Treating vascular complications of diabetes

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology, more specifically to cyclic peptides of TNF, and can be used in medicine. The cyclic peptide consisting of CGQRETPEGAEAKPWYC is used to prepare a therapeutic agent for preventing or treating vascular complications in the patients suffering diabetes, including as a part of a pharmaceutical composition.

EFFECT: invention enables preventing the vascular complications in the patients with diabetes effectively, herewith inducing no TNF-mediated pro-inflammatory reaction.

8 cl, 5 dwg, 1 tbl, 2 ex

 

The present invention relates to methods for prevention and treatment of vascular complications of diabetes.

Diabetes mellitus is a common metabolic disorder, which suffered about 150 million people in 2000, and is projected in 2010, the number of patients could reach 220 million. Diabetes and its associated complications have become a significant public health problem. Cardiovascular disease cause most cases of complications and mortality in diabetes mellitus. Adults with diabetes are 2-4 times more at risk of cardiac disease compared with those who do not suffer from diabetes. Cardiovascular disease accounts for 80% of premature deaths among patients with diabetes. Because of huge premature morbidity and mortality associated with this disease, prevention of its complications is a key issue.

In diabetes, dysfunction of the vascular endothelium is considered an important factor in the pathogenesis of diabetic micro - and macroangiopathy. There are three main sources that contribute to endothelial dysfunction in diabetes: (i) hyperglycemia and its immediate biochemical consequences directly alter endothelial function; (ii) high glucose level, indirectly influencing the functioning of endothelial cells via� the synthesis of growth factors and vasoactive agents in other cells and alters the permeability of the endothelial monolayer; (iii) components of the metabolic syndrome, which can affect the endothelium (Schalkwijk et al., Clin. Sci. 109(2005), 143-159).

The purpose of the present invention is to provide a means of reducing the pathological effects of vascular complications in patients with diabetes.

Accordingly, the present invention provides a peptide consisting of 7-17 amino acids and comprising adjacent hexamer TX1EX2X3E, where X1, X2and X3can be any natural or synthetic amino acid, wherein the peptide has no TNF-specific inflammatory activity (Hribar et al., Eur. J. Immunol. 1999; Elia et al., AJRCCM 2003; see also examples section) and is cyclic for the treatment and prevention of vascular complications in patients with diabetes.

X1, X2and X3preferably selected from the 20 natural standard amino acids of a core set of natural amino acids for protein synthesis; in a more preferred embodiment, X1, X2and X3choose from among natural amino acids with hydrophobic side chain (Ala, Ile, Leu, Met, Val, Pro and Gly). Especially preferred are (independently from each other) X1=Pro; X2and X3=Gly, Ala, Val, Leu or Ile; X2X3preferably is a dipeptide selected from Gly-Ala, Gly-Val, Ala-Gly or Ala-Val.

Preferably, the peptide according to the present Fig�structure consists of 7-17 amino acids and contains hexamer TPEGAE (SEQ ID NO. 4), wherein the peptide is cyclic and has activity of binding to TNF-receptor.

Especially preferred variant of the present invention relates to cyclic peptide consisting of a sequence of successive amino acids selected from the group

- QRETPEGAEAKPWY (SEQ ID NO. 5)

- PKDTPEGAELKPWY (SEQ ID NO. 6)

- CGQRETPEGAEAKPWYC (SEQ ID NO. 1), and

- CGPKDTPEGAELKPWYC (SEQ ID NO. 7)

and fragments of at least seven amino acids, which include hexamer TPEGAE, for the treatment and prevention of vascular complications in patients with diabetes.

The peptides according to the present invention are known, e.g. from European patent EP 1264599 B1 as applicable for the treatment of pulmonary edema.

Suddenly these peptides proved to be particularly favorable for the treatment and prevention of vascular complications in patients with diabetes. Thus, the present invention relates to the use of these peptides for the production of medicaments for the treatment and prevention of vascular complications in patients with diabetes.

The type of diabetes treatable or preventable according to the present invention, may be any common type of diabetes, especially type 1 diabetes, type 2 diabetes, gestational diabetes, congenital diabetes associated with cystic fibrosis, diabetes, steroid diabetes (caused by high doses glucocorti�ides), and may include several forms of monogenic diabetes; however, Type I and Type II are the predominant diseases that are the subject of the present invention, particularly diabetes Type II. The present invention provides a method for the treatment and prevention of vascular complications in patients with diabetes, with an effective amount of the peptide according to the present invention (or a mixture of such peptides) is administered to a patient with diabetes at risk of developing vascular complications, as described by the authors.

