WO2016196281A1

WO2016196281A1 - Mtap inhibitors for the treatment of sickle cemtap disease

Info

Publication number: WO2016196281A1
Application number: PCT/US2016/034625
Authority: WO
Inventors: Steven Isaacman; Andrew Mahon
Original assignee: Nanometics LLC
Current assignee: Nanometics LLC
Priority date: 2015-06-01
Filing date: 2016-05-27
Publication date: 2016-12-08
Anticipated expiration: 2017-12-01

Abstract

The present invention relates to treatments and therapies for anemia conditions and diseases of the blood, and more particular is a therapy for the acute and chronic treatment of sickle cell diseases and thalassemia by administration of a 5'-Methylthioadenosine phosphorylase inhibitor, or a pharmaceutically acceptable salt thereof, optionally in combination with an additional bioactive agent.

Description

TAP INHIBITORS FOR THE TREATMENT OF SICKLE CEMTAP DISEASE

FIELD OF THE INVENTION

The present invention relates to treatments and therapies for anemia conditions and diseases of the blood, and more particular is a therapy for the acute and chronic treatment of sickle cell diseases and thalassemia by administration of an inhibitor of 5'

methylthioadenosine phosphorylase (MTAP), or a pharmaceutically acceptable salt thereof, optionally in combination with an additional bioactive agent.

RELATED APPLICATIONS

This application claims the benefit of priority of US provisional application no.

62/169,010, entitled "MTAP Inhibitors for the Treatment of Sickle Cell Disease", filed June 1 , 2015, the entire contents of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

Sickle cell disease (SCD) is a global health issue that affects over 13 million people worldwide, including ~100,000 Americans. [1 , 2] SCD results from an autosomal recessive red blood cell (RBC) disorder and is most common in populations of African, Mediterranean or Asian ancestry. [1 , 2] Over 300,000 babies are born each year with the disease. [1] SCD is caused by an inherited hemoglobinopathy that weakens the oxygen binding affinity of hemoglobin (Hb), enabling polymers of deoxyhemoglobin (deoxyHb) to form in the erythrocyte. Consequently, the RBCs become rigid and assume distorted morphologies. [2] The distorted erythrocytes are prone to hemolysis, which disrupt nitric oxide (NO)

homeostasis and causes ischemia-reperfusion injury, and rapid clearance from the circulation, which results in anemia. The compromised erythrocytes can occlude the circulation and contribute to a cascade of events that culminate in vaso- occlusion (VOC) and painful episodes of sickle crisis. VOC can result in ischemia, infarction, hemolytic anemia, organ damage and other debilitating acute and chronic effects. [2] While the frequency, severity and duration of VOC can vary among individuals, the recurrent episodes are characteristically painful, and lead to a high rate of hospitalization. [3] Consequently, SCD exhibits significant financial stress on the United States Healthcare system, with annual costs exceeding $1.1 billion. [3] Current paradigm for treatment: SCD can be cured using hematopoietic stem cell transplants, and the severity of the disease can be reduced with repeated transfusions. However, these approaches are costly and associated with high levels of risk. The only FDA approved therapeutic for SCD is hydroxyurea (HU). HU induces fetal Hb (HbF) production, and has a strong clinical track record in children and adults.

Proper use of HU requires strict compliance; self-administration by the patient; and careful monitoring by the physician to ensure proper dosing schedules. [2] Unfortunately, approximately a third of adult patients do not respond to HU treatment. [4] Moreover, myeolsuppression, reproductive toxicity and possible carcinogenic affects are all serious side effects associated with long-term use. [5] Importantly, there is currently no commercial therapeutic to avert imminent episodes of crisis or to attenuate acute events of crisis once they have begun. Patients are typically prescribed medications that alleviate the pain associated with episodes of crisis.

Overview of the Present Invention

Sickle cell disease (SCD) is a global health issue that affects over 13 million people worldwide, including approximately 100,000 Americans.^6,7 SCD results from an autosomal recessive red blood cell (RBC) disorder and is most common in populations of African, Mediterranean or Asian ancestry.^6,7 Over 300,000 babies are born each year with the disease.⁶ The molecular basis for SCD is a 6Glu→ Val mutation, which weakens the affinity of oxygen for hemoglobin (Hb), and enables polymers of deoxyhemoglobin

(deoxyHb) to form in erythrocytes. DeoxyHb polymers cause RBCs to become rigid and assume distorted morphologies.⁷ The compromised erythrocytes are prone to hemolysis and rapid clearance from the circulation results in anemia. Elevated levels of extracellular heme disrupt nitric oxide (NO) homeostasis and cause elevated oxidative stress via heme- catalyzed radical generation.⁸ Damaged endothelium leads to leukocyte recruitment and adherence of compromised erthyrocytes, which can result in vaso-occlusion (VOC) and episodes of sickle crisis. While the frequency, severity and duration can vary among individuals, recurrent episodes of crises are characteristically painful, and lead to a high rate of hospitalization.⁹ Consequently, SCD places significant financial stress on the United States Healthcare system, with annual costs exceeding $1.1 billion.⁹

SCD displays pathophysiological characteristics that mimic those of ischemia / reperfusion injury.¹⁰ Chronic hemolytic anemia disrupts the oxidative stress equilibrium of patients, and can trigger inflammatory cascades that result in adhesion of leukocytes to vascular walls, recurrent and intermittent episodes of VOC and painful episodes of crisis.^8, Due to the importance of inflammatory cascades in propagating VOC, many forms of anti-inflammatory therapy are being actively pursued to treat the disease. Notable examples of antiinflammatory approaches include: Regadenoson¹², an ADORAA2 agonist; statins¹³; Zieluotn, a 5-lipoxygenase inhibitor ; fructose-1 ,6-phosphate, which reduces ischemia induced tissue damage¹⁵; pegylated carboxyhemoglobin¹⁶, which delivers non-toxic levels of carbon monoxide to reduce inflammation; and, monoclonal antibody therapy to facilitate iNKT cell depletion.¹⁷

SCD patients are in a constant state of inflammation where elevated levels of proinflammatory signaling molecules are key contributors in recurring vaso-occlusive events.¹⁸ 5'-Methylthioadenosine (MTA) is a naturally occurring nucleoside that is present in all mammalian tissues, and the immunomodulatory potential of MTA has been investigated in several pre-clinical models of inflammation.^1"3 The potent anti-inflammatory profile of MTA led the Nanometics team to postulate that MTA might have beneficial effects for SCD patients. However initial experiments in Berkley (BERK) mouse models of SCD with single doses of MTA administered by intraperitoneal (i.p.) injection did not convey any observable benefits. This was attributed to the rapid clearance and metabolism of MTA (see Preliminary Results). This SBIR project proposes a new anti-inflammatory approach to treat SCD that blocks MTA metabolism at 5'-methylthioadenosine phosphorylase (MTAP), enabling the sustained accumulation of endogenous MTA to th erapeutically beneficial levels.

SUMMARY OF THE INVENTION

The present invention overcomes deficiencies of the presently available treatments and therapies by providing a novel therapy for sickle cell diseases, Thalassemia and other related blood diseases (e.g. sickle cell trait) comprising oral administration of a

therapeutically effective amount of a small molecule MTAP inhibitor, or pharmaceutically acceptable salts of a small molecule MTAP inhibitor, to patients in need. The MTAP inhibitor can be delivered by any means, preferably orally, subcutaneously, or intravenously, and is useful as a chronic treatment and as an acute treatment. Accordingly, the present invention is directed to a method of treating and/or reducing the likelihood of the symptoms of a blood disease, including sickle cell disease, Thalassemia or a related blood discussed comprising administering to a patient in need an effective amount of a MTAP inhibitor or a

pharmacetucially acceptable salt therof.

This application is directed to the use of a small molecule transition state inhibitor of MTAP as therapy for sickle cell disease, including sickle crisis. The sole function of MTAP in mammals is to metabolize MTA, and the systemic inhibition of MTAP causes whole-body accumulation of endogenous MTA to therapeutically beneficial levels (up to 100x in plasma for >24 h). Methylthio-DADMe-lmmucillin-A (MTDIA), is a powerful MTAP inhibitor and was used as a tool to demonstrate the novelty of the Nanometics approach described herein. A single dose of an MTAP inhibitor significantly reduced levels of leukocyte recruitment and endothelial activation in venules to produce hemodynamic parameters that resembled wild type (C57-BL) values. BERK mice are transgenic models of SCD that display severe pathology and similar features to the human disease state including elevated vascular endothelial activation and impaired blood flow.

Chronic hemolytic anemia and vaso-occlusive ischemia-reperfusion like injury are the hallmark pathologies of sickle cell disease. [5] Oxidative stress and nitric oxide (NO) homeostasis are thought to play a major role in the severity of the disease, and agents that modulate these pathways are highly desirable. The multifactorial process, which leads to VOC involves oxidative stress, damages to red blood cells (RBC), inflammation, vascular leukocyte adhesion, coagulation and abnormal rheology, and vascular tone modulation. [6] Ameliorating oxidative stress by directly targeting free radicals, reactive carbonyls and other oxidizing species with non-toxic therapeutic agents has tremendous potential as a treatment for SCD. MTAP inhibitors also have potential as an acute therapy for sickle crisis, for which, there are no FDA approved therapeutic options.

