PHARMACEUTICAL COMPOSITION, PROCESS FOR PRODUCING THE SAME, USE OF A PEPTIDE, USE OF A PHARMACEUTICAL COMPOSITION AND METHOD FOR TREATING DISEASES ASSOCIATED WITH INTRAOCULAR HYPERTENSION OR GLAUCOMA

Abstract

The present invention describes a pharmaceutical composition of biologically active peptides, associated with a controlled release system using cyclodextrins or derivatives thereof, liposomes and biodegradable polymers and/or mixtures of said systems for increasing bioavailability, duration and intensity of the biological effects of the peptide. Specifically, the present invention comprises a pharmaceutical composition, a process for preparing same, and the use of a peptide in said composition for preparing medication for intraocular hypertension or glaucoma. The present invention falls within the field of Medical Science, more specifically of preparations for medical purposes, and even more specifically of medicinal preparations containing peptides.

Claims

1. Pharmaceutical composition comprising: at least one peptide comprising the amino acid sequence with at least 80% similarity or identity with SEQ ID NO: 1; and controlled release system comprising: at least one cyclodextrin or a natural polymer or a modified biopolymer or liposomes or mixture thereof.

2. Pharmaceutical composition according to claim 1, wherein the peptide comprises the amino acid sequence as defined in SEQ ID NO: 1.

3. Pharmaceutical composition according to claim 1, wherein the peptide consists the amino acid sequence as defined in SEQ ID NO: 1.

4. Pharmaceutical composition according to claim 1, wherein the composition further comprises at least one pharmaceutically acceptable excipient selected from the group consisting of pharmaceutically acceptable carriers, pharmaceutically acceptable additives or combinations thereof.

5. Pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable carrier is selected from the group comprising: water, saline solution, phosphate buffered solutions, a Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions containing or not polyethylene glycol, fixed oils, seed oil, ethyl-oleate, or triglyceride.

6. Pharmaceutical composition according to claim 4, wherein the additive is selected from the group comprising sodium carboxymethylcellulose, sorbitol, dextran, phosphate buffer, bicarbonate buffer, Tris thimerosal buffer, m-cresol or o-cresol, formalin and benzyl alcohol.

7. Pharmaceutical composition according to claim 1, wherein the controlled release system is in the form of capsules, microcapsules, nanocapsules, micro-particles or nano-particles.

8. Pharmaceutical composition according to claim 1, wherein the controlled release system comprises liposomes of lipid moiety selected from the group comprising phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, cholesterol, phosphatidic acid, sphingolipids, glycolipids, fatty acids, sterols, phosphatidylethanolamine, phospholipids.

9. Pharmaceutical composition according to claim 8, wherein the lipid moiety consists of distearoyl-phosphatidylcholine, cholesterol and distearoyl-phosphatidylethanolamine-polyethylene glycol.

10. Pharmaceutical composition according to claim 9, wherein the lipid moiety comprises a molar ratio of 4:3:0.2 to 6:5:0.5 of distearoyl-phosphatidylcholine:cholesterol:distearoyl-phosphatidylethanolamine-polyethylene glycol.

11. Pharmaceutical composition according to claim 10, wherein the lipid moiety comprises a molar ratio of 5:4:0.3 of distearoyl-phosphatidylcholine:cholesterol:distearoyl-phosphatidylethanolamine-polyethylene glycol.

12. Pharmaceutical composition according to claim 8, wherein the peptide/lipid moiety ratio comprises 0.01 (w/w) to 0.06 (w/w) and the mean diameter of the vesicles comprises between 0.1 m to 0.5 m.

13. Pharmaceutical composition according to claim 1, wherein the controlled release system comprises polymer microspheres selected from the group comprising poly (2-hydroxy-ethylmethacrylate)), polyacrylamide, lactic acid-based polymers (PLA), polymers based on glycolic acid (PGA), copolymers of lactic and glycolic acid, (PLGA), poly(anhydrides) polymers such as sebacic acid-based polymers PSA and copolymers with hydrophobic polymers.

