Methods for promoting hepatic regeneration

Abstract

The present disclosure provides a method for in vivo liver regeneration in a subject in need thereof, comprising: administering to the subject a compound of formula (I): (I), or a pharmaceutically acceptable salt thereof, or a co-crystal thereof. The present disclosure also provides a method for ex vivo liver regeneration, comprising: applying to an ex vivo liver graft the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co- crystal thereof. The present disclosure also provides a method of treating a liver disorder for a subject in need thereof, comprising: administering to the subject a therapeutically effective dose of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a cocrystal thereof. The present disclosure also provides a dihydrochloride salt form of the compound of formula (I). The present disclosure also provides a co-crystal including the dihydrochloride salt form of the compound of formula (I) and a co-crystal former selected from succinic acid and adipic acid.

Description

METHODS FOR PROMOTING HEPATIC REGENERATIONCross-Reference to Related Applications

[0001] This application claims the priority of U.S. Application No. 63 / 657,472, filed June 7, 2024, which is incorporated herein by reference in its entirety.Technical Field of the Invention

[0002] The present disclosure relates to methods for in vivo liver regeneration in a subject in need thereof. The present disclosure also relates to methods for ex vivo liver regeneration. The present disclosure also relates to methods for treating a liver disorder.Background of the Invention

[0003] Liver regeneration is a unique phenomenon characterized by the ability of the liver to restore its mass and functionality following injury or surgical resection. This regenerative capacity is primarily attributed to the proliferation and differentiation of hepatocytes, the major parenchymal cells of the liver. Understanding the mechanisms that govern liver regeneration has significant implications for the development of therapeutic strategies to enhance liver regeneration and promote functional recovery in patients with liver diseases and after surgical procedures.

[0004] Partial hepatectomy, the surgical removal of a portion of the liver, has long been used as a model to study liver regeneration in experimental settings. This well-established model allows researchers to investigate the regenerative response of the liver, including the cellular and molecular events involved in hepatocyte proliferation and tissue remodeling.Numerous studies have aimed to identify compounds or agents that can modulate liver regeneration and enhance the regenerative capacity of the liver.

[0005] Liver transplantation is a treatment option for end-stage liver disease and acute liver failure, although the limited availability of donor organs poses a significant challenge. Small- for-size syndrome occurs when there is an insufficient liver mass to sustain the metabolic demand of the patient after a living donor liver transplantation or partial orthotopic liver transplantation. Surgery is the sole curative option for primary and metastatic liver tumors. Liver resection is constrained by the necessity to preserve an adequate amount of functional liver tissue. Excessive resection can lead to post-hepatectomy liver failure, particularly in cirrhotic livers. Complete resection of hepatic colorectal cancer (CRC) metastases can offer a potential long-term cure for some patients. However, the majority of liver metastases are not suitable for such surgery, mainly due to the limited size of the remaining liver.

[0006] In recent years, the development of novel compounds with regenerative properties has gained considerable attention. One such compound is HZ-02, a promising candidate for promoting liver regeneration. HZ-02 is a novel agent with potential regenerative effects on liver tissue, but its specific mechanisms of action and therapeutic potential in liver regeneration remain under study. HZ-02 was previously known as a pan inhibitor of the a- ketoglutarate dependent oxygenases subfamily of Jumonji (JMJD) histone lysine demethylases (KDMs) that exerts antitumoral effects on several tumor types.Summary

[0007] Disclosed herein is a method for in vivo liver regeneration in a subject in need thereof. The method comprises: administering to the subject a compound of formula (I):or a pharmaceutically acceptable salt thereof, or a cocrystal thereof.

[0008] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0009] In some embodiments, the subject has liver cancer or metastasis. In some embodiments, the subject has small-for-size syndrome. In some embodiments, the subject has post-hepatectomy liver failure. In some embodiments, the subject has undergone liver donation and has a remaining liver volume less than 35% of an initial liver volume.

[0010] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject daily, every other day, every three days, every four days, every five days, every six days, weekly, biweekly, three times a month, or once a month.

[0011] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject for at least one week, or two weeks, or three weeks, or four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or three months, or four months, or five months, or six months, orseven months, or eight months, or nine months, or ten months, or eleven months, or one year, or two years, or three years, or four years, or five years, or more than 5 years.

[0012] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject in an amount of about 0.01 mg / kg to about 100 mg / kg per dose per day.

[0013] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject orally, intravenously, intramuscularly, or subcutaneously.

[0014] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is a dihydrochloride salt of the compound of formula (I).

[0015] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is a cocrystal comprising a dihydrochloride salt of the compound of formula (I) and a cocrystal former selected from succinic acid (SUA) and adipic acid (ADA).

[0016] Also disclosed herein is a method for ex vivo liver regeneration. The method comprises: applying to an ex vivo liver graft a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a cocrystal thereof.

[0017] In some embodiments, the ex vivo liver graft is from a mammal. In some embodiments, the ex vivo liver graft is from a human. In some embodiments, the ex vivo liver graft is from donation after circulation death.

[0018] Also disclosed herein is a method of treating a liver disorder for a subject in need thereof. The method comprises: administering to the subject a therapeutically effective dose of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a cocrystal thereof.

[0019] Also disclosed herein is a compound of formula (II):(II).

[0020] Also disclosed herein is a cocrystal comprising the compound of formula (II) and a cocrystal former selected from succinic acid and adipic acid.

[0021] Also disclosed herein is a pharmaceutical composition comprising the compound of formula (II).

[0022] Also disclosed herein is a pharmaceutical composition comprising the co-crystal comprising the compound of formula (II) and the cocrystal former selected from succinic acid and adipic acid.Brief Description of the Drawings

[0023] The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings.

[0024] FIG. 1 is a graphical representation showing C3H / HEN mice being treated intraperitoneal (i.p) with a total of 5 doses (50 mg / kg) administered every other day (eod) with HZ-02 (E isomer), the Z isomer, or vehicle. Mice were euthanized 48 hs after the last dose, and values of Liver Index expressed as ratio of liver and body weight is shown. ***p<0.001 vs. Other groups by Anova.

[0025] FIG. 2 is a graphical representation showing BALB / c mice being treated with a total of 5 doses (25mg / kg) administered every other day (eod) with HZ-02 (E isomer), the HZ-02 salt, or vehicle. The E isomer was administered by intraperitoneal injection (i.p.) and the salt by intravenous injection (i.v.). Mice were euthanized 48 hs after the last dose, and values of Liver Index expressed as ratio of liver and body weight is shown. *p<0.05 y ***p<0.001 vs. Vehicle by Anova.

[0026] FIG. 3 is a graphical representation showing BALB / c mice being treated intraperitoneally (i.p, 25mg / kg, a total of 5 doses administered eod), intravenously (i.v., 25mg / kg, a total of 5 doses administered eod) or by gavage (37.5 mg / kg, a total of 10 doses administered every day). Mice were euthanized 48 hs after the last dose and values of Liver Index expressed as ratio of liver and body weight were represented. **p<0.001 vs. vehicle by Anova.

[0027] FIG. 4A is a graphical representation showing wistar rats being treated i.p. with a total of 5 doses (25 mg / kg) administered eod with HZ-02 or vehicle as controls. Rats were euthanized 48 hs after the last dose and values of Liver Index expressed as ratio of liver and body weight were represented. FIG. 4B is a photograpic representation showing wistar rats being treated i.p. with a total of 5 doses (25 mg / kg) administered eod with HZ-02 or vehicleas controls. Rats were euthanized 48 hs after the last dose and values of Liver Index expressed as ratio of liver and body weight were represented.

[0028] FIG. 5 is a graphical representation showing C57BL / c mice being treated with a total of 5 doses administered eod of the different compounds at the dose indicated in Table I. Mice were euthanized 48 hs after the last dose and values of Liver Index expressed as ratio of liver and body weight were represented. *p<0.05, **p<0.01 and ***p<0.001 vs. Vehicle by ANOVA.

[0029] FIG. 6A is a schematic representation showing an experimental design. FIG. 6B is a graphical representation showing values of Liver Index expressed as ratio of liver and body weight at time of surgery (gray bars) and sacrifice (black bars) of mice treated with 10, 25 and 50 mg / kg of HZ-02, according to the schedule shown in FIG. 6A, compared with vehicle (*p<0.05 or **p<0.01 vs. Vehicle by ANOVA at the time of sacrifice). FIG. 6C is a photographic representation showing representative liver imagens of mice 7 days after hepatectomy.

[0030] FIG. 7 is a photographical representation showing representative paraffin liver section stained with Hematoxilin-Eosin (10X image). HZ-02 ml and m2 are two mice of the same treated group.

[0031] FIG. 8 is a photographical representation showing representative paraffin liver section of mice treated with a total of 3 doses of HZ-02 (10 mg / kg body weight) stained using an PCNA antibody. Upper and lower panels correspond to a 10X and 20X images, respectively. HZ-02 ml and m2 are two mice of the same treated group.

[0032] FIG. 9 is a graphical representation showing values of Liver Index expressed as ratio of liver and body weight at the time of surgery and sacrifice of C57BL / c mice treated with 25 mg / kg of HZ-02 (a total of 3 doses administered eod) compared with vehicle. *p<0.05 vs. Vehicle at surgery by ANOVA.

[0033] FIG. 10 is a graphical representation showing prothrombin time of C57BL / c mice treated with 3 doses of HZ-02 (25mg / kg, eod) and subjected to sub-lethal hepatectomy, and euthanized 72 h later. Healthy mice were used as control (SHAM), n.s. p>0.05 vs Sham by Mann- Whitney.

[0034] FIG. 11 A is a schematic representation of an experimental design of lethal hepatectomy. FIG. 1 IB is a photographic representation of liver of mice treated with vehicle or HZ-02 5 days after hepatectomy. FIG. 11C is a graphical representation showing liver Index expressed as ratio of liver (estimated) and body weight at the time of surgery. Mice treated with a total of 3 doses of 50 mg / kg of HZ-02, according to the schedule shown in FIG. 11A, compared with vehicle (**p<0.01 vs. Vehicle by Mann- Whitney test).

[0035] FIG. 12A is a graphical representation showing biochemical analysis. FIG. 12B is a graphical representation showing biochemical analysis. Both graphs represents the average + SEM of a set of standard parameters measured in blood.

[0036] FIG. 13 is a graphical representation showing dose-response curves for cell viability assessed in HCC cells (Hepal29 and BNL) and normal hepatocytes treated with HZ-02. Data were normalized to the untreated controls (100% viability) and dose response curves plotted using a non-linear regression model.

[0037] FIG. 14A is a graphical representation showing tumor volume in orthotopic HCC in mice treated 3 times per week for a total of 6 doses with vehicle or with HZ-02 either 25 mg / kg / dose or 50 mg / kg / dose. FIG. 14B is a graphical representation showing liver weight in mice treated 3 times per week for a total of 6 doses with vehicle or with HZ-02 either 25 mg / kg / dose or 50 mg / kg / dose. Graph represents the average ± SEM.

[0038] FIG. 15A is a graphical representation showing tumor volume of orthotopic HCC in mice treated 3 times per week for a total of 6 doses with vehicle or SD-70 at 10 mg / kg / dose. FIG. 15B is a graphical representation showing liver weight in mice treated 3 times per week for a total of 6 doses with vehicle or SD-70 at 10 mg / kg / dose. Graphs represent the average ± SEM.

