WO2011111066A2 - Composition and uses thereof - Google Patents

Abstract

The present invention relates generally to pharmaceutical compositions and their use for the prophylaxis or treatment of conditions associated with hyperglycemia or hypertriglyceridemia, including (but not limited to), diabetes (e.g., Type 2 diabetes) and obesity. The present invention also relates to methods of prophylaxis or treatment of such conditions, based on the finding that an inhibitor of a phosphodiesterase 3 activity and an inhibitor of angiotensin receptor activity provide a desirable synergistic effect on hyperglycemia and hypertriglyceridemia.

Description

COMPOSITION AND USES THEREOF

TECHNICAL FIELD

[0001] The present invention relates generally to compositions and their use for the prophylaxis and/or treatment of disorders such as diabetes and other disorders of this type.

BACKGROUND ART

[0002] Diabetes is typically a chronic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces to regulate blood glucose levels. Hyperglycaemia, or raised blood sugar, is a common outcome of uncontrolled diabetes and over time leads to adverse physiological changes to those suffering from the disease, especially to the nervous system and the cardiovascular system.

[0003] The World Health Organisation (WHO) estimates that more than 220 million people worldwide suffer from diabetes. In 2005, an estimated 1.1 million people died from diabetes (although the actual number is likely to be much larger as this figure does not include people who have died from diabetic complications such as heart disease or kidney failure). Of all diabetes deaths, almost 80% occur in low- and middle-income countries, almost 50% in people under the age of 70 years and approximately 55% in women. The WHO further predicts that diabetes deaths will have doubled between 2005 and 2030 unless urgent preventive steps are taken to curb or reverse this epidemic. Whilst at least a part of the diabetic epidemic can be attributed to genetic factors, the primary driver is the rapid epidemiological transition associated with changes in dietary patterns and decreased physical activity, as evident from the higher prevalence of diabetes in the urban population. [0004] Whilst a healthy diet, regular physical activity, maintaining a normal body weight and avoiding tobacco use can prevent or delay the onset of the disease, there are currently no effective therapeutic strategies for the prophylaxis or treatment of diabetes.

[0005] Over time, diabetes can lead to damage to the heart, blood vessels, eyes, kidneys and nerves. For instance, diabetes increases the risk of heart disease and stroke, with around 50% of people with diabetes dying of cardiovascular disease. Combined with reduced blood flow, neuropathy in the feet increases the chance of foot ulcers and eventual limb amputation.

[0006] Retinopathy is one of the most common causes of blindness in patients with diabetes and occurs as a result of long-term accumulated damage to the small blood vessels in the retina. Data suggests that approximately 10% of patients will develop at least some visual impairment after 15 years with diabetes and approximately 2% of patients will become blind over this time period.

[0007] Diabetes is also among the leading causes of kidney failure and as per the WHO Statistics, approximately 10-20% of people with diabetes die of kidney failure.

[0008] Diabetic neuropathy is typically defined by damage to the nerves as a result of diabetes, and affects up to 50% of people with diabetes. Although many different problems can occur as a result of diabetic neuropathy, common symptoms are tingling, pain, numbness, or weakness in the feet and hands.

[0009] Diabetes and its complications also have a significant economic impact on individuals, families and the health systems of both developed and developing countries. For example, the WHO estimates that from 2006 to 2015, China will lose US$558 billion in foregone national income due to heart disease, stroke and diabetes alone.

[0010] Simple lifestyle measures have been shown to be effective in preventing or delaying the onset of type 2 diabetes. To help prevent type 2 diabetes and its complications, it is recommended that people achieve and maintain healthy body weight, be physically active (at least 30 minutes of regular, moderate-intensity physical activity on most days, with more activity required for weight control), eat a healthy diet of between three and five servings of fruit and vegetables a day, reduce sugar and saturated fats intake and avoid tobacco use (which further increases the risk of cardiovascular diseases). [0011] Early diagnosis of diabetes can be accomplished through relatively inexpensive blood testing.

[0012] Current treatment regimes involve lowering blood glucose (with oral medication and/or insulin), blood pressure control and lowering the levels of other known risk factors that damage blood vessels. Tobacco cessation is also important to avoid diabetic complications, such as heart disease. Other cost saving interventions include screening for retinopathy and screening for early signs of diabetes-related kidney disease.

[0013] Type 1 diabetes (also known as insulin-dependent, juvenile or childhood-onset diabetes) is characterized by deficient insulin production and requires daily administration of insulin. Symptoms of Type 1 diabetes include excessive excretion of urine (polyuria), thirst (polydipsia), constant hunger, weight loss, vision changes and fatigue. [0014] Gestational diabetes is often referred to where a patient presents with hyperglycaemia during pregnancy and is most often diagnosed through prenatal screening, rather than reported symptoms, which are similar to the symptoms of Type 2 diabetes, below.

[0015] Impaired glucose tolerance (IGT) and impaired fasting glycaemia (IFG) are intermediate conditions in the transition between normality and diabetes. People with IGT or IFG are also at high risk of progressing to Type 2 diabetes. [0016] Type 2 Diabetes Mellitus (T2DM) is characterized by peripheral insulin resistance, defective insulin secretion and increased glucose output from the liver, leading to increased blood sugar levels (hyperglycemia). Long-standing hyperglycemia can lead to microvascular and macrovascular complications, which are primarily responsible for the morbidity and mortality associated with T2DM. Patients with impaired glucose tolerance (IGT) and impaired fasting glucose (IFG), often referred to as "pre-diabetic" states, are highly susceptible to progression to T2DM and cardiovascular disease (see Novoa FJ et al., DiabetesCare 2005; 28:2388-93).

[0017] T2DM affects about 90% of people with diabetes around the world and is largely the result of excess body weight and physical inactivity. The symptoms of T2DM may be similar to those of Type 1 diabetes, but are often less pronounced. As a result, the disease may remain undiagnosed for several years after onset and until complications have arisen. Until recently, T2DM was seen only in adults, but it is now also occurring in children. [0018] Frequently, patients with T2DM are also prone to associated risk factors like central obesity, dyslipidemia and hypertension. The most common pattern of dyslipidemia in T2DM patients is elevated triglyceride levels and decreased HDL cholesterol levels. Hypertriglyceridemia (HTG) is correlated with an increased risk of cardiovascular disease (CVD), particularly in the setting of low HDL cholesterol (HDL-C) levels, and/or elevated LDL cholesterol (LDL-C) levels. Clinical studies suggest that high triglyceride level is also an independent risk factor for cardiovascular diseases. Hyperglycemia is often associated with high triglyceride level in T2DM.

[0019] Reduction in triglyceride levels can significantly increase the HDL-C and marked reduction in cardiovascular events. According to NCEP/ATP III guidelines, the triglyceride levels are classified as normal (<150 mg/dL), borderline high (150-199 mg/dL), high (200-499 mg/dL), and very high (>500 mg/dL). Because metabolism of the triglyceride-rich lipoproteins (chylomicrons, VLDL) and metabolism of HDL are interdependent, as triglycerides are very labile, the independent impact of hypertriglyceridemia on cardiovascular disease risk is difficult to confirm. Hypertriglyceridemia could also be regarded as an independent risk factor for insulin resistance (see Zhonghua Nei Ke Za Zhi. 2001 May; 40(5):299-302).

[0020] Hyperglycemia and hypertriglyceridemia together contribute to the enormous disease burden associated with T2DM. Whilst lifestyle modifications may assist, at least in part, in the regression of these pre-diabetic states to normalcy (or close to normalcy), further interventions are certainly needed to curb the progression of disease. Currently, however, there are no drugs approved for the treatment of pre-diabetic states. Most of the currently existing therapies for T2DM address mainly the glucose metabolism component, while leaving the lipid metabolism unaddressed or actually may worsen it. Conversely, existing therapies for treating high triglycerides and low HDL do not adequately address the glucose component. For instance, drugs like fibrates have high risk of causing hepatotoxicity or myotoxicity. Furthermore, in a certain percentage of patients with T2DM, satisfactory control of the disease cannot be achieved with currently treatments (see Rutten, Ned. Tijdschr. Gen. 2001 ; 145:1547-1550). Hence, there is a need for a treatment regime that effectively and safely addresses hyperglycemia and/or hypertriglyceridemia. [0021] Patients susceptible to these conditions may also be found to be susceptible to other disorders of the liver such as non-alcoholic fatty liver disease (NALFD). It is related to insulin resistance and the metabolic syndrome and typically responds to treatments that are effective for other insulin resistant states. Non-alcoholic steatohepatitis (NASH) is the most extreme form of NAFLD and is regarded as a major cause of cirrhosis of the liver. NASH resembles alcoholic liver disease but occurs in people who drink little or no alcohol. The major feature of NASH is fat in the liver along with subsequent inflammation and damage. There are currently no specific therapies for NASH and it therefore represents an important therapeutic target.

