COFACTOR DEFICIENCY TYPE A

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 62/1.96,796, filed on July 24, 2015, the disclosure of which is incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to the use of cyclic pyranopterm monophosphate (cPMP, also known as molybdopterin derivative Precursor Z) for the treatment of diseases or disorders related to molybdenum cofactor deficiency (MoCD) type A.

BACKGROUND

[0003] Molybdenum cofactor deficiency (MoCD) is characterized by early, rapidly progressive, postnatal encephalopathy and intractable seizures, leading to severe disability and early childhood death. MoCD usually manifests during the first postnatal days with exaggerated startle reactions, alterations in muscle tone, lethargy, intractable seizures, and autonomic dysfunction. At the onset of clinical symptoms, brain imaging reveals global white matter and deep gray matter involvement, followed by rapidly evolving widespread subcortical necrosis. Multi cystic lesions appear within days, with subsequent brain atrophy and secondary microcephaly. Symptoms are predominantly- caused by sulfite toxicity due to a functional loss of sulfite oxidase, one of the

molybdenum cofactor-dependent enzymes in humans which also include xanthine oxidoreductase and aldehyde oxidase.

[0004] U.S. Patent No, 7,504,095 to Schwarz et al. discloses the structure of

molybdopterin derivative precursor Z (cPMP) with molecular weight of 363 Da

(CioHi ₄ sO P) made recombinantly in E. coli and administration of recombinant cPMP (rcPMP) to knockout mice.

[0005] International Publication No. WO 2012/1 12922 to Alexion Pharmaceuticals, Inc. discloses synthetic methods for preparing cPMP as an off-white solid compound. |Ό006] Pre-clinical studies using rcPMP have been performed in an animal model for MoCD type A (Schwarz et al., Hum Moi Gen., 2004, 13, 1249-1255). Initial treatment response to rcPMP administration has been reported for a single infant found to have MoCD type A (Veldman et al., Pediatrics, 2010, 125, el249-1254). Postnatal treatment of a neonate has been reported (Hitzert et al., Pediatrics, 2012, 130, el005-el010).

SUMMARY

[0007] In certain aspects, the present disclosure provides a method of treating

Molybdenum Cofactor Deficiency (MoCD) Type A in a human patient in need thereof, the method comprising administering to the patient a pharmaceutically effective amount of cPMP:

or of a pharmaceutically acceptable salt thereof, at least once per day, wherein the pharmaceutically effective amount is a dosage of from about 80 .ug/kg to about

3000 fig/kg body weight per day, particularly from 120 ^ig/kg to about 320 u u kg body weight per day, wherein the administering is started prior to detection of cerebral lesions in the patient and/or prior to significant cerebral encephalopathy in the patient.

[0008] In certain aspects, the present disclosure provides a method of treating MoCD Type A in a human patient in need thereof! the method comprising administering to the patient a pharmaceutically effective amount of cPMP, or of a pharmaceutically acceptabl e salt thereof, at least once per day, wherein the pharmaceutically effective amount is a dosage of from about 300 ,ug/kg to about 1500 ^tg/kg body weight per day, particularly from about 525 ^ig/kg to about 1200 μg/kg body weight per day, wherein the administering is started prior to detection of cerebral lesions in the patient and/or prior to significant cerebral encephalopathy in the patient.

? [0009] In certain other aspects, this disclosure provides a method of treating

Molybdenum Cofactor Deficiency (MoCD) type A in a human patient in need thereof, the method comprising: a) determining the patient has encephalopathy or is at risk for encephalopathy, b) optionally conducting at least one test to diagnose the human patient as having MoCD; c) administering to the human a pharmaceutically effective amount of cPMP, or of a pharmaceutically acceptable salt thereof, at least once per day, wherein the pharmaceutically effective amount is a dosage of from about about 300 ug/kg to about 1500 m*/kg body weight per day, particularly from about 325 ,ug/kg to about 1200 &'Τ¾ body weight per day, particularly from 120 _, ug/kg to about 320 μg/kg body weight per day; and d) conducting at least one test to diagnose the human patient as having MoCD type A, wherein if the human does not have MoCD type A, the administering ceases, wherein the administering is started prior to detection of cerebral lesions in the patient and/or prior to significant cerebral encephalopathy in the patient.

[0010] In certain aspects, the methods of this disclosure provide methods of treating MoCD type A in a human patient, wherein the patient is able to at least one of: feed orally at or before about three weeks of age, breathe unassisted by about one week of age; sit up unassisted at about 6 months of age, and sit up unassisted at about 12 months of age. In certain aspects, the methods of this disclosure provide methods of treating MoCD type A in a patient, where the patient survival is increased compared to untreated MoCD type A patients, wherein the increase is from about one day to about three years or more.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. DESCRIPTION OF DRAWINGS

[0012] FIG. 1 shows molybdenum cofactor synthesis and its incorporation into the four known human molybdenum enzymes. Shown are the starting molecule (GTP), cyclic pyranopterin monophosphate (cPMP) and molybdopterin (MPT) as intermediates, as well as the proteins involved in individual synthetic steps: MOCS1A, MOCS I AB, MOCS2A, MOCS2B, and gephyrin (GPNH).

[0013] FIG. 2 shows clinical details and treatment characteristics of 16 infants with molybdenum cofactor deficiency (MoCD) treated with recombinantly-produced cPMP (rcPMP). Combined data for patients 1-11 and 12-16 are median (range).

AUS=Austraiia. DEU=Germany. NLD=The Netherlands. NA=not applicable. F=female. M=male. OFC-occipital frontal circumference. MoCD=molybdenum cofactor deficiency. cPMP=cyclic pyranopterin monophosphate. P D=prenatal diagnosis. Cause of death of index patients: * Seizures and respiratory failure.† Seizures, cardiorespiratory failure. ^Encephalopathy, hydrocephalus, diagnosed with MoCD after the younger sibling had been diagnosed. §Patient alive with severe neurological disability. ^Seizures and cardiorespiratory arrest secondary to pneumonia, jjPregnancy terminated after prenatal diagnosis. Cause of treatment discontinuation: ** Treatment continued to date. ††Death from respirator}' failure. JJFamily moved away and abandoned treatment.

§§ Advanced brain injury despite detoxification with ECMO and dialysis prior to cPMP. fflf Advanced brain injur}' and co-morbidity (severe combined immune deficiency).

jjjjParental consent to treatment withdrawn, patient off cPMP and alive.

[0014] FIG. 3 shows clinical severity scoring for patients with MoCD type A.

[0015] FIG. 4A shows urinary concentration (micromole per millimole creatinine) of S- sulfocysteine (normal controls < 9 micromole per millimole creatinine) for 1 1 patients with MoCD type A on rcPMP treatment during the fi rst two weeks of treatm ent.

[0016] FIG. 4B shows urinary concentration (micromole per millimole creatinine) of xanthine (normal controls < 38 micromole per millimole creatinine) for 1 1 patients with MoCD type A on rcPMP treatment during the first two weeks of treatment. [0017] FIG. 4C shows urinary concentration (micromole per miliimoie creatinine) of urate (normal controls 820-2813 micromole per miliimoie creatinine) for 1 1 patients with MoCD type A on rcPMP treatment during the first two weeks of treatment.

[0018] FIG. 4D shows urinary S-sulfocysteine (SSC) normalization in patients during the first three months of treatment.

