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WO2016198687A1 - Méthodes et compositions pour le traitement de troubles métaboliques conduisant à une accumulation de sulfites - Google Patents

Méthodes et compositions pour le traitement de troubles métaboliques conduisant à une accumulation de sulfites Download PDF

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Publication number
WO2016198687A1
WO2016198687A1 PCT/EP2016/063462 EP2016063462W WO2016198687A1 WO 2016198687 A1 WO2016198687 A1 WO 2016198687A1 EP 2016063462 W EP2016063462 W EP 2016063462W WO 2016198687 A1 WO2016198687 A1 WO 2016198687A1
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Prior art keywords
sulfite
sulfite oxidase
variant
mammalian
seq
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Inventor
Günter Schwarz
Abdel Ali BELAIDI
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Colbourne Pharmaceuticals GmbH
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Colbourne Pharmaceuticals GmbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/03—Oxidoreductases acting on sulfur groups as donors (1.8) with oxygen as acceptor (1.8.3)
    • C12Y108/03001—Sulfite oxidase (1.8.3.1)

Definitions

  • This application relates to the field of metabolic disorders relating to sulfite accumulation.
  • Sulfite oxidase is responsible for the oxidation of sulfite to sulfate, a reaction that is the final step in the degradation of sulfur-containing metabolites including the amino acids cysteine and methionine.
  • S-sutfocysteine and thiosulfate accumulate in patients lacking active sulfite oxidase, while in healthy individuals levels of these markers are very Sow or not detectable. Elevated sulfite levels are also found in patients with pneumonia and in patients with chronic renal failure [Mitsuhashi, H. et al., (2004), Shock 21(2) 99-102; Kajiyama, H., et al., (2000) J Am Soc Nephrol. 1 , 923-927].
  • Affected individuals with sulfite accumulation usually present symptoms such as intractable seizures, metabolic acidosis, intracranial hemorrhage, exaggerated startle reactions, and feeding difficulties.
  • Neurological damage is severe and rapidly progressive as a result of accumulation of toxic levels of sulfite and its metabolites in the brain. Death commonly occurs in the neonatal period, and patients who survive that period usually develop encephalopathy and psychomotor retardation.
  • Sulfite oxidase deficiency is a rare autosomal inherited disease caused by mutations in the sulfite oxidase gene, resulting in reduced enzyme activity.
  • Molybdenum cofactor deficiency is a rare autosomal recessive disorder characterized by the loss of activity of all molybdenum-dependent enzymes, namely sulfite oxidase, xanthine oxidase, aldehyde oxidases and the mitochondria!
  • mARC amidoxide-reducing enzyme
  • MOCS1 Type A disorder, observed in about two thirds of patients with the MoCD disorder
  • MOCS2 Type B disorder
  • GPHN gephyrin gene
  • sulfite sensitivity include asthma, urticaria, angioedema, abdominal pain, nausea, diarrhea, seizures, and anaphylactic shock.
  • the mammalian sulfite oxidase variant is one that lacks the heme domain.
  • the mammalian sulfite oxidase variant is PEGylated.
  • the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSO A «VATV (SEQ ID NO: 10) and human sulfite oxidase variant HSC APTV (SEQ ID NO: 11).
  • the PEGylated sulfite oxidase is a plant sulfite oxidase, or a variant (i.e., a mutant) thereof, or a PEGylated vertebrate sulfite oxidase, or a variant thereof.
  • a method is provided of treating a sulfite oxidase deficiency, an excess sulfite accumulation, or reduced sulfite oxidase activity resulting from molybdenum cofactor deficiency in a patient, the method comprising administering a pharmaceutical composition disclosed herein or a pharmaceutical composition comprising sulfite oxidase to the patient.
  • the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSOAKVATV (SEQ ID NO: 10) and human sulfite oxidase variant (SEQ ID NO: 1 1 ).
  • a method is provided of reducing sulfite level in a subject, the method comprising administering a pharmaceutical composition disclosed herein or a pharmaceutical composition comprising sulfite oxidase to the subject.
  • the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSOAKVATV (SEQ ID NO: 10) and human sulfite oxidase variant HSO AK VAPTV (SEQ ID NO: 1 1 ).
  • a PEGylated mammalian oxygen-reactive sulfite oxidase variant in particular a PEGylated oxygen-reactive sulfite oxidase variant wherein the mammalian sulfite oxidase variant lacks part or all of the heme domain; more specifically, wherein the variant is at least one of human sulfite oxidase variant HSOAKVATV (SEQ ID NO: 10) and human sulfite oxidase variant HSO FIKVAPW (SEQ ID NO: 11 ).
  • FIG. 1 The heme domain impacts hydrogen peroxide formation in mammalian SO.
  • H 2 0 2 formation is shown after 30 min incubation of 1 ⁇ wt MSO (SEQ ID NO: 13) (A), wt HSO (SEQ ID NO: 9) (B), PSO (C), MSOi h eme (SEQ ID NO: 14) (D), HSC TV (SEQ ID NO: 10) (E) and HSOA VAPTV (SEQ ID NO: 11 ) (F).
  • FIG. 2 Heme deletion enables oxygen reactivity of mammalian
  • FIG. 3 Oxygen consumption by p!ant and mammalian SO
  • PEGylation of PSO increases its molecular weight in a time-dependent manner.
  • PSO was PEGylated with a 4.2 kDa (kilodalton) branched PEG and aliquots were taken after 0-30 min.
