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WO2025184395A1 - Méthodes de traitement de troubles congénitaux de glycosylation (cdg) et d'ataxie chez les sujets souffrant de cdg - Google Patents

Méthodes de traitement de troubles congénitaux de glycosylation (cdg) et d'ataxie chez les sujets souffrant de cdg

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Publication number
WO2025184395A1
WO2025184395A1 PCT/US2025/017678 US2025017678W WO2025184395A1 WO 2025184395 A1 WO2025184395 A1 WO 2025184395A1 US 2025017678 W US2025017678 W US 2025017678W WO 2025184395 A1 WO2025184395 A1 WO 2025184395A1
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WIPO (PCT)
Prior art keywords
cdg
composition
mannose
buffer
dose
Prior art date
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Pending
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PCT/US2025/017678
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English (en)
Inventor
Horacio Benjamin PLOTKIN
Peter MCWILLIAMS
Geoffrey Hird
Hicham Alaoui
Mercedes SERRANO GIMARE
Teppei SHIRAKURA
Drew Steven FOLK
Kevin Patrick HERLIHY
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Glycomine Inc
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Glycomine Inc
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Publication of WO2025184395A1 publication Critical patent/WO2025184395A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers

Definitions

  • the present invention relates generally to methods of treating congenital disorders of glycosylation (CDG), as well as methods of treating ataxia in a human suffering from CDG.
  • CDG congenital disorders of glycosylation
  • Glycosylation the enzymatic attachment of carbohydrates (glycans) to proteins and lipids, is a co-translational and post-translational modification (PTM) that is more common than any other PTM as it applies to a majority of proteins synthesized in the rough endoplasmic reticulum (ER).
  • PTM co-translational and post-translational modification
  • ER rough endoplasmic reticulum
  • Glycosylation plays a critical role in a variety of biological processes of membrane and secreted proteins. In the ER, glycosylation defines the protein structure and folding and acts as a quality control mechanism that dictates the export of properly folded proteins to Golgi or targets misfolded ones for degradation.
  • Glycan moieties may also act as ligands for cell surface receptors to mediate cell attachment or stimulate signal transduction pathways.
  • CDG syndromes Congenital disorders of glycosylation, also known as CDG syndromes, are a group of rare genetic diseases where tissue proteins and/or lipids carry defective glycosylation and/or lack of glycosylation. These diseases are linked to numerous enzymatic deficiencies and often times cause severe, sometimes fatal, impairments of the nervous system, muscles, intestines, and several other organ systems.
  • Common clinical symptoms in children with CDG include hypotonia, developmental delay, failure to thrive, hepatic dysfunction, coagulopathy, hypothyroidism, esotropia, abnormal fat pattern and inverted nipples, hypoglycemia, seizure, cerebellar hypoplasia, and stroke-like episodes in a developmentally delayed child.
  • the presentation may include ataxia, cognitive impairment, the absence of puberty in females, small testes in males, retinitis pigmentosa, scoliosis, joint contractures, and peripheral neuropathy.
  • CDG may be classified into two groups: CDG type I and CDG type II.
  • CDG type I is characterized by defects in the initial steps of N-linked protein glycosylation, z.e., biosynthesis of dolichol pyrophosphate linked oligosaccharide (DLO), which occur in the ER, or transfer of the DLO to asparagine residues of nascent polypeptides.
  • CDG type II involves defects in furtherf processing (synthetic or hydrolytic) of the protein-bound glycan.
  • DLO dolichol pyrophosphate linked oligosaccharide
  • CDG-Ia (approximately 70% of all CDG cases), which is characterized by loss or reduction of phosphomannomutase 2 (PMM) activity leading to the deficiency or insufficiency in intracellular N-glycosylation (Jaeken et al. J. of Inherit. Met. Disease. 2008, 31 : 669-672).
  • PMM phosphomannomutase 2
  • CDG-Ib is one known CDG for which a treatment is available, namely oral D-mannose administration.
  • a treatment is available, namely oral D-mannose administration.
  • such therapy may not be as effective in treating CDG-Ia patients and there are currently limited treatment options for other CDG type I subtypes and CDG type II diseases.
  • One of the reasons for the lack in established therapy for CDG-I disorders may be due to the plethora of heterogeneous clinical phenotypes presented that do not show a direct correlation to the PMM enzyme activity.
  • Patients suffering from a reduction in PMM activity have reduced productions of mannose- 1 -phosphate (M1P, which is also referred to in the art as Man-l-P), associated with symptoms of multivisceral impairments.
  • M1P mannose- 1 -phosphate
  • M6P mannose-6-phosphate
  • PMI phosphomannose isomerases
  • Another potential solution is to use a delivery vehicle (e.g., lipid particles) to encapsulate and deliver M1P (see WO 2015/053910).
  • a delivery vehicle e.g., lipid particles
  • M1P e.g., phosphorylated carbohydrates
  • the optimal delivery vehicle must overcome challenges of stability, phosphorylated carbohydrates loading rate and concentration, toxicity, and delivery efficiency.
  • Ataxia is a key driver of disease burden in PMM2-CDG. This condition affects over 95% of PMM2 patients from infancy through adulthood, causing impairment of ambulation, balance, speech, swallowing (choking), and vision. See Pettinato et al., 2021. Ataxia describes poor muscle control that causes clumsy movements. Ataxia can affect walking and balance, hand coordination, speech and swallowing, and eye movements. Ataxia usually results from damage to the part of the brain called the cerebellum or its connections. [0013] Ataxia is commonly quantified by the International Cooperative Ataxia Rating Scale (ICARS), for ages 4 and up.
  • ICARS International Cooperative Ataxia Rating Scale
  • ICARS evaluates: (1) Limb Coordination: Tremor and ataxia in upper & lower body; (2) Postural / Gait: Disturbances in posture and gait; (3) Speech: Fluency & clarity of speech; and (4) Oculomotor: Abnormal eye movements & eye tracking.
  • compositions and methods for delivering phosphorylated carbohydrates such as M1P and M6P, to treat disorders, such as a congenital disorder of glycosylation (CDG) and ataxia, to subjects (including, for example, humans) in need of such treatment.
  • CDG congenital disorder of glycosylation
  • a method of treating a congenital disorder of glycosylation in a subject in need thereof comprising administering to the subject a composition comprising mannose- 1 -phosphate (M1P).
  • M1P mannose- 1 -phosphate
  • the M1P is encapsulated in a lipid particle.
  • Lipid particles include, but are not limited to liposomes, micelles, solid lipid nanoparticles, niosomes, lipospheres, emulsomes and emulsions.
  • the composition is a liposomal composition.
  • the composition administered may be any of the liposomal compositions described herein.
  • the M1P is delivered in a nanoparticle formulation formed from one or more copolymers.
  • poly (D,L-lactide-co-glycolide) PLGA nanoparticles can be used to deliver the M1P.
  • the congenital disorder of glycosylation is CDG-Ia, CDG- le, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG.
  • the congenital disorder of glycosylation is CDG-Ia (also known as PMM2-CDG).
  • the disclosure provides a method of treating PMM2-CDG in a patient in need thereof, comprising administering to the patient weekly, or biweekly, or every two weeks an aqueous parenteral (e.g., intravenous) dose of a composition (e.g., a liposomal composition) comprising M1P or a pharmaceutically salt thereof, wherein the dose provides a steady state plasma AUC(o- «>) of M1P of from about 10,000 pg.hr/mL to about 35,000 pg.hr/mL.
  • a composition e.g., a liposomal composition
  • AUC(o- «>) of M1P of from about 10,000 pg.hr/mL to about 35,000 pg.hr/mL.
  • administration of a composition comprising M1P or a pharmaceutically acceptable salt thereof provides a steady state plasma AUC(o- «>) of M1P of from about 12,000 pg.hr/mL to about 30,000 pg.hr/mL.
  • administration of a composition e.g., a liposomal composition
  • administration of a composition comprising M1P or a pharmaceutically salt thereof provides a steady state plasma AUC(o- «>) of M1P of from about 20,000 pg.hr/mL to about 30,000 pg.hr/mL.
  • administration of a composition e.g., a liposomal composition
  • a method of treating ataxia in a human suffering from phosphomannomutase 2-congenital disorder of glycosylation comprising administering to the human a liposomal composition comprising mannose 1- phosphate or a pharmaceutically acceptable salt thereof, at a dose between 10 mg/kg and 30 mg/kg of mannose- 1 -phosphate.
  • the method further comprises improving the human’s (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders.
  • the method further comprises periodically measuring an International Cooperative Ataxia Rating Scale (ICARS) score for (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders during the treatment duration in the human; and assessing the human’s ICARS scores over the treatment duration.
  • ICARS International Cooperative Ataxia Rating Scale
  • the liposomal composition is administered to the human for a treatment duration over a plurality of weeks, wherein an International Cooperative Ataxia Rating Scale (ICARS) score is measured periodically for (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders during the treatment duration, and wherein the human’s (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders improves over the treatment duration based on the ICARS scores.
  • ICARS International Cooperative Ataxia Rating Scale
  • the subject is a human. In one variation, the subject is an adult. In some embodiments, the compositions are administered weekly, or biweekly, or every two weeks.
  • the M1P is administered as a pharmaceutically acceptable salt.
  • the salt is a di-potassium salt.
  • the salt is a hydrate or a dihydrate.
  • the dose of a composition comprising M1P or a pharmaceutically salt thereof can provide a Cmax of M1P in the plasma of from about 150 pg/mL to about 600 pg/mL.
  • the dose of a composition comprising M1P or a pharmaceutically acceptable salt thereof can provide a Cmax of M1P in the plasma of from about 250 pg/mL to about 500 pg/mL.
  • the dose of a composition comprising M1P or a pharmaceutically acceptable salt thereof can provide a Cmax of M1P in the plasma of from about 300 pg/mL to about 475 pg/mL.
  • the dose is administered weekly, or biweekly, or every two weeks to achieve the Cmax effects described.
  • the composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 10,000 pg.hr/mL to about 35,000 pg.hr/mL and a Cmax of from about 150 pg/mL to about 600 pg/mL.
  • the composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 12,000 pg.hr/mL to about 30,000 pg.hr/mL and a Cmax of from about 250 pg/mL to about 500 pg/mL.
  • the composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 10,000 pg.hr/mL to about 28,000 pg.hr/mL and a Cmax of from about 200 pg/mL to about 480 pg/mL.
  • the composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 15,000 pg.hr/mL to about 35,000 pg.hr/mL and a Cmax of from about 400 pg/mL to about 600 pg/mL.
  • the dose is administered weekly, or biweekly, or every two weeks to achieve the Cmax and AUC effects described.
  • the compositions are administered at a dose of M1P or a pharmaceutically salt thereof from about 10 mg/kg to about 40 mg/kg, from about 20 mg/kg to about 40 mg/kg, from about 30 mg/kg to about 30 mg/kg, from about 25 mg/kg to about 30 mg/kg.
  • the dose is administered weekly, or biweekly, or every two weeks at the doses (mg/kg) described.
  • the compositions are administered parenterally (e.g., intravenously). For instance, the compositions may be administered by intravenous infusion.
