CN101254299B - Treatment of endothelial dysfunction in diabetic patients - Google Patents

Abstract

The present invention relates to a method for treating endothelial dysfunction in a diabetic patient, including diabetes-induced macrovascular disorders and diabetes-induced microvascular disorders, comprising administering to the patient an effective amount of High Density Lipoprotein (HDL), preferably parenterally.

Description

Treatment of endothelial dysfunction in diabetic patients

technical field

The invention relates to a treatment method for diabetic endothelial dysfunction. In particular, the present invention relates to a method for improving endothelial function in the treatment of conditions involving endothelial dysfunction (macrovessels and microvessels) in diabetic patients. the

Background technique

In diabetes, the chronic disease, the body loses the ability to properly produce or respond to the hormone insulin, so peripheral tissue cells are unable to actively take up glucose from the blood for use or storage. In diabetic individuals, glucose levels in the peripheral blood can be elevated (hyperglycemia) and generally remain elevated unless some form of intervention (eg, administration of exogenous insulin) is employed to return glucose to the blood to normal levels. If left undetected, hyperglycemia in diabetic individuals can lead to shock, organ degeneration or failure (eg, renal failure, blindness, neuropathy, cardiovascular disease), tissue necrosis (eg, requiring amputation), and even death. the

The two main forms of diabetes are type I diabetes and type II diabetes. Type 1 diabetes, formerly known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, is an autoimmune disease in which the body damages the insulin-producing beta cells (islet cells) of the pancreas, resulting in the absolute need for daily administration of Exogenous insulin to maintain normal blood sugar levels. Type 1 diabetes is often diagnosed in children and young adults, but can appear at any age. Type I diabetes accounts for 5-10% of diagnosed diabetes cases. the

By far the more prevalent form of diabetes is type 2 diabetes, formerly known as non-insulin-dependent diabetes mellitus (NIDDM). Type 2 diabetes was also formerly known as adult-onset diabetes, however, this form of diabetes is becoming more prevalent in the growing population of overweight and clinically obese children and young adults. Type II diabetes accounts for 90-95% of all diagnosed diabetes cases. Type 2 diabetes typically begins with insulin resistance, the failure of the body's cells to respond appropriately to insulin, followed by a gradual loss of the ability of the pancreas to produce and secrete insulin. Type II diabetes is associated with various factors including increasing age, obesity, family history of diabetes, history of gestational diabetes, impairment of glucose metabolism, physical inactivity, and multiple races and ethnicities. Individuals with type 2 diabetes must attempt to control their blood sugar levels through careful diet, exercise and weight loss, and complementary medicines. the

Major factors contributing to the preatherogenic state of diabetes, especially type II diabetes (DM2), include dyslipidemia, hyperglycemia, hypertension, visceral obesity, and insulin resistance (1, 2). Observational studies have clearly demonstrated the importance of diabetic dyslipidemia in the development of diabetic atherosclerosis, demonstrated by the fact that low-density lipoprotein (LDL) and high-density lipoprotein (HDL) are associated with cardiovascular events The correlation between the two exceeds fasting plasma glucose (3). Statin intervention studies have revealed a clear benefit of statin therapy in reducing cardiovascular events in DM2 (4, 5); however, despite this impressive progress, the majority of DM2 patients remain suffer a cardiovascular event (6). the

In the last two decades, endothelial dysfunction has been exposed as one of the early stages of atherogenesis. Endothelial dysfunction, which is characteristic of all diabetic patients (ie type I and type II), has been shown to have predictive value for future cardiovascular events (7-9). Consistent with the multifactorial pathogenesis of diabetes-induced vascular disease (10, 11), a number of therapeutic interventions have been evaluated for their potential to improve endothelial function in DM2 patients (9, 12). Surprisingly, although complete restoration of endothelial function may be possible by statin therapy in patients with dyslipidemia (13), several studies have demonstrated that even intense statin intervention does not restore vascular function in DM2 Bad returns to normal (14, 15). The latter emphasizes the possibility of additional forms of treatment in this high-risk group. the