  • B PSO was PEGylated with a branched 4.2 kDa and linear 5 kDa PEG. 10 and 20 vg of non-modified and PEGylated PSO were separated by a 12 % SDS-PAGE.
  • FIG. 6 PEGylation of SO retains catalytic activity and oxygen reactivity.
  • PSO and HSO Mo were PEGylated with a linear 0.5 or 5 kDa PEG and the influence of PEGylation on catalytic activity (A-D) and H 2 0 2 formation (E-H) was investigated.
  • substrate inhibition fitting was used for the determination of the kinetic parameters of the PEGylated plant proteins (A: 0.5 kDa PEG and B, 5 kDa PEG), while ichaelis-Menten fitting was used for PEGylated HSOM O ((SEQ ID NO: 4) C: 0.5 kDa PEG and D, 5 kDa PEG).
  • FIG. 7. SO prevents suifite-dependent hydrogen peroxide toxicity in HEK cells.
  • FIG. 8 impact of heme binding and hinge deletions on the folding of mammalian SO.
  • FIG. i Impact of hinge deletions on activity and oxygen reactivity of human SO.
  • FIG. 10 Structure of the NHS-PEG molecules used in the study,
  • the structure of the 0.5 kDa linear PEG corresponds to a methyl- PEOs-NHS ester with a molecular weight of 509.54 Da and a spacer arm length of
  • FIG. 11 PEGyfation of SO increases is molecular weight without loss of Moco.
  • FIG. 12 Size exclusion chromatography of non-modified
  • FIG. 13 Impact of heme domain deletion and PEGylation on
  • the stability of the human SO wt, deletion and PEGylated variants was assessed at room temperature (25 °C) by measuring either the sutfiterferri cyanide activity (C) or by measuring cofactor saturation of the proteins through Form A analysis (D) over a time period of 10 hours.
  • FIG. 14 PEGylation of murine SO MO preserves catalytic activity and oxygen reactivity.
  • Murine SO Mo was PEGylated with either linear 0.5 or 5 kDa PEGs and the influence of PEGylation on catalytic activity (A, B) and H 2 0 2 formation (C, D) was investigated.
  • A, B ichaelis- enten plots of ferricyanide:sulfite activity of
  • FSG. 15 Hydrogen peroxide-dependent toxicity in the absence and presence of purified catalase.
  • a mammalian cell represents “one or more mammalian cells” or “at least one mammalian cell.”
  • Molybdenum Molybdenum
  • Moco molybdenum cofactor
  • Animal sulfite oxidase is a dimeric enzyme, harboring a cytochrome b 5 -type heme domain in addition to the pterin-based molybdenum cofactor domain.
  • the catalytic cycle of animal sulfite oxidase involves electron transfer from sulfite to pterin-based molybdenum cofactor, followed by two electron transfer steps via the cytochrome b 5 domain to the terminal electron acceptor cytochrome c.
  • the orthologue plant sulfite oxidase (PSO) lacks the heme domain and thereby constitutes the simplest eukaryotic Mo-enzyme.
  • PSO orthologue plant sulfite oxidase
  • Mammalian and plant SO are localized in different cellular compartments catalyzing the oxidation of sulfite by coupling electron transfer either to mitochondrial respiration or peroxidation, respectively.
  • mammalian SO requires a heme domain- mediating electron transfer to cytochrome c, while PSO consists only of a single catalytic domain, which passes electrons directly to molecular oxygen.
  • PSO uses molecular oxygen as electron acceptor for sulfite oxidation and consequently produces H 2 0 2 .
  • H 2 0 2 formation is very low.
  • Administration of PSO to animals would likely result in undesirable inflammatory or allergic reactions.
  • reaction mechanism of sulfite oxidase can be divided into a
  • sulfite binds at the Mo vl center and is oxidized to sulfate by the transfer of two electrons to the molybdenum center yielding the reduced Mo ⁇ species.
  • one electron is transferred via IET to the heme domain creating a paramagnetic Mo v intermediate state, which can be detected using electron paramagnetic resonance (EPR) spectroscopy.
  • EPR electron paramagnetic resonance
  • the oxidative half-reaction is initiated with the transfer of one electron from heme to the final electron acceptor cytochrome c.
  • the second electron can leave the Mo center by a second IET step via heme to a second cytochrome c yielding the fully oxidized form of the enzyme.
  • the absence of the heme domain in plant sulfite oxidase implicates a different oxidative half-reaction than in animal sulfite oxidase.
  • MoCD pterin-based molybdenum cofactor deficiency
  • SOD sulfite oxidase deficiency
  • Type A deficiency affects two-thirds of all patients and is caused by mutations in the MOCS1 gene.
  • Type B patients accumulate the first Moco intermediate cyclic pyranopterin monophosphate (cPMP) due to defects in the MOCS2 gene.
  • Type C deficiency affects the GPHN gene.
  • cPMP is the only reported stable pterin-based molybdenum cofactor intermediate and similar therapies for MoCD type B and C are not feasible.
  • a pharmaceutical composition comprising a mammalian oxygen-reactive sulfite oxidase variant.
  • the mammalian oxygen-reactive sulfite oxidase variant is a mammalian sulfite oxidase variant lacking the heme domain function, caused by partial or complete deletion, mutational change, or altering the linking peptide between the Moco and heme domain.
  • the mammalian oxygen-reactive sulfite oxidase variant is a mammalian sulfite oxidase variant that lacks a functional heme domain
  • a mammalian sulfite oxidase includes, for example, sulfite oxidase from at least one of human, mouse, monkey, rat, etc.
  • the mammalian oxygen-reactive sulfite oxidase variant is PEGylated.
