Peptides Des-[Asp1]-[Ala1], angiotensin-(1-7) agonist and pharmaceutical compositions for the treatment of diseases

Abstract

The present invention is related to the peptide Des-[Asp.sup.1]-[Ala.sup.1]-Angiotensin-(1-7) (Ala.sup.1-Arg.sup.2-Val.sup.3-Tyr.sup.4-Ile.sup.5-His.sup.6-Pro.sup.7) (SEQ ID NO: 1) and/or its related compounds as vasodilating and cardioprotective agents to be used in mammals. This invention also comprises the production of compounds containing Des-[Asp.sup.1]-[Ala.sup.1]-Angiotensin-(1-7) and/or its related compounds and its use in methods for treating and preventing diseases.

Claims

1. A peptide Des-[Asp.sup.1]-[Ala.sup.1]-Angiotensin-(1-7) consisting of the amino acid sequence SEQ ID NO: 1.

2. A pharmaceutical composition comprising the peptide Des-[Asp.sup.1]-[Ala.sup.1]-Angiotensin-(1-7) as defined in claim 1, in pharmaceutically and physiologically acceptable carriers and/or excipients.

3. The pharmaceutical composition according to claim 2, which is administered by an oral, intramuscular, intravenous, subcutaneous, topical, or a transdermal route or a device to be implanted or injected.

4. The pharmaceutical composition according to claim 2, which is included in a controlled release system comprising a cyclodextrin, biodegradable polymer, mucoadhesive polymer, gel, or liposome.

5. The peptide according to claim 1, which is formulated for treatment of a vascular or cardiovascular disease in a human or other mammal.

6. The peptide according to claim 1, which is formulated for treatment of a neurological disease or disorder in a human or other mammal.

7. The peptide according to claim 1, which is formulated for treatment of a renal, endocrinal, reproductive, dermatological, neoplastic, or blood disease or disorder in a human or other mammal.

8. A method for treatment of disease, comprising administration of an effective amount of Ala.sup.1-Angiotensin-(1-7) and/or related components as defined in claim 1 or of a pharmaceutical composition thereof for treating a disease selected from the group consisting of cardiovascular, renal, endocrinal, reproductive, dermatological, neoplastic, blood, and cerebral diseases.

9. The method according to claim 8, which is administered by an oral, intramuscular, intravenous, subcutaneous, topical, or transdermal route or a device to be implanted or injected.

10. The method according to claim 8, wherein a controlled release system comprising at least cyclodextrin, biodegradable polymer, mucoadhesive polymer, gel, or liposome is also administered.

11. A method for treatment of a vascular or cardiovascular disease or disorder, comprising administration of an effective amount of Ala.sup.1-Angiotensin-(1-7) as defined in claim 1 or of a pharmaceutical composition thereof to a patient in need thereof.

12. The method according to claim 11, the disease or disorder is selected from the group consisting of endothelium dysfunction, atherosclerosis, ischemic and reperfusion injury, acute myocardial infarction, high blood pressure, primary and secondary arterial hypertension, chronic and acute congestive cardiac insufficiency, left ventricular hypertrophy, vascular hypertrophy, primary and secondary hyperaldosteronism, diabetes, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, renal insufficiency, therapies for kidney transplants or diabetic retinopathy, and angioplasty.

13. The method according to claim 11, which is administered by oral, intramuscular, intravenous, subcutaneous, topical, or transdermal route or a device to be implanted or injected.

14. The method according to claim 11, wherein a controlled release system comprising at least cyclodextrin, biodegradable polymer, mucoadhesive polymer, gel, or liposome is also administered.

15. A method for treatment of a neurological disease or disorder, comprising administration of an effective amount of Ala.sup.1-Angiotensin-(1-7) as defined in claim 1 or of a pharmaceutical composition thereof to a patient in need thereof.

16. The method according to claim 15, wherein the disease or disorder is selected from the group consisting of peripheral diabetic neuropathy, pain, cerebral vascular accident, and cerebral ischemia.

17. The method according to claim 15, which is administered by oral, intramuscular, intravenous, subcutaneous, topical, or transdermal route or a device to be implanted or injected.

18. The method according to claim 15, wherein a controlled release system comprising at least cyclodextrin, biodegradable polymer, mucoadhesive polymer, gel, or liposome is also administered.