[0019] FIG. 4E shows long term monitoring of urinary SSC concentrations in patients #1-5, 7, and 9.

[0020] FIG. 4F shows long-term monitoring of urinary xanthine concentrations in patients #1-5, 7, and 9.

[0021] FIG. 5 shows a study profile for the observational prospective follow-up of 6 patients identified with MoCD in the neonatal period and treated with rcPMP on compassionate grounds.

[0022] FIG. 6A shows a representative axial T2 -weighted MRI brain image of patient

#3, prior to treatment with rcPMP (age ^:; = 3 days). Widespread increase in white matter signal but no necrosis, cavitation or gross white matter loss was observed.

[0023] FIG. 6B shows a representative axial T2-weighted MRI brain image of patient #3 under continued rcPMP substitution (age = 37 months). No abnormal parenchymal signal was observed; mild prominence of peri cerebral cerebrospinal fluid (CSF) spaces was observed.

[0024] FIG. 6C shows a representative axial T2~ weigh ted MRI brain image of patient #5 prior to treatment with rcPMP (age = 5 h). Diffuse brain swelling and mild volume loss were observed, but no signs of necrosis were observed.

[0025] FIG. 6D shows a representative axial T2 -weighted MRI brain image of patient #5 under continued rcPMP treatment (age = 47 months). Mild brain atrophy and mild white matter hyperintensity were observed.

[0026] FIG. 6E shows a representative axial T2-weighted MRI brain image of patient #1 prior to treatment with rcPMP (age = 4 h). Diffuse brain swelling and no signs of necrosis were observed. [0027] FIG. 6F shows a representative axial T2-weighted MRI brain image of patient #7 under continued rcPMP (age ^: = 24 months). Mild atrophy and white matter

hyperintensity were observed.

[0028] FIG. 7 shows clinical severity scores of the initial patients with MoCD type A treated with rcPMP from Examples 1 -3. Scores as described in Fig. 3.

[0029] FIG. 8 shows predicted mean (90% confidence interval) cPMP AUC (area under the curve) for the dosing regiment in pre-term and full-term neonates.

DETAILED DESCRIPTION

[0030] Molybdenum cofactor deficiency (MoCD) is a rare, life-threatening, autosomal recessive, inborn error of metabolism, characterized by disruption of the metabolic pathway for production of molybdenum cofactor (MoCo), resulting in a deficiency of activity of the three detoxifying enzymes, sulfite oxidase (SO), xanthine dehydrogenase, and aldehyde oxidase. Molybdenum cofactor (MoCo) is synthesized by a complex biosynthetic pathway involving three steps, as shown in FIG. 1.

[0031] Approximately two-thirds of patients with MOCD suffer from mutations in the MOCS1 gene, resulting in the inability to synthesize the first intermediate in the biosynthetic pathway, cyclic pyranopterin monophosphate (i.e., cPMP or Precursor Z), and are classified as MoCD type A. Almost all remaining patients suffer from a defect in the A40CS2 gene, resulting in the accumulation of cPMP that cannot be further utilized, and are classified as MoCD type B (Edwards et al., Meta Gene 3, 2015, 43-49). MoCD type A and type B, and the extremely rare MoCD type C, are autosomal recessiveiy transmitted. While MoCD type A and type B are clinically indistinguishable, type C patients show additional lack of synaptic inhibition with a more severe progression than MoCD type A or type B. Patients of all MoCD types may present with, e.g., elevation in exaggerated startle reactions, alterations in muscle tone (hypertonia, axial hypotonia, and/or limb hypertonia), lethargy, myoclonic twitching, intractable seizures, autonomic dysfunction, evolving brain edema and cysts, brain atrophy, enlargement of subarachnoid space and ventricles, loss of gray/white matter differentiation, abnormalities in feeding behavior, burst suppression or multifocal epileptic encephalogram, metabolic acidosis, and feeding difficulties. A retrospective natural history review confirms a median survival of 36 months (Mechler, et al., Genetics in Medicine, (2015) 1-6).

[0032] The present disclosure provides safety and efficacy data for the complete prospectively monitored cohort of the first 16 neonates and infants with classical presentation of MoCD (1 1 with type A and f ve with type B), who received

recombinantly prepared, intravenous cPMP for up to five years. Additionally, the present disclosure provides data on the treatment of a neonate identified with MoCD type A, starting on day zero of life, with synthetic cPMP.

[0033] Various methods of preparation of cPMP have been described, i.e. U.S. Patent No. 7,504,095 (recombinant) and WO 2012/1 2922 (synthetic). Pre-clinical studies using rcPMP have been performed in an animal model for MoCD (Schwarz et al., Hum Mol Gen., 2004, 13, 249-1255). Initial treatment response to rcPMP has been reported for a single infant found to have MoCD type A (Veldman et al., Pediatrics, 2010, 125, el 249- 1254), Postnatal treatment of a neonate has been reported (Hitzert et al,

Pediatrics, 2012, 130, el005-el010).

[0034] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0035] For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. As used herein, the term "about" is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about", whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0036] The term "pharmaceutically acceptable salt" is understood to include one or more stable, pharmaceutically acceptable, non-toxic counterions; particularly

"7 including but not limited to one or more of acetates, benzoates, bromides, carbonates, chlorides, bi tartrates, tartrates, stearates, and/or salicylates,

[0037] The following abbreviations may be used herein: aq. (aqueous); °C (degrees Celsius); ECG (electrocardiogram); EEG (electroencephalography), g (gram(s)); h (hour(s)); H ₃ P0 ₄ (phosphoric acid); HPLC (high performance liquid chromatography); HPLC-UV-vis (high performance liquid chromatography-uitra violet-mass spectrometry); kg (kilogram); KH ₂ P0 ₄ (potassium di hydrogen phosphate); M (molar), mARC

(mitochondrial amidoxime-reducing component); mM (millimolar); mg

(milligram(s)); min. (minutes(s)); mL (milliliters)); mmol (millimole(s)); MoCD

(molybdenum cofactor deficiency); MoCo (molybdenum cofactor); MoCS (molybdenum cofactor synthesis); MRI (magnetic resonance imaging); MPT (molybdopterin); NaCl (sodium chloride); Na ₂ HP0 ₄ (di sodium hydrogen phosphate); NaOH (sodium hydroxide); MR (nuclear magnetic resonance spectroscopy); cPMP or Precursor Z (cyclic pyranopterin monophosphate), SSC (S-sulfocysteine); _, ug (microgram(s)); μΐ- (microiiter(s)); μΜ (micromoiar); rcPMP (recombinant cPMP).

[0038] In this disclosure, the first day of life is considered to be day zero,

[0039] Methods of Use

[0040] The present application provides m ethods of treating or preventing a disease in a patient in need thereof. In some embodiments, the method comprises administering a pharmaceutically effective amount of cyclic pyranopterin monophosphate (i.e., cPMP or Precursor Z):

or a pharmaceutically acceptable salt thereof, to a patient in need thereof for the treatment or prevention of a disease or disorder associated with molybdenum cofactor deficiency (MoCD) type A.

