EP1307217A2 - Peptides associ s au peptide intestinal vasoactif, destin s au traitement de dermatoses - Google Patents

Peptides associ s au peptide intestinal vasoactif, destin s au traitement de dermatoses

Info

Publication number
EP1307217A2
EP1307217A2 EP01940960A EP01940960A EP1307217A2 EP 1307217 A2 EP1307217 A2 EP 1307217A2 EP 01940960 A EP01940960 A EP 01940960A EP 01940960 A EP01940960 A EP 01940960A EP 1307217 A2 EP1307217 A2 EP 1307217A2
Authority
EP
European Patent Office
Prior art keywords
lys
asn
tyr
leu
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01940960A
Other languages
German (de)
English (en)
Inventor
Illana Gozes
Ruth Granoth
Matityahu Fridkin
E. Douglas Brenneman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Government Of United States, asd
Ramot at Tel Aviv University Ltd
Yeda Research and Development Co Ltd
Original Assignee
Ramot at Tel Aviv University Ltd
Yeda Research and Development Co Ltd
US Department of Health and Human Services
US Government
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ramot at Tel Aviv University Ltd, Yeda Research and Development Co Ltd, US Department of Health and Human Services, US Government filed Critical Ramot at Tel Aviv University Ltd
Publication of EP1307217A2 publication Critical patent/EP1307217A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—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
    • A61K38/22—Hormones
    • A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
    • 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

