elevated Hcy levels and various ocular diseases (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Ajith TA, Ranimenon. Clin Chim Acta 2015); 450:316-321; Mulvihill et al. J AAPOS 2001; 5:311-315; Wright et al. Eye (Lond) 2008; 22:989-993; each of which is incorporated herein by reference in its entirety), including myopia and lens luxation (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [serial online] 2005; Mudd et al. Am J Hum Genet 1985; 37:1-31; Martinez-Gutierrez et al. Int Ophthalmol 2011; 31:227-232; Suri et al. J Neurol Sci 2014; 347:305-309; Mulvihill et al. J AAPOS 2001; 5:311-315; Lieberman et al. Am J Ophthalmol 1966; 61:252-255; Harrison et al. Ophthalmology 1998; 105:1886-1890; Ramsey et al. Am J Ophthalmol 1972; 74:377-385; Couser et al. Ophthalmic Genet 2017;38:91-94; each of which is hereby incorporated by reference in its entirety), iridodonesis (see Mulvihill et al. J AAPOS 2001; 5:311-315, which is hereby incorporated by reference in its entirety), retinal arteriosclerosis (see Ghorbanihaghjo et al. Mol Vis 2008;14:1692-1697, which is hereby incorporated by reference in its entirety), age-related macular degeneration (see Javadzadeh et al. Mol Vis 2010; 16:2578-2584; Seddon et al. Am J Ophthalmol 2006; 141:201-203; Coral et al. Eye (Lond) 2006; 20:203-207; Axer-Siegel et al. Am J Ophthalmol 2004;137:84-89, each of which is hereby incorporated by reference in their entireties), age-related maculopathy (AMD) (Heuberger et al. Am J Clin Nutr 2002; 76:897-902; Huang et al. Sci Rep 2015; 5:10585, which are hereby incorporated by reference in their entireties), cataracts (Sen et al. Indian J Clin Biochem 2008; 23:255-257; Yousefi et al. Protein Pept Lett 2013; 20:932-941, which are hereby incorporated by reference in their entireties), diabetic retinopathy (possibly due to kidney disease) (Looker et al. Diabetologia 2003; 46:766-772; Pusparajah et al. Front Physiol 2016; 7:200, which are each incorporated by reference in their entireties) and macular and optic nerve atrophy due to retinal vascular occlusion or non-arteritic ischemic optic neuropathy (see Gerth et al. J AAPOS 2008; 12:591-596; Stanger et al. Clin Chem Lab Med 2005; 43:1020-1025; Cahill et al. Am J Ophthalmol 2003; 136:1136-1150; Minniti et al. Eur J Ophthalmol 2014; 24:735-743, each of which is incorporated by reference in their entireties).

[0253] Studies examining the relationship between Hcy levels and glaucoma have yielded conflicting results. Some of them showed a positive association between Hcy levels and normotensive glaucoma, pseudoexfoliation glaucoma (PEXG), and primary open-angle glaucoma (POAG), while other studies showed no such results (see Lieberman et al. Am J Ophthalmol 1966;61:252–255; Turkcu et al. Medicina (Kaunas) 2013;49:214–218; Vessani et al. Am J Ophthalmol 2003;136:41–46; Leibovitch et al. J Glaucoma 2003;12:36–39; Leibovitzh et al. Relationship between homocysteine and intraocular pressure in men and women: A population-based study. Medicine (Baltimore) 2016;95:e4858; Micheal et al. Mol Vis 2009;15:2268-2278; Clement et al. J Glaucoma 2009;18:73-78; Cumurcu et al. BMC Ophthalmol 2006;6:6; Bleich et al. J Neural Transm (Vienna) 2002;109:1499-1504; Lee et al. Curr Eye Res 2017;1-6; Wang et al. Am J Ophthalmol 2004;137:401-406; each of which is hereby incorporated by reference in its entirety). However, loss of retinal ganglion cells (RGCs), commonly observed in patients with glaucoma, has been demonstrated in mice with endogenously elevated Hcy levels caused by deletion of the CBS gene, suggesting a possible link between glaucoma and elevated tHcy levels in patients with HCU (see Ganapathy et al., Invest Ophthalmol Vis Sci 2009;50:4460-4470, each of which is incorporated herein by reference in its entirety).

1. Mechanism

[0254] A number of mechanisms have been proposed to explain the effects of elevated Hcy levels on ocular health (see Ajith TA, Ranimenon; Clin Chim Acta 2015; 450:316-321, which is incorporated herein by reference in its entirety). Mechanisms to explain the effects of elevated tHcy include impaired vascular endothelial function, retinal ganglion cell apoptosis, extracellular matrix changes, decreased lysyl oxidase activity and oxidative stress, as well as direct cytotoxic and proinflammatory effects of Hcy that appear to contribute to lens opacification and optic nerve damage.

[0255] Potential mechanisms also include activation of the N-methyl-D-aspartate (NMDA) receptor, resulting in calcium influx into cells and increased production of reactive oxygen species (ROS), which contribute to cataract formation. These changes, along with the direct cytotoxic effects of Hcy, may cause endothelial injury that initiates thrombosis and RGC apoptosis, leading to retinopathy and glaucoma. Elevated Hcy levels have also been shown to promote increased asymmetric dimethylarginine (AMDA) levels and block nitric oxide synthase (NOS) activity, which causes vasoconstriction and optic nerve atrophy by decreasing nitric oxide (NO) levels. Finally, accumulation of homocysteinylated proteins on the vascular wall can trigger the production of anti-Hcy antibodies and inflammatory responses, which will lead to phagocytosis, oxidative stress, RGC apoptosis, and extracellular matrix (ECM) changes. Overall, such changes damage the vasculature, lens proteins, and optic nerve, ultimately causing visual impairment.

[0256] In patients with HCU, lens dislocation is primarily caused by degenerative changes in the ligament fibers, and in particular by Cys-rich multidomain ECM proteins such as fibrillin-1 (see Sadiq et al. Semin Ophthalmol 2013; 28:313-320; Hubmacher et al. Biochemistry 2011; 50:5322-5332; Hubmacher et al. J Biol Chem 2005; 280:34946-34955; Hubmacher et al. J Biol Chem 2010; 285:1188-1198; each of which is hereby incorporated by reference in its entirety).

[0257] In healthy individuals, the formation of multiple intradomain disulfide bonds in fibrillin-1 ensures the proper folding of the protein necessary for maintaining structural integrity and function. Fibrillin-1 strands can then form interchain disulfide bonds, resulting in the assembly of high molecular weight multidomain protein aggregates known as microfibrils (see Kinsey et al. J Cell Sci 2008; 121:2696-2704; Hubmacher et al. Proc Natl Acad Sci USA 2008; 105:6548-6553, each of which is hereby incorporated by reference in its entirety).

[0258] This process is largely dependent on the interaction between fibrillin-1 and fibronectin (see Hubmacher et al. Biochemistry 2011; 50:5322-5332, which is hereby incorporated by reference in its entirety). Microfibrils form a scaffold for tropoelastin deposition, which is an important step in the formation of elastic fibers such as those of the skin, lung, blood vessels/arteries, ligaments, and eye (see Hubmacher et al. J Biol Chem 2010; 285:1188-1198, which is hereby incorporated by reference in its entirety). The importance of fibrillin-1 has been demonstrated in patients with Marfan syndrome, a condition caused by mutation(s) in the fibrillin 1 gene, in which connective tissue dysfunction is associated with symptoms such as lens dislocation, organ prolapse, osteoporosis, and joint hypermobility (see Suk et al., J Biol Chem 2004, 279:51258-51265, Collod-Beroud et al., Hum Mutat, 2003, 22:199-208, which are incorporated herein by reference in their entireties).

[0259] In vitro studies have shown that addition of Hcy to fibrillin-1 disrupted disulfide bond formation, which in turn resulted in abnormal protein folding, increased susceptibility to proteolytic degradation, and abnormal formation of extracellular matrix and elastic fibers (see Hubmacher et al. J Biol Chem 2010; 285:1188-1198; Whiteman et al., Antioxid Redox Signal 2006; 8:338-346, which are hereby incorporated by reference in their entireties). Addition of Hcy to human dermal fibroblasts was also associated with the formation of reduced forms of fibronectin that suboptimally bound to fibrillin-1, thereby preventing microfibril formation (see Hubmacher et al. Biochemistry 2011; 50:5322-5332; Hubmacher et al. J Biol. Chem 2010; 285:1188-1198, which are incorporated herein by reference in their entireties).

[0260] In addition to lens dislocation, degeneration of the zonular fibers in patients with HCU can lead to increased lens curvature, lens myopia, astigmatism, retinal detachment, strabismus, cataracts, and iridodonesis (see Sadiq et al. Semin Ophthalmol 2013; 28:313-320, which is hereby incorporated by reference in its entirety). If untreated, anterior lens dislocation can cause acute glaucoma, leading to pupillary block. In extreme cases, complete lens displacement is associated with increased axial length of the eye, possibly as a compensation response to blurred vision (see Mulvihill et al. J AAPOS 2001; 5:311-315, which is hereby incorporated by reference in its entirety).

[0261] A retrospective study of 25 cases of HCU identified in Ireland between 1971 and 1996, either through the national NBS programme or clinical presentation, was conducted to examine the effect of Hcy-lowering therapy on outcome (see Yap et al. J Inherit Metab Dis. 1998; 21:738-747, which is incorporated by reference in its entirety). In the majority of cases (24/25), patients were unresponsive to pyridoxine. Therefore, treatment for most patients consisted of a Met-free diet with Cys supplementation, and vitamin B12 and folate supplementation if required. Treatment was started before the patients were 6 weeks old and was compared with another group where treatment was started after diagnosis and with treatment of one control patient who had never been treated. The mean follow-up was 14.3 years (range 2.5–23.4) in the groups treated before 6 weeks of age and 14.7 years (range 11.7–18.8) for the remaining patients, for a total of 365.7 patient-years of treatment. Of the 21 patients identified by NBS, 18 had no complications during treatment. Of these patients, 15/18 had 20:20 vision and 3/18 had progressive myopia over the previous 2 years.

[0262] According to the findings of Mudd et al. (see Mudd et al. Am J Hum Genet 1985; 37:1-31, which is incorporated by reference in its entirety), lens dislocation occurs at approximately 2 years of age in late-diagnosed individuals. Lens dislocation was not observed in any of the early-treated patients who were well compliant with therapy. Three of the "early-treated patients" (those with the highest fHcy levels) showed progression of myopia without lens dislocation, which was most likely related to the relatively high fHcy levels in this small group of patients. This led the authors to speculate that progressive myopia may be the first sign preceding lens dislocation and poor dietary compliance, despite the patient's insistence to the contrary. The progression of myopia in these patients highlights how fragile the balance is between neutral and negative clinical outcomes for these patients. All late-diagnosed patients developed ectopia lentis, suggesting that treatment may delay the onset of lens dislocation, but not prevent it.

[0263] The median lifetime plasma fHcy level was higher in myopic patients than in non-myopic patients (18, 18, and 48 μmol/L versus 11 μmol/L, respectively). In the group where treatment was initiated after diagnosis, of the three patients identified by NBS who developed complications, none were on the diet. Overall, six of the 24 patients had lens dislocation, two of whom were diagnosed early but were not on the diet, and four of whom were diagnosed late, including one patient who was never treated. Consistent with the findings of Mudd et al. (See Mudd et al. Am J Hum Genet 1985; 37:1-31, which is incorporated herein by reference in its entirety), lens dislocation in later-diagnosed individuals (i.e., patients with complications after age 2 years) occurred at approximately age 2 years. At the time of publication of the study, lens dislocation had not occurred in any of the previously treated patients with good compliance.

[0264] In compliant patients, fHcy was maintained at levels equivalent to tHcy, i.e., well below 120 μmol/L. However, at the time of publication, all patients were under 24 years of age, and many were still children. Compliance with a protein-restricted diet was shown to decline rapidly from adolescence into adulthood. The delicate balance described above suggests that the modest reduction in tHcy levels achieved in these patients may delay the onset of symptoms, but not prevent them, as these patients age.

[0265] These results (see Yap et al. J Inherit Metab Dis 1998; 21:738-747; Mudd et al. Am J Hum Genet 1985; 37:1-31; which are incorporated herein by reference in their entireties) were confirmed by data from a similar case-control study conducted in Qatar, which reported treatment outcomes, including visual impairment, in 32 HCU cases and 25 sibling controls (see El Bashir et al. JIMD Rep 2015; 21:89-95, which is incorporated herein by reference in its entirety). The mean age of the individuals was 11.2 years (range 0.6-29) and 56% were male. Overall, 9 of 32 cases (28%) were diagnosed by NBS and treated in the first month of life. The remainder were diagnosed between 14 and 240 months of age. tHcy and Met levels were significantly lower in those diagnosed by NBS compared with those diagnosed in the clinic, possibly reflecting better compliance with diet and medications early in life. None of the 9 cases diagnosed by NBS had visual problems at the time of publication of the study, compared with 18 (78%) in the late-diagnosed group (p<0.001 between groups). However, as in the Irish study, the 25 patients described above ranged in age from 0.6 to 29 years at the time of this study, and long-term sequelae were not yet known.

[0266] Comparison of the data from Yap and Naughten (see Yap et al. J Inherit Metab Dis 1998; 21:738-747, which is incorporated herein by reference in its entirety) with the Kaplan-Meier curves of Mudd et al. showed that the proportion of “early treated” patients with lens dislocation and osteoporosis was significantly lower than expected for untreated patients with HCU (p≤0.001).

[0267] Therefore, elevated Hcy levels are considered to be an important and independent risk factor for ocular complications, particularly lens dislocation, in patients with HCU and in the general population (e.g., as shown by Yap et al. and Mudd et al.). This highlights the need for early diagnosis and treatment of HCU, as well as patient compliance.

B. Skeletal complications

[0268] HCU is associated with an increased risk of osteoporotic fractures, which may be partly related to low bone mineral density (see Mudd et al. and Weber et al. Mol Genet Metab 2016; 117:351-354; each of which is incorporated herein by reference in its entirety).

[0269] A retrospective review of 19 patients with HCU over 8 years found that low bone mineral density (BMD) was common in both pediatric and adult patients with HCU (see Weber et al.). This study showed that bone mass accretion during childhood and adolescence, an important period for skeletal development, is deficient in Hcy and may negatively impact final bone mass formation. This study also showed that even diet-restricted patients with moderately elevated tHcy levels, only 5 times the normal range, already have clinical skeletal abnormalities in childhood.

[0270] According to Mudd et al., 80% of patients with HCU develop osteoporosis by age 30 years. Furthermore, elevated Hcy levels are associated with an increased risk of osteoporotic fractures even in patients without HCU (see van Meurs et al. N Engl J Med 2004; 350:2033-2041; McLean et al. N Engl J Med 2004; 350:2042-2049; each of which is incorporated herein by reference in its entirety).

[0271] A retrospective review of 19 patients (9 males, aged 3.5 to 49.2 years) who underwent clinical bone densitometry with DXA between 2002 and 2010 found that low BMD was common in both pediatric and adult patients with HCU (see Weber et al. Mol Genet Metab 2016; 117:351–354, which is incorporated by reference in its entirety). At the time of the first DXA scan, the mean lumbar spine (LS) BMD Z-score was -1.2±1.3 and the total hip BMD Z-score was -0.89±0.4, both of which were significantly below 0 (the expected mean Z-score in the general population) with p=0.002 and 0.02, respectively. BMD LS Z-score at diagnosis was -1.26±1.4 in patients under 21 years of age and -1.06±1.1 in adults. Overall, 38% of patients had low BMD for their age (Z-score ≤ -2). tHcy and Met levels were positively associated with BMD LS Z-score in multiple linear regression models (see Weber et al. Mol Genet Metab 2016; 117:351–354, which is incorporated by reference in its entirety). The mean tHcy levels for these 19 individuals were only 59.2 μmol/L, and most of the 19 patients were children. This study suggests that bone mass accrual during childhood and adolescence, a critical period for skeletal development, is deficient in Hcy and may negatively impact final bone mass formation. This study also showed that even diet-controlled patients with only moderately elevated tHcy levels, only 5 times the normal range, already have clinical skeletal pathologies in childhood.

[0272] Previous studies have demonstrated a strong association between Hcy levels and fracture risk in older adults (see van Meurs et al. N Engl J Med 2004;350:2033-2041; McLean et al. N Engl J Med 2004;350:2042-2049), each of which is incorporated by reference in its entirety). Results from two prospective cohort studies including 2,406 individuals aged ≥55 years showed that age- and sex-adjusted fracture risks increased by 30% for each standard deviation increase in tHcy levels (see van Meurs et al. N Engl J Med 2004;350:2033-2041, which is incorporated by reference in its entirety). Homocysteine levels in the highest age quartile were associated with a 1.9-fold increased risk of fracture. The association between homocysteine levels and fracture risk was independent of bone mineral density and other potential fracture risk factors. Elevated homocysteine levels were an important and independent risk factor for osteoporotic fractures in older men and women overall, where such factors were similar in magnitude to established risk factors for fracture and cardiovascular disease (see van Meurs et al. N Engl J Med 2004;350:2033–2041; which is incorporated by reference in its entirety). In addition, a prospective US study of 825 men and 1174 women (the HOPE-2 study) found that serum tHcy levels in the highest quartile were associated with a 1.9-fold increased risk of hip fracture among women and a four-fold increased risk of hip fracture among men compared with the risk observed with serum tHcy levels in the lowest quartile (see Sawka et al. Arch Intern Med. 2007 Oct 22;167(19):2136-9, which is incorporated herein by reference in its entirety). The association between tHcy levels and fracture risk was independent of BMD and other potential fracture risk factors (see van Meurs et al. N Engl J Med 2004; 350:2033–2041; McLean et al. N Engl J Med 2004, 350: 2042–2049; Sawka et al., Arch Intern Med., 2007 Oct 22;167(19):2136–9, each of which is incorporated by reference in its entirety).

[0273] In line with these results, a study of 433 stroke patients over the age of 65 years found that age-adjusted incidence rates, namely hip fractures per 1000 person-years, increased almost linearly from 2.89 in the lower quartiles of Hcy levels to 27.87 in the upper quartiles (see). Overall, these results suggest that elevated Hcy levels are an important and independent risk factor for osteoporotic fractures in older men and women.

[0274] Skeletal abnormalities are absent at birth and are uncommon in infants and very young children (see Mudd et al. Am J Hum Genet 1985; 37:1-31, which is hereby incorporated by reference in its entirety). The first signs of skeletal involvement are usually genu valgus and pes cavus with long bones, typical of Marfan syndrome, often developing near puberty (see Morris et al. J Inherit Metab Dis 2017; 40:49-74, which is hereby incorporated by reference in its entirety). Osteoporosis, particularly of the vertebrae and long bones, is common in patients with HCU and may result in scoliosis/kyphosis and/or vertebral collapse (see Mudd et al. Am J Hum Genet 1985;37:1-31; Weber et al. Mol. Genet Metab 2016;117:351-354; each of which is incorporated by reference in its entirety). Other skeletal abnormalities may include Marfan-like facial features caused by prominent upper teeth and a high palate, as well as anterior chest wall deformities such as sunken chest or pectus carinatum (see Morris et al. J Inherit Metab Dis 2017;40:49–74; Sweetser et al., N Engl J Med 2016;375:1879–1890; Brenton et al., J Bone Joint Surg Br 1972;54:277–298, each of which is incorporated by reference in its entirety). Because of these shared skeletal characteristics between Marfan syndrome and HCU, patients with HCU are sometimes mischaracterized as having Marfan disease.

[0275] A study of 25 Irish patients with HCU followed for 25 years found that the risk of osteoporosis was significantly lower in patients identified by newborn screening with good compliance with Hcy-lowering treatment (diet, vitamins, and/or betaine) compared with non-compliant patients or those diagnosed late (see Yap et al. J Inherit Metab Dis 1998; 21:738-747). Supporting evidence for these findings was provided by a small Korean study of five HCU patients with good long-term metabolic control. In this study, patients who had previously received Hcy-lowering therapy had fewer skeletal abnormalities than patients diagnosed later (see Lim et al. Osteoporos Int 2013, 24:2535–2538, which is incorporated herein by reference in its entirety). Finally, in a study using a mouse model of HCU, normalization of tHcy levels with CBS ET treatment was associated with prevention of osteoporosis (see Majtan et al. Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria, FASEB J 2017, which is incorporated herein by reference in its entirety).

[0276] The exact mechanisms leading to low BMD and skeletal fragility in patients with HCU are not fully understood (see Weber et al. Mol Genet Metab 2016; 117:351-354; Lim JS, Lee DH. Changes in bone mineral density and body composition of children with well-controlled homocystinuria caused by CBS deficiency. Osteoporos Int 2013; 24:2535-2538, which are each incorporated by reference in their entireties). However, many of the connective tissue disorders in patients with HCU resemble those of Marfan syndrome, a connective tissue disorder caused by mutations in the fibrillin-1 gene and characterized by features such as long bones and osteoporotic fractures (see Hubmacher et al. Biochemistry 2011; 50:5322–5332; Hubmacher et al. J Biol Chem 2010; 285:1188–1198, which are each incorporated by reference in their entireties). Elevated Hcy levels are thought to lead to bone fragility and fractures through two different mechanisms (see Behera et al. J Cell Physiol 2016, which is incorporated by reference in its entirety). The first mechanism results in reduced bone gain during childhood and adolescence due to impaired fibrillin assembly. The second mechanism involves impaired bone remodeling, resulting in bone fragility due to decreased collagen cross-linking (see Behera et al. J Cell Physiol 2016; Kang et al. J Clin Invest 1973; 52:2571–2578, which are each incorporated by reference in their entireties). Overall, these data suggest that bone mass accretion during childhood and adolescence, a critical period for skeletal development, is inadequate in patients with HCU and that this negatively impacts final bone mass formation.

