Methods and compositions for the treatment of primary sclerosing cholangitis, ulcerative colitis, and crohn's disease

Abstract

Methods comprising administering a therapeutically effective amount of H2S scavenger, and optionally butyrate, as described herein, to a subject who has an inflammatory gut condition, e.g., Ulcerative Colitis (UC), Primary Sclerosing Cholangitis (PSC), colonic Crohn's Disease (CD), pouchitis and inflammation after proctocolectomy with ileal pouch-anal anastomosis (j-pouch surgery) to treat UC, irritable bowel disease (IBD), and subjects who have those conditions who are at risk of developing colon / colorectal cancer.

Description

[0001] Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0002] Methods and Compositions for the Treatment of Primary Sclerosing Cholangitis, Ulcerative Colitis, and Crohn’s Disease

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 778,923, filed on March 27, 2025. The entire contents of the foregoing are hereby incorporated by reference.

[0005] TECHNICAL FIELD

[0006] Methods comprising administering a therapeutically effective amount of H2S scavenger and / or butyrate as described herein, to a subject who has an inflammatory gut condition, e.g.. Ulcerative Colitis (UC), Primary Sclerosing Cholangitis (PSC). colonic Crohn’s Disease (CD), pouchitis and inflammation after proctocolectomy with ileal pouch-anal anastomosis (j-pouch surgery) to treat UC, irritable bowel disease (IBD), and subjects who are at risk of developing colon / colorectal cancer.

[0007] BACKGROUND

[0008] Ulcerative Colitis (UC) is seen as an inflammatory disease of the colon though early initiating causes remain unknown. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammation and fibrosis of the biliary' tree, can lead to debilitating consequences such as end-stage liver disease and malignancies. Currently no therapy has been found to be effective for PSC other than liver transplant, which remains the mainstay of therapy. However, the disease can recur after transplant. PSC is intimately associated with inflammatory' bowel disease (IBD), particularly ulcerative colitis (UC). Approximately 70-80% of patients with PSC also have underlying IBD (Hov and Karlsen, Nat Rev Gastroenterol Hepatol. 2023 Mar;20(3): 135-154; Sayed et al.. Gastroenterology’, 2022. 162(3): p. S76), but only around 3- 5% of UC patients will develop PSC (Mertz et al., Ann Gastroenterol, 2019. 32(2): p. 124-13). Thus, the combination of the two creates a distinct disease phenoty pe, known as PSC-IBD, which is a prototy pical disease of the gut-liver axis (Hov and Karlsen, Nat Rev Gastroenterol Hepatol. 2023 Mar;20(3): 135-154; Hsu and Schnabl, Nat Rev Microbiol, 2023. 21(11): p. 719-733).Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0009] The pathophysiology of PSC remains poorly understood. The paradigm of IBD is relied on as a model to understand PSC, presuming an interaction between the environment, the microbiome and immune system with a genetic predisposition (Noble et al., Gastroenterology7. 2023 Jul;165(l):44-60.e2; Kuhnen, Surg Clin North Am. 2019 Dec;99(6): 1197-1207). Other hypotheses suggest a toxic bile acid or a cross reacting T cell response between the colonocytes and cholangiocytes (Hov and Karlsen, Nat Rev Gastroenterol Hepatol. 2023 Mar;20(3): 135-154; Vesterhus and Karlsen, J Gastroenterol. 2020 Jun;55(6):588-614). The immune modifying approaches that can be effective in IBD do not benefit PSC. A distinct dysbiotic fecal microbiome has been detailed in PSC which overlaps with that of IBD (Hov and Karlsen, Semin Liver Dis. 2017 Nov;37(4):314-331). While numerous studies have investigated changes in the gut microbiome in the context of IBD63’73, only a few have focused on PSC74'79.

[0010] SUMMARY

[0011] Provided herein are methods for treating Ulcerative Colitis (UC), Primary Sclerosing Cholangitis (PSC), colonic Crohn’s Disease (CD), pouchitis and inflammation after proctocolectomy with ileal pouch-anal anastomosis (j-pouch surgery) to treat UC, irritable bowel disease (IBD), and for reduction of risk of developing colon / colorectal cancer in a subject. The methods comprise administering to the subject a treatment comprising a therapeutically effective amount of (i) an H2S scavenger and optionally (ii) butyrate. In some embodiments, only the H2S scavenger is administered, e.g., the butyrate is not administered.

[0012] In some embodiments, the H2S scavenger is hydroxocobalamin, aquohydroxocobinamide, cobinamide, or a sulfonyl azide (e.g., SS20).

[0013] In some embodiments, the therapeutically effective amount decreases levels of fecal calprotectin, and / or decreases urinary' and / or plasma nitrate, nitrite, and / or nitrosothiol levels, in the subject.

[0014] In some embodiments, the methods further comprise measuring one or more of fecal calprotectin levels and / or urinary and / or plasma nitrate, nitrite, and / or nitrosothiol levels, prior to and after initiating the treatment, and determining that a therapy7is effective if it decreases one or more of fecal calprotectin levels and / or urinary and / or plasma nitrate, nitrite, and / or nitrosothiol levels.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0015] In some embodiments, the methods comprise comprising administering hydroxocobalamin at 1-5, 1-4, or 1-2 g daily. In some embodiments, an initial loading dose of 4-5 or 4-10 g is administered intravenously (IV), and subsequently oral doses of 1-5, 1-4, or 1-2 g daily are administered orally.

[0016] In some embodiments, the methods comprise administering hydroxocobalamin in 1-2500mg capsules twice a day or 3-4 300 mg capsules twice a day, optionally 3 300-500 mg capsules are administered wherein two are administered in the morning and one is administered at night.

[0017] In some embodiments, the methods comprise administering 20 mg -240 mg buty rate daily, preferably in a divided dose (e.g., 120 mg twice daily).

[0018] In some embodiments, the H2S scavenger, the butyrate, or both, are administered in a colonic release formulation.

[0019] In some embodiments, the subject has Primary' Sclerosing Cholangitis (PSC). In some embodiments, the subject does not have UC.

[0020] Also provided herein are oral dosage forms, e.g., capsules or tablets, comprising 240, 250. 300, 400, or 500 mg of hydroxocobalamin, and an excipient, e.g., for use in a method described herein.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials know n in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0022] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0023] DESCRIPTION OF DRAWINGS

[0024] Figures 1A-C. Overview of nutrition study design. Two dietary interventions (LPD and SCD) w ere compared and profiled fecal metagenomic and metabolomic data at four time points. 3-day dietary' records, co-diagnosis of IBD (UC, CD, or non-IBD), and clinical parameters were collected. After dietary interventionsAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0025] (A), the response outcomes and temporal trajectory were analyzed (B) and interpreted through multiple perspectives (C).

[0026] Figures 2A-F. LPD dietary intervention benefits patients with PSC, whereas SCD exacerbates the condition.

[0027] a) Protein consumption in LPD and SCD. Each line represents an individual. b) Percent change in alkaline phosphatase (ALP) from baseline before, during and after dietary intervention. Each line represents an individual.

[0028] c) Coefficients of selected nutrient features after Lasso regression, indicating that reduced protein intake significantly lowers ALP values. “Prof ’ is short for protein.

[0029] d-f) Variations in ALP values and the potential rates to respond indicated differing responses among three disease categories, d) PSC-UC, e) PSC-CD, and f) PSC-alone. The Mann- Whitney U test was employed to assess significance between LPD and SCD, with *** denoting strong significance (p-value < 0.001), ** very significant (0.001< p-value < 0.01), * significant ( 0.01< p-value < 0.05), and ‘ns’ indicating no significant difference (p-value > 0.05).

[0030] Figures 3A-D. Fecal metagenomics predicts response outcomes.

[0031] a) PCoAplot of the gut microbial compositions of LPD samples, differentiated by response outcomes (blue for responders: individuals showing decreased ALP, and pink fornon-responders: individuals showing increased ALP, shows that LPD responders have distinct microbial structure from LPD non-responders. Arrows were drawn to connect samples from the same individuals in chronological order.

[0032] b) Heatmap of bacterial species markers that can distinguish LPD responders from non-responders and the abundance distribution of these markers correlate with disease categories, as determined by multivariate linear regression models (target p-value < 0.05 and FDR < 0.2). Each column in the heatmap is a sample, and all the samples including both LPD responders and LPD non-responders are clustered based on the similarity of the bacterial composition. Each row is a species, and species are clustered based on the similarity of the distribution pattern across samples of the specified species.

[0033] c) PC A plot of the gut metabolite compositions of LPD samples, differentiated by response outcomes (blue and pink), with arrows to connect samples from the same individuals in chronological order.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0034] d) Heatmap of metabolite markers that can distinguish LPD responders from non-responders. The columns in this heatmap are arranged to correspond directly with the order of the columns in the microbial profile heatmap in Fig. 3b.

[0035] Figures 4A-J. The fecal metabolomics predicts the disease improvement trajectory under LPD in those LPD responders.

[0036] a) Volcano plot derived from multivariate linear regression models, using time series metabolite intensity profiles from WO, W4, and W8 and adjusting for covariates including disease categories. The metabolites were colored according to the class subclasses.

[0037] b) Metabolite set enrichment analysis (MSEA) of selected metabolites with significant diet-induced changes after the regression model in Fig.3a (p-value < 0.05).

[0038] c-f) Temporal changes in four key sulfur metabolism metabolites, including methionine, methionine sulfoxide, taurine, and cysteine. Bars are colored blue to represent metabolites that decreased after LPD, and orange for those that increased. The Mann-Whitney U test was employed to assess significance between sampling time points, with * denoting significance (0.01< p-value < 0.05), and 'ns’ indicating no significant difference (p-value > 0.05).