Definitions

  • the present invention relates to compounds, pharmaceutical compositions and methods for treating skin disorders and for inducing cell apoptosis. More particularly, the present invention relates the treatment of skin conditions associated with hyperproliferation of skin cells, which involves topical administration of lipophilized, vasoactive intestinal peptide (VIP), VIP- derived peptides and conjugates thereof (all referred to herein in the specification as "VIP-related peptides"), optionally with other substances traditionally used in the art for the treatment of such skin disorders. Additionally, a method for the induction of apoptosis in cells is disclosed, by subj ecting the cells to the VIP-related peptides .
  • VIP vasoactive intestinal peptide
  • Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP), are members of a family of regulatory peptides that includes secretin and glucagon [1].
  • VIP a basic 28-amino acid peptide, has been established as a modulator of growth, survival and differentiation in many cell systems, including the brain, the gastro-intestinal tract, lung and immune cells, of both primary origin and cancerous one [1].
  • IL Patent No. 87055 (corresponding to EP Patent No. 0354992 and U.S. Patent No. 5,147,855) and IL Patent No. 99924 (corresponding to EP Patent No. 0540969 and U.S. Patent No. 5,998,368) teach the treatment of male impotence by transdermal administration [13] of modified, shortened and/or lipophilically derivatized VIP, whereas EP Patent No. 0620008 and U.S. Patent No. 5,972,883 teach the treatment of neurodegenerative diseases by preferably intranasal administration of modified, shortened and/or lipophilically derivatized VIP.
  • Natural VIP is a hydrophilic amidated peptide characterized by a very short half life in the serum and having the following sequence: His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met- Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1)
  • the first is lipophilization, namely, the addition of a fatty acid moiety, designed to augment VTP's ability to penetrate biological membranes without loss of activity.
  • SEQ ID NO: 2 Stearoyl-VIP (SEQ ID NO: 2), a molecule combining
  • the second modification includes the replacement of native amino acids with unnatural amino acids, namely, a substitution of methionine (at position 17) by norleucine, aimed at stabilizing the molecule against oxidation, as well as at increasing its lipophiliciry.
  • SNV Stearoyl-Nle ⁇ -VIP
  • SEQ ID NO: 3 Stearoyl-Nle ⁇ -VIP
  • VHA VTP-associated activities [18,19]. More particularly, it has been found that VHA inhibits the growth of VIP receptor bearing tumor cells such as, for example, lung tumor cells (i.e., NSCLC cells). To this end see U.S. Pat. No. 5,217,953 and references [20-22].
  • U.S. Pat. No. 5,565,424 issued to Goz&y et al. discloses another family of lipophilized polypeptides, which are antagonists of VIP.
  • the VIP antagonists disclosed therein are 10-1000 times more efficacious, i.e., more potent in inhibiting VTP-associated activity, than previous VIP antagonists [14, 18].
  • These superactive VIP antagonists were shown to inhibit cancer growth of lung [23] and glioblastoma cells.
  • An example of such a superactive VIP antagonist is the conjugate referred to herein as SNH, Stearoyl-neurotensin 6. j j VIP 7 . 28 (SEQ ID NO: 5).
  • VIP effects have also been documented for skin cells [3], and VIP has been directly implicated in keratinocyte proliferation [6,7,8], the major cell type in the epidermis [4].
  • the epidermis is a continually renewing tissue, where homeostasis is maintained by factors that influence the interrelated processes of keratinocytic life cycle, with apoptosis which serves as a mechanism for cell number control.
  • apoptosis have all been linked to homeostatic dysregulation characteristic of various skin disorders [12], such as disorders of the hyperproliferative type, including, but not limited to, basal cell carcinomas, disorders of keratinization, e.g., keratosis, various dermatoses, dandruff and psoriasis.
  • disorders of the hyperproliferative type including, but not limited to, basal cell carcinomas, disorders of keratinization, e.g., keratosis, various dermatoses, dandruff and psoriasis.
  • Psoriasis for example, is a chronic skin condition of the epidermis, diagnosed by itchy, scaling, erythematous lesions. The disease is a major cause of disability and disfigurement for between 1 to 3% of the World's population.
  • Psoriasis conditions can range from mild to severe. In the United States, between 150,000 and 250,000 new cases of psoriasis are diagnosed each year, with about 40,000 of these cases classified as severe. This disease is characterized by a hyperproliferation of the basal cells (a several fold increase in the number of basal cells of the epidermis), thus reducing the turnover time of the epidermis from the normal 27 days to 3-4 days. This shortened interval prevents normal cell maturation and keratinization, reflected in an array of abnormal morphologic and biochemical changes. Numerous cytologic, histologic, histochemical, and biochemical alterations are known to be the result, rather than the cause, of the disease process.
  • corticosteroids e.g., triamcinolone and hydrocortisone creams
  • keratolytic/destractive agents such as anthralin or salicylic acid
  • lubricants such as hydrogenated vegetable oils and white petroleum
  • oral retinoids vitamin D analogs and tar based therapies.
  • systemic chemotherapeutic agents especially the antimetabolite methotrexate and the immunosuppressant cyclosporine. Photochemotherapy was introduced in 1974, the so-called PUVA treatment.
  • This treatment consists of administering psoralen prior to partial or whole body irradiation with a special light system that emits predominately long wavelength ultraviolet light (UV-A).
  • UV-A predominately long wavelength ultraviolet light
  • current psoriasis treatments, as well as other treatments known for hyperproliferative skin disorders, such as skin cancer suffer from one or more deficiencies, including potential toxic side effects and achieving only temporary relief.
  • the present invention fulfills these and other needs.
  • Vasoactive intestinal peptide is a recognized growth factor affecting many cell types.
  • VIP vascular endophilic VIP analogues containing an N-terminal covalently attached stearoyl moiety was previously described.
  • the current invention teaches of VIP derived conjugates, and specifically, Stearoyl- Nle 17 VIP (SNV), and Stearoyl-Nle 17 -neurotensin 6 .nVIP 7 . 28 (SNH), acting at ⁇ M concentrations, as cytotoxic agents for human keratinocytes.
  • SNV Stearoyl- Nle 17 VIP
  • SNH Stearoyl-Nle 17 -neurotensin 6 .nVIP 7 . 28
  • the core C- terminal active VTP-derived peptide Stearoyl-Lys-Lys-Tyr-Leu-NH2 (St- K YL-NH2, SEQ ID NO:6), is further described as being responsible for the observed cytotoxicity. Cytotoxicity coincided with marked reduction in intracellular cGMP and was abolished by co-treatment with the endonuclease inhibitor, aurintricarboxylic acid (ATA), indicating apoptotic mechanisms.
  • ATA aurintricarboxylic acid
  • a method for treating a hyperproliferative skin disorder of a patient comprising the step of administering to a skin area of the patient a therapeutically effective amount of a VIP-related peptide.
  • the method further comprising the step of administering a therapeutically effective amount of a substance traditionally used for treatment l o of the hyperproliferative skin disorder.
  • a method of inducing cell apoptosis comprising the step of subjecting cells to a VIP-related peptide.
  • compositions for treating a hyperproliferative skin disorder comprising, as active ingredients, therapeutically effective amounts of a VIP-related peptide and a substance traditionally used for treatment of the hyperproliferative skin disorder.
  • the VIP-related peptide includes an amino acid sequence identified by SEQ ID NO:7.
  • the VIP-related peptide includes at least one non-natural amino acid.
  • the at least one non-natural amino acid is selected from the group consisting of a D amino acid, norleucine, norvaline, ⁇ -aminobutyric acid, di- amino butyric acid, di-aminopropionic acid, -HN(CH2)nCOOH, wherein n is 3-5, -NH(CH 2 (R))n-COOH, wherein R is an alkyl, H 2 N(CH 2 ) m COOH,0 wherein m is 2-4, H 2 N-C(NH)-NH(CH2)kCOOH, wherein k is 2-3, hydroxy lysine, N-methyl lysine, ornitine, p-amino phenylalanine, TIC, naphthylelanine, a ring-methylated derivative of phenylalanine, a halogen
  • the VIP-related peptide includes at least one peptide-bond modification.
  • the VIP-related peptide includes a cyclic peptide moiety. According to still further features in the described preferred embodiments the VIP-related peptide includes a lipophilic moiety.
  • the lipophilic moiety is selected from the group consisting of a saturated alkyl a branched alkyl, a non-saturated alkyl, a non-branched alkyl, a saturated acyl, a branched acyl, a non-saturated acyl and a non-branched acyl.
  • VIP vasoactive intestinal peptide
  • composition comprises VIP, of the sequence;
  • the composition comprises a VIP antagonist, of the sequence: Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-
  • composition comprises an analog of VIP in which one or more amino acids has been replaced, of the sequence:
  • X 1 , X 2 and X 3 are the same or different and each is the residue of a natural or non-natural amino acid, provided that when both X 1 and X 3 are valine, X 2 is not methionine.
  • the composition comprises an analog of a
  • VIP antagonist in which one or more amino acids has been replaced, of the sequence:
  • Y 1 and Y 2 are the same or different and each is the residue of a natural or non-natural amino acid, provided that Y 2 is not methionine.
  • the groups X 1 , X 2 and X 3 , and Y l and Y 2 may be the same or different and each may be a natural amino acid selected from the group consisting of leucine, isoleucine, valine, tryptophan, phenylalanine and methionine, or a non- natural amino acid is selected from the group consisting of a D-amino acid, norleucine (Nle), norvaline, ⁇ -aminobutyric acid, di-amino butyric acid, di- aminopropionic acid, -HN(CH2) n COOH, wherein n is 3-5, -NH(C H 2(R))n- COON, wherein R is an alkyl, H2N(CH2)m .
  • the pharmaceutical composition comprises a physiologically active fragment of VIP or of an analog thereof, having a sequence selected from the group consisting of: (i) X 4 -X 5 -X 6 -X 7 -NH-R2
  • X 4 is a covalent bond, Ala, Val, Ala-Val, Val-Ala, Lys, D-Lys, Ala-Lys, Val-Lys, Ala-Val-Lys, Val-Ala-Lys or Orn;
  • X 5 is L-Lys, D-Lys or Orn;
  • X 6 is L-Tyr, D-Tyr, Phe, Trp or a residue of p-amino-phenylalanine;
  • X 9 is lie or Tyr
  • X 10 is a residue of a hydrophobic aliphatic amino acid
  • X 7 is X 10 , X 10 -Asn, X 10 -Ser, X 10 -Ile, X 10 -Tyr, X 10 -Leu, X 10 -Nle, X 10 -D- Ala, X 10 -Asn-Ser, X 10 -Asn-Ser-Ile, X 10 -Asn-Ser-Tyr, X 10 -Asn-Ser-Ile-Leu, X 10 -Asn-Ser-Tyr-Leu, X 10 -Asn-Ser-Ile-Leu-Asn or X 10 -Asn-Ser-Tyr-Leu-Asn;
  • X 8 is a covalent bond, Asn, X 10 , X 10 -Asn, Tyr-X 10 , Tyr-X 10
  • X 11 is a covalent bond, Asn, Ser, He, Tyr, Leu, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Tyr, Asn-Ser-Ile-Leu, Asn-Ser-Tyr-Leu, Asn-Ser-Ile-Leu-Asn or Asn- Ser-Tyr-Leu-Asn;
  • X 12 is a covalent bond or Asn
  • X 13 is a covalent bond, X 10 , Tyr, Lys, Tyr-X 10 , Lys-X 10 , Lys-Tyr-X 10 , Lys-Lys-Tyr-X 10 , Val-Lys-Lys-Tyr-X 10 , Ala-Lys-Lys-Tyr-X 10 , or Ala-Val-Lys- Lys-Tyr-X 10 ;
  • Z is -CONH-, -NHCO-, -S-S-, -S(CH 2 )tCO-NH- or -NH-CO(CH2)tS-; m is 1 or 2 when Z is -CONH-, -S-S- or -S(CH2)tCO-NH-, or m is 2, 3 or 4 when Z is -NH-CO- or -NH-CO(CH2)tS-; n is 1 or 2 when Z is -NH-CO-, -S-S- or -NH-CO(CH2)tS- or n is 2, 3 or 4 when Z is -CO-NH- or-S(CH 2 )tCO-NH-; and t is 1 or 2.
  • fragments of VIP or of an analog thereof are selected from the group consisting of:
  • the composition comprises a peptide consisting of a physiologically active fragment of VIP coupled to a fragment of pituitary adenylate cyclase activating peptide (PACAP) or an analog thereof, said peptide being selected from the group consisting of the novel peptides VIP I 5.23PACAP 24 . 2 7 and VIPi5. 23 Nle 17 PACAP 24 . 27 of the sequences:
  • PACAP pituitary adenylate cyclase activating peptide
  • th composition comprises a VIP- related peptide that is a conjugate selected from the group consisting of: 0) R1-Y1-X1-X1'-X1"-X2-NH-Y2-R2
  • Rl is selected from the group consisting of hydrogen and a saturated or unsaturated lipophilic group having at least 4 carbon atoms
  • R2 is selected from the group consisting of hydrogen, a saturated or unsaturated lipophilic group having at least 4 carbon atoms, a lipophilic group substituted by X3-Ser-X4-Leu-Asn-NHR2 or a spacer consisting of 1-3 residues of a non-charged amino acid coupled to X1-XP-XF'-X2-NH-Y2-R2, with the proviso that at least one of Rl and R2 is a lipophilic group; Yl and Y2 are each independently -CH2-, -CO-, or a covalent bond;
  • XI is a covalent bond, Ala, Val, Ala- Val, Val- Ala, Lys, D-Lys, Ala-Lys, Val-Lys, Ala-Val-Lys, Val-Ala-Lys or Orn;
  • XI ' is Lys, D-Lys or Orn;
  • XI is Tyr, D-Tyr, Phe, Trp or a residue of p-amino phenylalanine;
  • X4 is He or Tyr;
  • X5 is a residue of a hydrophobic aliphatic amino acid
  • X2 is X5, X5-Asn, X5-Ser, X5-Ile, X5-Tyr, X5-Leu, X5-Nle, X5-D- Ala, X5-Asn-Ser, X5-Asn-Ser-Ile, X5-Asn-Ser-Tyr, X5-Asn-Ser-Ile-Leu, X5- Asn-Ser-Tyr-Leu, X5-Asn-Ser-Ile-Leu-Asn or X5-Asn-Ser-Tyr-Leu-Asn; X3 is a covalent bond, Asn, X5, X5-Asn, Tyr-X5, Tyr-X5-Asn, Lys-X5,
  • Lys-X5-Asn Lys-Tyr-X5, Lys-Tyr-X5-Asn, Lys-Lys-Tyr-X5, Lys-Lys-Tyr- X5-Asn, Val-Lys-Lys-Tyr-X5, Val-Ala-Lys-Lys-Tyr-X5-Asn or Ala-Val-Lys- Lys-Tyr-X5-Asn;
  • X6 is a covalent bond, Asn, Ser, He, Tyr, Leu, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Tyr, Asn-Ser-Ile-Leu, Asn-Ser-Tyr-Leu, Asn-Ser-Ile-Leu-Asn or Asn- Ser-Tyr-Leu-Asn;
  • X7 is a covalent bond or Asn;
  • X8 is a covalent bond, X5, Tyr, Lys, Tyr-X5, Lys-X5, Lys-Tyr-X5, Lys- Lys-Tyr-X5, Val-Lys-Lys-Tyr-X5, Ala-Lys-Lys-Tyr-X5, or Ala-Val-Lys-Lys- Tyr-X5;
  • X9 is a residue of a natural or non-natural amino acid, a residue of a natural or non-natural amino acid attached to threonine or a residue of a natural or non-natural amino acid attached to phenylalanine;
  • X10 and XI 1 are each independently a residue of a natural or non- natural amino acid;
  • X12 is a covalent bond, Lys, D-Lys or Orn;
  • XI 3 is a covalent bond or Asn
  • X14 is Phe or Thr
  • XI 5 is Asn or Ala
