AU2005203228B2 - Synergistic Combinatorial Therapies for the Treatment of Vitiligo - Google Patents

Abstract

Abstract Synergistic therapies for the treatment of vitiligo are provided. The major therapies for the Treatment of vitiligo - a pigmentary disorder characterized by patchy depigmentation of ;kin are Psoralens plus UV-A, steroids. Basic fibroblast growth (bFGF) peptide lotion or surgical procedures. Psoralens plus UV-A is effective in about 50% of cases, steroids are limitedly effective only in fast spreading cases of vitiligo and often reoccurs on stoppage of treatment. Surgical treatment is the last resort for vitiligo therapy, when all other therapies failed. It is limitedly effective. Basicfibroblast growth factor peptide(s) lotion was developed as a new mode of therapy for the treatment of vitiligo. It went through successfully various phases of clinical trials in India and is allowed to be marketed by the drug controller general (India). It is effective in 80% of stable and in segmental vitiligo. Here data are provided and demonstrated that combinatorial treatment of vitiligo by local application of bFGF peptide(s) lotions in association with psoralens and UV-A, or steroids or surgical procedures produce synergistic response and that the rate of repigmentation increases synergistically and more efficacious results are obtained than with any of them alone. Any Combinatorial therapy comprising the local application of bFGF peptide lotion on the vitiligo patch in combination with any other therapies for the treatment of vitiligo also may act synergistically.

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicant: Abburi Ramaiah Address for Service: CULLEN & CO. Level 26 239 George Street Brisbane Qld 4000 Invention Title: Synergistic Combinatorial Therapies for the Treatment of Vitiligo The following statement is a full description of the invention, including the best method of performing it, known to us: 2 Field of the Invention: This invention relates to a method of treating vitiligo using synergistic Formulations. Background of the invention: Vitiligo / leucoderma is an acquired depigmentation of skin characterized by patchy loss of pigment that becomes progressive with time. This disorder affects about 1 % of the world population. Traditional therapies for vitiligo mainly include photo chemotherapy with topical/oral psoralens followed by exposure to ultra violet A radiation (PUV-A) or topical /oral steroids. PUV-A therapy is perhaps the main stay in the treatment of vitiligo. However only about 50% of cases get repigmentation. More over in a patient in response to PUV-A, many vitiligo patches may repigment partially only and the rest of the patches may remain unresponsive to PUV-A therapy even after long duration of treatment. The repigmentation in the above therapies is a result of multiplication of melanocytes, the cells, which produce the pigment melanin in the skin. The multiplication of melanocytes in response to the above therapies occur from the margins of the vitiligo patch or at the pigmented hair follicles and their migration /spread to the vitiligo patch. Basic fibroblast growth factor (bFGF) also known as FGF2 so named because it contains a high number of basic amino acid residues (Lysine, Arginine and Histidine) is a potent mitogen for a variety of call types including melanocytes. Both human and bovine bFGF were isolated and the gene expressing this product were sequenced and cloned .In addition bFGF was found to be expressed in a wide variety of tissue types including placenta, keratinocytes and fibroblasts. The bFGF or its agonist peptides were tested on human volunteers in the various phases of clinical trials in India and found to be successful in repigmenting about 80% of volunteers with stable generalised vitiligo and segmental vitiligo. Patents of interest describing bFGF or agonist peptides and the formulation for their penetration through intact skin include US patent 6,143,723, Australian patent 722626, Indian patents 185613,186437 and 185703. Vitiligo is a pigmentary disorder with patchy loss of skin pigment melanin, (Ramaiah.A,Puri.N,Mojamdar M,A new hypothesis for the etiology of vitiligo,Acta 3 Derm, Venerol (Stockholm), 1989, 69, 323-327) postulated that deprivation of a mitogen(s) like basic fibroblast growth factor (bFGF) for melanocytes or its decreased level in the skin of vitiligo patients for as at unknown reason could result in the loss of melanin producing cells melanocytes in skin resulting in vitiligo. Basic fibroblast growth factor (bFGF) also known as FGF2 is a potent mitogen for variety of cell types including melanocytes. Both human and bovine bFGF have been isolated and the gene expressing this product have been sequenced and cloned. In addition bFGF has been found to be expressed in a wide variety of tissue types including pituitary, brain and adrenal gland corpusluteum, retina, kidney, placenta and keratinocytes, fibroblasts. The above hypothesis that a mitogen like bFGF may be reduced in its levels in vitiligo patch resulting in loss of melanocytes and the pigment melanin in vitiligo skin was supported recently from studies by others (Moretti S et al., Insight in to the pathogenesis of vitiligo, Imbalance of epidermal cytokines at sites of lesions, Pig. Cell. Res 2002, 15). Description of Invention According to this invention there is provided a method for combinatorial synergistic therapy for treatment of generalized vitiligo and segmental vitiligo comprises local application of an effective amount of a composition comprising 0.02 to 5% w/w of at least one peptide selected from a group consisting of Seq ID Nos: 1-8 as described in US patent 6143723 in the formulation as described in the US patent 6143723 along with psoralens and UV-A (PUV-A therapy)/PUV-ASOL. According to an embodiment, there is provided a method for combinatorial synergistic therapy for treatment of generalised vitiligo and segmental vitiligo comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids I to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with psoralens and UV-A (PUV-A therapy)/PUV-ASOL.

