SS-31 FOR THE PREVENTION AND/OR TREATMENT OF ANEURYSM

Abstract

The present invention relates to SS-31 or composition comprising SS-31 for use in the treatment and/or prevention of aneurysm.

Claims

1. The peptide D-Arg-2′,6′-dimethyltyrosine-Lys-Phe-NH.sub.2 for use in the treatment and/or prevention of aneurysm.

2. The peptide according to claim 1, wherein the aneurysm is an aortic aneurysm.

3. The peptide according to claim 1, wherein the aneurysm is an abdominal aortic aneurysm.

4. A composition comprising a therapeutically effective amount of peptide D-Arg-2′,6′-dimethyltyrosine-Lys-Phe-NH.sub.2 for use in the treatment and/or prevention of aneurysm.

5. The composition according to claim 4, wherein the aneurysm is an aortic aneurysm.

6. The composition according to claim 4, wherein the aneurysm is an abdominal aortic aneurysm.

7. The composition according to claim 4, wherein said composition comprises at least one pharmaceutically acceptable excipient.

8. The composition according to claim 4, wherein said composition is administered orally, intravenously, subcutaneously, intramuscularly or by inhalation.

9. The composition according to claim 4, wherein said composition is administered by means of an endovascular device.

10. The composition according to claim 4, further comprising at least one of the active agents selected from a hypolipemiant agent, an antihypertensive agent selected from at least one of a betablocker, an angiotensin-converting enzyme inhibitor, a calcium channel blocker, an angiotensin receptor blocker and a diuretic.

11. The composition according to claim 10, wherein the hypolipemiant agent is a statin; the betablockers is selected from propranolol, bisoprolol and ometoprolol; the angiotensin-converting enzyme inhibitor is selected from benazepril, zofenopril, perindopril, trandolapril, captopril, enalapril, lisinopril and ramipril; the calcium channel blocker is selected from amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, fendiline, gallopamil, verapamil, diltiazem, mibefradil, bepridil, flunarizine and fluspirilene; the angiotensin receptor blocker is selected from losartan, candesartan, telmisartan, valsartan and fimasartan; and the diuretic is selected from furosemide, ethacrynic acid, torasemide, bendroflumethiazide, hydrochlorothiazide, acetazolamide and methazolamide.

12. The composition according to claim 4, for use in a human patient.

13. The composition according to claim 4, for use in a patient subjected to an endovascular repair.

14. The composition according to claim 4, wherein the peptide is in the form a pharmaceutically acceptable salt thereof.

15. The composition according to claim 11, wherein the statin is selected from simvastatin, atorvastatin, rosuvastatin, lovastatin, pitavastatin and pravastatin.

16. The composition according to claim 14, wherein the peptide is in the form of the hydrochloride salt.

Description

DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1. Treatment with SS-20 or SS-31 does not affect systolic pressure and treatment with SS-31 improves survival in Apo E.sup.−/− mice infused with saline or angiotensin II. A) Graph representing the systolic blood pressure of ApoE.sup.−/− mice (males and females) infused with saline or angiotensin II (Ang II; 1000 ng/kg/min for 28 days) analyzed with the CODA® tail-cuff blood pressure system at the end of treatment. Animals infused with Ang II were treated or not with SS-31 or with SS-20 (3 mg/kg/day, administered together with Ang II through osmotic minipump). The values correspond to the mean±SEM (n=7-14). *P<0.05 vs. animals infused with saline. B) Percentage of animal survival in the four study groups described in (A). **P<0.01 vs. saline at end time; $ P<0.05 vs. animals infused with Ang II (not treated with SS-31) and treated or not with SS-20 at end time.