[0039] g-j) The relationship between each of the four metabolites and ALP values tested using Spearman correlation. Cysteine shows a negative association with ALP values, whereas the other three metabolites have no significant association (p-value < 0.05).

[0040] Figures 5A-E. Microbial sulfur metabolism and associated bacterial species.

[0041] a) Interactions among microbial sulfur metabolism genes, related metabolites, and relevant microbial species. Nodes were colored blue to represent features that decreased after LPD and red for those features that increased. Edge gradients represent the coefficients of Spearman correlation between the abundance (or intensity) of nodes. Only associations deemed significant, with FDR < 0.05, are shown here.

[0042] b-c) Shifts in the stratified abundance in bacterial species for two represent genes (bsh and cysK) after dietary treatments. Each stacked bar is a sample, with the left panel in purple background representing LPD responders at W4 (dark purple), W8 (purple) and W14 (light purple). In contrast, the right panel in pink backgroundAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0043] corresponds to samples from the SCD treatments. Each bar is stacked with components representing the stratified abundance of annotated bacterial species identified as hosts of the specified genes. Species are only colored if their stratified abundances showed significant changes (either increases or decreases) at W4 (or W8) compared to the baseline in those LPD responders. Unit cpm represents counts per million.

[0044] d-e) Temporal changes of two key bacteria involved in sulfur metabolism: d) Eggerthella lenta and e) Roseburia intestincilis . Bars are colored blue to represent metabolites that decreased after LPD, and orange for those that increased. The Mann-Whitney U test was employed to assess significance between sampling time points, with ** denoting very significant changes (0.001< p-value < 0.01), * significant (0.0K p-value < 0.05), and ‘ns’ indicating no significant difference (p-value > 0.05).

[0045] Figure 6. Characterized and proposed sulfur-related metabolic pathways in response to LPD intervention benefiting patients with PSC. Metabolites and genes that increased are colored red, those that decreased are blue, while those without significant changes are gray (Mann-Whitney U test p-value < 0.05).

[0046] Abbreviations used are as follows: MSF: methionine-sulfoxide; Hyc: homocysteine; Ctt: cystathionine; LA: L-alanine; 3-MP: 3-Mercaptopyruvate; Tauro-CA: taurocholic acid; CA: cholic acid; 7-keto-CA: 7 -ketodeoxy cholate; 12-dehydro-CA: 12-dehydrocholate.

[0047] DETAILED DESCRIPTION

[0048] Therapeutic approaches to treating UC have been directed at treating the inflammatory' response with a variety of immune suppressants such as steroids, azathioprine and anti-TNF agents such as infliximab. The fundamental cause remains uncertain. Primary sclerosing cholangitis (PSC) is associated with UC and Crohn's disease (CD) with about 3-5% of individuals with UC developing PSC and about 70% of those with PSC have UC and 10% have Crohn's- with about 20% having PSC alone. No therapy exists for PSC, which consequently results in liver failure, cholangiocarcinoma, or colon cancer within 10-20 years of diagnosis of PSC. The therapies that can be reasonably effective in UC and CD show no benefit for PSC (Vesterhus and Karlsen, J Gastroenterol. 2020 Jun;55(6):588-614). Therapies for UC and CD are essentially all directed at suppressing the immune system; however, results have been unsatisfactory for many patients.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0049] Blockage of the butyrate oxidation pathway may be one route that activates the inflammatory response in ulcerative colitis. Efforts to test this conception of the disease as tire basis for treatment have been limited. Studies of butyrate enemas had some positive studies but larger studies were negative for a larger effect (Sheppach W. 1996). However, we believe there is another factor inhibiting the proper incorporation and use of butyrate that is contributing to tire inflammatory effects we see in UC flares: hydrogen sulfide.

[0050] Although acute overexposure to hydrogen sulfides is rare, it can be fatal and has been a focus of researchers looking into industrial production exposures. It blocks mitochondrial function and increases the oxidative substrates that will in turn produce an inflammatory response. Without wishing to be bound by theory, the present inventors hypothesize that PSC, UC, and Crohn’s disease all result at least in part from an impairment of mitochondrial function, caused by excessive production of bacterially produced H2S (possibly from bacterial digestion of sulfur-containing amino acids such as cysteine, methionine and taurine, e.g., increased bacterial degradation of mucin, w hich is rich in cysteine and methionine) and / or reduced detoxification of H2S and subsequent production of NO1’2,3.

[0051] Briefly, without wishing to be bound by theory, it is believed that there are three interrelated steps of bacterial function driving the inflammatory state in UC: 1) increased degradation with thinning of the mucous layer causing a release of cysteine, leading to 2) increased H2S production combined with reduced detoxification w hich then contributes to mucosal injury compounded by 3) increased NO production. Each of these steps initiate and amplify a mucosal inflammatory response.

[0052] Again, without wishing to be bound by theory, H2S production is believed to be in part coupled with increased degradation of mucin. Mucin is rich in cysteine and methionine, the two amino acids that serve as prime sources of sulfur for the sulfate reducing bacteria that generate H2S.5An impairment in the enzymatic degradation of H2S by mucosal enzymes enables H2S to penetrate the mucosa. Increased mucosal levels inhibit mitochondrial functions leading to a reduction in ATP generation and increased production of pro-inflammatory reactive oxygen species. H2S further reduces mucin production and impairs colonocyte uptake and P-oxidation of butyrate. The availability of H2S enables the activation of bacterial production of nitric oxide (NO) which contributes to the worsening of the inflammatory state. The inflammatory state is driven by (1) thinning of the mucous layer- causing a closer association of bacteria to the mucosa and subsequent activation of mucosal immunity, (2) ER stress and activation of innate andAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0053] adaptive immunity through impairment of mitochondrial function by FtyS and subsequent activation of the inflamniasome. Aspects of these intertwined mechanisms have been suggested in both UC and CD with H2S recently suggested to be critical in CD while all elements may be central to UC5,6. Consequently, it is hypothesized that NO levels measured via nitrite, nitrate, and / or nitrosothiol levels may reflect the inflammatory state and will thus parallel disease activity. See, e.g., Stummer et al., Antioxidants (Basel). 2023 Aug 6; 12(8): 1570; Avdagic et al., Bosn J Basic Med Sci. 2013 Feb; 13(l):5-9).

[0054] No therapies currently exist or have ever been studied to reduce levels of H2S in UC or PSC. However, as shown in Example 1, a low-protein diet that reduced intake of sulfur-containing foods provided therapeutic benefit in subjects with PSC. Thus, the methods described herein involve reducing hydrogen sulfide in the intestine, which is a significantly different mechanism of disease that appears relevant to treat subjects with UC, PSC, patients with colonic CD (e.g., who are perinuclear antineutrophil cytoplasmic antibody (pANCA) positive), pouchitis and inflammation after proctocolectomy with ileal pouch-anal anastomosis (j-pouch surgery) to treat UC, irritable bowel disease (IBD), and for reduction of risk of developing colon / colorectal cancer, comprising administering a combination of (i) an H2S scavenger such as hydroxocobalamin. cobinamide, or another analog of vitamin Bl 2, and (ii) butyrate, preferably in a delayed colonic release form that delivers most of both of the agents to the colon. The methods can be used in combination with recommending or following a low-protein / low-sulfur diet.

[0055] HjS Scavengers

[0056] The present methods use agents that can act as scavengers to remove H2S from the colon. H2S scavengers can include hydroxocobalamin. cobinamide, or sulfonyl azide compounds. Hydroxocobalamin is being utilized to treat cyanide toxicity, which can also bind to H2S and reverse acute H2S toxicity. Although acute overexposure to H2S is rare, it can be fatal and has been a focus of researchers investigating industrial accidents and potential terrorist threats. Mitochondrial blockage by H2S parallels cyanide poisoning. Researchers have attempted to treat H2S toxicity in animal models with similar therapies to those used for cyanide poisoning. Acute cyanide exposure can be treated with sodium thiosulfate and sodium nitrite44,45. Alternate approaches have been developed, including treatment with high-dose IV or intramuscular hydroxocobalamin, an analog of vitamin B12 known as vitamin B12a46. This analog has been tested and found to be more effective and non-toxic: hydroxocobalamin is now a standard therapy and was approved for the treatment of cyanide toxicity in the U.S. in 2006 by the FDA. This medication has beenAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0057] studied in sheep models of H2S toxicity, where sheep were exposed to a lethal dose of H2S and were subsequently rescued using vitamin B12a47. Additionally, case reports suggest that hydroxocobalamin may also be used to treat acute H2S poisoning in humans48. Hydroxocobalamin is sold as an oral supplement and is used in the United States and numerous other countries to aid in B12 deficiency. It is a precursor of vitamin B12 and is used to treat pernicious anemia as well as other causes of B12 deficiency, though at a low er dose than the present methods.

[0058] Currently, high-dose hydroxocobalamin is the standard treatment of choice for cyanide toxicity. Patients who present with cyanide poisoning are given 5 grams of hydroxocobalamin IV, and a repeat dose of 5 grams if no response is seen after the first dose. Common side effects seen at these high doses include chromaturia (red urine) and reddening of the skin, likely attributable to the red color of tire substance. When tested in H2S poisoning, similar side effects were seen. A placebo-controlled study was done to determine safety in healthy volunteers (n=136). Patients were treated with 2.5, 5, 7.5, and 10 grams of hydroxocobalamin, administered intravenously over approximately 7.5 to 30 minutes49, indicated a safety profile that is benign enough to recommend its use in tire pre-hospital setting. No serious adverse events were seen.

