WO1989009258A1 - Methods for increasing melanin content and for inducing melanocyte proliferation in-vivo and in-vitro - Google Patents

Abstract

The present invention provides a variety of unique methods and compositions for inducing melanocyte proliferation and increases of melanin production in-vivo and in-vitro. Alternate compositions for use in these novel methods include a diacylglycerol and a conditioned medium derived from keratinocytes.

Description

"METHODS FOR INCREASING MELANIN CONTENT AND FOR INDUCING MELANOCYTE PROLIFERATION IN-VIVO AND IN-VITRO"

RESEARCH SUPPORT The research investigations leading to the present invention were supported by grant s from U . S . D . A . Contract No. 53-3K-06-5-10 and N.I.H. Grant CA 45687.

FIELD OF THE INVENTION The present invention is concerned generally with the cells of and the cellular reactions within the skin of living humans and animals and is particularly directed to novel methods for increasing the melanin content of skin in living subjects and for inducing melanocyte proliferation and growth in-vivo and in-vitro.

BACKGROUND OF THE INVENTION Melanins are a class of structurally related compounds that serve as the principal pigments of mammalian skin and hair. Melanin pigmentation is largely responsible for normal skin color and hair color; and provides protection against ultraviolet light damage from sunlight. In the skin, melanins are synthesized exclusively by specialized cells termed "melanocytes" found in the skin and hair follicles. Once synthesized, melanin is transferred via the cellular dendrites of the melanocyte to the surrounding keratinocytes, the most abundant cell type in the epidermis. This anatomical relationship, often termed the epidermal melanin unit, is generally envisioned in-vivo as being one melanocyte in contact with an estimated 36 keratinocytes in the basal and suprabasal layers of the epidermis. The rates of melanin pigment synthesis and the subsequent transfer of melanin by melanocytes appear to be influenced by ultraviolet light exposure and by certain inflammatory processes. The net result can take a number of different forms, some of which are considered normal and some of which are considered pathological.

Darker skin pigmentation is considered desirable by many persons, socially and esthetically. In addition, a high melanin content in human skin has recognized medical value. Unfortunately, the only presently known means of darkening skin is sun-tanning using either natural sunlight or specially designed ultraviolet light sources (tanning lamps). Exposure of human skin is well known to have adverse long term and short term health consequences, specifically skin cancer and photoaging (long term) and the risk of painful sunburn and keratitis (short term). Light-skinned individuals are highly susceptible to sun-induced skin cancers; face a high risk of melanoma and carcinoma; and incur photoaging or dermatoheliosis, a condition characterized bywrinkling, irregular pigmentation, and surface roughness. However, even darker skinned individuals exposed to prolonged sunlight incur a high risk of skin cancer and unwanted aging changes.

Other individuals are unable to achieve even normal pigmentation due to their being afflicted with vitiligo, piebaldism, abinism, or other hypopigmentation disorders. The result of such abnormal conditions, in the extreme, is total depigmentation of both hair and skin. In less severe instances, some hypopigmentation disorders result in patchy white areas within the skin and hair. All of these conditions cause severe cosmetic problems and mental stress.

Melanocytes are also responsible for the pigmentation of hair and feathers. Graying of hair is due to a decrease in number or activity of the melano cytes residing in the hair bulb. In non-human instances, changes in melanocyte content and melanin synthesis rate result in changes in the color of pelage, fur, wool, and other kinds of animal hair; the ability to maintain melanin production would thus prevent discoloration in fur, wool, feathers, and other animal hair counterparts. Controlling pigment synthesis in animals would permit also production of biologically engineered fur, wool, and feathers.

With the evolution of tissue culture techniques in recent years, a variety of different research efforts and investigations utilizing in-vitro tissue culture methods have been undertaken. Many reports and publications now exist in the scientific and technological literature directed to morphological and biochemical examination of the cells within the skin and the mechanism of their interactions as these relate to the growth of melanocytes and the production of melanin. These publications and reports can be separated into four broad categories of investigations and information: First, in-vitro culture media and cell culture techniques for the growth and maintenance of keratinocytes and melanocytes. This category is represented by the following publications: Gilchrest and Friedmann, Structure And Function Of Melanin (Jimbow, K., editor), Fuji-shoin Company, Ltd., Sapporo, Japan, Volume 4, 1978, pages 1-13; Gilchrest et al., J. Cell. Physiol. 117:235-240 (1983); Gilchrest et al., J. Invest. Dermatol. 83 : 370-376 (1984); U.S. Patent Number 4,443,546; U.S. Patent Number 4,507,321; U.S. Patent Number 4,456,687; U.S. Patent Number 4,444,760; Gilchrest et al., J. Cell. Physiol. 112: 197 (1982); Groelke et al., In-vitro 17:247 (1981); Maciag et al., Science 211:1432 (1981); Rheinwald et al., Nature 265:421 (1977); and Rheinwald et al., Cell 6:331 (1975).

The second broad category includes investigations into simulating agents and mitogens for melanocytes. A representative listing of publications in this second category include the following: Gordon et al., J. Invest. Dermatol. 87:723-727 (1986); Wilkins et al., J. Cell. Physiol. 122:350-361 (1985); Levine et al., J . Invest. Dermatol. 89:269-273 (1987); International Application Number PCT/US87/00226 published as International Publication Number W087/04623 on 13 August 1987; Ogata et al ., Biochem. Biophys. Res. Comm. 146:1204-1211 (1987); Halaban et al., In-vitro 23.47-52 (1987); Bregman et al., Exp. Cell. Res. 157.419-428 (1985); Nordlund et al., J. Invest. Dermatol. 86:433-437 (1986); Hadley et al., Endoc. Res. 11:157-170 (1985); and Preston et al., Proc. Natl. Acad. Sci. USA 84:5247-5251 (1987).

