Diabetes mellitus (DM) is a major cause of morbidity and mortality. Chronically elevated blood glucose leads to debilitating complications: nephropathy, often necessitating dialysis or renal transplant; peripheral neuropathy; retinopathy leading to blindness; ulceration of the legs and feet, leading to amputation; fatty liver disease, sometimes progressing to cirrhosis; vulnerability to coronary artery disease and myocardial infarction, gastroparesis, diseases associated with the autonomic nervous-system, nerve condition abnormalities, i.v. contrast induced nephropathy, small vessel diseases (both within the brain and outside the brain), hypogonadism, and heart failure.
DM is a group of disorders characterized by high levels of blood glucose. Prevalence of DM is reaching epidemic proportions in the United States and the world. In 2005, approximately 21 million people in the U.S. had DM of which 90%-95% had type-2 DM (DM-2). Every hour, in the United States, approximately 4100 new cases of DM are diagnosed, and 810 people die from complications of DM. In 2002, DM was the sixth leading cause of death in the U.S. and cost $132 billion. In 2005, DM was responsible for 11.2 million deaths world wide. Contrary to the conventional wisdom, DM affects all socio-economic strata in the world. Cardiovascular complications are the most common causes of morbidity and mortality in DM-2, accounting for up to 70% of the mortality. Interestingly pre-diabetes, where people have high blood glucose but not sufficient to be classified as DM-2, affects 54 million in the U.S. with age greater than 20 years. These people are at increased risk of DM-2 and cardiovascular disease. Despite significant decline in the coronary heart disease mortality, the effects of such a decline are less significant in diabetics as compared to non-diabetics.
There are two primary types of diabetes. Type I, or insulin-dependent diabetes mellitus (IDDM), is due to autoimmune destruction of insulin-producing beta cells in the pancreatic islets. The onset of this disease is usually in childhood or adolescence. Treatment consists primarily of multiple daily injections of insulin, combined with frequent testing of blood glucose levels to guide adjustment of insulin doses, because excess insulin can cause hypoglycemia and consequent impairment of brain and other functions. Type II diabetes (DM2), or noninsulin-dependent diabetes mellitus (NIDDM), typically develops in adulthood. NIDDM is associated with resistance of glucose-utilizing tissues like adipose tissue, muscle, and liver, to the actions of insulin. Initially, the pancreatic islet beta cells compensate by secreting excess insulin. Eventual islet failure results in decompensation and chronic hyperglycemia. Conversely, moderate islet insufficiency can precede or coincide with peripheral insulin resistance.
Insulin resistance can also occur without marked hyperglycemia, and is generally associated with atherosclerosis, obesity, hyperlipidemia, and essential hypertension. This cluster of abnormalities constitutes the “metabolic syndrome” or “insulin resistance syndrome”. Insulin resistance is also associated with fatty liver, which can progress to chronic inflammation (NASH; “nonalcoholic steatohepatitis”), fibrosis, and cirrhosis. Cumulatively, insulin resistance syndromes, including but not limited to diabetes, underlay many of the major causes of morbidity and death of people over age 40.
DM-2, which accounts for 90%-95% of all DM, is characterized by insulin resistance and relative insulin deficiency. In the early stages, this may manifest as glucose intolerance with relatively non-specific symptoms and may not be diagnosed. However, these patients are at increased risk for continuing progression of the disease with associated clinical complications involving multiple organs. Attempts to delay the onset and progression of DM-2 have met with mixed success. Published in 2002, the Diabetes Prevention Study (DPP) demonstrated that lifestyle modification consisting of moderate exercise regimen and dietary modification can be effective in preventing/delaying the rate of onset of DM-2. However significant barriers like behavioral modification make the routine implementation of this strategy difficult. Pharmaceutical agents such as metformin have also demonstrated the effectiveness of preventing/delaying the onset of DM-2. Despite advances in the medical and lifestyle therapies, the incidence and prevalence of the DM-2 continues to increase. Even more interesting is the fact that cardiovascular disease in DM-2 is more aggressive with earlier onset. DM-2 demonstrates characteristic lipoprotein changes including lower high density lipoprotein (HDL) and higher triglycerides (TG) concentrations. Low density lipoproteins (LDL) in DM-2 may not be markedly elevated as compared to control cohort. However, small dense LDL is present in greater concentration. This characteristic diabetic dyslipidemia is associated with markedly increased cardiovascular disease mortality (MRFIT) as compared to non-diabetics. Statins are a class of drugs that predominantly lower LDL. These medications are effective in reducing cardiovascular disease risks in both DM and non-DM, however the residual CVD risk in DM despite LDL lowering remains higher than non-diabetics taking placebo. Elevated HDL may provide an additional mechanism of cardiovascular disease risk reduction in both diabetics and non-diabetics. Multiple trials are ongoing to evaluate the efficacy of increasing HDL in decreasing CVD risk in both diabetic and non-diabetic population.
