A method for the treatment of endothelial dysfunction in a diabetic patient, including both diabetes induced macrovascular disorders and diabetes induced microvascular disorders, comprises administration, preferably parenteral administration, to the patient of an effective amount of high density lipoprotein (HDL).

Description of the Invention

FIELD OF INVENTION

This invention relates to a method for the treatment of endothelial dysfunction in diabetic patients. In particular, this invention relates to a method for improving endothelial function in treatment of disorders which are related to endothelial dysfunction, both macrovascular and microvascular, in diabetic patients.

BACKGROUND OF THE INVENTION

In the chronic disease diabetes mellitus (diabetes), the body loses the ability to properly produce or respond to the hormone insulin so that cells of the peripheral tissues fail to actively take up glucose from the blood for use or storage. In the diabetic individual, the level of glucose in the peripheral blood can become elevated (hyperglycaemia) and typically remains so unless some form of intervention is employed (e.g., administration of exogenous insulin) to return glucose in the blood to normal levels. Left unchecked, the hyperglycaemia of diabetic individuals can result in shock, organ degeneration or failure (e.g., kidney failure, blindness, nerve disease, cardiovascular disease), tissue necrosis (e.g., requiring foot amputation), and even death.

Two major forms of diabetes are type 1 and type 2 diabetes. Type 1 diabetes, which was previously known as insulin-dependent diabetes mellitus (IDDM) or juvenile onset diabetes, is an autoimmune disease in which the body destroys the insulin-producing .beta. cells (islet cells) of the pancreas resulting in an absolute requirement for daily administration of exogenous insulin to maintain normal blood glucose levels. Type 1 diabetes usually is diagnosed in children and young adults, but can occur at any age. Type 1 diabetes accounts for 5-10% of diagnosed cases of diabetes.

By far the more prevalent form of diabetes is type 2 diabetes, which was previously known as non-insulin-dependent diabetes mellitus (NIDDM). Type 2 diabetes was also previously known as adult-onset diabetes, however, this form of diabetes is becoming increasingly prevalent in the growing population of overweight and clinically obese children and young adults. Type 2 diabetes accounts for approximately 90-95% of all diagnosed cases of diabetes. Type 2 diabetes typically begins with insulin resistance, a disorder in which the body's cells do not respond to insulin properly, followed by a gradual loss on part of the pancreas to produce and secrete insulin. Type 2 diabetes is associated with a variety of factors including older age, obesity, family history of diabetes, history of gestational diabetes, impaired glucose metabolism, physical inactivity, and various races or ethnicities. Individuals with type 2 diabetes must attempt to control their blood glucose level with careful diet, exercise and weight reduction, and additional medications.

Major factors contributing to the pro-atherogenic state in diabetes, particularly type 2 diabetes mellitus (DM2), include dyslipidemia, hyperglycemia, hypertension, visceral obesity and insulin resistance (1,2). Observational studies have clearly demonstrated the importance of diabetic dyslipidemia in contributing to atherogenesis in diabetes, illustrated by the fact that the correlation between low density lipoproteins (LDL) as well as high density lipoproteins (HDL) versus cardiovascular events outweighs that of fasting plasma glucose (3). Statin intervention studies have revealed a clear benefit of statin treatment on reduction of cardiovascular events in DM2 (4, 5); however, in spite of this impressive achievement, the majority of DM2 patients will still suffer from cardiovascular events even when using statins (6).

During the last two decades, endothelial dysfunction has emerged as one of the earliest stages of atherogenesis. Endothelial dysfunction, which is a hallmark in all diabetic patients (ie both type 1 and 2) has been shown to have predictive value for future cardiovascular events (7-9). In line with the multifactorial pathogenesis of diabetes-induced vascular disease (10, 11), numerous therapeutic interventions have been evaluated for their potential to improve endothelial function in DM2 patients (9, 12). Surprisingly, whereas endothelial function could be fully restored by statin therapy in dyslipidemic patients (13), several studies have demonstrated that even intensive statin treatment cannot normalize vascular dysfunction in DM2 (14, 15). The latter emphasizes possibilities for additional therapeutic modalities in this high risk group.