14. Pharmaceutical composition according to claim 13, wherein the microsphere comprises lactic and glycolic acid co-polymers.

15. Pharmaceutical composition according to claim 13, wherein the microsphere comprises lactic and glycolic acid co-polymers (PLGA 50:50 w/w).

16. Pharmaceutical composition according to claim 13, wherein the peptide/microsphere ratio comprises between 0.01 (w/w) to 0.06 (w/w).

17. Pharmaceutical composition according to claim 1, wherein the cyclodextrin is beta-cyclodextrin.

18. Process for producing the pharmaceutical composition as defined in claim 1, comprising the following steps: encapsulation of the peptide comprising sequence with at least 80% similarity or identity with SEQ ID NO: 1, or formation of inclusion compound.

19. Process according to claim 18, wherein the encapsulation comprises the following steps: sterically stabilized liposomes; extrusion of DRV suspension.

20. Process according to claim 19, wherein the extrusion of DRV suspension comprises 200 nm pore polycarbonate membranes.

21. Process according to claim 19, wherein the encapsulation comprises the following steps: multiple W/O/W emulsion of microspheres; solvent evaporation.

22. Process according to claim 18, wherein the encapsulation comprises between 10 and 50% efficiency.

23. Process according to claim 18, wherein the formation of inclusion compound comprises the following steps: mixture of cyclodextrin and peptide solutions; continuous stirring until cyclodextrin dissolution; lyophilization of the mixture.

24. Use of a peptide comprising an amino acid sequence with at least 80% similarity or identity with SEQ ID NO:1, for the preparation of a pharmaceutical composition for the treatment of diseases associated with intraocular hypertension or glaucoma.

25. Use of a pharmaceutical composition as defined in claim 1, for the preparation of a medicament for the treatment of diseases associated with intraocular hypertension or glaucoma.

26. Method for treating diseases associated with intraocular hypertension or glaucoma, comprising administering a pharmaceutical composition, as defined in claim 1, in a subject.

27. Method for treating diseases associated with intraocular hypertension or glaucoma, comprising administering a pharmaceutical composition obtained by the process, as defined in claim 19, wherein the release of the peptide in physiological conditions comprises between 50 and 70% in 8 hours and comprising between 80 and 95% in 48 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention describes a pharmaceutical composition of a biologically active peptide using cyclodextrins and derivatives thereof, liposomes and biodegradable polymers and/or mixtures of such systems as a release system for the purpose of increasing bioavailability, duration and intensity of the biological effects of the peptide. The present invention further describes the preparation and use of said composition.

[0062] As a first object, the present invention discloses a pharmaceutical composition comprising: [0063] at least one amino acid sequence with at least 80% similarity or identity with SEQ ID NO: 1; and [0064] controlled release system comprising: [0065] at least one cyclodextrin or a natural polymer or a modified biopolymer or liposomes or mixture thereof.

[0066] In one embodiment of the pharmaceutical composition, the peptide comprises the amino acid sequence of SEQ ID NO: 1

[0067] In one embodiment of the pharmaceutical composition, the peptide consists of the amino acid sequence of SEQ ID NO: 1

[0068] In one embodiment, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient selected from the group consisting of pharmaceutically acceptable carriers, pharmaceutically acceptable additives or combinations thereof.

[0069] In one embodiment of the pharmaceutical composition, the pharmaceutically acceptable carrier is selected from the group comprising: water, saline solution, phosphate buffered solutions, and Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions containing or not polyethylene glycol, fixed oils, seed oil, ethyl-oleate, or triglyceride.

[0070] In one embodiment of the pharmaceutical composition, the additive is selected from the group comprising sodium carboxymethylcellulose, sorbitol, dextran, phosphate buffer, bicarbonate buffer, Tris thimerosal buffer, m-cresol or o-cresol, formalin and benzyl alcohol.