The third broad category of publications is directed to identifying the biochemical events, the chemical entities, and the intracellular pathways and mechanism of action within melanocytes under varying conditions. Publications representative of this broad category include the following: Hadley et al., "Biological Actions of Melanocyte-Stimulating Hormone," in Peptides Of The Pars Intermedia, Pitman Medical Company, London, 1981, pages 244-262; Lerner and McGuire, N.E.J. Med. 270:539-546 (1964); Friedmann and Gilchrest, J. Cell Physiol. 133:88-94 (1987); Nordlund, J.J., Dermat. Clin. 4:407-418 (1986); Nordlund and Abdel-Malek, Prog. Clin. Biol. Res. 256:219-236 (1988); Castagna, M., Biol. Cell 59:3-14 (1987); Abdel-Malek et al., In-vitro 22:75-81 (1986); Rosen et al., J. Invest. Dermatol. 88:774-779 (1987); Halaben et al., Arch. Biochem. Biophys. 230:383-387 (1984); and Preston et al., Proc. Natl. Acad. Sci. USA 84: 5247-5251 (1987).

The fourth broad category is directed to techniques and methods for directly inducing or increasing melanin production by melanocytes. This fourth category is the least developed and investigators have often resorted to the use of murine skin and murine melanoma cell lines rather than using human melanocytes. Representative of this fourth broad category are the following publications: Friedmann and Gilchrest, J. Cell. Physiol. 133:88-94 (1987); Rosdahl and Szabo, J_. Invest. Dermatol. 70:143-148 (1978); Warren, R., Photochem. Photobiol. 43:41-47 (1986); Jimbow and Uesugi, J. Invest. Dermatol. 78:108-115 (1982); Rosen et al., J.. Invest. Dermatol. 84: 5247-5251 (1987); U.S. Patent Number 4,508,706; U.S. Patent Number 4,618,484; and U.S. Patent Number 4,695,449.

Because of the variety and the scope of the pertinent literature in general, a summary of the pertinent facts believed to be true and supported by empirical evidence can be stated as follows: pure cultures of melanocytes respond directly to simulated sunlight by an increased melanin content per cell; by morphological changes; and by cellular growth arrest. These responses are recognized as partially mimicking the tanning response of melanocytes in intact skin. This in-vitro tanning response is not attributed to an altered cAMP content or to the effects of vitamin D, its precursors, photoproducts, or metabolites. In addition, it has been demonstrated that a variety of different tissue extracts and derived growth factors can stimulate melanocyte proliferation. However, the use of many stimulating agents and mitogens such as choleragen and phorbol esters, although inducing cell proliferation, also render the melanocytes insensitive to many other physiologic stimuli. Moreover, as part of the response to most mitogens in-vitro, there is an inverse relationship between melanocyte growth rate and melanin, content per cell. This is in contrast to living skin which responds to ultraviolet irradiation with both an increase in the number of melanocytes and an increase in the melanin synthesis rate per melanocyte. Finally, while the relationship between human keratinocytes and melanocytes continues to be a subject of considerable interest, there remains a noticeable absence of information and empirical data regarding what, if any, soluble keratinocyte cellular products enhance or influence melanocyte growth rate, melanocyte dendricity, and/or melanin pigment production by melanocytes. It would be recognized and appreciated, therefore, by practitioners ordinarily skilled in this art, that the availability of specific in-vitro culture medium products generated by keratinocytes that can individually affect and control melanocyte proliferation, or melanocyte dendricity, or melanin synthesis by melanocytes would be a major advance and recognized improvement over all presently known methods and compositions.

SUMMARY OF THE INVENTION

The present invention provides unique methods for inducing melanocyte proliferation and increasing the melanin synthesis of melanocytes under both in-vivo and in-vitro conditions. One of the unique methods employs a supplemented preparation comprising: at least a fraction of the products generated and released by keratinocytes into the surrounding medium during in-vitro culture in a medium containing at least an isotonic salt solution at about neutral pH value for minimal maintenance of keratinocytes in culture, 0 - 20 mg/ml albumin,

0 - 100 ug/ml inositol, 0 - 50 ug/ml choline chloride; and at least one supplement selected from the group consisting of a fibroblast growth factor, a blood serum, and a cyclic adenosine monophosphate mediator. Another material useful in these methods is at least one member selected from the chemical class consisting of diacylglycerols, diacylglycerol analogues, and diacylglycerol derivatives. Each of these compositions can be combined in admixture with a fluid carrier compatible with the skin of a living subject to form a topical formulation able to increase the melanin content of skin in-vivo. These unique compositions may also be employed with conventionally known media and culture techniques for in-vitro culture of melanocytes whether for scientific research or commercial purposes. In each instance, the use of these compositions in-vivo and in-vitro will induce melanocyte proliferation; increase melanocyte dendricity; and increase melanin synthesis within melanocytes.

BRIEF DESCRIPTION OF THE FIGURES The present invention may be more easily and completely understood when taken in conjunction with the accompanying drawing, in which:

Figs. 1a and 1b are micrographs illustrating the effects of supplemented keratinocyte derived conditioned medium upon melanocytes in-vitro;

Fig. 2 is a graph illustrating the dose-dependent effect of supplemented keratinocyte derived condi tioned medium upon melanocyte proliferation and melanin production;

Fig. 3 is another graph illustrating the biological activity of supplemented keratinocyte derived conditioned medium to increase melanocyte number and melanin production;

Figs. 4a-4d are micrographs illustrating increased melanocyte dendricity and melanin synthesis during incubation for 24 hours and 7 days with supplemented keratinocyte derived conditioned medium;

Fig. 5 is a graph illustrating the effects of whole keratinocyte derived conditioned medium, the greater than 10,000 dalton retentate fraction, and the less than 10,000 dalton filtrate fraction on human melanocyte proliferation;

Fig. 6 is a graph illustrating the effect upon melanin content per human melanocyte for the same reactants seen in Fig. 5;

Fig. 7 is a graph illustrating the lack of effect of MSH on human melanocyte prolif eration and melanin production ;

Fig. 8 is a graph illustrating the effect of MGF on human melanocyte proliferation and melanin production; Fig. 9a and 9b are graphs illustrating the effects of a diacylglycerol upon human melanocyte proliferation and melanin content;

Fig. 10 is a graph illustrating the effects of a diacylglycerol upon the proliferation of human melanocytes when administered alone and after pretreatment with a phorbol ester;