[0277] In addition, there is a strong association between Hcy levels and fracture risk in the elderly (see van Meurs et al. N Engl J Med 2004; 350:2033-2041; McLean et al. N Engl J Med 2004; 350:2042-2049; each of which is incorporated by reference in its entirety). Results from two international prospective cohort studies including 2,406 individuals aged 55 years or older showed that homocysteine levels in the highest age quartile were associated with a 1.9-fold increased risk of fracture (see van Meurs et al. N Engl J Med 2004; 350:2033-2041, which is incorporated by reference in its entirety). Elevated homocysteine levels are an important and independent risk factor for osteoporotic fractures in most older men and women, where such factors are similar in magnitude to established risk factors for fractures (low bone mineral density, cognitive impairment, recent falls) and cardiovascular disease (see van Meurs et al. N Engl J Med 2004; 350:2033-2041, which is incorporated herein by reference in its entirety). In addition, a prospective study of 1999 individuals in the United States (the HOPE-2 study) found that serum tHcy levels in the highest quartile were associated with a 1.9-fold increased risk of hip fracture in women and a four-fold increased risk in men, compared with serum tHcy levels in the lowest quartile (see Sawka et al. Arch Intern Med. 2007 Oct 22;167(19):2136-9, which is incorporated herein by reference in its entirety). The association between tHcy levels and fracture risk was independent of BMD and other potential fracture risk factors (see van Meurs et al. N Engl J Med 2004; 350:2033–2041; McLean et al. N Engl J Med 2004; 350: 2042–2049; Sawka et al., Arch Intern Med., 2007 Oct 22;167(19):2136–9, each of which is incorporated by reference in its entirety). Consistent with these results, a study of 433 stroke patients aged >65 years found that age-adjusted hip fracture rates per 1000 person-years increased nearly linearly from 2.89 in the lowest quartiles of Hcy levels to 27.87 in the highest quartiles. Overall, these results suggest that elevated Hcy levels are an important and independent risk factor for osteoporotic fractures in older men and women.

[0278] The exact mechanisms leading to low BMD and skeletal fragility in patients with HCU are not fully understood (see Weber et al. Mol Genet Metab 2016; 117:351-354; Lim JS, Lee DH. Changes in bone mineral density and body composition of children with well-controlled homocystinuria caused by CBS deficiency. Osteoporos Int 2013; 24:2535-2538, which are each incorporated by reference in their entireties). However, many of the connective tissue disorders in patients with HCU resemble those of Marfan syndrome, which are connective tissue disorders caused by mutations in the fibrillin-1 gene and characterized by features such as long bone lengthening and osteoporotic fractures (see Brenton et al., J Bone Joint Surg Br 1972, 54:277-298, Hubmacher et al., Biochemistry, 2011, 50:5322-5332, Hubmacher et al., J Biol Chem, 2010, 285:1188-1198, each of which is hereby incorporated by reference in its entirety).

[0279] In healthy individuals, fibrillin-1, together with collagen and elastin polymers, forms the ECM, i.e., the structural scaffolds for bone formation, homeostasis, and repair (see Olivieri et al. Fibrogenesis Tissue Repair 2010; 3:24, which is hereby incorporated by reference in its entirety). Studies have shown that elevated tHcy levels can result in structural modifications of fibrillin-1 fragments that prevent multimerization and lead to fibrillin-1 degradation (see Hubmacher et al. J Biol Chem 2005; 280:34946-34955; Hubmacher et al. J Biol Chem 2010; 285:1188-1198, which are hereby incorporated by reference in their entireties). This process is also disrupted by homocysteinylation of fibronectin, which prevents the formation of fibronectin-fibrillin complexes required for fibrillin-1 multimerization (see Hubmacher et al. Biochemistry 2011; 50:5322-5332, which is incorporated by reference in its entirety). These data suggest that elevated Hcy levels have a negative impact on ECM formation.

[0280] In healthy humans, fibrillin (i.e., microfibril) assembly plays an important role in bone mineralization through the accumulation and activation of transforming growth factor beta (TGF-beta) and bone marrow proteins (BMPs) (see Nistala et al. Ann NY Acad. Sci 2010; 1192:253-256; Nistala et al., J Biol Chem 2010; 285:34126-34133, which are each incorporated by reference in their entireties). Impaired TGF-beta and BMP activation may potentially contribute to the skeletal phenotype seen in both Marfan syndrome and HCU and may also reduce bone mineral content, as seen in mild forms of HCU (see Herrmann et al. Clin Chem 2005; 51:2348-2353, which is hereby incorporated by reference in its entirety). Additionally, there is in vivo and in vitro evidence that Hcy may weaken bone strength by reducing collagen cross-linking (see Kang et al. J Clin Invest 1973; 52:2571-2578, which is hereby incorporated by reference in its entirety). Overall, these data suggest that bone mass accretion during childhood and adolescence, a critical period for skeletal development, is insufficient in patients with HCU and that this negatively impacts final bone mass formation.

[0281] Elevated Hcy levels, in addition to their effects on bone deposition, increase the rate of bone remodeling by increasing osteoclast (OC) activity and decreasing osteoblast (OB) activity (Behera et al. J Cell Physiol 2016; Herrmann et al. Clin Chem 2005; 51:2348-2353; Vacek et al. Clin Chem Lab Med 2013; 51:579-590; Vijayan et al. J Endocrinol 2017; 233:243-255, each of which is incorporated by reference in their entireties). An imbalance between OB and OC activity may result in bone fragility and increased fracture rates. The mechanisms leading to Hcy-mediated reduction in OB activity are thought to include decreased bone blood flow (due to decreased NO availability) (see Tyagi et al. Vasc Health Risk Manag 2011; 7:31-35, which is incorporated herein by reference in its entirety) and increased OB apoptosis (see Behera et al. J Cell Physiol 2016; Kim et al. Bone 2006; 39:582-590, which are incorporated herein by reference in their entirety). The mechanisms leading to increased OC activity are thought to include increased levels of intracellular ROS, which enhance both OC differentiation and OC activity by increasing matrix metalloproteinase (MMP) activity (see Vacek et al. Clin Chem Lab Med 2013; 51:579-590, which is hereby incorporated by reference in its entirety) and suppressing OC apoptosis (see Behera et al. J Cell Physiol 2016; Herrmann et al. Clin Chem 2005; 51:2348-2353; Koh et al. J Bone Miner Res 2006; 21:1003-1011, each of which is hereby incorporated by reference in its entirety). Indeed, a recent study in CD1 mice fed a high-Hcy diet showed that short-term (7 days) Hcy administration was associated with loss of tissue mineral density (TMD) and an increase in OC, whereas long-term Hcy administration (30 days) resulted in OC reprogramming, apoptosis, and mineralization that restored TMD but impaired tissue biomechanical properties (see Vijayan et al. J Endocrinol 2017;233:243–255, which is incorporated herein by reference in its entirety).

[0282] Thus, elevated Hcy levels may lead to bone fragility and fractures via two different mechanisms (see Behera et al. J Cell Physiol 2016, which is incorporated herein by reference in its entirety). The first mechanism results in decreased bone mass accretion during childhood and adolescence due to impaired ECM formation and suppressed activation of fibrillin-1-associated TGF-beta and BMP. The second mechanism results in impaired bone remodeling, leading to bone fragility, due to increased OC activity and decreased OB activity.

[0283] Elevated Hcy levels are associated with increased oxidative stress in the bone microenvironment. Increased ROS levels induce osteoblast apoptosis, thereby decreasing osteoblast formation. This increase in oxidative stress further decreases NO availability through the formation of superoxide anions, which can also decrease bone blood flow and angiogenesis. ROS produced by this process activate osteoclast formation via monocyte fusion, which further contributes to BMD loss and leads to osteoporosis.

[0284] A recent study in neonatal CBS knockout (KO) mice maintained on standard rodent chow without Met restriction showed that subcutaneous administration of CBS ET using recombinant PEGylated truncated human CBS (PEG-CBS) for 5 months prevented the decrease in bone mineral density in these animals and could also normalize these values in animals treated at a later age (see Majtan et al. Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria. FASEB J 2017, which is incorporated by reference in its entirety). In this study, body composition changes that are characteristic of both the knockout model and HCU patients were prevented. In both plasma and tissue, tHcy and Cys levels were normalized, Cth levels were increased, and SAM/SAH ratios were elevated.

[0285] Data supporting the effect of Hcy reduction on skeletal pathologies come from a 25-year study of 25 Irish patients with HCU (see Yap et al. J Inherit Metab Dis 1998; 21:738-747, which is incorporated by reference in its entirety). In this study, osteoporosis (diagnosed by radiography, not DXA) was present in one of three noncompliant patients identified by NBS and in one of four patients diagnosed late (at two years of age). None of the 18 patients treated early (from 6 weeks of age) had evidence of osteoporosis.

[0286] A small study was conducted in Korea involving five patients with HCU (3 boys and 3 girls), all diagnosed at a young age (3 during NBS and 2 at age 7 years) with good metabolic control for 3.4 years (see Lim et al. Osteoporos. Int 2013; 24:2535–2538, which is incorporated by reference in its entirety). The mean plasma tHcy at diagnosis was 34.3±52.6 (13 to 78.6) μmol/L. Plasma Met was 716±1347.6 (24.3 to 1566) μmol/L, and treatment included a low-Met diet with pyridoxine, betaine, and folate supplementation. In the Korean patient group, body composition and BMD were within normal limits for all patients, and no significant changes in skeletal morphology were observed over time. Three patients (60%) had mild T-L spine scoliosis (Cobb angles 7.3°, 7.6°, and 10.3°), and four fractures occurred in three patients. Of these, two were due to sports injury and one was due to a traffic accident. Two cases of mild lumbar compression fracture were detected by radiography, and a history of severe back pain was documented. Patients with early diagnosis had fewer skeletal abnormalities than those with later diagnosis. However, this study showed that even patients diagnosed early by NBS, who were treated with dietary therapy and had only mild to moderate elevations in tHcy levels, were found to have skeletal abnormalities and multiple fractures as early as childhood.

[0287] Overall, these data suggest a beneficial effect of early Hcy-lowering treatment on skeletal morbidity in patients with HCU. It should be noted that tHcy levels were reduced but not normalized in patients who were compliant with treatment, and although these patients had less skeletal morbidity, significant negative clinical outcomes (osteoporosis and fractures) were observed in this predominantly young patient group.

[0288] The present studies demonstrated a positive correlation between BMD and Z-scores, plasma tHcy, and dietary protein intake in patients with HCU, suggesting that inadequate protein intake may play a role in increasing skeletal frailty in these patients. Patients with plasma tHcy levels<100 μM had significantly greater skeletal frailty than patients with levels>100 μM (Example 6; Fig. 2). A positive correlation was found between plasma tHcy levels and BMD at all three bone sites examined (Pearson r=0.33-0.51; p<0.03; Table 8). Moreover, higher BMD correlated with higher total dietary protein intake. Indeed, chronic restriction of total protein intake to control tHcy levels may increase skeletal frailty in a patient.

[0289] BMD and Z-scores assessed by dual-energy X-ray absorptiometry (DXA) as described herein (see Example 6) can be a reliable endpoint for assessing the effectiveness of treatment in patients with HCU. Thus, treatment methods comprising enzyme therapy described herein can be used to alleviate dietary restrictions in patients with HCU, thereby increasing protein intake and improving outcomes in terms of reversing skeletal fragility in patients. ET described herein in combination with BMD and Z-score assessment can be performed to determine a patient's tolerance to a soft diet and/or the need for a restricted diet.

C. Vascular complications

[0290] The association between HCU and vascular disease was first demonstrated in 1985 in an epidemiologic study of patients with moderately to severely elevated Hcy levels due to homozygous HCU (see Mudd et al. Am J Hum Genet 1985; 37:1-31, which is incorporated herein by reference in its entirety).

[0291] Thromboembolism is a major cause of morbidity and premature death in patients with HCU (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Karaca et al. Gene 2014; 534:197-203; Yap S. J Inherit Metab. Dis 2003; 26:259-265, each of which is incorporated by reference in its entirety). The overall incidence of thromboembolic complications in patients not treated with HCU is approximately 10% per year (see Cattaneo M. Semin Thromb Hemost 2006; 32:716-723, which is incorporated herein by reference in its entirety), with the risk increasing after surgery and during or immediately following pregnancy (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Novy et al. Thromb Haemost 2010; 103:871-873, which are incorporated herein by reference in their entireties). Thromboembolism can affect any blood vessel, but venous thrombosis (and in particular CSVT) is more common than arterial thrombosis in patients with HCU (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Karaca et al. Gene 2014; 534:197-203; Eslamiyeh et al., Iran J Child Neurol 2015; 9:53-57; Saboul et al. J Child Neurol 2015; 30:107-112, each of which is incorporated by reference in its entirety). Cerebrovascular disorders, particularly CSVT, have been described in infants (see Mahhale et al. J Pediatr Neurosci. 2017 Apr-Jun; 12(2): 206–207, which is incorporated by reference in its entirety), although they are most common in young adults (see Yap et al. Arterioscler Thromb Vasc Biol 2001; 21:2080–2085, which is incorporated by reference in its entirety).

[0292] The risk of thromboembolic complications was approximately 25% at age 16 years and 50% at age 29 years. In 1999, Hankey et al. reported that all three genetic causes of HCU (HCU, MTHFR deficiency, and vitamin B12 deficiency) were associated with a high risk of premature cardiovascular (CV) disease, affecting half of all homozygotes by age 30 years (see Hankey et al. Lancet 1999; 354:407-413, which is incorporated herein by reference in its entirety). The only biochemical change common to all three disorders is elevated serum Hcy levels (often exceeding 100 μmol/L) (see Faeh et al. Swiss Med Wkly 2006; 136:745-756, which is incorporated by reference in its entirety). Several reports have described how treatments that lower tHcy levels significantly reduce the incidence of vascular pathologies, the main cause of morbidity, in patients with HCU (see Yap et al. J Inherit Metab Dis 2001; 24:437-447; Wilcken DE, Wilcken B. The natural history of vascular disease in homocystinuria and the effects of treatment. J Inherit Metab Dis 1997; 20:295-300, which are incorporated by reference in their entireties). Since then, a number of other studies have demonstrated an increased risk of vascular complications, particularly venous thrombosis, in patients with HCU (see Karaca et al. Gene 2014; 534:197–203; Kelly et al. Neurology 2003; 60:275–279; Lussana et al. Thromb Res 2013; 132:681–684; Magner et al. J Inherit Metab Dis 2011; 34:33–37, each of which is incorporated herein by reference in its entirety).

[0293] Elevated plasma tHcy levels are a risk factor for vascular disease and an important predictor of mortality in patients with coronary artery disease, with or without HCU (see Mudd et al. Am J Hum Genet 1985; 37:1–31; Karaca et al. Gene 2014; 534:197–203; Kelly et al. Neurology 2003; 60:275–279; Faeh et al. Swiss Med Wkly 2006; 136:745–756; Boushey et al. JAMA 1995; 274:1049–1057; Clarke R et al. JAMA 2002; 288:2015–2022; Hankey et al. Lancet 1999; 354:407–413; Khan et al. al. Stroke 2008; 39:2943-2949; Graham et al. The European Concerted Action Project. JAMA 1997; 277:1775-1781; Clarke et al. N Engl J Med 1991; 324:1149-1155; Clarke et al. Ir J Med Sci 1992; 161:61-65; Woodward et al. Blood Coagul Fibrinolysis 2006; 17:1-5; Refsum et al. Annu Rev Med 1998; 49:31-62; Yoo et al. Stroke 1998; 29:2478-2483; Selhub et al. N Engl J Med 1995; 332:286-291; Wald et al. BMJ 2002; 325:1202; Bautista et al. J Clin Epidemiol 2002; 55:882-887; Brattstrom et al. Atherosclerosis 1990; 81:51-60; Lussana et al. Thromb Res 2013; 132:681-684; Casas et al. Lancet 2005; 365:224-232; McCully KS. Am J Pathol 1969; 56:111-128; Magner et al. J Inherit Metab Dis 2011; 34:33-37; Wilcken et al. J Clin Invest 1976; 57:1079-1082; Nygard et al. N Engl J Med 1997; 337:230-236; each of which is incorporated herein by reference in its entirety). Although there is evidence for an association between tHcy levels and cardiovascular disease risk (see Boushey et al. JAMA 1995;274:1049–1057, which is incorporated by reference in its entirety), the association between tHcy and stroke/peripheral arterial disease is much stronger (see Clarke et al. JAMA 2002;288:2015–2022; Khan et al. Stroke 2008;39:2943–2949; Wald et al. BMJ 2002;325:1202; Casas et al. Lancet 2005;365:224–232; Brattstrom et al. Haemostasis 1989;19 Suppl 1:35–44, each of which (which are incorporated herein by reference in their entirety). Although large-scale studies (NORVIT, HOPE-2, VITATOPS) in the general population initially showed that lowering Hcy levels had little effect on major vascular events and cardiovascular disease recurrence, more recent, more specific data analyses have clearly demonstrated the clinical benefits of lowering tHcy in stroke.

[0294] There is strong evidence that lowering Hcy levels reduces the risk of stroke in the general population with moderately elevated tHcy levels (see Saposnik et al. Stroke 2009; 40:1365-1372; Huo et al. JAMA 2015; 313:1325-1335; Lonn et al. et al. N Engl J Med 2006; 354:1567-1577; Hankey et al. Lancet Neurol 2012; 11:512-520; Spence JD, Lancet Neurol. 2007 Sep;6(9):830-8, each of which is incorporated by reference in its entirety). In the HOPE-2 trial (see Saposnik et al. Stroke 2009; 40:1365–1372, which is incorporated by reference in its entirety) in 5,552 patients, a modest reduction in tHcy levels (3 mmol/L compared with placebo) resulted in a significant reduction in stroke (27% relative risk reduction, 1.3% absolute risk reduction), suggesting that even small reductions in tHcy levels may be beneficial. This effect was most pronounced in patients with baseline Hcy in the top quartile, who had an absolute risk reduction of 4.3%. Although it is unclear whether lowering Hcy has an impact on overall cardiovascular outcomes in patients with moderately elevated tHcy and without HCU (see Marti-Carvajal et al. Cochrane Database Syst Rev 2015;1:CD006612, which is incorporated by reference in its entirety), the benefits of lowering vascular Hcy have been consistently demonstrated in patients with HCU (see Yap et al. J Inherit Metab Dis 1998; 21:738–747; Eslamiyeh et al. Iran J Child Neurol 2015; 9:53–57; Saboul et al. J Child Neurol 2015; 30:107–112; Yap et al. Arterioscler Thromb Vasc Biol 2001; 21:2080–2085; Woods et al. BMJ Case Rep 2017; Wilcken et al. J Inherit Metab Dis 1997; 20:295–300; Yap et al. Semin Thromb Hemost 2000; 26:335–340; Ruhoy et al. Pediatr Neurol 2014; 50:108–111, each of which is incorporated herein by reference in its entirety).

1. Mechanism

[0295] Several studies have shown that elevated Hcy levels promote the development of atherosclerosis or thrombosis through mechanisms related to oxidative stress (Faverzani et al. Cell Mol Neurobiol 2017; Nowak et al. Arterioscler Thromb Vasc Biol 2017; 37:e41-e52; Vanzin et al. Mol Genet Metab 2011; 104:112-117; Vanzin et al. Gene 2014; 539:270-274; Vanzin et al. Cell Mol Neurobiol 2015;35:899-911; each of which is incorporated by reference in its entirety), including inflammatory and immune activation via NF-kB (Rodriguez-Ayala et al. Atherosclerosis 2005; 180:333-340; van Guldener et al. Curr Hypertens Rep 2003; 5:26-31, which are incorporated herein by reference in their entireties). Medial injury leading to thrombosis is thought to be caused by Hcy-mediated endothelial dysfunction (see Jiang et al. Arterioscler Thromb Vasc Biol 2005; 25:2515–2521; Hossain et al. J Biol Chem 2003; 278:30317–30327; Cai et al. Blood 2000; 96:2140–2148; Zhang et al. J Biol Chem 2001; 276:35867–35874; Papapetropoulos et al. Proc Natl Acad Sci USA 2009; 106:21972–21977; Szabo et al. Br J Pharmacol 2011; 164:853–865; Chiku et al. J Biol Chem 2009; 284:11601–11612; Wang et al. Antioxid Redox Signal 2010; 12:1065–1077; Saha et al. FASEB J 2016; 30:441–456; Ebbing et al. JAMA 2008; 300:795–804; Bonaa et al. N Engl J Med 2006; 354:1578–1588; Marti-Carvajal et al. Cochrane Database Syst Rev 2015;1:CD006612; Celermajer et al. J Am Coll Cardiol 1993; 22:854–858; Rubba et al. Metabolism 1990; 39:1191–1195; each of which in full incorporated herein by reference), activation of coagulation pathways (see Spence JD. Int J Stroke. 2016 Oct;11(7):744-7; Fryer et al. Arterioscler Thromb 1993;13:1327-1333; Lentz et al. J Clin Invest 1991;88:1906-1914; each of which is incorporated herein by reference in its entirety), and increased vascular dilation. These prothrombotic mechanisms are similar to those seen in patients with Marfan disease (see Kelly et al. Neurology 2003; 60:275-279; Tripathi P. International Cardiovascular Forum J 2016; 6:13; van Guldener et al. Curr Hypertens Rep 2003; 5:26-31; Hackam et al. JAMA 2003; 290:932-940; Baumbach et al. Circ Res 2002; 91:931-937; Evangelisti et al. Int J Cardiol 2009; 134:251-254; de Valk et al. Stroke 1996; 27:1134-1136, each of which is hereby incorporated by reference in its entirety).

[0296] A causal relationship between tHcy levels and cardiovascular disease risk, as demonstrated by a meta-analysis of data from 27 studies (over 4,000 patients), showed a high risk of coronary, cerebrovascular, and peripheral vascular atherosclerosis, with a 5 μM increase in Hcy resulting in an 80% increased risk for women and a 60% increased risk for men (see Boushey et al. JAMA 1995; 274:1049-1057, which is incorporated herein by reference in its entirety). A meta-analysis of data from 30 prospective or retrospective studies including 5073 cases of coronary heart disease (CHD) and 1113 stroke found that a 25% lower than normal (adjusted for regression dilution bias) Hcy level (approximately 3 μmol/L) was associated with an 11% (odds ratio (OR), 0.89; 95% CI, 0.83 to 0.96) lower risk of CHD and a 19% (OR, 0.81; 95% CI, 0.69 to 0.95) lower risk of stroke (see Clarke R, et al. JAMA 2002; 288:2015–2022, which is incorporated by reference in its entirety).

[0297] A study in patients with and without pre-existing vascular disease demonstrated an increase in plasma Hcy levels following Met loading (exceeding the highest values in matched healthy controls) in 1/21 subjects with MI (5%), 14/37 subjects with aortoiliac disease (38%), and 17/53 subjects with cerebrovascular disease (32%). This suggests that the association between Hcy levels and peripheral arterial disease (PAD) and stroke is significantly greater than the association between Hcy levels and MI (see Brattstrom et al. Haemostasis 1989; which is incorporated herein by reference in its entirety). An independent strong association between Hcy levels and stroke was described in a prospective study in the UK in a group of 457 stroke patients and 179 stroke controls (see Khan et al. Stroke 2008; 39:2943-2949). The highest Hcy levels were observed in patients with small vessel disease (CVD) (16.2 vs. 11.8 μmol/L in non-stroke controls, p<0.001 after adjustment for age, sex, vascular risk factors, vitamin levels and renal function). In CVD cases, the highest Hcy levels were observed in individuals with lacunar infarction with confluent leukorrhea. In addition, a correlation was found between Hcy levels and the severity of leukorrhea (r=0.225; p<0.001).

