Purine nucleoside phosphorylase inhibitors for treatment of metabolic syndrome and related conditions

Abstract

The present invention provides PNP inhibitors for the treatment and / or prevention of metabolic syndrome and their eliciting diseases and disorders. PNP inhibitors may also be used to treat and / or prevent diseases associated with decreased levels of nicotinamide dinucleotide (NAD +). Particular preferred salts for use in these methods and dosing regimens that avoid immunogenic side effects of these compounds are also disclosed.

Description

Field of the Invention

[0001] The present disclosure relates to the use of purine nucleoside phosphorylase (PNP) inhibitors, such as ulodesine and its salts, in treating and / or preventing metabolic syndrome and its induced diseases and conditions; and in treating or preventing diseases and conditions associated with decreased NAD+ metabolites. The present disclosure further identifies and characterizes preferred salt forms of ulodesine and pharmaceutical compositions for such treatment and prevention.

[0002] The present disclosure specifically describes the use of urodesine hemiglutarate and pharmaceutical compositions comprising the salt in the disclosed treatment and prevention.

[0003] Specifically, the inventors have discovered that treating subjects with PNP inhibitors, such as urodesine and its salts, results in a significant increase in both inosine and NAD+ in tissues. As further detailed herein, this result leads directly to potential treatment and / or prevention of a variety of diseases and conditions, including those associated with decreased tissue levels of the metabolite NAD+.

[0004] Thus, in various aspects, the present disclosure relates to pharmaceutical methods, compounds, and compositions for treating and / or preventing metabolic syndrome and metabolic syndrome-induced diseases. Background of the Invention

[0006] Metabolic syndrome is a group of conditions that occur together and increase the risk of heart disease, liver disease, stroke, and type 2 diabetes. This group of conditions includes high blood pressure, high blood sugar, excess body fat (especially around the waist), fatty liver, and abnormal cholesterol or triglyceride levels (dyslipidemia).

[0007] Recently, medications such as glucagon-like peptide 1 (GLP-1) agonists (usually delivered as an injectable protein) have shown some promise in addressing certain metabolic syndromes. These compounds act in the brain to suppress appetite, but weight regain is common once treatment is stopped. While actively using these drugs, patients experience nausea and reduce their food intake, resulting in approximately 15% weight loss. Lowering blood sugar and stimulating insulin secretion reduce energy expenditure, but after stopping such treatment, patients regain weight, likely because food intake increases and fat cells shrink but do not disappear.

[0008] There is now a large body of evidence that the role of brown adipose tissue (BAT) or the browning (conversion) of white adipose tissue (WAT) can prevent obesity (Harms M. and Seale P., Nature Medicine, 2013, 19:1252–1263; Bartelt A. and Heeren J., Nature Reviews Endocrinology, 2014, 10:24–36).

[0009] The molecular mechanisms responsible for the energy-dissipating properties of brown fat have been studied in detail (Cannon B. and Nedergaard J., Physiological Reviews, 2004, 84:277–3594; Rosen ED and Spiegelman BM, Cell, 2014, 156:20–44). Uncoupling protein-1 (UCP-1) has been identified as a key factor controlling the thermogenic capacity of brown adipocytes (Klingenberg, Journal of Bioenergetics and Biomembranes, 1999, 31:419–430). UCP-1 disrupts the electrochemical gradient across the mitochondrial membrane by allowing protons to reenter the mitochondrial matrix. As a result, mitochondrial fatty acid oxidation increases, and chemical energy is "wasted" through heat production (termed "adaptive thermogenesis"). This process was long believed to occur only in brown and beige adipocytes.

[0010] It has been previously reported that treatment of healthy subjects with PNP inhibitors results in elevated inosine levels (Viegas et al., J. Clin. Pharmacol, 2000, 40:410-420). Recently, it has been discovered that inosine (a purine nucleoside) stimulates energy expenditure in brown adipocytes via the cAMP / protein kinase A signaling pathway. Treatment of mice with inosine increases BAT-dependent energy expenditure and induces browning of white adipose tissue (also known as beige adipose tissue).

[0011] Daily treatment of diet-induced obese mice with inosine for 26 days demonstrated significant weight loss compared to vehicle. Furthermore, inosine is released during apoptosis in brown adipocytes and may have a "replacement me" signaling function, which can regulate thermogenic fat production and potentially combat obesity in mice (Niemann et al., Nature, 2022, 609:361-368).

[0012] Because the amount of metabolically active BAT appears to be particularly low in patients with obesity or diabetes, it is desirable to identify therapies that would increase BAT production and / or increase adaptive thermogenic capacity by activating BAT in these patients.

[0013] NAD+ levels continuously decline during aging. By middle age in mice or humans, NAD+ levels have fallen to half their youthful levels, leading to a loss of sirtuin (SIRT) activity. Recent studies have shown that treating aged mice with poly(ADP-ribose) polymerase (PARP) inhibitors (which increase NAD+ levels), NAD+ precursors, or inhibitors of NAD+ degradation can significantly improve health. Observed effects include improved insulin sensitivity, lower cholesterol and triglyceride levels, slower weight gain, slower stem cell aging, and extended lifespan (Bai et al., Cell Metab. 2011, 13:461–468; Gomes et al., Cell. 2013, 155:1624–1638; Yoshino et al., Cell Metab. 2011, 14:528–536; Zhang et al., Science. 2016, 26:2016). Improvements in these metabolic parameters essentially lead to "healthy longevity," an effect that has been demonstrated with NAD+ precursors and inhibitors of NAD+ degradation, such as CD38 (Peclat et al., Aging Cell. 2022, 21: e13589). Genetic mouse models with altered NAD+ biosynthesis phenotypes have shown similar results.

[0014] Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, encompassing a spectrum of conditions ranging from simple steatosis, in which lipids accumulate in the liver, to non-alcoholic steatohepatitis (NASH), characterized by fibrosis, inflammation, and liver cell death. If left uncontrolled, NASH may progress to end-stage liver disease, such as cirrhosis and hepatocellular carcinoma (HCC). Therefore, one of the goals of effectively treating NAFLD and NASH is to prevent or reduce the likelihood of these conditions progressing to cirrhosis and HCC. The prevalence of NAFLD is increasing worldwide and has reached epidemic proportions. Estimates of the prevalence of NAFLD suggest that as many as 1 billion people may be affected worldwide (Loomba R, Sanyal AJ. Nat Rev Gastroenterol Hepatol. 2013 Nov; 10(11): 686-9). Therefore, there is a need for new treatments that can be administered in a safe and cost-effective manner to treat these conditions.

[0015] Genetic and intervention models in mice suggest that regulating the NAD+ synthesis pathway may improve fatty liver disease. For example, treatment with NAD+ precursors has been shown to prevent the progression of NAFLD to NASH, and supplementation with NAD+ precursors has been associated with reduced hepatic stellate cell activation and reduced fibrosis. (Dall et al., J Physiol. 2022, 600.5:1135-1154). In addition, sirtuins, which cleave NAD+ into nicotinamide (NA) and O-acetyl-ADP-ribose, may also play a role in NAFLD. Reduced hepatic expression of SIRT1, SIRT3, SIRT5, and SIRT6 has been reported in patients with NAFLD (Wu et al. Ann Clin Lab Sci. 2014; 44(4):410-8), while liver-specific knockout of SIRT1 and SIRT6 in mice leads to hepatic lipid accumulation (Kim et al. Cell Metab. 2010; 12(3):224-36; Purushotam et al. Cell Metab. 2009; 9:327–338). Thus, overexpression of SIRT1 appears to prevent high-fat diet (HFD)-induced obesity (Pfluger et al., Proc Natl Acad Sci US A. 2008; 105(28): 9793-8), and increasing the liver NAD pool by inhibiting NAD+-consuming PARP reduces weight gain and the development of hepatic steatosis in high-fat, high-sucrose-fed mice through a SIRT1-dependent mechanism (Gariani et al., J Hepatol. 2017; 66(1): 132-141). Therefore, sufficient sirtuin activity appears to be crucial for preventing hepatic lipid accumulation.

[0016] PNP inhibitors have been available for many years. Exemplary compounds are described in US5565463, US5008270, US5985848, US7109331, US7553839, US7427624, WO08030119A1, US8283345, CZ20190667A3, and CZ34625U1, all of which are incorporated herein by reference. In particular, US7553839 describes the synthesis of urodesine (see compound 8). These documents also describe methods for preparing the compounds and various formulations containing these molecules.