  • the pharmaceutical composition further comprises a catalase.
  • the pharmaceutical composition may further comprise a hydrogen peroxide
  • Mammalian SO variants are able to transfer electrons from sulfite to oxygen, but only with reasonable rates in the absence of efficient heme reduction. Heme-deleted mammalian SO variants can catalyze the formation of H 2 0 2 .
  • oxygen-reactive mammalian SO variants preserve their catalytic activity.
  • the mammalian sulfite oxidase variant is at least one of human sulfite oxidase variant HSO AK VATV (SEQ ID NO: 10) and human sulfite oxidase variant HSC APTV (SEQ ID NO: 11 ).
  • a mammalian oxygen-reactive sulfite oxidase variant is a variant that, unlike its wild type counterpart, readily reacts directly with oxygen, leading to the formation of superoxide ions as the immediate product of the oxidative half- reaction, which is spontaneously dismutated to H 2 0 2 , H 2 0 2 (which can be present in a stoichiometric or near stoichiometric amount) is thus produced in this reaction, in an amount greater than the amount produced by its wild-type counterpart.
  • mammalian oxygen-reactive sulfite oxidase variant includes a SO variant that has the heme domain completely deleted or the heme domain mutated at a heme- coordinating residue or the hinge region (the surface exposed tether peptide connecting the Mo and heme domains) deleted by 5 or six residues, and an SO variant with one or more point mutations in the heme domain and/or in the hinge region, in particular one to 20, preferably 1 to 10, more preferably one to 5 point mutations; and a SO variant with a non-functional or low functioning heme domain: such variants have impaired, reduced, or abolished heme domain-mediated electron transfer (IET) to cytochrome c.
  • IET heme domain-mediated electron transfer
  • HSO AKVA TV SEQ ID NO: 10
  • HSO AKVAPTV SEQ ID NO: 11
  • the human SO hinge region is at amino acid residues 105-115 of SEQ ID NO: 9 or at amino acid residues 86-91 of SEQ ID NO: 2.
  • the mammalian sulfite oxidase can be from any mammalian species that has a sulfite oxidase.
  • the mammalian sulfite oxidase is from mouse, human, rat, or monkey. In certain embodiments, the mammalian sulfite oxidase is from a human.
  • the cataiase can be from any source that has a catalase, including from any mammal that has a catalase.
  • the mammalian catalase is from mouse, human, rat, or monkey.
  • the catalase can be wild-type or a variant that retains or shows altered function.
  • the catalase can be from the same species as the sulfite oxidase to be administered to a patient, or from different species.
  • the sulfite oxidase is a plant sulfite oxidase, or a variant thereof, or a vertebrate sulfite oxidase, or a variant thereof.
  • the pharmaceutical composition may further comprise catalase or a hydrogen peroxide reducing agent or sequester, i.e. a potvdentate (multiple bonded) ligand which is able to form two or more separate coordinate bonds to a single central metal atom.
  • a method of treating a sulfite oxidase deficiency, an excess sulfite accumulation (due to any reason), or reduced sulfite oxidase activity resulting from pterin-based molybdenum cofactor deficiency in a patient comprising administering an effective amount of a pharmaceutical composition comprising sulfite oxidase to a patient in need thereof.
  • the pharmaceutical composition is a pharmaceutical composition described herein.
  • the method further comprises administering a pharmaceutical composition comprising a catalase to said patient.
  • the catalase and the sulfite oxidase, or a variant thereof, can be in the same or in a different pharmaceutical composition.
  • the method further comprises administering a hydrogen peroxide reducing agent or a sequester.
  • Hydrogen peroxide is a byproduct of oxygen-reactive sulfite oxidase activity and such reducing agents and sequester can prevent or reduce hydrogen peroxide accumulation.
  • a method of reducing sulfite level in a patient comprising administering an effective amount of a pharmaceutical composition comprising sulfite oxidase to a patient in need thereof.
  • the pharmaceutical composition is a pharmaceutical composition as disclosed herein.
  • the method further comprises administering a pharmaceutical composition comprising a catalase to said patient.
  • the catalase and the sulfite oxidase, or a variant thereof, can be in the same or in a different pharmaceutical composition.
  • the method further comprises administering a hydrogen peroxide reducing agent or sequester.
  • Hydrogen peroxide is a byproduct of sulfite oxidase activity and reducing agents and sequester can prevent or reduce hydrogen peroxide accumulation.
  • Impaired sulfite oxidation is the major cause of neuronal cell death in MoCD and SOD. Furthermore, the majority of sulfite in MoCD and SOD originates from peripheral tissue. Sulfite is primarily generated in the liver and kidneys and is transported to the brain via the vascular system. Consequently, the methods and compositions for enzyme replacement therapies of the present disclosure targeting sulfite removal from the blood are effective in preventing sulfite toxicity.
  • the disclosed mammalian SO variants e.g., the mammalian oxygen-reactive SO variants
  • Vertebrate sulfite oxidase includes sulfite oxidase from any vertebrate source that has a sulfite oxidase, such as mammalian, human, bovine, and murine sulfite oxidase.
  • Mammalian sulfite oxidase includes sulfite oxidase from any mammalian source, such as human and mice.
  • Plant sulfite oxidase includes sulfite oxidase from any source, including Arabidopsis sulfite oxidase as described in Eilers et al., (2001) J.
  • sulfite oxidase includes: sulfite oxidase that is a hybrid between one or more species; a sulfite oxidase that is an enzymatically active fragment; and a sulfite oxidase that is part of an enzymatically active fusion protein, fused with an appropriate other protein(s), peptide(s), or polypeptide(s).
