Treatment of disease

Abstract

This disclosure relates to the use of Purine Nucleoside Phosphorylase (PNP) inhibitors such as ulodesine and its salts, in the treatment and / or prevention of diseases associated with nicotinamide adenine dinucleotide (NAD+) depletion, including diseases of mitochondrial dysfunction (including neurodegeneration and peripheral neuropathies), the preservation of cognitive function and in muscular disorders such as sarcopenia; and in metabolic syndrome and associated conditions. In particular the disclosure provides the use of PNP inhibitors such as ulodesine in the treatment of neurodegenerative conditions such as Parkinson's disease and amyotrophic lateral sclerosis.

Description

[0001] TREATMENT OF DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to United Kingdom Patent Application No. 2408787.6, filed on June 19, 2024, the entire contents of which are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] This disclosure relates to the use of Purine Nucleoside Phosphorylase (PNP) inhibitors such as ulodesine and its salts, in the treatment and / or prevention of diseases associated with nicotinamide adenine dinucleotide (NAD+) depletion, including diseases of mitochondrial dysfunction (including neurodegeneration and peripheral neuropathies), the preservation of cognitive function and in muscular disorders such as sarcopenia; and in metabolic syndrome and associated conditions.

[0006] SUMMARY OF THE INVENTION

[0007] The inventors have identified that treatment of subjects with a PNP inhibitor such as ulodesine and its salts, leads to a significant increase in tissue NAD+. Further, despite increasing tissue NAD+, treatment with PNP inhibitors does not lead to increases in potentially toxic products of the NAD+metabolic pathway. The inventors have further identified new treatment regimens that reduce or eliminate immune related side effects associated with the use of PNP inhibitors.

[0008] The present disclosure relates generally to the use of a PNP inhibitor to increase the level of NAD+in tissues of a subject and thereby prevent or treat diseases or conditions that benefit from such treatment. In some embodiments the diseases or conditions are those associated with a reduced level (i.e. below population normal range) of NAD+in tissues. In some embodiments the disease or condition is associated with an age related decline in NAD+in tissues.

[0009] In some embodiments the disclosure provides a PNP inhibitor for use in the treatment or prevention of a disease selected from diseases of mitochondrial dysfunction, age related cognitive decline, and muscular disorders. Diseases of mitochondrial dysfunction, include so called “mitochondrial diseases” and those conditions in which tissues have a compromised mitochondrial function. Treatment with a PNP inhibitor may lead to an improved mitochondrial and / or cellular function and an increased cell viability.

[0010] In some embodiments the disease of mitochondrial dysfunction is a neurodegenerative disease or a peripheral neuropathy. In some embodiments the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), vascular dementia, Lewy body dementia, frontotemporal dementia, primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), and hereditary spastic paraplegia (HSP), ataxias (such as spinocerebellar ataxia, Friedreich's ataxia, ataxia-telangiectasia, xeroderma pigmentosum group A (XPA), Cockayne syndrome, and fragile X-associated ataxia), dyskinesias (such as cerebral palsy), Batten’s disease, optical neurodegenerative diseases (such as glaucoma and inherited retinal degenerative diseases (e.g. Leber’s hereditary optic neuropathy (LHON)), tauopathies and prion diseases (such as Creutzfeldt- Jakob disease).

[0011] In some embodiments muscular disorders include without limitation, disorders of muscle mass or of muscle fatigue, including without limitation, neuromuscular degeneration, muscle atrophy cachexia sarcopenia, muscular dystrophies (MD - such as Duchenne MD, facioscapulohumeral MD and limb girdle muscular dystrophy), chronic fatigue syndromes (including ME / CFS and Long Covid syndrome) and fibromyalgia. In some embodiments the muscle disorder is sarcopenia.

[0012] In some embodiments the disclosure provides a PNP inhibitor for use in the treatment or prevention of metabolic syndrome or a metabolic syndrome-induced disease or condition. In some embodiments the metabolic syndrome induced disease or condition is selected from hypertension, hyperglycemia, excess body fat, fatty liver disease and dyslipidemia; optionally the fatty liver disease is selected from NAFLD and NASH.

[0013] In some embodiments the metabolic syndrome induced disease or condition is selected from cardiovascular disease, and type 2 diabetes. In some embodiments the cardiovascular disease is selected from heart disease, peripheral vascular disease and stroke.

[0014] In some embodiments, a PNP inhibitor is used in combination with one or more precursors of NAD+biosynthesis, to treat any of the conditions taught herein. In some embodiments the precursor of NAD+biosynthesis is, for example, trigonelline (TG), nicotinic acid (NA), nicotinamide (NAM), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) and / or dihydronicotinamide riboside (NRH). In some embodiments the precursor of NAD+biosynthesis is NR and / or NAM.

[0015] In some embodiment, a PNP inhibitor is used in combination with one or more inhibitors of NAD+metabolising enzymes in the treatment of any of the conditions taught herein. In some embodiments NAD+metabolising enzymes include CD38, PARP (poly-ADP ribose polymerase) and SARM1 (sterile alpha and TIR motif containing 1). In some embodiments, PNP inhibitors may be used in combination with one or more precursors of NAD+biosynthesis and one or more inhibitors of an NAD+metabolising enzyme.

[0016] In some embodiments the PNP inhibitor may be used in a dosing regimen in which the PNP inhibitor is administered at a dose that is therapeutically effective to provide a treatment for the disease or condition but does not reduce, or does not effectively reduce lymphocyte count. In some embodiments a PNP inhibitor is provided in low and / or intermittent dosing regimen. In some embodiments the dosing regimen includes providing the PNP inhibitor for at least one day; optionally at least two days; optionally at least three days; followed by a period, wherein the subject receives no drug for at least one day; and optionally repeating this treatment.

[0017] In some embodiments the dosing regimen includes, the steps of: (i) treating the patient with the PNP inhibitor continuously until the level of total lymphocytes and / or a sub population thereof in a first sample from the patient falls below a first predetermined level; (ii) pausing treatment to allow the level of total lymphocytes and / or a sub population thereof to recover at least partially; and (iii) optionally repeating steps (i) and (ii). In some embodiments the level of total lymphocytes and / or a sub population thereof in the sample is determined by counting the total lymphocytes and / or a sub population thereof, or by determining the level of a lymphocyte or lymphocyte subpopulation marker in the sample.

[0018] The disclosure further provides methods for the treatment of any disease or condition disclosed herein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a PNP inhibitor, optionally in combination with a precursor of NAD+biosynthesis.

[0019] The disclosure further provides methods for the treatment or prevention of a disease or condition disclosed herein (particularly a disease of mitochondrial dysfunction) in an individual in need thereof or at risk thereof, the method comprising administering to the individual a PNP inhibitor in an amount effective to increase NAD+levels in tissues. In some embodiments, the PNP inhibitor may be used in combination with a precursor of NAD+biosynthesis and / or an inhibitor of one or more NAD+metabolising enzymes.

[0020] The disclosure further provides use of a PNP inhibitor in the manufacture of a medicament for the treatment or prevention of any of the diseases or conditions disclosed herein; optionally wherein the PNP inhibitor is used in combination with a precursor of NAD+biosynthesis and / or an inhibitor of one or more NAD+metabolising enzymes.

[0021] DETAILED DISCLOSURE THE INVENTION

[0022] Purine nucleoside phosphorylase, (PNP - EC 2.4.2.1) is an enzyme involved in purine, pyrimidine and nicotinamide (NAM) metabolism. Individuals with genetic PNP deficiency suffer from a severe combined immunodeficiency including T-cell deficiency as well as recurrent severe infections, neurological dysfunction and autoimmune conditions.

[0023] Inhibitors of the enzyme, such as ulodesine and forodesine (also known as immucillin H) have been investigated as hypouricemic drugs for the treatment of gout (Hollister et al. (2012), Gandhi et al. (2005) and for use in the treatment of acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphocytic leukemia (B-ALL). Forodesine hydrochloride (Mundesine ) has been approved in Japan for the treatment of relapsed / refractory peripheral T-cell lymphoma.

[0024] Applicant’s co-pending application (WO2024126630) discloses certain aspects of the use of PNP inhibitors, including ulodesine and forodesine, for the treatment of metabolic syndrome and associated conditions.

[0025] The present inventors have demonstrated that PNP inhibitors can be used to increase the level of NAD+in tissues such as brain, blood and muscle and so these compounds can be used to treat a variety of conditions associated with a reduced level (i.e. below population norms) of NAD+and, since maintenance of physiological NAD+levels is critical to the proper functioning of mitochondria, the compounds can be used to treat diseases and conditions associated with mitochondrial dysfunction, (including “mitochondrial diseases” and those conditions displaying a compromised mitochondrial function).

[0026] Nicotinamide adenine dinucleotide (NAD+or NAD) is an essential coenzyme that mediates various redox reactions and plays a critical role in mitochondrial energy production through the TCA cycle and oxidative phosphorylation. NAD+is synthesised via three classical routes (i) de novo from tryptophan, (ii) via the Preiss-Handler pathway from nicotinic acid (NA), or (as per the majority of NAD+synthesis in humans) via a salvage pathway from nicotinamide (NAM), nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). Within this pathway, PNP converts NR to NAM (see figure 1).

[0027] NAD+levels steadily decline during aging. By the time a mouse or human is middle aged, levels of NAD+have fallen to half of youthful levels. Several studies in recent years have shown that treatment of old mice with poly (ADP-ribose) polymerase (PARP) inhibitors (which increase NAD+levels), with NAD+precursors or with inhibitors of NAD+degradation, can improve health. Depleted NAD+levels are further associated with cognitive decline and mitochondrial dysfunction and associated conditions as described further below.

[0028] Without wishing to be bound by any theory, the inventors believe that PNP inhibitors increase NAD+levels in tissues through two mechanisms:

[0029] (1) Phosphoribosyl pyrophosphate (PRPP) sparing: The conversion of NAM to NAD+is limited by nicotinamide phosphoryl transferase (NAMPT) as NAMPT uses PRPP as co-substrate which is limiting. Hypoxanthine guanine phosphoribosyl transferase (HGPRT) which converts hypoxanthine and guanine to their respective nucleotides also uses PRPP as substrate. HGPRT is normally the major consumer of PRPP within this system, when PNP is inhibited hypoxanthine and guanine levels decrease, thus PRPP is spared. It is believed therefore that a combination of the NAD+precursor NAM with PNP inhibitors will further elevates NAD+levels (figure 1)

[0030] NA and trigonelline are converted to the mononucleotide through nicotinic acid phosphoribosyl transferase (NAPRT) which also uses PRRP as a co-substrate. Thus the combination of PNP inhibitor with NA and / or trigonelline can also further elevate NAD+levels.

[0031] (2) Inhibition of NR breakdown: PNP catalyses the breakdown of NAD+precursors, NR and NMN to nicotinamide (NAM) thus, inhibition of PNP in combination with these precursors further elevates NAD+levels.

[0032] The inventors have demonstrated (see below) that a further advantage of the use of PNP inhibitors to raise NAD+levels is that an increase in methylated analogues of nicotinamide (which are known to be uremic toxins) is avoided which is not the case when the NAD+precursors NR and NMN are used to elevate NAD+These uremic toxins are also believed to impact methylation of DNA and proteins, leading to changes in cellular transcriptome and proteome.

[0033] In some embodiments the treatments and uses disclosed herein are effective to increase the level of NAD+in tissues. In some embodiments the treatments and uses are effective to increase the level of NAD+in tissues by at least 10%, 20%, 30%, 40%, 50%, 100% or more.