[0039] FIG. 16 is a graphical representation showing biological process and pathways related with genes upregulated in transformed hepatocytes by HZ-02 treatment.

[0040] FIG. 17 is a graphical representation showing biological process and pathways related with genes upregulated in transformed hepatocytes by SD-70 treatment.

[0041] FIG. 18 is a graphical representation showing a1H-NMR spectrum of HZ-02- 2HC1.

[0042] FIG. 19 is a graphical representation showing a13C-NMR spectrum of HZ-02- 2HC1.

[0043] FIG. 20 is a graphical representation showing aJH-15N HSQC NMR spectrum ofHZ-02 (DMSO-de).

[0044] FIG. 21 is a graphical representation showing aJH-15N HMBC NMR spectrum of HZ-02 (DMSO-de).

[0045] FIG. 22 is a graphical representation showing aJH-15N HMBC NMR spectrum ofHZ-02-2HC1 (D2O).

[0046] FIG. 23 is a graphical representation showing an FTIR spectrum of HZ-02-2HC1.

[0047] FIG. 24A is a graphical representation showing a potentiometric titration curve for HZ-02-2HC1. FIG. 24B is a graphical representation showing a second derivative plot of FIG. 24A. Arrows indicate pKa values.

[0048] FIG. 25 is a graphical representation showing a time-dependent solubility assay of HZ-02-2HC1.

[0049] FIG. 26 is a graphical representation showing stacked time-dependentJH NMR spectra of HZ-02-2HC1 solution at 37 °C. Arrows indicate signals of decomposition products.

[0050] FIG. 27 is a graphical representation showing time-dependent chemical stability of HZ-02-2HC1 at 37 °C.

[0051] FIG. 28 is a graphical representation showing differential scanning calorimetry diagram of HZ-02-2HC1.

[0052] FIG. 29 is a graphical representation showing thermogravimetric analysis of HZ- 02-2HC1.

[0053] FIG. 30 is a graphical representation showing a powder X-ray diffractogram HZ- 02-2HC1 crystalline powder.

[0054] FIG. 31 is a graphical representation showing an Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of HZ-02-2HC1 showing the atom numbering scheme used (hydrogens not included for clarity) and ellipsoids drawn at the 50% probability level.

[0055] FIG. 32A is a photographical representation showing a monocrystal used for X-ray diffraction with its dimensions. FIG. 32B is a photographical representation showing a monocrystal on a loop of a diffractometer. FIG. 32C is a photographical representation showing an image of the obtained crystals.

[0056] FIG. 33 is a graphical representation showing aJH NMR spectrum (500 MHz) of the HZ2-2HC1-SUA cocrystal in D2O.

[0057] FIG. 34A is a graphical representation showing a FT-IR spectra of the HZ2-2HC1- SUA cocrystal, SUA, and HZ2-2HC1. FIG. 34B is a graphical representation showing FT-IR spectra of the HZ2-2HC1-ADA cocrystal, ADA, and HZ2-2HC1.

[0058] FIG. 35 is a graphical representation showing an animal study where the animals were treated intraperitoneally (i.p) with a total of 4 doses (40 mg / kg) administered daily with the HZ-02-2HC1-SUA cocrystal. Animals were sacrificed on the fifth day for tissue analysis. **p<0.01 vs. Vehicle by ANOVA at the time of sacrifice.Detailed Description

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is intended to describe particular embodiments only, and is not intended to limit the scope of the invention.

[0060] As used herein, the following terms have the meaning indicated, unless otherwise specifically noted in context. Unless otherwise defined herein, all technical terms used hereinhave the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0061] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0062] Except where otherwise indicated, all numbers expressing quantities of ingredients, time periods, and so forth used in the disclosure and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not to be considered as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding conventions.

[0063] Additionally, the disclosure of numerical ranges within this specification is considered to be a disclosure of all numerical values and ranges within that range. For example, if a range is from about 1 to about 50, it is deemed to include, for example, 1, 7, 34, 46.1, 23.7, 50 or any other value or range within the range. Moreover, as used herein, the term “at least” includes the stated number, e.g., “at least 50” includes 50.

[0064] As used herein, “subject” or “patient” refers to an animal, preferably a mammal, including a human or a non-human animal including livestock animals and domestic animals including, but not limited to, cattle, horses, sheep, swine, goats, rabbits, cats, dogs, and other mammals in need of treatment. In some embodiments, the subject is a human.

[0065] As used herein, the term "subject in need thereof,” refers to a subject having a disease (to be treated) or having an increased risk of developing the disease (to be prevented). A subject in need thereof can be one who has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be one who has (e.g., is suffering from) a disease or disorder disclosed herein. Alternatively, a subject in need thereof can be one who has an increased risk of developing such disease or disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large). A subject in need thereof can have a refractory or resistant a disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that doesn't respond or hasn't yet responded to treatment). The subject may be resistant at start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof received and failed all known effective therapies for a disease or disorder disclosed herein. In some embodiments, the subject in need thereof received at least one prior therapy.

[0066] As used herein, “administering” or “administered to” refers to prescribing a medicine to a subject, directing others to administer a medicine to a subject, directing a subject to self-administer a medicine and / or the act of physically ingesting the medicine. A medicine containing the compound of formula (I) as its active pharmaceutical ingredient, can therefore be administered by a physician or other medical professional who writes prescriptions for a medicine(s) or otherwise directs a patient to self-administer a prescription, and / or by the subject who ingests the medicine and / or by a subject’s caretaker who provides the medicine to a subject.

[0067] As used herein, the term "preventing" or "prevent" describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.

[0068] As used herein, the term "pharmaceutically acceptable" refers to those compounds, anions, cations, materials, compositions, carriers, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0069] As used herein, the term "daily" means every day, where each day is defined by a 24 hour period. For the avoidance of doubt, the 24 hour period defining “daily” can bridge two weekly calendar days, for example, Sunday -Monday; Monday-Tuesday; Tuesday- Wednesday; etc.

[0070] As used herein, the term "dosage" or "daily dosage" refers to the weight of an active ingredient.

[0071] As used herein, a “pharmaceutical composition” is a formulation containing at least one active ingredient, such as at least one compound of the disclosure, in a form suitable for administration to a subject.

[0072] As used throughout this disclosure, “compound(s) disclosed herein”, “compound(s) described herein”, “compound(s) of the disclosure”, etc., are used interchangeably and include the compound, as well as a pharmaceutically acceptable salt, solvate, polymorph, stereoisomer, or prodrug thereof.

[0073] Any pharmaceutically acceptable pro-drug modification of a compound disclosed herein which results in conversion in vivo to a compound within the scope of this disclosure is also within the scope of this disclosure.

[0074] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that is useful in preparing a formulation that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable excipient" as used in the specification and claims includes both one and more than one such excipient.

[0075] It is to be understood that the compound of formula (I) or the pharmaceutically acceptable salt thereof, may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present disclosure, and the naming of the compounds does not exclude any tautomer form. It will be understood that certain tautomers may have a higher level of activity than others.

[0076] As used herein, the term "salt" or "pharmaceutically acceptable salt" refers to a derivative of the compounds of the present disclosure wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2- hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucohep tonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodic, hydroxy maleic, hydroxy naphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc. Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4- chlorobenzenesulfonic acid, 2 -naphthalene sulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo- [2.2.2] -oct-2-ene-l-carboxylic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present disclosure also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In the salt form, it is understood that the ratio of the compound to the cation or anion of the salt can be 1: 1, or any ratio other than 1:1, e.g., 3: 1, 2: 1, 1:2, or 1:3. It is to be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.

[0077] As used herein, the term "treating" or "treat" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. That is, “treating” or “treatment” of a state, disorder, or condition therefore includes: (1) delaying the appearance of clinical symptoms of the state, disorder, or conditiondeveloping in a human that may be afflicted with the state, disorder, or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder, or condition, (2) inhibiting the state, disorder, or condition, i.e., arresting or reducing the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder, or condition or at least one of its clinical or subclinical symptoms.

[0078] As used herein, the term "effective amount" refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0079] All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure.

[0080] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

[0081] Disclosed herein is a method for in vivo liver regeneration in a subject in need thereof. The method comprises: administering to the subject a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a cocrystal thereof.

[0082] In some embodiments, the compound of formula (I) or the pharmaceutically acceptable salt thereof is a dihydrochloric acid salt of the compound of formula (I). The dihydrochloric acid salt of the compound of formula (I) has a structure of formula (II).

[0083] In some embodiments, the subject is a mammal.

[0084] In some embodiments, the subject is a human.

[0085] In some embodiments, the subject has liver cancer or metastasis.

[0086] In some embodiments, the subject has small-for-size syndrome.

[0087] In some embodiments, the subject has post-hepatectomy liver failure.

[0088] In some embodiments, the subject has undergone liver donation and has a remaining liver volume less than 35% of an initial liver volume.

[0089] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject daily, every other day, every three days, every four days, every five days, every six days, weekly, biweekly, three times a month, or once a month.

[0090] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject for at least one week, or two weeks, or three weeks, or four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or three months, or four months, or five months, or six months, orseven months, or eight months, or nine months, or ten months, or eleven months, or one year, or two years, or three years, or four years, or five years, or more than 5 years.

[0091] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject in an amount of from about 0.01 mg / kg to about 100 mg / kg, or about 0.01 mg / kg to about 90 mg / kg, or about 0.01 mg / kg to about 80 mg / kg, or about 0.01 mg / kg to about 70 mg / kg, or about 0.01 mg / kg to about 60 mg / kg, or about 0.01 mg / kg to about 50 mg / kg, or about 0.01 mg / kg to about 40 mg / kg, or about 0.01 mg / kg to about 30 mg / kg, or about 0.01 mg / kg to about 20 mg / kg, or about 0.01 mg / kg to about 10 mg / kg, or about 0.01 mg / kg to about 5 mg / kg, or about 0.01 mg / kg to about 1 mg / kg, or about 0.01 mg / kg to about 0.5 mg / kg, or about 0.1 mg / kg to about 100 mg / kg, or about 0.1 mg / kg to about 90 mg / kg, or about 0.1 mg / kg to about 80 mg / kg, or about 0.1 mg / kg to about 70 mg / kg, or about 0.1 mg / kg to about 60 mg / kg, or about 0.1 mg / kg to about 50 mg / kg, or about 0.1 mg / kg to about 40 mg / kg, or about 0.1 mg / kg to about 30 mg / kg, or about 0.1 mg / kg to about 30 mg / kg, or about 0.1 mg / kg to about 20 mg / kg, or about 0.1 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 5 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 100 mg / kg, or about 20 mg / kg to about 100 mg / kg, or about 30 mg / kg to about 100 mg / kg, or about 40 mg / kg to about 100 mg / kg, or about 50 mg / kg to about 100 mg / kg, or about 60 mg / kg to about 100 mg / kg, or about 70 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 80 mg / kg, or about 1 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 30 mg / kg, or about 1 mg / kg to about 10 mg / kg, or about 10 mg / kg to about 80 mg / kg, or about 10 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, or a range or a number between any two of these values.

