Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
  • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
  • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/001—Acyclic or carbocyclic compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

• substituted amino-thiol and amino- disulfide bioprotective compounds are disclosed herein, pharmaceutical compositions made thereof, and methods thereof including the treatment of any disease or disorder in a subject that can benefit from one or more of the bioprotective effects of the compounds, including but not limited to, binding of cystine, reducing oxidative stress, increasing adiponectin levels and/or increasing brain-derived neurotrophic factors.
• diseases and disorders include but are not limited to, cystinosis, and fatty liver diseases including non alcoholic steatohepatitis (NASH) .
• NASH non alcoholic steatohepatitis
• Cysteamine is an amino thiol with the chemical formula
• cysteamine is derived from coenzyme A degradation, although its plasma concentrations are low. Most experience with cysteamine as a drug originates from the field of the orphan disease cystinosis, in which cysteamine is prescribed to decrease intra lysosomal cystine accumulation. However, over the years, the drug has been used for several other applications both in vitro and in vivo. Cystamine is an organic disulfide that is the oxidized and dimeric form of cysteamine. There are as yet no clinical uses/indications for cystamine.
• Cysteamine is available for clinical use only in the bitartrate salt form (MW 217) and is marketed as prolonged-release (ProcysbiTM) and an immediate release (CystagonTM) form. These formulations cause GI symptoms, halitosis and body odor due to the large amount of systemically circulating free cysteamine which occurs following intestinal absorption. This free circulating cysteamine most likely undergoes metabolism in organs such as the liver to form DMS and other volatile sulfur containing metabolites.
• Cysteamine is an aminothiol agent which has a number of potential therapeutic effects. It is FDA approved for the treatment of the rare metabolic disorder Cystinosis in which lysosomal storage of the amino acid cystine occurs. Although cysteamine (given as bitartrate salt) is effective in cystinosis it is associated with adverse effects such as halitosis, body odor and GI symptoms. These effects contribute to non-compliance to therapy and rapid deterioration of disease state.
• the deuterated compounds of the disclosure are isotopically enriched with deuterium.
• the deuterated compounds of the disclosure have improved properties (e.g., ADME-PK) over the non-deuterated forms of the compounds that arise from the strategic deployment of the deuterium atom(s) .
• ADME-PK improved properties
• These benefits include altered metabolism of the molecule which (1) prolongs the duration of action of the active drug and (2) alters the pathway of metabolism of cysteamine to less active or inactive metabolites (thereby diminishing the production of body odor and halitosis producing noxious and volatile agents such as dimethyl sulfide (DMS) and dimethyl disulfide) .
• the compounds of the disclosure can be used to treat numerous diseases and can be administered orally as immediate- release or enteric-release or sustained-release formulations and also as inhaled/nebulized pulmonary delivery or intranasal delivery or transcutaneous delivery. It is expected that the compounds of the disclosure would have better patient compliance, and improved pharmacokinetics in comparison to currently approved formulations of cysteamine.
• R 1 -R 12 are independently selected from H or D; wherein at least one of R 1 -R 4 is D, and wherein at least one of R 5 -R 12 is D, with the proviso that the compound is not selected from the group
• the compound independently has deuterium enrichment of no less than about 98%.
• the compound has a structural formula selected from the group consisting of:
• the compound has a structure of:
• each position represented as D has deuterium enrichment of no less than about 10%. In yet another embodiment, each position represented as D has deuterium enrichment of no less than about 50%. In a further embodiment, each position represented as D has deuterium enrichment of no less than about 90%. In yet a further embodiment, each position represented as D has deuterium enrichment of no less than about 98%. In a certain embodiment, the compound is a pharmaceutically acceptable bitartrate salt form of the compound.
• the compound in comparison to cysteamine or cystamine exhibits at least one effect selected from the group consisting of: (a) decreased inter-individual variation in plasma levels of said compound or a metabolite thereof as compared to cysteamine or cystamine; (b) increased average plasma levels of said compound per dosage unit thereof as compared to cysteamine or cystamine; (c) decreased average plasma levels of at least one metabolite of said compound per dosage unit thereof as compared to cysteamine or cystamine; (d) increased average plasma levels of at least one metabolite of said compound per dosage unit thereof as compared to cysteamine or cystamine; and (e) an improved clinical effect during the treatment in the subject per dosage unit thereof as compared to cysteamine or cystamine.
• the compound in comparison to cysteamine or cystamine exhibits at least two effects selected from the group consisting of: (a) decreased inter-individual variation in plasma levels of said compound or a metabolite thereof as compared to cysteamine or cystamine; (b) increased average plasma levels of said compound per dosage unit thereof as compared to cysteamine or cystamine; (c) decreased average plasma levels of at least one metabolite of said compound per dosage unit thereof as compared to cysteamine or cystamine; (d) increased average plasma levels of at least one metabolite of said compound per dosage unit thereof as compared to cysteamine or cystamine; and (e) an improved clinical effect during the treatment in the subject per dosage unit thereof as compared to cysteamine or cystamine.
• the compound affects a decreased metabolism of the compound per dosage unit thereof by at least one polymorphically-expressed cytochrome P 450 isoform in a subject, as compared to cysteamine or cystamine.
• the cytochrome P450 isoform is selected from the group consisting of CYP2C8 , CYP2C9 , CYP2C19, and CYP2D6.
• the compound is characterized by decreased inhibition of at least one cytochrome P450 isoform or monoamine oxidase isoform in the subject per dosage unit thereof as compared to cysteamine or cystamine.
• the cytochrome P450 isoform or monoamine oxidase isoform is selected from the group consisting of CYP1A1, CYP1A2, CYP1B1, CYP2A6,
• the diagnostic hepatobiliary function endpoint is selected from the group consisting of alanine aminotransferase (ALT) , serum glutamic- pyruvic transaminase ("SGPT) , aspartate aminotransferase (AST," “SGOT) , ALT/AST ratios, serum aldolase, alkaline phosphatase (ALP), ammonia levels, bilirubin, gamma-glutamyl transpeptidase ("GGTP "Y- GTP “GGT) , leucine aminopeptidase (“LAP) , liver biopsy, liver ultrasonography, liver nuclear Scan, 5 ' -nucleotidase , and blood protein .
• alanine aminotransferase ase
• SGPT serum glutamic- pyruvic transaminase
• AST aspartate aminotransferase
• ALT/AST ratios ALT/AST ratios
• the disclosure also provides for a pharmaceutical composition
• a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable carrier, diluent, and/or binder.
• the pharmaceutical composition is formulated for oral delivery.
• the pharmaceutical composition is formulated for oral delivery.
• composition is in the form of granules, tablet, capsule, or caplet. In yet another embodiment, the pharmaceutical composition is formulated for delayed release. In a further embodiment, the pharmaceutical composition comprises an enteric coating. In yet a further embodiment, the pharmaceutical
• composition comprises colloidal silicon dioxide, croscarmellose sodium, D&C yellow no. 10 aluminum lake, FD&C blue no. 1 aluminum lake, FD&C blue no. 2 aluminum lake, FD&C red no. 40 aluminum lake, gelatin, magnesium stearate, microcrystalline cellulose,
• the pharmaceutical glaze pregelatinized starch, silicon dioxide, sodium lauryl sulfate, synthetic black iron oxide and/or titanium dioxide.
• the pharmaceutical glaze pregelatinized starch, silicon dioxide, sodium lauryl sulfate, synthetic black iron oxide and/or titanium dioxide.
• the pharmaceutical glaze pregelatinized starch, silicon dioxide, sodium lauryl sulfate, synthetic black iron oxide and/or titanium dioxide.
• the pharmaceutical glaze pregelatinized starch, silicon dioxide, sodium lauryl sulfate, synthetic black iron oxide and/or titanium dioxide.
• the pharmaceutical glaze pregelatinized starch, silicon dioxide, sodium lauryl sulfate, synthetic black iron oxide and/or titanium dioxide.
• composition comprises microcrystalline cellulose, Eudragit® L 30 D- 55, Hypromellose , talc, triethyl citrate, sodium lauryl sulfate, purified water, gelatin, titanium dioxide, blue ink and/or white ink.
• the pharmaceutical composition comprises: (i) a core particle comprising a mixture of the compound of claim 12 and a binder, and (ii) an enteric membrane surrounding the core particle; wherein the particles have a distribution of particle sizes in a range of about 0.7 mm to about 2.8 mm; wherein the enteric membrane begins to dissolve within a pH range of about 4.5 to about 6.5; wherein the enteric membrane is present in an amount in a range of about 25% to about 35% by weight, based on the weight of the core particles.
• the pharmaceutical composition comprises: (i) a core tablet comprising a mixture of the compound of claim 12 and a binder, and (ii) an enteric membrane surrounding the tablet, wherein the thickness of the enteric coating increases from 60 pm to 130 pm relative to the compound's base dose from 50 mg to 300 mg, and/or wherein the enteric coating is present in an amount in a range of about 9 to about 15% by weight of the core tablet.
• the disclosure provides a method of treating a subject suffering from a disease or disorder in need of treatment thereof, comprising administering to the subject a therapeutically effective amount of a compound having the structure of Formula I or Formula II:
• R 1 -R 12 are independently selected from H or D; wherein at least one of R 1 -R 4 is D, and wherein at least one of R 5 -R 12 is D. In another embodiment, at least one of R 1 -R 12 of the compound
• At least one of R 1 -R 12 of the compound independently has deuterium enrichment of no less than about 90%.
• At least one of R 1 -R 12 of the compound independently has deuterium enrichment of no less than about 98%.
• the compound has a structural formula selected from the group consisting of:
• the compound has a structure selected from:
• each position represented as D has deuterium enrichment of no less than about 10%. In yet another embodiment, each position represented as D has deuterium enrichment of no less than about 50%. In a further embodiment, each position represented as D has deuterium enrichment of no less than about 90%. In yet a further embodiment, each position represented as D has deuterium enrichment of no less than about 98%.
• the compound is a pharmaceutically acceptable bitartrate salt form of the compound. In another embodiment, the subject suffers from cystinosis. In an alternate embodiment, the disease or disorder is a fatty liver disease.
• the fatty liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD) , non-alcoholic steatohepatitis (NASH) , fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from NASH.