  • Enzymatic activity means an activity to catalyse sulfite oxidation, i.e. activity in transferring electrons to oxygen, resulting in H 2 0 2 , and is measured by sulfite:ferricyanide assay as decribed below.
  • Standard method for the determination of sulfite oxidase activity in the full-length animal enzyme Sulfite oxidase activity of full-length sulfite oxidase can determined use the sulfitexytochrome c-dependent activity assay, which is based on the sulfite-dependent reduction of cytochrome c monitored at 560 rim. Sulfite-dependent reduction of cytochrome c results in increase of cytochrome c absorption at 560 nm. Sulfite oxidase variants with either defects in the catalytic molybdenum domain or the heme domain or the electron transfer between domains will show reduced or absent sulfitexytochrome c activity.
  • Sulfite oxidase activity of full-length sulfite oxidase as well as sulfite oxidase molybdenum domain or variants with impaired electron transfer can be monitored using the sulfite:ferricyanide activity assay, which is based on the sulfite-dependent reduction of ferricyanide monitored at 420 nm. Sulfite-dependent reduction of ferricyanide results in the reduction of ferricyanide absorption at 420 nm.
  • Sulfite oxidase variants with defects in the catalytic moSybdenum domain will show reduced or absent su!fite:ferricyanide activity, while variants with a defective heme domain or defect in the electron transfer will remain active in this assay.
  • Reference herein to "sulfite oxidase variant” or “sulfite oxidase mutant” includes a sulfite oxidase variant having one or more, in particular one to 20, preferably one to 10, more preferably one to 5 amino acid substitutions, deletions, or additions provided that the sulfite oxidase is active in transferring electrons to oxygen, resulting in H 2 0 2 .
  • sulfite oxidase variant or "sulfite oxidase mutant” includes a sulfite oxidase variant that is a hybrid between one or more species; an enzymatically active fragment; a part of an enzymatically active fusion protein, fused with an appropriate other protein(s) peptide(s), or
  • Catalase includes a catalase variant having one or more amino acid substitutions, deletions or additions provided that the catalase is active in catalyzing the reaction of H 2 0 2 to water and oxygen determined by a colorimetric method, Catalase activity can be determined by Hydrogen peroxide quantification, which is based on the quantification of the complex formation between xylenol orange and ferric ions, which is produced by the peroxide- dependent oxidation of ferrous iron. The method can be performed using a commercial kit (National Diagnostics) and detection can be carried out
  • catalase includes a catalase from any source, including from human or mouse.
  • catalase includes a catalase that is a hybrid between one or more species; a catalase that is an enzymatically active fragment, wherein catalytic activity refers to the before-mentioned activity, namely catalyzing the reaction of H 2 0 2 to water; a part of an enzymatically active fusion protein, fused with an appropriate other protein(s) peptide(s), or polypeptide(s); and covalently or non- covalently modified with an agent, such as PEG.
  • an agent such as PEG.
  • Proteins are obtained by standard techniques well known in the art, such as by recombinant DNA technology, expression and purification. Mutants are also obtained by standard techniques well known in the art, such as by
  • the sulfite oxidase and the catalase can be further modified covendedly, or non-covalently, by methods known in the art.
  • the proteins may be PEGylated.
  • PEG refers to polyethylene glycol, a water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art.
  • the number of ethylene glycol units in PEG is approximated for the molecular mass described in Daltons. For example, if two PEG molecules are attached to a linker where each PEG molecule has the same molecular mass of 10 kDa, then the 20 total molecular mass of PEG on the linker is about 20 kDa.
  • the molecular masses of the PEG attached to the linker can also be different, e.g., of two molecules on a linker one PEG molecule can be 5 kDa and one PEG molecule can be 15 kDa.
  • a PEG terminates on one end with hydroxy or methoxy (methoxy PEG, mPEG) and is, on the other end, covalently attached to a linker moiety via an ether oxygen bond.
  • the PEG polymer is either linear or branched.
  • Useful PEG reagents are, e.g., available from Nektar Therapeutics.
  • Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol.
  • a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide.
  • Branched PEGs usually have from 2 to 8 arms and are described in, for example, EP-A 0 473 084 and U.S. Pat. No. 5,932,462.
  • the composition comprises 2-12 PEG
  • the composition comprises methyl PEG8-NHS ester, linear NHS activated PEG with 5kDa in size (polydisperse), linear activated PEG with 10 kDA in size (polydisperse) or branched NHS-activated PEG with 4.2 kDa in size (mortodisperse).
  • the number of PEG molecules attached to sulfite oxidase is 4 moiecu!es per monomer (branched PEG) or 8 molecules per monomer (5 kDa PEG).
  • all of the surface exposed lysine of a sulfite oxidase protein or variant SO protein is
  • PEGylated sulfite oxidase can be carried out at pH 4-10, pH 6, 7, 8, or 9 using techniques known to persons skilled in the art.
  • a higher pH may be used.
  • PEGylation at lower pH may avoid pH induced denaturatton of sulfite oxidase.
  • the sulfite oxidase-amino acid sequence may be varied to include more surface lysine residues, for example by site-directed mutagenesis.
  • any suitable dosage(s) and frequency of administration are contemplated.
  • compositions can include a pharmaceutically acceptable carrier.
  • a “pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are
  • compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge et al. (1977) J Pharm Sci. 66:1-19).
  • a pharmaceutically acceptable salt e.g., an acid addition salt or a base addition salt (see e.g., Berge et al. (1977) J Pharm Sci. 66:1-19).