  • X16 is Ser or Ala; XI is He or Val;
  • Z is -CONH-, -NHCO-, -S-S-, -S(CH 2 ) t CO-NH- or -NH-CO(CH 2 ) t S-; m is 1 or 2 when Z is -CONH-, -S-S- or -S(CH 2 )tCO-NH-, or m is 2, 3 or 4 when Z is -NH-CO- or -NH-CO(CH2) t S-; n is 1 or 2 when Z is -NH-CO-, -S-S- or -NH-CO(CH2)tS- or n is 2, 3 or 4 when Z is -CO-NH- or-S(CH 2 ) t CO-NH-; and t is 1 or 2.
  • XI, XI ' are lysine
  • XI" is tyrosine
  • X2 and X5 are leucine.
  • X10 is norleucine
  • one of Rl-Yl and R2-Y2 is hydrogen.
  • X10 and XI 1 are each independently selected from the group consisting of leucine, isoleucine, norleucine, valine, tryptophan, phenylalanine, 01
  • X5 is a residue of a D- or L-amino acid selected from the group consisting of alanine, leucine, isoleucine, norleucine, valine, methionine and norvaline.
  • X9 is alanine or glycine.
  • Rl and R2 are each independently of a formula CH3(CH2)k O, where k is an integer from 2 to 16.
  • k 16
  • composition comprises a conjugate of VIP or of a VIP analog, of the sequence:
  • X 1 , X 2 and X 3 are the same or different and each is the residue of a natural or non-natural amino acid
  • R 1 and R 2 are the same or different and each is hydrogen, a saturated or unsaturated lipophilic group or a C 1 -C 4 hydrocarbyl or C ⁇ C 4 carboxylic acyl, with the proviso that at least one of R 1 and R 2 is a lipophilic group;
  • Y 1 and Y 2 may be the same or different and each is -CH 2 - or a bond in case the associated R 1 and R 2 is hydrogen and Y 1 may further be -CO-.
  • composition comprises a conjugate of a VIP antagonist or of an analog thereof, of the sequence:
  • R 1 and R 2 are the same or different and each is hydrogen, a saturate or unsaturated lipophilic group or a -C 4 hydrocarbyl or C C 4 carboxylic acyl, with the proviso that at least one of R 1 and R 2 is a lipophilic group; and
  • Y 1 and Y 2 are the same or different and each is the residue of a natural or non-natural amino acid.
  • composition comprises a conjugate of a peptide according to any one of claims 7 to 9, said conjugate having at the
  • R and R 1 amino and/or carboxy terminal radicals R and R , the same or different, each of them being hydrogen, a saturated or unsaturated lipophilic group or a C -C 4 hydrocarbyl or C 1 -C 4 carboxylic acyl, with the proviso that at least one of R 1 and R 2 is a lipophilic group.
  • the lipophilic moiety in said conjugates is selected from the group consisting of a saturated or unsaturated hydrocarbyl or carboxylic acyl radical having at least 5 carbon atoms, and is preferably selected from caproyl (Cap), lauroyl (Lau), palmitoyl, stearoyl (St), oleyl, eicosanoyl, docosanoyl, and the corresponding hydrocarbyl radicals hexyl, dodecyl, hexadecyl, octadecyl, eicosanyl, and docosanyl, and is preferably stearoyl.
  • conjugates used according to the invention are selected from the group consisting of: Stearoyl-VIP (St-VIP)
  • Stearoyl- norleucine 17 -VIP (St-Nle 17 -VIP ; SNV) Caproyl- norleucine 17 -VIP (Cap-Nle 17 -VIP) Stearoyl-leucine 5 , norleucine 17 -VIP (St-Leu 5 , Nle I7 -VIP) Stearoyl-leucine 5 , leucine 17 -VTP (St-Leu 5 , Leu 17 - VIP) Stearoyl-threonine 7 -VTP (St-Thr 7 -VIP)
  • Stearoyl- norleucine 17 -neurotensin 6 - ⁇ VTP 7 _ 28 (SNH) Stearoyl-VIP I6 . 285 St-VIP 7 . 28 and St- VIP ⁇ 6 . 28 Stearoyl-Lys-Lys-Tyr-Leu-NH 2 Stearoyl- VIPi 5 . 23 PACAP 24 . 27 and Stearoyl- VTP ⁇ 5 . 23 Nle 17 PACAP 24 .
  • the hyperproliferative skin disorder is selected from the group consisting of psoriasis, hyperproliferation caused by papilloma virus infection, dermatoses, warts, corns, calluses, dandruff and skin cancer.
  • the therapeutically effective amount of a vasoactive intestinal peptide derived conjugate is formulated into a pharmaceutical composition.
  • the pharmaceutical composition contains a pharmaceutically acceptable carrier.
  • the substance is selected from the group consisting of an immunosuppressant, an antimetabolite, a corticosteroid, vitamin D, a vitamin D analog, vitamin A, a vitamin A analog, tar, coal tar, a keratolytic agent, a keratoplastic agent, an anti-pruritic agent, an emollient, a lubricant, a disinfectant, an antiseptant, photosensitizer and UV irradiation.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing a novel use of potent lipophilic VIP derived conjugates for the treatment of hyperproliferative skin disorders
  • FIG. 1 illustrates starved HaCaT cell viability, as assessed by the metabolic dye, MTS, in the presence of the indicated concentrations of stearyl peptides: SNV (SEQ ID. NO:3), SNH (SEQ ID NO:5) and St-KKYL-NH 2 (SEQ ID NO:6).
  • FIG. 2 illustrates starved 4th passage (4°) human keratinocytes viability, as assessed by the metabolic dye, MTS, in the presence of the indicated concentrations of stearyl peptides: SNV (SEQ ID NO:3), SNH (SEQ ID NO:5) and St-KKYL-NH 2 (SEQ ID NO:6) and St-VTP(15-23)PACAP(24-27) (SEQ ID NO:3), SNV (SEQ ID NO:5) and St-KKYL-NH 2 (SEQ ID NO:6) and St-VTP(15-23)PACAP(24-27) (SEQ
  • FIG. 3 illustrates starved HT29 cell viability, as assessed by the metabolic dye, MTS, in the presence of 10 ⁇ M stearyl peptides: SNV (SEQ ID NO:3), SNH (SEQ ID NO:5) and St-KKYL-NH 2 (SEQ ID NO:6).
  • FIGs. 4A-E are phase contrast microscopy images of HaCaT cultures under different experimental conditions.
  • 4A Control cultures (10% FCS supplemented MEM).
  • 4B HaCaT cells following a 48 h growth in 0.1% BSA supplemented MEM (starved HaCaT).
  • 4C Starved HaCaT cells following a 24 h treatment with 10 ⁇ M SNH.
  • 4D Starved HaCaT cells following a 24 h treatment with ATA (25 ⁇ M).
  • 4E Starved HaCaT cells following a 24 h co- treatment with 10 ⁇ M SNH and 25 ⁇ M ATA.
  • FIG. 5 A illustrates HaCaT cell viability in the presence of 5 and 10 ⁇ M stearyl peptides, as evaluated by MTS. 0 indicates cells without treatment.
  • FIG. 5B illustrates the restoration of HaCaT cell viability by the simultaneous incubation of starved HaCaT with 25 ⁇ M ATA and SNV (SEQ ID NO:3), SNH (SEQ ID NO:5) and St- KYL-NH (SEQ ID NO:6), as evaluated by MTS. 0 indicates treatment with ATA only (without stearyl peptides).
  • FIG. 5C illustrates the restoration of HaCaT cell viability by the simultaneous incubation of starved HaCaT with 25 ⁇ M ATA and SNV (SEQ ID NO:3), SNH (SEQ ID NO:5) and St- KYL-NH (SEQ ID NO:6), as evaluated by MTS. 0 indicates treatment with ATA only (without stearyl peptides).
  • FIG. 5C illustrates the restoration of
  • SNH SEQ ID NO:5
  • St-KKYL-NH 2 SEQ ID NO:6
  • FIG. 6 illustrates SNV (SEQ ID NO:3) induced increases in cGMP production by HaCaT cells, as determined by enzyme immuno assay (EIA).
  • the present invention is of compounds, pharmaceutical compositions and methods for treating skin disorders and for inducing cell apoptosis. More particularly, the invention discloses a new treatment of skin conditions associated with hyperproliferation of skin cells, which treatment involves topical administration of lipophilized, vasoactive intestinal peptide (V ⁇ P)-derived conjugates, optionally with other substances traditionally used in the art for the treatment of such skin disorders.
  • V ⁇ P vasoactive intestinal peptide
  • a method for treating a hyperproliferative skin disorder of a patient is effected by administering to a skin area of the patient a therapeutically effective amount of a vasoactive intestinal peptide derived conjugate.
  • VIP-related peptides when referring to the conjugates, will be referred to herein also as “VIP derived conjugate”, “VIP related conjugates” and “conjugates”, which are interchangeably used herein and refer to the conjugates of the present invention, as described hereinbelow and exemplified by “stearoyl peptides” .
  • a VIP derived conjugate includes a peptidic portion derived from VIP attached to a hydrophobic moiety.
  • skin disorder or “skin disease” as used herein in the specification and in the claim section that follows, include to abnormal skin conditions, as manifested in, but not limited to, hyperproliferative skin disorders and disorders of keratinization, cornification and scaling skin conditions.
  • the former includes, for example, various dermatoses, such as atopical dermatitis, contact dermatitis, seborrheic dermatitis, chronic eczematous dermatitis, psoriasis, hyperproliferation caused by papilloma virus infection and skin cancer, such as squamous cell carcinoma and basal cell carcinoma.
  • various dermatoses such as atopical dermatitis, contact dermatitis, seborrheic dermatitis, chronic eczematous dermatitis, psoriasis, hyperproliferation caused by papilloma virus infection and skin cancer, such as squamous cell carcinoma and basal cell carcinoma.
  • Certain benign hyperproliferative of the skin result from excess keratin deposition (hyperkeratosis) of the corneous layer.
  • hyperproliferative disorders include, for example, epidermolytic hyperkeratosis and follicular keratosis.
  • hypertrophic scar formation a keloid
  • a sharply elevated, irregularly-shaped, progressively enlarging scar due to the formation of excessive amounts of collagen in the corium during connective tissue repair following surgical and traumatic lacerations. While such hypertrophic tissue repair is most evident at sites of 01
  • keloid-prone individuals also manifest hypertrophic scarring internally.
  • the major consequences of external keloid scarring are mainly cosmetic, although keloids can also result in varying degrees of psychological and social trauma for the afflicted individuals. In such cases, surgical or laser intervention is indicated because there is currently no generally effective topical or systemic treatment for this condition.
  • hyperkeratotic afflictions include, for example, corns (heloma), calluses (tyloma) and warts (condyloma). These are well defined, thickened lesions of the epidermis, which occur at skin sites that are normally involved in chronic mechanical stress (corns and calluses) or infected with papilloma virus (warts). Pain produced by the thickened tissue can cause these lesions to be debilitating. Traditionally, keratolytic agents, such as salicylic acid and resorcinol, have been applied topically to these lesions to solubilize intercellular bonds resulting in desquamation of the thickened, hyperkeratotic tissues.
  • keratolytic agents such as salicylic acid and resorcinol
  • hyperkeratinizing and/or hyperproliferative skin is associated in conditions such as ichthyosis, porokeratoses, palmoplantar keratodermas, eczema, dandruff and dry skin.
  • treatment should be understood in the context of the present invention as prevention, alleviation, amelioration, improvement, minimization or abolishment of the abnormal conditions or symptoms manifested or associated with a skin disorder.
  • patienf refers to a subject in need of such treatment, such as, but not limited to, a mammal, preferably a human.
  • the VIP derived conjugates of the present invention may be administered by any conventional method which is known in the art, such as, but not limited to, parenterally, orally, and via inhalation routes, with topical administration being the preferred mode of administration.
  • administered to a skin area refers to administering a VIP-related peptide/conjugate of the present invention to an afflicted skin or a lesion.
  • topical administration is used herein in its conventional sense to indicate delivery of a topical drug or pharmacologically active agent to the skin or mucosa, as in, for example, the treatment of various skin disorders.
  • drug or the phrases “active ingredienf and “pharmacologically active agent” is meant a chemical compound suitable for topical administration and which induces a desired therapeutic effect.
  • an effective amount of a drug is meant a sufficient amount of a compound, to provide the desired local effect and performance at a reasonable benefit/risk ratio attending any medical treatment.
  • “Therapeutically effective amount”, “an amount sufficient” or “an effective amount” is that amount of a given VIP-related peptide, which antagonizes or inhibits cell proliferation, induces differentiation or result in cell death of cells, such as keratinocytes, both in vivo and in vitro, or, which provides either a subjective relief of symptom(s) in a patient or an objectively identifiable improvement, as noted by a clinician or other qualified observer.
  • Vasoactive intestinal peptide related of the present invention include a peptide sequence, either full length or a fragment, linear or cyclic, derived from vasoactive intestinal peptide (VIP, SEQ ID NO:l) and/or VHA (SEQ ID NO:4) and modified at its C- and/or N-terminus by a hydrophobic moiety, as is further described herein.
  • VIP vasoactive intestinal peptide
  • SEQ ID NO:l vasoactive intestinal peptide
  • VHA VHA
  • the VIP-related peptide includes the sequence KKYL (SEQ ID NO:7) as part of its peptidic portion.
  • a therapeutically effective amount of a substance traditionally used for treatment of the hyperproliferative skin disorder is coadministered along with a VIP-related peptide.
  • the phrase "substances traditionally used for the treatment of the hyperproliferative skin disorder” refers to known therapeutic agents or treatment approaches, presently employed in the management of skin disorders.
  • Various modalities are known in the art for the treatment of hyperproliferative skin disorders, such as psoriasis, all of which are potentially formulated with the conjugates of the present invention for treating any one or more of the skin disorders described hereinabove.
  • combination therapy preferably employs combinations of non overlapping mechanisms, such as immunosuppression by corticosteroids, all as is further detailed hereinbelow.
  • a method of inducing cell apoptosis is effected by subjecting cells to a vasoactive intestinal peptide related peptide.
  • Apoptosis is an evolutionary conserved, gene-directed, active cell death that follows an orderly pattern of morphologic and biochemical changes. Common histologic observations have established that a strict sequence of events is shared by all apoptotic cells. These include loss of cell contact accompanied by cell rounding and smoothing of the cell surface, loss of cytoskeletal integrity, cell shrinkage with resultant compaction of the cytoplasm and organelles, nuclear and chromatin condensation, chromatin fragmentation, formation of apoptotic bodies and engulfment. Throughout the various tissues of the organism, apoptosis normally functions in developmental remodeling, regulation of cell numbers, and defense against damaged, virus- infected, auto-reactive and transformed cells [5, 9, 17].