• NAFLD non-alcoholic fatty liver disease
• NASH non-alcoholic steatohepatitis
• fatty liver disease resulting from hepatitis fatty liver disease resulting from obesity
• fatty liver disease resulting from NASH non-alcoholic steatohepatitis
• a method of the disclosure further comprises measuring one or more markers of liver function selected from the group consisting of alanine aminotransferase (ALT) , alkaline phosphatase (ALP) , aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and triglycerides.
• ALT alanine aminotransferase
• ALP alkaline phosphatase
• AST aspartate aminotransferase
• GTT gamma-glutamyl transpeptidase
• triglycerides an ALT level of about 60-150 units/liter is indicative of fatty liver disease.
• an ALP level of about 150-250 units/liter is indicative of fatty liver disease.
• an AST level of about 40-100 units/liter is indicative of fatty liver disease.
• a GGT level of 50-100 units/liter is indicative of fatty liver disease.
• a triglyceride level above 150 mg/dL and/or high LDL level is indicative of fatty liver disease.
• the disclosure also provides a method of reducing fibrosis or fat content or fat accumulation in a liver of a subject associated with non-alcoholic fatty liver disease (NAFLD)
• Figure 1 demonstrates that 50 mM, 100 mM, and 200 mM cystamine is effective in reducing intracellular cystine levels in a dose-response fashion. Cysteamine 200 mM is effective and more rapid in its response time than 50 uM cystamine and 100 uM cystamine in particular.
• Figure 2A-B shows that the conversion of intracellular cystine to mixed disulfide (MD) of cysteine-cysteamine is delayed when cystinotic fibroblasts are cultured in the presence of (A) cystamine (50, 100, and 200 mM) compared with (B) cysteamine (200 mM) . This is most likely due to the delay in intracellular reduction of cystamine and hence the availability of cysteamine (active form) .
• MD mixed disulfide
• Figure 3 shows intracellular re-accumulation of cystine over time.
• the figure shows the rise of intracellular cystine levels after the cystinotic fibroblast cell plates were washed following a 3 h incubation with 100 mM cystamine. The washed cells were then incubated in cystamine-free media for 10, 30, 60, 180 mins and 17 h. Cystine levels were extracted from 2 cystinotic fibroblast plates that were not exposed to cystamine and their cystine levels were identical to the 17 h cystine measurement. It is unclear exactly when the plateau was first achieved.
• Figure 4A-D shows intracellular levels of (A)
• Figure 5A-C compares cystine depletion (measuring cysteamine in the form of NEM-cysteamine , and also cystamine) in a time course of drug exposure in cystinotic fibroblasts, using either a 100 uM cystamine or 100 uM d 2 -cystamine exposure.
• A-C The rate of intracellular cystine depletion was slower for 100 uM d 2 -cystamine compared with 100 uM cystamine. This most likely correlates with the rate/degree of reduction of cystamine to NEM- cysteamine which seems faster/higher with cystamine versus d 2 - cystamine especially in the first 10-15 minutes. This erratic and high level of cysteamine may correlate with greater production of noxious and volatile metabolites.
• Figure 6 presents the results of a cystine depletion study with cystamine, d 2 -cystamine, and d 4 -cystamine in dermal fibroblasts isolated from a cystinotic patient "cystinotic fibroblasts". As shown, the difference in depletion rates is nearly identical between cystamine and d 4 --cystamine . d 2 -Cystamine showed slightly slower kinetics.
• Figure 7A-B presents the results of cystine re- accumulation in washed cystinotic fibroblasts that were previously administered cystamine, d2-cystamine , and d4-cystamine .
• One phase exponential association curve was fit to each set of data points (note: Tau, shown in the tables represents the inverse of K) .
• Cystine re-accumulation between cystamine and d 4 -cystamine are very similar (A) . Though d 2 -cystamine appears slightly faster (B) , this could be due to this latter experiment being performed on a different day than the other 2, and using more confluent cells. In both cases, however, the cystine levels took 10-17 h to
• Figure 8A-B shows studies conducted with NEM-cysteamine and NEM-cysteine during constant incubation.
• NEM-cysteamine or NEM-di-cysteamine or NEM-d 2 -cysteamine ) was measured for each respective plate and normalized against the signal for c 4 -cystine.
• Higher levels of cysteamine are again seen (e.g., see FIG. 5B above) at early timepoints following cystamine compared with either of the deuterated molecules and this may result more "controlled" intracellular cysteamine availability and less “free cysteamine” for conversion to noxious metabolites.
• Figure 9 shows the levels of NEM-cysteine after a 3 h incubation with drug and then a washout. As shown, the NEM- cysteine levels all increase after the washout period for
• cystamine d 2 -cystamine and d 2 -cystamine .
• Figure 10 shows the levels of cystamine and isotopically enriched cystamine in cystinotic fibroblasts during constant drug incubation. Cystamine levels deplete rapidly during constant incubation. At near saturation levels, a plateau is observed.
• n 8 per group
• ALT levels at 8 weeks There was a statistically significant difference for ALT levels at 8 weeks between the control group (gl) and the D2 (g2) therapy but not between the control group and D4 (g3) group.
• D2 and D4 ALT levels at the 8 weeks treatment p ⁇ 0.01) .
• Figures 12A-B show a statistical analysis of the group 1 control mice (gl) and either (A) group 2 receiving D2-cystamine (g2) or (B) group 3 mice receiving D4-cystamine (g3) ALT levels.
• A Shows the ratio of Body Weight
• BW Liver Weight
• LW Liver Weight
• Figure 14 shows results of a cystine depletion study of
• Figure 15 shows a cystine reaccumulation curve using D4n and D8 cystamine.
• Figure 16 shows the results of CDllb staining of liver tissue from mice treated with a CDAHFD diet for 8 weeks and then by 2 weeks of gavage with either D2-cystamine or D4 cystamine.
• Groups Cl - control; C2 - 2 weeks D2-cystamine 200 mg/kg/day; C3 - 2 weeks D4-cystamine 200 mg/kg/day.
• Figure 17 show liver images stained with H&E showing cells and fat globules.
• Cl - control C2 - 2 weeks D2-cystamine 200 mg/kg/day; C3 - 2 weeks D4-cystamine 200 mg/kg/day.
• C2 and C3 sections show a reduction in fatty globules, particularly around the veins.
• deuterated- cysteamine and/or cystamine is/are more robust/stable and is likely to deliver more drug to the site of target cell action.
• deuterated compounds of the disclosure have an effect on the CDAHFD induced mouse model for NASH by significantly reducing ALT, hepatic fibrosis, hepatic inflammation and also pericentric hepatic steatosis just after 2 weeks of treatment.
• the deuterated compounds disclosed herein maintain the beneficial aspects of the corresponding non- isotopically enriched molecules while substantially increasing the maximum tolerated dose, decreasing toxicity, increasing the half- life, lowering the maximum plasma concentration (Cmax) of the minimum efficacious dose (MED) , lowering the efficacious dose and thus decreasing the non-mechanism-related toxicity, and/or lowering the probability of drug-drug interactions.
• the deuterated compounds of the disclosure shows improved AUC.
• the AUC measurements for cystamine D4-derived D2-cysteamine were much higher than cystamine derived cysteamine and also D2-cystamine- derived Dl-cysteamine .
• the hepatocyte elimination studies show that D4-cystamine is the most stable of the dimeric compounds.
• the data further show that D4-cystamine results in a higher AUC and Cmax (for its D2-cysteamine monomer form) compared with D2-cystamine and cystamine and also has a reduced rate of elimination in the hepatocyte elimination studies.
• the data show that the deuterated compounds of the disclosure reduce macrophage and microglial cells in liver tissue having a fatty liver phenotype.
• the data below show there was a statistically significant reduction (p ⁇ 0.0001) in the number of CDllb positive cells in liver tissue in mice treated with D2 and D4-cystamine compared with the control group.
• active Substance refers to a compound, which is administered, alone or in combination with one or more pharmaceutically
• combination therapy means the administration of two or more therapeutic agents to treat a therapeutic disorder described in the present disclosure. Such administration
• deuterium enrichment refers to the percentage of incorporation of deuterium at a given position in a molecule in the place of hydrogen. For example, deuterium enrichment of 1% at a given position means that 1% of molecules in a given sample contain deuterium at the specified position. Because the naturally occurring distribution of deuterium is about 0.0156%, deuterium enrichment at any position in a compound synthesized using non- enriched starting materials are about 0.0156%. The deuterium enrichment can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass
• deuterium when used to describe a given position in a molecule such as R-R or the symbol "D.” when used to represent a given position in a drawing of a molecular structure, means that the specified position is enriched with deuterium above the naturally occurring distribution of deuterium.
• deuterium enrichment is no less than about 1%, in another no less than about 5%, in another no less than about 10%, in another no less than about 20%, in another no less than about 50%, in another no less than about 70%, in another no less than about 80%, in another no less than about 90%, or in another no less than about 98% of deuterium at the specified position.
• disorder as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disease”, “syndrome”, and “condition” (as in medical condition) , in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and Symptoms.
• drug and “therapeutic agent” refer to a compound, or a pharmaceutical composition thereof, which is administered to a subject for treating, preventing, or ameliorating one or more symptoms of a disease or disorder.
• non-deuterated when used to describe a compound refers to a compound that has not been manufactured to increase the level of deuteration beyond what may naturally occur without the process of active deuteration. In some instances a non-deuterated molecule lacks any deuterated atoms.
• isotopic enrichment refers to the percentage of incorporation of a less prevalent isotope of an element at a given position in a molecule in the place of the more prevalent isotope of the element.
• non-release controlling excipient refers to an excipient whose primary function does not include modifying the duration or place of release of the active substance from a dosage form as compared with a conventional immediate release dosage form.
• physiologically acceptable carrier refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or Solid filler, diluent, excipient, solvent, or encapsulating material. Each component must be
• pharmaceutically acceptable in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington. The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005, Handbook of
• the compounds disclosed herein can and do exist as therapeutically acceptable salts.
• pharmaceutically acceptable salt represents salts or Zwitterionic forms of the compounds disclosed herein which are therapeutically acceptable as defined herein.
• the salts can be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate compound with a suitable acid or base.
• Therapeutically accept able salts include acid and basic addition salts.