19. A process for making a pharmaceutical composition according to claim 2, comprising formulating the peptide Des-[Asp.sup.1]-[Ala.sup.1]-Angiotensin-(1-7) in at least a pharmaceutically and physiologically acceptable carrier and/or excipient, isolated or combined, or even associated at least to another pharmaceutically active principle to make the pharmaceutical composition.

Description

EXAMPLE 1

The Vasodilating Effect of Ala1-Ang-(1-7) Independent of the Mas Receptor

(1) This example describes the vasodilating effect of Ala.sup.1-Ang-(1-7), independently of the Mas receptor, on aorta rings of mice with genetic deletion of the Mas receptor (Mas/) and wild-type mice (Mas+/+) from two different lineages, C57/BI-6 and FVB/N.

(2) The original rings of the descending thoracic aorta (2 mm), free from adipose tissue and connective tissue, were incubated in gasified solution (95% O.sub.2 and 5% CO.sub.2) of Krebs-Henseleit (mmol/L): NaCl 110.8, KCl 5.9, NaHCO.sub.3 25.0, MgSO.sub.4 1.07, CaCl.sub.2 2.49, NaH.sub.2PO.sub.4 2.33 and glucose 11.51, under a temperature of 37 C. and a 0.5 g tension, being equilibrated for 1 hour. The functional presence of endothelium was tested by its relaxing capacity produced by acetylcholine [ACh] (10 M) on vessels precontracted with fenilefrine (0.3 M). Only the vessels showing relaxation above 70% of extended contraction produced by fenilefrine were taken into account. Data on mechanical activity were isometrically obtained by using an amplified strength transducer (Model TMB-4; World Precision Instruments, Inc. Sarasota, Fla., USA) and converted to digital signal (AD16JR; World Precision Instruments, Inc.). A specific software for data acquisition was also used (World Precision Instruments, Inc.).

(3) The aorta rings of mice Mas/ and Mas+/+ from both lineages (C57/BI-6 and FVB/N) were precontracted to achieve the same tension level (approximately a 1.0 g tension) at a submaximum concentration of fenilefrine (0.1 M). Ala.sup.1-Ang-(1-7) and Ang-(1-7) were added in increasing and cumulative concentrations after stabilized contraction response to fenilefrine was achieved. The results are presented as E.P.M. mean values. The Two-way (ANOVA) test of variance analysis was used as a comparative method of curves, followed by the Bonferroni post-test for comparison of the dependent concentration curves obtained in the aorta rings. The vasodilating effects of Ala.sup.1-Ang-(1-7) and Ang-(1-7) were expressed as a percentage of relaxation relative to the maximum contraction induced by fenilefrine. The statistical analyses were considered significant when the value of p was lower than 0.05.

(4) Both PEPTIDES, Ala.sup.1-Ang-(1-7) and Ang-(1-7), have produced vasorelaxation in aorta rings of Mas+/+ mice from both lineages (C57/BI-6 and FVB/N). However, the vasodilating response of Ala.sup.1-Ang-(1-7) was totally preserved in Mas/ mice (from both lineages, C57/BI-6 and FVB/N), and the absence of Ang-(1-7) (from both lineages, C57/BI-6 and FVB/N) response was verified. These results show the vasodilating effect of Ala.sup.1-Ang-(1-7), which is independent from the Mas receptor, as compared to the vasodilating effect of Ang-(1-7), which is Mas-dependent.

EXAMPLE 2

The Vasodilating Effect of Ala1-Ang-(1-7) is Independent of the AT2 Receptor

(5) This example describes the vasodilating effect of Ala.sup.1-Ang-(1-7) and Ang-(1-7)which is independent of the AT.sub.2 receptoron the aorta of mice with genetic deletion of AT.sub.2 (AT.sub.2/) receptor.

(6) The original rings of the descending thoracic aorta (2 mm), free from adipose tissue and connective tissue, were incubated in gasified solution (95% O.sub.2 and 5% CO.sub.2) of Krebs-Henseleit (mmol/L): NaCl 110.8, KCl 5.9, NaHCO.sub.3 25.0, MgSO.sub.4 1.07, CaCl.sub.2 2.49, NaH.sub.2PO.sub.4 2.33 and glucose 11.51, under a temperature of 37 C. and a 0.5 g tension, being equilibrated for 1 hour. The functional presence of endothelium was tested by its relaxing capacity produced by acetylcholine [ACh] (10 M) on vessels precontracted with fenilefrine (0.3 M). Only the vessels showing relaxation above 70% of extended contraction produced by fenilefrine were taken into account. Data on mechanical activity were isometrically obtained by using an amplified strength transducer (Model TMB-4; World Precision Instruments, Inc. Sarasota, Fla., USA) and converted to digital signal (AD16JR; World Precision Instruments, Inc.). A specific software for data acquisition was also used (World Precision Instruments, Inc.). A specific software for data acquisition was also used (World Precision Instruments, Inc.).