[0017] Previously, it was suggested that PNP inhibitors (such as urodesine and furodesine) and the well-known salts of urodesine (such as succinate) are used as therapies for reducing uric acid in conditions such as gout and hyperuricemia because they can inhibit uric acid synthesis by inhibiting the synthesis of both hypoxanthine (from inosine) and guanine (from guanosine), and hypoxanthine and guanine are each mediated by PNP. Urodesine has also been proposed as a novel immune checkpoint inhibitor for the treatment of leukemia because it can indirectly affect TLR7 activation by increasing the (deoxy) guanosine ligand pool (Abt et al., J.Clin.Invest.132,e1608 (2022). PNP inhibitors such as urodesine have also been proposed as compounds that reduce the dNTP pool for the treatment of cancer (WO21022105A1). There is no teaching in the literature that PNP inhibitors can be used to treat or prevent metabolic syndrome or its related conditions and diseases.

[0018] There remains a need to provide new treatment options for the treatment and prevention of metabolic syndrome and related disorders and diseases, such as obesity, fatty liver disease and cardiometabolic disorders, such as heart disease, stroke and type 2 diabetes. Prior to the present study by the applicant, the potential of PNP inhibitors to have therapeutic relevance in these conditions had not yet been identified, hypothesized or actually studied. It can now be inferred from the results provided and about the mechanisms currently considered to be crucial at the cellular level that the practical application of such PNP inhibitors or their specific stable salts (as defined in the present application), such as urodesin, will be extremely beneficial for these therapeutic indications. SUMMARY OF THE INVENTION

[0020] The present inventors have identified for the first time that inhibition of PNP (e.g., using urodesine or a salt thereof) results in significant modulation of metabolic indices, including weight gain, percentage of brown and white adipose tissue by weight, fatty acid synthesis, liver triglycerides, cholesterol, blood glucose, and plasma insulin, as well as reductions in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as liver AST. Therefore, PNP inhibitors may be useful in treating a variety of conditions, including metabolic syndrome and its related diseases and conditions, as further discussed herein.

[0021] Preferred PNP inhibitors comprise the glutarate salt of urodesine. In embodiments, the salt comprises the hemiglutarate salt of urodesine. Until recently, identifying and reliably producing a stable form of urodesine suitable for pharmaceutical production has been a technical challenge. Physically stable salts are highly desirable properties in pharmaceutical production and are therefore of particular interest in the medical applications and methods related to urodesine disclosed and discussed in this application.

[0022] A PCT application (published as WO2023001893) successfully prepared urodesine hemiglutarate for the first time, using a novel process and generating a stable novel salt that may be useful in drug development. For the avoidance of doubt, the disclosure of that application relating to urodesine hemiglutarate and its preparation method is incorporated herein by reference.

[0023] The authors of this application noted that typical salt variants prepared by conventional methods in the art did not consistently produce reliably stable compounds, which is required for large-scale production of urodesine in pharmaceutical development. The authors had attempted available methods (known in the art) for preparing other existing salts of urodesine, as well as methods known in the art, but these methods were unsuccessful in preparing the glutarate salt. For this reason, the glutarate salt was not an obvious candidate to try as a starting point compared to other salts available at the time.

[0024] Furthermore, salt selection requires consideration of several analytical parameters to determine useful chemical and physical properties, such as the presence of sharp, clear diffraction peaks in the pattern, any noticeable amorphous peaks, solvent weight loss, and the ability to obtain crystalline forms under a variety of conditions. Glutaric acid is not a practical choice for the production of urodesine under standard conditions, as preliminary analyses described by the authors indicated low product crystallinity and some significant solvent weight loss when production was attempted using known methods.

[0025] Nevertheless, to overcome these challenges, after extensive research and development of alternative production methods, a new method for making the hemiglutarate salt was identified and produced a crystalline salt that is surprisingly highly stable compared to other salts. In an embodiment, urodesine hemiglutarate for use in the present invention can be prepared by the same method, which comprises the following steps: (a) preparing an aqueous solution of urodesine free base, and optionally stirring at room temperature; (b) adding glutaric acid to the mixture of step (a), and optionally stirring at room temperature for 30 minutes; (c) freeze-drying the solution of step (b) to obtain a white solid product; (d) dissolving the solid product of (c) in water; optionally heating to 75°C; adding ethanol and optionally stirring at 75°C for 30 minutes to form a homogeneous solution; (e) adding acetonitrile dropwise to the solution of (d), optionally adding over a period of 60 minutes; (f) stirring the solution of (e) for 60 minutes, optionally stirring at 75°C; and optionally cooling the solution to 0°C over a period of 60 minutes; (h) filtering and washing with acetonitrile to obtain urodesine hemisalt glutarate.

[0026] The chemical structure of urodesine hemiglutarate is provided below:

[0027]

[0028] In the method, glutaric acid is added along with a certain amount of the desired final salt form to assist and optimize the crystallization process. The specific recrystallization step identified above, as well as the choice of solvent and amount, is critical to obtaining the hemi-salt ethanol (in combination with the other steps), i.e., using ACN (acetonitrile) / water to form the final hemiglutarate salt crystals, otherwise less favorable salts such as the monosalt are obtained.

[0029] The present application provides further analytical work that confirms the selected recrystallization process and hemiglutarate salt form, its stability and therefore suitability for the medical uses described in this application. This additional information is provided in the detailed description of the invention disclosure and the figures accompanying this disclosure.

[0030] In an embodiment, urodesine hemiglutarate is in a stable crystalline form A, as clearly defined by the data content of this application. Experimental evidence provides a stable and well-characterized crystal structure of urodesine hemiglutarate.

[0031] Therefore, in one aspect of the present disclosure, a PNP inhibitor for the treatment or prevention of metabolic syndrome or a disease or condition induced by metabolic syndrome is provided. The term "PNP inhibitor" includes compounds that inhibit PNP. Compounds having an in vitro inhibition constant (Ki) value of less than about 5x 10-7M, typically less than about 1x10-8M, and preferably less than 5x 10-9M are preferred for in vivo use.

[0032] In some embodiments, the present disclosure provides the use of a PNP inhibitor in the manufacture of a medicament for treating metabolic syndrome and metabolic syndrome-induced disorders.

[0033] As mentioned above, metabolic syndrome is a group of conditions that tend to occur together. This group of conditions includes high blood pressure, high blood sugar, excess body fat, especially around the waist (i.e., abdominal fat), fatty liver, and abnormal cholesterol or triglyceride levels (dyslipidemia).

[0034] Abnormal cholesterol or triglycerides include high fasting serum LDL cholesterol, low fasting serum HDL cholesterol, and high fasting serum triglycerides compared to normal subjects.

[0035] References in this application to the treatment or prevention of metabolic syndrome relate to the treatment or prevention of at least one, at least two, at least three or more of the underlying conditions mentioned.

[0036] In one embodiment, the treatment or prevention of metabolic syndrome in the present application includes at least one of the following, preferably at least two: treatment or prevention of hypertension, treatment or prevention of insulin resistance, normalization of blood sugar, reduction of body fat (e.g., waist fat), increase in brown fat as a percentage of body weight, treatment or prevention of fatty liver and / or normalization of abnormal serum cholesterol and / or triglycerides.

[0037] In one embodiment, the treatment or prevention of metabolic syndrome in the present application includes at least one of the following, preferably at least two: reduction of high blood pressure, reduction of insulin resistance, normalization of blood sugar, reduction of body fat (e.g., waist fat), increase in brown fat as a percentage of body weight, reduction of fatty liver, normalization of abnormal serum cholesterol and / or triglycerides.

[0038] In one embodiment, the treatment or prevention of metabolic syndrome includes at least reduction of insulin resistance and normalization of blood glucose; and optionally includes at least one of: reduction of high blood pressure, reduction of body fat (e.g., waist fat), increase in brown fat as a percentage of body weight, reduction of fatty liver, and normalization of abnormal serum cholesterol and / or triglycerides.

[0039] In one embodiment, the treatment or prevention of metabolic syndrome includes at least normalization of serum cholesterol and reduction of body fat (e.g., waist fat); and optionally includes at least one of: reduction of insulin resistance, normalization of blood glucose, reduction of hypertension, increase in brown fat as a percentage of body weight, reduction of fatty liver, and normalization of serum triglycerides.

[0040] In one embodiment, the treatment or prevention of metabolic syndrome includes at least normalization of serum cholesterol and reduction of insulin resistance, and optionally includes at least reduction of body fat, normalization of blood glucose, reduction of hypertension, increase in brown fat as a percentage of body weight, reduction of fatty liver, and normalization of serum triglycerides.