Fig. 11 is a graph illustrating the effects of a diacylglycerol upon melanin production with human melanocytes when administered alone and after pretreatment with a phorbol ester; Fig. 12 is a graph illustrating the effects of a diacylglycerol on the proliferation of S91 melanoma cells in culture; and

Fig. 13 is a graph illustrating the effects of a diacylglycerol upon the melanin content of S91 melanoma cells in culture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is a general methodology for controlling the growth and biological activity of human and animal melanocytes under both in-vivo and in-vitro conditions. The present invention, in all its aspects, is based and relies upon a fundamental premise that the ability to control and specifically regulate melanocyte growth is separate and distinguishable from the control and regulation of melanocyte dendricity, and that both of these events are separately controllable and distinguishable from regulating the rate of melanin synthesis in the melanocyte. The premise is, therefore, that several different factors and mediators regulate and modulate each of these specific activities independently, but in concert with the others. Thus, for example, while the rate of melanin synthesis is directly controlled by one or more specific factors or mediators, there will be some degree of interaction and cross-effects with the other specific factors or mediators individually responsible for regulation of melanocyte growth and melanocyte dendricity. This fundamental premise differs substantially from the conventional approach.

The conventional outlook documented and accepted by ordinary practitioners in this art follows and complies with traditional histological studies. In contrast a novel sequence of events occurring in primary in-vitro cultures of human epidermis have been observed between the two relevent types of cells, keratinocytes and melanocytes. Initially, the epidermal suspension maintained in nutritionally adequate culture medium contains more than 90% keratinocytes and only an estimated 2-4% melanocytes. In some culture systems, the culture medium is such that the keratinocyte population dwindles and is lost within a few days, thereby prohibiting any meaningful interaction with melanocytes. In other culture systems, the keratinocytes survive but the proportions of the cells gradually reverse after two or three weeks culture as the melanocytes proliferate and the keratinocytes terminally differentiate in a selective growth medium. At this time, a striking preferential clustering of dendritic melanocytes around the few remaining viable keratinocytes is readily apparent. Complete disappearance of keratinocytes from the cultures occurs after three or four weeks; and is accompanied by cessation of melanocyte proliferation, and a decrease in melanocyte dendricity with substantial alteration of these cells to a polygonal cell morphology. To our knowledge, there has been no common or generally accepted belief heretofore that a regulatory role for keratinocytes might exist in determining melanocyte morphology, growth, or melanization. Hence, even the concept that keratinocytes play a major role in new; and the premise that different soluble factors and/or mediators generated and released by keratinocytes in culture could specifically regulate individual parts of the process separately or in concert is entirely novel and unforeseen in view of existing knowledge. There is also a second major difference and distinction in fundamental premise and approach of the present invention from the conventionally held views and beliefs of practitioners in this art. A substantial portion of the published literature describing both in-vivo and in-vitro experiments and providing empirical data have utilized non-human, animal models and employed non-human keratinocytes and melanocytes. A favorite of many investigators is the use of mouse cells, the S91 mouse melanoma cells being perhaps the best known. The prevalent view is that the animal data and models are directly applicable to the human melanocyte condition; and that the stimulating agents, mitogens, and other mediators of melanocyte activity effective within the mouse and other animal cell systems are also operative and in existence for the human melanocyte and human skin. The present invention does not accept this view and in fact provides evidence that specific factors and intracellular pathways mediate pigmentation in human skin which are different from those present in animal skin. The factors that govern human melanocyte growth, human pigment production, and human pigment transfer are effective regulatory controls for human melanocytes; whereas factors and mediators derived from non-human keratinocytes are often ineffective and non-regulatory. The second fundamental difference is thus the realization that major differences and distinctions exist among the factors and mediators of melanocytes effective in human and non-human cellular conditions. The present invention provides a variety of keratinocyte derived preparations and a class of diacylglycerol compositions, analogues, and derivatives, each of which can be usefully employed in the methodology for a variety of different purposes and advantages. The keratinocyte derived preparations may be employed as follows: i. to induce melanocyte proliferation in-vitro; ii. as an in-vitro culture additive for experimental research investigations and purposes; iii. in a method for pigmenting skin grafts, allografts, and autografts; iv. for inducing melanocyte proliferation in-vivo; v. for treating hypopigmentation disorders such as vitiligo, albinism, piebaldism, and post-inflammatory hypopigmentation; vi. as a sun-light independent tanning agent in-vivo; vii. as a tanning accelerator in the presence of natural sun-light; viii. as a treatment for darkening or repigmenting hair in-vivo; ix. as a preventor of gray (depigmented) hair in-vivo; and x. for the production of darkly colored pelage, fur, and wool in—vivo by animals. The class of diacyglycerol compositions is particularly us eful f or humans and provides the following benefits and advantages:

(a) inducing tanning of the skin in the absence or presence of sunlight; (b) acting as an accelerator of skin tanning in the presence of natural sunlight; and

(c) providing a treatment for gray (depigmented) hair.

The keratinocyte derived preparations and the chemical class of diacylglycerols as a whole are materials which function independently of one another and interact with living melanocytes, in-vivo or in-vitro, in non-complementary ways using different biochemical pathways. Accordingly, each family of materials is unique and is intended to be employed separately from the other with specific methods which achieve similar if not identical results. For these reasons, each family of materials will be described and characterized individually in order that a complete understanding and appreciation of each may be had without regard to the other.

The Keratinocyte Derived Preparations And The Manner Of Their Preparation And Use The keratinocyte derived preparations are the direct result of reacting a pure culture of mature keratinocytes (of human or animal origin) with a specifically prepared culture medium; a keratinocyte product generating medium (hereinafter "KPGM"), such that a variety of different factors and/or mediators are generated and released by the keratinocytes into the surrounding culture medium. The KPGM is specifically formulated and restricted in composition to induce the generation of these factors and/or mediators by the keratinocytes in a form which permits their release into the surrounding medium over the predetermined cell culture period of time. The culture medium containing the released factors and/or mediators is broadly termed "keratinocyte derived conditioned medium" (hereinafter "KDCM"). The whole KDCM preparation is then supplemented with at least one supplement selected from the group consisting of a hypothalmic extract and a blood serum to form a supplemented preparation ready for admixture with melanocytes in-vivo or in-vitro. The effect of the supplemented preparation comprising whole KDCM and preferably both the hypothalmic extract and the blood serum at specified concentrations induces three cell phenomenon and events in living melanocytes. These are: an increase in melanocyte proliferation and growth; an increase in melanocyte dendricity; and a substantial increase in melanin synthesis by the cell.