[0092] In some embodiments, the methods of the disclosure comprise administering to the subject in need thereof or applying to an ex vivo liver graft the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, as the sole agent. In some embodiments, the methods of the disclosure comprise administering to the subject in need thereof or applying to an ex vivo liver graft the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, wherein no additional agents are co-administered. In some embodiments, the methods of the disclosure comprise administering to the subject in need thereof or applying to an ex vivo liver graft the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, wherein no additional agents are co-administered during the pendency of the liver regeneration process.

[0093] In some embodiments, the method can comprise administering to the subject in need thereof or applying to an ex vivo liver graft one or more additional agents. As used herein, an “additional agent(s)” is intended to mean a pharmaceutically active agent(s) that is active in the body, including pro-drugs that convert to pharmaceutically active form after administration or application, which are different from the compounds of the disclosure, and also includes pharmaceutically acceptable salts of the additional active agents. Generally, any suitable additional agent(s) may be used in any combination with the compounds of the disclosure in a single dosage form (a fixed dose drug combination) or may be administered to the subject in one or more separate dosage formulations, which allows for concurrent or sequential administration or application of the compounds of the disclosure and the additional therapeutic agent(s) (co-administration or co-application of the separate active agents). The sequence in which the agents are administered or applied can vary. Agents may also be administered or applied in alternation. In some embodiments, the additional agents can be co-administered to the subject or applied to an ex vivo liver graft with the compound of formula(I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof. In some embodiments, the additional agents can be administered to the subject or applied to an ex vivo liver graft before the administration or application of the composition comprising the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, after the administration or application of the composition comprising the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, or both. In some embodiments, the additional agent(s) can be concurrently administered to the subject or applied to an ex vivo liver graft with the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof. In some embodiments, a composition comprising the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, can comprise one or more additional agents.

[0094] The compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, may be administered by any appropriate route of administration. Potential routes of administration of the compounds of the disclosure include without limitation oral or parenteral (including for example, intravenous, intramuscular, and subcutaneous).

[0095] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject orally, intravenously, intramuscularly, or subcutaneously. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject orally. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject intravenously. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to thesubject intramuscularly. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject subcutaneously.

[0096] The compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, can be administered without food. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered at least about 1 or 2 hours before or after a meal. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered at least about 2 hours after an evening meal. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, can also be taken substantially concurrently with food (e.g., less than about 30 minutes or 1 hour before or after a meal, or with a meal).

[0097] Pharmaceutical compositions can take any suitable form for the desired route of administration. Proper formulation can depend on various factors, such as the mode of administration chosen.

[0098] Where a compound of the disclosure, or a pharmaceutical composition comprising a compound of the disclosure, is to be administered orally, any suitable orally deliverable dosage form can be used. The oral dosage forms according to the disclosure can be solid, semi-solid or liquid. Such oral dosage forms include, but are not limited to, powders, dispersible granules, mini-tablets, and beads (which can be used, for example, for tableting, encapsulation, or direct administration), pills, tablets, lacquered tablets, sugar-coated tablets, hard and soft capsules including gelatin capsules, lozenges, rapidly dissolving tablets, aqueous, alcoholic or oily solutions, gels, syrups, emulsions or suspensions. The oral dosageforms according to the disclosure may comprise additionally one or more coatings which modify release properties, for example, coatings which impart delayed release or formulations which have extended release properties. Also included in the present disclosure are formulations which are intended to be converted, shortly before use, to a suspension or a solution; examples include, but are not limited to, freeze-dried formulations and liquid formulations adsorbed into a solid absorbent medium, including without limitation, tablets, capsules (solid or liquid filled), powders, granules, syrups and other liquids, elixirs, troches, lozenges, gels, pastes, solutions or suspensions in an aqueous liquid or / and a non-aqueous liquid, or oil-in-water liquid emulsions or water- in-oil liquid emulsions.

[0099] Injectable compositions or i.v. infusions can be provided, for example, in the form of solutions, suspensions, and emulsions.

[0100] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered to the subject intramuscularly. In some embodiments, the subcutaneous administration is made with an injection device.

[0101] In some embodiments, subcutaneous administration may be performed by injection using a syringe, or using other injection devices, injector pens, or needleless devices.An injection device is usually a device that introduces a substance into the body of a patient via a parenteral route, e.g., intramuscular, subcutaneous, or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., compound of the disclosure or a pharmaceutical composition thereof), a needle for piecing skin and / or blood vessels for injection of the fluid; and a plunger for pushing thefluid out of the cylinder and through the needle bore. In an embodiment, an injection device is an autoinjector, a jet injector or an external infusion pump.

[0102] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, can also be formulated for parenteral administration by injection or infusion to circumvent gastrointestinal absorption and first- pass metabolism. In some embodiments, a representative parenteral route is intramuscular, subcutaneous, or intravenous.

[0103] Additional advantages of intramuscular, subcutaneous, or intravenous administration include direct administration of a therapeutic agent into systemic circulation to achieve a rapid systemic effect, and the ability to administer the agent continuously and / or in a large volume if desired.

[0104] For intramuscular, subcutaneous, or intravenous administration, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HC1, and citric acid. In some embodiments, the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7. Antioxidant excipients may include, for example, sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA. Other non-limiting examples of suitable excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Antimicrobial agents may also be included to achieve a bacteriostaticor fungistatic solution, including but not limited to phenyl mercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

[0105] In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, for intravenous administration may be provided in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration. In other embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is provided in solution ready to administer parenterally. In still other embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is provided in a solution that is further diluted prior to administration. In embodiments that include administering the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, and another agent, the combination may be provided as a mixture. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, and another agent may be mixed prior to administration, or may be administered separately.

[0106] Formulations for injection or infusion can be in the form of, e.g., solutions, suspensions or emulsions in oily or aqueous vehicles, and can contain excipients such as suspending agents, dispersing agents and / or stabilizing agents. For example, aqueous or nonaqueous (e.g., oily) sterile injection solutions can contain the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, along with excipients such as an antioxidant, a buffer, a bacteriostat and solutes that render the formulation isotonic with the blood of the subject. Aqueous or non-aqueous sterile suspensions can contain the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, or a pharmaceutical composition comprising the compound of formula (I), or thepharmaceutically acceptable salt thereof, or the cocrystal thereof, along with excipients such as a suspending agent and a thickening agent, and optionally a stabilizer and an agent that increases the solubility of the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, to allow for the preparation of a more concentrated solution or suspension. As another example, a sterile aqueous solution for injection or infusion (e.g., subcutaneously or intravenously) can contain the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, along with NaCl, a buffering agent (e.g., sodium citrate), a preservative (e.g., meta-cresol), and optionally a base (e.g., NaOH) and / or an acid (e.g., HC1) to adjust pH. In alternate embodiments, aqueous or non-aqueous (e.g., oily) sterile injection solutions can contain the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, do not contain excipients or carriers.

[0107] The pharmaceutical compositions can be manufactured in any suitable manner known in the art, e.g., by means of conventional mixing, dissolving, suspending, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes.

[0108] A pharmaceutical composition can be presented in unit dosage form as a single dose wherein all active and inactive ingredients are combined in a suitable system, and components do not need to be mixed to form the composition to be administered. The unit dosage form can contain an effective dose, or an appropriate fraction thereof, of a compound of the disclosure. Representative examples of a unit dosage form include a tablet, capsule or pill for oral administration, or a single use sterile vial for intravenous administration.

[0109] In some embodiments, for example where a more rapid establishment of a therapeutic level of the compound of formula (I), or the pharmaceutically acceptable saltthereof, or the cocrystal thereof, is desired, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is administered under a dosing schedule in which a first dosage amount is administered, followed by one or more therapeutically effective subsequent dosage amounts. In some embodiments, a first dosage amount is larger (e.g., about 1.5, 2, 3, 4 or 5 times larger) than a subsequent dosage amount and is designed to establish a therapeutic level of the drug more quickly. In some embodiments, the first dosage amount is about 1.5 times, or about two times, or about three times, or about four times, or about five times greater than the subsequent dosage amount.

[0110] The amount, the frequency of administration of, and / or the duration of treatment with, a compound of the disclosure may vary based on various factors, including, but not limited to, the nature and severity of the disease and / or at least one symptom thereof, the subject to be treated, the general health of the subject, the age, weight, gender, and / or diet of the subject, route of administration, drug interaction(s), reaction sensitivities, and tolerance / response to therapy, all of which can be determined by one of ordinary skill in the art such as a medical professional. The dosage amount and / or duration may be adjusted by a medical professional, such as a physician or veterinarian, including in the event of any complication. Dosage amounts and / or duration can adjusted to provide sufficient levels of the compounds of the disclosure or to maintain the desired effect. For example, the dosage amount may be increased or decreased.

[0111] The compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, may be administered or applied alone or in the form of a composition (e.g., pharmaceutical composition or formulation). In some embodiments, a pharmaceutical composition comprises the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, and one or more pharmaceutically acceptable excipients.Pharmaceutical compositions may be conveniently presented as one or more unit dose forms containing a predetermined amount of an active agent per dose.

[0112] In some embodiments, a pharmaceutical composition comprises the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, and one or more pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers and excipients include pharmaceutically acceptable materials, vehicles and substances, including for example any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included.

[0113] Non-limiting examples of excipients include liquid and solid fillers, diluents, binders, lubricants, glidants, solubilizers, surfactants, dispersing agents, disintegration agents, emulsifying agents, wetting agents, suspending agents, thickeners, solvents, isotonic agents, buffers, pH adjusters, stabilizers, preservatives, antioxidants, antimicrobial agents, antibacterial agents, antifungal agents, absorption- delaying agents, sweetening agents, flavoring agents, coloring agents, adjuvants, encapsulating materials and coating materials. For example, conventional vehicles and carriers include without limitation oils (e.g., vegetable oils, such as sesame oil), aqueous solvents (e.g., saline, phosphate-buffered saline [PBS] and isotonic solutions [e.g., Ringer's solution]), and solvents (e.g., dimethyl sulfoxide [DMSO] and alcohols [e.g., ethanol, glycerol and propylene glycol]). Except insofar as any conventional carrier or excipient is incompatible with the active ingredient, the disclosure encompasses the use of all conventional carriers and excipients in formulations containingthe NK1R antagonist (e.g., aprepitant), such as a compound of the disclosure or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

[0114] As will be appreciated by the ordinarily skilled artisan, a pharmaceutically acceptable excipient is any constituent which adapts the composition to a particular route of administration or aids the processing of a composition into a dosage form without itself exerting an active pharmaceutical effect. In general, compositions comprise more than one pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient(s) is selected based on the form of an oral dosage form. Examples of pharmaceutically acceptable excipients and methods of manufacture of oral dosage forms such as those mentioned above may be found in A. Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th Edition, (2000), Lippincott Williams & Wilkins, Baltimore, MD.

[0115] Pharmaceutically acceptable excipients suitable for use in the present disclosure include, without limitation, carriers (such as lactose, starch, starch derivatives, talc, stearic acid or its salts for, e.g., pills, tablets, sugar-coated tablets and hard gelatin capsules; such as fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc. for soft capsules; such as water, physiologically acceptable sodium chloride solution, alcohols, glycerol, polyols, sucrose, invert sugar, glucose, mannitol, vegetable oils, etc. for solutions, emulsions or syrups), fillers, disintegrants, binders, lubricants, pressing aids, wetting agents, stabilizers, emulsifiers, absorption enhancers, penetration enhancers, permeation enhancers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents and / or antioxidants. A particular pharmaceutically acceptable excipient(s), as well as an amount(s) thereof, is selected for use in an oral dosage form so as to provide the desired amount of the compound of formula (I), or thepharmaceutically acceptable salt thereof, or the cocrystal thereof, in an oral dosage form of acceptable volume such that it can provide a therapeutic serum level of the active for an acceptable period of time in the subject to whom the oral dosage form is administered and such that the oral dosage form will retain biological activity during storage within an acceptable temperature range for an acceptable period of time.