  • Agents and events that can activate the apoptotic pathway are many and varied, and include growth factors, hormones, cytokines, UV and gamma irradiation, disruption of oxidative pathways and cell matrix interactions [10].
  • growth factors include growth factors, hormones, cytokines, UV and gamma irradiation, disruption of oxidative pathways and cell matrix interactions [10].
  • cytokines include growth factors, hormones, cytokines, UV and gamma irradiation, disruption of oxidative pathways and cell matrix interactions [10].
  • the pattern of apoptosis itself is highly conserved, indicative of a limited number of shared effector mechanisms that trigger apoptosis [24].
  • the pathway is conceptually divided into three mechanistically distinct phases: induction (or initiation), effector and degradation [11].
  • induction or initiation
  • effector the heterogeneous stimuli
  • the heterogeneous stimuli activate receptor- and non- receptor-mediated signal transduction pathways and second messengers such as cAMP, inositol triphosphate, diacylglycerol and ceramides [17, 25].
  • the pathways are not reserved for apoptotic signaling, but are also used for other cellular functions, such as growth control.
  • the outcome of signaling depends on the particular inducing stimulus (which may affect one or more signal transduction pathways), the developmental or physiologic state of the cell, and the cell's lineage.
  • a common outcome of the different signal transduction pathways that impact on apoptosis is interference with the cell cycle.
  • a major control of apoptotic susceptibility is determined by the cell cycle and its G l and Q2 checkpoints, which prevent aberrant DNA synthesis and mitosis [26]. These checkpoints also control cell cycle arrest necessary for commitment to normal terminal differentiation.
  • the effector phase common to all cell types, is regulated by distinct classes of "cell death genes", e.g., the bcl-2 genes can suppress cell death at the transition from induction to effector phases [35].
  • Many viral proteins are also effective inhibitors of cellular proteins controlling the inductive phase of apoptosis.
  • Tumor promoters and oncogenes function analogously to viral proteins to inhibit apoptosis and promote proliferation.
  • effector pathway enzymes of the caspase family activate other effector proteins that will be involved in the degenerative phase, possibly including additional proteases, endonucleases and transglutaminases. The action of which result in the characteristic morphologic cascade, typifying apoptosis [38].
  • the epidermis is a continually renewing tissue in which homeostasis ismaintained by factors that influence the interrelated processes of keratinocyte W 01
  • apoptosis is a controlling factor of the cell birth to cell death ratio [39].
  • New cells are produced in the proliferative compartment, primarily the basal cell layer, and are balanced by cell death (terminal differentiation/apoptosis). Terminal differentiation, by itself, may be an elaborated apoptotic pathway [40].
  • apoptotic dysregulation contributes to the hyperplasia and aberrant differentiation typical of the psoriatic plaque [12].
  • ATA a triphenylmethane dye
  • ATA a triphenylmethane dye
  • Dysregulation of the apoptotic pathway has been implicated in the pathogenesis and promotion of skin diseases, such as skin cancer and psoriasis. It has been previously suggested that terminal differentiation of the keratinocyte is a specialized form of apoptosis [11]. Apoptosis was suggested as the default pathway taken in the absence of continued proliferation or normal differentiation. The time between commitment to death and execution of the apoptotic effector phase was suggested to be extended in normally differentiating keratinocytes, relative to rapid apoptosis of abnormal or unnecessary keratinocytes, to allow expression of proteins subserving the differentiated function. For normal function, the apoptotic pathway, as the foundation of keratinocyte terminal differentiation, must be completed.
  • the cytotoxic effect of the VIP-related peptides of the present invention may be a result of their induction of excessive differentiation in the keratinocytes, resulting in unavoidable death.
  • SNV SEQ ID NO:3
  • SNH SEQ ID NO:5
  • Stearoyl- VIP 15 PACAP 24 . 27
  • PACAP 24 . 27 SEQ ID NO:21
  • Stearoyl-KKYL- H2 SEQ ID NO:6
  • a pharmaceutical composition for treating a hyperproliferative skin disorder comprising, as active ingredients, therapeutically effective amounts of a VTP-related peptide and a substance traditionally used for treatment of the hyperproliferative skin disorder.
  • the pharmaceutically active ingredients of the present invention can be administered to an organism per se, or in a pharmaceutical composition mixed with suitable carriers and or excipients.
  • Pharmaceutical compositions suitable for use in context of the present invention include those compositions in which the active ingredients are contained in an amount effective to achieve an intended therapeutic effect.
  • a "pharmaceutical composition” refers to a preparation of one or more of the VIP-related peptides described herein, or physiologically acceptable salts or prodrugs thereof, with other chemical components such as traditional drugs, physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • physiologically suitable carrier and
  • “pharmaceutically acceptable carrier” are interchangeably used and refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered VIP-related peptide.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate processes and administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
  • the topical route is preferred, and is assisted by a topical carrier.
  • the topical carrier is one, which is generally suited for topical drug administration and includes any such materials known in the art.
  • the topical carrier is selected so as to provide the composition in the desired form, e.g., as a liquid or non-liquid carrier, lotion, cream, paste, gel, powder, ointment, solvent, shampoo, liquid diluent, drops and the like, and may be comprised of a material of either naturally occurring or synthetic origin.
  • the selected carrier does not adversely affect the active agent or other components of the topical formulation, and which is stable with respect to all components of the topical formulation.
  • suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like.
  • the composition of the invention may also be administered in the. form of a shampoo, in which case conventional components of such a formulation are included as well, e.g., surfactants, conditioners, viscosity modifying agents, humectants, and the like.
  • Ointments are colorless, odorless ointments, lotions, creams and gels.
  • Ointments are semisolid preparations, which are typically based on petrolatum or other petroleum derivatives.
  • the specific ointment base to be' used is one that will provide for optimum drug delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like.
  • an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed.
  • ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases.
  • Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum.
  • Emulsifiable ointment bases also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.
  • Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.
  • W/O water-in-oil
  • O/W oil-in-water
  • Preferred water- soluble ointment bases are prepared from polyethylene gly cols of varying molecular weight; again, reference may be made to Remington: The Science and Practice of Pharmacy for further information.
  • Lotions are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations, in which solid particles, including the active agent, are present in a water or alcohol base.
  • Lotions are usually suspensions of solids, and preferably, for the present purpose, comprise a liquid oily emulsion of the oil-in-water type.
  • Lotions are preferred formulations herein for treating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be finely divided.
  • Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethylcellulose, or the like.
  • Creams containing the selected VIP derived conjugates are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in- oil.
  • Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase.
  • the oil phase also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.
  • Gel formulations are preferred for application to the scalp.
  • gels are semisolid, suspension-type systems.
  • Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.
  • Shampoos for treating psoriasis and other skin conditions associated with hyperproliferation and/or a keratolytic disorder, such as dandruff may be formulated with the VIP derived conjugate and standard shampoo components, i.e., cleansing agents, thickening agents, preservatives, and the like, with the cleansing agent representing the primary ingredient, typically an anionic surfactant or a mixture of an anionic and an amphoteric surfactant.
  • Various additives known to those skilled in the art, may be included in the topical formulations of the invention. For example, solvents may be used to solubilize certain drug substances.
  • topical preparations for the treatment of skin disorders according to the present invention may contain other pharmaceutically active agents or ingredients, those traditionally used for the treatment of such disorders.
  • immunosuppressants such as cyclosporine, antimetabolites, such as methotrexate, corticosteroids, vitamin D and vitamin D analogs, vitamin A or its analogs, such etretinate, tar, coal tar, anti pruritic and keratoplastic agents, such as cade oil, keratolytic agents, such as salicylic acid, emollients, lubricants, antiseptic and disinfectants, such as the germicide dithranol (also known as anthralin) photosensitizers, such as psoralen and methoxsalen and UV irradiation.
  • Other agents may also be added, such as antimicrobial agents, antifungal agents, antibiotics and anti- inflammatory agents.
  • the topical compositions of the invention may also be delivered to the skin using conventional "transdermal"-type patches, wherein the drug composition is contained within a laminated structure, that serves as a drug delivery device to be affixed to the skin.
  • the drug composition is contained in a layer, or "reservoir", underlying an upper backing layer.
  • the laminated structure may contain a single reservoir, or it may contain multiple reservoirs.
  • the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery.
  • suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
  • the particular polymeric adhesive selected will depend on the particular drug, vehicle, etc., i.e., the adhesive must be compatible with all components of the drug-containing composition.
  • the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
  • the backing layer in these laminates which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility.
  • the material selected for the backing material should be selected so that it is substantially impermeable to the drug and to any other components of the drug-containing composition, thus preventing loss of any components through the upper surface of the device.
  • the backing layer may be either occlusive or nonocclusive, depending on whether it is desired that the skin become hydrated during drug delivery.
  • the backing is preferably made of a sheet or film of a preferably flexible elastomeric material. Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, and polyesters.
  • the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the drug reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin.
  • the release liner should be made from a drug/vehicle impermeable material.
  • Such devices may be fabricated using conventional techniques, known in the art, for example by casting a fluid admixture of adhesive, drug and vehicle onto the backing layer, followed by lamination of the release liner. Similarly, the adhesive mixture may be cast onto the release liner, followed by lamination of the backing layer.
  • the drug reservoir may be prepared in the absence of drug or excipient, and then loaded by "soaking" in a drug/vehicle mixture.
  • the drug composition contained within the drug reservoirs of these laminated system may contain a number of components. In some cases, the drug may be delivered "neat,” i.e., in the absence of additional liquid. In most cases, however, the drug will be dissolved, dispersed or suspended in a suitable pharmaceutically acceptable vehicle, typically a solvent or gel. Other components, which may be present, include preservatives, stabilizers, surfactants, and the like.
  • Both the topical formulations and the laminated drug delivery systems may in addition contain a skin permeation enhancer. While the conjugates of the present invention have been specifically designed so as to increase their biological membrane penetration, it may yet be desirable to enhance the inherent permeability of the skin to the drug. Thus, an adequate therapeutic levels of the drug, passing through a reasonably sized area of unbroken skin, can be assured by coadministration of a skin permeation enhancer with such drugs.
  • Suitable enhancers are well know in the art and include, for example, dimethylsulfoxide (DMSO), dimethyl formamide (DMF), N,N- dimethylacetamide (DMA), decylmethylsulfoxide (CIO MSO), C2-C6 alkanediols, and the 1 -substituted azacycloheptan-2-ones, particularly 1-n- dodecylcyclazacycloheptan-2-one (available under the trademark Azone® from Whitby Research Incorporated, Richmond, Va.), alcohols, and the like.
  • suitable carriers are olive oil, glycerin, lubricants, nitroglycerin and SefsolTM, and mixtures thereof. Sefsol is a trademark (Nikko Chemicals, Tokyo) for, 1-glyceryl monocaprylate, glyceryl tricaprylate and sorbitan monocaprylate.
  • compositions herein described may also comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
  • topical compositions and drug delivery systems of the invention can be used in the prevention or treatment of the skin conditions identified above.
  • susceptible skin is treated prior to any visible lesions on areas known to be susceptible to such lesions in a particular individual.
  • Dosing is dependent on the type, the severity and manifestation of the affliction and on the responsiveness of the subject to the VIP derived lipophilic conjugates, as well as the dosage form employed the potency of the particular conjugate and the route of administration utilized. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • dosing can be a single or repetitive administration, with course of 01 00523
