Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links 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
• • 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
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/04—Preparations for care of the skin for chemically tanning the skin

Definitions

• This application claims the benefit of European Patent Application EP19382674.0 filed on August 2 nd , 2019.
• Technical Field This invention generally relates to the field of melanocyte-related disorders.
• the present invention refers to peptides with the ability to regulate melanogenesis, either promoting or reducing the production of melanin, as well as to inhibit the proliferation of malignant melanocytes; to pharmaceutical and cosmetical compositions containing thereof as an active ingredients, as well as to their use in therapy and cosmetics.
• Background Art Through a process called melanogenesis, melanocytes produce melanin, which is a pigment found in the skin, eyes, hair, nasal cavity, and inner ear.
• This melanogenesis leads to a long-lasting pigmentation, which is in contrast to the pigmentation that originates from oxidation of already-existing melanin.
• melanogenesis There are both basal and activated levels of melanogenesis; in general, lighter-skinned people have low basal levels of melanogenesis.
• Exposure to UV-B radiation causes increased melanogenesis.
• the purpose of melanogenesis is to protect the hypodermis, the layer under the skin, from damage by UV-B radiation.
• the color of the melanin is black, allowing it to absorb a majority of the UV-B light and block it from passing through the epidermis.
• Vitiligo, pityriasis alba, tinea versicolor, and postinflammatory effect are common causes of pigment loss.
• the attenuation of the irregularities of pigmentation are either due to aging and/or photoaging, to hormonal disorders or to post-inflammatory processes and, particularly, the re-establishment of the pigmentation in the areas affected by vitiligo with topical applications is, therefore, of interest to the cosmetic and pharmaceutical sector.
• Pigmentary disorders are a global problem. Despite being common, pigmentary disorders remain difficult to treat.
• the main approaches for the treatment of hyperpigmentation disorders are mainly based on preventing the production of melanin by hyperactive melanocytes or promoting removal of melanin from the epidermis by increasing epidermal cell turnover.
• a number of agents that inhibit tyrosinase, the rate-limiting enzyme involved in melanin synthesis, are currently used to treat disorders of hyperpigmentation with moderate success. These agents include hydroquinone, kojic acid (also referred as “KA”), arbutin, mequinol and azelaic acid.
• KA kojic acid
• arbutin mequinol and azelaic acid.
• Contact dermatitis is a common side effect occurring in up to 15% of patients who use these agents, limiting their usage. The efficacy of these agents varies significantly from patient to patient.
• Topical retinoids reduce epidermal melanin via inhibition of tyrosinase and also increase epidermal turnover, thereby facilitating melanin dispersion and removal.
• irritant dermatitis can be a frequent adverse effect, and caution should be used when prescribing higher concentrations of topical retinoids in darker skin types, since resulting irritation may result in erythema, desquamation and further postinflammatory hyperpigmentation.
• Laser therapy may sometimes be effective for hyperpigmentation. This poses, however, a significant problem in darker skinned patients, who have increased baseline epidermal melanin that is also targeted at these wavelengths.
• disorders of depigmentation also involve an inflammatory and autoimmune pathogenesis, so topical corticosteroids and immunomodulators may be useful in the treatment regimen. Because postinflammatory hypopigmentation is usually clinically apparent after the inflammatory process has resolved, these same topical agents may not be beneficial at this late stage.
• Light therapy including but not limited to psoralens plus UVA (oral or topical), psoralens and natural sunlight, UVB, narrow-band UVB and 308 nm excimer lasers have been used to treat both hypopigmentation and depigmentation.
• Disorders of depigmentation e.g., vitiligo
• peptides which can efficiently regulate melanocyte activity and, consequently, can be efficient in the treatment of pigmentary disorders.
• Summary of Invention The present inventors have found that peptides of sequences SEQ ID NO: 1 and 2 efficiently regulate melanocyte activity.
• the present inventors have surprisingly found that the peptides of the invention are involved in melanogenesis pathway. Thus, some of the peptides stimulated the production of melanin, which can be an indicia of melanocyte activation, whereas others, such as F22 and F29, drastically reduced the amount of melanin.
• the present invention provides a peptide or a pharmaceutical or cosmetical acceptable salt thereof, the peptide having an amino acid sequence with at least 85% of identity with respect sequence SEQ ID NO: 1 or 2: (Trp)m-(Asn)n-Lys-Gly-Thr-Ile-Leu-Lys-Ala-Ser-Val-Asp-Tyr-Ile-Arg-Lys-(Leu)p-(Gln)q (SEQ ID NO: 1) (Arg)mx-(Arg)nx-Arg-Arg-Phe-Asn-Ile-Asn-Asp-Arg-Ile-Lys-Glu-Leu-Gly-Thr-Leu-(Ile)px-(Pro)qx (SEQ ID NO: 2) wherein “
• the present invention provides a cosmetical or pharmaceutical composition comprising a cosmetically or therapeutically effective amount of the peptide as defined in the first aspect of the invention together with acceptable cosmetical or pharmaceutical excipients and/or carriers.
• the present invention provides the peptide or pharmaceutical salt thereof as defined in the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention, for use as a medicament.
• This aspect can be alternatively formulated as the use of a peptide or pharmaceutical salt thereof as defined in the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention, in the manufacture of a medicament for the treatment of a disease.
• This aspect can also be alternatively formulated as a method for the treatment of a disease, the method comprising administering an effective therapeutic amount of a peptide or pharmaceutical salt thereof as defined in the first or the pharmaceutical composition of the second aspect of the invention, to a subject in need thereof.
• the present invention provides the peptide or pharmaceutical salt thereof as defined in the first aspect of the invention or the pharmaceutical composition as defined in the second aspect of the invention for use in the treatment of cancer.