[0116] Alternatively, a pharmaceutical composition can be presented as a kit, wherein the active ingredient, excipients and carriers (e.g., solvents) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles or syringes) and need to be combined to form the composition to be administered or applied. The kit can contain instructions for storing, preparing and administering or applying the composition (e.g., a solution to be injected intravenously).

[0117] A kit can contain all active and inactive ingredients in unit dosage form or the active ingredient and inactive ingredients in two or more separate containers, and can contain instructions for using the pharmaceutical composition.

[0118] In some embodiments, a kit contains the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, and instructions for administering or applying the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof. In some embodiments, the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, is contained or incorporated in, or provided by, a device or system configured for pulmonary delivery of the compound by oral inhalation, such as a metered-dose inhaler, a dry powder inhaler or a nebulizer.

[0119] An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a transparent plastic material.

[0120] It may be desirable to provide a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested. Another example of such a memory aid is a calendar printed on the card, e.g., as follows “First Week, Monday, Tuesday, etc. . . . Second Week, Monday, Tuesday, . . . ” etc. Other variations of memory aids will be readily apparent. A “daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day. Also, a daily dose of the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the cocrystal thereof, can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa. The memory aid should reflect this.

[0121] In some embodiments, a dispenser designed to dispense the daily doses one at a time in the order of their intended use is provided. For example, the dispenser is equipped with a memory aid, so as to further facilitate compliance with the regimen. An example of such a memory aid is a mechanical counter which indicates the number of daily doses that has been dispensed. Another example of such a memory aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and / or reminds one when the next dose is to be taken.

[0122] Also disclosed herein is a method for ex vivo liver regeneration. The method comprises: applying to an ex vivo liver graft a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a cocrystal thereof.

[0123] In some embodiments, the ex vivo liver graft is from a mammal.

[0124] In some embodiments, the ex vivo liver graft is from a human.

[0125] In some embodiments, wherein the ex vivo liver graft is from donation after circulation death.

[0126] Also disclosed herein is a method of treating a liver disorder for a subject in need thereof. The method comprises: administering to the subject a therapeutically effective dose of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a cocrystal thereof.

[0127] Non-limiting examples of liver disorders include Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D (Delta hepatitis), Hepatitis E, Cytomegalovirus (CMV) hepatitis, Epstein-Barr Virus (EBV) hepatitis, Herpes Simplex Virus (HSV) hepatitis, Yellow fever (viral hemorrhagic disease), Amebic liver abscess (Entamoeba histolytica), Pyogenic liver abscess (bacterial), Schistosomiasis, Echinococcosis (hydatid disease), Autoimmune hepatitis, Primary biliary cholangitis (PBC), Primary sclerosing cholangitis (PSC), IgG4- related autoimmune cholangitis, Hemochromatosis (iron overload), Wilson’s disease (copper accumulation), Alpha- 1 antitrypsin deficiency, Gilbert’s syndrome, Crigler-Najjar syndrome (Types I and II), Dubin-Johnson syndrome, Rotor syndrome, Progressive familial intrahepatic cholestasis (PFIC), Glycogen storage disease (hepatic types), Tyrosinemia (Type I and II), Alcoholic liver disease (ALD), Non-alcoholic fatty liver disease (NAFLD), Non-alcoholicsteatohepatitis (NASH), Acetaminophen (paracetamol) toxicity, Drug-induced liver injury (DILI), Mushroom poisoning (e.g., Amanita phalloides), Industrial / toxin-related liver injury, Budd-Chiari syndrome (hepatic vein thrombosis), Portal vein thrombosis, Congenital hepatic fibrosis, Sinusoidal obstruction syndrome (veno-occlusive disease), Ischemic hepatitis (shock liver), Peliosis hepatis, Reye’s syndrome, Lysosomal storage diseases affecting the liver, Malnutrition-related liver disease, Hepatocellular carcinoma (HCC), Cholangiocarcinoma (bile duct cancer), Fibrolamellar carcinoma, Hepatoblastoma, Angiosarcoma of the liver, Hepatic hemangioma, Hepatic adenoma, Focal nodular hyperplasia (FNH), Cystic liver lesions, Liver metastases from other cancers, Cholestasis of pregnancy, Neonatal cholestasis, Biliary atresia, Choledochal cysts, Intrahepatic cholestasis, Sarcoidosis involving the liver, Amyloidosis of the liver, Zieve’s syndrome, Alagille syndrome, Hepatic encephalopathy, Liver cirrhosis, Hepatopulmonary syndrome, Portopulmonary hypertension, Polycystic liver disease, Graft-versus-host disease (GVHD), Liver involvement in systemic lupus erythematosus (SLE), and HELLP syndrome.EXAMPLES

[0128] The following Examples are provided by way of illustration and not by way of limitation.

[0129] Example 1

[0130] General Methods

[0131] Solvents were distilled before use. Thin layer chromatography (TLC) was performed on silica gel 60 F254 plates (Macherey-Nagel). The compounds were detected under 254 nm UV light.XH,13C and15N nuclear magnetic resonance (NMR) spectra wererecorded at 25 °C at 500, 125.7 and 50.7 MHz, respectively, in a Bruker Avance Neo 500 spectrometer.1H and13C chemical shifts are reported in parts per million (ppm) relative to the residual solvent peak (DMSO-de:1H: 3 ppm,13C: d ppm). For13C spectra recorded in D2O, EtOH was used as standard (<5 56.9 ppm and 17.3 ppm).15N chemical shifts are reported in ppm relative to NO2CH3. Assignments ofXH,13C were determined by analysis of coupling constants and assisted by 2D1H COSY, 2D 'H TOCSY, 2DJH-13C HSQC, 2DJH-13C HMBC experiments. Assignments of15N were extracted from 2DJH-15N HSQC and 2DXH-15N HMBC experiments.

[0132] Synthesis of (E)-5-chloro-2-(2-(phenyl(pyridin-2- yl)methylene)hydrazineyl)pyridine dihydrochloride (HZ-02 • 2HC1)

[0133] To obtain HZ-02 • 2HC1, the free base HZ-02 was previously synthesized. The synthesis of HZ-02 was already reported in literature (see Wang, L. et al. A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth. Nat. Commun. 4:2035 (2013); see also Easmon J. et al. Azinyl and diazinyl hydrazones derived from aryl N-heteroaryl ketones: synthesis and antiproliferative activity. J Med Chem. 40(26):4420-5 (1997)).

[0134] The hydrochloride salt HZ-02- 2HC1 was obtained by adding a 6 N aqueous solution of HC1 to the solid free base HZ-02 at 0°C. The product was recovered by evaporation of the solvent at reduced pressure and drying in a vacuum oven at 40 °C untilconstant weight has been achieved. Keeping the product at 40 °C for prolonged periods led to decomposition of the salt. Presumably, loss of HC1 occurred as the decomposed product no longer dissolved in water, but did in organic solvents. Alternatively, an aqueous solution of the solventless product was lyophilized. A complete chemical and physical characterization of the product was performed by means of NMR and FTIR spectroscopy, argentometry, potentiometry, differential scanning calorimetry and thermogravimetry. To unambiguously confirm whether the product corresponds to the E or Z isomer, monocrystal X-ray crystallography was carried out. Solubility and time-dependent solubility in water, hygroscopicity, and stability studies were executed as well.

[0135] Methods

[0136] Preparation of the hydrochloride salt (HZ-02-2HCI). In a 25 mL round-bottomed flask equipped with a magnetic stirrer, HZ-02 (500 mg, 1.62 mmol) was placed and cooled in an ice bath. A solution of 6N HC1 (1.6 mL, 9.7 mmol) was added dropwise under magnetic stirring. The solution turned yellow. It was stirred for 10 minutes at 0 °C, then the ice bath was removed, and stirring continued for another 10 minutes at room temperature. The complete consumption of the starting material was verified by TLC (95:5 CH2Ch:MeOH). The solvent was evaporated under reduced pressure using a rotary evaporator equipped with a solid NaOH trap. The product was dried to constant weight in a vacuum oven at 40 °C (48 hours), yielding an orange crystalline solid (HZ-02- 2HC1, 609 mg, 98.5%). Alternatively, the solventless product was redissolved in water and lyophilized for 48 hours.

[0137] 1H NMR (500 MHz, D2O) 3 8.86 (dd, J = 5.8, 0.9 Hz, 1H, H-2), 8.51 (td, J = 8.1, 1.8 Hz, 1H, H-4), 8.37 (d, J = 2.4 Hz, 1H, H-6’), 8.13 (dd, J = 9.4, 2.4 Hz, 1H, H-4’), 8.05(ddd, 7 = 7.8, 5.8, 1.2 Hz, 1H, H-5), 7.76 (d, 7 = 8.0 Hz, 1H, H-3), 7.71 (m, 3H, H-9, H-10, H-12), 7.51 (m, 2H, H-9, H-13), 7.31 (d, 7 = 9.4 Hz, 1H, H-3’).

[0138] 13C NMR (125.7 MHz, D2O, std EtOH) 3 148.4 (C-5’), 147.0 (C-4), 145.9 (C-6), 144.8 (C-4’), 144.2 (C-7), 141.7 (C-2), 136.2 (C-6’), 131.4 (C-ll), 129.7 (C-10, C-12), 128.6 (C-9, C-13), 126.8 (C-8), 126.7 (C-3), 126.6 (C-5), 124.1 (C-2’), 113.3 (C-3’).

[0139] 15N NMR (50.7 MHz, D2O, std NO2CH3) 3 -192.1 (N-l), -203.7 (N-F), -231.7 (N- 15).

[0140] Identification of possible problems during the synthetic process:

[0141] During the purification of the free base HZ-02 by recrystallization from ethanol, an insoluble solid residue, corresponding to NaHCOs from the work-up of the reaction, may have remained. When this occured, it was filtered hot before crystallizing the product.

[0142] The free base HZ-02 should not be heated for prolonged periods in the recrystallization solvent or any other solvent, as this favors isomerization to the inactive isomer HZ2-Z. For this reason, the minimum amount of ethanol necessary should be used to purify (approximately 8 mL / g of crude product).

[0143] Several methods of synthesis of the dihydrochloride were evaluated. In general, it was observed that in the presence of organic solvents and acid, partial isomerization of the free base occurred and a mixture of the dihydrochloride of the E isomer and the free base of the Z was obtained.

[0144] The Z isomer could not be transformed to its hydrochloride under these conditions(it does not dissolve in 6 N HC1). The Z free base was dissolved in AcCl / AcOH and upon analysis the obtained product turned out to be the dihydrochloride of the E isomer.

