NEW GROUP OF PEPTIDES FOR TREATING FEMALE SEXUAL DYSFUNCTION

Abstract

The invention relates to the chemistry of peptides, pharmacology and medicine, and specifically to a new group of peptides having the property of stimulating sexual and genital function and having increased storage stability. For this purpose, peptides of general formula (I) are proposed: A-Thr-Lys-Hyp-B-C-D-X, in which A is 0, Met, Met(0), Thr, Ala, His, Phe, Lys, Gly; B is 0, Gly, Asp, Trp, Gin, Asn, Tyr, Hyp, Arg; C is 0, Arg, Phe, Tyr, Gly, His, Hyp, Lys; D is 0, Val, Gly, Tyr, Trp, Phe, His; X is OH, OCH.sub.3, NH.sub.2; where 0 is the absence of an amino acid residue, provided that if A0, then B and/or C and/or D0, if B0, then C and/or D0, excluding the tetrapeptides, as well as the peptides Phe-Thr-Lys-Hyp-Gly, Thr-Lys-Hyp-Hyp-Arg and Thr-Lys-Hyp-Arg-Gly. The invention also relates to pharmaceutical compositions containing the indicated peptides, as well as to the use of the above indicated compositions for the treatment of women with severely decreased libido or with totally absent libido, orgasmic dysfunction, sexual dysfunction not caused by organic disorders or diseases, HSDD, FSAD or FSIAD, and to a method of treating and/or preventing female sexual dysfunction.

Claims

1. A peptide with general formula (I):
A-Thr-Lys-Hyp-B-C-D-X (SEQ ID NO: 8)formula (I), or its pharmaceutically acceptable salt, in which A is 0, Met, Met(0), Thr, Ala, His, Phe, Lys, Gly; B is 0, Gly, Asp, Trp, Gln, Asn, Tyr, Hyp, Arg; C is 0, Arg, Phe, Tyr, Gly, His, Hyp, Lys; D is 0, Val, Gly, Tyr, Trp, Phe, His; X is OH, OCH.sub.3, NH.sub.2; where 0 is the absence of an amino acid residue, provided that if A0, then B and/or C and/or D0, if B0, then C and/or D0, excluding tetrapeptides, as well as the peptides Phe-Thr-Lys-Hyp-Gly (SEQ ID NO: 9), Thr-Lys-Hyp-Hyp-Arg (SEQ ID NO: 10) and Thr-Lys-Hyp-Arg-Gly (SEQ ID NO: 11).

2. A pharmaceutical composition comprising the peptide of claim 1 and at least one pharmaceutically acceptable excipient.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipient is a vehicle, a preservative, and/or a solvent.

4. The pharmaceutical composition of claim 2, wherein said composition is formulated in a liquid dosage form.

5. The pharmaceutical composition of claim 4, wherein the liquid dosage form is a solution for intranasal administration.

6. The pharmaceutical composition of claim 5, comprising 2 to 20 g/l of the peptide and 0.095 to 0.105 g/l of benzalkonium chloride.

7. The method of claim 11, wherein the sexual dysfunction is characterized by a decreased libido or a total absence of libido.

8. The method of claim 11, wherein the sexual dysfunction is an orgasmic dysfunction.

9. The method of claim 11, wherein the sexual dysfunction is characterized by an aversion to sexual intercourse and absence of sexual satisfaction.

10. The method of claim 11, wherein the sexual dysfunction is vaginism, dyspareunia or hypolibidemia.

11. A method of treating or preventing female sexual dysfunction, hypoactive sexual desire disorder (HSDD), female sexual arousal disorder (FSAD), or female sexual interest/arousal disorder (FSIAD) in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 2.

12. The method of claim 11, wherein the pharmaceutical composition is administered intranasally.

13. (canceled)

14. The pharmaceutical composition of claim 2, comprising 2 to 20 g/l of the peptide and 0.095 to 0.105 g/l of benzalkonium chloride.