  • the compositions are administered as a weekly dose of M1P or a pharmaceutically acceptable salt thereof of about 10 mg/kg, wherein said dose provides a steady state plasma AUC(o- «>) of M1P of from about from about 7,000 pg.hr/mL to about 11,000 pg.hr/mL.
  • the dose provides a Cmax of from about 120 pg/mL to about 160 pg/mL.
  • the compositions are administered as a weekly dose of M1P or a pharmaceutically acceptable salt thereof of about 20 mg/kg, wherein said dose provides a steady state plasma AUC(o- «>) of M1P of from about from about 17,000 pg.hr/mL to about 24,000 pg.hr/mL.
  • the dose provides a Cmax of from about 260 pg/mL to about 340 pg/mL.
  • the compositions are administered as a weekly dose of M1P or a pharmaceutically acceptable salt thereof of about 30 mg/kg, wherein said dose provides a steady state plasma AUC(o- «>) of M1P of from about from about 28,000 pg.hr/mL to about 36,000 pg.hr/mL.
  • the dose provides a Cmax of from about 380 pg/mL to about 400 pg/mL.
  • the dose provides a Cmax of from about 400 pg/mL to about 500 pg/mL
  • M1P plasma concentrations of M1P greater than 100 pg/mL (e.g., greather than 200 pg/mL, greather than 300 pg/mL, greater than 400 pg/mL or greater than 500 pg/mL) can be achieved following administration of the disclosed compositions, and that these concentrations result in significant cellular uptake of M1P, particularly in liver cells.
  • concentrations of M1P in the plasma are associated with significant restoration of GDP -Mannose in liver cells. For instance, in particular embodiments, the levels of GDP -Mannose in liver cells can be restored to normal levels observed in healthy humans.
  • the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 10 pg/mL to about 40 pg/mL, wherein the concentration of M1P is measured ar the trough level (i.e., immediately before the next dose is administered). In other embodiments, the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 20 pg/mL to about 40 pg/mL, wherein the concentration of M1P is measured ar the trough level.
  • the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 20 pg/mL to about 30 pg/mL, wherein the concentration of M1P is measured ar the trough level. In other embodiments, the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 25 pg/mL to about 30 pg/mL, wherein the concentration of M1P is measured ar the trough level.
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 20% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 30% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 40% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 50% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 60% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 70% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL from about 20% to about 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL from about 30% to about 85% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL from about 50% to about 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • a dosing cycle e.g., weekly, or biweekly, or every two weeks.
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 20% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 30% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 40% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 50% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 60% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 70% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL from about 20% to about 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL from about 30% to about 85% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL from about 50% to about 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • a dosing cycle e.g., weekly, or biweekly, or every two weeks.
  • the half-life (T1/2) of M1P following administration of a composition disclosed herein is from about 50 hous to about 175 hours. In some embodiments, the half-life of M1P is from about 75 hours to about 150 hours. In some embodiments, the half-life of M1P is from about 70 hours to about 120 hours.
  • compositions disclosed herein can be administered safely and efficaciously to patients in need thereof.
  • the compositions can effectively restore the levels of GDP -mannose in the patient by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.
  • the compositions restore the levels of GDP -mannose in the patient of from about 20% to about 80%.
  • the compositions restore the levels of GDP-mannose in the patient of from about 30% to about 70%.
  • the compositions restore the levels of GDP-mannose in the patient of from about 40% to about 60%.
  • the liposomal comprise comprising three or more lipids.
  • the MIPlipid ratio is from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 0.1 to about 0.4, from about 0.1 to about 0.3, from about 0.1 to about 0.2, or from about 0.5 to about 1.5. In some variations, the MIPlipid ratio is about 0.1, 0.2, or 0.3.
  • the liposomal compositions comprise a phosphoethanolamine lipid. In other embodiments, the liposomal compositions comprise a phosphocholine lipid. In other embodiments, the liposomal compositions comprise a phosphoethanolamine lipid and a phosphocholine lipid.
  • the liposomal compositions include a lipid membrane enclosing an intraliposomal compartment, in which the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P).
  • the composition further includes intraliposomal buffer comprising a buffer salt, and optionally acid.
  • the pKa of the buffer salt in the intraliposomal buffer is between 6 to 8.5.
  • the composition further includes extraliposomal buffer comprising a buffer salt and a tonicity modifier.
  • the pKa of the buffer salt of the extraliposomal buffer is between 6.0 to 8.5.
  • the compositions optionally further include a radical scavenging antioxidant, present in the lipid bilayer.
  • the lipid membrane comprises: (a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail; (b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and (c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, conjugated to polyethylene glycol (PEG).
  • the lipid membrane comprises DOPE, DOPC and DSPE conjugated to PEG.
  • the intraliposomal buffer comprises tris(hydroxymethyl)aminomethane (Tris).
  • the extraliposomal buffer comprises Tris and saline.
  • the extraliposomal buffer comprises Tris and sugars, such as sucrose.
  • the radical scavenging antioxidant comprises butylated hydroxytoluene (BHT). In some embodiments, the radical scavenging antioxidant is absent.
  • the liposomal compositions comprise: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment M1P, and wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG; intraliposomal buffer comprising Tris; extraliposomal buffer comprising Tris and saline; and BHT.
  • the liposomal compositions comprise: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment M1P, and wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG; intraliposomal buffer comprising Tris; and extraliposomal buffer comprising Tris and saline.
  • FIG.l shows plasma concentration of M1P following three different liposomal compositions comprising various doses of M1P (3 mg/kg, 10 mg/kg and 20 mg/kg) after the third dose of a weekly dosing schedule.
  • FIGS. 2A-2C show that Formulation A is an effective MIP-liposome formulation in PMM2-CDG patient-derived fibroblasts.
  • FIG. 2A shows GDP -mannose levels in healthy fibroblasts (Normal) and GM20942 (PMM2) fibroblasts derived from patients with phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG). The * indicates significant (P ⁇ 0.05) difference between groups as determined by t-test.
  • FIG. 2B shows Formulation A increases GDP-mannose levels in fibroblasts from PMM2-CDG cells from patients with different genotypes. Total GDP-mannose was quantified in cells treated for 24 hours with 0.5 mM Formulation A or vehicle (untreated).
  • FIG. 1A shows GDP -mannose levels in healthy fibroblasts (Normal) and GM20942 (PMM2) fibroblasts derived from patients with phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG
  • FIG. 2C shows Con A western blot analysis of total proteins from Formulation A-treated or untreated GM20942 cells. Untreated healthy fibroblasts (HDF) were used as control. Triplicate samples were run for each condition. Protein blots were probed with either Concanavalin A (Con A) to detect glycoproteins or anti -glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Molecular weight markers are shown in FIG. 2D, which provide western blot of total proteins from Formulation A-treated (increasing concentrations) GM20942 cells or untreated HDF. Blot was probed with either an anti-intercellular adhesion molecule- 1 (ICAM-1) or anti-GAPDH antibodies. Numbers at the left indicate molecular weight markers.
  • IAM-1 anti-intercellular adhesion molecule- 1
  • FIG. 3 shows N-gly comics profile of healthy control cell lines.
  • Cells were processed for N-glycan analysis as described in Materials and Methods. For each N-glycan( with predicted structure) detected, the relative abundance versus total N-glycan content in the sample was calculated and reported as a percentage. For maximum clarity, N-glycan structures were divided into non-fucosylated asialo glycans (red), non-fucosylated glycans (blue), high mannose glycans (black) and sialylated glycans (purple).
  • FIG. 4 shows N-glycomics profile of CDG patient-derived cell lines.
  • N 3 for each cell type and treatment.
  • the relative abundance versus total N-glycan content in the sample was calculated and reported as a percentage.
  • the fold change in relative abundance in CDG versus healthy control cells was calculated for each glycan.
  • the mean fold change for triplicate samples was calculated and represented as a heat map.
  • N-glycans structures are shown on the left and are grouped from top to bottom into fucosylated asialo (red), non- fucosylated asialo (blue), high mannose (black) and sialylated (purple). Separate fold change scales are shown for fibroblasts (left) and LCL (right) lines.
  • FIG. 5 shows Formulation A restores the high-mannose N-glycan profile of PMM2-CDG patient derived fibroblasts.
  • the percent of total N- glycans was calculated and reported for each high mannose glycan.
  • Significance is indicated with * (p ⁇ 0.05), ** (p ⁇ 0.01), or *** (p ⁇ 0.001).
  • FIG. 6 shows Formulation A treatment effect on CDG patient-derived cells in vitro.
  • N For each N-glycan structure detected, the relative abundance versus total N-glycan content in the sample was calculated.
  • the fold change in N-glycan abundance in Formulation A-treated versus vehicle -treated cells was calculated.
  • the mean fold change for triplicate samples from each cell type was caclucated and represented as a heat map.
  • N- glycans structures are shown on the left and are grouped from top to bottom into fucosylated asialo (red), non-fucosylated asialo (blue), high mannose (black) and sialylated (purple).
  • the scale for the fold change between Formulation A vs. vehicle (untreated) cells is shown on the right.
  • FIGS. 7A-7C show near infra-red fluorescence (NIRF) imaging of liposome- encapsulated sulfo-Cy5.5 (Cy5.5).
  • NIRF near infra-red fluorescence
  • FIG. 7A shows quantification of fluorescence from ex vivo imaging of different tissues at 4 hours showed the strongest fluorescence in liver and spleen, compared to other tissues. Mean fluorescence shown as photons/s/cm A2 /sr.
  • FIG. 7B shows comparison of ex vivo imaging in different tissues at 4 hours (lymph node (LN), heart, lung, brain, kidney, liver, spleen, muscle, small intestine (SI), and colon (C).
  • FIGS. 8A-8C shows in vivo pharmacokinetic characterization of M1P liposomes.
  • Pharmacokinetic profile of Formulation A (20 mg/kg) by measuring M1P in mouse, showing Formulation A has a half-life of 11.2 hours (FIG. 8B), and in dogs (FIG. 8C) by measuring M1P at different dose levels ( 4.8, 25.2 and 46.8 mg/kg, with half-lives of 8.2, 24.8 and 40.2 hours, respectively).
  • FIG. 9A depicts a graph showing the ICARS Score with Formulation A.
  • FIG. 9B and FIG. 9C depict graphs showing the change in ICARS Score with Formulation A.
  • FIG. 11 depicts a graph showing the change in ICARS Score in a comparison across studies in PMM2.
  • compositions comprising mannose- 1 -phosphate are described herein.
  • Congenital disorders of glycosylation include, for example, CDG-Ia, CDG- le, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG.
  • the methods herein treat phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG), formerly known as congenital disorder of glycosylation (CDG) type la.
  • compositions comprising M1P encapsulated by liposomes are used in the methods herein.
  • the method further comprises: periodically measuring an International Cooperative Ataxia Rating Scale (ICARS) score for (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders during the treatment duration in the human; and assessing the human’s ICARS scores over the treatment duration.
  • ICARS International Cooperative Ataxia Rating Scale
  • the liposomal composition is administered to the human for a treatment duration of at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 6 months, or at least 1 year.
  • ICARS International Cooperative Ataxia Rating Scale
  • the human when an International Cooperative Ataxia Rating Scale (ICARS) score is measured periodically over the treatment duration for (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders during the treatment duration, the human’s (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders improves over the treatment duration based on the ICARS scores.