The present invention relates to compounds according to the chemical structure:

Where X is S-R or R', where R is a d-₃ alkyl group (preferably methyl or ethyl) or a phenyl group and R' is a C C₃ alkyl group,

Or a pharmaceutically acceptable salt, solvate or polymorph thereof. In preferred embodiments, R is S-Me ("MTDIA") and the compound is in the form of a salt, often a phosphate salt.

Pharmaceutical compositions according to the present invention comprise an effective amount of a compound (an MTAP inhibitor) disclosed above in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with another bioactive agent. In certain embodiments, pyridoxamine is optionally included with the MTAP inhibitor, further optionally with another bioactive agent as otherwise disclosed herein. In alternative embodiments, the present invention relates to a method of treating sick cell disease, Thalassemia and other related blood diseases in a patient in need thereof comprising administering to said patient an effective amount of an MTAP inhibitor compound, preferably a compound as disclosed hereinabove, optionally in combination with an additional bioactive agent.

In one embodiment, the present invention provides pharmaceutical compositions comprising (a) a dosage unit of an effective amount ranging from about 1 mg to about 3000 mg of a MTAP inhibitor (often about 1 mg to about 2000mg), or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier, optionally in combination with an additional bioactive agent, preferably an active agent for treating sickle cell disease, Thalassemia and other related blood diseases as otherwise described herein. These additional bioactive agents can include anti-sickling agents, selectin inhibitors, fetal hemoglobin regulators (including fetal hemoglobin inducing agents), agents which inhibit a platelet ADP receptor, agents which increase the affinity of sickle hemoglobin's binding to oxygen, anti-oxidant agents, anti-inflammatory agents, agents that target leukocyte adhesion and anti-platelet agents, among others as described herein.

In certain embodiments, the present invention provides pharmaceutical compositions comprising: (a) MTAP inhibitor or a pharmaceutically acceptable salt thereof; and (b) one or more additional bioactive agents as described herein, preferably a compound selected from the group consisting of fetal hemoglobin-inducing agents, agents that target leukocyte adhesion, anti-inflammatory agents, anti-oxidant agents, anti-platelet agents and anti-sickling agents, among others.

In a preferred embodiment, the one or more additional bioactive agents are selected from the group consisting of hemoglobin-inducing agents, in particular fetal hemoglobin- inducing agents, but not limited to, hydroxyurea, sodium phenyl butyrate, sodium phenyl acetate, sodium phenyl propionate, or an alternative pharmaceutical salt thereof (as disclosed in United States patent number 5,712,307, which is incorporated by reference herein), cyclic peptides such as FK228 (depsipeptide) and analogs thereof, as disclosed in Anemia 2012; 2012: 428137 (published online May 14, 2012), benzamides (such as MS- 275); non cyclic and cyclic hydroxamates, for example SAHA (suberoylanilide hydroxamic acid) and TSA (Trichostatin A) among others. In a further embodiment, the present invention provides methods for treating sickle cell disease and/or complications arising from sickle cell disease including, comprising administering to the human patient an amount of an MTAP inhibitor, or a pharmaceutically acceptable salt thereof, effective to alleviate the physiological manifestations of the disease including, but not limited to, venular occlusion and sickle crisis and chronic and systemic vasculopathies including stroke, sickle retinopathy, open leg ulcers, multi-organ failure, aseptic bone necrosis, dactylitis, hepatopathy, splenic autoinfarction, pulmonary

hypertension, cognitive deficits, renal failure, cholecystitis, decreased fertility, increased susceptibility to infection and decreased opsonization, among others.

In yet another embodiment, the present invention provides methods for treating or averting episodes of sickle cell crisis whereby MTAP inhibitor, or a pharmaceutically acceptable salt, is optionally combined with an additional bioactive agent is administered (preferably, orally or by injection) to mitigate the crisis episode.

In addition to the acute events that are normally associated with sickle cell disease, secondary disease states and conditions such as vessel occlusion can also cause chronic and systemic vasculopathies that yield devastating results. These can include: stroke, sickle retinopathy, open leg ulcers, multi-organ failure, aseptic bone necrosis, dactylitis,

hepatopathy, splenic autoinfarction, pulmonary hypertension, cognitive deficits, renal failure, cholecystitis and decreased fertility. Increased susceptibility to infection and decreased opsonization capabilities have caused significant mortality in this population. To date, therapy has focused on blood transfusions and hydroxyurea. However, transfusion begets chronic iron overload and the necessity of chelation therapy (to avoid cirrhosis and cardiac failure due to hemosiderosis) and the exposure of the patient to a high likelihood of alloimmunization. Hydroxyurea has been of major benefit to patients, both in reducing overall mortality and in decreasing painful crises. In addition to increasing fetal hemoglobin (HbF) levels, hydroxyurea can also increase nitric oxide (NO) levels and has been shown to depress leukocyte counts; decreasing the local inflammatory reaction and increasing mean cell volume. The complexity of SCD dictates that a multimodal approach to treat patients will likely be needed.

The present invention which relies on the use of an MTAP inhibitor, preferably MTDIA, provides for a non-toxic, therapeutic, preferably including an oral therapeutic, that will attenuate ongoing oxidative stress in the microcirculation, improve perfusion to organs, and mitigate the damage to circulating RBC. It is anticipated that the present invention will be useful in both children and adults, as a single agent or as part of a combination therapy. As a prophylactic, it is expected that an MTAP inhibitor, preferably MTDIA, will reduce oxidative stress mediated complications in the microcirculation, hence improving vascular and organ health in SCD patients. While the prophylactic approach would have a positive impact on all SCD patients, it could have maximum impact in children where progressive damage to endothelium and organs could be significantly mitigated. [13, 14] From a purely cost perspective, episodes of crisis often require emergency room visits and extended hospital stays that on average exceed 5 days and cost between $5K and $10K per visit. [3] Medicaid and Medicare are the major payers, and responsible for covering up to 75% of the related hospital costs, resulting in annual total costs to the United States healthcare system in excess of $1 billion. [3] The low toxicity of the MTAP inhibitor, preferably MTDIA, could render the therapeutic useful as a complement or supplement to HU and other therapies. MTAP inhibitors are expected to have a broad impact on global public health, and be useful as an inexpensive oral medication to attenuate oxidative damage to RBC, normalize the microcirculatory system and ameliorate the likelihood of VOC.

The pathophysiology of SCD resembles many aspects of ischemia-repurfusion injury. Oxidative stress is a major driver of the pro-inflammatory state associated with SCD. [7] Higher rates of autoxidation of HbS in vivo, RBC damage and lysis, cell free hemoglobin, ischemia-reperfusion injury and inflammation all contribute to increased levels of oxidants, which perpetuate the pro-inflammatory state. Cell free hemoglobin, as a result of chronic hemolysis, reduces nitric oxide (NO) bioavailability, which has vasoconstrictive,

antithrombotic and anti-inflammatory properties. [20] It also causes non-enzymatic activation and damage of the endothelium by incorporation of free heme into endothelial cell membranes and generating reactive oxygen species (ROS). [21] Endothelial generation of ROS is also thought to damage erythrocyte cell walls, further promoting hemolysis. [7] Chronic and acute instances of VOC result in ischemia-reperfusion injury, which is characterized by generation of tissue damaging free radicals upon reperfusion with oxygen rich blood. [22] The ensuing inflammatory cascade results in recruitment of adhesive leukocytes and aggregation of SS erythrocytes and vascular blockage. [23] Therapies that disrupt the oxidative stress/inflammation feed back loop and have great potential as treatments for SCD. [24]

Other aspects of the Present Invention include:

Technological Innovation: The potent anti-inflammatory profile of MTA was demonstrated in a number of preclinical models. In lipopolysaccharide (LPS) challenged mice, MTA markedly reduced serum levels of the pro-inflammatory tumor necrosis factor-alpha (TNF-a) and stimulated production of the anti-inflammatory cytokine interleukin-10.¹ Cell based LPS inflammation models have shown MTA to inhibit induction of p38 Mitogen Activated Protein Kinase (MAPK), c-Jun phosphorylation and Inhibitors of Kappa B-alpha (IKBa) degradation.