[0041] A reduction in insulin resistance refers to a reduction in the HOMA-IR score, as described in detail below.

[0042] Normalization of blood glucose means lowering fasting blood glucose concentrations to levels closer to those found in subjects without metabolic syndrome.

[0043] Normalization of abnormal serum cholesterol and / or triglycerides means lowering one or more of fasting serum total cholesterol, lowering fasting serum LDL (low-density lipoprotein) cholesterol, and / or lowering fasting serum triglycerides to bring the levels closer to those found in subjects not suffering from metabolic syndrome.

[0044] Without wishing to be bound by any theory, the present inventors believe that the mechanism behind this effect is as follows: inosine is broken down by purine nucleoside phosphorylase (PNP) and converted to hypoxanthine. When this enzyme is inhibited, inosine levels increase. In the salvage pathway for the synthesis of inosine monophosphate (IMP), hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts inosine to IMP, consuming phosphoribophosphate diphosphate (PRPP) in the process. HGPRT is normally the main consumer of PRPP. When PNP is inhibited, hypoxanthine and guanine are not produced, thereby substantially sparing PRPP. Figure 1 、 Figure 2 ).

[0045] Normally, the rate at which nicotinamide (NAM) and nicotinic acid (NA) are converted to NAD+ is limited by the availability of PRPP. However, inhibition of PNP results in a larger pool of available PRPP, which in turn leads to a significant increase in NAD+ levels in whole blood, as confirmed in experimental results further detailed in this application.

[0046] In addition to the pathways mentioned above, it is also worth noting that the NAD+ precursor nicotinamide riboside (NR) is metabolized by PNP. Therefore, inhibiting PNP may lead to a larger pool of NAD+ precursors and a subsequent increase in NAD+ levels.

[0047] This group of conditions, known as metabolic syndrome, may, together or individually, lead to more serious conditions or diseases, such as heart disease, liver disease, stroke, and type 2 diabetes. Treating or preventing the parameters of metabolic syndrome can prevent the progression of underlying conditions, and thus addressing metabolic syndrome as described herein can lead to preventing (or reducing the risk of) peripheral vascular disease, cardiovascular disease, such as heart disease and stroke, preventing or treating type 2 diabetes, preventing or treating liver disease, including fatty liver disease such as NASH or NAFLD, and preventing or treating dyslipidemia.

[0048] Dyslipidemia refers to an imbalance of lipids such as cholesterol (i.e., serum total cholesterol), serum low-density lipoprotein cholesterol (LDL-C), serum high-density lipoprotein cholesterol (HDL-C), and serum triglycerides. In one embodiment, dyslipidemia can be prevented or treated using a PNP inhibitor. In one embodiment, one or more or all of the following effects can be produced using a PNP inhibitor: lowering serum LDL cholesterol, raising serum HDL cholesterol, lowering serum total cholesterol, and / or lowering serum total triglycerides. In another embodiment, serum total cholesterol can be lowered using a PNP inhibitor. In another embodiment, serum triglycerides can be lowered using a PNP inhibitor.

[0049] Diabetes is a chronic metabolic disease characterized by elevated blood sugar levels that, over time, cause serious damage to the heart, blood vessels, eyes, kidneys, and nerves. Type 2 diabetes usually develops in adults and is caused by insulin resistance, or decreased insulin production. Insulin resistance is a condition that is closely related to metabolic syndrome and is present in most people with metabolic syndrome. In insulin resistance, cells (such as those in muscle, fat, and the liver) are unable to respond to insulin by increasing their uptake of glucose. In these cases, blood sugar rises, and because the pancreas secretes higher levels of insulin, levels may also be high. A simple measure of insulin resistance is the homeostasis model assessment of insulin resistance (HOMA-IR) test.

[0050] HOMA-IR = [fasting insulin (μIU / mL)] × [fasting blood glucose (mmol / L)] / 22.5

[0051] A HOMA-IR score <1.0 is optimal, >1.9 indicates early insulin resistance, and >2.9 indicates severe insulin resistance.

[0052] In some embodiments, treatment with a PNP inhibitor can reduce a subject's fasting blood glucose and / or reduce a subject's fasting blood insulin. In some embodiments, treatment with a PNP inhibitor can treat or prevent type 2 diabetes in a subject. In some embodiments, treatment with a PNP inhibitor can treat or prevent insulin resistance in a subject. In some embodiments, treatment with a PNP inhibitor can reduce a subject's HOMA-IR score.

[0053] Obesity is a condition in which excess body fat accumulates to a level that can have a negative impact on health. The World Health Organization (WHO) defines overweight as a body mass index (BMI) of 25 or higher and obesity as a BMI of 30 or higher. The U.S. Centers for Disease Control and Prevention (CDC) subdivides obesity into: Grade 1 obesity is a BMI of 30 to 35; Grade 2 obesity is a BMI of 35 to 40, and Grade 3 obesity is a BMI of over 40. In some embodiments, treatment with a PNP inhibitor can reduce the weight of a subject. In some embodiments, treatment with a PNP inhibitor can prevent or treat weight gain in a subject; in some embodiments, treatment with a PNP inhibitor can prevent or treat obesity and / or overweight in a subject.

[0054] Fatty liver diseases such as NAFLD range from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH). In some embodiments, treatment with a PNP inhibitor can prevent or treat fatty liver disease; in other embodiments, treatment with a PNP inhibitor can prevent or treat NAFLD. In some embodiments, treatment with a PNP inhibitor can prevent or treat NASH. In some embodiments, treatment with a PNP inhibitor can alleviate hepatic steatosis.

[0055] Improvement of metabolic syndrome has been shown to reduce the incidence of cardio-metabolic diseases, such as cardiovascular diseases such as heart disease, stroke, and type 2 diabetes (Wilsone et al., Circulation 2005; 112: 3066-3072), and thus, in some embodiments, treatment with a PNP inhibitor can treat or prevent cardiometabolic diseases, and in some embodiments, treatment with a PNP inhibitor can treat or prevent cardiovascular disease. In some embodiments, treatment with a PNP inhibitor can treat or prevent heart disease. In some embodiments, treatment with a PNP inhibitor can treat high blood pressure. In some embodiments, treatment with a PNP inhibitor can lower blood pressure. In some embodiments, treatment with a PNP inhibitor can treat or prevent stroke.

[0056] Many PNP inhibitors are known. Exemplary compounds are described in US5565463, US5008270, US5985848, US7109331, US7553839, US7427624, WO08030119A1, US8283345, CZ20190667A3 and CZ34625U1, all of which are incorporated herein by reference. In particular, US7553839 describes the synthesis of urodesine (see compound 8). These documents also describe methods for preparing the compounds and various preparations comprising these molecules.

[0057] Other compounds are known from the literature, for example, Evans et al., Organic Letters, 2003, 5:3639; Taylor et al., Journal of American Chemical Society, 2007, 129:6984; Evans et al., Journal of Medicinal Chemistry, 2003, 46:5271; Castilho et al., Bioorganic and Medicinal Chemistry, 2006, 14:516; Schramm et al., Journal of Biological Chemistry, 2007, 282:28297; and Bantia et al., International Immunopharmacology, 2010, 784 and 2001, 1:1199-1210; Kicska et al., Proceedings of National Academy of Sciences, 2001, 98:4593-4598; Ho et al., Proc Natl Acad Sci USA. 2010, 107(11):4805-12). The disclosures of each of these references are incorporated herein by reference in their entirety.

[0058] Preferred compounds are those of Formula I, Formula II and Formula III below.

[0059]

[0060] Preferably, the PNP inhibitor is urodesine (Formula I) or a pharmaceutically acceptable salt thereof. Known salts of urodesine include hydrochloride, dihydrochloride, hydrobromide, hemisulfate, p-toluenesulfonate, phosphate, citrate, L-tartrate, L-lactate, stearate, maleate, succinate, fumarate, L-malate and L-aspartate (see, for example, WO2010 / 111381).

[0061] The present inventors have identified and further characterized a favorable salt, namely the hemiglutarate salt. Therefore, the PNP inhibitor preferably comprises urodesine hemiglutarate.

[0062] In one embodiment, the present disclosure provides a pharmaceutical composition comprising urodesine hemiglutarate, optionally comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

[0063] The therapeutic dose size of a PNP inhibitor or a pharmaceutically acceptable salt, solvate, hydrate or stereoisomer thereof in acute or chronic treatment will vary with the nature and severity of the disease and the route of administering it. The dosage, and in some cases the frequency of administration, will also vary depending on the disease being treated, the age, weight and response of the individual patient. The PNP inhibitor of the present disclosure can be used in a dose between 1 mg and 1000 mg, but more typically up to 500 mg, and even more typically up to 250 mg. In some embodiments, the dosage is 1 mg to 150 mg or 1 mg to 120 mg. In some embodiments, the dosage is 5 mg to 120 mg. The dosage identified in this application is a dosage suitable for an adult weighing 80 kg.