[0298] These results were further confirmed in a Mendelian randomization study demonstrating a genetic association between MTHFR polymorphisms regulating Hcy metabolism and stroke risk (see Casas et al. Lancet 2005; 365:224-232, which is incorporated by reference in its entirety). A literature search for all relevant studies on the association between Hcy levels and MTHFR TT and CC polymorphisms conferring stroke risk yielded 111 studies involving 15,635 individuals without cardiovascular disease (CVD). The weighted mean difference in Hcy levels between TT and CC homozygotes was 1.93 μmol/L (95% CI 1.38 to 2.47). Based on the results of a previous meta-analysis of prospective studies in which a 5 μmol/L increase in plasma Hcy was associated with an OR for stroke of 1.59 (1.29 to 1.96), a 1.93 μmol/L increase in Hcy in healthy individuals with the TT genotype yielded an expected OR for stroke of 1.20 (1.10 to 1.31) (see Wald et al. BMJ 2002; 325:1202, which is incorporated by reference in its entirety). Consistent with this result, Khan et al. reported a 1.26 (1.14 to 1.40) OR for stroke for TT homozygotes compared with CC homozygotes (p=0.29), regardless of age group, ethnicity, or geographic location. Overall, these results suggest a causal role for elevated Hcy levels in the pathogenesis of stroke in a large group of individuals.

[0299] Whether moderate elevations of serum Hcy levels are typical of patients with heterozygous HCU due to vascular disease has been determined in studies that are much smaller in size than those conducted in a large group of individuals. According to Mudd et al., the risk of vascular disease in patients with moderately elevated tHcy levels due to heterozygous HCU (<5% at age 50 years) is similar to that in a large group of individuals (see Mudd et al. Am J Hum Genet 1981; 33:883-893, which is incorporated herein by reference in its entirety). Consistent with this, ultrasound examination of individuals with homozygous and heterozygous HCU revealed impaired endothelial function in the systemic arteries of homozygous children at age four years, whereas in heterozygous adults, endothelial function was essentially unchanged (see Celermajer et al. J Am Coll Cardiol 1993;22:854-858, which is incorporated herein by reference in its entirety).

[0300] Although a similar study demonstrated evidence of premature arterial disease in both homozygous and heterozygous individuals, in individuals with homozygous disease, the signs appeared at a much younger age (19 years versus 45 years) and the severity of the disease was significantly greater (see Rubba et al. Metabolism 1990; 39:1191-1195, which is incorporated herein by reference in its entirety).

[0301] Overall, the observations in patients with HCU were consistent with those reported in previously reported studies, where the risk of vascular disease increased with increasing Hcy levels (see Boushey et al. JAMA 1995; 274:1049-1057; Clarke et al. JAMA 2002; 288:2015-2022; Khan et al. Stroke 2008; 39:2943-2949; each of which is incorporated herein by reference in its entirety).

[0302] These results indicate that elevated Hcy levels are a risk factor for cardiovascular disease. Furthermore, lowering Hcy levels has been shown to significantly reduce the risk of stroke in the general population and in patients with HCU.

[0303] Studies have shown that elevated Hcy levels induced by HCU may potentially contribute to the development of atherosclerosis and/or thrombosis through various mechanisms. These include molecular perturbations such as the induction of oxidative stress and its downstream effects such as the activation of NF-κB (activator of nuclear factor of activated B cells-kappa light chain), a transcription factor that regulates pro-inflammatory and other injury-related genes. Various Hcy-mediated effects modulating the physicochemical properties of the vascular wall, such as effects leading to endothelial dysfunction or arterial stiffness, may contribute to the development of hypertension, thrombosis or other vascular pathologies. Finally, there is evidence for direct induction of coagulation pathways by Hcy, i.e., a more direct pathway leading to thrombosis (see Faverzani et al. Cell Mol Neurobiol 2017; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008–1017; Ganguly et al. Nutr J 2015; 14:6; Tripathi P. Molecular and biochemical aspects of homocysteine in cardiovascular diseases. International Cardiovascular Forum J 2016; 6:13; Fryer et al. Arterioscler Thromb 1993; 13:1327–1333; each of which is incorporated by reference in its entirety). After a brief description of the molecular and biochemical mechanisms of atherosclerosis, the following subsections will discuss potential mechanisms leading to vascular disease in individuals with elevated tHcy levels, including HCU.

2. Atherosclerosis

[0304] Among the best-studied conditions leading to thrombosis and hence vascular occlusion is atherosclerosis, a progressive inflammatory disease affecting the coronary, cerebral, and peripheral circulation (see Libby et al. Circulation 2005; 111:3481-3488, which is incorporated by reference in its entirety). In the early stages, vascular injury leads to endothelial cell (EC) activation, recruitment of monocytes to the intima, and activation of macrophages. This results in the formation of an inflammatory atherosclerotic lesion (fatty streak), driven by macrophages derived from lipid-laden monocytes ("foam cells") and T lymphocytes. Progressive lipid accumulation forms a lipid core surrounded by a fibrous cap. In later stages, activated macrophages secrete enzymes that weaken the fibrous cap, leading to plaque rupture, hemorrhage or thrombosis, and ischemic attacks/acute coronary syndromes. When plaque ruptures, tissue factor is exposed to blood within the arterial lumen, allowing it to form complexes with coagulation factors VII/VIIa. This process initiates a coagulation cascade leading to thrombus formation. Disrupted plaques can lead to either mural or occlusive thrombosis, causing partial or complete occlusion, respectively. Mural thrombosis causes ischemic symptoms such as unstable angina, whereas occlusive thrombosis leads to acute coronary syndromes such as myocardial infarction (MI) and stroke. Cytokines are involved in all stages of atherosclerosis and have a significant impact on its pathogenesis (see Ramji et al. Cytokine Growth Factor Rev 2015; 26:673–685, which is incorporated by reference in its entirety). In addition to being secondary to atherosclerosis, thrombosis can also be activated in the absence of plaque formation, such as by atrial fibrillation or direct activation of the coagulation cascade.

3. Oxidative stress

[0305] Studies of patients with elevated Hcy levels have shown that treated patients, and particularly untreated patients, were susceptible to oxidative stress, as evidenced by altered biomarkers reflecting oxidative damage to lipids, proteins, and DNA in various tissues (see Vanzin et al. Mol Genet Metab 2011; 104:112-117; Vanzin et al. Gene 2014; 539:270-274; Vanzin et al. Cell Mol Neurobiol 2015; 35:899-911, each of which is incorporated herein by reference in its entirety). Oxidative stress, defined as an imbalance in redox homeostasis, plays a key role in vascular pathologies such as atherosclerosis and associated thrombosis, the pathogenesis of which involves oxidative modification of low-density lipoproteins, endothelial activation, and initiation of vascular inflammatory responses (see Nowak et al., Arterioscler Thromb Vasc Biol 2017;37:e41-e52, which is incorporated herein by reference in its entirety). Oxidative stress can be caused by elevated levels of ROS (e.g., superoxide (O ^2- ) and hydroxyl (HO ^- ) radicals and hydrogen peroxide (H ₂ O ₂ )) and/or decreased levels of tissue antioxidants (e.g., superoxide dismutase, catalase, and glutathione peroxidase) (see Faverzani et al. Cell Mol Neurobiol 2017; Nowak et al. Arterioscler Thromb Vasc Biol 2017; 37: e41-e52, which are each incorporated by reference in their entireties). In healthy individuals, ROS are produced as byproducts of normal oxidative metabolism. However, ROS production is also triggered by cardiovascular disease risk factors such as cigarette smoke, alcohol consumption, hypercholesterolemia, hypertension, diabetes, and elevated Hcy levels.

[0306] At the molecular level, there are multiple mechanisms by which Hcy may increase oxidative stress, some of which have been discussed previously. For example, accumulation of immunogenic homocysteinylated proteins in the vascular wall may promote inflammation and hence ROS (O ^2- ) production by activated phagocytes. Another potential mechanism is Hcy-induced activation of NMDA receptors, which triggers signaling pathways leading to ROS production. In cardiac microvascular ECs, Hcy induces elevated levels of NADPH oxidase, a cell surface enzyme that, especially in activated cells, produces high levels of O ^2- . A recent study (see Chen et al. Sci Rep. 2017 Jul 31;7(1):6932, which is incorporated herein by reference in its entirety) has shown that in the brain of emic rats, Hcy induces mitochondrial dysfunction with the expected result of increased ROS production. Hcy is also thought to reduce the bioavailability of the beneficial vasodilator NO. O ^2- reacts with NO to form the reactive nitrogen species peroxynitrite, and indeed, Hcy-induced increases in the degree of tyrosine nitration, an indicator of peroxynitrite-induced protein damage, have also been reported (see Tyagi et al. Vasc Health Risk Manag 2011;7:31-35, which is incorporated herein by reference in its entirety). More broadly, thiol-thiol interactions involving Hcy are expected to perturb cellular redox status, for example by potentially reducing the availability of reduced glutathione and even disrupting protein assembly and folding.

[0307] In a study of HCU patients before and after treatment, treatment with pyridoxine, folic acid, betaine supplements, and vitamin B12 supplements attenuated oxidative lipid damage in patients but did not alter sulfhydryl content or total antioxidant status, both measures of tissue antioxidant capacity. However, there was a significant negative correlation between sulfhydryl content and Hcy levels and a positive correlation between malondialdehyde levels, a lipid peroxidation product, and Hcy levels. This suggests a potential mechanistic role for Hcy in the oxidative damage observed in HCU (see Vanzin et al. Mol Genet Metab 2011; 104:112-117, which is incorporated herein by reference in its entirety). Altered lipid profiles, specifically reduced high-density lipoprotein levels and enrichment of proinflammatory lipid species, were observed in the plasma of treated and untreated HCU patients (see Vanzin et al. Cell Mol Neurobiol 2015;35:899–911, which is incorporated by reference in its entirety). Another study reported significantly more DNA damage in HCU patients than in healthy controls (see Vanzin et al. Gene 2014;539:270–274, which is incorporated by reference in its entirety). Collectively, these data indicate that oxidative stress is involved in the pathogenesis of vascular injury associated with elevated Hcy levels. However, no correlation was found between Met levels and any parameters related to oxidative stress, suggesting that Met and its derivatives make a minor contribution to oxidative damage in HCU (see Vanzin et al. Mol Genet Metab 2011; 104:112-117, which is incorporated herein by reference in its entirety).

[0308] Among many other molecular effects, oxidative stress is associated with the activation of NF-κB, a group of transcription factors that regulate the expression of proinflammatory genes such as cytokines, which are known to be involved in the initiation and progression of atherosclerosis and thrombosis (see Ayala et al., Atherosclerosis 2005; 180:333-340, which is hereby incorporated by reference in its entirety). In vitro studies have shown that Hcy treatment of ECs activates NF-kβ through the production of ROS (see van Guldener et al., Curr Hypertens Rep 2003; 5:26-31, which is hereby incorporated by reference in its entirety). In addition to modulating gene expression, chemical modification of cellular macromolecules during oxidative stress may directly influence the structure and function of the vasculature and have other local or systemic effects, as discussed in the following section.

4. Changes in the vascular wall

[0309] Endothelial dysfunction is generally defined as an imbalance between endothelial factors that modulate vascular contractility and relaxation. Among these factors, NO or "endothelial-derived relaxing factor" is the best known, while hydrogen sulfide ( _H2S ) is another factor that has been described more recently (see Jiang et al. Arterioscler Thromb Vasc Biol 2005;25:2515-2521, which is incorporated herein by reference in its entirety). Several in vitro studies have examined the effects of Hcy on endothelial function, albeit using very high levels of Hcy (see Jiang et al. Arterioscler Thromb Vasc Biol 2005; 25:2515-2521; Hossain et al. J Biol Chem 2003; 278:30317-30327; Cai et al. Blood 2000; 96:2140-2148; Zhang et al. J Biol Chem 2001; 276:35867-35874; each of which is incorporated by reference in its entirety). One such study reported an unfolded protein response and programmed cell death in human umbilical vein endothelial cells (HUVEC) treated with Hcy, although Hcy concentrations were several-fold higher than those seen in patients with highly elevated Hcy levels (see Zhang et al. J. Biol Chem 2001; 276:35867-35874, which is incorporated herein by reference in its entirety). In other reports, Cth gamma lyase (CGL), an enzyme involved in Cth metabolism, produced excess _H2S in patients with elevated Hcy levels (see Papapetropoulos et al. Proc Natl Acad Sci USA 2009; 106:21972-21977; Szabo C et al., Br J Pharmacol 2011, 164:853-865, Chiku et al., J Biol Chem, 2009, 284:11601-11612, each of which is hereby incorporated by reference in its entirety). This observation was important because elevated _H2S levels have been reported to significantly increase collateral vessel growth, capillary density, and blood flow in regional tissues (see Wang et al. Antioxid Redox Signal 2010; 12:1065–1077, which is incorporated herein by reference in its entirety). However, high levels of Hcy (0.002 to 2 mM) did not significantly affect EC proliferation or phospho-eNOS levels in vitro (see Saha et al. Cystathionine beta-synthase regulates endothelial function via protein S-sulfhydration. FASEB J 2016; 30:441–456, which is incorporated herein by reference in its entirety). Overall, these results suggest a possible role for Hcy in endothelial dysfunction, although it is not yet clear whether Hcy directly affects ECs in vivo due to the high Hcy levels tested in vitro.

[0310] In a recent pharmacological and genetic study, loss of CBS function in ECs was associated with a 50% decrease in cellular _H2S and a 400% decrease in glutathione, with a concomitant increase in cellular ROS levels (see Saha et al. FASEB J 2016;30:441–456, which is incorporated by reference in its entirety). CBS silencing in ECs compromised phenotypic and signaling responses to vascular endothelial growth factor (VEGF), an effect that was exacerbated by decreased transcription of vascular endothelial growth factor receptor-2 (VEGFR-2) and neuropilin-1 (NRP-1), key receptors regulating endothelial functions such as angiogenesis. Transcriptional downregulation of VEGFR-2 and NRP-1 was mediated by decreased stability of transcription factor-specific protein 1 (Sp1), a target of _H2S sulfhydration. Replenishment of _H2S , but not glutathione, in CBS-silenced ECs resulted in restoration of Sp1 levels and binding of Sp1 to the VEGFR-2 promoter, as well as increased expression of VEGFR-2 and NRP-1 and VEGF-dependent cell proliferation and migration. This indicates that CBS-mediated protein S-sulfhydration is an important factor in maintaining vascular health and function, which supports previous observations that HCU patients have endothelial dysfunction (see Celermajer et al. J Am Coll Cardiol 1993; 22:854-858; Rubba et al. Metabolism 1990; 39:1191-1195, which are incorporated herein by reference in their entirety), and raises the possibility that HCU patients will suffer additional and different types of vascular injury in addition to the type of vascular injury seen in individuals with Hcy levels elevated for other reasons.

[0311] Dysregulation of endothelial function or other effects affecting vascular contractility can lead to blood pressure (BP) abnormalities. Plasma Hcy levels have been directly related to BP, and Hcy reduction by folate has been associated with decreased BP (see Tripathi P. Molecular and biochemical aspects of homocysteine in cardiovascular diseases. International Cardiovascular Forum J 2016; 6:13; Hackam et al. JAMA 2003; 290:932-940, which are each incorporated by reference in their entireties). Although the mechanisms leading to these effects are unclear, Hcy levels are more closely related to systolic than diastolic BP, suggesting that elevated Hcy levels increase arterial stiffness. The degree of arterial stiffness is largely determined by the number and function of smooth muscle cells (SMC), the ratio of collagen to elastin in the extracellular matrix (ECM), collagen quality, and endothelial function (see Tripathi P. Molecular and biochemical aspects of homocysteine in cardiovascular diseases. International Cardiovascular Forum J 2016; 6:13, which is incorporated herein by reference in its entirety).

[0312] Potentially high Hcy levels are associated with increased arterial stiffness due to increased SMC proliferation, collagen production, and elastin fiber formation (see van Guldener et al. Curr Hypertens Rep 2003; 5:26-31, which is incorporated herein by reference in its entirety). However, it is also possible that Hcy decreases arterial stiffness by impairing collagen cross-linking. In a study in piglets, diet-induced elevation of Hcy resulted in a "megaartery syndrome" with arterial hyperpulsatility, systolic (but not diastolic) hypertension, and prolonged reactive hyperemia of the conduction arteries with aortic dilation (see van Guldener et al. Homocysteine and blood pressure. Curr Hypertens Rep 2003; 5:26-31, which is incorporated by reference in its entirety). Fragmentation of the elastic lamina of the arterial wall, correlated with aortic stiffness, was also observed.

[0313] Consistent with these findings, a study in mice with and without HCU showed that vessel wall cross-sectional area was significantly greater in CBS ^+/- mice fed a control diet (437 ± 22 ^μm2 ), and in CBS ^+/+ mice (442 ± 36 ^μm2 ), and in CBS ^+/- mice (471 ± 46 ^μm2 ) fed a high Met diet, compared to CBS ^+/+ mice (324 ± 18 ^μm2 ) fed a control diet (p < 0.05) (see Baumbach et al. Structure of cerebral arterioles in cystathionine beta-synthase-deficient mice. Circ Res 2002; 91:931-937, which is incorporated herein by reference in its entirety).

[0314] During maximal vasodilation, the stress-strain curves in cerebral arterioles of control-fed CBS ^+/- mice and high-Met-fed CBS ^{+/+ mice} were shifted to the right of the curve for control-fed CBS ^+/+ mice, indicating that cerebral arteriolar compliance was greater in mice with elevated plasma tHcy levels. These results suggest that elevated Hcy levels induce cerebral vascular hypertrophy and alter cerebral vascular mechanics, both of which potentially contribute to increased thrombotic events such as stroke, even in the absence of atherosclerosis (see Baumbach et al. Circ Res 2002; 91:931-937, which is incorporated herein by reference in its entirety).

[0315] Further support for the effect of elevated Hcy on vasodilation was provided by studies of 5 Italian patients with HCU and tHcy levels ranging from 193.6 to 342 μmol/L (see Evangelisti et al. Int J Cardiol 2009; 134:251-254, which is incorporated by reference in its entirety). The patients had evidence of mild valve prolapse and/or regurgitation and connective tissue symptoms.

5. Thrombosis

[0316] Elevated Hcy levels are associated with a higher risk of deep vein thrombosis, cerebral sinus thrombosis, and retinal vein thrombosis (see Spence JD. Lancet Neurol. 2007 Sep;6(9):830-8, which is incorporated herein by reference in its entirety), although several studies have failed to find an association with the risk of myocardial infarction. Consistent with these results, additional, albeit small, studies have shown that HCU is associated with thrombosis but not necessarily with atherosclerosis. Vascular imaging in patients with familial hypercholesterolemia (FH) and HCU showed that although patients with FH had diffuse and focal carotid thickening and endothelial dysfunction resulting in reduced blood flow, patients with HCU rarely had carotid plaque and were similar to healthy controls with respect to both mean intimal thickness (IMT) and middle cerebral artery flow velocity (see Rubba et al. Stroke 1994; 25:943-950, which is incorporated by reference in its entirety). This study suggests that typical atherosclerotic lesions may not necessarily precede thrombosis in HCU and that medial damage leading to thrombosis may also be caused by arterial dilation.

[0317] Supporting this observation is a study that compared the prevalence of carotid and femoral artery atherosclerosis (as determined by IMT and ankle-brachial index) in 13 patients with enzymatically confirmed heterozygous HCU compared with 12 healthy individuals with normal Met stress test results (see de Valk et al., Stroke 1996, 27:1134-1136, which is incorporated by reference in its entirety). There were no significant differences between the groups in mean IMT values, IMT frequency distribution, or IMT in each of the five arterial segments. These results may be explained by the fact that the heterozygous individuals were too young (all were <50 years old) to have developed structural vascular changes. However, these data also suggest that elevated Hcy levels may primarily affect the coagulation cascade, at least in younger patients. Indeed, a report of three unrelated patients with HCU showed that one patient had a stroke due to intraluminal thrombosis and another patient developed cardiac or arterial thromboembolism, also without evidence of craniocervical atherosclerosis (see Kelly et al. Neurology 2003;60:275-279, which is incorporated herein by reference in its entirety).

[0318] Consistent with these observations, addition of Hcy to HUVEC and CV1 ECs irreversibly inactivated the anticoagulants protein C and thrombomodulin (see Lentz et al. J Clin Invest 1991; 88:1906-1914, which is hereby incorporated by reference in its entirety). Moreover, addition of Hcy to cultured human ECs increased procoagulant tissue factor activity in a time- and concentration-dependent manner (see Fryer et al. Arterioscler Thromb 1993; 13:1327-1333, which is hereby incorporated by reference in its entirety).

[0319] In both studies, Hcy enhanced coagulation pathways via a mechanism involving the free thiol group. Overall, these data support the hypothesis that abnormalities in vascular coagulation mechanisms contribute to the increased risk of vascular disease in patients with HCU, and that this may play an earlier role in patients with HCU, before the effects of Hcy on atherosclerosis become apparent.

[0320] Reducing plasma Hcy levels reduces the risk of vascular complications, especially stroke, in patients with HCU and in the general population.

[0321] Recent analyses have found a significant association between elevated homocysteine levels and stroke risk in all individuals (see Saposnik et al. Stroke 2009; 40:1365–1372; Spence JD. Homocysteine lowering for stroke prevention: Unravelling the complexity of the evidence. Int J Stroke. 2016 Oct; 11(7):744–7; Hankey et al. Lancet Neurol 2012; 11:512–520, each of which is incorporated by reference in its entirety).