15. A method for stimulating sexual and genital function in a subject in need thereof, said method comprising administering to the subject an effective amount of the peptide of claim 1.

16. A method of treating or preventing female sexual dysfunction, HSDD, FSAD, or FSIAD in a subject in need thereof, comprising administering to the subject an effective amount of the peptide of claim 1.

17. The pharmaceutical composition of claim 2, wherein said composition is formulated in a spray for intranasal administration.

18. The method of claim 16, wherein the sexual dysfunction is characterized by decreased libido or complete absence of libido.

19. The method of claim 16, wherein the sexual dysfunction is an orgasmic dysfunction.

20. The method of claim 16, wherein the sexual dysfunction is characterized by an aversion to sexual intercourse and absence of sexual satisfaction.

21. The method of claim 16, wherein the sexual dysfunction is vaginism, dyspareunia or hypolibidemia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1. Diagram for the synthesis of the tripeptide Thr-Lys-Hyp.

[0042] FIG. 2. Diagram for the synthesis of the pentapeptide Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12).

[0043] FIG. 3. Diagram for the synthesis of the hexapeptide Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13).

[0044] FIG. 4. Comparison of the influence of the peptide Thr-Lys-Hyp with the peptide Thr-Lys-Pro on the proceptive behavior of the rat.

[0045] FIG. 5. Comparison of the influence of the peptide Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12) with the peptide Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6) on the proceptive behavior of the rat.

[0046] FIG. 6. Comparison of the influence of the peptide Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13) with the peptide Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7) on the proceptive behavior of the rat.

DETAILED DISCLOSURE OF THE INVENTION

Definition and Terms

[0047] For a better understanding of the present invention, several terms used in the present invention are presented below.

[0048] Female sexual dysfunction is a disturbance in the course of the sexual responses at the stage of arousal, in the primary phase, the stage of orgasm or release, and painfulness during intercourse. The disorder, including subjective and objective factors, prevents the obtaining of satisfaction. Furthermore, it sometimes leads to infertility or is more or less connected with it.

[0049] Libido (from the Latin: attraction, desire, passion) means sexual desire or sexual instinct. It is the psychological component of sexual attraction.

[0050] Hypolibidemia, anaphrodisia is the absence or loss of sexual attraction. Hypolibidemia is one of the sexual dysfunctions not caused by organic disorders or diseases (code F52.0 of MKB-10).

[0051] HSDD is hypoactive sexual desire disorder.

[0052] FSAD is female sexual arousal disorder.

[0053] FSIAD is female sexual interest/arousal disorder.

[0054] Compliance is adherence to the treatment, the degree of correspondence between the patient's behavior and the recommendations given by the doctor.

[0055] Met(0) in the present document means methionine sulfoxide.

Implementing of the Invention

[0056] The possibility of objective manifestation of the technical result when using the invention is confirmed by reliable data presented in the examples, containing information of an experimental nature. It should be understood that these and all the examples presented in the materials of the application are not limiting and are given only as an illustration of the present invention.

Survey of Methods of Obtaining the Peptides Per the Invention

Example 1. Synthesis of the Tripeptide Thr-Lys-Hyp

[0057] The synthesis of the tripeptide Thr-Lys-Hyp is done according to the diagram presented in FIG. 1. The synthesis of the tripeptide Thr-Lys-Hyp is done using modern protective groups and methods of creating a peptide bond in solution that are known from the prior art. To create the peptide bond, the method of activated esters and the method of TBA (tetrabutylammonium) salts are used. The peptide chain is grown in stages.

Example 2. Synthesis of the Pentapeptide Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12)

[0058] The synthesis of the pentapeptide Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12) is done according to the diagram presented in FIG. 2. The synthesis of the pentapeptide Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12) is done using modern protective groups and methods of creating a peptide bond in solution that are known from the prior art. To create the peptide bond, the method of activated esters and the carbodiimide method are used. Both a stagewise growth of the peptide chain and a block method are used.

