WO2022040110A2 - Alkylating agent and combinations in the treatment of immune-related and liver diseases, alzheimer's diseases and fibrosis diseases - Google Patents

Abstract

This application provides compositions and methods useful in the treatment of immune-related diseases, inflammation related diseases, Non-Alcoholic Fatty Liver Disease (NAFLD), Nonalcoholic Steatohepatitis (NASH), liver cirrhosis, Alzheimer's diseases, fibrosis diseases and Drug Induced Liver Injury (DILI). Additionally, this invention related to novel compositions and methods to screen drugs for the treatment of the diseases. The invention contemplates that methoxyamine (MOA), alone or with other drugs shall be used as a treatment for aldehyde and ketone related immune-related and inflammation diseases, NAFLD, NASH, liver cirrhosis, Alzheimer's diseases, fibrosis diseases and DILL Specifically, the invention contemplates that methoxyamine, alone or with other potential drugs in sequence or in combination, shall be used as a treatment for NAFLD, NASH and liver cirrhosis Alzheimer's diseases, fibrosis diseases, and the invention contemplates that methoxyamine with obeticholic acid in sequence or in combination, shall be used as a treatment for NAFLD, NASH and liver cirrhosis.

Description

ALKYLATING AGENT AND COMBINATIONS IN THE TREATMENT OF IMMUNE-RELATED AND LIVER DISEASES, ALZHEIMER’S DISEASES AND FIBROSIS DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Serial No. 63/067,209, filed August 18, 2020, U.S. Serial No. 63/067,288, filed August 18, 2020, U.S. Serial No. 63/067,225, filed August 18, 2020, and U.S. Serial No. 63/109,843, filed November 4, 2020, the entire contents of which are incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing PDF file, name Sequence_Listing.PDF, was created on August 16, 2021, and is 16 kb.

FIELD

[0003] The present disclosure generally related to compositions and methods for the treatment of immune-related diseases, Alzheimer diseases, fibrosis disease, inflammation related diseases, NAFLD, NASH, liver cirrhosis and DILI. Additionally, this disclosure related to novel compositions and methods to screen drugs for the treatment of the diseases. More specifically, described and provided herein compositions comprising methoxamine alone or with other agent(s), and methods of treating certain liver diseases, such as NAFLD, NASH, liver cirrhosis and DILI, Alzheimer diseases, and fibrosis diseases. Additionally, this disclosure generally related to compositions and methods for the treatment of allysine related fibrosis diseases such as lung fibrosis, kidney fibrosis, liver fibrosis and cirrhosis. Additionally, this disclosure related to novel compositions and methods to screen effective anti-allysine related fibrosis compounds. More specifically, described and provided herein methoxamine to treat all allysine related fibrosis, scaring, and cirrhosis.

BACKGROUND

[0004] Immune system includes innate immune system and adaptive immune system. The adaptive immune system, also known as the acquired immune system is a subsystem of the overall immune system that is composed of highly specialized, systemic cells and processes that eliminate pathogens or prevent their growth. Acquired immunity creates immunological memory after an initial response to a specific pathogen, and leads to an enhanced response to subsequent encounters with that pathogen. The acquired immune system is highly specific to a particular pathogen, and provides long-lasting protection. The acquired system response destroys invading pathogens and any toxic molecules they produce. Antigens are any substances that elicit the acquired immune response. The cells that carry out the acquired immune response are white blood cells known as lymphocytes. Acquired immunity is triggered in vertebrates when a pathogen evades the innate immune system and generates a threshold level of antigen. The major functions of the acquired immune system include recognition of specific "non-self antigens in the presence of "self, during the process of antigen presentation; generation of responses that are tailored to maximally eliminate specific pathogens or pathogen-infected cells; and development of immunological memory, in which pathogens are "remembered".

[0005] Endogenous antigens are produced by intracellular bacteria and viruses replicating within a host cell. The host cell uses enzymes to digest virally associated proteins, and displays these pieces on its surface to T-cells by coupling them to MHC protein molecules. Endogenous antigens are typically displayed on MHC class I molecules, and activate CD8+ cytotoxic T- cells. With the exception of non-nucleated cells (including erythrocytes), MHC class I protein molecules are expressed by all host cells. Cytotoxic T cells are a sub-group of T cells that induce the death of cells that are infected with viruses and other pathogens, or are otherwise damaged or dysfunctional. Naive cytotoxic T cells are activated when their T-cell receptor (TCR) strongly interacts with a peptide-bound MHC class I molecule. This affinity depends on the type and orientation of the antigen/MHC complex, and is what keeps the cytotoxic T lymphocyte (CTL) and infected cell bound together. Once activated, the CTL undergoes a process called clonal selection, in which it gains functions and divides rapidly to produce an army of “armed” effector cells. Activated CTL then travels throughout the body searching for cells that bear that unique MHC Class I + peptide. When exposed to these infected or dysfunctional somatic cells, effector CTL release perforin and granulysin: cytotoxins that form pores in the target cell's plasma membrane, allowing ions and water to flow into the infected cell, and causing it to burst or lyse. CTL release granzyme, a serine protease that enters cells via pores to induce apoptosis (cell death). This process may cause temporary inflammation or permanent tissue damages in the body. To limit extensive tissue damage during an infection, CTL activation is tightly controlled and in general requires a very strong MHC-antigen activation signal. On resolution of the infection, most effector cells die and phagocytes clear them away — but a few of these cells remain as memory cells. On a later encounter with the same antigen, these memory cells quickly differentiate into effector cells, dramatically shortening the time required to mount an effective response. [0006] Therefore, unexpected covalent binding of a complex molecules with a protein or peptide in the body may lead to the formation of unfamiliar peptide conjugate molecules being presented by the HMC to the cell surface, where T cells perceive as antigens, and activate immune response of the adaptive immune system. This mechanism is widely hypothesized in drug allergic response. Drug or its reactive metabolites are considered as foreign antigens that bind to T cell receptors (TCR) and further activate immune response. Referred as “hapten/prohapten” theory, a drug or its reactive metabolite may bind covalently to an endogenous peptide to form an antigenic hapten-carrier complex. In this model, the covalent bonds are established among the drug (or its metabolite), self-peptides, and HLA (human leukocyte antigen complex, which is another name of MHC) molecule. It then results in the induction of drug-specific immune responses. Abacavir hypersensitivity is a well-studied drug- induced adverse drug reactions (ADRs). The mechanism is that short peptide fragments and derivatives from either the drug or its metabolites form a peptide-HLA complex specifically with HLA-B*57:01. This complex activates CD8+ T cells, which release inflammatory cytokines and start the hypersensitivity response. More recently, it has been shown that Abacavir might occupy a space below the region of HLA that presents peptides, which leads to an altered peptide presentation and trigger an autoimmune reaction.

[0007] The farnesoid X receptor (FXR) belongs to the nuclear receptor family and is activated by bile acids. Bile acids are responsible for effective absorption of fats and fatsoluble vitamins, facilitate digestion and are important regulators of cholesterol, triglyceride homeostasis and inflammation. Several of these metabolic actions of bile acids involve the activation of the FXR. FXR is used as a target for new drug therapies against metabolic dysregulation associated with obesity, including type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), and atherosclerosis as well as for the cholestatic liver disease primary biliary cholangitis (PBC). Pharmacological activation of FXR with specific agonists has shown promising results in treatment of the diseases. FXR is a member of the nuclear receptor family of ligand-activated transcription factors that includes receptors for the steroid, retinoid, and thyroid hormones. The bile acids that serve as FXR ligands include chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and the taurine and glycine conjugates of these bile acids. Obeticholic acid (OCA) is a semi -synthetic bile acid analogue. It is used as a drug to treat liver diseases. Activation of FXR by OCA increased insulin sensitivity and reduced markers of liver inflammation and fibrosis in patients with type 2 diabetes and NAFLD and NASH. Furthermore, OCA treatment in PBC patients significantly decreased bilirubin and Alkaline phosphatase (ALP) serum levels. However, it also increased the risk for pruritus and induced high cholesterol as serious side effects to the OCA drug.

[0008] Recently, a plethora of studies have shown the dynamic interaction between the intestinal microbiota and host innate and adaptive immune system. The dysbiosis of gut microbiota could jeopardize host immune responses and promote the development of various inflammatory disorders. This also appears to be the case for Alzheimer’s Disease (AD)- associated inflammation. Emerging evidence from both animal and human studies supports the association between dysbiosis of the gut microbiota and the microglia activation during AD development, such as perturbations in gut microbial diversity influenced neuroinflammation and amyloidosis. All the evidence suggests that gut microbiota is likely involved in regulating microglia activation and neuroinflammation in AD. In addition to the microglia activation, the role of infiltrating peripheral immune cells, such as CD4+ and CD8+ T cells, etc., in AD- associated neuroinflammation is increasingly appreciated. For example, peripheral type 1 and type 17 T-helper (Thl, Thl7) cells have been reported to be associated with releasing of inflammatory cytokines in multiple AD mouse models. Peripheral infiltrated lymphocytes were observed in the brain of both transgenic mouse models and AD patients. Additionally, in the post-mortem brains of AD patients, both CD4+ and CD8+ T cells were detected. Moreover, as the importance of microbiota metabolites is starting to unveil, it will be imperative to comprehend the specific metabolites that are involved in linking gut microbiota and brain neuroinflammation in AD progression. This disclosure pinpoints the molecular biochemistry mechanistic linkage between gut microbiota and AD progression, and provides the drug intervention strategies for AD.

SUMMARY

[0009] This application provides new compositions and methods useful in the treatment of immune-related diseases, inflammation related diseases, NAFLD, NASH, liver cirrhosis and DILI. Alkylating agents treat immune related diseases for mammals, which caused by aldehydes or reactive ketones in the mammalian body. Aldehydes and ketones may be reactive towards proteins and peptides with the 1,2-amino, thiol group of the N-terminal cysteine to form thiazolidine five-member ring structure under physiological condition. This modified protein or peptide in the mammalian body can lead to immune response and immune caused system damages. The response of immune system toward the protein or peptide conjugate can lead to immune related diseases in mammal. Alkylating agents can react with aldehydes and reactive ketones to block the formation of thiazolidine ring with the N-terminal cysteinylpeptide or -protein to prevent the formation of protein or peptide conjugate structure. In case the aldehyde or ketone conjugate is already formed, the alkylating agents can release the proteins or peptide from the aldehyde and ketone N-terminal cysteinyl-peptide conjugates by formation of alkylating conjugate with the aldehyde or ketone. In both cases, the alkylating agents can prevent or break down the formation of protein and peptide conjugation with aldehyde or ketones to prevent the activation of the immune responses of the mammal. The invention contemplates that methoxyamine (abbreviation: MOA), an alkylating agent, alone or with other drugs shall be used as a treatment for aldehyde and ketone related immune-related and inflammation diseases, NAFLD, NASH, liver cirrhosis and DILI. Specifically, the invention contemplates that methoxyamine, alone or with other potential NASH drugs in sequence or in combination, shall be used as a treatment for NAFLD, NASH and liver cirrhosis. Furthermore, the invention contemplates that methoxyamine and obeticholic acid in sequence or in combination, shall be used as a treatment for NAFLD, NASH and liver cirrhosis. Additionally, this invention related to novel compositions and methods to detect the aldehyde and reactive ketone that cause the diseases, and to screen drug candidates which have the potential to be metabolized to aldehydes and reactive ketones.

[0010] This application provides compositions and methods useful in the treatment of Alzheimer’s disease based on a new biochemistry linkage between gut microbiota and Alzheimer’s disease progression. Alkylating agents can be used to treat Alzheimer’s disease, by forming conjugate with 3-ketone form of the bile acids. There is a dynamic interaction between the intestinal microbiota and host innate and adaptive immune system. The dysbiosis of gut microbiota could jeopardize host immune responses and promote the development of various inflammatory disorders including AD associated inflammation. There is a significant positive correlation of AD progression to the higher ratio of secondary bile acid to primary bile acid in the serum of AD patients. The bile acids are converted into 3k-bile acids in the human body. The 3-position ketone on the 3k-bile acids is reactive towards the 1,2-amino, thiol group of the N-terminal cysteinyl-peptide or -protein to form thiazolidine five-member ring structure under physiological condition. This modified peptide or protein in the mammalian body can lead to immune response and immune caused system damages in the brain, leading to AD. Alkylating agents can react with 3k-bile acid either to reduce its cytotoxicity or to block the formation of thiazolidine ring with the N-terminal cysteinyl-peptide or -protein to prevent the formation of protein or peptide conjugate structure. In case the ketone conjugate is already formed, the alkylating agents can release the proteins or peptide from the 3k-bile acid-N- terminal cysteinyl-peptide conjugates by formation of alkylating conjugate with the 3k-bile acid. In both cases, the alkylating agents can prevent or break down the formation of protein and peptide conjugation with 3k-bile acids and form alkylating agent conjugated 3k-bile acids to reduce the cytotoxicity of the bile acids and prevent the activation of the immune responses to damage brain. The invention contemplates that methoxyamine (abbreviation: MOA), an alkylating agent, alone or with other Alzheimer’s treatment drugs shall be used as a treatment for Alzheimer’s disease. Additionally, this invention related to novel compositions and methods to screen drugs for the treatment of Alzheimer’s disease.

[0011] Additionally, this application provides new compositions and methods useful in the treatment of fibrosis diseases with alkylating agents. Alkylating agents treat the said diseases for mammals, which caused by allysine related fibrosis. The disclosure contemplates that methoxyamine (abbreviation: MOA), an alkylating agent, alone or with other drugs shall be used as a treatment for allysine related fibrosis diseases, such as lung, kidney, and liver fibrosis. Specifically, the disclosure contemplates that methoxyamine, alone or with other potential anti-fibrosis drugs in sequence or in combination, shall be used as a treatment for all allysine related fibrosis. Furthermore, the disclosure contemplates that methoxyamine and other anti-fibrosis drug in sequence or in combination, shall be used as a treatment allysine related fibrosis.

[0012] In some embodiments, disclosed herein is a method of treating a disease in a subject, the method comprising administering therapeutically effective amount of a composition comprising an alkylating agent. The disease is selected from aldehyde and reactive ketone caused diseases, immune related diseases, inflammation related diseases, non- alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver cirrhosis, drug related liver injury (DILI), farnesoid X receptor (FXR)-mediated disease, metabolic disease, fibrosis-related disease, and Alzheimer’s disease. In some embodiments, the disease is a liver disease selected from primary biliary cholangitis (PBS), NASH, NAFLD, portal hypertension, bile acid diarrhea, and primary sclerosing cholangitis (PSC). The fibrosis-related disease is selected from the group consisting of lung fibrosis, kidney fibrosis, and liver fibrosis, or brain fibrosis, and skin tissue scarring disease.

[0013] In some embodiments, the composition reduces symptoms, slows down, stops, or cures the disease. The alkylating agent is selected from methoxyamine (MOA), aminoguanidine, a pharmaceutically-acceptable salt thereof, or a combination thereof. The alkylating agent is administered in a dose of between about 0.001 μg/kg and 200 mg/kg body weight per day. In some embodiments, the alkylating agent is used in combination with a drug selected from obeticholic acid (OCA), OCA amino acid conjugate, and a pharmaceutically acceptable salt thereof. [0014] In some embodiments, The OCA amino acid conjugate is OCA glycine conjugate (GOCA) or OCA taurine conjugate (TOCA). The composition achieves an enhanced therapeutic effect for liver disease, and the therapeutic effect comprising reduction of adverse side effects of OCA, enhancement of the therapeutic effect of OCA, synergic effect of MOA with OCA, and combination therapeutic effect of MOA with OCA. In some embodiments, the side effects of OCA comprise pruritus and induced high cholesterol, paradoxical worsening of the liver disease, persistent worsening of serum enzyme elevations and hepatic decompensation, jaundice, fatigue ascites, hypersensitivity reactions, depression, liver failure and other severe liver injury.

[0015] In some embodiments, another drug is used in combination with the alkylating agent. In some embodiments, the other drug is GV-971. In some embodiments, the alkylating agent is administered in an amount effective to reduce the secondary bile acids concentration in the subject’s serum. The composition is administered orally, intravenously, intraperitoneally, intramuscularly, or transdermally.

[0016] In some embodiments, the alkylating agent is Formula (I) with the following structure:

Formula (I) or a pharmaceutically-acceptable salt, solvate, stereoisomer thereof, wherein X is O or NH;

Y is O, S, or NH;

Z is a bond, O, S, or NH; and

R is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl.

[0017] In some embodiments, the alkylating agent is Formula (II) with the following structure:

Formula (II) or a pharmaceutically-acceptable salt, solvate, stereoisomer thereof, wherein X is O, S, or NH; and R is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl.

[0018] In some embodiments, the alkylating agent is selected from a group consisting of methoxyamine; O-benzylohydroxylamine; ethyl aminooxyacetate; aminooxyacetic acid; H₂NOCHMeCO₂H; carboxymethoxyamine; aminooxyacetic acid;

HN=C(NH₂)SCH₂CH₂ONH₂; H₂NO(CH₂)3SC(NH₂)=NH; MeOC(O)CH(NH₂)CH₂ONH₂; H₂NOCH₂CH(NH₂)CO₂H; canaline; H₂NO(CH₂)4ONH₂; O-(p-nitrobenzyl)hydroxylamine; 2- amino-4-(aminooxymethyl)thiazole; 4-(aminooxymethyl)thiazole; O,O’ -(o- phenylenedimethylene)dihydroxylamine; 2,4-dinitrophenoxyamine; O,O’ -(m- phenylenedimethylene)dihydroxylamine; O,O’-(p-phenylenedimethylene)dihydroxylamine; H₂C=CHCH₂ONH₂; H₂NO(CH₂)4ONH₂; H₃C-(CH₂)₁₅-O-NH₂; 2,2’-(l,2-ethanediyl)bis(3- aminooxy)butenedioic acid dimethyl diethyl ester; and a pharmaceutically-acceptable salt thereof.

[0019] In some embodiments, the alkylating agent is selected from

[0020] In some embodiments, the composition comprises two or more alkylating agents. The composition comprises a pharmaceutically-acceptable carrier.

[0021] In some embodiments, disclosed herein is a method of reducing scar tissue or improving an appearance of scar tissue comprising topically applying to the scar tissue an effective amount of a composition comprising a therapeutically effective amount of an alkylating agents. The composition comprises a pharmaceutically-acceptable topical carrier. The composition comprises about 0.001% to 99.9 wt% of the alkylating agent. A daily dose of the composition comprises about 0.001 μg/kg to 200 mg/kg of the alkylating agent.

[0022] In some embodiments, disclosed herein is a method of treating a liver disease in a subject, the method comprising administering a first formulation comprising MOA or aminoganidine and a second formulation comprising OCA or GOCA or TOCA. The MOA is administered in a daily dose of about 0.01 μg/kg and 200 mg/kg body weight. The OCA is administered in a daily dose of about 0.001 μg/kg and 20 mg/kg body weight. The first formulation comprising MOA or aminoganidine is administered to reduce side effects associated with OCA. [0023] In some embodiments, disclosed herein is a method for selecting an alkylating agents for the treatment of a disease, the method comprising: (a) mixing (i) aldehyde-N- terminal cysteinyl-peptides conjugate, aldehyde-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal cysteinyl-peptides conjugate, or a combination thereof and (ii) an alkylating agent in a solvent; (b) incubating said mixture; and (c) detecting within the incubated mixture (i) the amount of the aldehyde-N-terminal cysteinyl-peptides conjugate, aldehyde-N-terminal homocysteinyl- peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, or reactive ketone-N-terminal cysteinyl-peptide conjugate, or a combination thereof (ii) the amount of N- terminal-peptide released from the aldehyde-N-terminal cysteinyl-peptides conjugate, aldehyde-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, or reactive ketone-N-terminal cysteinyl-peptide conjugate, and/or (iii) the amount of conjugate formed between the alkylating agent and the aldehyde or reactive ketone.

[0024] In some embodiments, the aldehyde-N-terminal cysteinyl-peptide conjugate, aldehyde-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, or reactive ketone-N-terminal cysteinyl-peptide conjugate is 3k-CDCA-Cys conjugate, 3k-CDCA-N-terminal cysteinyl-peptides conjugate, 3k-CDCA- HCys conjugate or 3k-CDCA-N-terminal homocysteinyl-peptide conjugate. The aldehyde comprises sugars, and the reactive ketone comprises 3k-bile acids, which comprising 3k-CA, 3k-DCA, 3k-CDCA, 3k-LCA, 3k-GCA, 3k-GDCA, 3k-GCDCA, 3k-GLCA, 3k-TCA, 3k- TDCA, 3k-TCDCA, 3k-TLCA, 3k-UDCA, 3k-GUDCA, and 3k-TUDCA. The alkylating agent can be MOA. The solvent is selected from dimethyl sulfoxide (DMSO), acetonitrile (ACN), phosphate buffered saline (PBS), and a combination thereof. The incubation step (b) is perform for about 0.1 second to 100 days and at a temperature of about -20 °C and 100 °C. The detecting step (c) is performed by liquid chromatography (LC)-UV, LC-mass spectrometry (MS), and Nuclear magnetic resonance (NMR).

[0025] In some embodiments, disclosed herein is a method for screening aldehyde or reactive ketone of a drug candidate and said drug candidate’s metabolites, which cause adverse drug effects, comprising: (a) a mixture comprising i) cysteine, homocysteine, N-terminal homocysteinyl derivatives or N-terminal cysteinyl-derivatives; ii) the said drug; iii) metabolizing enzymes, including the entities of microsomes, cells, or tissues which contain said metabolizing enzymes, (b) incubating said mixture in physiological solution and condition, and (c) detecting the formation of thiazolidine or 1,3-thiazinane derivatives of said cysteine, homocysteine, N-terminal homocysteinyl derivative or N-terminal cysteinyl derivative conjugates with said aldehyde and ketone of said drug candidate and said metabolites.