[0322] In the past, the benefit of Hcy-lowering treatments in patients with elevated Hcy levels has been mixed, with some studies showing a reduced risk of cardiovascular disease (see Yap et al. J Inherit Metab Dis 1998; 21:738–747; Yap et al. Arterioscler Thromb Vasc Biol 2001; 21:2080–2085; Wilcken et al. J Inherit Metab Dis 1997; 20:295–300; Yap et al. Semin Thromb Hemost 2000; 26:335–340; Saposnik et al. Stroke 2009; 40:1365–1372; Huo et al. JAMA 2015; 313:1325–1335; Hankey et al. al. Lancet Neurol 2012; 11:512–520, each of which is incorporated by reference in its entirety), while others have shown no benefit (see Marti-Carvajal et al. Cochrane Database Syst Rev 2015;1:CD006612; Ebbing et al. JAMA 2008; 300:795–804; Bonaa et al. N Engl J Med 2006; 354:1578–1588; Liem et al. Heart 2005; 91:1213–1214; Toole et al. JAMA 2004; 291:565–575; B vitamins in patients with recent transient ischemic attack or stroke in the VITAmins TO Prevent Stroke (VITATOPS) trial: a randomized, double-blind, parallel, placebo-controlled trial (see Lancet Neurol 2010; 9:855–865; Albert et al. JAMA 2008; 299:2027–2036, each of which is incorporated by reference in its entirety). Many of these trials have examined treatment with vitamins, primarily B vitamin supplements (B6 and B12) and folate supplements, to lower HCG levels. Consequently, study results are subject to confounding factors such as residence in areas where folate-fortified foods are commonly consumed; the presence of renal dysfunction, which makes individuals more susceptible to cobalamin toxicity; vitamin B12 deficiency due to poor absorption, which is relatively common in older individuals; or the use of antiplatelet drugs. These recent analyses have taken these confounding factors into account and concluded that, when accounting for these key abnormalities, elevated tHcy increases stroke risk in individuals overall (see Mudd et al. Arterioscler Thromb Vasc Biol 2000; 20:1704–1706; Spence JD. Int J Stroke. 2016 Oct;11(7):744–7; Spence JD, Clin Chem Lab Med. 2013 Mar 1;51(3):633–7, each of which is incorporated by reference in its entirety).

[0323] Therapy that lowers Hcy levels reduces the risk of stroke even in individuals without HCU. In the Heart Disease Prevention Experiment 2 (HOPE-2) trials, 5,522 adults aged ≥55 years with vascular disease or a history of diabetes and at least one additional risk factor for cardiovascular disease were randomized to receive a vitamin supplement (folic acid, vitamin B6, and vitamin B12) or placebo for 5 years (see Saposnik et al. Stroke 2009; 40:1365-1372, which is incorporated by reference in its entirety). Mean baseline Hcy concentrations were 11.5 μmol/L in both groups, and patients taking a daily vitamin supplement containing >0.2 mg folic acid were excluded from the study. Overall, a reduction in Hcy (mean 3.0 μmol/L compared with placebo) was associated with a significant 27% relative risk reduction in stroke (1.3% absolute risk reduction) (HR 0.75; 95% CI 0.59 to 0.97) and nonsignificant reductions in ischemic stroke (HR 0.81; 95% CI 0.60 to 1.09) and hemorrhagic stroke (HR 0.80; 95% CI 0.32 to 2.02). In subgroup analyses, the relative risk of stroke was significantly reduced in patients with baseline Hcy levels in the highest quartile (4.3% absolute risk reduction). The greatest treatment effect was observed in patients aged <69 years, and the trial included patients living in areas where folic acid was not added to food and patients not taking antiplatelet or lipid-lowering drugs. Thus, the HOPE-2 trial reported a reduction in the incidence of stroke in individuals receiving B vitamins compared with placebo (hazard ratio (HR) 0.75; 95% CI, 0.59 to 0.97), whereas the risk of myocardial infarction was similar in both treatment groups (RR 0.98; 95% CI, 0.85 to 1.14) (see Saposnik et al. Stroke 2009; 40:1365–1372; Lonn E, Yusuf S, Arnold MJ et al. Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 2006; 354:1567–1577, which are hereby incorporated by reference in their entireties).

[0324] Consistent with the HOPE-2 study results, a subanalysis of the VITATOPS study, which enrolled 8164 patients with a recent stroke or transient ischemic attack and randomized to double-blind treatment with B vitamins or placebo for a mean period of 3.4 years, showed that B vitamins significantly reduced the primary composite outcome (stroke, myocardial infarction (MI), or vascular death) in patients not receiving antiplatelet therapy at baseline (17% versus 21% in the placebo group; HR 0.76, 0.60-0.96). However, no significant effect of B vitamins was observed in individuals receiving antiplatelet therapy (see Hankey et al. Antiplatelet therapy and the effects of B vitamins in patients with previous stroke or transient ischemic attack: a post-hoc sub-analysis of VITATOPS, a randomized, placebo-controlled trial. Lancet Neurol 2012; 11:512-520, which is incorporated herein by reference in its entirety). In this study, tHcy levels were significantly reduced from 12.4 to 13.7 μmol/L at baseline to 9.9–10.5 μmol/L after vitamin therapy, regardless of whether patients were receiving antiplatelet therapy or not (p<0.0001 for both treatment groups) (see Hankey et al., Lancet Neurol 2012; 11:512–520, which is incorporated herein by reference in its entirety).

[0325] Inflammatory cascades are thought to contribute to the pathogenesis of ischemic stroke. In the Framingham Offspring Study of 3,224 individuals (see Shoamanesh et al. Neurology. 2016 Sep;87(12):1206-11, which is incorporated by reference in its entirety), elevated levels of tHcy and three other inflammatory markers were found to be strongly associated with the risk of ischemic stroke and improved the predictive ability of the Framingham Stroke Risk Profile score.

[0326] A meta-analysis of data from the VISP and VITATOPS trials showed that patients with normal renal function who had not previously been exposed to high doses of cyanocobalamin benefited significantly from vitamin therapy including high doses of cyanocobalamin (0.78, 0.67-0.90; probability of interaction p=0.03), whereas vitamin therapy including high doses of cyanocobalamin (in the form of vitamin B) did not affect the risk of stroke in individuals with impaired renal function (RR 1.04, 95% CI 0.84 to 1.27) (see Spence JD. Lancet Neurol. 2007 Sep;6(9):830-8, which is incorporated herein by reference in its entirety). These results highlight the potential confounding effects of cyanocobalamin, which is known to cause nephrotoxicity due to cyanide accumulation in patients with significant renal impairment (see Spence JD, Clin Chem Lab Med. 2013 Mar 1; 51(3):633-7, which is incorporated by reference in its entirety). Consistent with this, in the DIVINe (Diabetes Treatment with Vitamins Given in Nephropathy) trial, high doses of B vitamins, including 1000 mcg cyanocobalamin, were toxic and exacerbated the decline in eGFR (see Spence JD. Int J Stroke. 2016 Oct; 11(7):744-7; House et al. JAMA 2010; 303: 1603-1609, which are incorporated by reference in their entireties). Overall, these data support the use of non-cyanide containing B vitamins such as methylcobalamin instead of cyanocobalamin to lower Hcy levels in individuals at high risk of stroke, particularly those with renal failure.

[0327] Cyanocobalamin and cyanide toxicity have also been mentioned in previous studies as confounding factors in the presence of renal failure in the CSPPT (China Stroke Primary Prevention Trial (see Huo et al. JAMA. 2015 Apr 7;313(13):1325-35), which is incorporated herein by reference in its entirety). The benefit of treatment demonstrated in renal failure in the CSPPT was in contrast to the lack of benefit observed in the DIVINe, VISP, and VITOPS trials, likely due to cyanocobalamin treatment in the latter trials.

[0328] Since high-dose folic acid supplementation has not yet been used in China, the effect of folic acid supplementation on tHcy reduction could be studied there in a larger group of individuals. The CSPPT was a randomized, double-blind trial in 20,702 adults with hypertension but no history of stroke or MI, and this study demonstrated that folic acid significantly reduced the risk of first stroke (2.7 vs 3.4% without folic acid, HR 0.79; 95% CI 0.68 to 0.93), first ischemic stroke (2.2 vs 2.8% without folic acid, HR 0.76; 95% CI 0.64 to 0.91), and composite cardiovascular events (cardiovascular death, MI, and stroke; 3.1% vs 3.9% without folic acid, HR 0.80; 95% CI 0.69 to 0.92). In contrast, there was no significant difference in the risk of hemorrhagic stroke, all-cause mortality, or adverse event rates between the two groups. In an extension study of the CSPPT (see Xu et al. JAMA Intern Med. 2016 Oct 1;176(10):1443–1450, which is incorporated by reference in its entirety), both individuals with and without renal impairment (eGFR <60 mL/min/1.73 ^m2 ) benefited from folate supplementation, and the extension study also confirmed that the folate-treated group had a significantly greater reduction in serum Hcy than the no-folate group (1.9 vs. 0.2 μmol/L, respectively, p<0.001).

[0329] A 2017 Cochrane review (see Martí-Carvajal et al. Cochrane Database Syst Rev. 2017 Aug 17;8:CD006612, which is incorporated herein by reference in its entirety) that analyzed 15 randomized controlled trials involving 74,422 individuals reported a small difference in the effect of Hcy lowering with B vitamins on stroke, but no effect on MI, mortality from any cause, or adverse events. Compared with placebo/standard care, Hcy-lowering treatment was associated with a reduction in nonfatal or fatal stroke (4.33% vs 5.1% for control; RR 0.90, 95% CI 0.82-0.99) but had no effect on nonfatal or fatal MI (7.1% vs 6.0% for placebo; relative risk (RR) 1.02, 95% CI 0.95 to 1.10) or death from any cause (11.7% vs 12.3% for placebo; RR 1.01, 95% CI 0.96 to 1.06) in the overall group. This review has revised three earlier versions (2009, 2013 and 2015) which had previously concluded that there was no evidence to support an effect of Hcy-lowering treatments on cardiovascular disease, although the 2015 review did suggest a non-significant trend towards a reduction in stroke (see Marti-Carvajal Cochrane Database Syst Rev 2015;1:CD006612, which is incorporated by reference in full). The certainty of the evidence for Hcy-lowering treatments in stroke has improved as additional studies have become available.

[0330] The studies included in the review used different vitamin supplementation regimens as therapy to lower Hcy levels (see Martí-Carvajal et al. Cochrane Database Syst Rev. 2017 Aug 17;8:CD006612, which is incorporated by reference in its entirety). The 2017 Review added three new trials to the 2015 Review. Of the total trials, 10 trials used pyridoxine plus vitamins B9 (folate) and B12, five used vitamin B9 alone, and one of the 10 used 5-methyltetrahydrofolate instead of folate. Some trials included concomitant medications (in both the control and vitamin groups), seven trials used lipid-lowering drugs, and one trial used antihypertensive agents. Overall, Hcy-lowering treatments resulted in relatively small reductions in mean tHcy levels. Furthermore, three studies were conducted with a group receiving a folate-fortified diet and two studies were conducted with a mixed group (some individuals receiving the fortified diet and others not), which may have masked the Hcy-lowering effects. Given the confounding factors that can influence such studies, it is not surprising that earlier reviews failed to find any significant cardiovascular effects from vitamin treatments, and the most recent review reported only a modest effect on stroke. It should also be noted that many potential causes of elevated Hcy levels were not examined in the studies included in the Cochrane review (see Marti-Carvajal et al. Cochrane Database Syst Rev 2015; which is incorporated by reference in its entirety). However, there may be additional factors influencing the pathophysiology associated with vascular risk in HCU patients and at a younger age than the general population. Notably, even modest reductions in Hcy levels are significantly associated with a reduced risk of stroke.

[0331] After performing some of these analyses, an interesting question arises as to why some studies show an effect of Hcy levels on stroke but not on myocardial infarction (MI). In this regard, Spence (see Spence JD. Lancet Neurol. 2007 Sep;6(9):830-8, which is incorporated by reference in its entirety) pointed out the key differences between myocardial infarction and cerebral infarction, whereby cerebral infarction is closely associated with thrombosis/embolism, but thrombosis in situ is a consequence of plaque rupture in the coronary artery in almost all cases of MI. Thus, Spence concluded that a significant proportion of strokes are associated with thrombotic processes that may be associated with elevated tHcy levels.

[0332] Elevated tHcy levels may be important not only in cardioembolic stroke but also in atheroembolic and lacunar infarction. A study in elderly patients with atrial fibrillation receiving anticoagulant therapy showed that high tHcy levels (>90th percentile) were associated with a 4.7-fold increase in ischemic complications (see Poli et al. J Am Coll Cardiol 2009; 54:999-1002, which is incorporated herein by reference in its entirety). Another study in patients with cryptogenic ischemic stroke showed that patients with foramen ovale (a risk factor for cerebral infarction) had significantly higher plasma tHcy levels than those without it (8.9±3 vs. 7.9±2.6 μmol/L, respectively; p=0.021) (see Ozdemir et al. J Neurol Sci 2008;275:121–127, which is incorporated herein by reference in its entirety). In reviewing these results, Spence (see Spence, JD. "Homocysteine lowering for stroke prevention: Unravelling the complexity of the evidence" Int J Stroke (2016) Oct; 11(7):744–747, which is incorporated by reference in its entirety) suggested that Hcy primarily influences the formation of red thrombus (a fibrin polymer mesh of entrapped red blood cells that forms under stasis), but that lacunar infarctions and carotid plaques are also significantly associated with tHcy, since tHcy levels were also significantly higher in patients with microemboli on transcranial Doppler (16.2 vs. 10.1 mmol/L), and most of these microemboli are thought to represent platelet aggregates that are reduced by dual antiplatelet therapy (see Spence, JD. Homocysteine lowering for stroke prevention: Unravelling the complexity of the evidence. Int J Stroke. 2016 Oct; 11(7):744–747, which is incorporated herein by reference in its entirety).

[0333] In a study of 32 patients with HCU (aged 9 to 66 years) receiving pyridoxine, folic acid, and hydroxocobalamin, for a total of 539 patients over several years of therapy, two vascular events (fatal pulmonary embolism and MI) were reported during treatment (see Wilcken et al. J Inherit Metab Dis 1997, 20:295-300, which is incorporated herein by reference in its entirety). In an epidemiologic study by Mudd et al., 21 events would have been expected over the same time period without treatment (RR 0.09 (95% CI 0.02 to 0.38); p=0.0001). A second study of 84 patients from three countries aged 2.5 to 70 years found five venous embolisms (VE) in 1314 patients over several years of therapy, i.e., one pulmonary embolism, two MIs, and two abdominal aneurysms (see Yap et al. Semin Thromb Hemost 2000; 26:335–340, which is incorporated by reference in its entirety). According to Mudd et al. (see Mudd et al. Am J Hum Genet 1985; 37:1–31, which is incorporated by reference in its entirety), there were 53 VEs, as would be expected in untreated patients (RR 0.091 (95% CI 0.043 to 0.190); p<0.001).

[0334] Evidence supporting these findings was obtained from a large, international, multicenter pilot study of 158 patients with HCU (see Yap et al. Arterioscler Thromb Vasc Biol 2001; 21:2080-2085, which is incorporated herein by reference in its entirety), where the majority of patients were between 10 and 30 years of age. In this study, 17 vascular events were observed in 12 treated patients, i.e., three cases of pulmonary embolism, two cases of MI, five cases of deep vein thrombosis, three cases of cerebrovascular accident, one case of transient ischemic attack, one case of sagittal sinus thrombosis, and two cases of abdominal aortic aneurysm. Without treatment, 112 vascular events would be expected in a similar group (RR 0.09 (95% CI 0.036 to 0.228); p<0.0001). This study also showed that even treated and closely monitored young adult patients with HCU have poor clinical outcomes compared with the overall group.

[0335] Elevated plasma tHcy is a risk factor for vascular disease and an important predictor of mortality in patients with CAD, with or without CBSD (see Mudd et al. Am J Hum Genet 1985; 37:1–31; Karaca et al. Gene 2014; 534:197–203; Kelly et al. Neurology 2003; 60:275–279; Faeh et al. Swiss Med Wkly 2006; 136:745–756; Boushey et al. JAMA 1995; 274:1049–1057; Clarke et al. JAMA 2002; 288:2015–2022; Hankey et al. Lancet 1999; 354:407–413; Khan et al. al. Stroke 2008; 39:2943-2949; Graham et al. JAMA 1997; 277:1775-1781; Clarke et al. N Engl J Med 1991; 324:1149-1155; Clarke et al. Ir J Med Sci 1992; 161:61-65; Woodward et al. Blood Coagul Fibrinolysis 2006; 17:1-5; Refsum et al. Annu Rev Med 1998; 49:31-62; Yoo et al. Stroke 1998; 29:2478-2483; Selsun et al. N Engl J Med 1995; 332:286-291; Wald et al. BMJ 2002; 325:1202; Bautista et al. J Clin Epidemiol 2002; 55:882-887; Brattstrom et al. Atherosclerosis 1990; 81:51-60; Lussana et al. Thromb Res 2013; 132:681-684; Casas et al. Lancet 2005; 365:224-232; McCully KS. Am J Pathol 1969; 56:111-128; Magner et al. J Inherit Metab Dis 2011; 34:33-37; Wilcken et al. J Clin Invest 1976; 57:1079-1082; Nygard et al. N Engl J Med 1997; 337:230-236, each of which is hereby incorporated by reference in its entirety).

[0336] Of the data on the causal relationship between tHcy levels and cardiovascular disease risk (see Boushey et al. JAMA 1995;274:1049–1057, which is incorporated by reference in its entirety), the strongest association has been demonstrated between tHcy and stroke or PAD (see Clarke et al. JAMA 2002;288:2015–2022; Khan et al. Stroke 2008;39:2943–2949; Wald et al. BMJ 2002;325:1202; Casas et al. Lancet 2005;365:224–232; Brattstrom et al. Haemostasis 1989;19 Suppl 1:35-44, each of which is incorporated herein by reference in its entirety), including patients with homozygous and heterozygous CBS mutations (see Rubba et al. Metabolism 1990; 39:1191-1195, which is incorporated herein by reference in its entirety).

[0337] The mechanisms potentially linking elevated Hcy levels to vascular injury are diverse and highly complex. Several studies have identified oxidative stress (see Faverzani et al. Cell Mol Neurobiol 2017; Nowak et al. Arterioscler Thromb Vasc Biol 2017; 37: e41-e52; Vanzin et al. Mol Genet Metab 2011; 104:112-117; Vanzin et al. Gene 2014; 539:270-274; Vanzin et al. Lipid, Cell Mol Neurobiol 2015; 35:899-911, each of which is incorporated by reference in its entirety), including oxidative stress through inflammatory/immune activation via NF-κB (see Rodriguez-Ayala et al. Atherosclerosis 2005; 180:333-340; van Guldener et al. Curr Hypertens Rep 2003; 5:26-31, which are hereby incorporated by reference in their entireties). Although such inflammatory processes are characteristic of atherosclerosis, typical atherosclerotic lesions do not necessarily precede thrombotic disorders in patients with HCU (see Rubba et al. Stroke 1994; 25:943-950; de Valk et al. Stroke 1996; 27:1134-1136, which are hereby incorporated by reference in their entireties). Instead, medial injury leading to thrombosis is thought to be caused by Hcy-mediated endothelial dysfunction (see Marti-Carvajal et al. Cochrane Database Syst Rev 2015; 1:CD006612; Celermajer et al. J Am Coll Cardiol 1993; 22:854–858; Rubba et al. Metabolism 1990; 39:1191–1195; Jiang et al. Arterioscler Thromb Vasc Biol 2005; 25:2515–2521; Hossain et al. J Biol Chem 2003; 278:30317–30327; Cai et al. Blood 2000, 96:2140–2148; Zhang et al., J Biol Chem, 2001, 276:35867-35874, Papapetropoulos et al., Proc Natl Acad Sci USA, 2009, 106:21972-21977, Szabo et al., Br J Pharmacol, 2011; 164:853-865; Chiku et al., J Biol Chem 2009; 284:11601-11612; Wang et al., Antioxid Redox Signal, 2010; 12:1065-1077; Saha S, et al., FASEB J 2016; 30:441-456; Ebbing et al. JAMA 2008; 300:795-804; Bonaa et al. N Engl J Med 2006; 354:1578–1588, each of which is hereby incorporated by reference in its entirety), enhanced coagulation pathways (see Spence JD, Int J Stroke, 2016 Oct;11(7):744–7, Fryer et al. Arterioscler Thro mb 1993;13:1327–1333; Lentz et al. J. Clin. Invest 1991; 88:1906–1914, each of which is incorporated herein by reference in its entirety) and increased vascular dilation similar to the thrombotic processes characteristic of patients with Marfan disease (see Kelly et al. Neurology 2003; 60:275–279; Tripathi P. Molecular and biochemical aspects of homocysteine in cardiovascular diseases. International Cardiovascular Forum J 2016; 6:13; van Guldener et al. Curr Hypertens Rep 2003; 5:26–31; Hackam et al. JAMA 2003; 290:932–940; Baumbach et al. Circ Res 2002; 91:931–937; Evangelisti et al. Int J Cardiol. 2009; 134:251–254; de Valk et al. Stroke 1996; 27:1134–1136, each of which is incorporated by reference in its entirety). Consistent with the observations of endothelial dysfunction in patients with HCU, pharmacologic and genetic studies have implicated CBS-mediated protein S-sulfhydration in the maintenance of vascular health and function (see Celermajer et al. J Am Coll Cardiol 1993; 22:854–858; Rubba et al. Metabolism 1990; 39:1191–1195, which are each incorporated by reference in their entireties). Thus, it is likely that the mechanisms of vascular injury in patients with HCU are more diverse than in the general population of patients with cardiovascular disease.

[0338] There is compelling evidence that lowering Hcy levels is beneficial in reducing the risk of stroke in individuals with and without HCU (see Saposnik et al. Stroke 2009; 40:1365-1372; Huo et al. JAMA 2015; 313:1325-1335; Lonn et al. N Engl J Med 2006; 354:1567-1577; Hankey et al. Lancet Neurol 2012; 11:512-520; Spence JD. Lancet Neurol. 2007 Sep;6(9):830-8, each of which is incorporated by reference in its entirety). Although mean baseline Hcy levels were relatively low in studies conducted in individuals in the overall population, stroke reduction was significantly correlated with Hcy-lowering therapy, indicating benefits from even small reductions in tHcy levels.