[0064] It is known that genetic deficiency of PNP leads to severe combined immunodeficiency (Markert ML. Immunodefic Rev. 1991; 3(1): 45-81). Similarly, long-term high-dose administration of PNP inhibitors in humans is known to cause a decrease in various lymphocyte subsets (Gomes et al., Blood ASH Annual Meeting Abstracts, 2008, 112: Abstract 2583). Therefore, long-term treatment with high-dose PNP inhibitors may have immunogenic effects.

[0065] However, despite the technical prejudice against the use of these compounds in therapy, applicants have shown in this application that a therapeutic approach may in fact be possible.

[0066] To offset the immunogenic side effects observed in these drugs, the present disclosure provides low-dose and / or intermittent administration of PNP inhibitors (e.g., urodesine hemiglutarate), which allows for the production and increase of endogenous metabolites inosine and NAD+, which can promote key changes at the cellular level and lead to the alleviation of obesity and metabolic syndrome. This low and / or intermittent dose utilizes the natural endogenous metabolite inosine as a new tool to activate BAT to reduce adipose tissue and improve obesity, while utilizing the natural endogenous metabolite NAD+ to activate sirtuins and other enzymes that improve metabolic risk factors, but does not cause significant immune side effects, such as decreased lymphocyte levels.

[0067] Thus, in one embodiment, the inventors have provided a novel regimen for administering the drug, which provides beneficial therapeutic effects with low or minimal side effects. This dosing regimen constitutes a further embodiment of the present invention.

[0068] Thus, in some embodiments, the present disclosure provides a PNP inhibitor for the uses described elsewhere herein, wherein the PNP inhibitor is provided in an intermittent dosing regimen, a low-dose regimen, or a combination of both, as further described below.

[0069] PNP inhibitors of the present disclosure can be used at a dosage between 1 mg and 1000 mg / day. In one embodiment, they are used at a dosage of up to 500 mg / day. In one embodiment, they are used at a dosage of up to 250 mg / day.

[0070] When a lower dosage regimen is needed, for example, to reduce the incidence of immunogenic side effects, a dosage of 1 mg to 150 mg / day; optionally 1 mg to 100 mg / day; optionally 1 mg to 50 mg / day or 1 mg to 40 mg / day can be provided. In some embodiments, the lower dose within these ranges is 10 mg, for example 10 mg to 40 mg. These doses represent the daily dose provided and can be provided in 1, 2, 3 or more sub-doses throughout the day as needed. When the goal of administration is to reduce the incidence of immunogenic side effects, the goal of administration is to reduce the number of lymphocytes in the blood by <10% or <20%.

[0071] In some embodiments, the intermittent treatment regimen comprises or consists of repeating cycles of treatment periods, in which the subject receives a daily dose of the drug (dosing), followed by a rest period, in which the subject does not receive the drug.

[0072] In one embodiment, a treatment cycle includes administration for 1 to 6 days, followed by at least 1 day of drug withdrawal or consists of administration for 1 to 6 days, followed by at least 1 day of drug withdrawal. In one embodiment, a treatment cycle includes administration for 2 to 4 days, followed by at least 1 day of drug withdrawal (optionally 2 days, optionally 3 days, optionally 4 days) or consists of administration for 2 to 4 days, followed by at least 1 day of drug withdrawal (optionally 2 days, optionally 3 days, optionally 4 days). In one embodiment, a treatment cycle includes administration for 3 days, followed by at least 1 day of drug withdrawal (optionally at least 2 days, optionally at least 3 days, optionally at least 4 days) or consists of administration for 3 days, followed by at least 1 day of drug withdrawal (optionally at least 2 days, optionally at least 3 days, optionally at least 4 days). Typically, the drug withdrawal period is no more than 5, 6 or 7 days. In one embodiment, a treatment cycle includes administration for 2 to 4 days, preferably 3 days, followed by 3 to 4 days of drug withdrawal or consists of administration for 2 to 4 days, preferably 3 days, followed by 3 to 4 days of drug withdrawal.

[0073] The intermittent dosing regimen is thought to be sufficient to induce metabolic changes but, crucially, not significantly affect lymphocyte numbers (<10% or <20% change) and therefore not have an immunogenic response.

[0074] Immunogenic side effects can also be mitigated by treating subjects with both lower doses and intermittent dosing regimens, and thus in some embodiments, the lower dose regimens described above can be provided as intermittent dosing regimens. Thus, in some embodiments, a dose of 1 mg to 90 mg can be provided daily as an intermittent dosing regimen, wherein the subject receives repeated dosing cycles, wherein the cycles comprise or consist of 1 to 6 days of dosing followed by at least 1 day, at least 2 days, at least 3 days, at least 4 days and no more than 7 days, 6 days, or 5 days of rest.

[0075] Treatment can last for multiple cycles, and the cycles can be changed as needed.

[0076] For compliance purposes, it may be useful to administer treatment in 7-day cycles, so that in one embodiment, the subject receives treatment on days 1 to 6 but not on day 7, or on days 1 to 5 but not on days 6 and 7, or on days 1 to 4 but not on days 5 to 7, or on days 1 to 3 but not on days 4 to 7, or on days 1 and 2 but not on days 3 to 7, or on day 1 but not on days 2 to 7.

[0077] A subject is any subject to be treated according to the disclosed treatments. The subject is typically a mammalian subject, particularly a human subject, for whom treatment is required for the condition being treated, but may be any other subject, including, for example, a companion animal or domesticated animal.

[0078] The compositions of the present invention can be formulated as oral dosage forms, parenteral dosage forms or topical dosage forms. In specific embodiments, oral dosage forms can be formulated to provide sustained release of the PNP inhibitor.

[0079] In some embodiments, the present disclosure provides a method for treating a subject suffering from metabolic syndrome or a disease, disorder or condition induced by metabolic syndrome, the method comprising administering a pharmaceutically effective amount of a PNP inhibitor to the subject. In some embodiments, the PNP inhibitor is provided with an intermittent dosing regimen, a low-dose regimen, or a combination thereof, as described elsewhere herein.

[0080] In some embodiments, the present disclosure provides methods of treating a subject having a metabolic syndrome, disorder, disease or condition, [particularly where increasing the amount of extracellular inosine or NAD+ is desirable], comprising administering to a subject in need thereof a pharmaceutically effective amount of a PNP inhibitor.

[0081] In some embodiments, the present disclosure provides a method for increasing the percentage of brown adipose tissue (BAT) to body weight in a subject in need thereof, the method comprising administering a pharmaceutically effective amount of a PNP inhibitor to the subject. In some embodiments, the method can increase the percentage of BAT to body weight. In some embodiments, the method can increase the percentage of BAT to body volume. In some embodiments, the method can increase the total ratio of BAT to WAT, particularly iWAT.

[0082] In some embodiments, the present disclosure provides a method for improving the lipid profile of a subject in need thereof, the method comprising administering a pharmaceutically effective amount of a PNP inhibitor to the subject. Improving lipid profile means reducing the plasma concentration of one or more of cholesterol, triglycerides, and low-density lipoprotein (LDL). In particular, cholesterol.

[0083] In some embodiments, the present disclosure also provides a method for treating fatty liver disease in a subject in need thereof, the method comprising administering a pharmaceutically effective amount of a PNP inhibitor to the subject. The fatty liver disease may be NASH and / or NAFLD. In some embodiments, such treatment has one or more of the following effects: reducing lipid and / or liver enzyme abnormalities and reducing fatty degeneration in the liver. In some embodiments, the treatment comprises reducing the subject's liver total cholesterol.

[0084] In some embodiments, the present disclosure also provides a method of reducing the activity of the liver enzymes AST and / or ALT in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a PNP inhibitor.

[0085] In some embodiments, use of a PNP inhibitor to treat the conditions and diseases described herein comprises administering the PNP inhibitor according to a low and / or intermittent dosing regimen as described herein.

[0086] In some embodiments, the treatments disclosed herein include providing a PNP inhibitor in combination with one or more additional active agents. Examples of other active agents include, but are not limited to, ENT1 inhibitors (e.g., ), GLP-1 agonists (e.g., exenatide, liraglutide, albiglutide, and semaglutide), weight loss drugs (e.g., bupropion-naltrexone), antihypertensive drugs (e.g., amlodipine, captopril, enalapril, and furosemide), and antidiabetic drugs (e.g., metformin, rosiglitazone, and insulin analogs such as insulin glargine or insulin detemir), or other active agents known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Regulation of various purine metabolites by PNP inhibition is shown.