High-density lipoproteins (HDL) represent a large group of primarily spherical-like plasma lipoproteins that exhibit considerable variability in size, apolipoprotein (apo) and lipid composition. HDL particles have a density range of 1.063-1.21 g/ml (16), and because they are smaller than other lipoproteins, HDL is able to permeate between endothelial cells, thereby accumulating more easily in tissue fluids in relatively high concentrations (17) . The major apolipoprotein of almost all plasma HDL is apo A-I, which is associated with phospholipids and cholesterol surrounding a cholesteryl ester core (16). Nascent (ie, newly synthesized) HDL secreted by the liver and intestine does not contain cholesteryl esters and is discoid in shape (16). The inverse association of plasma HDL concentration with coronary artery disease has been well documented in epidemiological studies (18). Although animal experiments have demonstrated the antiatherogenic activity of HDL (19), it is not known whether this protective effect is related to the reverse cholesterol transport function of lipoproteins or is related to a different mechanism. The mechanism/multiple mechanisms by which HDL confers these cardioprotective effects is unclear, but may include the role of HDL in reverse transport of cholesterol from peripheral tissues to the liver, inhibition of LDL oxidation, or through changes in cycloprostaglandin production and mediate regulation of vasodilation and platelet activation (20). HDL is also able to activate endothelial nitric oxide (NO) synthase following interaction with scavenger receptor-B1 (SR-B1). the

Compounds with HDL increasing capacity are of particular interest in view of the emerging data on the NO priming effect of HDL (21-24). Indeed, in DM2 patients, HDL was positively associated with endothelium-dependent vasomotor response (8). In work leading up to the present invention, the inventors have assessed whether and to what extent increases in HDL after infusion of exogenous recombinant HDL (rHDL) translate into improvements in vascular function. ApoA-I levels and endothelial function were examined in the acute phase (4 hours post infusion) and 7 days post infusion of rHDL in DM2 and matched controls. the

Bibliographic details of publications mentioned in this specification are listed at the end of the specification. Citation of any prior art document in the specification is not and should not be considered as an acknowledgment or implication that the document forms part of the common general knowledge. the

Contents of the invention

In this specification and the appended claims, unless the context requires otherwise, the word "comprise" and/or variations such as "comprises" or "comprising" will be understood to mean It is meant to include the stated integer or step or group of integers or steps but not to exclude any other integer or step or group of integers or steps. the

In one aspect, the present invention provides a method for treating endothelial dysfunction in diabetic patients, comprising administering (preferably parenterally) an effective amount of high density lipoprotein (HDL) to the patient. the

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

In a further aspect, the present invention provides a formulation for administration, preferably parenteral administration, in the treatment of endothelial dysfunction in diabetic patients comprising high density lipoprotein (HDL). the

Description of drawings

Figure 1 shows that intra-arterial infusion of the endothelium-dependent vasodilator serotonin increases forearm blood flow (FBF) in healthy volunteers and DM2 patients in a dose-dependent manner. At baseline, the FBF response to serotonin was attenuated in DM2 compared to controls (p<0.001). After rHDL infusion, the FBF response to serotonin increased significantly, but not to a level comparable to that of the control group (p<0.01). The improvement in endothelium-dependent vasodilation persisted for 7 days after rHDL infusion (p<0.01). rHDL infusion did not affect the serotonin response in the control group. the

Figure 2 shows that at baseline the vasoconstrictor response to L-NMMA (reflecting basal nitric oxide (NO) activity) was blunted in DM2 patients compared to controls (p<0.001). Responses to L-NMMA inotropes improved after rHDL infusion and were still present 7 days post-infusion (p<0.01). Corresponding to the serotonin data, rHDL infusion did not affect L-NMMA responses in control subjects. the

Figure 3 shows that endothelium-independent vasodilation in response to sodium nitroprusside was lower in DM2 compared to controls (p<0.01), and rHDL infusion showed no effect on SNP in both patients and controls Response to vasodilators affects. the

Detailed ways

Patients with diabetes, especially type 2 diabetes (DM2), are characterized by a markedly increased cardiovascular risk. Systemic endothelial dysfunction, a hallmark of DM2, indicates the risk of future cardiovascular events. Considering the relationship between HDL and the NO pathway, the present inventors have evaluated the effect of rHDL infusion on the endothelial function of DM2. Specifically, endothelial function was assessed using venous occlusion plethysmography in seven DM2 patients and seven normallipidemic controls. Forearm blood flow (FBF) responds to intraarterial infusions of the endothelium-dependent and endothelium-independent vasodilators 5-hydroxytryptamine (5HT) and sodium nitroprusside, respectively, and infusions of rHDL (80 mg/kg protein) An inhibitor of nitric oxide synthase NG-monomethyl-1-arginine (L-NMMA) was assayed before, 4 hours after and 1 week after. the