[0026] In some embodiments, the cysteine, homocysteine, N-terminal homocysteinyl derivatives, N-terminal cysteinyl derivatives, thiazolidines derivatives and 1,3-thiazinane derivatives compound structures are: a)

i) cysteine: n=l, R₃=OH; ii) homocysteine: n=2, R₃=OH; iii) N-terminal cysteinyl derivative: n=l, R₃ is a peptide; iv) N-terminal homocysteinyl derivative: n=2, R₃ is a peptide,

i) thiazolidines derivatives: n=l, each R₁ and R₂ is independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl, R₁ and R₂ may also form connected ring structures, R₃ is a hydroxyl group or a peptide; ii) 1,3-thiazinane derivatives: n=2, each R₁ and R₂ is independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl, R₁ and R₂ may also form connected ring structures, R₃ is a hydroxyl group or a peptide, c) The elements of said compounds may be isotopes labeled. d) The elements of said compounds may be radioactive isotopes labeled.

[0027] In some embodiments, for detecting said thiazolidine and 1,3-thiazinane derivatives, comprising, LC-MS, NMR, Ion Chromatography, Florescence Spectroscopy, Radioactive Detection. [0028] Other features and advantages can become apparent from the following detailed drawings and description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to facilitate a full understanding of the present disclosure, reference is now made to the accompanying drawings, in which like elements are referenced with like numerals. These drawings should not be construed as limiting the present disclosure, but are intended to be illustrative only. The drawings are not necessarily to scale, or inclusive of all elements of a system, emphasis instead generally being placed upon illustrating the concepts, structures, and techniques sought to be protected herein.

[0030] FIGURE 1: 1, 2 -Amino, thiol group on the N-terminal cysteinyl-peptide and/or protein (1) forms conjugate (3) with a reactive ketone or aldehyde (2) through the thiazolidine (3) formation in physiological condition as shown in Pathway 1-Step 1. When methoxyamine or its salts presents in the system, the conjugate (3) goes down the Pathway 1-Step 2 to release the N-terminal cysteinyl-peptide and/or protein (1) and form epimers of O-methyl oxime ketone or aldehyde (4). Free reactive ketone or aldehyde (2) goes through Pathway 2 to directly forming O-methyl oxime of the ketone or aldehyde (4) to preserve the free N-terminal cysteinyl-peptide and/or protein (1). Note: AA_n represent amino acid unit. In compound 1, it is a N-terminal cysteinyl peptide. N-terminal cysteine is AAi. AAi to AA_n can be as few as just one cysteine amino acid, or can be as many as 100,000 amino acid units protein molecule with N-terminal cysteine (AAi).

[0031] FIGURE 2: Simplified diagram of protein degradation by the proteasome into peptides, the peptides’ transporting into endoplasmic reticulum by TAP complex, the peptides’ loading on MHC/HLA protein molecules, and the peptides’ transporting to the surface of cells for presentation. The reactive ketones and aldehydes form conjugates with N-terminal cysteinyl-peptides, which are free or associate with other molecules, in any locations both in and outside of the cells. The N-terminal cysteinyl-peptides are from any sources, not limited to the ones produced by proteasome. The conjugated peptides and non-conjugated peptides are all being presented by the MHC/HLA molecules on the cell surface. When the MOA is presented in the system, MOA can directly form O-methyl oxime with the reactive ketone or aldehyde (4) to preserve the free N-terminal cysteinyl-peptides (1), and the methoxyamine (MOA) can release the N-terminal cysteinyl-peptides from their conjugate structures. The MOA can react with reactive ketone and aldehyde both in and outside of the cells. The MOA can also release the N-terminal cysteinyl-peptides from their conjugations in the cells (arrows a), outside of the cells (arrow b), on the MHC/HLA molecules and being transported to the surface (arrows c), or on the MHC/HLA molecules on the surface (arrow d). (The numbers both in this figure and caption are corresponding to the compounds in FIG. 1).

[0032] FIGURE 3 : Bile acids (BA) can convert to the 3-position ketone of the 3-oxo-Bile Acid (3-keto-bile acid or 3k-BA) in physiological condition at the present of 3α-HSD enzyme and NAD+ co-factor. 3k-BAs are the major Phase 1 metabolites of BAs in human liver. The major enzyme responsible for 3k-BA metabolites is CYP3A4. 3k-BA (5) is a reactive ketone. The 1, 2-amino, thiol group on terminal N-cysteinyl-peptide and/or protein (1) react with the 3- position ketone of 3k-BA (5) to form a five-member ring thiazolidine in physiological condition. It is a simple chemical reaction without enzyme catalyst. This simple chemical reaction produces 3k-BA-N-terminal cysteinyl-peptide and/or protein conjugate (6) as shown in Pathway 1-Step 1. When methoxyamine or its salts (MO A) presents in the system, the conjugate (6) goes down the Pathway 1-Step 2 to release the N-terminal cysteinyl-peptide and/or protein (1) and form epimers of O-m ethyl oxime 3k-BA-t-MOA and 3k-BA-c-MOA conjugates (7). Free 3k-BA goes through Pathway 2 to directly forming epimers of o-m ethyl oxime 3k-BA-t-MOA and 3k-BA-c-MOA conjugates (7) to preserve the free N-terminal cysteinyl-peptide and/or protein (1). Note: AA_n represent amino acid unit. In compound 1, it is a N-terminal cysteinyl peptide. N-terminal cysteine is AAi. AAi to AA_n can be as few as just one cysteine amino acid, or can be as many as 100,000 amino acid units protein molecule with N-terminal cysteine (AAi).

[0033] FIGURE 4: Cysteine (Cys) and isotope labeled cysteine (cysteine with one ¹⁵N and three ¹³C, Cys4) of 1 : 1 molar mixture reacts with 3-ketone chenodeoxycholic acid (3k-CDCA). 1, 2-Amino, thiol groups on cysteine reacts with the 3-position ketone of 3k-CDCA to form a five-member ring thiazolidine in physiological condition. It is a simple chemical reaction without enzyme catalyst. This simple chemical reaction produces 3k-CDCA-Cys and 3k- CDCA-Cys4 conjugates mixture. The molecular weight of 3k-CDCA-Cys4 is about 4 Daltons (exact mass: 4.0071) higher than that of 3k-CDCA-Cys. Introducing methoxyamine (MOA) to the 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates mixture solution releases the Cys and Cys4 from the 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates, and forms 3k-CDCA-MOA conjugates. The 3k-CDCA-MOA has two epimers due to the rigidity of the C=N double bond. Both cis and trans epimers of ketone oxime exist simultaneously: 3k-CDCA-c-MOA and 3k- CDCA-t-MOA. The two epimers can also be labeled separately without differentiating their stereochemistry as 3k-CDCA-MOAl and 3k-CDCA-MOA2, or labeled combined as 3k- CDCA-MOA. [0034] FIGURE 5: LC-MS results of 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates in positive ion mode (PM). PM of 3k-CDCA&Cys+Cys4 sample shows 3k-CDCA-Cys and 3k- CDCA-Cys4 conjugates retention time (RT) at 14.01 and 13.99 minutes. A: Extracted ion chromatogram of ion C27H43O5NS +/- 10 ppm to show RT of 3k-CDCA-Cys at 14.01 minutes. B: Extracted ion chromatogram of ion C24H43OsS¹³C3¹⁵N +/- 10 ppm to show RT of 3k- CDCA-Cys4 at 13.99 minutes. C: Total ion spectrum of full scan at 14.01 minutes (RT of 3k- CDCA-Cys and 3k-CDCA-Cys4) to show ion masses of 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates are 494.2950 (theoretical ion mass: 494.2935, difference is 1.5 mDa [millidalton], which is 3.0 ppm [calculated formula: 1,000,000* {actual ion mass - theoretical ion massj/theoretical ion mass]) and 498.3016 (theoretical: 498.3006, 1.0 mDa, 2.0 ppm) and patterns of their corresponding isotope ions. D: Product scan of 494.3 (ion mass of 3k-CDCA- Cys) at 13.99 minutes (RT of 3k-CDCA-Cys). Product ion clusters of 50-250 Da are signature ions for all bile acids in PM. They are from 3k-CDCA part of the conjugate. Ion 337.2531 (theoretical: 337.2526, 0.5 mDa, 1.5 ppm), 355.2633 (theoretical: 355.2632, 0.1 mDa, 0.3 ppm), 387.2354 (theoretical: 387.2353, 0.1 mDa, 0.3 ppm), and 476.2834 (theoretical: 476.2829, 0.5 mDa, 1.0 ppm) are signature ions for 3k-CDCA-Cys. Their proposed structures are in FIG. 6. A. Major fragmentations of 3k-CDCA-Cys in PM. E: Product scan of 498.3 (ion mass of 3k-CDCA-Cys4) at 13.99 minutes (RT of 3k-CDCA-Cys4). Product ion clusters of 50-250 Da are signature ions for all bile acids in PM. They are from 3k-CDCA part of the conjugate. Ion 337.2529 (theoretical: 337.2526, 0.3 mDa, 0.9 ppm), 355.2633 (theoretical: 355.2632, 0.1 mDa, 0.3 ppm), 387.2353 (theoretical: 387.2353, 0.0 mDa, 0.0 ppm), and 480.2904 (theoretical: 480.2900, 0.4 mDa, 0.8 ppm) are signature ions for 3k-CDCA-Cys4. Their proposed structures are in FIG. 6. B. Major fragmentations of 3k-CDCA-Cys4 in PM.

[0035] FIGURE 6: Major fragmentations of both 3k-CDCA-Cys and 3k-CDCA-Cys4 in positive ion modes (PM). A: Proposed structures of major fragmentations of 3k-CDCA-Cys. The theoretical exact masses of the major fragmentations calculated in neutral form are 475.2756, 386.2280, 354.2559, and 336.2453. One proton (mass: 1.0073) is gained in PM, resulting in theoretical ion mass 476.2829, 387.2353, 355.2632, and 337.2526. B: Proposed structures of major fragmentations of 3k-CDCA-Cys4. The theoretical exact masses of the major fragmentations calculated in neutral form are 479.2827, 386.2280, 354.2559, and 336.2453. One proton (mass: 1.0073) is gained in PM, resulting in theoretical ion mass 480.2900, 387.2353, 355.2632, and 337.2526.

[0036] FIGURE 7: LC-MS results of 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates in negative ion mode (NM). NM of 3k-CDCA&Cys+Cys4 sample shows 3k-CDCA-Cys and 3k- CDCA-Cys4 conjugates retention time (RT) at 14.02 and 14.00 minutes. A: Extracted ion chromatogram of ion C₂₇H₄₃O₅NS +/- 10 ppm to show RT of 3k-CDCA-Cys at 14.02 minutes. B: Extracted ion chromatogram of ion C₂₄H₄₃O₅S¹³C₃ ¹⁵N +/- 10 ppm to show RT of 3k- CDCA-Cys4 at 14.00 minutes. C: Total ion spectrum of full scan at 14.02 minutes (RT of 3k- CDCA-Cys and 3k-CDCA-Cys4) to show ion masses of 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates are 492.2795 (theoretical: 492.2789, 0.6 mDa, 1.2 ppm) and 496.2865 (theoretical: 496.2860, 0.5 mDa, 1.0 ppm) and patterns of their corresponding isotope ions. D: Product scan of 492.3 (ion mass of 3k-CDCA-Cys) at 14.01 minutes (RT of 3k-CDCA-Cys). Product ion 414.3015 is the signature ion, which is 3k-CDCA-Cys conjugate with neutral loss of both H₂S and CO₂ (molecular formula: C26H41O3N, theoretical: 414.3013, 0.2 mDa, 0.5 ppm). E: Product scan of 496.3 (ion mass of 3k-CDCA-Cys4) at 14.00 minutes (RT of 3k-CDCA-Cys4). Product ion 417.3058 is the signature ion, which is 3k-CDCA-Cys4 conjugate with neutral loss of both H₂S and ¹³CO₂ (molecular formula: C₂₄H₄₁O3¹³C₂ ¹⁵N, theoretical: 417.3051, 0.7 mDa, 1.8 ppm).

[0037] FIGURE 8: LC-MS results of 3k-CDCA-MOA conjugates in positive ion mode (PM). PM of 3k-CDCA&Cys+Cys4&MOA sample shows 3k-CDCA-MOA conjugates retention time (RT) at 19.73 and 19.91 minutes. A: Extracted ion chromatogram of ion C25H41O4N +/- 10 ppm to show RT of 3k-CDCA-MOAl and 3k-CDCA-MOA2 at 19.73 and 19.91 minutes. B: Total ion spectrum of full scan at 19.73 minutes (RT of 3k-CDCA-MOAl) to show ion of 3k-CDCA-MOAl is 420.3110 (theoretical: 420.3109, 0.1 mDa, 0.2 ppm). Ion 402.3005 is the dehydration form of 3k-CDCA-MOAl (3k-CDCA-MOA - H₂O). C: Total ion spectrum of full scan at 19.91 minutes (RT of 3k-CDCA-MOA2) to show ion of 3k-CDCA- M0A2 is 420.3112 (theoretical: 420.3109, 0.3 mDa, 0.7 ppm). Ion 402.3008 is the dehydration form of 3k-CDCA-MOA2 (3k-CDCA-MOA - H₂O). D: Product scan of 420.3 (ion mass of 3k-CDCA-MOA) at 19.74 minutes (RT of 3k-CDCA-MOAl). Product ion clusters from 50 to 250 are bile acid common ion peaks from 3k-CDCA part of the conjugate. Signature ions are 352.2629 (theoretical: 352.2635, -0.6 mDa, -1.7 ppm), 370.2740 (theoretical: 370.2741, -0.1 mDa, -0.3 ppm), and 402.3004 (theoretical: 402.3003, 0.1 mDa, 0.2 ppm). Proposed structures of the signature ions are in FIG. 10. A. Major fragmentations of 3k-CDCA-MOA in PM. E: Product ion clusters from 50 to 250 are bile acid common ion peaks from 3k-CDCA part of the conjugate. Signature ions are 352.2633 (theoretical: 352.2635, -0.2 mDa, -0.6 ppm), 370.2742 (theoretical: 370.2741, 0.1 mDa, 0.3 ppm), and 402.3000 (theoretical: 402.3003, -0.3 mDa, -0.7 ppm). Proposed structures of the signature ions are in FIG. 10. A. Major fragmentations of 3k-CDCA-MOA in PM. [0038] FIGURE 9: LC-MS results of 3k-CDCA-MOA conjugates in negative ion mode (NM). NM of 3k-CDCA&Cys+Cys4&MOA sample shows 3k-CDCA-MOA conjugates retention time (RT) at 19.74 and 19.89 minutes. A: Extracted ion chromatogram of ion C₂₅H₄₁O₄N +/- 10 ppm to show RT of 3k-CDCA-MOAl and 3k-CDCA-MOA2 at 19.74 and 19.89 minutes. B: Total ion spectrum of full scan at 19.74 minutes (RT of 3k-CDCA-MOAl) to show ion of 3k-CDCA-MOAl is 418.2965 (theoretical: 418.2963, 0.2 mDa, 0.5 ppm) and its formic acid (HCOOH, FA) adduct ion 464.3018, sodium formate (HCOONa) adduct ion 486.2839, dimer ion 837.6005, and dimer sodium ion 859.5822. C: Total ion spectrum of full scan at 19.89 minutes (RT of 3k-CDCA-MOA2) to show ion of 3k-CDCA-MOA2 is 418.2965 (418.2963, 0.2 mDa, 0.5 ppm) and its FA adduct ion 464.3019, HCOONa adduct ion 486.2840, dimer ion 837.6006, and dimer sodium ion 859.5823. D: Product scan of 418.3 (ion mass of 3k-CDCA-MOA) at 19.74 minutes (RT of 3k-CDCA-MOAl). Product ions 94.0300 (theoretical: 94.0298, 0.2 mDa, 2.1 ppm) and 319.2282 (theoretical: 319.2278, 0.4 mDa, 1.3 ppm) are the signature ions, proposed structures of the signature ions are in FIG. 10. B. Major fragmentations of 3k-CDCA-MOA in NM. E: Product scan of 418.3 (ion mass of 3k-CDCA- MOA) at 19.91 minutes (RT of 3k-CDCA-MOA2). Product ions 94.0300 (theoretical: 94.0298, 0.2 mDa, 2.1 ppm) and 319.2282 (theoretical: 319.2278, 0.4 mDa, 1.3 ppm) are the signature ions, proposed structures of the signature ions are in FIG. 10. B. Major fragmentations of 3k-CDCA-MOA in NM.

[0039] FIGURE 10: Major fragmentations of 3k-CDCA-MOA in both positive and negative ion mode (PM&NM). A: Proposed structures of major fragmentations of 3k-CDCA- MOA in PM. The theoretical exact masses of the major fragmentations calculated in neutral form are 351.2562, 369.2668 and 401.2930. One proton (mass: 1.0073) is gained in PM, resulting in theoretical ion mass 352.2635, 370.2741, and 402.3003. B: Proposed structures of major fragmentations of 3k-CDCA-MOA in NM. The theoretical exact masses of the major fragmentations calculated in neutral form are 95.0371 and 320.2351. One proton (mass: 1.0073) is lost in NM, resulting in theoretical ion mass 94.0298, and 319.2278.

[0040] FIGURE 11: 1, 2 -Amino, thiol group on the N-terminal cysteinyl-peptide of oxytoceine (H₂0xy) react with the 3-position ketone of the 3k-CDCA to form a five-member ring thiazolidine in physiological condition. It is a simple chemical reaction without enzyme catalyst. This simple chemical reaction produces 3k-CDCA-Oxytoceine (3k-CDCCA-H20xy) conjugate. The acronym is located underneath the molecular structure. The exact mass of the molecule of interest is located below the molecular structure. Introducing methoxyamine hydrochloride (CH₃ONH₂ HCI, MOA) to the 3k-CDCA-H20xy conjugate solutions can release the H20xy from the 3k-CDCA-H20xy conjugate, form 3k-CDCA-MOA conjugates, and free H2Oxy.

[0041] FIGURE 12: LC-MS results of 3k-CDCA-H20xy conjugate in both positive and negative ion modes (PM&NM). PM of 3k-CDCA&Oxy+DTT sample shows 3k-CDCA- H20xy retention time (RT) at 13.97 minutes. A: Extracted ion chromatogram of doubly charged ion of C57H106O15N12S2 +/- 10 ppm to show RT of 3k-CDCA-H20xy at 15.47 minutes. B: Total ion spectrum of full scan at 15.47 minutes (RT of 3k-CDCA-H20xy) to show major ion of 3k-CDCA-H20xy is 691.3676 (doubly charged, theoretical m/z: 691.3666, 1.0 mDa, 1.4 ppm). Singly charged ion 1381.7288 (theoretical ion mass: 1381.7258, 3.0mDa, 2.2ppm) is in much lower ion intensity than doubly charged. C: Product scan of 691.4 (m/z of doubly charged 3k-CDCA-H20xy) at 15.47 minutes (RT of 3k-CDCA-H20xy). Low ion mass range 50-200 Daltons, major ions are 70.0651 (Proposed structure of the fragmentation ion is Pro - H2O - CO, theoretical: 70.0651, 0 mDa, 0 ppm) and 183.1492 (PL - H2O - CO, 183.1492, 0.0 mDa, 0.0 ppm). D: Product scan of 691.4 (m/z of doubly charged 3k-CDCA- H20xy) at 15.47 minutes (RT of 3k-CDCA-H20xy). High ion mass range 200-1100 Daltons, major ions are 285.1920 (PLG-NH₂, 285.1921, -0.1 mDa, -0.4 ppm), 355.2624 (3k-CDCA - 2H₂O, 355.2632, -0.8 mDa, 2.3 ppm), 549.2750 (doubly charged 3k-CDCA-CYIQNC - H₂O, 549.2742, 0.8 mDa, 1.5 ppm), 593.3391 (3k-CDCA-CY - 2H₂O - CO, 593.3408, -1.7 mDa, - 2.8 ppm), 752.4301 (3k-CDCA-CYI - H₂O, 752.4303, -0.02 mDa, -0.3 ppm), and 880.4866 (3k-CDCA-CYIQ - H₂O, 880.4889, -2.3 mDa, -2.6 ppm). NM of 3k-CDCA&Oxy+DTT sample shows 3k-CDCA-H20xy conjugates retention time (RT) at 15.44 minutes. E: Extracted ion chromatogram of ion mass of C57H106O15N12S2 +/- 10 ppm to show RT of 3k- CDCA-H20xy at 15.44 minutes. F: Total ion spectrum of full scan at 15.44 minutes to show major ions of 3kCDCA-H20xy is 1379.7131 (theoretical ion mass: 1379.7113, 1.8 mDa, 1.3 ppm). The intensity of ion 689.3524 (doubly charged, theoretical m/z: 689.3520, 0.4 mDa, 0.6 ppm) is low. This is different from PM. In PM, major ion is doubly charged. In NM, major ion is singly charged. The conjugate favors PM.

[0042] FIGURE 13: 1, 2 -Amino, thiol group on the N-terminal-cysteinyl peptide of Oxytoceine (H20xy) react with the 3-position ketone of the 3k-OCA to form a five-member ring thiazolidine in physiological condition. It is a simple chemical reaction without enzyme catalyst. This simple chemical reaction produces 3k-OCA-Oxytoceine (3k-OCA-H2Oxy) conjugate. Introducing methoxyamine hydrochloride (CH₃ONH₂ HCI, MOA) to the 3k-OCA- H20xy solution can release the H20xy from the 3k-OCA-H2Oxy conjugate, and forms 3k- OCA methoxy amine (3k-OCA-MOA) conjugates and free H2Oxy. The 3k-OCA-MOA has two epimers due to the rigidity of the C=N double bond. Both cis and trans epimers of ketone oxime exist simultaneously: 3k-OCA-c-MOA and 3k-OCA-t-MOA. The two epimers are labeled separately without differentiating their stereochemistry as 3k-OCA-MOAl and 3k- OCA-MOA2, or labeled combined as 3k-OCA-MOA in this document.