Despite the existence of drugs to treat such disorders, diabetes and other insulin-resistant disorders remain a major and growing public health problem. Late stage complications of diabetes consume a large proportion of national health care resources. There is a need for new active therapeutic agents which effectively address the primary defects of insulin resistance and islet failure with fewer or milder side effects than existing drugs. What is needed in the art are compositions and methods for treating insulin resistance.
Apolipoprotein E is a protein that binds lipid and has two major domains (Mahley, R. W., et al. J. Lipid Res. 1999, 40:622-630). The 22 kDa amino terminal domain has been shown by X-ray crystallographic studies to be a 4-helix bundle (Wilson, C., et al. Science 1991; 252: 1817-1822) and to contain a positively-charged receptor binding domain. For this region to mediate very low-density lipoprotein (VLDL) binding to its receptors, the apolipoprotein must associate with the lipoprotein surface; this is enabled by the C-terminal amphipathic helical region. If the 4-helix bundle that contains the positively charged receptor-binding domain does not open up on the lipoprotein surface, then the VLDL is defective in binding to receptors. Thus, the positively charged arginine (Arg)-rich cluster domain of the Apo E and the C-terminal amphipathic helical domain, are both required for the enhanced uptake of atherogenic Apo E-containing lipoproteins.
Apo E is secreted as a 299 amino acid residue protein with a molecular weight of 34,200. Based on thrombin cleavage of apo E into two fragments, a two-domain hypothesis was initially suggested to explain the fact that the C-terminal region of apo E (192-299) is essential for its binding to hypertriglyceridemic VLDL, and the N-terminal 22 kDa domain (1-191) binds to the LDL-R (Bradley, W. A., et al., (1986) J. Lipid Res. 27, 40-48). Additional physical-chemical characterization of the protein and its mutants have extended this concept and have shown that the region 192-211 binds to phospholipid while the amino terminal domain (1-191) is a globular structure that contains the LDL receptor binding domain in the 4-helix bundle (Wilson, C., et al., (1991) Science 252, 1817-1822). Studies with synthetic peptides (Sparrow et al.) and monoclonal antibodies pinpointed the LDL receptor binding domain of apo E between residues 129-169, a domain enriched in positively charged amino acids, Arg and Lys (Rall, S. C., Jr., et al., (1982) PNAS USA 79, 4696-4700; Lalazar, A., et al., (1988) J. Biol. Chem. 263, 3542-2545; Dyer, C. A., et al., (1991) J. Biol. Chem. 296, 22803-22806; and Dyer, C. A., et al., (1991) J. Biol. Chem. 266, 15009-15015).