[0071] In one embodiment of the pharmaceutical composition, the controlled release system is in the form of capsules, microcapsules, nanocapsules, micro-particles or nano-particles.

[0072] In one embodiment of the pharmaceutical composition, the controlled release system comprises liposomes of lipid moiety selected from the group comprising phosphatidylcholine, phosphatidyl serine, phosphatidylglycerol, cardiolipin, cholesterol, phosphatidic acid, sphingolipids, glycolipids, fatty acids, sterols, phosphatidylethanolamine, phospholipids.

[0073] In one embodiment of the pharmaceutical composition, the lipid moiety consists of distearoyl-phosphatidylcholine, cholesterol and distearoyl-phosphatidylethanolamine-polyethylene glycol.

[0074] In one embodiment of the pharmaceutical composition, the lipid moiety comprises a molar ratio of 4:3:0.2 and 6:5:0.5 distearoyl-phosphatidylcholine:cholesterol:distearoyl-phosphatidylethanolamine-polyethylene glycol.

[0075] In one embodiment of the pharmaceutical composition, the lipid moiety comprises a molar ratio of 5:4:0.3 distearoyl-phosphatidylcholine:cholesterol:distearoyl-phosphatidylethanolamine-polyethylene glycol.

[0076] In one embodiment of the pharmaceutical composition, the peptide/lipid moiety ratio comprises between 0.01 (w/w) and 0.06 (w/w) and the mean diameter of the vesicles comprises between 0.1 m and 0.5 m.

[0077] In one embodiment of the pharmaceutical composition, the controlled release system comprises polymer microspheres selected from the group comprising poly (2-hydroxy-ethylmethacrylate), polyacrylamide, lactic acid-based polymers (PLA), polymers based on glycolic acid (PGA), copolymers of lactic and glycolic acid, (PLGA), poly (anhydrides) polymers such as sebacic acid-based polymers PSA and copolymers with hydrophobic polymers.

[0078] In one embodiment of the pharmaceutical composition, the microsphere comprises lactic and glycolic acid co-polymers.

[0079] In one embodiment of the pharmaceutical composition, the microsphere comprises lactic and glycolic acid co-polymers (PLGA 50:50 w/w).

[0080] In one embodiment of the pharmaceutical composition, the peptide/microsphere ratio comprises between 0.01 (w/w) and 0.06 (w/w).

[0081] In one embodiment of the pharmaceutical composition, the cyclodextrin is -cyclodextrin.

[0082] As a second object, the present invention discloses a process for the production of said pharmaceutical composition comprising the following steps: [0083] encapsulation of the peptide comprising sequence with at least 80% similarity or identity of SEQ ID NO: 1, or [0084] formation of inclusion compound.

[0085] In one embodiment of the process, the encapsulation comprises the following steps: [0086] sterically stabilized liposomes [0087] extrusion of the DRV suspension

[0088] In one embodiment of the process, the extrusion of the DRV suspension comprises 200 nm pore polycarbonate membranes.

[0089] In one embodiment of the process, the encapsulation comprises the following steps: [0090] Multiple W/O/W emulsion of microspheres [0091] solvent evaporation.

[0092] In one embodiment of the process, encapsulation comprises between 10 and 50% efficiency.

[0093] In one embodiment of the process, the formation of the inclusion compound comprises the following steps: [0094] mixture of cyclodextrin and peptide solutions [0095] continuous stirring until cyclodextrin dissolution [0096] lyophilization of the mixture

[0097] As a third object, the present invention discloses a use of a peptide comprising an amino acid sequence with at least 80% similarity or identity with SEQ ID NO: 1 in the preparation of a pharmaceutical composition for the treatment of diseases associated with intraocular hypertension or glaucoma.

[0098] In one embodiment of use, the pharmaceutical composition is in the preparation of a medication for the treatment of diseases associated with intraocular hypertension or glaucoma.

[0099] As a fourth object, the present invention discloses a method for treating diseases associated with intraocular hypertension or glaucoma comprising administering said pharmaceutical composition in a subject.