• pharmaceutically acceptable salts include, but are not limited to, acetic acid, 2 , 2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+) - camphoric acid, camphorsulfonic acid, (+) - (IS) -camphor-10-Sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid,
• dodecylsulfuric acid ethane-1 , 2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, C-OXO-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, ( + ) -L-lactic acid, (t) -DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-) -L-malic acid, malonic acid,
• inorganic bases such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide
• organic bases such as primary, secondary, tertiary, and
• quaternary, aliphatic and aromatic amines including L-arginine, benethamine, benzathine, choline, deanol, diethanolamine,
• diethylamine dimethylamine , dipropylamine, diisopropylamine, 2- (diethylamino) -ethanol, ethanolamine , ethylamine, ethylenediamine , isopropylamine, N-methyl-glucamine , hydrabamine, lH-imidazole , L- lysine, morpholine, 4- (2-hydroxyethyl) -morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1— (2—
• hydroxyethyl ) -pyrrolidine pyridine, quinuclidine , quinoline, isoquinoline, secondary amines, triethanolamine, trimethylamine , triethylamine,N-methyl-D-glucamine , 2-amino-2- (hydroxymethyl) -1, 3- propanediol, and tromethamine .
• prevent refers to a method of delaying or precluding the onset of a
• prodrug refers to a compound functional derivative of the compound as disclosed herein and is readily convertible into the parent compound in vivo. Prodrugs are often useful because, in some situations, they may be easier to
• the prodrug may also have enhanced solubility in
• a prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. See Harper, Progress in Drug Research 1962, 4, 221-294; Morozowich et al. in
• release controlling excipient refers to an excipient whose primary function is to modify the duration or place of release of the active substance from a dosage form as compared with a conventional immediate release dosage form.
• subject refers to an animal, including, but not limited to, a primate (e.g., human, monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, and the like), lagomorphs, Swine (e.g., pig, miniature pig), equine, canine, feline, and the like.
• a primate e.g., human, monkey, chimpanzee, gorilla, and the like
• rodents e.g., rats, mice, gerbils, hamsters, ferrets, and the like
• lagomorphs e.g., Swine (e.g., pig, miniature pig)
• Swine e.g., pig, miniature pig
• equine canine
• feline feline
• substantially refers to a majority of, or mostly, as in at least about 51%, 60%, 70%, 80%,
• the term "therapeutically acceptable” refers to those compounds (or salts, prodrugs, tautomers, Zwitterionic forms, etc.) which are suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, immunogenicity, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
• therapeutically effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder being treated.
• terapéuticaally effective amount also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, Veterinarian, medical doctor, or clinician.
• treat are meant to include alleviating or abrogating a disorder or one or more of the symptoms associated with a disorder; or alleviating or eradicating the cause (s) of the disorder itself.
• treatment of a disorder is intended to include prevention .
• Cysteamine is a small aminothiol molecule that is easily transported across cellular membranes. Cysteamine markedly reduces intralysosomal cysteine accumulation and is currently approved as a treatment for cystinosis. Cysteamine can increase the cellular thiol and free thiol tripeptide glutathione pool, and thus modulate reactive oxygen species (ROS) scavenging, and decreased
• ROS reactive oxygen species
• cysteamine also increases adiponectin levels.
• Cysteamine is an attractive candidate for the treatment of fatty liver disease including NASH, as it reacts with cystine to produce cysteine, which can further be metabolized into
• Cysteamine is a precursor to the protein glutathione (GSH) precursor, and is currently FDA approved for use in the treatment of cystinosis, an intra-lysosomal cystine storage disorder.
• GSH protein glutathione
• cysteamine acts by converting cystine to cysteine and cysteine- cysteamine mixed disulfide which are then both able to leave the lysosome through the cysteine and lysine transporters respectively (Gahl et al . , N Engl J Med 2002 ; 347 (2) : 111-21) .
• the mixed disulfide can be reduced by its reaction with glutathione and the cysteine released can be used for further GSH syntheses.
• cysteamine In a recent study in children with cystinosis, enteral administration of cysteamine resulted in increased cysteamine absorption, which subsequently caused prolonged efficacy in the lowering of leukocyte cystine levels (Dohil et al . , J Pediatr 2006 ; 148 ( 6) : 764-9 ) . This may have been due to "re-cycling" of cysteamine when adequate amounts of drug reached the lysosome. If cysteamine acts in this fashion, then GSH production may also be significantly enhanced.
• Cysteamine is a potent gastric acid-secretagogue that has been used in laboratory animals to induce duodenal ulceration. Studies in humans and animals have shown that cysteamine-induced gastric acid hypersecretion is most likely mediated through hypergastrinemia . In previous studies performed in children with cystinosis who suffered regular upper gastrointestinal symptoms, a single oral dose of cysteamine (11-23 mg/kg) was shown to cause hypergastrinemia and a 2 to 3-fold rise in gastric acid- hypersecretion, and a 50% rise in serum gastrin levels. Symptoms suffered by these individuals included abdominal pain, heartburn, nausea, vomiting, and anorexia. U.S. Patent application No.
• cysteamine, cystamine, and glutathione are among the most important and active intracellular antioxidants. Cysteamine protects animals against bone marrow and gastrointestinal radiation syndromes. The rationale for the importance of SH compounds is further supported by observations in mitotic cells. These are the most sensitive to radiation injury in terms of cell reproductive death and are noted to have the lowest level of SH compounds. Conversely, S-phase cells, which are the most resistant to radiation injury using the same criteria, have demonstrated the highest levels of inherent SH compounds. In addition, when mitotic cells were treated with cysteamine, they became very resistant to radiation. It has also been noted that cysteamine may directly protect cells against induced mutations. The protection is thought to result from scavenging of free radicals, either directly or via release of protein-bound GSH. An enzyme that liberates cysteamine from coenzyme A has been reported in avian liver and hog kidney.
• radioprotectant has been found to alleviate tremors and prolong life in mice with the gene mutation for Huntington's disease (HD) .
• the drug may work by increasing the activity of proteins that protect nerve cells, or neurons, from degeneration. Cystamine appears to inactivate an enzyme called transglutaminase and thus results in a reduction of huntingtin protein (Nature Medicine 8, 143-149, 2002) .
• cystamine was found to increase the levels of certain neuroprotective proteins.
• due to the current methods and formulation of delivery of cystamine due to the current methods and formulation of delivery of cystamine,
• cysteamine is FDA approved only for the treatment of cystinosis. Patients with cystinosis are normally required to take cysteamine every 6 hours or use an enteric form of cysteamine (PROCYSBI®) every 12 hours. Subjects with cystinosis are required to ingest oral cysteamine (CYSTAGON®) every 6 hours day and night or use an enteric form of cysteamine (PROCYSBI®) every 12 hours. When taken regularly, cysteamine can deplete intracellular cystine by up to 90% (as measured in circulating white blood cells) , and reduces the rate of progression to kidney failure/transplantation and also to obviate the need for thyroid replacement therapy. Because of the difficulty in taking
• CYSTAGON® reducing the required dosing improves the adherence to therapeutic regimen.
• International Publication No. WO 2007/089670 demonstrates that delivery of cysteamine to the small intestine reduces gastric distress and ulceration, increases C max and increases AUC. Delivery of cysteamine into the small intestine is useful due to improved absorption rates from the small intestine, and/or less cysteamine undergoing hepatic first pass elimination when absorbed through the small intestine. A decrease in leukocyte cystine was observed within an hour of treatment.
• the animal body expresses various enzymes, such as the cytochrome Paso enzymes (CYPs), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases, to react with and convert these foreign substances to more polar intermediates or metabolites for renal excretion.
• CYPs cytochrome Paso enzymes
• esterases cytochrome Paso enzymes
• proteases ases
• reductases reductases
• dehydrogenases dehydrogenases
• monoamine oxidases monoamine oxidases
• C-H carbon-hydrogen
• C-O carbon-oxygen
• C-C carbon-carbon
• the transition state in a reaction is a short-lived state along the reaction pathway during which the original bonds have stretched to their limit.
• the activation energy E a for a reaction is the energy required to reach the transition state of that reaction. Once the transition state is reached, the molecules can either revert to the original reactants, or form new bonds giving rise to reaction products.
• a catalyst facilitates a reaction process by lowering the activation energy leading to a transition state. Enzymes are examples of biological catalysts.
• the magnitude of the DKIE can be expressed as the ratio between the rates of a given reaction in which a C H bond is broken, and the same reaction where deuterium is substituted for protium.
• the DKIE can range from about 1 (no isotope effect) to very large numbers, such as 50 or more. Substitution of tritium for hydrogen results in yet a stronger bond than deuterium and gives numerically larger isotope effects.
• Deuterium (D) is a stable and non-radioactive isotope of hydrogen which has approximately twice the mass of protium (H) , the most common isotope of hydrogen.
• Deuterium oxide (DO) or deuterium dioxide (D 2 O) or "heavy water” looks and tastes like 3 ⁇ 40, but has different physical properties. When pure D 2 O is given to rodents, it is readily absorbed. The quantity of deuterium required to induce toxicity is extremely high. When about 0-15% of the body water has been replaced by heavy water, animals are healthy but are unable to gain weight as fast as the control (untreated) group.
• Metabolic switching is enabled by the relatively vast size of binding pockets in many Phase I enzymes and the promiscuous nature of many metabolic reactions. Metabolic switching can lead to different proportions of known metabolites as well as altogether new metabolites. This new metabolic profile may impart more or less toxicity. Such pitfalls are non-obvious and are not predictable a priori for any drug class.
• Cysteamine is a small aminothiol molecule that is easily transported across cellular membranes. Cystamine is a small disulfide molecule that can be reduced into 2x cysteamine molecules. The carbon-hydrogen bonds of cysteamine and cystamine contain a naturally occurring distribution of hydrogen isotopes, namely H or protium (about 99.984.4%), H or deuterium (about
• KIE Kinetic Isotope Effect
• Cysteamine is metabolized in vivo by first being converted to hypotaurine by the action of the cysteamine
• compositions and methods of the disclosure such as NAFLD, NASH or cystinosis, to produce symptoms that are best medicated around the clock for extended periods of time.
• a medicine with a longer half-life may result in greater efficacy and cost savings.