  • ICARS International Cooperative Ataxia Rating Scale
  • a method of treating ataxia in a human suffering from phosphomannomutase 2-congenital disorder of glycosylation comprising administering to the human a liposomal composition comprising mannose 1- phosphate or a pharmaceutically acceptable salt thereof, at a dose between 10 mg/kg and 30 mg/kg of mannose- 1 -phosphate.
  • the method further comprises improving the human’s (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders.
  • the method further comprises periodically measuring an International Cooperative Ataxia Rating Scale (ICARS) score for (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders during the treatment duration in the human; and assessing the human’s ICARS scores over the treatment duration.
  • ICARS International Cooperative Ataxia Rating Scale
  • the liposomal composition is administered to the human for a treatment duration over a plurality of weeks, wherein an International Cooperative Ataxia Rating Scale (ICARS) score is measured periodically for (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders during the treatment duration, and wherein the human’s (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders improves over the treatment duration based on the ICARS scores.
  • ICARS International Cooperative Ataxia Rating Scale
  • the liposomal compositions described herein are useful for delivering M1P to a subject in need thereof.
  • the subject is a mammal, such as a human, domestic animal, such as a feline or canine subject, farm animal (e.g., bovine, equine, caprine, ovine, and porcine subject), wild animal (whether in the wild or in a zoological garden), research animal, such as mouse, rat, rabbit, goat, sheep, pig, dog, and cat, and birds.
  • the subject is a human.
  • the subject is an adult human.
  • the subject may be at risk.
  • the subject at risk is a human.
  • a subject at risk of developing a particular disease, disorder, or condition, such as a congenital disorder of glycosylation may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein.
  • an individual “at risk” is an individual having risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art.
  • a subject having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition such as a congenital disorder of glycosylation, than a subject without one or more of these risk factors.
  • congenital disorders of glycosylation is a group of genetic disorders that result in errors of metabolism in which glycosylation of a variety of tissue proteins and/or lipids is deficient or defective.
  • Congenital disorders of glycosylation may also be known as CDG syndromes.
  • CDG syndromes may often cause serious, occasionally fatal, malfunction of several different organ systems, such as the nervous system, brain, muscles, and intestines, in affected infants.
  • Manifestations of CDG syndromes may range from severe developmental delay and hypotonia beginning in infancy, to hypoglycemia and protein-losing enteropathy with normal development. Developmental delay can be a common initial indication for a CDG diagnosis.
  • CDG-Ia also known as PMM2-CDG
  • phosphomannomutase 2 which is the enzyme responsible for the conversion of mannose-6-phosphate into mannose- 1 -phosphate
  • CDG syndromes may be classified as type I (CDG-I) and type II (CDG-II). Such classification may depend on the nature and location of the biochemical defect in the metabolic pathway relative to the action of oligosaccharyltransferase. Methods for screening for CDG subtype may include the analysis of transferrin glycosylation status by, for example, isoelectric focusing or ESI-MS.
  • CDG type I include, for example, la (PMM2-CDG), lb (MPI- CDG), Ic (ALG6-CDG) , Id (ALG3-CDG), le (DPM1-CDG), If (MPDU1-CDG), Ig (ALG12-CDG), Ih (ALG8-CDG), li (ALG2-CDG), Ij (DPAGT1-CDG), Ik (ALG1-CDG), IL (ALG9-CDG), Im (DOLK-CDG), In (RFT1-CDG), Io (DPM3-CDG), Ip (ALG11-CDG), Iq (SRD5A3-CDG), Ir (DDOST-CDG), DPM2-CDG, TUSC3-CDG, MAGT1-CDG, DHDDS- CDG, and I/IIx.
  • CDG type II include, for example, Ila (MGAT2-CDG), lib (GCS1-CDG), lie (SLC335C1-CDG), lid (B4GALT1-CDG), lie (COG7-CDG), Ilf (SLC35A1-CDG), Ilg (COG1-CDG), Ilh (COG8-CDG), Hi (COG5-CDG), Ilj (COG4-CDG), IIL (COG6-CDG), ATP6V0A2-CDG, MAN1B1-CDG, and ST3GAL3-CDG.
  • CDG glycosylation
  • la PMM2-CDG
  • MPI- CDG Ic
  • AG6-CDG Id
  • AG3-CDG Id
  • le DPMI -CDG
  • compositions and methods described herein are suitable to treat CDG-Ia, CDG-Ib, CDG-Ie, CDG-Ig, CDG-Ii, CDG-Ik, CDG-Io, CDG-Ip, or DPM2-CDG.
  • treatment includes an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results may include one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and/or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and/or preventing or delaying the spread of the disease or condition); and/or c) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
  • a) inhibiting the disease or condition e.g., decreasing one or more symptoms resulting from the disease or condition, and/or diminishing the extent of the disease
  • prevention includes any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop.
  • Compounds may, in some embodiments, be administered to a subject (including a human) who is at risk or has a family history of the disease or condition.
  • an “effective amount” is at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • An effective amount can be provided in one or more administrations.
  • a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease, disorder, or condition, such as a congenital disorder of glycosylation.
  • a therapeutically effective amount herein may vary according to factors such as the disease state, genotype, age, sex, and weight of the subject, and the ability of the lipid compositions of the present disclosure to elicit a desired response in the subject.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the lipid compositions of the present disclosure are outweighed by the therapeutically beneficial effects.
  • the compositions are administered at a dose of M1P or a pharmaceutically salt thereof from about 10 mg/kg to about 40 mg/kg, from about 20 mg/kg to about 40 mg/kg, from about 30 mg/kg to about 30 mg/kg, from about 25 mg/kg to about 30 mg/kg.
  • the dose is administered weekly, or biweekly, or every two weeks.
  • the subject has a total ICARS score of greater than about 20. In some embodiments, the subject has a total ICARS score of less than about 80. In some embodiments, the subject has a total ICARS score of between about 20 and about 80. In some embodiments, the subject has a total ICARS score of greater than 20. In some embodiments, the subject has a total ICARS score of less than 80. In some embodiments, the subject has a total ICARS score of between 20 and 80. In some embodiments, the subject has a total ICARS score of > 20 and ⁇ 80.
  • the disclosure provides a method of treating PMM2-CDG in a patient in need thereof, comprising administering to the patient weekly or biweekly or every 2 weeks an aqueous parenteral (e.g., intravenous) dose of a composition (e.g., a liposomal composition) comprising M1P or a pharmaceutically salt thereof, wherein the dose provides a steady state plasma AUC(o- «>) of M1P of from about 10,000 pg.hr/mL to about 35,000 pg.hr/mL.
  • the dose is administered weekly, or biweekly, or every two weeks to the patient to achieve the AUC effects described.
  • steady state plasma AUC(o- «>) or Cmax of M1P refers to the total amount of M1P present in the plasma, which constitutes M1P encapsulated in the liposome and free M1P not associated with the liposome.
  • administration of a composition e.g., a liposomal composition
  • M1P or a pharmaceutically acceptable salt thereof provides a steady state plasma AUC(o- «>) of M1P of from about 12,000 pg.hr/mL to about 30,000 pg.hr/mL.
  • administration of a composition comprising M1P or a pharmaceutically salt thereof provides a steady state plasma AUC(o- «>) of M1P of from about 15,000 pg.hr/mL to about 28,000 pg.hr/mL.
  • administration of a composition e.g., a liposomal composition
  • administration of a composition e.g., a liposomal composition
  • a composition comprising M1P or a pharmaceutically salt thereof provides a steady state plasma AUC(o- «>) of M1P of from about 15,000 pg.hr/mL to about 25,000 pg.hr/mL.
  • the M1P is administered as a pharmaceutically acceptable salt.
  • the salt is a di-potassium salt.
  • the salt is a hydrate or a dihydrate.
  • the weekly or biweekly or every 2 weeks dose of a composition comprising M1P or a pharmaceutically acceptable salt thereof can provide a Cmax of M1P in the plasma of from about 300 pg/mL to about 475 pg/mL.
  • the weekly or biweekly or every 2 weeks dose of a composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 10,000 pg.hr/mL to about 35,000 pg.hr/mL and a Cmax of from about 150 pg/mL to about 600 pg/mL.
  • the weekly or biweekly or every 2 weeks dose of a composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 12,000 pg.hr/mL to about 30,000 pg.hr/mL and a Cmax of from about 250 pg/mL to about 500 pg/mL.
  • the weekly or biweekly or every 2 weeks dose of a composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 10,000 pg.hr/mL to about 28,000 pg.hr/mL and a Cmax of from about 200 pg/mL to about 480 pg/mL.
  • the weekly or biweekly or every 2 weeks dose of a composition comprising M1P or a pharmaceutically salt thereof can provide a steady state plasma AUC(o- «>) of M1P of from about 15,000 pg.hr/mL to about 35,000 pg.hr/mL and a Cmax of from about 400 pg/mL to about 600 pg/mL.
  • the compositions are administered as a weekly dose of M1P or a pharmaceutically acceptable salt thereof of about 10 mg/kg, wherein said dose provides a steady state plasma AUC(o- «>) of M1P of from about from about 7,000 pg.hr/mL to about 11,000 pg.hr/mL.
  • the dose provides a Cmax of from about 120 pg/mL to about 160 pg/mL.
  • the compositions are administered as a weekly dose of M1P or a pharmaceutically acceptable salt thereof of about 20 mg/kg, wherein said dose provides a steady state plasma AUC(o- «>) of M1P of from about from about 17,000 pg.hr/mL to about 24,000 pg.hr/mL.
  • the dose provides a Cmax of from about 260 pg/mL to about 340 pg/mL.
  • the compositions are administered as a weekly dose of M1P or a pharmaceutically acceptable salt thereof of about 30 mg/kg, wherein said dose provides a steady state plasma AUC(o- «>) of M1P of from about from about 28,000 pg.hr/mL to about 36,000 pg.hr/mL.
  • the dose provides a Cmax of from about 380 pg/mL to about 400 pg/mL.
  • the dose provides a Cmax of from about 400 pg/mL to about 500 pg/mL
  • M1P plasma concentrations of M1P greater than 100 pg/mL (e.g., greather than 200 pg/mL, greather than 300 pg/mL, greater than 400 pg/mL or greater than 500 pg/mL) can be achieved following administration of the disclosed compositions, and that these concentrations result in significant cellular uptake of M1P, particularly in liver cells.
  • concentrations of M1P in the plasma are associated with significant restoration of GDP -Mannose in liver cells. For instance, in particular embodiments, the levels of GDP -Mannose in liver cells can be restored to normal levels observed in healthy humans.
  • the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 10 pg/mL to about 40 pg/mL, wherein the concentration of M1P is measured ar the trough level (i.e., immediately before the next dose is administered). In other embodiments, the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 20 pg/mL to about 40 pg/mL, wherein the concentration of M1P is measured ar the trough level.
  • the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 20 pg/mL to about 30 pg/mL, wherein the concentration of M1P is measured ar the trough level. In other embodiments, the concentration of M1P in the plasma following administration of a composition disclosed herein is from about 25 pg/mL to about 30 pg/mL, wherein the concentration of M1P is measured ar the trough level.
