The present invention provides comprehensive compositions for treating problems associated with hair graying and balding via the incorporation of: (i) the cell growth factor of HSCF to induce the migration of melanocyte stem cells and keratinocyte stem cells and then to increase the growth of melanocytes and keratinocytes in hair follicles, (ii) a formula of amino acids and vitamins to provide the nutritional factors for hair growth and pigmentation, and (iii) minoxidil to enhance the function of HSCF on hair re-growth. The compositions comprising at least one of (i), (ii) or (iii) are administered on skin and/or scalp through liposome in the follicular delivery systems, including penetration enhancers and suitable carrier bases. The compositions packaged in liposome in the follicular delivery systems in this invention has been proven to reach the dermis from the skin surface within 15-30 min.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides comprehensive compositions for treating The problems associated with hair graying and balding via the incorporation of: (i) Cell growth factor of HSCF to induce the migration of melanocyte stem cells and keratinocyte stem cells and then to increase the growth of melanocytes and keratinocytes in hair follicles, (ii) a formula of amino acids and vitamins to provide the nutritional factors for hair growth and pigmentation, and (iii) minoxidil to enhance the function of HSCF on hair re-growth. The compositions comprising at least one of (i), (ii) or (iii) are administered on skin and/or scalp through the liposome in the follicular delivery systems, including penetration enhancers and suitable carrier bases. The composition packaged in liposome in the follicular delivery systems of this invention has been proven to reach the dermis from the skin surface within 15-30 min. The essential function of the liposome is to maintain the activity of the growth factor of HSCF for at least one to three years.

2. Description of the Prior Art

Dual therapies of hair graying and balding in topical delivery systems have never been claimed in the market. When a graying hair derives in large part from a mixture of pigmented and white hair, it can clearly be noted that individual hair follicles indeed exhibit pigment dilution or true graying. This dilution could be due to a reduction in tyrosinase activity of hair bulbar melanocytes, sub-optimal melanocyte-cortical keratinocyte interaction, or defective migration of melanocytes from a reservoir in the upper outer root sheath to the pigment-permitting microenvironment close to dermal papilla of the hair bulb.sup.(12). Some studies have reported that stem cell factor (SCF) has the functions to proliferate and migrate to the melanoblasts and keratinoblasts restored in the upper outer root sheath, called hair bulge.sup.(1, 2). These melanoblasts and keratinoblasts, called melanocyte and keratinocyte stem cells, respectively, in hair bulge, could proliferate and migrate to dermal papilla in hair bulb and then differentiate to become melanocytes and keratinocytes.sup.(12, 13, 14). Hair pigmentation is mostly produced by melanin accumulation, which is secreted from melanocytes.sup.(12, 16). And hair shaft is mostly produced by keratin accumulation, which is secreted from keratinocytes. This growth factor of SCF has been proven to stimulate stem cells in hair bulge.sup.(3, 6, 9). However, mice with homozygous SCF gene mutation (SL.sup.-/SL.sup.-) show the symptoms of white hair, infertility, and anemia.sup.(1, 5). It suggests the importance of SCF in hair pigmentation.sup.(7, 13), but effects on hair growth are limited because the mutated mice still have hair. In addition, many growth factors and signals through different pathways control the hair growth. Especially, insulin-like growth factor-I (IGF-I) has been reported to induce growth of hair in many papers.sup.(4, 15). Based on our previous data, the same results have also been obtained. Although the amino acid sequence and protein structure of recombinant human SCF has been clearly studied.sup.(17) and also been applied as a patent (U.S. Patent Publication No.: 20050080250, application Ser. No. 10/620,642, filed: Jul. 16, 2003. Methods of stimulating growth of stromal cells in a human.), SCF and other ingredients are incorporated to claim the dual therapies for hair aging in our experimental data.