At baseline, HDL was the same in DM2 and controls (1.1±0.2 vs. 1.2±0.3 mmol/L, ns). 5-HT-induced vasodilation (max 17±10%) and L-NMMA-induced vasoconstriction (max-17±15%) were decreased in DM2 compared to controls (5-HT 114±22 and L-NMMA-48±5%, both p<0.05). rHDL infusion increased apoA-I levels in DM2 and controls, respectively (1.2±0.2 to 2.8±0.4 vs. 1.2±0.2 to 2.7±0.4 g/L, p<0.01), and restored the effect of FBF on 5HT in DM2 (86±22%, p<0.05) and L-NMMA (-45±9%, p<0.01) responses. This effect persisted for 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 the control group. Thus, this work demonstrates that a dramatic increase in HDL improves endothelial function in DM2 and that this improvement persists for at least 7 days, despite the return of HDL concentrations to baseline. the

In one aspect, the present invention provides a method for treating endothelial dysfunction in diabetic patients, comprising administering to the patient an effective amount of high-density lipoprotein (HDL). the

Preferably, the mode of administration is parenteral. the

"Treatment", as used herein, is considered to have its broadest meaning. The term "treatment" does not necessarily mean that the subject receives treatment until full recovery. Thus, treatment includes amelioration of the symptoms of a particular condition or disorder as well as lessening of the severity of a particular condition or disorder, or elimination of a particular condition or disorder. the

As used herein, references to "treatment of endothelial dysfunction" shall be taken to mean the improvement of endothelial function in the treatment of disorders related to endothelial dysfunction. Such conditions include macrovascular disorders (pertaining to large vessels) such as transient ischemic attack, stroke, angina, myocardial infarction, heart failure, and peripheral vascular disease, and microvascular disorders (pertaining to small vessels) Vascular) such as diabetic retinopathy (non-proliferative, proliferative, macular edema), microalbuminuria, macroalbuminuria, end-stage renal disease, erectile dysfunction, autonomic neuropathy, peripheral neuropathy, Osteomyelitis and lower extremity ischemia. the

"Diabetes" patients mentioned herein should be understood as referring to type I diabetes (DM1) or type II diabetes (DM2) patients. the

According to the present invention, HDL is administered to diabetic patients. The term "HDL", as used herein, refers to all forms of high-density lipoproteins and includes mature HDL, nascent HDL or recombinant HDL (rHDL) or any mixture thereof, as well as recombinant apolipoproteins or recombinant apolipoproteins. rHDL made from peptides or other analogues. Such analogs include functional peptides derived from the structure of apolipoprotein (Apo), such as those described in International Patent Applications WO99/16459 and WO99/16408, the contents of which are hereby incorporated by reference. the

HDL includes protein components and lipids. The protein is preferably a human apolipoprotein such as an apolipoprotein such as apolipoprotein A-I (apoA-I), apolipoprotein A-II (apoA-II) or apolipoprotein A-IV (apoA-IV) or a recombinant apolipoprotein , or a functional homologous peptide with similar properties. Suitable lipids are phospholipids, preferably phosphatidylcholines, optionally mixed with other lipids (cholesterol, cholesteryl esters, triglycerides, sphingolipids or other lipids). Lipids can be synthetic lipids, naturally occurring lipids, or combinations thereof. the

Preferably, HDL is recombinant HDL. the

The generation of recombinant HDL is described eg in US 5652339 and by Matz and Jonas (25) and Lerch et al. (26). Generation of rHDL with recombinant apolipoproteins is eg described in EP469017 (in yeast), US 6559284 (in E.coli) and International Patent Publication WO87/02062 (in E.coli, yeast and Cho cells) and WO88/ 03166 (in E. coli) is described. The contents of each of these documents are hereby incorporated by reference. the

HDL is administered in an effective amount. "Effective amount" means that amount necessary to achieve at least in part the desired response, or to delay the onset or inhibit the development or abort altogether of the particular condition or disorder being treated. This amount will vary depending on the health and physical condition of the individual to be treated, the ethnic background of the individual to be treated, the degree of protection desired, the formulation of the composition, the evaluation of the medical condition, and other relevant factors. This amount is expected to be within a fairly broad range that can be determined by routine experimentation. the