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 20% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 30% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 40% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 50% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 60% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 70% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL for at least 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL from about 20% to about 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL from about 30% to about 85% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 20 pg/mL from about 50% to about 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • a dosing cycle e.g., weekly, or biweekly, or every two weeks.
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 20% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 30% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 40% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 50% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 60% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 70% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL for at least 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL from about 20% to about 90% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks). In other embodiments, the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL from about 30% to about 85% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • the disclosed composotions of M1P are administered at a dose that achieves a plasma concentraion of greater than 40 pg/mL from about 50% to about 80% of time over a dosing cycle (e.g., weekly, or biweekly, or every two weeks).
  • a dosing cycle e.g., weekly, or biweekly, or every two weeks.
  • the half-life (T1/2) of M1P following administration of a composition disclosed herein is from about 50 hous to about 175 hours. In some embodiments, the half-life of M1P is from about 75 hours to about 150 hours. In some embodiments, the half-life of M1P is from about 70 hours to about 120 hours.
  • compositions disclosed herein can be administered safely and efficaciously to humans in need thereof.
  • the compositions can effectively restore the levels of GDP -mannose in the human by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.
  • the compositions restore the levels of GDP -mannose in the human of from about 20% to about 80%.
  • the compositions restore the levels of GDP-mannose in the human of from about 30% to about 70%.
  • the compositions restore the levels of GDP-mannose in the of from about 40% to about 60%.
  • the compositions are administered to a human, including specifically an adult human, weekly or every other week.
  • Administration of the liposomal compositions provided herein can be continuous or intermittent, depending, for example, on the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. It is within the scope of the present disclosure that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue.
  • dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
  • the liposomal compositions provided herein may be chronically or intermittently administered to a subject (including, for example, a human) in need thereof.
  • chronic administration is administration of the medicament(s) in a continuous as opposed to acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • the liposomal compositions used in the methods herein comprise: at one liposome, wherein each liposome has a liposomal layer and a central core.
  • the M1P or a pharmaceutically acceptable salt thereof is encapsulated within the central core of each liposome.
  • each liposome has a size between about 70 nM and about 130 nM, or about 100 nM.
  • the liposome layer comprises at least three phospholipids.
  • the liposome layer comprises 1- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DSPE-PEG2000).
  • DOPE 1- dioleoyl-sn-glycero-3 -phosphoethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3- phosphocholine
  • DSPE-PEG2000 l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]
  • the liposomes in the liposomal compositions used in the methods herein may be described in other ways.
  • the liposomes have a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment M1P or a pharmaceutically acceptable salt thereof; intraliposomal buffer; extraliposomal buffer; and optionally a radical scavenging antioxidant.
  • the liposomes have an interior surface in contact with the intraliposomal buffer and an external surface in contact with the extraliposomal buffer.
  • the interior and exterior surfaces are in both in contact with a buffer solution of neutral pH.
  • a pharmaceutically acceptable salt of MIP is present in the liposomes of the liposomal compositions.
  • the pharmaceutically acceptable salt is a dipotassium salt.
  • the liposomes comprise a-D(+)mannose 1-phosphate or a pharmaceutically acceptable salt thereof. In one variation, the liposomes comprise substantially pure a-D(+)mannose 1-phosphate or a pharmaceutically acceptable salt thereof.
  • the liposomes comprise a-D(+)mannose 1-phosphate dipotassium salt. In certain variations, the liposomes comprise substantially pure a- D(+)mannose 1-phosphate dipotassium salt.
  • the liposomes has less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of other isomers of MIP, including the beta isomer of MIP.
  • the lipid membrane comprises: (a) at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail; (b) at least one phospholipid having a choline group and at least one unsaturated fatty acid tail; and (c) at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail, conjugated to polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the unsaturated fatty acid tail independently comprises at least one Cio-28 carbon chain.
  • the unsaturated fatty acid tail is an optionally substituted Cio-28 alkenyl or an optionally substituted Cio-28 alkynyl.
  • each of the fatty acid chains is an unsubstituted Cio-28 alkenyl.
  • the alkenyl is linear or branched.
  • each of the fatty acid chains has one or more double bonds.
  • each double bond has cis configuration. In some embodiments, each double bond has trans configuration.
  • each of the fatty acid chains is a Cio-28 alkenyl substituted by a substituent selected form the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl and heterocyclyl.
  • the phospholipid having an ethanolamine head group in (a) of the lipid membrane has oleoyl tail groups.
  • the phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) or a salt thereof.
  • DOPE dioleoyl-sn-glycero-3 -phosphoethanolamine
  • the phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is: or a salt thereof.
  • the unsaturated fatty acid tail independently comprises at least one Cio-28 carbon chain.
  • the unsaturated fatty acid tail is an unsubstituted Cio-28 alkenyl.
  • the alkenyl is linear or branched.
  • the unsaturated fatty acid tail has one or more double bonds.
  • each double bond has cis configuration.
  • each double bond has trans configuration.
  • the unsaturated fatty acid tail is a Cio-28 alkenyl substituted by a substituent selected form the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl and heterocyclyl.
  • the phospholipid having choline group in (b) of the lipid membrane has oleoyl tail groups.
  • the phospholipid having a choline group and at least one unsaturated fatty acid tail is l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), or a salt thereof.
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • the phospholipid having a choline group and at least one unsaturated fatty acid tail is: or a salt thereof.
  • the saturated fatty acid tail independently comprises at least one C4-28 carbon chain.
  • the saturated fatty acid tail is an optionally substituted alkyl.
  • the saturated fatty acid tail is an unsubstituted C4-28 alkyl.
  • the saturated fatty acid tail is a C4-28 alkyl substituted by a substituent selected form the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl and heterocyclyl.
  • the phospholipid having an ethanolamine head group in (c) of the lipid membrane has stearoyl tail groups.
  • the phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), or a salt thereof.
  • the phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is: or a salt thereof.
  • the phospholipid conjugated to PEG is DSPE or a salt thereof.
  • PEGylated phospholipid is DSPE-PEG.
  • PEGylated phospholipid is DSPE-PEG2000.
  • the DSPE- PEG is further conjugated to a carbohydrate.
  • the DSPE-PEG is further conjugated to a monosaccharide.
  • the DSPE-PEG is further conjugated to a galactose moiety.
  • the DSPE-PEG-galactose has the following structure: or a salt thereof.
  • the lipid membrane comprises DOPC, DOPE and DSPE conjugated to PEG.
  • the lipid membrane comprises DOPC:DOPC:DSPE- PEG2000 at a molar ratio of from about 58 : 38 : 3 to 75 : 22 : 3; or from about 48.5 : 48.5 : 3 to about 80 : 17 : 3.
  • the lipid membrane comprises DOPC:DOPE:DSPE- PEG2000 at a molar ratio of about 58 : 39 : 3.
  • the lipid membrane comprises DOPC:DOPE:DSPE-PEG2ooo at a molar ratio of about 67 : 30 : 3.
  • PEG is present in the composition at a concentration that ranges from about 0.5 molar percent to about 20 molar percent. In some embodiments, PEG is present in the composition at a concentration of about 0.5 molar percent, about 1 molar percent, about 2 molar percent, about 3 molar percent, about 4 molar percent, about 5 molar percent, about 6 molar percent, about 7 molar percent, about 8 molar percent, about 9 molar percent, about 10 molar percent, about 11 molar percent, about 12 molar percent, about 13 molar percent, about 14 molar percent, about 15 molar percent, about 16 molar percent, about 17 molar percent, about 18 molar percent, about 19 molar percent, or about 20 molar percent.
  • PEG is present in the composition at a concentration of at least about 0.5 molar percent, at least about 1 molar percent, at least about 2 molar percent, at least about 3 molar percent, at least about 4 molar percent, at least about 5 molar percent, at least about 6 molar percent, at least about 7 molar percent, at least about 8 molar percent, at least about 9 molar percent, at least about 10 molar percent, at least about 11 molar percent, at least about 12 molar percent, at least about 13 molar percent, at least about 14 molar percent, at least about 15 molar percent, at least about 16 molar percent, at least about 17 molar percent, at least about 18 molar percent, at least about 19 molar percent, or at least about 20 molar percent; or between 0.5 molar percent and 50 molar percent, between 0.5 molar percent and 40 molar percent, between 0.5 molar percent and 30 molar percent, or between 0.5 molar percent
  • PEG is present in the composition at a molecular weight that ranges from about 200 Da to about 40,000 Da. In some embodiments, PEG is present in the composition at a molecular weight that ranges from about 200 Da to about 10,000 Da.
  • PEG is present in the composition at a molecular weight of about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1,000 Da, about 1,500 Da, about 2,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about 6,000 Da, about 6,500 Da, about 7,000 Da, about 7,500 Da, about 8,000 Da, about 8,500 Da, about 9,000 Da, about 9,500 Da, or about 10,000 Da; or between about 200 Da and about 10,000 Da.
  • compositions that include liposomes having a lipid membrane enclosing an intraliposomal compartment, in which the liposomes encapsulate a mannose phosphate, such as M1P, in the intraliposomal compartment.
  • the compositions further include an intraliposomal buffer comprising a buffering gent, and optionally an acid or a base.
  • the pH of the intraliposomal buffer is from about 6.0 to about 7.9. In some embodiments, the pH of the intraliposomal buffer is from about 6.2 to about
  • the pH of the intraliposomal buffer is from about 6.4 to about
  • the pH of the intraliposomal buffer is from about 6.5 to about
  • the pH of the intraliposomal buffer is from about 6.8 to about
  • the pH of the intraliposomal buffer is about 6.5. In some embodiments, the pH of the intraliposomal buffer is about 6.8. In some embodiments, the pH of the intraliposomal buffer is about 7.0. In some embodiments, the pH of the intraliposomal buffer is about 7.2. In some embodiments, the pH of the intraliposomal buffer is about 7.3. In some embodiments, the pH of the intraliposomal buffer is about 7.4. In some embodiments, the pH of the intraliposomal buffer is about 7.5. [0106] In some embodiments, the intraliposomal buffer comprises tri s(hydroxymethyl)aminom ethane (Tris).
  • Tris tri s(hydroxymethyl)aminom ethane
  • the intraliposomal buffer comprises 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES).
  • the intraliposomal buffer comprises bicarbonate.
  • the Tris or HEPES buffer maintain the pH of the intraliposomal compartment at from about 6.8 to about 7.6, or from about 7.0 to about 7.4.
  • the pH of the intraliposomal buffer is about 7.0.
  • the pH of the intraliposomal buffer is about 7.2.
  • the pH of the intraliposomal buffer is about 7.3.
  • the pH of the intraliposomal buffer is about 7.4.
  • the pH of the intraliposomal buffer is about 7.5.
  • the intraliposomal buffer comprises a histidine or a citrate buffer.
  • the histidine or citrate buffer maintains the pH of the intraliposomal compartment from about 6 to about 7.2, or at a pH of from about 6.2 to about 7.0, or from about 6.4 to about 6.8.
  • the intraliposomal buffer comprises an acetate, ascorbate, benzoate, diethanolamine, phosphate, succinate, tartrate or tromethamine buffering agent.