[0022] The present invention seeks to overcome, or at least alleviate, some of the aforementioned problems in the art by providing compositions and their use for preventing and/or treating a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis. [0023] Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps. SUMMARY OF THE INVENTION

[0024] The present invention is based, at least in part, on the Applicant's finding that the combination of (i) an inhibitor of phosphodiesterase 3 (PDE3; 3',5'-cyclic-nucleotide phosphodiesterase; EC 3.1.4.-) activity and (ii) an inhibitor of angiotensin receptor activity can reduce the severity of hyperglycemia and/or hypertriglyceridemia, particularly in a subject with diabetes and prevent macrovascular and/or microvascular complications typically associated with diabetes, including (but not limited to) cardiovascular disease (CVD), cerebrovascular disease, diabetic nephropathy, neuropathy and retinopathy. The Applicant has shown that the combination of an inhibitor of PDE3 activity and an inhibitor of angiotensin receptor activity provides a synergistic effect on such parameters, both in vitro and in vivo. The Applicant has also shown, for the first time, that the combination of an inhibitor of phosphodiesterase 3 activity and an inhibitor of angiotensin receptor activity inhibits diet- induced weight gain.

[0025] Thus, in one aspect, the present invention provides a composition including (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity.

[0026] In some embodiments, the composition according to the present invention further includes a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.

^" [0027] In another aspect of the present invention there is provided a use of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity in the prophylaxis or treatment of a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis. In some embodiments, the condition is Type 2 diabetes.

[0028] In another aspect of the present invention there is provided a use of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity in the manufacture of a medicament for the prophylaxis or treatment of a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis. In some embodiments, the condition is Type 2 diabetes. [0029] In another aspect of the present invention there is provided a method of preventing or treating a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis, the method including administering to a subject in need thereof a therapeutically effective amount of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity. In some embodiments, the condition is Type 2 diabetes.

[0030] In some embodiments, the inhibitor of phosphodiesterase 3 activity is selected from the group consisting of Enoximone, Cilostazol, Milrinone, Vesnarinone and Pimobendan, or a prodrug, analogue or biologically-active variant thereof. [0031] In some embodiments, the inhibitor of angiotensin receptor activity is an angiotensin receptor antagonist, including, but not limited to, those selected from the group consisting of Losartan, Telmisartan, Valsartan, Irbesartan, Olmesartan, Eprosartan and Candesartan, or a prodrug, analogue or biologically-active variant thereof. [0032] In some embodiments, the inhibitor of angiotensin receptor activity is an angiotensin converting enzyme inhibitor, including, but not limited to, those selected from the group consisting of alacepril, alindapril, altiopril, benazepril, benazeprilat, captopril, ceronapril, cilazapril, cilazaprilat, delapril, enalapril, enalaprilat, fosinopril, imidapril, indolapril, libenzapril, lisinopril, moexepril, moveltipril, pentopril, perindopril, quinapril, quinaprilat, ramipril, rentiapril, spirapril, temocapril, teprotide, trandolapril, zofenopril, omapatrilat, fasidotril, mixanpril, sampatrilat, gemopatrilat (BMS-189921 ), MDL-100240 and Z13752A (GW660511), or a prodrug, analogue or a biologically-active variant thereof.

[0033] In some embodiments, the inhibitor of phosphodiesterase 3 activity inhibits phosphodiesterase 3 expression. Similarly, in other embodiments, the inhibitor of angiotensin receptor activity inhibits angiotensin receptor expression.

[0034] In some embodiments, the inhibitor of phosphodiesterase 3 expression or the inhibitor of angiotensin receptor expression is an antisense nucleic acid molecule.

[0035] In some embodiments, methods or use of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity in accordance with the present invention is in the form of a composition as herein described. [0036] In another aspect of the present invention there is provided a kit including (i) an inhibitor of phosphodiesterase 3 activity, as herein described and (ii) an inhibitor of angiotensin receptor activity, as herein described. [0037] In some embodiments, the kit further includes written instructions as to the use of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity in the prophylaxis or treatment of a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis.

[0038] In another aspect of the present invention there is provided a use of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity to modulate the differentiation of adipocytes, in vitro or in vivo.

[0039] In one further aspect of the present invention there is provided a method of modulating the differentiation of adipocytes, in vitro or in vivo, the method including contacting adipocytes with (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity.

BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 shows triglyceride (TAG) accumulation in HePG2 cells in vitro. HepG2 cells were grown in DMEM containing excess glucose (33mM) supplemented with 10% fetal bovine serum (FBS) and high FFA (200 μΜ palmitate) to induce TAG accumulation. Cells treated with different concentrations (100nm to 3μΜ) of Telmisartan (T) and Cilostazol (C) alone and in combination were observed for inhibition of TAG accumulation. At lower concentrations, the combination of Telmisartan and Cilostazol showed inhibition of excess glucose and FFA-induced TAG accumulation; n=6, N=3; Data are represented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. Dunnet's test was used as a post measure ("^*P<0.001 , ^**P<0.01 , ^*P<0.05).

[0041] Figure 2 shows the effect of Cilostazol (50 mg/kg p.o.) and Telmisartan (10 mg/kg p.o.), in combination, on body weight. All values are expressed as mean +/- SEM; statistical analysis was performed by unpaired f test, Control (n=5); Cafeteria diet Control (n=10); FDC- II treatment (n=10); P value summary: ^*<0.05, ^**<0.01 , ^***<0.001 when compared with cafeteria diet control.

[0042] Figure 3 shows the effect of Cilostazol (50 mg kg p.o.) and Telmisartan (10 mg kg p.o.), in combination, on triclyceride (TG) levels in vivo. All values are expressed as mean +/- SEM, statistical analysis was performed by unpaired t' test, Control (n=5); Cafeteria diet Control (n=10); FDC-II treatment (n=10); P value summary: ^*<0.05, ^**<0.01 , ^***<0.001 when compared with cafeteria diet control.

[0043] Figure 4 shows the effect of Cilostazol (50 mg/kg p.o.) and Telmisartan (10 mg/kg p.o.), in combination, on adipocyte size in vivo. All values are expressed as mean +/- SEM, statistical analysis was performed by Bonferroni's Multiple Comparison Test, Control (n=5); Cafeteria diet Control (n=10); FDC treatment (n=10); P value summary: ^*<0.05, "<0.01 , ^***<0.001 when compared with cafeteria diet control. [0044] Figure 5 shows the effect of Cilostazol (12.5 mg/kg p.o.) and Telmisartan (2.5mg/kg p.o.), in combination, on liver in vivo. All values are expressed as mean +/- SEM, statistical analysis was performed by Bonferroni's Multiple Comparison Test, Control (n=4); Choline deficient diet (n=4); FDC treatment (n=4); P value summary: ^*<0.05, ^**<0.01 , ^*"<0.001 when compared with cafeteria diet control.

[0045] Figure 6 shows the effect of Cilostazol (12.5 mg/kg p.o.) and Telmisartan (2.5mg/kg p.o.), in combination, on adipose in vivo. All values are expressed as mean +/- SEM, statistical analysis was performed by Bonferroni's Multiple Comparison Test, Control (n=4); Choline deficient diet (n=4); FDC treatment (n=4); P value summary: ^*<0.05, ^**<0.01 , ^***<0.001 when compared with cafeteria diet control.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The Applicant has shown for the first time that inhibiting phosphodiesterase 3 activity and angiotensin receptor activity can effectively treat hyperglycemia and/or hypertriglyceridemia in patients with diabetes and prevent macrovascular and/or microvascular complications of diabetes, such as cardiovascular diseases (CVD), cerebrovascular diseases, diabetic nephropathy, neuropathy and retinopathy. Furthermore, the combination of inhibiting phosphodiesterase 3 activity and angiotensin receptor activity has been shown by the Applicant to provide a synergistic effect on parameters related to hyperglycemia and hypertriglyceridemia, both in vitro and in vivo, as compared to the effect of inhibiting either phosphodiesterase 3 activity or angiotensin receptor activity, alone.

Compositions

[0047] Thus, in one aspect of the present invention, there is provided a composition including (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity. [0048] Without limiting the present invention, such compositions have been found by the Applicant to have particular beneficial properties in reducing the severity of hyperglycemia and hypertriglyceridemia in subjects with T2DM and minimising the macrovascular and microvascular complications of T2DM.

[0049] Phosphodiesterase 3 (PDE3; a phosphoric diester hydrolase) belongs to a class of intracellular enzymes (EC 3.1.4.-), including PDE3A and PDE3B, involved in the metabolism of the second messenger nucleotides cAMP and cGMP. For instance, phosphodiesterases degrade the phosphodiester bond in the second messenger molecules cAMP and cGMP and thus regulate the localization, duration and amplitude of cyclic nucleotide signaling within subcellular domains. Phosphodiesterases are therefore important regulators of signal transduction mediated by these second messenger molecules.

[0050] The amino acid sequence of human phosphodiesterase 3A (SEQ ID NO:1), the nucleic acid sequence of human phosphodiesterase 3A (SEQ ID NO:2), the amino acid sequence of human phosphodiesterase 3B (SEQ ID NO:3) and the nucleic acid sequence of human phosphodiesterase 3B (SEQ ID NO:4) are disclosed herein, with particular reference to the sequence listing. [0051] Phosphodiesterase 3 inhibitors, like Cilostazol (a quinolinone derivative) have been used as antiplatelet agents and vasodilators for the treatment of peripheral arterial diseases. The mechanism of action of cilostazol, for example, for this indication is not fully understood. Cilostazol and several of its metabolites are cyclic AMP phosphodiesterase III inhibitors, leading to an increase in cAMP in platelets (decreased aggregation) and blood vessels (vasodilatation) and an increase in interstitial adenosine in the heart (thus attenuating the cardiotonic effects of cAMP).