[0145] Preparation of the free base (HZ-02). In a 250 mL round-bottomed flask, 5 g of 2- benzoyl pyridine (1, 27.3 mmol) and 3.89 g of 5-chloro-2-hydrazinyl pyridine (2, 27.3 mmol) were dissolved in 156 mL of isopropanol. The solution was maintained at room temperature with vigorous magnetic stirring, and 7.5 mL of concentrated H2SO4 was added slowly using a dropping funnel. Once the addition was complete, a condenser was attached, and the mixture was heated to 80 °C under a nitrogen atmosphere for 3 hours. The progress of the reaction was monitored by TLC (95:5 CH2Ch:MeOH, R / = 0.65). Then, the mixture was transferred to a 500 mL Erlenmeyer flask and cooled in an ice bath. It was neutralized by slow addition of saturated NaHCOs solution. The mixture was left at 4 °C for 16 hours to promote product precipitation, then filtered under vacuum and washed with water (150 mL) and cold Et2O (50 mL). The product was purified by two consecutive recrystallizations from absolute ethanol, yielding a white needle-like solid (HZ-02, 5.39 g, E:Z ratio 99.7:0.3, 64.2%).

[0146] ' H NMR (500 MHz, DMSO-d6) b 8.82 (sa, NH), 8.44 (ddd, 8.51, J = 5, 1.5, 1 Hz, 1H, H-2), 8.20 (dt, J = 8, 1 Hz, 1H, H-5), 8.12 (dd, J = 2.5, 0.5 Hz, 1H, H-6’), 7.87 (td, J = 8, 1.5 Hz, 1H, H-4), 7.82 (dd, J = 9, 2.5 Hz, 1H, H-4’), 7.57 (tt, J = 7.5, 6, 1.5 Hz, 2H, H-9, H- 13), 7.51 (tt, 7 = 7.5, 1.5 Hz, H-l l), 7.49 (dd, 7 = 9, 0.5 Hz, H-3’), 7.33 (m, 3H, H-3, H-10, H-12).

[0147] 13C NMR (125.7 MHz, DMSO-d6) b 155.5 (C-6), 154.6 (C-2’), 148.6 (C-2), 147.0 (C-7), 145.9 (C-6’), 138.1 (C-4’), 136.6 (C-4), 131.8 (C-8), 129.1 (C-9, C-13), 129.0 (C-l l), 129.0 (C-10, C-12), 123.2 (C-3), 122.0 (C-5’), 120.6 (C-5), 108.6w (C-3’).

[0148] 15N NMR (50.7 MHz, DMSO-d6, std NO2CH3) d -65.9 (N-14), -68.1 (N-l), -107.3 (N-F), -231.5 (N-15).

[0149] Analysis of HZ-02* 2HC1 by NMR spectroscopy

[0150] As the free base HZ-02 contains three different protonable positions, the protonation state of the hydrochloride was investigated. NMR characterization of hydrochloride salts and its free bases may inform on the protonated nitrogens by comparison of the chemical shifts of the surrounding nuclei. A complete assignment of the signals in1H (FIG. 18) and proton-decoupled13C (FIG. 19) NMR spectra was performed on the basis of 2D1H-COSY,1H-TOCSY,JH-13C HSQC andJH-13C HMBC experiments. To finally determine the protonation state of the molecule,15N NMR experiments were performed.

[0151] To determine the protonation state of the molecule,15N NMR experiments were performed. These experiments were useful to discern between protonated and not protonated atoms by studying the magnetic environment of the nitrogen nuclei of the molecule.JH-15N HSQC (FIG. 20) and HMBC (FIG. 21) NMR spectra for the free base HZ-02 in DMSO-d6andJH-15N HMBC (FIG. 22) spectra for the hydrochloride salt HZ-02- 2HC1 in D2O, were performed using NO2CH3 as external reference. A complete assignment of the15N atoms in HZ-02 could be done through identification of relevant couplings. In the case of HZ-02- 2HC1, as the compound rapidly isomerized in DMSO, water was used as a solvent and the loss of interchangeable protons precluded the identification of N-14. Despite this, the study was conclusive to confirm the dihydrochloride form of the molecule, which is protonated at nitrogens N-l and N-F exclusively. This was derived from comparison of the chemical shifts of nitrogens N-15, N-l and N-F of the free base and the salt, observing solely a strong change in the chemical shifts of N-l and N-F (Table 1).

[0152] Table 1: Comparison of15N-NMR chemical shifts of HZ-02 and HZ-02- 2HC1

[0153] Methods

[0154] JH, proton-decoupled13C and15N nuclear magnetic resonance (NMR) spectra were recorded at 25 °C at 500, 125.7 and 50.7 MHz, respectively, in a Bruker Avance Neo 500 spectrometer.1H and13C chemical shifts are reported in parts per million (ppm) relative to the residual solvent peak (DMSO-de:1H: d 2.49 ppm,13C: <5 39.5 ppm, D2O:1H: d 4.79 ppm). For13C spectra recorded in D2O, EtOH was used as standard (<5 56.9 ppm and 17.3 ppm).15N chemical shifts are reported in ppm relative to NO2CH3 which was used as standard.Assignments of1H and13C were determined by analysis of coupling constants and assisted by 2D1H COSY, 2D1H TOCSY, 2DJH-13C HSQC, 2DJH-13C HMBC experiments using standard pulse sequences. Assignments of15N were extracted from 2DJH-15N HSQC and 2DJH-15N HMBC experiments.

[0155] Observation: chemical shift values may vary depending on the concentration of the sample, likely due to changes in non-covalent interactions between molecules.

[0156] Argentometric determination

[0157] To experimentally confirm the molecular formula of the hydrochloride salt HZ-02-2HC1, an argentometric titration of the chlorides present in the molecule was performed using the Fajans method. This determination included the titration of the chlorides in the sample and in this way the degree of protonation of the compound and its molecular mass were indirectly determined. A molecular weight of (381.77 ± 0.4) g / mol was determined (calculated MW = 381.69 g / mol).

[0158] Methods

[0159] A determination of the chlorides present in HZ-02-2HC1 was performed by argentometry using the Fajans method. A solution of dichlorofluorescein 0.1% in EtOH was used as an adsorption indicator. The titration solution of 0.05 M AgNOs was previously valorated using analytical grade NaCl 99.99%. 60 mg of hydrochloride were weighed in a 125 mL Erlenmeyer flask and dissolved in 30 mL of milliQ H2O. Excess of AcONa (100 mg) was added to release the chloride ions. The free base precipitated which did not perturb the determination. Two drops of indicator were added and then the titration solution was added from a 25 mL burette. The molecular weight of the hydrochloride was calculated using the eq 1.MW HZ02.2HCI =2xmHZ2 2HCl(Eq 1) mAgNO3vAgNO3

[0160] Infrared spectroscopy

[0161] Infrared spectra (FIG. 23) were acquired in a Jasco 4700 FTIR spectrometer using the potassium bromide (KBr) pellet method. 32 scans were recorded per sample in the wavenumber range of 4000-400 cm'1.

[0162] Acid-constant (pKa) potentiometric determination

[0163] Regarding pharmaceutical salt formation, the pKa rule states that a pKa difference of ApKa > 4 should exist between the free base and the acid (in this case, HC1) to consider the product a salt, and ApKa < 1 to consider it a cocrystal.

[0164] A potentiometric titration was performed in order to determine the acid constants (pKa) of the molecule. Three different pKa values (pKal 2.56, pKa2 4.03, pKa3 11.17) were derived from the titration using the second derivative method (FIGS. 24A and 24B). The pKa difference between the lowest pKa in HZ-02-2HC1 (pKal = 2.56) and HC1 (pKa = -6) is greater than 4 units, which confirm the product as a salt.

[0165] Methods

[0166] A potentiometric titration was performed to determine acidic constant (pKa) values of HZ-02-2HC1. A 0.05 M NaOH solution was prepared and valorated with oxalic acid using phenolphthalein as indicator. A pHmeter was previously calibrated using standard solutions of pH 4.00, 7.00 and 10.00. An amount of 100 mg of the hydrochloride HZ-02-2HC1 was weighed in a 100 mL Erlenmeyer flask and dissolved with milliQ H2O. Magnetic stirring was maintained during the whole process. The initial pH of the solution was registered and the titrant was added from a burette. After each addition of titrant, the pH lecture was registered letting it stabilize for at least 30 seconds. The pKa values were determined using the second- derivative (A2pH / AV2) method (see Qiang, Z. et al., Water Research 38:2874-2890 (2004)).

[0167] Solubility

[0168] The dihydrochloride salt HZ-02-2HC1 showed a much higher solubility than the free base, which is essentially insoluble. A solubility value of (45.3 ± 1.2) mg / ml was determined at 25 °C.

[0169] Methods

[0170] Solubility in water was determined as follows. UV spectrum of HZ-02-2HC1 was recorded with a PheraStar microplate reader. A calibration curve was constructed with stock solutions of different concentrations ranging 0.5-125 pg / mL by measuring absorbance at Amax = 390 nm. 12 mg of HZ-02-2HC1 were weighed in a 1.5 mL microcentrifuge tube and 200 pL of milliQ water were added. The mixture was vortexed for 10 min and centrifuged at 13000 rpm for 10 min. 100 pL of supernatant were transferred to another tube and diluted appropriately. The determination was performed by triplicate.

[0171] Time-dependent solubility

[0172] To determine whether HZ-02-2HC1 increases its solubility in time, a timedependent determination was performed. From FIG. 25 it can be seen that the compound progressively increases its solubility in water at 37 °C until it reaches a maximum and slightly decreases at the end of the experiment (3 h). It was noticed that following the experiment some amount of the excess solid no longer solubilized in water which was likely related to a loss of HC1 after long periods at 37 °C in the super- saturation condition.

[0173] Methods

[0174] The time-dependent solubility of the HZ-02-2HC1 hydrochloride salt was measured in water at 37 °C. 50 mg of the salt was weighed in a 5 mL tube and 1 mL milliQH2O was added. The tube was kept at 37 °C under magnetic stirring. Aliquots of 50 pL weretaken out at 10, 20, 40, 60, 80, 90, 120, 150 and 180 min and centrifuged at 13000 rpm. Appropriately dilutions of the supernatants were performed and absorbance was determined in a 96-well plate. Solubility values were derived from a calibration curve as described in the Solubility determination. Triplicates were performed.

[0175] Chemical Stability

[0176] To check the chemical stability of the hydrochloride salt HZ-02-2HC1 the crystalline solid, lyophilized solid, and solution were kept for one month at 37 °C. FIG. 26 shows that following one week, low intensity signals for decomposition products start to appear for the compound in solution and slightly for the crystalline and lyophilized solid forms. To quantify the stability, qNMR assays were performed using dimethylsulfone (DMSO2) as an internal standard. After 4 weeks, 92%, 92% and 97% of HZ-02-2HC1 remains stable in solution, crystalline and lyophilized solid, respectively (FIG. 27).

[0177] Methods

[0178] Chemical stability of the hydrochloride salt HZ-02-2HC1 was assessed by qNMR spectroscopy for the vacuum oven dried solid, lyophilized solid and aqueous solution.Briefly, several vials containing the weighed amounts of vacuum oven dried solid, lyophilized powder or D2O solution (500 pL) of HZ-02-2HC1 were stored at 37 °C for four weeks. Weekly, each sample was dissolved in 500 pL of D2O, 100 pL of a stock solution of DMSO2 in D2O was added and qNMR spectra were recorded in a Bruker Avance Neo 500 spectrometer (TD = 64k, DI = 30 s, NS = 16). The chemical stability was determined by plotting the percentage abundance of HZ-02-2HC1 versus time. The percentage abundance ofHZ-02-2HC1 was calculated using Eq 2.