[0034] Mitochondria are responsible for, inter alia, ATP production, cellular metabolism, signal transduction, calcium homeostasis, and immune responses. Maintenance of the mitochondrial NAD’ pool is of crucial importance for mitochondrial function and, in turn, the stability of mitochondrial function plays a crucial role in many tissues. Mitochondrial dysfunction can lead to pathological changes in cells, such as an impaired calcium buffer period, excessive generation of free radicals, increased mitochondrial membrane permeability, and oxidative stress (OS). These mitochondrial dysfunctions are associated with pathological changes and lead to a variety of disease states (diseases of mitochondrial dysfunction). Among these disease states are various neurological and neurodegenerative diseases (Zhao et al 2022).

[0035] The group of diseases known as “mitochondrial diseases” are caused by mutations in either mitochondrial DNA, or in nuclear DNA coding for mitochondrial proteins. In some cases they are the result of acquired mitochondrial dysfunction due to drug side effects, infections, or other environmental factors.

[0036] Mitochondrial diseases include primary and secondary mitochondrial diseases. Such diseases include (but are not limited to) mitochondrial myopathies, maternally inherited diabetes mellitus and deafness (MIDD), Leber's hereditary optic neuropathy (LHON), Leigh syndrome, retinitis pigmentosa, and ptosis (NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with ragged red fibres syndrome (MERRF), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS syndrome) and mitochondrial DNA depletion syndrome (MDDS).

[0037] Primary mitochondrial diseases are caused by pathogenic variants in genes coding for the mitochondrial respiratory chain and related proteins. They therefore characterised by impaired oxidative phosphorylation and are typically neuro-metabolic disorders. Primary mitochondrial diseases include (but are not limited to) Kearns-Sayre syndrome, Pearson syndrome, Leigh syndrome, NARP, ataxia neuropathy syndrome, Alpers-Huttenlocher syndrome, Mitochondrial complex IV deficiency, Cytochrome c oxidase (COX) deficiency, (Complex IV deficiency), MNGIE, MELAS, MERRF and LHON.

[0038] Secondary mitochondrial disease (SMD) are caused by variants of nuclear-encoded genes that do not influence (or indirectly influence) oxidative phosphorylation (OXPHOS). These include (but are no to limited to) Friededreich’s ataxia, and various other conditions such as schizophrenia, sepsis, cardiovascular diseases, cancer, diabetes and metabolic syndrome.

[0039] Additional conditions whose cause and / or symptoms are believed to include mitochondrial related features such as a compromised mitochondrial function (for example leading to increased oxidative stress in tissues) and which also benefit from treatment as described herein, include neurodegenerative disorders, neuronal activation disorders, and muscle disorders.

[0040] Neurodegenerative diseases are those that result in a progressive loss of structure or function of neurons. Neurodegenerative disorders include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), vascular dementia, Lewy body dementia frontotemporal dementia, primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), hereditary spastic paraplegia (HSP), ataxias (such as spinocerebellar ataxia, Friedreich's ataxia, ataxiatelangiectasia, xeroderma pigmentosum group A (XPA), Cockayne syndrome and fragile X-associated ataxia), dyskinesias (such as cerebral palsy), Batten’s disease; optical neurodegenerative diseases (such as glaucoma and inherited retinal degenerative diseases (e.g. Leber’s hereditary optic neuropathy (LHON)), tauopathies and prion diseases (such as Creutzfeldt- Jakob disease).

[0041] There is strong evidence that mitochondrial dysfunction and oxidative stress play a central role in neurodegenerative disease pathogenesis, including in AD, PD, HD, and in ALS (Linn and Flint (2006) and supplementation with NAD+has been demonstrated to ameliorate disease phenotype by activating mitochondrial function (Bresque et al.,( 2023). Further, NAD+metabolism is also believed to play an important role in axonal degeneration, a characteristic feature of both neurodegenerative diseases and peripheral neuropathy (Hikosaka et al (2021), Eautrup et al. (2019)). Further, there is increasing evidence of the involvement of NAD+in the aetiology of neurodegenerative diseases of the eye including glaucoma (Tribble et al. 2023).

[0042] Muscle disorders include disorders of muscle mass or of muscle fatigue, including without limitation, neuromuscular degeneration, muscle atrophy, cachexia, sarcopenia, muscular dystrophies (such as Duchenne MD, facioscapulohumeral MD and limb girdle MD); chronic fatigue syndrome and fibromyalgia. In some embodiments the muscle disorder is sarcopenia.

[0043] Parkinson's disease (PD) significantly affects muscles, causing stiffness, rigidity, and tremors, leading to a range of movement and posture problems. While there isn't a specific class of drugs that directly improves muscle strength in Parkinson's disease, some medications can help manage symptoms that affect muscle strength, and exercise, particularly resistance training, is crucial for maintaining and improving it. Research suggests that NAD+supplementation, may offer therapeutic benefits for PD by improving mitochondrial function, reducing neuroinflammation, improving muscle strength and potentially slowing disease progression.

[0044] ALS, directly damages motor neurons, causing progressive muscle weakness and eventual paralysis. While there is currently no cure for ALS, some drugs and treatments can help improve muscle strength and function, and potentially slow disease progression. Studies indicate that NAD+levels are reduced in ALS patients, potentially contributing to disease progression. Boosting NAD+levels through pharmacologic or genetic manipulation shows promise in ALS models. NAD+ can improve motor neuron function, reduce oxidative stress, improve muscle strength and decrease mitochondrial dysfunction, all of which are implicated in ALS pathology.

[0045] In some embodiments the PNP inhibitor may be used in the preservation of cognitive function. The preservation of cognitive function by increasing NAD+levels has been identified as a promising treatment strategy for example in age-related cognitive decline, in dementias such as AD and vascular dementia, particularly early stage dementias where cognitive decline is, as yet, limited, and in traumatic brain injury.

[0046] Treating APP / PS1 -mutant mice with NR improved learning and memory, and NA treatment of mice following intracerebroventricular injection of AP1-42 prevents memory deficits. Nicotinamide mono nucleotide (NMN) ameliorated cognitive impairment in the APPswe / PSldE9 mouse model of AD (Hou et al (2021), Rehman et al. (2021), Yao et aZ.(2017)). In a rat model of chronic cerebral hypoperfusion (a major cause of vascular dementia) direct supplementation with NAD+ameliorated the impairment of learning and memory.

[0047] In some embodiments, the PNP inhibitor may be used to treat metabolic syndrome and associated conditions. Metabolic syndrome is a cluster of conditions that occur together, increasing risk of heart disease, liver disease, stroke and type 2 diabetes. This cluster of conditions includes increased blood pressure (hypertension), high blood sugar (hyperglycemia), excess body fat, particularly around the waist, fatty liver and abnormal cholesterol or triglyceride levels (dyslipidemia).

[0048] Several studies in recent years have shown that treatment of old mice with poly (ADP-ribose) polymerase (PARP) inhibitors (which increase NAD+levels), with NAD+precursors or with inhibitors of NAD+degradation can greatly improve health. Observed effects include increased insulin sensitivity, decreased cholesterol and triglyceride levels, decreased weight gain, reduced stem cell senescence, and extension of lifespan (Bai et al. (2011); Gomes et al. (2013); Yoshino etal. (2011); Zhang etal. (2016)). Improvement of these metabolic parameters essentially leads to “healthy longevity”, an effect that has been demonstrated with NAD+precursors and also with inhibitors of NAD+degraders such as CD38 (Peclat et al (2022)). Genetic mouse models with altered NAD+biosynthesis phenotypes show similar results.

[0049] There is now a body of evidence that the actions of brown adipose tissue (BAT) or the browning (conversion) of white adipose tissue (WAT) can protect against obesity (Harms and Seale (2013); Bartelt and Heeren (2014). The molecular mechanisms that are responsible for the energy-dissipating qualities of brown fat have been studied in detail (Cannon and Nedergaard (2004); Rosen and Spiegelman (2014). Uncoupling protein-1 (UCP-1) has been identified as the key factor controlling the thermogenic capacity of brown adipocytes (Klingenberg (1999). UCP-1 disrupts the electrochemical gradient across the mitochondrial membrane by allowing protons to re-enter the mitochondrial matrix. Consequently, mitochondrial fatty acid oxidation is increased and chemical energy is “wasted” through heat production (known as “adaptive thermogenesis”). This process has long been believed to occur exclusively in brown and brown-like (beige) adipocytes.

[0050] As the amount of metabolically active BAT seems to be particularly low in patients with obesity or diabetes mellitus, it would be desirable to identify therapies that would increase the production of BAT and / or increase the capacity for adaptive thermogenesis through activation of BAT in these patients.

[0051] Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, and covers a spectrum of conditions, from simple steatosis, where lipid accumulates in the liver, to nonalcoholic steatohepatitis (NASH) characterized by fibrosis, inflammation and hepatocyte cell death. If left unchecked, NASH can progress to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). One goal of effectively treating NAFLD and NASH is therefore to prevent or reduce the likelihood of these conditions progressing to cirrhosis and HCC.

[0052] Genetic and interventional models in mice suggest that modulating the NAD+synthetic pathway has the potential to ameliorate fatty liver disease For example, treatment with NAD+precursors has been suggested to prevent the progression of NAFLD to NASH and NAD+precursor supplementation is associated with decreased hepatic stellate cell activation, and decreased fibrosis. (Dall et al (2022)). Furthermore, sirtuins, which cleave NAD+to NAM and O-acetyl-ADP-ribose, may also play a role in NAFLD. Decreased hepatic expression of SIRT1, SIRT3, SIRT5, and SIRT6 has been reported in patients with NAFLD (Wu et al (2014)), while liver-specific knockout of SIRT1 and SIRT6 in mice results in hepatic lipid accumulation (Kim et al., (2010); Purushotam et al. (2009)). Thus overexpression of SIRT1 appears to protect against high fat diet (HFD)-induced obesity (Pfluger et al (2008)), and increasing the hepatic NAD+pool by inhibition of NAD+-consuming PARPs decreases weight gain and hepatic steatosis development by a SIRT1 -dependent mechanism in high-fat high-sucrose-fed mice (Gariani et al (2017)). Hence, sufficient sirtuin activity appears important for prevention of hepatic lipid accumulation.

[0053] The term “PNP inhibitor” includes those compounds that inhibit the enzymatic conversion of a purine nucleoside to the corresponding purine and alpha-D-ribose 1 -phosphate by purine nucleoside phosphorylase (EC2.4.2.1), in the presence of phosphate; for example the enzymatic conversion of inosine into hypoxanthine and / or guanosine into guanine. Compounds having in-vitro inhibitory constant (Ki) values of less than about 5 x 107M, typically less than about 1 x 108M, and preferably less than 5 x 109M are preferred for in vivo use.

[0054] Many examples PNP inhibitors are known. In some embodiments the PNP inhibitor is a guanine analogue, a guanosine analogue, a hypoxanthine analogue or an inosine analogue. In some embodiments, in each case, the analogue may comprise a substituent at the 8-position. In some embodiments the PNP inhibitor is a PNP transition state analogue.

[0055] 8-substituted guanines include, but are not limited to 8-aminoguanine (8-AG), 8 -hydroxy guanine (8- OH) and or 8 -nitroguanine (8-NO2G). 8-substituted guanosines include, but are not limited to, 8- aminoguanosine (8-AGN) and 8 -hydroxy guanosine (8-OHGN). 8-substituted inosines include, but are not limited to, 8-aminoinosine (8-AI). 8-substituted hypoxanthines include, but are not limited to, 8- aminohypoxanthine (8-AHX). Further PNP inhibitors are described in the following disclosures:

[0056] US5565463 describes PNP inhibitors of the formula: Wherein

[0057] (a) — CH? Ar represents: in which Ri represents hydrogen, halogen, Ci-Cg-atkyl, Ci-Cg-alkoxy, benzyloxy, hydroxy or trifluoromethyl; and R2 represents hydrogen, halogen, Ci-Cs-alkyl, Ci-Cg-alkoxy, benzyloxy, hydroxy or trifluoromethyl, provided R2 represents hydrogen or Ci-Cs-alkyl if Ri represents trifluoromethyl, or that Ri represents hydrogen or Ci-Cs-alkyl if R2 represents trifluoromethyl; or

[0058] (b) — CH2Ar represents in which X represents sulfur or oxygen and in which attachment to the thiophene or furan ring is at the 2- or 3 -position; and tautomers thereof.