• Various deuteration patterns can be used to (a) reduce or eliminate unwanted metabolites, (b) increase the half-life of the parent drug, (c) decrease the number of doses needed to achieve a desired effect, (d) decrease the amount of a dose needed to achieve a desired effect, (e) increase the formation of active metabolites, if any are formed, (f) decrease the production of deleterious metabolites in specific tissues, and/or (g) create a more effective drug and/or a safer drug for polypharmacy, whether the polypharmacy be intentional or not.
• the deuteration approach has the potential to slow the metabolism and/or selectively shunt the metabolism of cysteamine and/or cystamine to more favorable enzymatic pathways.
• the deuteration approach presented herein could potentially prevent or reduce the production of odiferous cysteamine metabolites that can lead to patient
• the disclosure provides bioprotective amino-thiol and amino-disulfide compounds and pharmaceutical compositions have been discovered, together with methods of synthesizing and using the compounds, including methods for the treatment of liver diseases and disorders in a patient by administering a compound of the disclosure .
• R 4 -R 4 are independently selected from H or D; wherein at least one of R 4 -R 4 is D.
• the disclosure demonstrates that deuterated forms of Formula I have characteristics that are different compared to non-deuterated forms of a compound of formula I (i.e., wherein Rl- R4 do not contain any deuterated atoms) .
• the deuterated form of Formula I has a half-life that is longer than the half-life of a non-deuterated form. This is useful in the treatment of various disease and disorder to reduce dosing
• the disclosure provides that the compound having the structure of Formula I does not have a structure selected from:
• R 5 -R 12 are independently selected from H or D; wherein at least one of R 5 -R 12 is D.
• deuterated forms of Formula II also show longer half-life compared are to non- deuterated forms. As mentioned above, such increase half-lives can have effects on dosing and dosing frequencies.
• a compound disclosed herein is substantially a single enantiomer, a mixture of about 90% or more by weight of the (-)-enantiomer and about 10% or less by weight of the (+)-enantiomer, a mixture of about 90% or more by weight of the (+)-enantiomer and about 10% or less by weight of the (-)- enantiomer, Substantially an individual diastereomer, or a mixture of about 90% or more by weight of an individual diastereomer and about 10% or less by weight of any other diastereomer.
• the compound of disclosure is selected from:
• the compound of disclosure is selected from:
• compositions of the disclosure can contain a deuterated cysteamine or deuterated
• the active agents in the composition i.e., deuterated cysteamine or deuterated cystamine, may be administered in the form of a pharmacologically acceptable salt, ester, amide, prodrug or analog or as a combination thereof.
• Salts, esters, amides, prodrugs and analogs of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure," 4th Ed. (New York: Wiley-Interscience , 1992) .
• basic addition salts are prepared from the neutral drug using conventional means, involving reaction of one or more of the active agent's free hydroxyl groups with a suitable base.
• the neutral form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the base is added thereto.
• the resulting salt either precipitates or may be brought out of solution by addition of a less polar solvent.
• Suitable bases for forming basic addition salts include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine , or the like.
• Preparation of esters involves functionalization of hydroxyl groups which may be present within the molecular structure of the drug.
• the compounds as disclosed herein may also contain less prevalent isotopes for other elements, including, but not limited to, 13 C or 14 C for carbon, 33 S, 34 S, or 36 S for sulfur, 15 N for nitrogen, and 17 0 or 18 0 for oxygen.
• the compound disclosed herein may expose a patient to a maximum of about 0.000005% DO or about 0.00001% DHO, assuming that all of the C-D bonds in the compound as disclosed herein are metabolized and released as DO or DHO.
• the levels of DO shown to cause toxicity in animals is much greater than even the maximum limit of exposure caused by administration of the deuterium enriched compound as disclosed herein.
• the deuterium- enriched compound disclosed herein should not cause any additional toxicity due to the formation of DO or DHO upon drug metabolism.
• the deuterated compounds disclosed herein maintain the beneficial aspects of the
• the deuterated compounds of the disclosure shows improved AUC.
• the AUC measurements for cystamine D4-derived cysteamine D2 were much higher than cystamine derived cysteamine and also cysteamine D2-derived cysteamine D1.
• the compounds of the disclosure exhibit a reduced rate of metabolism by at least one
• polymorphically-expressed cytochrome P450 isoform in a subject per dosage unit thereof in comparison to non-isotopically enriched cysteamine and cystamine.
• polymorphically-expressed cytochrome P450 isoforms include, but are not limited to, CYP2C8, CYP2C9, CYP2C19, and CYP2D6.
• the compounds of the disclosure exhibit a reduced rate of metabolism by at least one cytochrome P450 isoform or monoamine oxidase isoform in a subject per dosage unit thereof in comparison to non-isotopically enriched cysteamine and cystamine.
• cytochrome P450 isoforms and monoamine oxidase isoforms include but are not limited to, CYP1A1, CYP1A2 , CYP1B1, CYP2A6, CYP2A13, CYP2B6,
• the compounds of the disclosure exhibit an improvement in a diagnostic hepatobiliary function end point, as compared to the corresponding non-isotopically enriched cysteamine and cystamine.
• diagnostic hepatobiliary function endpoints include, but are not limited to, alanine aminotransferase (ALT) , serum glutamic pyruvic transaminase
• SGPT aspartate aminotransferase
• AST aspartate aminotransferase
• ALT/AST ratios serum aldolase
• alkaline phosphatase (ALP) alkaline phosphatase
• ALP alkaline phosphatase
• ammonia levels serum aldolase
• alkaline phosphatase (ALP) ammonia levels
• bilirubin gamma glutamyltranspeptidase
• GGTP gamma glutamyltranspeptidase
• GGT gamma glutamyltranspeptidase
• LAP leucine aminopeptidase
• deuterated compounds of the disclosure e.g., D2 and D4-cystamine
• a deuterated compound of the disclosure can reduce ALT, reduce fibrosis, reduce Collagen 1 and TIMP expression, reduce steatosis (pericentral) and reduce inflammatory infiltrate (CDllb) of the liver.
• the disclosure provides method of treating a subject to reduce ALT, reduce fibrosis, reduce Collagen 1 and TIMP expression, reduce steatosis (pericentral) and/or reduce inflammatory infiltrate (CDllb) comprising administering a deuterated compound of the disclosure.
• processes for preparing a compound as disclosed herein or other pharmaceutically acceptable derivative thereof such as a salt, solvate, or prodrug, as an antioxidant, and a treatment for cystinosis and fatty liver disorders, such as NAFLD and NASH.
• compositions which comprise one or more of certain deuterated compounds disclosed herein, or one or more pharmaceutically acceptable salts, prodrugs, or solvates thereof, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients.
• Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences.
• the pharmaceutical compositions disclosed herein may be manufactured in any manner known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
• compositions may also be formulated as a modified release dosage form, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release , and gastric retention dosage forms.
• modified release dosage form including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release , and gastric retention dosage forms.
• a deuterated cysteamine and/or cystamine can be enterically coated (e.g., enterically coated beads or capsules) .
• enterically coated e.g., enterically coated beads or capsules
• non-deuterated enteric formulations of cysteamine bitartrate have been shown to provide improved drug compliance, reduce frequency of administration and prolonged reduction of cystine levels in cystinosis patients.
• an enterically coated formulation comprising a deuterated compound of formula I and/or II can have improved administration and longer biological activity.
• an enterically coated formulation of compound of formula I and/or II can be administered at lower doses than a non- deuterated enteric formulation and/or may be administered less frequently .
• compositions include those suitable for oral, parenteral (including Subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal , transmucosal , transdermal, rectal and topical (including dermal, buccal, Sublingual and intraocular) administration.
• parenteral including Subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary
• intraperitoneal including transmucosal
• transdermal rectal
• topical including dermal, buccal, Sublingual and intraocular
• the compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
• these methods include the step of bringing into association a compound of the disclosure or a pharmaceutically salt, prodrug, or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients.
• active ingredient a compound of the disclosure or a pharmaceutically salt, prodrug, or solvate thereof
• the carrier which constitutes one or more accessory ingredients.
• the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
• Formulations of the compounds disclosed herein suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules (including enterically coated granules) ; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
• the active ingredient may also be presented as a bolus, electuary or paste.
• compositions which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents.
• Moulded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added.
• Dragee cores are provided with suitable coatings.
• concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer Solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• the compounds may be formulated for parenteral
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried
• sterile liquid carrier for example, saline or sterile pyrogen-free water
• injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
• Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which may contain antioxidants, buffers,
• bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents.
• Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt .
• compositions may take the form of tablets, lozenges, pastilles, or gels formulated in conventional manner.
• Such compositions may comprise the active ingredient in a flavored basis such as sucrose and acacia or tragacanth.
• Certain compounds disclosed herein may be administered topically, that is by non-systemic administration. This includes the application of a compound disclosed herein externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream.
• systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
• Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as gels, liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
• the powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
• Typical unit dosage formulations are those containing an effective dose, as herein below recited, or an appropriate fraction thereof, of the active ingredient.
• Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of one or more compounds which is effective at such dosage or as a multiple of the same, for instance, units containing 1 mg to 1000 mg of the compounds disclosed herein, usually around 100 mg to 500 mg of the compound .
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration .
• the compounds can be administered in various modes, e.g. orally, topically, or by injection.
• the precise amount of compound administered to a patient will be the responsibility of the attendant physician.
• the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diets, time of administration, route of administration, rate of excretion, drug combination, the precise disorder being treated, and the severity of the disorder being treated. Also, the route of administration may vary depending on the disorder and its severity.
• formulated formulation comprising at least one deuterated form of cystamine and/or cysteamine.
• the deuterated forms comprise pharmacokinetic and pharmacodynamic changes related to non-deuterated forms.
• prior formulations comprising enterically coated cysteamine and/or cystamine have also showed improved pharmacokinetic and pharmacodynamic data relative to non- enterically formulated formulations.
• the combination of enterically coated and deuterated forms of cystamine and/or cysteamine are expected to further modulate the pharmacokinetics and pharmacodynamics of cysteamine and/or cysteamine delivery including, for example, both the delayed and extended release of the active ingredient as reflected by modulation of the AUC and Cmax and/or Tmax.
• the administration of the compounds may be administered chronically, that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disorder.
• the administration of the compounds may be given continuously or temporarily suspended for a certain length of time (i.e., a "drug holiday') . Once improvement of the patient's conditions has occurred, a maintenance dose is
• the dosage or the frequency of administration, or both can be reduced, as a function of the symptoms, to a level at which the improved disorder is retained.
• Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.
• This disclosure identifies patient populations that can benefit from the compounds disclosed herein, particularly juvenile patients.
• the disclosure provides composition of the compounds disclosed herein that can be used in the treatment of various diseases including cystinosis, Huntington's disease, and NAFLD (including NASH) .
• Cystinosis is a rare disease that is typically diagnosed prior to age 2. Cystinosis is a genetic metabolic disease that causes an amino acid, cystine, to accumulate in various organs of the body. Cystine crystals accumulate in the kidneys, eyes, liver, muscles, pancreas, brain, and white blood cells. Without specific treatment, children with cystinosis develop end stage kidney failure at approximately age nine. Cystinosis also causes complications in other organs of the body. The complications include muscle wasting, difficulty swallowing, diabetes, and hypothyroidism. It is estimated that at least 2,000 individuals worldwide have cystinosis, though exact numbers are difficult to obtain because the disease is often undiagnosed and/ or
• Cystinosis There are three forms of Cystinosis. Infantile Nephropathic Cystinosis is the most severe form of the disease. Children with Cystinosis appear normal at birth, but by 10 months of age, they are clearly shorter than others their age. They urinate frequently, have excessive thirst, and often seem fussy. At 12 months, they haven't walked and bear weight only gingerly.
• One of the major complications of Cystinosis is renal tubular Fanconi Syndrome, or a failure of the kidneys to reabsorb nutrients and minerals. The minerals are lost in the urine. The urinary losses must be replaced. Generally, they are picky eaters, crave salt, and grow very slowly. If left untreated, this form of the disease may lead to kidney failure by 10 years of age. In people with
• Cysteamine (Cystagon®) has been approved by the Food and Drug Administration (FDA) for standard treatment of Cystinosis. Cysteamine is a cystine-depleting agent that lowers cystine levels within the cells. Cysteamine has proven effective in delaying or preventing renal failure. Cysteamine also improves growth of children with Cystinosis. In view of the harmful effects of chronic cystine accumulation, and the indications of the
• Cysteamine should be used by post-transplant Cystinosis patients.
• Procysbi® cysteamine bitartrate delayed release capsules
• Cystaran cysteamine ophthalmic solution
• 0.44% is an ophthalmic solution approved by the FDA for the treatment of corneal cystine crystal accumulation in patients with cystinosis.
• D4 cystamine is eliminated at a slower rate than D2 or non-deuterated cystamine (i.e., D4 is slower than D2 which is slower than non- deuterated cystamine) .
• D4 is slower than D2 which is slower than non- deuterated cystamine
• This correlates with the higher AUC for D2- cysteamine (when D-4 cystamine is administered to rats.
• Table 1 also shows that D-cysteamine and D2-cysteamine are eliminated at a slower rate than cysteamine bitartrate.
• the data shows that the deuterated compounds of the disclosure have higher Cmax and AUC compared to non- deuterated compounds.
• a study of rats receiving non- deuterated cytamine and D4-cystamine shows that the Cmax of the deuterated compound was ⁇ 2x that of the non-deuterated compounds and that the AUC was at least 1.6x greater than non-deuterated cystamine (Table 2 ) .
• Table 2 Average Pharmacokinetic data from rats receiving cystamine and D4-cystamine .
• the monomer cysteamine or D2-cysteamine was measured.
• a subject having cystinosis is administered a compound of the disclosure or pharmaceutically acceptable salt thereof in an amount to obtain about 10-100 mmol (e.g., 10, 20, 30, 40, 50, 60, 70, 80 or any value there between) of the compound in the plasma.
• the dose is up to about 10-40 mg/kg. In another embodiment, the dose is
• the compound of the disclosure is administered 2-4 times per day at about 300 mg to 1 gram per dose.
• the compound of the disclosure is administered in multiple doses that do not exceed 2.0 g/m 2 /day or 95 mg/kg/day.
• the dose is changed over time to reach the highest tolerable dose for the subject, typically between about 30-50 mmol plasma of a compound disclosed herein.
• an initial dose may provide a circulating level of about 10 mmol of the compound of the disclosure, which will be adjusted up to the highest tolerable dose, typically about 40 mmol.
• an initial dose may result in a circulating level of 80 mmol, which will be adjusted down to about 40 mmol .
• the goal of therapy is to keep leukocyte cystine levels below 1 nmol/1 ⁇ 2 cystine/mg protein five to six hours following administration of the compound of the disclosure. Patients with poorer tolerability still receive significant benefit if white cell cystine levels are below 2 nmol/1 ⁇ 2 cystine/mg protein.
• the dose of the compound disclosed herein can be increased to a maximum of 2.0 grams/m 2 /day to achieve this level.
• the recommended maintenance dose of 1.30 grams /m 2 /day can be approximated by administering the compound according to the following table, which takes surface area as well as weight into consideration.
• the dose should be raised if the leukocyte cystine level remains > 2 nmol/1 ⁇ 2
• cystine/mg/protein cystine/mg/protein
• compositions and methods of the disclosure can also be used to treat NAFLD and NASH as well as liver fibrotic diseases.
• NAFLD Non-alcoholic fatty liver disease
• NASH represents a spectrum of disease occurring in the absence of alcohol abuse. It is
• steatosis fat in the liver
• metabolic syndrome including obesity, diabetes and hypertriglyceridemia
• NAFLD is linked to insulin resistance, it causes liver disease in adults and children and may ultimately lead to cirrhosis (Skelly et al . , J Hepatol 2001; 35: 195-9; Chitturi et al . , Hepatology
• nonalcoholic fatty liver or NAFL relatively benign isolated predominantly macrovesicular steatosis (i.e., nonalcoholic fatty liver or NAFL) to non-alcoholic
• Hepatic fibrosis resulting from NASH may progress to cirrhosis of the liver or liver failure, and in some instances may lead to hepatocellular carcinoma.
• the degree of insulin resistance correlates with the severity of NAFLD, being more pronounced in patients with NASH than with simple fatty liver (Sanyal et al . , Gastroenterology 2001 ; 120 ( 5 ) : 1183-92 ) .
• insulin- mediated suppression of lipolysis occurs and levels of circulating fatty acids increase.
• Two factors associated with NASH include insulin resistance and increased delivery of free fatty acids to the liver. Insulin blocks mitochondrial fatty acid oxidation. The increased generation of free fatty acids for hepatic re- esterification and oxidation results in accumulation of
• Glutathione gammaglutamyl-cysteinyl-glycine ; GSH
• GSH gammaglutamyl-cysteinyl-glycine
• Glutathione itself is does not enter easily into cells, even when given in large amounts.
• glutathione precursors do enter into cells and some GSH precursors such as N-acetylcysteine have been shown to be effective in the treatment of conditions such as acetaminophen toxicity by slowing or preventing GSH depletion (Prescott et al . , Annu Rev Pharmacol Toxicol 1983;23:87-101) .
• GSH precursors include cysteine, N-acetylcysteine, methionine and other sulphur-containing compounds such as cysteamine (Prescott et al . , J Int Med Res 1976;4(4 Suppl ) : 112-7 ) .
• Cysteine is a major limiting factor for GSH synthesis and that factors (e.g., insulin and growth factors) that stimulate cysteine uptake by cells generally result in increased
• N-acetylcysteine has been administered to patients with NASH.
• obese individuals with NASH treated with N-acetylcysteine for 4-12 weeks exhibited an improvement in aminotransferase levels and gamma-GT even though there was no reported change in subject body mass index (Pamuk et al . , J
• triglyceride During periods of increased calorie intake, triglyceride will accumulate and act as a reserve energy source. When dietary calories are insufficient, stored triglycerides (in adipose) undergo lipolysis and fatty acids are released into the circulation and are taken up by the liver. Oxidation of fatty acids will yield energy for utilization.
• Steatosis also may develop into steatohepatitis through oxidative stress due to reactive oxygen species (ROS) and decreased anti-oxidant defense (Sanyal et al . , Gastroenterology 2001,
• ROS can be generated in the liver through several pathways including mitochondria, peroxisomes, cytochrome P450,
• NADPH oxidase and lipooxygenase (Sanyal et al., Nat Clin Pract Gastroenterol Hepatol, 2005;2(1) : 46-53) .
• Insulin resistance and hyperinsulinism has been shown to increase hepatic oxidative stress and lipid peroxidation through increased hepatic CYP2EI activity (Robertson et al., Am J Physiol Gastrointest Liver Physiol, 2001 281 (5) : G1135-9; Leclercq et al . , J Clin Invest 2000, 105(8) :1067- 75) .
• NAFLD Newcastle disease virus
• the medical conditions most commonly associated with NAFLD are obesity, Type II diabetes and dyslipidemia . These conditions can be induced by feeding mice and rats with high fat or sucrose diets. Rats fed with a >70% fat-rich diet for 3 weeks developed pan-lobular steatosis, patchy
• NASH mice have been induced through intragastric overfeeding. Mice were fed up to 85% in excess of their standard intake for 9 weeks. The mice became obese with 71% increase in final body weight; they
• mice demonstrated increase white adipose tissue, hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance and insulin resistance.
• the livers of the overfed mice were about twice as large expected, beige in color with microscopic evidence of lipid droplets, cytoplasmic vacuoles and clusters of inflammation.
• Mouse models of NASH can be used to study various therapies. Mouse models are created through specific diets
• NASH mice are useful in screening and measuring the effects cysteamine on NASH related disease and disorders. For example, the effect of treatment can be measured by separating the NASH mice into a control group where animals will continue to receive MCD diet only and three other treatment groups where mice will receive MCD diet as well as anti-oxidant therapy.
• the three therapy groups for example, can receive cysteamine 50mg/kg/day, lOOmg/kg/day and SAME .
• NASH is a disease subset falling under the umbrella of NAFLD and is characterized by various biomarkers and histological examination. NASH has been
• Type 1 and Type 2 NASH are typically identified in juvenile patients .
• Type 1 NASH demonstrates a prevalent lobular inflammation in the liver in contrast with a prevalent portal inflammation in Type 2 NASH.
• differentiating factors that can be used in the methods disclosed herein is identifying, by histological examination, the presence of Type 1 vs. Type 2 NASH.