[0179] Hygroscopicity

[0180] The hygroscopicity of a drug is determinant for its chemical stability and so it was aimed to determine whether HZ-02-2HC1 is hygroscopic or not. The amount of moisture absorbed by a compound may vary depending on the morphology of the solid, i.e. its surface area, its stability and also on atmospheric conditions. The European Pharmacopoeia classifies hygroscopicity into four classes ordered by the percentage increase in weight after 24 h exposure of the compound to an 80% relative humidity (RH) atmosphere. Non Hygroscopic compounds belong to Class 1 (<0.2 %w / w), slightly hygroscopic compounds belong to Class 2 (0.2-2 %w / w), hygroscopic compounds belong to Class 3 (2-15 %w / w), and very hygroscopic compounds belong to Class 4 (>15 %w / w).

[0181] The hygroscopicity determination of HZ-02-2HC1 was assessed both for its lyophilized form and its crystalline form. The lyophilized solid showed 2.8 % w / w after exposing it to 80% RH for 24 h, which means that this form can be classified as hygroscopic (Class 3). On the other hand, the increase in weight for the crystalline form of HZ-02-2HC1 was 16.1 % w / w leading this solid form to the Class 4, very hygroscopic.

[0182] This difference in hygroscopicity for both forms of the same compound was also noticeable macroscopically. While the lyophilized solid retained its orange color and flowability, the crystalline form turned yellow and remained stuck to the bottom of the vial after the experiment.

[0183] Methods

[0184] 100 mg of HZ-02-2HC1 (vacuum oven dried crystalline solid or lyophilized) was weighed using a microbalance. The vial containing the sample was placed into a desiccator under 80% relative humidity (generated with saturated ammonium chloride aqueous solution) at 25 °C. A hygrometer was placed into the desiccator as well to ensure maintenance of the correct humidity atmosphere. The samples were weighed after 24 h and the percentage mass increase was calculated.

[0185] Elemental analysis

[0186] CHNS elemental analysis was performed on Sample A and compared to a sulfanilic acid standard.

[0187] Nitrogen (N): 15.5% in A.

[0188] Carbon (C): 56.7%.

[0189] Hydrogen (H): 4.3%.

[0190] Sulfur (S): Not detected.

[0191] These proportions were consistent with the proposed molecular formulas for HZ- 02-2HC1: C17H14CI2N4, C17H15CI3N4 with some small amount (not a whole molecule) of water.

[0192] Methods

[0193] Elemental analysis was performed in a Carlo Erba EA 1108 apparatus previously calibrated with a sulfanilic acid standard.

[0194] Differential scanning calorimetry

[0195] As can be seen in FIG. 28, several endothermic processes were identified between 30 and 130 °C with a maximum peak at 73 °C. An intense exothermic process was observed at around 240 °C.

[0196] Methods

[0197] Differential scanning calorimetry (DSC) curves were acquired with a Shimadzu DSC-50 apparatus. The study was carried out under N2 atmosphere (constant flow rate of 30 mL / min) in temperatures ranging room temperature to 250 °C with a heating gradient of 10 °C / min. A mass of 1.06 mg of HZ-02-2HC1 was weighed in an aluminum capsule. The apparatus was previously calibrated using In and Zn standards.

[0198] Thermogravimetric analysis

[0199] As can be seen in FIG. 29, a mass loss of 13% between 50 and 206 °C was registered, which suggests that an elimination of hydration or adsorbed water and / or the loss of HC1 occurs. A second mass loss of 79% occurs between 223 and 357 °C, and a third one of 7% occurs between 460 and 750 °C.

[0200] Methods

[0201] Thermogravimetric analysis (TGA) was performed in a TGA 51H-SHIMADZU thermobalance operating under N2 atmosphere (constant flow rate of 30 mL / min). A mass of 9.57 mg of HZ-02-2HC1 was weighed in a platinum capsule. The measurements were performed in temperatures ranging room temperature to 950 °C with a heating gradient of 10 °C / min.

[0202] Powder X-ray diffraction

[0203] The powder X-ray diffraction (PXRD) analysis showed differences between different solid forms of HZ-02-2HC1. As can be seen in FIG. 30, the crystalline powder showed high crystallinity with narrow, sharp and intense signals. On the other hand, the monocrystal of HZ-02-2HC1 also showed a high degree of crystallinity but a different pattern in its diffractogram, suggesting the presence of different polymorphs / solvates of the same compound.

[0204] Methods

[0205] Powder X-ray diffraction (PXRD) patterns were obtained in a Panalytical Empyrean powder diffractometer (Malvern Panalytical Ltd., Malvern, UK) using a Cu Ka radiation source. The study was carried out registering over the 20 range of 4-50° with a step of 0.02° and a counting time of 200 s.

[0206] Single crystal X-ray diffraction

[0207] As can be seen in FIG. 31, the results of the monocrystal XRD confirm that the molecule crystallizes as a methanol solvate of the hydrochloride, and that protonation occurred at both pyridinic nitrogens N-l and N-T. The stabilization of the dictation is observed through N-H—Cl and C-H—Cl electrostatic intermolecular bonds between the chlorides and the pyridinium, the hydrazone NH and C-H groups. Intermolecular bonds O- H—Cl and C-H—O can also be observed between the solvent, chloride C12 and the molecule. The structure of the molecule unambiguously coincides with the E isomer of HZ-02-2HC1.

[0208] Methods

[0209] Monocrystal samples of HZ-02- 2HC1 were obtained as follows. In a vial, 8 mg ofHZ-02- 2HC1 was dissolved in a mixture of methanol / isopropanol and then cyclohexane was slowly added and let it stand until the appearance of crystals as small yellow needles.

[0210] As can be seen in FIGS. 32A-32C, a monocrystal of 68.4 pm x 265.1 pm was analyzed with a Gemini E (Oxford Diffraction-Rigaku) monocrystal diffractometer equipped with a CCD detector, operated with Mo (Kai = 0.71 A) radiation. The collected images were analyzed with Micrometrics SE Premium 4 and CrysAlisPRO 171.41_64.95al software. Data reduction and absorption correction were done with a multiscan routine in CrysAlisPRO. The structure was solved using Olex2 and SHELXT3 softwares using intrinsic phases and refined with SHELXL3 using minimum square minimizations. Mercury was used for visualization of the structure.

[0211] Example 2

[0212] In this study, the regenerative potential of HZ-02 in mice and rats, and in murine models of hepatectomy was investigated. The effects of HZ-02 on liver mass recovery, hepatocyte proliferation, and histoarchitecture preservation were assessed. Additionally, the safety profile of HZ-02 was evaluated through preliminary toxicology analysis. Furthermore, considering the clinical relevance, the selective antiproliferative effect of HZ-02 on liver tumor cells was examined while preserving normal hepatocytes. Finally, the regenerative effects of HZ-02 were compared with another histone demethylase JMJD2 inhibitor, SD-70, in the context of liver hypertrophy, as well as with other JMJD members.

[0213] To provide a comprehensive understanding of the regenerative potential of HZ-02, histological analyses, immunohistochemistry, and transcriptome profiling were performed to unravel the molecular mechanisms underlying the observed regenerative effects. The findingsfrom this study may contribute to the development of innovative therapeutic strategies for liver regeneration and offer potential applications in the treatment of liver diseases and postresection liver failure.

[0214] Material and Methods

[0215] Animals

[0216] BALB / c,C57BL / 6J or C3H / HeJ mice (six to eight weeks) or Wistar Rats, were housed at our Animal Resources Facilities in compliance with the guidelines of the experimental ethical committee and the NIH regulations concerning the ethical treatment of animals. The experimental protocol was approved by the Animal Care Committee at the School of Biomedical Sciences, Universidad Austral, and adhered to the essential principles outlined in the ARRIVE guidelines.

[0217] Mice and Rat models of liver hypertrophy induction

[0218] Mice were treated with a total of 5 doses every other day (eod) of HZ-02 (E isomer), Z isomer, the (E)-5-chloro-2-(2-(phenyl(pyridin-2- yl)methylene)hydrazineyl)pyridine dihydrochloride HZ-02 salt and the compounds listed in the table I. Mice were euthanized 48 h after the last dose and liver weighted.

[0219] Animal models of sub-lethal and lethal hepatectomy

[0220] Sub-lethal hepatectomy was performed by removing 66% of the liver, following the protocol described by Mitchell et al., “A reproducible and well -tolerated method for 2 / 3 partial hepatectomy in mice,” Nat. Protoc. 3, 1167-1170 (2008). Briefly, mice wereanesthetized with isoflurane inhalation, a midline abdominal incision was made to expose the liver, and the right and left lobes were removed after ligating their hepatic artery branches.

[0221] Lethal hepatectomy was performed by removing 85% of the liver, as described by Hori et al., “Simple and sure methodology for massive hepatectomy in the mouse,” Ann. Gastroenterol. Hepatol. 24, 307-318 (2011). In addition to the right and left lobes, the right posterior and omental lobes were ligated and subsequently removed.

[0222] Animal model of orthotopic HCC

[0223] Six-to-eight-week-old male C3H / HeJ were utilized in the study. To induce fibrosis, thioacetamide (TAA) was administered intraperitoneally (i.p.) at a dosage of 200 mg / kg (Sigma- Aldrich, St. Louis) three times per week for a duration of 42 days. On day 30, orthotopic tumors were established by subcapsular inoculation of 1.25 x 105Hepal29 cells into the left liver lobe via laparotomy. Seven days after tumor implantation, a group of mice received HZ-02 or SD-70 (at 10 mg / kg / dose) or vehicle by intraperitoneal injection in a 12.5% Cremophor EL and 12.5% DMSO aqueous suspension, administered three times per week for a period of two weeks. Twelve days after tumor implantation, the mice were euthanized, and the tumor volume (in mm3) was calculated using the formula 7t / 6 x larger diameter x (smaller diameter)2based on caliper measurements.

[0224] Liver histopathology

[0225] Liver samples were fixed in 4% formalin for 48 h and embedded in paraffin to prepare 5 pm sections. Paraffin sections were deparaffinized and rehydrated, and then used for hematoxylin and eosin (H&E) staining or PCNA immunostaining.

[0226] PCNA immuno staining

[0227] To block the endogenous peroxidase activity, the preparations were incubated with3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed using pH 6.0 citrate buffer by heating the samples in a microwave. Avidin and endogenous biotin blocking was achieved by overnight incubation at 4°C with the respective blocking solutions (Vector Laboratories, Inc., USA). Monoclonal anti-PCNA antibody (1 / 100; Santa Cruz Biotechnology, USA) was used. Following washing, the slides were incubated at room temperature for 2 hours with biotinylated goat anti-mouse secondary antibodies (1 / 100, Vector Laboratories, Inc., USA). After additional washing, the samples were incubated with an avidin-peroxidase enzyme conjugate (Extravidin-peroxidase, Sigma- Aldrich, MO, USA) diluted 1: 100 in PBS for 30 minutes at room temperature. Subsequently, the samples were washed in PBS and 0.1 M sodium acetate. Staining was performed using a mixture of 0.1% diaminobenzidine in distilled water and a solution of 5% ammonium-nickel sulfate, 0.08% C1NH4, and 0.4% glucose in 0.2 M ammonium acetate. Finally, the samples were washed in 0.1 M acetate and PBS, the tissue was dehydrated through sequential passages of 70%, 96%, and 100% alcohol, as well as xylene, and mounted with Canada balsam. In the control samples, incubation with primary antibodies was omitted.