• This aspect can alternatively be formulated as the use of the peptide or pharmaceutical salt thereof as defined in the first aspect of the invention or the pharmaceutical composition as defined in the second aspect of the invention in the manufacture of a medicament for the treatment of cancer.
• This aspect can also be alternatively formulated as a method for the treatment of cancer, the method comprising administering an effective therapeutic amount of the peptide or pharmaceutical salt thereof as defined in the first aspect of the invention or the pharmaceutical composition as defined in the second aspect of the invention, to a subject in need thereof.
• the present invention also provides the non-therapeutic use of a peptide or cosmetical salt thereof as defined in the first aspect of the invention or a cosmetical composition as defined in the second aspect of the invention for regulating melanogenesis, particularly for reducing or increasing the production of melanin.
• the present invention provides a cosmetical method for the treatment of the hyper- or hypopigmentation in a subject in need thereof, the method comprising the application of the peptide or cosmetical salt thereof as defined in the first aspect or the cosmetical composition as defined in the second aspect of the invention to the affected area suffering from the hyper- or hypopigmentation.
• the present invention provides: (a) a combination comprising the peptide or pharmaceutical salt thereof as defined in the first or second aspect of the invention; and a therapeutic agent, particularly an anti-cancer agent; (b) a combination comprising the peptide or pharmaceutical salt thereof as defined in the first or second aspect of the invention; and a therapeutic agent, particularly an anti-cancer agent, for use as a medicament, more particularly for use in the treatment of cancer; (c) a peptide or pharmaceutical salt thereof as defined in the first or second aspect of the invention for use in combination therapy for the prevention or treatment of cancer, wherein the therapy comprises its administration to a subject simultaneously, sequentially or separately with an anti-cancer agent; and (d) an anti-cancer agent for use in combination therapy with the peptide or pharmaceutical salt thereof as defined in the first or second aspect of the invention, wherein the use comprises the prevention or treatment of cancer.
• FIG.1 shows the percentage of melanin in treated melanoma cells with respect to that of untreated melanoma cells.
• Kojic Acid KA
• FIG.1 shows the percentage of melanin in treated melanoma cells with respect to that of untreated melanoma cells.
• KA Kojic Acid
• the present invention provides peptides comprising sequences with at least 85% degree identity with SEQ ID NO: 1 or 2, as it has been stated above.
• identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. If, in the optimal alignment, a position in a first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the sequences exhibit identity with respect to that position.
• a number of mathematical algorithms for rapidly obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include the MATCH-BOX, MULTAIN, GCG, FASTA, and ROBUST programs for amino acid sequence analysis, among others.
• Preferred software analysis programs include the ALIGN, CLUSTAL W, and BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof).
• a weight matrix such as the BLOSUM matrixes (e.g., the BLOSUM45, BLOSUM50, BLOSUM62, and BLOSUM80 matrixes), Gonnet matrixes, or PAM matrixes (e.g., the PAM30, PAM70, PAM120, PAM160, PAM250, and PAM350 matrixes), are used in determining identity.
• the BLAST programs provide analysis of at least two amino acid sequences, either by aligning a selected sequence against multiple sequences in a database (e.g., GenSeq), or, with BL2SEQ, between two selected sequences.
• BLAST programs are preferably modified by low complexity filtering programs such as the DUST or SEG programs, which are preferably integrated into the BLAST program operations. If gap existence costs (or gap scores) are used, the gap existence cost preferably is set between about -5 and -15. Similar gap parameters can be used with other programs as appropriate.
• the BLAST programs and principles underlying them are further described in, e.g., Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215, pages 403-410.
• the CLUSTAL W program can be used. The CLUSTAL W program desirably is run using "dynamic" (versus "fast") settings.
• Amino acid sequences are evaluated using a variable set of BLOSUM matrixes depending on the level of identity between the sequences.
• CLUSTAL W program and underlying principles of operation are further described in, e.g., Higgins et al., “CLUSTAL V: improved software for multiple sequence alignment”, 1992, CABIOS, 8(2), pages 189-191.
• pharmaceutical or cosmetical acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutical and cosmetical acceptable salts are well known in the art.
• Examples of pharmaceutical and cosmetical acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
• inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
• organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
• compositions include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pec
• Salts derived from appropriate bases include alkali metal, alkaline earth metal, and ammonium.
• Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
• Further pharmaceutical and cosmetical acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
• the term (C 1 -C 10 )alkyl refers to a saturated straight or branched alkyl chain having from 1 to 10 carbon atoms.
• Illustrative non-limitative examples are: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, neo-pentyl and n-hexyl.
• the term (C1-C20)alkyl refers to a saturated straight or branched alkyl chain having from 1 to 20 carbon atoms.
• the term (C 2 -C 10 )alkenyl refers to a saturated straight, or branched alkyl chain containing from 2 to 10 carbon atoms and also containing one or more double bonds.
• Illustrative non-limitative examples are ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
• the term (C 2 -C 10 )alkynyl refers to a saturated straight, or branched alkyl chain containing from 2 to 20 carbon atoms and also containing one or more triple bonds. Examples include, among others, ethynyl, 1-propynyl, 2- butynyl, 1,3-butadinyl, 4-pentynyl, and 1-hexynyl.
• halogen refers to the group in the periodic table consisting of five chemically related elements: fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At).