Example 3. Synthesis of the Hexapeptide Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13)

[0059] The synthesis of the hexapeptide Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13) is done according to the diagram presented in FIG. 3. The synthesis of the hexapeptide Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13) is done using modern protective groups and methods of creating a peptide bond in solution that are known from the prior art. To create the peptide bond, the method of TBA salts, the method of activated esters, the carbodiimide method and the method of mixed anhydrides are used. Both a stagewise growth of the peptide chain and a block method are used.

[0060] For the synthesis of the peptides presented in examples 1-3, derivatives of both protected and free L-amino acids are used. The solvents used during the synthesis of the peptides are dehydrated in corresponding manner. The boiling down of the solutions is done on a vacuum evaporator at 40 C. The obtained intermediate compounds and the synthesized peptides are checked with the aid of TLC analysis on plates of silica gel from the Silufol company (Czech Republic). The compounds obtained are detected by spraying the plate with a solution of ninhydrin and (or) o-tolidine. Verification of the homogeneity of the obtained peptides is done with the aid of high-efficiency liquid chromatography (HELC) on a liquid microcolumn chromatograph Millichrom A-02. The structure of the synthesized peptides is characterized with the aid of mass spectrometry on a mass spectrometer from the firm ThermoElectron LCQ Advantage MAX.

[0061] The results of the chromatographic and mass spectrometry analysis of the peptides according to the invention, presented in examples 1-3, are shown in table 1. The form of the gradient for separating the synthesized peptides by the HELC method is shown in table 2.

TABLE-US-00001 TABLE1 Primarychromatographicandmassspectrometryparametersofthepeptides oftheinvention. Chromatographic Massspectrometry Molecular characteristics characteristics weight,MW, Retention **Fragmenting No. Peptide g/mol time,Tr,min Purity,% *[M+H].sup.+ ofmol.peak 1 Thr-Lys-Hyp 360 3.02 98.1 361 343(100), 230(44), 260(16) 2 Thr-Lys-Hyp-Arg-Hyp 629 4.56 98.5 630 612(100), (SEQIDNO:12) 230(63), 499(32) 3 Thr-Lys-Hyp-Arg-Hyp- 776 10.31 97.9 777 759(100), Phe(SEQIDNO:13) 612(71), 499(43) Note: *Molecular peak corresponding to the ion [M+H].sup.+ **The most intensive ions forming upon fragmenting of the molecular peak at energy of ion collisions with helium atoms of 35 eV.

[0062] Table 1 presents the data (HELC) obtained on the liquid microcolumn chromatograph Millichrom A-02 and the mass spectrometry characteristics of the synthesized peptides obtained with the aid of the mass spectrometer ThermoElectron LCQ Advantage MAX.

[0063] The chromatography conditions that were developed make it possible to easily obtain chromatographically pure products.

[0064] Chromatography Conditions for Analysis of the Peptides:

[0065] Chromatograph Millichrom A-02

[0066] Column: Prontosil 120-5C18aq, 2*75 mm

[0067] Eluent A: 0.2M LiClO.sub.4+5 mM HClO.sub.4

[0068] Eluent B: methanol

TABLE-US-00002 TABLE 2 Form of the gradient for separation of the synthesized peptides. Time Eluent B, % 0 5 16.5 80

[0069] Flow rate: 150 mcl/min;

[0070] Detector wavelength set: 210, 220, 230, 240 nm.

[0071] Mass Spectrometry Analysis Conditions:

[0072] Instrument: ThermoElectron LCQ Advantage MAX;

[0073] Ion source: electrospray; direct feeding to the source of a peptide solution with concentration of 10 mcg/ml in 0.1% acetic acid at a rate of 5 mcl/min;

[0074] Fragmenting of the molecular ion at 35 eV by the method of ion collisions (He). Temperature of source 250 C., ionization potential +3.5 kV.

[0075] The Method of Therapeutic Use of the Compounds

[0076] The subject matter of the present invention also includes the administering to a subject in need of the corresponding treatment of a therapeutically effective quantity of a peptide of the general formula (I).