Concomitant reduction of Nuclear Factor-KB (NFKB) activation was also observed. Each of these pathways is associated with the modulation of inflammatory mediators and supports the role of MTA as promoting an anti-inflammatory state. In a rat model of

ischemia/reperfusion injury following liver transplant, the LPS results are emulated where notable suppression of NFKB and MAPK pathways was observed.³ A similar reduction in pro-inflammatory cytokines was observed in murine models of inflammation induced colon cancer.²

The use of MTA alone as a therapeutic for SCD has limited utility because of pharmacokinetics (PK).²¹ MTAP rapidly metabolizes MTA in vivo with excess MTA cleared renally, as demonstrated by high urine concentrations of MTA in exploratory PK experiments (data not shown). The present inventors' approach utilizes a single dose of

methylthioDADMe-lmmucillin-A (MTDIA) to block MTA metabolism at MTAP, and allow for the whole-body accumulation of MTA. MTDIA inhibits MTAP, the sole enzyme responsible for MTA recycling, with picomolar affinity and a high level of specificity. An oral or i.p. dose of MTDIA inhibits MTAP activity in vivo for >24 h and results in whole-body accumulation of MTA in plasma to >100-fold higher than baseline levels (Preliminary Results). A single i.p. dose of MTDIA given to BERK mice improved endothelial function and ameliorated the endothelial activation that is associated with venular occlusion and episodes of sickle crisis. It is proposed that a single oral dose of MTDIA will convey similar therapeutic benefits for an extended duration (> 24 h) and experiments to validate this hypothesis are proposed in this SBIR. As one of the most powerful MTAP inhibitors ever reported, (K_m/K,^*= 58,000) ⁴'⁵, the phosphate salt of MTDIA is being developed as an oral therapeutic for acute sickle crisis.

Potential Impact

Many of the emerging therapies for acute crisis target the VOC cascade, and are predominantly anti-inflammatory approaches. The MTDIA approach is unique because it blocks the metabolism of MTA, which has downstream anti-inflammatory immunomodulatory effects that persist for > 24h. BRIEF SUMMARY OF THE FIGURES

Figure 1 shows the schematic representation of intravital microscopy protocols, (a) In normoxia protocol, a single oral dose of MTDIA (15mg/kg, gavage) was administered to BERK sickle mice 21 hours (time -21 hr) prior to the surgical preparation of cremasteric muscle for intravital microscopy (time 0). Images of the cremasteric venules under intravital microscopy were recorded between the time points of 1 and 2 hr. During the filming, the hemodynamic parameters, including centerline velocity, venular diameter, and shear rate, were measured, (b) Hypoxia/Reoxygenation protocol was designed to assess the therapeutic effect of MTDIA on sickle mice with ongoing acute vaso-occlusive crisis induced by hypoxia and reoxygenation. A single oral dose of MTDIA (15mg/kg, gavage) was administered to BERK sickle mice 16 hours (time -16hr) prior to placing the mice in 8% 0₂ (balanced with N₂) hypoxia chamber (time 0). After 3 hours in hypoxia chamber, mice were returned to ambient air for a total of 4 hours (reoxygenation). Exteriorization of cremasteric muscle and imaging/recording of cremasteric microcirculation were carried out during the 4hr-reoxygenation period.

Figure 2 shows the results of intravital microscopic analysis of cremasteric microcirculation. Results are presented as mean ± SEM (n=3 for each group of C57BL/6 mice; n=5 for each group of BERK sickle mice). Student's ests. *p<0.05; **p < 0.01 , ^***p<0.001. A single dose of MTDIA (15mg/kg, oral gavage, 24 hr prior) improves blood flow rate (a) and reduces leukocyte adhesion (b) in BERK mice (normoxia) and in BERK mice challenged with hypoxia/reoxygenation (H/R). H/R challenge; however, did not further reduce blood flow rate or further increase leukocyte adhesion in BERK mice (both blood flow rate and number of adherent leukocyte are comparable between normoxia and H/R-challenged BERK mice). MTDIA treatment did not alter (c) the systemic white blood cell (WBC) and (d) did not significantly change the number of emigrated leukocytes in the area adjacent to the postcapillary venules in the cremaster muscle of BERK mice during the time frame of our experiment.

Figure 3 shows the values of red blood cell (RBC) count, hemoglobin concentration (Hgb) and hematocrit (Hct) of vehicle- and MTDIA-treated (1 dose, 15mg/kg, oral gavage, 24hr prior) BERK sickle mice during steady state or challenged with hypoxia/reoxygenation (H/R). Results are presented as mean ± SEM (n=6 for vehicle- and MTDIA-treated BERK mice challenged with H/R, n=5 for steady state BERK mice treated with vehicle, n=7 for steady state BERK mice treated with MTDIA 24hr prior to blood collection). Student's f-tests.

*p<0.05; ^**p < 0.01 , **^*p<0.001. During steady state, MTDIA treatment did not change RBC, Hgb and Hct of BERK mice. However, MTDIA treatment protected BERK mice from H/R- mediated acute hemolysis of RBC and significantly increased RBC count, Hgb and Hct of treated BERK mice.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used throughout the specification to describe the present invention. Where a term is not specifically defined herein, that term shall be understood to be used in a manner consistent with its use by those of ordinary skill in the art.

Where a range of values is provided in the present application, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. In instances where a substituent is posited for use in a present compound, it is understood that only those substituents which form stable bonds or stable compounds are to be embraces.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those

described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set out below.

The term "patient" or "subject" is used throughout the specification within context to describe an animal, generally a mammal, especially including a human, to whom treatment, including prophylactic treatment (prophylaxis), with the compounds or compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a human patient of either or both genders. In certain embodiments, the patient is resistant to therapy with hydroxyurea. In other embodments, hydroxyurea may be co-administered with the MTAP inhibitor (preferably MTDIA) to effect therapy in the patient. The term

"prophylactic" or "prophylaxis" shall mean preventing or reducing the likelihood that a disease, condition or event will occur.

The term "effective" is used herein, unless otherwise indicated, to describe an amount of a compound or component which, when used within the context of its use, produces or effects an intended result, whether that result relates to the prophylaxis and/or therapy of a disease state, a secondary disease state or condition thereof or as otherwise described herein. The term effective subsumes all other effective amount or effective concentration terms

(including the term "therapeutically effective") which are otherwise described or used in the present application.

The term "compound" is used herein to describe any specific compound or bioactive agent disclosed herein, including any and all stereoisomers (including diasteromers) if applicable, individual optical isomers (enantiomers) or racemic mixtures, pharmaceutically acceptable salts, prodrug forms, including hydrates and solvates of these compounds. The term compound herein refers to stable compounds. Within its use in context, the term compound may refer to a single compound or a mixture of compounds as otherwise described herein.

The term "bioactive agent" or "additional bioactive agent" refers to any biologically active compound or drug which may be formulated for use in an embodiment of the present invention. Exemplary bioactive agents include the compounds according to the present invention which are used to treat sick cell anemia, Thallesemia or a disease state or condition which occurs secondary to sick cell anemia, thallesemia or and other related blood diseases as well as other compounds or agents which are otherwise described herein.

Bioactive agents for use in the present invention include anti-sickling agents, selectin and adhesion inhibitors, fetal hemoglobin regulators, agents which inhibit a platelet ADP receptor and other anti-platelet agents, agents which increase the affinity of sickle hemoglobin's binding to oxygen, antioxidants, nitric oxide generating agents, vascular tone agents and anti-inflammatory agents and agents that target leukocyte adhesion, among others. A fetal hormone regulator includes hemoglobin-inducing agents, in particular fetal hemoglobin- inducing agents.

The following presents specific representative bioactive agents which can be coadministered with the MTAP inhibitor (preferably MTDIA) or a pharmaceutically acceptable salt thereof. anti-sickling agents: 5-hydroxymethylfurfural, 4-Hydroxy-3-methoxybenzaldehyde. selectin and adhesion inhibitors: GMI-1070, GMI-1271 , intravenous immunoglobulin, tinzaparin, propranolol, SelG1 (humanized anti p-selectin antibody), heparin. fetal hemoglobin regulators include hemoglobin inducing agents: hydroxyurea, decitabine, sodium dimethylbutyrate, pomalidomide, (2E)-N-hydroxy-3-[4-({[2-(2-methyl-1 H- indol-3-yl)ethyl]amino}methyl)phenyl]acrylamide, sodium phenyl butyrate, sodium phenyl acetate, sodium phenyl propionate, or an alternative pharmaceutical salt thereof (as disclosed in United States patent number 5,712,307, which is incorporated by reference herein), cyclic peptides such as FK228 (depsipeptide) and analogs thereof, as disclosed in Anemia 2012; 2012: 428137 (published online May 14, 2012), benzamides (such as MS- 275); non cyclic and cyclic hydroxamates, for example SAHA (suberoylanilide hydroxamic acid) and TSA (Trichostatin A) among others. agents which inhibit a platelet ADP receptor: (f?,S)-5-[2-cyclopropyl-1-(2-fluorophenyl)-2- oxoethyl] 4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate, N-hydroxy-N'-phenyl- octanediamide. agents which increase the affinity of sickle hemoglobin's binding to oxygen: 5- hydroxymethylfurfural, pyridoxal-5-phosphate, 4-hydroxy-3-methoxybenzaldehyde. antioxidants including, but not limited to, para-aminobenzoic acid (PABA), pyridoxine, pyridoxine-5-phosphate, pyridoxal-5-phosphate, ascorbic acid, W-acetyl cysteine, a-linolenic acid, eicosapentaenoic acid, docosahexanoc acid, glutamine, acetyssl-L-carnitine.