[0052] An "inhibitor of phosphodiesterase 3 activity, as herein described, is typically a compound that is capable of at least partially inhibiting or reducing, selectively or non- selectively, the activity or expression (e.g., gene expression) of phosphodiesterase 3.

[0053] Phosphodiesterase 3 activity and/or expression can be at least partially inhibited or reduced by any means known to the skilled person. Inhibitors of phosphodiesterase 3 activity include, but are not limited to Enoximone, Cilostazol, Milrinone, Vesnarinone and Pimobendan, or a prodrug, analogue or biologically-active variant thereof. The phosphodiesterase 3 inhibitor can be highly specific for phosphodiesterase 3 or it may display activity across several PDE isoforms or other substrates. Other examples of suitable inhibitors of phosphodiesterase 3 activity are described, e.g., in WO2009/097406. Inhibitors of phosphodiesterase 3 activity used in the present invention may also include prodrugs, analogues {e.g., pharmaceutically acceptable salts) or biologically-active variants thereof. [0054] One of the preferred inhibitors of phosphodiesterase 3 activity for use in the present invention is Cilostazol which is sold under the brand name Pletal. The drug is currently used as a medication for the treatment of intermittent claudication, a condition caused by the narrowing of the arteries that supply the legs with blood. [0055] Angiotensin receptors are expressed on the surface of a number of cell types and interact with several ligands, including angiotensin II. At least four receptor subtypes are known, termed ATi, AT₂, AT₃ and AT₄, with the A^"P| and AT₂ subtypes having been well characterised as compared to the AT₃ and AT₄ subtypes. In recent times, great efforts have been made to identify substances that inhibit angiotensin receptor activity, including (but not limited to) angiotensin receptor antagonists and angiotensin converting enzyme inhibitors.

[0056] Inhibition of angiotensin receptor activity in vivo, in particular Α1Ί receptor activity, leads to vasodilation, reduced secretion of vasopressin, reduced production and secretion of aldosterone and other actions, the combined effect of which is generally the reduction of blood pressure. Angiotensin II receptor antagonists are primarily used for the treatment of hypertension and heart failure, particularly where the patient in question becomes intolerant of angiotensin converting enzyme (ACE) inhibitor therapy.

[0057] Examples of angiotensin receptor antagonists that may be used in the present invention include, but are not limited to, Losartan, Telmisartan, Valsartan, Irbesartan, Olmesartan, Eprosartan and Candesartan. The angiotensin receptor antagonists that may be used in the present invention also include prodrugs, analogues (e.g., pharmaceutically acceptable salts) or biologically-active variants thereof. [0058] One of the preferred angiotensin receptor agonist that is used in this invention is Telmisartan (It is marketed under the trade names Micardis (Boehringer Ingelheim), Targit (Pfizer) and Temax (Wockhardt). It is well known in prior art that Telmisartan is an angiotensin II receptor blocker that shows high affinity for the angiotensin II receptor type 1. Furthermore, telmisartan also acts as a selective modulator of peroxisome proliferator- activated receptor gamma (PPAR-γ), a central regulator of insulin and glucose metabolism and hence is preferred by the inventors of this invention. [0059] Whilst any combination of an inhibitor of phosphodiesterase 3 activity and an inhibitor of angiotensin receptor activity is envisaged in the present invention, in some embodiments, the composition of the present invention includes Cilostazol and Telmisartan. [0060] Alternatively, or in addition to angiotensin receptor antagonists, inhibitors of the renin- angiotensin system can also be included in the compositions of the present invention. Inhibitors of the renin-angiotensin system are used in the clinical setting to lower blood pressure in hypertension and in congestive heart failure as described, for example, in N. Eng. J. Med. 316, 23 (1987) p1429-1435. A large number of peptide and non-peptide inhibitors of the renin angiotensin system are known, the most widely studied being the angiotensin converting enzyme (ACE) inhibitors. ACE inhibitors, which include captopril, enalapril, lisinopril, benazepril and spirapril, inhibit the proteolytic conversion of the precursor peptide angiotensin I to angiotensin II. [0061] Examples of ACE inhibitors that may be used in the present invention include, but are not limited to, alacepril, alindapril, altiopril, benazepril, benazeprilat, captopril, ceronapril, cilazapril, cilazaprilat, delapril, enalapril, enalaprilat, fosinopril, imidapril, indolapril, libenzapril, lisinopril, moexepril, moveltipril, pentopril, perindopril, quinapril, quinaprilat, ramipril, rentiapril, spirapril, temocapril, teprotide, trandolapril, zofenopril, omapatrilat, fasidotril, mixanpril, sampatrilat, gemopatrilat (BMS-189921), DL-100240 and Z13752A (GW660511), including prodrugs, analogues (e.g., pharmaceutically acceptable salts) or biologically-active variants thereof.

[0062] The inhibitors of phosphodiesterase 3 activity and angiotensin receptor activity according to the present invention include, but are not limited to, small molecules, peptides, antibodies, ribozymes, nucleic acid molecules and antisense oligonucleotides.

[0063] In some embodiments, the inhibitors of phosphodiesterase 3 activity and angiotensin receptor activity may be the same compound. Alternatively (or in addition), the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity may be different compounds present in the compositions of the present invention either as separate components or linked in such a way {e.g., conjugated to one other) that they would retain at least some of their respective therapeutic activities. [0064] As used herein, the term "prodrug" typically refers to a pharmacological substance that is administered in an inactive or significantly less active form. Once administered, the prodrug is metabolised in vivo or in vitro into an active metabolite. [0065] As used herein, the term "analogue" is typically used to denote a compound that has a chemical structure that is substantially similar to the structure of the parent compound, whilst retaining at least some of the biological function of the parent compound. Analogues also include pharmaceutically acceptable salts.

[0066] The term "substantially similai", as used herein, typically denotes a substitution or addition of any one or more chemical substituents of the chemical structure such that the resulting analogue has at least some of the biological activity of the parent compound.

[0067] The term "compouncf, used interchangeably herein with terms such as "active compound^*, "agent, "active agent', "ingredient', "active ingredient', "substance", "active substance" and the like, typically refers to an inhibitor of phosphodiesterase 3 activity, as herein described, and an inhibitor of angiotensin receptor activity, as herein described.

[0068] The compounds that may be used in the present invention, as herein described, are not intended to be limited to synthetic compounds, manufactured by processes known to those skilled in the art. Thus, in some embodiments, the compounds are naturally-occurring compounds that have been extracted from natural sources (e.g., plant or animal material) by methods known to those skilled in the art and, optionally, at least partially purified as required. In some embodiments, the compounds can be purified to a substantially pure form as required. The term "substantially pure", as used herein, typically means a compound which is substantially free of other compounds with which it may normally be associated in nature. [0069] Combinations of naturally-occurring and synthetic compounds are also envisaged in some embodiments of the present invention.

[0070] As used herein, the phrase "at least partially purified' typically means a compound, or an analogue thereof or biologically-active fragment or variant thereof, which has been partially purified from its natural state. In some embodiments, the at least partially purified compounds are substantially free of proteins, nucleic acids, lipids, carbohydrates or other materials with which it is naturally associated. Optionally, the compounds that may be used in the present invention can be further purified using routine and well-known methods. The term "substantially free", as used herein, typically refers to a preparation of the compounds that may be used in the present invention, or analogues thereof or biologically-active fragments or variants thereof, having less than about 90%, 70%, 50%, 30%, 20%, 10% or 5% (by dry weight) of a molecule with which it is naturally associated. [0071] In some embodiments, pharmaceutically acceptable salts and solvates of the compounds that may be used in the present invention are also included. The term "pharmaceutically acceptable salts? typically refers to salts prepared from pharmaceutically acceptable substantially non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids, as well as salts that can be converted into pharmaceutically acceptable salts. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, Ν,Ν'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. [0072] When a compound that may be used in the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. In some embodiments, the acids are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[0073] A solvate, as used herein, typically refers to a compound (or a salt thereof), in association with a solvent, such as water. Representative examples include hydrates, hemihydrates, trihydrates and the like.

[0074] As used herein, "variants of the compounds that may be used in the present invention may exhibit chemical structures that are at least 80% identical to a known inhibitor of phosphodiesterase 3 activity or a known inhibitor of angiotensin receptor activity, such as those described herein. In some embodiments, a variant will exhibit a chemical structure that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to known inhibitors of phosphodiesterase 3 and angiotensin receptor activity. Percent identity may be determined by visual inspection and mathematical calculation. Among the variants (and fragments thereof) provided are variants of known phosphodiesterase 3 inhibitors and angiotensin receptor antagonists that retain the same or substantially equivalent biological activity thereof.