  • Conjugates of the present invention contain a peptide constituent.
  • “Peptides” and “polypeptides” are chains of amino acids (typically L-amino acids) whose ⁇ -carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the ⁇ -carbon of one amino acid and the amino group of the ⁇ -carbon of another amino acid.
  • the terminal amino acid at one end of the chain i.e., the amino terminal
  • the terminal amino acid at the other end of the chain i.e., the carboxy terminal
  • amino terminus refers to the free ⁇ -amino group on the amino acid at the amino terminal of the peptide or to the ⁇ -amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide.
  • carboxy terminus refers to the free carboxyl group on the amino acid at the carboxy terminus of a peptide or to the carboxyl group of an amino acid at any other location within the peptide.
  • amino acids making up a polypeptide are numbered in order, starting at the amino terminal and increasing in the direction of the carboxy terminal of the polypeptide.
  • that amino acid is positioned closer to the carboxy terminal of the polypeptide than the "preceding" amino acid.
  • the term "residue” as used herein refers to an amino acid or an amino acid mimetic that is incorporated into a peptide by an amide bond or an amide bond mimetic.
  • the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics).
  • the vasoactive intestinal peptide derived conjugate includes at least one non-natural amino acid.
  • the at least one non-handed ⁇
  • PCT/IL01/00523 natural amino acid is preferably selected from the group consisting of a D amino acid, norleucine, norvaline, ⁇ -aminobutyric acid, di-amino butyric acid, di-aminopropionic acid, -HN(CH2) n COOH, wherein n is 3-5, -NH(CH 2 (R))n- COOH, wherein R is an alkyl, H2N(CH2) m COOH, wherein m is 2-4, H2N- C(NH)-NH(CH2)kCOOH, wherein k is 2-3, hydroxy lysine, N-methyl lysine, ornitine, p-amino phenylalanine, TIC, naphthylelanine, a ring-methylated derivative of phenylalanine, a halogenated derivative of phenylalanine or o- methyl-tyrosine.
  • the peptidic moiety of the VIP derived conjugates of the present invention may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, i.e., to increase biological activity, bioavailability, stability and biological membrane penetration.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as, for example, Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Residues which can be modified without loosing the biological activity of the VIP derived conjugate can be identified by single amino acid substitutions, deletions, or insertions using conventional techniques known to those of ordinary skill in the art, this especially true of the VIP derived conjugates of the present invention, being that they are relatively short in length.
  • the contributions made by the side chains of the residues can be probed via a systematic scan with a specified amino acid (e.g., Ala).
  • the VIP derived conjugates of the present invention are relatively short in length and are typically no more than 28 amino acids in length. As such, it is feasible to prepare such conjugates using any of a number of chemical peptide synthesis techniques well known to those of ordinary skill in the art, including both solution methods and solid phase methods, with solid phase synthesis being presently preferred
  • solid phase synthesis in which the C-terminal amino acid of the peptide sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for preparing the VIP antagonists of the present invention.
  • Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology (Gross and Meienhofer (eds.), Academic press, New York, vol. 2, pp. 3-284 (1980)); Merrifield, et al., J. Am. Chem. Soc. 85, 2149-2156 (1963); and Stewart, et al., Solid Phase Peptide Synthesis (2nd ed., Pierce Chem. Co., Rockford, 111. (1984)), the teachings of which are hereby incorporated by reference.
  • Solid phase synthesis is started from the carboxy-terminal end (i.e., the C-terminus) of the peptide by coupling a protected amino acid via its carboxyl group to a suitable solid support.
  • the solid support used is not a critical feature of the present invention provided that it is capable of binding to the carboxyl group while remaining substantially inert to the reagents utilized in the peptide synthesis procedure.
  • a starting material can be prepared by attaching an amino-protected amino acid via a benzyl ester linkage to a chloromethylated resin or a hydroxymethyl resin or via an amide bond to a benzhydrylamine (BHA) resin or p-methylbenzhydrylamine (MBHA) resin.
  • halomethyl resins such as chloromethyl resin or bromomethyl resin
  • hydroxymethyl resins such as phenol resins, such as 4-( ⁇ -[2,4-dimethoxyphenyl]-Fmoc- aminomethyl)phenoxy resin
  • tert-alkyloxycarbonyl-hydrazidated resins such as tert-alkyloxycarbonyl-hydrazidated resins, and the like.
  • resins are commercially available and their methods of preparation are known by those of ordinary skill in the art.
  • the acid form of the peptides of the present invention may be prepared by the solid phase peptide synthesis procedure using a benzyl ester resin as a solid support.
  • the corresponding amides may be produced by using benzhy rylamine or methylbenzhydrylamine resin as the solid support.
  • benzhy rylamine or methylbenzhydrylamine resin as the solid support.
  • treatment with anhydrous hydrofluoric acid to cleave the polypeptide from the solid support produces a polypeptide having a terminal amide group.
  • the ⁇ -amino group of each amino acid used in the synthesis should be protected during the coupling reaction to prevent side reactions involving the reactive ⁇ -amino function.
  • Certain amino acids also contain reactive side- chain functional groups (e.g., sulfhydryl, amino, carboxyl, hydroxyl, etc.), which must also be protected with appropriate protecting groups, to prevent chemical reactions from occurring at those sites during the polypeptide synthesis.
  • Protecting groups are well known to those of skill in the art. See, for example, The Peptides: Analysis, Synthesis, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Gross and Meienhofer (eds.), Academic Press, New York (1981)), the teachings of which are incorporated herein by reference.
  • a properly selected ⁇ -amino protecting group will render the ⁇ -amino function inert during the coupling reaction, will be readily removable after coupling under conditions that will not remove side chain protecting groups, will not alter the structure of the peptide fragment, and will prevent racemization upon activation immediately prior to coupling.
  • side- chain protecting groups must be chosen to render the side chain functional group inert during the synthesis, must be stable under the conditions used to remove the ⁇ -amino protecting group, and must be removable after completion of the polypeptide synthesis under conditions that will not alter the structure of the polypeptide.
  • protecting groups for an ⁇ -amino group include, but are not limited to, the following: aromatic urethane-type groups such as, for O 01/93889 example, fluorenylmethyloxycarbonyl (Fmoc), carbobenzoxy (Cbz), and substituted benzyloxycarbonyls including p-chlorobenzyloxycarbonyl, o- chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2,6- dichlorobenzyloxycarbonyl, etc.; aliphatic urethane-type groups such as, for example, butyloxycarbonyl (Boc), t-amyloxycarbonyl, isopropyloxycarbonyl, 2-(p-bi ⁇ henylyl)-isopropyloxycarbonyl, allyloxycarbonyl, etc.; and cycloalkyl urethane-type groups such as, for example, cyclopentyl
  • any of the protecting groups described above for the protection of the ⁇ -amino group are suitable.
  • other suitable protecting groups include, but are not limited to, the following: butyloxycarbonyl (Boc), p-chlorobenzyloxycarbonyl, p-brorhobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,6- dichlorobenzyloxy- carbonyl, 2,4-dichlorobenzyloxycarbonyl, o- bromobenzyloxycarbonyl, p-nitro- benzyloxycarbonyl, t-butyloxycarbonyl, isopropyloxycarbonyl, t-amyloxy- carbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxy- carbonyl, adamantyloxycarbonyl, p- tol
  • suitable protecting groups include, but are not limited to, the following: nitro, tosyl (Tos), carbobenzoxy (Cbz), adamantyloxycarbonyl (Adoc), butyloxycarbonyl (Boc), 4-metnoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) and 2,2,5,7,8-pentamethylchloroman-6-sulfonyl (PMC).
  • 4-methoxy-2,3,6-trimethylbenzenesulfonyl and 2,2,5,7,8-penta- methylchloroman-6-sulfonyl are the protecting groups used for Arg.
  • hydroxyl group on the side chains of serine (Ser), threonine (Thr) or tyrosine (Tyr) can be protected by a C1-C4 alkyl such as, for example, methyl, O 01/93889
  • ethyl and t-butyl or by a substituted benzyl such as, for example, p- methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl and 2,6- dichlorobenzyl.
  • the preferred aliphatic hydroxyl protecting group for Ser, Thr and Tyr is t-butyl.
  • the carboxyl group of aspartic acid (Asp) may be protected by, for example, esterif ⁇ cation using groups such as benzyl, t-butyl, cyclohexyl, cyclopentyl, and the like. For Asp, t-butyl is the presently preferred protecting group.
  • the basic imidazole ring in histidine may be protected by, for example, t-butoxymethyl (Bom), butyloxycarbonyl (Boc) and fluorenylmethyloxycarbonyl (Fmoc).
  • t-butoxymethyl (Bom) is the protecting group used.
  • Coupling of the amino acids may be accomplished by a variety of chemistries known to those of skill in the art. Typical approaches involve either the conversion of the amino acid to a derivative that will render the carboxyl group more susceptible to reaction with the free N-terminal amino group of the polypeptide fragment, or use of a suitable coupling agent such as, for example, N,N'-dicyclohexylcarbodimide (DCC) or N,N'-diisopropylcarbodiimide (DIPCDI). Frequently, hydroxybenzotriazole (HOBt) is employed as a catalyst in these coupling reactions. Appropriate synthesis chemistries are disclosed in The Peptides: Analysis, Structure, Biology, Vol.
  • Fmoc-Asn can, for example, be coupled to the 4-( ⁇ -[2,4- dimethoxy ⁇ henyl]-Fmoc-amino-methyl) ⁇ henoxy resin using N,N'- dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole (HOBt) at about 25 °C, for about two hours with stirring.
  • DCC N,N'- dicyclohexylcarbodimide
  • HOBt hydroxybenzotriazole
  • the ⁇ -amino protecting group is removed using 20% piperidine in DMF at room temperature. After removal of the ⁇ -amino protecting group, the remaining Fmoc- protected amino acids are coupled stepwise in the desired order.
  • Appropriately protected amino acids are commercially available from a number of suppliers (e.g., Nova (Switzerland) or Bachera (California)).
  • appropriately protected peptide fragments consisting of more than one amino acid may also be coupled to the "growing" polypeptide. Selection of an appropriate coupling reagent, as explained above, is well known to those of skill in the art. It should be noted that since the VIP derived conjugates of the present invention are relative short in length, this latter approach (i.e., the segment condensation method) is not the most efficient method of peptide synthesis.
  • Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in excess and the coupling is carried out in a medium of dimethylformamide (DMF), methylene chloride (CH2CI2) or, mixtures thereof. If coupling is incomplete, the coupling reaction may be repeated before deprotection of the ⁇ -amino group and addition of the next amino acid. Coupling efficiency may be monitored by a number of means well known to those of skill in the art. A preferred method of monitoring coupling efficiency is by the ninhydrin reaction. Polypeptide synthesis reactions may be performed automatically using a number of commercially available peptide synthesizers (e.g., Biosearch 9500, Biosearch, San Raphael, California).
  • the peptide can be cleaved and the protecting groups removed by stirring the insoluble carrier or solid support in anhydrous, liquid hydrogen fluoride (HF) in the presence of anisole and dimethylsulfide at about 0 °C. for about 20 to 90 minutes, preferably 60 minutes; by bubbling hydrogen bromide (HBr) continuously through a 1 mg/10 mL suspension of the resin in trifiuoroacetic acid (TFA) for 60 to 360 minutes at about room temperature, depending on the protecting groups selected; or, by incubating the solid support inside the reaction column used for the solid phase synthesis with 90% trifiuoroacetic acid, 5% water and 5% triethylsilane for about 30 to 60 minutes.
  • HF liquid hydrogen fluoride
  • TFA trifiuoroacetic acid
  • Other deprotection methods well known to those of skill in the art may also be used.
  • the conjugates of the present invention can be isolated and purified from the reaction mixture by means of peptide purification well known to those of skill in the art.
  • the polypeptides may be purified using known chromatographic procedures such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent distribution. See, the Example Section, infra, for a detailed description of the methods and protocols used to synthesize and purify the VIP antagonists of the present invention.
  • the VIP derived conjugates of the present invention are preferably prepared or produced using chemical peptide synthesis techniques such as described above, it will be understood by those of ordinary skill in the art that they can also be prepared by other means including, for example, recombinant techniques.
  • the amino acids referred to herein are described by shorthand designations as follows:
  • the peptidic moiety is further derivatized by a lipophilic moiety, with, for example alkyl or acyl groups as described hereinabove, by methods commonly used in the art.
  • the vasoactive intestinal peptide derived conjugate includes at least one peptide-bond modification.
  • An amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
  • peptide bonds (-CO-NH-) within the peptide may be substituted by N-methylated bonds (-N(CH3 CO-), ester bonds (-C(R)H-C-0- 0-C(R)-N-), ketomethylene bonds (-CO-CH2-), ⁇ -aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefinic double bonds (- CH-CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO- ), wherein R is the natural side chain on the carbon atom.
  • R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), o