[0043] FIGURE 14: LC-MS results of 3k-OCA oxytoceine conjugate (3k-OCA-H2Oxy) in both positive and negative ion modes (PM&NM). PM of OCA&3α-HSD&Oxy+DTT sample shows 3k-OCA-H2Oxy conjugates retention time (RT) at 17.10 minutes. A. Extracted ion chromatogram of doubly charged ion of 3k-OCA-H2Oxy (formula: C69H110O15N12S2) +/- 10 ppm to show RT of 3k-OCA-H2Oxy at 17.10 minutes. B. Total ion spectrum of full scan at 17.10 minutes (RT of 3k-OCA-H2Oxy) to show monoisotope ion of 3k-OCA-H2Oxy is 705.3832 (doubly charged, theoretical m/z: 705.3822, 1.0 mDa, 1.4 ppm) and its corresponding isotope ion pattern (every half Dalton interval, doubly charged ion). NM of OCA&3a- HSD&Oxy+DTT sample shows 3k-OCA-H2Oxy conjugates retention time (RT) at 17.08 minutes. C. Extracted ion chromatogram of 3k-OCA-H2Oxy (formula: C₅₇H₁₀₆O₁₅N₁₂S₂) +/-10 ppm to show RT of 3k-OCA-H2Oxy at 17.08 minutes. D. Total ion spectrum of full scan at 17.08 minutes (RT of 3k-OCA-H2Oxy) to show monoisotope ion of 3k-OCA-H2Oxy is 1407.7468 (theoretical: 1407.7426, 4.2 mDa, 3.0 ppm) and its corresponding isotope ion pattern. The ionization patterns of 3k-OCA-H2Oxy in both PM and NM are very similar as 3k-CDCA-H20xy since the chemical structure of these two compounds are very similar.

[0044] FIGURE 15: LC/MS results of allysine (Aly) and methoxyamine (MOA) conjugates (Aly-MO A, A-MOA) in Aly&MOA sample with 1-2 hours incubation in both positive and negative ion modes (PM&NM). PM of Aly&MOA sample shows Aly-MOA retention time (RT) at 5.15 minutes. A: Extracted ion chromatogram of ion C₇H₁₄N₂O₃ +/- 10 ppm to show RT of Aly-MOA at 5.15 minutes. B: Total ion spectrum of full scan at 5.15 minutes (RT of Aly-MOA) to show major ions of Aly-MOA is 128.0708 (theoretical: 128.0706, 0.1 mDa, 0.8 ppm, in-source fragmentation of Aly-MOA) and 175.1079 (175.1077, 0.2 mDa, 1.1 ppm). C: Total ion spectrum of product scan of 175.1(ion mass of Aly-MOA) at 5.14 minutes (RT of Aly-MOA). Major ions are 53.0390 (53.0386, 0.4 mDa, 7.3 ppm), 54.0342 (54.0338, 0.4 mDa, 7.3 ppm), 55.0546 (55.0542, 0.4 mDa, 7.3 ppm), 56.0498 (56.0495, 0.3 mDa, 5.4 ppm), 59.0493 (59.0491, 0.2 mDa, 3.4 ppm), 70.0652 (70.0651, 0.1 mDa, 1.4 ppm), 80.494 (80.0495, -0.1 mDa, -1.3 ppm), 82.0653 (82.0651, 0.2 mDa, 2.4 ppm), 86.0599 (86.0600, -0.1 mDa, -1.2 ppm), 97.0760 (97.0760, 0.0 mDa, 0.0 ppm), 112.0753 (112.0757, -0.4 mDa, -3.5 ppm), and 126.0548 (126.0550, -0.2 mDa, -1.6 ppm). NM of Aly&MOA sample shows Aly-MOA retention time (RT) at 5.13 minutes. D: Extracted ion chromatogram of ion C7H14N2O3 +/- 10 ppm to show RT of Aly-MOA at 5.13 minutes. E: Total ion spectrum of full scan at 5.13 minutes (RT of Aly-MOA) to show major ions of Aly- MOA is 173.0931 (theoretical: 173.0932, -0.1 mDa, -0.6 ppm). C: Total ion spectrum of product scan of 173.1 (ion mass of A-MOA) at 5.15 minutes (RT of Aly-MOA). Ion intensities are low. Major ion is 123.0568 (theoretical 123.0564, 0.4 mDa, 3.2 ppm). The rest of ions may come from the fragmentation of parent ions of 173.1 +/- 0.4 Da.

[0045] FIGURE 16: LC/MS results of allysine aldol and MOA conjugates (Aly aldol- MOA, aldol -MO A, Aly-Aly-MOA, AA-MOA) in Aly&MOA sample with 1-2 hours incubation in both positive and negative ion modes (PM&NM). PM of Aly&MOA sample shows Aly aldol-MOA retention time (RT) at 3.36, 4.03, 4.18, and 4.65 minutes. A: Extracted ion chromatogram of ion C₁₃H₂₃N₃O₅ +/- lOppm to show RT of Aly aldol-MOAs at 3.36, 4.03,

4.18, and 4.65 minutes. B: Total ion spectrum of full scan at 4.18 minutes (RT of third Aly aldol-MOA) to show major ions of 3^rd Aly aldol-MOA is 302.1712 (theoretical: 302.1711, 0.1 mDa, 0.3 ppm). C: Total ion spectrum of product scan of 302.2 (ion mass of Aly aldol-MOA) at 4.21 minutes (RT of 3^rd Aly aldol-MOA). Major ions are 55.0546 (55.0542, 0.4 mDa, 7.2 ppm), 56.0498 (56.0495, 0.3 mDa, 5.4 ppm), 82.0651 (82.0651, 0.0 mDa, 0.0 ppm), 97.0760 (97.0760, 0.0 mDa, 0.0 ppm), 128.0706 (128.0706, 0.0 mDa, 0.0 ppm), 135.0916 (135.0917, - 0.1 mDa, -0.7 ppm), 143.0816 (143.0815, 0.1 mDa, 0.7 ppm), 161.1073 (161.1073, 0.0 mDa, 0.0 ppm), and 179.1181 (179.1179, 0.2 mDa, 1.1 ppm). NM of Aly&MOA sample shows Aly aldol-MOA retention time (RT) at 3.32, 4.00, 4.19, 4.62, and 4.83 minutes. A: Extracted ion chromatogram of ion C₁₃H₂₃N₃O₅ +/- lOppm to show RT of Aly aldol-MOAs at 3.32, 4.00,

4.19, 4.62, and 4.83 minutes. B: Total ion spectrum of full scan at 4.19 minutes (RT of third Aly aldol-MOA) to show major ions of 3^rd Aly aldol-MOA is 300.1566 (theoretical: 300.1565, 0.1 mDa, 0.3 ppm). C: Total ion spectrum of product scan of 300.2 (ion mass of Aly aldol- MOA) at 4.20 minutes (RT of 3^rd Aly aldol-MOA). Major ions are 52.0190 (52.0193, -0.3 mDa, 5.8 ppm), 66.0350 (66.0349, 0.1 mDa, 1.5 ppm), 70.0299 (70.0298, 0.1 mDa, 1.4 ppm), 72.0091 (72.0091, 0.0 mDa, 0.0 ppm), 80.0506 (80.0506, 0.0 mDa, 0.0 ppm), 96.0456 (96.0455, 0.1 mDa, 1.0 ppm), 123.0565 (123.0564, 0.1 mDa, 0.8 ppm), 141.0671 (141.0670, 0.1 mDa, 0.7 ppm), 169.0984 (169.0983, 0.1 mDa, 0.6 ppm), and 197.1296 (197.1296, 0.0 mDa, 0.0 ppm).

[0046] FIGURE 17: Fibrosis formation by cross-links and degradation by MMPs and antifibrosis mechanism of MOA. Pl, P2,...P10 are polypeptide chains within collagens and/or elastin. They can be the same or can be different. LOX: Lysyl oxidase enzyme can oxidize primary amine of peptidyl lysine side chain to aldehyde (Aly). MMPs: Matrix metalloproteinases can degrade all kinds of ECM proteins including fibrosis tissue. Desmosine and isodesmosine are fibrosis biomarkers and can be detected and quantified in plasma of both animals and humans. Mechanism of collagen and elastin lysine oxidation by LOX, fibrosis formation based on chemical reactions among Aly and Lys, and anti-fibrosis reactions from MOA chemical reaction with aldehyde functionalities. LOX can oxidize Lys to Aly. Aly can spontaneously react with other Aly or Lys within the same protein or other proteins to form Aly aldol or dehydrolysinonorleucine. At the end to form desmosine or isodesmosine with proteins crosslinking. MOA can react with Aly to form Aly-MOA to block Aly aldol formation. MOA can also react with Aly aldol to form Aly aldol-MOA to block desmosine or isodesmosine formation. Furthermore, MOA can also react with dehydrolysinonorleucine to form Aly-MOA and release lysine. Fibrosis tissue is removed by MMPs in ECM.

[0047] FIGURE 18: Advanced glycation pathways and products that contribute to Alzheimer's disease, inflammation, and aging. MOA can form alkylating products with the aldehyde reactants or intermediate to prevent glycation. MOA can also react with the advanced glycation end-products (AGEs) to release the proteins or peptides. A. 3 -Deoxy glucosone (3DG) is a sugar in human body. 3DG reacts with protein to form AGEs, which may contribute to Alzheimer's disease, inflammation, and aging. In the dotted square, MOA can react with the 3DG to form alkylating product with the aldehyde functional groups. This can effectively prevent the glycation pathway with arginine containing peptides and proteins. The MOA can also break the 4-imidazolone ring to release the arginine based peptides and proteins from glycation. B. Glycations occur to simple sugars: glucose, fructose, and galactose. Glycation can occur through Amadori rearrangement reactions (or Schiff base reactions, and Maillard reactions) which lead to AGEs. In the dotted square, MOA can react with the aldehyde intermediate. This can effectively cut off the glycation Amadori rearrangement pathway. The MOA can also break the Amadori rearrangement end product six member ring to release the peptides and proteins from glycation.

DESCRIPTION

[0048] Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0049] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0050] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0051] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide molecule" includes a plurality of such peptides, and so forth.

[0052] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.

[0053] As used herein, the term “FGFR inhibitor” or “FGFRi” refers to any compound capable of inhibiting the enzymatic of FGFR, including its own auto-phosphorylation and the kinase activity. Such inhibitors efficiently inhibit FGFRs, and are said to “inhibit”, “decrease”, or “reduce” the biological activity of FGFRs. The FGFR inhibitors of the disclosure can be “pan-inhibitor” and present a broad efficiency at inhibiting one or more of FGFRI -FGFR₄ , or present a specific efficiency at inhibiting only one FGFR, FGFR₄ for example.

Definitions [0054] Bile acid (abbreviated BA): Bile acids are steroid acids found predominantly in the bile of mammals and other vertebrates. Different molecular forms of bile acids can be synthesized in the liver by different species. Chemical structures of BAs are shown in FIG. 3. Bile acids are conjugated with taurine or glycine in the liver, and the sodium and potassium salts of these conjugated bile acids are called bile salts. Primary bile acids are those synthesized by the liver (BA in FIG. 3., when R₃=0H and R₆=H). Secondary bile acids result from bacterial actions in the colon (BA in FIG. 3., when R₃=H and R₆=H). In humans, taurocholic acid and glycocholic acid (derivatives of cholic acid) and taurochenodeoxycholic acid and glycochenodeoxy cholic acid (derivatives of chenodeoxy cholic acid) are the major bile salts in bile and are roughly equal in concentration. The conjugated salts of their 7-alpha- dehydroxylated derivatives, deoxycholic acid and lithocholic acid, are also found, with derivatives of cholic, chenodeoxycholic and deoxycholic acids accounting for over 90% of human biliary bile acids.

[0055] 3k-bile acids (abbreviated 3k-BA): Bile acid metabolic products, which their corresponding 3α-hydroxyl group (-OH) converted to 3-oxo group (C=O). 3k-BAs is the major Phase 1 metabolites of BAs in human liver by CYP3A4 enzymes. The taurine (T) and glycine (G) conjugate of 3k-BA are abbreviated as 3k-TBA and 3k-GBA, respectively. Chemical structures of 3k-BAs are shown in FIG. 3.

[0056] Chenodeoxycholic acid (abbreviated CDCA): Also known as chenodesoxycholic acid, chenocholic acid and 3a,7a-dihydroxy-5P-cholan-24-oic acid is a bile acid. Salts of this carboxylic acid are called chenodeoxycholates. CDCA is one of the main bile acids produced by the liver. Chemical structure of CDCA is shown in BA of FIG. 3. when R₃=0H, R₄=H, R₅=0H, and R₆=H.

[0057] 3k-CDCA: 3-Oxo-7α-hydroxy-5P-cholanoic acid, a major Phase 1 metabolite of CDCA in human liver by its CYP3A4 enzyme. Chemical structure of 3k-CDCA is shown in FIG. 4.

[0058] C4: 7α-Hydroxy-4-cholesten-3-one, is an intermediate in the biochemical synthesis of bile acids from cholesterol. Its precursor, 7α-hydroxycholesterol, is produced from cholesterol by hepatic cholesterol 7α-hydroxylase (CYP7A1). It is metabolized by the enzyme 7α-hydroxycholest-4-en-3-one 12α-hydroxylase to 7α,12α-dihydroxycholest-4-en-3-one and then to cholic acid, the major primary bile acid in humans. Alternatively, it can be converted into 5P-cholestane-3a,7a-diol and then to CDCA, the other major primary bile acid in humans. Serum C4 concentrations reflect the activity of the bile acid synthetic pathway. Serum C4 values vary during the day as bile acid synthetic rates have a diurnal rhythm.

[0059] N-terminal cysteinyl-peptide: A peptide with N-terminal amino acid is cysteine without modification on both amino and thiol groups. 1,2-Amino, thiol groups on N-terminal cysteine are both without modifications. Chemical structure of N-terminal cysteine peptide is shown compound (1) in FIG. 1.

[0060] N-terminal cysteinyl-proteins: Proteins which N-terminal amino acid is cysteine with free thiol (-SH) and free amino (-NH₂) groups.

[0061] 3a-HSD: 3 α-Hydroxy steroid dehydrogenase is an enzyme in humans known to catalyze the conversion of aldehydes and ketones to their corresponding alcohols by utilizing NADH and/or NADPH as cofactors.

[0062] Thiazolidine: Thiazolidine is a heterocyclic organic compound with the formula (CH₂)3(NH)S. It is a saturated 5-member ring with a thioether group and an amine group in the 1- and 3-positions. Chemical structure of thiazolidine ring is shown in FIG. 1.

[0063] Cys4: Isotopes labeled L-Cysteine of three ¹³C and one ¹⁵N as shown in FIG. 4.

[0064] HCys: Homocysteine, is a non-proteinogenic a-amino acid. It is biosynthesized from methionine by the removal of its terminal Cε methyl group. HCys can be recycled into methionine or converted into cysteine with the aid of certain B-vitamins.

[0065] 3k-CDCA&Cys+Cys4: The reaction mixture with starting materials of 3k-CDCA, Cys and Cys4 compound of reaction Step 1 in FIG. 4.

[0066] 3k-CDCA&Cys+Cys4&MOA: The reaction mixture with starting materials of 3k- CDCA, Cys, Cys4 and then with MOA compound of reaction Step 2 in FIG. 3.

[0067] 3k-CDCA&Cys-Gly: The reaction mixture with starting materials of 3k-CDCA and Cys-Gly compound of reaction Step 1 in FIG. 1.

[0068] 3k-CDCA&Cys-Gly&MOA: The reaction mixture with starting materials of 3k- CDCA, Cys-Gly and MOA compound of reaction Step 2 in FIG. 1.

[0069] 3k-CDCA&HCys: The reaction mixture with starting materials of 3k-CDCA and HCys.

[0070] 3k-CDCA&HCys&MOA: The reaction mixture with starting materials of 3k- CDCA, HCys, and then with MOA.

[0071] 3k-CDCA-Cys: 3k-CDCA and Cys conjugate. 1, 2-Amino, thiol group on cysteine react with the 3-position ketone of 3k-CDCA to form a five-member ring thiazolidine. For chemical structure, please see 3k-CDCA-Cys in FIG. 4. Step 1. [0072] 3k-CDCA-Cys4: 3k-CDCA and Cys4 conjugate. 1, 2-Amino, thiol group on isotope labeled cysteine react with 3 -position ketone of 3k-CDCA to form a five-member ring thiazolidine. For chemical structure, please see 3k-CDCA-Cys4 in FIG. 4 Step 1.

[0073] C4-Cys: C4 and Cys conjugate. 1, 2-Amino, thiol group on cysteine react with the 3 -position ketone of C4 to form a five-member ring thiazolidine.

[0074] C4-Cys4: C4 and Cys4 conjugate. 1, 2-Amino, thiol group on isotope labeled cysteine react with 3 -position ketone of C4 to form a five-member ring thiazolidine.

[0075] 3k-CDCA-Cys+Cys4: 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates mixture.

[0076] C4-Cys+Cys4: C4-Cys and C4-Cys4 conjugate mixture.

[0077] 3k-CDCA-Cys-Gly: 3k-CDCA and Cys-Gly conjugate. 1, 2-Amino, thiol groups on cysteine react with 3-position ketone of 3k-CDCA to form a five-member ring thiazolidine.

[0078] 3k-CDCA-HCys: 3k-CDCA-HCys conjugate. 1, 3-Amino, thiol groups on HCys react with 3-position ketone of 3k-CDCA to form a six-member ring 1, 3-thiazinane.

[0079] 3k-CDCA-cysteinyl-peptide: 3k-CDCA and N-terminal cysteinyl-peptides conjugate. 1, 2-Amino, thiol groups on N-terminal cysteine react with the 3-position ketone of 3k-CDCA to form a five-member ring thiazolidine. Representative chemical structure of 3k- CDCA-cysteinyl -peptides is shown compound (3) in FIG. 1. R₁ and R₂ form the CDCA rings structure.

[0080] 3k-bile acid-cysteinyl-peptides (3k-BA-cysteinyl-peptides): 3k-bile acid and N- terminal cysteinyl-peptide conjugates. 1, 2-Amino, thiol groups on N-terminal cysteine reacts with the 3-position ketone of 3k-BA to form a five-member ring thiazolidine. Representative chemical structure of 3k-BA-cysteinyl-peptides is shown compound (3) in FIG. 1. R₁ and R₂ form the BA rings structure.

[0081] 3k-CDCA-MOA: 3k-CDCA and MOA conjugate. The 3k-CDCA-MOA has two epimers due to the rigidity of the C=N double bond. Both cis and trans epimers of ketone oxime exist simultaneously including 3k-CDCA-c-MOA and 3k-CDCA-t-MOA. The two epimers can also be labeled separately without differentiate their stereochemistry as 3k-CDCA- M0A1 and 3k-CDCA-MOA2, or labeled combined as 3k-CDCA-MOA. Chemical structure of 3k-CDCA-MOA is shown in FIG. 4.

[0082] C4-M0A: C4 and MOA conjugates. The C4-M0A has two epimers due to the rigidity of the C=N double bond.

[0083] GUA: aminoguanidine, Chemical structure is NH₂-(C=NH)-NH-NH₂

[0084] 3k-CDCA-GUA: 3k-CDCA and GUA conjugates. The 3k-CDCA-GUA has two epimers due to the rigidity of the C=N double bond. Both cis and trans epimers of hydrazone exist simultaneously including 3k-CDCA-c-GUA and 3k-CDCA-t-GUA. The two epimers can also be labeled separately without differentiate their stereochemistry as 3k-CDCA-GUAl and 3k-CDCA-GUA2, or labeled combined as 3k-CDCA-GUA.

[0085] C4-GUA: C4 and GUA conjugates. The C4-GUA has two epimers due to the rigidity of the C=N double bond.

[0086] 3k-bile acid-MOA (3k-BA-MOA): 3k-bile acid and MOA conjugates, 3k-BA-MOA has two epimers due to the rigidity of the C=N double bond. Chemical structure of 3k-BA- MOA is compound (4) in FIG. 1. R₁ and R₂ form the BA rings structure.

[0087] Aldehyde-N-terminal-cysteinyl-peptides: Aldehyde and N-terminal cysteinylpeptide conjugates. 1,2-Amino, thiol groups on N-terminal cysteine react with the aldehyde to form a five-member ring thiazolidine. Chemical structure of aldehyde-cysteinyl-peptides is compound (3) in FIG. 1. R₁ is organic moiety, and R₂ is hydrogen.

[0088] Aldehyde-MOA: Aldehyde and MOA conjugates, aldehyde-MOA has two epimers due to the rigidity of the C=N double bond. Chemical structure of aldehyde-MOAs is compound (4) in FIG. 1. R₁ is an organic moiety, and R₂ is hydrogen.

[0089] Ketone-N-terminal-cysteinyl-peptides: Ketone and N-terminal cysteinyl-peptide conjugates. 1, 2-Amino, thiol group on N-terminal cysteine react with the ketone to form a five-member ring thiazolidine. Chemical structure of aldehyde-cysteinyl-peptides is compound (3) in FIG. 1. R₁ and R₂ are organic moieties, and can also form rings.

[0090] Ketone-MOA: Ketone and MOA conjugates, ketone-MOA has two epimers due to the rigidity of the C=N double bond. Chemical structure of ketone-MOA is compound (4) in FIG. 1. R₁ and R₂ are organic moieties, and can also form rings.