Preferably the dosage range of HDL is 0.1-200 mg, more preferably 10-80 mg HDL (based on the weight of apolipoprotein)/kg body weight/per treatment. For example, the dose of HDL administered may be about 0.2-100 mg HDL/kg body weight (based on apolipoprotein weight) for a clinically necessary period of time (e.g., minutes to hours, such as up to 24 hours) for interventional injection and/or administered by infusion. HDL administration can be repeated once or several times if necessary. The actual amount administered will be determined by the nature of the condition or disorder being treated and the rate of administration of HDL. the

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

According to the present invention, HDL is preferably administered to a patient by the parenteral route of administration. Parenteral administration includes any route of administration that does not pass through the digestive tract (ie, parenteral), such as including injection, infusion, and the like. Administration by injection includes, for example, intravenous (intravenous), intraarterial (intraarterial), intramuscular (intramuscular) and under the skin (subcutaneous) injections. HDL may also be administered by subcutaneous, intradermal or intramuscular injection in a depot or sustained release formulation in doses sufficient to achieve the desired pharmacological effect. the

Compositions suitable for convenient parenteral administration include sterile aqueous formulations of the active ingredient which are 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, such as polyethylene glycol and lactic acid solution. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, suitable carbohydrates (eg, 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 diglycerides. In addition, fatty acids such as oleic acid find use in injectables. the

The formulation of these therapeutic compositions is well known to those skilled in the art. 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 is described by way of example in: Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Unless any conventional media or agents are incompatible with the active ingredients, they should be considered usable in the pharmaceutical compositions of the present invention. Supplementary active ingredients can also be incorporated into the compositions. the

Other delivery systems may include sustained release delivery systems. Preferred sustained release delivery systems are those used for the release of the active ingredient of the invention in the form of sustained release pellets or capsules. Many types of sustained release delivery systems are available. These systems include, but are not limited to: (a) corrosion systems where the active ingredient is contained within a matrix, and (b) dispersed systems where the active ingredient permeates the polymer at a controlled rate. the

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

In a further aspect, the present invention provides a medicament for administration, preferably parenterally, in the treatment of endothelial dysfunction in diabetic patients comprising high density lipoprotein (HDL). the

The invention is further illustrated by the following non-limiting examples. the

Example

I. Method

Seven non-smoking uncomplicated DM2 patients (4 males, 3 females) and 7 non-smoking age- and sex-matched normal blood lipid controls (4 males, 3 females) were recruited. The inclusion criteria for DM2 patients are as follows: (1) fasting blood glucose > 7.0mmol/L, (2) under diet and metformin treatment; (3) without exogenous insulin; (4) with blood glucose less than 2.0 and 3.5mmol/L, respectively Mild dyslipidemia in plasma triglyceride and LDL cholesterol levels. The presence of macrovascular disease, defined as ECG abnormalities, abnormal ankle-brachial index or cardiac, cerebral history of peripheral vascular events, and the presence of autonomic neuropathy were exclusion criteria for patients or control subjects. All female patients were postmenopausal and not on hormone replacement therapy. The median duration of diabetes was 5.2±1.2 [mean±SD] years. Evaluate at least 4 weeks after discontinuation of vasoactive drugs (eg, ACE inhibitors, angiotensin receptor blockers, calcium channel blockers, aspirin, NSAIDs, and vitamin supplements). None of the patients were treated with statins. Alcohol, caffeine, and metformin were discontinued within 12 hours of the study. Written informed consent was given by all subjects and approved by the internal review board of the Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands. The research was done in accordance with the principles of the Statement of Helsinki. the