[0059] US5008270 describes PNP inhibitors that are derivatives of 2-amino-7-(substituted methyl)-3H,5H- pyrrolo[3,2-d]-pyrimidin- 4-one (I) where substituted methyl is — CH2 — R, where the R group is an optionally substituted heteroalicyclic group. Preferably, the heteroalicyclic group is a 5 or 6 membered saturated ring having oxygen, nitrogen, or sulfur as the heterocyclic atom; wherein Ri is H, NH2, or OCH3, R2 is an optionally substituted cyclic group optionally containing one or more heteroatoms, R3 and R4 are independently H or C1-4 alkyl, m is 0-4, n is 0-6, p is 0-1, X is CN, CSNH2, PO(OH)2, COOH, SO2NH2, NH2, OH, CNHNH2, tetrazole, triazole or COR5 where R5 is CM alkyl, CF3, NH2, or OCi 4 alkyl, and Y is O or NH.

[0060] US5726311 describes PNP inhibitors comprising a compound of the formula:

[0061]

[0062] Wherein R1is H, NH2, or OCH3, R2is an optionally substituted cyclic group optionally containing one or more heteroatoms, R3and R4are independently H or C1-4 alkyl, m is 0-4, n is 0-6, p is 0-1, X is CN, CSNH2, PO(OH)2, COOH, SO2 NH2, NH2, OH, CNHNH2, tetrazole, triazole or COR5where R5is CM alkyl, CF3, NH2, or OCi 4 alkyl, and Y is O or NH.

[0063] US5985848 describes PNP inhibitors of the formula wherein A is CH or N; B is chosen from OH, NH2, NHR, H or halogen; D is chosen from OH, NH2, NHR, H, halogen or SCH3; R is an optionally substituted alkyl, aralkyl or aryl group; and X and Y are independently selected from H, OH or halogen except that when one of X and Y is hydroxy or halogen, the other Is hydrogen; and Z is OH or, when X is hydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ, Q is an optionally substituted alkyl, aralkyl or aryl group; or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof; or a prodrug thereof.

[0064] US7109331 describes PNP inhibitors of the formula: wherein:

[0065] A is selected from N, CH and CR, where R is selected from halogen, optionally substituted alkyl, aralkyl and aryl, OH, NH2, NHR1, NR’R2and SR3, where R1, R2and R3are each optionally substituted alkyl, aralkyl or aryl groups; B is selected from OH, NH2, NHR4, H and halogen, where R4 is an optionally substituted alkyl, aralkyl or aryl group; D is selected from OH, NH2, NHR5, H, halogen and SCH3, where R5is an optionally substituted alkyl, aralkyl or aryl group; X and Y are independently selected from H, OH and halogen, with the proviso that when one of X and Y is hydroxy or halogen, the other is hydrogen; Z is OH, or, when X is hydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ and OQ, where Q is an optionally substituted alkyl, aralkyl or aryl group; and W is OH or H, with the proviso that when W is OH, then A is CR where R is as defined above; or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof; or a prodrug thereof.

[0066] US7553839 describes PNP inhibitors of the formula:

[0067] V is selected from CH2 and NH, and W is selected from NR1and NR2; or V is selected from NR1and NR2, and W is selected from CH2 and NH; X is selected from CH2 and CHOH in the R or S- configuration; Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR1and NR2then Y is hydrogen; Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q is an optionally substituted alkyl, aralkyl or aryl group; R1is a radical of the formula (II)

[0068] R2is a radical of the formula (III)

[0069] A is selected from N, CH and CR, where R is selected from halogen, optionally substituted alkyl, aralkyl or aryl, OH, NH2, NHR3, NR3R4and SR5, where R3, R4and R5are each optionally substituted alkyl, aralkyl or aryl groups; B is selected from OH, NH2, NHR6, SH, hydrogen and halogen, where R6is an optionally substituted alkyl, aralkyl or aryl group; D is selected from OH, NH2, NHR7, hydrogen, halogen and SCH3, where R7is an optionally substituted alkyl, aralkyl or aryl group; E is selected from N and CH; G is selected from CH2 and NH, or G is absent, provided that where W is NR1or NR2and G is NH then V is CH2, and provided that where V is NR1or NR2and G is NH then W is CH2; or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.

[0070] US7427624 provides conjugates of various PNP inhibitors.

[0071] WO13057722A1 describes 4-[2-(5-Amino-lH-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N- (1, 3-thiazol-4-yl)benzenesulfonamide and its tosylate salt.

[0072] W008030119A1 provides PNP inhibitors of the formula:

[0073] Wherein R1is H or NR3R4; R2is H or is an alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, or aryl group each of which is optionally substituted with one or more hydroxy, alkoxy, thiol, alkylthio, arylthio, aralkylthio, halogen, carboxylic acid, carboxylate alkyl ester, nitro, or NR3R4groups, where each alkylthio, arylthio and aralkylthio group is optionally substituted with one or more alkyl, halogen, amino, hydroxy, or alkoxy groups; provided that when R1is H, R2is an alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, or aryl group which is substituted with at least one NR3R4group; R3and R4, independently of each other, is H or is an alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, or aryl group each of which is optionally substituted with one or more hydroxy, alkoxy, thiol, alkylthio, arylthio, aralkylthio, halogen, carboxylic acid, carboxylate alkyl ester, nitro, or NR3R4groups, where each alkylthio, arylthio and aralkylthio group is optionally substituted with one or more alkyl, halogen, amino, hydroxy, or alkoxy groups; A is N or CH; B is OH or alkoxy; and D is H, OH, NH2, or SCH3; provided that when R1is NR3R4, R2is H, A is CH, B is OH, and D is H, then R3is not hydroxyethyl or hydroxypropyl when R4is hydroxyethyl; and provided that when R1is NR3R4, R2is H, A is CH, B is OH, and D is NH2, then R3is not hydroxyethyl when R4is H, methyl, ethyl, or hydroxyethyl, and R4is not hydroxyethyl when R3is H, methyl, ethyl, or hydroxyethyl; or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester prodrug form thereof.

[0074] US8283345 provides PNP inhibitors of the formula: wherein: W and X are each independently selected from hydrogen, CH2OH, CH2OQ and CH2SQ; Y and Z are each independently selected from hydrogen, halogen, CH2OH, CH2OQ, CH2SQ, SQ, OQ and Q; Q is an alkyl, aralkyl or aryl group each of which may be optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy; R1is a radical of the formula: or R1is a radical of the formula:

[0075] A is selected from N, CH and CR2, where R2is selected from halogen, alkyl, aralkyl, aryl, OH, NH2, NHR3, NR3R4and SR5, where R3, R4and R5are each alkyl, aralkyl or aryl groups optionally substituted with hydroxy or halogen, and where R2is optionally substituted with hydroxy or halogen when R2is alkyl, aralkyl or aryl; B is selected from hydroxy, NH2, NHR6, SH, hydrogen and halogen, where R6is an alkyl, aralkyl or aryl group optionally substituted with hydroxy or halogen; D is selected from hydroxy, NH2, NHR7, hydrogen, halogen and SCH3, where R7is an alkyl, aralkyl or aryl group optionally substituted with hydroxy or halogen; E is selected from N and CH; G is a C1-4 saturated or unsaturated alkyl group optionally substituted with hydroxy or halogen, or G is absent; or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.

[0076] WO21083438A1 provides PNP inhibitors of the formula,

[0077] Wherein: X is independently -NH- or -S- in combination with Ar, Z, G and R; Ar is independently aryl or heteroaryl in combination with X, Z, G and R; Z is omitted or it is independently -CH2-, -CH2O-, - OCH2- -CH2OCH2- and -CH=CH- attached independently to the position 2- or 3- of the Ar relative to the X, in combination with X, Ar, G and R; G is a group selected from -COA1, -SO2A1, -P(O)(A’)(A2), -OH and -H in combination with X, Ar, Z and R, wherein: A1and A2are identical or different groups and consist independently of -OH, -OR1and -NHR2, wherein R1is independently linear or branched Ci-Ce alkyl chain, C6-C12 aryl, arylalkyl and -CH2OC(O)R2and R2is independently linear or branched C1-C10 alkyl chain and -CHR3COOR4, wherein R3is independently linear or branched C1-C10 alkyl chain in which any -CH2- group can be replaced independently by -S-, -O-, -NH-, C6-C12 aryl and Ce- C12 arylalkyl, and R4is independently linear or branched C1-C10 alkyl chain; R is -H or a group selected from -OH, -O(aryl), -O(arylalkyl), -C(O)-, -CN and halogen in combination with X, Ar, Z and G; with the proviso that when Ar is phenyl and Z is not present, then R and G are not both a hydrogen atom, and their pharmaceutically acceptable salts.

[0078] In particular US7553839 describes the synthesis of ulodesine (see compound 8). The above disclosures also describe methods of manufacturing the compounds and various formulations comprising these molecules which have utility in carrying out the present invention.

[0079] Evans et al (2003a), Taylor et al. (2007), Evans et al (2003b), Castilho et al (2006), Schramm et al (2007), Bantia et al (2001), Bantia et al (2010), Kicska et al (2001) and Ho et al (2010) provide further PNP inhibitors. The disclosure of each of the above documents is hereby incorporated in its entirety by reference.

[0080] Preferred PNP inhibitors are those of the formulas I, II and III below.

[0081]

[0082] Formula I Formula II

[0083] Formula III or pharmaceutically acceptable salts thereof.

[0084] In some embodiments the PNP inhibitor is ulodesine (formula I) or a pharmaceutically acceptable salt thereof. Known salts of ulodesine include hydrochlorides, dihydrochorides, hydrobromides, hemisulfate, p-tosylate, phosphate, citrate, L-tartrate, L-lactate, stearate, maleate, succinate, fumarate, and L-malate and L-aspartate (see for example WO2010 / 111381). In some embodiments the PNP inhibitor is ulodesine hemiglutarate, the synthesis of which is described in W02023 / 001893 and co pending application WO2024 / 126630. Ulodesine hemiglutarate may also be referred to herein as MS- 001.

[0085] The dose of PNP inhibitor (or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomer thereof) used in the disclosed treatments will vary, with the nature and severity of the disease and the route by which it is administered. The dose, and in some cases the dose frequency will also vary according to disease treated, the age, body weight and response of the individual patient. For clarity, when referring to the dose of the PNP inhibitor the figure refers to the free base and appropriate adjustments should be made for the salt component. The PNP inhibitors of the present disclosure may be used at a dose of between 0.1 mg to 1000 mg (equivalent to 1.25ug / kg to 12.5mg / kg) or Img and lOOOmg, (equivalent to 125ug / kg to 12.5mg / kg) but more typically at up to 500mg (6.25mg / kg) and yet more typically at up to 250mg (3.125mg / kg) . In some embodiments the dose is O.lmg to 150mg (1.25ug / kg to 12.5mg / kg) or Img to 150mg (125ug to 1.875 mg / kg). In some embodiments the dose may be or O.lmg to 120mg (1.25ug / kg to 1.5mgs / kg) or Img to 120 mg (125ug / kg to 1.5mgs / kg). In some embodiments the dose is 5mg to 120mg (625ug to 1.5mgs / kg). Doses identified herein are those appropriate for an adult 80kg human, doses for use in other subjects in need to such treatments will be apparent to the skilled person.