[0091] Reactive ketone: Any ketone reacts with Cys (or HCys) in physiological condition to form a five-member ring thiazolidine (or six-member ring 1, 3-thiazinane) ketone-Cys (or ketone-HCys) conjugate.

[0092] Oxytocin: Abbreviated as Oxy. Oxy is a human endogenous peptide of nine amino acids (a nonapeptide) in the sequence cysteine-tyrosine-isoleucine-glutamine-asparagine- cysteine-proline-leucine-glycine-amide (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂ or CYIQNCPLG-NH₂); its C-terminus has been converted to a primary amide and a disulfide bridge joins the cysteine moieties. Ox has a molecular mass of 1007.2 Da.

[0093] Oxytoceine: Abbreviated as H20xy. Oxytoceine is reduced form of Oxy with 2 free thiol groups in its two Cys. H20xy has a molecular mass of 1009.2 Da. Please see structure of Oxytoceine in FIG. 11. [0094] 3k-CDCA-H20xy: 3k-CDCA and H20xy conjugate. N-terminal 1, 2-amino, thiol groups of H20xy react with the 3-position ketone of 3k-CDCA to form a five-member ring thiazolidine. For chemical structure, please see 3k-CDCA-H20xy in FIG. 11.

[0095] k-bile acid-H2Oxy: Abbreviated as 3k-BA-H20xy. 3k-BA and H20xy conjugate. 1,

2 -Amino, thiol group on H20xy react with the 3-position ketone of the 3k-BA to form a five- member ring thiazolidine. For chemical structure, please see example of 3k-CDCA-H20xy in FIG. 11.

[0096] 3k-bile acid-GUA (3k-BA-GUA, 3k-BA-G): 3k-BA and GUA conjugate. The 3k- BA-GUA has two epimers due to the rigidity of the C=N double bond.

[0097] Obeticholic acid: Abbreviated to OCA. OCA is a semi -synthetic bile acid analogue which has the chemical structure 6a-ethyl-chenodeoxycholic acid. It is used as a drug to treat primary biliary cholangitis. OCA is the most highly potent FXR agonist. OCA is the first FXR agonist to be used in human drug studies. Chemical structure of OCA is shown in BA of FIG. 3, when R₃=0H, R₄=H, R₅=0H, R₆=C₂H₅.

[0098] OCA glycine conjugate (GOCA): carboxylic group of OCA conjugates with amine of glycine to form an amide. It is a major metabolite of OCA in liver by the enzymes. Chemical structure of GOCA is shown in FIG. 3. (BA, when R₃=0H, R₄=H, R₅=Glycine, R₆=C₂H₅).

[0099] OCA taurine conjugate (TOCA): carboxylic group of OCA conjugates with amine of taurine to form an amide. It is a major metabolite of OCA in liver by the enzymes. Chemical structure of TOCA is shown in BA of FIG. 3. when R₃=0H, R₄=H, R₅=Taurine, R₆=C₂H₅.

[00100] 3k-OCA: 3α-hydroxy group (-OH) of OCA converted to 3-oxo group (C=O) as 3- keto-OCA. It is a metabolite of OCA in liver by the CYP3 A4 enzyme. Chemical structure of 3k-OCA is shown in FIG. 13.

[00101] 3k-GOCA: 3α-hydroxy group (-OH) of GOCA converted to 3-oxo group (C=O) as

3-keto-GOCA. It is a metabolite of GOCA in liver by the CYP3A4 enzyme. Chemical structure of 3k-GOCA is shown in 3k-BA of FIG. 3. when R₃=0H, R₄=H, R₅= Glycine, R₆=C₂H₅.

[00102] k-TOCA: 3α-hydroxy group (-OH) of TOCA converted to 3-oxo group (C=O) as 3- keto-TOCA. It is a metabolite of TOCA in liver by the CYP3 A4 enzyme. Chemical structure of 3k-TOCA is shown in 3k-BA of FIG. 3 when R₃=0H, R₄=H, R₅=Taurine, R₆=C₂H₅.

[00103] OCA&3a, OCA&3a-HSD: The reaction mixture with starting reagents of OCA, 3a- HSD enzyme and co-factor NAD+. Final solution is the mixture of OCA and 3k-OCA. The chemical reaction and structures of OCA and 3k-OCA are in BA and 3k-BA of FIG. 3. when R₃=OH, R₄=H, R₅=OH, R₆=C₂H₅.

[00104] OCA&3a-HSD&Cys+Cys4: OCA&3a-HSD&CC4, OCA&3a&CC4, The reaction mixture of OCA, 3α-HSD enzyme and co-factor NAD+, and Cys+Cys4 mixture. The reaction is Step 1 in FIG. 1.

[00105] OCA&3a-HSD&Cys+Cys4&MOA: OCA&3a&CC4&MOA. The reaction mixture with starting reagents of OCA, 3α-HSD enzyme and co-factor NAD+, and Cys+Cys4 mixture after Step 1 in FIG. 1, then mixed with MOA. The reaction is Step 2 in FIG. 1.

[00106] OCA&3a-HSD&CG: OCA&3a&CG. The reaction mixture with starting reagents of OCA, 3α-HSD enzyme and co-factor NAD+, and Cys-Gly. The reaction is Step 1 in FIG. 1.

[00107] OCA&3a-HSD&CG&MOA: OCA&3a&CG&MOA. The reaction mixture with starting reagents of OCA, 3α-HSD enzyme and co-factor NAD+, and CG after Step 1 in FIG. 1, then mixed with MOA. Reaction is Step 2 in FIG. 1.

[00108] OCA&3a-HSD&Oxy&DTT: OCA&3a&Oxy&DTT. The reaction mixture with starting materials OCA, 3α-HSD enzyme and co-factor NAD+, Oxytocine and DTT. The conversion of Oxytoceine (H20xy) from Oxytocine (Oxy) by DTT and their structures are in FIG. 13. The conversion of 3k-OCA from OCA by 3α-HSD enzyme and co-factor NAD+ and their structures are in FIG. 13. The reaction of 3k-OCA-H2Oxy conjugates and their structures are in Step 1 in FIG. 13.

[00109] OCA&3a-HSD&Oxy+DTT&MOA: OCA&3a&Oxy+DTT&MOA. The reaction mixture with starting materials of OCA&3a&Oxy+DTT and MOA. The reaction is Step 2 in FIG. 13.

[00110] 3k-OCA-Cys: 3k-OCA and Cys conjugate. 1, 2-Amino, thiol groups on cysteine react with the 3-position ketone of the 3k-OCA to form a five-member ring thiazolidine.

[00111] 3k-OCA-Cys4: 3k-OCA and Cys4 conjugate. 1, 2-Amino, thiol groups on isotope labeled cysteine react with 3-position ketone of 3k-OCA to form a five-member ring thiazolidine.

[00112] 3k-OCA-Cys+Cys4: 3k-OCA-CC4, 3k-OCA-Cys and 3k-OCA-Cys4 conjugate mixture.

[00113] 3k-OCA-Cys-Gly: 3k-OCA and Cys-Gly conjugate. 1, 2-Amino, thiol groups on cysteine react with 3-position ketone of 3k-OCA to form a five-member ring thiazolidine. [00114] 3k-OCA-H2Oxy: 3k-0CA and H20xy conjugate. 1, 2-Amino, thiol groups on N- terminal cysteine react with 3-position ketone of 3k-OCA to form a five-member ring thiazolidine. For chemical structure, please see 3k-OCA-H2Oxy in product of FIG. 13 Step 1. [00115] 3k-OCA-cysteinyl-peptide: 3k-OCA and N-terminal cysteinyl-peptides conjugate. 1, 2-Amino, thiol groups on N-terminal cysteine react with the 3-position ketone of 3k-OCA to form a five-member ring thiazolidine. Representative chemical structure of 3k-OCA-cysteinyl- peptides is shown compound (3) in FIG. 1. R₁ and R₂ form the OCA rings structure.

[00116] 3k-OCA-MOA: 3k-OCA and MOA conjugate. The 3k-OCA-MOA has two epimers due to the rigidity of the C=N double bond. Both cis and trans epimers of ketone oxime exist simultaneously including 3k-OCA-c-MOA and 3k-OCA-t-MOA. The two epimers can also be labeled separately without differentiating their stereochemistry as 3k-OCA-MOAl and 3k- OCA-MOA2, or labeled combined as 3k-OCA-MOA. Chemical structure of 3k-OCA-MOA is shown in the products of FIG. 13. Step 2.

[00117] CA: Cholic acid, representative chemical structure of CA is shown compound (BA) in FIG. 3. when R₃=0H; R₄=0H; R₅=0H; R₆=H.

[00118] DCA: Deoxycholic Acid, representative chemical structure of DCA is shown compound (BA) in FIG. 3. when R₃=H; R₄=OH; Rs=OH; R₆=H.

[00119] TCDCA: Taurochenodeoxy cholic Acid, representative chemical structure of TCDCA is shown compound (BA) in FIG. 3. when R₃=OH; R₄=H; R₅=taurine; R₆=H.

[00120] GCA: Glycocholic acid, representative chemical structure of GCA is shown compound (BA) in FIG. 3. when R₃=OH; R₄=OH; R₅=glycine; R₆=H.

[00121] GDCA: Glycodeoxycholic Accid, representative chemical structure of GDCA is shown compound (BA) in FIG. 3. when R₃=H; R₄=OH; R₅=glycine; R₆=H.

[00122] GCDCA: Glycochenoceoxycholic Acid, representative chemical structure of GCDCA is shown compound (BA) in FIG. 3. when R₃=OH; R₄=H; R₅=glycine; R₆=H.

[00123] 3k-CA: 3-keton CA, representative chemical structure of 3k-CA is shown compound (3k-BA) in FIG. 3. when R₃=OH; R₄=OH; R₅=OH; R₆=H.

[00124] 3k-DCA: 3-keton DCA, representative chemical structure of 3k-DCA is shown compound (3k-BA) in FIG. 3. when R₃ H; R₄=OH; Rs=OH; R₆=H.

[00125] 3k-TCDCA: 3-keton TCDCA, representative chemical structure of 3k-TCDCA is shown compound (3k-BA) in FIG. 3. when R₃=OH; R₄=H; R₅=taurine; R₆=H.

[00126] 3k-GCA: 3-keton GCA, representative chemical structure of 3k-GCA is shown compound (3k-BA) in FIG. 3. when R₃ OH; R₄=OH; R₅=glycine; R₆=H. [00127] 3k-GDCA: 3-keton GDCA, representative chemical structure of 3k-GDCA is shown compound (3k-BA) in FIG. 3. when R₃=H R₄=OH; R₅=glycine; R₆=H.

[00128] 3k-GCDCA: 3-keton GCDCA, representative chemical structure of 3k-GCDCA is shown compound (3k-BA) in FIG. 3. when R₃ OH; R₄=H; R₅=glycine; R₆=H.

[00129] 3k-bile acid-cysteinyl-peptides (3k-BA-cysteinyl-peptides): 3k-bile acid and N- terminal cysteinyl-peptide conjugates. 1,2-Amino, thiol groups on N-terminal cysteine reacts with the 3-position ketone of 3k-BA to form a five-member ring thiazolidine. Representative chemical structure of 3k-BA-N-terminal cysteinyl-peptides is shown compound (6) in FIG. 3.

[00130] 3k-bile acid-MOA (3k-BA-MOA): 3k-bile acid and MOA conjugates, 3k-BA-MOA has two epimers due to the rigidity of the C=N double bond. Chemical structure of 3k-BA- MOA is compound (7) in FIG. 3.

[00131] When abbreviate for an analyte or a compound name, the dash line (-) and underline (_) are used interchangeably in this document, for example, the abbreviation of 3k-DCA-C and 3k- DCA C refer to the same compound of 3k-DCA and cysteine conjugate. When abbreviate for a sample name, “&” represents “and” and “+”, for example, abbreviation of 3k- CDCA&Oxy&DDT and 3k-CDCA+Oxy+DDT refer to the same starting sample mixture of 3k- CDCA, oxytocin, and DDT.

[00132] Subject, individual or patient: For purposes of the specification and claims, to mean a human or other animal, such as farm animals or laboratory animals (e.g., guinea pig or mice) capable of having immune related diseases, either naturally occurring or induced, including but not limited to NAFLD, NASH, liver cirrhosis and DILI.

[00133] Therapeutically effective amount: the amount of the subject compound that will elicit a desired response, from example, a biological or medical response of a tissue, system, animal, or human that is sought, for example, by a researcher, veterinarian, medical doctor, or other clinician.

[00134] Pharmaceutically acceptable salt: Refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid and the like. Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as di cyclohexylamine, N-methyl-D-Glucamine, tris(hydroxymethyl)methylamine, C1-C7, alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine, lysine, and the like.

[00135] Fibrosis: also known as fibrotic scarring, is a pathological wound healing in which connective tissue replaces normal parenchymal tissue to the extent that it goes unchecked, leading to considerable tissue remodeling and the formation of permanent scar tissue.

[00136] Collagen: is the main structural protein in the extracellular matrix in the various connective tissues in the body. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole-body protein content. [00137] Extracellular matrix (ECM): is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, which provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM.

[00138] Matrix metallopeptidases (MMPs), also known as matrix metalloproteinases or matrixins, are metalloproteinases that are calcium-dependent zinc-containing endopeptidases; other family members are adamalysins, serralysins, and astacins. The MMPs belong to a larger family of proteases known as the metzincin superfamily. Collectively, these enzymes are capable of degrading all kinds of ECM proteins.

[00139] Lysyl oxidase (LOX): also known as protein-lysine 6-oxidase, is an enzyme that, in humans, is encoded by the LOX gene. It catalyzes the conversion of lysine molecules into highly reactive aldehydes (allysine) that form cross-links in ECM proteins.

[00140] Allysine (Aly): α-aminoadipidic-δ-semialdehyde, is a derivative of lysine, used in the production of elastin and collagen. It is produced by the actions of the enzyme LOX in the ECM and is essential in the crosslink formation that stabilizes collagen and elastin. Fibrous tissue containing oxidized collagen may result in a condition known as fibrosis. The oxidation of lysine resides present in collagen creates the aldehyde, Aly. Increased Aly concentration in tissues has been correlated to the presence of fibrosis. The general structure of Aly is in PIG. 17. [00141] A(l)-piperideine-6-carboxylate (P6C, Aly - H20, A - H20): P6C is an equilibrium of Aly in physiological condition.

[00142] Allysine aldol (Aly aldol, Aly-Aly, AA): is an aldol condensation of 2 allysines. The general structure of Aly aldol in fibrosis proteins is in FIG 17.

[00143] Allysine-MOA (Aly-MOA, A-MOA): Aly and MOA conjugates. Aly covalently binds with MOA. The general structure of Aly-MOA is in FIG 17.

[00144] Allysine aldol-MOA (Aly aldol-MOA, Aly-Aly-MOA, AA-MOA): Aly aldol and MOA conjugates. Allysine aldol covalently binds with MOA. The general structure of Aly aldol-MOA is in FIG 17

[00145] Allysine-GUA (Aly-GUA, A-GUA): Aly and GUA conjugates, Aly covalently binds with GUA.

[00146] Allysine aldol-GUA (Aly aldol-GUA, Aly-Aly-GUA, AA-GUA): Aly aldol and GUA conjugates, Aly aldol covalently binds with GUA.

[00147] Liver fibrosis: is the excessive accumulation of ECM proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation.

[00148] Liver cirrhosis: Cirrhosis occurs when the liver sustains substantial damage, and the liver cells are gradually replaced by scar tissue, which results in the inability of the liver to work properly. It’s a complication of liver disease that involves loss of liver cells and irreversible scarring of the liver. Some patients who develop cirrhosis may eventually require a liver transplant.

General description of the disclosure

[00149] This invention generally relates to novel compositions and methods for the treatment of certain immune response diseases, inflammation related diseases, Alzheimer’s diseases and fibrosis diseases. Additionally, this invention relates to novel compositions and methods to screen drug candidates, which may be metabolized to harmful aldehydes and/or reactive ketones

[00150] Certain endogenous and exogenous reactive compounds can bind covalently to protein and DNA resulting in organ toxicity and carcinogenesis, respectively. A number of withdrawn drugs are known to undergo bioactivation by a range of drug metabolizing enzymes to become reactive metabolites. An important goal in drug discovery is to identify structural sites of bioactivation within discovery molecules for providing strategic modifications that eliminate or minimize reactive metabolite formation. Techniques currently used to detect reactive drug metabolites are in vitro radiolabeled drug covalent binding to protein and reactive metabolite trapping with reagents such as glutathione, cyanide, semicarbazide and DNA bases. A framework hypothesis introduced during the 1960s and 1970s and used to research drug- mediated toxicity proposed that some drugs cause organ toxicity or carcinogenesis after becoming metabolized to chemically reactive metabolites that react with and covalently bind to tissue macromolecules, including proteins and DNA. The bioactivation of drugs having varied structures by CYP450s leads to varied types of chemically reactive species such as epoxide-, α,β-unsaturated ketone-, quinone-, quinone imine-, quinone methide-, imine methide-, and nitroso- intermediates that lead to covalent binding (CVB) to cellular macromolecules and potentially resulting in organ toxicity or carcinogenesis.

[00151] Drug bioactivation is an important issue in drug discovery where a major concern is not only the ability of reactive metabolites to mediate acute mechanism-based toxicity but also that reactive drug metabolites might form immunogenic drug-protein conjugates that lead to a triggering of immune-based idiosyncratic toxicities. There have been significant advances towards understanding of drug bioactivation mechanisms and subsequent reactions with cellular macromolecules since the 1960s and 1970s. These advancements were made possible due to drug metabolism and bioanalytical scientists becoming increasingly capable at rapidly characterizing chemical structures of reactive intermediates, adducts and conjugates with cellular nucleophiles, using advanced instrumentation such as liquid chromatography-tandem mass spectrometry (LC-MS).

[00152] Aldehyde can react with N-terminal cysteinyl-peptides under physiological condition through the formation of covalently bond five-member ring thiazolidine derivative. Proteins in cells can be proteolyzed to small peptides through chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH) protein digestive enzymes in proteasome. Within these proteolyzed small peptides, some of them are N-terminal cysteinyl-peptides. These N-terminal cysteinyl-peptides covalently bind with endogenous or exogenous aldehydes to form aldehyde-N-terminal cysteinyl-peptide conjugates (complexes) through thiazolidine derivative formation between the aldehyde functional group and 1,2-amino thiol groups of N- terminal cysteine. Certain aldehyde-N-terminal cysteinyl-peptide conjugates can cause immune response and immune activation to finally cause the cell damages of those cells. Ketone has less tendency to react with N-terminal cysteine to form thiazolidine derivative compared to that of the aldehyde. The reaction of the 3-position ketone of 3-Oxo-7α-hydroxy-5β-cholanoic Acid (acronym: 3 -ketone-chenodeoxy cholic acid or 3k-CDCA) with N-terminal cysteinyl- peptide to form thiazolidine have not been reported and verified before in both in vitro and in vivo conditions.

[00153] This invention discloser reports the discovery of certain ketone (3k-bile acids, for example, the ketone at the 3-position of 3k-CDCA) can covalently bind to 1,2-amino thiol groups of N-terminal cysteinyl-peptides under physiological condition to form thiazolidine five-member ring structure as shown in FIG. 1. This reaction is specific to certain types of ketone (Example 1, 2, 5, and 7, the ketone at the 3-position of 3k-CDCA, 3k-BAs, and 3k- OCA). The formation of thiazolidine by condensation of aldehyde (or reactive ketone) with 1,2-amino thiol groups of N-terminal cysteine can be chemically reversed, and the free N- terminal cysteine can be regenerated by covalent trapping of the aldehyde (or reactive ketone) derivative compound. Both MOA and GUA can break the covalent bond of thiazolidine under physiological condition to release N-terminal cysteinyl-peptides by MOA or GUA covalently binding with the aldehyde (or reactive ketone). Therefore, MOA and GUA can be used to release N-terminal cysteinyl-peptides and -proteins from their peptides and proteins conjugates of the aldehydes or reactive ketones. This release reaction induced by MOA or GUA can happen inside or outside of the cell, such as plasma or lymphatic fluid. This release reaction can also happen to the peptides conjugates either free or complexed with HMC/HLA molecules. MOA and GUA can also directly covalently bind with the reactive ketone, which form thiazolidines with the N-terminal cysteine. The direct binding of MOA or GUA with reactive ketone or aldehyde prevents or reduces the formation of the N-terminal cysteinylpeptide and -protein conjugation with aldehyde or reactive ketone molecules. The nucleophilic amino group of methoxyamine or aminoguanidine reacts with aldehydes, reactive ketones or thiazolidine by a Schiff-base reaction mechanism leading to the formation of stable oxime or hydrazone. A whole class of the alkylating agents can perform the similar functions described here as that of methoxyamine.

[00154] Bile acids are mainly synthesized in liver, transported to and stored in bile, and used in the digestive track, and collected back to liver through enterohepatic circulation system. Bile acids are involved in several important physiological functions, including the excretion of excess hepatic cholesterol and phospholipid, as well as the solubilization and absorption of lipid-soluble nutrients from the diet. Bile acids also serve as hepatic signaling molecules through activation of nuclear receptors such as famesoid X receptor (FXR), vitamin D receptor (VDR), pregnane X receptor (PXR), and constitutive androstane receptor, which function in the transcriptional regulation of genes involved in bile acid synthesis, transport, and metabolism. The 3-keto-bile acids are the major metabolic products of bile acids by the acts of 3α-hydroxysteroid dehydrogenase (3α-HSD) and human liver microsomes CYP3A4 enzymes in the body. We discover that 3k-CDCA can react with 1,2-amino, thiol groups of the N- terminal cysteinyl-peptides under physiological condition as shown in FIG. 4 and FIG. 11. Since the major components of bile is bile acids and their major metabolites are 3k-bile acids in the liver, we hypothesize that the existence in the liver of broad ranges of 3k-bile acids and peptides conjugates via the formation of thiazolidine five-member ring structure between 3- position ketone of 3k-bile acids and 1,2-amino thiol groups of the N-terminal cysteinylpeptides. 3k-bile acid-cysteinyl-peptide conjugates when presented by the HMC/HLA molecules to the cell (hepatocyte) surface, may be identified as harmful foreign antigens by the T cells to activate the adaptive immune response as shown in FIG. 2. The immune responses may lead to formation of permanent hepatocyte, tissue, and organ damage such as steatosis and cirrhosis of the liver. Therefore, we propose the immune response to the 3k-bile acids with N- terminal cysteinyl-peptides and -proteins conjugates be a leading cause of the liver diseases of NAFLD, NASH, and liver cirrhosis.