  • the VIP-related includes a cyclic peptide moiety, as is further described hereinbelow.
  • cyclic peptides containing intramolecular amide bonds i.e., -CONH- or -NHCO- may be prepared through conventional solid phase synthesis.
  • peptide chains may be assembled on the solid support while incorporating suitable amino and carboxyl side chain protected amino acid derivatives at the positions selected for cyclization.
  • the protecting groups can be selectively removed from the corresponding amino and carboxyl functions, leaving other protecting groups and the peptide-support bond intact.
  • Cyclization can then be accomplished using known peptide coupling agents.
  • the cyclic peptide may be cleaved from the support along with deprotection of side chain moieties using known procedures, and purification of the desired cyclic peptide can be achieved by chromatographic techniques.
  • Cyclic peptides containing intramolecular disulfide bond may be prepared through conventional solid phase synthesis while inco ⁇ orating S-protected cysteine or homocysteine residues at the positions selected for cyclization.
  • two possible routes for cyclization can be performed: 1. Selective removal of S- protecting groups with a consequent on-support oxidation of free corresponding two SH-functions, to form S'-S bonds. This may be followed by conventional removal of the product from the support and appropriate chromatographic purification. 2. Removal of the peptide from the support along with complete side-chain deprotection, followed by oxidation of free SH- functions in highly dilute aqueous solution. Both routes lead to the same final desired product. Cyclic peptides containing intramolecular S-alkyl bonds, i.e., -
  • S(CH2)tCO-NH or -NH-CO(CH2)tS- may be prepared through conventional solid phase synthesis.
  • an amino acid residue with suitable amino- protected side chain, and a suitable S-protected cysteine or homocysteine residue may be incorporated during peptide chain assembly at positions selected for cyclization.
  • the blocked side-chain amino function is selectively deprotected followed by bromoacylation.
  • the peptide can then be detached from the support, along with side-chain deprotection, under acidic conditions. Under neutral or slightly basic conditions, the corresponding free SH and bromoacylated moieties may then selectively interact at high dilution to afford the desired cyclic peptide.
  • the vasoactive intestinal peptide derived conjugate includes a lipophilic moiety, such as, saturated alkyl, branched alkyl, non-saturated alkyl, non-branched alkyl, saturated acyl, branched acyl, non-saturated acyl and non-branched acyl.
  • a lipophilic moiety such as, saturated alkyl, branched alkyl, non-saturated alkyl, non-branched alkyl, saturated acyl, branched acyl, non-saturated acyl and non-branched acyl.
  • alkyl is used herein to refer to substituents that are monovalent aliphatic hydrocarbon radicals.
  • the alkyl groups may be straight- chain or branched-chain, with for example, straight-chain alkyl groups of C4- C20.
  • alkyl radicals include, but are not limited to, the following: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and icosyl.
  • acyl is used herein to refer to an organic radical derived from an organic acid by removal of the hydroxyl group.
  • the acyl radical or group "butyryl” is derived from butanoic acid by removal of the hydroxyl group.
  • the acyl group "stearyl” is derived from stearic acid by removal of the hydroxyl group.
  • the acyl group may be saturated or unsaturated, with acyl groups having, for example, four to twenty carbon atoms.
  • a “saturated” acyl or alkyl group is one which has no double or triple bonds, whereas an "unsaturated” acyl or alkyl group is one which has double or triple bonds.
  • Suitable acyl groups include, but are not limited to, the following: butyryl, caproyl, octanoyl, lauryl, myristyl, palmityl, stearyl, aracidyl, linoceryl, etc.
  • butyryl caproyl
  • octanoyl lauryl
  • myristyl palmityl
  • stearyl aracidyl
  • linoceryl etc.
  • vasoactive intestinal peptide derived conjugate of the present invention is preferably of any of the formulae listed below:
  • Rl is selected from the group consisting of hydrogen and a saturated or unsaturated lipophilic group having at least 4 carbon atoms;
  • R2 is selected from the group consisting of hydrogen, a saturated or unsaturated lipophilic group having at least 4 carbon atoms, a lipophilic group substituted by X3-Ser-X4-Leu-Asn-NH .2 or a spacer consisting of 1-3 residues of a non-charged amino acid coupled to Xl-Xr-Xl"-X2-NH-Y2-R2, with the proviso that at least one of Rl and R2 is a lipophilic group;
  • Yl and Y2 are each independently -CH2-, -CO-, or a covalent bond;
  • XI is a covalent bond, Ala, Val, Ala- Val (SEQ ID NO: 8), Val-Ala (SEQ ID NO: 9), Lys, D-Lys, Ala-Lys (SEQ ID NO: 10), Val-Lys (SEQ ID NO: 11), Ala-Val-Lys (SEQ ID NO: 12), Val-Ala-Lys (SEQ ID NO: 13), or Orn;
  • XI ' is Lys, D-Lys or Orn;
  • XI is Tyr, D-Tyr, Phe, Trp or a residue of p-amino phenylalanine;
  • X4 is He or Tyr;
  • X5 is a residue of a hydrophobic aliphatic amino acid
  • X2 is X5, X5-Asn, X5-Ser, X5-Ile, X5-Tyr, X5-Leu, X5-Nle, X5-D- Ala, X5-Asn-Ser, X5-Asn-Ser-Ile, X5-Asn-Ser-Tyr, X5-Asn-Ser-Ile-Leu, X5- Asn-Ser-Tyr-Leu, X5-Asn-Ser-Ile-Leu-Asn or X5-Asn-Ser-Tyr-Leu-Asn;
  • X3 is a covalent bond, Asn, X5, X5-Asn, Tyr-X5, Tyr-X5-Asn, Lys-X5,
  • Lys-X5-Asn Lys-Tyr-X5, Lys-Tyr-X5-Asn, Lys-Lys-Tyr-X5, Lys-Lys-Tyr- X5-Asn, Val-Lys-Lys-Tyr-X5, Val-Ala-Lys-Lys-Tyr-X5-Asn or Ala-Val-Lys- Lys-Tyr-X5-Asn; 01
  • X6 is a covalent bond, Asn, Ser, He, Tyr, Leu, Asn-Ser (SEQ ID NO: 14), Asn-Ser-Ile (SEQ ID NO: 15), Asn-Ser-Tyr (SEQ ID NO: 16), Asn-Ser- Ile-Leu (SEQ ID NO: 17), Asn-Ser-Tyr-Leu (SEQ ID NO: 18), Asn-Ser-Ile- Leu-Asn (SEQ ID NO: 19), or Asn-Ser-Tyr-Leu-Asn (SEQ ID NO: 20);
  • X7 is a covalent bond or Asn;
  • X8 is a covalent bond, X5, Tyr, Lys, Tyr-X5, Lys-X5, Lys-Tyr-X5, Lys- Lys-Tyr-X5, Val-Lys-Lys-Tyr-X5, Ala-Lys-Lys-Tyr-X5, or Ala-Val-Lys-Lys- Tyr-X5;
  • X9 is a residue of a natural or non-natural amino acid, a residue of a natural or non-natural amino acid attached to threonine or a residue of a natural or non-natural amino acid attached to phenylalanine;
  • XI 0 and XI 1 are each independently a residue of a natural or non- natural amino acid
  • XI 2 is a covalent bond, Lys, D-Lys or Orn;
  • X13 is a covalent bond or Asn;
  • X14 is Phe or Thr
  • XI 5 is Asn or Ala
  • XI 6 is Ser or Ala
  • XI 7 is He or Val;
  • Z is -CONH-, -NHCO-, -S-S-, -S(CH 2 ) t CO-NH- or -NH-CO(CH ) t S-;
  • m is 1 or 2 when Z is -CONH-, -S-S- or -S(CH 2 )tCO-NH-, or m is 2, 3 or 4 when Z is -NH-CO- or -NH-CO(CH2)tS-;
  • n is 1 or 2 when Z is -NH-CO-, -S-S- or -NH-CO(CH )tS- or n is 2, 3 or 4 when Z is -CO-NH- or-S(CH 2 ) t CO-NH-; and
  • t is 1 or 2.
  • XI, XI' are lysine
  • XI" is tyrosine
  • X2 and X5 are leucine, thus yielding the KKYL (SEQ ID NO: 7), identified as the core active fragment within the VTP peptide.
  • XI 0, the amino acid residue at position 17 of the full length VIP peptide, is norleucine. Additionally and preferably, one of Rl-Yl and R2-Y2 is hydrogen.
  • XI 0 and XI 1 are each independently selected from the group consisting of leucine, isoleucine, norleucine, valine, tryptophan, phenylalanine, methionine, octahydroindole-2-carboxylic acid, cyclohexylglycine and cyclopentylglycine.
  • X5 is a residue of a D- or L-amino acid selected from the group consisting of alanine, leucine, isoleucine, norleucine, valine, methionine and norvaline.
  • X9 is alanine or glycine.
  • the Rl and R2 are each independently of a formula CH3(C ⁇ " 2)kCO, where k is an integer from 2 to 16, preferably k equals 16, i.e., stearyl.
  • conjugates including a lipophilic moiety and peptides of characterized by a core sequence Lys-Lys-Tyr-Leu (SEQ ID NO:7) which is derived from positions 20-23 of the VIP sequence, and is shared by the VIP related peptide, PACAP. Additional or alternative sequences are of peptides including the core sequence Asn-Ser-Ile-Leu-Asn (SEQ ID NO: 19), which is derived from positions 24-28 of the VIP sequence, and 24-27 of the PACAP peptide including the core sequence Ala-Ala- Val-Leu (SEQ ID NO:23).
  • Superior activity of SNV (SEQ ID NO:3) and SNH (SEQ ID NO:4) in the keratinocyte model may also involve a parameter of preference to specific VIP receptors on characterizing these cells, and may bear significance in targeting specific VTP- related drugs toward therapeutics, and affecting VIP receptor bearing cells.
  • Peptides were prepared by solid phase synthesis using the Fmoc chemistry as described [28]. Additional detailed solid phase synthetic and purification procedures were described in U.S. Patent Nos. 5,565,424, 5,147,855 and EP Patent No. 0620008B1 issued to Gozes et al, which are inco ⁇ orated herein by reference. Small scale automated synthesis was achieved using an ABIMED AMS-422 multiple peptide synthesizer (Abimed Analyses -technik, Langenfeld, Germany) with 25 ⁇ mols of corresponding amino acid-bound-polymer (Rinkamide, Novabiochem, Laufelfingen, Switzerland) in each reaction vessel.
  • Cyclic VIP derived conjugates synthesis was achieved as described in PCT application. No. WO 97/40070 by Gozes et al. which is inco ⁇ orated herein in its entirety by reference.
  • RP-HPLC reversed-phase high performance liquid chr ⁇ atography
  • the isolated peptides were subjected to analytical RP-HPLC to confirm their purity and to amino acid analysis to verify their composition. Mass spectrometry was employed for molecular weight determinations.
  • Keratinocyte Serum Free medium Keratinocyte Serum Free medium (K- SFM, Gibco-BRL, Gaithersburg MD, USA) supplemented with 25 ⁇ g/ml bovine pituitary extract (BPE) and 0.1 ng ml human recombinant epidermal growth factor (EGF).
  • K- SFM Keratinocyte Serum Free medium
  • BPE bovine pituitary extract
  • EGF epidermal growth factor
  • This medium is based on MCDB 153 medium, enriched by various hormones (insulin 5 ⁇ g/ml, tri-iodothyronine, transferrin, hydrocortisone 0.5 ⁇ g/ml, cholera toxin, adenine).
  • Subculturing was achieved under serum free conditions, using trypsin: EDTA solution (0.025:0.01%), and neutralizing by soybean trypsin inhibitor (0.05 mg/ml, Biological Industries, Beit Haemek, Israel).
  • HaCaT Human HaCaT keratinocytes were a kind gift from Prof. Fusenig [30]. Cells were routinely propagated in minimal Eagle's Essential medium, MEM supplemented with 1% Pen-Strep-Nystatin, 2 mM L-Gln and 10% fetal calf serum (FCS, Biological Industries, Beit Haemek, Israel) under humidified atmosphere with 10% CO2 at 37 °C. Subculturing was achieved by trypsinization with Versene solution (trypsin:EDTA 0.25:0.02%), and neutralizing by serum containing medium.
  • FCS fetal calf serum
  • HT29 The human colon carcinoma cell line was routinely propagated in RPMI-1640 supplemented with 1% Pen-Strep-Nystatin, 2 mM L-Gln and 10% FCS under humidified atmosphere with 10% CO2 at 37 °C. Subculturing was achieved by trypsinization with Versene solution and neutralizing by serum containing medium.
  • Cell viability assays :
  • Neonatal keratinocytes Cells were seeded 10,000/well in 96 well microtiter plates (Nunc, Roskilde, Denmark), in K-SFM supplemented with bovine pituitary extract (BPE) and recombinant EGF (rEGF). The following day, medium was changed to K-SFM lacking EGF and BPE (basal medium), in order to achieve a quiescent state. Following 48 h, the medium was replaced by fresh basal medium supplemented with peptides at the indicated concentrations for an additional 22-24 h. Cells were used in their third or forth passage.
  • BPE bovine pituitary extract
  • rEGF recombinant EGF
  • MTS reagent CellTiter 96 AQueous cell proliferation kit, #G5430, Promega, Madison WI, USA
  • oxidation of which by active mitochondria results in color development at 490 nm.
  • HaCaT Cells were seeded 4,000/well in 96 well microtiter plates, in 5% FCS supplemented MEM. The following day, cells were starved by changing the medium to MEM supplemented with 0.1% bovine serum albumin (BSA) for a 48 h period, after which the peptides were added, in fresh medium, at the specified concentrations and incubation proceeded for 22-24 h period. Cell viability was monitored by 1.5-3 h incubation with the MTS reagent.
  • BSA bovine serum albumin
  • HT29 Cells were seeded 4,000/well into 96 well plates in 10% supplemented RPMI-1640 (Biological Industries, Beit Haemek, Israel). Medium was changed to 0.1% BSA supplemented RPM the following day, for a 48 h period. Stearyl peptides were added, in fresh 0.1% BSA medium for additional 22-24 h. Cell viability was monitored by addition of the MTS reagent during the last 3 h of incubation. 2. Lactate dehydrogenase (LDH) determination:
  • HaCaT cells were seeded, 4x10 ⁇ per 35 mm dish (Corning, MA, USA), in 5% FCS supplemented MEM, as above. On the following day, cells were starved by changing the medium to MEM supplemented with 0.1% BSA for a 48 h period. On the forth day, the medium was changed to fresh MEM (without supplements). SNV was added for a stimulation period of 15 min. Control cultures received saline. Following stimulation, the cultures were washed three times with cold PBS, and the cyclic nucleotides extracted by 30 min incubation at 4 °C in 80% ethanol and mechanical removal of the cells. The resulting suspension was pelleted by centrifugation at 2000xg for 15 min at 4 °C.
  • the lipophilic VIP antagonist, SNH was tested for its antiproliferative activity on HaCaT cells following a 24 h incubation period ( Figure 1). Results indicated that at 7.5 ⁇ M, SNH produced essentially complete cell killing. In order to estimate the time dependence of cytotoxic effect manifestation, SNH was applied for a shorter exposure period. Thus, activity of SNH was already evident after a 2 h exposure, when only 32% of the cells maintained their viability, as compared to control cultures (not shown).
  • St StearoyI; Inactive indicates +10% of control values (assigned to 100%). All peptides are in their C-terminal amidated form.
  • ATA endonuclease inhibitor
  • apoptosis marker commonly utilized to distinguish between these two types of cell death [31] was applied, at 25 ⁇ M concentration, together with the cytotoxic peptides.
  • ATA interferes with the final degradation phase of the apoptotic process. While ATA itself did not dramatically change cellular morphology ( Figure 4D), a remarkable restoration in cell viability was observed in cultures co-treated with SNH and ATA ( Figure 4E).
  • CHI cycloheximide