The combination of the two growth factors, SCF and IGF-I, has been studied to have the more advanced functions on the hair follicles in our study, because it significantly stimulates the hair growth and melanin synthesis at the same time. We gave the definition of the two growth factors of SCF and IGF-I as HSCF (hair stem cell factor), meaning either the combination, named HSCF-I, or the recombinant protein, named HSCF-II. The recombinant protein of HSCF-II is first established by our group, which is produced by a cloning vector inserted with a combination of human SCF and IGF-I sequence together. The medium containing HSCF results in a higher increase of shaft length of mouse hair follicles cultured in vitro, when compared with SCF or IGF-I alone. After the above compositions are administered along with a hair follicular delivery vehicle and/or device on the skin of mice, longer hair length and darker pigmentation of hair shaft are obtained.

Minoxidil, being a potassium channel opener, has been proven to induce human hair growth from many papers and is popular in the market to treat patients with androgenic alopecia. But the effects of minoxidil on treating hair balding are still limited.sup.(10). The functions of minoxidil could reduce continuous hair loss, but couldn't increase hair amount and density or recover the pigment of graying hair. We tried to amplify the function of minoxidil by adding the above compositions, because it has been proven by the inventors of this invention that HSCF could stimulate the stem cells in hair bulge to migrate into hair bulb, and then differentiate to melanocyte and keratinocyte to regenerate a newly pigmented hair in a hair follicle.

The results showed that compositions of (i) HSCF, (ii) a formula of some amino acids and vitamins, and (iii) minoxidil could achieve the best efficiency on hair growth and melanin synthesis. It also has been proven to treat the graying and balding problems by the in vitro culture of hair follicle organ and also by the in vivo animal models of graying and balding mice. The invention could be further applied to treat the hair aging of human beings.