• (C 1 -C 10 )haloalkyl refers to a group resulting from the replacement of one or more hydrogen atoms from a (C 1 -C 10 )alkyl group with one or more, preferably from 1 to 6, halogen atoms, which can be the same or different.
• Examples include, among others, trifluoromethyl, fluoromethyl, 1-chloroethyl, 2-chloroethyl, 1- fluoroethyl, 2-fluoroethyl, 2-bromoethyl, 2-iodoethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 3-fluoropropyl, 3- chloropropyl, 2,2,3,3-tetrafluoropropyl, 2,2,3,3,3-pentafluoropropyl, heptafluoropropyl, 4-fluorobutyl, and nonafluorobutyl.
• ring system refers to a ring system which is chemically feasible and is known in the art and so intends to exclude those ring systems that are not chemically possible.
• ring system is formed by “isolated” rings means that the ring system is formed by two, three or four rings and said rings are bound via a bond from the atom of one ring to the atom of the other ring.
• isolated also embraces the embodiment in which the ring system has only one ring.
• Illustrative non-limitative examples of known ring systems consisting of one ring are those derived from: cyclopropyl, cyclobutyl, cyclopentyl, cyclhexyl, cycloheptyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, phenyl, and cycloheptenyl.
• rings “totally fused” means that the ring system is formed by two, three or four rings in which two or more atoms are common to two adjoining rings.
• Illustrative non-limitative examples are 1,2,3,4-tetrahydronaphthyl, 1-naphthyl, 2-naphthyl, anthryl, or phenanthryl.
• the ring system when “partially fused” it means that the ring system is formed by three or four rings, being at least two of said rings totally fused (i.e. two or more atoms being common to the two adjoining rings) and the remaining ring(s) being bound via a bond from the atom of one ring to the atom of one of the fused rings.
• the amino acids forming the peptides of the invention can have L- or D-configuration.
• Amino acids used in the construction of peptides of the present invention may be prepared by organic synthesis, or obtained by other routes, such as, for example, degradation of or isolation from a natural source.
• the peptide is other than one of sequence SEQ ID NO: 24, 25 or 26.
• the peptide is one wherein m and n are the same.
• the peptide is one wherein p and q are the same.
• m and n represent 1 and p and q represent 0.
• m and n represent 0 and p and q represent 1.
• the peptide is a lineal peptide.
• the term “lineal peptide” means that it does not include the “L” biradical.
• the peptide is a lineal peptide consisting of SEQ ID NO: 1, and is selected from the peptides wherein: - at least three of m, n, p and q are the same; or, alternatively, - when “m” is 0 and “n” is 1, then “p” is 0 and “q” is 0 or 1; or, alternatively - when “m” is 0 and “n” is 1, then “p” is 0 or 1, and “q” is 1.
• the peptide is one wherein mx and nx are the same. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the peptide is one wherein px and qx are the same. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, mx and nx represent 1 and px and qx represent 0. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, mx and nx represent 0 and px and qx represent 1.
• the peptide or salt thereof is one comprising a “L” linker wherein R1 and R3 are biradicals independently selected from the group consisting of: (C 1 -C 10 )alkyl; (C 2 -C 10 )alkenyl; and (C 2 -C 10 )alkynyl.
• the peptide or salt thereof is one comprising a “L” linker wherein R1 and R3 are the same or different and represent (C 1 -C 10 )alkyl.
• the peptide or salt thereof is one comprising a “L” linker wherein R2 is a biradical selected from the group consisting of: (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, and (C 2 -C 10 )alkynyl.
• R2 is a biradical selected from the group consisting of: (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, and (C 2 -C 10 )alkynyl.
• the peptide or salt thereof is one comprising a “L” linker wherein R2 is (C2- C10)alkenyl.
• the peptide or salt thereof is one comprising a “L” linker wherein R1 and R3 are the same or different and represent (C 1 -C 10 )alkyl; and R2 is (C 2 -C 10 )alkenyl.
• the peptide or salt thereof is one wherein R19 is selected from the group consisting of: (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, and (C 2 -C 10 )alkynyl.
• the peptide or salt thereof is one wherein R19 is a (C 1 -C 10 )alkyl monoradical.
• the peptide or salt thereof is one comprising a “L” linker wherein R1, R3 and R19 are the same or different and represent (C 1 -C 10 )alkyl; and R2 is (C 2 -C 10 )alkenyl.
• the peptide or salt thereof is one wherein R 4 is -OH (i.e., the C- terminal end is –C(O)OH).
• the peptide or salt thereof is one wherein R 4 is -NR17R18, R17 and R18 meaning the same.
• the N-terminal end corresponds to —NH2.
• the C-terminal and N-terminal ends of the peptide of the invention are, respectively, –C(O)OH and -NH2.
• the C-terminal and N- terminal ends of the peptide of the invention are, respectively, -C(O)NH2 and –NH2.
• the C-terminal and N-terminal ends of the peptide of the invention are, respectively, –C(O)OH and -NHR 5 , wherein R 5 is selected from (C1-C20)alkyl and (C 1 -C 10 )alkyl substituted by one or more radicals selected from the group consisting of: halogen, (C 1 -C 10 )alkyl, -OR 6 , -NR7R 8 , -SR 9 , - SOR 10 , -SO 2 R 11 , and -CO 2 R 12 .
• the C-terminal and N-terminal ends of the peptide of the invention are, respectively, –C(O)R 4 and -NH2, wherein R 4 means -NHR18, and R18 is a radical selected from the group consisting of: (C 1 -C 10 )alkyl and (C 1 -C 10 )alkyl substituted by one or more radicals selected from the group consisting of: halogen, (C 1 -C 10 )alkyl, -OR 6 , -NR7R 8 , -SR 9 , -SOR 10 , -SO 2 R 11 , and -CO 2 R 12 .
• the C-terminal and N-terminal ends of the peptide of the invention are, respectively, –C(O)R 4 and -NH2, wherein R 4 means -NR17R18, and R17 and R18 are radicals independently selected from the group consisting of: (C 1 -C 10 )alkyl and (C 1 -C 10 )alkyl substituted by one or more radicals selected from the group consisting of: halogen, (C 1 -C 10 )alkyl, -OR 6 , -NR7R 8 , -SR 9 , -SOR 10 , -SO 2 R 11 , and -CO 2 R 12 .
• the linker biradical of formula (I) is between an alpha carbon atom of an amino acid located at position “i” in the peptide sequence of sequence SEQ ID NO: 1 and an alpha carbon atom of an amino acid located at position “i+7” in the peptide sequence SEQ ID NO: 1.
• the peptide or salt thereof is selected from the group consisting of amino acid sequences SEQ ID NO: 3 to 8:
• the linker biradical of formula (I) is between an alpha carbon atom of an amino acid located at position “i” in the peptide sequence of SEQ ID NO: 2 and an alpha carbon atom of an amino acid located at position “i+7” in the peptide sequence of SEQ ID NO: 2.
• the peptide or salt thereof is selected from the group consisting of amino acid sequences SEQ ID NO: 9 to 16:
• the peptide or salt thereof is one with at least 85% of identity with a sequence SEQ ID NO: 3 to 8, wherein R1, R3 and R19 are the same or different and represent (C 1 -C 10 )alkyl; and R2 is (C 2 -C 10 )alkenyl.
• the peptide or salt thereof is one with at least 85% of identity with a sequence SEQ ID NO: 3 to 8, wherein R1, R3 and R19 are the same or different and represent (C 1 -C 10 )alkyl; R2 is (C 2 -C 10 )alkenyl; the C-terminal is selected from –C(O)OH; and –CONH2 and the N-terminal end is -NH2.
• the peptide or salt thereof is one with at least 85% of identity with a sequence SEQ ID NO: 9 to 16, wherein R1, R3 and R19 are the same or different and represent (C 1 -C 10 )alkyl; and R2 is (C 2 -C 10 )alkenyl.
• the peptide or salt thereof is one with at least 85% of identity with a sequence SEQ ID NO: 9 to 16, wherein R1, R3 and R19 are the same or different and represent (C 1 -C 10 )alkyl; R2 is (C 2 -C 10 )alkenyl; the C-terminal is selected from –C(O)OH and –CONH2 and the N-terminal end is -NH2.
• the peptide is one having at least 85% of identity with an amino acid sequence, or pharmaceutical or cosmetical salt thereof, selected from the group consisting of sequence SEQ ID NO: 17 to 23:
• the peptide or salt thereof is a peptide which has an identity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with respect to any of the sequences SEQ ID NO: 1 to 23, and “m”, “n”, “p”, “q”, “m x ”, “n x ”, “p x ”, “q x ”, L and R 19 are as defined in any of the above embodiments.
• the peptide or salt thereof is a peptide which has an identity of 100% with respect to any of the sequences SEQ ID NO: 1 to 23, and “m”, “n”, “p”, “q”, “m x ”, “n x ”, “p x ”, “q x ”, L and R 19 are as defined in any of the above embodiments.
• the two aminoacidic residues connected by the linker are not considered when performing the alignment to determine the identical positions for confirming the identity degree.
• the peptide of the first aspect is conjugated to a moiety, such as a label, a drug or a cell penetration peptide.
• a moiety such as a label, a drug or a cell penetration peptide.
• the peptide is conjugated to a label.
• the label is conjugated to the N- or C-terminal of the peptide.
• a “label” as used herein is a molecule or compound that can be detected by a variety of methods including fluorescence, electrical conductivity, radioactivity, size, and the like.
• the label may be intrinsically capable of emitting a signal, such as for example fluorescent label that emits light of a particular wavelength following excitation by light of another lower, characteristic wavelength.
• the label may not be capable of intrinsically emitting a signal but it may be capable of being bound by another compound that does emit a signal.
• An example of this latter situation is a label such as biotin which itself does not emit a signal but which when bound to labeled avidin or streptavidin molecules can be detected.
• Other examples of this latter kind of label are ligands that bind specifically to particular receptors. Detectably labeled receptors are allowed to bind to ligand labeled unit specific markers in order to visualize such markers.
• Labels that may be used according to the invention include but are not limited to electron spin resonance molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, an enzyme, a biotin molecule, an avidin molecule, an electrical charge transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic molecule, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, nucleic acid, a carbohydrate, a hapten, an antigen, an antibody, an antibody fragment, and a lipid.
• Radioisotopes can be detected with film or charge coupled devices (CCDs), ligands can be detected by binding of a receptor having a fluorescent, chemiluminescent or enzyme tag, and microbeads can be detected using electron or atomic force microscopy.
• CCDs charge coupled devices
• ligands can be detected by binding of a receptor having a fluorescent, chemiluminescent or enzyme tag
• microbeads can be detected using electron or atomic force microscopy.
• the conjugation of the label to the peptide can be performed following routine protocols well-known for the skilled in the art.
• the peptide of the first aspect of the invention optionally in combination with any of the embodiments provided above or below, is conjugated to a drug.
• the drug is conjugated to the N-terminal end of the peptide.
• the peptide is conjugated to a cell penetrating peptide.
• cell penetrating peptide refers to short peptides that facilitate cellular uptake of various molecular cargo (from nanosize particles to small chemical molecules and large fragments of DNA).
• the "cargo” is associated to peptides via the C(t) or N(t)-end, either through chemical linkage via covalent bonds or through non-covalent interactions.
• the function of the CPPs are to deliver the cargo into cells, a process that commonly occurs through endocytosis.
• CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively.
• a third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake.
• the conjugation of the CPP to the peptide provided in the present invention can be performed following well- known routine protocols, such as solid phase synthesis or solution selective capping. (cf. Copolovici D. M. et al., “Cell-Penetrating Peptides: Design, Synthesis, and Applications”, 2014, ACS Nano, 2014, 8 (3), pp 1972– 1994).
• the cell penetrating peptide is a polycationic CPP, polyArg or, alternatively, penetratine.
• the process for the preparation of the peptides according to the first aspect of the invention comprises: (1.a) the coupling, by condensation, of the corresponding amino acids of the peptide with a compound of formula (III) and a compound of formula (IV), which correspond to the amino acids referred as “i” and “i+4” or “i+7”.
• Compounds (III) and (IV) will be those suffering a subsequent cyclization step to generate the “L” biradical”: wherein R19 is as defined above, Z1 and Z2 are the same or different and represent (C 2 -C 10 )alkenyl; and (1.b) a cyclization step comprising the ring-closed metathesis of Z1 and Z2 (cf.
• the process for the preparation of the peptide according to the second aspect of the invention comprises the coupling, by condensation, of the carboxylic group or C-terminus of one amino acid with the amino group or N-terminus of another, this coupling reaction being repeated the number of times required until the desired peptide is obtained.