[0339] Updated reviews of relevant studies support the beneficial effect of Hcy-lowering treatments in stroke (see Martí-Carvajal et al. Cochrane Database Syst Rev. 2017 Aug 17;8:CD006612, which is incorporated by reference in its entirety). Confounding factors such as folate supplementation, vitamin B12 deficiency, or renal dysfunction making patients vulnerable to high doses of cyanocobalamin, or individuals taking antiplatelet drugs, have confounded the trial results. Recent analyses have shown that when accounting for such factors, the association between tHcy and stroke risk was strongest in the overall group (see Spence JD, Clin Chem Lab Med. 2013 Mar 1;51(3):633-7, which is incorporated by reference in its entirety). However, it is important to note that HCU patients with much higher tHcy levels than the general population have consistently demonstrated benefits of Hcy lowering in the treatment of vascular disease (see Yap et al. J Inherit Metab Dis 1998; 21:738–747; Eslamiyeh et al. Iran. J Child Neurol 2015; 9:53–57; Saboul et al. J Child Neurol 2015; 30:107–112; Yap et al. Arterioscler Thromb Vasc Biol 2001; 21:2080–2085; Woods et al. BMJ Case Rep 2017; 2017; Wilcken et al. J Inherit Metab Dis 1997; 20:295–300; Yap et al. Semin Thromb Hemost 2000; 26:335-340; Ruhoy et al. Pediatr Neurol 2014; 50:108-111, each of which is incorporated herein by reference in its entirety).

[0340] These data suggest that elevated Hcy levels are a risk factor for cardiovascular disease, particularly stroke, in patients with and without HCU, and that the risk of cardiovascular or cerebrovascular disease can be reduced by long-term Hcy-lowering therapy.

[0341] Thromboembolism is a major cause of morbidity and premature death in patients with HCU (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Karaca et al. Gene 2014; 534:197-203; Yap S. J Inherit Metab Dis 2003; 26:259-265, each of which is hereby incorporated by reference in its entirety). The overall incidence of thromboembolic events in untreated patients with HCU is approximately 10% per year (see Cattaneo M. Semin Thromb Hemost 2006; 32:716-723, which is hereby incorporated by reference in its entirety), with the risk increased following surgery. and during or immediately after pregnancy (see Mudd et al. Am J Hum Genet 1985; 37:1-31; Novy et al. Thromb Haemost 2010; 103:871-873, which are hereby incorporated by reference in their entireties). Thromboembolism can affect any blood vessel, but venous thrombosis (particularly cerebral sinovenous thrombosis (CSVT)) is more common than arterial thrombosis in patients with HCU (see Mudd et al. Am J Hum Genet 1985; 37:1–31; Karaca et al. Gene 2014; 534:197–203; Eslamiyeh et al., Iran J Child Neurol 2015; 9:53–57; Saboul et al. J Child Neurol 2015; 30:107–112, each of which is hereby incorporated by reference in its entirety).

[0342] A study of 629 untreated patients with HCU found that of the 253 patients with vascular disease (158 patients), 81 (32%) had cerebrovascular accident, 130 (51%) had peripheral venous disease (32 of which resulted in pulmonary embolism), 10 (4%) had myocardial infarction (MI), 28 (11%) had peripheral arterial disease, and four patients (2%) did not fall into any of these categories (see Mudd et al. Am J Hum Genet 1985; 37:1-31, which is incorporated herein by reference in its entirety). Cerebrovascular disorders, particularly CSVT, have been described in infants (see Mahhale et al. J Pediatr Neurosci. 2017 Apr-Jun; 12(2):206–207, which is incorporated by reference in its entirety), although they most commonly occur in young adults (see Yap et al. Arterioscler Thromb Vasc Biol 2001; 21:2080–2085, which is incorporated by reference in its entirety). Cerebrovascular disorders have been reported to be only marginally associated with the pyridoxine-responsive patient population.

[0343] The risk of vascular complications was approximately 30% in patients under 20 years of age, increasing to 50% by age 30. However, such symptoms can occur at any age, and fatal thrombosis has been described in infants as young as 6 months of age (see Cardo et al. Dev Med Child Neurol 1999; 41:132-135, which is incorporated by reference in its entirety). After age 10, one vascular event is expected every 25 years. Typically, the first signs of HCU in children are cognitive symptoms, manifested by developmental delay during the first or second year of life and/or lens luxation/high myopia. In contrast, adults have a high risk of vascular complications.

D. Effect of diet on phenotypic traits

[0344] In some embodiments, I278T mice, a mouse model of HCU, were used to evaluate the long-term effects of enzyme therapy for the treatment of HCU using 20NHS PEG-CBS on clinical endpoints relevant to human patients. The efficacy of 20NHS PEG-CBS was assessed in the context of normal methionine intake (REG) and a Met-restricted diet (MRD), as well as in MRD monotherapy. Treatment with 20NHS PEG-CBS can result in a 90% reduction in plasma homocysteine concentrations and correction of learning/cognition, endothelial dysfunction, hemostasis, bone mineralization, and body composition phenotypes associated with HCU. In some embodiments, treatment with 20NHS PEG-CBS in the context of MRD normalized plasma Hcy levels. It was observed that MRD alone reduced plasma Hcy by 67% and corrected the HCU phenotype in I278T mice. However, MRD increased anxiety and decreased bone mineral content in both I278T mice and wild-type controls. Thus, 20NHS PEG-CBS was very effective in treating HCU in individuals on REG or Met-restricted diet. In fact, enzyme therapy (ET) with 20NHS PEG-CBS in the presence of normal Met intake produced similar or better results than Met-restricted diet.

[0345] In summary, MRD alone is effective in correcting multiple symptoms of HCU, although it fails to reduce plasma Hcy concentrations below the recommended level and results in increased anxiety and decreased bone mineralization. On the other hand, enzyme therapy using 20NHS PEG-CBS as described herein reduced plasma Hcy concentrations below 100 μM and corrected all observed symptoms of HCU. In addition, 20NHS PEG-CBS maintains its efficacy in restricting Met, providing a fully normalized plasma biochemical profile. When extrapolated to patients, the results showed that 20NHS PEG-CBS as a single lifelong therapy may be effective in preventing and correcting the clinical symptoms of HCU. In addition, treatment with 20NHS PEG-CBS allows for a relaxation of dietary Met restriction and thus significantly improves the quality of life of HCU patients and their families.

E. Neurological complications

[0346] Studies have shown that early reduction of Hcy levels induced by a low-Met diet, folate/B vitamin supplementation, and/or pyridoxine and betaine therapy can prevent and sometimes reverse the progression of various neurological disorders and ensure normal IQ in patients with HCU (see El El Bashir et al. JIMD Rep 2015; 21:89–95; Yap et al. J Inherit Metab Dis 2001; 24:437–447; Mech AW, Farah A. Correlation of clinical response with homocysteine reduction during therapy with reduced B vitamins in patients with MDD who are positive for MTHFR C677T or A1298C polymorphism: a randomized, double-blind, placebo-controlled study. J Clin Psychiatry 2016; 77:668–671; Grobe H. Eur J Pediatr 1980; 135:199-203; each of which is incorporated herein by reference in its entirety). Further evidence comes from case studies of patients with HCU, in whom significant reduction, even normalization of Hcy levels, resulted in complete or partial correction of CNS disorders (see Yap et al. J Inherit Metab Dis 2001; 24:437–447; Brenton et al. J Child Neurol 2014; 29:88–92; Rezazadeh et al. Child Neurol Open 2014; 1:2329048X14545870; Kaeser et al. J Neurol Neurosurg Psychiatry 1969; 32:88–93; Colafrancesco et al. Eur J Pediatr 2015; 174:1263–1266; Yokoi et al. Pediatr Int 2008; 50:694–695; Li et al. Pathology 1999; 31:221-224, each of which is incorporated herein by reference in its entirety).

[0347] The association between elevated Hcy levels and CNS symptoms, including cognitive symptoms, neurodegenerative diseases, seizures, dystonia, psychosis, cognitive impairment, dementia, and depression, is well documented in patients with HCU and in the general population (see Morris et al. J Inherit Metab Dis 2017; 40:49–74; Abbott et al. Am J Med Genet 1987; 26:959–969; Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7 ed. New York: McGraw Hill; 2001;1279–1327; Hidalgo et al. Eur Child Adolesc Psychiatry 2014; 23:235-238; Smith et al. PLoS One 2010;5:e12244; Seshadri et al. N Engl J Med 2002; 346:476-483; Bottiglieri et al. J Neurol Neurosurg Psychiatry 2000; 69:228-232; Bjelland et al. Arch Gen Psychiatry 2003; 60:618-626; Tolmunen et al. Am J Clin Nutr 2004; 80:1574-1578; Kaeser et al. J Neurol Neurosurg Psychiatry 1969; 32:88-93; Golimbet et al. Psychiatry Res 2009; 170:168-171; Clarke et al. Arch Neurol 1998; 55:1449-1455; Permoda-Osip et al. Neuropsychobiology 2014; 69:107-111; Oliveira et al. BMJ Case Rep 2016;2016; Troen et al. Proc Natl Acad Sci U S A 2008; 105:12474-12479; Sudduth et al. J Cereb Blood Flow Metab 2013; 33:708-715; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008-1017; Herrmann et al. Clin Chem Lab Med 2011; 49:435-441; Kim et al. J Nutr 2007; 137:2093-2097; Selhub et al. Am J Clin Nutr 2000; 71:614S-620S; McCaddon et al. Dement Geriatr Cogn Disord 2001; 12:309-313; Smallwood et al. Neuropathol Appl Neurobiol 2012; 38:337-343; Beydoun et al. BMC Public Health 2014; 14:643; Gortz et al. J Neurol Sci 2004; 218:109-114; Health Quality O. Vitamin B12 and cognitive function: an evidence-based analysis. Ont.Health Technol.Assess.Ser.13 (23), 1e45. 2013. Ref Type: Online Source; Salagre et al. Eur Psychiatry 2017; 43:81-91, each of which is hereby incorporated by reference in its entirety). The mechanisms leading to CNS disorders in individuals with elevated Hcy levels are thought to include tHcy-mediated neuronal injury (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7 ed. New York: McGraw Hill; 2001;1279–1327; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008–1017; Stefanello et al. Metab Brain Dis 2007; 22:172–182; Toborek et al. Atherosclerosis 1995; 115:217–224, each of which is incorporated by reference in its entirety), endothelial damage, and cellular dysfunction (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7 ed. New York: McGraw Hill; 2001;1279–1327; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008–1017; Stefanello et al. Metab Brain Dis 2007; 22:172–182; Toborek et al. Atherosclerosis 1995; 115:217–224, each of which is hereby incorporated by reference in its entirety), endothelial damage, and cellular dysfunction (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7 ed. New York: McGraw vascular dysfunction caused by Hcy-mediated oxidative stress (see Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004; 287: R39-R46, which is hereby incorporated by reference in its entirety), neuronal death (see Yeganeh et al., J Mol Neurosci 2013; 50:551-557; Heider et al. J Neural Transm Suppl 2004; 1-13, which are hereby incorporated by reference in their entireties), and impaired neural network activity (see Gortz et al. J Neurol Sci 2004; 218:109-114, which is hereby incorporated by reference in its entirety). Depression and seizures are thought to be caused, at least in part, by Hcy-mediated reductions in cerebral adenosine levels, followed by reductions in norepinephrine and dopamine levels (see Mech et al. J Clin Psychiatry 2016; 77:668–671; Domagala et al. Thromb Res 1997; 87:411–416; Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004;287:R39–R46; Folstein et al., Am J Psychiatry 2007:164:861–867, each of which is hereby incorporated by reference in its entirety).

[0348] These results demonstrated a significant correlation between Hcy levels and an increased risk of CNS disorders in patients with HCU. Early therapy aimed at reducing Hcy levels is necessary for normal development of children with early-onset HCU, as well as for correction or elimination of CNS disorders in patients diagnosed with HCU at a later age.

[0349] A study of 63 patients with HCU found that 51% of patients had psychiatric disorders, including anxiety and episodic depression (10%), chronic behavioral disorders (eg, aggression and drug or alcohol abuse) (17%), chronic obsessive-compulsive disorder (5%), and personality disorders (19%) (see Abbott et al. Am J Med Genet 1987; 26:959-969, which is incorporated herein by reference in its entirety). Psychosis may appear in adolescence (see Hidalgo et al. Eur Child Adolesc Psychiatry 2014; 23:235-238, which is incorporated herein by reference in its entirety).

[0350] In the absence of treatment, approximately 90% of pyridoxine non-responders develop learning disabilities (see Mudd et al. Am J Hum Genet 1985; 37:1-31, which is incorporated herein by reference in its entirety), with IQs typically ranging from 10 to 138, with a mean of 57 in pyridoxine non-responders, compared to 79 in untreated pyridoxine responsive patients and 105 in treated pyridoxine responsive and compliant patients (see Yap et al. J Inherit Metab Dis 2001; 24:437-447, which is incorporated herein by reference in its entirety). Seizures occur in 20% of refractory patients by age 12 years, and several cases of movement disorders unrelated to basal ganglia infarction have been reported, including eye-dancing syndrome, dystonia, and Parkinson's disease (see Morris et al. J Inherit Metab Dis 2017; 40: 49-74; Rezazadeh et al. Child Neurol Open 2014; 1:2329048X14545870, which are incorporated herein by reference in their entireties).

[0351] The association between HCU and neuropsychiatric symptoms was first described by Schimke et al. in 1965 (see Schimke et al. JAMA 1965; 193:711-719, which is incorporated herein by reference in its entirety). This association was later confirmed in a study reporting psychopathology in over 50% of patients with CBS deficiency (see Abbott et al. Am J Med Genet 1987; 26:959-969, which is incorporated herein by reference in its entirety). Since then, numerous epidemiological studies have shown a positive dose-dependent association between even modest elevations of plasma tHcy and the risk of CNS disorders, including cognitive symptoms and neurodegenerative diseases (see Morris et al. J Inherit Metab Dis 2017; 40:49-74); Seshadri et al. N Engl J Med 2002; 346:476-483; Clarke et al. Arch Neurol 1998; 55:1449-1455; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008-1017; Herrmann et al. Clin Chem Lab Med 2011; 49:435-441; Kim J et al. J Nutr 2007; 137:2093-2097; Selhub et al. Am J Clin Nutr 2000; 71:614S-620S; McCaddon et al. Dement Geriatr Cogn. Discord 2001; 12:309-313; Smallwood et al., Neuropathol Appl Neurobiol 2012; 38:337-343; Beydoun et al., BMC Public Health 2014; 14:643; each of which is incorporated herein by reference in its entirety). In general, patients with highly elevated Hcy levels (50 to 200 μM/L) experience acute neuronal dysfunction, including seizures and psychosis, whereas more moderate Hcy levels (15 to 50 μM/L) are associated with cognitive impairment and dementia (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7 ed. New York: McGraw Hill; 2001; 1279-1327; Gortz et al., J Neurol Sci 2004; 218:109-114, each of which is incorporated by reference in its entirety).

[0352] Elevated Hcy levels are widely recognized as a reliable and independent risk factor for cognitive impairment (see Smith et al. PLoS One 2010;5:e12244; Seshadri et al. N Engl J Med 2002;346:476-483, each of which is hereby incorporated by reference in its entirety), the onset of dementia (see Health Quality O. Vitamin B12 and cognitive function: an evidence-based analysis. Ont. Health Technol.Assess.Ser.13 (23), 1e45. 2013. Ref Type: Online Source, which is hereby incorporated by reference in its entirety), and Alzheimer's disease (see Seshadri et al. N Engl J Med 2002;346:476-483, which is hereby incorporated by reference in its entirety). Furthermore, elevated Hcy levels (>15 μmol/L) have been shown to be present in 90% of patients with depression (see Bottiglieri et al. J Neurol Neurosurg Psychiatry 2000; 69:228-232; Bjelland et al. Arch Gen Psychiatry 2003; 60:618-626, which are each incorporated by reference in their entireties), with men in the top tertile of tHcy levels more than twice as likely to be depressed as men in the lowest tertile (see Tolmunen et al., Am J Clin Nutr 2004; 80:1574-1578, which is incorporated by reference in their entireties). Elevated Hcy levels are commonly reported in cases of schizophrenia, multiple sclerosis, Parkinson's disease, fibromyalgia/chronic fatigue syndrome (see Kaeser et al. J Neurol Neurosurg Psychiatry 1969; 32:88-93; Golimbet et al. Psychiatry Res 2009; 170:168-171; Clarke et al., Arch Neurol 1998; 55:1449-1455; each of which is hereby incorporated by reference in its entirety), and relapsing dystonia without cerebrovascular disease (see Sinclair et al. Mov Disord 2006; 21:1780-1782, which is hereby incorporated by reference in its entirety). A possible association between the T833C polymorphism of the CBS gene and bipolar disorder has been described (see Permoda-Osip et al. Neuropsychobiology 2014; 69:107–111, which is incorporated herein by reference in its entirety), and a recent meta-analysis showed an association between elevated Hcy levels and mania/euthymia in individuals with bipolar disorder (see Salagre et al. Eur Psychiatry 2017; 43:81–91, which is incorporated herein by reference in its entirety). The first reported case of peripheral neuropathy associated with HCU was recently described in an 18-year-old man with HCU (see Oliveira et al. BMJ Case Rep 2016, which is incorporated herein by reference in its entirety), and a fatal case of psychosis was described in a 17-year-old adolescent with previously undiagnosed HCU (see Hidalgo et al. Eur Child Adolesc Psychiatry 2014;23:235-238, which is incorporated herein by reference in its entirety).

[0353] Direct evidence for a relationship between Hcy levels and dementia has been provided by animal studies, in which Hcy administration has been associated with the development of brain lesions (see Troen et al. Proc Natl Acad Sci USA 2008; 105:12474-12479; Sudduth et al. J Cereb. Blood Flow Metab 2013; 33:708-715, each of which is incorporated herein by reference in its entirety). In the first such study, male C57BL6/J mice with elevated Hcy levels (induced by a vitamin B-deficient diet) had severely impaired spatial learning and memory, with significant hippocampal microvascular rarefaction, without concomitant gliosis or neurodegeneration (see Proc Natl Acad Sci USA 2008; 105:12474–12479, which is incorporated by reference in its entirety). Total hippocampal capillary length was inversely correlated with Morris water maze escape latency (r=0.757, p<0.001) and with plasma tHcy (r=0.631, p<0.007). Mice fed a Met-rich diet had similar but less pronounced effects. These data suggest that elevated Hcy levels are associated with cerebral microvascular rarefaction leading to cognitive dysfunction in the absence of neurodegeneration or prior neurodegeneration. This may explain the association between elevated Hcy levels and cognitive decline in humans.

[0354] In a second study, healthy mice were fed a diet deficient in folate, vitamins B6 and B12, and supplemented with Met to induce a modest increase in Hcy levels (plasma tHcy 82.93±3.56 μmol/L). These mice exhibited spatial memory deficits as assessed by a radial-arm water maze for 2 days (see Sudduth et al. J Cereb Blood Flow Metab 2013; 33:708-715, which is incorporated herein by reference in its entirety). MRI and histology revealed a significant incidence of microbleeds. Neuroinflammation and increased expression and activity of MMP2 and MMP9, both enzymes involved in the pathogenesis of cerebral hemorrhage, were also observed, suggesting a link between elevated Hcy levels and vascular dementia, such as in Alzheimer's disease.

[0355] In humans, white matter changes (a sign of vascular injury) are often associated with elevated Hcy levels in individuals with HCU (see El Bashir et al. JIMD Rep 2015; 21:89–95; Vatanavicharn et al. J Inherit Metab. Dis 2008; 31 Suppl 3:477–481; Brenton et al. J Child Neurol 2014; 29:88–92; Ruhoy et al. Pediatr Neurol 2014; 50:108–111; (each of which is hereby incorporated by reference in its entirety) and without (see Hogervorst et al. Arch Neurol 2002; 59:787-793; Kloppenborg et al. Neurology 2014; 82:777-783; each of which is hereby incorporated by reference in its entirety). However, these changes are not always associated with features of stroke. In addition, brain imaging studies in patients with HCU often show features of atrophy or venous occlusion (see Vatanavicharn et al. J Inherit Metab Dis 2008; 31 Suppl 3:477-481, which is hereby incorporated by reference in its entirety).

[0356] The scan revealed diffuse, symmetrical, abnormal signal enhancement in the subcortical white matter and, to a lesser extent, in the deeper white matter of the cerebral hemispheres, predominantly in the parieto-occipital regions.

1. Mechanism

[0357] The exact mechanisms by which elevated Hcy levels affect neurological health are unknown. Several animal studies have shown an association between elevated tHcy levels and neurotoxicity, as well as associated neurological and psychiatric disorders.

[0358] A previous study showed that very high intraperitoneal doses of Hcy induced generalized convulsive status epilepticus in rats with cobalt-induced lesions in the motor cortex (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D., eds. The Metabolic and Molecular Basis of Inherited Diseases, 7 ed., New York: McGraw Hill, 2001, 1279-1327, which is incorporated herein by reference in its entirety). Seizures were enhanced by the addition of Met and vitamin B, and there was some evidence for a synergistic effect of pyridoxal 5'-phosphate and Hcy in blocking the postsynaptic gamma-aminobutyric acid receptor. Furthermore, treatment of rodent neocortical tissue with Hcy resulted in the uptake of adenosine in the form of AdoHcy (see Heinecke et al. J Biol Chem 1987; 262:10098-10103, which is incorporated herein by reference in its entirety). The authors stated that adenosine is predominantly a depressant in cerebral functions and that the seizure states and mental changes associated with high Hcy levels may be mediated by decreased cerebral adenosine levels.

[0359] Hcy has been associated with both neurotoxicity and morphological changes in the brain (see Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008-1017, which are hereby incorporated by reference in their entireties). For example, studies in rats and rabbits have shown that neuronal damage was caused by Hcy-mediated increases in thiobutyric acid-reactive substances (TBARS), indicators of oxidative stress (see Stefanello et al. Metab Brain Dis 2007; 22:172-182; Toborek et al., Atherosclerosis, 1995, 115:217-224, which are hereby incorporated by reference in their entireties). A similar increase in plasma TBARS has also been observed in humans after an oral Met load (see Domagala et al. Thromb Res 1997;87:411-416, which is incorporated herein by reference in its entirety). Furthermore, a study in a mouse model of elevated Hcy levels showed that oxidative stress-induced cellular damage may be enhanced in patients with HCU because decreased Cys levels result in decreased levels of neuronal glutathione, an important antioxidant synthesized from glutamate, Cys, and glycine (see Vivitsky et al., Am J Physiol Regul IntegrComp Physiol 2004;287:R39-R46, which is incorporated herein by reference in its entirety).

[0360] In vivo injection of Hcy into the left ventricle of rats caused a dose-dependent loss of neurons (see Yeganeh et al. J Mol Neurosci 2013; 50:551-557, which is incorporated herein by reference in its entirety), and incubation of rat mesencephalic tegmental neurons with Hcy resulted in lesser and shorter-term dopaminergic neurites (see Heider et al. J Neural Transm Suppl 2004; 1-13, which is incorporated herein by reference in its entirety). In both studies, the effects of Hcy were attenuated by co-administration of Hcy with NMDA antagonists and metabotropic glutamate receptors, suggesting a glutamate receptor-mediated pathway for Hcy-induced neuronal injury. Further evidence for this pathway comes from a study in which Hcy administration resulted in dose-dependent lipid peroxidation in rat brain synaptosomes (see Jara-Prado et al. Neurotox Res 2003; 5:237-243, which is incorporated herein by reference in its entirety). Again, the effects were inhibited by administration of an NMDA receptor antagonist.