research proposal

Vascular function was assessed at baseline and after rHDL infusion using venous occlusive tensioner plethysmography as previously published (EC-4; Hokanson Inc, Bellevue, USA). Measurements were performed in a quiet room at a constant temperature (22°C-24°C), and started at 8:00 in the morning. Subjects remained in a supine position throughout the study. A 20 gauge flexible polyurethane catheter (Arrow Inc, Reading, USA) was inserted into the brachial artery of the nondominant arm. A 30-minute saline infusion was performed after insertion to re-establish baseline conditions. Thereafter, forearm blood flow (FBF), expressed in ml/min/100 mL forearm tissue volume (FAV), was measured simultaneously on both arms. On-line monitoring is performed using a microcomputer-based R-wave trigger system. During each measurement, a blood pressure cuff around the upper arm was inflated (40 mm Hg) using a quick cuff inflator. Simultaneously, both wrist cuffs were inflated to above systolic blood pressure to exclude hand circulation (200 mm Hg). Continuous monitoring of intra-arterial blood pressure and heart rate. Second, the effect of FBF on cumulative doses of the endothelium-dependent vasodilator serotonin (5HT, Sigma; 0.6, 1.8, and 6 ng·100mL FAV ^-1 ·min ^-1 ), the endothelium-independent vasodilator sodium nitroprusside ( SNP, Spruyt Hillen; 6, 60, 180 and 600ng·100mL FAV ^-1 ·min ^-1 ) and a competitive inhibitor of endothelial NO synthase (eNOS) ^NG -monomethyl-L-arginine (L- NMMA, Kordia; 50, 100, 200 and 400 μg·100 mL FAV ⁻¹ ·min ⁻¹ ). Serotonin and nitroprusside infusions were administered in random order, followed by L-NMMA infusions. All infusions were prepared in AMC's pharmacy following Good Manufacturing Practice (GMP) guidelines. Each dose was administered intraarterially over 6, 4 and 8 minutes using a constant rate infusion pump. The 6 measurements obtained during the last 2 minutes of each infusion were averaged to determine the mean FBF. These 3 different infusions were given after a 15 min rest period or until FBF returned to baseline. Subsequently, an intravenous catheter was inserted into the contralateral arm for administration of rHDL (CSL, Behring, Bern, Switzerland) at a dose of 80 mg/kg body weight over 4 hours (26, 27). Thereafter, the infusion procedure was repeated. Patients were then asked not to restart medications (except metformin) and to return 7 days after rHDL infusion to repeat endothelial function measurements.

Laboratory Evaluation

Blood samples were drawn from subjects immediately after rHDL infusion, 4 hours later and 7 days after a 12 hour overnight fast. After centrifugation within 1 hour of blood collection, aliquots were snap frozen in liquid nitrogen and stored at -80°C until assayed. All assays were performed at the University Hospital of Amsterdam, Academic Medical Centre, Vascular and Clinical Laboratory. ALAT and ASAT were determined using the pyridoxal phosphate activation assay (Rose Diagnostics, Basel, Switzerland). HbA1c of variant II (Bio-Rad Laboratories) was determined by HPLC (Reagens Bio-Rad Laboratories B.V., The Netherlands). Blood glucose was assessed twice using the hexokinase method (Gluco-quant, in Hitachi 917; Hitachi). Plasma triglycerides, total cholesterol, LDL and HDL levels were determined using standard enzymatic methods (Rose Diagnostics, Basel, Switzerland). Plasma levels of apoA-I and apoB in stored plasma were assessed by rate nephelometric method. the

Statistical Analysis

All clinical parameter results, including volume scan data, are expressed as mean ± SD. Descriptive statistics were compared between two groups by means of a 2-tailed independent Student's t test. Six consecutive recordings of FBF were averaged over the last 2 min of each infusion step. FBF recordings made during the first 30 s after cuff inflation were not used for analysis. Statistical analysis of FBF measurements, HDL mass, and inflammatory markers for each subject between the two groups was performed using 2-way ANOVA for replicate measurements. A probability value of P<0.05 was considered significant, and P<0.01 was considered highly significant. the

II. Results

Object characteristics are listed in Table 1. Baseline FBF was not significantly different between patients and control subjects (Table 1). As expected, plasma levels of fasting glucose (P<0.01), hemoglobin A _1c (P<0.01), triglycerides (P<0.01) and apoB (P<0.01) were observed in DM2 patients. 0.05) is higher. HDL-C and apoA-I levels were comparable between DM2 patients and controls. After rHDL infusion, plasma apoA-I increased in DM2 and control group after 4 hours (1.2±0.2 to 2.8±0.4vs.1.2±0.2 to 2.7±0.4g/L, p<0.01), A slight increase remained in DM2 patients 7 days after injection (1.5±0.3 g/L, p<0.05 compared to baseline). Plasma apoB levels were higher in DM2 patients compared with controls and were not affected by rHDL infusion (1.0±0.3 to 0.9±0.3 vs. 0.7±0.2 to 0.7±0.2 g/L, ns ).