[0077] The term therapeutically effective quantity is understood to be that quantity of a peptide which, when administered as a monotherapy or a combined therapy, elicits a therapeutic effect sufficient for the treatment of female sexual dysfunction. When the peptide of the invention is used in combined therapy, the term therapeutically effective quantity refers to the combination of the quantity of active ingredients the taking of which leads to the preventive or therapeutic effect when taken in succession or at the same time. The exact quantity required may vary from one subject to another depending on the age and the general condition of the patient, the severity of the illness, the technique of administering the drug, combined treatment with other drugs, and so on.

[0078] The invention also relates to pharmaceutical compositions which contain peptides according to the invention and at least one pharmaceutically acceptable excipient, especially a vehicle, a preservative, and/or a solvent, which can be given to the patient along with the peptides constituting the essence of the present invention, and not disturbing the biological activity of said peptide, and being nontoxic when given in doses adequate to deliver the effective quantity of the peptide.

[0079] Pharmaceutically Acceptable Peptide Derivatives

[0080] The compounds of the present invention may exist in the free form in the course of their processing or, if so required, in the form of a pharmaceutically acceptable salt or other derivative. The term pharmaceutically acceptable salt used here pertains to those salts which, in the context of a medical evaluation, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, and so forth, and which have a reasonable risk/benefit ratio. The salts may be obtained in the course of the synthesis, the separation or the purification of the compounds according to the invention, and they may also be obtained separately, by the reacting of the free acid or the free base of the compound of the invention with a suitable base or acid, respectively. Examples of pharmaceutically acceptable, nontoxic salts of acids are, in particular, the acetate, the hydrochloride, the citrate, and others.

[0081] Pharmaceutical Compositions

[0082] The invention also relates to pharmaceutical compositions containing at least one of the compounds described here (or a prodrug form, a pharmaceutically acceptable salt or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable vehicles, solvents, and/or fillers. Said compositions may also contain one or more additional therapeutic agents. On the other hand, a compound of the present invention may be given to a patient requiring the corresponding therapy in combination with one or more other therapeutic regimens.

[0083] The pharmaceutical compositions proposed in the present invention contain compounds of the present invention together with pharmaceutically acceptable vehicles, which may include any given solvents, diluents, dispersions or suspensions, surfactants, isotonic agents, preservatives, and so forth, suitable for the specific dosage form. Except for such cases where the medium of the usual vehicles is incompatible with the compound of the invention, for example upon appearance of any unwanted biological effects or other unwanted interactions with any other component(s) of the pharmaceutical composition, the use of such compositions is within the scope of the present invention.

[0084] Investigations of the Stability of Pharmaceutical Forms Containing Peptides Having the Property of Stimulating the Genital and Sexual Functions

[0085] This experiment investigated the stability of compositions containing peptides according to the invention. The results of the investigations are illustrated by the example of several peptides according to the invention containing hydroxyproline, in particular Thr-Lys-Hyp (SEQ ID NO: 12), Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 13), Thr-Lys-Hyp-Arg-Hyp-Phe and Thr-Lys-Hyp-Arg-Hyp-Gly-Hyp (SEQ ID NO: 14).

[0086] The peptides described in RU2507212 were also obtained, in particular Thr-Lys-Pro, Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6), Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7) and Thr-Lys-Pro-Arg-Pro-Gly-Pro (SEQ ID NO: 1).

[0087] From the compounds obtained, a liquid pharmaceutical form (a solution) was prepared, including the peptide and the preservative benzalkonium chloride with the following ratio of components: 0.2 and 0.01 mass %, respectively, water the rest. Samples were placed in storage at two temperature regimes, namely, +4 C. and +25 C. Investigation of the stability of the peptides was carried out after 1, 3, 6, 12, and 24 months of storage. In the investigation of the samples for stability, the quantity of single impurities and the total quantity of impurities were evaluated, testifying to the degradation of the peptides. The pharmaceutical requirements of the company for impurities are: any single impurity, not more than 1.5%; total content of impurities, not more than 3.0%. If the content of impurities exceeded the above indicated values, the pharmaceutical form was deemed to have failed the test for stability. The results of the investigation are presented in table 3.