[00155] OCA is a semi-synthetic bile acid analogue. OCA metabolizes differently from nature bile acids in the mammalian body. OCA transforms into OCA taurine (TOCA) and glycine (GOCA) conjugate in human body. The 3k-OCA and its two glycine (3k-GOCA) and taurine (3k-TOCA) conjugates are the major metabolic products of OCA and its two glycine (GOCA) and taurine (TOCA) conjugates by the acts of 3α-hydroxysteroid dehydrogenase (3a- HSD) and human liver microsomes CYP3A4 enzymes in the body as shown in FIG. 3. We discover that 3k-OCA can react with 1,2-amino, thiol groups of the N-terminal cysteinyl- peptides under physiological condition as shown in FIG. 13. Since the major phase 2 metabolites of OCA is GOCA and TOCA conjugates and their major phase 1 metabolites are 3k-TOCA and 3k-GOCA in the liver, we hypothesize that the existence in the liver of broad ranges of 3k-OCA and its two glycine and taurine conjugates and peptides conjugates via the formation of thiazolidine five-member ring structure between 3-position ketone of 3k-OCA, 3k-GOCA and 3k-TOCA and 1,2-amino thiol groups of the N-terminal cysteinyl-peptides. 3k- OCA-cysteinyl-peptide conjugates (along with 3k-GOCA-cysteinyl-peptide conjugates and 3k- TOCA-cysteinyl-peptide conjugates) when presented by the HMC/HLA molecules to the cell (hepatocyte) surface, may be identified as harmful foreign antigens by the T cells to activate the adaptive immune response as shown in FIG. 2. Since OCA is a semi-synthetic bile acid, 3k-OCA(3k-TOCA and 3k-GOCA)-N-terminal cysteine piptide/protein conjugates may be even stronger foreign antigens to activate adaptive immune response of the mammalian body. The immune responses may lead to pruritus, hypersensitivity reactions, and the formation of permanent hepatocyte, tissue, and organ damage such as liver toxicity and liver enzyme elevations. Therefore, we propose the immune response to the 3k-OCA and its two glycine and taurine conjugates (3k-TOCA and 3k-GOCA) with N-terminal cysteinyl-peptides and proteins conjugates be a leading cause of adverse effects of OCA, such as pruritus, induced high cholesterol, paradoxical worsening of the liver disease, persistent worsening of serum enzyme elevations and hepatic decompensation, jaundice and fatigue, liver enzyme elevations, and severe hypersensitivity reactions, and other severe liver injury.

[00156] Since MOA can not only release N-terminal cysteinyl-peptides from their 3k-bile acids conjugates, but also directly react with the 3k-bile acids in physiological condition to prevent the bile acids and N-terminal cysteinyl-peptides or proteins conjugation reactions from happening, MOA can be used as a drug to eliminate or reduce the concentration of antigens (such as N-terminal cysteinyl-peptides and protein conjugate with 3k-bile acids). This will reduce, stop, or revise the symptoms of immune response in the liver; therefore MOA and its physiological accepted salts can be used as an agent to treat NAFLD, NASH and liver cirrhosis. Since MOA can not only release N-terminal cysteinyl-peptides from their 3k-OCA (3k-GOCA and 3k-TOCA) related conjugates, but also directly react with the 3k-OCA (3k- GOCA and 3k-TOCA) in physiological condition to prevent the 3k-OCA and its two glycine and taurine conjugates and N-terminal cysteinyl-peptides or proteins conjugation reactions from happening, MOA can be used as a drug to eliminate or reduce the concentration of antigens (such as N-terminal cysteinyl-peptides and -protein conjugate with 3k-bile acids or 3k-OCA). This will reduce, stop, or revise the symptoms of immune response in the liver; therefore MOA and its physiological accepted salts could be used as an agent alone or with other drugs combination to treat NAFLD, NASH and liver cirrhosis.

[00157] Homocysteine thiolactone, an intramolecular thioester of homocysteine, is synthesized by methionyl-tRNA synthetase in an error-editing reaction that prevents translational incorporation of homocysteine into proteins. The synthesis of thiolactone occurs in all human cell types investigated. An increase in homocysteine levels leads to elevation of thiolactone levels in human cells. In cultured human cells and in human serum, homocysteine thiolactone reacts with proteins and peptides by a mechanism involving homocysteinylation of primary amines of proteins and peptides, which are N-terminal amine and lysine residues, to form N-terminal homocysteinyl-peptides or -proteins. Like N-terminal cysteinyl-peptides, N- terminal homocysteinyl-peptides can also react with 3k-CDCA to form 3k-CDCA-N-terminal homocysteinyl-peptides conjugates. These conjugates can also activate the immune system. MOA can reacts with reactive ketone-N-terminal homocysteinyl-peptides conjugates to form reactive ketone-MOA conjugates and release homocysteinyl-peptides from reactive ketone-N- terminal homocysteinyl-peptides conjugates.

[00158] Like MOA, GUA can not only release N-terminal cysteinyl-peptides from their 3k- bile acids conjugates, but also directly react with the 3k-bile acids in physiological condition to prevent the bile acids and N-terminal cysteinyl-peptides or -proteins conjugation reactions from happening, GUA can be used as a drug to eliminate or reduce the concentration of antigens (such as N-terminal cysteinyl-peptides and -protein conjugate with 3k-bile acids). This will reduce, stop or revise the symptoms of immune response in the liver; therefore GUA and its physiological accepted salts can be used as an agent to treat NAFLD, NASH and liver cirrhosis.

[00159] NAFLD, NASH, and liver cirrhosis are the most prevalent liver disease worldwide, and there is no approved pharmacotherapy. The current NASH therapies include four main pathways. The first approach is targeting hepatic fat accumulation. Medications in this approach include modulation of peroxisome proliferator-activator receptors (e.g., pemafibrate, elafibranor), medications targeting farnesoid X receptor axis (obeticholic acid, abbreviated OCA), inhibitors of de novo lipogenesis (aramchol, ACC inhibitor), and fibroblast growth factor-21 analogues. A second target is oxidative stress, inflammation, and apoptosis. This class of drug includes apoptosis signaling kinase 1 (ASK1) inhibitor and emricasan (an irreversible caspase inhibitor). A third target is intestinal microbiomes and metabolic endotoxemia. Several agents are in ongoing trials, including IMMel24, TLR₄ antagonist, and solithromycin (macrolide antibiotics). The final target is hepatic fibrosis, which is strongly associated with all-cause or liver-related mortality in NASH. Antifibrotic agents are a cysteinecysteine motif chemokine receptor-2/5 antagonist (cenicriviroc; CVC) and galectin 3 antagonist. This invention disclosure provides a different pathway to address the NAFLD, NASH, and liver cirrhosis diseases. This invention can be used independently to treat liver diseases, or it can be used as a combination drug treatment along with one or more of the current four pathway drugs.

[00160] The MOA agent can either release N-terminal cysteinyl-peptides from their 3k-bile acids conjugates, or directly react with the 3k-bile acids in physiological condition to prevent the conjugation reactions from happening. The agents, which can perform this task, are not limited to MOA. Agents such as GUA, semi carb azide, and alkylating compounds as defined in the embodiment (Detailed description of preferred embodiments, 1. The choices of alkylating agents) used for the treatment of NAFLD, NASH, and liver cirrhosis. [00161] The MOA agent not only can release N-terminal cysteinyl-peptides from their 3k- bile acids and 3k-OCA (also include 3k-GOCA and 3k-TOCA) conjugates, but also can directly react with the 3k-bile acids and 3k-OCA in physiological condition to prevent the conjugation reactions from happening. The agents, which can perform this task, are not limited to MOA. Agents such as aminoguanidine (GUA), semi carb azide, and alkylating compounds as defined in the embodiment (Detailed description of Preferred embodiments: 1. The choices of alkylating agents) used for the treatment of primary biliary cholangitis (PBC), nonalcoholic steatohepatitis (NASH), NAFLD, portal hypertension, bile acid diarrhea and primary sclerosing cholangitis (PSC).

[00162] Since MOA and GUA can potentially treat reactive ketone and aldehyde caused diseases, alkylating agents which can react with ketones and aldehydes can be used for treatment of all potential ketone and aldehyde caused diseases.

DILI

[00163] Drug-induced liver injury (DILI) is an uncommon, but potentially fatal, cause of liver disease that is associated with prescription medications, over the counter (OTC) drugs, and herbal and dietary supplements (HDS). Drugs or its reactive metabolites are considered as foreign antigens that bind to T cell receptors (TCR) and further activate immune response. In case of the Abacavir hypersensitivity, the aldehyde metabolites from its hydroxyl group react with a short peptide and form a peptide-HLA complex. This complex activates immune response, which release inflammatory cytokines and start the hypersensitivity response. More recently, it has been shown that Abacavir might occupy a space below the region of HLA that presents peptides, which leads to an altered peptide presentation and trigger an autoimmune reaction. MOA and other alkylating agents can release the peptide from the drug or its metabolites peptide conjugation, therefore the immune response will not be triggered. MOA and alkylating agents can be used to treat DILI.

AZ

[00164] Bile acids are mainly synthesized in liver, transported to and stored in bile, and used in the digestive track, and collected back to liver through enterohepatic circulation system. In the terminal ileum and colon, BAs are reabsorbed by the enterocytes and released into the portal vein for return to the liver where they are conjugated to produce their glycine and taurine forms. Primary bile acids are those synthesized by the liver. Secondary bile acids result from bacterial actions in the colon. Small amount of primary bile acids is also synthesized in the central nerve system such as in the human brain. Beyond BAs’ role in cholesterol clearance, BAs are major regulators for maintaining energy homeostasis through binding to nuclear receptors, including FXR and LXR among others. BAs also modulate the gut microbiome and are indicators of gut dysbiosis. Both primary and secondary BAs are present in the brains of mice and possibly humans with evidence that they cross the blood-brain barrier. Some BAs such as ursodeoxycholic acid exert beneficial effects while others are known to be cytotoxic. In particular, DCA’s toxicity has been associated with modulating apoptosis involving mitochondrial pathways in a variety of tissues and cell types. In recent pilot human studies, BA profiles have strong correlation with AD. When compared BA profile in AD to control group, a significant decrease in levels of the primary BA, CA, is detected. In contrast, a significant increase of bacterially produced secondary BA, DCA, was noted along with several secondary conjugated BAs, GDCA, TDCA, and GLCA. GDCA and GLCA were significantly associated with ADAS-Cogl3 where higher levels indicated worse cognition. The significant increase in ratios of secondary to primary BAs suggest altered activity of bacterial 7α-dehydroxylases leading to excess production of secondary BAs many of which are cytotoxic.

[00165] The 3-keto-bile acids (3k-bile acids) are the major metabolic products of bile acids (both primary and secondary) by the acts of 3α-hydroxysteroid dehydrogenase (3α-HSD) and human liver microsomes CYP3A4 enzymes in the body. We discover that 3k-bile acids can react with 1,2-amino, thiol groups of the N-terminal cysteinyl-peptides under physiological condition as shown in FIG. 3. Since the major components of bile is bile acids and their major metabolites are 3k-bile acids in the liver, we hypothesize that the existence in the brain of broad ranges of 3k-bile acids and peptides conjugates via the formation of thiazolidine five- member ring structure between 3-position ketone of 3k-bile acids and 1,2-amino thiol groups of the N-terminal cysteinyl-peptides. 3k-bile acid-cysteinyl-peptide conjugates when presented by the HMC/HLA molecules to the brain cell surface may be identified as harmful foreign antigens by the T cells to activate the adaptive immune response as shown in FIG. 2. In AD patients, the over-supply of secondary bile acids leads to over conversion to the secondary 3k- bile acids. Secondary 3k-bile acids are particularly liable to induce host adaptive immune responses as they are made by gut bacteria rather than synthesized by the host. The immune responses may lead to inflammation of related organs, especially the liver and brain, where are the origins of the bile acid synthesis. Therefore, we propose the immune response in the brain to the 3k-bile acid-N-terminal cysteinyl-peptides and -proteins conjugates be a leading cause of the Alzheimer’s disease. The 3k-bile acid-N-terminal cysteinyl-peptides and -proteins conjugates are molecular biochemistry mechanistic connection among the gut bacteria, liver and brain.

[00166] Since MOA can not only release N-terminal cysteinyl-peptides from their 3k-bile acids conjugates, but also directly react with the 3k-bile acids in physiological condition to prevent the bile acids and N-terminal cysteinyl-peptides or -proteins conjugation reactions from happening, MOA can be used as a drug to eliminate or reduce the concentration of antigens (such as N-terminal cysteinyl-peptides and -protein conjugate with 3k-bile acids). This will reduce, stop or revise the symptoms of inflammation of the brain tissues; therefore MOA and its physiological accepted salts can be used as an agent to treat Alzheimer’s disease. The agents, which can perform this task, are not limited to MOA. Agents such as Semicarbazide (SCZ), and alkylating compounds as defined in the embodiment 1 (The choices of alkylating agents) can also be used for the treatment of AD. The alkylating agents can be used independently to treat AD, or they can be used as a combination drug treatment along with one or more of the current drugs.

Testing

[00167] Due to the unique reactivity of the cysteine (Cys) towards reactive ketone and aldehyde to form five-member ring thiazolidines in physiological condition, cysteine can be used as a maker for reactive ketone and aldehyde detection. We have demonstrated using cysteine mixed with isotopes labeled cysteine-¹³C3,¹⁵N (Cys4) to react with 3k-CDCA to form the 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugate, and using LC-MS to verified the formation of the Cys and 3k-CDCA conjugate (3k-CDCA-Cys) and Cys4 and 3k-CDCA (3k-CDCA- Cys4) conjugate. This simple method uses basic triple Qua MS to detect the signature Cys and 3k-CDCA and Cyc4 and 3k-CDCA conjugates. This invention disclosure also describes using cysteine, N-terminal cysteinyl-peptides and -proteins to detect aldehyde and reactive ketone. This method can be used in detecting aldehydes and reactive ketones in physiological condition for drug reactive metabolite screening, and for endogenous aldehyde and reactive ketone detection.

[00168] Like Cys, homocysteine (HCys) towards reactive ketone and aldehyde to form six- member ring 1, 3-thiazinane in physiological condition, homocysteine and isotope labeled homocysteine can be used as a maker for reactive ketone and aldehyde detection.

[00169] 3k-CDCA-N-terminal cysteinyl-peptide conjugates (or other reactive ketone and aldehyde and N-terminal cysteinyl-peptide or -protein conjugates) are covalently bond with the thiazolidine ring in physiological condition. Methoxyamine (MOA) can release the N-terminal cysteinyl-peptide in physiological condition, and form 3k-CDCA-MOA conjugates. The 3k- CDCA-Cys or 3k-CDCA-N-terminal cysteinyl-peptide can be used to screen for the potency of alkylating agents to release N-terminal Cys-peptide from 3k-CDCA-N-terminal cysteinyl- peptide conjugates (or other reactive ketone and aldehyde conjugates).

Fibrosis

[00170] This disclosure generally related to compositions and methods for the treatment of allysine-related fibrosis diseases, Additionally, this disclosure related to novel compositions and methods to detect the aldehyde that cause the diseases, and to screen drugs for the treatment of the diseases.

[00171] Collagen in ECM can be oxidized by lysyl oxidase. Some lysines in oxidized collagens converted to allysines. Allysines in the collagens can spontaneous condense to allysine aldol from 2 allysines, and further condense to desmosines from 3 allysines and one lysine. Collagens condensed by desmosine structures can cause fibrosis in different tissues, such as lung, kidney, liver, and brain. In physiological condition, MMPs enzymes in ECM can remove fibrosis tissue in ECM. If fibrosis structures are over the remove ability of MMPs, it will last in normal tissue. The fabric connective tissue will take the space used to occupied by normal tissue and decrease the function of normal tissue or organ, Such as liver fibrosis and cirrhosis.

[00172] Alkylating agent such as MOA and GUA can react with allysine to form aldehyde alkylating agent conjugates to prevent allysine condensation reaction from becoming allysine aldol. Alkylating agent can also react with allysine aldol to form aldehyde alkylating agent conjugates to block further condensation of allysine aldol with lysine from forming desmosine, which is the final structure of collagen condensation. Alkylating agent can also react with dehydrolysinonorleucine to form allysine alkylating agent conjugates and release normal lysine. Because alkylating agent (such as MOA or GUA) can react with allysine, allysine aldol, and dehydrolysinonorleucine in physiological condition, and the reaction rate between the aldehyde functional group and alkylation agent is high, certain alkylating agent can be used for treatment of allysine related fibrosis diseases.

[00173] In treatment, the dose of agent optionally ranges from about 0.0001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.15 mg/kg to about 3 mg/kg, 0.5 mg/kg to about 2 mg/kg and about 1 mg/kg to about 2 mg/kg of the subject's body weight. In other embodiments the dose ranges from about 100 mg/kg to about 5 g/kg, about 500 mg/kg to about 2 mg/kg and about 750 mg/kg to about 1.5 g/kg of the subject's body weight. For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of agent is a candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage is in the range from about 0.001 μg/kg to 200 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. Unit doses can be in the range, for instance of about 5 mg to 500 mg, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg and 300 mg.

[00174] In some embodiments, an agent is administered to a human patient at an effective amount (or dose) of less than about 1 μg/kg, for instance, about 0.35 to about 0.75 μg/kg or about 0.40 to about 0.60 μg/kg. In some embodiments, the dose of an agent is about 0.35 μg/kg, or about 0.40 μg/kg, or about 0.45 μg/kg, or about 0.50 μg/kg, or about 0.55 μg/kg, or about 0.60 μg/kg, or about 0.65 μg/kg, or about 0.70 μg/kg, or about 0.75 μg/kg, or about 0.80 μg/kg, or about 0.85 μg/kg, or about 0.90 μg/kg, or about 0.95 μg/kg or about 1 μg/kg. In various embodiments, the absolute dose of an agent is about 2 μg/subject to about 45 μg/subject, or about 5 to about 40, or about 10 to about 30, or about 15 to about 25 μg/subject. In some embodiments, the absolute dose of an agent is about 20 μg, or about 30 μg, or about 40 μg.

[00175] In various embodiments, the dose of an agent may be determined by the human patient’s body weight. For example, an absolute dose of an agent of about 2 μg for a pediatric human patient of about 0 to about 5 kg (e.g. about 0, or about 1, or about 2, or about 3, or about 4, or about 5 kg); or about 3 μg for a pediatric human patient of about 6 to about 8 kg (e.g. about 6, or about 7, or about 8 kg), or about 5 μg for a pediatric human patient of about 9 to about 13 kg (e.g. 9, or about 10, or about 11, or about 12, or about 13 kg); or about 8 μg for a pediatric human patient of about 14 to about 20 kg (e.g. about 14, or about 16, or about 18, or about 20 kg), or about 12 μg for a pediatric human patient of about 21 to about 30 kg (e.g. about 21, or about 23, or about 25, or about 27, or about 30 kg), or about 13 μg for a pediatric human patient of about 31 to about 33 kg (e.g. about 31, or about 32, or about 33 kg), or about 20 μg for an adult human patient of about 34 to about 50 kg (e.g. about 34, or about 36, or about 38, or about 40, or about 42, or about 44, or about 46, or about 48, or about 50 kg), or about 30 μg for an adult human patient of about 51 to about 75 kg (e.g. about 51, or about 55, or about 60, or about 65, or about 70, or about 75 kg), or about 45 μg for an adult human patient of greater than about 114 kg (e.g. about 114, or about 120, or about 130, or about 140, or about 150 kg). [00176] In certain embodiments, an agent in accordance with the methods provided herein is administered subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intranasally or topically. Administration of an agent described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the human patient. The dosage may be administered as a single dose or divided into multiple doses. In some embodiments, an agent is administered about 1 to about 3 times (e.g. 1, or 2 or 3 times).

Detailed description of certain embodiments

Embodiment 1 : The choices of alkylating agents

[00177] Aldehydes and reactive ketones may be reactive towards proteins and peptides with 1,2-amino, thiol groups of the N-terminal cysteine to form thiazolidine five-member ring structure under physiological condition. These protein or peptide aldehyde or ketone conjugates in the mammalian body may lead to immune response and immune caused system damages. The response of immune system toward the protein and peptide conjugate can lead to immune related diseases in mammal. Alkylating agents such as methoxyamine (MOA) and aminoguanidine (GUA) and their salts can react with aldehydes and reactive ketones to block the formation of thiazolidine ring with N-terminal Cys to prevent the formation of protein or peptide conjugate structure. In case the aldehyde (or ketone) and peptide (or protein) conjugates are already formed, the alkylating agents can release the peptide (or protein) from the aldehyde (or ketone) conjugates by formation of alkylating conjugate with the aldehyde (or ketone). The alkylating agents can block the formation of protein (or peptide) conjugation with aldehyde (or ketone), and can break down the protein (or peptide) and aldehyde (or ketone) conjugation to prevent the activation of the immune responses of the mammal, therefore prevent or stop the progress of immune related diseases or reduce and relieve the symptoms of the immune related diseases. The immune related diseases include but not limited to metabolic diseases, NAFLD, NASH, liver cirrhosis, Alzheimer’s disease, fibrosis diseases, and/or DILI (drug induced liver injury, idiosyncratic toxicity) of certain drugs, including but not limited to toxicity of HIV drug Abacavir.