[0088] Figure 2 Schematic diagram of the role of PNP in purine metabolism, illustrating the possible relationship between PNP inhibition and increased inosine and NAD+ levels.

[0089] Figure 3 The effects of urodesine hemiglutarate on body weight, food intake, body mass, blood glucose, and serum cholesterol in a diet-induced obesity (DIO) mouse model are shown.

[0090] Figure 4 Shown are the effects of urodesine hemiglutarate on serum cholesterol, blood glucose, plasma insulin, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and iWAT / body mass percentage in the DIO mouse model.

[0091] Figure 5 Shown are the effects of urodesine hemiglutarate on the expression of fatty acid synthase markers in iWAT and thermogenesis marker (UPC-1 expression) in BAT tissue in a DIO mouse model.

[0092] Figure 6 Shown are the effects of urodesine hemiglutarate on plasma inosine and plasma NAD+ levels in a DIO mouse model.

[0093] Figure 7 Shown are the effects of urodesine hemiglutarate on liver parameters (hepatic total cholesterol (TC) and hepatic AST) in the DIO mouse model.

[0094] Figure 8 The effects of increasing doses of urodesine hemiglutarate on lymphocytes in a diet-induced obesity (DIO) mouse model are shown.

[0095] The following figures provide a graphical analysis resulting from various analytical tests showing the physical and chemical properties of the hemiglutarate produced as disclosed in the context of the present disclosure.

[0096] Figure 10 An XRPD overlay of Form A glutarate is shown; this scaled-up sample was identified as the hemiglutarate salt of urodesine provided according to the present disclosure;

[0097] Figure 11 The TGA and DSC results of Form A glutarate are shown; this scaled-up sample was identified as the hemiglutarate of urodesin provided according to the present disclosure;

[0098] Figure 12 The 1H NMR spectrum of the glutarate form A is shown; this was a scaled-up sample and was identified as the hemiglutarate salt of urodesine provided according to the present disclosure.

[0099] Figure 13 The PLM crystal structure of glutarate form A is shown; this scaled-up sample is identified as the hemiglutarate salt of urodesin provided according to the present disclosure;

[0100] Figure 14 A DVS graph showing the hygroscopic properties of glutarate form A is shown; this scaled-up sample was identified as the hemiglutarate salt of urodesin provided according to the present disclosure;

[0101] Figure 15 Shows about Figure 14 Overlay of XRPD patterns of the same Form A glutarate before and after DVS as described in;

[0102] Figure 16 Shown is an XRPD overlay of drying stability data for the same Form A glutarate salt;

[0103] Figure 17 Shown is an XRPD overlay characterizing Form A of the glutarate salt, provided by polymorph screening of a previously characterized urodesine hemiglutarate starting (reference) material;

[0104] Figure 18 TGA and DSC data are shown, which further characterize and confirm the hygroscopic properties of Form A glutarate provided by the polymorph screening experiments;

[0105] Figure 19 H NMR spectra are shown, which further characterize and confirm the stoichiometry of Form A glutarate provided by the polymorph screening experiments;

[0106] Figure 20 An XRPD overlay characterizing Form B glutarate is shown;

[0107] Figure 21 An overlay of XRPD patterns characterizing Form C glutarate and its possible conversion to Form A glutarate is shown; and

[0108] Figure 22 Shown is an overlay of XRPD patterns characterizing glutarate Form C and its possible conversion to glutarate Form A.

[0109] Hemiglutarate confirmation and characterization

[0110] In WO2023001893, the authors disclosed that after several technical changes to the crystallization process studied, an alternative recrystallization method was needed to produce the hemi-salt. The chemical structure of urodesine hemiglutarate is provided below:

[0111]

[0112] A new crystallization method specific for the hemiglutaric acid salt form was established using a mixed solvent method using several different solvents. For clarity, this method is repeated here: Glutaric acid (278.36 mg, 1.89 mmol) was added to a solution of urodesine (1000.00 mg, 3.78 mmol) in water (50 mL). The mixture was stirred at room temperature for 30 minutes and then lyophilized to yield 1155.00 mg of a white solid, a 90% yield. 1055 mg of urodesine hemiglutaric acid salt was dissolved in 3 mL of water, heated to 75°C, and 15 mL of ethanol was added. The mixture was stirred at this temperature for 30 minutes to form a homogeneous solution. 30 mL of acetonitrile was then added dropwise over 1 hour. The mixture was then stirred at this temperature for 1 hour. The mixture was cooled to 0°C over 1 hour. The mixture was filtered, the filter cake washed with acetonitrile, and dried to yield 810 mg of urodesine hemiglutarate as a white solid, a 76.8% yield.

[0113] Analysis identified the solid as the crystalline hemiglutarate salt and demonstrated that a novel alternative process (including several steps and the addition of ethanol) is necessary for the efficient recrystallization of urodesine hemiglutarate.

[0114]

[0115] Furthermore, during this work, the authors of the application noted that, in order to confirm that the disclosed complete preparation method yielded the same usable salt product, it was crucial to use the free base product to prepare the desired salt (rather than starting with simple urodesine free base). Using the free base (rather than hydrochloric acid) as the reaction partner eliminated the need for subsequent addition of a base to the reaction. This reaction was more efficient and yielded a higher product content than previously used mechanisms. Importantly, this modification also avoided the hydrochloride salt dissociation phenomenon previously observed in those existing methods. The urodesine free base obtained using this method had high HPLC purity, and analytical testing in the application confirmed that, by modifying this early step, the hemiglutarate salt could be reliably and consistently produced from urodesine in the complete urodesine preparation method.

[0116] As shown below, urodesine hemiglutarate was obtained in good batch yields by this new method and was verified to be chemically pure. The properties of the resulting salt were consistent with the original salt selection studies in WO23001893.

[0117] batch number quantity HPLC purity ee Total yield CP-0031535-13 34.9 grams 99.12％ 98.6％ 8.2％

[0118] Analysis of exemplary batch (35 g) CP-0031535-13:

[0119] Test items Analytical methods Observation results Appearance visual White crystalline solid powder Identification <![CDATA[ 1 H-NMR]]> Consistent with the structure purity HPLC 99.12% (a / a) Enantiomeric purity HPLC 98.6％ Content determination <![CDATA[ 1 H-NMR]]> 96.0-103.0% (w / w) Residual solvents GC EtOH: 0.03%, MeCN: 0.02% water KF 0.147% (w / w) Peak temperature DSC Peak temperature of the salt selection batch Polymorph XRPD XRPD of salt selected batches

[0120] Verify data

[0121] Additional confirmatory studies were included in this application to verify the appropriate salt form of crystalline urodesine hemiglutarate and its stability to reaffirm its suitability for use in this pharmaceutical application.

[0122] Furthermore, this data supports the rationale for the specific selection of urodesine hemiglutarate for its use as a medicament (or in a pharmaceutically acceptable composition). This particular salt choice provides a suitable solution if other salts are unsatisfactory in terms of both stability and potential for commercial scale-up. Given the reaffirmation of the relevant properties in this application, it is considered highly advantageous for the described uses, particularly for the treatment and / or prevention of various defined diseases and conditions, including those associated with decreased tissue levels of the metabolite: NAD+.

[0123] In this study, urodesine free base and glutaric acid were used in several salt formation experiments using different solvent systems: EtOH, acetone, EtOAc, THF, ACN / H2O (9:1), and DCM.

[0124] Consistent with the findings in publication WO2023001893, only one crystalline form with high crystallinity was observed, which was designated Form A and recrystallized from an acetonitrile and water solvent mixture ACN / H2O (9:1) as originally detailed in the methods and experiments in WO2023001893. Other salt formation experiments only produced free base or had low crystallinity.

[0125] The following method was then used to scale up the Form A glutarate to 500 mg to obtain sufficient material for evaluation: Form A glutarate was re-prepared on a 500 mg scale in acetonitrile / water (9:1, v / v). Specifically, 499.34 mg of Form A free base and 137.43 mg of glutaric acid (feed ratio 0.55:1, acid / base) were weighed in a 20 ml vial, and 7 ml of the corresponding solvent was added to the solid to form a suspension. After magnetic stirring at room temperature (about 25°C) for 1 day, the solid was isolated by vacuum filtration and dried in vacuo at room temperature for 5 hours. 600.67 mg of solid was obtained with a yield of 91.3%.