  • the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 15 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 35 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 50 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 50 mM.
  • the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is at least 50 mM.
  • the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is from about 15 mM to about 75 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is from about 30 mM to about 60 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is about 40 mM. In other embodiments, the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is about 50 mM.
  • the concentration of the buffering agent (e.g., Tris) in the intraliposomal compartment of the liposomes is about 50 mM.
  • the pH of the intraliposomal buffer changes to a minimal extent or does not change upon storage of the liposomal composition for 6 months or 2 years at 5°C or at room temperature.
  • the pH of the intraliposomal buffer changes by less than 0.4 units (e.g., less than 0.3 units, less than 0.2 units, or less than 0.1 unit) upon storage of the liposomal composition for six month or for 2 years at 5°C or at room temperature. For instance, if the starting pH of the intraliposomal buffer is 7.2, the pH of the intraliposomal buffer will be no less than 6.8 and no more than 7.6 following storage of the liposomal composition for up to 2 years at 5 °C or at room temperature.
  • the buffering agent comprises a buffer salt.
  • the extraliposomal buffer comprising a buffer salt and a tonicity modifier.
  • the pKa of the buffer salt is between 6.5 to 7.5.
  • the extraliposomal buffer is in a physiological pH range.
  • the buffer salt is tri s(hydroxymethyl)aminom ethane (Tris).
  • the extraliposomal buffer comprises bicarbonate, Tris, or HEPES, or any combination thereof.
  • the concentration of the buffering agent in the extraliposomal buffer is from about 10 mM to about 20 mM. In some embodiments, the concentration of the buffering agent in the extraliposomal buffer is from about 15 mM to about 20 mM.
  • the pH of the extraliposomal buffer changes to a minimal extent or does not change upon storage of the liposomal composition for 6 months or 2 years at 5°C or at room temperature. In some embodiments, the pH of the extraliposomal buffer changes by less than 0.4 units (e.g., less than 0.3 units, less than 0.2 units, or less than 0.1 unit) upon storage of the extraliposomal composition for six month or for 2 years at 5°C or at room temperature.
  • the pH of the extraliposomal buffer will be no less than 6.8 and no more than 7.6 following storage of the liposomal composition for up to 2 years at 5 °C or at room temperature.
  • the tonicity modifier comprises sugar or saline, or a combination thereof.
  • Suitable sugars that may be used for tonicity include, for example, sucrose and dextrose.
  • the tonicity modifier is an ionic tonicity modifier.
  • the tonicity modifier comprises saline.
  • the osmolality of the liposomal compositions is isotonic.
  • the osmolality of the liposomal compositions is from about 270 to about 320 mOsm/kg.
  • the osmolality of the liposomal compositions is from about 290 to about 320 mOsm/kg.
  • concentration of the tonicity modifier in the extraliposomal buffer is from about 5 mM to about 25 mM, from about 5 mM to about 15 mM, from about 10 mM to about 20 mM, from about 10 mM to about 15 mM, or from about 15 mM to about 20 mM.
  • the M1P and Tris, and optionally an acid are present in the composition at a ratio suitable to maintain a neutral pH.
  • the buffer capacity of the intraliposomal solution may be increased to maintain a neutral pH in the presence of mannose- 1 -phosphate (M1P).
  • M1P mannose- 1 -phosphate
  • the concentration of the intraliposomal buffer is increased to maintain the neutral pH.
  • the composition further comprises a radical scavenging antioxidant.
  • a radical scavenging antioxidant Any suitable radical scavenging antioxidants may be used in the liposomal compositions provided herein.
  • the radical scavenging antioxidant is butylated hydroyanisole (BHA), butylated hydroxytoluene (BHT), or alpha tocopherol.
  • the radical scavenging antioxidant is BHT.
  • the compositions comprises more than one radical scavenging agent. Any combinations of the radical scavenging antioxidants described herein may be used.
  • the compositions comprise BHT and alpha tocopherol. It should be understood that, in certain variations, certain of these radical scavenging antioxidants may be present in the liposomal compositions due to their presence in the lipids used (that are commercially available).
  • compositions described herein are optimized for treating diseases and disorders such as congenital disorders of glycosylation (CDG).
  • CDG congenital disorders of glycosylation
  • the composition has a drug-to-lipid (D/L) ratio of at least 0.01, at least 0.1, or at least 0.2.
  • the composition has a D/L ratio from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 0.1 to about 0.4, from about 0.1 to about 0.3, from about 0.1 to about 0.2, or from about 0.5 to about 1.5.
  • the D/L ratio is about 0.1, 0.2, or 0.3. It should be understood that the drug-to-lipid (D/L) ratio refers to the mass ratio of drug to total lipids in a given sample.
  • the total mannose phosphate (e.g., MlP)concentration in the sample is between 1 mg/ml and 50 mg/ml, between 1 mg/ml and 25 mg/ml, 1 mg/ml and 15 mg/ml, between 5 mg/ml and 25 mg/ml, between 5 mg/ml and 12 mg/ml, or between 6 mg/ml and 10 mg/ml.
  • the encapsulated D/L ratio refers to the mass ratio of drug encapsulated in the liposome to total lipids in a given sample. In some variations, the encapsulated D/L ratio does not exceed 0.15. In other variations, the encapsulated D/L ratio is between 0.001 and 0.15, between 0.01 and 0.15, between 0.1 to 0.15, between 0.1 and 0.14, between 0.1 and 0.13, between 0.1 and 0.12, or between 0.1 and 0.11. In other variations, the encapsulated D/L ratio is about 0.1 +/- 0.25%, about 0.1 +/- 0.20%, about 0.1 +/- 15%, or about 0.1 +/- 0.1%.
  • the total encapsulated mannose phosphate (e.g., MlP)concentration in the sample is between 1 mg/ml and 15 mg/ml, between 5 mg/ml and 12 mg/ml, or between 6 mg/ml and 10 mg/ml.
  • the composition has a mannose phosphate (e.g., MlP)concentration (based on the free acid) from about 1 mg/ml to about 10 mg/ml.
  • the mannose phosphate (e.g., M1P) concentration is from about 1 mg/ml to about 10 mg/ml, from about 1 mg/ml to about 9 mg/ml, from about 1 mg/ml to about 8 mg/ml, from about 1 mg/ml to about 7 mg/ml, from about 1 mg/ml to about 6 mg/ml, from about 1 mg/ml to about 5 mg/ml, or from about 1 mg/ml to about 4 mg/ml, from about 1 mg/ml to about 3 mg/ml, or from about 1 mg/ml to about 2 mg/ml.
  • the mannose phosphate (e.g., M1P) concentration is at least 1 mg/ml. In some variations, the mannose phosphate (e.g., M1P) concentration is about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, or 10 mg/ml.
  • the composition has a mannose phosphate (e.g., MlP)concentration (based on the free acid) from about Img/mL to 250 mg/ml, between 1 mg/mL and 200 mg/ml, 1 mg/ml to about 100 mg/ml, from about 1 mg/ml to about 75 mg/ml, from about 1 mg/ml to about 50 mg/ml, from about 25 mg/ml to about 75 mg/ml, from about 30 mg/ml to about 55 mg/ml, or from about 30 mg/ml to about 50 mg/ml, or from about 40 mg/ml to about 55 mg/ml, or from about 50 mg/ml to about 55 mg/ml.
  • the mannose phosphate (e.g., MlP)concentration is the maximum concentration of mannose phosphate (e.g., MlP)in the liposome.
  • the composition minimizes both lipid degradation and liposomal agglomeration.
  • the lipid degradation of the composition is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.1%.
  • the liposomal compositions of the disclosure show minimal or no degradation of the individual lipids comprising the liposome.
  • the buffered liposomal compositions are far less prone to lipid degradation compared to compositions formulated in unbuffered solutions (e.g., unbuffered saline solutions).
  • each of the individual lipids of the liposomal composition degrade less than 10% when stored at 5 °C or room temperature for up to 2 years.
  • each of the individual lipids of the liposomal composition degrade less than 1% when stored at 5°C or room temperature for up to 2 years.
  • each of the individual lipids of the liposomal composition degrade less than 0.5% when stored at 5°C for up to 2 years. In some embodiments, each of the individual lipids of the liposomal composition degrade less than 0.25% when stored for up to 2 years.
  • the composition has less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.1% of total lipid impurities.
  • the buffered liposomal compositions of the disclosure show high purity levels upon storage for 6 months or more.
  • the total impurity level observed in the liposomal compositions of the disclosure is less than 1%, less than 0.5%, less than 0.25%, or less than 1% following storage at 5°C or at room temperature for up to 2 years.
  • the composition maintains a pH range between 6.5 and 7, 7 and 7.4 or between 7.35 and 7.45. In certain embodiments, the composition maintains a physiological pH range. [0130] In some embodiments, the composition has a Z-average between 80 nm and 130 nm, between 80 nm and 120 nm, between 80 nm and 110 nm, between 80 nm and 100 nm, between 90 nm and 130 nm, between 90 nm and 120 nm, between 90 nm and 110 nm, or between 90 nm and 100 nm.
  • the composition has a poly dispersity index of less than 0.2 or less than 0.1.
  • no free mannose phosphate e.g., MlP
  • MlP mannose phosphate
  • percentage (%) of encapsulated M1P is the percent of encapsulated mannose phosphate (e.g., M1P) divided by the total mannose phosphate in the finished drug product. It should be understood that the finished drug product refers to the liposomal composition.
  • the percentage of encapsulated mannose phosphate (e.g., M1P) is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or between 90% and 99%, or between about 90% and 95%, or close to 100%.
  • percentage (%) encapsulation efficiency is the efficiency of mannose phosphate (e.g., M1P) encapsulation in the liposomes e.g., of the finished drug product) relative to the starting amount of mannose phosphate. In some variations, the %EE is between about 5% and 50%, between about 10% and 30%, or between about 10% and 25%.
  • compositions provided herein may have any one or more of the properties described above.
  • the composition provided herein has all of the following properties:
  • composition provided herein has all of the following properties:
  • Stability of the liposomal compositions provided herein may be measured over a time period over a range of temperatures, such as 5°C, 25°C and 40°C.
  • the time period is 1-3 months. In other variations, the time period is at least 6 months, at least 1 year, or at least 2 years.
  • the liposomal compositions of the disclosure also display negligible leakage of the M1P from the intraliposomal component, even following storage of the liposomal composition for up to 2 years. For instance, in some embodiments, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.25%, or less than 0.1% of the intraliposomal mannose phosphate (e.g., M1P) is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature.
  • M1P intraliposomal mannose phosphate
  • Liposomal compositions used in the methods described herein can be incorporated into a variety of formulations for therapeutic use (e.g., by administration) or in the manufacture of a medicament (e.g., for delivering M1P to a subject in need thereof and/or delivering M1P to the cell interior of a subject in need thereof and/or for treating or preventing a disease or disorder such as a congenital disorder of glycosylation (CDG) in a subject in need thereof and/or for treating ataxia in a human suffering from PMM2-CDG) by combining the composition with appropriate carriers (including, for example, pharmaceutically acceptable carriers or diluents), and may be formulated, for example, into preparations in liquid form.