[0059] In addition, hydroxocobalamin is used w orldwide for treatment of B12 deficiency. Doses used orally are usually in the range of 1 mg daily but sometimes more. Doses of 1 mg IM weekly for up to 6 months are also commonly utilized. Receptors in the ileum would be saturated with the oral doses used in the present methods, such that not much of the oral dose of hydroxocobalamin would be absorbed in the ileum. A maximum of 1-2 mg could be absorbed orally as the receptors are saturated for 3-4 hours. In some embodiments, a delayed release formulation is used to minimize absorption as well, so that only about 1% is absorbed passively. Even at the massive doses used for cyanide toxicity, hydroxocobalamin has proven not to be dangerous. As a vitamin requiring a receptor to be absorbed, most of the hydroxocobalamin delivered in a large dose goes to the colon without being absorbed. As a w ater-soluble vitamin, most of what is absorbed is excreted, though there is some element of enterohepatic circulation. The amount absorbed beyond capacity7for storage is excreted in the urine. In some embodiments, the total maximum absorption over tire course of 30 days would be 150-180 mg, e.g., 168 mg total w ith excretion of excess hydroxocobalamin. In the present methods a delayed delivery formulation can be used resulting in a smaller percentage being absorbed.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0060] Butyrate

[0061] In some embodiments, the methods include administering butyrate in combination with an H2S scavenger. Butyrate has been sold as a supplement for oral delivery in the United States since the 1970s and is an endogenous compound produced by bacteria in the intestine. Butyrate is Generally Recognized as Safe (GRAS) and is safe as a food additive, as an animal food additive, and as a supplement. In addition, tributyrin which delivers butyrate to the duodenum is also GRAS. Oral delivery of butyrate has been utilized in a variety of human clinical studies including 4 grams orally for Crohn’s disease in an enteric coated capsule for 8 weeks50and for ulcerative colitis 4 grams in enteric coated capsules daily for 6 weeks at51. Neither study identified any significant adverse events. Butyrate is currently available as a supplement in the United States. Butyrate enemas up to 4 grams have been studies in IBD as well and numerus studies have aimed to delivery complex carbohydrates to deliver butyrate to the colon.

[0062] Methods of Treatment

[0063] The present methods can be used to treat UC, PSC, colonic CD, pouchitis and inflammation after proctocolectomy with ileal pouch-anal anastomosis (j -pouch surgery) to treat UC. irritable bowel disease (IBD). and for reduction of risk of developing colon / colorectal cancer; as described herein, it is believed that the production of hydrogen sulfide in the intestines is relevant to the central elements of the pathophysiology of each of these diseases.

[0064] In the present methods, a high dose of an H2S scavenger, e.g., hydroxocobalamin, cobinamide, aquoh droxocobmamide, or a sulfonyl azide (e.g., SS20), is given, e.g.. twice a day. For example, hydroxocobalamin can be administered orally at a dose of 1-5 g daily (e.g., 500 mg, 1 g, 1.2 g, 1.24g, l-2g, 2g, or 2.5 g twice a day); optionally at least 50, 100, 250, 400, 500, 1000, 1200, 1240, 1250, or 2000 mg are administered once or twice or three times a day. Cobinamide can be administered orally, e.g., at a dose of 10 mg - 4g per day (e.g.. 25 mg - 2 grams twice a day); optionally at least 10, 20, 40, 50, 100, 250, 500, or 1000 mg are administered once or twice a day. In some protocols, an initial loading dose of 4-5 or 4-10 g hydroxocobalamin is administered intravenously (IV), and subsequently oral doses of 1-4 or 1-5 g daily are administered orally.

[0065] In addition, subjects can be dosed with butyrate, e.g., in one or a plurality of capsules once or twice daily, for a total dose of 100-500 mg, e.g., 200-250 mg, e.g., about 240 mg daily.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0066] In preferred embodiments, a colonic release formulation is used. An exemplary formulation is ProButyrate™ (Tesseract Medical Research), which has about 24 mg of butyrate per capsule, formulated utilizing a cyclodextrin-based molecular trap; see, e.g., WO / 2021 / 191803 and WO / 2017 / 136775. Other colonicrelease formulations can also be used, e.g., pH-sensitive methacry late polymers (EUDRAGIT), time-based formulations, and microbiota-dependent systems, among others, or a combination thereof; see, e.g.. McCoubrey et al., J Control Release. 2023 Jan:353:1107-1126; Philip and Philip, Oman Med J. 2010 Apr; 25(2): 79-87; Singh, Recent Pat Drug Deliv Formul. 2007;l(l):53-63; Doggwiler et al., Int J Pharm. 2023 Jan 25;631:122499. Numerous probiotics are also being developed that deliver butyrate producing probiotics to the colon.

[0067] In some embodiments, the H2S scavenger and butyrate are administered in a single dosage form, e.g., combined together in a tablet or capsule. In some embodiments, the H2S scavenger and butyrate are administered in separate dosage forms, e.g., in separate tablets or capsules, e g., at substantially the same time (e.g., within 1, 2, 5, 10, or 15 minutes of each other).

[0068] Generally, the methods include administering a therapeutically effective amount of H2S scavenger, and optionally buty rate, as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.

[0069] Subjects include mammals, e.g., humans ornon-human veterinary’ subjects, who have an inflammatory gut condition, e.g., UC, PSC, colonic CD, pouchitis and inflammation after proctocolectomy with ileal pouch-anal anastomosis (j -pouch surgery) to treat UC, irritable bowel disease (IBD), and subjects with those conditions who are at risk of developing colon / colorectal cancer. "‘Subject’' and “patient” are used interchangeably herein.

[0070] As used in this context, to “treat” means to ameliorate at least one symptom of the inflammatory’ gut condition. Often, inflammatory’ gut conditions such as the above result in symptoms including abdominal pain; diarrhea, sometimes with blood; urgency to have a bowel movement and / or fecal incontinence; rectal bleeding; weight loss; fever; anemia; and for PSC, itchy skin, diarrhea, j aundice, and feeling tired or weak. A treatment as described herein can result in a reduction in severity' and / or frequency of one or more of the symptoms. In addition, administration of a therapeutically effective amount of a compound described herein for the treatment ofAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0071] an inflammatory gut condition may result in decreased levels of fecal calprotectin, and / or decreased urinary and / or plasma nitrate, nitrite, and / or nitrosothiol levels. Thus the methods can include measuring one or more of fecal calprotectin levels and / or urinary and / or plasma nitrate, nitrite, and / or nitrosothiol levels, prior to and after initiating a therapy (eg a therapy as described herein), and determining that a therapy is effective if it decreases one or more of fecal calprotectin levels and / or urinary and / or plasma nitrate, nitrite, and / or nitrosothiol levels.

[0072] Oral Dosage Forms

[0073] Also provided herein are oral dosage forms, e.g., for colonic delivery of hydroxocobalamin, e.g., for use in the methods described herein. The dosage forms can be, e.g., capsules or tablets, optionally enteric-coated for colonic delivery.

[0074] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy , 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0075] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Other earners and excipients as known in the art can also be used.

[0076] Preferably the oral dosage forms are enteric-coated for colonic delivery. Often enteric coatings comprise one or more pH-dependent polymers that remain insoluble in the stomach and proximal small intestine, dissolving in the less-acidic distal ileum and colon. Exemplary polymers, such as methacrylic acid copolymers (e.g., EUDRAGIT S, L, and FS) and Cellulose Acetate Phthalate (CAP), can be applied asAtorney Docket No. 29618-0428WO1 / BWH 2023-135

[0077] thick coatings. See, e.g., McCoubrey et al., J Control Release. 2023 Jan:353:1107-1126; Gazzaniga et al.. Pharmaceutics. 2022 Dec 9;14(12):2762. The polymers can be used in combination with microbiota-degradable coatings (e.g., PHLORAL) or time-controlled systems (e.g., EUDRACOL).

[0078] An exemplary formulation is shown in Table A.

[0079] TABLE A: Exemplary Hydroxocobalamin 400mg, Enteric coated Capsule Weight (mg) per Component %w / w %w / w (w / capsule)

[0080] capsules Hydroxocobalamin

[0081] 89.87 68.72 400.0 Base (API)

[0082] CAB-O-SIL® M5-P

[0083] (Silicon Dioxide) 1.18 0.90 5.25 USP / NF / EP / JP

[0084] Microcrystalline

[0085] Cellulose, NF (Avicel 5.40 4.13 24.03 PH 102)

[0086] Magnesium Stearate,

[0087] 2.37 1.81 10.55 Powdered, NF / EP / JP

[0088] Polyplasdone XL-10,

[0089] Micropulverized, 1.18 0.90 5.25 Pharmaceutical Grade

[0090] Total (blend) 100.0 - 445.1 Capsules:

[0091] Capsuline Size 00 - 23.54 137.0 (1 ea.) Vegetarian Enteric

[0092] Capsules White / White

[0093]

[0094] Total - 100.0 541.1

[0095] EXAMPLES

[0096] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0097] Example 1. A randomized clinical trial examining a Low-Protein Diet Versus the Specific Carbohydrate Diet in Patients with Primary Sclerosing Cholangitis

[0098] Methods

[0099] The following methods and materials were used in Example 1.

[0100] Study setting and design. This was a decentralized randomized controlled trial across the United States. Participant enrollment occurred between August 28, 2020 and June 9. 2021. Recruitment occurred in-person and by video remotely, and participants were able to conduct the study remotely. Recruitment was facilitated viaAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0101] physician referral, patient advocacy organizations (PSC Partners Seeking a Cure and Consortium for Autoimmune Liver Disease), and self-referral.