[0041] In certain aspects, the present disclosure provides a method of treating

Molybdenum Cofactor Deficiency (MoCD) Type A in a human patient in need thereof, the method comprising administering to the patient a pharmaceutically effective amount of cPMP:

or of a pharmaceutically acceptable salt thereof, at least once per day, wherein the pharmaceutically effective amount is a dosage of from about 80 .ug/kg to about 3000 μ^/kg body weight per day, particularly from about 120 μg/kg to about 320 μg/kg body weight per day.

[0042] In certain embodiments, the administering is started prior to detection of cerebral lesions in the patient. In certain embodiments, the administering is started prior to significant cerebral encephalopathy in the patient. In certain embodiments, the dosage is from about 240 μg/kg to about 320 μg/kg body weight per day. In certain embodiments, the dosage is from about 525 g/kg to about 1300 body weight per day. The cPMP can be, for example, recombinant or synthetic. The route of administration can be any route, for example, is at least one of intravenous (including intravenous infusion), oral, subcutaneous, intramuscular, and peritoneal route. The patient can be any patient, including a neonate, and can be human. In certain embodiments, the administering is started when a MoCD is suspected but not yet confirmed. In certain embodiments, the administering is started after a strongly positive sulfite dipstick test from a fresh urine sample of a symptomatic newborn baby,

[0043] In certain embodiments, the patient does not have encephalopathy in the brain at the start of administration, and does not have encephalopathy in the brain at about two years or more (e.g., about two to about four years) into cPMP treatment. The lack of irreversible or cystic lesions in the brain can be determined by, for example, a brain scan, such as MRI. [0044] In certain other aspects, this disclosure provides a method of treating

Molybdenum Cofactor Deficiency (MoCD) type A in a human patient in need thereof, the method comprising: a) determining the patient has encephalopathy or is at risk for encephalopathy, b) optionally conducting at least one test to diagnose the human patient as having MoCD; c) administering to the human a pharmaceutically effective amount of cPMP, or of a pharmaceutically acceptable salt thereof, at least once per day, wherein the pharmaceutically effective amount is a dosage of from about 120 ₍ u,g/kg to about 320 μίί/kg body weight per day, or is a dosage from about about 525 to about 1300 μξ/kg body weight per day, and d) conducting at least one test to diagnose the human patient as having MoCD type A, wherein if the human does not have MoCD type A, the administering ceases.

[0045] In certain embodiments, the administering is started prior to detection of cerebral lesions in the patient. In certain embodiments, the administering is started prior to significant cerebral encephalopathy in the patient. In certain embodiments, the dosage is from about 240 ^ig/kg to about 320 μg/kg body weight per day. In certain embodiments, the administering is started prior to significant cerebral encephalopathy in the patient, and the dosage is from about 80 μg/kg to about 3000 μg/kg body weight per day. In certain embodiments, the administering is started prior to significant cerebral encephalopathy in the patient, and the dosage is from about 525 μg/kg to about 1300 μg/kg body weight per day. The cPMP can be, for example, recombinant or synthetic, and may be any pharmaceutically acceptable said. The route of administration can be any route, for example, is at least one of intravenous (including intravenous infusion), oral,

subcutaneous, intramuscular, and peritoneal route. In certain embodiments, the route of administration is intravenous or oral. The patient can be any patient, including a neonate, and can be human.

[0046] The at least one test can be a sulfite dipstick test, a brain scan, a urine test, a sulfite test, an SSC test, a xanthine test, or a urate test, in plasma or in urine. In certain embodiments, the at least one test to diagnose the patient as having MoCD type A identifies at least one mutation on two alleles at a locus for MOCS1. Also, the at least one mutation can be identified in the patient, or can be identified in at least one parent or both parents of the patient, in certain embodiments, at least one of sulfite metabolite, urine S-sulfocysteine, thiosulfate, and xanthine level decreases in the human patient. In certain embodiments, the administering is started when a MoCD is suspected but not yet confirmed. In certain embodiments, the administering is started after a strongly positive sulfite dipstick test from a fresh urine sample of a symptomatic newborn baby.

[0047] In certain embodiments, the patient does not have encephalopathy in the brain at the start of administration, and does not have encephalopathy in the brain at about two years or more (e.g., about two to about four years) into cPMP treatment. The lack of irreversible or cystic lesions in the brain can be determined by, for example, a brain scan, such as MRI.

[0048] In some embodiments, the disease or disorder is not MoCD type B. As used herein, the term "patient" or "human patient" refers to a human. A patient who has or is at risk for having MoCD, specifically type A, may be identified by having parents of at least one already affected sibling, having at least one affected immediate family member (cousins, etc.), previously diagnosed by genetic or phenotypic testing.

[0049] In some embodiments, at least one or both parents are identified as heterozygote carriers of at least one mutation at the locus on chromosome 6 for MOCS1. A patient may have at least one mutation at the locus for MOCS1, e.g. including but not limited to mutations as identified in Reiss and Hahnewald, Human Mutation, 32(1): 10-18 (2011) and/or as identified in Leimkuhler et al., Human Genetics, 117(6): 565-570 (2005).

[0050] In addition, a patient may be identified by early signs of encephalopathy, blood levels of elevated sulfite, brain scans, urine tests (including but not limited to SSC, xanthine, and/or urate concentrations), and urine sulfite dipstick tests (i.e., a strongly positive sulfite dipstick test from a fresh postnatal urine sample). A patient may be identified by genetic analyses in utero or following birth (e.g., immediately, one hour, one day, two days, three days, four days, five days, six days, one week, etc.). In some embodiments, the testing (whether phenotypic or genetic) is performed immediately following birth. A patient may be identified by jerkiness, twitching, poor sucking, altered consciousness, and/or recurrent seizures following birth. [0051] In particular embodiments, the patient is a neonate. As used herein the term "neonate" refers to a newborn human child that was born (whether by vaginal or by caesarean delivery) less than about 4 weeks ago. In some embodiments, the neonate is from about zero days (birth date) to about 4 weeks old, from about two days to about 4 weeks old, from about three days to about 4 weeks old, from about four days to about 4 weeks old, from about five days to about 4 weeks old, from about six days to about 4 weeks old, from about 1 week to about 4 weeks old, from about 2 weeks to about 4 weeks old, or from about 3 weeks to about 4 weeks old. In some embodiments, the neonate is from about 1 day to about 1 week old, from about 2 days to about I week old, from about 3 days to about 1 week old, from about 4 days to about 1 week old, from about 5 days to about 1 week old, or from about 6 days to about one week old. In some embodiments, the neonate is less than about 1 week old. In some embodiments, the patient is treated on day zero, i.e., on the same day as the patient's birth.

[0052] In some embodiments, provided herein is a method for treating a patient diagnosed with MoCD (e.g., MoCD type A), comprising administering to the patient a pharmaceutically effective amount of cPMP, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a method for treating a patient presenting one or more symptoms of MoCD type A or a patient having an elevated risk of developing symptoms of MoCD type A, and administering a pharmaceutically effective amount of cPMP, or a pharmaceutically acceptable salt thereof to a subject determined to have MoCD. Such methods can include performing an assay (e.g., blood, urine, or cerebral scans) on the patient or on a sample provided from the patient, and identifying one or more symptoms of MoCD and/or diagnosing the patient as having MoCD,

[0053] Indicators of MoCD can include one or more of the gene or protein sequence variants (see, e.g., Table 2 in Reiss, J. and Hahnewald, R. Human Mutation 32(1): 10-18 (2011)).

