Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

• the invention relates to the prophylactic or therapeutic treatment of skin tumors in early stages. More particularly, the invention relates to the use of a TGF- ⁇ 1 inhibitor peptide for the prophylactic or therapeutic treatment of early-stage skin tumors, preferably by topical application.
• Skin cancer is a malignant growth of certain cell types of the skin that can have many causes, with solar radiation induction being one of the best known.
• Skin cancer usually develops in the epidermis, the outermost layer of the skin, and can also be located in the dermis.
• the most common types of skin cancer are basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and melanoma.
• Basal cell carcinoma tends to be slow growing and rarely spreads.
• Squamous cell carcinoma is usually more aggressive than basal cell cancer, being more likely to spread to other parts of the body.
• the most dangerous type of skin cancer is melanoma, especially malignant melanoma, which can be fatal if not treated early.
• Skin cancer is one of the fastest growing cancers and outnumber lung, breast, colorectal and prostate cancer.
• TGF- ⁇ 1 The transforming growth factor ⁇ 1 (TGF- ⁇ 1) is a multifunctional cytokine that regulates a variety of cellular processes, such as cell proliferation, differentiation, apoptosis, tissue remodeling and angiogenesis.
• TGF- ⁇ 1 has a double function in the carcinogenesis of the skin, acting as a suppressor of the formation of benign tumors in the early stages of tumor development and as a stimulator of malignant progression in the last stages of carcinogenesis, promoting invasion and metastasis (RJ. Akhurst et al., "Genetic events and the role of TGF beta in epithelial tumour progression ", J. Pathol. 1999, vol. 187, pp. 82-90). Therefore, it is expected that the inhibition of said factor in the last stages of carcinogenesis has a suppressive effect on malignant progression, while that its inhibition in the initial stages of tumor development should have a stimulating effect on the formation of benign tumors and on their progression to malignant carcinoma.
• peptide P144 is described for the first time (whose DCI is distinguished, SEQ ID NO: 1). It is also described that said peptide is useful for the treatment of liver diseases, more specifically for the treatment of liver fibrosis.
• WO07048857 describes the use of disitertide as a modulating agent of the immune response and for the treatment of cancer.
• the use of disitertide for the treatment of cancer is in accordance with the studies mentioned above that suggest the stimulating effect of the factor on the malignant progression in the last stages of carcinogenesis.
• TGF- ⁇ 1 inhibitor peptides inhibit on the one hand the formation of skin tumors in the early stages and, on the other, their subsequent progression to carcinoma.
• disitertide inhibits the formation of papillomas and retards its progression to carcinoma in mice.
• disitertide could be useful for the treatment of cancer in advanced stages, nothing related or suggested is related to the use of disitertide for the treatment of skin tumors in early stages.
• one aspect of the invention refers to a TGF- ⁇ 1 inhibitor peptide for use in the preventive or therapeutic treatment of early skin tumors, where the peptide is disitertid or a derivative thereof.
• Another aspect of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of the peptide defined above, together with at least one pharmaceutically acceptable excipient, diluent or vehicle, for use in the preventive or therapeutic treatment of skin tumors in initial stages
• Another aspect of the invention relates to the use of the peptide defined above for the manufacture of a medicament for the preventive or therapeutic treatment of early skin tumors in a mammal, including a human.
• This last aspect of the invention relates to a method for preventing or treating in a mammal, including a human, a tumor of the skin in an initial stage, in particular a papilloma or actinic keratosis, where the method comprises applying to the skin. of the mammal a composition comprising a therapeutically effective amount of the peptide defined above, together with pharmaceutically acceptable diluents or carriers.
• the mammal has a tumor of the skin in an initial stage and the method comprises applying the composition on the skin of the mammal to treat the tumor and prevent its progression to carcinoma.
• the mammal does not have a tumor of the skin and the method comprises applying the composition on the skin of the mammal to prevent the tumor.
• the topical application of a peptide according to The invention on the skin of mice used as an animal model protects the development of both benign papillomas and CCEs induced by DMBA / TPA. Therefore, said results demonstrate that the peptides of the present invention are useful in the preventive or therapeutic treatment of early-stage skin tumors, such as papillomas or actinic keratosis.
• the peptides of the invention are preferably applicable in the skin of a human, although if desired they can also be applied in the skin of mammals in general.