  • the protein compositions can be stabilized and formulated as a solution, microemulsion, dispersion, liposome, lyophilized (freeze- dried) powder, or other ordered structure suitable for stable storage at high
  • Sterile injectable solutions can be prepared by incorporating an enzyme in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by sterile filtration.
  • dispersions are prepared by incorporating an enzyme into a sterile vehicle that contains a basic dispersion medium.
  • methods for preparation include vacuum drying and freeze-drytng that yield a powder of a an enzyme plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin.
  • treatingTM and treatment refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or
  • the present method of "treating" a disorder encompasses both prevention of the disorder in a predisposed individual and treatment of the disorder in a clinically symptomatic individual.
  • TreatingTM covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human, and includes; inhibiting the condition, i.e., arresting its development; or relieving or ameliorating the effects of the condition, i.e., causing regression of the effects of the condition.
  • “Prophylaxis” or “prophylactic” or “preventative” therapy as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it.
  • composition may be administered by any suitable route, such as orally, topically, or parenterally, parenterally being particularly preferred.
  • compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of
  • the route can be, e.g., intravenous (“IV”) injection or infusion, subcutaneous (“SC”) injection, intraperitoneal (“IP”) injection, pulmonary delivery such as by intrapulmonary injection, intraocular injection, intraarticular injection, or intramuscular (“IM”) injection.
  • IV intravenous
  • SC subcutaneous
  • IP intraperitoneal
  • pulmonary delivery such as by intrapulmonary injection, intraocular injection, intraarticular injection, or intramuscular (“IM”) injection.
  • the composition can be administered by intra-hepatic injection.
  • parenteral as used herein includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, subconjunctival, intracavity, transdermal and subcutaneous injection, aerosol for administration to lungs or nasal cavity or administration by infusion by, for example, osmotic pump.
  • Preparations for parenteral administration include, for example, sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors, and inert gases and the like.
  • a suitable dose can depend on a variety of factors including, e.g., the age, gender, and weight of a subject to be treated. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the disease and/or the extent of the sulfite accumulation. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will depend upon the judgment of the treating medical practitioner (e.g., doctor or nurse).
  • the treatment may involve administration of one or more other agents.
  • Such agents can provide supplemental, additional, or enhanced function for sulfite oxidase.
  • agents include, without limitation, mo!ybdate, a composition of a cofactor required for sulfite oxidase activity, cyclic pyranopterin monophosphate (cP P), molybdopterin precursor Z and derivatives (see U.S. Patent No. 7,504,095 and WO2012/112922), molybdopterin or molybdenum cofactor, catalase, IV fluids, or cytochrome C.
  • Such agents can be formulated with the composition or may be administered simultaneously or sequentially to a subject being treated.
  • subject is used interchangeably with the term “patient” and includes adults, neonates, and infants, including, for example, those aged less than
  • a neonate is considered to be a baby from birth to 4 weeks and an infant is considered a baby under 12 months old.
  • the subject may also be aged less than one week at the time of diagnosis and aged less than one week at the start of treatment.
  • the subject may also be older.
  • a "subject,” as used herein, can be a human.
  • a “patient” is used herein interchangeably with a “subject.” In certain embodiments, the patient (or the subject) is a human patient (or human subject).
  • an effective amount or "a therapeutically effective amount” is a dosage that is sufficient to reduce sulfite levels and/or S-sulfocysteine levels in a subject.
  • a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition in the subject. The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the term “an effective amount” or “a therapeutically effective amount” can also be a dosage that is sufficient to elicit a desired medical outcome, such as improved symptoms of MoCD or SOD in a patient, and improved survival of the patient, by any amount of time, including one day.
  • a therapeutic treatment includes a series of doses, which will usually be administered concurrently with the monitoring of clinical endpoints with the dosage levels adjusted as needed to achieve the desired clinical outcome,
  • Appropriate dosages for administering the composition may range from
  • compositions can be administered in one dose, or at intervals such as once daily, once every second day, once weekly, and once monthly or for a substantial part or the whole of the lifetime of the patient. Dosage schedules can be adjusted depending on the half-life of the composition, or the severity of the patient's condition.
  • the compositions are administered as a bolus dose or continuous infusion, to maximize the circulating levels of the composition of the seventh aspect for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
  • a typical dosage regime can involve daily to weekly administration of a composition to a subject at a dose of 10-640 pg/kg, including a dose of 10, 50, 100, 150, 160, 200, 250, 300, 320, 350, 400, 450, 480, 500, 550, 600, or 640 pg/kg.
  • Safety and tolerability assessments may be made during the dosage regime.
  • a physical examination including head circumference, neurological examination and imaging (including EEG and MRI), ECG, vital signs (heart rate, non-invasive blood pressure, respiratory rate, and temperature), adverse events, blood gas analysis, blood chemistry (including urea, electrolytes, creatinine, uric acid, and liver function tests), hematology, and urinalysis (including creatinine), s- sulfocysteine [SSC], S-sulfonated transthyretin, homocysteine, cystine, thiosulfate, and dipstick testing for sulfite in urine.
  • SSC s- sulfocysteine
  • proteins and mutants may be constructed, produced, and purified by standard techniques, such as by recombinant DNA technology.