(7) The aorta rings of AT.sub.2/ mice were precontracted (approx. 1.0 g of tension) with a felinefrine submaximum concentration (0.1 M). Ala.sup.1-Ang-(1-7) and Ang-(1-7) were added in increasing and cumulative concentrations after the contraction response to felinefrine has been stabilized. The results were shown as a E.P.M. mean. The Two-way (ANOVA) variance analysis was used as a comparative method of curves, which was followed by the Bonferroni post-test so as to compare the dependent-concentration curves obtained in the aorta rings. The vasodilating effects of Ala.sup.1-Ang-(1-7) and Ang-(1-7) were expressed as a relaxation percentage relative to the maximum contraction induced by fenilefrine. The statistical analyses were considered significant when the value of p was lower than 0.05.

(8) The vasodilating effects produced by Ala.sup.1-Ang-(1-7) and Ang-(1-7) were preserved in the aorta rings of AT.sub.2/ mice. This proves that the vasodilating effects of Ala.sup.1-Ang-(1-7) and Ang-(1-7) are receptor-independent.

EXAMPLE 3

The Vasodilating Effect of Ala1-Ang-(1-7) is Endothelium-Dependent

(9) This example describes the endothelium-dependence of the Ala.sup.1-Ang-(1-7) vasorelaxing activity.

(10) The original rings of the descending thoracic aorta (2 mm), free from adipose tissue and connective tissue, were incubated in gasified solution (95% O.sub.2 and 5% CO.sub.2) of Krebs-Henseleit (mmol/L): NaCl 110.8, KCl 5.9, NaHCO.sub.3 25.0, MgSO.sub.4 1.07, CaCl.sub.2 2.49, NaH.sub.2PO.sub.4 2.33 and glucose 11.51, under a temperature of 37 C. and a 0.5 g tension, being equilibrated for 1 hour. The functional presence of endothelium was tested by its relaxing capacity produced by acetylcholine [ACh] (10 M) on vessels precontracted with fenilefrine (0.3 M). In accordance with experimental protocols for its absence, the endothelium was removed with a slight friction on the vessel internal surface. Only the vessels showing relaxation by acetylcholine above 70% of extended contraction produced by fenilefrine were taken into account. Data on mechanical activity were isometrically obtained by using an amplified strength transducer (Model TMB-4; World Precision Instruments, Inc. Sarasota, Fla., USA) and converted to digital signal (AD16JR; World Precision Instruments, Inc.). A specific software for data acquisition was also used (World Precision Instruments, Inc.).

(11) The Ala.sup.1-Ang-(1-7) vasorelaxing activity was measured in vessels (Mas+/+ and Mas/, of lineages C57/BI-6 and FVB/N) in the presence or absence of precontracted functional endothelium (with approximately a 1.0 g tension) and a submaximum concentration of fenilefrine (0.1 M). Ala.sup.1-Ang-(1-7) was added in increasing and cumulative concentrations after stabilization of contraction response to felinefrine had been achieved. The results were presented as a E.P.M. mean. The two-way (ANOVA) variance analysis was used as a comparative method of curves, which was followed by the Bonferroni post-test so as to compare the dependent-concentration curves obtained in the aorta rings. The vasodilating effects of Ala.sup.1-Ang-(1-7) and Ang-(1-7) were expressed as a relaxation percentage relative to the maximum contraction induced by fenilefrine. The statistical analyses were considered significant when the value of p was lower than 0.05.

(12) The vasodilating effect of Ala.sup.1-Ang-(1-7) was abolished in vessels not presenting functional endothelium (FIGS. 1-4). These results show that the vasodilating effect produced by Ala.sup.1-Ang-(1-7) is endothelium-dependent.

EXAMPLE 4

Ala1-Ang-(1-7) Inhibits the Angiotensin Converter Enzyme

(13) This example describes the inhibiting activity of the angiotensin converter enzyme (ACE) produced by Ala.sup.1-Ang-(1-7).