Nitric oxide generating agents: L-Arginine.

Vascular tone agents: intravenous magnesium.

Anti-inflammatory agents: 2-{4-[(methylamino)carbonyl]- 1 H-pyrazol-1-yl}adenosine, N-[1- (1-benzothien-2-yl)ethyl]-N-hydroxyurea, Fructose-1 ,6-diphosphate, oxooxan-2-yl]ethyl}-3,7- dimethyl-1 ,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate, 2-acetoxybenzoic acid.

The terms "treat", "treating", and "treatment", are used synonymously to refer to any action providing a benefit to a patient at risk for or afflicted with a disease state or condition as described herein, including improvement in the disease state or condition through lessening, inhibition, suppression or elimination of at least one symptom, delay in progression of the disease, prevention, delay in or inhibition of the likelihood of the onset of the disease or condition, etc.

Treatment, as used herein, encompasses both prophylactic and therapeutic treatment, principally of sickle cell diseases, Thalassemia and other related blood diseases, as well as secondary disease states and conditions such as vessel occlusion, chronic and systemic vasculopathies, as well as stroke, sickle retinopathy, open leg ulcers, multi-organ failure, aseptic bone necrosis, dactylitis, hepatopathy, splenic autoinfarction, pulmonary

hypertension, cognitive deficits, renal failure, cholecystitis, decreased fertility, increased susceptibility to infection and decreased opsonization, as well as other disease states and conditions. Compounds according to the present invention can, for example, be

administered prophylactically to a patient in advance of the occurrence of a disease state or condition to reduce the likelihood of that disease state or condition. Prophylactic

administration is effective to reduce or decrease the likelihood of the subsequent occurrence of disease in the patient, or decrease the severity of the disease state or condition that subsequently occurs, especially including secondary disease states or conditions.

Alternatively, compounds according to the present invention can, for example, be

administered therapeutically to a patient that is already afflicted by disease. In one embodiment of therapeutic administration, administration of the present compounds is effective to eliminate the disease and substantially eliminate the likelihood of further manifestations of disease. Administration of the compounds according to the present invention is effective to decrease the severity of the disease or lengthen the lifespan of the mammal so afflicted, or inhibit or even eliminate the causative agent of the disease.

The term "pharmaceutically acceptable" as used herein means that the compound or composition is suitable for administration to a subject, including a human patient, to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

The term "inhibit" as used herein refers to the partial or complete elimination of a potential effect, while inhibitors are compounds/compositions that have the ability to inhibit. The term "prevention" or "prophylactic" when used in context shall mean "reducing the likelihood" of preventing a disease, condition or disease state from occurring as a

consequence of administration or concurrent administration of one or more compounds or compositions according to the present invention, alone or in combination with another agent. It is noted that prophylaxis will rarely be 100% effective; consequently the terms prevention and reducing the likelihood are used to denote the fact that within a given population of patients or subjects, administration with compounds according to the present invention will reduce the likelihood or inhibit a particular condition or disease state (in particular, the worsening of a disease state such as the growth or metastasis of cancer) or other accepted indicators of disease progression from occurring.

The terms "coadminister" and "coadministration" are used synonymously to describe the administration of MTAP inhibitor, preferably MTDIA and at least one additional bioactive agent (as otherwise described herein), which are administered in amounts or at

concentrations which would be considered to be effective amounts at or about the same time. While it is preferred that coadministered compositions/agents be administered at the same time, agents may be administered at different times such that effective concentrations of both (or more) compositions/agents appear in the patient at the same time for at least a brief period of time. Alternatively, in certain aspects of the present invention, it may be possible to have each coadministered composition/agent exhibit its inhibitory effect at different times in the patient, with the ultimate result being the inhibition and treatment of sickle cell disease, Thalassemia or a related blood disease or disorder or a secondary disease state or condition thereof. Of course, when more than disease state or condition is present, the present compounds may be combined with other agents to treat that other disease state or condition as required.

The term "sickle cell disease" (SCD) as used herein refers to a hereditary blood disorder in which red blood cells assume an abnormal, rigid, sickle shape. Sickling of erythrocytes decreases the cells' flexibility and results in a risk of various life-threatening complications. Sickle cell anaemia is a form of sickle cell disease.

Sickle-cell disease may lead to various acute and chronic complications, several of which have a high mortality rate. These include sickle cell crisis, vaso-occlusive crisis, splenic sequestration crisis, acute chest syndrome (ACS), aplastic crisis, haemolytic crisis, dactylitis, increased risk of severe bacterial infections, especially Streptococcus pneumonia and Haemophilus influenza, due to loss of functioning spleen tissue, stroke, cerebral infarction in children, cerebral haemorrhage in adults, silent stroke, causing no outward symptoms but associated with damage to the brain, cholelithiasis (gallstones) and cholecystitis, avascular necrosis (aseptic bone necrosis) of the hip and other major joints, decreased immune reactions due to hyposplenism, priapism, osteomyletis (bacterial bone infection, often from Salmonella), acute papillary necrosis (kidneys), leg ulcers, eye complications (background retinopathy, proliferative retinopathy, vitreous haemorrhages and retinal detachments), pregnancy complications (intrauterine growth retardation, spontaneouos abortion, pre- eclampsis, chronic pain (even in the absence of acute vaso-occlusive pain), pulmonary hypertension, strain on the right ventricle and risk of heart failure, nephropathy, chronic renal failure due to nephropathy and cognitive deficits. SCD patients often suffer from kidney disease and/or kidney related conditions similar to those of diabetes patients. These disease states and/or conditions may be treated using the present invention as well.

The term "Thalassemia" is used herein to describe an inherited blood disorder in which the body makes an abnormal form of hemoglobin resulting in less hemoglobin than normal and far fewer circulating red blood cells, resulting in a mild or severe anemia. Thalassemia is often present as microcytic anemia. Thalassemia can cause significant complications, including iron overload, an enlarged spleen, susceptible to illness, bone deformities and cardiovascular illness, each of which may be improved and/or resolved by treatment of principal disease state. Thalassemia may confer a level of protection against malaria.

The term "sickle cell trait" is used herein to describe a condition in which a person has one abnormal allele of the hemoglobin beta gene (is heterozygous), but does not display the severe symptoms of sickle cell disease that occur in a person who has two copies of that allele (is homozygous).

Sickle cell disease (SCD) can be cured using hematopoietic stem cell transplants, and the severity of the disease can be reduced with repeated transfusions. However, these approaches are costly and associated with high levels of risk. The only FDA approved therapeutic for SCD is hydroxyurea (HU). HU induces fetal Hb (HbF) production, and has a strong clinical track record in children and adults. Proper use of HU requires strict compliance; self-administration by the patient; and careful monitoring by the physician to ensure proper dosing schedules. [2] Unfortunately, approximately a third of adult patients do not respond to HU treatment. [4] Moreover, myeolsuppression, reproductive toxicity and possible carcinogenic affects are all serious side effects associated with long-term use. [5] Importantly, there is currently no commercial therapeutic to avert imminent episodes of crisis or to attenuate acute events of crisis once they have begun. Patients are typically prescribed medications that alleviate the pain associated with episodes of crisis.

While SCD is typically characterized as a molecular disease; its pathophysiological consequences mimic those of ischemic reperfusion injury. [6] The chronic hemolytic anemia disrupts the oxidative stress equilibrium of patients, and can trigger inflammatory cascades that result in adhesion of leukocytes to vascular walls, recurrent and intermittent episodes of vaso-occlusion (VOC) and painful episodes of crisis. [4, 7] The pain associated with crisis can be debilitating, and lead to hospitalization. [3] Due to the importance of oxidative stress events in propagating VOC, many forms of antioxidant therapy are being actively pursued as novel approaches to treat the disease. Examples include oral supplementation of arginine [8], N-acetyl cysteine [9] and treatment with omega-3 fatty acids. [10]

Chronic hemolytic anemia and vaso-occlusive ischemia-reperfusion like injury are the hallmark pathologies of sickle cell disease. [5] Oxidative stress and NO homeostasis are thought to play a major role in the severity of the disease, and agents that modulate these pathways are highly desirable. The multifactorial process, which leads to VOC involves oxidative stress, damages to red blood cells (RBC), inflammation, vascular leukocyte adhesion, coagulation and abnormal rheology, and vascular tone modulation. [6]

Ameliorating oxidative stress by directly targeting free radicals, reactive carbonyls and other oxidizing species with non-toxic therapeutic agents has tremendous potential as a treatment for SCD. Surprisingly, Nanometics (in collaboration with the Albert Einstein College of Medicine) has discovered that MTAP inhibitors are useful to ameliorate the effects of SCD in transgenic mouse models of disease.