[0100] In one embodiment of the method of treatment, the release of the peptide in physiological conditions comprises between 50 and 70% in 8 hours and comprises between 80 and 95% in 48 hours.

[0101] The principal advantage of this invention is related to the use of the biologically active peptide of SEQ ID NO: 1 and analogs thereof, which has a great potential for controlling intraocular pressure by regulating local arterial pressure, in a conventional way, orally or by eye drops.

EXAMPLESEMBODIMENTS

[0102] The examples set forth herein are meant to exemplify one of numerous ways of carrying out the invention, but do not limit the scope thereof.

Example 1. Preparation of the Peptide of SEQ ID NO: 1 in Liposomes

[0103] This example describes the preparation of the peptide of SEQ ID NO: 1 in encapsulated form in sterically stabilized liposomes and improving the bioavailability of the peptide of SEQ ID NO: 1 when administered in this form.

[0104] The preparation of peptide of SEQ ID NO: 1 in encapsulated form in liposomes was carried out according to the Kirby and Gregoriadis Method [Biotechnology 2: 979-984, 1984] and followed by extrusion of the DRV suspension (acronym in English for dehydration-rehydration vesicles, through 200 nm diameter pore polycarbonate membranes [Nayar et al. Biochim. Biophys. Acta. 986:200-206 (1989)]. Liposomes containing encapsulated peptide were separated from the non-encapsulated peptide by means of dialysis and were sterilized by filtration through 0.22 micrometer sterile membranes. A lipid composition of distearoyl-phosphatidylcholine, cholesterol and distearoyl-phosphatidylethanolamine-polyethylene glycol (MW 2,000) and a molar ratio of 5:4:0.3 were chosen. The amount of encapsulated peptide was determined using the intrinsic fluorescence of SEQ ID NO: 1. Encapsulation efficiency was 12% and a peptide/lipid ratio of 0.03 (w/w). The size of liposomes was determined by quasi-elastic light scattering technique. The mean diameter of the vesicles was 0.19 micrometers. Additionally, the present invention can be optimized for up to 50% encapsulation efficiency.

[0105] Liposomes containing SEQ ID NO: 1 (Lang) were unilaterally micro-injected (35 ng of Ang-(1-7) in 200 nL) in the rostro-ventrolateral bulb (RVLB) with a needle (30G) which was slowly inserted into the brain tissue by the dorsal surface using the stereotaxis coordinates: 1.8 mm anterior, 1.8 mm lateral to obex, and only on the pia mater. Empty liposomes (Lvaz) were micro-injected similarly at the same dose of lipid. The arterial pressure was recorded through telemetry for 10 seconds, every 10 minutes, starting 4 previous days and ending 12 days later, in non-disturbed rats with freedom of movement.

[0106] Microinjection of Lang produced a significant pressor effect during the daytime period which was maintained for five days. The highest mean arterial pressure (MAP) was obtained on day 3 (1144 mmHg) which was significantly different from that recorded on day 0 (1003 mmHg). As expected, Lvaz did not produce significant change in MAP (945 mmHg on day 3 vs 905 mmHg on day 0). Furthermore, daytime MAP was significantly higher in Lang group than in Lvaz group on days 1, 2, and 3. Night MAP, in contrast to daytime MAP, was not affected significantly by Lang micro-injection.

[0107] Previous studies have established that microinjection of Free (non-encapsulated) SEQ ID NO: 1 in the RVLB, at a similar dose (25-50 ng), produces an increase of 15 mmHg for approximately 10 min. The short duration of this effect was assigned to increased metabolism of peptide in vivo.

[0108] Therefore, the present technology is characterized by allowing to establish, in chronic conditions, the pressor effect of SEQ ID NO: 1 at RVLB level. It is further characterized by the ability to increase the bioavailability of peptide.