TABLE-US-00003 TABLE3 Resultsoftheinvestigationofthestabilityofpharmaceuticalformscontaining peptideswiththepropertyofstimulatinggenitalandsexualfunctionat+4 C.and +25 C. Storedfor1 Storedfor3 Storedfor6 Storedfor12 Storedfor24 Peptideformula month months months months months andstorage Single/total Single/total Single/total Single/total Single/total temperature impurities(%) impurities(%) impurities(%) impurities(%) impurities(%) Thr-Lys-Pro,at 0.051/0.13 0.053/0.135 0.3/0.9 0.9/1.7 1.2/2.2 +4 C. Thr-Lys-Pro,at 0.06/0.1 0.13/0.25 1.7/3.1 +25 C. Thr-Lys-Pro-Arg- 0.042/0.09 0.042/0.93 0.09/1 0.8/1.5 1.1/1.9 Pro(SEQIDNO: 6),at+4 C. Thr-Lys-Pro-Arg- 0.067/0.54 0.19/0.9 1.4/3.0 4/6 Pro(SEQIDNO: 6),at+25 C. Thr-Lys-Pro-Arg- 0.054/0.17 0.09/0.18 0.5/1.1 0.9/1.3 1.6/2,9 Pro-Phe(SEQID NO:7),at+4 C. Thr-Lys-Pro-Arg- 0.11/0.19 0.9/1.1 1.9/3.5 Pro-Phe(SEQID NO:7),at+25 C. Thr-Lys-Pro-Arg- 0.05/0.17 0.07/0.2 0.8/1.4 1/1.7 2/3.7 Pro-Gly-Pro (SEQIDNO:1), at+4 C. Thr-Lys-Pro-Arg- 0.065/0.13 0.9/1.2 1.7/3.1 Pro-Gly-Pro (SEQIDNO:1), at+25 C. Thr-Lys-Hyp,at 0.034/0.09 0.035/0.11 0.036/0.2 0.09/0.3 0.1/0.9 +4 C. Thr-Lys-Hyp,at 0.052/0.11 0.052/0.9 0.2/1 0.3/1.2 0.8/1.8 +25 C. Thr-Lys-Hyp-Arg- 0.03/0.09 0.03/0.09 0.04/0.1 0.07/0.2 0.1/0.7 Hyp(SEQID NO:12),at+4 C. Thr-Lys-Hyp-Arg- 0.046/0.14 0.047/0.15 0.1/0.7 0.2/0.9 0.9/1.2 Hyp(SEQID NO:12),at+25 C. Thr-Lys-Hyp-Arg- 0.067/0.15 0.068/0.15 0.08/0.2 0.1/0.3 0.9/1.9 Hyp-Phe(SEQ IDNO:13),at +4 C. Thr-Lys-Hyp-Arg- 0.032/0.09 0.033/0.09 0.09/0.6 0.4/0.8 1.3/2.7 Hyp-Phe(SEQ IDNO:13),at +25 C. Thr-Lys-Hyp-Arg- 0.049/0.12 0.05/0.12 0.06/0.14 0.09/0.3 0.1/1.1 Hyp-Gly-Hyp (SEQIDNO:14), at+4 C. Thr-Lys-Hyp-Arg- 0.069/0.14 0.07/0.5 0.09/0.9 0.3/1.1 1.2/2.6 Hyp-Gly-Hyp (SEQIDNO:14), at+25 C.

[0088] It emerges from table 3 that the peptides containing hydroxyproline instead of proline were much more successful in passing the test for stability during storage (content of impurities in the samples with peptides containing hydroxyproline is several times less than that in the samples with peptides containing proline), and this at both temperatures. Thus, the peptides containing hydroxyproline are more stable to degradation than their peptide analogues (RU2507212), which makes it possible to keep the finished pharmaceutical forms based on the peptides of the invention at temperature up to +25 C. for at least 24 months.