There are several emerging therapies that are under clinical evaluation and these include: fetal hemoglobin-inducing agents, agents that target leukocyte adhesion, anti-inflammatory agents, anti-oxidant therapies, anti-platelet therapies and anti-sickling approaches. [17] Another therapy, pyridoxamine is unique in that it is a well-tolerated vitamer of the B6 family, and has been studied extensively in humans. Preliminary results suggest that pyridoxamine is multimodal; disrupting molecular mechanisms of the vaso-occlusion (VOC) cascade, and simultaneously facilitating oxygen delivery to the brain. Many of the emerging therapies target the VOC cascade, and are predominantly anti-inflammatory approaches. It is anticipated that the MTAP inhibitor, preferably MTDIA, as its pharmaceutically acceptable salt, will be useful as a single agent, or as part of a combination therapy (co-administration approach) to treat sickle cell disease.

Such co-administration of current or emerging therapeutics with a MTAP inhibitor may also permit administration of lower dosages of these other therapeutics, thus minimizing potential side effects. Thus, in a further aspect, the present invention provides pharmaceutical compositions comprising (a) MTAP inhibitor, or a pharmaceutically acceptable salt thereof; and (b) one or more compounds that can provide benefit in a human patient, or

pharmaceutically acceptable salts thereof. In a preferred embodiment, such compounds are selected from the group consisting of fetal hemoglobin-inducing agents as otherwise described herein, agents that target leukocyte adhesion, anti-inflammatory agents, antioxidant therapies, anti-platelet therapies or anti-sickling therapeutics. In another preferred embodiment, pyridoxamine is co-administered with hydroxyurea.

Pharmaceutically acceptable salts in accordance with the present invention, include salts with physiologically acceptable bases and/or acids well known to those skilled in the art of pharmaceutical technique. Suitable salts with physiologically acceptable bases include, for example, alkali metal and alkaline earth metal salts, such as sodium, potassium, calcium and magnesium salts, and ammonium salts and salts with suitable organic bases, such as methylamine, dimethylamine, trimethylamine, piperidine, morpholine and triethanolamine. Suitable salts with physiologically acceptable acids include, for example, salts with inorganic acids such as hydrohalides (especially hydrochlorides or hydrobromides), sulphates and phosphates, and salts with organic acids. Phosphate salts of MTDIA are preferred. The pharmaceutical compositions of this aspect of the invention include admixtures of the MTAP inhhibitor, or pharmaceutically acceptable salt thereof, and the one or more other

compounds, as well as separate unit dosages of each that are manufactured for

combinatorial use. Such separate unit dosages may be administered concurrently or sequentially as determined by the clinician.

In all aspects of the pharmaceutical compositions of the present invention, the compounds are combined with one or more pharmaceutically acceptable carriers, excipients and/or additives appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art. In a preferred embodiment of each of the above aspects of the invention, the pharmaceutical compositions of the invention are prepared for oral administration. As such, the pharmaceutical composition can be in the form of, for example, a tablet, a hard or soft capsule, a lozenge, a cachet, a dispensable powder, granules, a suspension, an elixir, a liquid, or any other form reasonably adapted for oral administration. The pharmaceutical compositions can further comprise, for example, buffering agents. Tablets, pills and the like additionally can be prepared with enteric coatings. Unit dosage tablets or capsules are preferred. Pharmaceutical compositions suitable for buccal administration include, for example, lozenges comprising a MTAP inhibitor or a pharmaceutically acceptable salt thereof and a flavored base, such as sucrose, acacia tragacanth, gelatin, and/or glycerin. Liquid dosage forms for oral administration can comprise pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions can also comprise, for example, wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

Pharmaceutical compositions according to the present invention comprise an effective amount of MTAP inhibitor or a pharmaceutically acceptable salt, optionally in combination with an additional bioactive agent as otherwise described herein (which may include pyridoxamine) formulated to effect an intended result (e.g. therapeutic and/or prophylactic result) formulated in combination with a pharmaceutically acceptable carrier, additive or excipient. Pharmaceutical compositions according to the present invention may also comprise an addition bioactive agent or drug as otherwise described herein.

Generally, dosages and routes of administration of the compound are determined according to the size and condition of the subject, according to standard pharmaceutical practices. Dose levels employed can vary widely, and can readily be determined by those of skill in the art. Typically, amounts in the milligram or less up to gram quantities or more are employed. The composition may be administered to a subject by various routes, e.g. orally, transdermal^, topically, perineurally or parenterally, that is, by intravenous, subcutaneous, intraperitoneal, intrathecal or intramuscular injection, among others, including buccal, rectal and transdermal administration. Compositions may be administered by inhalation dosage form to the lungs, trachea, nasal passages, mouth, etc. Subjects contemplated for treatment according to the method of the invention include humans, companion animals, laboratory animals, and the like. The invention contemplates immediate and/or sustained/controlled release compositions, including compositions which comprise both immediate and sustained release formulations. This is particularly true when one or more different bioactive agents are used in the pharmaceutical compositions in combination with MTAP inhibitor or its pharmaceutically acceptable salt as otherwise described herein.

Administration of these formulations once or twice a day is preferably contemplated.

Formulations containing the compounds according to the present invention may take the form of liquid, solid, semi-solid or lyophilized powder forms, such as, for example, solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, patches or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

Pharmaceutical compositions according to the present invention typically include a conventional pharmaceutical carrier, excipient and/or additive and may additionally include other medicinal agents, adjuvants and the like. Preferably, the composition is about 0.1 % to about 85%, about 0.5% to about 75% by weight of an MTAP inhibitor or its salt and optionally, an additional bioactive agent, with the remainder of the composition consisting essentially of suitable pharmaceutical excipients.

An injectable composition for parenteral administration (e.g. intravenous, intramuscular or intrathecal) will typically contain the compound in a suitable i.v. solution, such as sterile physiological salt solution. The composition may also be formulated as a suspension in an aqueous emulsion. Liquid compositions can be prepared by dissolving or dispersing the active agent(s) (about 0.5% to about 20% by weight or more), and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension. For use in an oral liquid preparation, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.

For oral administration as discussed above, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like, as described. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.

When the composition is employed in the form of solid preparations for oral administration, the preparations may be tablets, granules, powders, capsules or the like. In a tablet formulation, the composition is typically formulated with additives, e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.

Methods for preparing dosage forms for use in the present invention are known or are apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co., 1985). The composition to be administered will contain a quantity of the selected compound in a pharmaceutically effective amount for therapeutic use in a patient according to the present invention. These pharmaceutical compositions can be prepared by any suitable method that includes the step of bringing into association pyridoxamine, or a pharmaceutically acceptable salt thereof (and optionally the other compounds) and the pharmaceutically acceptable carrier. In general, the compositions are prepared by uniformly and intimately admixing the MTAP inhibitor, or a pharmaceutically acceptable salt thereof, with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, preparation of tablets can comprise compressing or molding a powder or granule of the compound. Compressed tablets can be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binding agent, lubricant, inert diluent and/or surface active/dispersing agent(s). Molded tablets can be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid diluent.

In another aspect, the present invention provides methods for limiting the progression of end organ disease and/or complications in a human patient with sickle cell disease by administering to the patient an amount of a MTAP inhibitor, or a pharmaceutically acceptable salt thereof, effective to limit the progression of end organ disease or complications in the sickle cell disease patient. In a preferred embodiment, the methods comprise administering the pharmaceutical compositions of the invention to the patient. Thus, one embodiment of the method comprises administering between about 500 pg and 2500mg, more often about 1 mg to about 2000 milligrams of a MTAP inhibitor (preferably MTDIA), or a pharmaceutically acceptable salt thereof, to the patient, more preferably between about 5 and 1000 milligrams of a MTAP inhibitor, preferably MTDIA, or a pharmaceutically acceptable salt thereof, optionally in combination with an additional bioactive agent. EXAMPLES

Preliminary Studies: Preliminary studies tested the hypothesis that systemic MTAP inhibition, and the subsequent increase in whole-body MTA levels, would be non-toxic in mice and convey therapeutic benefits in BERK models of SCD. All studies were conducted with the phosphate salt of MTDIA. i. A single oral dose of MTDIA alleviates endothelial activation in BERK mouse models of SCD: Knockout Berkley (BERK) mice express 100% human hemoglobin a-subunit, >99% human hemoglobin ^s-subunit and similar pathologies to humans with SCD. Characteristic pathologies include anemia, hemolysis, reticulocytosis, low hematocrit, and extensive multiple organ damage.^22,23 Vascular obstruction is thought to occur primarily as a result of interactions between adherent leukocytes, in postcapillary and collecting venules, and circulating RBCs.²⁴ BERK mice display elevated levels of leukocyte recruitment to the vascular endothelium, which is detrimental to blood rheology and a key contributor to disease pathology. Intravital microscopic analysis of cremastic venules 4h after a single i.p. dose of MTDIA was used to determine the effect of MTDIA on leukocyte recruitment and hemodynamic properties in BERK mice. Importantly, a single i.p. dose of MTA (15 - 100 mg/kg) did not induce any measurable changes in these parameters (data not shown). A single i.p. dose of MTDIA (15 mg/kg) significantly decreased the number of rolling leukocytes, as defined by the total number of rolling leukocytes (stationary on vessel walls f or <40 sec) per 100 μηι of vessel observed. Sickle red blood cells (sRBCs) primarily interact with adherent leukocytes and the number of adherent leukocytes was dramatically reduced to near wild-type levels. Leukocytes were considered adherent when observed stationary on vessel walls >40 sec. Leukocyte emigration, was also significantly reduced in these studies, which indicates a reduced inflammatory response. Improvements in RBC velocity, and shear rate were also noted. Venule diameter remained constant, indicating that

improvements in shear rate were a result of attenuation of endothelial activation.