4 The method of combinatorial synergistic therapy for treatment of vitiligo patches not responding to PUV-A therapy comprises local application of an effective amount of a composition comprising 0.02-5% w/w of at least one peptide selected from a group consisting of Seq ID Nos: 1-8 as described in US patent 6143723 in the formulation as described in US patent 6143723 along with continuing the oral intake of psoralens and exposure to UV-A. According to an embodiment, there is provided a method of combinatorial synergistic therapy for treatment of vitiligo patches not responding to PUV-A therapy comprises local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids I to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with continuing the oral intake of psoralens and exposure to UV-A. The method of combinatorial synergistic therapy for treatment of fast spreading vitiligo comprises local application of an effective amount of a composition comprising 0.02-5% w/w of at least one peptide selected from a group consisting of Seq ID Nos: 1-8, as described in the US patent 6143723 in the formulation as described in the US patent 6143723 along with steroid therapy. According to an embodiment, there is provided a method of combinatorial synergistic therapy for treatment of fast spreading vitiligo comprises local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids 1 to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with steroid therapy.

5 The method for combinatorial synergistic therapy for treatment of vitiligo comprises local application of an effective amount of composition comprising 0.02% to 5% w/w of at least one peptide selected from a group consisting of SEQ ID Nos: 1-8, as described in the US patent 6143723 in the formulation as described in the US patent 6143723 along with surgical procedures. The method for combinatorial synergistic therapy for treatment of vitiligo comprises local application of the composition further comprises 10-50% w/w of solvent, 10 40% w/w of glycols, and 10-40% w/w of at least one penetration enhancing agent. According to an embodiment, there is provided a method of combinatorial synergistic therapy for treatment of vitiligo comprises location application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids I to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with surgical procedures. As described in the US patent 6143723, the solvent suitable for the present composition may comprise alcohols selected from isopropyl alcohol, propyl alcohol or ethyl alcohol. As described in the US patent 6143723, the glycols for the present invention may comprise propylene glycol or polyethylene glycol. As described in the US patent 6143723, the penetration enhancing agent may comprise propyl, isopropyl or ethyl myristate or propyl or ethyl stearate or propyl, isopropyl or ethyl palmitate. The local application of bFGF peptide(s) in the formulation described in the US patent 6,143,723 is effective in more than 80% of cases of stable generalised vitiligo or segmental vitiligo. It is felt that the speed of repigmentation and even better outcomes than 80% success rate may be accomplished if it is combined with traditional therapies like PUV-A therapy for vitiligo. In addition bFGF peptide lotion therapy is not effective to prevent the fast spread of vitiligo and perhaps a synergistic combinatorial therapy may emerge if bFGF lotion therapy is used in combination with steroid therapy traditionally used in the case of fast spreading vitiligo.