  • hexa-histidine-tagged Arabidopsis sulfite oxidase (“hexa- histidine” disclosed as SEQ ID NO: 7) was made and purified from E. colt. Plant sulfite oxidase (PSO) was expressed as described in Eilers et a!., 2001, J. Biol, Chem. 276, 48989-46994 using the plasmid pQE80-AtPSO. The protein was purified using its C-terminal histidine tagged by metal ion chelate chromatography and ion exchange chromatography (see Schrader et ai., (2003) Structure 11 , 1251- 1263).
  • PSO was PEGylated in order to protect PSO from rapid clearance and degradation and to avoid an immunogenic response to the plant protein.
  • N- hydroxysuccinimide-(NHS) activated PEG molecules were used to modify PSO by PEGyiation: Methyl-PEG8-NHS ester; Linear NHS-activated PEG with 5 kDa in size (polydisperse); Linear NHS-activated PEG with 10 kDa in size (polydisperse); and Branched NHS-activated PEG with 4.2 kDa in size
  • PAGE, size exclusion chromatography, and mass spectrometry The number of attached PEG molecules as determined by ALDI-TOF were; branched PEG: 4 molecules per monomer and 5 kDa PEG: 8 molecules per monomer. Note that the use of SDS-PAGE and size exclusion chromatography cannot accurately determine the number of added PEGs, but does illustrate that the protein still runs as a single band and at a larger, PEGylated size.
  • HSO AKVATV (SEQ ID NO: 10) has been shown in previous studies to exhibit an almost 100-fold reduction in IET rate, which in contrast resulted in only three-fold decreased sulfite ytochrome c activity (FIG. 9A).
  • An additional deletion of Pro 111 (conserved residue in animal SO) [residue number 111 of SEQ ID NO: 9 and residue number 89 of SEQ ID NO: 2] further reduced the steady state activity in HSOAKVAPTV (SEQ ID NO: 11), suggesting a further diminished IET, which correlated well with the increased reactivity towards oxygen.
  • PEGylated mammalian SO MO variants appear as homogenous dimeric proteins in contrast to the non-modified proteins, which showed a high degree of oligomeric heterogeneity, probably due to the lack of heme domain and subsequent exposure of hydrophobic surface patches. PEGylated SO proteins showed only minor changes in their activity as demonstrated by su!fate:ferricyanide steady state kinetics. More importantly, H 2 0 2 production and thereby oxygen reactivity was preserved for all PEGylated proteins. [00108] Example 3. Electron transfer between Moco and heme determines
  • H 2 0 2 production was determined as a function of sulfite oxidation in both animal and plant SOs. For this purpose, a colorimetric method that quantifies all organic peroxides including superoxide ions and H 2 0 2 was used and the exclusive production of H 2 0 2 was probed by the addition of recombinantly expressed and purified human catalase. Sulfite-dependent H 2 0 2 production (for 30 min) of wt murine ( SO) and human SO (HSO) was determined and compared to that of PSO. H 2 0 2 formation was low in the presence of MSO, showing 15 ⁇ H 2 0 2 formation with 75 ⁇ sulfite (FIG.
  • a heme-defictent murine SO (M so ) variant (MSO ⁇ ) (SEQ ID NO: 14) was generated by replacing both heme-coordinating histidines to alanines (H119A, H144A), thus resulting in a loss of heme binding [Kiein, J. M. and Schwarz, G. (2012) Cofactor-dependent maturation of mammalian sulfite oxidase links two mitochondrial import pathways. Journal of Cell Science, 125, 4676-4885].
  • HSO human SO
  • 1ET between the Mo and heme domains is essentia! to complete the catalytic cycle.
  • the 1ET process was extensively investigated in HSO using laser flash photolysis, which enables the measurement of the IET rate constants between both redox centers in HSO and identified the importance of the tether linking Mo and heme during the IET process [Johnson- Winters, K., Tollin, G. and Enemark, J. H.
  • HSO deletion variant HSO VATV
  • HOAKVAPTV a second deletion variant
  • HSOA VATV which is not deficient in heme but shows a reduced IET rate constant [Johnson-Winters, K., Tollin, G. and Enemark, J. H. (2010) Biochemistry 49, 7242- 7254] between Moco and heme, again a linear H 2 0 2 production was observed (FIG. 1E). However, the molar ratio of H 2 0 2 formed per sulfite dropped from 0.72 to 0.41 as compared to MSO AHEME (FIG. 1D). The additional deletion of Pro111 in
  • HSOAKVAPTV further reduced sulfite:cytochrome c activity (FIG. 9A), while the rate of H 2 0 2 formation per mole sulfite increased to 0.54 (FIG. 1F).
  • Example 4 Mammalian SO molybdenum domains show oxygen reactivity similar to PSO
  • Mammalian SO Mo variants revealed two major differences to PSO. First, SO Mo variants showed no inhibition at high substrate concentration, which was in contrast to PSO. Second, the determined Jc cat values for MSO MO (SEQ ID NO: 16) and HSO MO (SEQ ID NO; 4) were 2.4 and 4.7-fold lower, respectively, as compared to PSO (FIG. 2 A-B and FSG. 9B).
  • H 2 0 2 was almost not detected at low sulfite concentrations (below 10 ⁇ ) using the mammalian SO Mo variants (FIG. 2 C-D). This is due to the fact that under aerobic conditions and low concentrations, sulfite is susceptible to air oxidation, which combined with the non-catalytic sulfite oxidation mediated by H 2 0 2l might explain the detection limit of H 2 0 2 at low sulfite concentrations.
  • Example 5 Reduced IET increases oxygen consumption in
  • FIG. 3B The activity of MSO and HSO remained very low (1-2 ⁇ s "1 ) supporting the previous results of H 2 0 2 formation (FIG. 3B).