High-density lipoproteins (HDLs) represent a broad group of mostly spheroidal plasma lipoproteins, which exhibit considerable diversity in their size, apolipoprotein (apo) and lipid composition. HDL particles fall into the density range of 1.063-1.21 g/ml (16) and as they are smaller than other lipoproteins, HDLs can penetrate between endothelial cells more readily allowing relatively high concentrations to accumulate in tissue fluids (17). The major apolipoprotein of almost all plasma HDLs is apo A-I, which in association with phospholipids and cholesterol, encloses a core of cholesteryl esters (16). Nascent (i.e. newly synthesised) HDLs secreted by the liver and intestine contain no cholesteryl esters and are discoidal in shape (16). The negative association of plasma HDL concentration with coronary artery disease has been well documented in epidemiological studies (18). Although experiments in animals have demonstrated an anti-atherogenic activity of HDLs (19), it is not yet known whether this protective effect is related to the role of the lipoprotein in reverse cholesterol transport or to a different mechanism. The mechanism/mechanisms via which HDLs provide these cardioprotective actions are not clearly understood, but may include a role for HDLs in reverse transport of cholesterol from peripheral tissues to the liver, inhibition of the oxidation of low-density lipoproteins, or modulation of vasodilatation and platelet activation mediated by changes in the production of prostacyclin (20). HDLs can also activate endothelial nitric oxide (NO) synthase subsequent to its interaction with scavenger receptor-B1 (SR-B1).

In view of the emerging data on the NO promoting effects of HDL, compounds with HDL-increasing capacity are of particular interest (21-24). Indeed, in DM2 patients HDL is positively associated with endothelium-dependent vasomotor responses (8). In work leading to the present invention, the inventors have evaluated whether and to what extent HDL increase upon infusion of exogenous reconstituted HDL (rHDL) would translate into an improvement of vascular function. ApoA-I levels and endothelial function were assessed both acutely (4 hours after infusion) as well as 7 days after infusion of rHDL in DM2 and matched controls.

Bibliographic details of the publications referred to in this specification are referenced at the end of the description. The reference to any prior art document in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that the document forms part of the common general knowledge.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

In one aspect, the present invention provides a method for the treatment of endothelial dysfunction in a diabetic patient, which comprises administration (preferably parenteral administration) to the patient of an effective amount of high density lipoprotein (HDL).

In another aspect, the present invention provides the use of high density lipoprotein (HDL) in the manufacture of a medicament for administration (preferably parenteral administration) to a diabetic patient for the treatment of endothelial dysfunction in the patient.

In yet another aspect, the present invention provides an agent for administration (preferably parenteral administration) in the treatment of endothelial dysfunction in a diabetic patient, which comprises high density lipoprotein (HDL).


DETAILED DESCRIPTION OF THE INVENTION

Patients with diabetes, particularly type 2 diabetes mellitus (DM2), are characterized by a marked increase in cardiovascular risk. Systemic endothelial dysfunction, a hallmark in DM2, predicts future risk for cardiovascular events. In view of the relation between HDL and the NO pathway, the present inventors have evaluated the effect of rHDL infusion on endothelial function in DM2. Specifically, in 7 DM2 patients and 7 normolipidemic controls, endothelial function was assessed using venous occlusion plethysmography. Forearm blood flow (FBF) responses to intra-arterial infusion of the endothelium-dependent and independent vasodilators serotonin (5HT) and sodium nitroprusside, respectively, and the inhibitor of nitric oxide synthase NG-monomethyl-l-arginine (L-NMMA) were measured, both before, 4 hours after and 1 week after infusion of rHDL (80 mg/kg based on protein).

At baseline HDL was similar in DM2 versus controls (1.1.+-.0.2 vs. 1.2.+-.0.3 mmol/L, ns). 5HT-induced vasodilation (max 17.+-.10%) and L-NMMA induced vasoconstriction (max -17.+-.15%) were reduced in DM2 versus controls (5-HT 114.+-.22 and L-NMMA -48.+-.5%, both p<0.05). rHDL infusion raised apoA-I levels (1.2.+-.0.2 to 2.8.+-.0.4 vs. 1.2.+-.0.2 to 2.7.+-.0.4 g/L, p<0.01) in DM2 and controls, respectively and restored FBF responses to 5HT (86.+-.22%, p<0.05) and L-NMMA (-45.+-.9%, p<0.01) in DM2. This effect persisted 7 days after infusion (5HT; 80.+-.25%, p<0.05 and L-NMMA -37.+-.7%, p<0.01 compared to baseline). rHDL infusion had no effect in controls. Accordingly, this work demonstrates that acute HDL increase improves endothelial function in DM2 and that the improvement persists for at least 7 days in spite of return-to-baseline of HDL concentration.

In one aspect, the present invention provides a method for the treatment of endothelial dysfunction in a diabetic patient, which comprises administration to the patient of an effective amount of high density lipoprotein (HDL).

Preferably, the administration is parenteral administration.

Reference herein to "treatment" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Accordingly, treatment includes amelioration of the symptoms of a particular condition or disorder as well as reducing the severity of, or eliminating a particular condition or disorder.

As used herein, references to "treatment of endothelial dysfunction" are to be considered as references to improvement of endothelial function in treatment of disorders which are related to endothelial dysfunction. Such disorders include both macrovascular disorders (relating to the large blood vessels) such as transient ischaemic attack, stroke, angina, myocardial infarction, cardiac failure, and peripheral vascular disease, as well as microvascular disorders (relating to the small blood vessels) such as diabetic retinopathy (non-proliferative, proliferative, macular oedema), microalbuminuria, macroalbuminuria, end stage renal disease, erectile dysfunction, autonomic neuropathy, peripheral neuropathy, osteomyelitis and lower limb ischaemia.