Diabetes is characterized by recurrent or persistent hyperglycemia, and is diagnosed through the manifestation of any of the following features:

- The level of glucose in fasting plasma 7.0 mmol/l or above (126 mg/DL according to (who) definition, two measurements of fasting glucose levels, showing more than 126 mg/DL (7.0 mmol/l) are considered as symptomatic for diabetes mellitus).

- The level of plasma glucose 11.1 mmol/l (200 mg/DL) or higher two hours after oral load of 75 g of glucose, as in the test for glucose tolerance.

- Symptoms of hyperglycemia and a random level of plasma glucose 11.1 mmol/l or above (200 mg/DL).

- Glycosylated hemoglobin (hemoglobin A1C) level of 6.5 or above (this criterion was recommended by the American Association of diabetologists in 2010; this paper reviews�UNT he is awaiting approval from who).

Patients with fasting glucose levels from 100 to 125 mg/DL (5.6 to 6.9 mmol/l) are considered to be patients with impaired fasting glucose levels. Patients with the level of plasma glucose 140 mg/DL (7.8 mmol/l) or higher but not exceeding 200 mg/DL (11.1 mmol/l) two hours after an oral load of 75 g of glucose are considered as patients with impaired glucose tolerance. From these two prediabetic States the latter, in particular, is a major risk factor for progression to full-blown diabetes mellitus and cardiovascular disease (as serious vascular complications in patients with diabetes).

Vascular complications in patients with diabetes can be caused by micro - and macroangiopathy. Retinal and renal microangiopathy causes of diabetic retinopathy and nephropathy, respectively, and microangiopathy of the small blood vessels plays an important role in the pathogenesis of neuropathy. Macroangiopathy in diabetes is mainly of an accelerated form of atherosclerosis and affects coronary, carotid and peripheral arteries, thus increasing the risk of myocardial infarction, stroke and diabetic foot syndrome. Large-scale clinical studies of diabetes Type I and Type II demonstrated that hyperglycemia plays an important role in the pathogenesis of microvascular ologne�rd, such as increased permeability of cardiac capillaries. Hypertension, Smoking, hypercholesterolemia, dyslipidemia, obesity and hyperhomocysteinemia are more serious causes microangiopathy.

The risk of macroangiopathy apparently, not much is associated with hyperglycemia, however, is associated with common risk factors of atherothrombosis, such as age, Smoking, hypertension, hypercholesterolemia, dyslipidemia, obesity and hyperhomocysteinemia. All these factors create a state of permanent and progressive damage to blood vessels, manifested in the process low grade inflammation and endothelial dysfunction. As mentioned above (Schalkwijk et al., 2005) dysfunction of the vascular endothelium is considered an important factor in the pathogenesis of micro - and macroangiopathy. Parameters involved in vascular complications during diabetes, are dysfunctional vasorelaxation and hyperpermeability heart vessels.

Thus, the present invention is particularly applicable to the prevention or treatment of micro - and macrovascular disease, myocardial infarction, increased permeability of cardiac capillaries, stroke, neuropathy, retinopathy, nephropathy or diabetic foot syndrome in patients with diabetes.

The present invention provides the ability to treat or prevent suck�East of complications in patients with diabetes. Because diabetes currently is a disease that cannot be completely cured, "treating" in the context of the present invention should be understood as including the provision of attenuation of vascular disease in patients with diabetes compared with the normal progression of these complications. So "prevention" according to the present invention also implies that vascular complications in patients with diabetes occur at a later stage of the disease thanks to the present invention in comparison with the regular occurrence of these complications in patients with diabetes.

Particularly preferred peptide according to the present invention consists of the amino acid sequence CGQRETPEGAEAKPWYC and cyclizine through With residues (at positions 1 and 17).