  • Example 8 Mouse brain lacks capacity for su!fite oxidation
  • mammalian SO is not feasible due to the requirement for mammalian SO translocation into mitochondria where its native electron acceptor, oxidized cytochrome c, is localized.
  • the ability of mammalian SO Mo domain variants to use oxygen as electron acceptor offers the possibility for the development of an enzyme replacement therapy towards MoCD and SOD, in which SO variants can use dissolved oxygen in blood for sulfite oxidation.
  • PSO was PEGylated and parameters such as incubation time and structure of PEG molecules on the efficacy of modification were investigated (FIG. 5 A and B).
  • a 4.2 kDa PEG molecule approximately 50 % of PSO was PEGylated within 2 minutes (min), as depicted by a shift in MW and within 30 min, PEGyiation was nearly completed (FIG. 5A).
  • two PEG molecules differing in size and structure were used to PEGylate PSO: a branched (4.2 kDa) and a linear PEG (5 kDa) (FIG. 10).
  • PEGylated proteins did not correlate with the MW and number of added PEG molecules as previously determined by mass spectrometry (see Table 2).
  • PEG is a highly soluble amphiphilic polyether diol that can be linear or branched.
  • the increase in MW of PEGylated proteins is mainly due to the large hydrodynamic volume of the PEG and not only due to its MW as depicted by differences in the MW determined for PEGylated PSO by SDS-PAGE and mass spectrometry.
  • PSO modified with a 5 kDa linear PEG showed in SDS-PAGE an apparent MW of 170 kDa, while by mass spectrometry the determined MW was 85 kDa, which corresponds to the addition of eight PEG molecules per monomer.
  • the number of attached PEG molecules to the protein largely depended on the chemistry of the PEG molecule used, as depicted by the differences between PSO PEGylated with either the branched or the linear PEG (see Table 2).
  • the number of added PEG molecules was twice as much when using the linear 5 kDa PEG as compared to the branched one, while both PEG molecules had a similar MW. This observation can be explained by steric hindrance resulting from the large surface occupied by branched PEGs as compared to linear PEGs.
  • all other PEG molecules to the protein were twice as much when using the linear 5 kDa PEG as compared to the branched one, while both PEG molecules had a similar MW
  • PEGylated PSO, MSO Mo and HSO Mo were determined. PEGylation of SO proteins did not result in major changes in the corresponding kinetic parameters (FIG. 6 A- D, Table 3 and FIG. 14 A and B). PEGylation of PSO with 5 kDa PEG did not alter its catalytic turn over (k of 483 min "1 versus 511 min "1 ), whereas a slight increase in Jc cat was observed when the 0.5 kDa PEG was used (Jr cat of 650 min "1 versus 511 min "1 ). On the other hand, an approximately two-fold increase in K m was determined for both PEGylated PSO proteins as compared to native PSO. See Table 3.
  • Example ⁇ SO is able to catalyze sulfite oxidation using dissol ed oxygen in cell culture
  • Previous examples showed that mammalian SO variants are able to react with oxygen in vitro. Furthermore, it was shown that PEGylation as a masking method, consisting of shielding the protein by the cova!ent attachment of PEG molecules to the surface-exposed lysine residues of the protein, did not cause loss of activity.
  • the capacity of both plant and human SOs to use oxygen as an electron acceptor, thus generating H 2 0 2 were evaluated in a cell-based assay.
  • human embryonic kidney cells HEK 293 were exposed to SO-dependent H 2 0 2 toxicity. Cell viability was determined using the MTT (3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide) cell
  • HSOMO as well as the PEGylated proteins were equally effective in inducing cell death resulting in 20 % cell survival as compared to control (FIG. 7D), and toxicity was again prevented if purified catalase was added (FIG. 7E).
  • full length HSO was not able to induce cell toxicity, which again confirms its inability to accept oxygen as a suitable co-substrate (FIG. 7D).
  • HSO deletion variants HSOAKVATV and HSC AFTV were generated by fusion PGR and cloned into the pQE80L vector using Sac! and Sal! restriction sites.
  • MSO (wt) and MSO A heme were generated as previously described [Klein, J. M. and Schwa rz, G. (2012) Journal of Cell Science 125, 4876-4885].
  • catalase expression the coding sequence of human catalase (GenBank ® accession number BC110398.1 ) was PCR-cloned into pQE80L using Sail and Hindll! restriction sites.
  • PSO the previously described rAt-SO construct was used [Eilers, T deliberately Schwarz, G., Brinkmann, H., Witt, C, Richter, T., Nieder, J., Koch, B., Hille, R., Hansch, R. and Mendel, R. R. (2001) J Biol Chem. 276, 46989-46994].
  • Moco saturation was determined by denaturing 500 pmol of protein using acid iodine oxidation and alkaline phosphatase treatment resulting in the formation of the stable Moco oxidation product FormA-dephospho, which was further quantified using HPLC reverse phase chromatography as described [Klein, E. L, Belaidi, A. A., Raitsimring, A. M., Davis, A. C, Kramer, T deliberately Astashkin, A. V., Neese, F,, Schwa rz, G. and Enemark, J. H. (2014) inorganic Chemistry 53, 961- 971].
  • Activities were measured at an enzyme concentration of 50 n and 500 nM for the plant and mammalian SO proteins, respectively.
  • the assay buffer mixture contained 50 mM Tris/acetate pH 8; 0.2 mM deoxycholic acid; 0.1 mM potassium cyanide and 0.5 mM sodium sulfite. All activities were measured at room temperature (25 °C) using a 96 well-plate reader (BioTeK, Germany).