Alternatively, the whole KDCM can be fractionated, preferably by ultrafiltration, to provide two distinct and different fractions of KDCM based upon the differences in nominal molecular weight of the soluble products within the fluid medium. It has been empirically demonstrated that the meaningful differences in biological activity between the fractions occur at about the 10,000 dalton molecular weight level. The fractionated KDCM containing products greater than about 10,000 daltons in nominal molecular weight, arbitrarily designated herein as Fraction A, are combined with at least one supplement selected from the group consisting of a fibroblast growth factor, a blood serum, and a cyclic adenosine monophosphate (hereinafter "cAMP") mediator to yield a supplemented Fraction A preparation. When combined with living melanocytes in-vivo or in-vitro, the predominant and overwhelming result is only an increased melanocyte proliferation and growth; there is comparably little if any increase in melanocyte dendricity or increase of melanin synthesis. On the other hand, if the fraction of KDCM containing those soluble products of keratinocyte culture having a nominal molecular weight of less than about 10,000 daltons, arbitrarily designated herein as Fraction B, is combined with at least one supplement selected from the group consisting of a hypothalmic extract, a blood serum, and choleragen, a supplemented Fraction B preparation is formed. When the supplemented Fraction B preparation is combined with living melanocytes in-vivo or in-vitro, a distinctly different and unique phenomenon occurs: there is a substantial increase in melanocyte proliferation and growth similar to that provided by supplemented Fraction A preparations. In addition to cell proliferation, however, there is also a substantial increase in both melanocyte dendricity and a significant increase in melanin synthesis within the existing number of melanocytes. The cellular events and phenomena induced by the supplemented Fraction B preparation are thus distinctly different and distinguishable from the cellular events and phenomenon caused by supplemented Fraction A upon identical types of melanocytes. A useful summary of each reaction and result is provided by Reaction Schemes I, II, IIIA, and IIIB respectively provided below.

To allow the prospective user to employ each of the preparations and each type of cell most effectively in accordance with Reaction Schemes I,

II, IIIA, and IIIB respectively, a detailed description for each is provided.

Keratinocytes and Melanocytes

The source for both kinds of human cells is preferably neonatal foreskins obtained within two hours of elective circumcision. The epidermis is separated from the dermis after overnight incubation in 0.25% trypsin [GIBCO, Grand Island, New York; Gilchrest et al., J. Invest. Dermatol. 83 : 370-376 (1984)]. Primary cultures of epidermal keratinocytes are established using the method of Rheinwald and Green [Cell 6:331-334 (1975)], modified by using a 4:1 ratio of Dulbecco's modified Eagles's medium (hereinafter "DMEM") and Ham's F12 medium supplemented with 10 mM adenine and 10% fetal bovine serum [MA Bioproducts, Walkersfield, MR]. The confluent primary cultures of keratinocytes are then washed with 0.5 mM ethylenediamine tetracetic acid (hereinafter "EDTA") and incubated with 1 millileter (hereinafter "ml") of 0.25% trypsin for approximately 15 minutes. The resulting cell suspension is then mixed with an equal volume of 10% FBS in M199 culture medium [GIBCO]. The cells were then pelleted by centrifugation; resuspended in BITIC medium; and seeded at 1 x 10⁶ cells into 60 millimeter (hereinafter "mm") dishes containing 4 ml of BITIC medium. BITIC medium is prepared as follows : BITIC MEDIUM (500 ml)

ADDxml STOCK FINAL Medium 199 (Gibco 400-1100) 491

Insulin* (INS) 0.5 10 mg/ml 10 ug/ml Epidermal Growth Factor (EGF) 0.5 10 ug/ml 10 ng/ml Triiodothyronine (T3) 0.5 1 uM 1 nM

Transferrin (Tf) 0.5 10 mg/ml 10 ug/ml

Hydrocortisone (HC) 0.5 1.4 mM 1.4 uM

Inositol (Ino) 0.5 10 mg/ml 10 ug/ml Choline Chloride (CC) 0.5 10 mg/ml 10 ug/ml

Bovine Serum Albumin (BSA) 5 200 mg/ml 2 mg/ml Penicillin/Streptomycin** (P/S) 5 1% v/v

(optional)

* Insulin is added first as it is acidic ** If Pen/Strep is used, reduce M199 volume to 486.

Before plating, the culture dishes were pretreated with 30 micrograms (hereinafter "ug") per dish of affinity purified human fibronectin. The cultures were then incubated in 8% carbon dioxide/air at 37 C and provided with fresh medium twice weekly.

Melanocytes were established in primary culture from similarly prepared epidermis following the procedure of Gilchrest et al. [J. Invest. Dermatol. 83: 370-376 (1984)]. In brief, operative specimens were cut into 4 mm² fragments; rinsed in calcium-free phosphate-buffered saline (hereinafter "PBS"); and incubated in 0.25% trypsin (GIBCO) overnight at 4 C.

The epidermal portions of the fragments were separated from the dermis with forceps; incubated for 10 minutes in 0.02% EDTA at 37 C; vortexed to yield a single cell suspension; inoculated at a concentration of

10 cells per 35-mm dish in melanocyte growth medium; and maintained at 37 C in 8% carbon dioxide and 92% air. Cultures were provided with fresh melanocyte growth medium three times weekly.