[0361] A study in spontaneously active neurons of the rat embryonic cortex showed that Hcy levels above the acute Hcy range produced a dose-dependent suppression of neuronal network activity (see Gortz et al. J Neurol Sci 2004; 218:109-114, which is incorporated by reference in its entirety). The effects observed in this study were not clinically significant because such elevated Hcy levels are never reached in HCU patients. However, moderate elevations of homocysteine sulfinic acid and homocysteine acid (oxidized forms of Hcy often found in patients with elevated Hcy levels) had a similar effect. In each case, neuronal network damage was inhibited by 2-amino-5-phosphonovaleric acid, again implicating the NMDA receptor as a mediator of this Hcy-induced neuronal dysfunction. These results suggest that neuronal dysfunction associated with elevated Hcy levels is likely caused by oxidized forms of Hcy rather than Hcy itself.

[0362] The absence of cognitive symptoms, seizures, and other CNS abnormalities in patients with Marfan syndrome and other connective tissue disorders suggests that the neurologic disorders in patients with HCU are not caused by defects in either fibrillin or collagen (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D., eds. The Metabolic and Molecular Basis of Inherited Diseases, 7 ed., New York: McGraw Hill, 2001, 1279-1327, which is incorporated herein by reference in its entirety). In untreated HCU patients, elevated SAM levels and decreased SAH levels inhibit the transmethylation reactions required for myelin synthesis, leading to further neuronal damage (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D., eds. The Metabolic and Molecular Basis of Inherited Diseases, 7 ed., New York: McGraw Hill, 2001, 1279-1327, which is incorporated herein by reference in its entirety). Decreased myelin synthesis may also be caused by low serine levels in HCU patients due to increased remethylation rates (see Orendac et al. J Inherit Metab Dis 2003; 26:761-773, which is incorporated herein by reference in its entirety). Finally, Hcy metabolism plays a key role in monoamine synthesis, providing methyl groups for the production of norepinephrine and dopamine (see Mech et al. J Clin Psychiatry 2016; 77:668–671; Folstein et al. Am J Psychiatry 2007; 164: 861–867, each of which is hereby incorporated by reference in its entirety). Indeed, the “homocysteine hypothesis of depression” (see Folstein et al. Am J Psychiatry 2007; 164:861–867, which is hereby incorporated by reference in its entirety) posits that low norepinephrine and dopamine levels caused by elevated Hcy levels are the primary cause of depression.

[0363] Electron microscopy of rat brain biopsies revealed cerebrovascular structural changes in animals fed a high-Hcy diet for 8 weeks. These changes were associated with high plasma tHcy levels (see Lee et al. J Nutr 2005; 135:544-548, which is incorporated by reference in its entirety). Dietary folic acid supplementation for a further 8 weeks reduced plasma tHcy levels to normal levels and significantly reduced the incidence of vascular injury. This suggests that Hcy reduction by folic acid supplementation may reduce the adverse effects of experimentally induced elevated Hcy levels on the vascular endothelium.

[0364] In a study conducted to evaluate the efficacy and safety of reduced B vitamins as monotherapy in adults with major depressive disorder (MDD) and HCU due to at least one MTHFR polymorphism (N=330), treatment with a combination of reduced B vitamins significantly reduced tHcy levels in 131 treated patients (82.4%) (mean reduction in this subgroup was 25%; p<0.001), whereas tHcy levels showed a small increase in patients receiving placebo (see Mech et al. J Clin Psychiatry 2016; 77:668-671, which is incorporated herein by reference in its entirety). Treated patients had a mean 12-point reduction in the Montgomery-Åsberg Depression Rating Scale (MADRS) score by week 8, with 42% achieving complete remission (p<0.001). Further clinical improvement was correlated with significant reductions in tHcy levels in the majority of responders. Although this study was not performed in patients with HCU, it did demonstrate a clear benefit of Hcy reduction in depressed individuals, supporting the “Hcy-depression hypothesis” (see Folstein et al. Am J Psychiatry 2007;164:861-867, which is incorporated by reference in its entirety).

[0365] The benefits of Hcy-lowering therapy in individuals with psychiatric symptoms associated with HCU were first demonstrated in a study of 12 late-diagnosed patients (see Grobe H. Eur J Pediatr 1980; 135:199-203, which is incorporated herein by reference in its entirety). Three of these patients were never effectively treated and had severe psychiatric disorders with premature death. The remaining 8 patients ranged in age from 1 to 26 years (mean 13 years) and all had psychiatric symptoms including irritability, ADHD, apathy, and psychosis. Treatment with pyridoxine or a low-metabolite diet supplemented with L-cystine for 2 to 9 years was associated with striking improvements in behavior and intellectual development, which correlated with biochemical normalization. The authors emphasized the need to treat all patients, regardless of age at diagnosis and previous treatment, due to the reversibility and resolution of HCU-related sequelae observed in this study.

[0366] More recent data from the Irish Screening Programme were used to compare the intelligence of 23 patients with pyridoxine-unresponsive HCU (339 patients over several years of treatment) with that of 10 healthy control siblings (see Yap et al. J Inherit Metab Dis 2001, 24:437-447, which is incorporated by reference in its entirety). Of the 23 patients identified, 19 were diagnosed with HCU by NBS and were treated early in life (within 6 weeks of birth), two were diagnosed late (at 2.2 and 2.9 years of age), and two were untreated at the time of assessment. All patients were treated with a Met-free synthetic amino acid mixture with added cysteine and with vitamin B12 and folate supplementation as needed. Betaine has been used for the past 5 years as an adjunct to treatment in patients treated early in life who have become poor dietary compliant, as well as in all patients with late-onset HCU.

[0367] Overall, 13 of 19 patients in the early treatment group (mean age 14.4 years; range 4.4 to 24.9) were compliant with treatment (defined as lifetime median plasma fHcy <11 μmol/L) and had no complications, while the remaining 6 patients (mean age 19.9 years; range 13.8 to 25.5 years) who were poorly compliant had complications. The mean full scale IQ (FIQ) was 105.8 (range 84 to 120) in the compliant group compared with 80.8 (range 40 to 103) in the poorly compliant group. The control group (n=10) with a mean age of 19.4 years (range 9.7–32.9) had a mean FIQ of 102 (range 76–116). The two late-diagnosed patients aged 18.9 and 18.8 years had FIQs of 80 and 102, respectively, while the two untreated patients aged 22.4 and 11.7 years had FIQs of 52 and 53, respectively. However, there were no significant differences between the compliant early-treated patients and their healthy siblings (controls), except for FIQ, which was significantly higher in the affected siblings (p=0.0397). Although relatively small, these results suggest that early treatment with good biochemical control prevents cognitive symptoms.

[0368] Similar results were obtained in a case-control study that reported neurodevelopmental, educational, and cognitive outcomes in 32 HCU cases and 25 control siblings in Qatar (see El Bashir et al. JIMD Rep 2015;21:89–95, which is incorporated by reference in its entirety). The mean age of the participants in this study was 11.2 years (range 0.6–29 years) and 56% were male. Compared with healthy siblings, cases had lower overall IQ (especially in terms of short-term memory, quantitative reasoning, and visuospatial domains), and a significant number of adolescent and adult patients had comorbidities and behavioral and emotional disorders. Of these, 9 cases (28%) had HCU diagnosed by NBS and were treated in the first month of life. The remainder were diagnosed between 14 and 240 months of age. tHcy and Met levels during treatment were significantly higher in patients diagnosed by NBS than in those diagnosed in the clinic, possibly due to better compliance with diet and medication at an early age. A significant difference in IQ was observed between the early and clinically diagnosed patients. Although differences in language areas, attendance at special schools, and access to additional classroom support were not statistically significant between the groups, the “clinically diagnosed” group clearly had more problems.

[0369] Although the number of patients studied here was small, there was a marked difference between children diagnosed at birth and those diagnosed at an early age. A mean tHcy level of 115 μmol/L in the clinically diagnosed group was associated with poor clinical outcomes and very low IQ.

[0370] Additional evidence for the benefit of Hcy-lowering treatment for psychopathology in patients with HCU comes from a retrospective review of all patients with HCU admitted to Boston Children's Hospital since 1963 (unpublished data courtesy of M. Almuqbil et al.). A total of 19 patients with HCU were identified, three of whom were excluded from the analysis because of the possible presence of methylmalonic acidemia (also associated with psychological defects) in addition to HCU. Of the remaining 16 patients, 7 (6 with HCU and one with cobalamin (Cbl) deficiency) had good compliance with treatment early in life (four on diet, two on diet plus betaine, and one on Cbl). Six of these patients had no overt psychiatric symptoms other than mild cognitive deficits. In contrast, 9 patients (7 with pyridoxine-unresponsive HCU and 2 with CblG deficiency) had poor or inconsistent compliance (two on betaine and diet, one on diet alone, three on B vitamins, and two on folic acid and betaine). All 7 patients with HCU and poor compliance had psychiatric disorders including depression (n=4), paranoid experiences (n=2), paranoia and delusional psychosis (n=1), anxiety and mood variability (n=1), and ADHD, which were attenuated with good metabolic control (n=1). In two cases, the CblG-deficient patients were markedly anxious or agitated. Age, gender, and cognitive levels did not appear to differ significantly between psychiatric patients and healthy individuals. These results suggest that good metabolic control (reduced Hcy and/or Met) may delay and possibly prevent the onset of mental and behavioral disorders in patients with HCU. However, the study did not establish whether poorly controlled HCU leads to psychopathology or whether comorbidity with psychopathology itself hinders good patient compliance.

[0371] The association between elevated Hcy levels and CNS symptoms, including cognitive symptoms, neurodegenerative diseases, seizures, dystonia, psychosis, cognitive impairment, dementia, and depression, is well documented in patients with and without HCU (see Morris et al. J Inherit Metab Dis 2017; 40:49–74; Abbott et al. Am J Med Genet 1987; 26:959–969; Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7 ed. New York: McGraw Hill; 2001;1279–1327; Hidalgo et al. Eur Child Adolesc Psychiatry 2014; 23:235-238; Schimke et al. JAMA 1965; 193:711-719; Smith et al. PLoS One 2010;5: e12244; Seshadri et al. N Engl J Med 2002; 346:476-483; Bottiglieri et al. J Neurol Neurosurg Psychiatry 2000; 69:228-232; Bjelland et al. Arch Gen Psychiatry 2003; 60:618-626; Tolmunen et al. Am J Clin Nutr 2004; 80:1574-1578; Kaeser et al. J Neurol Neurosurg Psychiatry 1969; 32:88-93; Golimbet et al. Psychiatry Res 2009; 170:168-171; Clarke et al. Arch Neurol 1998; 55:1449-1455; Sinclair et al. Mov Disord 2006; 21:1780-1782; Permoda-Osip et al. Neuropsychobiology 2014; 69:107-111; Oliveira et al. BMJ Case Rep 2016;2016; Troen et al. Proc Natl Acad Sci USA 2008; 105:12474-12479; Sudduth et al. J Cereb Blood Flow Metab 2013; 33:708-715; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008-1017; Herrmann et al. Clin Chem Lab Med 2011; 49:435-441; Kim et al. J Nutr 2007; 137:2093-2097; Selhub et al. Am J Clin Nutr 2000; 71:614S-620S; McCaddon et al. Dement Geriatr Cogn Disord 2001; 12:309-313; Smallwood et al. Neuropathol Appl Neurobiol 2012; 38:337-343; Beydoun et al. BMC Public Health 2014; 14:643; Gortz et al. J Neurol Sci 2004; 218:109-114; Health Quality O. Vitamin B12 and cognitive function: an evidence-based analysis. Ont.Health Technol.Assess.Ser.13(23), 1e45. 2013. Ref Type: Online Source; Salagre et al. Eur Psychiatry 2017;43:81–91, each of which is incorporated herein by reference in its entirety). The mechanisms leading to CNS disorders in individuals with elevated Hcy levels are thought to include tHcy-mediated neuronal injury (see Mudd et al. Disorders of transsulfuration. In: Scriver CL, Beaudet AL, Sly WS, Valle D., eds. The Metabolic and Molecular Basis of Inherited Diseases, 7 ed., New York: McGraw Hill, 2001, 1279–1327; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008–1017; Stefanello et al. 182; Toborek et al. Atherosclerosis 1995; 115:217–224, each of which is incorporated by reference in its entirety), vascular endothelial injury caused by Hcy-mediated oxidative stress ... stress (see Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004; 287: R39-R46, which is hereby incorporated by reference in its entirety), neuronal death (see Yeganeh et al., J Mol Neurosci 2013; 50:551-557; Heider et al. J Neural Transm Suppl 2004; 1-13, which are hereby incorporated by reference in their entireties), and impaired neural network activity (see Gortz et al. J Neurol Sci 2004; 218:109-114, which is hereby incorporated by reference in its entirety). Depression and seizures are thought to be caused, at least in part, by Hcy-mediated reductions in cerebral adenosine levels, followed by reductions in norepinephrine and dopamine levels (see Mech et al. J Clin Psychiatry 2016; 77:668–671; Domagala et al. Thromb Res 1997; 87:411–416; Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004;287:R39–R46; Folstein et al., Am J Psychiatry 2007: 164:861–867, each of which is hereby incorporated by reference in its entirety).

[0372] Numerous studies in animal models of HCU (see Lee et al. J Nutr 2005; 135:544–548, which are each incorporated by reference in their entireties) and in patients with HCU (see El Bashir et al. JIMD Rep 2015; 21:89–95; Yap et al. J Inherit Metab Dis 2001; 24:437–447; Mech et al. J Clin Psychiatry 2016; 77:668–671; Grobe H. Eur J Pediatr 1980; 135:199–203, each of which is incorporated by reference in their entireties) have shown that early reductions in Hcy levels induced by a low-Met diet, folate/vitamin B supplementation, and/or pyridoxine/betaine therapy may prevent and sometimes reverse the progression of various nervous disorders. Further evidence is provided by a series of 6 case studies involving patients with HCU, in whom significant reduction and even normalization of Hcy levels resulted in complete or partial correction of CNS abnormalities (see Yap et al. J Inherit Metab Dis 2001; 24: 437–447; Brenton et al. J Child Neurol 2014; 29:88–92; Rezazadeh et al. Child Neurol Open 2014; 1:2329048X14545870; Kaeser et al. J Neurol Neurosurg Psychiatry 1969; 32:88–93; Colafrancesco et al. et al. Eur J Pediatr 2015; 174:1263–1266; Yokoi et al. Pediatr Int 2008; 50:694–695; Li et al. Pathology 1999; 31:221-224, each of which is incorporated herein by reference in its entirety).

[0373] These results demonstrated a significant correlation between Hcy levels and an increased risk of CNS disorders in patients with HCU and in the overall group. Early therapy aimed at reducing Hcy is necessary for normal development of children with early-onset HCU, as well as for the correction or elimination of CNS disorders in patients diagnosed with HCU at a later age.

IX. Definitions

[0374] The articles “a,” “an,” and “the,” as used in this description, when used with singular nouns, may also refer to plural nouns unless the context of the description otherwise requires.

[0375] When a range of values is specified, it is understood that each intermediate value between the upper and lower limits of this range and any other stated or intermediate value in the specified range is included within the scope of the present invention and is specifically specified. Thus, for example, if a range from 1 μm to 8 μm is specified, it is understood that the values 2 μm, 3 μm, 4 μm, 5 μm, 6 μm and 7 μm are also included in this range, as well as values in the range of values greater than or equal to 1 μm and less than or equal to 8 μm.

[0376] As used herein, the term "co-administered" or "co-administering" means the administration of two or more therapeutic components, including a pharmaceutical composition.

[0377] As used herein, the term "drug" refers to a dosage form of a pharmaceutical composition comprising a human PEGylated truncated CBS protein drug substance with the amino acid sequence of SEQ ID NO: 1 (e.g., 20NHS PEG-CBS).

[0378] As used herein, the term "drug substance" refers to a PEGylated CBS protein having the amino acid sequence of SEQ ID NO: 1 (e.g., 20NHS PEG-CBS).

[0379] As used herein, the term "adverse clinical outcome" refers to an undesirable phenotypic outcome resulting from a disease, disorder, or condition.

[0380] As used herein, the term "recombinant" when referring to, for example, a cell, nucleic acid, polypeptide, expression cassette, or vector, means a material or material that corresponds to the natural or native form of the product that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto, but has been obtained or produced from synthetic materials. Thus, for example, recombinant cells express genes that are not present in the native (non-recombinant) form of the cell (i.e., "exogenous nucleic acids"), or express native genes that are otherwise expressed at a different level, and are normally expressed at an insufficient level or not at all.

[0381] Recombinant methods may include, for example, using a recombinant nucleic acid, such as a cDNA encoding a protein or an antisense sequence, to insert into an expression system, such as an expression vector; followed by introducing the resulting construct into a cell, such that the cell expresses the nucleic acid and protein, if desired. Recombinant methods also include ligating nucleic acids with coding or promoter sequences from different sources into a single expression cassette or vector for fusion protein expression, constitutive protein expression, or inducible protein expression.

[0382] As used herein, the terms "subject," "individual," or "patient" are synonymous and refer to a vertebrate, and preferably a mammal. As used herein, the term "mammal" refers to, but is not limited to, a human.

[0383] As used herein, the term "non-genetically inherited" when used with respect to an individual, patient, and/or group of patients refers to one or more individuals who lack or have not been diagnosed with a genetically inherited cystathionine beta synthase deficiency (e.g., lack of a missense mutation or loss-of-function mutation in one or more alleles of the CBS gene). For example, individuals with genetically non-heritable elevated tHcy levels are individuals who have tHcy levels above the normal range expected based on age, sex, diet, and other factors, but who lack or have not been diagnosed with a genetic deficiency in one or more alleles of the CBS gene; that is, lack of genetically inherited HCU.

[0384] As used herein, the term "associated" refers to a relationship with the development or manifestation of a disease, condition, or phenotype. The association may be due to, but is not limited to, genes responsible for housekeeping functions, the alteration of which may serve as the basis for the development of various diseases and conditions, namely, conditions that are part of a pathway involved in a particular disease, condition, or phenotype, and conditions that indirectly contribute to the manifestation of a disease, condition, or phenotype.

[0385] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient that may optionally be included in the compositions of the invention and that does not cause significant adverse toxicological effects in a subject. In particular, in this case, it refers to an excipient that can be administered to a mammalian subject in combination with an active compound (in this case, PEGylated htCBS or "20NHS PEG-CBS") without significant adverse toxicological effects in the subject.

[0386] As used herein, the terms "adjuvant," "diluent," or "carrier" mean any substance that, while not itself a therapeutic agent, is used as a vehicle for delivering a therapeutic agent and is suitable for administration to an individual, such as a mammal, or that is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the preparation of a unit dosage form of the composition as a separate product, such as a capsule or tablet, suitable for oral administration. The terms "adjuvant," "diluent," or "carrier" encompass "excipients," including "pharmaceutically acceptable excipients," "carriers," "solvents," and the like, which are also used herein. Excipients and carriers include any substances known to those skilled in the art, such as any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which are non-toxic and do not adversely affect other components of the composition. Administration may mean oral administration, inhalation, enteral administration, feeding, or inoculation by intravenous injection. Excipients may include standard pharmaceutical excipients, and may also include any components that can be used to prepare foods and beverages for human and/or animal consumption, feed or bait compositions, or other food products.

[0387] As used herein, the term "drug" or "active agent" or any other similar term means any chemical or biological material or compound, including peptides, suitable for administration by methods already known to those skilled in the art and/or by the methods described herein, wherein said agents induce a desired biological or pharmacological effect, which may include, but is not limited to: (1) prophylactic action on the body and prevention of an undesirable biological effect, such as prevention of infection, (2) alleviation of a condition caused by a disease, such as relief of pain or inflammation caused by a disease, and/or (3) alleviation, reduction or complete elimination of a disease in the body. Such an effect may be local, for example, it may provide a local anesthetic effect, or it may be systemic.

[0388] As used herein, the term "approximately" in particular with respect to a given quantity encompasses variations of plus or minus five percent.

[0389] As used herein, the term "therapeutically effective amount" in relation to a composition of the invention refers to a non-toxic but sufficient amount of the active agent (or a composition containing the active agent) to provide a desired level in the bloodstream or at the site of its action in the body (e.g., inside a cell) of the individual being treated and/or to provide a desired physiological, biophysical, biochemical, pharmacological, or therapeutic response, such as a reduction in the severity of homocystinuria. The exact amount required may vary from individual to individual and will depend on a variety of factors such as the active agent, the potency of the composition, the delivery device used, the physical characteristics of the composition, the intended use by the patient (i.e., the number of doses administered per day), as well as patient characteristics such as the species, age, and general health of the individual, the severity of the condition being treated, additional medications taken by the individual, the route of administration, and the like. These factors and characteristics can be readily determined by one of skill in the art based on the information provided herein. The appropriate "effective amount" in any particular case can be determined by one of skill in the art by routine experimentation based on the information provided herein.

[0390] As used herein, the term "nucleic acid" may mean RNA or DNA, and includes messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA. DNA may be double-stranded or single-stranded, and if single-stranded, it may be a coding strand or a non-coding (antisense, complementary) strand.

[0391] As used herein, the term "mutant" is a mutant protein that is produced or engineered to alter properties or functions associated with glycosylation, protein stabilization, and/or ligand binding.

[0392] As used herein, the terms "native" or "wild type" with respect to a given cell, polypeptide, nucleic acid, trait, or phenotype refer to the form in which it is normally found in nature.

[0393] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" have their generally accepted meanings and are used synonymously herein to mean a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). Furthermore, the polypeptides described herein are not limited to any particular length. Included within this definition are D- and L-amino acids and mixtures of D- and L-amino acids. This term does not include or excludes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications that are known to those skilled in the art and may or may not occur in nature. A polypeptide may be an entire protein or a subsequence thereof. Polypeptides may also be amino acid subsequences that include epitopes, that is, antigenic determinants that are largely responsible for the immunogenic properties of the polypeptide and are capable of eliciting an immune response.