• the compounds of formula (III), (IV), (V), (VI), (VII), and (VIII) are commercially available and are coupled by condensation to the already formed portion of peptide sequence. These compounds can carry beads for the appropriate solid phase synthesis of the peptide, as well as protecting groups of the carboxy, amino or side- chain.
• Illustrative non-limitative examples of compounds are: 2-(2'-propenyl)alanine, 2-(3'-butenyl)glycine, 2- (4'-pentenyl)alanine, 2-(6'-heptenyl) alanine, 2-(7'-octenyl)alanine, allyl–glycine, 5-azido-norvaline, and alpha- propargyl-alanine, among others.
• the “coupling” step can be performed in solid phase, following the protocol “deprotection-wash-coupling- wash”, by condensation of the carboxylic group of one amino acid with the amino group of another amino acid residue, using amino acids as defined above as well as alpha-alpha di-substituted amino acids of formula (II) to (VII).
• the general principle of solid phase peptide synthesis is to repeat cycles of deprotection-wash-coupling-wash.
• the free N-terminal amine of a solid-phase attached peptide is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
• Amino acids have reactive moieties at the N- and C-termini, which facilitates amino acid coupling during synthesis. Many amino acids also have reactive side chain functional groups, which can interact with free termini or other side chain groups during synthesis and peptide elongation and negatively influence yield and purity. To facilitate proper amino acid synthesis with minimal side chain reactivity, chemical groups have been developed to bind to specific amino acid functional groups and block, or protect, the functional group from nonspecific reactions. These protecting groups, while vast in nature, can be separated into three groups, as follows: N-terminal protecting groups, C-terminal protecting groups (mostly used in liquid-phase synthesis), and side chain protecting groups. For coupling the peptides the carboxyl group is usually activated. This is important for speeding up the reaction.
• activating groups There are two main types of activating groups: carbodiimides and triazolols. However, the use of pentafluorophenyl esters (FDPP, PFPOH]) and BOP-Cl are useful for cyclising peptides. Purified, individual amino acids are reacted with these protecting groups prior to synthesis and then selectively removed during specific steps of peptide synthesis.
• FDPP pentafluorophenyl esters
• PFPOH PFPOH
• Exemplary resins which may be employed by the present invention include, but are not limited to: (1) alkenyl resins (e.g., REM resin, vinyl sulfone polymer-bound resin, vinyl-polystyrene resin); (2) amine functionalized resins (e.g., amidine resin, N-(4-Benzyloxybenzyl)hydroxylamine polymer bound, (aminomethyl)polystyrene, polymer bound (R)-(+)-a-methylbenzylamine, 2—Chlorotrityl Knorr resin, 2-N-Fmoc-Amino- dibenzocyclohepta-1,4-diene, polymer-bound resin, 4-[4-(1-Fmoc-aminoethyl)-2-methoxy-5- nitrophenoxy]butyramidomethyl-polystyrene resin, 4-Benzyloxybenzylamine, polymer-bound, 4- Carboxybenzenesulfonamide,
• PG Protecting group
• Suitable amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2- phenylethyl carbamate (hZ), 1-(
• suitably protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids.
• suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like.
• suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl.
• suitable alkenyl groups include allyl.
• suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl.
• suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6- dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.
• MPM p-methoxybenzyl
• MPM 3,4-dimethoxybenzyl
• O-nitrobenzyl p-nitrobenzyl
• p-halobenzyl 2,6- dichlorobenzyl
• p-cyanobenzyl 2,6- dichlorobenzyl
• 2- and 4-picolyl 2- and 4-picolyl.
• the present invention provides a pharmaceutical or cosmetical composition.
• the expression "therapeutically effective amount” as used herein, refers to the amount of a compound that, when administered passes to blood stream and is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
• the particular dose of the peptide administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.
• the pharmaceutical composition is a parenteral composition.
• the pharmaceutical composition is for oral administration.
• cosmetically effective amount in the context of the present invention, refers to the amount of a compound that when it is applied (for example topically) is retained within the skin tissue (such as at subcutaneous level), without significantly reaching, or not reaching, the blood stream, thus exerting a local effect within skin structure.
• the particular dose of the peptide administered according to this invention will of course be determined by the particular circumstances surrounding the case.
• the “cosmetically effective amount” of the peptide of the invention is higher, equal or lower than the amount needed to achieve a therapeutic effect when it is administered by parenteral route, particularly by intravenous route.
• compositions or vehicles refers to acceptable materials, compositions or vehicles. Each component must be pharmaceutically or cosmetically acceptable in the sense of being compatible with the other ingredients of the composition. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
• Suitable acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical or cosmetical composition, its use is contemplated to be within the scope of this invention.
• the formulations of the pharmaceutical and cosmetical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and cosmetics.
• Such preparatory methods include the step of bringing the active ingredient (the peptide) into association with a excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
• a pharmaceutical composition of the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
• a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
• the relative amounts of the active ingredient (i.e., the peptide as defined in any of the previous aspects and embodiments), the acceptable excipients, and/or any additional ingredients in the composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
• Acceptable excipients used in the manufacture of these compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the inventive formulations.
• Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents can be present in the composition, according to the judgment of the formulator.
• Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and combinations thereof.
• Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.
• crospovidone cross-linked polyvinylpyrrolidone
• sodium carboxymethyl starch sodium starch glycolate
• Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
• natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
• colloidal clays e.g. bentonite [aluminum silicate]
• stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters ⁇ e.g., polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyce
• binding agents include, but are not limited to, starch (e.g., cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol
• Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
• Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
• Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate.
• EDTA ethylenediaminetetraacetic acid
• citric acid monohydrate disodium edetate
• dipotassium edetate dipotassium edetate
• edetic acid fumaric acid, malic acid
• phosphoric acid sodium edetate
• tartaric acid tartaric acid
• trisodium edetate trisodium edetate.
• antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
• Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
• Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
• Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
• preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.
• the preservative is an anti-oxidant.
• the preservative is a chelating agent.
• buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, al
• Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.
• oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury
• Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.
• Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable liposomes emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzy
• the conjugates of the invention are mixed with solubilizing agents such as polyethoxylated castor oil (e.g. CREMOPHORTM), alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.
• injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
• the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid are used in the preparation of injectables.
• the preparation can be in the form of liposomes.
• the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
• peptides of the invention can be in micro-encapsulated form with one or more excipients as noted above. In one embodiment, the peptides of the invention are formulated in liposomes.
• peptides and pharmaceutical compositions of the present invention can be employed in combination therapies.
• the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved.
• the therapies employed may achieve a desired effect for the same purpose (for example, an inventive conjugate useful for detecting tumors may be administered concurrently with another agent useful for detecting tumors), or they may achieve different effects (e.g., control of any adverse effects).
• the pharmaceutical composition of the present invention may be administered either alone or in combination with one or more other therapeutic agents.
• compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
• each agent will be administered at a dose and/or on a time schedule determined for that agent.
• the invention encompasses the delivery of the peptide or pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
• the particular combination of therapies to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and/or the desired therapeutic effect to be achieved.
• the therapies employed may achieve a desired effect for the same disorder (for example, a peptide of the invention may be administered concurrently with another biologically active agent used to treat the same disorder), and/or they may achieve different effects (e.g., control of any adverse effects).
• biologically active agents utilized in this combination may be administered together in a single composition or administered separately in different compositions.
• the expression “in combination with” also encompasses the possibility of conjugating (by chemical-physical interactions) the peptide of the invention to any of the further agents mentioned above and below, which can be either a therapeutic agent or an agent for improving the profile of the peptide (such as bioavailability), among others.
• the peptides of the invention are administered in combination with one or more anti- cancer agents.
• An anti-cancer agent may be, for instance, methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12- 9566, RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin,
• the peptides of the first aspect of the invention are useful in the therapeutic treatment of melanoma, particularly metastatic melanoma by reducing the proliferation of malignant melanocytes.
• the peptide or pharmaceutical composition when used with a therapeutic purpose, it is orally or parenterally administered.
• the present invention also provides the non-therapeutic use of the peptide or cosmetical salt thereof for regulating melanogenesis, particularly for reducing or increasing the production of melanin. All the embodiments provided above regarding the peptide and cosmetical composition are also embodiments of this aspect.
• the peptide is for use in the treatment of hyperpigmentation (by reducing the amount of melanin) or hypopigmentation (by increasing the amount of melanin).
• it is regulated the melanogenesis in skin or hair, particularly in skin.
• the subject does not suffer skin cancer.
• the present invention also provides a cosmetical method for the treatment of the hyper or hypopigmentation comprising the application of the peptide or cosmetical salt thereof as defined in the first aspect or the cosmetical composition as defined in the second aspect of the invention.
• This cosmetic method does not encompass the therapeutic method. All the embodiments provided above regarding the peptide and cosmetic composition are also embodiments of this aspect.
• the peptide or cosmetical composition is applied on skin and/or hair.
• the peptide or cosmetical composition is applied on skin.
• the present invention also provides a pigmenting composition, such as a topical cosmetical or pharmaceutical pigmenting composition, comprising the peptide or salt thereof as defined in any of embodiments provided above.
• a depigmenting composition such as a topical cosmetical or pharmaceutical depigmenting composition, comprising the peptide or salt thereof as defined in any of the embodiments provided above.
• the peptide was cleaved from the resin and deprotected by exposure to solution F (95% TFA, 2,5% water, 2.5% TIS) and lyophilized.