[0126] The product was urodesine hemiglutarate, presumably a monohydrate with high crystallinity. The water content (wt%) measured by Karl Fischer titration was 6.0, and the stoichiometry (hemiglutarate / free base) was 0.5. The HPLC purity was 97.9 area%. The product was further characterized to confirm the following properties, which are summarized below and in conjunction with the accompanying figures:

[0127] XRPD diffractogram of the re-prepared sample ( Figure 10) showed that the solid obtained was glutarate type A according to the reference substance.

[0128] The TGA results showed that the weight loss was 6.1 wt% before 160.0 ° C (the theoretical value is 5.2% for water), and the DSC results showed three endothermic peaks at 120.7 ° C, 126.8 ° C and 145.0 ° C (peak). Figure 11 ).

[0129] 1H NMR spectrum of the re-prepared A-type glutarate ( Figure 12 ) showed a glutaric acid / free base stoichiometry of 0.5 with no residual ACN.

[0130] PLM Image( Figure 13 ) showed that the A-type glutarate sample was small crystals and agglomerates, which was consistent with the expected crystal structure.

[0131] To understand the hygroscopicity of glutarate type A, DVS was used to measure the mass change at 25 °C as a function of relative humidity ( Figure 14 ). Form A glutaric acid salt showed a water absorption rate of 1.1% at 25℃ / 80%RH (since no dehydration was observed at 0%RH, it was calculated based on 0-80%RH), indicating that Form A glutaric acid salt has slight hygroscopicity. At 25℃ / 95%RH, the water absorption rate increased to 10.4%. XRPD results showed that the morphology did not change after DVS treatment ( Figure 15 ).

[0132] Kinetic solubility: (37°C / 24 hours) exceeds 16.8 mg / mL at all pH values ​​(water / SGF / FaSSIF / FeSSIF)

[0133] Physicochemical stability

[0134]

[0135] (2 weeks, open condition): No crystal form change was observed under all conditions (25°C / 60% RH, 40°C / 75% RH and 60°C) for up to 2 weeks.

[0136] Drying stability (vacuum, room temperature / 50 / 70℃): After vacuum drying overnight at room temperature and 50℃, the crystallinity did not change, except for a slight decrease at 70℃ ( Figure 16 ).

[0137] The term "mono" means that the ratio of API to acid in the crystal structure of the salt of the compound urodesine is 1:1, respectively. The term "semi" means that the ratio of API to acid in the crystal structure of the salt of the compound urodesine is 2:1, respectively. The term "inert organic solvent" refers to a solvent that does not chemically interfere with the reaction.

[0138] The term "isostructural" is used to describe crystalline substances that have the same type of crystal structure, such as when a new molecular entity replaces another in the crystal structure without significantly disturbing the unit cell.

[0139] The "crystalline state" of the salts is determined by standard techniques and these are defined below. The crystals and their crystal structure were characterized using a variety of techniques including single crystal X-ray crystallography, X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC).

[0140] The behavior of the crystals under different humidity conditions can be analyzed by gravimetric vapor sorption studies and XRPD. These techniques help characterize the resulting salt and confirm whether the product is optimized. Specifically, X-ray crystallography involves the analysis and interpretation of X-ray diffraction patterns from single crystals. In amorphous solids, the three-dimensional structure typically present in crystals is absent, and the positions of the molecules relative to each other are essentially random.

[0141] In the experimental formation process, hemiglutarate is recrystallized from water and other organic solvents. The present disclosure provides a solvate formed by incorporating a nontoxic pharmaceutically acceptable solvent into the solid state structure (e.g., crystal structure) of the compound provided in the present application. Examples of such solvents include water, alcohol (e.g., ethanol, isopropanol, and butanol), acetonitrile (ACN), and dimethyl sulfoxide (DMSO) as described above. Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and X-ray crystallography help to determine whether a solvate has been formed. Solvate can be a stoichiometric or non-stoichiometric solvate and can include hydrates, such as hemihydrates, monohydrates, and dihydrates. Alternatively, the resulting compound can be anhydrous (e.g., anhydrous crystalline form). However, it is presumed that stable Type A is a monohydrate.

[0142] The following figures describe various analytical techniques to aid in the discussion of this application:

[0143] Polarized Light Microscopy (PLM)

[0144] Micrographs were taken at room temperature using a Nikon ECLIPSE Ci-POL polarizing microscope equipped with a 10x eyepiece and objectives with 5x, 20x, 50x, and 100x magnifications. Micrographs were taken after spreading the sample on a glass slide with a drop of oil.

[0145] X-ray powder diffraction (XRPD)

[0146] X-ray powder diffraction data were collected on a Malvern Panalytical Aeris diffractometer under ambient conditions. A few milligrams of sample were spread flat on a silicon sample holder, compacted with a glass slide, and protected with a piece of weighted paper. A 300 W X-ray generator equipped with a Cu (Kα) X-ray tube with a Kα2 / Kα1 intensity ratio of 0.50 was used at 40 kV and 7.5 mA. Data were collected from 3° to 40° (2θ) with a scan rate of 0.14° / s and a step size of 0.02° (2θ). X-ray powder diffraction data were collected under ambient conditions on a Malvern Panalytical Empyrean diffractometer. A few milligrams of sample were spread flat on a silicon sample holder, pressed with a glass slide, and protected with a piece of weighted paper. An 1800 W X-ray generator equipped with a Cu (Kα) X-ray tube with a Kα2 / Kα1 intensity ratio of 0.50 was used at 45 kV and 40 mA. Data were collected from 3° to 40° (2Θ) with a step size of 0.026° (2Θ) and a step time of 49.7 seconds.

[0147] Differential Scanning Calorimetry (DSC)

[0148] DSC is a thermal analysis technique in which the difference in the amount of heat required to raise the temperature of a sample and a reference is measured as a function of temperature. Differential Scanning Calorimetry was performed using a TA Discovery 2500 Series DSC, using several milligrams of material in a Tzero aluminum pan sealed with a Tzero seal lid. The sample was analyzed at a heating rate of 10°C per minute under a nitrogen flow rate of 50 ml / min. Heating Test: Differential Scanning Calorimetry was performed using a TA Discovery Series DSC2500. Several milligrams of material were placed in a Tzero aluminum pan and covered with a Tzero seal lid. The sample was analyzed at a heating rate of 10°C per minute under a nitrogen flow rate of 50 ml / min, from room temperature to the target temperature, and then cooled to room temperature.

[0149] Thermogravimetric analysis (TGA)

[0150] TGA is a test that is performed on a sample to determine the relationship between weight change and temperature. Thermogravimetric analysis data was collected using a TADiscovery 5500 Series TGA. A few milligrams of sample were placed in a Tzero aluminum pan and heated from room temperature to the target temperature at a heating rate of 10°C per minute under a nitrogen blanket (flow rate of 25 mL / min).

[0151] Dynamic Vapor Sorption (DVS)

[0152] Dynamic vapor sorption was performed at 25°C under a nitrogen stream using an ADVENTURE series DVS and an Intrinsic-1 DVS. Approximately 30 mg of adsorption material was used. The anhydrous form was analyzed using the following method: at 10% RH, 0% RH to 95% RH to 0% RH (5% RH from 90% RH to 95% RH to 90% RH). After reaching equilibrium under ambient conditions (50% RH), the hydrate sample was analyzed using the following method: 1. at 10% RH, ambient relative humidity to 95% RH (5% from 90% RH to 95% RH). 2. at 10% RH, 95% RH to 0% RH (5% from 95% RH to 90% RH). 3. at 10% RH, 0% RH to 95% RH (5% RH from 90% RH to 95% RH).

[0153] Water sorption curves were generated at 25°C using a DVS moisture balance flow system (Model Advantage 1.0) under the following conditions: sample weight approximately 10 mg, drying at 25°C for 60 min, adsorption range 0% to 95% RH, desorption range 95% to 0% RH, with a step interval of 5%. The equilibrium criterion was a weight change of <0.01% within 5 min, with a maximum of 120 min.

[0154] Water content (KF)

[0155] The water content of the samples was collected using a Metrohm 870 / 803 Karl Fischer titrator. The volumetric method was used, using HYDRAN / AL Composite 2 (FLUKA) as the titrant, anhydrous methanol as the solvent, and a stirring time of 400 seconds. 0.2 g of sample was weighed (accurate to 0.0001 g) and the water content was measured using the Karl Fischer titrator.