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Endocrinology (AREA)
  • Vascular Medicine (AREA)
  • Dermatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des compositions pharmaceutiques destinées au traitement de dermatoses, lesdites compositions contenant des peptides associés au peptides intestinal vasoactif en tant qu'agent actif. Les compositions selon l'invention provoquent l'apoptose cellulaire, et peuvent par conséquent être utilisées en particulier pour le traitement de dermatoses associées à l'hyperprolifération de cellules dermiques. La présente invention concerne également un procédé destiné au traitement de dermatoses hyperprolifératives et/ou à provoquer l'apoptose cellulaire, ledit procédé consistant à administrer une quantité thérapeutique efficace de peptides associés au peptides intestinal vasoactif au niveau de la zone dermique devant être traitée.
EP01940960A 2000-06-07 2001-06-07 Peptides associ s au peptide intestinal vasoactif, destin s au traitement de dermatoses Withdrawn EP1307217A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IL13663100 2000-06-07
IL13663100A IL136631A0 (en) 2000-06-07 2000-06-07 Vip-related peptides for treatment of skin disordes
PCT/IL2001/000523 WO2001093889A2 (fr) 2000-06-07 2001-06-07 Peptides associés au peptide intestinal vasoactif, destinés au traitement de dermatoses

Publications (1)