6 Similarly, the local application of bFGF peptide(s) lotion therapy may be advantageously used with any other therapy including the surgical or other therapies involving immune modulators. Definitions of the specific embodiments of the invention as claimed herein follow. According to a first embodiment of the invention, there is provided a method for combinatorial synergistic therapy for treatment of generalized vitiligo and segmental vitiligo comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids 1 to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with psoralens and UV-A (PUV-A therapy)/PUV-ASOL. According to a second embodiment of the invention, there is provided a method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids I to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with PUV-A therapy. According to a third embodiment of the invention, there is provided a method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids 1 to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 7 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with oral steroid therapy. According to a fourth embodiment of the invention, there is provided a method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids 1 to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with surgical procedures. According to a fifth embodiment of the invention, there is provided a method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids I to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with oral/topical application of psoralens/others that act like psoralens plus UV-A/UV-B or any other radiation that help in pigmentation or oral/local steroids or any immuno modulators. Synergistic combinatorial therapy for treatment of fast spreading vitiligo cases comprising bFGF peptide(s) in the formulation described in the US patent 6,143,723 and steroid therapy. The peptide(s) lotion therapy is effective for stable vitiligo or segmental vitiligo but not for fast spreading vitiligo.

8 Steroid therapy is the only therapy available in the presently known methods of treatment to treat fast spreading vitiligo (Kendel E, Vitiligo response to 02% betamethasone 17-valerate in flexible collodin. Dermetologica, 1970, 141, 277-281). However prolonged therapy with steroid produce many side effects (Geraldiz CB, Gutierrez GT, A clinical trial of clobetasol propionate in Filipino vitiligo patients. Clinical Therapy, 1987, 9, 474-482, Ortonne J, Clinical Dermatol 1989, 7, 120-135). In addition after stoppage of treatment, the disease reoccurs. The mode of action of steroids in stopping the fast spread of vitiligo and repigmentation was thought to be brought about by its inhibitory effect on production of auto antibodies to melanocytes which were shown to be responsible for the fast spread of vitiligo macules (Han K, Chen D, Bystryn JC, Systemic steroids suppress antimelonocyte antibodies in vitiligo, J. Cutan. Med. Surg. 1997, 1, 193-195). It was thought therefore possible that the combinatorial therapy for treatment of vitiligo with local application of bFGF peptide(s) lotion along with oral steroids may accomplish dual goals, that is: 1. Arrest of past spread of vitiligo; and 2. Faster repigmentation of vitiligo macules. The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required. Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia. Description of the Accompanying drawings: Fig. 1 (a) is the representation of vitiligo before treatment with topical bFGF peptide lotion. Fig. 1 (b) is the representation of vitiligo after treatment with topical bFGF peptide lotion. Fig.2 (a) is the representation of fast spreading vitiligo before treatment with combination of steroid and topical bFGF peptide lotion. Fig.2 (b) is the representation of fast spreading vitiligo after treatment with combination of steroid and topical bFGF peptide lotion.

9 Fig.3 (a) is the representation of vitiligo before the treatment with combination of topical bFGF peptide lotion and PUVASOL. Fig.3 (b) is the representation of vitiligo after the treatment with combination of topical bFGF peptide lotion and PUVASOL. Fig.4 (a) is the representation of vitiligo before the treatment with topical bFGF peptide lotion and PUVASOL. Fig.4 (a) is the representation of vitiligo before the treatment with topical bFGF peptide lotion and PUVASOL. Fig.4 (b) is the representation of vitiligo after the treatment with topical bFGF peptide lotion and PUVASOL. Fig.5 (a) is the representation of vitiligo resistant for more than a year for PUV-A before treatment with topical peptide bFGF lotion and PUV-A. Fig.5 (b) is the representation of vitiligo resistant for more than a year for PUV-A after the treatment with topical peptide bFGF lotion and PUV-A. Fig.6 (a) is the representation of vitiligo resistant for more than a year for PUV-A before the treatment with topical peptide bFGF lotion and PUV-A. Fig.6 (b) is the representation of vitiligo resistant for more than a year for PUV-A after the treatment with topical peptide bFGF lotion and PUV-A. Fig.7 (a) is the representation of vitiligo resistant for more than a year for PUV-A before treatment with topical peptide bFGF lotion and PUV-A. Fig.7 (b) is the representation of vitiligo resistant for more than a year for PUVA after the treatment with topical peptide bFGF lotion and PUVA. Fig.8 (a) L is the representation of vitiligo after punch grafting but before start of the treatment with bFGF peptide lotion. R is the control. Fig.8 (b) L is the representation of vitiligo after punch grafting and after the treatment with bFGF peptide lotion for 2 months. R is the control.