[0394] As used herein, the term "a position corresponding to..." and the like refers to a position of interest (i.e., a base number or a residue number) in a nucleic acid molecule or protein relative to a position in another reference nucleic acid molecule or protein. Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, such that the identity between the sequences is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. The position of interest is then assigned a number corresponding to the reference nucleic acid molecule. Thus, for example, if a particular polymorphism in Gene-X occurs at nucleotide 2073 of SEQ ID NO: X, then to identify the corresponding nucleotide in another allele or isolate, the sequences are aligned and then the position that matches position 2073 is identified. Since different alleles can have different lengths, the position representing 2073 may not be nucleotide 2073, but instead may be a position that “matches” a position in the reference sequence.

[0395] As used herein, the term "long-term administration" refers to administration of the CBS enzyme, htCBS, or htCBS mutant (e.g., with a C15S mutation) conjugated to a PEG moiety over a period of 6 weeks or more. The term "long-term continuous treatment" refers to repeated administration of the CBS enzyme, htCBS, or htCBS mutant (e.g., with a C15S mutation) conjugated to a PEG moiety throughout the course of the study via subcutaneous injection or an implanted osmotic pump.

[0396] The present application describes methods for treating homocystinuria and/or CBS deficiency using enzyme therapy (ET) using a medicament described herein comprising a PEGylated truncated human CBS protein with a substitution of cysteine at amino acid position 15 to serine.

[0397] A detailed description of one or more embodiments of the invention is given below. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are those described herein. Other features, objects, and advantages of the present invention will be apparent from the description. In the description of the present invention, the singular forms "a" and "the" include the plural forms unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of any conflict, the description set forth herein controls.

[0398] The present invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1. Review of the study of pathogenesis and patient characteristics

[0399] CBS-HCY-NHS-01 is an ongoing, multicenter (8 centers), International Prospective Study of the Pathogenesis (“NHS”) of HCU, recruiting 55 pediatric (5-17 years) and adult (>18 years) patients to characterize the clinical course of HCU in patients treated for their current disease in the clinic over 3 years, to understand how homocystinuria progresses over time, and to develop new treatments for patients with homocystinuria. CBS-HCY-NHS-01 evaluates the range of plasma concentrations of total Hcy (tHcy) and related sulfur metabolites, as well as variability in the clinical outcome of the disease. Interim analyses were conducted assessing patient characteristics, cognitive impairment, and skeletal pathologies. The observed patient characteristics are presented in Table 1.

Table 1. Patient demographics and baseline characteristics Patient characteristics Prevalence in all patients Age at entry into group, median [range]
Children (<18 years), N (%)
Adults (≥18 years), N (%) 21.0 [5-53]
23 (42%)
32 (58%) Gender: male/female, % 55%/45% Race/Ethnicity, N (%)
White
African American
Hispanic or Latino
Not a Spaniard 51 (93%)
3 (6%)
6 (11%)
46 (84%) Adult human weight, median [range], kg 80.5 [55-138] Adult height, median [range], cm 176 [148-200] BMI, median [range], kg/ ^m2 21.5 [15.0-49.5] Diagnosed before 1 year of age, N (%)
Yes
No 28 (51%)
25 (46%) Family history of HCU, N (%)
Yes
No 20 (36%)
35 (64%) Higher education in adult patients
Completion of high school
Graduation from high school
College/special college graduation
Master's degree
Degree of professionalism
Unknown 14%
21%
41%
14%
3%
7% tHcy at diagnosis, ^† median [range], μM
Adults (≥18 years)
Children (<18 years) 100 [5-364]
144 [35-298] Diet with restriction of natural protein, N (%)
Adults (≥18 years)
Children (<18 years) 38 (93%)
20 (91%)
18 (95%) L-amino acid mixture without Met, N (%)
Adults (≥18 years)
Children (<18 years) 32 (58%)
15 (47%)
17 (74%) Addition of vitamin B, N (%)
Adults (≥18 years)
Children (<18 years) 40 (83%)
19 (76%)
21 (91%) Betaine addition, N (%)
Adults (≥18 years)
Children (<18 years old) 41 (85%)
21 (84%)
20 (87%) Time to study, median [range], months 12.2 [0.03 ^$ -24.8] † Self-reported tHcy history data were available for only 33 patients.
$ One individual made only 1 visit.

[0400] This pathogenesis study included 55 patients with HCU. The median study time was 12.2 months at the time of the interim analysis. The disease affects both sexes almost equally (55% male, 45% female). In this study, patients with HCU were young (median age: 21 years); 42% were children (<18 years) and 58% were adult patients. In this group, the median BMI was within the normal range (21.5 kg/m ² ). Half of the patients (51%; 65% children; 41% adults) were diagnosed in the first year of life (<1 year). 36% of patients had a family history of HCU. The median plasma tHcy level was 95 μM (74 μM for children; 104 μM for adults), consistent with undertreated underlying disease. Ninety-five percent of children had tHcy levels above the upper limit of normal (ULN), and 82% had tHcy levels >50 μM (82%). Almost half (45%) of children had tHcy levels >100 μM. All (100%) adult patients had tHcy levels above the ULN, 93% had tHcy levels >50 μM, and 77% had tHcy levels >100 μM (77%). Based on 3-day diet diaries recorded before each clinic visit, most patients (93%; 95% children; 91% adults) were on a usual protein-restricted diet, and most were taking a Met-free L-amino acid mixture (58%; 74% children; 47% adults). Most patients (83%; 91% children; 76% adults) were taking vitamin B supplements. Most patients (85%; 87% children; 84% adults) were taking betaine (Table 1).

Example 2. NHS - Ophthalmic defects

[0401] Patients often had a history of ectopia lentis. In the largest retrospective study to date, 85% of patients with HCU developed the condition by age 20 (Mudd SH, et al. The natural history of homocystinuria due to cystathionine beta-synthase deficiency. Am J Hum Genet. 1985;37(1):1-31). In contrast, only 19% of adults and 9% of children in this treatment group had the condition (see Table 2), suggesting that a restricted diet, vitamin B supplementation, and betaine supplementation may be effective in attenuating ocular defects, although the disease was not completely cured as indicated by mean plasma tHcy levels.

Table 2. Ophthalmological defects All patients
N=55 Children
(5-17 years)
N=23 Adults
(≥18 years)
N=32 Any eye diseases 38 (69%) 16 (70%) 22 (69%) Ectopia lentis/lens dislocation 8 (15%) 2 (9%) 6 (19%) Cataract 6 (11%) 2 (9%) 4 (13%) Retinal degeneration 1 (2%) 0 (0%) 1 (3%) Retinosis pigmentosa 1 (2%) 1 (4%) 0 (0%) Farsightedness 11 (28%) 7 (30%) 4 (13%) Myopia 25 (63%) 9 (39%) 16 (50%)

Example 3. NHS - CBS gene mutations

[0402] Analysis of mutations in the CBS gene of study participants revealed 26 unique mutations identified in 48 patients, of which 16 patients were homozygotes and 32 patients were mixed heterozygotes. The DNA mutations and resulting mutant CBS proteins are presented in Table 3 according to the standard nomenclature of genetic mutations described in Ogino, Shuji, et al. "Standard mutation nomenclature in molecular diagnostics: practical and educational challenges," The Journal of molecular diagnostics 9.1 (2007): 1-6, which is incorporated herein by reference in its entirety. Patients with genetically inherited HCU are patients with a mutation in the CBS gene, including, but not limited to, any of the mutations shown in Table 3.

Table 3. Mutations in the CBS gene in the patient DNA Protein c.689del p.Leu230Argfs*39 c.209+1G>A p.(?) c.1126G>A p.Asp376Asn c.752T>A p.Leu251Gln c.808_810del p.Glu270del c.442G>A p.Gly148Arg c.536_553del p.Asp179_Leu184del c.700G>A p.Asp234Asn c.1106G>C p.Arg369Pro c.325T>C p.Cys109Arg c.361C>T p.Arg121Cys c.1006C>T p.Arg336Cys c.785C>T p.Thr262Met c.1058C>T p.Thr353Met c.1039G>A p.Gly347Ser c.770C>T p.Thr257Met c.153_165del p.Arg51Serfs*27 c.1224-2A>C p.(?) c.919G>A p.Gly307Ser c.1330G>A p.Asp444Asn c.833T>C p.Ile278Thr c.1152G>C p.Lys384Asn c.1339C>T p.Pro447Ser c.488A>G p.Tyr163Cys c.624G>A p.Trp208* c.738del p.Lys274Serfs*2 c.1136G>A p.Arg379Gln c.1397C>A p.? c.162G>A p.Trp54Ter c.19dupC p.Gln7ProfsX30 c.302T>C p.Leu101Pro c.362G>A Arg121His c.667-14_667-7delCTCTTTCT p? c.685C>A p.Pro229Thr c.816T>A pCys272ter c.829-78_1146-273delins469 p.? c.982G>A p.?

Example 4. NHS - tHcy Levels

[0403] tHcy levels were determined at each visit (Table 4). Most patients (96%) had plasma tHcy levels 5-40 times the ULN of 14 μM for children over 12 years of age and the ULN of 9.6 μM for children 0-12 years of age. Interindividual variability was moderate. Overall, patients with low tHcy levels at Visit 1 tended to have low tHcy levels throughout the study (and vice versa) (Pearson's R=0.6294). The even greater intervisit variability observed in some patients may be related to changes in diet/adherence between visits, highlighting the challenge of adequate patient compliance with restricted diet and supplementation.

Table 4. tHcy levels during the study Patients N Median (µM) Minimum (µM) Maximum (µM) All 52 95 2 402 Children (<18 years) 22 74 2 361 Adults (≥18 years) 30 104 10 402

Example 5. NHS - Other metabolic indicators

[0404] Various laboratory values were determined during the study (see Fig. 1 and Table 5). High levels of tHcy and Met and low levels of cystathionine and total cysteine were found, which are hallmarks of the disease, despite the fact that most patients were following standard treatment (protein-restricted diet and vitamin B and betaine supplements). Methionine levels >1000 μM were observed in 11% of patients (14% of children, 10% of adults) and ≥600 μM in 33% of patients (36% of children, 33% of adults). As expected, high levels of betaine, vitamins B12 and B6 were observed, since most patients were taking these supplements. Dimethylglycine (DMG) levels above the ULN were observed in 76% of patients, which may lead to a higher risk of acute myocardial infarction in some patients (Svingen et al., 2013). ALT levels above the ULN were observed in 37% of patients (52% of children, 28% of adults). Creatinine levels below the lower limit of normal (LLN) were observed in 43% of patients (74% of children, 21% of adults), and this may be related to low muscle mass caused by protein restriction. hsCRP levels above the ULN were observed in 35% of patients tested (N=40; only patients aged ≥13 years were tested and only patients aged ≥18 years were observed). Low levels of protein C activity were observed in 28% of patients, and low levels of fibrinogen were observed in 31% of patients (N=29; both tests were performed only in patients ≥13 years old, and only patients ≥18 years old were observed).

[0405] Normal levels were observed for: AST (89% of patients) (all patients tested); antithrombin III (83% of patients) and apolipoprotein A (93% of patients) (these laboratory parameters were tested only in patients ≥13 years); bone-specific alkaline phosphatase (97% of patients), serum CTX (95% of patients), and P1NP (87% of patients) (these laboratory parameters were tested only in patients ≥18 years).

[0406] Additional laboratory values are shown in Fig. 1, where the columns indicate the number of patients with a high/normal/low laboratory value.

Table 5. Selected abnormal laboratory values (all laboratory ranges are age-adjusted) All patients Children (<18 years) Adults (≥18 years) tHcy>ULN 51 (98%) 21 (95%) 30 (100%) tHcy>50 µM 46 (88%) 18 (82%) 28 (93%) tHcy>100 µM 33 (63%) 10 (45%) 23 (77%) Methionine>ULN 45 (88%) 20 (91%) 23 (85%) Methionine>1000 µM 6 (11%) 3 (14%) 3 (10%) Methionine ≥600 µM 18 (33%) 8 (36%) 10 (33%) Cystathionine<LLN 48 (94%) 20 (95%) 26 (93%) DMG>ULN 41 (79%) 19 (86%) 20 (71%) ALT>ULN 20 (37%) 12 (52%) 8 (28%) Creatinine<LLN 23 (43%) 17 (74%) 6 (21%) hsCRP*>ULN
hsCRP*>2x ULN 14 (35%)
10 (25%) 0 (0%)**
0 (0%)** 13 (45%)**
9 (31%)** Protein activity C*<LLN 8 (28%) 0 (0%) 8 (28%) Fibrinogen *<LLN 9 (31%) 0 (0%) 9 (31%) * hsCRP, protein C activity, and fibrinogen were tested only in patients aged ≥13 years.
**Age for one patient is not specified.

[0407] Table 5 and Fig. 1 demonstrate that, in addition to tHcy, methionine, cystathionine, DMG, ALT, creatinine, hsCRP, protein C activity, fibrinogen, ALT-SGPT, betaine, cystathionine, plasma vitamin B6, total cysteine, and vitamin B12, etc., may be useful as metabolic indicators of disease severity or progression or otherwise indicate disease severity or progression in an individual. These patient characteristics indicate that despite the fact that these patients were followed and treated at Centers of Excellence and were prescribed a normal protein-restricted diet and/or Met-free L-amino acid mixture and supplements, many patients had plasma tHcy values 5-40 times the ULN for tHcy and these patients also had hypermethioninemia (≥6000 μM) (33% of patients: 36% of children, 33% of adults), visual impairment, signs of inflammation, abnormal protein metabolism, and/or liver dysfunction. These data indicate that current dietary and therapeutic treatments are of limited effectiveness and/or that most patients are unable to comply with treatment, resulting in elevated tHcy levels even in patients who are frequently followed at Centers of Excellence. In particular, these data also indicate that betaine does not adequately reduce tHcy levels in HCU and, therefore, betaine alone is not an adequate treatment for HCU, contrary to popular belief. Furthermore, this study identified novel mutations in the CBS gene (Table 3) and new laboratory markers or metabolic indicators of disease severity or progression (Table 5 and Fig. 1), which are expected to be useful for diagnosing HCU, monitoring disease progression, monitoring patient compliance, monitoring treatment response, assessing quality of life, tailoring treatments to specific patient profiles, and for clinical trials conducted to determine the efficacy of new treatments.

Example 6. NHS - Skeletal fragility

[0408] Bone mass density (BMD) was assessed at baseline and 1 year at three sites (hip, lumbar spine, and total body) using dual-energy X-ray absorptiometry (DXA) using Hologic or General Electric/Lunar densitometers. BMD and Z-scores (number of standard deviations from the mean BMD for the control group, normalized for age and sex) were calculated for each site. A Z-score of -1 to -2.5 indicates osteopenia, and a Z-score below -2.5 indicates osteoporosis. tHcy levels were determined in plasma, and mean total protein intake (g/day) was calculated from 3-day food intake records. Total protein intake was defined as the sum of total natural protein from the diet and protein from a Met-free L-amino acid mixture if the patient was taking such a mixture. Correlations between BMD and tHcy or dietary protein were calculated using Pearson's correlation coefficient (R). Table 6 shows the percentage ranks associated with the different Z-scores.

Table 6. Z-score and percentile rank Percentile Z-score 5 -1.65 10 -1.29 15 -1.04 20 -0.84 25 -0.68 30 -0.53 25 -0.39 40 -0.26 45 -0.13 50 0 55 0.13 60 0.26 65 0.39 70 0.53 75 0.68 80 0.84 85 1.04 90 1.29 95 1.65

[0409] BMD data were available for 43 patients. The median Z-score was negative for both adults and children at all body sites (Table 7), indicating skeletal fragility. 46% of adults and 53% of children had bone density below the 15th percentile (Z-score ≤-1) at at least one site (Table 7). BMD results and Z-scores were stable over 12 months for each patient (N=15-20; correlation between Visit 1 and Visit 3 for BMD at each site: Pearson R=0.94 to 0.97; p<0.0001).

Table 7. Systematized Z-score results by DXA scan site Hip Spine Body All patients Adults Children All patients Adults Children All patients Adults Children N 43 24 19 42 23 19 37 19 18 Average -0.76 -0.60 -0.97 -0.52 -0.63 -0.39 -0.72 -0.81 -0.62 SD 1.32 1.38 1.26 1.30 1.44 1.15 1.37 1.36 1.20 Median -0.9 -0.75 -1.00 -0.65 -0.80 -0.50 -0.8 -1.00 -0.65 Range from -2.6 to 3.0 from -2.4 to 3.0 from -2.6 to 1.9 from -3.2 to 3.2 from -3.2 to 3.2 from -2.3 to 1.9 from -3.0 to 2.2 from -3.0 to 2.2 from -2.9 to 1.3 N (%) of patients with Z-score ≤-1.0 21 (48.8%) 11
(45.8%) 10
(52.6%) 14
(33.3%) 10
(43.5%) 4
(21.1%) 17
(45.9%) 10
(52.6%) 7
(38.9%)

[0410] Patients with plasma tHcy levels <100 μM had significantly greater skeletal frailty than patients with levels >100 μM (Figure 2). There was a positive correlation between plasma tHcy levels and BMD at all three sites (Pearson's coefficient 0.33 to 0.51; p < 0.03; Table 8). These data showed that BMD improves with higher total dietary protein intake, as evidenced by the positive correlation between BMD at all three sites and total dietary protein intake (Pearson's coefficient 0.55 to 0.76; p < 0.011; Table 8). There was a positive correlation between BMD at all three sites and C-reactive protein (hsCRP) (Pearson's coefficient=0.36-0.39; p<0.051; Table 8), a marker of inflammation, suggesting that inflammation may be associated with high tHcy levels or increased protein intake.

Table 8. Correlations between BMD and other parameters BMD N (all patients, adults, children) All patients Adults Children Pearson correlation with tHcy Hip 45, 26, 19 0.3340 (p=0.0249) 0.1545 (p=0.4510) 0.4963 (p=0.0307) Spine 41, 24, 17 0.4784 (p=0.0016) 0.5001 (p=0.0128) 0.5005 (p=0.0408) Body 38, 19, 19 0.5095 (p=0.0011) 0.4772 (p=0.0388) 0.3939 (p=0.0952) Pearson correlation with hsCRP Hip 33, 26, 7 0.3597 (p=0.0398) 0.3398 (p=0.0894) 0.3334 (p=0.4649) Spine 30, 24, 6 0.3611 (p=0.0500) 0.2875 (p=0.1732) 0.6717 (p=0.1440) Body 26, 19, 7 0.3925 (p=0.0473) 0.2999 (p=0.2122) 0.2123 (p=0.6476) Pearson correlation with total dietary protein content Hip 24, 11, 13 0.7566 (p<0.0001) 0.7268 (p=0.0113) 0.6843 (p=0.0099) Spine 22, 10, 12 0.6870 (p=0.0004) 0.7073 (p=0.0221) 0.5532 (p=0.0621)

[0411] These data showed that most patients with HCU have skeletal fragility despite their relatively young age, confirming previous studies (Parrot F, et al. J Inherit Metab Dis. Osteoporosis in late-diagnosed adult homocystinuric patients. 2000;23:338-40; Weber DR, et al. Low bone mineral density is a common finding in patients with homocystinuria. Med Genet Metab. 2016;117:351-4). For each patient, BMD and Z-scores were stable over 12 months. Thus, BMD and Z-scores assessed by DXA can be considered a reliable endpoint to assess the efficacy of the study treatment in future clinical trials in patients with HCU. Bone health in this group correlated with higher total dietary protein intake and higher tHcy levels, suggesting that chronically restricting total protein intake to regulate tHcy levels may increase skeletal fragility in patients.

[0412] In general, patients with HCU have greater skeletal fragility than healthy individuals. However, as discussed above, patients with higher tHcy levels showed a positive correlation with bone mass. Accordingly, skeletal symptoms in individuals with elevated tHcy levels, including individuals with non-genetically inherited CBS deficiency, may be treated using the methods described herein to alleviate skeletal symptoms associated with elevated tHcy levels.

Example 7. NHS - Cognitive abilities

[0413] Previous studies have demonstrated an association between lifetime total Hcy (tHcy) as a key indicator of intellectual function in HCU (Al-Dewik N, et al. Natural history, with clinical, biochemical and molecular characterization, of classical homocystinuria in the Qatari population. J Inherit Metab Dis. 2019; April 10; Yap S, et al. The intellectual abilities of early-treated individuals with pyridoxine-nonresponsive homocystinuria due to cystathionine β-synthase deficiency. J Inher Metabol Dis. 2001;24(4):437-47). The current literature lacks a description of profiles of strong or weak cognitive functions, and there are no data on studies of the relationship between proximal HCU biomarkers and cognitive abilities.

[0414] Executive functions refer to a set of top-down mental processes that are effortful and necessary for paying attention (Diamond A. Executive functions Annu Rev Psychol. 2013; 64:135-68). Executive function is particularly sensitive to changes in physical health and is a good candidate for assessing associations with biomarkers of HCU severity. Unlike general intelligence, executive functions can be improved through therapy (Diamond, 2013). Cognitive function was assessed at baseline and every 6 months using the age-normalized NIH Toolbox Cognitive Battery (NIHCB), which assesses language, working memory, episodic memory, processing speed, set shifting, and inhibitory control (Weintraub S, et al. Cognition assessment using the NIH Toolbox. Neurol 2013:80(11):S54-64; Weintraub et al. NIH Toolbox Cognition Battery (CB): introduction and pediatric data. Monogr Soc Res Child Dev. 2013;78(4):1-15). Mean visit scores were used in the analysis. Data were available for patients with visits 1 through 5. tHcy levels were measured in plasma. Correlations between cognitive function and tHcy, Cys, and Met were calculated using Pearson's correlation coefficient (r).

[0415] Cognitive data were available for 51 patients. The overall cognitive function of patients with HCU was severely impaired (median Composite of General Intelligence at the 20th percentile; 21st percentile for adults and 14th percentile for children). The domains of cognition that were affected included the Fluid Intelligence Composite, executive function, memory, and processing speed. The Fluid Intelligence Composite is a summary score of memory, executive function, and processing speed. This composite includes assessments of new learning and information processing ability. In the present study, the median Composite of Fluid Intelligence was found to be in the 10th percentile. Executive function represents the inhibition of automatic response tendencies and the ability to shift behavior based on task demands. It includes set shifting and inhibitory control. The median executive function in the present study was found to be at the 18th percentile for set shifting and the 9th percentile for inhibitory control. Memory is the ability to hold and manipulate information in a short-term buffer and the ability to access, store, and retrieve information. It includes working memory and episodic memory. The median memory in the present study was found to be at the 24th percentile for working memory and the 32nd percentile for episodic memory. Processing speed is the mental efficiency in processing information. The median processing speed in the present study was found to be at the 24th percentile.