[0027] FIG. 2. Treatment with SS-31 inhibits the development of aortic aneurysm in angiotensin II-infused ApoE.sup.−/− mice. A) Graphic representing the aortic diameter of ApoE.sup.−/− mice infused with saline (Saline) or angiotensin II (Ang II; 1000 ng/kg/min for 28 days; males and females) analyzed by ultrasonography at the end of the study (28 days). Animals infused with Ang II were treated or not with SS-31 or with SS-20 (3 mg/kg/day, administered together with Ang II through osmotic minipumps). The values correspond to the mean±SEM (n=15-22). P<0.01: ** vs. saline; $$ vs. animals infused with Ang II (not treated with SS-31 or SS-20); *P<0.05 vs. saline; #P<0.05 vs. animals infused with Ang II and treated with SS-31. B) Representative photographs of the aortas of the mice of the 4 study groups at the end of the study period (28 days). C) Representative images obtained by ultrasonography of the transversal vision of the aorta of each one of the experimental groups indicated in (A) at the end of the 4 weeks of treatment.

[0028] FIG. 3. Treatment with SS-31 decreases the incidence of AAA in ApoE.sup.−/− mice infused with angiotensin II for 28 days. ApoE.sup.−/− mice were infused with saline (Saline) or angiotensin II (Ang II; 1000 ng/kg/min for 28 days; males and females). Animals infused with Ang II were treated or not with SS-31 or with SS-20 (3 mg/kg/day, administered together with Ang II through osmotic minipumps). A representative graph of the incidence of aneurysm formation in the animals of the four study groups is shown.

[0029] FIG. 4. Treatment with SS-31 limits structural alterations in the abdominal aorta of ApoE.sup.−/− mice infused with Ang II. ApoE.sup.−/− mice were infused with saline (Saline) or Ang II (Ang II; 1000 ng/kg/min for 28 days; males and females). Animals infused with Ang II were treated or not with SS-31 or with SS-20 (3 mg/kg/day, administered together with Ang II through osmotic minipumps). (A and B) Representative images of histological analysis by Masson's trichrome (A) and orcein (B) staining in sections of the abdominal aorta of the indicated study groups. In (B) the elastic ruptures are indicated with arrows. Bar: 200 μm. (C) Histogram showing the quantification of the number of ruptures in the elastic fibers per aortic ring. The results are shown as mean±SEM (n=5; *P<0.05, **P<0.01 vs. Saline; $ P<0.05 vs. animals infused with Ang II (not treated with SS-31 or SS-20); #P<0.05 vs. animals infused with Ang II and treated with SS-31).

[0030] FIG. 5. Treatment with SS-31 reduces the expression of metalloproteinases MMP2 and MMP9 in the abdominal aorta of ApoE.sup.−/− mice infused with angiotensin II. MMP2 and MMP9 expression levels analyzed by real-time PCR in the abdominal aorta of ApoE.sup.−/− mice infused with saline (Saline) or angiotensin II (Ang II; 1000 ng/kg/min for 28 days; males and females) treated or not with SS-31 or with SS-20 (3 mg/kg/day, administered together with Ang II through osmotic minipumps). The results, normalized by the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) mRNA level, are shown as mean±SEM (n=10-15; *P<0.05, **P<0.01, vs. Saline; $ P<0.05, $$ P<0.01 vs. Ang II (without additional treatments); #P<0.01 vs. animals infused with Ang II and treated with SS-31).

EXAMPLES

[0031] The invention is illustrated below by the examples showing the effectiveness of SS-31 treatment in reducing the incidence and severity of aortic aneurysms developed in response to Ang II infusion in Apo E.sup.−/− mice (Daugherty A et al. J Clin Invest. 2000; 105:1605-1612).

Example 1: Analysis of the Impact of SS-31 and SS-20 on the Development of Ang II-Induced Aneurysms in ApoE.SUP.−/− .Mice

[0032] The studies were developed in ApoE.sup.−/− mice bred in the facility. The animals were infused with Ang II dissolved in saline (1000 ng/kg/min) by means of osmotic minipumps (model 1004, Alzet) implanted subcutaneously in the interscapular space of the mice previous anesthesia with isoflurane. The procedure lasted approximately 15 minutes per animal. Immediately after surgery, antibiotics (penicillin, 22,000 u/Kg, i.m.) and analgesics (buprenorphine 0.05 mg/Kg, i.m.) were administered to prevent infections and limit discomfort in the animals. Further, mice were kept on a heating pad until they woke up after surgery and were carefully monitored during the post-surgery period.