[0086] The present inventors have identified that a PNP inhibitor may also be used in combination with one or more precursors of NAD+biosynthesis, which leads to a further increase in tissue NAD+. The present disclosure therefore also relates to treatments in which a PNP inhibitor is used in combination with a precursor of NAD+biosynthesis. Precursors of NAD+biosynthesis include trigonelline (TG), nicotinic acid (NA), nicotinamide (NAM), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) and / or dihydronicotinamide riboside (NRH). In some embodiments the precursor of NAD+biosynthesis is NR or NAM. It is to be noted however, that PNP inhibitors are effective at increasing NAD+levels in tissues on their own and so may also be used in the absence of NAD+biosynthesis precursors. The NAD+precursor may be provided prior to, during or after treatment with the PNP inhibitor.

[0087] In addition to boosting NAD+levels, when these precursors are provided exogenously, they may be metabolised to methylated analogues of NAM, such as methylnicotinamide, N-methyl-2-pyridone-5- carboxamide (2-PY), and N-methyl-4-pyridone-5-carboxamide (4-PY). Of these, 2-PY and 4-PY are considered uremic toxins (Lenglet et (2016)).

[0088] It is believed that this occurs because, although following conversion to NAM a small proportion of the precursor is salvaged back to NAD+through the NAMPT pathway, within this pathway PRPP is limiting and so excess NAM is diverted through nicotinamide methyl transferase (NNMT) to the methylated NAM compounds.

[0089] For example the NAD+precursor NMN given at 1000 mg for 28 days in human trials leads to an increase in NAD+(2-fold) but 1 -methyl nicotinamide was increased approximately 200 fold, 2-PY was increased approximately 4000 fold and NAM is increased approximately 4- fold (Pencina et al (2023)).

[0090] The present inventors have also identified that treatment with a PNP inhibitor leads to an increase in NAD+in tissues, in the absence of an increase in methylated metabolic products of NAM as demonstrated further herein.

[0091] In some embodiments suitable doses for the NAD+biosynthetic precursor (particularly NR), when used in combination with a PNP inhibitor are from 0.1 to lOOOmg / kg; optionally 1 to lOOOmg / kg. In some embodiments from 0.1 or 1 to 500mg / kg or 0.1 or 1 to 250 mg / kg. In some embodiments the precursor may be used at 0.1 or 1 to 100 mg / kg and particularly about 10 mg / kg to about lOOmg / kg.

[0092] PNP inhibitors may be used in combination with an inhibitor of NAD+metabolism, that is to say that the PNP inhibitor may be used in combination with one or more inhibitors of NAD+metabolising enzymes to treat any of the conditions taught herein. PNP inhibitors may be used in this manner with or without the addition of one or more NAD+biosynthesis precursors as described elsewhere herein. Further. Since PNP inhibitors are effective at increasing NAD+levels in tissues on their own, they may be used in the absence of an inhibitor of NAD+metabolism, including in the absence of those disclosed further herein.

[0093] NAD+degrading enzymes include, inter alia, CD38, PARP (poly-ADP ribose polymerase) and SARM1 (sterile alpha and TIR motif containing- 1).

[0094] CD38 (cyclic ADP ribose hydrolase) is a major regulator of NAD+levels. Inhibitors of CD38 include inhibitory antibodies such as daratumumab, isatuximab and felzartamab as well as small molecule inhibitors such as CD38-IN-78c, crysanthemin, apigenin, luteolinidin, MK-0159, TNB-738 and those disclosed in W013 / 002879, W023 / 039170, WO22 / 170265, WO22 / 165114, WO21 / 207168 each of which is incorporated herein by reference in its entirety

[0095] Poly-ADP ribose polymerases (PARP) is involved in single strand DNA break repair. Synthesis of its substrate, ADP-ribose monomer, requires NAD+; high activity of PARP therefore leads to depletion of NAD+reserves. Inhibitors of PARP are well known and several are approved for use as drugs. PARP inhibitors include, without limitation, olaparib, veliparib, rucaparib, niraparib, talazoparib, and pamiparib.

[0096] Inhibitors of SARM1 enzyme activity include, but are not limited to, DSRM-3716 (Disarm Therapeutics), NB-4746 (Nura Bio), ASHA 264, LY38783862 (Eli Lily) and those disclosed in WO24077273, CN116059367, WO23240084, WO23235719, WO23193809, WO23168298 and WO23025244.

[0097] In some embodiments, the treatments disclosed herein comprise providing a PNP inhibitor in combination with one or more additional active agents. Examples of other active agents include, but are not limited to Equilibrative Nucleoside Transporter 1 (ENT-1) inhibitors such as dilazep, anti-obesity drugs (such as bupropion-naltrexone), antihypertensives (such as amlodipine, captopril, enalaopril and furosemide) and anti diabetic drugs (such as metformin, rosiglitazone and insulin analogues such as insulin glargine or insulin detamir). Particularly GLP-1 agonists (such as exenatide, liraglutide, albiglutide tirzepetide and semaglutide) used as anti diabetic drugs and anti obesity drugs, may be used in combination with PNP inhibitors, such combinations can lead to enhanced weight loss when used to treat excess body fat (for example in obesity or overweight) as illustrated further herein and have additional benefits in addressing co-morbidities associated with obesity and over weight including those of metabolic syndrome or a metabolic syndrome-induced diseases or conditions.

[0098] Inherited deficiencies in PNP, are known to lead to a severe combined immunodeficiency (Markert 1991). Likewise, it is known that the use of PNP inhibitors in humans, for extended periods at high doses, can lead to a significant decrease in lymphocyte count and certain classes of lymphocyte are particularly affected such as CD4, CD8 and CD20 (Gomes et al 2008). These effects have contributed to the difficulty in obtaining regulatory approval for drugs in this class, except for their use to treat certain cancers where this effect is central to their utility (see above). However, despite the technical prejudice against the use of these compounds in other therapies, the applicant has now demonstrated that certain dosing regimens can provide useful therapeutic effects without causing undesirable immunological effects, opening up their use in other areas of therapy.

[0099] This disclosure now provides low and / or intermittent dosing regimens of PNP inhibitors, such as ulodesine hemi-glutarate, Such approaches lead to elevation of NAD+in tissues but does not lead to significant immune side effects such as depletion of total lymphocytes or lymphocyte sub populations (eg CD4, CD8 and CD20). These dosing regimens comprises a further embodiment of the invention. Where dosing targets reduced incidence of immunological side effects, dosing targets a <10% or <20% reduction in total lymphocyte and / or lymphocyte sub populations (e.g. CD4) in blood.

[0100] The PNP inhibitors of the present disclosure may in general, be used at a dose of between Img and lOOOmg / day. In one embodiment they are used at a dose of up to 500mg / day. In one embodiment they are used at a dose of up to 250mg / day

[0101] Where lower dose regimens are required, for example in order to reduce the incidence of immunological side effects, doses of Img to 150mg / day; optionally Img to lOOmg / day; optionally Img to 50mg / day or Img to 40mg / day may be provided. In some embodiments the lower dose in these ranges is lOmg for example lOmg to 40 mg or lOmg to 30mg.

[0102] In some embodiments the lower dose is 0. Img / day; for example 0. Img to 150mg / day; optionally 0. Img to lOOmg / day; optionally O.lmg to 50mg / day or O.lmg to 40mg / day may be provided

[0103] Such doses represent the daily dose provided and may be provided in 1 , 2, 3 or more sub doses over the day as appropriate.

[0104] Where dosing targets reduced incidence of immunological side effects, dosing targets a <10% or <20% reduction in total blood lymphocyte and / or lymphocyte sub population numbers. The PNP inhibitor is provided for at least one day; optionally at least two days; optionally at least three days; followed by a period, wherein the subject receives no drug for at least 1 day.

[0105] In some embodiments, the intermittent treatment regimen comprises or consists of repeated cycles (e.g. at least two cycles) of periods of treatment wherein the subject receives the daily dose of drug (dosing) e.g. for at least one day, optionally at least two days; optionally at least 3 days, followed by a break period, for example wherein the subject receives no drug for at least 1 day (24 hours).

[0106] In one embodiment one cycle of treatment comprises or consists of dosing for 1 to 6 days, followed by a break of at least 1 day. In one embodiment one cycle of treatment comprises or consists of dosing for

[0107] 2 to 4 days, followed by a break of at least 1 day (optionally 2, optionally 3, optionally 4 days, optionally at least 5 days).

[0108] In one embodiment one cycle of treatment comprises or consists of dosing for 3 days, followed by a break of at least 1 day (optionally at least 2, optionally at least 3, optionally at least 4 days). Typically break periods are no longer than 5, 6 or 7 days. In one embodiment, the cycle of treatment comprises or consists of dosing for 2 to 4 days, preferably 3 days, followed by a break of 3 to 4 days.

[0109] An intermittent dosing regimen of this nature is thought to be sufficient to induce the necessary metabolic changes to provide treatment but without significantly impacting the total lymphocyte and / or lymphocyte sub population numbers (<10% or <20% change) and therefore does not impact immune response.

[0110] Immunological side effects may also be mitigated by beating the subject with both a lower dose and an intermittent dosing regimen in combination, thus in some embodiments, the lower dose regimen described above may be provided in the intermittent regimen described above.

[0111] Thus in some embodiments, for example, a dose of Img to 150mg or 0. Img to 150mg may be provided daily in an intermittent dosing regime in which the subject receives repeated cycles of dosing wherein a cycle comprises or consists of dosing for at least 1 day, optionally at least two days, optionally at least

[0112] 3 days, followed by a break period wherein the subject receives no drug for at least 1 day (24 hours) as described above.

[0113] Cycles may be continued for the period of treatment, or may be varied according to need.

[0114] For compliance purposes, it may be useful for the treatment to operate on a 7 day cycle, so in one embodiment, the subject is treated on days 1 to 6 and not heated on day 7, or treated on days 1 to 5 and not treated on days 6 and 7, or treated on days 1 to 4 and not treated on days 5 to 7, or treated on days 1 to 3 and not heated on days 4 to 7, or heated on days 1 and 2 and not heated on days 3 to 7 or treated on day 1 and not treated on days 2 to 7. In a further embodiment, a dosing regimen may comprise, or consist of, the steps of: (i) treating the patient with the PNP inhibitor until the level of lymphocytes and / or a sub population thereof , such as CD4 in a first sample from the patient falls below a first predetermined figure; (ii) pausing treatment to allow the level of lymphocytes (and / or a sub population thereof, such as CD4) to recover at least partially; and (iii) optionally repeating steps (i) and (ii).

[0115] Thus a subject may be treated continuously until the level of lymphocytes (and / or a sub population thereof, such as CD4) cells in a first sample from the patient falls below a first predetermined figure. In this context treating continuously means providing the PNP inhibitor for a period of time according to a dosing regime, for example once daily. First and second samples from the patient are typically blood samples, which may be further processed, for example by removal of RBCs if required. The first predetermined figure represents the preferred limit to which lymphocytes or selected lymphocyte populations (e.g. CD4, CD8 and / or CD20) may safely be allowed to fall. Typically this may be 95% 90% 80%, 70% 60% or 50% or even lower according to the patient’s circumstances. This level may be measured against pre-treatment levels for the individual patient or against a control level set by an average of non-treated individuals, for example.

[0116] The treatment may then be paused to allow lymphocyte (and / or a sub population thereof, such as CD4) levels to recover, at least partially. This may be to pretreatment levels or some level below that considered appropriate for the patient. In each case, the level of lymphocytes or CD4 may be determined or the period may be a previously determined to be sufficient, for example by reference to data obtained during previous treatment cycles from the patient, or from data obtained from other individuals. Where the level of lymphocytes (or a subpopulation thereof) is determined this may be achieved by cell counting or for example by measuring the level of a marker associated with either total lymphocytes or the appropriate sub population.