Embodiment 2: MOA and alkylating agents as a drug to treat liver diseases, Alzheimer’s diseases and/or fibrosis diseases. [00178] SSAO (primary-amine oxidase, also known as Semicarbazide-Sensitive Amine Oxidase) converts lysine residues to a-aminoadipidic-δ-semialdehydes, generally referred to as allysines. Increased allysine concentration in tissues has been correlated to the presence of fibrosis. Both MOA and GUA can react directly with aldehyde of allysine to stop the crosslinking. MOA, GUA and alkylating agents can react with aldehyde of allysine and forms stable allysine-MOA (-GUA, -alkylating agents) conjugates to block cross-link formation. MOA can also react with dehydrolysinonorleucine (Schiff Base) bifunctional cross-link, forms stable allysine-MOA conjugates, and releases normal lysine residue from the bifunctional cross-link. MOA, GUA and alkylating agents can further stop or slow down fibrosis for NAFLD, NASH, liver cirrhosis, and/or other fibrosis related diseases.

[00179] There is a newly discovered biochemistry linkage between gut microbiota and Alzheimer’s disease progression. The 3k-bile acid-N-terminal cysteinyl -peptide (or -protein) conjugate is molecular biochemistry mechanistic connection among the gut bacteria, liver and brain. Increasing evidence suggests a role for the gut microbiome in central nervous system disorders and a specific role for the gut-brain axis in neurodegeneration. Bile acids (BAs), products of cholesterol metabolism and clearance, are produced in the liver and are further metabolized by gut bacteria. They have major regulatory and signaling functions and dysregulated in Alzheimer’s disease (AD). Serum levels of 15 primary and secondary BAs and their conjugated forms were measured in 1464 subjects including 370 cognitively normal older adults, 284 with early mild cognitive impairment, 505 with late mild cognitive impairment, and 305 AD cases enrolled in the AD Neuroimaging Initiative. There are strong associations of BA profiles including selected ratios with diagnosis, cognition, and AD-related genetic variants, adjusting for confounders and multiple testing.

[00180] In AD compared to cognitively normal older adults, it is observed significantly lower serum concentrations of a primary BA, cholic acid (CA), and increased levels of the bacterially produced, secondary BA, deoxycholic acid (DCA), and its glycine and taurine conjugated forms. An increased ratio of DCA:CA, which reflects 7α-dehydroxylation of CA by gut bacteria, strongly associated with cognitive decline. Several genetic variants in immune response-related genes implicated in AD showed associations with BA profiles. It is clearly demonstrated an association between altered BA profile, genetic variants implicated in AD, and cognitive changes in disease through a large multicenter study.

[00181] One preferred embodiment is to use methoxyamine or pharmaceutically acceptable salts (MOA) (and/or aminoguanidine, and/or alkylating agent) to a patient diagnosed with NAFLD, NASH, liver cirrhosis, Alzheimer’s disease, and/or fibrosis diseases. Therapeutically effective amount of MOA is administered to the patient orally in an amount and duration sufficient to slowing down, stopping progress of said diseases or reversing the symptoms of said diseases.

[00182] Another preferred embodiment is to use methoxyamine or pharmaceutically acceptable salts (and/or aminoguanidine, and/or alkylating agent) and another potential NASH, Alzheimer and/or fibrosis drug(s) as a combination drugs to a patient diagnose with NAFLD, NASH, liver cirrhosis, Alzheimer’s disease, and/or fibrosis diseases. Therapeutically effective amount of MOA and the potential NASH, Alzheimer and fibrosis drug(s) are administered orally to the patient in the amount and duration sufficient to slowing down, stopping progress of said diseases or reversing the symptoms of said diseases.

[00183] Another preferred embodiment is to use methoxyamine or pharmaceutically acceptable salts (and/or aminoguanidine, and/or alkylating agent) and Obeticholic acid (OCA) or OCA pharmaceutically acceptable salt (or OCA 24-position carboxylic acid and amino acid conjugates) as a combination drugs to a patient diagnose with NAFLD, NASH, and liver cirrhosis. Therapeutically effective amount of MOA and OCA are administered orally to the patient in the amount and duration sufficient to slowing down, stopping progress of NAFLD, NASH, and liver cirrhosis or reversing the liver damage. One function of the MOA is to reduce the adverse drug effects of OCA.

[00184] Another preferred embodiment is to use MOA (and/or aminoguanidine, and/or alkylating agent) for the treatment of allysine related fibrosis, include, but not limited to, lung fibrosis, kidney fibrosis, liver fibrosis and cirrhosis.

[00185] Another preferred embodiment is to use a topical composition include MOA or GUA or alkylating agents for the reduction of old and new scar tissue and improving the appearance of scar tissue after it has formed. The topical composition includes one or more alkylating agents such as MOA, GUA formulated in a pharmaceutically acceptable topical carrier. Topically applying a composition including one or more said agents formulated in a pharmaceutically acceptable topical carrier to scar tissue to reduce the amount of, or improve the appearance of the scar tissue. The topical compositions and methods of the present disclosure reduce scar tissue after it has formed and improving the appearance of the remaining scar tissue.

Embodiment 3 : MOA and alkylating agents for the treatment of aldehyde and reaction ketone induced adverse drug response [00186] Since MOA can potentially treat reactive ketone and aldehyde caused immune related diseases, alkylating agents which can react with ketones and aldehydes can be used for treatment of all reactive ketone and aldehyde caused immune related diseases.

[00187] Certain drug-induced liver injury (DILI) is a reactive ketone and aldehyde caused immune related disease. Alkylating agents can be used in the treatment of DILI. DILI is an uncommon, but potentially fatal, cause of liver disease that is associated with prescription medications, OTC drugs, and herbal and dietary supplements (HDS). Drugs or its reactive metabolites are considered as foreign antigens that bind to T cell receptors (TCR) and further activate immune response. In case of the Abacavir hypersensitivity, the aldehyde metabolite from its hydroxyl group may react with a short peptide and form a peptide-HLA complex. This complex activates immune response, which release inflammatory cytokines and start the hypersensitivity response. More recently, it has been shown that Abacavir might occupy a space below the region of HLA that presents peptides, which leads to an altered peptide presentation and trigger an autoimmune reaction. MOA and other alkylating agents can release the peptide from the drug or its metabolites peptide conjugation. Therefore, the immune response will not be triggered. MOA and alkylating agents can be used to treat aldehyde and ketone caused DILI.

[00188] One preferred embodiment is to use methoxyamine or its pharmaceutically acceptable salts to a patient diagnosed with early, middle or late stage of DILI. Therapeutically effective amount of MOA is administered to the patient orally or intravenously in an amount and duration sufficient to slowing down, stopping progress or reversing the liver damage.

[00189] Another preferred embodiment is to use methoxyamine or its pharmaceutically acceptable salts and a second drug(s), which can cause DILI, as a combination drugs to a patient. The second drug(s) are effective to the disease but can cause DILI of the patients. Therapeutically effective amount of MOA and the second drug(s) are administered orally to the patient in the amount and length sufficient to prevent or reduce the DILI, and effectively treat the disease.

Embodiment 4: Cysteine and isotope labeled cysteine or homocysteine and isotope labeled homocysteine and alkylating agents to screen for reactive metabolites, which cause adverse immune response

[00190] Due to the unique reactivity of the cysteine (Cys) towards reactive ketone and aldehyde to form five-member ring thiazolidines, cysteine can be used as a maker for reactive ketone and aldehyde detection. This invention disclosure also describes using cysteine, N- terminal cysteinyl-peptides and -proteins to detect aldehyde and reactive ketone. This method can be used in detecting aldehydes and reactive ketones in physiological condition, drug reactive metabolite screening, and endogenous aldehyde and reactive ketone detection.

[00191] One preferred embodiment is to use cysteine mixed with isotopes labeled cysteine- ¹³C₃,¹⁵N (Cys4) to react with 3k-CDCA to form the 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates, and using LC-MS to verified the formation of the Cys and 3k-CDCA conjugate (3k-CDCA-Cys) and Cys4 and 3k-CDCA (3k-CDCA-Cys4) conjugate. This simple method used basic triple Qua MS to detect the signature Cyc and 3k-CDCA and Cyc4 and 3k-CDCA conjugates. The molecular ion mass of 3k-CDCA-Cys and 3k-CDCA-Cys4 are 4 Daltons apart (exact mass is 4.0071), making aldehydes and reactive ketone and cysteine conjugates very easy to track and identify. Cysteine can be used to detect the existence of aldehyde and reactive ketones from both endogenous and exogenous sources, and can be used to conduct metabolite screening for drug candidates.

[00192] Like cysteine, homocysteine and isotope labeled homocysteine towards reactive ketone and aldehyde to form six-member ring 1, 3-thiazinane, homocysteine can be used as a maker for reactive ketone and aldehyde detection.

[00193] 7k-CDCA, C4, 3k-CDCA have the similar structure, but their reaction rate with Cys are different. No reaction for 7k-CDCA, low reaction rate for C4 (α, β-unsaturated ketone), and high reaction rate for 3k-CDCA. These three compounds can be used as a control samples for reactive ketone screening.

Embodiment 5: Use 3k-CDCA-cysteinyl-peptide conjugates (or other reactive ketone and aldehyde and Cysteinyl-peptide or-protein conjugates) to screen for the alkylating agents to release cysteinyl-peptide from 3k-CDCA (or other reactive ketones and aldehydes).

[00194] One preferred embodiment is to use Cys-Gly with 3k-CDCA to form the 3k-CDCA- Cys-Gly conjugate, and using LC-MS to verified the formation of the Cys-Gly and 3k-CDCA conjugate (3k-CDCA-Cys-Gly). The alkylating agent MOA is used to treat the 3k-CDCA-Cys- Gly conjugate to release the Cys-Gly in the following step. This simple method used basic triple Qua MS to detect the signature 3k-CDCA and 3k-CDCA-Cys-Gly conjugates, and 3k- CDCA-MOA conjugates. A N-terminal Cysteinyl-peptide can form 3k-CDCA conjugate as 3k- CDCA-cysteinyl-peptide. 3k-CDCA-Cys or 3k-CDCA-Cys-peptide can be used as a screening agent to search for the alkylating agents to release Cys or N-terminal-cysteinyl-peptides from the conjugate. The alkylating agents that can effectively release Cys and N-terminal-cysteinyl- peptide in physiological condition, and have low biological toxicity are the best candidates for treating the immune related diseases such NAFLD, NASH, liver cirrhosis or DILI.

[00195] Like 3k-CDCA-N-terminal cysteinyl-peptides, 3k-CDCA-N-terminal homocysteinyl-peptides can be used to measure the effectiveness of alkylating reagents for N- terminal homocysteinyl-peptides releasing test.

[00196] Like MOA, GUA can also react with reactive ketone-N-terminal cysteinyl-peptide, to form reactive ketone-GUA conjugates and release N-terminal cysteinyl-peptides from reactive ketone-N-terminal cysteinyl-peptides. GUA can also directly react with reactive ketones to form reactive ketone-GUA conjugates. But the reaction rate is much lower than MOA. GUA and MOA can both use as a standard to measure the reaction rate of alkylating agents for drug discovery.

Materials and methods

Chemicals and Reagents

[00197] L-Cysteine (Cys), L-Cysteine-¹³C3, ¹⁵N (Cys4), Cysteine-Glycine (Cys-Gly), L- HomoCysteine (HCys), Oxytocin (Oxy), DL-Dithiothreitol (DTT), 7α-Hydroxy-4-Cholesten- 3 -one (C4), Cholic acid (CA), Deoxy cholic acid (DC A), Sodium taurochenodeoxy cholate (TCDCA), Glycocholic acid hydrate (GCA), Sodium glycodeoxycholate (GDCA), Sodium glycochenodeoxy cholate (GCDCA), Obeti cholic Acid (OCA), 3 α-Hydroxy steroid Dehydrogenase (3α-HSD), β-Nicotinamide adenine dinucleotide hydrate (NAD+), Methoxyamine hydrochloride (MOA), Aminoguanidine (GUA), L-Lysine (Lys), L-AHysine ethylene acetal (AEA), Hydrochloric acid (HC1), Sodium bicarbonate (NaHCOQ, Dimethyl sulfoxide (DMSO), and Acetonitrile (ACN) were purchased from MilliporeSigma Company (St. Louis, Mo., USA). 3-Oxo-7α-hydroxy-5P-cholanoic Acid (3k-CDCA), 3α-Hydroxy-7-oxo- 5 β-cholanic Acid (7k-CDCA) were purchased from Toronto Research Chemicals (TRC, North York, ON M3J 2K8, Canada). Phosphate Buffered Saline, PBS (IX), pH 7.4 was purchased from Quality Biological (Gathersburg, MD 20879, USA).

Stock Solutions

[00198] 100 mM of each 3k-CDCA, 7k-CDCA, C4, CA, DC A, TCDCA, GCDCA, GCA, GDCA, OCA, Lys, and AEA, 1 M Cys, 1 M Cys4, 1 M Cys+Cys4 (by 1 : 1 mix of 1 M Cys and 1 M Cys4), 1 M Cys-Gly, 1 M HCys, and 10 M of each MOA and GUA stock solutions were prepared by dissolving in DMSO. 100 mM NAD+, 1 unit/20pl 3α-HSD, I M Oxy, and 1 M DTT stock solutions were prepared by dissolving in PBS. Stocks solution added to PBS at final concentration of <1% DMSO. Final solutions were incubated at 37°C. All sock solutions were kept at -24°C.

Sample Incubation and Preparation

[00199] The incubation samples are 2 mL PBS in volume at 37°C. The incubation time was 2-4 hours. Control samples were carried out in the same fashion. All incubated samples were kept in -24°C after incubation. All samples were loaded to injection plate before LC/MS sample analysis. An aliquot of 5 pL was analyzed by LC-MS. Details composition and procedures are included below.

[00200] All 6 bile acid samples (CA, DCA, TCDCA, GCDCA, GCA, GDCA) are incubated individually at 100 μM with 2.5 unit/ml 3α-HSD and 2.5 mM NAD+ at 37 °C for 1.5 hours first. After incubation, add one part of acetonitrile to one part of each sample and mix well to kill the 3α-HSD enzyme and then spin down to get each supernatant. Add one part of each supernatant to 4 part of PBS as 3k-bile acid mixture solution (3k-BA solution, final concentration of each bile acid is 5 μM, total bile acids concentration is 30 μM).

[00201] 3k-BA (5 μM for each bile acid) solution and 20 μM 3k-CDCA solution are incubated individually with 200 μM Cys+Cys4 or 200 μM Oxytocin with 1 mM DTT for 1.5 hours, and then aliquot part of each solution out as 3k-CDCA&Cys+Cys4, 3k- CDCA&Oxy+DTT, 3k- BA&Cys+Cys4, and 3k-BA&Oxy+DTT.

[00202] The rest of part of each sample were incubated with 1 mM MO A for another 1.5 hours as 3k-CDCA&Cys+Cys4&MOA, 3k-CDCA&Oxy+DTT&MOA, 3k- BA&Cys+Cys4&MOA, and 3k-BA&Oxy+DTT&MOA.

[00203] 20 μM 3k-CDCA solution are incubated individually with 200 μM Cys+Cys4 or 200 μM Oxytocin with 5 mM DTT for 1.5 hours, and then aliquot part of each solution out as 3k- CDCA&Cys+Cys4, 3k-CDCA&Oxy+DTT.

[00204] The rest of part of each sample were incubated with 1 mM MOA or GUA for another 1.5 hours as 3k-CDCA&Cys+Cys4&MOA, 3k-CDCA&Oxy+DTT&MOA, 3k- CDCA&Cys+Cys4&GUA, and 3k-CDCA&Oxy+DTT&GUA.

[00205] 200 μM Oxytocin is incubated individually with and without 1 mM DTT for 1.5 hours as Oxy+DTT and Oxy only (negative control) samples.

[00206] OCA is incubated at 100 μM with and without 2.5 unit/ml 3α-HSD and 2.5mM NAD+ at 37 °C for 1.5 hours first. After incubation, add one part of acetonitrile to one part of each sample and mix well to kill the 3α-HSD enzyme and then spin down to get the supernatant as OCA&3a-HSD (OCA&3a, final concentration is 50 μM of mixture of OCA and 3k-OCA) and OCA only (OCA only, negative control) samples.

[00207] Each 3k-OCA (OCA&3a-HSD sample, 3k-OCA and OCA mixture) solution are incubated individually with 200 μM Cys+Cys4, 200 uM Cys-Gly, or 200 μM Oxytocin with 5 mM DTT for 1.5 hours, and then aliquot part of each solution out as OCA&3a- HSD&Cys+Cys4, OCA&3a-HSD&CG, and OCA&3a-HSD&Oxy+DTT samples.

[00208] The rest of part of each sample were incubated with ImM MO A for another 1.5 hours as OCA&3a-HSD&Cys+Cys4&MOA, OCA&3a-HSD&CG&MOA, and OCA&3a- HSD&Oxy+DTT&MOA samples.

[00209] Make 30 mM ally sine (Aly) stock solution by adding 1 part of 100 mM AEA stock to 1 part of 1 N HC1 incubate at 37C for 1-2 hours and vertex every 15 min. And then add 1 pat of 1 N NaHCOs to adjust pH to 7-8. Using PBS to adjust the total volume to make the final concentration of Aly stock to be 30 mM.

[00210] Make 5 ml 200 uM Lysine and 200 uM Aly PBS mixture solution and incubate at 37°C overnight as 200 uM Aly+Lys solution.

[00211] Make individual 5 ml each of 200 uM Aly and 200 uM Lys PBS solutions using Aly and Lys stock solutions.

[00212] Aliquot 500 ul out from each of Aly, Lys, and Aly+Lys solutions as Aly only, Lys only, and Aly+Lys only solution.

[00213] Split each of leftover solutions to half. First half incubate with 1 mM MOA and the second half incubate with 1 mM GUA at 37°C and aliquot 500 ul each solution out at 1.5, 3, and 4.5 hours as sample Aly&MOAl, Aly&MOA2, Aly&MOA3, ... Aly+Lys&GUA2, and Aly+Lys&GUA3 solutions.

Instrumentation

[00214] The LC-MS analysis was conducted on Vanquish Horizon UPLC pumps and Vanquish autosampler coupled with Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). The separation was carried on an ACQUITY UPLC HSS C18 column (Waters, 2.1 x 150 mm, 1.8 μm, 186003534).

[00215] For bile acid related samples, mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The normal gradient was: 2% B at Omin, 2%B at 0.5min, 90%B at 25 min, 90%B at 27 min, 2% B at 27.1 min, 2% B at 30.0 min, stopped at 30.1 min. The gradient for C4 and GUA sample setting was: 2% B at Omin, 2%B at 0.5min, 90%B at 15 min, 90%B at 27 min, 2% B at 27.1 min, 2% B at 30.0 min, stopped at 30.1min. Flow rate was 0.45 mL/min. Column temperature is 40°C.

[00216] For allysine related samples, mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in methanol. The normal gradient was: 1% B at Omin, 90%B at 14 min, 90%B at 16 min, 1% B at 16.1 min, 1% B at 20.0 min, stopped at 20.1 min. Flow rate was 0.2 mL/min. Column temperature was 25°C.

[00217] MS acquisition in both positive and negative mode. Full MS: Runtime 2.9-27 min for bile acid related samples and runtime 0.6-16 min for allysine related samples. Resolution is 70,000. AGC target is IM. Max IT is 50 ms. Scan range is 300 to 1000 m/z for bile acid related samples and scan range is 100 to 700 m/z for allysine related samples. Spectrum data type is profile. dd-MS2 (data dependent product scan): Resolution is 17,500. AGC target is 100K. Max IT is 50 ms. Loop count is 2. MSX count is 1. TopN is 2. Isolation window is 0.4 m/z. Isotope offset is 0. Scan range is 200 to 2000 m/z. Fixed first mass is 50 m/z. CE / stepped CE ce: 30, 45, 60. Spectrum data type is profile. Minimum AGC target is 4K. Intensity threshold 80K. Exclude isotopes is on. Dynamic exclusion is 2.0 s.

Data Mining

[00218] The LC-MS data acquired on Q-Exactive Plus were processed using Thermo Scientific Xcalibur 4.2.28.14. Extract ion chromatogram ion extraction setting is theoretical ion mass (or m/z for multiple charged ions) +/- 10 ppm.

[00219] Presented below are examples discussing the design and evaluation of efficacy of certain small molecule pharmaceuticals, contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present disclosure, but are not intended to limit the scope of the disclosure. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES EXAMPLE 1

The 3k-CDCA-Cys+ 3k-CDCA-Cys4 conjugate formation between Cys and Cys4 (1:1 molar ratio) amino acids and 3k-CDCA, and the formation of 3k-CDCA-MOA and the release of Cys and Cys4 by methoxyamine (MO A) from the conjugate shown in FIG. 4-10 and Table 1-2. [00220] 10μM 3k-CDCA with and without (as a negative control) 100μM Cys, 100μM Cys4, or 100μM Cys+Cys4 (1/1 molar ratio of Cys/Cys4) in 2 mL PBS solution are incubated at 37°C for 1.5 hours (Step 1). The incubated each solution of 3k-CDCA&Cys, Cys4, or Cys+Cys4 is split into two halves (1 mL each). The first half of the solution is stored for analysis. Continue to incubate the second half of the solution with ImM MOA for 1.5 hours (Step 2).