Conclusion: A single i.p. dose of MTDIA (15 mg/kg) mitigates vascular endothelial activation in BERK mice, which results in improved hemodynamics.

Pursuant to this example, Figure 1 shows the schematic representation of intravital microscopy protocols, (a) In normoxia protocol, a single oral dose of MTDIA (15mg/kg, gavage) was administered to BERK sickle mice 21 hours (time -21 hr) prior to the surgical preparation of cremasteric muscle for intravital microscopy (time 0). Images of the cremasteric venules under intravital microscopy were recorded between the time points of 1 and 2 hr. During the filming, the hemodynamic parameters, including centerline velocity, venular diameter, and shear rate, were measured, (b) Hypoxia/Reoxygenation protocol was designed to assess the therapeutic effect of MTDIA on sickle mice with ongoing acute vaso- occlusive crisis induced by hypoxia and reoxygenation. A single oral dose of MTDIA

(15mg/kg, gavage) was administered to BERK sickle mice 16 hours (time -16hr) prior to placing the mice in 8% 0₂ (balanced with N₂) hypoxia chamber (time 0). After 3 hours in hypoxia chamber, mice were returned to ambient air for a total of 4 hours (reoxygenation). Exteriorization of cremasteric muscle and imaging/recording of cremasteric microcirculation were carried out during the 4hr-reoxygenation period.

Figure 2 shows the results of intravital microscopic analysis of cremasteric microcirculation. Results are presented as mean ± SEM (n=3 for each group of C57BL/6 mice; n=5 for each group of BERK sickle mice). Student's -tests. *p<0.05; **p < 0.01 , **^*p<0.001. A single dose of MTDIA (15mg/kg, oral gavage, 24 hr prior) improves blood flow rate (a) and reduces leukocyte adhesion (b) in BERK mice (normoxia) and in BERK mice challenged with hypoxia/reoxygenation (H/R). H/R challenge; however, did not further reduce blood flow rate or further increase leukocyte adhesion in BERK mice (both blood flow rate and number of adherent leukocyte are comparable between normoxia and H/R-challenged BERK mice). MTDIA treatment did not alter (c) the systemic white blood cell (WBC) and (d) did not significantly change the number of emigrated leukocytes in the area adjacent to the postcapillary venules in the cremaster muscle of BERK mice during the time frame of our experiment.

*p<0.05; **p < 0.01 , ***p<0.001. During steady state, MTDIA treatment did not change RBC, Hgb and Hct of BERK mice. However, MTDIA treatment protected BERK mice from H/R- mediated acute hemolysis of RBC and significantly increased RBC count, Hgb and Hct of treated BERK mice. References

1 WHO. (Geneva, 2005).

2 Kauf, T. L, Coates, T. D., Huazhi, L, Mody-Patel, N. & Hartzema, A. G. The cost of health care for children and adults with sickle cell disease. Am J Hematol 84, 323-327

(2009) .

3 Piatt, O. S. Sickle cell anemia as an inflammatory disease. J Clin Invest 106, 337-338 (2000).

Smith, W. R. Pain in sickle cell disease: the future of acute treatment. Expert Rev Hematol 4, 237-239 (2011 ).

5 Kaul, D. K. ef al. Robust vascular protective effect of hydroxamic acid derivatives in a sickle mouse model of inflammation. Microcirculation 13, 489-497 (2006).

Taylor, L. E., Stotts, N. A., Humphreys, J., Treadwell, M. J. & Miaskowski, C. A review of the literature on the multiple dimensions of chronic pain in adults with sickle cell disease. J Pain Symptom Manage 40, 416-435 (2010).

Smith, W. R. et al. Daily assessment of pain in adults with sickle cell disease. Ann Intern Me / 148, 94-101 (2008).

Brandow, A. M., Brousseau, D. C. & Panepinto, J. A. Postdischarge pain, functional limitations and impact on caregivers of children with sickle cell disease treated for painful events. Br J Haematol 144, 782-788 (2009).

Lanzkron, S. et al. Systematic review: Hydroxyurea for the treatment of adults with sickle cell disease. Ann Intern Med 148, 939-955 (2008).

10 Brousseau, D. C, Owens, P. L, Mosso, A. L, Panepinto, J. A. & Steiner, C. A. Acute care utilization and rehospitalizations for sickle cell disease. Jama 303, 1288-1294

(2010) .

11 Mayer, M. L, Konrad, T. R. & Dvorak, C. C. Hospital resource utilization among patients with sickle cell disease. J Health Care Poor Underserved 14, 122-135 (2003).

2 Rees, D. O, Williams, T. N. & Gladwin, M. T. Sickle-cell disease. Lancer 376, 2018-2031 (2010).

13 Quinn, C. T., Rogers, Z. R., McCavit, T. L. & Buchanan, G. R. Improved survival of

children and adolescents with sickle cell disease. Blood 115, 3447-3452 (2010).

4 Piel, F. B. et al. Global epidemiology of sickle haemoglobin in neonates: a contemporary geostatistical model-based map and population estimates. Lancer 381, 142-151 (2013). 5 Steinberg, M. H. et al. Fetal hemoglobin in sickle cell anemia: determinants of response to hydroxyurea. Multicenter Study of Hydroxyurea. Blood 89, 1078-1088 (1997).

6 Manwani, D. & Frenette, P. S. Vaso-occlusion in sickle cell disease: pathophysiology and novel targeted therapies. Blood 19, 19 (2013). Chang, J. et al. GMI-1070, a novel pan-selectin antagonist, reverses acute vascular occlusions in sickle cell mice. Blood 116, 1779-1786, doi:10.1182/blood-2009-12-260513 (2010).

Qari, M. H. et al. Reduction of painful vaso-occlusive crisis of sickle cell anaemia by tinzaparin in a double-blind randomized trial. Thromb Haemost 98, 392-396 (2007). Zhang, C. et al. Anti-sickling effect of MX-1520, a prodrug of vanillin: an in vivo study using rodents. Br J Haematol MS, 788-795 (2004).

Hevia, H. et al. 5'-methylthioadenosine modulates the inflammatory response to endotoxin in mice and in rat hepatocytes. Hepatology Z9, 1088-1098 (2004).

Li, T. W. ei al. Effects of S-adenosylmethionine and methylthioadenosine on

inflammation-induced colon cancer in mice. Carcinogenesis 33, 427-435 (2012).

Tang, Y. et al. 5'-Methylthioadenosine Attenuates Ischemia Reperfusion Injury After Liver Transplantation in Rats. Inflammation 37, 1366-1373 (2014).

Schramm, V. L. Enzymatic transition states: thermodynamics, dynamics and analogue design. Archives of biochemistry and biophysics 433, 13-26,

doi:10.1016/j.abb.2004.08.035 (2005).

Singh, V. et al. Picomolar transition state analogue inhibitors of human 5'- methylthioadenosine phosphorylase and X-ray structure with MT-immucillin-A.

Biochemistry 43, 9-18 (2004).

Nur, E., Biemond, B. J., Otten, H.-M., Brandjes, D. P. & Schnog, J. J. Oxidative stress in sickle cell disease; pathophysiology and potential implications for disease management. Am J Hematol 86, 484-489 (2011 ).

Sparkenbaugh, E. & Pawlinski, R. Interplay between coagulation and vascular inflammation in sickle cell disease. Br J Haematol 162, 3-14 (2013).

Conran, N. & Costa, F. F. Hemoglobin disorders and endothelial cell interactions. Clin Biochem 42, 1873-2933 (2009).

Field, J. J. ei al. Sickle cell vaso-occlusion causes activation of iNKT cells that is decreased by the adenosine A2A receptor agonist regadenoson. Blood 17, 3329-3334 (2013).

Adam, S. S. & Hoppe, C. Potential role for statins in sickle cell disease. Pediatr Blood Cancer 60, 550-557 (2013).

Kuvibidila, S. ei al. Differential effects of hydroxyurea and zileuton on interleukin-13 secretion by activated murine spleen cells: implication on the expression of vascular cell adhesion molecule-1 and vasoocclusion in sickle cell anemia. Cytokine 30, 213-218 (2005). Marangos, P. J., Fox Aw Fau - Riedel, B. J., Riedel Bj Fau - Royston, D., Royston D Fau - Dziewanowska, Z. E. & Dziewanowska, Z. E. Potential therapeutic applications of fructose-1 ,6-diphosphate. Expert Opin Investig Drugs 7, 615-623 (1998).

Belcher, J. D. et al. MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1 , inflammation, and vaso-occlusion in transgenic sickle mice. Blood 122, 2757-2764 (2013).