References herein to a "diabetic" patient are to be understood as a reference to a patient suffering from either type 1 diabetes (DM1) or type 2 diabetes (DM2).

In accordance with the present invention, HDL is administered to a diabetic patient. The term "HDL" as used herein relates to all forms of high density lipoproteins and includes mature HDL, nascent HDL or reconstituted HDL (rHDL) or any mixture thereof, as well as rHDL produced from recombinant apolipoprotein or an analogue thereof with functional relationship to nascent or reconstituted HDL. Such analogues include functional peptides derived from the apolipoprotein (Apo) structure such as those described in International Patent Publications Nos. WO 99/16459 and WO 99/16408, the contents of which are incorporated herein by reference.

The high density lipoproteins comprise a protein component, and lipid. The proteins are preferably apolipoproteins, e.g. human apolipoproteins such as apolipoprotein A-I (apoA-I), apolipoprotein A-II (apoA-II) or apolipoprotein A-IV (apoA-IV) or recombinant apolipoproteins, or functionally homologous peptides with similar properties. Suitable lipids are phospholipids, preferably phosphatidyl choline, optionally mixed with other lipids (cholesterol, cholesterol esters, triglycerides, sphingolipids, or other lipids). The lipids may be synthetic lipids, naturally occurring lipids or combinations thereof.

Preferably, the HDL is reconstituted HDL.

Production of reconstituted HDL is described, by way of example, in U.S. Pat. No. 5,652,339 and by Matz and Jonas (25) and Lerch et al. (26). Production of rHDL with recombinant apolipoproteins is described, by way of example, in European Patent No. EP 469017 (in yeast), U.S. Pat. No. 6,559,284 (in E. coli), and International Patent Publications Nos. WO 87/02062 (in E. coli, yeast and Cho cells) and WO 88/03166 (in E. coli). The contents of each of these documents are incorporated herein by reference.

The HDL is administered in an effective amount. An "effective amount" means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of the particular condition or disorder being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the racial background of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Preferred HDL dosage ranges are from 0.1-200 mg, more preferably 10-80 mg, HDL (weight based on apolipoprotein) per kg body weight per treatment. For example, the dosage of HDL which is administered may be about 0.2-100 mg HDL per kg body weight (weight based on apolipoprotein) given as an intravenous injection and/or as an infusion for a clinically necessary period of time, e.g. for a period ranging from a few minutes to several hours, e.g. up to 24 hours. If necessary, the HDL administration may be repeated one or several times. The actual amount administered will be determined both by the nature of the condition or disorder which is being treated and by the rate at which the HDL is being administered.

Preferably, the patient is a human, however the present invention extends to treatment and/or prophylaxis of other mammalian patients including primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals.

In accordance with the present invention, the HDL is preferably administered to a patient by a parenteral route of administration. Parenteral administration includes any route of administration that is not through the alimentary canal (that is, not enteral), including administration by injection, infusion and the like. Administration by injection includes, by way of example, into a vein (intravenous), an artery (intraarterial), a muscle (intramuscular) and under the skin (subcutaneous). The HDL may also be administered in a depot or slow release formulation, for example, subcutaneously, intradermally or intramuscularly, in a dosage which is sufficient to obtain the desired pharmacological effect.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in a polyethylene glycol and lactic acid. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, suitable carbohydrates (e.g. sucrose, maltose, trehalose, glucose) and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The formulation of such therapeutic compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Other delivery systems can include sustained release delivery systems. Preferred sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules. Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the active component is contained within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer.

The present invention also provides the use of high density lipoprotein (HDL) in the manufacture of a medicament for administration, preferably parenteral administration, to a diabetic patient for the treatment of endothelial dysfunction in the patient.

In yet another aspect, the invention provides an agent for administration, preferably parenteral administration, in the treatment of endothelial dysfunction in a diabetic patient, which comprises high density lipoprotein (HDL).

Claim 1 of 11 Claims

1. A method for the treatment of endothelial dysfunction in a Type 2 diabetic patient, which comprises administering to a Type 2 diabetic patient suffering from endothelial dysfunction associated with a diabetes induced microvascular disorder selected from the group consisting of diabetic retinopathy (non-proliferative, proliferative, macular oedema), microalbuminuria, macroalbuminuria, end stage renal disease, erectile dysfunction, autonomic neuropathy, peripheral neuropathy, osteomyelitis and lower limb ischaemia, an amount of high density lipoprotein (HDL) effective to treat said endothelial dysfunction and improve endothelial function.
 

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