[0054] In some embodiments, the parents of a patient are tested for the presence of one or more gene or protein sequence variants associated with MoCD, particularly MoCD type A. In some embodiments, the patient is tested for one or more gene or protein sequence variants associated with MoCD, particularly MoCD type A. The patient can be tested, for example, in utero, before birth, or following birth. In some embodiments, the patient is tested less than one hour following birth, less than two hours following birth, less than six hours following birth, less than 12 hours foliowing birth, less than 24 hours following birth, less than 3 days following birth, less than 5 days following birth, less than 10 days following birth, less than 20 days following birth, or less than 30 days following birth. In some embodiments, testing is performed foliowing birth by genetic testing of DNA extracted from blood or fibroblasts or other DNA sampling sources. In some

embodiments, testing is performed prior to birth, via in utero amniotic sampling and/or chorionic villi sampling.

[0055] MoCD can also be diagnosed using phenotypic symptoms or indicators. For example, blood tests, urine tests, and/or cerebral scans can be performed to identify one or more abnormalities associated with MoCD in the patient. S-sulfocysteine (SSC) is a non-enzymatic reaction product of cysteine and sulfite which is highly abundant in MoCD patient urine. Increased blood or urine levels of sulfite, S-sulfocysteine (SSC), and xanthine can be associated with MoCD in a patient.

[0056] Measurement of blood or urine levels of sulfite, SSC, and xanthine can be performed by direct dip-stick methods (e.g., sulfite) or HPLC methods as needed.

Neurological examinations measuring consciousness, quality of movements, feeding patterns, and seizure activity can be useful in diagnosing a patient with MoCD.

Electroencephalograms (EEGs) (measuring rhythmic elements and/or epileptiform discharges), brain imaging, and developmental testing using known procedures can also be used to determine whether encephalopathy or cerebral lesions are present in the patient.

[0057] These methods can also be used to determine any changes to the status of the patient over time (e.g., increase in cerebral lesions, decrease in abnormal encephalopathy results). Phenotypic symptoms and indicators may include, for example, improvement according to cognitive and/or motor scales from one or more accepted developmental scales, including Bayley Scale of Infant Development (BSID-III or Bayley-ΠΙ), improvement of quality of life (QoL), improvement in MRI severity (particularly cranial imaging), developmentally appropriate milestone attainment, appropriate increase in head circumference, or increase in survival rate. Successful treatment outcomes include but are not limited to at least one of improvement and/or lack of decline according to cognitive and/or motor scales from one or more accepted developmental scales, including Bayley Scale of Infant Development (BSID-III or Bayley-III), improvement of quality of life (QoL), improvement in MRI severity (particularly cranial imaging), developmental ly appropriate milestone attainment, appropriate increase in head circumference and growth, lack of brain necrosis, lack of brain cystic lesions on MRI imaging, lack of or decrease of seizures, decrease of dosing of anticonvulsive drugs, increase in survival rate, and improvement of life expectancy. In certain embodiments, the patient is able to at least one of feed orally at or before about three weeks of age; breathe unassisted by about one week of age; and/ or sit up unassisted at about 12 months of age. The embodiments include where the patient is able to do one, two, or all three of these tasks.

[0058] In certain embodiments, a therapeutically effective amount of cPMP can include an amount (or various amounts in the case of multiple administrations) that improves the patient's chance of survival, increases the patient's survival rate, or increases the patient's life expectancy. In certain embodiments, a disclosed method improves the life expectancy of a patient by any amount of time, including at least one day, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least six months, at least one year, at least 18 months, at least two years, at least 30 months, or at least three years, or the duration of treatment.

[0059] A patient may also be identified based on familial history (e.g., a sibling or close relative having MoCD or previously having tested positive for a MoCD type A mutation). Additional assays, non-limiting assays, and phenotypic symptoms that may be used in these methods are described herein. Additional assays are also known in the art.

[0060] As used herein, the term "treating" or "treatment" refers to one or more of (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or

symptomatology of the disease, conditi on or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease.

[0061] In some embodiments, the method comprises administering to the patient a therapeutically effective amount of cPMP, or a pharmaceutically acceptable salt thereof. As used herein, the expressions "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, or patient by a researcher, veterinarian, medical doctor or other clinician. In particular, therapeutically effective amounts of cPMP will elicit improvements in the phenotypic markers of MoCD type A.

[0062] Compounds, Pharmaceutical Compositions and Formulations

[0063] The term "compound" as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified. All compounds, and salts thereof (e.g., pharmaceutically acceptable salts), can be found together with other substances such as water and solvents (e.g., hydrates and solvates).

[0064] The compound provided herein (i .e., cPMP) also includes tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include, for example, ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system. Tautomeric forms can be in equilibrium or stericaliy locked into one form by appropriate substitution. In some cases, cPMP may have the following tautomeric form:

[0065] In some embodiments, cPMP, or a pharmaceutically acceptable salt thereof, is substantially isolated. As used herein, the expression "substantially isolated" refers to a compound that is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%o, at least about 97%>, or at least about 99% by weight of the compound provided herein, or pharmaceutically acceptable salt thereof.

[0066] As used herein, chemical structures that contain one or more stereocenters depicted with dashed and bold bonds {i.e., ^■ are meant to indicate absolute

stereochemistry of the stereocenter(s) present in the chemical structure. As used herein, bonds symbolized by a simple line do not indicate a stereo-preference. Unless otherwise indicated to the contrary, chemical structures, which include one or more stereocenters, illustrated herein without indicating absolute or relative stereochemistry encompass all possible stereoisomenc forms of the compound {e.g., diastereomers and enantiomers) and mixtures thereof Structures with a single bold or dashed line, and at least one additional simple line, encompass a single enantiomeric series of all possible diastereomers.

[0067] When employed as a pharmaceutical, cPMP can be administered in the form of a pharmaceutical composition. As used herein, the expression "pharmaceutical

composition" comprises at least one compound, i.e., cPMP, or a pharmaceutically acceptable salt thereof, as a single compound or in a mixture, in combination with at least one excipient. These compositions can be prepared as described herein or elsewhere, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be oral or parenteral. In some embodiments, administration is oral. In some embodiments, administration is parenteral.

[0068] As used herein, the expression "parenteral admini stration" includes intravenous, intraarterial, subcutaneous, peritoneal, intramuscular, or injection or infusion; or intracranial (e.g., intrathecal or intraventricular administration). Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. In some embodiments, administration is selected from the group consisting of intravenous, oral, subcutaneous, intramuscular, and peritoneal

administration. Conventional pharmaceutical excipients such as carriers, aqueous, powder or oily bases, thickeners, and the like, may be necessary or desirable.

[0069] Some examples of suitable excipients include, without limitation, lactose, dextrose, sucrose, sorbitol, raannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and raethyi cellulose. The formulations can additionally include, without limitation, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; flavoring agents, or combinations thereof. In some embodiments, the suitable excipient comprises at least one

pharmaceutically acceptable acid, including but not limited to ascorbic acid, acetic acid, and citric acid, sodium salts including Na ₂ HP0 ₄ -H ₂ 0, and hydroxide compounds including NaOH.