[0156] 1H NMR

[0157] 1H NMR data were acquired using a Bruker AVANCE NEO 400 MHz in DMSO-d6 or D2O.

[0158] High performance liquid chromatography (HPLC)

[0159] Chemical purity was determined using the SHIMADZU LC-20AD, using an HPLC method for purity testing.

[0160]

[0161] Polymorph screening

[0162] To confirm the stability and suitability of urodesine hemiglutarate form A, polymorph screening was performed to search for other potential crystalline forms and identify the most suitable form to confirm previous findings.

[0163] First, the urodesine hemiglutarate raw material (E0007-A01-02) was characterized by X-ray powder diffractometry (XRPD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), polarizing microscopy (PLM), proton nuclear magnetic resonance (1HNMR) and high performance liquid chromatography (HPLC) using the methods described previously.

[0164] Characterization results showed that the raw material was consistent with Type A glutarate, i.e., the monohydrate of hemiglutaric acid.

[0165] The solubility of the starting material (E0007-A01-02) was estimated at room temperature (~27°C).

[0166] Specifically, approximately 2 mg of solid was added to a HPLC glass vial. The following solvents were then added stepwise (50 / 50 / 200 / 700 μL) to the vial until the solid dissolved or the total volume reached 1.0 mL.

[0167] Solubility data is used to guide solvent selection for polymorph screening. The approximate solubility of the starting material (E0007-A01-02) at room temperature is as follows:

[0168]

[0169]

[0170] Using Form A glutarate as the starting material, polymorph screening was performed under 100 conditions by antisolvent addition, solid vapor diffusion, liquid vapor diffusion, slow evaporation, polymer-induced crystallization, temperature cycling, room temperature (RT) / 50°C slurry, and slow cooling.

[0171] The following is a summary of the polymorph screening results for urodesine hemiglutarate:

[0172]

[0173] Two new crystalline forms of the hemiglutarate salt (Form B and Form C glutarate) and two new free base forms (Form B / Form C free base) were observed.

[0174]

[0175] The hemiglutarate salt crystalline form was characterized using previously described analytical techniques coupled with variable temperature X-ray powder diffractometry (VT-XRPD):

[0176] X-ray powder diffraction data were collected at variable temperature using a Malvern Panalytical Empyrean diffractometer. A few milligrams of sample were spread flat on a sample holder, compacted with a glass slide, and protected with a sheet of weighing paper. An 1800 W X-ray generator equipped with a Cu (Kα) X-ray tube with a Kα2 / Kα1 intensity ratio of 0.50 was used at 45 kV and 40 mA. Data were collected from 3° to 40° (2Θ) with a step size of 0.026° (2Θ) and a step time of 49.7 seconds.

[0177] By XRPD / PLM / TGA / DSC / 1 H NMR / HPLC characterized the type A glutarate. XRPD diffraction pattern ( Figure 17 ) showed that it was consistent with the raw material type A hemiglutarate, consisting of small crystals and agglomerates. TGA results showed a weight loss of 5.7 wt% before 160 ° C (theoretical monohydrate is 5.2%), and DSC data showed three endothermic peaks at 117.8 ° C, 129.9 ° C and 142.3 ° C (peak) ( Figure 18 ). 1 H NMR spectrum ( Figure 19 ) showed that the stoichiometry of glutaric acid / free base was 0.5 and no residual ACN was present. The HPLC purity of Form A glutarate was 98.0 area %.

[0178] Glutarate Form B (E0007-A01-06) was only observed using X-ray powder diffractometer in variable temperature mode (VT-XRPD) by heating glutarate Form A to 130°C. Upon cooling to room temperature, it rapidly converted to glutarate Form A ( Figure 20 Based on the characterization results, type B glutarate is metastable at room temperature.

[0179] Form C glutarate was obtained from a temperature-cycled slurry in anisole / MeOH (1:2, v / v). XRPD diffractograms showed no change in form after drying overnight at room temperature. TGA / DSC results showed a 3.5% weight loss (2.7% theoretical hemihydrate) up to 140°C, and four endothermic peaks at 71.4°C, 105.7°C, 137.9°C, and 155.9°C (peak). 1 The HNMR spectrum showed an acid / free base stoichiometry of 0.5 and no residual solvent.

[0180] Form C glutarate was further heated to 110°C to investigate its thermal events, and XRPD results revealed no form change. TGA / DSC results of the heated sample revealed a 3.4% weight loss (theoretical value for hemihydrate is 2.7%) up to 140°C, as well as two endothermic peaks at 61.4°C and 156.4°C (peak). Based on these characterization results, Form C glutarate was presumed to be a channel-type hydrate (water molecules in channel-type hydrates exist adjacent to other water molecules in adjacent unit cells, forming channels in the crystal).

[0181] Form C was re-prepared in triplicate at 100 mg via temperature cycling slurry in anisole / MeOH (1:2, v / v). XRPD results (see Figure 21 and Figure 22 Figure 2 (in the figure) shows that two experiments produced glutarate Form C wet cake and one produced glutarate Form A+C. However, after drying under ambient conditions for approximately 6 hours (approximately 20°C / 32% RH), both of these glutarate Form C wet cakes converted to glutarate Form A. In addition, after drying under ambient conditions (approximately 25°C / 30% RT), two batches of glutarate Form C wet cake obtained by screening (partially) converted to glutarate Form A. Based on these results, glutarate Form C readily converts to glutarate Form A during drying under ambient conditions, reflecting its lower stability compared to glutarate Form A.

[0182] Based on the polymorph screening results, Form A glutarate exhibited good solid-state properties and stability and was therefore identified as a lead form for stable drug development.

[0183] Test Examples

[0184] The following further test examples are described to illustrate certain aspects and features of the present disclosure and should not be construed as limiting the full scope as defined by the appended claims.

[0185] Example 1 demonstrates that administration of the PNP inhibitor uluodesin hemiglutarate significantly reduced body weight gain, serum cholesterol, and blood glucose in a diet-induced obesity (DIO) mouse model without any reduction in food consumption.

[0186] Example 2 demonstrates that administration of urodesin hemiglutarate in a DIO mouse model reduces serum cholesterol, serum AST and ALT levels, blood glucose and insulin, and improves insulin sensitivity. Furthermore, urodesin treatment demonstrated a significant decrease in inguinal white adipose tissue (iWAT) / body weight (%), a decrease in fatty acid synthase, and an increase in thermogenic markers without any decrease in food consumption.

[0187] Example 3 demonstrated that the PNP inhibitor urodesin hemiglutarate significantly reduced total cholesterol and AST in the liver in the DIO mouse model.

[0188] Example 4 demonstrates that low-dose and intermittent administration of urodesine hemiglutarate (2 mg / kg) did not significantly affect lymphocytes in the DIO mouse model.

[0189] Example 1: Effects of low-dose and intermittent administration of urodesine hemiglutarate in a diabetic iodine (DIO) mouse model.

[0190] The purpose of this pilot study was to determine the effects of low-dose and intermittent administration of urodesine hemiglutarate on the following parameters: weight gain, food intake, fasting blood glucose concentration, and serum cholesterol. This study evaluated the efficacy of urodesine hemiglutarate on the listed parameters in the DIO mouse model of obesity and metabolic dysfunction.

[0191] Methods: C57 / BL6J mice were fed the obesogenic diet D12492 (60 kcal, % fat) starting at 6 weeks of age. 18-week-old mice (approximately 40 g) were treated with either vehicle (control) or 2 mg / kg of urodesin hemiglutarate orally three days a week (days 1, 2, and 3) for a 28-day period. On days -2 and 28, the animals were fasted for 6 hours by withdrawing food, and blood was collected for blood glucose and cholesterol measurements.

[0192] Results: As Figure 3 As shown in Figure 2, the group treated with urodesine hemiglutarate exhibited a significant decrease in weight gain compared to the control group. In addition to the decrease in weight gain, blood glucose and serum cholesterol also decreased. Food intake in the urodesine hemiglutarate-treated animals was similar to that of the control group. There were no signs of drug toxicity or adverse effects.

[0193] Example 2. Effects of low-dose and intermittent administration of urodesine hemiglutarate in the DIO mouse model.

[0194] The purpose of this study was to determine the effects of low-dose and intermittent administration of urodesine hemiglutarate on the following parameters: weight gain, food intake, fasting blood glucose concentration, serum insulin, serum cholesterol, plasma liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT), inguinal white adipose tissue (iWAT), BAT; inosine in plasma and NAD+ in whole blood; and expression of markers of lipogenesis (fatty acid synthesis) in iWAT and thermogenic markers in BAT. This study will evaluate the efficacy of urodesine hemiglutarate on the listed parameters in the DIO mouse model of obesity and metabolic dysfunction.