Melanocyte growth medium is prepared as follows:

MELANOCYTE GROWTH MEDIUM (500 ml)

ADDxml STOCK FINAL Medium 199 (Gibco 400-1100) 467 Insulin* (INS) 0.5 10 mg/ml 10 ug/ml Triiodothyronine (T3) 0.5 1 uM 1 nM Epidermal Growth Factor (EGF) 0.5 10 ug/ml 10 ng/ml Transferrin (Tf) 0.5 10 mg/ml 10 ug/ml Hydrocortisone (HC) 0. 5 1 .4 mM 1.4 uM Cholera Toxin (CT) 5 10^-7 M 10^-9 M dialyzed Brain Extract (dBE) 25 2 mg/ml 100 ug/ml Penicillin/Streptomycin** (P/S) 5 1% v/v (optional)

* Insulin is added first as it is acidic. ** If Pen/Strep is used, reduce M199 volume to 462.

Regarding keratinocytes and melanocytes from non-human sources, it is expected that the origin of the cells and the manner in which each is isolated and maintained in culture will follow those procedures previously established in the literature for these specific purposes.

Keratinocyte Product Generating Medium (KPGM)

It is intended that a variety of different formulations and compositions in admixture can be utilized as KPGM for purposes of the present invention. KPGM is thus not a single formulation or mixture of specific ingredients, but includes a variety of differently prepared culture media suitable for in-vitro maintenance of grown keratinocytes for the specific purpose and goal of inducing the keratinocytes in culture to generate and release a variety of different factors and/or mediators as products into the surrounding medium. KPGM therefore may be most broadly defined as a variety of specifically prepared maintenance media for continuing in-vitro culture of grown keratinocytes. A At a minimum, this medium comprising the following: at least an isotonic salt solution at about neutral pH values for minimal maintenance of grown keratinocytes in culture ;

0 - 20 mg/ml albumin;

0 - 100 ug/ml inositol; and

0 - 50 ug/ml choline chloride. ▲ An alternative minimal formulation will comprise the following: essential amino acids, vitamins, nutrients, and salts for minimal maintenance of keratinocytes in culture; 1 - 100 ug/ml insulin;

0 - 10 nM of a iodothyronine, preferably triiodothyronine;

0 - 100 ug/ml transferrin;

0 - 15 M of a cortisone, preferably hydrocortisone; and

0 - 100 ng/ml epidermal growth factor.

▲ A preferred Formula I for KPGM comprises: Dulbecco's Minimal Eagle's medium containing: 10 ug/ml insulin; 1 nM triiodothyronine;

10 ug/ml transferrin; 1.4 x 10 M hydrocortisone;

10 ng/ml epidermal growth factor; and

10^-9 M choleragen (chlorea toxin). ▲ An alternate preferred Formula II for KPGM comprises:

Medium 199 (GIBCO) containing 0.3 uM myo-inositol, 3.6 uM choline, 1.8 uM calcium, and 0.5 mM chloride;

10 ug/ml insulin;

10^-9 M transferrin;

1.4 x 10^-6 M hydrocortisone; 10 ng/ml epidermal growth factor; and 2 mg/ml bovine serum albumin.

▲ An alternate preferred Formula III for KPGM includes the following:

1 part Dulbucco's Minimal Eagle's medium; 4 parts Ham's F12 medium; 10 mM adenine;

10% fetal calf serum;

10 ug/ml insulin;

10^-9 M triiodothyroni:

10 ug/ml transferrin; 1.4 x 10^-6 M hydrocortisone; and

10 ng/ml epidermal growth factor.

▲ Within these alternative definitions of KPGM, a number of additives may be optionally added for increasing the longevity of the keratinocytes in culture or increasing the concentration of soluble factors and/or mediators released into the medium during keratinocyte culture. These include:

20 - 300 ug/ml of bovine hypothalmic extract (Gilchrest et al., J. Invest. Dermatol. 83 : 370-376 (1984); Wilkins et al., J . Cell. Physiol. 122:350-361 (1985)];

0.5 - 500 uM myo-inositol; 0.4 - 40 uM choline and 0.5 - 500 uM myo-inositol in combination;

0.5 - 500 uM ethanolamine ; 0.5 - 500 uM phosphoethanolamine ; 0 - 5 mM serine;

0 - 1 uM selenium;

0 - 10 uM prostaglandin E₂, E₁ ; and 10^-8 - 10^-13 M cholera toxin.

▲ Compounds to be avoided as additives to KPGM generally include the following: stearic acid in either dilute or concentrated form; methionine in either dilute or concentrated form; cyanocobalamine in either dilute or concentrated form; and folic acid in either dilute or concentrated form.

Culture Conditions for KPGM An Keratinocytes In-Vitro The incubation conditons for keratinocytes and the KPGM follow conventional routine. All cultures are preferably incubated at 37 C ±4 C in an atmosphere containing 8 ±4% carbon dioxide. Fresh medium is added to each dish at intervals ranging from 1 to 4 times per week. Use of sterile technique and sterile precautions is presumed throughout.

The Keratinocyte Derived Conditioned Medium (KDCM)

Properly obtained KDCM contains specific factors and/or mediators derived from keratinocytes directly as a result of in-vitro culture with KPGM exclusively.

The true identity and exact chemical composition of these factors and meditors, however, has not yet been fully analyzed and chemically characterized. Nevertheless, the resulting KDCM is clearly definable as a product-by-process composition which relies upon the mode of preparation described herein concurrent with a showing of specific biological activity for melanocytes as the basis for demonstrating its presence.

Moreover, while the chemical identity of KDCM is not yet known, a number of other previously identified keratinocyte derived factors and cell mediators reported in the literature have been specifically excluded as being responsible for the activity observed in any preparation of KDCM. All of the following have been empirically demonstrated and proven not to be responsible for KDCM activities in every instance; i. basis fibroblast growth factor [Halaban et al.,

In-vitro 23:47-52 (1987); Ogata et al.., Biochem.

Biophys. Res. Comm. 146:1201-1211 (1987)]; ii. interleukin 1, interkeukin 1a, and interleukin 1b

[Gospodarowics et al., Endochrine Rev. 82 : 95-114

(1987); Kupper et al., J., Exp. Med.