• the sequence SEQ ID NO: 1 (disitertide) or a derivative thereof is included.
• Disitertide (DCI of peptide P144) is described for the first time in WO 00/31135.
• the disitertide comprises amino acids 730-743 of the type III receptor of TGF- ⁇ 1 (accession number Q03167, SwissProt).
• initial stage tumor refers to tumors confined in the place of development that have not invaded surrounding tissues, or to those that have invaded but in which the area of the invasion is confined to a localized area. This definition is applicable to the vast majority of cancers, including skin cancer. Actinic or solar keratosis is considered the most initial stage for the development of skin cancer. More specifically, actinic keratosis is considered a pre-cancer and is the main precursor of the CCE. Skin papillomas are considered another type of early stage tumor, constituting benign non-cancerous tumors.
• the term "disitertide derivative” includes mammalian fragments, analogs and homologues of the sequence SEQ ID NO: 1, as long as they maintain their ability to inhibit the biological activity of TGF- ⁇ 1.
• the ability of the peptides of the invention to inhibit the biological activity of TGF- ⁇ 1 can be evaluated and, if desired, quantified, by means of the growth inhibition assay of the cell line Mv-1 -Lu, a cell line derived from mink pulmonary epithelium whose proliferation is inhibited by TGF- ⁇ 1, as described in WO 00/31135.
• fragment means any amino acid sequence shorter by at least one amino acid than the sequence of origin SEQ ID NO: 1 and which comprises a length of consecutive amino acids, preferably of at least 6 residues, of the sequence of origin.
• analog includes peptides in which one or more amino acids of SEQ ID NO: 1 have been replaced by a different amino acid.
• Conservative amino acid substitutions are preferred. Conservative substitutions are those where an amino acid residue is replaced by another biologically similar residue. Examples of conservative substitutions include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine with another, or the replacement of a polar residue with another such as between arginine and lysine, between glutamic and aspartic or between glutamine and asparagine.
• Conservative substitutions also include the use of a substituted amino acid instead of an unsubstituted amino acid, provided that the ability of the peptide to inhibit the biological activity of TGF- ⁇ 1 is maintained.
• TGF- ⁇ 1 inhibitor peptides of the present invention also include peptides that have at least 75% homology with the amino acid sequence of the disitertide, preferably at least 85% homology and, more preferably, at least 90% homology with said peptide, provided that the ability to inhibit the biological activity of TGF- ⁇ 1 is maintained.
• the peptides of the invention may have chemical modifications in the amino acids.
• hybrid fusion peptides or dimers comprising the sequence SEQ ID NO: 1 or its fragments.
• mammalian homologue includes peptides with the same biological function of species other than human.
• a homologous mammalian peptide of the disitertide is the P54 peptide, with sequence SEQ ID NO: 2, which comprises amino acids 731-742 of the rat type III receptor of TGF- ⁇ 1 (accession number P26342, SwissProt).
• sequence SEQ ID NO: 2 which comprises amino acids 731-742 of the rat type III receptor of TGF- ⁇ 1 (accession number P26342, SwissProt).
• Ia disitertide is equivalent to peptide P54.
• the ability for both the disitertide and the P54 peptide to act as inhibitors of TGF- ⁇ 1 has been previously demonstrated, as described in WO 00/31135.
• the peptide for use in the preventive or therapeutic treatment of early-stage skin tumors is different (SEQ ID NO: 1).
• the peptide of the present invention is useful as an inhibitor of the generation of papillomas of the skin and its progression to carcinoma. More preferably, the peptide of the present invention is useful for use in the preventive or therapeutic treatment of actinic keratosis.
• the peptides of the invention can be obtained by conventional methods, for example, by solid phase chemical synthesis techniques and purified by high performance liquid chromatography (HPLC), for example, as described in WO 00/31135. If desired, they can be analyzed by conventional techniques, for example, by sequencing and mass spectrometry, amino acid analysis, nuclear magnetic resonance, etc. Alternatively, the peptides of the invention can be obtained by means of recombinant DNA technology.
• HPLC high performance liquid chromatography
• pharmaceutically acceptable salts of the peptide of the invention are pharmaceutically acceptable salts of the peptide of the invention.