• NASH NAFLD Activity Score
• the NAS is the sum of the separate scores for steatosis (0-3), hepatocellular ballooning (0-2) and lobular inflammation (0-3), with the majority of patients with NASH having a NAS score of 3 5 (Kleiner DE, Brunt EM, Van Natta M et al . Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41(6), 1313-1321 (2005)).
• cytokeratin 18 is a useful indicator of inflammation in NASH, due to
• Normal cytokeratin 18 levels are typically characterized as being less than 200 units per liter.
• subjects with liver disease, including NALFD and NASH have a statistically significant elevation in cytokeratin 18 (e.g., above 200 U/L; 200-300 U/L) .
• cytokeratin 18 levels can be used as a marker to determine whether a treatment is being effective. For example, a reduction in cytokeratin 18 levels of greater than 10% (e.g., 20-
• ALT/AST aspartate aminotransferase
• gamma-GT gamma-glutamyltrans ferase
• ALT levels have been shown to be indicative of liver function.
• normal ALT levels are about 7 to 55 units (e.g., 10-40 units) per liter has been shown to correlate with normal liver function. This value is somewhat varied in children and adolescents. Thus, in some instances ALT levels less than 25 units per liter are "normal" in children and adolescents.
• AST may be measured alone, but preferably the determination should be made in combination with one or more other markers of liver function or dysfunction. For example, a subject having AST levels above about 50 is indicative of liver disease or dys function .
• ALP may be measured alone, but preferably the determination should be made in combination with one or more other markers of liver function or dysfunction. For example, a subject having ALP levels above about 150 is indicative of liver disease or dys function .
• Triglycerides levels have been shown to be indicative of liver function. For example, triglyceride levels less than about 150 mg/dL (e.g., 100-150 mg/dL) has been shown to correlate with normal liver function. Increased levels of triglycerides have been shown to correlate with liver disease and disorders. For example, NAFLD and NASH subjects typically show triglyceride levels of between 150 to 200 (e.g., 155-195, 160-190, 165-185, 170-180, or any number between any two values thereof) .
• 150 mg/dL e.g., 100-150 mg/dL
• Increased levels of triglycerides have been shown to correlate with liver disease and disorders.
• NAFLD and NASH subjects typically show triglyceride levels of between 150 to 200 (e.g., 155-195, 160-190, 165-185, 170-180, or any number between any two values thereof) .
• triglycerides may be measured alone, but preferably the determination should be made in combination with one or more other markers of liver function or dysfunction. For example, a subject having triglyceride levels above about 150 mg/dl is indicative of liver disease or
• Triglycerides are the form in which fat moves through the bloodstream. Triglycerides can be metabolized by various organs, including the liver, to form phospholipids (LDLs and HDLs), cholesterol and oxidized forms thereof. Oxidized phospholipids (OxPL) including OxLDL are known inflammatory mediators and strongly correlated with cardiovascular diseases. For example, Bieghs et al. (Hepatology, 65(3) : 894-903,
• Adiponectin circulates as trimer (low molecular weight adiponectin) , hexamer (medium molecular weight adiponectin) and higher order multimer (high molecular weight adiponectin) in serum and isoform-specific effects have been demonstrated.
• adiponectin is believed to have a hepatoprotective effect due to protective effects against oxidative damage.
• Normal levels of adiponectin vary by age and sex. For example, females have a higher baseline adiponectin level compared to males. A normal weight female typically has an adiponectin level of between about 8.5 and 11 mg/ml and males typically have an adiponectin level of between about 6 and 8 mg/ml .
• adiponectin levels that are about 50-90% of normal levels (e.g., decreased by 10-50% from normal, or any value there between) (see, e.g., Merl et al . Int. J. Obes (Lond) , 29(8), 998-1001, 2005).
• the resistin protein is increased in NASH subjects compared to normal subjects.
• Human resistin is a cysteine-rich, 108-amino-acid peptide hormone with a molecular weight of 12.5 kDa. In adult humans, resistin is expressed in bone marrow. Moreover, in adipocytes of subjects having a low or healthy BMI, resistin mRNA is almost undetectable. Consistent with this, body mass index (BMI) is appears to correlate with resistin concentrations in serum, and women may have higher resistin concentrations than men. Resistin mRNA expression in human peripheral mononuclear cells is increased by proinflammatory cytokines. Serum resistin is significantly elevated in both NASH and simple steatotic subjects. Hepatic resistin is significantly increased in NASH patients in both mRNA and protein levels than those in simple steatosis and normal control subjects. Because of the cysteine-rich structure of resistin changes in sulfur
• cysteamine and cystamine can modulated cysteine and/or glutathione levels in subjects taking cysteamine or cystamine.
• Subjects afflicted with NAFLD or NASH tend to be in a higher percentile of weight for their age group (e.g., above the 97 th percentile for BMI for their age group) . Treating pediatric patients at an early stage may have lifelong benefits in the management of liver function and obesity.
• the deuterated compounds reduced the risk of
• the disclosure demonstrates that following treatment to induce fatty liver disease administration of deuterated compounds of the disclosure resulted in an improvement in both ALT markers of liver function as well as a reduction in inflammatory infiltrate, liver fibrosis and markers of fibrosis such as collagen 1 and TIMP.
• deuterated cystamine and/or cysteamine can be used to prevent and/or treat NAFLD, NASH and liver fibrosis resulting from these diseases.
• the disclosure provides populations of subject with NASH that that have high probability of responding to treatment with a deuterated cysteamine or cystamine composition.
• the disclosure provides a method of treating a subject suffering from fatty liver disease, such as NASH, comprising administering a therapeutically effective amount of a compound of the disclosure.
• the fatty liver disease is selected from the group consisting of non-alcoholic fatty acid liver disease (NAFLD) , non- alcoholic steatohepatitis (NASH) , fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy, and lipodystrophy.
• NAFLD non-alcoholic fatty acid liver disease
• NASH non-alcoholic steatohepatitis
• fatty liver disease resulting from hepatitis fatty liver disease resulting from obesity
• fatty liver disease resulting from diabetes fatty liver disease resulting from insulin resistance
• fatty liver disease resulting from hypertriglyceridemia Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolmans disease, acute
• pediatric and juvenile patients are treated with a deuterated compound of the disclosure.
• a subject having NASH is administered a formulation comprising a deuterated compound of the disclosure for oral administration in an amount to obtain about 10-80 mmol (e.g., 10, 20, 30, 40, 50, 60, 70, 80 or any value there between) of a deuterated compound disclosed herein in the plasma.
• the formulation is a delayed release oral formulation.
• the dose is about 10-40 mg/kg.
• the dose is administered 2-4 times per day at about 300 mg to 1 gram per dose.
• the dose is changed over time to reach the highest tolerable dose for the subject, typically between about 30-50 mmol of the compound in plasma.
• an initial dose may provide a circulating level of about 10 mmol of the compound, which will be adjusted up to the highest tolerable dose, typically about 40 mmol.
• an initial dose may result in a circulating level of 80 mmol, which will be adjusted down to about 40 mmol.
• subjects less than 15 years of age e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 years of age
• BMI body mass index
• the subject has a BMI above 97 th percentile for the age and weighs less than 65kg. In some embodiment, these same subjects have high triglyceride levels, low LDH, and low or low normal adiponectin levels. In still another embodiment, the subjects have high or high normal resistin levels. In various embodiments of any of the foregoing, the patient weighs less than 65 kg. In various embodiments, the patient weighs from about 35-65 kg, or from about 40-60 kg, or from about 45-55 kg, or about 35,
• the patient weighing less than 65 kg receives 600 to 1200 mg/day of a
• the subject has Type I NASH or NASH with Type 1 histological pattern.
• the subject has lobular inflammation of the liver.
• the subject has low adiponectin and high triglycerides characteristic of NASH.
• the subject has a marker (e.g., AST, ALT, GGT or other liver marker) having a level consistent with NASH as described herein.
• the patient weighs 65-80 kg, and may receive 750 to about 1500 mg/day of a compound of the
• the subject has Type I NASH.
• the subject has lobular inflammation of the liver.
• the subject has low adiponectin and high triglycerides characteristic of NASH.
• the patient weighs more than 65 kg and receives 900 to about 2000 mg/day of a deuterated compound of the disclosure or an amount to obtain circulating plasma levels of the deuterated compound of about 10-80 mmol (typically about 30-
• the subject has Type I NASH.
• the subject has lobular inflammation of the liver.
• the subject has low adiponectin and high triglycerides characteristic of NASH.
• the subject can be an adult, adolescent or child.
• the patient is from 2 to 7 years old, from 8 to 11 years old, from 9 to 12 years old, or from 13 to 18 years old.
• an adolescent is from 10 to 19 years old as described in the National Institutes of Health standards.
• the administration results in a decrease in NAFLD Activity Score of two or more points, no worsening or an improvement of fibrosis, reduction in serum aminotransferases and gammaglutamyl transpeptidase (GGT) ; reduction in MRI-determined hepatic fat fraction; changes to markers of oxidation and anti-oxidant status; changes in fasting insulin and glucose; an increase in circulating adiponectin levels; a decrease in circulating resistin levels; a decrease in triglyceride levels; a decrease in oxidized phospholipids; changes in weight, height, body mass index (BMI) and waist circumference; changes in the Pediatric Quality of Life score; changes to any symptoms that patient may have experienced; proportion with a change from a histological diagnosis of definite NASH or indeterminate for NASH to not NASH at end of treatment; individual histological
• steatosis fatty liver
• lobular inflammation portal chronic inflammation
• ballooning fibrosis score and stage la versus lb fibrosis
• a deuterated compound of the disclosure is administered at a daily dose ranging from about 10 mg/kg to about 2.5 g/kg, or from about 100 mg/kg to about 250 mg/kg, or from about 60 mg/kg to about 100 mg/kg or from about 50 mg/kg to about 90 mg/kg, or from about 30 mg/kg to about 80 mg/kg, or from about 20 mg/kg to about 60 mg/kg, or from about 10 mg/kg to about 50 mg/kg.