Example 2.Preparation of Peptide of SEQ ID NO: 1 in PLGA Microspheres

[0109] This example describes the preparation of peptide of SEQ ID NO: 1 in PLGA microspheres and the sustained release of the peptide from the resulting formulation.

[0110] Polymer particles were prepared from lactic and glycolic acid co-polymers (PLGA 50:50), by the method of the multiple W/O/W emulsion with later evaporation of the solvent [Jeffery et al. Int. J. Pharm. 77:169-175 (1991)]. Such a method was employed for the encapsulation of Ang-(1-7) with the following steps: 100 mg of PLGA polymer (50:50 w/w) was dissolved in 1 mL of dichloromethane. Next, 1.8 mg of SEQ ID NO: 1 was added, previously dissolved in 200 l of deionized water, and the mixture was undergone to sonication for obtaining a water/oil (W/O) emulsion. The resulting W/O emulsion was added to 50 mL of a 1% PVA solution (w/v) in deionized water. The mixture was undergone to sonication (5000 revolutions/minute) for approximately 1 minute. Thus, the second water/oil/water emulsion (W/O/W) is formed. The emulsion was maintained at continuous stirring for 2 hours at room temperature for evaporating dichloromethane. Next, formed microspheres were subjected to 3 centrifugation/wash cycles with deionized water. The microspheres were then lyophilized and stored at 20 C.

[0111] To determine the amount of incorporated peptide, the peptide was extracted from the polymer particles after polymer dissolution in dichloromethane. The dosage of peptide was carried out by radioimmunoassay [Neves et al., Biochem. Pharmacol. 50:1451-1459 (1995)]. The incorporated amount was 1.9 mg of peptide per g of microspheres, representing a 15% incorporation percentage.

[0112] The kinetics of peptide release was evaluated after re-suspending the microspheres in buffered saline solution (pH 7.2) and incubation at 37 C. These experimental conditions represent model physiological conditions. The released peptide was dosed by radioimmunoassay at intervals of 8 hours, 24 hours, and 48 hours. The percentage of peptide released from the microspheres at standard physiological conditions was about 60% in 8 hours, and about 90% at 48 hours.

[0113] Therefore, this example illustrates the ability of polymeric microspheres to incorporate the peptide and promote an extended release of the peptide.

Example 3. Preparation of the Inclusion Compound of -Cyclodextrin and Derivatives Thereof, and the Peptide of SEQ ID NO: 1

[0114] The preparation is made in equimolar ratios of -cyclodextrin and derivatives thereof, and SEQ ID NO: 1 and/or analogs in aqueous solutions. The mixture of solutions is subjected to continuous stirring until full dissolution of the -cyclodextrin.

[0115] Thereafter the mixture is frozen at liquid nitrogen temperature and subjected to the lyophilization process for 24 hours. Solid, thus obtained, was characterized by physico-chemical analysis techniques. The art that provided major features of the host:guest interaction was the fluorescence and absorption spectroscopy in the ultraviolet-visible region.

[0116] The absorption and biological stability tests were performed with solutions of the peptide-cyclodextrin inclusion compound. To carry out the experiments, 12 normal Wistar rats were used, which had previously cannulated the left femoral artery. Animals were divided into 3 experimental groups and subjected to gavage using saline solution (0.9%/50 L)), SEQ ID NO: 1 (10 g/50 l)) and SEQ ID NO: 1 CD (10 g/50 L). Four blood draws (1 mL) were carried out, being the first prior to gavage, and the three others within 2, 6 and 24 hours after gavage

[0117] The obtained results demonstrated that SEQ ID NO: 1 CD is largely absorbed in the TGI, reaching its maximum blood concentration about 6 hours (620194 pg/mL), returning to near basal values following 24 hours of the gavage (308 pg/mL vs 2510 prior to gavage). Administration of SEQ ID NO: 1 alone also increased the plasma concentration of this peptide 6 hours following its administration (8613 pg/ml) but this increase was about 8 times less than observed with SEQ ID NO: 1 cyclodextrin. Administration of saline did not alter the plasma levels of SEQ ID NO: 1. These results show that SEQ ID NO:1 cyclodextrin may be used for administering SEQ ID NO:1 and likely analogs thereof orally.