• solution F 95% TFA, 2,5% water, 2.5% TIS
• the lyophilized peptides were purified by reverse phase HPLC using a C18 column (see compounds characterization for details).
• the peptides were identified by LC-MS-ESI. All the mass spectral data for all the compounds are shown below in Table 1.
• Fmoc-protected a-amino acids include than the olefinic amino acids Fmoc-[(S)-2-(4 pentenyl)alanine]OH, Fmoc-[(R)-2-(4 pentenyl)alanine]OH, Fmoc-[(S)-2-(7 octenyl)alanine]OH, Fmoc-[(R)-2-(4 pentenyl)alanine]OH, 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (TBTU), resins, dimethylformamide (DMF), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), Grubbs Ru(IV) catalyst and piperidine were purchased from different suppliers.
• DMF dimethylformamide
• linear polypeptides were synthesized with automatic synthesizer using Fmoc solid phase peptide chemistry. Only the coupling with olefinic amino acids was performed manually after removing the resins from the reactor vessel, as disclosed in the previous section.
• the ring-closing metathesis reaction was performed in solution with a first generation Grubbs catalyst after cleaving the linear peptide from the resin, as disclosed by Scott J. M. and colleagues (Scott J.M. et al., “Application of Ring-Closing Metathesis to the Synthesis of Rigidified Amino Acids and Peptides”,1996, J. Am. Chem. Soc., 1996, 118 (40), pp 9606–9614).
• IGR-39 skin, human melanoma derived from metastatic site: groin lymph node.
• B16-F10 skin, mouse melanoma, ATCC-CRL-6475TM.
• 501-MEL skin, human malignant melanoma.
• Cell culture Cell lines SK-MEL-28, IGR-37, IGR-39, B16-F10, 501-MEL and A-375 were cultured in incubator at 37oC in Dulbecco ⁇ s modified Eagle ⁇ s medium (Invitrogen Life Technologies, Foster City, CA) containing 10% fetal bovine serum inactivated (FBS, Dutscher, France), penicillin (50 U/ml), and streptomycin sulphate (50 mg/ml) (Gibco BRL, Germany).
• adherent cells were rinsed with DPBS (Dulbecco’s Phosphate Buffered Saline, Sigma D1283) three times and afterward treated for 5 minutes with trypsin ([0,5 g / ml] / EDTA [0.2 g/ml]) (Gibco-BRL, 15400054) in solution of DPBS at 37 o C, and, once detached, transferred in the culturing medium. Cells were counted in a Neubauer chamber after labelling with Tripan-Blue. Each assay was performed only when the viability was superior to 90%.
• DPBS Denbecco’s Phosphate Buffered Saline, Sigma D1283
• Viability assay Cell lines SK-MEL-28, IGR-37, B16-F10, 501-MEL and A-375 were seeded at a density of 1,000 cells/well in 96 well plates. After 24h, the compounds to be tested were added to calculate the efficacy at the concentration of 10 ⁇ M with serial dilutions. Controls are the untreated cells. Each experiment was performed in triplicate. Cells were incubated during 72h in incubator under CO 2 atmosphere at 37°C. Cell viability was measured by colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. MTT was incubated for a further 4 h, and the medium was then discarded.
• colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
• DMSO was added to dissolve the formazan product, and absorbance at 570 nm was measured with a microplate reader (MULTISKAN, Labsystems). Cell viability percentages were calculated by dividing the absorbance value of cells treated with a given compound by the absorbance of untreated cells. Statistics Data analysis was performed calculating the percentage of cell viability normalized vs. the values of negative control, which was considered equal to 100%.
• Pigment formation during zebrafish development starts at about 24hpf by the retinal pigment epithelium (RPE) and then progresses towards the dorsolateral skin, where melanocytes are distributed.
• RPE retinal pigment epithelium
• Biochemical melanin quantification performed in untreated embryos to compare to embryos treated with compound F22.
• F22 was dissolved in 1X PBS to stock solution.
• F22 was serially diluted in E3 medium to obtain: 2.5 ⁇ M in a final volume of 1 ml.
• the table shows the quantification of melanin as percentage compared to the negative control (E3 medium) at 72 hrs of incubation.
• Asterisks indicate statistical significance after a One-way ANOVA: * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
• Table2 Melanin quantification by imaging analysis. Melanin content is calculated as the mean of the sum of pixels related to the body of each zebrafish larvae (E3 medium) at 96 hpf.
• Asterisks indicate statistical significance after a One-way ANOVA: * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
• a peptide or a pharmaceutical or cosmetical acceptable salt thereof having an amino acid sequence with at least 85% of identity with respect sequence SEQ ID NO: 1 or 2: (Trp)m-(Asn)n-Lys-Gly-Thr-Ile-Leu-Lys-Ala-Ser-Val-Asp-Tyr-Ile-Arg-Lys-(Leu)p-(Gln)q (SEQ ID NO: 1) (Arg)mx-(Arg)nx-Arg-Arg-Phe-Asn-Ile-Asn-Asp-Arg-Ile-Lys-Glu-Leu-Gly-Thr-Leu-(Ile)px-(Pro)qx (SEQ ID NO: 2) wherein “m”, “n”, “p”, and “q” represent integers and are selected from 0 and 1; “mx”, “nx”, “px”, and “qx” represent integers and are selected from 0 and 1; a C-
• R1 and R3 are biradicals independently selected from the group consisting of: (C 1 -C 10 )alkyl; (C 2 -C 10 )alkenyl; and (C 2 -C 10 )alkynyl;
• R2 is a biradical selected from the group consisting of: (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, and (C 2 -C 10 )alkynyl;
• R19 is a monoradical selected from the group consisting of: (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, and (C 2 -C 10 )alkynyl.
• the peptide according to any one of the previous clauses which has at least a 85%, at least 90%, at least 95% or it is 100% identical to a peptide sequence SEQ ID NO: 3 to 16, wherein “L”, “R19”, “m”, “n”, “p”, “q”, “mx”, “nx”, “px” and “qx” are as defined in any of the preceding clauses.
• the peptide according to any one of the previous clauses which is a peptide with an amino acid having at least 85%, at least 90%, at least 95% or is 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 17 to 23. 10.
• a pharmaceutical or cosmetical composition comprising a therapeutically or cosmetically effective amount of the peptide as defined in any one of the clauses 1-9, together with acceptable pharmaceutical or cosmetical excipients and/or carriers.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Epidemiology (AREA)
• Dermatology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Birds (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Immunology (AREA)
• Gastroenterology & Hepatology (AREA)
• Oncology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Cosmetics (AREA)
• Medicinal Preparation (AREA)