• the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/ 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 425 mg/kg, 450 mg/kg, 475 mg/kg, 500 mg/kg, 525 mg/kg , 550 mg/kg, 575 mg/kg, 600 mg/kg, 625 mg/kg, 650 mg/kg, 675 mg/kg, 700 mg/kg, 725 mg/kg, 750 mg/kg, 775 mg//
• the deuterated compound of the disclosure is administered at a total daily dose of from approximately 0.25 g/m 2 to 4.0 g/m 2 body surface area, about 0.5-2.0 g/m 2 body surface area, or 1-1.5 g/m 2 body surface area, or 1-1.95g/m 2 body surface area, or 0.5-1 g/m 2 body surface area, or about 0.7-0.8 g/m 2 body surface area, or about 1.35 g/m 2 body surface area, or about 1.3 to about 1.95
• grams/m 2 /day or about 0.5 to about 1.5 grams /m 2 /day, or about 0.5 to about 1.0 grams /m 2 /day, e.g., at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m 2 , or up to about 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 3.25, 3.5 or 3.75 g/m 2 or may range between any two of the foregoing values.
• the delayed and extended release formulation comprises an enteric coating that releases a deuterated compound disclosed herein when the formulation reaches the small intestine or a region of the gastrointestinal tract of a subject in which the pH is greater than about pH 4.5.
• the formulation releases at a pH of about 4.5 to 6.5, 4.5 to 5.5, 5.5 to 6.5 or about pH 4.5, 5.0, 5.5, 6.0 or 6.5.
• the deuterated compound of the disclosure or a pharmaceutically acceptable salt, prodrug or solvate thereof is formulated for oral administration (e.g., as a capsule, table, caplet, solution, etc. ) .
• the disclosure provides for capsules, tablets, or caplets, comprising 50 mg to 200 mg of a compound of disclosure or a pharmaceutically acceptable salt (e.g., a bitartrate salt), prodrug or solvate thereof.
• the capsules, tablets, or caplets further comprise inactive ingredients, such as colloidal silicon dioxide, croscarmellose sodium, D&C yellow no. 10 aluminum lake, FD&C blue no. 1 aluminum lake, FD&C blue no. 2 aluminum lake, FD&C red no. 40 aluminum lake, gelatin, magnesium stearate, microcrystalline cellulose, pharmaceutical glaze, pregelatinized starch, silicon dioxide, sodium lauryl sulfate, synthetic black iron oxide and/or titanium dioxide.
• a compound disclose herein is administered at a frequency of 4 or less times per day (e.g., one, two or three times per day) .
• the frequency of 4 or less times per day e.g., one, two or three times per day.
• composition is a delayed or controlled release dosage form that provides increased delivery of a compound disclosed herein to the small intestine.
• the deuterated compound of the disclosure or a pharmaceutically acceptable salt, prodrug or solvate thereof is formulated for oral administration (e.g., as a capsule, table, caplet, solution, etc.) that provides for delayed release.
• the disclosure provides for delayed release capsules, tablets, or caplets, comprising 25 mg to 75 mg of a deuterated compound of disclosure or a pharmaceutically acceptable salt (e.g., a bitartrate salt), prodrug or solvate thereof.
• the delayed release capsules, tablets, or caplets further comprise inactive
• ingredients such as microcrystalline cellulose, Eudragit® L 30 D- 55, Hypromellose , talc, triethyl citrate, sodium lauryl sulfate, purified water, gelatin, titanium dioxide, blue ink and/or white ink.
• the delay or controlled release form can provide a C max of a deuterated compound disclosed herein, or a biologically active metabolite thereof, that is at least about 10%, 20%, 30% or higher than the C max provided by an enterically coated non-deuterated cystamine and/or cysteamine (e.g., Procysbi®) dosage form
• the delay and extended release formulation comprising a deuterated cysteamine and/or cystamine provides an improved AUC compared to non-deuterated delayed and/or extended release forms of the compound.
• the AUC is increased compared to non-deuterated delayed and/or extended release formulation.
• the delayed or controlled release dosage form comprising deuterated cystamine and/or cysteamine comprises an enteric coating that releases a compound disclosed herein when the composition reaches the small intestine or a region of the gastrointestinal tract of a subject in which the pH is greater than about pH 4.5.
• the pH is between 4.5 and 6.5.
• the pH is about 5.5 to 6.5.
• the compound of the disclosure is delivered throughout the small intestine providing an extended release in the small intestine.
• formulation comprising a deuterated compound of the disclosure is granulated and the granulation is compressed into a tablet or filled into a capsule.
• the granules are enterically coated prior to compressing into a tablet or capsule.
• Capsule materials may be either hard or soft, and are typically sealed, such as with gelatin bands or the like. Tablets and capsules for oral use will generally include one or more commonly used excipients as discussed herein.
• a suitable pH-sensitive polymer is one which will dissolve in intestinal environment at a higher pH level (pH greater than 4.5), such as within the small intestine and therefore permit release of the pharmacologically active substance in the regions of the small intestine and not in the upper portion of the GI tract, such as the stomach.
• PCT/US2014/042607 and PCT/US2014/042616 (the disclosure of which are incorporated herein by reference) .
• the dosage form i.e., the tablet or capsule comprising the enterically coated deuterated compound of the disclosure
• a total weight in the range of approximately 50 mg to 1000 mg is used.
• the tablet or capsule comprises 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400 or 500 mg deuterated active ingredient, and multiple tablets or capsules are administered to reach the desired dosage.
• the dosage form is orally administered to a subject in need thereof.
• a tablet core comprises about 50 mg of a deuterated compound of the disclosure that is encapsulated in an enteric coating material having a thickness of about 60-100 pm (e.g., about 71, 73, 75, 77, or 79 pm or any value there between) and/or about 10-13% (e.g., about 10.5, 11.0, 11.2, 11.4, 11.6,
• a tablet core comprises about 150 mg of a deuterated compound of the disclosure about that is encapsulated in an enteric coating material having a thickness of about 90-130pm (e.g., about 97, 99, 101, 103, 105,
• the enteric coating material can be selected from the group comprising polymerized gelatin, shellac, methacrylic acid copolymer type C NF, cellulose butyrate phthalate, cellulose hydrogen phthalate, cellulose proprionate phthalate, polyvinyl acetate phthalate (PVAP) , cellulose acetate phthalate (CAP) , cellulose acetate trimellitate (CAT) , hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulose succinate, carboxymethyl ethyl cellulose (CMEC) , hydroxypropyl methylcellulose acetate succinate (HPMCAS) , and acrylic acid polymers and
• copolymers typically formed from methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate with copolymers of acrylic and methacrylic acid esters.
• composition can be administered orally or
• the method results in improvement in liver fibrosis compared to levels before
• the method results in a reduction in fat content of liver, a reduction in the incidence of or progression of cirrhosis, or a reduction in the incidence of hepatocellular carcinoma.
• the method results in a decrease in hepatic aminotransferase levels compared to levels before administration of the deuterated compound of the disclosure.
• the administering results in a reduction in hepatic transaminase of between approximately 10% to 70%, e.g. 10, 20, 30, 40, 50, 60 or 70% or any value between these numbers, compared to levels before treatment.
• the administering results in a reduction in alanine or aspartate aminotransferase levels in a treated patient to approximately 50%, 40%, 30%, 20% or 10% above normal ALT levels, or at normal ALT levels.
• the administering results in a reduction in serum ferritin levels compared to levels before treatment with a compound of the disclosure.
• the administration results in a lowering of NAS score.
• prodrugs can be "activated” by use of the enterically coated deuterated compound of the disclosure.
• Prodrugs are pharmacologically inert, they themselves do not work in the body, but once they have been absorbed, the prodrug decomposes.
• the prodrug approach has been used successfully in a number of therapeutic areas including antibiotics, antihistamines and ulcer treatments.
• the advantage of using prodrugs is that the active agent is chemically camouflaged and no active agent is released until the drug has passed out of the gut and into the cells of the body.
• a number of prodrugs use S-S bonds. Weak reducing agents, such as cysteamine, reduce these bonds and release the drug. Accordingly, the deuterated compositions of the disclosure are useful in combination with pro-drugs for timed release of the drug.
• a pro-drug can be
• formulations for use in the methods described herein can comprise a pharmaceutically acceptable salt of the deuterated compound of the disclosure, such a bitartrate salt, instead of free base compound.
• the methods and composition of the disclosure can also include administering a second agent in combination with a deuterated compound of the disclosure to treat a disease or disorder.
• a second agent in combination with a deuterated compound of the disclosure to treat a disease or disorder.
• the subject in another embodiment of any of the foregoing methods or composition, can be treated with a
• the combination includes a deuterated compound of the disclosure and one or more of metformin, statins, anti-oxidants, and/or antibodies against oxidized phospholipids.
• metformin a compound of the disclosure
• statins a compound of the disclosure
• anti-oxidants a compound of the disclosure
• antibodies against oxidized phospholipids Such a combination can have unexpected synergy due to a multifaceted approach to modulating inflammation and
• Such a combination would increase the anti-oxidant effects of adiponectin by increasing adiponectin levels, reduce triglyceride levels thereby reducing circulating phospholipids, reduce insulin resistance, and block the
• the crude reaction was purified by silica gel column chromatography (0 - 20% of ethyl acetate in hexanes) to give both the tert-butyl (2-mercaptoethyl-2-d) carbamate (8) as a colorless oil in 61% yield (167 mg) and the di-tert-butyl (disulfanediylbis (ethane-2 , l-diyl-2-d) ) dicarbamate ( 9 ) as an orange solid in 19% yield (53 mg) .
• N-Boc-glycine methyl ester (0.100 g, 0.528 mmol, 1.00 equiv) was slowly added to a stirred solution of lithium aluminum deuteride (0.027 g, 0.634 mmol, 1.20 equiv) in anhydrous tetrahydrofuran (1 mL) at 0 °C. The mixture was heated at reflux for 3 h and then cooled at 0 °C. Ethyl acetate (2 mL) was first added, followed by addition of Rochelle's salt (1 mL) . The reaction mixture was stirred at ambient temperature for 1 h, then water was added and the mixture was extracted with ethyl acetate (x3) .
• reaction was concentrated and purified by silica gel column chromatography (hexanes /ethyl acetate 80:20) to afford the desired compound as a colorless oil in 51 % yield (0.192 g, 0.605 mmol) .
• disulfanediylbis (ethane-2, 1-diyl-l, l-d2) bis (4- methylbenzenesulfonate) 800 mg, 1.71 mmol, 1 eq
• sodium diformamide 407 mg, 4.29 mmol, 2.5 eq
• the reaction was cooled to r.t. and water was added (4 ml) and the mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried with MgSOi and concentrated in vacuo.