[00221] The first halves of the solutions are analyzed by LC-MS. There are substantial equal amount of 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates in the incubated solution of 3k- CDCA&Cys+Cys4. Because Cys and Cys4 are isotopes, Cys and Cys4 and their conjugates chemical properties, such as reactivity with 3k-CDCA, ionization rate on MS, and hydrophobicity on column (ion retention time), and etc., are exact the same except 4 Daltons difference of ion mass. The initial concentrations of total cysteine (Cys and/or Cys4) are the same for all 3 3k-CDCA&Cys, 3k-CDCA&Cys4, and 3k-CDCA&Cys+Cys4 samples, which is 100μM. The only difference is the ratio for Cys and Cys4. We get perfect match ratio of conjugates from the 3 samples. There is no 3k-CDCA-Cys4 conjugate and about 2 fold of 3k- CDCA-Cys conjugate in the incubated 3k-CDCA&Cys sample compare to 3k- CDCA&Cys+Cys4 sample. There is no 3k-CDCA-Cys conjugate and about 2 fold of 3k- CDCA-Cys4 conjugate in the incubated 3k-CDCA&Cys4 sample compare to 3k- CDCA&Cys+Cys4 sample. There is no detectable amount of 3k-CDCA-Cys or 3k-CDCA- Cys4 conjugate in 3k-CDCA only negative control solution. Both negative and positive ion summaries show the consistent result. It is concluded that 3k-CDCA and cysteine can form 3k-CDCA-cysteine (Cys or Cys4) conjugate in a physiological condition.

[00222] After incubation, the 2^nd half of the solution with MOA is analyzed by LC-MS. There is very low concentration of 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugates in 3k- CDCA&Cys+Cys4 solution. There is also very low concentration of 3k-CDCA left in the solution. In the reference samples of 3k-CDCA&Cys&MOA and 3k-CDCA&Cys4&MOA, there are also very low concentrations of 3k-CDCA-Cys and 3k-CDCA-Cys4 conjugate respectively in the solutions. Similar high concentration of 3k-CDCA-MOA conjugates and similar low concentration of 3k-CDCA are observed in 2^nd half of the solution with MOA incubation. There are two epimers of 3k-CDCA-MOA conjugate, 3k-CDCA-t-MOA and 3k- CDCA-c-MOA, in the solution. 3k-CDCA-Cys+Cys4 conjugates and 3k-CDCA completely react with MOA to replace Cys and Cys4 with MOA to become 3k-CDCA-MOA conjugates in physiological condition. Both negative and positive ion summaries show the consistent result. It is concluded that 3k-CDCA and 3k-CDCA-cysteine (Cys or Cys4) conjugates completely react with MOA to become 3k-CDCA-MOA conjugates, and release cysteine amino acid.

Table 1. MS negative ion mode ion intensity summary of each analyte of 7 samples.

Sample Name:

3k-CDCA only: 3k-CDCA with PBS sample, a blank control.

3k-CDCA&Cys: 3k-CDCA PBS with Cys sample, Step 1 products of FIG. 4.

3k-CDCA&Cys4: 3k-CDCA PBS with Cys4 sample, Step 1 products of FIG. 4.

3k-CDCA&Cys+Cys4: 3k-CDCA PBS with Cys+Cys4 (1:1) sample, Step 1 products ofFIG. 4.

3k-CDCA&Cys&MOA: 3k-CDCA&Cys with MOA sample, Step 2 products ofFIG. 4.

3k-CDCA&Cys4&MOA: 3k-CDCA&Cys4 with MOA sample, Step 2 products of FIG. 4.

3k-CDCA&Cys+Cys4&MOA: 3k-CDCA&Cys+Cys4 with MOA sample, Step 2 products ofFIG. 4.

The NA entries of ion intensity summary have no detectable ion intensity based on the experimental and data analyses setup.

Analyte Name: 3k-CDCA: 3-keto ChenoDeoxyCholic Acid, 3k-CDCA-Cys: 3k-CDCA and cysteine conjugate, 3k-CDCA-Cys4: 3k-CDCA and isotope labeled cysteine conjugate, 3k-CDCA-MOAl: the first epimer of 3k- CDCA and methoxyamine conjugates, 3k-CDCA-MOA2: the second epimer of 3k-CDCA and metho xyamine conjugates.

Table 2. MS positive ion mode ion intensity summary of each analyte of 7 samples.

EXAMPLE 2 The 3k-CDCA-H20xy (Oxytoceine) conjugate formation between N-terminal cysteinyl oxytoceine (H20xy) and 3k-CDCA, and the formation of 3k-CDCA-MOA and the release of oxytoceine by methoxyamine (MO A) from the conjugate shown in FIG. 11-12 and Table 3-4.

[00223] 20 μM 3k-CDCA with 200 μM Oxytocin and 5 mM DTT in 2 mL PBS solution are incubated at 37°C for 1.5 hours (Step 1). The incubated solution is split into two halves (1 mL each). The first half of the solution is stored for analysis. Continue to incubate the second half of the solution with ImM MOA hydrochloride (CH3ONH₂ HCI) for 1.5 hours (Step 2).

[00224] The first half of the solution is analyzed by LC-MS. There is substantial amount of 3k-CDCA-H20xy conjugate in the incubated 3k-CDCA&Oxy+DTT solution. There is no detectable amount of 3k-CDCA-H20xy conjugate in 2 negative control blank solutions. It is concluded that 3k-CDCA and N-terminal cysteinyl Oxytoceine (H20xy) can form 3k-CDCA- H20xy conjugate in a physiological condition.

[00225] After incubation, the 2^nd half of the solution with MOA are analyzed by LC-MS. There is very low amount of 3k-CDCA-H20xy conjugate in the 3k-CDCA&Oxy+DTT&MOA solution. There is also very low amount of 3k-CDCA left in the solution. There are two epimers of 3k-CDCA-MOA conjugate, 3k-CDCA-t-MOA and 3k-CDCA-c-MOA, in the solution. 3k-CDCA-H20xy conjugates and 3k-CDCA completely react with MOA to replace N-terminal cysteinyl Oxytocieine with MOA to become 3k-CDCA-MOA conjugate in a physiological condition. It is concluded that 3k-CDCA and 3k-CDCA-H20xy conjugates completely react with MOA to become 3k-CDCA-MOA conjugates, and release Oxytoceine peptide.

Table 3. MS negative ion mode ion intensity summary of each analyte of 4 samples.

Sample Name:

Oxy only: Oxytocin with PBS sample,

Oxy+DTT: Oxy&DTT, Oxytocin PBS with DTT sample,

3k-CDCA&Oxy+DTT: 3k-CDCA with Oxy+DTT sample,

3k-CDCA&Oxy+DTT&MOA: 3k-CDCA and Oxy+DTT with MOA sample.

The NA entries of ion intensity summary have no detectable ion intensity based on the experimental and data analyses setup.

Analyte Name: Oxy: Oxytocin, H2Oxy: Oxytoceine, 3k-CDCA: 3-keto ChenoDeoxyCholic Acid, 3k-CDCA- H2Oxy: 3k-CDCA and Oxytoceine conjugate, 3k-CDCA-MOAl: the first epimer of 3k-CDCA and methoxyamine conjugates, 3k-CDCA-MOA2: the second epimer of 3k-CDCA and methoxyamine conjugates.

Table 4. MS positive ion mode ion intensity summary of each analyte of 4 samples.

EXAMPLE 3

The 3k-CDCA-Cys+ 3k-CDCA-Cys4 and 3k-CDCA-H20xy conjugates formation between 3k- CDCA and Cys+Cys4 (1:1 molar ratio) amino acids or Oxy+DTT, and the formation of 3k- CDCA-MOA or 3k-CDCA-GUA and the release of Cys+Cys4 or H20xy by methoxyamine (MOA) or aminoguanidine (GUA) from the conjugates shown in Table 5-6. Used 3k to represent 3k-CDCA to save space in Table 5-6.

[00226] In order to save space, using 3k to represent 3k-CDCA for both Table 5 and Table 6.

Analyte Name: 3k: 3k-CDCA, 3-keto ChenoDeoxyCholic Acid. 3k-Cys: 3k-CDCA-Cys, 3k-CDCA and cysteine conjugate. 3k-Cys4: 3k-CDCA-Cys4, 3k-CDCA and cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate. 3k-H20xy: 3k-CDCA-H20xy, 3k-CDCA and oxytoceine conjugate. 3k-MOAl: 3k-CDCA-MOAl, the first epimer of 3k- CDCA and methoxyamine conjugates. 3k-MOA2: 3k-CDCA-MOA2, the second epimer of 3k-CDCA and methoxyamine conjugates. 3k-GUA12: mixture of 3k-CDCA-GUAl (the first epimer of 3k-CDCA and aminoguanidine conjugates) and 3k-CDCA-GUA2 (the second epimer of 3k-CDCA and aminoguanidine conjugates).

Table 6. MS Positive ion mode ion intensity summary of each analyte of 7 samples.

[00227] 20 μM 3k-CDCA with and without (as a negative control 3k_only) 200 μM Cys/Cys4 (1/1 molar ratio Cys/Cys4. Cys4 is isotope labeled Cys of three ¹³C and one ¹⁵N) or 200 μM Oxy (Oxytocin) and 5 mM DTT in 2 mL PBS solution are incubated at 37°C for 1.5 hours (Step 1). The incubated 3k-CDCA&Cys+Cys4 and 3k-CDCA&Oxy+DTT solutions are split into three equal parts (666 uL each). The first part of the solution is stored for analysis. Continue to incubate the other two parts with ImM MOA hydrochloride (CH3ONH₂ HCI) or GUA for 1.5 hours (Step 2).

[00228] The first parts of the solutions are analyzed by LC-MS. There is substantial amount of 3k-CDCA-Cys and 3k-CDCA-Cys4 or 3k-CDCA-H20xy conjugates in the incubated solutions. There is no detectable amount of 3k-CDCA-Cys and 3k-CDCA-Cys4 or 3k-CDCA- H20xy conjugates in 3k-CDCA only negative control solution. It is concluded that 3k-CDCA and cysteine or Oxytoceine (H2Oxy, generated from Oxy+DTT) can form 3k-CDCA-Cys or 3k-CDCA-H20xy conjugates in a physiological condition.

[00229] After first incubation, one of the three parts of the solution with MOA is analyzed by LC-MS. There is no or very low concentration of 3k-CDCA-Cys+Cys4 or 3k-bile acid-H2Oxy conjugates in the solutions. There is also no or very low concentration of 3k-CDCA left in the solution. High concentration of 3k-CDCA-MOA conjugates and no or low concentration of 3k- CDCA are observed in Table 5-6, the solutions with MOA incubation. There are two epimers of 3k-CDCA-MOA conjugate, 3k-CDCA-t-MOA and 3k-CDCA-c-MOA, in the solution. 3k- CDCA-Cys+Cys4 conjugates or 3k-CDCA-H20xy and 3k-CDCA completely react with MOA to replace Cys and Cys4 or H20xy with MOA to become 3k-CDCA-MOA conjugate in physiological condition. It is concluded that 3k-CDCA and 3k-CDCA-Cys or 3k-CDCA- H20xy conjugates completely react with MOA to become 3k-CDCA-MOA conjugate, and release Cys amino acid or H20xy peptide.

[00230] After first incubation, one of the three parts of the solution with GUA is analyzed by LC-MS. There is decrease of 3k-CDCA-Cys+Cys4 or 3k-bile acid-H2Oxy conjugates in the solutions. There is still a lot of 3k-CDCA left in the solutions with GUA. High concentration of 3k-CDCA-GUA conjugates and decreased concentration of 3k-CDCA are observed in Table 5-6, the solutions with GUA incubation. There are two epimers of 3k-CDCA-GUA conjugate, 3k-CDCA-t-GUA and 3k-CDCA-c-GUA in the solution. There is no good baseline separation of the two epimers. They are quantified together as 3k-CDCA-GUA12. 3k-CDCA-Cys+Cys4 conjugates or 3k-CDCA-H20xy and 3k-CDCA can react with GUA to replace Cys and Cys4 or H20xy with GUA to become 3k-CDCA-GUA conjugate in physiological condition. It is concluded that 3k-CDCA and 3k-CDCA-Cys or 3k-CDCA-H20xy conjugates can react with GUA to become 3k-CDCA-GUA conjugate, and release Cys amino acid or Oxytoceine peptide.

EXAMPLE 4

The conversion of bile acids to corresponding 3k-bile acids by 3o -HSI) in physiological condition shown in FIG. 3 and Table 7-8.

[00231] All 6 bile acid samples (CA, DCA, TCDCA, GCDCA, GCA, GDCA) are incubated individually at 100 μM with and without (As negative control, 6 BAs only samples) 2.5 unit/ml 3α-HSD and 2.5mM NAD+ at 37 °C for 1.5 hours first. After incubation, add one part of acetonitrile to one part of each sample and mix well to denature 3α-HSD enzyme and then spin down to get each supernatant. Add one part of each supernatant to 4 part of PBS as 3k- bile acid mixture solution (3k-BA, final concentration of each bile acid and its corresponding 3k-BA is 5μM). Do the same for 6 BAs only samples as one BA only mixture sample (negative control sample without 3α-HSD enzyme).

[00232] Both samples are analyzed by LC-MS. For each bile acid, there is more than one third of the bile acid converted to 3k-bile acid. There is more conversion for secondary bile acids to their corresponding 3k-bile acids, than the conversion of primary bile acids. Bile acid leftover of each bile acid in both positive and negative ion modes after 1.5 hours incubation with 3α-HSD: GCA 67% (positive ion mode) and 62% (negative ion mode), GCDCA 59% and 59%, CA 58% and 62%, TCDCA 54% and 53%, GDCA 34% and 34%, DCA 24% and 22%.

EXAMPLE 5

The 3k-bile acid-Cys and 3k-bile acid-Cys4 or 3k-bile acid-H20xy conjugates formation between Cys and Cys4 (1:1 molar ratio) amino acids or H20xy (Oxytoceine, N-cysteinyl 9 amino acid) peptide and 3k-bile acids, and the formation of 3k-bile acid-MOA and the release of Cys and Cys4 or H20xy by methoxyamine (MO A) from the conjugate shown in FIG. 3 and Table 7-8.

[00233] 3k-bile acid mixture solution (3k-BA from sample BA&3a) with and without (as a negative control BA&3a, 5 μM of each bile acid) 200 μM Cys+Cys4 (1 : 1 molar ratio Cys:Cys4. Cys4 is isotope labeled Cys of three ¹³C and one ¹⁵N) or 200 μM Oxy (Oxytocin) and 1 mM DTT in 2 mL PBS solution are incubated at 37°C for 1.5 hours (Step 1). The incubated BA&3a&Cys+Cys4 and BA&3a&Oxy+DTT solutions are split into two halves (1 mL each). The first half of the solution is stored for analysis. Continue to incubate the second half of the solution with ImM MOA hydrochloride (CHsONFF-HCl) for 1.5 hours (Step 2).

[00234] The first half of the solutions are analyzed by LC-MS. There is substantial amount of 3k-bile acid-Cys+3k-bile acid-Cys4 or 3k-bile acid-H2Oxy conjugates in the incubated solutions. There is no detectable amount of 3k-bile acid-Cys and 3k-bile acid-Cys4 or 3k-bile acid-H2Oxy conjugates in both BA only and BA&3a negative control blank solutions. It is concluded that 3k-bile acids (from BA&3a) and cysteine or Oxytoceine (generated from Oxy+DTT) can form 3k-bile acid-Cys or 3k-bile acid-H2Oxy conjugates in a physiological condition. [00235] After incubation, the 2^nd half of the solution with MOA is analyzed by LC-MS. There is no or very low concentration of 3k-bile acid-Cys+3k-bile acid-Cys4 or 3k-bile acid- H2Oxy conjugates in the solutions. There is also no or very low concentration of 3k-bile acids left in the solution. High concentration of 3k-bile acid-MOA conjugates and no/low concentration of 3k-bile acid are observed in the 2^nd half of the solution with MOA incubation. There are two epimers of 3k-bile acid-MOA conjugate, 3k-bile acid-t-MOA and 3k-bile acid-c- MOA, in the solution. 3k-bile acid-Cys and 3k-bile acid-Cys4 conjugates or 3k-bile acid- H20xy and 3k- bile acid completely react with MOA to replace Cys and Cys4 or H20xy with MOA to become 3k-bile acid-MOA conjugates in physiological condition. It is concluded that 3k-bile acid and 3k-bile acid-Cys or 3k-bile acid-H2Oxy conjugates completely react with MOA to become 3k- bile acid-MOA conjugate, and release Cys amino acid or oxytoceine peptide.

Table 7. MS negative ion mode ion intensity summary of each analyte of 6 samples.

BA only: 6 bile acids with PBS sample,

BA&3a: 6 bile acids PBS with 3α-HSD sample (in order to save space, using 3a to represent 3a-HSD),

BA&3a&Cys+Cys4: 6 bile acids PBS and 3α-HSD with Cys+Cys4 (1:1 molar) sample,

BA&3a&Cys+Cys4&MOA: 6 bile acids PBS,3α-HSD, and Cys+Cys4 (1:1 molar) with MOA sample,

BA&3a&Oxy+DTT: 6 bile acids and 3α-HSD with Oxytocin+DTT sample,

BA&3a&Oxy+DTT&MOA: 6 bile acids, 3a-HSD, and Oxytocin+DTT with MOA sample.

The NA entries of ion intensity summary have no detectable ion intensity based on the experimental and data analyses setup.

Analyte Name: CA: Cholic Acid, 3k-CA: 3-keto Cholic Aicd, 3k-CA-Cys: 3k-CA cysteine conjugate, 3k-CA- Cys4: 3k-CA cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate, 3k-CA-H20xy: 3k-CA oxytoceine conjugate, 3k-CA- MOA12: mixture of 2 3k-CA methoxyamine conjugates, 2 epimers quantified together since there is no LC separation.

Analyte Name: DCA: DeoxyCholic Acid, 3k-DCA: 3 -keto Deoxy Cholic Aicd, 3k-DCA-Cys: 3k-DCA cysteine conjugate, 3k-DCA-Cys4: 3k-DCA cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate, 3k-DCA-H20xy: 3k-DCA oxytoceine conjugate, 3k-DCA-MOAl: first epimer of 3k-DCA methoxyamine conjugates, 3k-DCA-MOA2: second epimer of 3k-DCA methoxyamine conjugates.

Analyte Name: TCDCA: TauroChenoDeoxyCholic Acid, 3k-TCDCA: 3-keto TauroChenoDeoxyCholic Acid, 3k- TCDCA-Cys: 3k-TCDCA cysteine conjugate, 3k-TCDCA-Cys4: 3k-TCDCA cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate, 3k-TCDCA-H20xy: 3k-TCDCA oxytoceine conjugate, 3k-TCDCA-MOAl: first epimer of 3k- TCDCA methoxyamine conjugates, 3k-TCDCA-MOA2: second epimer of 3k-TCDCA methoxyamine conjugates.

Analyte Name: GCDCA: GlycoChenoDeoxyCholic Acid, 3k-GCDCA: 3-keto GlycoChenoDeoxyCholic Acid, 3k-GCDCA-Cys: 3k-GCDCA cysteine conjugate, 3k-GCDCA-Cys4: 3k-GCDCA cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate, 3k-GCDCA-H20xy: 3k-GCDCA oxytoceine conjugate, 3k-GCDCA-MOAl: first epimer of 3k- GCDCA methoxyamine conjugates, 3k-GCDCA-MOA2: second epimer of 3k-GCDCA methoxyamine conjugates.

Analyte Name: GCA: GlycoCholic Acid, 3k-GCA: 3-keto GlycoCholic Acid, 3k-GCA-Cys: 3k-GCA cysteine conjugate, 3k-GCA-Cys4: 3k-GCA cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate, 3k-GCA-H20xy: 3k-GCA oxytoceine conjugate, 3k-GCA-MOA12: mixture of 2 3k-GCA methoxyamine conjugates, 2 epimers quantified together since there is no LC separation.

Analyte Name: GDCA: GlycoDeoxyCholic Acid, 3k-GDCA: 3-keto GlycoDeoxyCholic Acid, 3k-GDCA-Cys: 3k-GDCA cysteine conjugate, 3k-GDCA-Cys4: 3k-GDCA cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate, 3k- GDCA-H2Oxy: 3k-GDCA oxytoceine conjugate, 3k-GDCA-MOAl: first epimer of 3k-GDCA methoxyamine conjugates, 3k-GDCA-MOA2: second epimer of 3k-GDCA methoxyamine conjugates. Table 8. MS positive ion mode ion intensity summary of each analyte of 6 samples.

EXAMPLE 6

The conversion of OCA to 3k-0CA by 3α-HSD in physiological condition shown in FIG. 3. and Table 9-10.

[00236] Both human liver microsomes CYP3A4 and 3 α-HydroxySteroid Dehydrogenase (3α-HSD) enzymes can convert bile acids to 3k-bile acids. Current experiment used 3α-HSD for its high conversion rate.

[00237] OCA is incubated at 100 μM with and without (as negative control) 2.5 unit/ml 3a- HSD and 2.5 mM NAD+ at 37 °C for 1.5 hours first. After incubation, add one part of acetonitrile to one part of the sample and mix well to denature 3α-HSD enzyme and then spin down to get each supernatant as OCA&3a and OCA only sample solutions.

[00238] Both samples are analyzed by LC-MS. There is more than one third of the OCA converted to 3k-OCA. Leftover OCA after 1.5 hours incubation with 3α-HSD: 58% (positive ion mode) and 65% (negative ion mode). There is no 3k-OCA in OCA only sample.