• pharmaceutically acceptable salts includes salts commonly used to form metal salts or acid addition salts. The nature of the salt is not critical as long as it is pharmaceutically acceptable.
• Pharmaceutically acceptable salts of the peptide of the invention can be obtained from acids or bases, organic or inorganic. Said salts can be obtained by conventional methods well known to those skilled in the art.
• Peptide administration can be carried out by any route of administration.
• the administration of the peptide of the invention is carried out topically.
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of the peptide of the invention together with at least one pharmaceutically acceptable excipient, diluent or vehicle for use in the preventive or therapeutic treatment of skin tumors in early stages.
• the pharmaceutical composition of the present invention may contain one or more peptides of the invention, optionally, together with one or more alternative TGF- ⁇ 1 inhibitor compounds.
• Said pharmaceutical composition is useful for administration in the body of a mammal, preferably in the human body.
• the pharmaceutical composition of the present invention is a topical composition, and can be presented, for example, in the form of lotion, ointment, gel, cream, patch or spray.
• the amount of pharmaceutically acceptable peptide or salt thereof that may be present in the pharmaceutical composition of the present invention may vary within a wide range.
• the dosage for the preventive or therapeutic treatment of skin tumors in early stages with the peptides and / or pharmaceutical compositions of the invention will depend on numerous factors, including the age, condition of the patient, the severity of the disease or pathological alteration, Ia route and frequency of administration and of the peptide of the invention to be administered.
• the invention relates to the use of a peptide of the invention for the manufacture of a medicament to inhibit the generation of papillomas and their progression to carcinoma. In another preferred embodiment, the invention relates to the use of a peptide of the invention for the manufacture of a medicament for the preventive or therapeutic treatment of actinic keratosis.
• Fig. 1 represents images of the dorsal skin of treated mice representative of different groups, as described in the examples.
• EXAMPLE 1 Preparation of a topical formulation
• a vehicle topical formulation and another containing disitertide 300 ⁇ g / g were prepared.
• the carrier formulation was prepared by mixing the following components: 10% dimethicone, 43.5% liquid paraffin, 0.02% methylparaben, 0.01% propylparaben, 0.5% cetrimide and a 1.5% ketostearyl alcohol. This mixture was heated to 50-60 0 C and emulsified with purified water to 100% (also at 50-60 0 C).
• the disitertide formulation was prepared in the same manner, except that the water was replaced by a mixture of water plus 0.03% of disitertide previously dissolved in 0.2 ml of dimethylsulfoxide (0.22% w / w).
• EXAMPLE 2 Effect of the disitertide on the formation of papillomas and on the development and progression of papillomas is established.
• mice Female Swiss albino mice (4-6 weeks old) purchased from Har ⁇ an SL (Barcelona, Spain) were used. Before being treated, the animals were subjected to an adaptation period of 1-2 weeks under conventional temperature and humidity conditions. They were provided with sterile water and a standard ad libitum diet. All animal experiments were approved and performed according to institutional guidelines for the care and use of animals for experimentation.
• the two-stage chemical mouse carcinogenesis protocol was used in the mouse.
• Mouse skin is an epithelial tissue with certain advantages as an experimental model to study the changes associated with neoplastic development.
• This protocol involves the treatment of mice with a single dose of a carcinogenic initiator, specifically 7,12-dimethylbenz (a) anthracene (DMBA), followed by repeated applications of the tumor promoter 12-O-tetradecanoyl forbol-13-acetate (TPA) .
• DMBA 7,12-dimethylbenz
• TPA tumor promoter 12-O-tetradecanoyl forbol-13-acetate
• This treatment results in the appearance of benign tumors (papillomas). Most benign papillomas disappear or return, but a small fraction of the system (5-10%) progress to malignant squamous cell carcinomas (SCC), which cross the basement membrane and progressively invade the underlying dermis, subcutaneous tissue and the muscle.
• SCC malignant squamous cell carcinomas
• Tumors were induced on the shaved dorsal skin of mice 6-8 weeks of age by a single topical application of DMBA (32 ⁇ g in 200 ⁇ l of acetone) followed by treatment twice a week with 12.5 ⁇ g of TPA in 200 ⁇ l of acetone (10 4 M) for 12 weeks. Groups of mice were further treated with a topical formulation in the form of a cream containing or not disitertide, as specified below. Each week the number of tumors with a diameter greater than 2 mm in each mouse was recorded. At the end of the experiment, the animals were anesthetized and sacrificed (32 weeks after initiation with DMBA), and the tumors were fixed in 10% formaldehyde and embedded in paraffin for histological studies.