• N (disulfanediylbis (ethane-2 , 1- diyl-1, l-d2) ) bis (N-formylformamide) (107 mg, 0.399 mmol, 1 eq) in methanol (10 mL) was added hydrogen chloride (1.99 mL, 4 molar, 7.97 mmol, 20 eq) and the mixture was stirred at r.t. for lh before it was concentrated to dryness to furnish (D4n- cystamine) : 2 , 2 ' -disulfanediylbis (ethan-1 , l-d2-l-aminium) chloride
• cystamine non-labeled
• d2-cystamine d2-cystamine
• d4-cystamine d4-cystamine
• cystine NEM-cysteamine , NEM-cysteine , cystamine, and 2- (methylthio) ethylamine . All data fit well to single exponential curves (either decay or association) , except where mentioned. [00189] Cystine measurement during constant cystamine exposure:
• Fibroblast cultures were exposed to lOOuM cystamine/d2- cystamine/d4-cystamine for the following timepoints : 0 min, 10 min, 30 min, 1 h, 3 h, and 5 h. Following incubation, cells were washed 2x in PBS containing N-ethylmaleimide . Wash was removed, and cells were harvested on ice using 80% acetonitrile/1% formic acid, containing stable isotope internal standards for cystine (d4- cystine) and cysteamine (d4-cysteamine and d8 cystamine) .
• cystine re-accumulation between cystamine and d4-cystamine are very similar. Though d2-cystamine appears slightly faster, this could be due to this latter
• Fibroblast cultures were exposed to drug (either cystamine, d2-cystamine , or d4-cystamine) and harvested as described on page 2 for the cystine depletion curve.
• drug either cystamine, d2-cystamine , or d4-cystamine
• NEM-cysteamine or NEM-dl-cysteamine or NEM-d2-cysteamine was measured for each respective plate and normalized against the signal for d4-cystine.
• NEM-cysteamine appear to differ between drug types. Higher levels are seen at early timepoints, with the highest seen after non-labeled cystamine exposure (e.g., see FIG. 8A) . Levels decrease at 3 hours, but then rise again for all drug types during the 5-hour exposure (e.g., see FIG. 8A) . The levels of cystine (bottom graph) also show a spike from 3 h to 5 h (e.g. , see FIG. 8B) .
• NEM-cysteamine during washout Fibroblast cultures were exposed to drug (either cystamine, d2-cystamine , or d4-cystamine) for 3 h were then exposed to media for various timepoints and harvested as previously described.
• NEM-cysteine during washout Fibroblast cultures were exposed to drug (either cystamine, d2-cystamine , or d4-cystamine) for 3 h were then exposed to media for various timepoints and harvested as previously described.
• cysteine levels all rise during course of washout period for cystamine, d2-cystamine and d4- cystamine. D2 porduced higher cysteine levels suggesting that it is more effective in reducing intracellular cystine to cysteine and cysteine-D-cysteamine mixed disulfide.
• Cystamine during constant drug incubation Fibroblast cultures were exposed to drug (either cystamine, d2-cystamine, or d4-cystamine) for various timepoints following harvesting as described earlier. Cystamine could be measured in the non-labeled cystamine incubation as well as the d4-cystamine incubation. d2- cystamine could not be accurately measured due to a strong background interference signal corresponding to that mass. No cystamine could be measured in the washout experiments.
• cystamine depletes rapidly during washout. This implies that the drug is getting reduced rapidly in the cells or forming a mixed disulfide. At near saturation levels, additional drug may be slow to enter the cell, hence the plateau after several hours.
• Fibroblasts from a de-identified cystinotic patient were split 3x from 2 plates into 18 plates.
• Two different drugs were used for the experiments: D4n-cystamine , and D8-cystamine .
• the other series included a washout after 3hours of drug incubation, using fresh media following a PBS wash, and included the time- points: 0, lhr, 3hr, and 16hr.
• a direct quench and extraction technique was used for harvesting all cells to minimize further oxidation and metabolism.
• cystine NEM-cysteamine, NEM-cysteine , and cystamine. All data fit well to single exponential curves (either decay or association) , except where mentioned.
• Fibroblast cultures were exposed to 50 mM cystamine (D8 or D4n) for 3 hours. Following incubation, cells were washed with PBS and then incubated with regular mediate containing FBS for lh, 3h, and 16h. Cells were harvested as above. A one phase
• Cystine reaccumulation between D4n-cystamine and D8-cystamine are very similar, indicating similar levels of drug remaining during the washout timepoints (FIG. 15) .
• CDllb Liver Inflammatory Cells.
• CDllb stains macrophages and microglia. CDllb staining of liver tissue was taken after 10 weeks of CDAHFD in three groups of mice. Group 1 control mice (“Cl”) represents the control group that did not receive drug. At 8 weeks, treatment group 2 (“C2") mice started gavage feeding with D2-
• cystamine and cysteamine in hepatocytes were assessed for in vitro metabolic stability of each of the deuterated and non-deuterated cysteamine and cystamine compounds via hepatocyte stability studies.
• liver microsomes primarily assess cytochrome P450 system-mediated metabolism (phase I metabolism)
• hepatocytes represent a complete, undisrupted metabolic system, including cofactors, and thus assess both phase I and phase II metabolism.
• the metabolic stability of test compounds was determined by monitoring, via LC/MS/MS, the percent parent compound remaining versus time of incubation in human hepatocytes.
• hepatocytes were washed by centrifugation and replated in Williams' Medium E supplemented with Heptocyte Plating Supplement Pack, Serum-containing.
• Cells were diluted to 1,000,000 per ml in maintenance media (Williams' E Media supplemented with Hepatocyte Maintenance Supplement Pack, Serum-free) .
• 500 m ⁇ of cell suspension was added to each well of a microtiter plate containing 500 m ⁇ of the deuterated test agent and placed on an orbital shaker at 120 rpm at 37 C.
• 50 m ⁇ aliquots were removed from the well and added to 50 m ⁇ of ice cold ethyl acetate and vortexed for 5 min and froze until chromatography analysis.
• D4-Cystamine is the most stable of the dimeric compounds. This is why D4 results in a higher AUC and Cmax (for its monomer form) compared with D2 and cystamine and also reduced rate of elimination in the hepatocyte elimination studies .
• Table 1 shows that compared to non- deuterated cysteamine and cystamine, the deuterated forms have a slower hepatocyte clearance.
• NASH models from normal C57BL/6 male mice on CDAHFD diet (choline deficient, L-amino acid defined, high fat diet with restricted amount of methionine) were used. Two deuterated cystamine molecules were tested:
• Drug (D2 or D4) was administered in drinking water at a concentration to deliver animals about 200mg/kg/day of drug. Daily weight. Volume of fluid drunk/ day was also measured.
• Group 1 Control mice on CDAHFD for 8 weeks. Blood drawn at 0, 4, and 8 weeks.
• Group 2 Mice on CDAHFD plus D2 at 200mg/kg/day for 8 weeks. Blood drawn at 0, 4, and 8 weeks.
• Group 3 Mice on CDAHFD plus D4 at 200mg/kg/day for 8 weeks. Blood drawn at 0, 4, and 8 weeks.
• FIG. 11 shows ALT levels of the mice in the group.
• Group 1 (gl) control mice ALT levels were elevated by week 8 compared to group 2 (g2; D2) and group 3 (g3; D4) mice.
• FIG. 12 provides a statistical analysis of group 1 controls compared to group 2 (D2) treated mice. Compared with baseline there were similar changes in weight for the three groups.
• the prevention study it is important to recognize that the drug delivery was not "controlled” but was rather delivered via water consumption by the animals and would vary depending upon the amount of water each animal drank throughout the day.
• mice received 8 weeks of CDAHDF diet before beginning treatment with deuterated compounds of the disclosure. During the treatment phase the mice continued to receive CDAHDF diet. Four mice were set in each group: control (Cl) not receiving any drug; treatment (C2) receiving 200 mg/kg of cystamine D2 by once daily gavage; and treatment (C3) receiving 200mg/kg cystamine D4 by once daily gavage .
• FIG. 13A-D show results obtained from this treatment study.
• FIG. 13C shows Sirius Red staining of liver sections. Sirius Red stains for collagen and therefore provides a measure of fibrosis.
• MMPs matrix metalloproteinases
• TRIP metalloproteinases
• Collagen 1 is a more useful marker of fibrosis than TIMP. In all treatment groups there was a reduction in expression of col 1 and TIMP.
• the treatment groups showed significant reduction in the amount of positive liver staining in the D2 and D4 treated groups. There appears to be less pericentral fibrosis in the D4 treated liver (see, FIG. 13C) .
• the data from this staining, as well as reduced expression of collagen 1 (col 1) and TIMP (FIG. 13D) show that the deuterated compounds are reducing established hepatic fibrosis despite continuous CDAHFD.
• H&E staining showed that the treatment groups had reduced fatty deposits compared to control (see, FIG. 17) . This reduction is particularly apparent in D4 group.
• a pilot study was performed with 3 animals to determine whether the methodology was acceptable.
• 2 rats were used for pilot study, each animal received a different dose of cystamine (200mg/kg, and 300mg/kg) .
• Plasma was obtained at 4 time-points per rat, all taken on the same day as drug administration (20min, 40min, 60min, and 2hrs) .
• the plasma concentration of both cyteamine and cystamine were measured. The experiment showed that the pK profile for cystamine and its cysteamine counterpart are very similar, thus providing sufficient findings that the measurements were effective.
• Blood was sampled at 0, 20 min, 40 min, 1, 2, 4 and 6 hours (7 total samples per rat) . Approximately 160 m ⁇ of whole blood was collected into an EDTA retro-orbital blood collection tube and briefly mixed to insure uniform mixing of EDTA. The blood was quickly transferred into a clean ependorf tube containing 40 m ⁇ of fresh 150 mM NEM, mixed and incubated at room temperature for 10 minutes. The Eppendorf tube was centrifuged at 3000 rmp for 5 minute sto pellet RBC and collect plasma into a fresh Eppendorf tube. The volume of the plasma was measured by pipette. Formic acid was added to the plasma to a concentration of 1% and mixed. Table 3 provides the results.

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