[0070] The active compound can be effective over a wide dosage range and is generally administered in a therapeutically effective amount. In some embodiments, the dosage of the compound, cPMP, or a pharmaceutically acceptable salt thereof, administered to a patient or individual is from about 80 μg kg patient body weight to about 320 ,ug/kg patient body weight, for example from about 100 μg/kg or about 120 ,ug/kg to about 320 ^ig/kg, from about 200 ^ig/kg to about 320 ^ig kg, or from about 300 _, ug/kg to about 320 .g/kg patient body weight. In some embodiments, the dosage of the compound, cPMP, or a pharmaceutically acceptable salt thereof, administered to a patient or individual is from about 120 ^ig/kg to about 320 ^ig/kg patient body weight. In some embodiments, the dosage of the compound, cPMP, or a pharmaceutically acceptable salt thereof, is from about 240 g/kg to about 320 ^ig/kg patient body weight.

[0071] In some embodiments, the dosage of the compound, cPMP, or a pharmaceutically acceptable salt thereof, is from about 80 ,ug/kg to about 3000 ^ig/kg patient body weight. In some embodiments, the dosage of the compound, cPMP, or a pharmaceutically acceptable salt thereof, is from about 300 to about 1300 m*/kg patient body weight.

[0072] The administration of the drug can be done by, for example, TV infusion and the dosage can be, for example, once per per day, twice per day, three times per day, or more often. In some embodiments, the administration is by IV infusion twice daily followed by once daily administration.

[0073] EXAMPLES

[0074] General Methods

[0075] Recombinant cPMP (Examples 1-3) was obtained according to the method described in U.S. Patent No. 7,504,095 by Orphatec/Col bourne Pharmaceuticals GmbH. Recombinant cPMP was produced in 24 L expression cultures of E. coli and purified by two-step HPLC chromatography according to previously reported procedures, forming an amorphous solid upon freeze-drying (Santamaria-Araujo et al ., J. Biol Inorg. Chem., 2012, 17, 113-122. Different amounts of rcPMP (10 to 15 mg), purified on a 250 mm x 21 mm 15 μτη 100 A PS-DVB strong anion-exchange HPLC column and eluted in 30 mM citrate buffer pH=3.0, were loaded into a basic spacer 250 mm xlO mm 5 urn 100 A CI 8 reversed phase/anion exchange column equilibrated in 10 mM formic acid at pi I 3 Under those conditions, rcPMP eluted at 4-6 column volumes, which was dependent on the loading volume and the initial rcPMP concentration. Total volume resulting from lOx 250mm x 10 mm 5 μηι 100 A C18 reverse phase/anion exchange purifications yielded 1 157 ml rcPMP solution in 10 mM formic acid at pH=3. UV-Vis absorption spectra was recorded and an A267=0.75 was obtained. The 1157 ml rcPMP-solution was maintained under an argon atmosphere until the entire volume was transferred into two 1L flat- bottomed freeze-drying flasks. [0076] The two flasks were submerged into liquid nitrogen until the rcPMP solution was completely frozen. The two flasks were then transferred into a Christ Alpha 1-4 freeze- dryer. The total solution was freeze-dried for 48 hours till an off-white powder was obtained. The resulting powder was transferred into a 2 ml screw-cap polypropylene tube and washed three times with 1 ml Mi Hi Q water. In between each washing step, the tube was centrifuged at 13,000 rpm for 2 min and the supernatant was discarded. The washing steps were aimed to wash out remaining trace amounts of citric acid. The resulting powder was transferred into the freeze-dryer in order to remove any residual water. After 48 hours, the resulting freeze-dried powder was weighed in an analytical balance, resulting in 52.64 mg of rcPMP. An extinction coefficient at 267 nm at pH=3.0 of 6.000 was obtained based on an A267=0.75 of a 1 157 ml rcPMP-solution which yielded 52.64 mg of rcPMP.

[0077] Recombinant cPMP from different purifications was pooled, stabilized with the addition of a 10-fold excess of ascorbic acid (5 mmol/L), pH-adjusted to 7,2 ± 0.2 with the addition of NaOH and Na ₂ HP0 ₄ , and finally tonicity-adjusted by NaCl, then aliquoted and stored at -80°C, in concentrations ranging from 160 to 205 g/niL of rcPMP (acid form). The concentration in solution was quantitated according to the extinction coefficient of rcPMP and volumes were adjusted to reach the desired dosage. Storage at -80°C was required to maintain stability. The precise salt composition (if any) of the isolated rcPMP was not determined, HPLC-UV-MS and 1H-NMR spectroscopy indicated a purity of rcPMP of greater than 98% with respect to other organic

components. Endotoxin and microbiological assays providing acceptable results were performed by Chemical Analysis (Melbourne, Australia) or at EML (Surry Hills, Sydney, Australia).

[0078] Sulfite was semi -quantified at the bedside in fresh urine using a colorimetric dipstick test (Merckoquant Sulfite Test, Merck KGaA, Darmstadt, Germany). Urinary cPMP concentrations were determined by HPLC analysis. Urinary SSC, thiosulfate, taurine, urate and xanthine for patient # 1 were determined by direct injection electrospray tandem mass spectrometry. For all other patients, SSC was quantified by HPLC analysis after pre-column derivatization. Xanthine and uric acid were separated on a 250 x 3 mm Nucleodur-100 C18 5 μιη column (Machery-Nagel, Diiren, Germany), with an isocratic elution at 30°C and J rnL/min in a 10 mM KH ₂ P0 ₄ buffer adjusted to pH ^:=: 3.0 with H ₃ PO ₄ and measured by HPLC-DAD (HPLC with diode-array detection) analysis (thiosulfate and taurine were not measured). Urine samples were diluted in water, 100- fold for samples having creatinine concentrations of 0.1 - 5 mmol L, or 200-fold for those above 5 mmol/L.

[0079] Molecular genetic studies were performed at the University Institutes for Human Genetics of either Gottingen or Cologne. DNA was extracted from blood, fibroblasts or chorionic villi, according to standard procedures. Individual exons were amplified and sequenced as previously described (see Reiss et al ., Hum. Genet 1998, 103, 639-644 and Reiss et al., Am. J. Hum. Genet. 1999, 64, 706-71 1).

[0080] Patients

[0081] Patients were newly presented neonates and young infants with confirmed MoCD in whom treating physicians requested treatment with rcPMP on compassionate grounds between 2008 and 2013 (FIG. 2). Each medical team sought approval from the respective local ethics committee, according to national guidelines, and obtained consent from parents.

[0082] MoCD was suspected based on either symptoms or previous index cases in the family and initially diagnosed by demonstrating increased concentrations of sulfite, S- sulfocysteine (SSC), and xanthine, and decreased urate, as well as absence of urothione (the catabolic excretion product of MoCo) in body fluids. Every effort was made to start cPMP substitution as early as possible. Confirmatory diagnosis of MoCD type A or type B was confirmed through measurement of Compound Z (the cPMP oxidation product) concentration in pre-treatment urine, and by sequencing the MOCS1 and MOCS2 genes.

[0083] Sixteen infants diagnosed with MoCD were started on rcPMP substitution in the UK (6), Australia (3), Germany (3), the Netherlands (2), and the USA (2). The age at onset of treatment was day zero (0, day of birth) to 68 days (median 7 days)(Examples 1- 3). Three patients were siblings of children previously diagnosed with MoCD type A. Four neonates were started on rcPMP prior to subtyping. Three additional children previously diagnosed with oCD type B were also treated despite a low probability of response, in all five neonates ultimately identified as having MoCD type B, rcPMP treatment was ineffective, and rcPMP administration was subsequently discontinued. An additional patient was identified prenatally and treated as described in Example 4.