10 EXAMPLE: 1 As a typical illustration of efficacy of the peptide(s) in repigmentation of vitiligo patch of human volunteers (Fig la, lb) were depicted. Fig la was the size of the vitiligo patch before start of treatment with the peptide lotion and lb was of the same patch after local application of the peptide(s) lotion for a period of 6 months. However the above treatment was effective in 80% of stable vitiligo and in segmented vitiligo cases (See Table -1, 2 for more details). Patents of interest describing the peptides for use as pigmentary agents include US Patent 6,143,723, Australian Patent No: 722626, and Indian Patents 185613, 186437 and 186703. The local application of bFGF peptide(s) in the formulation described in the US patent 6,143,723 is effective in more than 80% of cases of stable generalised vitiligo or segmental vitiligo. It is felt that the speed of repigmentation and even better out come than 80% success rate may be accomplished if it is combined with the traditional therapies like PUV-A therapy for vitiligo. In addition the peptide(s) lotion therapy is not effective to prevent the fast spreading of vitiligo and perhaps a synergistic combinatorial therapy may emerge if the peptide lotion therapy is used in combination with the steroid therapy traditionally used in the case of fast spreading vitiligo. Similarly the local application the peptide(s) lotion therapy may be advantageously used with any other therapy including the surgical therapies. EXAMPLE: 2 The mode of action of steroids in stopping the fast spread of vitiligo and repigmentation was thought to be brought about by its inhibitory effect on production of auto anti bodies to melanocytes which were shown to be responsible for the fast spread of vitiligo macules (Han K, Chen D, Bystryn JC, Systemic steroids suppress antimelonocyte antibodies in vitiligo, J. Cutan. Med. Surg. 1997, 1, 193-195). It was thought therefore possible that the combinatorial therapy for treatment of vitiligo with local application of the peptide(s) lotion along with oral steroids may accomplish dual goals, that is: 1. Arrest of past spread of vitiligo; and 2. Faster repigmentation of vitiligo macules. That this happens was shown in a clinical trial on human volunteers. The above twin objectives were accomplished. A typical case of such treatment was shown in Fig 2a, 2b. Fig 2a describes two vitiligo patches at the start of treatment of oral steroids, while 2b describes the same 10a patches at the end of 3 months of treatment with oral steroids while at the same time the peptide(s) lotion was applied locally once a day on the lower vitiligo patch for the duration of intake of oral steroids. It can be clearly seen that there was faster repigmentation where the peptide(s) lotion was applied as compared to the upper vitiligo patch where there was no application of the peptide(s) lotion. The arrest of the fast spread of the disease was accomplished to the same extent of 88% at the end of 6 months of treatment with oral steroids and the result was not altered by the local application of the peptide(s) lotion (See Table 3 for more details). At the end of 3 months of combinatorial treatment with steroids and local application of the peptide(s) lotion more than 3 fold more patients got pigmented to a marked extent than with steroid alone (See Table 4 for more details). The percentage of volunteers who had moderate and marked rate of repigmentation with combinatorial therapy at the end of three months of treatment is twice than with the steroid therapy alone. Synergistic combinatorial therapy for treatment of fast spreading vitiligo cases comprising bFGF peptide(s) in the formulation described in the US patent 6,143,723 and oral psoralens plus application of UV-A. The major or main stay of treatment of stable vitiligo all over the world is at present psoralens and UV-A therapy (Mofty EL, A preliminary clinical report on the treatment of leucoderma with ami Majus Linn. Jroy. Egypt. Med. Assoc, 1948, 651-660, Mofty EL, In Vitiligo, and psoralens 1968, Pregman Press Oxford, Ortonne JP, Bose Sk, Vitiligo, Where do we stand, Pig. Cell. Res. 1993, 6, 61-72). However it is only partially effective (Westerhof Wiete, Ludmila Nieuwebor-krobotova, Treatment of vitiligo with UV-B radiation VS topical psoralen Plus UV A. Arch. Dermatol 1997, 1525-1528) and requires long duration of treatment. In addition it has many side effects particularly on liver and the risk of cancer in the Caucasian skin type 11 and 111 population (Henseleer T, Cristopher E, Honingsmann H, Wolff K, Skin tumors in European PUV-A study, 8 year follow up of 1643 patients treated with PUV-A for psoriasis J. Am. Acad. Dermatol. 1997, 16, 108-116, Stem RS, Lard M, The carcinogenic risk of treatment for severe psoriasis photochemotherapy follow up study. 1994, 73, 2759-2764). Therefore there is indeed a need to reduce the: a) Duration of the treatment; b) To increase success rate; and 10b c) To treat cases non responsive to PUV-A therapy. The mechanism of action of psoralens and UV-A in repigmentation of vitiligo patches is still an enigma. Psoralens by themselves are ineffective in treating vitiligo unless followed by exposure of skin to UV-A (Gupta AK, Anderson TF, Psoralen photochemotherapy. J. Amer. Acad. Dermatol. 1987, 17, 703-34). The absorption maxima of psoralens lie in the 210 - 330 nm range (Harbar LC, Bickeers. DR, "Photosensitivity Diseases" I" edition Philadelphia: W. B. Saundeers 1981, PP45-55) and the action spectrum of ingested psoralens is probably in the range of 320 - 335 nm (Flemming MG, Brody N, Biological effectiveness of black light fluorescent lamps for PUV-A. J. Amer. Acad. Dermatol., 1985, 12, 894). The psoralens induce skin photosensitization and involves inter-chelation of psoralens with the DNA of the cell, which leads eventually to the death of that cell. That this is true in the case of melanocytes also was shown by exposure of melanocytes in culture to PUV-A (Kao C, Yu H, Comparison of the effect 8-methoxy psoralen plus UV-A on human melonocytes in vitiligo vulgaris in-vitro. J. Invest. Dermatol. 1992, 98, 734-40). How to explain then the beneficial effects of PUV-A in increasing the proliferation of melanocytes, their migration to vitiligo patches and thus repigment vitiligo macules? The main target of PUV-A is the epidermis, which receives UV-A radiation. This could kill keratinocytes of the epidermis. The cellular contents of keratinocytes can then leak and stimulate the proliferation of the adjacent melonacytes. The cellular contents of keratinocytes include many mitogens to melanocytes in addition to bFGF (Halban R, Tyrrell L, Longely J, Yanden Y, Rubin J, Ann. NY. Acad. Sci. 1993, 680, 290-300). That the serum from successfully treated vitiligo patients with PUV-A stimulates the proliferation of melonacytes from normal untreated vitiligo patients was shown in 1989 (Ramaiah. A, Puri. N, Mojamdar. M, A new hypothesis for the etiology of vitiligo, Acta Derm, Venerol (Stockholm), 1989, 69, 323-327). In addition it was shown that the melonacytes from successfully treated vitiligo patients grow with less generation time than the melanocytes from the untreated normal individuals (Ramaiah. A, Puri. N, Mojamdar. M, A new hypothesis for the etiology of vitiligo, Acta Derm, Venerol (Stockholm), 1989, 69, 323-327). These results indicated that the combinatorial treatment of vitiligo comprising local application of the peptide(s) lotion with oral psoralens plus UV-A might act synergistically and produce faster repigmentation than with either of them alone.