  • appropriate carriers including, for example, pharmaceutically acceptable carriers or diluents
  • compositions intended for in vivo use are sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process.
  • compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
  • compositions of the present disclosure may be used in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time.
  • Dosages and desired concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles described in Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp.42-46.
  • the present disclosure also provides articles of manufacture and/or kits containing any of the liposomal compositions described herein for use according to the methods to treat CDG as described herein.
  • Articles of manufacture and/or kits of the present disclosure may include one or more containers comprising a purified liposomal composition of the present disclosure. Suitable containers may include, for example, bottles, vials, syringes, and IV solution bags. The containers may be formed from a variety of materials such as glass or plastic.
  • the articles of manufacture and/or kits further include instructions for use in accordance with any of the methods of the present disclosure.
  • these instructions comprise a description of administration of any of the liposomal compositions described herein to treat a congenital disorder of glycosylation (CDG) to a subject in need thereof, according to any of the methods of the present disclosure.
  • the instructions comprise a description of how to detect a congenital disorder of glycosylation (CDG), for example in a subject, in a tissue sample, or in a cell.
  • the article of manufacture and/or kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether that subject has the disease and the stage of the disease.
  • the instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the articles of manufacture and/or kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the article of manufacture and/or kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the label or package insert indicates that the composition is used for treating a congenital disorder of glycosylation (CDG). Instructions may be provided for practicing any of the methods described herein.
  • CDG congenital disorder of glycosylation
  • the articles of manufacture and/or kits of the present disclosure may be in suitable packaging.
  • suitable packaging includes, for example, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags).
  • packages for use in combination with a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
  • An article of manufacture and/or kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (e.g, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a carbohydrate such as M1P capable of treating a congenital disorder of glycosylation (CDG) and/or improving one or more symptoms thereof.
  • the container may further comprise a second pharmaceutically active agent.
  • Articles of manufacture and/or kits may optionally provide additional components such as buffers and interpretive information. Normally, the article of manufacture and/or kit comprises a container and a label or package insert(s) on or associated with the container.
  • a method of treating a congenital disorder of glycosylation (CDG) in a human in need thereof comprising: administering to the human a liposomal composition comprising mannose 1- phosphate or a pharmaceutically acceptable salt thereof, at a dose between 10 mg/kg and 40 mg/kg.
  • CDG congenital disorder of glycosylation
  • A6 The method of embodiment A5, wherein the liposomal composition is administered via intravenous infusion.
  • A9 The method of any one of embodiments Al to A8, wherein the liposomal composition comprises a-D(+)mannose 1 -phosphate or a pharmaceutically acceptable salt thereof.
  • A10 The method of embodiment A9, wherein the composition comprises substantially pure a-D(+)mannose 1 -phosphate or a pharmaceutically acceptable salt thereof.
  • the liposomal composition comprises: at one liposome, wherein each liposome has a liposomal layer and a central core;
  • each liposome has a size between about 70 nM and about 130 nM.
  • each liposome has a size of about 100 nM.
  • A15 The method of any one of embodiments A12 to A14, wherein the liposome layer comprises at least three phospholipids.
  • A16 The method of any one of embodiments A12 to A15, wherein the liposome layer comprises l-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DSPE-PEG2000).
  • DOPE l-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3- phosphocholine
  • DSPE-PEG2000 l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]
  • the liposomal composition comprises: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P), and wherein the lipid membrane comprises:
  • At least one phospholipid having a choline group and at least one unsaturated fatty acid tail is l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), or a salt thereof.
  • DOPC l,2-dioleoyl-sn-glycero-3- phosphocholine
  • A20 The method of any one of embodiments Al 7 to Al 9, wherein at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), or a salt thereof.
  • DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • lipid membrane comprises DOPC, DOPE and DSPE conjugated to PEG.
  • lipid membrane comprises DOPC, DOPE and DSPE-PEG2000.
  • A26 The method of any one of embodiments Al 7 to A25, wherein the intraliposomal comprises Tris.
  • the extraliposomal buffer comprises Tris and saline.
  • intraliposomal buffer comprises about 50 mM of Tris
  • extraliposomal buffer comprises 15 mM of Tris and at least 145 mM of saline.
  • each liposome further comprises at least one radical scavenging antioxidant.
  • each liposome further comprises butylated hydroyanisole (BHA), butylated hydroxytoluene (BHT), or alpha tocopherol, or any combination thereof.
  • BHA butylated hydroyanisole
  • BHT butylated hydroxytoluene
  • alpha tocopherol or any combination thereof.
  • each liposome further comprises butylated hydroxytoluene (BHT).
  • BHT butylated hydroxytoluene
  • PMM2-CDG phosphomannomutase 2- congenital disorder of glycosylation
  • invention B2 further comprising: improving the human’s (i) postural and gait disturbances, (ii) limb ataxia, (iii) dysarthria, and/or (iv) oculomotor disorders.
  • ICARS International Cooperative Ataxia Rating Scale
  • ICARS International Cooperative Ataxia Rating Scale
  • BIO The method of any one of embodiments B 1 to B8, wherein the human is a child.
  • B14 The method of any one of embodiments B 1 to B13, wherein the pharmaceutically acceptable salt is a di-potassium salt.
  • Bl 5 The method of any one of embodiments B 1 to B14, wherein the liposomal composition comprises: at one liposome, wherein each liposome has a liposomal layer and a central core; and
  • each liposome has a size between about 70 nM and about 130 nM.
  • each liposome has a size of about 100 nM.
  • the liposomal composition comprises: liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P), and wherein the lipid membrane comprises:
  • PEG polyethylene glycol
  • lipid membrane comprises DOPC, DOPE and DSPE conjugated to PEG.
  • lipid membrane comprises DOPC, DOPE and DSPE-PEG2000.
  • B31 The method of any one of embodiments B20 to B30, wherein M1P and Tris, and optionally acid, are present in the composition at a ratio suitable to maintain a neutral pH.
  • B32 The method of any one of embodiments B20 to B31, wherein the intraliposomal buffer comprises about 50 mM of Tris; and the extraliposomal buffer comprises 15 mM of Tris and at least 145 mM of saline.
  • each liposome further comprises at least one radical scavenging antioxidant.
  • each liposome further comprises butylated hydroyanisole (BHA), butylated hydroxytoluene (BHT), or alpha tocopherol, or any combination thereof.
  • BHA butylated hydroyanisole
  • BHT butylated hydroxytoluene
  • alpha tocopherol or any combination thereof.
  • each liposome further comprises butylated hydroxytoluene (BHT).
  • BHT butylated hydroxytoluene
  • a composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P), and wherein the intraliposomal compartment comprises a buffering agent and optionally an acid or base, wherein the pH of the intraliposomal compartment is from about 6.0 to about 7.9.
  • M1P mannose- 1 -phosphate
  • composition of embodiment Cl, wherein the pH of the intraliposomal compartment is from about 6.2 to about 7.4.
  • composition of embodiment Cl or C2, wherein the buffering agent in the intraliposomal compartment is tri s(hydroxymethyl)aminom ethane (Tris).
  • composition of any one of embodiments C1-C3, wherein the buffering agent in the intraliposomal compartment is 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES).
  • HEPES 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid
  • the liposomes further comprise an extraliposomal component, wherein the extraliposomal compartment comprises a buffering agent and a tonicity modifier, wherein the pH of the extraliposomal compartment is from about 6.0 to about 7.9.
  • composition of embodiment C7, wherein the pH of the extraliposomal compartment is from about 6.2 to about 7.4.
  • composition of embodiment C7 or C8, wherein the buffering agent in the intraliposomal compartment is tri s(hydroxymethyl)aminom ethane (Tris).
  • composition of any one of embodiments C1-C9, wherein the lipid membrane comprises:
  • composition of embodiment CIO wherein at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), or a salt thereof.
  • DOPE 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine
  • composition of embodiment CIO or Cl 1, wherein at least one phospholipid having a choline group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), or a salt thereof.
  • DOPC 1,2-dioleoyl-sn-glycero- 3 -phosphocholine
  • composition of any one of embodiments C10-C12, wherein at least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail is l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), or a salt thereof.
  • DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • composition of any one of embodiments C1-C23 wherein, over a period of time at temperatures between 5°C and 40°C, the composition has a mean osmolality within the range of 290 mOsm/kg to 320 mOsm/kg.
  • C25 The composition of any one of embodiments C1-C24, further comprising at least one radical scavenging antioxidant.
  • composition of embodiment C25, wherein at least one radical scavenging antioxidant is butylated hydroyanisole (BHA), butylated hydroxytoluene (BHT), or alpha tocopherol, or any combination thereof.
  • composition of embodiment C27 further comprises alpha tocopherol.
  • a composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment mannose- 1 -phosphate (M1P), and wherein the intraliposomal compartment comprises a buffering agent and optionally an acid or base, wherein the pH of the intraliposomal compartment is from about 6.0 to about 7.9.
  • M1P mannose- 1 -phosphate
  • composition of embodiment DI wherein the pH of the intraliposomal compartment is from about 6.2 to about 7.4.
  • composition of embodiment DI or D2 wherein the buffering agent in the intraliposomal compartment comprises tri s(hydroxymethyl)aminom ethane (Tris).
  • Tris tri s(hydroxymethyl)aminom ethane
  • D4 The composition of embodiment DI or D2, wherein the buffering agent in the intraliposomal compartment comprises 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES).
  • HEPES 4-(2 -Hydroxy ethyl)- 1 -piperazineethanesulfonic acid
  • composition of embodiment DI or D2 wherein the buffering agent in the intraliposomal compartment comprises a histidine or citrate buffer.
  • composition of embodiment DI or D2 wherein the buffering agent in the intraliposomal compartment comprises an acetate, ascorbate, benzoate, diethanolamine, phosphate, succinate, tartrate or tromethamine.
  • composition of embodiment D7, wherein the pH of the extraliposomal compartment is from about 6.2 to about 7.4.
  • Tris tri s(hydroxymethyl)aminom ethane
  • composition of any one of embodiments DI to D9, wherein the lipid membrane comprises:
  • At least one phospholipid having an ethanolamine head group and at least one saturated fatty acid tail conjugated to polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Dl l The composition of embodiment DIO, wherein at least one phospholipid having an ethanolamine head group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE), or a salt thereof.
  • DOPE 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine
  • composition of embodiment DIO or DI 1 wherein at least one phospholipid having a choline group and at least one unsaturated fatty acid tail is 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), or a salt thereof.
  • DOPC 1,2-dioleoyl-sn-glycero- 3 -phosphocholine
  • DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • composition of any one of embodiments DIO to D13, wherein the lipid membrane comprises DOPC, DOPE, and DSPE conjugated to PEG.
  • composition of embodiment DI 4 wherein the lipid membrane comprises
  • DOPC DOPC
  • DOPE DOPE
  • DSPE-PEG2000 DOPC, DOPE, and DSPE-PEG2000.
  • DI 6 The composition of any one of embodiments DI to DI 5, wherein all of the lipids comprising the lipid membrane degrade less than about 1% when stored at 5°C or room temperature for up to 2 years.
  • DI 7 The composition of any one of embodiments DI to DI 5, wherein less than 10% of the intraliposomal M1P is released from the intraliposomal component of the liposomal composition following storage for up to 2 years at 5°C or at room temperature.