[0084] Example 1. rcPMP Intravenous Infusions

[0085] Recombinant cPMP infusions typically started at a daily dose of 80 ₍ u,g/kg body weight in the first 11 patients (#1-7 and #12-15). This dose was extrapolated from a dose that most effectively abolished symptoms and lethality in a mouse model (Schwarz et al., Hum. Mol Gen. 2004, 13. 1249-1255) and was adjusted and administered using a standardised treatment protocol derived from successful treatment of the initial case (Veldman et al., Pediatrics, 2010, 125, el249-e!254). In patients #1 and #2, prior to use, rcPMP aqueous stock solution (dissolved in 30 mmol/L citrate buffer, pH 3.0) was neutralized by NaOH and phosphate-buffered saline. In all subsequent patients, frozen pre-formulated and neutralized aqueous rcPMP solution was used as described herein. Frozen aqueous solutions were thawed and intravenously administered through a 0,22 um filter line.

[0086] Patients #1-7 and #12-15 received the following administration of cPMP: On administration day 1, three test doses of 10% of the total daily dose were administered over 30 min, each followed by a 30 min observation period. Next, the remaining 70% of the first dose was infused over 3 h. On subsequent administration days 2-12, the total daily dose was infused over 1 h. After 12 days, the daily dose was increased to 120 g kg, increased to 160 u u kg after 35 days, and finally increased to 240 g/kg per day after 75 days of treatment.

[0087] Five additional patients diagnosed after August 2011 (#8-1 1 and #16) were treated according to a modified protocol, starting with a total daily dose of 240 μg/kg rcPMP. On days I and 2, a test dose of 24 g/kg rcPMP was administered over 30 min, followed by a 30 min observation period after which the remaining dose was infused over 1 h. The test dose was omitted and the total dose was administered over I h from days 3- 6 after which the infusion time was reduced to 30 min, and later reduced to 20 min. All patients were maintained on a daily dose of 240 ^ig/kg throughout their treatment, except for patient #1 who was transiently treated with up to 320 ^ig/kg. [0088] Example 2. Treatment Monitoring

[0089] Patient safety was evaluated clinically and biochemically. Vital signs and blood gases were carefully monitored during and after infusions during the first five weeks. Blood samples were obtained at frequent intervals (initially daily), to monitor full blood count, lactate dehydrogenase, glucose, electrolytes, calcium and magnesium, liver and renal function. Urine samples were also analyzed regularly. Internal organs were routinely assessed by ECG, echocardiography, and abdominal ultrasound imaging.

Adverse events were documented and reviewed for any potential relationship to rcPMP, according to International Conference on Harmonization Good Clinical Practice guidelines.

[0090] Biochemical Efficacy

[0091] Biochemical efficacy was determined using urinary SSC as a marker for sulfite oxidase activity and urinary xanthine and urate for xanthine oxidoreductase activity. During the first 90 days of treatment, samples were collected daily to determine treatment response and to inform dose adjustment. Samples were then collected every week or every two weeks.

[0092] Clinical Efficacy

[0093] Clinical efficacy was assessed daily for the first two weeks, at least weekly during the first three months, and regularly thereafter by clinical and neurological examination, which recorded consciousness, quality of movements, feeding pattern, and seizure activity. A scoring system ranging from 0 (asymptomatic) to 12 (severe encephalopathy) was developed to quantify clinical symptoms. The total score (obtained by combining scores from 0-2 for each tested indication) ranges from 0 (asymptomatic) to 12 (severely compromised) according to FIG. 3. Electroencephalography, brain imaging, and developmental testing were done as clinically indicated.

[0094] Scores were obtained immediately prior to treatment, after 6 months (where applicable), and at the latest available evaluation. Pati ent #8 was scored at 7 days of age, prior to ECMO and haemodialysis for sulfite detoxification; rcPMP treatment was started 4 days later. Patient #1 1 had rcPMP treatment discontinued after 5 days, and patient surviving. EEG, brain imaging, and developmental testing were performed as clinically indicated. Results are shown in FIG. 7,

[0095] Safety

[0096] All 16 patients tolerated daily intravenous rcPMP infusions well. No serious adverse drug-related reactions were observed over within the 6000 daily infusions administered over periods of up to five years. As previously described, (Hitzert et al., Pediatrics, 2012, 130, el 005-e 1.010) one preterm neonate (#5) experienced a transient decrease in blood pressure during initial rcPMP infusions, all other patients showed no changes in vital signs during or after the infusions. Most patients developed transient iatrogenic anemia due to initial extensive laboratory monitoring but required no blood transfusion. Biochemical and hematological laboratory parameters remained otherwise normal.

[0097] Clinical adverse events were mostly related to intercurrent diseases such as airway infections. Seven of the eight patients on long-term rcPMP treatment suffered from at least one episode of pneumonia, and two patients (#4, #5) required short-term mechanical ventilation. Two patients temporarily suffered from mild asthma (#3, #4), and three from infantile eczema (#2, #3, #4). One patient (#6) was diagnosed with oral leukoplakia and mild left ventricular myocardial hypertrophy two weeks into treatment, unlikely related to rcPMP and not requiring intervention. Patient #3 was diagnosed with asymptomatic cholelithiasis during the third year. All patients on long-term treatment had permanent central venous access surgically inserted, and experienced one or more serious line-related complications such as site infection, septicaemia, or rupture.

[0098] Example 3, Clinical Outcomes

[0099] Infants with MoCD type A showed a dramatic decline in urinary concentrations of SSC (FIG, 4A) and thiosulfate (patient #\) within two days of rcPMP substitution.

Sulfite dipsticks urine tests became negative between day 2 and 4 of treatment and remained negative throughout continued rcPMP substitution. Xanthine normalized within approximately one or two weeks of treatment (FIG. 4B). Urinary urate was more variable and remained slightly below or in the low normal range (FIG. 4C). Following the initial decline, urinary SSC concentrations varied considerabl between individuals during the first 90 days (range 0 - 80 mmol/mol creatinine) with no correlation to clinical symptoms (FIG. 4D), Long-term SSC and purine concentrations remained persistently in the normal range for up to five years, apart from a temporary unexplained increase in SSC in patients #3 and #4 between 18 and 24 months of age (FIG. 4E and 4F). None of the patients with MoCD type B showed any changes in these biomarkers.

[00100] Clinical outcomes for all patients under treatment are presented with an overview of the study profile, shown in FIG. 5, and in FIG. 2 and FIG. 7, Clinical deterioration was halted upon starting rcPMP treatment in eight patients with MoCD type A, and symptoms rapidly improved within approximately 1 - 2 days from beginning treatment. Patients became more alert and responsive, were less distressed, experienced decreased seizure activity, and had improved breathing and oral feeding. Three other patients were found to be already too ill to recover: patient #8 was initially treated with hemodialysis and supported by extracorporeal membrane oxygenation until rcPMP became available. Patient #10 suffered from severe combined immune deficiency.

Patients #8, #10, and #11 showed signs of extensive necrosis in their initial cerebral MRI scan and treatment was therefore withdrawn after 12, 3, and 5 days, respectively, in agreement with their parents.