10c EXAMPLE: 3 That indeed the combinatorial therapy for treatment of vitiligo comprising local application of the peptide(s) lotion and PUV-A works synergistically could be seen in the typical pictures shown in Fig 3a, 3b and 4a, 4b. The 3a and 4a are the pictures of two volunteers before the combinatorial treatment, while 3b and 4b are the pictures of the same volunteers respectively at the end of 3 months of combinatorial treatment where the right hand of each volunteer was in addition treated with the local application of the peptide(s) lotion. As can be seen from the pictures, the right hands of both the volunteers treated in addition with the local application of the peptide(s) lotion repigmented many times more than that of the left hands of both the volunteers, which were treated with the PUV-A therapy only. The data presented in Table 4 clearly indicate that 7 volunteers treated with combinatorial treatment with the peptide(s) lotion and PUV-A had marked repigmentation in 3 months compared to only one with PUV-A. The percentage of volunteers who had moderate to marked rate of repigmentation with the combinatorial therapy at the end of three months is about twice than the number with PUV-A therapy alone (See Table 5 for details). Synergistic combinatorial therapy for treatment of vitiligo not responding to PUV-A comprising local application of the peptide(s) and PUV-A therapy. In response to PUV-A therapy, less than 20% of vitiligo patches repigment fully, 30-40% of vitiligo patches repigment partially (Westerhof Wiete, Ludmila Nieuwebor-krobotova, Treatment of vitiligo with UV-B radiation VS topical psoralen Plus UV-A. Arch. Dermatol. 1997, 1525-1528). The vitiligo patches of volunteers, who did not respond PUV-A therapy even after prolonged treatment for a year or more were selected for the combinatorial therapy of local application of the agonist peptide(s) lotion with the continued PUV-A therapy. EXAMPLE: 4 The combinatorial therapy was very successful in repigmenting vitiligo patches with in 3 months which were unresponsive for more than year to PUV-A therapy. The typical results were shown in fig 5a, 5b and 6a, 6b and 7a, 7b. The rate of repigmentation is indeed remarkable in 5a, 5b and 6a, 6b. It is also remarkable that the vitiligo patch in the palm which normally never responds to PUV-A therapy even after prolonged duration responded well to combinatorial therapy of the peptide(s) lotion and PUV-A. The response to combinatorial therapy was shown in more than 90% of cases (See Table 6 for details).