[0117] The compounds used in the treatments disclosed herein are generally known in the art and appropriate formulations are provided in the documents in which they were originally disclosed as exemplified above. Typically, PNP inhibitors may be administered via a number of routes, including orally, parenterally, by inhalation, topically, rectally, nasally, buccally or using an indwelling reservoir of compound.

[0118] Oral formulations are well known in the art and include both solid and liquid based formulations. These include, but are not limited to tablets, capsules, powders, solutions, suspensions dispersions rapidly dissolving sub-lingual formulations. Tablets include conventional tablet bases such as lactose, sucrose and corn starch; binders, such as corn starch or gelatin; disintegrants such as alginate; and lubricants such as stearates For oral administration in the form of capsules, diluents such as lactose and dried cornstarch may be employed. Aqueous suspensions may include carriers, such as water, optionally including a small amount of ethanol; as well as additional components such as emulsifying agents, suspending agents and / or surfactants.

[0119] Injectable formulations include a physiologically acceptable diluent such as water or saline, and optionally one or more of solubilising agents (e.g. ethanol or a pharmaceutically acceptable surfactant).

[0120] Topical formulations include creams and lotions. Such formulations typically include a carrier such as mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, and water, as well as additional ingredients such as sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol.

[0121] Forodesine, for example, is approved for use in Japan as an orally administered capsule comprising 113.6 mg of forodesine hydrochloride, crystalline cellulose, hypromellose, croscarmellose sodium, and magnesium stearate as excipients.

[0122] By subject, is meant any subject to be treated according to the disclosed treatments. The subject is typically a mammalian subject and particularly a human subject in need of treatment for the condition to be treated but may also be any other subject including, for example companion or farmed animals.

[0123] Compositions of the invention may be formulated as oral dosage forms, parenteral dosage form or topical dosage form. In specific embodiments, the oral dosage form may be formulated to provide slow release of the PNP inhibitor.

[0124] The terms "treat", "treating" and "treatment" include alleviating, ameliorating or reducing the severity or frequency of, inhibiting the progress of, reversing or abrogating a medical condition or one or more symptoms or complications associated with the condition, and alleviating, ameliorating or eradicating one or more causes of the condition. The term "prevent' in relation to a disease or condition means, reducing the risk or delaying the onset of developing the disease or condition.

[0125] Throughout this specification unless the context requires otherwise, the words "comprise" "comprises" and "comprising", should be understood to mean has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. FIGURES

[0126] Figure 1 illustrates the NAD+salvage pathway and its relationship to hypoxanthine / guanine metabolism and the effect of PNP inhibition.

[0127] Figure 2 illustrates the effect of ulodesine hemi glutarate (MS-001) in a diet induced obesity (DIO) mouse model on body weight (% change) and cumulative food intake. Mice were either treated with vehicle (control) or ulodesine hemi glutarate at 2mg / kg, 3 days a week (days 1 , 2 and 3) given orally.

[0128] Figure 3 illustrates the effect of MS-001 in a DIO mouse model, on serum cholesterol (A) and blood glucose (B).

[0129] Figure 4 illustrates the effect of MS-001 in a DIO mouse model on plasma inosine and blood NAD+levels.

[0130] Figure 5 Illustrates the effect of MS-001 in a DIO mouse model on UCP-1 gene expression in brown adipose tissue (BAT).

[0131] Figure 6 Illustrates the effect of MS-001 in a DIO mouse model on steroyl CoA desaturase-1 (Scd-1) and Fatty acid synthase (Fas) gene expression in inguinal white adipose tissue (iWAT).

[0132] Figure 7 Illustrates the effect of MS-001 in a DIO mouse model on blood parameters and on iWAT as a percent of body weight. A - serum cholesterol; B - change in blood glucose; C - plasma insulin; D - serum ALT; E - serum AST; and F - iWAT as a percent of body weight.

[0133] Figure 8 shows the effect of MS-001 on liver parameters (A - liver total cholesterol (TC) and B - liver AST) in a DIO mouse model.

[0134] Figure 9 illustrates the effect of MS-001 in a DIO mouse model on weight over a 58 day period (A), between day 46 and 58 expressed as % body weight increase (B) and food intake. Mice were either treated with vehicle (control) or MS-001 at Img / kg, 3 days a week (days 1, 2 and 3) given orally.

[0135] Figure 10 illustrates the effect of MS-OOlon the change in % fat mass and % lean mass over the study illustrated in figure 9.

[0136] Figure 11 illustrates the effect of MS-001 on serum cholesterol during the study of figure 9 (A), on liver total cholesterol (TC) at the end of the study (B) and on liver alanine transaminase (ALT) (C) and aspartate transaminase (AST) (D) at the end of the study.

[0137] Figure 12 illustrates the percentage of lymphocytes in total live cells in a blood sample following 28 day treatment with ulodesine hemi glutarate (for 3 days a week ) at 1, 2, 5 and 10 micrograms per kg, in a DIO mouse model.

[0138] Figure 13 illustrates the concentration of NAD+in whole mouse brains following 8 weeks of daily treatment with MS-001 (for 3 days a week ) at 1, 2 and 5 mg / kg, in a DIO mouse model. n=8. Figure 14 Illustrates the relative expression of the oxidative phosphorylation related genes cycloxygenase 1 (cox 7) and succinate dehydrogenase (mitochondrial complex II) subunit C (sdhc) in white adipose tissue (iWAT - A) and brown adipose tissue (BAT - B) in DIO mice treated with MS- 001 at 1 , 2 or 5 mg / kg.

[0139] Figure 15 illustrates (A) the level of NAD+in gastrocnemius muscle of mice (C57 / BL6J) treated with 5mg / kg ulodesine hemi glutarate for 3 days and (B) the weight of gastrocnemius muscle as a percentage of total body weight in the same mice.

[0140] Figure 16 illustrates the levels of nicotinamide metabolites in plasma of C57 / BL6J mice following treatment with MS-001 at 5 mg / kg for 3 days, NR at 50 mg / kg and the combination of the two. A - 1- methyl nicotinamide; B - Nl-methyl 2-pyridone 5-carboxamide (2PY).

[0141] Figure 17 illustrates the levels of NAD+in blood, brain, gastrocnemius muscle and iWAT of mice treated with a combination of nicotinamide riboside at 50mg / kg and ulodesine hemiglutarate (MS-001) at a dose of either 2 or 5 mg / kg.

[0142] Figure 18 illustrates the levels of NAD+in (A) - HeLa cells treated with either (A) MS-001 (5uM), nicotinamide riboside (NR- 25uM ) or a combination of the two for 24 hrs; and (B) - HeLa cells treated with either nicotinamide (NAM - 2mM) or NAM plus MS-001 (5uM) for 24 hrs.

[0143] Figure 19 illustrates the level of 1 -methyl nicotinamde in C57 / BL6J mouse blood samples following treatment with either 2 or 5 mg / kg ulodesine hemi glutarate in the DIO model (Example 10).

[0144] Figure 20 illustrates the effect of NAD+precursors, MS-001, FK866 or combinations of these agents on NAD+levels in HeLa cells following incubation for 24 hours. NR = nicotinamide riboside; NMN = nicotinamide mononucleotide; NA = nicotinic acid; Trigo. = trigonelline; and FK = FK866 (also known as Daporinad). Significance of the difference between data points is represented by * = p<0.05; ** = p<0.01; *** = p<0.005 and **** = p<0.0001.

[0145] Figure 21 illustrates the effect of MS-001 and nicotinamide riboside (NR) on NAD+levels in SH-SY5Y human neuroblastoma cells. NR was used at 25uM (A) or 50uM (B). Each data point is the mean of three measurements. Significance of the difference between data points is represented by ns = not significant; * = p<0.05; ** = p<0.01; ***= p<0.005.

[0146] Figure 22 illustrates the effect of nicotinamide riboside (NR), MS-001 (MS), FK (FK866 - Daporinad) and a combination of 2-deoxyglucose and oligomycin (2DGO) on ATP levels in SH-SY5Y human neuroblastoma cells. Each data point is the mean of four measurements. Significance of the difference between data points is represented by ns = not significant; = p<0.01; **** = p<0.0001 . Figure 23 illustrates the effect of nicotinic acid, MS-OOland a combination of NA and MS-001 on liver weight as a percentage of body weight (A); on liver NAD+levels (as % change following treatment) (B); and on kidney NAD+levels (as percent change following treatment) (C). C57 / BL6J mice were treated with vehicle (control); MS-001 (5 mg / kg) or MS-001 in combination with NA (50 mg / kg) each given orally for 3 days. Significance of the difference between control and test data points is represented * = p < 0.05; **= p < 0.01 ; and **** = p < 0.0001.

[0147] Figure 24 illustrates the effect of MS 001, semaglutide and a combination of semaglutide and MS-001 on the body weight (A); as percent change in body weight (B); cumulative food intake (C); and exercise performance test (D) of C57 / BL6J mice in a DIO mouse model. C57 / BL6J mice were treated with vehicle (control); MS-OOlat Img / kg, 3 days a week (days 1, 2 and 3) given orally, or semaglutide at 10 nmole / kg Q3D given subcutaneously (SC) or the combination of semaglutide and MS-001 at these doses. Food intake was measured every 2 days and body weight was measured twice a week for first 4 weeks followed by three times a week. Number of mice in each group is 7 and the error represents standard error of mean. The significance of the difference between semaglutide and semaglutide +MS001 data points is ** = p<0.01

[0148] Figure 25 illustrates the effect of MS-001 co-incubated with FK866, NR or both in SHSY-5Y cells on mitochondrial potential. The TMRM / Mitotracker™ fluorescence ratio represents the ratio of mitochondrial membrane potential to the mass of mitochondria to provide a report of mitochondrial function.

[0149] Figure 26 illustrates the effect of NR (50uM) or a combination of NR and MS001 (5uM) (MS) on transcripts of 4 genes (YME1L, BNIP3, BECN1 and COX5A) associated with the mitochondrial stress response in SHSY-5Y cells.

[0150] Figure 27 illustrates the effect of doxorubicin (DOX), DOX plus NR and DOX plus MS-001 (MS) on expression of 3 transcripts (IL-6, CCL-20 and CXCL1) associated with the senescence-associated secretory phenotype.

[0151] EXAMPLES

[0152] Example 1: Effect of MS-001 in a DIO mouse model - study 1 (2mg / kg).

[0153] The aim of this study was to determine the effect of low and intermittent dosing of ulodesine hemi glutarate (MS-001) on weight gain, food intake, fasting blood glucose concentration and serum cholesterol in a diet induced obesity (DIO) mouse model of obesity and metabolic dysfunction.

[0154] C57 / BL6J mice were fed an obesogenic diet (D12492 - 60 kcal % fat) from the age of 6 weeks. Eighteen week old mice (approximately 40 g) were either treated with vehicle (control) or ulodesine hemi glutarate at 2mg / kg for 3 days a week (days 1, 2 and 3) given orally, over a 28 day period. Two days before treatment (day minus 2) and 28 days post treatment (day 28), animals were fasted for 6 hr by withdrawing food only, and blood was collected for both blood glucose and cholesterol measurements.

[0155] Figure 2 illustrates the weight gain (figure 2A) and cumulative food intake (figure 2B) for control and treated groups. The ulodesine treated group exhibited a significant decrease in weight gain compared to the control group. In addition to decrease in weight gain there was also decrease in blood glucose and serum cholesterol (figure 3). Food intake of ulodesine treated animals was similar to the control group. There were no signs of any toxicity or adverse effects of the drug.

[0156] Example 2. Effect of MS-001 in a DIO mouse model - study 2 (2mg / kg).

[0157] The aim of this study was to determine the effect of a low and intermittent ulodesine (MS -001) dosing regimen on plasma inosine and whole blood NAD+, as well as on several additional parameters.