[0075] Where the inhibitor of phosphodiesterase 3 and/or angiotensin receptor activity is a naturally-occurring peptide, suitable variants can include polypeptides that are substantially homologous to the naturally-occurring inhibitors, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions. In some embodiments, polypeptide variants may include from one to ten deletions, insertions or substitutions of amino acid residues when compared to a native sequence. A given sequence may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative substitution of one aliphatic residue for another, such as He, Val, Leu or Ala for one another; substitution of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known in the art. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, an amino acid residue of a naturally-occurring polypeptide is replaced with another amino acid residue from the same side chain family. In some embodiments, mutations can be introduced randomly along all or part of the amino acid sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for phosphodiesterase 3 inhibitory and angiotensin receptor antagonistic activity.

[0076] Variants may also be generated by the truncation of naturally-occurring inhibitors of phosphodiesterase 3 and angiotensin receptor activity. Suitable variants may also include deglycosylated polypeptides, or fragments thereof, or those polypeptides demonstrating increased glycosylation when compared to the native molecules. Also encompassed are variants with increased hydration. [0077] As used herein, the term "fragment' typically refers to a portion of an inhibitor of phosphodiesterase 3 activity or an inhibitor of angiotensin receptor activity that may be used in the present invention, as herein described (or variants thereof). Where such compounds are peptides, fragments may comprise at least 1 amino acid residue, at least 5 amino acid residues, at least 10 amino acid residues, or at least 20 amino acid residues of the naturally- occurring peptide molecule (or a variant thereof).

[0078] In some embodiments, the inhibitor of phosphodiesterase 3 activity and/or the inhibitor angiotensin receptor activity is the same compound, although in most cases, they will be different compounds present in combination in the compositions of the present invention.

[0079] In some embodiments, the inhibitor(s) of phosphodiesterase 3 and angiotensin receptor activity will at least partially inhibit phosphodiesterase 3 and angiotensin receptor activity by competitively modulating their respective activities. T erefore, in some embodiments, the inhibitor(s) of phosphodiesterase 3 and angiotensin receptor activity may include soluble forms of phosphodiesterase 3 and angiotensin receptor ligands (or fragments thereof) that have little or no inherent activity of their own. Such competitors may include, for example, fragments of angiotensin II that are able to bind to an angiotensin receptor to inhibit binding of the native molecule, but have no inherent activity of their own.

[0080] As used herein, the terms "activity', "biological activity and the like typically refer to the activity of phosphodiesterase 3 and/or angiotensin receptor activation. For example, the activity of a phosphodiesterase 3 inhibitor can be identified by the person skilled in the art by its ability to:

• Increase in. cA P in platelets (decreased aggregation) and blood vessels (vasodilatation); and/or

• Increased interstitial adenosine in the heart (attenuating cardiotonic effects of cAMP).

[0081] The activity of angiotensin receptor activation can be identified by the person skilled in the art, for example, by its ability to increase nitric oxide production in endothelial cells.

Nucleic Acid Molecules

[0082] Alternatively, or in addition to the inhibitors of phosphodiesterase 3 and angiotensin receptor activity, as herein described, the compositions of the present invention may also include nucleic acid molecules capable of inhibiting the expression (e.g., gene expression) of phosphodiesterase 3, angiotensin receptors and/or an angiotensin receptor ligand (e.g., Ang II). The term "nucleic acid molecule" includes DNA molecules (e.g., a cDNA or genomic D A) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0083] As used herein, the terms "gene" typically refer to nucleic acid molecules which include an open reading frame encoding phosphodiesterase 3, an angiotensin receptor and/or an angiotensin receptor ligand, and may further include non-coding regulatory sequences and introns.

[0084] For example, a nucleic acid molecule that may be used in the present invention may include a nucleotide sequence which is at least about 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the naturally-occurring nucleotide sequence of the target gene (e.g.., phosphodiesterase 3, angiotensin receptor and/or angiotensin receptor ligand such as Ang I or Ang II). The sequence of the nucleic acid molecule that may be used in the present invention may be derived from any organism, including, but not limited to, plants, animals (including humans) and lower organisms such as bacteria.

[0085] In some embodiments, oligonucleotides (or fragments thereof) capable of inhibiting the expression of phosphodiesterase 3, angiotensin receptor and/or an angiotensin receptor ligand may also be used in the present invention.

[0086] Where the inhibitor of phosphodiesterase 3 and angiotensin receptor activity is a nucleic acid molecule (or molecules), as herein described, the nucleic acid molecules can be inserted into vectors and used as gene therapy vectors. In some embodiments, the nucleic acid molecules are inserted into retroviral vectors, such as retroviral vector pLXSN. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Gene Therapy

[0087] Gene therapy may also be employed to inhibit phosphodiesterase 3 and/or angiotensin receptor activity and or expression. For example, cells comprising a retroviral vector driving the expression of a recombinant phosphodiesterase 3 inhibitor and/or a recombinant angiotensin receptor antagonist may be administered to a subject for engineering cells in vivo to express the recombinant molecule(s) in vivo. For overview of gene therapy, see, for example, Chapter 20, Gene Therapy and other Molecular Genetic- based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, Strachan T. and Read A. P., BIOS Scientific Publishers Ltd (1996).

[0088] Further, antisense and ribozyme molecules that inhibit expression of the target molecules {i.e., phosphodiesterase 3, an angiotensin receptor and/or an angiotensin receptor ligand) may also be used in accordance with the invention to inhibit the expression of the target molecule in vivo or in vitro. Still further, triple helix molecules can be utilized in reducing the level of target gene expression.

[0089] As used herein, the term "antisense" preferably refers to a nucleotide sequence that is complementary to a nucleic acid encoding phosphodiesterase 3 and/or a angiotensin receptor, as hereinbefore described, e.g., complementary to the coding strand of the double- stranded cDNA molecule or complementary to the mRNA sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand (e.g., Ang II). The antisense nucleic acid may be complementary to an entire coding strand, or to only a portion thereof. In some embodiments, the antisense nucleic acid molecule is antisense to a non-coding region of the coding strand of a nucleotide sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or an angiotensin receptor ligand {e.g., the 5' and 3' untranslated regions).

[0090] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand, but can be an oligonucleotide which is antisense to only a portion of the coding or non-coding region of phosphodiesterase 3 mRNA and/or the angiotensin receptor mRNA and/or angiotensin receptor ligand mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of phosphodiesterase 3 mRNA and/or the angiotensin receptor mRNA and/or angiotensin receptor ligand mRNA. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. [0091] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation {i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

[0092] In some embodiments of the present invention, short interfering nucleic acid molecules (siRNA) that inhibit expression of the target gene can also be used in accordance with the present invention to reduce the level of target gene expression, as herein described.

[0093] The term "short interfering nucleic acid', "siNA", "short interfering RNA", "siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically-modified short interfering nucleic acid molecule^1*, as used herein, typically refers to any nucleic acid molecule capable of inhibiting or down-regulating gene expression of phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand, for example, by mediating RNA interference ("RNAi") or gene silencing in a sequence-specific manner. Chemical modifications can also be applied to any siNA sequence that may be used in the present invention. For example, the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof) and the sense region having a nucleotide sequence corresponding to a nucleotide sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof). The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example, wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence encoding phosphodiesterase 3 and/or the angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof) and the sense strand comprises nucleotide sequence corresponding a nucleotide sequence encoding phosphodiesterase 3 and/or the angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof). Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having a nucleotide sequence corresponding to a nucleotide sequence encoding phosphodiesterase 3 and/or the angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof). The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence encoding phosphodiesterase 3 and/or the angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof) and the sense region having a nucleotide sequence corresponding to a nucleotide sequence encoding phosphodiesterase 3 and/or the angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof) and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof; for example, where such siNA molecule does not require the presence within the siNA molecule of a nucleotide sequence corresponding to a nucleotide sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand, or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'- phosphate or a 5',3'-diphosphate. In some embodiments, the siNA molecule that may be used in the present invention includes separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non- nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. In some embodiments, the siNA molecule that may be used in the present invention includes a nucleotide sequence that is complementary to a nucleotide sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand (or a portion thereof). In some embodiments, the siNA molecule that may be used in the present invention interacts with a nucleotide sequence encoding phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand in a manner that causes inhibition of expression of the target genes. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses molecules comprising chemically-modified nucleotides or those in combination with non-nucleotides. In some embodiments, the siNA molecule that may be used in the present invention lacks 2'-hydroxy (2'-OH) containing nucleotides. Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can, however, have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups. Optionally, siNA molecules that may be used in the present invention can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified siNA molecules of the invention can also be referred to as short interfering modified oligonucleotides "siMON." As used herein, the term siNA is typically meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), translational silencing, and others. In addition, as used herein, the term RNAi is typically meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post- transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of phosphodiesterase 3 and or angiotensin receptor and/or angiotensin receptor ligand gene expression by siNA molecules that may be used in the present invention can result from siNA-mediated modification of the chromatin structure to alter gene expression of phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand.