10d The results presented here were not exhaustive. The combinatorial therapy with application of bFGF peptide(s) in the formulation described in US patent 6,143,723 can be applied to almost any other therapy that are now in the market for the treatment of various types of vitiligo with synergistic effects similar to what was observed in the cases described above. The peptide(s) in the formulation may be applied locally not only in the form of a lotion but also in any other form like gel/ointment/cream with similar results. Synergistic combinatorial therapy for the treatment of vitiligo comprising punch graft surgical procedure/or other surgical procedures followed by the local application of the peptide(s). Surgical procedures as mentioned earlier are resorted to as the last chance for the treatment of vitiligo patches. They include thrish grafting, expansion of autologus melanocytes in culture and their application on to derm-abraded vitiligo patch, autologus expansion of keratinocytes and melaonocytes in culture in the form of a multi cell layered sheets and their application on the derm-abraded vitiligo patches, or punch grafting of pigmented autologus skin on to the derm-abraded vitiligo patches. In all these cases, after the surgical procedures, the vitiligo patches were surgical bandaged with appropriate antibiotic regimen for the healing of the wounds. The wounds heal with in week or so. Then the local application of the bFGF peptide(s) in the formulation described in US patent 6,143,723 was done for 3 months. This results in faster repigmentation uniformly. EXAMPLE: 5 The photographs 8a and 8b describe the synergistic effect on increasing the rate of repigmentation in 8b where the local application of the peptide(s) in the formulation was done following the punch graft surgical procedure. [Text continues on page 11] 11 i0 ** ~ 4 a 'S C eo C U: 0I .0 UE 0 0 0 0 12 00 4. a? I.. I.CIO CC L ---- - a 13 * EF761U ii le II aftV 14 gg Sa -ILO CLe C 0 C oo

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Claims (2)

1. A method for combinatorial synergistic therapy for treatment of generalised vitiligo and segmental vitiligo comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids 1 to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8;

10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with psoralens and UV-A (PUV-A therapy)/PUV-ASOL. 2. A method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids I to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with PUV-A therapy. 3. A method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids I to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, 18 wherein said composition is administered along with oral steroid therapy. 4. A method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 15 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids 1 to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with surgical procedures. 5. A method for combinatorial synergistic therapy of vitiligo patches not responding to PUV-A therapy comprising local application of an effective amount of a composition comprising: 0.02-0.5% w/w of at least one peptide selected from a group consisting of bFGF amino acids 106 to 115 (Seq ID No: 1), bFGF amino acids 106 to 120 (Seq ID No: 5), bFGF amino acids 1 to 24 (Seq ID No: 6) and Seq ID Nos: 2, 3, 4, 7 and 8; 10-50% w/w solvent; 10-40% w/w glycols; and 10-40% w/w of at least one penetration enhancing agent, wherein said composition is administered along with oral/topical application of psoralens/others that act like psoralens plus UV-A/UV-B or any other radiation that help in pigmentation or oral/local steroids or any immuno modulators. Date: 5 March 2012