Cyclization according to the present invention can be achieved by direct intramolecular cyclization functional groups of amino acid residues, preferably via C-C bond (via a disulfide bond between two residues). The peptide also may be connected (e.g., using two tanks) with a substance carrier. Thus, the peptides according to the present invention preferably have cysteine residues at the beginning and at the end of the molecule. Other functional groups capable of cyclization of the peptide, can use�from, for example, an amine or an alcohol group, amino acid balance, which leads to the closure of the amide or ester ring (these include, for example, amino acids aspartic acid and glutamic acid to serine, threonine, tyrosine, asparagine, glutamine or lysine, which can preferably be collisioni intramolecular). Thus, other preferred peptides according to the invention are, for example, CGQKETPEGAEAKPWYC (SEQ ID NO. 8), CGQRETPEGAEARPWYC (SEQ ID NO. 9), CGQRETPEGAEAKPC (SEQ ID NO. 10), CQRETPEGAEAKPWYC (SEQ ID NO. 11) or CGQRETPEGAEAKFWYC (SEQ ID NO. 12).

Acceptable carriers are all traditionally used pharmaceutical carriers, having suitable binding group for binding with the peptides according to the present invention, for example the media that react with SH-groups cysteine for the formation of covalent bonds. Other suitable carriers are adjacent bifunctional groups (e.g., acidic group close to an amine or alcohol group). In this context, it is important to note that the "cyclization" according to the present invention includes both intramolecular cyclization and participating media (represented by the associated peptide (N - and C-end peptide binds to a carrier for the formation of loops on the media)). In both embodiments, the cyclic peptide demonstrated�should cyclical spatial structure and therefore stabilizing. In some embodiments, preference is given to intramolecular cyclization, in particular in the case where no molecules of the medium is preferred for reasons of solubility or from considerations of molecular weight or molecular size.

According to another aspect of the present invention relates to a pharmaceutical composition containing the peptide according to the present invention (or a mixture of peptides according to the present invention and a pharmaceutical carrier. This pharmaceutical composition is used for the treatment or prevention of vascular complications in patients with diabetes.

The term "pharmaceutical composition" refers to any composition or preparation containing a peptide as defined above, which allows to alleviate, cure or prevent the above-described condition. In particular, the expression "pharmaceutical composition" refers to compositions comprising a peptide according to the present invention and a pharmaceutically acceptable carrier or formative (both terms are used interchangeably). Suitable carrier materials or shaping known to specialists in this field, such as saline solution, ringer's solution, dextrose solution, buffers, Hank's solution, vesiculopapular compounds (e.g., lipids), fatty oils, ethyloleate, 5% dextrose � saline solution, substances that increase izotonichnost and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not cause the generation of patient antibodies that are harmful to the patient. Examples can be well-tolerated proteins, polysaccharides, polylactic acid, polyglycol acid, polymeric amino acids and amino acid copolymers. As has been described above, the peptides according to the present invention can be cyclosiloxane with such a device via a direct covalent bond. This pharmaceutical composition (medicament) may be administered by appropriate means known to specialists in this field. Preferred route of administration is parenteral administration, in particular through inhalation (aerosol), or intravenous. For parenteral administration the pharmaceutical composition according to the present invention is provided in intended for injection of a unit dosage form, for example in the form of a solution, suspension or emulsion, receptional in combination with defined above, pharmaceutically acceptable formative. However, the dosage and method of administration depend on the individual patient under treatment. Typically, the peptide according to the present image�meniu administered in a dose from 1 μg/kg to 10 mg/kg, more preferably from 10 μg/kg to 5 mg/kg, most preferably from 0.1 to 2 mg/kg. Preferably the composition is administered as an intraperitoneal bolus dose. Can also be used a continuous infusion. In this case, the peptide is administered by infusion at a dose of 5 to 20 mcg/kg/min, more preferably from 7 to 15 mcg/kg/min.

According to the present invention is particularly preferred peptide according to the present invention (also referred to as "AR 301") has the following amino acid sequence:

SEQ ID NO:1

(NH2)Cys-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Cys(COOH).

The invention is further described by the following examples and figures, which are merely explanatory and do not limit the scope of the invention.

Fig.1 shows caused by acetylcholine narrowed extension using U46619 septal coronary arteries from control and treated with streptozocin (STZ) or treated with TIP peptide + STZ rats 4 weeks after STZ injection (n=3);

Fig.2 shows that the treatment TIP peptide does not affect the level of glucose in the blood of treated STZ rats (n=3);

Fig.3 shows that the treatment of the TIP peptide in a dose-slightly reduces the volume of urine in treated STZ rats (n=3);

Fig.4 shows obtained by fluorescence microscopy image perfoirmance hearts of rats (Langendorff) using FITC-BSA;

�IG.5 shows the effect of the TIP peptide (125 μg/rat for 4 weeks intraperitoneally) on cardiac permeability (defined by the introduction of albumin-FITC with the application of the method of Langendorf).