[0158] C57 / BE6J mice were raised and maintained according to Example 1. Nineteen week old mice (approximately 50 g) were either treated with vehicle (control) or ulodesine hemi glutarate at 2 mg / kg for 3 days a week (days 1, 2 and 3), given orally, over a 28 day period. On day minus 2 and on day 28, animals were fasted for 6 hr (only food withdrawn) and blood was collected for both blood glucose and cholesterol. On day 28 plasma liver enzymes (AST and AFT) and insulin were determined and additional plasma and whole blood was frozen and used for inosine and NAD+measurement. At the end of the study BAT and iWAT were collected, weighed and quantitative PCR was performed for the lipogenesis markers stearoyl CoA desaturase 1 (SCD-1) and fatty acid synthetase (FAS) and for the thermogenesis marker uncoupling protein-1 (UCP-1). Inosine and NAD+levels in stored plasma and whole blood respectively were determined by EC / MS / MS.

[0159] For NAD+, EC was caried out on a Atlantis Premier BEH Z-HIEIC E7um 2.1* 100mm column with

[0160] 10 mM ammonium acetate in water and acetonitrile mixture as the mobile phase. For inosine a Waters Acquity™ BEH HILIC 1.7um 2.1 *50mm column was used with 5 mM ammonium acetate in water and acetonitrile mixture as the mobile phase.

[0161] Plasma inosine and total blood NAD+are illustrated in figure 4. The effect on UPC-1 and on fatty acid synthesis enzymes are illustrated in figure 5 and figure 6 respectively.

[0162] The ulodesine treated group showed decreases in blood glucose, plasma cholesterol, AST and ALT and insulin levels compared to the control group (figure 7A - E). There was significant decrease in iWAT / Body weight % in the ulodesine treated group compared to the control group (Figure 7F).

[0163] The ulodesine treated group demonstrated an increase in expression of the thermogenic marker, UCP- 1 in BAT tissue (figure 4) and decrease in lipogenesis markers (SCD-1 and FAS) in iWAT tissue compared to the vehicle treated group (Figure 5). As for example 1, the food intake of ulodesine treated animals was similar to the control group. The decrease in weight gain in the ulodesine group was not significantly different from the control group unlike in the previous study. One possible reason is the starting weight of these animals was about 50 g in this study which is 25% more than the previous study and it may take longer treatment to realize significant decrease in weight gain. Both inosine and NAD+ were significantly elevated in plasma and whole blood respectively (Figure 4).

[0164] These studies demonstrate that the PNP inhibitor ulodesine is suitable for use in the treatment of diseases associated with metabolic syndrome such as obesity, fatty liver diseases and cardio-metabolic diseases. There were no signs of any toxicity or adverse effects of the drug.

[0165] Example 3. Effect of MS-001 on liver parameters in a DIO mouse model (2mg / kg).

[0166] The aim of this study is to determine the effect of low and intermittent dosing of ulodesine on the liver parameters: liver weight, total cholesterol, triglycerides, AST and AFT.

[0167] C57 / BE6J mice were raised and maintained according to Example 1. Eighteen week old mice (approximately 40 g) were either treated with vehicle (control) or ulodesine hemi glutarate (2mg / kg orally) for 3 days a week (days 1 , 2 and 3) for 8 weeks. At the end of eight weeks, the liver was removed and total cholesterol, triglycerides, AST and AFT were determined.

[0168] The ulodesine treated group exhibited a significant decrease in total cholesterol (TC) and AST. Although AFT and triglycerides showed a tendency to a reduction (figure 8), the decreases did not achieve statistical significance.

[0169] This study supports the use of PNP inhibitors such as ulodesine for the treatment of fatty liver diseases. Example 4: Effect of 8-week treatment of MS-001 on body weight and other metabolic parameters in DIO mouse model - study 3 (Img / kg)

[0170] The aim of this study was to determine the effect of ulodesine hemi glutarate given at 1 mg / kg, 3 days a week on weight gain, food intake, fasting blood glucose concentration, serum cholesterol, liver cholesterol and liver enzymes and inguinal white adipose tissue in a diet induced obesity (DIO) mouse model of obesity and metabolic dysfunction.

[0171] C57 / BL6J mice were fed an obesogenic diet (D12492 - 60 kcal % fat) from the age of 6 weeks. Eighteen week old mice (approximately 40 g) were either treated with vehicle (control) or ulodesine hemi glutarate at Img / kg, 3 days a week (days 1, 2 and 3) given orally, over a 8-week period. Two days before treatment (day minus 2) and 56 days post treatment (day 56), and end of study animals were fasted for 6 hours by withdrawing food only, and blood was collected for cholesterol measurements. MRI was performed to measure fat and lean mass pretreatment and end of the study. At the end of the study iWAT was collected and weighed. Liver was also collected to measure cholesterol, and liver enzymes AST and ALT.

[0172] Figure 9 illustrates the weight gain (figure 9A and B) and cumulative food intake (figure 9C ) for control and treated groups. The ulodesine treated group exhibited no decrease in weight gain compared to the control. However, the weight gain in the MS-001 treatment arm for the last 2 weeks (figure 9B) was lower than the control group. The control group weight increased by 3.1 g in week 7 and 8 whereas treatment group gained only 1.7 g. The food intake in both groups was similar.

[0173] There was significant decrease in iWAT / Body weight % in the ulodesine treated group compared to the control group (figure 10A), also decrease in fat mass and increase in lean mass compared to control group was observed (figure 10B) although the levels did not reach statistical significance. In addition, other metabolic parameters which includes serum cholesterol (figure 11 A), Liver total cholesterol (figure 1 IB) liver enzymes ALT (figure 11C) and AST (figure 1 ID) improved. There were no signs of any toxicity or adverse effects of the drug.

[0174] Example 5. Effect of MS-001 on lymphocyte population in a DIO mouse model.

[0175] C57 / BL6J mice were raised and maintained according to Example 1. Eighteen week old mice (approximately 40 g) were treated with either vehicle (control) or ulodesine hemi glutarate at 1, 2, 5, or 10 mg / kg for 3 days a week (days 1, 2 and 3) given orally for 4 weeks. At the end of four weeks, flow cytometry was used to enumerate total lymphocytes. Ulodesine hemi glutarate at 1 and 2 mg / kg did not impact the number of total lymphocytes. Doses of 5 mg and 10 mg / kg appeared to lead to an observable decrease in total lymphocytes, this was not significant (figure 12). This study demonstrates that a dosing regimen that improves various cardio metabolic parameters does not significantly impact total lymphocytes.

[0176] Example 6. Effect of MS-001 on NAD+levels in the brain and OXPHOS gene expression in iWAT and BAT tissue in DIO mouse model.

[0177] C0X1 (cytochrome C oxidase subunit 1) is a gene that encodes a protein component of the cytochrome c oxidase (COX) complex, also known as Complex IV and is a critical component of the mitochondrial electron transport chain. This complex is responsible for the final step of oxidative phosphorylation, generating energy for the cell in the form of ATP. COXI is the main subunit of the COX complex and is encoded by mitochondrial DNA (mtDNA).

[0178] SDHC (Succinate Dehydrogenase Complex Subunit C) is a gene that encodes a protein component of the succinate dehydrogenase (SDH) complex, also known as Complex II of the mitochondrial electron transport chain. This complex plays a crucial role in the process of oxidative phosphorylation, which generates energy for the cell in the form of ATP.

[0179] C57 / BL6J mice were raised and maintained according to Example 1. Eighteen weeks old mice (approximately 40 g) were either treated with vehicle (control) or ulodesine hemi glutarate at 1 , 2, and 5 mg / kg for 3 days a week (days 1, 2 and 3) given orally for 8 weeks.

[0180] BAT and iWAT was collected at the end of the study, weighed and quantitative RT-PCR was performed for transcripts of cyclooxygenase 1 (COXI) and succinate dehydrogenase subunit C (SDHC). Whole brains were collected at the end of the study for determination of NAD+ levels by LC / MS / MS.

[0181] Brain NAD+levels in vehicle and treated mouse brains are shown in figure 13. The NAD+levels in brain at 5 mg / kg dose group was significantly higher than the vehicle treated group. The ulodesine treated group also demonstrated dose dependant increase in OXPHOS genes, COXI and SDHC, in both iWAT ( Figure 14A) and BAT (figure 14B) tissue. Effects were significantly higher than the vehicle group in 5 mg / kg dose group in each case (P< 0.05 - 0.001 vs vehicle). Example 7. Effect of MS-001 on NAD+levels in mouse gastrocnemius muscle and on NAD+metabolites in plasma.

[0182] Twenty six week old C57 / BL6J mice were treated with vehicle (control) or MS-001 (5 mg / kg) given orally for 3 days. Four hours after the third dose, the mice were euthanized and gastrocnemius muscle was collected, weighed, and approximately 80 mg was flash frozen in liquid nitrogen. The frozen samples were used for NAD+measurement, carried out by LC / MS / MS.

[0183] NAD+was significantly increased in gastrocnemius muscle in the treated group (figure 15A). A significant increase in gastrocnemius muscle weight / BW(%) was also noted (figure 15B).

[0184] Example 8. Effect of a combination of MS-001 and nicotinamide riboside (NR) on NAD+levels in a selection of tissues and methyl nicotinamide analogues in plasma.

[0185] C57 / BL6J mice were treated with vehicle (control) or MS-001 (5 mg / kg) with or without NR (50 mg / kg) each given orally for 3 days. Four hours after the third dose, the mice were euthanized and tissues (blood, brain iWAT and gastrocnemius muscle) were collected, weighed and flash frozen in liquid nitrogen. NAD+determination was carried out according to example 6. Methyl nicotinamide analogues were determined in blood samples by LC / MS / MS.

[0186] Methyl nicotinamide analogues which include 1 -methyl nicotinamide (Me-NAM) and N1 -methyl 2- pyridone 5-carboxamide (2PY) were elevated in plasma with NR treatment and decreased when NR is used in combination with MS 001 (Figure 16) because MS 001 inhibits the breakdown of NR into methyl nicotinamide analogues.

[0187] NR treatment on its own did not lead to a significant increase in NAD+in any of the tissues tested. Ulodesine alone lead to an increase in NAD+in blood and muscle as seen in earlier examples. The combination of ulodesine and NR led to much higher levels of NAD+compared to any of the groups in all the tissues tested (see figure 17).

[0188] Example 9. Combination of MS 001 with NR and nicotinamide (NAM) in HeLa cells:

[0189] 100,000 HeLa cells were seeded into each well with 600 pL of complete media. Treatment with the relevant compounds occurred 24 to 36 hours post-seeding, when the cells reached approximately 50% confluency and were fully adhered to the plate. After 24 hrs of treatment, the complete media was aspirated, and 100 pL of 0.6M Perchloric Acid (PCA) was added for cell lysis. The lysates were centrifuged at 13,000 x g for 2 minutes at 4 °C. The supernatant was then transferred to new 1.5 mL microcentrifuge tubes for the NAD+ fluorescence assay, according to the protocol adapted from the literature (Dall et al., 2018). The concentration of MS-001 was 5uM, NR concentration was 25 uM and NAM concentration was 2 mM. MS 001 by itself showed no increase in NAD+however the combination of MS 001 with either NR or NAM showed further increase in NAD+(figure 18).

[0190] Example 10. Effect of treatment with MS-001 on endogenous levels of 1-methyl nicotinamide and Nl-methyl 2-pyridone 5-carboxamide (2-PY)

[0191] Plasma samples from vehicle and from the 2 and 5 mg / kg dose MS 001 treated groups of Example 6 were evaluated for methylated NAM analogues. Mice were treated with MS-001 at 2 or 5 mg / kg for 3 days a week (days 1, 2 and 3) given orally for 4 weeks. At the end of four weeks plasma was analysed by LC / MS / MS to determine levels of metabolites as per Example 7

[0192] Ulodesine treated mice showed a significant decrease in blood 1-methyl nicotinamide in comparison to vehicle treated group (figure 19) suggesting that nicotinamide is being salvaged into NAD+through the nicotinamide phosphoryl transferase (NAMPT) pathway to a greater extent than being converted to methyl nicotinamide. Comparable to the shorter duration treatment of Example 7, no change in 2-PY levels were observed (data not shown). This further confirms that PNP inhibitors increase NAD+through phosphoribosyl pyrophosphate (PRPP) sparing and boosting of the NAMPT pathway.