Priority Applications (11)

Applications Claiming Priority (2)

Publications (1)

Family

ID=67658460

Family Applications (1)

Country Status (14)

Families Citing this family (1)

Citations (3)

Family Cites Families (3)

• 2020
  • 2020-07-30 AR ARP200102140A patent/AR119529A1/es unknown
  • 2020-07-30 TW TW109125814A patent/TWI889695B/zh active
  • 2020-07-31 KR KR1020227002877A patent/KR20220041094A/ko active Pending
  • 2020-07-31 JP JP2022506027A patent/JP2022542512A/ja active Pending
  • 2020-07-31 AU AU2020325636A patent/AU2020325636A1/en active Pending
  • 2020-07-31 MX MX2022001114A patent/MX2022001114A/es unknown
  • 2020-07-31 IL IL290019A patent/IL290019B2/en unknown
  • 2020-07-31 WO PCT/EP2020/071629 patent/WO2021023646A1/en not_active Ceased
  • 2020-07-31 EP EP20747418.0A patent/EP4007563A1/en active Pending
  • 2020-07-31 CN CN202080056109.0A patent/CN114245800A/zh active Pending
  • 2020-07-31 US US17/632,463 patent/US12492225B2/en active Active
  • 2020-07-31 CA CA3148785A patent/CA3148785A1/en active Pending
  • 2020-07-31 BR BR112022001729A patent/BR112022001729A2/pt unknown
• 2022
  • 2022-01-26 CL CL2022000208A patent/CL2022000208A1/es unknown
• 2025
  • 2025-05-20 JP JP2025083931A patent/JP2025131623A/ja active Pending

Patent Citations (3)

Non-Patent Citations (10)

Also Published As

Similar Documents

Legal Events