[0102] Eligible participants were randomized in 1 : 1 fashion to the LPD and SCD at a screening visit (week 0, W0, referred to as “baseline”) during which eligibility was assessed and bloodwork, stool, baseline Food Frequency Questionnaire (FFQ) and Patient-reported Outcome Measures (PROMs) were collected. Thereafter, participants were given two weeks for education on appropriate guidelines for the assigned dietary intervention as w ell as procurement of food. The dietary’ intervention began at week 2 (W2) and lasted for 8 consecutive weeks until week 10 (W10) under dietitian supervision, with an option to continue self-directed for an additional 4 weeks (W14). There was a total of 7 video visits with a research dietitian. Additional dietary’ counselling was also offered between screening and baseline visits to provide further instruction and support. Three-day’ food diaries, Woodw ork, stool samples and PROMs w ere collected at 7 points throughout the study (Fig. la).

[0103] Eligibility criteria. Adults between 18 and 70 years of age with large-duct PSC diagnosed by typical cholangiogram findings with no evidence of a secondary cause of sclerosing cholangitis and with serum ALP >1.5 times the upper limit of normal (ULN) were potentially eligible for enrollment. Participants with concomitant UC or CD were eligible if the Simple Clinical Colitis Activity Index or Harvey-Bradshaw Index, respectively, were <5. Additional inclusion criteria included platelet count 2yl50.00() / mm\ serum albumin > 3.3 g / dL, serum creatinine < ULN, and stable dose or no use of ursodeoxycholic acid (UDCA) for at least 3 months prior to enrollment for those taking and not taking UDCA respectively. Proficiency in English and ability to complete PROMs independently was also a requirement. Exclusion criteria included pregnancy or lactation, ALT above 10 times the ULN, total bilirubin at least twice the ULN, INR >1.2, decompensated cirrhosis, small duct PSC, other etiologies of liver disease, positive AMA, history’ of liver transplantation, history’ of hepatocellular carcinoma or cholangiocarcinoma, ascending cholangitis within 90 days of enrollment, antibiotic use within 6 weeks prior to enrollment or planned during the study period, current vegetarian or adherence to the SCD, nut allergy given that nut flour is a dietary’ staple of many SCD recipes and nut allergy could compromise diet adherence, celiac disease, history' of malignancy w ithin 5 years with the exception of adequately treated cervical carcinoma in situ and basal or squamousAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0104] cell carcinoma, inability to complete a dietary log. or concurrent participation in another therapeutic clinical trial. Medical records of all individuals were reviewed at the screening visit to determine eligibility.

[0105] Dietary interventions. The LPD was developed according to 2015-2020 USDA Dietary7Guidelines of a vegan diet which state the diet should be rich in grains, legumes, nuts and other plant-based proteins, with a focus on increased consumption of fruits, vegetables and healthy fats (U.S. Department of Health and Human Services and U.S. Department of Agriculture. 2015-2020 Dietary’ Guidelines for Americans.

[0106] 8th ed. Washington, DC: US Government Printing Office; 2015). In addition to these guidelines, the diet restricted high-sulfate items, defined as containing >100mg sulfur per 100g of food item. A ty pical vegan diet contains approximately 2.3g / day of sulfur-containing amino acids, falling within the estimated range of appropriate sulfur content of 2.1-3.0 g / day (Tuttle SG, Bassett SH, Griffith WH, Mulcare DB, Swendseid ME. Further Observations on the Amino Acid Requirements of Older Men: II. Methionine and Lysine. Am J ClinNutr 1965;16:229-31 ). The SCD followed the dietary guidelines detailed in the book Breaking the Vicious Cycle by Elaine Gottschall (Gottschall, EG. Breaking the Vicious Cycle: Intestinal Health Through Diet. Kirkton (ON): Kirkton Press; 1994.). Participants were provided with education materials, recipes and a food procurement stipend for their assigned diet at the screening visit. Total daily energy intake was calculated by the Mifflin St. Jeor equation using an activity factor of 1.3 reflecting light activity' and exercise level (Fig. la).

[0107] Assessment of diet composition and adherence. Habitual diet was recorded using a validated FFQ at the screening visit (week 0) that queried dietary habits over the preceding year, as well as a 3-day food diary at the baseline visit (week 2) prior to the intervention phase. During the intervention phase, 3-day food diaries were recorded at four time points (weeks 4, 6, 8, 10) (Fig. la). One of these days included a weekend. Participants recorded diet in real-time via photodocumentation of each meal, snack and beverage using a smartphone application that was customized for this study. The application transmitted time-stamped photographs taken during the recording period, along with a reference-sized study ruler to assess relative sizes of food items. Caloric intake and micronutrient composition of each meal was calculatedAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0108] and analyzed using Food Processor, a program with the ability- to quantify protein intake, as well as cysteine, methionine and taurine content as a proxy for sulfur.

[0109] In order to optimize compliance, the research team reviewed dietary records in real-time and discussed barriers to compliance with participants when appropriate. Dietary- analysis was also performed at the conclusion of each diet recording period to further assess compliance. Feedback was provided to participants via the application if there were fewer than 2 entries per day or if a 24-hour period elapsed without any recording. The study coordinator followed up by email and / or telephone if no confirmation was received from the participant within 24 hours of the communication.

[0110] Outcome measures. The outcome measures were response in alkaline phosphatase (ALP), Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) (Fig. lb).

[0111] Whole Metagenomic Sequencing. Stool samples were frozen w ithin 15 minutes of collection and maintained at -80 °C. DNA extraction, whole-genome shotgun library preparation, and Illumina sequencing were performed. The metagenomics sequencing targeted approximately 5 Gb of sequences per sample, utilizing 151 base pair paired-end reads.

[0112] Read-level quality control and metagenomic profiling. Raw sequencing reads underwent quality control (QC) using KneadData version 0.12.0 (huttenhower.sph.harvard.edu / kneaddata / ). Briefly, the first step of this process utilizes Trimmomatic version 0.39 for forward / reverse adapter removal, low-quality reads trimming and tandem repeats removal using the default parameters.

[0113] Contaminants from the host (human) genomes w ere subsequently identified and removed by mapping against the reference database (hg37_and_human_contamination) using Bowtie2 version 2.5.1.

[0114] After QC, each dataset contained over 2.8 Gb of clean, paired-end reads. Clean reads were then taxonomically profiled using MetaPhlAn4 version 4.0.6, which includes an updated database significantly larger than the previous version 3.1.

[0115] Species-level relative abundances were considered in most of this study, except the F / B ratio calculation. Species that failed to exceed 0.1% average relative abundance or were detected in less than 4 samples were excluded. Functional profiling was performed using HUMAnN3 version 3.6 under default parameters according to the UniRef90 definition to get the relative abundance of gene families in the unit of readsAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0116] per kilobase (RPK). And MetaCyc provides pathway definitions to group gene families to get pathway abundance and coverage. The abundances of gene families were further transformed into the unit of copies per million (CPM), and we regrouped the gene families to KEGG Orthogroups (KOs), to filter out 74 gene families that were involved in the microbial sulfur transformation.

[0117] Global untargeted metabolomics. Stool samples were sent for global untargeted metabolomics following the standard procedure. Briefly, samples were pretreated to remove proteins and recover chemically diverse metabolites. The resulting extract was divided into five fractions: two for analysis by two separate reverse phases (RP) / UPLC-MS / MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP / UPLC-MS / MS with negative ion mode ESI. one for analysis by HILIC / UPLC-MS / MS with negative ion mode ESI, and one sample was reserved for backup. All methods utilized a Waters ACQUITY ultraperformance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution / accurate mass spectrometer interfaced with a heated electrospray ionization (HES1-1I) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Raw data w ere extracted, peak-identified and QC processed using the contractor’s hardware and software. Compounds w ere identified by comparison to library entries of purified standards or recurrent unknow n entities. The intensity of each compound was quantified using area-under-the-curve.

[0118] Metabolite-level quality control and pretreatment. Metabolite intensities w ere normalized as z-scores:

[0119] Xj - fi

[0120] (j

[0121] where,*; represents the original metabolite intensity, is the mean value of that metabolite intensity across all samples, and cr is the standard deviation of that metabolite intensity7across all samples. Subsequently, missing values were imputed using the minimum normalized value for each compound. The normalized and imputed values were used in the association and multivariate linear regression analyses throughout the context. With the exception of the estimation of temporal changes of metabolite intensity' in each individual, the original intensities of specified metabolites in W0 were subtracted from their original intensity in W4 (or W8, W14).

[0122] Statistical analyses. Prior to downstream analysis, metagenomic and metabolomic w ere combined. Samples with complete profiles of both types were usedAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0123] in the downstream analysis, totalling 64 samples. Three samples with only metabolomics datasets were excluded. The intake of 91 nutrient features, measured in grams, was adjusted for the weight of each patient followed by Lasso regression utilizing the 'glmnef (ref) library in RStudio to select significant dietary features (predictors) that are non-zero. The response variable was the corresponding ALP value. Subsequently, we incorporated the selected features and included patient ages as an additional variable to construct a linear model. The coefficients and p-values were extracted for statistical significance. For each patient, the Change of ALP value is estimated by subtracting the baseline ALP (WO) from the ALP at W4 (or W8, W14), then dividing by the baseline ALP (WO). Then for each disease category (PSC-UC / PSC-CD / PSC-alone), the Potential to Response was further calculated as the number of samples with reduced ALP value divided by the total number of samples. Alpha diversity7of gut microbiota was estimated using the Shannon index. Principal coordinates analysis was performed using the bray-curtis distance of the relative abundance of species in all samples. Metabolite set enrichment analysis (MSEA) was conducted quantitatively through the online platform of MetaboAnalyst 5.0.