Acute and long-term effects of rHDL infusion on NO bioavailability

Intraarterial infusion of the endothelium-dependent vasodilator serotonin increased FBF in a dose-dependent manner in both groups. At baseline, the FBF response to serotonin was attenuated in DM2 compared to controls (p<0.001, Figure 1 ). After rHDL infusion, the response of FBF to serotonin was significantly enhanced, but did not reach the level comparable to that of the control group (p<0.01, Figure 1). Interestingly, the improvement in endothelium-dependent vasodilation was maintained for 7 days after rHDL infusion (p<0.01, Figure 1 ). rHDL infusion did not affect the serotonin response in the control group (Figure 1). the

At baseline, the vasoconstrictor response to L-NMMA (reflecting basal NO activity) was impaired in DM2 patients compared to controls (p<0.001, Figure 2). Following rHDL infusion, the L-NMMA inotropic response was improved and persisted for 7 days after infusion (p<0.01, Figure 2). Consistent with the serotonin data, rHDL infusion did not affect the L-NMMA response in control subjects (Figure 2). the

Finally, endothelium-independent vasodilation in response to SNP was lower in DM2 patients compared with controls (p<0.01, Figure 3), and rHDL infusion showed no effect on SNP in either patients or controls. Vasodilator response is affected. the

III. Discussion

The present study demonstrates that basal and stimulated NO activity is severely compromised in DM2 patients compared with age- and sex-matched controls. Interestingly, rHDL infusion significantly improved endothelial function despite near-normal HDL in DM2 patients. This improvement persisted up to 7 days after rHDL infusion, during which time HDL levels had returned to baseline values. These data suggest that HDL-increasing strategies may provide efficacy in DM2, even though HDL-C levels were not significantly reduced in these patients. the

Vascular function at baseline

Consistent with previous studies (8, 9, 12), impaired basal NO activity and impaired NO release were demonstrated in DM2 following receptor-mediated stimulation. Several mechanisms have been suggested for diabetic endothelial dysfunction. The reduction in the bioavailability of the essential cofactor tetrahydrobiopterin (BH4) is associated with the uncoupling of endothelial NO synthase, as this leads to the direct generation of oxygen radicals from eNOS instead of NO (28- 30). Other sources of increased free radical production include NADPH oxidase and mitochondrial uncoupling (10). Several interventional studies have highlighted the major role of ROS in diabetic vascular dysfunction, reporting complete restoration of endothelial function after intra-arterial infusion of high concentrations of antioxidants (9, 12). the

Effect of rHDL infusion on vascular function

rHDL infusion was associated with a rapid improvement in basal NO activity as well as receptor-stimulated NO activity within hours of infusion. The first explanation is that rHDL increases NO production. Nitric oxide (NO) is synthesized by eNOS by converting L-arginine to L-citrulline. Its activity is regulated by complex signal transduction pathways, including the activation of kinases that alter the phosphorylation of eNOS, i.e., the signaling of MAP kinase and akt-kinase, or the increase in intracellular Ca2 ⁺ levels, followed by eNOS Ca-Ca Heregulin-dependent activation (33). Yuhanna showed that binding of apoA-I to the endothelial scavenger receptor B-1 was accompanied by an enhanced endothelium-dependent release response in the aorta (24), mainly due to the activation of akt and MAP-kinase (32). In addition, HDL also has the ability to positively regulate eNOS membrane content in endothelial cells by preserving the stability of eNOS protein and by preventing the transport of eNOS from the cell membrane to intracellular organelles (23). All of these effects were due to increased basal NO availability after rHDL infusion, assessed as increased vasoconstrictor response to the competitive NO inhibitor L-NMMA. In contrast, the aforementioned mechanisms do not fully explain the serotonin-dependent, receptor-stimulated increase in NO availability, which is dependent on calcium-calmodulin activation of eNOS (31). Since serotonin binding to endothelial 5HT _-2A receptors (33) is unlikely to be altered after rHDL infusion, the reduction in NO degradation by oxygen free radicals provides a second mechanism that would enable increased NO bioavailability. main path. Indeed, the potent antioxidant properties of HDL are in no way due to the presence of enzymes such as diethyl-p-nitrophenyl phosphatase (paraoxonase) and platelet-activating factor hydrolase on HDL particles (23).