EXAMPLES

1. Impact taken from TNF TIP peptide on endothelial relaxation during streptozocin-induced diabetes in rats

Streptozocin (STZ) is an antibiotic that can cause the destruction of β-cells of the pancreas, so it is widely used in experiments as an agent capable of causing insulin-dependent diabetes mellitus (IDDM), also known as diabetes mellitus type 1 (T1DM).

In this experiment male rats Sprague-Dawley (from 240 to 265 g, n=3) were divided into three groups: 1. Control rats receiving only the injection of the filler 2. Rats with STZ induced diabetes (50 mg/kg, intraperitoneally), and 3. Treated STZ rats, which are in parallel I got a TIP peptide (125 μg, intraperitoneally) once every three days, for the first time - two days before STZ injection, for four weeks. Rats whose blood level was >350 mg/DL were considered diabetic. Interventricular coronary artery, rats were prepared as described previously (Romero et al., 2008). In General, the segments of the coronary arteries were fixed in the myograph for small vessels (Danish Myo Technology), compressed using the analogue of thromboxane A2 (U46619 9,11-dideoxy-9a,11a-mechanoactivation F2a

(Chemical name: (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid)

and were tested on their ability to expansion� in response to increasing concentrations of acetylcholine. Vasodilator reactions are expressed as percentage of maximum control of the. As shown in Fig.1, interventricular coronary artery from rats with STZ induced diabetes demonstrated significantly reduced ability to expand in comparison with vessels of control rats. Parallel treatment of diabetic rats taken from TNF TIP peptide AR has greatly improved shown through vasodilatation response in septal coronary arteries. This model of an animal with confirmed diabetes shows that the TIP of the TNF peptide OR can improve endothelial vasorelaxation during diabetes.

Also examined the impact of treatment TIP peptide according to the present invention in these rats on the level of blood glucose, blood pressure, urine volume and mass (Fig.2 and 3). Treated TIP peptide showed a comparable level of glucose in STZ-group, however, they observed a lower urine volume and higher blood pressure compared to STZ group. They also showed increased weight loss. TIP peptide according to the present invention also prevents associated with STZ cardiac permeability in vivo.

According to the same Protocol as described above, heart control, treated and STZ-treated TIP/STZ rats were separated in 4 weeks. The hearts were perfesional, applying �] of Langendorf with BSA-FITC (Di Napoli et al., Nitric Oxide 16 (2007), 228-236). Then the hearts were frozen in liquid nitrogen, and sliced into layers with the help of microtome. Then fluorescence was determined in 4 layers each heart and recorded totals of the fluorescent reaction. As shown in Figures 4 and 5, the TIP peptide OR significantly prevents increased permeability of the heart vessels: fluorescence microscopy of ventricular perfoirmance FITC-albumin hearts of rats revealed significantly less fluorescent dyes in the presence of the TIP peptide (Fig.5) in comparison with experiments without TIP peptide (Fig.4).

Presented at the animal model demonstrated that STZ induced diabetes Type I in rats causes vascular dysfunction, characterized by impaired endothelial-dependent vasodilatation and increased permeability of blood vessels of the heart.

The data presented for this model, show that the treatment with the use of a typical representative of the peptides according to the present invention ("TIP peptide"; "AR"; SEQ ID No.1) can effectively treat and prevent diabetic vascular complications in these animals. These data confirm that treatment with the peptides according to the present invention represents a promising approach to new therapies for the treatment of diabetic vascular complications and also people.

2. Ex vivo evaluation of PR�inflammatory properties of peptide OR in whole human blood

Ex vivo pharmacological safety studies of peptide OR in whole human blood was performed to determine whether the peptide OR to the release of Pro-inflammatory marker interleukin-6 (IL-6) from viewsate whole human blood (i.e., does the APN 301 TNF-specific inflammatory activity).

This study used viewsat whole human blood, since it is a predictive modeling system to assess the inflammatory response in vivo.

Brief description of methodology

The purpose of this study was to determine proinflammatory signaling ability of the peptide AR. Used culture of whole blood and the secretion of interleukin-6 (IL-6), a highly sensitive marker of proinflammatory stimulation was quantitatively determined by ELISA analysis.