164:2095-2100 (1986)]; iii. prostaglandin E₂ [Tomita et al.. J. Invest. Dermatol. 89:299-301 (1987); Pentland and

Needleman, J., Clin. Invest. 77:246-251 (1986)]; iv. 12-hydroxy-eicosatetraenoic acid [Hammarstrom et al., Proc. Soc. Natl. Acad. Sci. USA

72:5130-5134 (1975)]; v. leukotriene B4 [Goldyne, Prog. Dematol. 202 : 1-7

(1986)]; vi. nerve growth factor [Wilkins et al., Cold Spring

Harbor Conferences On Cell Proliferation, Vol. 9,

1982, pp 929-926]; and vii. transforming growth factor alpha or epidermal growth factor [Carpenter, G., Ann. Rev. Biochem. 56:881-914 (1987)].

Supplements To Keratinocyte Derived Conditioned Medium The KDCM preparation is intended to be supplemented with at least one class of supplement selected from the group consisting of: a fibroblast growth factor, a blood serum preparation, and a cyclic adenosine monophosphate (cAMP) mediator. Each will be described individually.

A fibroblast growth factor: This class of supplement can be utilized in purified, semi-purified, and crude extract forms as needed or desired by the user. Included as members of this class of supplement are: acid and basic fibroblast growth factors; brain extracts; brain portion extracts, particularly of the hypothalmus; and, most desirably, the bovine hypothalmic extract prepared according to Gilchrest et al. [ J . Invest. Dermatol. 83:370-376 (1984)]. The bovine hypothalmic extract (hereinafter "BHE") is added at final concentrations of 0 - 100 ug/ml. Basic fibroblast growth factor is preferably present at final concentrations of 0.05 - 10.0 ng/ml.

A blood serum preparation: This class of supplement can be used as whole serum or as a prepared serum fraction such as dialyzed serum and heat-treated serum. Serum rather than plasma is preferred and serum from calves, horses, and any other animal serum can be employed. Fetal bovine serum (hereinafter "FBS") is most desirable and is used at final concentrations ranging from 0 - 50% by volume [Gilchrest et al., J. Invest. Dermatol. 83 :370-376 (1984)]. A cyclic adenosine monophosphate (cAMP) mediator: This class of supplement includes a wide range of compounds which act as cAMP modifiers. The members of this class of supplement include: cholera toxin (choleragen) at 10^-6 - 10^-13 M final concentrations; methyl xanthines such as isobutyl methyl xanthine

(hereinafter "IBMX") at final concentrations of 0.01 ¬

1.0 mM and theophylline at final concentrations of

0.1 - 110.0 mM ; forskolin and its analogues at final concentrations of 0.1 - 50 mM ; and active analogues of cAMP such as dibutyl cyclic adenosine monophosphate and 8-bromo cyclic adenosine monophosphate at final concentrations of 0.1 - 200 mM.

Whole KDCM is preferably combined with bovine hypothalmic extract to a final concentration of 100 ug/ml BHE and fetal bovine serum (FBS) to a final concentration of 2.0%. Alternatively, either class of supplement may be utilized alone in combination with whole KDCM to yield the supplemented preparation of choice.

If the user prefers to choose among the various biological activities provided by supplemented whole KDCM, in order to specifically gain increases in melanocyte proliferation without concomittant increases of melanocyte dendricity and melanin production (or the converse), it is required that whole KDCM be separated into at least two different fractions. Fractionation is preferably achieved via the conventionally known technique of ultrafiltration using exclusion membranes of known pore size, porosity, and materials. A variety of such exclusion membranes are available commercially, some of which are provided by Amicon Corporation (Danvers, Massachusetts). In brief, whole KDCM is passed through a porous membrane able to exclude molecules having a nominal molecular weight greater than 10,000 daltons using nitrogen gas at 50 psi. It is preferred that the retentate - containing molecules greater than 10,000 daltons - be concentrated to a volume about 10% of the original. The filtrate, containing molecules less than 10,000 daltons in molecular weight, is preferably concentrated by lyophilization and reconstituted to up to 50% of the original volume. Both the retentate and filtrate are desirably sterilized by filtration through 0.2 micron filters prior to use. Clearly, the retentate corresponds to Fraction A of Reaction Scheme IIIA previously described and is preferably supplemented with 2% FBS, 100 ug/ml BHE, and 10^-9 M choleragen before use. If desired, the Fraction A retentate may be supplemented with only one class of supplement. Similarly, the filtrate corresponds to Fraction B of Reaction Scheme IIIB and is desirably supplemented also with 2% FBS, 100 ug/ml BHE, and 10^-9 M choleragen before use. If desired, any one class of supplement may be employed alone with Fraction A.

It will be recognized and appreciated that the preferred member of each supplement class may be substituted by any other member of that class in each separated fraction. Accordingly, FBS may be substituted by any other animal serum; BHE may be substituted for by any other material containing a fibroblast growth factor; and choleragen may be substituted by any other cAMP modifier. All of these substitutions may be freely made as the need or desire of the user dictates.

It will be appreciated also that the ability to separate the different biological functions of whole KDCM lies substantially in separating soluble products in the whole fluid at about the 10,000 mole cular weight level. Investigations employing other fractions of KDCM containing soluble products of less than 2,000 daltons and less than 500 daltons molecular weight were found to be substantially equivalent in biological activity to those fractions less than 10,000 daltons. Each fraction containing soluble products of about 10,000 daltons or less provides substantial melanin synthesis activity and increases in melanocyte dendricity; and provides an equivalent increase in melanocyte proliferation to the fraction containing products greater than 10,000 daltons in weight. Alternatively, Fraction A containing those soluble products of whole KDCM greater than about 10,000 daltons in molecular weight retains only the ability to induce substantial melanocyte proliferation but has little ability to induce changes in melanocyte dendricity or melanin synthesis.