Long-term effects of rHDL on vascular function

Strikingly, endothelium-dependent vasodilation remained significantly improved 1 week after rHDL infusion. In contrast, apoAI and HDL levels had almost returned to pre-infusion levels. Strikingly, HDL levels in DM2 were also not significantly different from control subjects at baseline. In contrast, rHDL infusion had no effect whatsoever on vascular function in the control group. These data suggest that, regardless of normal HDL concentrations, HDL mass in DM2 can be impaired. In fact, the loss of HDL protection in DM2 is partly due to non-enzymatic glycation of the major leucine chains in HDL. Glycation of ApoAI-HDL compromises the ability of HDL to protect LDL from oxidative loss through the loss of PON-1 activity therein (34). Furthermore, glycated HDL reduces eNOS expression in the endothelium, resulting in reduced NO production capacity (35). Indeed, levels of HDL antioxidant activity in DM2 patients are closely linked to levels of oxidative stress and glycation control (36). the

Study Limitations

Vascular function studies were not repeated after 7 days in the control group since the effect of rHDL was not apparent 4 hours after infusion in the control group. Accordingly, the vascular function data of DM2 patients on day 7 were compared with the observation data of baseline and control group on day 1. However, since the main conclusions on vascular function relate to sustained improvement in DM2 patients compared to baseline, the absence of a Day 7 study in the control group does not affect the conclusions drawn in this study. Second, although only a relatively small group of DM2 patients has been studied, the fact that significant improvements have been found in a limited group of patients and that this improvement is reproduced after 4 hours and 1 week strongly supports the role of HDL in DM2 patients. There is an obvious conclusion that vasculature has an effect, despite the small sample size. the

Clinical implications for patients with DM2

Inhibins are a central paradigm of cardiovascular protective strategies. However, given the large number of events that were not avoided during statin therapy, the search for optimal combination therapy is an overall advance. The recognition of HDL augmentation strategies is rapidly expanding. The strong inverse relationship between HDL-C and cardiovascular events was consistent in non-diabetic and diabetic patients. Unfortunately, there are insufficient reliable data providing evidence of reduced cardiovascular risk following HDL increasing interventions, mainly due to the lack of selective and potent HDL increasing compounds (37). the

Recent data have shown that a 5-week infusion of rHDL derived from a variant apolipoprotein (apoA-I Milano) slows the progression of, and even induces regression of, coronary atherosclerotic volumes in patients with recent myocardial infarction (38 ). Consistent with this rapid action, both laboratory and in vivo studies have shown that the anti-atherogenic potential of HDL is not limited to its role in anti-cholesterol transport. The observation of acute and sustained recovery of endothelial dysfunction in DM2 further supports the role of HDL over its role in anti-cholesterol transport. This supports a role for HDL enhancement in DM2, even though HDL levels were not significantly reduced. the

Table 1

^* p<0.05, #p<0.01

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Claims (12)

CLAIMS 1. Use of high density lipoprotein HDL in the manufacture of a medicament for administration to a diabetic patient for the treatment of endothelial dysfunction in the patient. 2. The use according to claim 1, wherein the patient is a type II diabetic patient. 3. The use according to claim 1, wherein the treatment is a treatment of diabetes-induced macrovascular disorders selected from the group consisting of transient ischemic attack, stroke, angina, myocardial infarction, heart failure and peripheral vascular disease. 4. purposes as claimed in claim 1, wherein, described treatment is to the treatment of the microvascular disorder that diabetes induces, and described microvascular disorder is selected from the retinopathy that diabetes causes, microalbuminuria, macroalbuminuria, End-stage renal disease, erectile dysfunction, autonomic neuropathy, peripheral neuropathy, osteomyelitis, and lower extremity ischemia. 5. The use according to claim 1, wherein the HDL is selected from: mature HDL, nascent HDL, recombinant HDL, rHDL made from recombinant apolipoprotein or recombinant apolipoprotein functional peptide or other analogues thereof. 6. The use according to claim 5, wherein said HDL is recombinant HDL. 7. The use according to claim 1, wherein the dosage range of said HDL is 0.1-200 mg/kg body weight of the patient per treatment, preferably 10-80 mg/kg body weight per treatment. 8. The use according to claim 1, wherein said administration is parenteral administration. 9. The use according to claim 8, wherein said parenteral administration is selected from the group consisting of intravenous, intraarterial, intramuscular and subcutaneous injection or infusion. 10. The use according to claim 8, wherein said parenteral administration is intravenous injection or infusion. 11. The use according to claim 4, wherein said diabetic retinopathy is non-proliferative, proliferative or macular edema. 12. A formulation for administration in the treatment of endothelial dysfunction in diabetic patients comprising rHDL.

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