[0094] The antisense and short interfering RNA molecules that may be used in the present invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridise with or bind to cellular mRNA and/or genomic DNA encoding phosphodiesterase 3 and/or angiotensin receptor and/or angiotensin receptor ligand to thereby inhibit expression of said phosphodiesterase 3 and/or an angiotensin receptor and/or angiotensin receptor ligand, e.g., by inhibiting transcription and/or translation. Alternatively, the molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense or siRNA molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the molecules to peptides or antibodies that bind to cell surface receptors or antigens. The molecules can also be delivered to cells using vectors, or by viral mechanisms (such as retroviral or adenoviral infection delivery). To achieve sufficient intracellular concentrations of the molecules, vector constructs in which the molecule is placed under the control of an appropriate promoter. [0095] In some embodiments, the antisense nucleic acid molecule that may be used in the present invention is an α-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual a-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0096] In some embodiments, the antisense nucleic acid that may be used in the present invention is a ribozyme. A ribozyme having specificity for phosphodiesterase 3- and/or angiotensin receptor- and/or angiotensin receptor ligand-encoding nucleic acid molecules can include one or more sequences complementary to the nucleotide sequence of phosphodiesterase 3 and/or angiotensin receptor and/or angiotensin receptor ligand cDNA and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591 ). For example, a derivative of a Tetrahymena L- 9 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a phosphodiesterase 3- and/or angiotensin receptor- and/or angiotensin receptor ligand- encoding mRNA (see, e.g., U.S. Pat. No. 4,987,071 ; and U.S. Pat. No. 5,116,742). Alternatively, phosphodiesterase 3 and/or angiotensin receptor and/or angiotensin receptor ligand mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261 :141 1-1418).

[0097] In a further embodiment, phosphodiesterase 3 and/or angiotensin receptor and/or angiotensin receptor ligand expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the target gene {e.g., promoters and/or enhancers) to form triple helical structures that prevent transcription of the target gene in cells (see generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15). The potential sequences that can be targeted for triple helix formation can be increased by creating a so- called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[0098] The antisense molecules may also be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecule can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acid' or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93:14670-675.

[0099] In some embodiments, the antisense molecules may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, antisense molecules can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[00100] It is possible that the use of antisense, siRNA, ribozyme, and/or triple helix molecules to reduce or inhibit target gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy methods. [00101] Another method by which nucleic acid molecules may be utilized in preventing or treating hyperglycemia and hypertriglyceridemia (such as in a subject with Type 2 diabetes mellitus) is through the use of aptamer molecules specific for phosphodiesterase 3 and/or angiotensin receptor and/or angiotensin receptor ligand. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem. Biol. 1 (1):5-9; and Patel, D. J. (June 1997) Curr. Opin. Chem. Biol. 1 (1):32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which phosphodiesterase 3 and/or angiotensin receptor and/or angiotensin receptor ligand activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects. Antibodies

[001O2] In some embodiments, the inhibitor of phosphodiesterase 3 activity and/or the inhibitor of angiotensin receptor activity that may be used in accordance with the present invention include antibodies or antigen-binding fragments thereof. Examples include, but are not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, FAb, F(ab')₂ and FAb fragments, scFV molecules, and epitope-binding fragments thereof.

Formulations

[00103] In the compositions of the present invention the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity may be present at a molar ratio ranging from 99:1 to 1 :99. In some embodiments, the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity may be used in accordance with the present invention at a molar ratio ranging from about 25:5 to about 5:25. In some embodiments the molar range is from 4:1 to 1 :4. In some embodiments the molar range is from 3:1 to 1 :3. In some embodiments the molar range is from 2:1 to 1 :2.

[00104] Viewed another way the compositions of the present invention may contain any of a large number of possible weight ratios of active ingredients. In one embodiment the weight ratio of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity is from 20:1 to 1 : 20. In another embodiment the ratio is from 10:1 to 1 :10. In yet an even further embodiment the ratio is from 8:1 to 1 :1. In yet an even further embodiment the ratio is from 2:1 to 6:1. In an even further embodiment the ratio is from 3:1 to 5:1. In a most preferred embodiment the weight ratio of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity is about 4:1.

[00105] The concentration of the actives in the composition may also range from as low as 0.1% of the total amount of the composition up to as high as 100%. In some embodiments the concentration of actives in the composition is from 1 % to 90% by weight. In some embodiments the concentration of actives in the composition is from 1% to 90% by weight. In some embodiments the concentration of actives in the composition is from 5% to 80% by weight. In some embodiments the concentration of actives in the composition is from 10% to 70% by weight. The exact amount will depend upon the identity of the two actives and any additional materials chosen.

[00106] In another aspect, the composition of the present invention further includes a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.

[00107] As used herein, the term "pharmaceutically acceptable carrier" typically includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions of the present invention.

[00108] The compounds that may be used in the present invention can be formulated as compositions in such a way as to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00109] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition is preferably sterile and should be fluid to the extent that easy syringability exists. In some embodiments, it will be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, or liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion or by the use of surfactants. Prevention of the action of microorganisms can be achieved by incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, or sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[00110] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00111] Oral compositions generally comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

[00112] Formulations for oral use may be in the form of tablets which may be obtained by mixing active ingredients with known excipients. The tablet may also comprise of bilayer or film or gelatin coated by coating cores produced analogously to the tablets with substances normally used for coating. To achieve immediate release, the tablet may contain a suitable excipient, as herein described, along with the active ingredients. [00113] Suitable tablets may be obtained for example, by mixing at least one of the compounds that may be used in the present invention with known excipients, for example diluents such as microcrystalline cellulose, calcium carbonate, calcium phosphate or lactose, disintegrants such as croscaramellose sodium, HPMC, sodium starch glycolate, binders such as starch or gelatine, guar gum, xanthum gum, lubricants such as magnesium stearate or talc and/or agents. The shapes include round, caplet, flat, oval and bevelled edges with and without embossing.

[00114] Capsules like hard or soft gelatin containing the compounds that may be used in the present invention may, for example, be prepared by mixing the active compounds with inert carriers such as lactose or sorbitol and packing them into gelatine capsules. Capsules may be with or without imprinting.

[00115] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredients are mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin; or as soft gelatin capsules wherein the active ingredients are mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[00116] Alternatively (or in addition), formulations for oral use may be presented as capsules, tablets and the like wherein the active ingredients are substantially separated, for example, by an inert barrier. In some embodiments, the formulation for oral use is a capsule or tablet comprising a first compartment comprising an inhibitor of phosphodiesterase 3 activity, as herein described, and a second compartment comprising an inhibitor of angiotensin receptor activity, as herein described. In other embodiments, the formulation for oral use can be a container (including a capsule) that comprises a first bead (or other suitable carrier known to the skilled person) at least partially coated with an inhibitor of phosphodiesterase 3 activity, as herein described, and a second bead at least partially coated with an inhibitor of angiotensin receptor activity, as herein described. The first and second beads may be contained in the same compartment or in separate compartments, as required. [00117] For administration by inhalation, the compounds that may be used in the present invention may be delivered in the form of an aerosol spray from a pressurised container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[00118] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished with nasal sprays or suppositories. The compounds can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00119] For transdermal administration, the compounds that may be used in the present invention, as herein described, can be formulated into ointments, salves, gels, or creams as generally known in the art. The compounds may also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal patches, wherein the compounds are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the compounds are typically contained in a layer, or "reservoir," underlying an upper backing layer. The laminated device may contain a single reservoir, or it may contain multiple reservoirs, in one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, poiyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug- containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. [00120] The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing material should be selected so that it is substantially impermeable to the active ingredient and any other materials that are present. The backing layer may be either occlusive or non-occlusive, depending on whether it is desired that the skin become hydrated during drug delivery. The backing is preferably made of a sheet or film of a preferably flexible elastomeric material. Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, polyesters, and the like.

[00121] During storage and prior to use, the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the drug reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin. The release liner should be made from a drug/vehicle impermeable material. [00122] Transdermal drug delivery devices may be fabricated using conventional techniques, known in the art, for example by casting a fluid admixture of adhesive, drug and vehicle onto the backing layer, followed by lamination of the release liner. Similarly, the adhesive mixture may be cast onto the release liner, followed by lamination of the backing layer. Alternatively, the drug reservoir may be prepared in the absence of drug or excipient, and then loaded by "soaking" in a drug vehicle mixture.

[00123] The laminated transdermal drug delivery systems may in addition contain a skin permeation enhancer. That is, because the inherent permeability of the skin to some compounds may be too low to allow therapeutic levels of the compounds to pass through a reasonably sized area of unbroken skin, it is necessary to co-administer a skin permeation enhancer with such drugs.

[00124] In some embodiments, the compounds that may be used in the present invention are prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyan hydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 , the contents of which are incorporated herein by reference in their entirety.

[00125] The compounds that may be used in the present invention may also be incorporated in excipients conventionally to use in pharmaceutical compositions such as for example, diluents like microcrystalline cellulose, binders, disintegrates, lubricants/glidents, wetting agents, coating and miscellaneous materials, such as those exemplified in Table 1 , below.

Table 1 :

[00126] Pharmaceutical compositions of the present invention may be employed alone or in conjunction with other compounds, including other therapeutic compounds. 4

30

Dosages

[00127] As used herein, the terms "an effective amount¹ or "therapeutically effective amount', typically refers to an amount of a compound that may be used in the present invention that is sufficient to effect beneficial or desired results as described herein when administered to a subject such as a mammal, preferably a human, in need of such therapy; for example, a subject who is suffering from a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis. The dosage of compound that may be used in the present inventions will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other therapeutic compounds being administered, the age, size, and species of the subject, e.g., human patient, and like factors. In general, the dosage of a compound that may be used in the present invention will be an amount which is the lowest dose effective to produce the desired effect with no or minimal side effects.