(14) The plasmatic activity of ACE was measured by the fluorimetric method using Hip-His-Leu as substrate, as previously described (Santos, R. A. S. et al., Hypertension, 7:244-52, (1985)). Plasma aliquots (10 L) of Wistar rats were incubated with a 500 mL solution containing a 1 mM substrate (Hip-His-Leu) and 0.4 M of sodium borate, 0.9 M NaCl (pH=8.3) for 15 minutes at 37 C. The reaction was halted by adding 1.2 mL of NaOH at 0.34 M and 100 mL of orthoftaldehyde (20 mg/mL in methanol). After 10 minutes at environment temperature, 200 mL of HCl at 3 N was added. Later on, after a 5-minute 800g centrifugation, the floating solution fluorescence was measured (a 365 nm excitation and a 495 nm emission). The blank was prepared by inverting the addition order of plasma and NaOH. A 0.5 to 20 nmol curve, containing the product (His-Leu) resulting of the substrate break (Hip-His-Leu) by ACE plasmatic activity, was prepared in each assay. In order to test the inhibition effect of ACE by the PEPTIDEs Ang-(1-7) and Ala.sup.1-Ang-(1-7), 3.310.sup.7 or 3.310.sup.6 M of each PEPTIDE was added before the plasma addition. The assay specificity was demonstrated by a 98% inhibition of the ACE activity with the use of 5 mM of enalaprilate. The percentage of enzymatic activity inhibition was estimated in function of the maximum activity obtained.

(15) The PEPTIDEs Ang-(1-7) and Ala.sup.1-Ang-(1-7) have inhibited the ACE activity, although the inhibiting effect of Ala.sup.1-Ang-(1-7) (3.310.sup.7 M: 76.3% of ACE inhibition, 3.310.sup.6 M: 98.3% of inhibition) was higher than that of Ang-(1-7) (3.310.sup.7 M: 42.4% of ACE inhibition, 3.310.sup.6 M: 85.8% of inhibition).

EXAMPLE 5

Preparing and Characterizing the Inclusion Complexes of Peptide Ala1-Ang-(1-7) in Cyclodextrins

(16) Preparation of the inclusion complex between -cyclodextrin and its derivatives and Ala.sup.1-Ang-(1-7) and its related compounds.

(17) The preparation is accomplished in equimolar proportions of -cyclodextrin and its derivatives and Ala.sup.1-Ang-(1-7) and its related compounds in aqueous solutions. The solution mixture is constantly agitated up to the complete -cyclodextrin dissolution.

(18) Later on the combination is frozen at the liquid nitrogen temperature and submitted to lyophilization for 24 hours. The solid thus obtained was characterized by means of the physico-chemical techniques of analysis. The technique used, which provided important characteristics of the host/guest interaction, was that of fluorescence and spectroscopy of absorption in the ultraviolet-visible region.

(19) The absorption and biological stability tests were carried out with solutions of the peptide-cyclodextrin inclusion complex. Devices of controlled peptide release were prepared as well as those of their peptide-cyclodextrin inclusion complexes.

(20) Therefore, the peptide Ala.sup.1-Ang-(1-7) and its related compounds, combined with cyclodextrin, results in an oral or systemic formulation with longer effect duration.

DESCRIPTION OF FIGURES

(21) FIG. 1 shows the endothelium dependence for the vasodilating effect of Ala.sup.1-Ang-(1-7) and Ang-(1-7) in aorta rings of Mas /mice of FVB/N lineage. Each dot represents the +E.P.M. mean.

(22) FIG. 2 shows the endothelium dependence for the vasodilating effect of Ala.sup.1-Ang-(1-7) and Ang-(1-7) in aorta rings of Mas +/+ mice of FVB/N lineage. Each dot represents the +E.P.M. mean.

(23) FIG. 3 shows the endothelium dependence for the vasodilating effect of Ala.sup.1-Ang-(1-7) and Ang-(1-7) in aorta rings of Mas /mice of C57/BL-6 lineage. Each dot represents the +E.P.M. mean.

(24) FIG. 4 shows the endothelium dependence for the vasodilating effect of Ala.sup.1-Ang-(1-7) and Ang-(1-7) in aorta rings of Mas+/+ mice of C57/BL-6 lineage. Each dot represents the +E.P.M. mean.

(25) FIG. 5 shows the inhibiting activity of ACE by PEPTIDEs Ala.sup.1-Ang-(1-7) and Ang-(1-7). Each dot represents the E.P.M. mean.