  • the assay is based on the formation of a complex between xylenol orange and ferric ions, which is produced by the peroxide-dependent oxidation of ferrous iron.
  • the method was performed using a commercial kit (National
  • Quantification was carried out after an incubation time of 30 min at room temperature (25 °C) by measuring the absorption at 560 nm using a 96 well-plate reader (BioTeK, Germany).
  • Oxygen consumption was measured using an Oroboros Oxygraph 2k Instrument (Oroboros Instruments GmbH, Austria). First, a 2 ml solution containing
  • HEK 293 human embryonic kidney cells
  • MTT 3-(4 5-dimethy!thiazo!-2-yl)-2 5-diphenyltetrazolium bromide
  • 80 ⁇ of HEK cell suspension (containing 2 * 10 4 cells) were dispensed into each well of a 96-well tissue culture plate and incubated overnight at 37 °C in a humidified, 5 % C0 2 atmosphere.
  • 10 ⁇ ! of SO and/or catalase proteins were added to each well in a final
  • a sulfite oxidase-deficient rat mode! system can also be used to study sulfite-dependent H 2 0 2 formation. See, e.g., Gunnison et a!. Fd. Cosmet. Toxicol. Vol. 19, pp. 209-220 (1981).
  • a sulfite oxidase-deficient whole body or organ-specific knock-out mode! could be used in mice.
  • HEK cells were used as a cellular model system to study sulfite-dependent H 2 0 2 formation. While sulfite (up to 500 ⁇ ) is well tolerated, SO- dependent H 2 0 2 formation severely impacted cell survival. By using this assay it was confirmed that mammalian heme-deleted SO variants catalyzed sulfite oxidation by using oxygen from the culture medium as electron acceptor.
  • catalase was required to efficiently remove produced H 2 0 2 .
  • the clearance rate of sulfite would be low, which in turn would enable the non-enzymatic oxidation of sulfite by H 2 0 2 , formed in the first case.
  • HSOAKVATV (Deletion of the amino acid residues: 108, 109, 110, 112, 113 of WT HSO)
  • HSOA VAPTV (Deletion of the amino acid residues: 108, 109, 110, 111, 112, 113 of WT HSO) MGTLLGLGAVLAYQDHRCRAAQESTHIYTKEEVSSHTSPETGIWVTLGSEVFDVTEFVDLHP GGPSKLMLAAGGPLEPFWALYAVHNQSHVRELLAQYKIGELNPEDETSDPYADDPVRHPAL KVNSQRPFNAEPPPELLTENYITPNPiFFTRNHLPVPNLDPDTYRLHWGAPGGQSLSLSLD DLHNFPRYEITVTLQCAGNRRSE TQVKEVKGLEWRTGAISTARWAGARLCDVLAQAGHQ LCETEAHVCFEGLDSDPTGTAYGASIPLARAMDPEAEVLLAYEMNGQPLPRDHGFPVRVW PGWGARHVKWLGRVSVQPEESYSHWQRRDYKGFSPSVDWETVDFDSAPSIQELPVQSA!

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Abstract

La présente invention concerne, entre autres, une composition pharmaceutique comprenant une sulfite oxydase, y compris une sulfite oxydase pégylée, un variant de la sulfite oxydase de mammifère ne possédant pas le domaine de l'hème, et un variant de la sulfite oxydase de mammifère réactive à l'oxygène, ainsi que des méthodes d'utilisation de ceux-ci pour traiter une déficience en sulfite oxydase ou une diminution de l'activité de la sulfite oxydase provenant d'une déficience du cofacteur molybdène chez le patient, ou pour faire baisser le niveau des sulfites chez le patient.
PCT/EP2016/063462 2015-06-12 2016-06-13 Méthodes et compositions pour le traitement de troubles métaboliques conduisant à une accumulation de sulfites Ceased WO2016198687A1 (fr)

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CN115711927A (zh) * 2022-11-25 2023-02-24 大唐环境产业集团股份有限公司 一种亚硫酸盐生物传感器及其制备方法和测量方法

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Title
ERKAN KARAKAS ET AL: "Structural analysis of missense mutations causing isolated sulfite oxidase deficiency", DALTON TRANSACTIONS: THE INTERNATIONAL JOURNAL FOR INORGANIC, ORGANOMETALLIC AND BIOINORGANIC CHEMISTRY, no. 21, 1 January 2005 (2005-01-01), GB, pages 3459, XP055301966, ISSN: 1477-9226, DOI: 10.1039/b505789m *
KAYUNTA JOHNSON-WINTERS ET AL: "Effects of large-scale amino acid substitution in the polypeptide tether connecting the heme and molybdenum domains on catalysis in human sulfite oxidase", METALLOMICS, vol. 2, no. 11, 1 January 2010 (2010-01-01), GB, pages 766, XP055302287, ISSN: 1756-5901, DOI: 10.1039/c0mt00021c *
KAYUNTA JOHNSON-WINTERS ET AL: "Elucidating the Catalytic Mechanism of Sulfite Oxidizing Enzymes Using Structural, Spectroscopic, and Kinetic Analyses", BIOCHEMISTRY, vol. 49, no. 34, 31 August 2010 (2010-08-31), US, pages 7242 - 7254, XP055302295, ISSN: 0006-2960, DOI: 10.1021/bi1008485 *
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CN115711927A (zh) * 2022-11-25 2023-02-24 大唐环境产业集团股份有限公司 一种亚硫酸盐生物传感器及其制备方法和测量方法

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