[00128] In some embodiments, the inhibitor of phosphodiesterase 3 activity, as herein described, will be administered at dosages from about 1 ng/kg to about 1000 mg/kg, such as from about 0.1-1.0 pg/gm, including 0.20-0.80 pg/gm, as well as e.g., about 1 mg kg to about 100 mg/kg, including from about 5 mg/kg to about 50 mg kg. Based on these ratios representative dosages of the inhibitor of phosphodiesterase 3 activity according to the present invention include about 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg per day or those dosages described herein and with reference to the Examples.

[00129] In some embodiments, the inhibitor of angiotensin receptor activity that may be used in the present invention will be administered at dosages from about 1 ng/kg to about 1000 mg/kg, such as from about 0.1-1.0 pg/gm, including 0.20-0.80 pg/gm, as well as e.g., about 1 mg kg to about 100 mg/kg, including from about 5 mg/kg to about 50 mg/kg. Based on these ratios representative dosages of the inhibitor of angiotensin receptor activity include about 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg per day or those dosages described herein and with reference to the Examples. [00130] In use in the methods and uses of the present invention the weight ratio of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity is typically from 20:1 to 1: 20. in another embodiment the ratio is from 10:1 to 1:10. In yet an even further embodiment the ratio is from 8:1 to 1 :1. In yet an even further embodiment the ratio is from 2:1 to 6:1. In an even further embodiment the ratio is from 3:1 to 5:1. In a most preferred embodiment the weight ratio of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity is about 4:1.

[00131] In some embodiments, the therapeutically effective amount of an inhibitor of phosphodiesterase 3 activity or an inhibitor of angiotensin receptor activity will range from about 25 to 50 mg and from about 5 to 10 mg, respectively.

[00132] The inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity may be used in accordance with the present invention at a molar ratio ranging from 99:1 to 1:99. In some embodiments, the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity may be used in accordance with the present invention at a molar ratio ranging from about 25:5 to about 5:25.

[00133] The effective dose of a compound that may be used in the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[00134] In some embodiments, the compounds that may be used in the present invention are administered once per day for between about 1 week to greater than 12 months. In some embodiments, the compounds that may be used in the present invention are administered once per day for more than 6 months.

[00135] Without being bound by theory, it is expected that the duration of treatment required in order to affect a change in triglyceride and glucose levels could be about 13 weeks. However, this duration may vary depending, for example, on the severity of the disorder and the dosage of the compounds administered to the subject.

[00136] In some embodiments, the compounds that may be used in the present invention are administered at dosages of (ratio of inhibitor of PDE3 activity:inhibitor of angiotensin receptor activity) 25:5 mg/ day, 50:10 mg/ day or 100:20 mg /day, or any combination thereof. [00137] In some embodiments, the compounds that may be used in the present invention, as herein described, are advantageously formulated in dosage units, each dosage unit being adapted to supply a single dose of the active compound for ease of administration and uniformity of dosage. "Dosage unit fornf, as used herein, typically refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of the compound calculated to produce the desired therapeutic effect, either alone or in association with a pharmaceutical carrier.

[00138] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00139] The data obtained from the ceil culture assays and animal studies can be used in formulating a range of dosages for use in humans. T e dosage typically lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in accordance with the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00140] Another example of determination of effective dose for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate phosphodiesterase 3 and/or angiotensin receptor activity is used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix that contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al. (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such "imprinted" affinity matrices are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrices in this way can be seen in Vlatakis, G. et al. (1993) Nature 361 :645-647. Through the use of isotope-labelling, the "free" concentration of compound which modulates the expression or activity of phosphodiesterase 3 and/or an angiotensin receptor can be readily monitored and used in calculations of IC₅₀. Such "imprinted" affinity matrices can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fibre-optic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC₅₀. A rudimentary example of such a "biosensor" is discussed in Kriz, D. et al. (1995) Analytical Chemistry 67:2142-2144.

[00141] The specific weight ratio of the respective compounds that may be used in the present invention may be varied when necessary and will depend upon the effective dose of each compound or the effective dose of the combination of compounds in a formulation (although a therapeutically- or prophylactically-effective dose of each will typically be used). For example, the skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, the degree of expression or activity to be modulated, the severity of the disease or disorder, previous treatments and other diseases present.

[00142] The skilled artisan will also appreciate that each of active compounds that may be used in the present invention, as herein described, can be administered in combination (i.e., present in a single composition) or individually [i.e., in separate compositions), depending on the preferred mode of use.

[00143] Where the inhibitor of phosphodiesterase 3 activity or angiotensin receptor activity is an antibody, typical dosages may be from about 0.1 to 20 mg/kg of body weight per day. Generally, partially or fully human (or humanised) antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration are often possible. Modifications such as lipidation can be used to stabilize [00150] In relation to simultaneous administration, the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor expression and/or activity may be administered as a combined preparation or composition, such as the composition of the present invention, as herein described. They may also be administered as separate preparations to a subject at the same time. For example, if the administration is made orally, the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity could be administered in the form of two tablets or the like at the same time or one after the other, in no particular order. For instance, a first tablet comprising an inhibitor of phosphodiesterase 3 activity can be administered first, followed some time later by a second tablet comprising an inhibitor of angiotensin receptor activity. The amount of time that lapses between the administration of the first and second tablets can vary (e.g., from between a few seconds to several days). In a similar manner, where the compounds that may be used in the present invention are to be administered intravenously, the subject can have two drip lines inserted wherein the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity, as herein described, are administered simultaneously through two separate drip lines.

[00151] The administration of the compounds that may be used in the present invention, as herein described, may also be administered sequentially and in any order. For example, an inhibitor of phosphodiesterase 3 activity may be administered, followed by administration of an inhibitor of angiotensin receptor activity. Alternatively, an inhibitor of angiotensin receptor activity may be administered followed by an inhibitor of phosphodiesterase 3 activity. In relation to sequential administration, the period of time between administrations of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity can vary considerably and the compounds that is administered second may be administered as little as a few seconds up to a significant number of days (e.g., 1 , 7, 14 or 21 days) after administration of the compound that is administered first.

[00152] Administration of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity may also be such that there is a partial concurrent administration. Thus, for example, an inhibitor of phosphodiesterase 3 activity may be delivered slowly such as by infusion and before completion of this administration, the administration of an inhibitor of angiotensin receptor activity is commenced such that for a period of time there is concurrent administration such that both compounds are being administered at the same time. [00153] In some embodiments, the prophylactic and/or therapeutic methods of the present invention comprise the steps of administering the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity to a subject who has hyperglycemia and/or hypertriglyceridemia, a symptom of or predisposition toward hyperglycemia and/or hypertriglyceridemia, for the purpose to cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect hyperglycemia and/or hypertriglyceridemia, the symptoms of hyperglycemia and/or hypertriglyceridemia, or the predisposition towards hyperglycemia and/or hypertriglyceridemia. [00154] In some embodiments, the prophylactic and/or therapeutic methods of the present invention comprise the steps of administering the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity to a subject who has Type 2 diabetes mellitus, a symptom of or predisposition toward Type 2 diabetes mellitus, for the purpose to cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect Type 2 diabetes mellitus, the symptoms of Type 2 diabetes mellitus, or the predisposition towards Type 2 diabetes mellitus.

[00155] In some embodiments, the prophylactic and/or therapeutic methods of the present invention comprise the steps of administering the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity to a subject who is obese, or has a predisposition toward obesity, for the purpose to cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect obesity, the symptoms of obesity, or the predisposition towards obesity.

[00156] In some embodiments, the prophylactic and/or therapeutic methods of the present invention comprise the steps of administering the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity to a subject who has Non-alcoholic fatty liver disease, or has a predisposition toward Non-alcoholic fatty liver disease, for the purpose to cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect Non-alcoholic fatty liver disease, the symptoms of Non-alcoholic fatty liver disease, or the predisposition towards Non-alcoholic fatty liver disease.

[00157] In some embodiments, the prophylactic and/or therapeutic methods of the present invention comprise the steps of administering the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity to a subject who is Non-alcoholic steatohepatitis, or has a predisposition toward Non-alcoholic steatohepatitis, for the purpose to cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect Non-alcoholic steatohepatitis, the symptoms of Non-alcoholic steatohepatitis, or the predisposition towards Non-alcoholic steatohepatitis. [00158] In the context of the present invention, the term "therapy" typically also includes "prophylaxis?. Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of a condition (e.g., hyperglycemia, hypertriglyceridemia, diabetes, obesity), particularly in association with (but not limited to) diabetes. Persons at risk of developing the particular condition or disorder generally include those having a family history of the condition or disorder, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition or disorder.