[0124] Linear regression models. We used multivariate linear regression models to identify potential microbial markers distinguishing responders from non-responders after LPD. Abundances were fitted with the following species-specific linear mixed-effects model:

[0125] feature ~ response outcomes + diagnostic categories + gender + age +BMI + (1 | individual) + 8 (Formula 1)

[0126] In each species-specific multivariate model, the abundance of each species was modeled as the function of the binary7response outcomes (responders / non-responders, with non-responder as reference) within each individual (as random effect), while adjusting for diagnostic categories (PSC-UC / PSC-CD / PSC-alone, with PSC-alone as reference), genders (male / female, with male as reference), age (continuous variable) and BMI (continuous variable). MaAsLin2 in RStudio was the package used to fit the model, with p-values adjusted for multiple hypothesis testing and a target FDR of 0.2.

[0127] Separately, another series of multivariate linear regression models were fitted to find a differential abundance of features after LPD versus baseline using microbial profile, metabolic profile, and microbial functional profile, respectively. The model is as below:Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0128] feature ~ treatment stage + diagnostic categories + gender + age +BMI + (1 | individual) + e (Formula 2)

[0129] The difference between this model and the previous one is that the abundance of each feature is modeled as the function of the treatment stage (baseline / after LPD, with baseline as reference) instead of response results, while the rest fixed effect and random effect are not changed. The baseline corresponds to samples in WO, while after LPD corresponds to samples in W4 and W8. Only those with a response of ALP were considered to fit these models to explain the dietary effects of the LPD.

[0130] Example 1.1 Patient characteristics

[0131] We screened -100 individuals with PSC, and 25 met eligibility criteria and 5 eligible individuals chose not to participate prior to randomization. Ten participants were randomly assigned to the LPD and ten to the SCD (Figs. 1A-C). Participant characteristics were balanced between the groups, however, the LPD group had male predominance (70% LPD vs. 40% SCD). Two participants withdrew due to cholangitis, and their data until withdrawal were incorporated into the analysis.

[0132] Participants in the LPD group demonstrated lower protein consumption (grams per kcal per kg body weight) in 3-day food recordings at all study time points during dietary intervention compared to SCD group participants (Mann- Whitney U test, p-value < 2.2e-16). supporting the compliance with the dietary interventions (Fig. 2a).

[0133] Example 1.2 LPD benefits PSC patients, but SCD does not Comparing the ALP levels at W4 and W8 with that at W0, we found that LPD led to improvement (i.e.. decrease in ALP) in eight of the ten individuals (80%), with six of them (75%) returning to their baseline ALP level when dietary interventions w ere removed at W10 (Fig. 2b). In contrast, only tw o of the ten participants (20%) in the SCD group responded to SCD (i.e., with lower ALP levels than their baseline levels). Moreover, for most of the participants in the SCD group, their ALP values did not appear to be affected after the stop of the dietary intervention, suggesting that SCD may not be a potentially effective treatment for PSC. For those eight participants who responded to LPD, hereafter referred to as LPD-responders, their ALP values were reduced by 18.5% ± 10.4% at W4 (i.e.. after two w eeks of treatment), and by 22.6% ± 15.6% at W8 (i.e., after another four weeks of treatment). This result suggests that LPD significantly affects the progression of PSC.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0134] When accounting for an ALP decrease of at least 10%, LPD showed effectiveness in 60% of patients, compared to a 20% effectiveness rate in SCD. With a reduction of at least 20%, LPD demonstrated effectiveness in 30% of patients, while SCD showed a 10% rate. Considering a decrease of at least 30%, LPD maintained its effectiveness in 30% of patients, with no cases of effectiveness in SCD. Finally, with an ALP decrease of at least 50%, LPD is effective in 10% of patients, with SCD also showing no cases of effectiveness.

[0135] Direct evidence obtained from Lasso Regression analysis revealed a significant positive correlation between the protein levels and the ALP levels (Fig.2c). This finding indicates that lower protein intake is associated with lower ALP levels compared to baseline values. However, it is crucial to note that this relationship is correlative rather than causative.

[0136] In addition to protein, levels of saturated fat, monounsaturated fat, and vitamin B3 were also positively associated with the ALP levels (Fig. 2c). By contrast, the levels of total fat, omega 6 and sucrose were negatively associated with the ALP levels. At the same time, we observed that carbohydrate consumptions (grams per kcal per kg body weight) exhibited significant differences between LPD and SCD, with LPD being higher than SCD at all time points during dietary7intervention compared to baseline (Mann-Whitney test, p-value < 2.2e-16).

[0137] Example 1.3 Three disease categories respond differently to dietary intervention

[0138] We performed a stratified analysis to determine if comorbid IBD influences ALP response to dietary interventions. Though the numbers of individuals in each subgroup were small, marked differences were evident for certain disease categories. For instance, for PSC-UC patients (n= 10 in total), LPD (n=6) was significantly more effective in lowering ALP than SCD (n-4), (Mann-Whitney U test, p-value = 0.0003). Moreover, all the PSC-UC patients in the LPD arm responded to LPD in the sense that their ALP levels decreased, see Fig.2d. For PSC-CD patients (n= 3 in total), there was only one patient in the LPD arm (n=l), and this patient responded to LPD. But for PSC-CD patients, LPD (n=l) was only slightly better than SCD (n=2) in reducing the ALP level (Fig. 2e) and the statistical significance remains unclear due to the small sample size in this disease category. Interestingly, for PSC patients without IBD (PSC-alone) (n=7 in total), neither LPD (n=3) nor SCD (n=4) reducedAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0139] patients’ ALP levels. Moreover, the two interventions didn't demonstrate any significant difference in reducing ALP levels (Fig. 2f).

[0140] Example 1.4 Gut microbial features distinguish LPD responders from non-responders

[0141] Given the effectiveness of LPD, we aimed to determine distinguishing microbial biomarkers between LPD responders and non-responders using shotgun metagenomic sequencing data. We found that the alpha diversity (measured by the Shannon index and species richness) of stool samples in the PSC-UC group was significantly lower than that in the PSC-CD and PSC-alone groups. Samples in each category were clustered together and distinct from the other two groups (PERMANOVA p-value=0.001, F=3.53). Interestingly, the clusters of LPD responders (individuals showing reduced ALP after LPD) were separated from LPD non-responders (individuals showing increased ALP after LPD) in the PCoA plot based on the Bray-Curtis dissimilarity, suggesting that gut microbiota is key in differentiating response outcomes (Fig. 3a). Similar results were revealed by UMAP clustering.

[0142] To account for three disease categories as covariates and to eliminate random effects of inter-individual variation due to temporal changes, we employed MaAsLin2 to perform a multivariate linear regression analysis on the taxonomic profiles of the stool samples (see Methods, Formula 1). We aimed to find microbial features that significantly differentiate LPD responders from LPD non-responders (with cutoff p-value < 0.05 and q-value < 0.2). We identified 21 (or 15) microbial species as significantly positively (or negatively) associated with responders, respectively (Fig.

[0143] 3b). Among these, Ruminococcus gnavus, Blautia caecimuris, Blautia producta, Enterocloster bolteae, and Flavonifractor plautii, were more abundant in responders across samples of all three disease categories. Conversely, Clostridium bacteria, Roseburia bacteria, Senegalimassilia anaerobia, and others were found in much lower abundance in responders.

[0144] In the heatmap of microbial profiles, samples are clustered based on the similarity7of their bacterial structures (Fig. 3b). Interestingly, we found that the selected bacterial markers not only differentiate responders from non-responders but also distinguish between disease categories. For instance, PSC-UC samples are clustered together, as are PSC-CD samples, while non-responders, who were all PSC-Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0145] alone, also form a distinct group. Samples from the same individuals are clustered together on both the heatmap (Fig.3b) and the PCoA plot (Fig.3a), indicating a personalized gut microbiome, i.e., intra-individual differences are smaller than interindividual ones. Therefore, disease categories can also be considered as a general predictor of LPD response.

[0146] Example 1.5 Gut metabolome are affected by multiple variables

[0147] To better understand metabolomic parameters of LPD response, we conducted the global untargeted metabolomic analysis of all stool samples using the ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS) methods (see Methods). A total of 1,428 metabolites were quantified from at least one sample in this study, and a subset of 1 ,057 metabolites with known chemical names and corresponding identities in at least one of the following three databases: the Human Metabolome Database (HMDB)97, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database98, and the PubChem database99. Metabolite compositions exhibited greater temporal variations within individuals than microbial profiles, as indicated by ordination analysis (Fig. 3a versus Fig. 3c), revealing that after dietary interventions samples from the same individuals at different time points w ere interspersed with samples from others, leading to a mixed clustering pattern. Using the previously described multivariate linear regression model, we identified 29 metabolomic markers associated with clinical outcomes (p-value < 0.05 and q-value < 0.2) (see Methods, Formula 1). Of these, 27 metabolites w ere positively associated with LPD responders, while 2 metabolites were negatively associated (Fig. 3d). The intensity of metabolite markers in LPD responders versus LPD non-responders was less distinctive than that observed with microbial markers (Fig. 3d). Additionally, in contrast to microbial markers, clustering based on metabolite markers composition did not show a clear correlation with disease category. Moreover, the obser able intra-individual variation in these markers after dietary intervention indicates baseline metabolic profile cannot serve as a reliable predictor of response outcome to diet intervention, unlike microbial markers.