Manner Of KDCM Administration And Use In-Vivo The properly supplemented preparation, be it supplemented whole KDCM or supplemented Fraction A or supplemented Fraction B, is intended to be used in-vivo as a topical formulation. The purposes and uses of such topical formulations in humans and animals have been previously identified herein. Accordingly, these supplemented preparations are intended to be admixed in a pharmacological topical carrier such as a gel, an ointment, a lotion, or a cream; and will include such fluid carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglyerides, fatty acid esters, or mineral oils. Other possible topical carriers are liquid petrolatum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolauriate (5%) in water, sodium lauryl sulfate (5%) in water, and the like. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. In addition, in certain instances, it is expected that the supplemented preparations as described herein may be disposed within devices placed upon, in, or under the skin; such devices include patches, implants, and injections which release the supplemented preparation into the skin either by passive or active release mechanisms.

Manner Of KDCM Administration And Mode Of Use In-Vitro

It is clearly intended that the supplemented preparations, be it supplemented whole KDCM or supplemented Fraction A or supplemented Fraction B, be employed in a variety of different in-vitro applications and uses. A representative listing of such in-vitro applications have been identified previously herein. Under these circumstances, it is preferred that the supplemented preparation by lyophilyzed and stored at -20 C until required for use. Each supplemented preparation may then be reconstituted using the fluid most suitable for use under the given circumstances. Accordingly, if further research investigations were the purpose of the intended application, it is desirable that the lyophilyzed supplemented preparation be reconstituted directly into the prepared culture medium intended to be employed within the cell culture system. The supplemented preparation may be reconstituted to 100% of its original volume if so desired; alternatively, the supplemented preparation may be reconstituted to only a fraction of its original volume; provided that, the degree of concentration not be so great as to cause artifacts or undesirable disturbances within the cell culture medium and/or system. If KDCM is fractionated, the high molecular weight fraction of greater than 10,000 daltons can be used at the 10 fold concentration. If concentrated further, it should be useful in even greater concentration. The low molecular weight fraction, less than 500 daltons can be used at 50% of the original volume, which is twice the original concentration if reconstituted in distilled water. The melanization activity of this fraction appears to be unstable when stored at 4 C. Thus, the material should be used fresh or stored frozen until use. Alternatively, lyophilyzed whole KDCM can be used at twice original concentration if reconstituted in distilled water at half the original volume. Fractions prepared from such preparations may be used at higher or lower concentrations with improved effectiveness.

DIACYLGLYCEROLS (DAG) Chemical Structure And Occurrence Diacylglycerols (hereinafter "DAG") are a class of organic chemical compositions having the chemical structure and formulation of Compounds A, B, or C as follows :

in which R and R' are carbon containing moieties. R and R' are usually long-chain (greater than 14 carbon atoms) carboxylic acids and may contain one or more carbon-carbon double bonds. In most instances, R and R' are chemically different in composition. In comparison, R" can be any chemical entity which does not form a carbon ester linkage with the adjoining carbon atom in the glycerol structure. Accordingly, R" may include a hydroxyl group, a phosphate group, a sulfur atom, an ether group, a halide, a nitrogen containing entity, or hydrogen.

Diacylglycerols are a class of naturally occurring substances. One known role for diacylglycerols is in an intracellular signal transduction pathway in which DAGs are recognized as physiological activators of protein kinase C (hereinafter "PKC"); and phorbol esters can act through similar biochemical pathways [Berridge, N.J., Ann. Rev. Biochem. 56:159-193 (1987); Nishizuka, Y., Science 233:305-312 (1986)]. The synthesis of DAG and many DAG analogues is conventionally known [Ganong and Bell, Methods In Enzymology, Volume 141, 1987, pages 313-320].

Diacylglycerols Of The Present Invention The diacylglycerols of the present invention are those DAG compositions and analogues which are biologically active to produce an increase in cellular melanin content in melanocytes under in-vivo and/or in-vitro conditions. The naturally occurring DAGs are derivatives of phosphatidylinositol; usually contain a long-chain mono-unsaturated fatty acid acylated to the number one carbon position in the glycerol structure; and also typically contain a highly unsaturated fatty acid, primarily arachidonic acid, acylated to the number two carbon position in the glycerol structure. The most preferred member of this chemical class for use in the present methodology is 1-oleoyl-2-acetyl-glycerol because of its solubility in water and its ability to produce a dose-dependent response in melanin content with no concommitant effect upon proliferation and/or growth of melanocytes. In addition, other preferred embodiments for DAG contain a free hydroxyl group at the number three carbon position in the glycerol structure; and are limited to the traditional three carbon backbone structure of glycerol. A representative, but incomplete listing of DAG molecules believed to be biologically active and potent for stimulation of melanin synthesis within melanocytes are those provided by Table II below.

PREFERRED DAG ANALOGUES

1,2-diformylglycerol

1,2-diacetylglycerol

1,2-dibutanoylglycerol 1,2-dihexanoylglycerol

1,2-dioctanoylglycerol

1,2-didecanoylglycerol

1,2-didodecanoylglycerol

1,2-diteradecanoylglycerol 1,2-dihexadecoylglycerol

1,2-dioctadecanoylglycerol

1,2-dieicosanoylglycerol

1,2-didocosanoylglycerol

1,2-ditetracosanoylglycerol 1,2-dipalmitoylglycerol

1,2-dioleoylglycerol

1,2-dilinoleoylglycerol

1,2-dilinolenoylglycerol

1,2-arachidonoylglycerol 1-octanoyl-2-formyl-glycerol

1-octanoyl-2-acetyl-glycerol

1-octanoyl-2-butanoyl-glycerol

1-octanoyl-2-hexanoyl-glycerol

1-octanoyl-2-decanoyl-glycerol 1-octanoyl-2-dodecanoyl-glycerol

1-octanoyl—2-tetradecanoyl-glycerol

1-o ctanoyl-2-hexadecanoyl-g lycerol

1-octanoyl-2-octadecanoyl-glycerol

1-octanoyl-2-eicosanoyl-glycerol 1-octanoyl-2-docosanoyl-glycerol

1-octanoyl-2-tetracosanoyl-glycerol

1-octanoyl-2-palmitoyl-glycerol

1-octanoy1-2-oleoyl-glycerol

1-palmitoyl-2-formyl-glycerol PREFERRED DAG ANALOGUES (CONT'D)