[00101] Recombinant cPMP (rcPMP) was later discontinued in two of the eight

MoCD type A patients on long-term treatment. The parents of patient #6 decided to abandon treatment after 111 days and the patient was lost to follow-up. Patient #2 exhibited significant cerebral lesions via MRI prior to rcPMP treatment, had been treated late and suffered from severe disability. When the patient became terminally ill from aspiration pneumonia, treatment was reevaluated. Recombinant cPMP was discontinued after 446 days, and patient #2 eventually succumbed to respiratory failure. Both patients #6 and #2 had shown a complete biochemical response and partial clinical improvement regarding seizure control, feeding pattern, and behavior.

[00102] Six of the 11 patients with MoCD type A have been continuously treated with daily intravenous infusions of rcPMP for periods of 289 to 1778 days (about more than 5 years) at their last evaluation. Three patients (#1, #4, and #9) who already showed signs of advanced encephalopathy upon initiation of rcPMP intravenous infusions only partially recovered, and suffer from severe neurodevelopmental disability. They have shown no progress in motor skills, but evolved tetraplegic cerebral palsy, with frequent myoclonic jerks, and with cognitive and visual impairment. They require tube feeding and medication including muscle relaxants and anticonvulsants. Their follow-up cerebral MRI scans demonstrate loss of brain tissue with secondary microcephaly.

[00103] Three patients (#3, #5, and #7) in whom rcPMP treatment was initiated prior to onset of severe encephalopathy were spared from clinically significant disability. These patients exhibited no signs of necrosis in their initial brain MRI scans. Follow-up MRI scans two to four years into rcPMP treatment were normal or demonstrated mild atrophy and hypomyelination, but no irreversible or cystic lesions, as shown in FIG. 6A- 6F, and head growth has been normal throughout subsequent follow-up. The patients acquired motor milestones appropriately and have exhibited nearly normal long-term cognitive development, without sensory deficits or seizures. All three patients were affected with speech delay (persisting in patient #3) and show mild central muscular hypotonia. Using Bayiey's Scales of Infant and Toddler Development (Bayley, 3 edition 2006, Harcourt Assessment, San Antonio, TX) patients #3, #5, and #7 scored in the low- normal range for cognitive function, respectively, at age 26.5 months (patient #3, centile 9), 40.5 months (patient #5, centile 16), and 24.5 months (patient #7, centile 16).

[00104] Recombinant cPMP is well -tolerated and safe in treatment of neonates and infants suffering from MoCD type A and type B. Neither tachyphylaxis nor any serious adverse drag reaction were observed over a period of up to five years. Long-term daily intravenous infusions however may be challenging for patients, their families and health care systems and they may carry a risk of bacteriemia and severe infection. Serious adverse events unsurprisingly arose from the frequent and long-term use of central venous lines. The industrial large-scale synthesis of cPMP may allow the further development of safer and more cost-effective and convenient administration routes.

[00105] Administration of rcPMP led to a rapid and sustained biochemical response and clinical improvement in children with MoCD type A. Even patients with severe pre-existing brain lesions responded favorably with decreased seizure activity, an improved level of consciousness, and clearly reduced irritability. For some patients treatment came too late to prevent significant morbidity or death. Cerebral functional impairment relating to a MoCD severity score above 6 was already associated with poor long-term outcome despite an excellent biochemical response (FIG. 7).

[00106] However, three neonates who were treated prior to manifesting recurrent seizures and decreased consciousness have been spared from epilepsy and significant disability. Of note, ail three had previous siblings diagnosed with severe MoCD. Siblings of Patients #3 and #5 had succumbed to MoCD during infancy while the older sibling of Patient #7 is still alive with severe microcephaly, neurodevelopmental disability, and intractable seizures. This suggests that the favorable outcome was caused by timely treatment and not due to a milder disease and that there is inter-individual variation of disease progression in untreated children. The outcome depends on whether there are already irreversible cerebral lesions present prior to starting rcPMP supplementation, and not merely on postnatal age. The continued absence of signs of cytotoxic edema or necrotic lesions in follow-up MRI images of pre-symptomatically treated patients also indicates that the current intravenous dosing regime is sufficient to maintain the treatment effect for at least five years,

[00107] MoCD can cause prenatal symptoms such as facial dysmorphism and, rarely, intrauterine brain malformations and connatal microcephaly (Carmi-Nawi, N. et al., J Child Neurol. 26:460-64 (2010)). Patient #5 started convulsing within one hour after birth. Only one of the patients was born mildly microcephalic and none was encephalopathic immediately after birth. Their rapid postnatal deterioration supports the notion that fetal sulfite could potentially be cleared through transplacental transport or maternal metabolism in utero (Sie, SD et al., J Inherit Metab Dis 2010 33(suppl 3):401 - 07). The predominantly postnatal onset of acute toxicity in MoCD may leave a small window of opportunity for treatment. While general newborn screening is a well- established process to detect treatable genetic disease at an early presymptomatic stage, this could not be applied to MoCD due to the short interval between birth and

manifestation. Selective screening for MoCD of newborn babies with early signs of encephalopathy, however, is feasible. To use this opportunity effectively, every newborn with early signs of encephalopathy should be immediately investigated for MoCD. It might be justifiable to start treatment with cPMP in suspected cases even before the diagnosis of MoCD type A has been established. In the absence of other readily available rapid diagnostic approaches, a strongly positive sulfite dipstick test from a fresh urine sample of a symptomatic newborn baby can suffice to raise suspicion of MoCD.

[00108] MoCD type A has become a treatable disease. Restoration of molybdenum cofactor-dependent enzyme activities with rcPMP before the onset of significant encephalopathy results in good long-term developmental outcomes. Later treatment alleviates symptoms. The possibility of MoCD type A needs to be urgently explored in every neonate presenting with altered consciousness or recurrent seizures to avoid any delay in rcPMP substitution, and to ensure maximize possible treatment benefit.

[00109] Example 4 Treatment of Neonate

[00110] A patient was identified prior to birth by a combination of prenatal genetic testing and medical history (deceased affected sibling) as having MoCD type A. A predelivery brain MRI and ultrasound (33 weeks, 5 days) showed very minor abnormalities, for example, mild dysmorphic signs manifested by hypertelorism; very mild hypoplasia of the cerebellar hemispheres, potentially the result of increased retro-cerebellar fluid spaces, mild thickening of the cavum septum peilucidum (CSP), but otherwise, no abnormal findings, and no cysts, cystic lesions, or ischemic or edematous signs.

[00111] Delivery was induced between 36-37 weeks (36 weeks and 3 days) gestation for reasons of safety and to minimize potential subsequent cerebral damage. Birth weight was 2,88kg at delivery. Some evidence of brain edema was found on ultrasound prior to cPMP administration. The dosing schedule was performed according to Example 5.

[00112] Synthetic cPMP was manufactured according to previously published methods, i.e. WO 2012/1 12922. The initial dose of synthetic cPMP was administered via intravenous syringe pump at a rate of 1.5ml/minute approximately 2 hours post delivery (day zero) at a concentration of 525 μο/kg body weight (concentration 0.5mg/ml). The second dose was administered (525 μ§/¾¾) about 21 hours after the initial dose. During the first 24 hours of life, the patient experienced episodes of apnea that were possibly due to seizure activity and required intubation. Feeding was supplemented via nasogastric tube (10-20cc each feeding) in addition to oral feedings because oral feeding was insufficient.