Scheuplein, F. et al. A humanized monoclonal antibody specific for invariant Natural Killer T (iNKT) cells for in vivo depletion. PLoS One 27, e76692 (2013).

Rees, D. C. & Gibson, J. S. Biomarkers in sickle cell disease. British Journal of

Hematology 156, 433-445 (2013).

Lanzkron, S., Carroll, C. P. & Haywood, C. The Burden of Emergency Department Use for Sickle Cell Disease: An Analysis of the National Emergency Department Sample Database. American journal of hematology 85, 797-799, doi:10.1002/ajh.21807 (2010). Stallworth, J. R., Jerrell, J. M. & Tripathi, A. Cost-effectiveness of hydroxyurea in reducing the frequency of pain episodes and hospitalization in pediatric sickle cell disease. Am J Hematol 85, 795-797 (2010).

Moore, R. D., Charache, S., Terrin, M. L, Barton, F. B. & Ballas, S. K. Cost-effectiveness of hydroxyurea in sickle cell anemia. Investigators of the Multicenter Study of

Hydroxyurea in Sickle Cell Anemia. Am J Hematol 64, 26-31 (2000).

Moreno, B., Fernandez-Diez B Fau - Di Penta, A., Di Penta A Fau - Villoslada, P. & Villoslada, P. Preclinical studies of methylthioadenosine for the treatment of multiple sclerosis. Mult Scler 16, 1102-1108 (2010).

Reiter, C. D. et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 8, 1383-1389 (2002).

Jeney, V. et al. Pro-oxidant and cytotoxic effects of circulating heme. Blood 100, 879-887 (2002).

Zweier, J. L. & Talukder, M. A. H. The role of oxidants and free radicals in reperfusion injury. Cardiovascular Research 70, 181-190, doi:10.1016/j.cardiores.2006.02.025 (2006).

Hebbel, R. P., Osarogiagbon, R. & Kaul, D. The endothelial biology of sickle cell disease: inflammation and a chronic vasculopathy. Microcirculation 11, 129-151 (2004).

Karaman, M. W. et al. A quantitative analysis of kinase inhibitor selectivity. Nat

Biotechnol 26, 127-132 (2008).

Ryan, T. M., Ciavatta, D. J. & Townes, T. M. Knockout-transgenic mouse model of sickle cell disease. Science 278, 873-876 (1997).

Paszty, C. et al. Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 278, 876-878 (1997). Manci, E. A. et al. Pathology of Berkeley sickle cell mice: similarities and differences with human sickle cell disease. Blood 107, 1651-1658 (2006).

Turhan, A., Weiss, L. A., Mohandas, N., Coller, B. S. & Frenette, P. S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm. Proc Natl Acad Sci U S A 9S, 3047-3051 , doi: 10.1073/pnas.052522799 (2002).

Colerangle, J. B. in A Comprehensive Guide to Toxicology in Preclinical Drug

Development (ed Ali S. Faqi) 517-542 (Academic Press, 2013).

Kannan, K. & Jain, S. K. Effect of vitamin B6 on oxygen radicals, mitochondrial membrane potential, and lipid peroxidation in H202-treated U937 monocytes. Free Radic Biol Med 36, 423-428 (2004).

Pell, G. S., Lewis, D. P. & Branch, C. A. Pulsed arterial spin labeling using TurboFLASH with suppression of intravascular signal. Magn Reson Med 49, 341-350 (2003).

Cain, D. M., Vang, D., Simone, D. A., Hebbel, R. P. & Gupta, K. Mouse models for studying pain in sickle disease: effects of strain, age, and acuteness. Br J Haematol 156, 535-544 (2012).

Freireich, E. J., Gehan, E. A., Rail, D. P., Schmidt, L. H. & Skipper, H. E. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep 50, 219-244 (1966).

Chen, G. et al. Heme-induced neutrophil extracellular traps contribute to the

pathogenesis of sickle cell disease. Blood 123, 3818-3827, doi:10.1 182/blood-2013-10- 529982 (2014).

Zhang, Y. et al. Detrimental effects of adenosine signaling in sickle cell disease. Nat Med 17, 79-86 (201 1 ).

Scheiermann, C. et al. Adrenergic nerves govern circadian leukocyte recruitment to tissues. Immunity 37, 290-301 (2012).

Lim, Y. C. & Luscinskas, F. W. Isolation and culture of murine heart and lung endothelial cells for in vitro model systems. Methods Mol Biol 341 , 141 -154 (2006).

Zhang, D. et al. Neutrophil ageing is regulated by the microbiome. Nature 525, 528-532 (2015).

Lowell, C. A., Fumagalli, L. & Berton, G. Deficiency of Src family kinases p59/61 hck and p58c-fgr results in defective adhesion-dependent neutrophil functions. J Cell Biol 133, 895-910 (1996).

Belbin, T. J. er al. Molecular profiling of tumor progression in head and neck cancer. Arch Otolaryngol Head Neck Surg 131 , 10-18 (2005).

Zhou, D., Xue, J., Gavrialov, O. & Haddad, G. G. Na+/H+ exchanger 1 deficiency alters gene expression in mouse brain. Physiol Genomics 18, 331 -339 (2004). Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001). Kaul, D. K., Fabry, M. E., Windisch, P., Baez, S. & Nagel, R. L. Erythrocytes in sickle cell anemia are heterogeneous in their Theological and hemodynamic characteristics. The Journal of Clinical Investigation 72, 22-31 (1983).

lacobas, D. A., lacobas, S., Urban-Maldonado, M. & Spray, D. C. Sensitivity of the brain transcriptome to connexin ablation. Biochim Biophys Acta 1711 , 183-196 (2005).

Claims

We Claim:

1. A method for treating sickle cell disease, thalassemia, sickle cell trait or a secondary

disease state or condition thereof in a human patient comprising administering to a human patient in need comprising an effective amount of an MTAP inhibitor, or a pharmaceutically acceptable salt thereof, optionally in combination with an additional bioactive agent.

2. The method of claim 1 , wherein the MPAP inhibitor is a compound according to the

chemical structure:

Where X is S-R or R', where R is a alkyl group (preferably methyl or ethyl) or a phenyl group and R' is a C C₃ alkyl group,

or a pharmaceutically acceptable salt, solvate or polymorph thereof.

3. The method according to claim 1 or 2 wherein said MTAP inhibitor is MTDIA

(methylthio-DADMe-lmmucillin-A, R is methyl).

4. The method according to any one of claims 1 -3 wherein said patient is treated for

thalassemia.

5. The method according to any of claims 1 -3 wherein said patient is treated for sickle cell trait.

6. The method according to any of claims 1 -3 wherein said secondary disease state or condition is venular occlusion, sickle crisis, chronic and/or systemic vasculopathies, stroke, sickle retinopathy, open leg ulcers, multi-organ failure, aseptic bone necrosis, dactylitis, hepatopathy, splenic autoinfarction, pulmonary hypertension, cognitive deficits, renal failure, cholecystitis, decreased fertility, increased susceptibility to infection and decreased opsonization.

7. The method according to any of claims 1-3 wherein said secondary disease state or condition is sickle cell crisis, vaso-occlusive crisis, splenic sequestration crisis, acute chest syndrome (ACS), aplastic crisis, haemolytic crisis, dactylitis, increased risk of severe bacterial infections, hyposplenism, stroke, cerebral infarction in children, cerebral haemorrhage in adults, silent stroke, cholelithiasis (gallstones) and

cholecystitis, avascular necrosis of the hip and other major joints, decreased immune reactions, priapism, osteomyletis, acute papillary necrosis, leg ulcers, background retinopathy, proliferative retinopathy, vitreous haemorrhages and retinal detachments, intrauterine growth retardation, spontaneouos abortion, pre-eclampsis, chronic pain, pulmonary hypertension, strain on the right ventricle and risk of heart failure, nephropathy, chronic renal failure due to nephropathy and cognitive deficits.

8. The method according to any of claims 1-7 wherein said MTAP inhibitor or a

pharmaceutically acceptable salt thereof and optional additional bioactive agent is formulated in parenteral dosage form or sustained or controlled release oral dosage form.

9. The method according to claim 8 wherein said dosage form is administered to said patient once or twice a day.

10. The method according to any of claims 1-9 wherein said MTAP inhibitor, or a

pharmaceutically acceptable salt thereof, is administered in oral dosage form in an amount ranging from about 1 mg to about 300 mg.

11. The method according to claim 10 wherein said MTAP inhibitor or a pharmaceutically acceptable salt thereof, is administered in a 50 mg. oral dosage form.

12. The method according to claim 10 wherein said MTAP inhibitor or a pharmaceutically acceptable salt thereof, is administered in a 100-250 mg. oral dosage form.

13. The method according to claim 10 wherein said MTAP inhibitor or a pharmaceutically acceptable salt thereof, is administered in a 300 mg. oral dosage form.

14. The method according to any of claims 10-13 wherein said oral dosage form is an immediate or sustained release dosage form administered to said patient once or twice a day.