Publication Number Publication Date
EP1307217A2 true EP1307217A2 (fr) 2003-05-07

Family

ID=11074239

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01940960A Withdrawn EP1307217A2 (fr) 2000-06-07 2001-06-07 Peptides associ s au peptide intestinal vasoactif, destin s au traitement de dermatoses

Country Status (4)

Country Link
EP (1) EP1307217A2 (fr)
AU (1) AU2001274446A1 (fr)
IL (1) IL136631A0 (fr)
WO (1) WO2001093889A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2730615A1 (fr) * 2008-07-18 2010-01-21 Galderma Research & Development Modulateurs de voie de signalisation pacap pour traiter des maladies inflammatoires de la peau avec une composante neurogene et plus particulierement l'acne rosacee et composition contenant ceux-ci
CA3042620A1 (fr) 2011-02-02 2012-11-29 Emory University Antagonisme de la voie de signalisation piv

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL99924A (en) * 1991-10-31 1995-12-31 Yeda Res & Dev Derivatives of structurally modified vip and pharmaceutical compositions containing them

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0193889A2 *

Also Published As

Publication number Publication date
AU2001274446A1 (en) 2001-12-17
WO2001093889A3 (fr) 2003-02-27
WO2001093889A2 (fr) 2001-12-13
IL136631A0 (en) 2001-06-14

Similar Documents

Publication Publication Date Title
AU2018255294B2 (en) Parenteral non-systemic administration of buffering agents for inhibiting metastasis of solid tumors, hyperpigmentation and gout
TW487711B (en) Conjugates of lipophilic moieties and peptides
JP5449673B2 (ja) 対象者におけるメラニン形成を誘導するための組成物および方法
US7659252B2 (en) Transdermal delivery peptides and method of use thereof
AU677603B2 (en) Anti-inflammatory peptide analogs and treatment to inhibit vascular leakage in injured tissues
US10766927B2 (en) Peptides that stimulate subcutaneous adipogenesis
WO2007035474A2 (fr) Peptides d'administration transdermique et procede d'utilisation de ceux-ci
US7214655B2 (en) Peptides and the use thereof in darkening the skin
CA2182795C (fr) Antagonistes superactifs du polypeptide intestinal vasoactif
JP2006528645A (ja) ざ瘡の処置のための、ｘｍｐ−６２９の使用
JP2014177464A (ja) Ｍｎｔｆペプチドの美容および皮膚科用製剤
US7081442B2 (en) Composition containing a peptide and a pigment and the use thereof in darkening the skin
EP0510063A1 (fr) Peptides anti-inflammatoires et traitement inhibitoire de fuites vasculaires dans des tissus traumatises
JP2007508297A (ja) 経皮の高分子量化合物及び低分子量化合物
WO2015184125A1 (fr) Peptides, compositions et procédés pour stimuler l'adipogenèse sous-cutanée
WO2007002594A1 (fr) Compositions cosmetiques et procedes faisant appel a des mimetiques de transformation du facteur de croissance beta
EP1307217A2 (fr) Peptides associ s au peptide intestinal vasoactif, destin s au traitement de dermatoses
KR101688696B1 (ko) 엔케팔린 변이체를 함유하는 상처 치료용 조성물
CA2654956C (fr) Peptides a activite anti-proliferative
US10188704B2 (en) Enhanced melanoma cancer prevention by novel melanotropins

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20021220

AK Designated contracting states

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: THE GOVERNMENT OF THE UNITED STATES OF AMERICA,ASD

Owner name: RAMOT AT TEL-AVIV UNIVERSITY LTD.

Owner name: YEDA RESEARCH & DEVELOPMENT COMPANY, LTD.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20030703