[0416] Language processing function (receptive vocabulary and reading) and cognitive domains reflecting past learning and knowledge (Composite Analysis of Crystallized Intelligence) were within normal limits.

[0417] NIH Toolbox scores were consistent across visits (CV<10% for most patients). Intraclass correlations (ICCs) ranged from 0.73 (inhibitory control) to 0.89 (General Intelligence Composite) with the exception of episodic memory with an ICC of 0.64. Initial validation of the NIH Toolbox considers ICCs between 0.4 and 0.74 adequate and those above 0.75 excellent. Figure 3 shows the median and quartile NIH Toolbox scores for the cognitive functions tested.

[0418] Correlations between cognition and other parameters were determined. tHcy levels were negatively correlated with general cognitive process (Composite Analysis of General Intelligence) (r=-0.32; p=0.023; Table 9); that is, the higher the tHcy level, the lower the cognitive function score. Inhibitory control is the area most affected by tHcy levels (r=-0.33; p=0.019; Table 9).

[0419] Although the overall cognitive function of HCU patients was generally severely impaired (r=-0.32; p=0.023; Table 9), these impairments increased with increasing tHcy levels. Patients with tHcy levels>100 μM had significantly poorer overall cognitive function than patients with tHcy ≤100 μM across all cognitive domains. When comparing patients with tHcy ≤100 μM with patients with tHcy>100 μM, the inhibitory control and receptive vocabulary domains showed the most statistically significant differences (Figure 4). In contrast, plasma Met levels were negatively correlated with global cognition (r=-0.28; p=0.049; Table 9), suggesting that both tHcy and Met levels should be maintained as close to normal as possible. Cys levels were positively correlated with global cognition (r=0.37; p=0.008; Table 9).

Table 9. Correlation between cognitive functions and other parameters Areas of knowledge Pearson parameters tHcy in plasma Cys in plasma Met in plasma Shifting attitudes r -0.2787 0,1547 -0.2001 p 0,0476 0.2783 0.1591 n 51 51 51 Brake control r -0.3288 0.2223 -0.1987 p 0,0185 0.117 0.1622 n 51 51 51 Analysis of information processing r -0.1526 0.0565 0,0620 p 0.2852 0.6939 0.6659 n 51 51 51 Episodic memory r -0.1372 0.3245 -0.3374 p 0.337 0,0202 0.0155 n 51 51 51 Composite analysis of fluid intelligence r -0.28554 0.24182 -0.23209 p 0.042 0.087 0.101 n 51 51 51 Composite Analysis of General Intelligence r -0.3181 0.36741 -0.27767 p 0.0229 0,008 0,0485 n 51 51 51

[0420] Patients with lower plasma tHcy levels performed better on measures of executive function, extending previous studies showing that regulation of tHcy levels is necessary to maintain intellectual function in the mid-range (Walter JH, et al. Strategies for the treatment of cystathionine β-synthase deficiency: the experience of the Willink Biochemical Genetics Unit over the past 30 years. Eur. J. Pediatr. 1998;157(2):S71-6; Yap et al. 2001, Al-Dewik et al. 2019).

[0421] This study demonstrated that the NIH Toolbox is a reliable tool, illustrating its potential value for analyzing cognitive function over time in patients with HCU, including monitoring treatment response.

[0422] The results of this study have practical implications for the treatment of HCU. Neuropsychological assessment has been shown to be an important component of the care of patients with HCU, and assessment of executive functions (including inhibitory responses) should be included in this assessment. For children with HCU, supports similar to those used for children with other executive function problems, such as ADHD, should be considered.

[0423] The identification of specific cognitive impairments in HCU provides clues for future studies focusing on the neural systems affected by HCU. For example, activation of the dorsal anterior cingulate cortex has been associated with the monitoring of conflicting information, which is a central element of the inhibitory response during arm-maze task-setting tests (Botvinick MM, et al. Conflict Monitoring and anterior cingulate cortex: update. Trends Cog Sci. 2004;8(12):539-46).

[0424] The significant correlation between tHcy levels and cognitive symptoms indicates that tHcy levels are a reliable indicator of cognitive impairment. Accordingly, cognitive symptoms in individuals with elevated tHcy levels, including individuals with non-genetically inherited CBS deficiency, can be treated using the methods described herein to reduce cognitive symptoms associated with elevated tHcy levels.

Example 8. Validation of the efficiency of methods for quantitative determination of tHcy

[0425] Existing procedures for the quantitative determination of metabolites such as homocysteine, methionine, cysteine, etc., associated with CBS deficiency, including the procedures described in the previous examples as well as in Table 5 and Fig. 1, require the separation of plasma from whole blood samples prior to analysis. This procedure requires sophisticated equipment and expert technical knowledge to analyze any given metabolite starting from an individual's blood sample. Therefore, there is a need to develop inexpensive and efficient validated methods for collecting and preparing patient blood samples for quantitative analysis. Such validated methods would reduce the cost and time required to diagnose patients with CBS deficiency, for example, by allowing blood samples to be collected from patients at home without the need for them to visit a Test Center. These methods would also allow individuals and health care professionals to more effectively monitor the disease status in real time and monitor treatment by sending multiple samples directly from home. In addition, such validated diagnostics and diagnostic methods may allow individuals to reliably collect blood samples at home to monitor metabolite levels without the assistance of a healthcare professional. Home sampling involves collecting and processing a blood sample for quantitative analysis, such as using devices such as a finger prick needle and a plasma separation device (PSD). The current standard for quantifying total homocysteine in plasma is based on tHcy levels measured by LC-MS/MS. Accordingly, alternative diagnostic methods are needed to collect and prepare whole blood samples for LC-MS/MS analysis. One such alternative method involves collecting dried blood spots ("DBS"). Alternatively or additionally, PSDs may be used. Methods for collecting and analyzing metabolite levels using DBS and PSD approaches are described, for example, in WO2014150900 and US20120318971, the disclosures of which are incorporated herein by reference in their entireties. However, these alternative methods have never been validated and their reliability and value for home applications such as home sampling or home testing by individuals have not been assessed. Therefore, total homocysteine levels in individuals were estimated using the PSD method as described in WO2014150900 and compared with the conventional plasma-separated LC-MS/MS method. The results are presented in Table 10, where “PSD method” refers to the PSD approach described in WO2014150900 and “LC-MS/MS method” refers to the conventional plasma-separated LC-MS/MS approach.

Table 10. Validation of tHcy quantification using the PSD method Plasma tHcy (µM) PSD Method LC-MS/MS method 173 118 20.6 31.6 10.7 8.9 86.6 139 18.2 13.2 169 94.7 13.4 10.2 91.1 82.4 55.8 30.1 17.2 12.1 53.4 52.6 88.8 61.1 104 58.9 140 133 373 652 44 31.7 104 83.9 402 491 299 203 391 364 104 81.1 37.4 32 127 133 66.7 69.2

[0426] A simple linear regression analysis was performed to assess the correlation between samples tested using the PSD method and the conventional LC-MS/MS method. The optimal line had a slope of 1.096 (1/slope=0.912) with a standard error of the slope of 0.0804 (95% confidence intervals for the slope range from 0.9302 to 1.263). These results showed a significant correlation and confirmed the reliability of the PSD method for quantifying tHcy in blood samples from individual individuals.

Equivalents and scope of application

[0427] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention as described herein. The scope of the present disclosure is not limited by the foregoing description, but is merely set forth in the appended claims.

[0428] Articles used in claims, such as "a," "an," and "the," may be used to refer to one or more than one subject matter unless otherwise stated or clearly contradicted by the context of the invention. A claim or description that includes the conjunction "or" between one or more members of a group satisfies the criteria if one, more, or all of the members of the group are present, used, or otherwise related to the preparation or process, unless otherwise stated or clearly contradicted by the context. The disclosure includes embodiments of the invention in which exactly one member of the group is present, used, or otherwise related to the preparation or process. The disclosure includes embodiments of the invention in which more than one or all of the group members are present, used, or otherwise related to the preparation or process.

[0429] It should also be noted that the term "comprising" is "open-ended" and allows for, but does not require, the inclusion of additional elements or steps. If the term "comprising" is used, the scope of that term also includes the concept of "consisting of."

[0430] When ranges are specified, endpoints are included. It should also be noted that unless otherwise indicated or unless the context and the skill of one skilled in the art dictate otherwise, values expressed as ranges may take any specific value or include a subrange within the stated ranges in various embodiments of the invention down to tenths of the lower limit of the range, unless the context clearly dictates otherwise.

[0431] It should also be noted that any particular embodiment of the invention that is known in the art may be expressly excluded from any one or more of the claims. Since such embodiments are believed to be known to those skilled in the art, they may be excluded even if the exclusion is not expressly stated in this application. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient, any method of production, any method of use, etc.) may be excluded from any one or more of the claims for any reason, regardless of whether it is known in the art or not.

[0432] It should be noted that the words used herein are used for description only and not for limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

[0433] Although the present invention has been described in some detail and with some particularity with respect to several embodiments of the invention described, it is not intended to be limited to any such details or such embodiments or to any particular embodiment of the invention, but it should be construed with reference to the appended claims so as to ensure that such claims are given the broadest possible interpretation in view of the state of the art and, therefore, to effectively cover the intended scope of the invention.

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LIST OF SEQUENCES

<110> SELLOS-MOURA, MARCIA

BUBLIL, EREZ M.

GLAVIN, FRANK

<120> CYSTATHIONINE BETA SYNTHASE THERAPY FOR

TREATMENT OF DISEASES CAUSED BY ELEVATED HOMOCYSTEINE LEVELS

<130> 2089.1006PCT

<140> PCT/USXXXX/XXXXXX

<141> 2020-09-03

<150> 62/983,862

<151> 2020-03-02

<150> 62/895,230

<151> 2019-09-03

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 412

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Pro Ser Glu Thr Pro Gln Ala Glu Val Gly Pro Thr Gly Ser Pro His

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Arg Ser Gly Pro His Ser Ala Lys Gly Ser Leu Glu Lys Gly Ser Pro

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Glu Asp Lys Glu Ala Lys Glu Pro Leu Trp Ile Arg Pro Asp Ala Pro

35 40 45

Ser Arg Cys Thr Trp Gln Leu Gly Arg Pro Ala Ser Glu Ser Pro His

50 55 60

His His Thr Ala Pro Ala Lys Ser Pro Lys Ile Leu Pro Asp Ile Leu

65 70 75 80

Lys Lys Ile Gly Asp Thr Pro Met Val Arg Ile Asn Lys Ile Gly Lys

85 90 95

Lys Phe Gly Leu Lys Cys Glu Leu Leu Ala Lys Cys Glu Phe Phe Asn

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Ala Gly Gly Ser Val Lys Asp Arg Ile Ser Leu Arg Met Ile Glu Asp

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Ala Glu Arg Asp Gly Thr Leu Lys Pro Gly Asp Thr Ile Ile Glu Pro

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Thr Ser Gly Asn Thr Gly Ile Gly Leu Ala Leu Ala Ala Ala Val Arg

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Gly Tyr Arg Cys Ile Ile Val Met Pro Glu Lys Met Ser Ser Glu Lys

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Val Asp Val Leu Arg Ala Leu Gly Ala Glu Ile Val Arg Thr Pro Thr

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Asn Ala Arg Phe Asp Ser Pro Glu Ser His Val Gly Val Ala Trp Arg

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Leu Lys Asn Glu Ile Pro Asn Ser His Ile Leu Asp Gln Tyr Arg Asn

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Ala Ser Asn Pro Leu Ala His Tyr Asp Thr Thr Ala Asp Glu Ile Leu

225 230 235 240

Gln Gln Cys Asp Gly Lys Leu Asp Met Leu Val Ala Ser Val Gly Thr

245 250 255

Gly Gly Thr Ile Thr Gly Ile Ala Arg Lys Leu Lys Glu Lys Cys Pro

260 265 270

Gly Cys Arg Ile Ile Gly Val Asp Pro Glu Gly Ser Ile Leu Ala Glu

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Pro Glu Glu Leu Asn Gln Thr Glu Gln Thr Thr Tyr Glu Val Glu Gly

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Ile Gly Tyr Asp Phe Ile Pro Thr Val Leu Asp Arg Thr Val Val Asp

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Lys Trp Phe Lys Ser Asn Asp Glu Glu Ala Phe Thr Phe Ala Arg Met

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Leu Ile Ala Gln Glu Gly Leu Leu Cys Gly Gly Ser Ala Gly Ser Thr

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Cys Val Val Ile Leu Pro Asp Ser Val Arg Asn Tyr Met Thr Lys Phe

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His His His Thr Ala Pro Ala Lys Ser Pro Lys Ile Leu Pro Asp Ile

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Arg Cys Val Val Ile Leu Pro Asp Ser Val Arg Asn Tyr Met Thr Lys

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Phe Leu Ser Asp Arg Trp Met Leu Gln Lys Gly Phe Leu Lys Glu Glu

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Asp Leu Thr Glu Lys Lys Pro Trp Trp Trp His Leu Arg Val Gln Glu

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Leu Gly Leu Ser Ala Pro Leu Thr Val Leu Pro Thr Ile Thr Cys Gly

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His Thr Ile Glu Ile Leu Arg Glu Lys Gly Phe Asp Gln Ala Pro Val

435 440 445

Val Asp Glu Ala Gly Val Ile Leu Gly Met Val Thr Leu Gly Asn Met

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Leu Ser Ser Leu Leu Ala Gly Lys Val Gln Pro Ser Asp Gln Val Gly

465 470 475 480

Lys Val Ile Tyr Lys Gln Phe Lys Gln Ile Arg Leu Thr Asp Thr Leu

485 490 495

Gly Arg Leu Ser His Ile Leu Glu Met Asp His Phe Ala Leu Val Val

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His Glu Gln Ile Gln Tyr His Ser Thr Gly Lys Ser Ser Gln Arg Gln

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<213> Homo sapiens

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atgccgtcag aaaccccgca ggcagaagtg ggtccgacgg gttgcccgca ccgtagcggt 60

ccgcattctg caaaaggcag tctggaaaaa ggttccccgg aagataaaga agccaaagaa 120

ccgctgtgga ttcgtccgga cgcaccgtca cgctgtacct ggcagctggg tcgtccggca 180

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ctgaagaaaa ttggtgacac cccgatggtt cgtatcaaca aaatcggcaa aaaattcggt 300

ctgaaatgcg aactgctggc taaatgtgaa tttttcaatg cgggcggttc cgtgaaagat 360

cgtatctcac tgcgcatgat tgaagatgct gaacgcgacg gcaccctgaa accgggtgat 420

acgattatcg aaccgacctc tggcaacacg ggtatcggtc tggcactggc ggcggcagtc 480

cgtggttatc gctgcattat cgtgatgccg gaaaaaatga gctctgaaaa agttgatgtc 540

ctgcgtgctc tgggcgcgga aattgttcgt accccgacga atgcccgctt cgacagtccg 600

gaatcccatg tgggtgttgc atggcgcctg aaaaacgaaa tcccgaattc gcacattctg 660

gatcagtatc gtaacgctag caatccgctg gcgcattacg ataccacggc cgacgaaatc 720

ctgcagcaat gtgatggcaa actggacatg ctggtcgctt ctgtgggtac cggcggtacc 780

attacgggca tcgcgcgtaa actgaaagaa aaatgcccgg gctgtcgcat tatcggtgtg 840

gatccggaag gcagtattct ggcggaaccg gaagaactga accagaccga acaaaccacg 900

tatgaagttg aaggcatcgg ttacgatttt attccgaccg tcctggatcg cacggtggtt 960

gacaaatggt tcaaaagcaa tgacgaagaa gcctttacct tcgcacgtat gctgatcgct 1020

caggaaggtc tgctgtgcgg tggttcagca ggttcgacgg tcgcagtggc agttaaagct 1080

gcgcaggaac tgcaagaagg tcaacgttgt gtcgtgattc tgccggattc tgttcgcaac 1140

tacatgacca aatttctgag tgaccgttgg atgctgcaaa aaggcttcct gaaagaagaa 1200

gatctgaccg agaaaaaacc gtggtggtgg cacctgcgcg tgcaggaact gggtctgtcc 1260

gcaccgctga ccgttctgcc gaccatcacg tgcggccata cgattgaaat cctgcgtgaa 1320

aaaggttttg atcaggcccc ggttgtcgac gaagcaggcg tgattctggg tatggttacc 1380

ctgggtaaca tgctgagttc cctgctggcg ggcaaagtgc aaccgagcga tcaggttggt 1440

aaagtcatct acaaacaatt caaacagatt cgtctgaccg atacgctggg ccgcctgtcg 1500

cacatcctgg aaatggacca tttcgcgctg gttgtgcacg aacagattca ataccatagc 1560

accggcaaat catcgcagcg ccaaatggtc tttggtgtcg tgacggccat tgatctgctg 1620

aatttcgtgg ccgcacaaga acgtgaccag aaataa 1656

<210> 4

<211> 413

<212> Protein

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 4

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Pro Glu Asp Lys Glu Ala Lys Glu Pro Leu Trp Ile Arg Pro Asp Ala

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Thr Asn Ala Arg Phe Asp Ser Pro Glu Ser His Val Gly Val Ala Trp

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245 250 255

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305 310 315 320

Asp Lys Trp Phe Lys Ser Asn Asp Glu Glu Ala Phe Thr Phe Ala Arg

325 330 335

Met Leu Ile Ala Gln Glu Gly Leu Leu Cys Gly Gly Ser Ala Gly Ser

340 345 350

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355 360 365

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370 375 380

Phe Leu Ser Asp Arg Trp Met Leu Gln Lys Gly Phe Leu Lys Glu Glu

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Asp Leu Thr Glu Lys Lys Pro Trp Trp Trp His Leu Arg

405 410

<210> 5

<211> 413

<212> Protein

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

human mutant CBS C15S truncated polypeptide

<400> 5

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His Arg Ser Gly Pro His Ser Ala Lys Gly Ser Leu Glu Lys Gly Ser

20 25 30

Pro Glu Asp Lys Glu Ala Lys Glu Pro Leu Trp Ile Arg Pro Asp Ala

35 40 45

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50 55 60

His His His Thr Ala Pro Ala Lys Ser Pro Lys Ile Leu Pro Asp Ile

65 70 75 80

Leu Lys Lys Ile Gly Asp Thr Pro Met Val Arg Ile Asn Lys Ile Gly

85 90 95

Lys Lys Phe Gly Leu Lys Cys Glu Leu Leu Ala Lys Cys Glu Phe Phe

100 105 110

Asn Ala Gly Gly Ser Val Lys Asp Arg Ile Ser Leu Arg Met Ile Glu

115 120 125

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130 135 140

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145 150 155 160

Arg Gly Tyr Arg Cys Ile Ile Val Met Pro Glu Lys Met Ser Ser Glu

165 170 175

Lys Val Asp Val Leu Arg Ala Leu Gly Ala Glu Ile Val Arg Thr Pro

180 185 190

Thr Asn Ala Arg Phe Asp Ser Pro Glu Ser His Val Gly Val Ala Trp

195 200 205

Arg Leu Lys Asn Glu Ile Pro Asn Ser His Ile Leu Asp Gln Tyr Arg

210 215 220

Asn Ala Ser Asn Pro Leu Ala His Tyr Asp Thr Thr Ala Asp Glu Ile

225 230 235 240

Leu Gln Gln Cys Asp Gly Lys Leu Asp Met Leu Val Ala Ser Val Gly

245 250 255

Thr Gly Gly Thr Ile Thr Gly Ile Ala Arg Lys Leu Lys Glu Lys Cys

260 265 270

Pro Gly Cys Arg Ile Ile Gly Val Asp Pro Glu Gly Ser Ile Leu Ala

275 280 285

Glu Pro Glu Glu Leu Asn Gln Thr Glu Gln Thr Thr Tyr Glu Val Glu

290 295 300

Gly Ile Gly Tyr Asp Phe Ile Pro Thr Val Leu Asp Arg Thr Val Val

305 310 315 320

Asp Lys Trp Phe Lys Ser Asn Asp Glu Glu Ala Phe Thr Phe Ala Arg

325 330 335

Met Leu Ile Ala Gln Glu Gly Leu Leu Cys Gly Gly Ser Ala Gly Ser

340 345 350

Thr Val Ala Val Ala Val Lys Ala Ala Gln Glu Leu Gln Glu Gly Gln

355 360 365

Arg Cys Val Val Ile Leu Pro Asp Ser Val Arg Asn Tyr Met Thr Lys

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Phe Leu Ser Asp Arg Trp Met Leu Gln Lys Gly Phe Leu Lys Glu Glu

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Asp Leu Thr Glu Lys Lys Pro Trp Trp Trp His Leu Arg

405 410

<---

Claims (13)

1. A method of treating CBS deficiency in an individual, comprising: a. determining the level of a metabolic indicator of disease severity or disease progression in an individual, where the metabolic indicator of disease severity or disease progression is total homocysteine (tHcy); and where the tHcy level is slightly higher than low and ranges from 10 μmol/L to 50 μmol/L, tHcy levels slightly above average range from 50 μmol/L to 100 μmol/L, and tHcy level slightly above high is from 100 μmol/L to 500 μmol/L; and b. administering to an individual a therapeutically effective amount of a pharmaceutical composition comprising: i. a medicinal substance comprising an isolated cystathionine β-synthase (CBS) protein comprising SEQ ID NO: 1, covalently linked to at least one 20 kDa PEG molecule functionalized with an NHS ester; and ii. a pharmaceutically acceptable excipient, diluent or adjuvant, wherein the individual is a patient with a non-genetically inherited disease associated with elevated tHcy levels or a patient with a non-genetically inherited disease associated with CBS deficiency, and wherein the therapeutically effective amount of the pharmaceutical composition comprises a low dose containing approximately 0.25 mg/kg to approximately 1.0 mg/kg of drug administered to the subject in accordance with a tHcy level slightly above the low dose, an average dose containing approximately 0.5 mg/kg to approximately 1.5 mg/kg of drug administered to a subject corresponding to a tHcy level slightly above average, or a high dose containing approximately 1 mg/kg to approximately 10 mg/kg of drug administered to the subject in accordance with a tHcy level slightly above the high dose. 2. The method of claim 1, wherein determining the level of a metabolic indicator of disease severity or disease progression in an individual comprises taking a blood sample or a plasma sample from the individual; assessing the level of one or more metabolic indicators of disease severity or disease progression in the sample and comparing the level of the one or more metabolic indicators of disease severity or disease progression with the level of the same metabolic indicator in a control sample taken from a healthy individual.

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