[00159] The term "disease", as used herein, typically has the same meaning as "condition" and "disorder" and are used interchangeably throughout

[00160] The skilled addressee will appreciate that the uses and methods of the present invention are applicable to any conditions in which hyperglycemia or hypertriglyceridemia is implicated, including (but not limited to) diabetes and obesity. In some embodiments, the condition is Type 2 diabetes. Other conditions may include Mixed Dyslipidemia, Hypertriglyceridemia, Non-alcoholic Steatosis, Diabetic Nephropathy, Diabetic Neuropathy, Diabetic Retinopathy, Diabetic Hypertension, Diabetic Dyslipidemia, Essential Hypertension, Hypertensive Nephropathy, Hypertensive Retinopathy, Congestive heart failure, Hypertensive Cardiomyopathy, Atherosclerosis, Cardiovascular Diseases-myocardial infarction and cerebral stroke, Inflammatory Diseases and Cushing's Syndrome. Such conditions may be identified by those skilled in the art by any or a combination of diagnostic or prognostic assays known in the art. For example, a biological sample obtained from a subject {e.g. blood, serum, plasma, urine and/or saliva) may be analysed for glucose and triglyceride levels, as herein described (see, e.g., the Examples).

[00161] The Applicant has also shown, for the first time, that the combination of an inhibitor of phosphodiesterase 3 activity and an inhibitor of angiotensin receptor activity modulates the differentiation of adipocyte differentiation (see Examples).

[00162] Thus, in another aspect of the present invention there is provided a use of (i) an inhibitor of phosphodiesterase 3 activity (as herein described) and (ii) an inhibitor of angiotensin receptor activity (as herein described) to modulate the differentiation of adipocytes, in vitro or in vivo. W

38

[00163] In another aspect of the present invention there is provided a method of modulating the differentiation of adipocytes, in vitro or in vivo, the method including contacting adipocytes with (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity.

5

[00164] The Applicant has also shown, for the first time, that the combination of an inhibitor of phosphodiesterase 3 activity and an inhibitor of angiotensin receptor activity, when administered to a subject, reduces body weight in that subject (see Examples). Without being bound by theory, the Applicant postulates that modulation of adipocyte differentiation with the0 combination of an inhibitor of phosphodiesterase 3 activity and an inhibitor of angiotensin receptor activity promotes storage of fat in adipocytes and the decrease in body weight seen in vivo is probably due to enhanced lipolysis and increased fatty acid oxidation in adipose tissue, liver and muscle. 5 [00165] Thus, it is also an aspect of the present invention to provide a use of (i) an inhibitor of phosphodiesterase 3 activity (as herein described) and (ii) an inhibitor of angiotensin receptor activity (as herein described) to reduce body weight in a subject.

[00166] In another aspect of the present invention there is provided a method of reducing0 body weight in a subject, the method including contacting adipocytes with (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity.

[00167] It would also be well appreciated by one skilled in the art that the methods of treatment hereinbefore described could be used in any number of combinations with each5 other, or with other treatment regimes currently employed in the art.

Kits

[00168] In another aspect of the present invention there is provided a kit including (i) an inhibitor of phosphodiesterase 3 activity, as herein described and (ii) an inhibitor of0 angiotensin receptor activity, as herein described.

[00169] In some embodiments, the kit further includes written instructions as to the use of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity in the prophylaxis or treatment of a condition selected from the group consisting of5 hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis. For example, where the kit includes a first container, pack or dispenser that includes an inhibitor of phosphodiesterase 3 activity and a second container, pack or dispenser that includes an inhibitor of angiotensin receptor activity, the kit may further include instructions for simultaneous administration and/or sequential administration in any order, or a combination thereof, of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity. The instructions may be attached to, or printed on, one of the containers, may be printed on a separate sheet of paper inside the package, or may be attached to or printed on the package.

[00170] In some embodiments, the kit includes an inhibitor of phosphodiesterase 3 activity in a first container, pack or dispenser and an inhibitor of angiotensin receptor activity in a second container, pack or dispenser. In other embodiments, the kit includes an inhibitor of phosphodiesterase 3 activity and an inhibitor of angiotensin receptor activity in the same container, pack or dispenser. Where the compounds are in the same container, pack or dispenser, they may be present as an admixture or separated by a barrier. In other embodiments, the kit includes the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity in the same container formulated as a composition according to the present invention, as herein described.

[00171] Suitable containers include tubes, ampules, vials, bottles, foil packets, the wells of a tray and the molded depressions of blister packs. The kit will also comprise instructions for administration of the compounds of the present invention, as the container(s) may be contained in a package, such as a box.

[00172] It is to be noted that "a" or "an" entity refers to one or more of that entity. For example, "a container" refers to one or more containers.

[00173] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of each claim of this application.

[00174] Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein. [00175] Future patent applications may be filed on the basis of or claiming priority from the present application. [00176] Examples of the procedures used in the present invention will now be more fully described. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES

[00177] Cilostazol and Telmisartan were selected as representative therapeutic inhibitors of phosphodiesterase 3 and angiotensin receptor activity, respectively. [00178] A battery of cell-based assays was conducted and the results from these assays unequivocally demonstrate that the combination of compounds tested has a synergistic effect on the parameters related to the endpoints of hyperglycemia and hypertriglyceridemia.

Example 1 : Effect of combination on ApoB-100 secretion from HePG2 cells

[00179] The efficacy of Cilostazol (1μ ) and Telmisartan (1μΜ) individually and in combination was tested for ApoB-100 secretion in HepG2 cells. It was observed that combination of Cilostazol and Telmisartan at 1 μΜ showed significant decrease in ApoB-100 secretion and was more prominent when compared with individual drug. Example 2: Triglycerides (TAG) accumulation assay in HePG2 cells

[00180] HepG2 cells were grown in DMEM containing excess glucose (33mM) supplemented with 10% fetal bovine serum (FBS) and high FFA (200 μΜ palmitate) to induce TAG accumulation. Cells treated with different concentrations (100nm to 3μΜ) of Telmisartan(T) and Cilostazol(C) alone and in combination were observed for inhibition of TAG accumulation. At lower concentrations, the combination of Telmisartan and Cilostazol showed inhibition of excess glucose and FFA-induced TAG accumulation (see Figure 1).

Example 3: Effect of combination drug on adipocytes differentiation

[00181] The adipogenic cell line 3T3F44 was used to study the effect of combination drug on adipocyte differentiation. The cells were differentiated to adipocytes in presence of the drugs. Telmisartan and Cilostazol were used at a concentration range from 1-1 ΟμΜ individually and in combination.

[00182] At lower concentrations of Telmisartan and Cilostazol combination showed statistically significant (P<0.001) increase in adipogenesis compared individual drugs and vehicle control. The reference standard used for the assay also showed significant increase in adipogenesis compared to vehicle control (P<0.001). Example 4: Acute toxicity

[00183] Acute toxicity studies were conducted in Wistar rats and Swiss Albino mice. The combination of Cilostazol and Telmisartan at a ratio of 9:1 was found to be safe in animals, with a maximum tolerated dose (MTD) of 2,000 mg/kg.

Example 5: Efficacy - cafeteria diet-fed Wistar rats

[00184] The purpose of this study was to evaluate the effect of the combination of Cilostazol and Telmisartan administered to Standardized Cafeteria Diet-fed Wistar rats for a period of 2 weeks, looking at any improvements in hypertriglyceridemia. Daily doses of Cilostazol (25 mg/kg, po) and Telmisartan (5 mg/kg, po) were administered to the Wistar rats for 2 weeks. The three groups were evaluated at the end of 2 weeks [normal control (n=9), cafeteria (n=6) and treatment (n=7)]. [00185] The results show that the cafeteria diet-fed group exhibited around 100% increase in the triglyceride levels when compared with the normal control group. The treatment group showed decrease in the triglyceride levels around 40-50% when compared with the cafeteria group. Example 6: Efficacy - cafeteria diet-fed cafeteria diet fed C57 mice

[00186] The purpose of this study was to evaluate the effect of the combination of Cilostazol and Telmisartan administered to cafeteria diet-fed C57 mice for a period of 24 weeks on glucose tolerance, body weight and biochemical parameters (Triglyceride, Cholesterol, Glucose, Insulin).

[00187] No. of animals: Control (n=5), Cafeteria diet control (n=10), Treatment group (n=10).

[00188] Doses: Cilostazol (50 mg/kg, po) and Telmisartan (10 mg/kg, po) [00189] Conditions: Control group, Cafeteria diet-fed group, Treatment group (cafeteria diet + treatment; FDC-II)

[00190] The objective of this study was to evaluate the effect of treatment on body weight and biochemical parameters. The treatment group (FDC-II) showed significant reduction in the body weight (approx. 12.5% reduction; see Figure 2) and also a reduction in serum and liver triglyceride levels (see Figure 3) when compared with the cafeteria diet-fed control groups. Treatment did not show any effect on cholesterol and insulin levels as compared to the control groups.

Example 7: Effect of Cilostazol and Telmisartan on adipocytes in vivo

[00191] No. of animals: Control (n=5), Cafeteria diet control (n=10), Treatment group (n=10).

[00192] Doses: Cilostazol (50 mg/kg, po) and Telmisartan (10 mg/kg, po). [00193] Conditions: Control group, Cafeteria diet-fed group, Treatment group (cafeteria diet + treatment; FDC-II).