[0148] Example 1.6 Sulfur-related metabolites predict the LPD-induced changes Diet-induced temporal changes in metabolite profiles were further analyzed to determine the primary mechanism underlying the effect of LPD. We employedAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0149] multivariate linear regression models, which are different from those described earlier, to distinguish metabolite markers at W4 and W8 from WO, incorporating the individual as a random effect and disease category7, age, gender, and BMI as fixed effects (see Methods, Formula 2). We identified 49 metabolite markers that significantly changed throughout LPD compared to baseline (p-value <0.05 and q-value < 0.2), 34 of which have known chemical annotations. Of these, 22 (or 12) metabolites’ abundances increased (or decreased) with LPD. Notably, the majority of depleted metabolites (8 / 12) were either amino acids or fatty acids. Among the 22 enriched metabolites, eight were classified as carboximidic acids (n=2), beta-hydroxy acids (n=2), carbohydrates (n=2), or tricarboxylic acids (n=2) (Fig. 4a).

[0150] To identify metabolic pathways that are significantly altered, we performed metabolite set enrichment analysis (MSEA) using all the 34 metabolite features that significantly changed throughout treatment and with known chemical names. We found significant changes in glycerolipid metabolism (p-value=0.0065), fatty acid biosynthesis (p-value=0.02), purine metabolism (p-value=0.04), and sulfur metabolism (p-value=0.04) with LPD. Notably, sulfur metabolism had the highest enrichment ratio (enrich=13.8), suggesting it may be a primary mechanism behind the effect of LPD (Fig. 4b & 6).

[0151] To extract metabolites potentially converted from or to sulfur-containing chemicals, out of the 1,043 detected metabolites, we identified 40 sulfur-related metabolites referenced in the SMILES database. Four key metabolites were found to have significantly changed (Fig.4c-f & Fig. 6). Methionine (p-value=0.01 at W4, p-value=0.02 at W8) and its oxide methionine sulfoxide (p-value=0.01 at W4, p-value=0.02 at W8) were significantly depleted compared to baseline, as analyzed by the Wilcoxon test adjusted for individual random effects. This suggests that reduced protein intake led to a continual decrease in methionine and methionine sulfoxide in the gut. Taurine significantly enriched by W8 (p-value=0.03) compared to baseline. Cysteine level varied over time, increasing at W4 but decreasing at W8. Notably, cysteine intensify changes showed a significant negative association with ALP changes, indicating that cysteine accumulation correlates with lower ALP values (Spearman correlation: R = -0.42, p-value= 0.0172) (Fig. 4j). Furthermore, in association with taurine accumulation, significant changes in luminal bile acids were observed. Notably, we found the levels of cholic acid. 7 -ketodeoxy cholate and 12-Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0152] dehydrocholate were significantly elevated with LPD (Fig. 6) (pathways referred to KEGG database96,99).

[0153] Example 1.7 Changes in functional metagenomic features with LPD intervention

[0154] To explain the microbial contributions to the metabolite variations and to understand the temporal changes of functional consequences of the microbial community leading to response, we profiled gene families in all stool samples. Based on the shotgun metagenomic sequencing data, we analyzed the functional gene profiles using HUMAnN3.6 and identified 2,427 enzymes (KOs) from different bacterial species. Although the functional pathways were relatively stable across individuals and across times, due to microbial functional redundancy, we then selected enzy mes that significantly changed throughout dietary intervention in LPD responders by fitting a multivariate linear model to each enzyme, adjusting for the random effect of each individual and other covariates (see Methods, Formula 2). In total, 99 enzymes significantly increased and 11 decreased at W4 and W8 compared to baseline (WO) (p-value <0.05).

[0155] We focused on enzy mes involved with sulfur metabolism to decipher the relationship between sulfur-related metabolite changes and microbial functional changes. We referred to a comprehensive list of 74 enzymes involved in microbial sulfur metabolism in humans100,101. Of these, 27 were detected in at least one sample in this study, with 6 (or 5) of these enzymes significantly decreasing (or increasing) during intervention in LPD responders (Mann-Whitney U n test, p-value < 0.05) (Fig.

[0156] 5a), and 4 genes increasing with marginal significance (0.05< p-value < 0.06).

[0157] Interestingly, LPD led to a reduced amount of cysJ, a critical enzy me for producing hydrogen sulfide (PLS) from sulfite, and a reduced amount of dsrD, key for PLS production from cysteine (Fig. 6)100-101. Conversely, the cysK gene, involved in metabolizing H2S in the biosynthesis of cysteine, increased throughout LPD (Fig. 6)100’101. Similarly, there was an increase in the bsh gene that metabolizes taurine and aids in the deconjugation of tauro-conjugated bile acids excreted to the gut from the bi 1 i ary tree (Fig. 6)96. These findings highlight the relationship between microbial functional profile with metabolite changes, supporting the potential mechanistic role of altered sulfur metabolism in attenuating luminal inflammation and enhancing ALP reduction in PSC.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0158] Example 1.8 Bacterial species associations with functional genes Given the significant changes in microbial functional profiles, we explored the bacterial species connected to these enzymes. We constructed a large-scale association network among functional genes, metabolites, and bacterial species. To identify covariations strictly linked to diet change, we first residualized each feature in either measurement type using the same multivariate model employed to determine differential changes in responders during LPD intervention (see Methods, Formula 2). This residualization process, using longitudinal measurements, minimizes interindividual variation and highlights within-person associations over time, considering diagnostic categories as covariates. The resulting network contained 74 edges linking genes and metabolites, 906 edges between genes and species, and 57 edges linking gene pairs, where at least one connected gene was sulfur-related and the paired correlation was significant (Spearman correlation FDR < 0.05) (Fig. 5a). The network encompassed 331 nodes spanning features from three measurement types. tauD, a gene converting taurine to sulfite (further convertible to H2S) (Fig. 6), was central in the network with the most connections (n=174) to different bacterial species.

[0159] However, for most of these species (90.2%), the abundance change of a single species was not significant (multivariate model p-value < 0.05). Eggerthella lenta, significantly positively associated with tauD (Spearman correlation FDR < 0.05), showed a marked decrease in abundance during LPD intervention (Wilcoxon test p-value = 8.7e-3; multivariate model: p-value = 0.016) (Fig. 5d). This species is critical in diet-induced changes, because it is also linked to dmsC (K00185), involved in the conversion from methionine to sulfite (Fig. 6), estimating via the taxonomic stratified functional profile by HUMAnNv3.6, and carries 10 other genes associated with sulfur metabolism, including iscS, and ahcY, which are genes significantly reduced with LPD (Mann-Whitney U test, p-value < 0.05) as well. Genes cysJand dcyD carried the second and fourth most connections, contributed by 92 and 74 different species, respectively, including E. coli and K. pneumoniae (Fig. 5a). These two genes were significantly interrelated (Spearman correlation coefficient = 0.95, R = 5. Odell) and associated with other reduced genes including tauD. dmsC, aspC, and iscC. The stratified abundance of cysJ in E. coli and K. pneumoniae did not significantly change between W4 and baseline but notably decreased at W4 LPD compared to W4 after SCD. Similar results were observed for the dcyD gene in K. pneumoniae throughAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0160] either large-scale association network analysis or significant tests of taxonomic stratified gene abundance changes, bsh, a gene that metabolizes taurine and aids in the deconjugation of tauro-conjugated bile acids, did not exhibit positive correlations in any species in the complex association network. However, the taxonomic stratified analysis showed a significant increase in the abundance of bsh in Roseburia inleslinalis in LPD responders (Wilcoxon test p-value = 1.7e-3), a decrease in the SCD group and no change in LPD non-responders (Fig. 5b). Notably, Roseburia intestinalis is a key bacterium in sulfur metabolism, containing 10 relevant genes that significantly changed, including cysK, metK. dem, metB, metY, luxS, and others (Fig.

[0161] 5c). Not only gene abundances, the bacterial abundance of Roseburia intestinalis showed a marked increase in LPD responders (Mann- Whitney U test p-value = 3.8e-3; multivariate model: p-value = 4.4e-4) (Fig. 5d).

[0162] Example 2. Ulcerative Colitis Clinical Trial

[0163] This study is being performed to determine a dose of hydroxocobalamin in a pilot study in patients with UC and determine if this approach can reduce urinary measures of sulfur metabolism including reduction in calprotectin, a > 50% reduction from pretreatment levels sulfate, S-nitrosothiol, and thiosulfate, and clinical symptoms of UC, and if the combination of hydroxocobalamin and butyrate is safe in patients with UC. A combination of hydroxocobalamin and butyrate may be preferable to address the underlying etiology' of UC.

[0164] A pilot study is conducted to assess a preferable dose of hydroxocobalamin based on reduction of fecal calprotectin, and to determine if this reduction is sustained over time and is correlated to changes in clinical disease activity and urinary and plasma nitrite, nitrate, and nitrosothiol levels.

[0165] A dose-ranging pilot study of hydroxocobalamin as a potent scavenger of both H2S as well as NO, optionally provided in combination with butyrate, is conducted to assess the benefit in the treatment of UC. Without wishing to be bound by theory’, butyrate may serve to reduce inflammation, enhance mucin production and improve mucosal repair with uptake of buty rate by colonocytes enhanced by' the reduction of H2S.