[0228] Isolation of primary hepatocytes

[0229] Livers were perfused through the portal vein and digested with type I collagenase (Sigma-Aldrich, St. Louis, USA). Then, hepatocytes were isolated by a 30 min decantation followed by two steps of centrifugation (50 g for 10 min) and plated onto 6-well plates at a density of 200.000 living cells per well in DMEM supplemented with 2 pM glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin and 10% heat- inactivated fetal bovine serum (FBS).

[0230] Tumoral hepatic cell lines

[0231] HepG2 and BNL cells were maintained in supplemented DMEM media as described previously. Cell lines were confirmed to be mycoplasma-free using e-Myco kit (Boca Scientific). All cells were cultured at 37 °C in a 5% CO2 atmosphere.

[0232] Viability assay

[0233] For cell viability assays, cells were plated at 500-2000 cells / well in 96 well plates and treated the next day with increasing doses of HZ-02. Standard MTT assays were performed after 4 days of treatment. Dose-response curves were plotted using a non- linear regression model and IC50 values were determined from the fitted curves using GraphPad prism.

[0234] Toxicology analysis and liver transaminases quantification

[0235] Healthy C57BL / 6J were treated 3 times per week for 2 weeks by intraperitoneal (i.p.) injection with 50 mg / kg body weight (n=6) per day of HZ-02 or vehicle (n=6) in 12.5% Cremophor EL, 12.5% DMSO. One day after the last dose, mice were sacrificed and blood samples collected for serum quantification of biochemical studies.

[0236] RNA-Seq

[0237] For RNA-Seq analysis 2xl06HuH7 cells were seeded in pl50 plates. After 24 h, cells were treated with HZ-02, SD-70 or DMSO for 24 h and total RNA was extracted using RNeasy Plus Mini kit (Qiagen), with a gDNA elimination step. RNA quality check was performed using the Agilent 2100 Bioanalyzer to ensure that only high-quality RNA was used (RIN Score 8 or higher). The Qubit fluorometer (Invitrogen) was used to determine RNA concentration prior to library preparation with the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina). Samples were run on the Illumina HiSeq 2500, at the McDermottSequencing Core at UT Southwestern. For RNA-Seq analysis, TopHat was used for transcript assembly, and the edge R package from Bioconductor software was used for differential expression calling and calculation of Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Genes were considered differentially expressed when log2 (fold change) was greater than 0.3 or lower than -0.3 with a false discovery rate (FDR). Gene ontology and pathway analysis were done using the ToppGene.

[0238] Statistical analyses

[0239] Data are reported as arithmetic means ± SEM. Statistical analysis was performed using PRISM V.8.0 software (GraphPad, Carlsbad, USA). Statistical significance was determined with the appropriate test. Graphs were prepared in Prism (GraphPad, USA) and figures compiled in Adobe Photoshop (Adobe Systems, USA).

[0240] Results

[0241] HZ-02 but not the inactive isomer Z induce liver hypertrophy.

[0242] To assess the induction of liver hypertrophy by HZ-02 (E isomer), mice were treated with a total of 5 doses (50 mg / kg) administered every other day (eod) of HZ-02. Vehicle and an inactive HZ-02 isomer (Z Isomer) were used as controls. As observed in FIG.1, HZ-02 but not the vehicle or the Z isomer is able to induce liver hypertrophy.

[0243] To study whether the salt of HZ-02 also induces liver hypertrophy we compared the salt of HZ-02 administered intravenously (i.v.) with the E isomer administered intraperitoneally (i.p.) (FIG. 2).

[0244] To study whether the administration route could affect the result HZ-02 was compared by 3 different routes: intraperitoneally (i.p.), intravenously (i.v.) and by gavage. Remarkably, HZ-02 pro-hypertrophy effect was conserved either if the compound is administered by i.v. or by gavage (FIG. 3).

[0245] To validate if the observed effect replicates in another species, the effect of a total of 5 doses (administered eod, 25 mg / kg) of HZ-02 in Wistar rats were compared. As expected, HZ-02 was also able to induce liver hypertrophy in rats (FIGS. 4 A and 4B).

[0246] A set ofJMJD inhibitors induce liver hypertrophy.

[0247] It was demonstrated herein that the Jumonji demetilase pan-inhibitor HZ-02 has the ability to induce liver hypertrophy. Next, whether other Jumonji inhibitors could have similar effect (Table 2) was questioned. In addition, inhibitors of HIF prolylhydroxylases (PHDs), another subfamily of a-ketoglutarate oxygenases, were also tested (Table 2).

[0248] Table 2: List of Compounds TestedAbbreviations: KDM = Lysine demethylase.

[0249] Remarkably, treatment with either IOX4, ML324, TC-E 5002 or CPI-455 (5 doses eod) showed liver hypertrophy induction (FIG. 5).

[0250] Effect of EZ-02 on liver mass recovery and regeneration after partial hepatectomy

[0251] To assess the regenerative effect of HZ-02 on liver regeneration after sub-lethal hepatectomy, C57BL / c mice were pre-treated with three doses of 10, 25, or 50 mg / kg body weight of HZ-02 according to the schedule shown in FIG. 6A before undergoing surgery. Three days after the last dose, the surgery was performed, and two days later, the treatment was resumed with two additional doses of HZ-02. After 7 days, the animals were euthanized, and the liver index was evaluated (FIG. 6A).

[0252] As depicted in FIG. 6B, HZ-02 treatment resulted in a dose-dependent increase in the hepatic index. Notably, the highest dose led to a doubling of liver mass compared to the control group (FIG. 6C).

[0253] The histology of mice liver sections was analyzed at the time of surgery and at the time of sacrifice after administration of 3 or 5 doses of 10 mg / kg body weight of HZ-02, respectively. FIG. 7 demonstrates that not only was the histoarchitecture preserved, but also hepatocyte hyperplasia was observed in the treated mice at both time points.

[0254] In addition, to assess whether HZ-02 induces hepatocyte proliferation, it was decided to analyze the expression levels of the proliferation cell marker PCNA in liver tissue. As shown in FIG. 8, a significant increase in PCNA-positive cells was found at the time of surgery after three doses of HZ-02 (10 mg / kg body weight) compared to the vehicle group. However, at the time of sacrifice, PCNA-positive cells were at the same levels as the vehiclegroup. These results indicate that pre-treatment with HZ-02 induces an enhanced proliferative response and promotes liver regeneration at the time of surgery.

[0255] It was then hypothesized whether HZ-02 could enhance liver mass recovery after surgery decreasing the time to reach the liver size as it happens when partial hepatectomy is performed. To assess this, mice were treated with a total of 3 doses administered eod of HZ- 02 and then performed sub-lethal hepatectomy was performed. As observed in FIG. 9, 72 h after surgery HZ-02 treated mice almost reached the liver size of a healthy mice, while vehicle-treated mice were still recovering.

[0256] To determine if the recovery of the liver mass is accompanied by a preserved liver function, prothrombin time (PT) was measured. Remarkably, it was found herein that treated mice have a PT similar to those observed in healthy controls (FIG. 10).

[0257] Effects of HZ-02 on liver mass recovery after lethal hepatectomy

[0258] To evaluate the regenerative potential of HZ-02 in cases of excessive and lethal liver resection, mice were pre-treated with a total of 3 doses of 50 mg / kg body weight or the vehicle before undergoing an 85% hepatectomy (as shown in FIG. 11A). Three days after the final dose, surgery was performed, and animal survival was monitored for 5 days. Remarkably, all animals survived at the five-day mark. Furthermore, the liver mass of the HZ-02 treated mice was significantly higher in comparison with control group, hepatic index of 4.6 vs. 3.7, respectively (FIG. 11B). Additionally, the HZ-02 treated mice experienced a body weight loss of 10%, while the control group exhibited a weight loss of 20%.

[0259] Preliminary toxicology in healthy mice treated with HZ-02

[0260] HZ-02 (50 mg / kg) or vehicle was intraperitoneally injected into healthy C57BL6 mice for a period of 2 weeks, resulting in a total of 6 doses. One day after the final dose, animals were sacrificed, and blood samples were collected to analyze biochemical markers of liver and kidney function. As depicted in FIGS. 12A and 12B, no significant differences were observed in the biochemical analysis between HZ-02-treated mice and control group.

[0261] HZ-02 induces liver hypertrophy in tumor-bearing mice.

[0262] Considering that the use HZ-02 for the prevention of post-resection liver failure (small for size syndrome) in the context of hepatic cancer (primary or secondary) was proposed herein, the sensitivity of normal hepatocytes and HCC cells (Hepal29 and BNL) to HZ-02 was evaluated first. Remarkably, HZ-02 treatment showed a selective antiproliferative effect on cancer cells while preserving normal hepatocytes (FIG. 13).

[0263] Next, tests to assess the tumor and liver size after HZ-02 treatment (FIGS. 14A and 14B) and SD-70 treatment (FIGS. 15A and 15B) in vivo were conducted.

[0264] To further investigate the potential mechanisms involved in the liver growth induced by HZ-02, a transcriptome analysis was conducted in transformed hepatocytes using the HuH7 cell line. The analysis revealed upregulated genes by HZ-02, and gene ontology analysis (FIG. 16) of these genes demonstrated terms such as “vascular development”, “response to hypoxia,” "Wound Healing," "Regeneration," "Liver development," "Embryonic liver development," and "Regulation of cell size." These results indicate that HZ-02 has the ability to induce the expression of genes associated with the observed phenotype in the mice treated with HZ-02. A similar outcome was observed with SD-70 (FIG. 17).

[0265] Targeted Population

[0266] Patients with primary liver cancer or metastases that cannot be treated with curative resection due to the limited size of the remaining liver.

[0267] Patients experiencing small-for-size syndrome or post-hepatectomy liver failure.

[0268] Healthy patients who are suitable candidates for living donation when the size of the liver remnant is less than 35% of the initial volume.

[0269] Ex vivo liver perfusion in order to induce liver regeneration in marginal livers, or to increase the use of donation after circulation death liver grafts.

[0270] Example 3

[0271] Pharmaceutical cocrystals are multicomponent crystalline solids formed by the interaction of an active pharmaceutical ingredient (API) with a cocrystal former via non- covalent forces, typically hydrogen bonds. Cocrystallization has emerged as a versatile approach in pharmaceutical development for modifying the physicochemical properties of APIs without changing their pharmacological activity. Key attributes that can be improved through cocrystal formation include solubility, dissolution rate, hygroscopicity, mechanical properties, and thermal stability. Dicarboxylic acids such as succinic acid and adipic acid are among the most commonly employed cocrystal formers due to their GRAS (Generally Recognized As Safe) status, hydrogen-bonding capacity, and compatibility with multiple APIs. Notable examples include:• Carbamazepine-succinic acid cocrystals, which improve moisture stability and flowability.• Meloxicam-succinic acid cocrystals, enhancing dissolution rate and bioavailability.• Caffeine-adipic acid cocrystals, improving compressibility and processing.