REFERENCE

1. Broudy, V. C. (1997) Stem cell factor and hematopoiesis. Blood 90:1345-1364. 2. Botchkareva, N. V., M. Khlgatian, B. J. Longley, V. A. Botchkarev, B. A. Gilchrest, 2001. SCF/c-KIT signaling is required for cyclic regeneration of the hair pigmentation unit. FASEB J. 15(3):645-658. 3. Botchkareva, N. V., V. A. Botchkarev, B. A. Gilchrest. 2003. Fate of melanocytes during development of the hair follicle pigmentary unit. J. Investig. Dermatol. Symp. Proc. 8(1):76-79. 4. Frankel, S. K., B. M. Moats-Staats, C. D. Cool, M. W. Wynes, A. D. Stiles, D. W. Riches. 2005. Human insulin-like growth factor-IA expression on transgenic mice promotes adenomatous hyperplasia but not pulmonary fibrosis. Am. J. Physiol. Lung Cll Mol. Physiol. 288(5):L805-812. 5. Guyonneau, L., F. Murisier, A. Rossier, A. Moulin, F. Beermann. 2004. Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice. Mol. Cell Biol. 24:3396-3403. 6. Hemesath, T. J., E. R. Price, C. Takemoto, T. Badalian, D. E. Fisher. 1998. MAP kinase links the transcription factor microphthalmia to c-kit signaling in melanocytes. Nature 391:298-301. 7. Ito, M., Y. Kawa, H. Ono, M. Okura, T. Baba, Y. Kubao, S. I. Nishikawa, M. Mizoguchi, 1999. Removal of stem cell factor or addition of monoclonal anti-c-kit antibody induces apoptosis in murine melanocyte precursors. J. Invest. Dermatol. 112(5):796-801. 8. Jiang, X., O. Gurel, E. A. Mendiaz, G W. Stearns, C. L. Clogston, H. S. Lu, T. D. Osslund, R. S. Syed, K. E. Langley, W. A. Hendrickson. 2000. Structure of the active core of human stem cell factor and analysis of binding to its receptor kit. EMBO J. 19:3192-3203. 9. Nishimura, E. K., S. A. Jordan, H. Oshima, H. Yoshida, M. Noriyama, I. J. Jackson, Y. Barrandon, Y. Miyachi, S. I. Nishikawa. 2002. Dominant role of the niche in melanocyte stem-cell fate determination. Nature 416:854-860. 10. Mager, M., R. Pause, N. Farjo, S. Muller-Rover, E. M. J. Peters, K. Foitzik, D. J. Tobin. 2004. Limitations of human occipital scalp hair follicle organ culture for studying the effects of minoxidil as a hair growth enhancer. Exp. Dermatol. 13:635-642. 11. Mol, C. D., K. B. Lim, V. Sridhar, H. Zou, E. Y. T. Chien, B. C. Sang, J. Nowakowski, D. B. Kassel, C. N. Cronin, D .E. McRee. 2003. Structure of a c-kit product complex reveals the basis for kinase transactivation. J. Biol. Chem. 278:31461-31464. 12. Panteleyev, A. A., C. A. B. Jahoda, A. M. Christiano. 2001. Hair follicle predetermination. J. Cell Sci. 114:3419-3431. 13. Peters. E. M. J., D. J. Tobin, N. Botchkareva, M. Maurer, R. Paus. 2002. Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit expression. Histochem. Cytochem. 50:751-766. 14. Peters, E. M. J., M. Maurer, V. A. Botchkarev, K. deM. Jensen, P. Welker, G. A. Scott, R. Paus. 2003. Kit is expressed by epithelial cells in vivo. J. Invest. Dermatol. 121:976-984. 15. Philpott, M. P., D. A. Sander, T. Kealey. 1994. Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro. J. Invest. Dermatol. 102(6):857-861. 16. Sulaimon, S. S.,B. E. Kitchell. 2003. The biology of melanocytes. Vet. Dermatol. 14(2):57-65. 17. Zhang, Z., R. Zhang, A. Joachimiak, J. Schlessinger, X. P. Kong. 2000. Crystal structure of human stem cell factor: Implication for stem cell factor receptor dimerization and activation. Proc. Natl. Acad. Sci. USA 97:7732-7737. 18. Park, W. S., C. H. Lee, B. G Lee, I. S. Chang. 2003. The extract of Thujae occidentalis semen inhibited 5.alpha.-reductase and androchronogenetic alopecia of B6CBAF1/j hybrid mouse. J. Dermatol. Sci. 31:91-98.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides the compositions for dual hair therapies for balding and graying. The best composition of (i) the recombinant growth factor of HSCF, (ii) a formula of some amino acids and vitamins, and (iii) minoxidil significantly increases the degree of hair pigmentation and the length of hair shaft, not only in in vivo but also in in vitro models. In the base formula (ii), the effects of the mixture of HSCF and minoxidil are much stronger than those of HSCF (i) or minoxidil (iii) alone. It provides strong evidence for the effects of these compositions and methods for dual therapies of hair graying and balding in the follicular delivery systems.

In the invention, each of the recombinant human growth factors, including SCF, IGF-I and HSCF-II, has been cloned, expressed, and purified (FIG. 1-6, see Original Patent). The activities of these proteins have been tested in different cell lines, respectively (FIGS. 3, 5, see Original Patent). The artificial recombinant protein of HSCF-II is first established in this invention by integrating the human SCF and IGF-I sequence together into the expressing vector. When the pET24a vector was inserted with the sequence of HSCF-II, the expressed protein also has the combined functions of both SCF and IGF-I. Moreover, it appears that the combined functions on hair follicles are even better than each of SCF and IGF-I (FIG. 7, see Original Patent). The HSCF-II possesses the benefits of being efficient, economical, and convenient. The major functions of HSCF-II on hair follicles are to induce the proliferation of melanocyte stem cells and keratinocyte stem cells, their migration from the bulge to the hair bulb, and also their differentiation into melanocytes and keratinocytes. It could revitalize the hairless follicles to regenerate pigmented hair shaft (FIG. 8, see Original Patent).