• the TPA was replaced by a treatment with a topical formulation in the form of a cream containing disitertide (group 2, formed by 5 animals) or only with vehicle (group 1, composed of 5 mice) during the phase of promotion of chemical carcinogenesis.
• group 2 formed by 5 animals
• vehicle group 1, composed of 5 mice
• the possible carcinogenic activity of the disitertide on a group of 5 mice was analyzed by repeated applications of disitertide every two days for 23 weeks.
• mice initiated with DMBA and promoted with TPA were further treated with disitertide every 2 days for 4 and 12 weeks (groups of 5 and 6, respectively, 10 animals each one), in order to evaluate if the disitertide could prevent against the carcinogenesis of the skin.
• the treatment with disitertide began in the same week in which the mice started with DMBA (week 0) and applications were made on alternate days with respect to the TPA.
• a group of mice (group 4, composed of 5 animals) was treated only with vehicle (control cream) for 12 weeks.
• a group of animals with tumors (group 7, composed of 10 animals) was treated with disitertide every 2 days for 20 weeks from week 12 to week 32 after initiation with DMBA. This group of mice was included to evaluate a possible anti-tumor effect of the peptide.
• the disitertide was applied at a dose of 60 ⁇ g in 200 ⁇ l of the topical formulation in the form of cream, except for the mice of group 7 in which the dose of disitertide was increased to 120 ⁇ g (in 400 ⁇ l of cream).
• a 30-60% reduction in the appearance of benign papillomas was observed with respect to untreated mice (group 3) or mice treated only with vehicle (group 4), as can be seen at 10 and 14 post-initiation weeks (Table 1, Fig. 1).
• mice initiated with DMBA were promoted with the cream containing disitertide or only with vehicle for 12 weeks (groups 2 and 1, respectively).
• groups 2 and 1 mice initiated with DMBA were promoted with the cream containing disitertide or only with vehicle for 12 weeks (groups 2 and 1, respectively).
• group 1 mice initiated with DMBA were promoted with the cream containing disitertide or only with vehicle for 12 weeks (groups 2 and 1, respectively).
• group 1 did not initiate or promote the carcinogenesis.
• the disitertide also showed no harmful effect on normal skin after repeated applications every 2 days for more than 20 weeks (data not shown).
• mice In order to analyze the effect of the disitertide on the development and progression of papillomas already established, a group of mice (group 7) was included in which the application of the disitertide was started in week 12 after initiation with DMBA, when each mouse had approximately 7-8 papillomas on average (Table 1), and lasted for 20 weeks. No significant reduction in the number or size of papillomas was observed in this group. In fact, after 14 weeks post-initiation, the average number of tumors larger than 2 mm in diameter per mouse tended to be matched in all groups (Table 1). The same happened with the average number of tumors larger than 5 mm in diameter (Table 2).
• the initial papillomas are generally small (see Fig. 1), but as they grow they adopt a cauliflower-like structure with a narrow base or a broad base.
• CCEs are normally endophytic tumors that present as plaques with an ulcerated surface. Based on these characteristics, the presence of carcinomas in each mouse was examined every week. As shown in Table 3, the first carcinoma observable in control mice (groups 3 and 4) and in treated mice with disitertide for 4 weeks (group 5) occurred in weeks 17-18 post-initiation. Remarkably, mice treated with disitertide during weeks 12 to 32 (group 7, who had papillomas at the beginning of treatment, showed a delay of about 14 weeks in the first occurrence of carcinomas. Mice treated with disitertide for 12 weeks post-initiation (group 6) had a delay of about 6 weeks. These results show that the application of disitertide on the skin not only prevents tumor formation but also postpones its malignant conversion.
• the time of appearance of the carcinomas was estimated by visual observation
• mice show that the disitertide can protect against the carcinogenesis of the chemical skin.
• the topical application of disitertide on the skin during the promotion of tumors with APT reduced the early onset of benign papillomas.
• the topical formulation comprising disitertide was applied directly on the surface of the tumors, their conversion from papillomas to malignant carcinomas was delayed.

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