[0272] The formation of cocrystals of HZ02-2HC1, a highly hygroscopic API, with succinic acid and adipic acid, was studied to improve its solid-state stability. Lyophilization was also explored using PVP40 as a stabilizing excipient.

[0273] Materials and Methods

[0274] Cocrystal formation

[0275] In a crystallizer, HZ-02-2HC1 and succinic acid (SUA) were combined in a 2: 1 molar ratio and dissolved in 1.8 mL of methanol. The solution was sonicated for 1 minute and left to evaporate at room temperature for 4 days to yield cocrystals (HZ-02-2HC1-SUA).

[0276] The same method was applied for adipic acid (ADA) to yield HZ-02-2HC1-ADA.

[0277] Lyophilization with PVP40

[0278] A solution containing 150 mg of HZ-02-2HC1 and 47.5 mg of PVP40 in 15 mL of Milli-Q water was frozen at -80 °C for 1 hour. The system was then subjected to the following lyophilization steps:• 0.05 mBar at -80 °C for 2 hours• 0.05 mBar at 10 °C for 30 hours• Lowest achievable pressure for 39 hours

[0279] Hygroscopicity Assessment

[0280] Hygroscopicity was determined by measuring the mass change of a vacuum-dried sample after exposure to 80% relative humidity at 25 °C for 24 hours.

[0281] Accelerated Stability Testing

[0282] Samples were exposed to 75% RH at 60 °C for 1, 3, and 5 days. Quantification of the remaining compound was performed using quantitativeJH NMR (qNMR):• For lyophilized salt + PVP40: Maleic acid as internal standard, D2O as solvent• For cocrystals: Dimethyl sulfone as internal standard

[0283] Spectroscopic Characterization of Cocrystals

[0284] Cocrystals with succinic acid were characterized byJH NMR at 500 MHz in D2O and (FIG. 33) FT-IR using KBr as the attenuating matrix (FIGS. 34A-34B). Cocrystals with adipic acid were characterized solely by FT-IR.

[0285] FIG. 33 is a graphical representation showing a1H NMR spectrum (500 MHz) of the HZ-02-2HC1-SUA cocrystal in D2O. 'H NMR (500 MHz, D2O) 5 8.22 (dd, J = 5.8, 1.0 Hz, 1H), 7.88 (td, J = 8.0, 1.5 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.46 (dd, J = 9.3, 2.4 Hz, 1H), 7.43 - 7.40 (m, 1H), 7.12 (d, J = 8.1 Hz, 1H), 7.11 - 7.06 (m, 3H), 6.89 - 6.87 (m, 2H), 6.66 (d, J = 9.3 Hz, 1H), 2.01 (s, 2.5H).

[0286] FIG. 34A shows the FT-IR spectra of the obtained cocrystals alongside the spectra of the individual components. Notably, the spectrum corresponding to the HZ-02-2HC1-SUA co-crystal presents a band at 3632 cm-1, which is absent in the spectra of the starting materials. Additionally, several characteristic bands are shifted, supporting the formation of a new solid phase.• FT-IR HZ-02- 2HC1-SUA (cm1): 3632.27, 3444.24, 3376.26, 3039.75, 1715.85, 1649.8, 1619.91, 1606.9, 1583.75, 1560.13, 1455.03, 1265.07• FT-IR SUA (cm1): 3043.12, 2931.75, 2652.12, 2537.86, 1730.32, 1698.98,1420.8, 1310.88, 1201.43, 1177.33, 917.47, 893.84, 802.72, 637.84, 583.36, 546.72• FT-IR HZ-02 2HC1 (cm1): 3426.89, 3363.25, 3056.62, 2969.84, 1631.48, 1617.5, 1597.25, 1559.65, 1533.61, 1503.24, 1473.83, 1451.65, 1427.55• FT-IR HZ-02- 2HC1-ADA (cm1): 3483.78, 3421.58, 3321.78, 3302.02, 3050.35, 2951.04, 1697.05, 1636.79, 1613.64• FT-IR ADA (cm1): 3033.48, 2962.13, 2918.73, 2877.27, 2671.41, 1695.6,1463.22, 1428.51, 1408.26, 1402.57

[0287] Results

[0288] Hygroscopicity

[0289] The following table (Table 3) summarizes the percentage of mass gain observed after exposure to 80% relative humidity at 25 °C for 24 hours. This test was used to evaluate the hygroscopic behavior of the different solid forms of HZ-02- 2HC1.

[0290] Table 3

[0291] The native HZ-02-2HC1 salt exhibited a high hygroscopicity value of 16.1%, confirming its sensitivity to ambient humidity and highlighting the need for solid-state stabilization strategies. Among the evaluated cocrystals, the HZ-02-2HCl-succinic acid cocrystal (SUA) showed the lowest hygroscopicity, with only 1.4% mass gain under test conditions. This indicates that cocrystallization with succinic acid effectively reduces moisture uptake. The adipic acid cocrystal (ADA) also provided an improvement, decreasing hygroscopicity to 7.5%. Based on established classification criteria, compounds with <2% water uptake are categorized as slightly hygroscopic (Class II), whereas values above 10% correspond to very hygroscopic materials (Class IV). Accordingly, the SUA cocrystal is classified as Class II and represents a significant improvement in the physicochemical stability of HZ-02-2HC1, making it a promising candidate for long-term storage and pharmaceutical development.

[0292] Accelerated Stability

[0293] The chemical stability of the API under accelerated stress conditions was assessed by storing samples at 75% relative humidity and 60 °C for 1, 3, and 5 days. The percentage of remaining HZ-02-2HC1 was quantified by quantitativeJH NMR (qNMR), using maleic acid or dimethyl sulfone as internal standards depending on the sample type. The results are summarized below in Table 4.

[0294] Table 4

[0295] All formulations demonstrated high initial stability after one day of exposure to accelerated conditions. By the third day, a gradual decline in the amount of intact compound was observed in both the lyophilized salt (PVP40) and the native HZ-02- 2HC1, with approximately 94% and 93% of the API remaining, respectively. In contrast, the succinic acid cocrystal (SUA) retained 98.5% of the compound, showing superior resistance to degradation. After five days, differences in stability became more pronounced. The PVP40 formulation exhibited a substantial decrease to 74% remaining, accompanied by high variability, indicating lower robustness under prolonged stress. The native salt showed moderate degradation, with 95% of the compound remaining. In comparison, the SUA cocrystal maintained excellent stability, with 98.1% of the API preserved and minimal variability (±0.3%).

[0296] These results clearly demonstrate that cocrystallization with succinic acid significantly enhances the chemical stability of HZ-02- 2HC1, effectively protecting it from degradation under harsh conditions of elevated temperature and humidity. Consequently, the SUA cocrystal emerges as a highly stable solid form, suitable for long-term storage and continued pharmaceutical development.

[0297] Example 4

[0298] To assess whether the cocrystal of HZ-02 dihydrochloride salt and succinic acid (2: 1 molar ratio) induces liver hypertrophy, the cocrystal was administered intraperitoneally (i.p.) at a dose of 40 mg / kg, corresponding to 25 mg / kg of HZ-02, once daily for four consecutive days. Animals were sacrificed on the fifth day for tissue analysis. Results are shown in FIG. 35.

Claims

CLAIMS1. A method for in vivo liver regeneration in a subject in need thereof, comprising: administering to the subject a compound of formula (I):or a pharmaceutically acceptable salt thereof, or a co-crystal thereof.

2. The method of claim 1, wherein the subject is a mammal.

3. The method of claim 1 or 2, wherein the subject is a human.

4. The method of any one of claims 1-3, wherein the subject has liver cancer or metastasis.

5. The method of any one of claims 1-3, wherein the subject has small- for-size syndrome.

6. The method of any one of claims 1-3, wherein the subject has post- hepatectomy liver failure.

7. The method of any one of claims 1-3, wherein the subject has undergone liver donation and has a remaining liver volume less than 35% of an initial liver volume.

8. The method of any one of claims 1-7, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, isadministered to the subject daily, every other day, every three days, every four days, every five days, every six days, weekly, bi-weekly, three times a month, or once a month.

9. The method of any one of claims 1-8, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is administered to the subject for at least one week, or two weeks, or three weeks, or four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or three months, or four months, or five months, or six months, or seven months, or eight months, or nine months, or ten months, or eleven months, or one year, or two years, or three years, or four years, or five years, or more than 5 years.

10. The method of any one of claims 1-9, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is administered to the subject in an amount of about 0.01 mg / kg to about 100 mg / kg per dose per day.

11. The method of any one of claims 1-10, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is administered to the subject orally, intravenously, intramuscularly, or subcutaneously.

12. The method of any one of claims 1-11, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is a dihydrochloride salt of the compound of formula (I).

13. The method of any one of claims 1-11, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is a cocrystal comprising a dihydrochloride salt of the compound of formula (I) and a co-crystal former selected from succinic acid and adipic acid.

14. A method for ex vivo liver regeneration, comprising:applying to an ex vivo liver graft a compound of formula (I):or a pharmaceutically acceptable salt thereof, or a co-crystal thereof.

15. The method of claim 14, wherein the ex vivo liver graft is from a mammal.

16. The method of claim 14 or 15, wherein the ex vivo liver graft is from a human.

17. The method of any one of claims 14-16, wherein the ex vivo liver graft is from donation after circulation death.

18. The method of any one of claims 14-17, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is a dihydrochloride salt of the compound of formula (I).

19. The method of any one of claims 14-17, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is a cocrystal comprising a dihydrochloride salt of the compound of formula (I) and a co-crystal former selected from succinic acid and adipic acid.

20. A method of treating a liver disorder for a subject in need thereof, comprising administering to the subject a compound of formula (I):or a pharmaceutically acceptable salt thereof, or a co-crystal thereof.

21. The method of claim 20, wherein the subject is a mammal.

22. The method of claim 20 or 21, wherein the subject is a human.

23. The method of any one of claims 20-22, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is administered to the subject daily, every other day, every three days, every four days, every five days, every six days, weekly, bi-weekly, three times a month, or once a month.

24. The method of any one of claims 20-23, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is administered to the subject for at least one week, or two weeks, or three weeks, or four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or three months, or four months, or five months, or six months, or seven months, or eight months, or nine months, or ten months, or eleven months, or one year, or two years, or three years, or four years, or five years, or more than 5 years.

25. The method of any one of claims 20-24, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is administered to the subject in an amount of about 0.01 mg / kg to about 100 mg / kg per dose per day.

26. The method of any one of claims 20-25, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is administered to the subject orally, intravenously, intramuscularly, or subcutaneously.

27. The method of any one of claims 20-26, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is a dihydrochloride salt of the compound of formula (I).

28. The method of any one of claims 20-27, wherein the compound of formula (I), or the pharmaceutically acceptable salt thereof, or the co-crystal thereof, is a cocrystal comprising a dihydrochloride salt of the compound of formula (I) and a co-crystal former selected from succinic acid and adipic acid.

29. A compound of formula (II):(II).

30. A co-crystal comprising the compound of formula (II) of claim 29 and a co-crystal former selected from succinic acid and adipic acid.

31. A pharmaceutical composition comprising the compound of formula (II) of claim 29.

32. A pharmaceutical composition comprising the co-crystal of claim 30.

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