  • DI 8 The composition of any one of embodiments DI to D17 wherein, over a period of time at temperatures between 5 °C and 40°C, the composition has a Z-average between 80 nm and 130 nm.
  • DI 9 The composition of any one of embodiments DI to DI 8, wherein, over a period of time at temperatures between 5°C and 40°C, the composition has a poly dispersity index of less than 0.2 or less than 0.1.
  • D20 The composition of any one of embodiments DI to DI 9, wherein, over a period of time at temperatures between 5°C and 40°C, no free M1P is detected in the composition.
  • D21 The composition of any one of embodiments DI to D20, wherein, over a period of time at temperatures between 5 °C and 40°C, the composition has a mean osmolality within the range of 290 mOsm/kg to 320 mOsm/kg.
  • a composition comprising liposomes having a lipid membrane enclosing an intraliposomal compartment, wherein the liposomes encapsulate in the intraliposomal compartment a mannose phosphate, and wherein the intraliposomal compartment comprises tri s(hydroxymethyl)aminom ethane (Tris), wherein the liposomes further comprise an extraliposomal component, wherein the extraliposomal component comprises Tris and saline, wherein the concentration of Tris in the intraliposomal compartment is higher than the concentration of Tris in the extraliposomal compartment.
  • Tris tri s(hydroxymethyl)aminom ethane
  • composition of embodiment D26, wherein the mannose phosphate is M1P.
  • composition of any one of embodiments D26 to D28, wherein the molar ratio of Tris in the intraliposomal compartment to extraliposomal compartment is between about 6: 1 and 2 : 1.
  • D30 The composition of any one of embodiments D26 to D29, wherein the saline present in the extraliposomal component is at least about 125 mM.
  • Example 1 A Study Assessing Pharmacodynamics (PD) Activity, Safety, Tolerability, and Pharmacokinetics (PK) of Formulation A Administered Intravenously to Adult Patients with Formulation A
  • Formulation A includes M1P encapsulated within the central core of liposomes (also known as small unilamellar vesicles) approximately 100 nm in diameter.
  • M1P belongs to the class of chemical entities known as monosaccharide phosphates.
  • the liposome layer is composed of three phospholipids: 1 -di oleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000).
  • DOPE 1 -di oleoyl-sn-glycero-3 -phosphoethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • DOPE enhances the fusion of liposomes to the cellular membranes
  • DOPC stabilizes the structure of the liposomes
  • DSPE-PEG2000 prolongs the half-life and inhibits non-specific protein binding to the liposomes.
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • the study includes a screening period, a twelve-week treatment period, and a thirty-day follow-up period.
  • Adult patients are injected with intravenous infusions of Formulation A at a dose of 10 mg/kg given weekly or 20 mg/kg given weekly.
  • Half of the patients are given the dose of 10 mg/kg given weekly, and the other half are given the dose of 10 mg/kg given weekly.
  • the patients are diagnosed with PMM2- CDG with a genetic test confirmation.
  • the treatment period in this study is twelve weeks.
  • the primary outcome measure is to evaluate changes in coagulation and antithrombosis factors, and specifically, changes in antithrombin (AT-III) and Factor XI activity levels are assessed.
  • Secondary outcomes are also measured, including, for example:
  • RR respiratory rate
  • body temperature body temperature
  • blood pressure systolic and diastolic
  • pulse pulse
  • plasma concentrations of total M1P are also evaluated to measure Cmax, tmax, AUCo-iast, AUCo-oo, AUCo-tau, CL, Vz, Vss, and ti/2.
  • a core battery of GLP safety pharmacology studies was conducted to evaluate the effects of liposomal M1P on the central nervous system (CNS) (Sprague Dawley rats), respiratory system (Sprague Dawley rats), and cardiovascular system in vivo (telemetered beagle dogs) and in vitro hERG channel inhibition in human cells). All in vivo studies were conducted with liposomal M1P administered IV, which is the intended clinical route of administration.
  • Cardiovascular safety in vivo- Telemetered dogs exhibited an immediate transient hypotensive response (lower systolic, diastolic, mean, and pulse pressure) consistent with (CARP A), which resolved within 1-hour post-dose. No direct effects on body temperature, electrocardiogram (ECG) waveforms, or heart rate were observed, except increases in heart rate compensatory to hypotension.
  • ECG electrocardiogram
  • FIG. 1 shows plasma concentration of M1P following three different liposomal compositions comprising various doses of M1P (3 mg/kg, 10 mg/kg and 20 mg/kg) after the third dose of a weekly dosing schedule.
  • Table 1 Pharmacokinetics of M1P in Phase 1 Study (Formulation A)
  • Cmax and AUC over the dosing interval were slightly greater than proportional over the range of 3 mg/kg to 20 mg/kg.
  • Accumulation of Cmax ranged from approximately 1.1 -fold to 1.4-fold whereas AUC ranged from approximately 1.4-fold to 1.6-fold as doses increased from 3 mg/kg to 20 mg/kg.
  • Example 4 Effect of Formulation A treatment on protein N-glycosylation of PMM2- CDG patient-derived fibroblasts
  • PMM2-CDG is the most common congenital disorder of glycosylation (CDG). Patients with this disease often carry a compound heterozygote mutation of the gene encoding the phosphor-mannose mutase 2 (PMM2) enzyme. PMM2 converts mannose-6- phosphate (M6P) to mannose- 1 -phosphate (M1P), which is a critical upstream metabolite for proper protein N-glycosylation. Therapeutic options for PMM2-CDG patients are limited to management of the disease symptoms as no drug is currently approved by the FDA to treat this disease. Formulation A is a MIP-loaded liposomal formulation being developed as a candidate drug to treat PMM2-CDG.
  • This example describes the effect of Formulation A treatment on protein N-glycosylation of PMM2-CDG patient-derived fibroblasts. It also characterizes the effect of Formulation A on N-glycomics profiles of cell lines derived from six additional individual CDG patients: ALG1-, ALG3-, ALG6-, ALG12-, DPMI-, and DPAGT1-CDG.
  • Liposomes were prepared using the thin-film hydration method. Briefly, the lipids were suspended in chloroform and warmed until completely dissolved. The mixture was then dried to a thin-film coating on the flasks. The thin films were hydrated by addition of a solution of neutral buffer containing the M1P, or Sulfo-Cy5.5-COOH (Lumiprobe, Hunt Valley, MD) payload. The lipid/MlP suspension was warmed, vortexed, and sonicated to achieve complete suspension of lipids. The liposome suspension was subsequently extruded to obtain liposomes of an average size of -100 nm. If necessary, the pH of the suspension was adjusted to the target. Unencapsulated payload was removed from the formulation by dialysis. All liposomes were stored at 2-8°C until use.
  • a panel of CDG patient-derived fibroblast cell lines including GM20942, GM20945, GM20956, GM27226, GM27386, GM20944, GM20949, GM20950, GM20101, and the patient derived lymphoblastoid cell line (LCL), GM20941, were obtained.
  • cHealthy dermal fibroblasts, and healthy lymphoblastoid cell line (LCL) were purchased from commercially available sources. All cell lines were cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM L-glutamine, and maintained at 37°C in a 5% CO2 atmosphere until use.
  • FBS fetal bovine serum
  • penicillin-streptomycin 1% penicillin-streptomycin
  • 2 mM L-glutamine 2 mM L-glutamine
  • ALG1 mutant human fibroblast cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM L-glutamine and maintained at 37°C in a 5% CO2 atmosphere until use.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin-streptomycin 1% penicillin-streptomycin
  • 2 mM L-glutamine maintained at 37°C in a 5% CO2 atmosphere until use.
  • GM20942 cells were treated in triplicate with concentrations of 0.15, 0.3, 0.5, 0.7, and 1.4 mM Formulation A.
  • Control cells were incubated with phosphate-buffered saline (PBS), and empty wells with cell culture media only served as controls for baseline absorbance values. The plates were incubated for 18 hours at 37°C, after which the culture medium was removed, and the cells rinsed with fresh medium.
  • PBS phosphate-buffered saline
  • Metabolites were extracted by adding 60% methanol (MeOH) in water pre-chilled to -20°C (1.5 mL per dish) and incubating for 15 minutes on dry ice, centrifuged at 13,000 rpm for 5 minutes at 3 °C, and the supernatants were collected. The samples were stored at -80°C until high performance lipid chromatographymass spectrometry (HPLC-MS/MS) analysis.
  • MeOH 60% methanol
  • HPLC-MS/MS high performance lipid chromatographymass spectrometry
  • PMM2-deficient fibroblasts (GM20942) were seeded in replicate 10-cm tissue culture dishes. At confluency, the growth medium was exchanged for treatment medium described above except glucose concentration was changed to 0.5 mM and 10 ng/mL tumor necrosis factor-a was added.
  • Formulation A was diluted in the treatment medium to give final concentrations of 0 (control), 0.015, 0.16, 0.3, 0.5, and 0.7 mM, and added to GM20942 fibroblasts. After a 24- hour incubation, cells were scraped, and washed with cold PBS. Cells were lysed with radioimmuno-precipitation assay (RIP A) buffer (0.3 mL), then centrifuged at 14,000 x g for 15 minutes at 4°C. The supernatants were collected, and the protein concentration measured using a BCA protein assay kit (Thermo Scientific, catalog # 23225). Proteins in the lysates were resolved by SDS-PAGE followed by western blotting using either anti-ICAM-1 or anti- GAPDH primary antibody. Blots were imaged using an Odyssey CLx Imaging System.
  • PMM2-deficient fibroblasts were treated with Formulation A as described above. Following a 24-hour treatment, cells were lysed as described above and cell lysates were resolved by SDS-PAGE followed by western blotting using either anti-mouse glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) antibody or biotinylated Con A. The membrane was then washed and incubated with IRDye 800CW-coupled goat anti-mouse secondary antibody and IRDye 680RD-streptavidin in Intercept antibody diluent buffer, washed and imaged.
  • GPDH anti-mouse glyceraldehyde 3 -phosphate dehydrogenase
  • N- glycan preparation and Quadrupole Time of Flight (QTOF) analysis was performed as described in Chen et al. (Chen et al. 2016).
  • QTOF Quadrupole Time of Flight
  • the percentage of total glycans in the sample was calculated.
  • the Glycomics profile for each CDG cell line was compared to its healthy counterpart (fibroblasts or LCL cells).
  • Formulation A modulation of N-glycans was calculated and reported as the fold change versus healthy fibroblasts.
  • BALB/c male mice were purchased from Charles River Laboratory (Wilmington, MA, USA) and used between 20 - 25 grams in weight. After an acclimation period, the animals were injected with 5 nmol free Sulfo-Cy5.5 or GLR0052, a liposome formulation with encapsulated Sulfo-Cy5.5-COOH. Before administration, free sulfo-Cy5.5 and GLR0052 were diluted using the dilution buffer (15 mM Tris-HCl + 145 mM NaCl (pH 7.0)) supplied with the test article.