[00113] On day 3, the dose of cPMP was increased, and the third dose of 700 μξ/ g body weight was administered in two aliquots of 350ug/kg body weight delivered 12 hours apart, and repeated for three days (days 3, 4, and 5).

[00114] After three days of twice daily administration, once daily administration was continued at 700 g/kg until approximately day 28.

[00115] The breathing tube was able to be removed on day 2, and no seizures were observed after the first day.

[00116] After one week, the patient was stable, as measured by both hemodynamic parameters and by respiratory function. Neurological state showed ongoing and persistent improvement; including good eye contact, muscle tone, and spontaneous movements as well as primitive reflexes. No apnea episodes were observed, oxygen saturation was normal, and respiratory support was no longer needed. Clinical observations improved, such as weight gain, eye contact and following, improved muscle tone, oral feeding, good sucking ability, ability to breathe unassisted except for supplemental oxygen, etc., and observations were normal for the patient's adjusted age. Nasogastric feeding became unnecessaiv as the patient completed multiple oral feedings of 50cc without assistance. Patient was able to be discharged from the neonatal intensive care unit after 28 days.

[00117] After three weeks, the patient was receiving cPMP once daily, every morning around 11 :00am at 700^ig/kg body weight per day. The patient's brain ultrasound at three weeks showed further improvement, Sulcation appeared fine, brain parenchyma seemed normal with only mild lateral ventricular enlargement (bilateral). No adverse events were reported, and no seizures and no apnea were reported. The patient had been feeding orally without assistance for more than 10 days and the sucking was very good. The patient was gaining weight appropriately, and formula amounts were increasing appropriately. The neurological examination seemed normal for patient's age, and there was improvement in muscle tone (previously mild hypotonia) and in eye contact and following of objects (age appropriate). The patient's physical and

neurological examinations were normal for patient's age.

[00118] On approximately day 28, the daily dosing was increased to 800 g/kg administered intravenously once daily.

[00119] The patient's Bayley-III and GMFM examinations at 28 days showed normal responses of a typically developing newborn. Central hypotonicity was not observed; patient was able to hold head upright for 15 seconds in a supported carry, was able to lift and turn head in prone position to clear airway, showed anti gravity arm and leg movement, was able to flex hip and knee off of surface through partial range of movement, and was able move head to midline in a supine position, hi contrast, a newborn with neurological impairment would potentially have reduced head control, dominance of head to one side, leg kicking that favored extension, and an inability to lift and turn its head in a prone position.

[00120] At three months of age, the daily dosing of cPMP will be increased to about 1000 μg/kg; at six months the daily dosing will be increased to about 1200 ,ug/kg, and at nine months the daily dosing will be increased to about 1300 ^ig/kg and maintained at this dose.

[00121] An MRI will be performed regularly and will show no cystic lesions and no cerebral encephalopathy.

[00122] At about one year or earlier (perhaps as early as about six months), patient will be able to sit up unassisted (without external support) for at least approximately 60 seconds.

[00123] Example 5 Dosing Scheme for Neonate Treatments

[00124] Previous data in juvenile knockout mice had suggested that plasma SSC levels and liver sulfite oxidase levels required different doses of cPMP to achieve optimal responses. Therefore, higher exposures than those expected to reduce plasma SSC levels were proposed to achieve optimal clinical outcomes in neonates. Additionally, variable renal clearance would be a function of maturity of the neonatal kidney and would change with age and weight. [00125] A standard 2-compartment PPK model was developed based on clinical data from healthy adults and three of the previously treated MoCD type A patients from the previous Examples (fit to the data using NONMEM 7.2). Parameters are given in Table 1.

Table 1: Population PK Mode! Parameters

[00126] In Table 1, additive and proportional residual error were estimated to be 1.5 ng/mL and 19.2% respectively. *PK parameters were normalized to a 70-kg healthy adult subject **Between-subject variability terms were expressed as the square root of the variance of normal random variables x 100%, e.g. CLi below. The index i refers to subject. Post-menstrual age (PMA) or gestational age ^;=: time in weeks from the last menstrual period until delivery; CLi = (CL in 70 kg) (WTi/70 kg)01 ^χ (ΡΜΑϊγ / (ΡΜΑίγ + Τ50γ) x exp(nCLi); Vci = (Vc in 70 kg) (WTi/70 kg) )2 < exp(r}Vi)

Qi = (Q in 70 kg) χ (WTi/70 kg)01 ; Vpi = (Vp in 70 kg) χ (WTi/70 kg)02.

[00127] Allometric PK scaling equations were derived (Holford, N., et al., J.

Pharm. Sci. 102, 2941 -2952 (2013)), while changes in clearance due to renal maturation in early age used a saturable function with parameter values. The renal maturation factor was calculated by inserting gestational age into the Rhodin equation (Rhodin, M.M., et al., Pediatr. Nephrol. 24: 67-76 (2009)). Age-specific PK parameters were calculated using estimated population parameters (available from, for example, the World Health Organization and/or the Centers for Disease Control), the renal maturation factor, and body weight. Then AUC was calculated for doses ranging from 100 to 2,000 _, ug/kg in increments of 100 ^ig/kg. The dose achieving an AUC matching the NOAEL (no- adverse-effect-level) (5,490 ng*h/mL) was determined using linear interpolation between the available doses. Table 2 illustrates the dosing nomogram for neonates bom at 34 weeks gestational age to achieve NOAEL and Table 3 illustrates the dosing nomogram for neonates born at 40 week gestational age (full term) to achieve NOAEL.

Table 3: Dosing nomogram for neonates born at 40-week gestational age to achieve NOAEL

7 days 3.53 0.374 0.48 1.03 738

14 days 3.77 0.393 0.52 1.1 764

1 mo 4.33 0.438 0.65 1.26 821

2 mo 5.35 0.517 0.9 1.56 920

3 mo 6.1 1 0.589 1.13 1.78 1010

4 mo 6.71 0.651 1.34 1.96 1090

6 mo 7.62 0.751 1.7 Ζ.Δ5 1220

9 mo 8.56 0.848 2.09 2.5 1340

12 mo 9.3 0.903 2.37 2.72 1400

[00128] Additional neonates will be identified and will be treated according to Table 4, the dosing regimen based on Tables 1 and 2, The proposed dosing scheme is expected to yield mean cPMP exposures (area under the curve, AUC) between 4,000- 5,490 ng*h/rnL. The dosing regimen has been defined for full-term (> 37 weeks gestational age (GA)) and pre-term neonates (<37 weeks GA; Table 3). Figure 8 shows the predicted AUC in both populations over the first year of dosing. The peaks and troughs are minimized to maintain near-maximal exposures relative to the NOAEL by increasing the dose over the first year of life.

[00129] The patients and the treatment success will be evaluated by previously described testing, including but not limited to, Bayley-III, QoL evaluations, cranial imaging, MRI, increase in survival, increase in lifespan, etc, particularly compared to untreated patients with MoCD type A. Patients will be able to sit up unassisted by 12 months, or be able to sit up unassisted by about 6 months.

Table 4. Dosing Schedule for Neonates

[00130] OTHER EMBODIMENTS

[00131] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.