Example 4Stability Study of Peptide of SEQ ID NO: 1

[0118] The results obtained during the Long-Term and accelerated stability study demonstrate that the raw material is stable for 36 months in condition of 5 C.3 C. and for 6 months in condition of 25 C.2 C. with 60% relative humidity Example 5Residual solvents in peptide of SEQ ID NO:1

[0119] In the manufacture of pharmaceutical formulations and the chemical synthesis of excipients and drugs, the use of a high number of organic solvents is required, which are not always completely removed during manufacturing processes. These solvents in addition to have no therapeutic value, represent a risk of toxicity to the consumer and carry with them possible adverse effects, making its analysis essential. The ideal is that the presence of these undesirable solvents is the smallest possible (RDC Resolution #57, 2009; United States Pharmacopeia, 2009; International Conference on Harmonization, 1997).

[0120] The test of residual solvents is performed to evaluate the amount of organic solvent present in a given formulation and to verify whether this product has the concentration allowed by law. These tests are generally not mentioned in specific monographs, since the solvents employed vary from one manufacturer to the other. (United States Pharmacopeia, 2009).

[0121] According to developed methodology, it has been found that solvents used in the synthesis pathway, Dimethylformamide, Methanol, Acetonitrile, Dichloromethane, Diethylether, Acetic Acid and Trifluoroacetic acid are controlled by the manufacturer through techniques developed by GC (for dimethylformamide, Methanol, Acetonitrile, Dichloromethane, Diethylether solvents) and HPLC for Acetic Acid and Trifluoroacetic Acid solvents. They assume specifically the values according to table 2:

TABLE-US-00001 Solvent Source Specification Limit Dimethylformamide Reactive Solvent 880 ppm Methanol Reactive Solvent 3000 ppm Acetonitrile Reactive Solvent 410 ppm Dichloromethane Reactive Solvent 600 ppm Diethylether Reactive Solvent 600 ppm Acetic Acid Reactive Solvent It determines and reports Trifluoroacetic Acid Reactive Solvent 10000 ppm

Example 6Physical-Chemical Tests of Peptide of SEQ ID NO: 1

[0122] To ensure the quality of the active substance, in addition to the tests mentioned above, the following tests were proposed for evaluating raw material quality: mass spectrometry in order to characterize the molecule, quantification of amino acids content of liquid peptide and peptide content (HPLC), appearance of sample, solubility, content (purity), water content (HPLC), and microbiological tests.

Example 7Stability Study of Peptide of SEQ ID NO: 1

[0123] Stability is defined as the time during which pharmaceutical expertise or even raw material considered alone is maintained within specified limits and throughout the period of storage and use, the same conditions and characteristics that had upon the time of their manufacture. It can also be defined as the time period comprised between the time at which the product is being manufactured to that when its potency is reduced to not more than 10%, since the alteration products are all securely identified and previously recognized their effects (Taboranski, 2003; Vehabovic et al. 2003; Stulzer & Silva, 2006).

[0124] Stability study was carried out in two conditions, the condition A being at 5 C.3 C. with no humidity and condition B 25 C.2 C. with the relative humidity of 60%5%. The study demonstrated that for all tests that the methodology proposes and from time 0 to time of 36 months in the long-term condition and was also stable in the accelerated condition for 6 months.

[0125] Therefore, the present technology, based on the association of the peptide to the cyclodextrin, allows to increase the peptide bioavailability orally, as well as in the form of intravitreal or intraocular injection and/or by topical use, for example, eye drops.

[0126] Additional tests were performed in order to demonstrate that excellent results were obtained in the association of peptide in the mixture of cyclodextrins, polymers and liposomes.

[0127] Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the disclosed embodiments and in other variants, encompassed within the scope of the appended claims.