[0166] Patients with active UC are enrolled into this dose ranging, crossover, pilot study with a 2: 1 randomization scheme in the first 4 weeks, active treatment (hydroxocobalamin with butyrate) vs placebo (placebo with butyrate).Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0167] a. Inclusion Criteria

[0168] 1) Age 18-75

[0169] 2) Ability to give consent

[0170] 3) Patients with a confirmed diagnosis of UC for > 3 months

[0171] 4) History of > 15 cm of colonic involvement as confirmed by colonoscopy

[0172] 5) Disease activity' based on calprotectin > 200

[0173] 6) Allowed medications: mesalamine and sulfasalazine

[0174] 7) Partial Mayo score of > 4 for phase 1 or a total Mayo score > 5 in phase 2

[0175] 8) Patients with primary sclerosing cholangitis are eligible to enroll b. Exclusion Criteria

[0176] 1) History of uncontrolled hypertension with systolic BP > 140 and systolic BP > 90

[0177] 2) Chronic kidney disease as defined by GFR <60mL / min

[0178] 3) Impaired hepatic function (transaminases elevated > 2.5 x ULN) unless due to PSC

[0179] 4) Evidence of C. difficile (Negative test result within 1 month is acceptable)

[0180] 5) Infectious Colitis or drug induced colitis

[0181] 6) Crohn’s Disease or Indeterminate colitis

[0182] 7) Decompensated liver disease

[0183] 8) Patients who are pregnant or breastfeeding

[0184] 9) Use of rectal therapies

[0185] 10) Patients who have a confirmed malignancy or cancer within 5 years 11 ) Congenital or acquired immunodeficiencies

[0186] 12) Other comorbidities including: Diabetes mellitus, systemic lupus 13) Patients with a history of kidney stones

[0187] 14) High likelihood of colectomy in the next 2 months

[0188] 15) Participation in a therapeutic clinical trial in the preceding 30 days or simultaneously during this trial

[0189] 16) Patients with a history' or risk of cardiovascular conditions, including arrhythmia, long QT syndrome, congestive heart failure, stroke, or coronary’ arteryAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0190] disease

[0191] 17) Prohibited medications: Vitamin C, prednisone, immune modulators (including but not limited to azathioprine, 6-mercaptopurine, my cophenolate mofetiL tacrolimus, cyclosporine, thalidomide, interleukin- 10 and interleukin-11) and anti-TNF agents within the past six weeks

[0192] Vital signs, blood tests, stool tests, urine tests, and a series of questionnaires (outlined below) will take place at visits as outlined in the schedule below. The patient will be sent home with 5 stool & urine kits for collection on Day -21, Day 0, Day 28 and Day 56. Subjects will be asked to collect first stool of the day. Subjects will also be asked to provide a stool sample at dropout.

[0193] Labs including CBC, CRP, complete metabolic panel, liver function tests, and B12 will be draw n at Day -21, Day 0, Day 14 and Day 28, along with collection at the safety follow' up visit. Folate will be drawn at Day -21. First of the morning urine sample will also be collected on Day -21, Day 0, Day 14 and at Day 28, along with collection at the safety follow up visit. Four 5 cc aliquots will be obtained and saved from the collection. An additional purple top tube of blood will also be collected at each visit for plasma nitrite, nitrate, and nitrosothiol measurement. Plasma should be collected and frozen.

[0194] Study drugs:

[0195] Hydroxocobalamin: Capsules containing 400 or 500 mg of hydroxocobalamin will be manufactured using the Vcaps® Enteric capsules from Capsugel, which are two-piece hard capsules manufactured with pharmaceutical-grade cellulosic derivatives (HPMCAS, HPMC).

[0196] Butyrate: Butyric acid was reacted with Wacker W-6 cyclodextrin, which is a food grade alpha-cyclodextrin from Wacker Chemie AG. This forms buty ric inclusion powder. The release agent is an admixture of E4M and KI OOM Coloron METHOCEL (hydroxypropyl methylcellulose), which is manufactured by The Dow' Chemical Company. This provides an approximate 12-hour release of the product independent of gastric pH or digestive enzymes. The release agent to break down the cyclodextrin is Enzeco® Fungal Amylase H-MG from Enzyme Development Corporation. To help the capsule flow', a small amount of leucine is included. For additional flow', 5 mg of Calcium Laurate is also used, which is a dry slippery' form of lauric acid bound to calcium.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0197] Study drugs will be distributed to each subject at Day 0, Visit 2 and they will be instructed to begin taking the drug on Day 1. A second set of doses will be distributed at Day 14, Visit 3 for the second half of dosing. Day 28 will be the last day of dosing.

[0198] Patient Groups:

[0199] Patients will be enrolled in one of 4 groups. In Group 1, 5 patients will receive 1.2 g of hydroxocobalamin orally daily for 4 weeks. In Group 2, 5 patients will receive 2.4 g orally daily (1.2 g twice a day) for 4 weeks. In Group 3, 5 patients will be dosed with 2.5 grams IV on day 0 and receive 2.4 g orally daily (1.2 g twice a day) for 4 weeks.

[0200] Group 1) The first group will take hydroxocobalamin at 1.2 g daily, PO (2 capsules in the morning and 1 capsule in the evening). Butyrate will be 240 mg daily in a divided dose (120 mg twice daily) which is 5 pills twice a day. All patients will receive the active drug open label. All patients will take buty rate through week 4.

[0201] Patients will have a flexible sigmoidoscopy with biopsies at screening and at week 4 (± 3 days). The flexible sigmoidoscopy will include visual inspection and biopsies before treatment and after treatment. Four biopsies will be collected from the most inflamed areas; an additional four biopsies will be collected if any non-inflamed areas are present.

[0202] Group 2) The first group will take hydroxocobalamin at 2.4 g daily (1.2g twice a day), PO. Butyrate will be 240 mg daily in a divided dose (120 mg twice daily) which is 5 pills twice a day. All patients will receive the active drug open label. All patients will take buty rate through week 4.

[0203] Patients will have a flexible sigmoidoscopy with biopsies at screening and at week 4 (± 3 days). The flexible sigmoidoscopy will include visual inspection and biopsies before treatment and after treatment. Four biopsies will be collected from the most inflamed areas; an additional four biopsies will be collected if any non-inflamed areas are present.

[0204] Group 3) The second group dose of hydroxocobalamin will involve a one-time dose of 2.5 g hydroxocobalamin administered via IV at Week 0 followed by a dosage of the oral form of hydroxocobalamin at 2.4g daily (1.2g twice a day) for 4 weeks. All patients will take 240mg of butyrate daily for the duration of the study.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0205] Patients will have a flexible sigmoidoscopy with biopsies at screening and at week 4 (± 3 days). The flexible sigmoidoscopy will include visual inspection and biopsies before treatment and after treatment. Four biopsies will be collected from the most inflamed areas; an additional four biopsies will be collected if any non-inflamed areas are present.

[0206] Group 4) At the conclusion of 15 patients, the study team will decide if there appears to be a signal of improvement in colitis symptoms and inflammation and a fourth dosing arm might be done enrolling 5 patients at a dose which appeared to be safest and will a higher response rate without buty rate.

[0207] Patients will have a flexible sigmoidoscopy with biopsies at screening and at week 4 (± 3 days). The flexible sigmoidoscopy will include visual inspection and biopsies before treatment and after treatment. Four biopsies will be collected from the most inflamed areas; an additional four biopsies will be collected if any non-inflamed areas are present.

[0208] In addition to lab tests, CBC, complete metabolic panel, B12 level, and LFTs, we will be collecting disease activity scores and disease history as outlined in patients’ medical records. We will also be collecting an additional purple top tube of blood.

[0209] Stool studies will be done for calprotectin and 16S microbial assessment and depending on results possibly whole genome sequencing. Collected plasma will be utilized for assessment of mitochondrial function and collected plasma and urine will be utilized to measure nitrate, nitrite levels, and nitrosothiol levels.

[0210] Biostatistical Analysis

[0211] Primary endpoint: The primary measure of dose response will be urinary measures of sulfur metabolism including a > 50% reduction from pretreatment levels sulfate, S-nitrosothiol, and thiosulfate.

[0212] Secondary Endpoints: Secondary7endpoints include incidence of treatment-emergent adverse events (AE) as assessed by Common Terminology Criteria for Adverse Events (CTCAE). A change in Total Mayo Score with improvement being a reduction in 3 points and remission being a score of 0 or 1 from screening to end of week 4 (at second flexible sigmoidoscopy). Clinical symptoms assessed by Simple Clinical Colitis Activity7Index (SCCAI). Additional assessments include urinary and plasma nitrite, nitrate levels and nitrosothiol levels, and change from baseline fecalAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0213] calprotectin at week 4 and proportion of patients with reductions in fecal calprotectin. In addition, we aim to explore the correlation between urinary, plasma nitrite, nitrate or nitrosothiol levels and fecal calprotectin. A secondary clinical assessment will be determined as well utilizing a reduction in calprotectin to < ULN at the end of week 4. We will also collect Mayo scores and compare partial Mayo in part 1 and complete Mayo in part 2 baseline scores to week 4.

[0214] Preliminary Results

[0215] Table 1 shows preliminary results in the first 4 patients treated in an open labeled trial for those on biologies or other immune suppression and not responding (SHARC) and a crossover trial for patients on mesalamine only or nothing that is called TURTLE. The endpoint of this study is calprotectin, a reliable marker of inflammation in the stool and how we track inflammation in ulcerative colitis. (A calpro level of over 120 is indicative of inflammation; 50-120 is a gray zone and less than 50 is considered remission).

[0216] Treatment was given over a 4 week period and a surprisingly durable response was seen after such a short period on treatment. No significant adverse events were seen. Patient #1, who had the highest level of calpro at the start of the study, had some response but went on a different therapy at the end of their involvement in the trial so the reduction in calpro at week 12 may not be related to hydroxocobalamin. The other 3 patients had an excellent response.