Test system
Test systemFor tests used 25 ml viewsate heparinized blood of 5 healthy volunteers (HV).
The investigated sample
SymbolPeptide AR (dose: 1 ng/ml to 10 μg/ml; od�Otradnoe introduction in solution)
DescriptionWhite powder, purity 96%

Culture whole blood

The cultivation of whole blood (WB) was carried out by way of the transfer pipette 1 ml of WB into the wells of 24-well plates. In each experiment used restimulative and control-stimulated culture.

Possible substances and stimulants, be the research, each experiment was added in equal volume to each well, no more than 10% of the total volume contained in the hole. For restimulating control used PBS. Adjustment of the dose and dilution to different modes of treatment were also carried out using PBS.

The contents of each well were mixed and the plates were incubated at 37°C and 5% CO2in the next 24 hours. After incubation content of each well was transferred into a clean 1.5 ml of microtrace and centrifuged at 8.000 to 9.000 x g for 15 minutes. The supernatant of each sample was separately transferred into two 1.5 ml micro-tubes and stored at -20°C until use.

Detection of interleukin-6

Interleukin-6 was quantitatively determined by specific ELISA analysis of Human IL-6 ELISA Set, BD Biosciences, Cat. No. 555220) using antibodies against human IL-6 as an immobilized antibody, biotinylating identifying antibodies against cel�human IL-6, conjugate the avidin - horseradish peroxidase as the enzyme reagent and recombinant IL-6 as standard. Measurement of absorbance was performed at 450 nm using a Packard FusionReader.

Data analysis

The results for each tablet was stored and evaluated using the program FusionDataAnalysis.

Brief description of the study results

The purpose of this study was to determine proinflammatory signaling ability of the peptide AR. Used culture of whole blood and the secretion of IL 6, a highly sensitive marker of proinflammatory stimulation was quantitatively determined by ELISA analysis.

Samples of whole blood of five healthy volunteers were left estimulante (negative control), stimulated high and low doses of LPS (positive control) or were incubated with peptide in nine degrees semi-logarithmic dilutions from 10 μg/ml to 1 ng/ml.

Table
The release of interleukin-6 from viewsate whole human blood after addition of the peptide A and LPS
OR peptideThe positive control (LPS)
The concentration of IL-6 (PG/ml, n=5)
Concentration
0 (negative control)less than 0.5less than 0.5
10 mg/mlless than 0.5195,640
1 mg/mlless than 0.5108,370
3 ng/mlless than 0.534,867
1 ng/mlless than 0.5is not defined

The results clearly show that the peptide OR not cause any significant level of secretion of IL-6 in any of the tested concentrations. The positive control (LPS) as a result caused a strong secretion of IL-6.

Discussion

The experiments were carried out in order to determine whether the peptide OR a mediator in the appearance of proinflammatory reaction. Parameter read was induced secretion of IL-6 in cultures of whole blood of five healthy donors. The results clearly demonstrated that the peptide OR did not cause significant levels of IL-6 in any of the donor cultures. Thus, it was demonstrated that the peptide OR ne�the TTI does not cause inflammatory reactions in selected ex vivo model.

1. The use of a cyclic peptide consisting of a sequence of successive amino acids CGQRETPEGAEAKPWYC, to obtain drugs for prevention or treatment of vascular complications in patients with diabetes.

2. The use of a cyclic peptide according to claim 1, characterized in that said complications are selected from the group including micro - and macrovascular disease, myocardial infarction, increased permeability of cardiac capillaries, stroke, neuropathy, retinopathy, nephropathy or diabetic foot syndrome in patients with diabetes.

3. The use of a cyclic peptide according to claim 1 or claim 2, characterized in that it cycletour via cysteine residues.

4. The use of a cyclic peptide according to claim 3, characterized in that it cyclist through disulfide bonds between cysteine residues.

5. Pharmaceutical composition comprising a cyclic peptide consisting of a sequence of successive amino acids CGQRETPEGAEAKPWYC, and a pharmaceutical carrier, for obtaining a medicinal product for the prevention or treatment of vascular complications in patients with diabetes.

6. Pharmaceutical composition according to claim 5, characterized in that said complications are selected from the group including micro - and macrovascular disease, myocardial infarction, increased permeability of cardiac capillaries, stroke, neuropathy, retinopathy, nephropathy or diabetic foot syndrome in patients with diabetes.

7. Pharmaceutical composition according to claim 5 or claim 6, characterized in that the peptide cycletour via cysteine residues.

8. Pharmaceutical composition according to claim 7, characterized in that the peptide cyclist through disulfide bonds between cysteine residues.



 

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