[0033] A group of mice infused with Ang II were treated with SS-31 in the form of chloride and dissolved in saline (3 mg/kg/day). Another group of animals infused with Ang II were treated with the SS-20 peptide also in the form of chloride, dissolved in saline and at the same dose. Both were administered together with Ang II by osmotic mini pump during the whole study (28 days). The control group consisted of ApoE.sup.−/− mice infused with saline.

[0034] Blood pressure was evaluated at the end of treatment (28 days) using the tail plethysmography method (CODA®tail-cuff blood pressure system).

[0035] The diameter of the aorta was evaluated weekly by abdominal echography using an ultrasound equipment (Vevo2100 Imaging systems; Visualsonics) with a 30 MHz transducer. The mice were anesthetized by inhalation of 1.5% isofluorane and secured in supine position on a thermal platform. After shaving the precord region, the transducer was applied to the abdominal wall to measure the abdominal aorta. Those abdominal aortas with external diameters greater than or equal to 1.5 mm were considered aneurysmal. All measurements were made from the captured images using the analysis software provided by Visualsonics.

[0036] After 4 weeks, the animals were euthanized under intraperitoneal terminal anesthesia with a mixture of medetomidine (1 mg/kg) and ketamine (75 mg/kg) in saline (final volume of 200 μl), the aortas were collected immediately, examined to determine the presence of aneurysms and fixed in paraformaldehyde or frozen in liquid nitrogen for subsequent RNA extraction.

[0037] These analyses showed that infusion with Ang II increased blood pressure and that this parameter was not significantly altered as a consequence of treatment with SS-31 or SS-20 (FIG. 1).

[0038] Infusion with Ang II caused the death of approximately 20% of the animals, while treatment with SS-31 significantly improved the survival rate (FIG. 1B). SS-20 did not modify the survival rate (FIG. 1B).

[0039] Ultrasound studies showed that infusion with Ang II in ApoE.sup.−/− mice significantly increased the diameter of the aorta compared to control animals infused with saline, and that this effect was attenuated by treatment with SS-31 (FIG. 2A). The administration of SS-31 reduced the increase in aortic diameter significantly, as can also be determined through macroscopic visualization of the aorta (FIG. 2B). However, SS-20 could not attenuate this increase in the aortic diameter (FIG. 2B).

[0040] Accordingly, we observed that in animals infused with Ang II, treatment with SS-31 significantly decreased the incidence of AAA (FIG. 3). On the contrary, the administration of SS-20 in animals infused with Ang II did not alter the aortic diameter nor the incidence of aneurysms, parameters that were similar to those of the group exclusively infused with Ang II (FIGS. 2 and 3).

[0041] Histological analyses using Masson's trichrome stain of sections of 5 μm of the fixed and paraffin-embedded aortic samples showed that in ApoE.sup.−/− mice, infusion with Ang II produced an important vascular remodeling characterized by an increase in collagen deposition that was attenuated by treatment with SS-31 but not by treatment with SS-20 (FIG. 4A). Likewise, orcein staining showed that SS-31 limited the disorganization and rupture of elastic fibers induced by Ang II infusion, an improvement in elastin structure that was not observed in animals treated with SS-20 (FIGS. 4B and 4C).

[0042] The expression levels (mRNA) of the metalloproteinases MMP2 and MMP9 in the abdominal aortas of the different study groups were also analyzed. For this purpose, the total RNA was extracted from the tissues with the RNeasy Micro kit (Qiagen) system following the manufacturer's instructions. The RNA (1 μg) was retrotranscribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in the presence of random hexamers, and a real time PCR analysis was performed using specific Taqman® probes for MMP2 and MMP9. These analyses showed that Ang II-infusion in ApoE.sup.−/− mice significantly increased aortic expression of MMP2 and MMP9 with respect to control animals infused with saline solution. Treatment with SS-31 attenuated this effect, unlike SS-20, which did not modify the increase in the mRNA level of these MMPs induced by Ang II (FIG. 5).