[0193] These studies indicate that the combination of a PNP inhibitor with NAM can further increase NAD+ levels. They also suggest that the NAD+precursors trigonelline and NA, which through NAPRT (uses PRPP co-substrate) is eventually converted to NAD+, may also be used in combination with a PNP inhibitor to further increase NAD+levels in tissue.

[0194] Example 11. Effect of NAD+precursors and MS-001 on NAD+in HeLa cells

[0195] HeLa cells (IxlO5cells per well) were cultured as described in example 9 in the presence of 5uM MS- 001 as a single agent, in combination with NAD+precursors, in the presence of FK866 (InM; an NAMPT inhibitor), or in the presence of both the precursor and the NAMPT inhibitor (FK866; InM). NAD+was then measured by enzymatic assay according to Dall et al (2018). Precursors used were nicotinamide riboside (NR; 25 - 50uM), nicotinamide mono nucleotide (NMN; 20uM), nicotinic acid (NA; 500uM) and trigonelline (Trig.; ImM). MS-001 by itself showed no increase in NAD+in this cell line, but when combined with precursors of NAD+biosynthesis (NR, NMN, NA, and Trigonelline) a significant increase in NAD+is seen (figure 20).

[0196] When cells are treated with FK866, they experience stress primarily due to a significant depletion of NAD+levels. NA and trigonelline are converted to the mononucleotide through nicotinic acid phosphoribosyl transferase (NAPRT) which also uses PRRP as a co-substrate. Thus, the combination of PNP inhibitor with NA and / or trigonelline leads to an elevation of NAD+levels.

[0197] Example 12. Determination of NAD+levels and evaluation of mitochondrial function in SHSY- 5Y cells treated with MS-001 as single agent and in combination with NR.

[0198] SH-SY5Y human neuroblastoma cells are one of the most widely used cellular models to study neurodegenerative diseases. SH-SY5Y cells can proliferate continuously and as undifferentiated cells they present a neuroblast-like morphology and express immature neuronal markers.

[0199] SH-SY5Y cells (100, 000 cells) were seeded into each well with 600 pL of complete media. Treatment with the relevant compounds occurred 24 to 36 hours post-seeding, when the cells reached approximately 50% confluency and were fully adhered to the plate. After 24 hours of treatment, the complete media was aspirated, and 100 pL of 0.6M Perchloric Acid (PCA) was added for cell lysis. The lysates were centrifuged at 13,000 x g for 2 minutes at 4 °C. The supernatant was then transferred to new 1.5 mL microcentrifuge tubes for the NAD+ fluorescence assay, according to literature (Dall et al., 2018). The cells were were incubated with MS-001 as single agent (5uM) and in combination with the NAD+precursor NR (25uM or 50uM) for 24 hours. NAD+was measured by enzymatic assay as per Example 11.

[0200] MS-001 (5uM) by itself caused no significant increase in NAD+levels, either at 25uM (figure 21A) or at 50uM (figure 2 IB) but when combined with NR a significant increase in NAD+was observed.

[0201] Example 13: Effect of MS-001 in combination with NR, in the presence and absence of FK866 and 2DGO, on ATP levels in SH SY5Y cells.

[0202] Mitochondria are the primary organelles responsible for producing ATP, the cell's primary energy currency; therefore, higher ATP levels signify that the mitochondria are actively generating more energy through oxidative phosphorylation. To determine whether the increase in NAD+seen in SH SY5Y cells in the presence of MS-001 and NR also leads to an increase in ATP levels, SH SY5Y cells were incubated with MS-001 (5uM) as a single agent or in combination with NR (5 uM) in the presence or absence of FK866 (lOnM) or 2-deoxy glucose (2DG0; 20 mM) and oligomycin (2 uM).

[0203] Promega CellTiter-Glo® Luminescence Cell Viability Assay was used for ATP measurement. 10,000 cells were seeded per well in lOOul or 150pL and treatments with respective compounds was administered 24 to 36 hours post-seeding, once cells had reached 50% confluence and had fully adhered to the wells.

[0204] After 24 to 36 hours post-treatment, the complete medium was aspirated for wells designated for ATP measurement (4-5 wells per condition), while medium was retained in separate wells designated for downstream protein quantification (3-4 wells per condition). A 1:1 master mix of fresh complete medium and CellTiter-Glo® Luminescent Cell Viability Reagent was prepared. An equal volume of this master mix (100 pL or 150 pL, matching the initial seeding volume) was added to each well using a multi-channel pipette with the reverse pipetting technique. All steps involving the luminescent reagent were performed in the dark.

[0205] After adding the luminescent assay, the plate was covered with its lid and wrapped in aluminium foil to protect it from light. It was then shaken on an orbital shaker at 115 to 130 rpm for 2 minutes, followed by a 10-minute incubation at room temperature on the benchtop. Luminescence was then measured using the Tecan Spark® microplate reader (SciMed, NA).

[0206] Following luminescence recording, the medium was aspirated from the wells designated for protein quantification and protein quantification was performed using the Pierce BCA Protein Assay Kit.

[0207] Figure 22 shows that the combination of MS-001 with NR does not lead to an increase in ATP levels. FK866 significantly suppressed ATP levels, which are restored when cells are cultured with NR. When cells are cultured in the presence of both MS-001 and NR, this leads to a further increase ATP levels. This increase in ATP levels in the presence of MS 001 and NR is lost when the mitochondria are treated with 2-deoxy glucose and oligomycin (2DGO), which effectively deprives the mitochondria of the ability to synthesize ATP.

[0208] This confirms that MS-001 in combination with NR increases NAD+and ATP levels through an increase in mitochondrial function when cells are put under stress due to treatment with FK866. Example 14: Effect of a combination of MS-001 and nicotinic acid (NA) on NAD+levels in a selection of tissues.

[0209] Twenty six C57 / BL6J mice were treated with vehicle (control) or ulodesine hemi glutarate (5 mg / kg) with or without NA (50 mg / kg) each given orally for 3 days. Four hours after the third dose, the mice were euthanized and tissues were collected, weighed and flash frozen in liquid nitrogen. NAD+determination was carried out using LC / MS / MS (figure 23).

[0210] Mice treated with NA demonstrated a significant increase in NAD+in both liver (figure 23B) and kidney (figure 23C) tissue. The combination of NA and MS-001 treatment further increased NAD+in both liver and kidney compared to vehicle, NA and MS-001 alone. The NAD+increase in liver was associated with a decrease in liver weight (figure 23 A). In the combination treatment, liver weight was significantly lower than the vehicle treatment group. No change in the kidney weight was observed compared to vehicle treatment groups. These data suggest that the combination of MS-001 with NA may be used to treat fatty liver disease.

[0211] Example 15: Effect of MS-001 as a single agent and in combination with semaglutide on body weight and other metabolic parameters in a DIO mouse model.

[0212] C57 / BL6J mice were fed an obesogenic diet (D12492 - 60 kcal % fat) from the age of 6 weeks. Eighteen week old mice (approximately 36 g) were either treated with vehicle (control) or MS-001 at Img / kg, 3 days a week (days 1, 2 and 3) given orally, or semaglutide at 10 nmole / kg Q3D given subcutaneously (SC) or the combination of semaglutide and MS-001 at the doses mentioned above. Food intake was measured every 2 days and body weight was measured twice a week for the first four weeks and then three times a week. Semaglutide administration was stopped on day 40 in both semaglutide and the combination group and Day 43 Exercise performance test was performed on all groups to assess muscle function.

[0213] Exercise Performance test: An exercise performance test was performed on Day 43. Exercise performance was assessed on a 4-lane metabolic treadmill (1 mouse from each group, total 4 mice each round ). Mice are acclimatized to the treadmill environment the day before the formal experiment. The treadmill is set to an angle of 10° for treadmill acclimatization. The running belt does not move, and the shock grid is turned off. Mice were adapted to motor noise for 15 minutes then the treadmill was accelerated to 15 m / min within 3 minutes and the shock grid is turned on. The treadmill was then accelerated to 20 m / min within 20 minutes and then to 25 m / min within 20 minutes and maintained at this speed. If the mouse cannot maintain the speed of the treadmill it receives a small electric shock until it starts running again. The time at which the mouse receives an electric shock lasting 3 seconds is recorded as the “exhaustion time”.

[0214] Figure 24 illustrates both the body weight (figure 24A) and percentage weight gain (figure 24B) and figure 24C illustrates cumulative food intake for all the groups. The MS-001 treated group exhibited no decrease in weight gain compared to the control group. The semaglutide group as expected demonstrated decrease in body weight and food intake. The combination of MS-001 and semaglutide demonstrated further decrease in body weight compared to semaglutide alone.

[0215] There was no decrease in food intake between the vehicle and the MS-001 group. There was significant decrease in food intake in the semaglutide group compared to vehicle group as expected. Compared to the semaglutide group, there was no change in the food intake in the combination (MS001+ semaglutide) treatment group. There was a trend towards increase in exhaustion time in the exercise performance test in the combination (MS 001 -(-Semaglutide) group compared to semaglutide alone which indicates improvement in muscle strength. Both semaglutide and the combination groups demonstrated an increase in exhaustion time compared to vehicle group.

[0216] These data suggests that further decrease in weight in the combination (MS -001 + semaglutide) group compared to the semaglutide group is not related to food intake but highly likely related to increased energy expenditure through improved mitochondrial function and browning of adipose tissue. This is also consistent with the results of the exercise performance test which shows a trend towards improved muscle strength in the combination group compared to semaglutide alone group. There were no signs of any toxicity or adverse effects of the drugs.

[0217] Example 16: Effect of MS 001 and NR on mitochondrial membrane potential in SHSY-5Y cells

[0218] Tetramethylrhodamine methyl ester (TMRM) is a cell-permeant dye that accumulates in active mitochondria with intact membrane potentials. It is widely used for monitoring mitochondrial function and measuring mitochondrial membrane potential ( A m), which is crucial for assessing cellular health and ATP generation. TMRM is particularly sensitive to changes in mitochondrial function.

[0219] SH-SY5Y cells were treated with FK866 alone (10 nM), FK866 plus MS-001 (5 pM), FK866 plus NR (5 pM), or FK866, NR and MS-001. Mitochondrial membrane potential (Aym) and mitochondrial mass were assessed using TMRM and MitoTracker™ dye (ThermoFisher) respectively.

[0220] Briefly, 50,000 cells were seeded per well in 500 pL complete media using a 24-well cell culture plate Treatment with respective compounds was performed 24 to 36 hours after seeding where cells reached 50% confluence and have completely adhered to the plate. Wells were washed twice with HyClone™ Phosphate-Buffered Saline (PBS) solution free of calcium and magnesium. Hanks' Balanced Salt Solution (HBSS) buffer without Phenol Red was used to dilute 1 mol / L-HEPES buffer solution to a final concentration of 10 mM. The diluted HEPES buffer solution was used to dilute the respective immunofluorescence dyes to their respective concentration. Complete media was aspirated 24 to 36 hours after treatment, and the immunofluorescence master mix was added prior to a 15-minute incubation in the dark at 37°C with 5% CO2 in a Forma Steri-Cycle CO2 incubator.

[0221] Imaging was performed using AXIO Observer Immunofluorescence microscopy (Zeiss, NA). Immunofluorescence intensity and mitochondrial number were quantified using Image J™ software (https: / / imagej.net / ij / index.html). Fluorescence intensities (TMRM and MTG) were normalized to cell number using Hoechst staining (Thermofisher), quantified with Image J™ software.