Further studies with synthetic peptides were used to characterize the structural features of the binding domain of apo E that mediates its interaction with the LDL receptor (Dyer, C. A., et al., (1991) J. Biol. Chem. 296, 22803-22806; Dyer, C. A., et al., (1991) J. Biol. Chem. 266, 15009-15015; and Dyer, C. A., et al., (1995) J. Lipid Res. 36, 80-8). Residues 141-155 of apo E, although containing the positively charged residues, did not compete for binding of LDL in a human skin fibroblast assay, but did so only as tandem covalent repeats [i.e., (141-155)2]. N-acetylation of the (141-155)2 peptide, on the other hand, enhanced LDL binding to fibroblasts (Nicoulin, I. R., et al., (1998) J. Clin Invest. 101, 223-234). The N-acetylated (141-155)2 analog selectively associated with cholesterol-rich lipoproteins and mediated their acute clearance in vivo (Nicoulin, I. R., et al., (1998) J. Clin Invest. 101, 223-234). Furthermore, these studies indicated that the prerequisite for receptor binding is that the peptides be helical (Dyer, C. A., et al., (1995) J. Lipid Res. 36, 80-88). Enhanced LDL uptake and degradation were also observed (Mims, M. P., et al., (1994) J. Biol. Chem. 269, 20539-20647) using synthetic peptides modified to increase lipid association by N,N-distearyl derivation of glycine at the N-terminus of the native 129-169 sequence of Apo E (Mims, M. P., et al., (1994) J. Biol. Chem. 269, 20539-20647). Although LDL binding is mediated by the cationic sequence 141-155 of human Apo E, Braddock et al. (Braddock. D. T., et al., (1996) Biochemistry 35, 13975-13984) have shown that model peptides of the highly conserved anionic domain (41-60 of human Apo E) also modulate the binding and internalization of LDL to cell surface receptors. However, these peptides do not enhance LDL degradation.
Chylomicron is a lipoprotein found in blood plasma, which carries lipids from the intestines into other body tissues and is made up of a drop of triacylglycerols surrounded by a protein-phospholipid coating. Chylomicron remnants are taken up by the liver (Havel, R. J., 1985, Arteriosclerosis. 5:569-580) after sequestration in the space of Disse, which is enriched with Apo E (Kwiterovich, P. O., Jr., 1998; Deedwania, P. C., 1995; and Watts, G. W., et al., 1998). Apo E is the major mediator of hepatic remnant lipoprotein uptake by the LDL receptor or LRP. Lipolysis of normal VLDL Sf (subfraction) of more than 60 permit binding of the lipolytic remnant to the LDL receptor (Catapano, A. L. et al. 1979, J. Biol. Chem. 254:1007-1009; Schonfield, G., et al. 1979. J. Clin. Invest. 64:1288-1297). Lipoprotein lipase (LpL) may facilitate uptake through localization of Apo B-containing lipoproteins to membrane heparan sulphate proteoglycan (HSPG) (Eisenberg, et al. 1992. J. Clin. Invest. 90:2013-2021; Hussain, M., et al., J. Biol. Chem. 2000, 275:29324-29330) and/or through binding to the LDL-receptor-related protein (LRP) (Beisiegel, U., et al., 1989, Nature 341:162-164). Cell-surface HSPG may also function as a receptor and has variable binding affinities for specific isoforms of Apo E. In particular, Apo E is synthesized by the liver and also by monocyte/macrophages, where it exerts its effect on cholesterol homeostasis. In vivo evidence for the local effect of lack of Apo E comes from the observations of Linton and Fazio, who showed accelerated atherosclerosis in C57BL/6 mice transplanted with bone marrow from Apo E-deficient mice (Linton, M. F. and Fazio, S. Curr. Openi. Lipidol. 1999, 10:97-105). Apo E-dependent LDL cholesteryl ester uptake pathway has been demonstrated in murine adrenocortical cells (Swarnakar, S., et al. J. Biol. Chem. 2001, 276:21121-21126). This appears to involve chondroitin sulphate proteoglycan (CSPG) and a 2-macroglobulin receptor.
U.S. Pat. No. 6,506,880 denotes the first effort to synthesize apolipoprotein E-mimicking peptides based on the hypothesis that since lipid binding is essential for surface localization of the peptide on lipoproteins and for the receptor binding domain of apo E to be appropriately accessible to bind to the LDL receptor, joining a well-characterized, lipid-associating peptide such as the model class A amphipathic helix, 18A, to the 141-150 peptide sequence of apo E should be sufficient to confer biological activity.
The present invention provides novel synthetic ApoE-mimicking peptides wherein the receptor binding domain of ApoE is covalently linked to 18A, the well characterized lipid-associating model class A amphipathic helical peptide as well as possible applications of the synthetic peptides in lowering human plasma glucose levels.