[0195] Methods: C57 / BL6J mice were fed the obesogenic diet D12492 (60 kcal% fat) from 6 weeks of age. 19-week-old mice (approximately 50 g) were treated with vehicle (control) or 2 mg / kg of urodesin hemiglutarate orally three days a week (days 1, 2, and 3) for a 28-day period. On days -2 and 28, animals were fasted for 6 hours (food only) and blood was collected for both blood glucose and cholesterol. Plasma liver enzymes (AST and ALT) and insulin were measured on day 28, and additional plasma and whole blood were frozen and used for inosine and NAD+ assays. At the end of the study, BAT and iWAT were collected, weighed, and subjected to quantitative PCR for the lipogenic markers stearoyl-CoA desaturase 1 (SCD-1) and fatty acid synthase (FAS), as well as the thermogenic marker uncoupling protein 1 (UCP-1). Inosine and NAD+ levels were determined in stored plasma and whole blood, respectively, by LC / MS / MS.

[0196] Results: The results are as follows Figure 4 Compared with the control group, the group treated with urodesin showed a decrease in blood glucose, plasma cholesterol, AST, ALT and insulin levels ( Figure 4 Compared with the control group, the iWAT / body weight% in the urodesin-treated group was significantly decreased ( Figure 4 F).

[0197] Compared with the vehicle-treated group, the urodesine-treated group showed increased expression of the thermogenic marker UCP-1 in BAT tissue and decreased expression of adipogenic markers (SCD-1 and FAS) in WAT tissue ( Figure 5 As in Example 1, the food intake of animals treated with urodesin hemiglutarate was similar to that of the control group. Unlike previous studies, the reduction in weight gain in the urodesin group was not significantly different from that in the control group. One possible reason is that the starting weight of the animals in this study was approximately 50 grams, which was 25% greater than in previous studies, and a longer treatment period may be required to achieve a significant reduction in weight gain. Both inosine and NAD+ in plasma and whole blood were significantly increased ( Figure 6 ).

[0198] These studies demonstrate that the PNP inhibitor urodesine hemiglutarate can potentially be used to treat conditions associated with metabolic syndrome, such as obesity, fatty liver disease, and cardiometabolic disease. There were no signs of any toxic or adverse effects of the drug.

[0199] Example 3. Effects of low-dose and intermittent administration of urodesin on liver parameters in the DIO mouse model.

[0200] The aim of this study was to determine the effects of low-dose and intermittent administration of urodesine hemiglutarate on liver parameters: liver weight, total cholesterol, triglycerides, AST, and ALT. This study evaluated the efficacy of urodesine hemiglutarate on the listed parameters in the DIO mouse model of obesity and metabolic dysfunction.

[0201] Methods: C57 / BL6J mice were fed the obesogenic diet D12492 (60 kcal% fat) from 6 weeks of age. 18-week-old mice (approximately 40 g) were treated with either vehicle (control) or 2 mg / kg of urodesin hemiglutarate orally three days a week (days 1, 2, and 3) for 8 weeks. At the end of the 8-week period, livers were removed and the following parameters were assessed: total cholesterol, triglycerides, AST, and ALT.

[0202] Results: As Figure 7 As shown in Figure 2, the group treated with urodesin showed a significant decrease in total cholesterol (TC) and AST. Although triglycerides and ALT decreased with treatment, they did not reach statistical significance.

[0203] This study supports the use of PNP inhibitors, such as urodesin, for the treatment of fatty liver disease.

[0204] Example 4. Effects of increasing doses of urodesine on lymphocyte populations in a DIO mouse model.

[0205] The purpose of this study was to determine the effects of increasing doses of urodesine hemiglutarate on lymphocytes in whole blood.

[0206] Methods: C57 / BL6J mice were fed the obesogenic diet D12492 (60 kcal / % fat) starting at 6 weeks of age. 18-week-old mice (approximately 40 g) were treated with vehicle (control) or 1 mg / kg, 2 mg / kg, 5 mg / kg, and 10 mg / kg of urodesin hemiglutarate orally, only three days a week (days 1, 2, and 3), for four weeks. At the end of the four weeks, total lymphocytes were counted by flow cytometry.

[0207] Results: As Figure 8 As shown in the , 1 mg / kg and 2 mg / kg doses of urodesin hemiglutarate had no effect on lymphocyte counts. Although the effects were not significant at 5 mg / kg and 10 mg / kg doses, a decrease in lymphocyte counts was observed. This study confirms that doses that improve various cardiometabolic parameters have no significant effect on lymphocytes.

Claims

1. A PNP inhibitor for use in the treatment or prevention of metabolic syndrome or a disease or condition induced by metabolic syndrome.

2. The PNP inhibitor for use according to claim 1, wherein the treatment or prevention of metabolic syndrome comprises treating or preventing at least one condition selected from the group consisting of elevated blood pressure, hyperglycemia, excess body fat, fatty liver, and dyslipidemia.

3. The PNP inhibitor for use according to claim 1 or 2, wherein the metabolic syndrome-induced disease or disorder is obesity or overweight.

4. PNP for use according to claim 1 or 2, wherein the metabolic syndrome induced disease or condition is selected from cardiovascular disease and type 2 diabetes; optionally wherein the cardiovascular disease is selected from heart disease, peripheral vascular disease and stroke.

5. The PNP inhibitor for use according to claim 1 or 2, wherein the metabolic syndrome-induced disease or disorder is fatty liver disease, and optionally the fatty liver disease is selected from NAFLD and NASH.

6. A PNP inhibitor for use according to any preceding claim, wherein the PNP inhibitor is selected from compounds of formula I, formula II or formula III: or a pharmaceutically acceptable salt thereof.

7. The PNP inhibitor for use according to claim 5, which is a compound of formula I or a pharmaceutically acceptable salt thereof; optionally, wherein the pharmaceutically acceptable salt is hemiglutarate.

8. A PNP inhibitor for use according to any preceding claim, wherein the PNP inhibitor is provided in a dose of 1 mg to 1000 mg / day; optionally in a dose of 1 mg to 250 mg / day.

9. A PNP inhibitor for use according to any one of claims 1 to 7, wherein the PNP inhibitor is provided in a dose of 1 mg to 150 mg; preferably in a dose of 10 mg to 40 mg.

10. A PNP inhibitor for use according to any preceding claim, wherein the PNP inhibitor is provided on an intermittent dosing regimen.

11. A PNP inhibitor for use according to claim 10, wherein the intermittent dosing regimen comprises or consists of repeated treatment cycles, wherein one treatment cycle comprises or consists of 1 to 6 days of treatment followed by a pause of at least 1 day; optionally wherein one treatment cycle comprises or consists of a period of treatment of 2 to 4 days followed by a pause of at least 2 days.

12. A PNP inhibitor for use according to any one of claims 8 to 11, wherein the dosing regimen results in a reduction in the blood lymphocyte population of less than 20%.

13. A PNP inhibitor for use according to any preceding claim, wherein the PNP inhibitor is provided in combination with at least one additional active agent selected from an ENT1 inhibitor, a GLP-1 agonist, an anti-obesity agent and an anti-diabetic drug.

14. A method for treating a subject suffering from metabolic syndrome or a metabolic syndrome-induced disease, disorder or condition, the method comprising administering to the subject a pharmaceutically effective amount of a PNP inhibitor.

15. The method of claim 14, wherein the treatment of metabolic syndrome comprises treating or preventing at least one condition selected from the group consisting of elevated blood pressure, hyperglycemia, excess body fat, fatty liver, and dyslipidemia.

16. The method of claim 14, wherein the metabolic syndrome induced disease or condition is obesity or being overweight.

17. The method of claim 14, wherein the metabolic syndrome induced disease or condition is selected from cardiovascular disease and type 2 diabetes; optionally wherein the cardiovascular disease is selected from heart disease, peripheral vascular disease and stroke.

18. The method of claim 14, wherein the metabolic syndrome induced disease or disorder is fatty liver disease, and optionally the fatty liver disease is selected from NAFLD and NASH.

19. A method of increasing the percentage of brown adipose tissue (BAT) to body weight in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a PNP inhibitor.

20. A method of improving the lipid profile of a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a PNP inhibitor.

21. The method of any one of claims 12 to 17, wherein the PNP inhibitor is provided in an intermittent dosing regimen.

22. The method according to any one of claims 14 to 21, wherein the PNP inhibitor is urodesine, optionally provided as the hemiglutarate salt.

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