EXAMPLE 7

The 3k-OCA-Cys+Cys4, The 3k-OCA-Cys-Gly, or 3k-OCA-H2Oxy conjugates formation between Cys and Cys4 (1:1 molar ratio) amino acids, cysteinyl-glycine dipeptide, or H20xy (Oxytoceine, N-cysteinyl 9 amino acid) peptide and 3k-OCA, and the formation of 3k-OCA- MOA and the release of Cys and Cys4, Cys-Gly or H20xy by methoxyamine (MO A) from the conjugates shown in FIG. 13-14 and Table 9-10.

[00239] OCA&3a solution (3k-OCA and OCA mixture at final concentration of 50 uM) with 200 μM Cys/Cys4 (1/1 molar ratio Cys/Cys4. Cys4 is isotope labeled Cys of three ¹³C and one ¹⁵N), 200 μM Cys-Gly (Cysteinyl-Glycine dipeptide, CG), or 200 μM Oxy (Oxytocin) and 5 mM DTT in 2 mL solution are incubated at 37°C for 1.5 hours (Step 1) as samples OCA&3a&Cys+Cys4, OCA&3a&Cys-Gly, and OCA&3a&Oxy+DTT. The incubated 3 solutions are split into two halves (1 mL each). The first halves of 3 solutions are stored for analysis. Continue to incubate the second half of 3 solutions with ImM MOA hydrochloride (CH3ONH₂ HCI) for 1.5 hours (Step 2) as samples OCA&3a&Cys+Cys4&MOA, OCA&3a&Cys-Gly&MOA, and OCA&3a&Oxy+DTT&MOA.

[00240] The first halves of the solutions are analyzed by LC-MS. There are substantial amount of 3k-OCA-Cys+3k-OCA-Cys4, 3k-OCA-Cys-Gly, or 3k-OCA-H2Oxy conjugates in the incubated solutions. There is no detectable amount of conjugates in both OCA only and OCA&3a negative control solutions. It is concluded that 3k-OCA and cysteine, Cys-Gly, or Oxytoceine (generated from Oxy+DTT) can form 3k-OCA-Cys, 3k-OCA-Cys-Gly, or 3k-bile acid-H2Oxy conjugates in a physiological condition.

[00241] After incubation, the 2^nd half of the solution with MOA is analyzed by LC-MS. There is no or low concentration of 3k-OCA-Cys+3k-OCA-Cys4, 3k-OCA-Cys-Gly, or 3k- OCA-H2Oxy conjugates in the solutions. There is also no or very low concentration of 3k- OCA left in the solution. High concentration of 3k-OCA-MOA conjugates and no/low concentration of 3k-OCA are observed in the 2^nd half of the solution with MOA incubation. There are two epimers of 3k-OCA-MOA conjugate, 3k-OCA-t-MOA and 3k-OCA-c-MOA in the solution. 3k-OCA-Cys+Cys4, 3k-OCA-Cys-Gly, or 3k-OCA-H2Oxy conjugates and 3k- OCA react with MOA to replace Cys and Cys4, Cys-Gly, or H20xy with MOA to become 3k- OCA-MOA conjugates in physiological condition. It is concluded that 3k-OCA and 3k-OCA- Cys, 3k-OCA-Cys-Gly, or 3k-OCA-H2Oxy conjugates react with MOA to become 3k-OCA- MOA conjugate, and release Cys amino acid, Cys-Gly dipeptide, or Oxytoceine peptide.

Table 9. MS negative ion mode ion intensity summary of each analyte of 8 OCA samples.

OCA only: OCA with PBS sample,

OCA&3a: OCA PBS with 3a-HSD sample (in order to save space, using 3a to represent 3a-HSD),

OCA&3a&Cys+Cys4: OCA and 3oc-HSD with Cys+Cys4 (1: 1 molar) sample,

OCA&3a&Cys+Cys4&MOA: OCA, 3oc-HSD, and Cys+Cys4 (1:1 molar) with MOA sample,

OCA&3a&Cys-Gly: OCA and 3oc-HSD with Cys-Gly sample,

OCA&3a&Cys-Gly&MOA: OCA, 3oc-HSD, and Cys-Gly with MOA sample,

OCA&3a&Oxy+DTT: OCA and 3oc-HSD with Oxy+DTT sample,

OCA&3a&Oxy+DTT&MOA: OCA, 3oc-HSD, and Oxy+DTT with MOA sample.

The NA entries of ion intensity summary have no detectable ion intensity based on the experimental and data analyses setup.

Analyte Name: OCA: obeticholic acid, 3k-OCA: 3-keto obeticholic aicd, 3k-OCA-Cys: 3k-OCA cysteine conjugate, 3k-OCA-Cys4: 3k-OCA cysteine isotope (3 ¹³C and 1 ¹⁵N) conjugate, 3k-OCA-Cys-Gly: 3k-OCA cysteinyl-glyceine dipeptide conjugate, 3k-OCA-H2Oxy: 3k-OCA Oxytoceine conjugate, 3k-OCA-MOAl: first epimer of 3k-OCA metho xyamine conjugates, 3k-OCA-MOA2: second epimer of 3k-OCA methoxyamine conjugates.

Table 10. MS positive ion mode ion intensity summary of each analyte of 8 OCA samples.

Analyte Name OCA 3k-OCA 3k-OCA-Cys 3k-OCA-Cys4 3k-OCA-Cys-Gly 3k-OCA-H2Oxy 3k-OCA-MOA1 3k-OCA-MOA2

Retention Time (min) 19 73 20.04 15.97 15.96 17.16 17.10 22.28 22.67 Steps

Sample Name Unit: million positive ions

OCA only 1 NA NA NA NA NA NA Blank

OCA&3a 1 ,564 1 ,529 NA NA NA NA 1 2 Blank2

OCA&3a&Cys+Cys4 3)673: )) :s377: 277 279 NA NA NA NA Stepl

OCA&3a&Cys+Cys4&MOA 1 ,540 13 6 6 NA NA 7,749 8,710 Step2

OCA&3a&Cys-Gly 694 1 .980 0 0 541 NA 0 0 Stepl

OCA&3a&Cys-Gly&MOA 894 24 NA NA 207 NA 10,830 12,317 Step2

OCA&3a&Oxy+DTT 3)5 2: 1 448 NA NA 0 5 8 1 1 Stepl

OCA&3a&Oxy+DTT&MOA 1 ,487 6 NA NA 0 1.9 8,060 8,823 Step2

EXAMPLE 8

The conversion of allysine ethylene acetal (AEA) to allysine (Aly) at acidic condition and the equilibrium between Aly and \-l-Piperideine-6-carboxylale (P6C) and condensation of Aly to allysine aldol (Aly-aldol, Aly-Aly, AA) shown in Table 11-12.

[00242] There is no detectable AEA in all Aly related sample solutions in both positive and negative ion modes. This result is consistent with literature reports regarding AEA to Aly conversion in acidic solutions. There are high amount of Aly and P6C in Aly only solution. The result matches with literature reports. There are significant amount of alysine aldol (Aly- aldol, Aly-Aly, AA) in Aly only solutions. The result matches with literature reports. There is nonenzymic spontaneous condensation between Aly to Aly-aldol in physiological conditions. There is no detectable Aly+Lys Shiff-Base condensation product of dehydrolysinonorleucine in Aly+Lys only sample solution due to the unstable nature of the Schiff base. There is no detectable desmosine in Aly+Lys only sample solution due to the short incubation time and conditions.

Table 11. MS positive ion mode ion intensity summary of each analyte of 3 primary samples.

Sample Name: Lys only: Lys in PBS. Aly only: Aly in PBS, (allysine from AEA incubation in acidic condition). Lys+Aly only: Lys and Aly in PBS incubate at 37 C overnight. Analytes Lys Aly P6C Aly-aldol

RT 1.66 2.52 2.52 2.38

Sample Name Unit: Million Positive Ions

Lys only 263 2 34 1

Aly only 0 54 1,687 1,080

Lys+Aly only 270 6 163 73

Analytes: Lys: lysine. Aly: allysine. P6C: A-l-Piperideine-6-carboxylate. Aly-aldol: allysine aldol.

Table 12. MS negative ion mode ion intensity summary of each analyte of 3 primary samples.

Analytes Lys Aly P6C Aly-aldol

RT 1.66 2.51 2.51 2.37

Sample Name Unit: Million Negative Ions

Lys only 3.97 1 0.0 0

Aly only 0.00 35 5.5 93

Lys+Aly only 4.10 4 0.0 7

EXAMPLE 9

The allysine methoxyamine conjugates (Aly-MO A, A-MOA) formation between allysine (Aly) and methoxyamine (MOA) and the allysine aldol methoxyamine conjugates (Aly-aldol-MOA, Aly-Aly-MOA, AA-MOA) formation between allysine aldol (Aly-aldol, Aly-Aly, AA) and methoxyamine (MOA) shown in FIG. 15-17 and Table 13-14.

[00243] Each primary sample (Lys only, Aly only, or Aly+Lys only) incubated with 1 mM MOA in PBS for 1.5 (samplel), 3 (sample2), or 4.5 (sample3) hours. There is more than 95% of Aly reacted with MOA and more than 99% of Aly-aldol reacted with MOA. The corresponding MOA conjugates can be detected in the MOA samples. There is no difference among different incaution time. 1.5 hours incubation is good enough. AA-MOA ion intensity is from 5 different retention time from 3.35-4.83 minutes.

Table 13. MS positive ion mode ion intensity summary of each analyte of 3 primary and 9 MOA samples.

Sample Name: Lys only: Lys in PBS. Aly only: Aly sample in PBS. Lys+Aly only: Lys+Aly sample in PBS. Lys&MOAl, 2, 3: Lys with MOA in different incubation time. Aly& MOA 1, 2, 3: Aly sample with MOA in different incubation time. Lys+Aly& MOA 1, 2, 3: Lys+Aly sample with MOA in different incubation time.

Analytes: Lys: lysine. Aly: sum of ion of allysine + ion of A-l-Piperideine-6-carboxylate. A-MOA: allysine and methoxyamine conjugates. Aly-aldol: allysine aldol. AA-MOA1-5: sum of ion of 5 allysine aldol methoxyamine conjugates from retention time (RT) 3.35-4.83 minutes.

Table 14. MS negative ion mode ion intensity summary of each analyte of 3 primary samples and 9 MOA samples.

EXAMPLE 10

The allysine aminoguanidine conjugates (Aly-GUA, A-GUA) formation between allysine (Aly) and aminoguanidine (GUA) and the allysine aldol aminoguanidine conjugates (Aly-aldol- GUA, Aly-Aly-GUA, AA-GUA) formation between allysine aldol (Aly-aldol, Aly-Aly, AA) and aminoguanidine (GUA) shown in Table 15-16.

[00244] Each primary sample (Lys only, Aly only, or Aly+Lys only) incubated with 1 mM GUA in PBS for 1.5 (samplel), 3 (sample2), or 4.5 (sample3) hours. There is only little amount (less than 10%) of Aly reacted with GUA and a small amount (20-30%) of Aly-aldol reacted with GUA. The corresponding GUA conjugates can be detected in the GUA samples. There is no difference among different incaution time. 1.5 hours incubation is good enough. AA-GUA ion intensity are from 2 different retention time from 1.97-2.19 minutes.

Table 15. MS positive ion mode ion intensity summary of each analyte of 3 primary and 9 GUA samples.

Sample Name: Lys only: Lys in PBS. Aly only: Aly sample in PBS. Lys+Aly only: Lys+Aly sample in PBS. Lys&GUAl, 2, 3: Lys with GUA in different incubation time. Aly&GUAl, 2, 3: Aly sample with GUA in different incubation time. Lys+ Aly&GUAl, 2, 3 : Lys+Aly sample with GUA in different incubation time.

Analytes: Lys: lysine. Aly: sum of ions of allysine + ions of A-l-Piperideine-6-carboxylate. A-GUA: allysine and aminoguanidine conjugates. Aly-aldol: allysine aldol. AA-GUA1-2: sum of ion of 2 allysine aldol aminoguanidine conjugates from retention time (RT) 1.97-2.19 minutes.

Table 16. MS negative ion mode ion intensity summary of each analyte of 3 primary samples and 9 GUA samples.

[00245] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A method of treating a disease in a subject, the method comprising administering therapeutically effective amount of a composition comprising an alkylating agent.

2. The method of claim 1, wherein the disease is selected from aldehyde and reactive ketone caused diseases, immune related diseases, inflammation related diseases, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver cirrhosis, drug related liver injury (DILI), farnesoid X receptor (FXR)-mediated disease, metabolic disease, fibrosis- related disease, and Alzheimer’s disease.

3. The method of claim 1, wherein the disease is a liver disease selected from primary biliary cholangitis (PBS), NASH, NAFLD, portal hypertension, bile acid diarrhea, and primary sclerosing cholangitis (PSC).

4. The method of claim 2, wherein the fibrosis-related disease is selected from the group consisting of lung fibrosis, kidney fibrosis, and liver fibrosis, or brain fibrosis, and skin tissue scarring disease.

5. The method of claim 1, wherein the composition reduces symptoms, slows down, stops, or cures the disease.

6. The method of claim 1, wherein the alkylating agent is selected from methoxyamine (MO A), aminoguanidine, a pharmaceutically-acceptable salt thereof, or a combination thereof.

7. The method of claim 1, wherein the alkylating agent is administered in a dose of between about 0.001 μg/kg and 200 mg/kg body weight per day.

8. The method of claim 1, wherein the alkylating agent is used in combination with a drug selected from obeticholic acid (OCA), OCA amino acid conjugate, and a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the OCA amino acid conjugate is OCA glycine conjugate (GOCA) or OCA taurine conjugate (TOC A).

10. The method of claim 9, wherein the composition achieves an enhanced therapeutic effect for liver disease, and the therapeutic effect comprising reduction of adverse side effects of OCA, enhancement of the therapeutic effect of OCA, synergic effect of MOA with OCA, and combination therapeutic effect of MOA with OCA.

11. The method of claim 10, wherein said side effects of OCA comprise pruritus and induced high cholesterol, paradoxical worsening of the liver disease, persistent worsening of serum enzyme elevations and hepatic decompensation, jaundice, fatigue ascites, hypersensitivity reactions, depression, liver failure and other severe liver injury.

12. The method of claim 1, wherein another drug is used in combination with the alkylating agent.

13. The method of claim 12, wherein the other drug is GV-971.

14. The method of claim 1, wherein the alkylating agent is administered in an amount effective to reduce the secondary bile acids concentration in the subject’s serum.

15. The method of claim 1, wherein said composition is administered orally, intravenously, intraperitoneally, intramuscularly, or transdermally.

16. The method of claim 1, wherein the alkylating agent is Formula (I) with the following structure:

Formula (I) or a pharmaceutically-acceptable salt, solvate, stereoisomer thereof, wherein X is O or NH;

Y is O, S, or NH;

Z is a bond, O, S, or NH; and R is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl.

17. The method of claim 1, wherein the alkylating agent is Formula (II) with the following structure:

R-X-NH₂

Formula (II) or a pharmaceutically-acceptable salt, solvate, stereoisomer thereof, wherein X is O, S, or NH; and

R is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl.

18. The method of claim 1, wherein the alkylating agent is selected from a group consisting of methoxyamine; O-benzylohydroxylamine; ethyl aminooxyacetate; aminooxyacetic acid; H₂NOCHMeCO₂H; carboxymethoxyamine; aminooxyacetic acid;

HN=C(NH₂)SCH₂CH₂ONH₂; H₂NO(CH₂)3SC(NH₂)=NH; MeOC(O)CH(NH₂)CH₂ONH₂;

H₂NOCH₂CH(NH₂)CO₂H; canaline; H2NO(CH₂)4ONH₂; O-(p-nitrobenzyl)hydroxylamine; 2- amino-4-(aminooxymethyl)thiazole; 4-(aminooxymethyl)thiazole; O,O’ -(o- phenylenedimethylene)dihydroxylamine; 2,4-dinitrophenoxyamine; 0,0’ -(m- phenylenedimethylene)dihydroxylamine; O,O’-(p-phenylenedimethylene)dihydroxylamine; H₂C=CHCH₂ONH₂; H₂NO(CH₂)4ONH₂; H₃C-(CH₂)₁₅-O-NH₂; 2,2’-(l,2-ethanediyl)bis(3- aminooxy)butenedioic acid dimethyl diethyl ester; and a pharmaceutically-acceptable salt thereof. The method of claim 1, wherein the alkylating agent is selected from

20. The method of claim 1, wherein the composition comprises two or more alkylating agents.

21. The method of claim 1, wherein the composition comprises a pharmaceutically- acceptable carrier.

22. A method of reducing scar tissue or improving an appearance of scar tissue comprising topically applying to the scar tissue an effective amount of a composition comprising a therapeutically effective amount of an alkylating agents.

23. The method of claim 22, wherein the composition comprises a pharmaceutically- acceptable topical carrier.

24. The method of claim 22, wherein the composition comprises about 0.001% to 99.9 wt% of the alkylating agent.

25. The method of claim 22, wherein a daily dose of the composition comprises about 0.001 μg/kg to 200 mg/kg of the alkylating agent.

26. A method of treating a liver disease in a subject, the method comprising administering a first formulation comprising MOA or aminoganidine and a second formulation comprising OCA or GOCA or TOCA.

27. The method of claim 26, wherein the MOA is administered in a daily dose of about 0.01 μg/kg and 200 mg/kg body weight.

28. The method of claim 26, wherein the OCA is administered in a daily dose of about 0.001 μg/kg and 20 mg/kg body weight.

29. The method of claim 26, wherein the first formulation comprising MOA or aminoganidine is administered to reduce side effects associated with OCA.

30. A method for selecting an alkylating agents for the treatment of a disease, the method comprising:

(a) mixing (i) aldehyde-N-terminal cysteinyl-peptides conjugate, aldehyde-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal cysteinyl-peptides conjugate, or a combination thereof and (ii) an alkylating agent in a solvent;

(b) incubating said mixture; and

(c) detecting within the incubated mixture (i) the amount of the aldehyde-N-terminal cysteinyl-peptides conjugate, aldehyde-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, or reactive ketone-N- terminal cysteinyl-peptide conjugate, or a combination thereof (ii) the amount of N- terminal-peptide released from the aldehyde-N-terminal cysteinyl-peptides conjugate, aldehyde-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, or reactive ketone-N-terminal cysteinyl-peptide conjugate, and/or (iii) the amount of conjugate formed between the alkylating agent and the aldehyde or reactive ketone.

31. The method of claim 30, wherein the aldehyde-N-terminal cysteinyl-peptide conjugate, aldehyde-N-terminal homocysteinyl-peptide conjugate, reactive ketone-N-terminal homocysteinyl-peptide conjugate, or reactive ketone-N-terminal cysteinyl-peptide conjugate is 3k-CDCA-Cys conjugate, 3k-CDCA-N-terminal cysteinyl-peptides conjugate, 3k-CDCA- HCys conjugate or 3k-CDCA-N-terminal homocysteinyl-peptide conjugate.

32. The method of claim 30, wherein the aldehyde comprises sugars, and the reactive ketone comprises 3k-bile acids, which comprising 3k-CA, 3k-DCA, 3k-CDCA, 3k-LCA, 3k- GCA, 3k-GDCA, 3k-GCDCA, 3k-GLCA, 3k-TCA, 3k-TDCA, 3k-TCDCA, 3k-TLCA, 3k- UDCA, 3k-GUDCA, and 3k-TUDCA.

33. The method of claim 30, wherein the alkylating agent is MOA.

34. The method of claim 30, wherein the solvent is selected from dimethyl sulfoxide (DMSO), acetonitrile (ACN), phosphate buffered saline (PBS), and a combination thereof.

35. The method of claim 30, wherein the incubation step (b) is perform for about 0.1 second to 100 days and at a temperature of about -20 °C and 100 °C.

36. The method of claim 30, wherein the detecting step (c) is performed by liquid chromatography (LC)-UV, LC-mass spectrometry (MS), and Nuclear magnetic resonance (NMR).

37. A method for screening aldehyde or reactive ketone of a drug candidate and said drug candidate’s metabolites, which cause adverse drug effects, comprising a) a mixture comprising i) cysteine, homocysteine, N-terminal homocysteinyl derivatives or N-terminal cysteinyl-derivatives; ii) the said drug; iii) metabolizing enzymes, including the entities of microsomes, cells, or tissues which contain said metabolizing enzymes, b) incubating said mixture in physiological solution and condition, c) detecting the formation of thiazolidine or 1,3-thiazinane derivatives of said cysteine, homocysteine, N-terminal homocysteinyl derivative or N-terminal cysteinyl derivative conjugates with said aldehyde and ketone of said drug candidate and said metabolites.

38. The method of claim 37, wherein said cysteine, homocysteine, N-terminal homocysteinyl derivatives, N-terminal cysteinyl derivatives, thiazolidines derivatives and 1,3- thiazinane derivatives compound structures are:

v) cysteine: n=l, R₃=OH; vi) homocysteine: n=2, R₃=OH; vii)N-terminal cysteinyl derivative: n=l, R₃ is a peptide; viii) N-terminal homocysteinyl derivative: n=2, R₃ is a peptide, b)

iii) thiazolidines derivatives: n=l, each R₁ and R₂ is independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl, R₁ and R₂ may also form connected ring structures, R₃ is a hydroxyl group or a peptide; iv) 1,3-thiazinane derivatives: n=2, each R₁ and R₂ is independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C8 aryl, and C6-C10 heteroaryl, each of which is optionally substituted by halogen, CN, NO₂, CF₃, methoxy, hydroxyl, amine, and C1-C3 alkyl, R₁ and R₂ may also form connected ring structures, R₃ is a hydroxyl group or a peptide, c) The elements of said compounds may be isotopes labeled. d) The elements of said compounds may be radioactive isotopes labeled.

39. A method of claim 37, for detecting said thiazolidine and 1,3-thiazinane derivatives, comprising, LC-MS, NMR, Ion Chromatography, Florescence Spectroscopy, Radioactive Detection.