The graying animal model to mimic human hair graying is first established by our invention. It is derived from the hybrid of the C3H and BALB/C mice. The hybrid possessing the heterologous tyrosinase gene (Tyr+/Tyr-) appears in light brown color, which is the mimic to the genotype of aging human in hair follicles because of tyrosinase depletion. In fact, tyrosinase is the most important enzyme to produce melanin. If any reason causes the inactivation of tyrosinase during aging, the hair would lose its color and becomes gray. We used the whiting gel (containing 1-20% hydroquinone and 1-20% glycolic acid) on the dorsal skin of the hybrid to cause the inhibition of the tyrosinase activity. After 7 days of treatment, the hair color of the re-grown hair in the hybrid becomes white. Actually, it is very easy to observe the hair color from the graying animal model to check if these treatments indeed work. In fact, it has been proven in this invention. The compositions significantly change the hair color (FIG. 8) and increase the melanin level (FIG. 9, see Original Patent) of the hair shaft in the graying animal model.

Minoxidil has been popular in the market to treat patients with androgenic alopecia for many years. It has been theoretically explained that minoxidil could be a potassium-ion channel opener on cell membrane to help with the entrance of amino acids and other nutrients. But the effects of minoxidil on treating hair balding are still limited because it can reduce continuous hair loss but not increase the hair amount, density, or the pigment of the graying hair. The compositions in this invention can indeed amplify the function of minoxidil, by the extra additions of the growth factor of HSCF and a formula of some amino acids and vitamins (FIG. 7 and FIG. 10d, see Original Patent). The function of HSCF on hair follicles is to stimulate the proliferation and migration of stem cells. And the formula supports rich nutrients to hair follicles. Since the best combination consists of three parts, it enhances not only the pigmentation of graying hair but also the regeneration of hairless follicles.

To prove the therapeutic function of these compositions on hair balding, we established the androchronogenetic alopecia (AGA) animal model, which was modified from a paper by Park et al..sup.(18). It is derived from the hybrid of C57BL/6J female.times.CBA/J male, and is called B6CBAF1/j hybrid. It has been proved that hair loss of the female of the hybrid can be induced by the subcutaneous injection of testosterone (1-100 mg/mice/day) for 3 weeks. Thus, the AGA animal model is the human AGA mimic. From the in vivo data (FIG. 10, see Original Patent), it strongly suggests that even HSCF alone has the function to improve hair re-growth in the AGA mice. Moreover, the combination of HSCF and minoxidil has more efficacy in treating hair loss in the AGA mice, which re-grows hair more quickly. The result also shows that the mixture has the best efficacy in the hair growth of the hair follicle cultured in vitro (FIG. 7, see Original Patent). In summary, the effect of the mixture on hair re-growth in the follicles is significantly better than minoxidil alone.

These compositions which are topically applied to the graying and AGA animal models through the follicular delivery systems should be packaged in liposome to preserve their activities, especially for the HSCF protein. The follicular delivery systems include penetration enhancers and suitable carrier bases. The term "penetration enhancers" as used herein means a compound that facilitates the movement of substances into and/or through the epidermis of skin. Examples of penetration enhancers include, but are not limited to, lipids, lipoproteins, fatty acids and fatty alcohol, detergents, alcohols, glycols, mineral oils, liposome, and transdermal delivery vehicles or devices. And the term "suitable carrier" as used herein means a carrier suitable for the topical application to mammalian skin without causing undue toxicity, irritation, allergic response, and the like. The addition of penetration enhancers and suitable carrier bases in the follicular delivery systems contribute to the most effectiveness on the dermis through the topical applications. We have proven that the penetration rate is limited to 15-30 min to reach the dermis from the skin application by the liposome of the follicular delivery system. It showed on the immunofluorescence of the liposome (FIG. 11a, see Original Patent), the sizes of which are limited to 50 nm-2 .mu.m, and the frozen specimen of mice skin after 15-30 min of the topical application of the liposome packaged with these compositions (FIG. 11b, see Original Patent).

In summary, the present invention provides the compositions and methods for dual therapies of hair graying and balding. The compositions comprise (i) the growth factor of HSCF, (ii) a formula of amino acids and vitamins, and (iii) minoxidil in the topical delivery systems. The invention also provides two experimental animal models for testing the functions of the compositions on hair graying and balding in vivo. The invention further relates to dual therapeutic methods useful for treating disorders of human hair graying and balding due to aging.
 

Claim 1 of 12 Claims

1. An isolated recombinant polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:5.

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