[0222] Figure 22 illustrates TMRM signal normalised to MitoTracker fluorescence intensity. At least three biological replicates were analyzed per treatment group. When normalized for mitochondrial content, the TMRM signal appears significantly higher following treatment with FK866 and NR in combination with MS-001 compared to FK866 in combination with NR alone, further confirming improvement in mitochondrial function.

[0223] The mitochondrial membrane potential (ATm) is a crucial component of cellular energy production, acting as a proton gradient that drives ATP synthesis. TMRM readily enters cells and accumulates in the negatively charged interior of healthy mitochondria with a maintained A m. When TMRM accumulates in mitochondria, it exhibits orange fluorescence, indicating a normal A m. If the mitochondrial membrane potential is lost (i.e. the mitochondria are depolarized), TMRM no longer accumulates, and the fluorescence either diminishes or disappears, signaling mitochondrial dysfunction or stress.

[0224] Figure 22 illustrates TMRM signal normalised to MitoTracker fluorescence intensity. At least three biological replicates were analyzed per treatment group. When normalized for mitochondrial content, the TMRM signal is significantly higher following treatment with FK866 and NR in combination with MS-001 compared to FK866 in combination with NR alone, further confirming improvement in mitochondrial function and maintenance of mitochondrial membrane potential.

[0225] Example 17: Effect of MS 001 and NR on mitochondrial stress response (MSR) genes in SHSY- 5Y cells

[0226] In this experiment, the level of 4 transcripts coding for proteins involved in mitochondrial unfolded protein response (UPRmt), mitophagy, and oxidative phosphorylation (OXPHOS) pathways were determined in response to treatment with NR or with NR in combination with MS-001. The results are illustrated in figure 26

[0227] SHSY-5Y cells were incubated in the presence of 50uM NR or 50uM NR and 5uM MS-001 as described in the previous experiment. Cells were harvested and expression levels of YME1L, BNIP3, BECN1 and C0X5A gene products were measured by RT-PCR. Briefly 1 mL cold TRIzol™ reagent (Invitrogen, Cat. No. 15596018) was added to each well, and the cell lysates collected were incubated on ice for 5 minutes. Downstream RNA extraction was conducted following the Invitrogen TRIzol RNA extraction protocol. RNA concentration (ng / pL) and purity for each sample was assessed using a NanoDrop One™ spectrophotometer. RNA (1000 ng / pl) was reverse transcribed with HiScript III All- in-one RT Supermix™ (Vazyme, Cat. No. R333-01). A 1:1 conversion of RNA to cDNA was assumed. cDNA was diluted to 1 ng / pL (50-fold dilution) using RNAse free ddH2O water for SYBR-Green quantitative Polymerase Chain Reaction (qPCR). Fourty PCR cycles and melt curve were performed for each run using QuantStudio™ 5 qPCR systems (Thermofisher Scientific, Cat. No. A34322). Betaactin was used as the housekeeping reference gene. Data represent the mean + S.E.M. from three replicates per treatment group.

[0228] Significant increases were seen in the expression of BNIP3 and COX5A genes when exposed to NR plus MS-001.

[0229] BNIP3 regulates mitochondrial dysfunction, mitochondrial fragmentation, mitophagy, cell apoptosis, and the development of lipid disorder diseases via numerous cellular signaling pathways and is presently being investigated as a potential therapeutic target for diseases involving mitochondrial dysfunction.

[0230] The COX5A gene encodes the nuclear-encoded subunit Va of the human mitochondrial respiratory chain enzyme, which is crucial for the function of the mitochondrial electron transport chain. The COX5A protein, weighing 17 kDa and composed of 150 amino acids, is a subunit of Complex IV, the last enzyme in the mitochondrial electron transport chain. It plays a vital role in the reduction of molecular oxygen to water, contributing to the ATP synthesis via protonmotive force. The COX5A gene is also associated with diseases such as Mitochondrial Complex IV Deficiency (COX deficiency) and Benign Infantile Mitochondrial Myopathy (infantile reversible cytochrome c oxidase (COX) deficiency myopathy).

[0231] Treatment of SHSY-5Y cells with the combination of NR and MS-001 demonstrated an increase in COX5A and BNIP3 expression which further supports that MS 001 in combination with NR improves mitochondrial function. Note that unlike in vivo, where NAD+precursors such as NAM, NR and NMN are produced due to a breakdown of NAD+, in in-vitro studies NR has to be added as there is a limited source of NAD+precursors for synthesis of NAD+in this system.

[0232] Example 18: Effect of MS-001 on cellular senescence.

[0233] Senescence is a multi-functional cell fate, characterized by an irreversible cell-cycle arrest and a pro- inflammatory phenotype. Senescent cells can secrete high levels of pro-inflammatory cytokines, chemokines, and extra-cellular matrix degrading proteins, which are collectively known as the senescence-associated secretory phenotype (SASP).

[0234] Emerging evidence indicates that accumulation of senescent cells in multiple tissues drives tissue dysfunction and several age-related conditions. Mitochondrial dysfunction is a hallmark of cellular senescence which plays important roles not only in the senescence growth arrest but also in the development of SASP and resistance to cell-death.

[0235] Mouse embryonic fibroblasts (MEFs) were seeded at a density of 80,000 cells in 600 uL complete media in Costar® 12-well cell culture plates or 100,000 cells per well in 1.2 mL of complete media in sterile 6-well cell culture plate. After 24 hours, cell morphology was imaged with using an inverted microscope and the cells were treated with 0.025% DMSO (vehicle control), 250 nM doxorubicin, 50 pM NR and 5 pM MS-001. Medium containing treatment compounds was replaced every day for three consecutive days (Day 1-3). On Day 4, the treatment media were replaced with fresh drug-free complete media to allow recovery of non-senescent or senescence-escaped cells. On Day 5, fresh drug-free media was used to replace the media used the previous day. On day 6 RT-qPCR was performed to measure a selection of SASP associated transcripts (IL-6, CCL20, CXCL1). Doxorubicin is used to induce senescence in normal cells and increase SASP.

[0236] The reduction in IL-6, CCL20 and CXCL1 mRNA in the presence of MS-001 indicates a suppression of SASP (also known as a senomorphic effect). Results are illustrated in figure 27.

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[0274] Zhao et al., Cells 2022, 11, 2049

Claims

CLAIMS1. A method for the treatment or prevention of a disease selected from diseases of mitochondrial dysfunction, age related cognitive decline, and muscular disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a PNP inhibitor.

2. A method according to claim 1 , wherein the PNP inhibitor has an in-vitro inhibitory constant of less than 5 x 107M.

3. A method according to claim 1 or claim 2, wherein the PNP inhibitor, is selected from ulodesine, forodesine, the compound of formula III; or a pharmaceutically acceptable salt of any of the foregoing; optionally wherein the PNP inhibitor is ulodesine hemiglutarate.

4. The method according to any preceding claim, wherein the disease of mitochondrial dysfunction is selected from neurodegenerative diseases.

5. The method according to any preceding claim, wherein the neurodegenerative disease is selected from Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS).

6. The method according to claim 1 , wherein the disease is peripheral neuropathy.

7. The method according to claim 1, wherein the disease is a muscular disorder selected from neuromuscular degeneration, muscle atrophy, cachexia, sarcopenia, muscular dystrophy, chronic fatigue syndromes and fibromyalgia; particularly wherein the muscular disorder is sarcopenia.

8. A method for the treatment or prevention of metabolic syndrome or a metabolic syndrome- induced disease or condition in a subject comprising administering to the subject a therapeutically effective amount of a PNP inhibitor.

9. The method according to claim 8, wherein the metabolic syndrome induced disease or condition is selected from hypertension, hyperglycemia, excess body fat, fatty liver disease and dyslipidaemia; optionally wherein the fatty liver disease is selected from NAFLD and NASH.

10. The method according to claim 8, 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.

11. The method according to any preceding claim, wherein the therapeutically effective amount of a PNP inhibitor is effective to increase the level of NAD+in the blood of the subject.12 The method according to any preceding claim, wherein the therapeutically effective amount of a PNP inhibitor is effective to increases the level of NAD+in the blood of the subject , but does not deplete total lymphocytes in the blood of the subject by more than 20%.

13. The method according to any preceding claim, wherein the PNP inhibitor is used at a dose of between O.lmg and lOOOmg per day.

14. The method according to any preceding claim, wherein the PNP inhibitor is used at a dose of between O.lmg and 150mg per day.

15. The method according to any preceding claim, wherein the PNP inhibitor is provided in a regimen comprising repeated cycles in which the PNP inhibitor is provided for at least one day; optionally at least two days; optionally at least three days; followed by a period, wherein the subject receives no drug for at least 1 day.

16. The method according to claim 15, wherein the PNP inhibitor is provided in a regimen comprising repeated cycles in which the PNP inhibitor is provided for 2 to 4 days, followed by a break of at least 1 day; optionally a break of 2 days; optionally a break of 3 days; optionally a break of 4 days.

17. The method according to any preceding claim, wherein the PNP inhibitor is provided in combination with a precursor of NAD+biosynthesis selected from trigonelline (TG), nicotinic acid (NA), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) and dihydronicotinamide riboside (NRH).

18. The method according to claim 17, wherein the PNP inhibitor is provided in combination with nicotinic acid (NA).

19. The method according to any preceding claim, wherein the PNP inhibitor is provided in combination with one or more inhibitors of NAD+metabolising enzymes.

20. The method according to claim 19, wherein the NAD+metabolising enzyme is selected from the group consisting of CD38, poly-ADP ribose polymerase (PARP) and sterile alpha and TIR motif containing-1 (SARM1).

21. The method according to any preceding claim, wherein the PNP inhibitor is provided in combination with one or more additional drugs selected from the group consisting of ENT1 inhibitors, anti obesity drugs, antihypertensives and anti diabetics.

22. The method according to claim 21, wherein the PNP inhibitor is provided in combination with one or more GLP-1 agonists.

23. A method according to any of claims 1 to 22, comprising the steps of:(i) treating the subject with the PNP inhibitor until the level total lymphocytes and / or a sub population thereof in a first sample from the patient falls below a first predetermined figure;(ii) pausing treatment to allow the level of total lymphocytes and / or a sub populationthereof to recover at least partially; and(iii) optionally repeating steps (i) and (ii).

24. The method according to claim 23, comprising(i) treating the subject with the PNP inhibitor until the level of at least one lymphocyte sub pollution selected from CD4, CD8 and CD20 lymphocytes in a first sample from the patient falls below a first predetermined figure;(ii) pausing treatment to allow the level of the at least one lymphocyte sub population to recover at least partially.

25. A PNP inhibitor for use in the treatment or prevention of a disease selected from diseases of mitochondrial dysfunction, age related cognitive decline, and muscular disorders.

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

27. Use of a PNP inhibitor in the manufacture of a medicament for the treatment or prevention of a disease or condition selected from diseases of mitochondrial dysfunction, age related cognitive decline, and muscular disorders.

28. Use of a PNP inhibitor in the manufacture of a medicament for the treatment or prevention of metabolic syndrome or a metabolic syndrome-induced disease or condition.

29. A PNP inhibitor for use according to claims 25 or 26, or use of a PNP inhibitor according to claims 27 or 28, wherein the PNP inhibitor has an in-vitro inhibitory constant of less than 5 x 107M.

30. A PNP inhibitor for use according to claims 25 or 26, or use of a PNP inhibitor according to claims 27 or 28, wherein the PNP inhibitor, is selected from ulodesine, forodesine, the compound of formula III; or a pharmaceutically acceptable salt of any of the foregoing; optionally wherein the PNP inhibitor is ulodesine hemiglutarate.

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