15. The method according to any of claims 1-14 wherein said MTAP inhibitor or a

pharmaceutically acceptable salt thereof is coadministered with an additional bioactive agent.

16. The method according to any of claims 1-15 wherein said composition includes at least one additional bioactive agent selected from the group consisting of anti-sickling agents, selectin inhibitors, fetal hemoglobin regulators, inhibitors of a platelet ADP receptor and agents which increase the affinity of sickle hemoglobin's binding to oxygen.

17. The method according to any of claims 1-15 wherein said composition includes at least one additional bioactive agent selected from the group consisting of anti-sickling agents, selectin and adhesion inhibitors, fetal hemoglobin regulators, agents which inhibit a platelet ADP receptor, agents which increase the affinity of sickle hemoglobin's binding to oxygen, anti-oxidants, nitric oxide generating agents, vascular tone agents, anti-inflammatory agents, agents that target leukocyte adhesion and anti-platelet agents.

18. The method according to any of claims 1 -15 wherein said composition includes at least one additional bioactive agent selected from the group consisting of pyridoxamine, 5- hydroxymethylfurfural, 4-Hydroxy-3-methoxybenzaldehyde, GMI-1070, G I-1271 , intravenous immunoglobulin, tinzaparin, propranolol, SelG1 (humanized anti p-selectin antibody), heparin, hydroxyurea, decitabine, sodium dimethylbutyrate, pomalidomide, (2E)-N-hydroxy-3-[4-({[2-(2-methyl-1 H-indol-3-yl)ethyl]amino}methyl)phenyl]acrylamide, (R, S)-5-[2-cyclopropyl-1 -(2-fluorophenyl)-2-oxoethyl] 4,5,6,7-tetrahydrothieno[3,2- c]pyridin-2-yl acetate, N-hydroxy-N'-phenyl-octanediamide, 5-hydroxymethylfurfural, pyridoxal-5-phosphate, 4-hydroxy-3-methoxybenzaldehyde, para-aminobenzoic acid (PABA), pyridoxine, pyridoxine-5-phosphate, pyridoxal-5-phosphate, ascorbic acid, N- acetyl cysteine, a-linolenic acid, eicosapentaenoic acid, docosahexanoc acid, glutamine, acetl-L-carnitine, L-Arginine, intravenous magnesium, 2-{4- [(methylamino)carbonyl]- 1 H-pyrazol-1 -yljadenosine, N-[1 -(1 -benzothien-2-yl)ethyl]-N- hydroxyurea, Fructose-1 ,6-diphosphate, oxooxan-2-yl]ethyl}-3,7-dimethyl-1 ,2,3,7,8,8a- hexahydronaphthalen-1 -yl (2S)-2-methylbutanoate and 2-acetoxybenzoic acid.

19. The method according to any of claims 1-15 wherein said additional bioactive agent is at least one agent selected from the group consisting of pypridoxamine, hydroxyurea, sodium phenyl butyrate, sodium phenyl acetate, sodium phenyl propionate, or an alternative pharmaceutical salt thereof (as disclosed in United States patent number 5,712,307, which is incorporated by reference herein), FK228 (depsipeptide) or an analog thereof, a benzamide, a non-cyclic or cyclic hydroxamate, and TSA

(Trichostatin A).

20. A method of treating kidney disease and/or a kidney related condition or disorder secondary to sickle cell disease, thalassemia, sickle cell trait or diabetes (I or II) in a patient in need comprising administering to said patient an effective amount of a MTAP inhibitor (preferably MTDIA), or a pharmaceutically acceptable salt thereof, optionally in combination with an additional bioactive agent.

21. The method according to claim 20 wherein said kidney disease and/or kidney related condition or disorder is nephropathy or renal failure.

22. The method according to claim 21 wherein said renal failure is chronic renal failure and said disease state occurs secondary to sickle cell disease, thalassemia or sickle cell trait and diabetes.

23. The method according to any of claims 20-22 wherein said MTAP inhibitor or a

pharmaceutically acceptable salt thereof and optional additional bioactive agent is formulated in parenteral (preferably intravenous) dosage form or sustained or controlled release oral dosage form.

24. The method according to claim 23 wherein said oral dosage form is administered to said patient once or twice a day.

25. The method according to claim 24 wherein said oral dosage form comprises

pyridoxamine, or a pharmaceutically acceptable salt thereof, and is administered in an amount ranging from about 50 mg to about 750 mg.

26. The method according to claim 25 wherein said oral dosage form comprises

pyridoxamine, or a pharmaceutically acceptable salt thereof, and is administered in an amount of about 100-750 mg.

27. A pharmaceutical composition comprising an effective amount of a MTAP inhibitor

(preferably MTDIA) or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, additive and/or excipient, optionally in combination with at least one additional bioactive agent.

28. The composition according to claim 27 wherein said additional bioactive agent is at least one agent selected from the group consisting of anti-sickling agents, selectin inhibitors, fetal hemoglobin regulators, agents which inhibit a platelet ADP receptor, agents which increase the affinity of sickle hemoglobin's binding to oxygen, anti-oxidant agents, anti-inflammatory agents, agents that target leukocyte adhesion and anti-platelet agents.

29. The composition according to claim 27 wherein said additional bioactive agent is at least one agent selected from the group consisting of pyridoxamine, 5-hydroxymethylfurfural,

4- Hydroxy-3-methoxybenzaldehyde, GMI-1070, GMI-1271 , intravenous

immunoglobulin, tinzaparin, propranolol, SelG1 (humanized anti p-selectin antibody), heparin, hydroxyurea, decitabine, sodium dimethylbutyrate, pomalidomide, (2E)-N- hydroxy-3-[4-({[2-(2-methyl-1 H-indol-3-yl)ethyl]amino}methyl)phenyl]acrylamide, (R, S)-

5- [2-cyclopropyl-1 -(2-fluorophenyl)-2-oxoethyl] 4,5,6, 7-tetrahydrothieno[3,2-c]pyridin-2- yl acetate, N-hydroxy-N'-phenyl-octanediamide, 5-hydroxymethylfurfural, pyridoxal-5- phosphate, 4-hydroxy-3-methoxybenzaldehyde, para-aminobenzoic acid (PABA), pyridoxine, pyridoxine-5-phosphate, pyridoxal-5-phosphate, ascorbic acid, /V-acetyl cysteine, a-linolenic acid, eicosapentaenoic acid, docosahexanoc acid, glutamine, acetl-L-carnitine, L-Arginine, intravenous magnesium, 2-{4-[(methylamino)carbonyl]-

1 H-pyrazol-1 -yl}adenosine, N-[1 -(1 -benzothien-2-yl)ethyl]-N-hydroxyurea, Fructose- 1 ,6-diphosphate, oxooxan-2-yl]ethyl}-3,7-dimethyl-1 ,2,3,7,8,8a-hexahydronaphthalen- 1 -yl (2S)-2-methylbutanoate and 2-acetoxybenzoic acid.

30. Use of an effective amount of a MTAP inhibitor (preferably, MTDIA), or a

pharmaceutically acceptable salt thereof, optionally in combination with at least one additional bioactive agent in the manufacture of a medicament for the treatment of sickle cell disease, thalassemia or sickle cell trait or a secondary disease state or condition thereof in a human patient.

31. Use according to claim 30, wherein the patient is resistant to hydroxyurea therapy.

32. Use according to claim 30, wherein said patient is treated for sickle cell disease.

33. Use according to claim 30, wherein said patient is treated for thalassemia.

34. Use according to claim 30, wherein said patient is treated for sickle cell trait.

35. Use according to claim 30, wherein said secondary disease state or condition is venular occlusion, sickle crisis, chronic and/or systemic vasculopathies, stroke, sickle

retinopathy, open leg ulcers, multi-organ failure, aseptic bone necrosis, dactylitis, hepatopathy, splenic autoinfarction, pulmonary hypertension, cognitive deficits, renal failure, cholecystitis, decreased fertility, increased susceptibility to infection and decreased opsonization.

36. Use according to claim 30, wherein said secondary disease state or condition is sickle cell crisis, vaso-occlusive crisis, splenic sequestration crisis, acute chest syndrome (ACS), aplastic crisis, haemolytic crisis, dactylitis, increased risk of severe bacterial infections, hyposplenism, stroke, cerebral infarction in children, cerebral haemorrhage in adults, silent stroke, cholelithiasis (gallstones) and cholecystitis, avascular necrosis of the hip and other major joints, decreased immune reactions, priapism, osteomyletis, acute papillary necrosis, leg ulcers, background retinopathy, proliferative retinopathy, vitreous haemorrhages and retinal detachments, intrauterine growth retardation, spontaneouos abortion, pre-eclampsis, chronic pain, pulmonary hypertension, strain on the right ventricle and risk of heart failure, nephropathy, chronic renal failure due to nephropathy and cognitive deficits.

37. Use according to any of claims 30-36 wherein said MTAP inhibitor or a pharmaceutically acceptable salt thereof and optional additional bioactive agent is formulated in parenteral (preferably intravenous) or sustained or controlled release oral dosage form.