[00194] The purpose of this study was to evaluate the effect of the combination of Cilostazol and Telmisartan administered to cafeteria diet-fed C57 mice for a period of 24 weeks on adipocyte size (cross-sectional area) and adipocyte numbers.

[00195] At the end of the 24 week period of treatment, the animals were sacrificed and adipose tissue collected for adipocytes analysis. [00196] The results showed that cafeteria-diet fed animals had an increase in adipocyte size and number (p<0.05) when compared with the chow-diet fed control animals. Treatment with Cilostazol and Telmisartan in combination (FDC-II) showed a significant decrease in adipocyte size and number (p<0.05; see Figure 4). Example 8: Effect of Cilostazol and Telmisartan on liver and adipose in vivo

[00197] No. of animals: Control (n=4), Choline deficient diet (CDD) group (n=4), Treatment group (n=4).

[00198] Doses: Cilostazol (12.5 mg/kg, po) and Telmisartan (2.5 mg/kg, po), daily once.

[00199] Conditions: Control group, Choline deficient diet (CDD) group, Treatment group (CDD + treatment; FDC-II).

[00200] The objectives of the study were to evaluate the treatment regimen (FDC-II) for its effects on liver and adipose in the Choline deficient diet (CDD) treated Swiss albino mice. [00201] At the end of the 6 week period of treatment, the animals were sacrificed, liver and adipose tissue collected. Later total RNA was prepared and converted to cDNA. The expressions of tissue specific markers were analyzed by qPCR method. [00202] The results showed that FDC-II treated CDD model animals had an increase in liver insulin sensitivity, moderate increase in fat oxidation (see Figure 5). In adipose, FDC-II treated animals showed an increase in insulin sensitivity, fat oxidation and reduction in inflammatory adipokines. This will lead to reduction in ectopic fat storage and an overall improvement in peripheral insulin sensitivity (see Figure 6 ).

[00203] The details of specific embodiments described in this invention are not to be construed as limitations. Various equivalents and modifications may be made without departing from the essence and scope of this invention, and it is understood that such equivalent embodiments are part of this invention.

Claims

Claims

1. A composition including (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity.

2. The composition according to claim 1 , wherein the inhibitor of phosphodiesterase 3 activity is selected from the group consisting of Enoximone, Cilostazol, Milrinone, Vesnarinone and Pimobendan, or a prodrug, analogue or biologically-active variant thereof.

3. The composition according to claim 1 or claim 2, wherein the inhibitor of angiotensin receptor activity is an angiotensin receptor antagonist.

4. The composition according to claim 3, wherein the angiotensin receptor antagonist is selected from the group consisting of Losartan, Telmisartan, Valsartan, Irbesartan,

Olmesartan, Eprosartan and Candesartan, or a prodrug, analogue or biologically- active variant thereof.

5. The composition according to claim 1 , wherein the inhibitor of angiotensin receptor activity is an angiotensin converting enzyme inhibitor.

6. The composition according to claim 5 wherein the angiotensin converting enzyme inhibitor is selected from the group consisting of alacepril, alindapril, altiopril, benazepril, benazeprilat, captopril, ceronapril, cilazapril, cilazaprilat, delapril, enalapril, enalaprilat, fosinopril, imidapril, indolapril, libenzapril, lisinopril, moexepril, moveltipril, pentopril, perindopril, quinapril, quinaprilat, ramipril, rentiapril, spirapril, temocapril, teprotide, trandolapril, zofenopril, omapatrilat, fasidotril, mixanpril, sampatrilat, gemopatrilat (BMS-189921 ), MDL-100240 and Z13752A (GW660511 ), or a prodrug, analogue or a biologically-active variant thereof.

7. The composition according to claim 1 , wherein the inhibitor of phosphodiesterase 3 activity inhibits phosphodiesterase 3 expression.

8. The composition according to claim 1 , wherein the inhibitor of angiotensin receptor activity inhibits angiotensin receptor expression.

9. The composition according to claim 7 or claim 8, wherein the inhibitor of phosphodiesterase 3 expression or the inhibitor of angiotensin receptor expression is an antisense nucleic acid molecule.

10. The composition according to claim 1 , 7 or 8, further including a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.

11. Use of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity in the prophylaxis or treatment of a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis

12. Use of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity in the manufacture of a medicament for the prophylaxis or treatment of a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis.

13. The use according to claim 11 or claim 12, wherein the inhibitor of phosphodiesterase 3 activity is selected from the group consisting of Enoximone, Cilostazol, Milrinone,

Vesnarinone and Pimobendan, or a prodrug, analogue or biologically-active variant thereof.

14. The use according to any one of claims 11 to 13, wherein the inhibitor of angiotensin receptor activity is an angiotensin receptor antagonist.

15. The use according to claim 14, wherein the angiotensin receptor antagonist is selected from the group consisting of Losartan, Telmisartan, Valsartan, Irbesartan, Olmesartan, Eprosartan and Candesartan, or a prodrug, analogue or biologically- active variant thereof.

16. The use according to any one of claims 11 to 13, wherein the inhibitor of angiotensin receptor activity is an angiotensin converting enzyme inhibitor.

17. The use according to claim 16 wherein the angiotensin converting enzyme inhibitor is selected from the group consisting of alacepril, alindapril, altiopril, benazepril, benazeprilat, captopril, ceronapril, cilazapril, cilazaprilat, delapril, enalapril, enalaprifat, fosinopril, imidapril, indolapril, libenzapril, lisinopril, moexepril, moveltipril, pentopril, perindopril, quinapril, quinaprilat, ramipril, rentiapril, spirapril, temocapril, teprotide, trandolapril, zofenopril, omapatrilat, fasidotril, mixanpril, sampatrilat, gemopatrilat (B S-189921), MDL-100240 and Z13752A (GW660511 ), or a prodrug, analogue or a biologically-active variant thereof.

18. The use according to claim 11 or claim 12, wherein the inhibitor of phosphodiesterase 3 activity inhibits phosphodiesterase 3 expression.

19. The use according to any one of claims 11 , 12 and 18, wherein the inhibitor of angiotensin receptor activity inhibits angiotensin receptor expression.

20. The use according to claim 18 or claim 19, wherein the inhibitor of a phosphodiesterase 3 expression or the inhibitor of angiotensin receptor expression is an antisense nucleic acid molecule.

21. The use according to claim 11 or claim 12, wherein the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity are used in the form of a composition according to any one of claims 1 to 10.

22. The use according to any one of claims 11 to 21 , wherein the condition is Type 2 diabetes.

23. A method of preventing or treating a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Nonalcoholic fatty liver disease and Non-alcoholic steatohepatitis, the method including administering to a subject in need thereof, a therapeutically effective amount of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity.

24. The method according to claim 23, wherein the inhibitor of phosphodiesterase 3 activity is selected from the group consisting of Enoximone, Cilostazol, Milrinone, Vesnarinone and Pimobendan, or a prodrug, analogue or biologically-active variant thereof.

25. The method according to claim 23 or claim 24, wherein the inhibitor of angiotensin receptor activity is an angiotensin receptor antagonist.

The method according to claim 25, wherein the angiotensin receptor antagonist is selected from the group consisting of Losartan, Telmisartan, Valsartan, Irbesartan, Olmesartan, Eprosartan and Candesartan, or a prodrug, analogue or biologically- active variant thereof.

The method according to claim 23 or claim 24, wherein the inhibitor of angiotensin receptor activity is an angiotensin converting enzyme inhibitor.

The method according to claim 27 wherein the angiotensin converting enzyme inhibitor is selected from the group consisting of alacepril, alindapril, altiopril, benazepril, benazeprilat, captopril, ceronapril, cilazapril, cilazaprilat, delapril, enalapril, enalaprilat, fosinopril, imidapril, indolapril, libenzapril, lisinopril, moexepril, moveltipril, pentopril, perindopril, quinapril, quinaprilat, ramipril, rentiapril, spirapril, temocapril, teprotide, trandolapril, zofenopril, omapatrilat, fasidotril, mixanpril, sampatrilat, gemopatrilat (BMS-189921), MDL-100240 and Z13752A (GW660511), or a prodrug, analogue or a biologically-active variant thereof.

The method according to claim 23, wherein the inhibitor of phosphodiesterase 3 activity inhibits phosphodiesterase 3 expression.

The method according to claim 23 or claim 24, wherein the inhibitor of angiotensin receptor activity inhibits angiotensin receptor expression.

The method according to claim 29 or claim 30, wherein the inhibitor of a phosphodiesterase 3 expression or the inhibitor of angiotensin receptor expression is an antisense nucleic acid molecule.

A kit including (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity .

The kit according to claim 34, further including written instructions as to the use of the inhibitor of phosphodiesterase 3 activity and the inhibitor of angiotensin receptor activity in the prophylaxis or treatment of a condition selected from the group consisting of hyperglycemia, hypertriglyceridemia, diabetes, Type 2 diabetes, obesity, Non-alcoholic fatty liver disease and Non-alcoholic steatohepatitis.

34. Use of (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity to modulate the differentiation of adipocytes.

35. A method of modulating the differentiation of adipocytes, the method including contacting adipocytes with (i) an inhibitor of phosphodiesterase 3 activity and (ii) an inhibitor of angiotensin receptor activity.