[0089] Investigation of the Efficacy of the Peptides of the Invention as a Means of Stimulating the Genital and Sexual Functions

[0090] In order to confirm the efficacy as a means of stimulating the genital and sexual functions, peptides according to the invention were synthesized, in particular the tripeptide Thr-Lys-Hyp, the pentapeptide Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12), and the hexapeptide Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13), and tests were performed as to the efficacy in vivo on the relevant preclinical model (Lordoz test). Furthermore, the efficacy of the group of peptides Thr-Lys-Pro, Thr-Lys-Pro-Arg-Pro (SEQ ID NO: 6), and Thr-Lys-Pro-Arg-Pro-Phe (SEQ ID NO: 7) was also investigated.

[0091] The efficacy of the aforementioned groups of peptides was investigated in a dose of 100 mcg/rat with respect to the sexual behavior of female rats. The sexual behavior was registered over the course of 0-16 days in ovarioectomized, hormonally stimulated females in the case of direct contact with sexually active males and in the case when no such contact was possible. It was found that the peptides of the group Thr-Lys-Hyp, Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12), and Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13) increase the intensity of proceptive behavior of the females from 124 to 244-366 acts during the time of the registration (p=0.028, Wilkinson's criterion). The results testify to an increase in sexual motivation under the action of the peptides Thr-Lys-Hyp, Thr-Lys-Hyp-Arg-Hyp (SEQ ID NO: 12), and Thr-Lys-Hyp-Arg-Hyp-Phe (SEQ ID NO: 13). The results of the test are presented in table 4.

TABLE-US-00004 TABLE4 Resultsofinvestigatingtheefficacyofthepeptidesoftheinventionasameans ofstimulatingthegenitalandsexualfunctionsandthepeptidesdescribedin RU2507212,onarelevantpreclinicalmodel(Lordoztest). Pro- In- Numberofproceptivebehavioracts gesterone, vestigated Dayofgivingthedrug Group mg/rat drug,pig/rat 0 5 11 16 Thr-Lys-Pro 0.5 100 14 4 15 4 20 4 28 4 Thr-Lys-Hyp 0.5 100 12 4 25 4 24 4 26 4 Thr-Lys-Pro-Arg-Pro 0.5 100 12 4 15 4 24 4 36 6 (SEQIDNO:6) Thr-Lys-Hyp-Arg-Hyp 0.5 100 13 4 38 4 36 4 36 6 (SEQIDNO:12) Thr-Lys-Pro-Arg-Pro-Phe 0.5 100 14 4 16 4 19 4 26 4 (SEQIDNO:7) Thr-Lys-Hyp-Arg-Hyp- 0.5 100 14 4 24 4 23 4 24 4 Phe(SEQIDNO:13)

[0092] Moreover, if one compares the data obtained in dynamic manner (comparing of the proceptive behavior of the female rats on day 5, 11 and 16 of administering the peptides), It is evident that the effect from the action of the peptides of the invention develops twice as fast as that from the action of the peptides described in RU2507212 (FIG. 1-3).

[0093] Thus, the data obtained testify to the fact that the peptides of the invention containing hydroxyproline not only have a greater stability as compared to the peptide analogues from RU2507212, but also have a higher rate of achieving a positive (therapeutic) effect, which in turn makes it possible to shorten the treatment course of the patient by using a pharmaceutical based on the peptides of the invention. Moreover, the effect is specific and is manifested in an adequate behavioral situation.

[0094] Even though the invention has been specified with reference to the variant embodiments disclosed, it will be evident to the person skilled in the art that the specific experiments described in detail are given merely for purposes of illustrating the present invention and should not be considered as limiting the scope of the invention in any way. It should be understood that it is possible to make various modifications without departing from the essence of the present invention.