  • dilution buffer 15 mM Tris-HCl + 145 mM NaCl (pH 7.0)
  • mice were anesthetized briefly under isoflurane and scanned in ventral and dorsal positions prior to dosing and at 30 minutes, 2, 4, 8, 24, and 48 hours postdose (Spectral Instruments Imaging, SPECTRAL Ami HTX Advanced Molecular Imager) to achieve live whole body imaging. They were allowed to regain consciousness and returned to their cage until the next scheduled imaging timepoint. Fluorescence was analyzed using Aura software (Total fluorescence was shown as Photons/s; data not shown).
  • mice were dosed with vehicle control (15mM Tris-HCl +145 mM NaCl (Ph 7.0), 5 nmol of free Sulfo-Cy5.5 or GLR0052, at a dose volume of 100 L, and euthanized at 4 hours after injection. Animals were anesthetized as stated above and live imaged before euthanasia. All animals were anesthetized under isoflurane and perfused with lx Dulbecco's phosphate-buffered saline (DPBS) until the fluid exiting the right atrium was clear.
  • DPBS Dulbecco's phosphate-buffered saline
  • tissue Immediately after perfusion and harvest, tissues were placed into individual wells of a 12-well plate (lymph nodes x 2, heart, lung, brain, kidneys, liver, spleen, skeletal muscle, small intestine, and colon) and imaged immediately after removal to minimize tissue degradation.
  • Aura software was used to analyze the numerical pixel value and image information (Fluorescence (mean radiance) data shown as Photons/ s/ cm A 2/ sr) .
  • mice Twenty-seven male CD-I mice (3/timepoint) were administered a single intravenous (IV) dose of Formulation A at 20 mg/kg. At times 0, 1, 2, 4, 8, 12, 24, 48, and 72 hours after dosing, mice were euthanized, and blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes. Samples were processed to plasma and assessed for M1P concentrations using liquid chromatography-mass spectrometry (LC/MS- MS) analysis at Alturas Analytics. Pharmacokinetic parameters were determined for composite concentration-time data using WinNonlin.
  • IV intravenous
  • PK parameters Male beagle dogs, 3 per group, were administered with three different doses of Formulation A for evaluation of PK parameters, via intravenous infusion using a calibrated infusion pump, followed by a 1ml sterile saline flush. Doses evaluated were 4.8, 25.2, or 46.8 mg/kg, respectively. Blood samples were collected at pre-dose, and then 1.5, 4, 12, 24, 48, and 72 hours after administration of Formulation A. PK parameters were estimated using Phoenix pharmacokinetics software, and with WinNonLin. A non-compartmental approach consistent with the intravenous route of administration was used for parameter estimation.
  • Formulation A treatment restores (iDP-Mannose, total glycoprotein expression and KAM-1 in PMM-2 patient-derived fibroblasts [0182]
  • To increase production of GDP-mannose in PMM2-deficient cells we sought to deliver activated mannose, in the form of mannose 1- phosphate (M1P) via liposome encapsulation.
  • M1P mannose 1- phosphate
  • Formulation A was investigated the ability of Formulation A to modulate the N- glycosylation machinery downstream of GDP-mannose synthesis.
  • Formulation A s effect on overall glycoprotein levels was assessed by concanavalin A (Con A) binding to total proteins.
  • Con A concanavalin A
  • HDF healthy dermal fibroblasts
  • Formulation A increased cellular glycoprotein levels to approximately 94% (quantification data not shown) of that present in normal fibroblasts (FIG. 2C).
  • ICAM-1 intracellular adhesion molecule- 1
  • N-glycan pathway restoration Following Formulation A treatment of PMM2 -deficient fibroblast, N-gly comics profiling was conducted. Additionally, given the beneficial impact of Formulation A treatment on glycoprotein synthesis in PMM2-deficient fibroblasts, N-glycomics profiling was extended to patient derived cells from CDGs other than PMM2.
  • HDF Healthy dermal fibroblasts
  • parental LCL parental LCL
  • asialo-biantennary glycan Hex5HexNAc4
  • FuclHex5HexNAc4 fucosylated counterpart
  • sialylated complex glycan was the mono-sialo fucosylated Neu5AclFuclHex5NAc4 (5.86%), followed by its non-fucosylated version as well as the di-sialo biantennary complex glycan at 3.36% and 4.27%, respectively (FIG. 3).
  • the inter-flask glycosylation variation in LCL was smaller than that in cultured skin fibroblasts, the overall abundance of complex glycans was low ( ⁇ 5% of total glycan amount).
  • ALG12-CDG showed the most significant relative depletion of sialylated glycans, including the mono-sialo fucosylated Neu5AclFuclHex5NAc4, its non-fucosylated version, and the di-sialo biantennary complex glycan Neu5Ac2Hex5HexNAc4.
  • PMM2-CDG cells appear to contain comparable levels to those found in HDF. Having examined all the complexed glycans, including non-fucosylated, fucosylated, and sialylated glycans, similar to the asialo glycan, no consistent pattern was identified.
  • N-glycans from a DPAGT1-CDG line were compared to the normal line derived from one of the unaffected parents (FIG. 4). While the abundance of fucosylated di -sialylated, mono-sialylated and a-galactosylated glycans was reduced in DPAGT1-CDG cells, the abundance of fucosylated a-sialo-biantennary glycan was increased. Thus, the overall under-glycosylation of complexed glycans was difficult to evaluate in cultured type 1 CDG cells. As expected, no generalized change in Golgi associated glycosylation including glcNAcylation, galactosylation, sialylation or fucosylation was observed.
  • Formulation A corrects the abnormal glycosylation pattern in PMM2-CDG cells
  • the liposome formulation used for Formulation A distributes primarily to the liver
  • lipid nanoparticle (including liposomes) biodistribution is driven primarily by their lipid content, rather than their payload.
  • a surrogate formulation (GLR0052), with encapsulated M1P substituted with Sulfo-Cy5.5-COOH, was administered to wild type mice in order to enable live animal imaging followed by ex-vivo imaging of dissected tissues at necropsy.
  • mice were administered free Sulfo-Cy5.5 (control) or GLR0052 via intravenous injection and whole body live near infra-red fluorescence (NIRF) imaging was performed at various time points post-injection (data not shown).
  • NIRF near infra-red fluorescence
  • Optimal NIRF signal could be observed at 4 hours when assessed ventrally (FIG. 7A) for GLR0052. While measurable signal was measurable for free Sulfo-Cy5.5, it was at a lower intensity, compared to that in GLR0052 animals.
  • Formulation A s pharmacokinetics profile supports its further development as a drug candidate for PMM2-CDG
  • M1P levels decreased slowly in a monophasic manner with an estimated terminal half-life (t 1/2) of approximately 11.2 hours.
  • the volume of distribution was 0.2 L/kg and was lower than the total body of water in mice ( ⁇ 0.7 L/kg), suggesting that Formulation A was not extensively distributed in tissues.
  • Systemic clearance of total M1P was 11.2 mL/min/kg and was lower than hepatic blood flow ( ⁇ 90 mL/min/kg) suggesting Formulation A was slowly cleared from the body.
  • the Cmax was 142 pg/mL and AUCiast was 1270 hr*pg/mL (FIG. 8A).
  • Peak plasma M1P concentrations were observed over a range from 1 to 4 hours post-start of infusion.
  • Systemic clearance ranged from approximately 2 to 7 mL/h/kg and volume of distribution ranged from approximately 80 to 130 mL/kg, resulting in terminal elimination half-life that generally ranged from approximately 8 at the lowest dose to 40 hours at the highest.
  • the PK profile of Formulation A across three different species shows a relatively long systemic exposure with dose-proportional increases in circulating concentrations that reach several fold the estimated in vitro efficacy concentration observed in PMM-2 CDG fibroblasts (FIG. 2).
  • Example 5 Phase 2 Dose Escalation Study - PMM2-CDG Patients with Ataxia
  • a 30 mg/kg dose was selected for a Phase 2, randomized, open-label, 24-week study to assess clinical effects related to ataxia of multiple doses of Formulation A administered intravenously to adult, adolescent, and pediatric (2-11 years) participants with PMM2-CDG. Participants received weekly infusions of Formulation A, or at least 9 infusions in the first 12 weeks of the treatment period (including the first infusion and the infusion at week 12), or at least 18 infusions over 24 weeks (including the first infusion and the week 24 infusion). Fexofenadine was administered approximately 12 ( ⁇ 2) hours and 3 ( ⁇ 1) hours before Formulation A infusion.
  • the International Cooperative Ataxia Rating Scale was used to quantify impairment levels resulting from ataxia.
  • the ICARS is an outcome measure created to standardize the quantification of impairment due to cerebellar ataxia.
  • the scale is scored out of 100 with 19 items and 4 subscales of postural and gait disturbances, limb ataxia, dysarthria, and oculomotor disorders. Higher scores indicate higher levels of impairment.
  • the ICARS has been validated for use in patients with focal cerebellar lesions and hereditary spinocerebellar and Friedrich's ataxia. In the present study, the ICARS was scored from 0 (best score/normal) to 100 (worst score/most severe impairment) with scores calculated based on the 4 subscales as described in Table 4 below.
  • FIG. 9B Changes in ICARS scores at week 12, week 24 and week 27 (washout) for each subject are shown in FIG. 9B and FIG. 9C.
  • Clinical improvements in ICARS were observed at 12 weeks and maintained or further improved at 24 weeks.
  • FIGS. 10A-10B depict the baseline (FIG. 10A) and week 12 (FIG. 10B) results of an Archimedes Spiral test to assess upper-limb coordination in Subject 2007.
  • FIGS. 10C-10D depict the baseline (FIG. 10C) and week 12 (FIG. 10D) results of an Archimedes Spiral test to assess upper-limb coordination in Subject 2008.
  • FIG. 11 shows a comparison of changes in ICARS scores across previously reported studies in PMM2-CDG patients having undergone Formulation A treatment over 12 weeks and 24 weeks. Standard of Care (SOC) treatment averaged an improvement in ICARS scores of 2.6 points over 12 months in patients 5-18 yrs.
  • SOC Standard of Care
  • Acetazolamide clinical studies averaged an improvement in ICARS score of 6 points over 25 weeks in patients 5-21 yrs. Average ICARS score improvement after 12 and 24 weeks of Formulation A treatment was significantly greater than the previously reported results in PMM2-CDG (i.e., SOC and acetazolamide treatments).
  • the total changes in ICARS scores and change in ICARS scores were measured in each of the 4 subscales: postural/gait; limb coordination; dysarthria; and oculomotor. The most significant impact of Formulation A was observed in limb coordination and postural/gait section of ICARS.

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Abstract

La divulgation concerne des thérapies de remplacement de glucides (CRT) phosphorylés qui comprennent des compositions de glucides phosphorylés, tels que le mannose-1-phosphate (M1P), et des phospholipides, ainsi que des procédés de préparation de telles compositions. De telles compositions sont appropriées pour l'administration pharmaceutique de glucides phosphorylés, tels que M1P, pour le traitement de maladies de CDG de type I et de CDG de type II, comprenant la phosphomannomutase 2-trouble congénital de glycosylation (PMM2-CDG), ainsi que l'ataxie.
PCT/US2025/017678 2024-02-29 2025-02-27 Méthodes de traitement de troubles congénitaux de glycosylation (cdg) et d'ataxie chez les sujets souffrant de cdg Pending WO2025184395A1 (fr)

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