[0217] Table 1. Preliminary Results - Calprotectin Levels SID Age Gender Baseline Week 4 Week 8 Week 12

[0218] SHARC

[0219] 1 42 F 4840 5830 2570 71*

[0220] (Day -5) (Dav 55) (Dav 55) (Dav 84)

[0221] 3 49 F 656 67 16 —

[0222] (Day -13) (Dav 29) (Dav 58)

[0223] 4 43 F 397 52 349 —

[0224] (Day -1) (Dav 30) (Dav 56)

[0225] TURTLE

[0226] 2 48 F 935 22 53 504

[0227]

[0228] (Day 0) (Day 28) (Day 55) (Day 83)

[0229] SID, subject ID

[0230] response potentially unrelated to study treatmentAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0231] Example 3. Primary Sclerosing Cholangitis (PSC) Clinical Trial This is a randomized controlled trial to assess the benefit of hydroxocobalamin in the treatment of PSC. We aim to test two different oral doses and then supplement the oral dosing with a single IV dose combined with oral dosing.

[0232] a. Inclusion Criteria

[0233] 1. Age 18-75

[0234] 2. A diagnosis of PSC for at least 6 months based upon cholangiography (ERCP or MRCP)

[0235] 3. demonstrating intrahepatic and / or extrahepatic biliary strictures, beading or irregularity consistent with PSC.

[0236] 4. ALP > 1.5 times the upper limit of normal (ULN) at screening.

[0237] 5. Subject must either be on a stable dose of ursodeoxycholic acid for > 6 months prior to screening or have been discontinued > 4 weeks prior to screening (enrollment of patients who are on UDCA will be limited to 60% of all enrolled patients).

[0238] b. Exclusion Criteria

[0239] 1. Anticipated need for liver transplant within one year as determined by Mayo PSC risk score

[0240] 2. Evidence of decompensated liver disease such as variceal bleeding, ascites, or hepatic encephalopathy.

[0241] 3. Evidence of advanced liver disease including MELD score > 10, bilirubin > 3.0, platelet count < 100,000; or INR > 1.4

[0242] 4. Concomitant chronic liver disease including alcohol related liver disease, chronic hepatitis B or C infection, haemochromatosis, Wilson’s disease, alphal -antitry psin deficiency, non-alcoholic steatohepatitis, autoimmune hepatitis, or primary biliary’ cholangitis

[0243] 5. Secondary causes of sclerosing cholangitis

[0244] 6. Patients who have a confirmed malignancy or cancer within 5 years except non-melanoma skin cancers

[0245] 7. Treatment with any investigational agents, w ithin two months or 5 half-lives of the investigational product, whichever is longer.

[0246] 8. Active illicit drug or more than moderate alcohol consumption.Attorney Docket No. 29618-0428WO1 / BWH 2023-135

[0247] a. Moderate alcohol consumption is defined as 1 standard drink per day for women and 2 drinks per day for men; whereby 1 standard drink is equivalent to: 12 oz beer (5% alcohol); 5 ounces of wine (12% alcohol), and 1.5 ounces of 80 proof (40% alcohol).

[0248] 9. Evidence of bacterial cholangitis within 6 months of enrollment

[0249] 10. In patients with Ulcerative Colitis, or, if Crohn’s disease, a need for additional therapy at time of screening.

[0250] 11. Chronic kidney injury (eGFR < 60)

[0251] 12. Pregnancy or lactation

[0252] 13. Uncontrolled hypertension with a systolic BP > 140 and a systolic BP > 90 14. Prohibited medications: current use of vitamin C and prednisone

[0253] 15. Patients with ahistory or risk of cardiovascular conditions, including arrhythmia, long QT syndrome, congestive heart failure, stroke, or coronary' artery disease

[0254] 16. Congenital or acquired immunodeficiencies

[0255] 17. Other comorbidities including: diabetes mellitus, systemic lupus

[0256] 18. An episode of acute cholangitis within 4 weeks of screening

[0257] Tw enty-eight patients will be enrolled into four successive groups in a crossover, placebo-controlled design. Groups 1, 2, 3, and 4 will all contain 7 subjects each, with each subject receiving active study drug and placebo in a randomized order; half will receive the placebo first for four weeks followed by active treatment for four weeks, while the other half will receive active treatment for four weeks followed by placebo for four w eeks. Patients in Group 1 w ill receive an initial oral dosing of 1.2 gram twice daily for 4 weeks in the active treatment arm.

[0258] If patients tolerate the Group 1 dosing well without any significant adverse events, the dosing for patients in Group 2 would be increased to 2 grams twice daily for 4 weeks. The DSMB will unblind the data at the end of group 2 to assess response. If there is a normalization in alkaline phosphatase in more than 80% of Group 2 patients, Group 3 will enroll 7 patients to receive the same dose of 2 grams twice daily for 4 weeks in the active treatment arm. If there is not a normalization in alkaline phosphatase in more than 80% of Group 2 patients, patients in Group 3 will receive an IV dose of either placebo or hydroxocobalamin. The active treatment arm will be hydroxocobalamin using Cyanokit® with subsequent oral dosing at 2g twiceAttorney Docket No. 29618-0428WO1 / BWH 2023-135

[0259] daily of hydroxocobalamin. In the placebo arm, they will receive an IV dose (2mg) of cyanocobalamin followed by oral dosing with placebo twice daily for four weeks. We expect a dropout rate of approximately 10%. Group 4 will enroll 7 patients to receive the preferred dose found based on the results of Groups 1-3, either 1.2g twice daily, 2g twice daily, or the IV dosage followed by 2g twice daily.

[0260] If patients do not tolerate dosing in Group 1 , the dosing for patients in Group 2 would be decreased to 400mg twice daily for 4 weeks. If patients do not tolerate the dosing increase in Group 2, further oral dosing in Group 3 would be decreased to 1 ,2g twice daily for 4 weeks while continuing with an IV dose. The results will be assessed by the DSMB after Group 1 and 2. If there are no significant severe adverse events (grade 3 or above) the next dosing group will be initiated. If there are 3 or more SAEs in a dosing group, the subsequent lower dosing regimen will be utilized.

[0261] Patients who have ahistory of UDCA use will fall into two categories. One, those who have been on a stable dose for at least six months prior to enrollment in the study and two, those whose have discontinued their UDCA and have been off the medication for at least 4 weeks prior to enrollment. Enrollment of patients currently taking UDCA will be limited to 60% of the enrollment target.

[0262] The goal is to determine the preferable dosing of hydroxocobalamin in an 8-week pilot study in patients with PSC. The primary endpoint for this study is the capacity of hydroxocobalamin to normalize alkaline phosphatase levels from baseline (week 0) compared to week 8. Secondary endpoints include: 1) reduction in mean ALP to < 1.5 ULN and normalization of ALP 2) proportion of patients with ALP to < 1.5 x ULN at end of active treatment (4 weeks). 2) Change in alkaline phosphatase at end of active treatment (4 weeks); 3) change in mean AST, ALT, total bilirubin, GGT, Mayo PSC risk score, CRP, FFS, PSC PRO, and pruritus VAS score from baseline to end of active treatment (4 weeks), and 4) AEs anytime during treatment. 5) Change in cytokine levels measure in peripheral blood from baseline to end of active treatment (4 weeks).

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[0382] OTHER EMBODIMENTS

[0383] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Atorney Docket No. 29618-0428WO1 / BWH 2023-135WHAT IS CLAIMED IS:

1. A method of treating Ulcerative Colitis (UC), Primary' Sclerosing Cholangitis (PSC), colonic Crohn’s Disease (CD), pouchitis and inflammation after proctocolectomy with ileal pouch-anal anastomosis (j -pouch surgery) to treat UC, irritable bowel disease (IBD), and for reduction of risk of developing colon / colorectal cancer, in a subject, the method comprising orally administering to the subject a treatment comprising a therapeutically effective amount of (i) an H2S scavenger and optionally (ii) butyrate.

2. The method of claim 1, wherein the H2S scavenger is hydroxocobalamin, aquohydroxocobinamide, cobinamide, or a sulfonyl azide, optionally SS20.

3. The method of claim 1 or 2, wherein the therapeutically effective amount decreases levels of fecal calprotectin, and / or decreases urinary' and / or plasma nitrate, nitrite, and / or nitrosothiol levels, in the subject.

4. The method of claim 3, further comprising measuring one or more of fecal calprotectin levels and / or urinary' and / or plasma nitrate, nitrite, and / or nitrosothiol levels, prior to and after initiating the treatment, and determining that a therapy is effective if it decreases one or more of fecal calprotectin levels and / or urinary and / or plasma nitrate, nitrite, and / or nitrosothiol levels.

5. The method of claim 2, comprising administering hydroxocobalamin at 1-5 OR 1- 4 g daily.

6. The method of claim 5, comprising administering hydroxocobalamin in 1-2500mg capsules twice a day, or 3-4300 mg capsules twice a day, optionally 3 capsules wherein two are administered in the morning and one is administered at night.

7. The method of claims 1-6, comprising administering 20 mg -240 mg buty rate daily, preferably in a divided dose, optionally 120 mg twice daily.

8. The method of any of claims 1-7, wherein the H2S scavenger, the butyrate, or both, are administered in a colonic release formulation.Atorney Docket No. 29618-0428WO1 / BWH 2023-1359. The method of any of claims 1-8, wherein the subject has Primary Sclerosing Cholangitis (PSC).

10. The method of any of claims 1-8, wherein the subject does not have UC.

11. A capsule comprising 300, 400, or 500 mg of hydroxocobalamin, and an excipient.

12. The capsule of claim 11, for use in a method of any of claims 1-10.

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