1-palmitoyl-2-acetyl-glycerol

1-palmitoyl-2-butanoyl-glycerol

1-palmitoyl-2-hexanoyl-glycerol 1-palmitoyl-2-octanoyl-glycerol

1-palmitoyl-2-decanoyl-glycerol

1-palmitoyl-2-dodecanoyl-glycerol

1-palmitoyl-2-tetradecanoyl-glycerol

1-palmitoyl-2-hexadecanoyl-glycerol 1-palmitoyl-2-octadecanoyl-glycerol

1-palmitoyl-2-eicosanoyl-glycerol

1-palmitoyl-2-dodcosanoyl-glycerol

1-palmitoyl-2-oleoyl-glycerol

1-palmitoyl-2-linoleoyl-glycerol 1-palmitoyl-2-arachidonoyl-glycerol

1-oleoyl-2-formyl-glycerol

1-oleoyl-2-acetyl-glycerol

1-oleoyl-2-butanoyl-glycerol

1-oleoyl-2-hexanoyl-glycerol 1-oleoyl-2-octanoyl-glycerol

1-oleoyl-2-decanoyl-glycerol

1-oleoyl-2-dodecanoyl-glycerol

1-oleoyl-2-tetradecanoyl-glycerol

1-oleoyl-2-palmitoyl-glycerol 1-oleoyl-2-linoleoyl-glycerol

1-oleoyl-2-arachidonoyl-glycerol

1-hexanoyl-2-formyl-glycerol

1-hexanoyl-2-acetyl-glycerol

1-hexanoyl-2-butanoyl-glycerol 1-hexanoyl-2-octanoyl-glycerol

1-hexanoyl-2-decanoyl-glycerol

1-hexanoyl-2-dodecanoyl-glycerol

1-hexanoyl-2-tetradecanoyl-glycerol

1-hexanoyl-2-hexadecanoyl-glycerol PREFERRED DAG ANALOGUES (CONT'D)

1-hexanoyl-2-octandecanoyl-glycerol

1-hexanoyl-2-eicosanoyl-glycerol

1-hexanoyl-2-palmitoyl-glycerol

1-hexanoyl-2-oleoyl-glycerol

1-hexanoyl-2-linoleoyl-glycerol

1-hexanoyl-2-arachidonoyl-glycerol

Diacylglycerol Administration And Manner Of Use

As will be empirically demonstrated hereinafter, DAG analogues are able to produce increases in cellular melanin content without altering melanocyte proliferation. Moreover, DAG analogues do not increase dendricity of melanocytes. DAGs are therefore selective to their biological action and cellular function; and are generally limited to being able to induce melanin synthesis and production specifically within preexisting melanocytes.

It is intended that the DAG analogues be employed within in-vivo and in-vitro conditions. For in-vivo use, it is desirable that topical administration of one or more DAG analogues be made directly to the skin of the living subject. For this purpose, a fluid carrier is intended to be combined in admixture with the chosen DAG analogue in the manner previously described herein for supplemented preparations of keratinocyte derived conditioned medium. For in-vivo use, it is preferred that the DAG analogue be present in a final concentration range from 0.10 20.0 mM in a fluid carrier material. Alternatively, for in-vitro use as a laboratory reagent with cultures of melanocytes, the chosen DAG analogue may be added directly to the culture media surrounding the living cells at any desired concentration in accordance with the purposes and goals of the experimental design. It is clearly expected and intended that empirical determinations of inhibitory and toxic concentrations for the chosen DAG analogue will be made; accordingly, there is no effective limit on the concentration of the chosen DAG analogue within these in-vitro applications and uses.

As will be empirically demonstrated hereinafter, the preferred DAG analogue, 1-oleoyl-2-acetyl-glycerol is selective in its activity with melanocytes from human skin; in contrast, this DAG analogue is demonstratably inactive and does not induce increases in melanin production when administered to S91 mouse melanoma cells, the most commonly tested animal melanocyte model.

To demonstrate the unique biological activity and operability of the compositions and methods comprising the present invention as a whole, a variety of different experiments performed in the laboratory will be described. These in-vitro experiments serve merely to illustrate the diversity of applications and conditions with which the methods of the present invention may be usefully employed. While the experimental design is solely in laboratory scale terms, it is clear that the empirical parameters can be expanded at will to meet different in-vivo and in-vitro applications. Moreover, the described empirical data and experiments will be clearly understood to be only representative of the number, variety, and diversity of compositions and culture media which can be advantageously employed.

Experiment 1; Effects of Supplemented Keratinocyte Conditioned Medium This experimental series demonstrates the biological activity and cellular effects of supplemented keratinocyte derived conditioned medium (KDCM) preparations upon melanocytes in-vitro.

Cells Neonatal foreskins obtained within two hours of elective circumcision were the source of human keratinocytes and melanocytes. The epidermis was separated from the dermis after overnight incubation in 0.25% trypsin. Primary cultures of epidermal keratinocytes were established using the method of Rheinwald and Green [Cell 6:331-334 (1975)] modified by using Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 (4:1 ratio) supplemented with 10 mM adenine and 10% fetal bovine serum. Melanocytes were established in primary culture from similarly prepared epidermis according to the procedure of Gilchrest et al. [J. Invest. Dermatol. 83_:370-376 (1984)].

Keratinocyte Derived Conditioned Medium

For the production of keratinocyte derived conditioned medium (KDCM), a second passage keratinocytes were seeded at 1 x 10 cells per 60 mm dish in a completely defined culture medium and permitted to grow to approximately 80% confluence. Each keratinocyte culture was then provided with fresh medium containing the following: Dulbecco's modified

Eagle's medium (DMEM) supplemented with 10 ug/ml insulin, 10^-9 M triiodothyronine, 10 ug/ml transferrin, 1.4 x 10^-6 M hydrocortisone, 10 ng/ml epidermal growth factor, and 10^-9 M choleragen. This keratinocyte product generating medium was combined with the keratinocytes and allowed to incubate at

37 C for 24 hours. The resulting conditioned medium, KDCM, was aspirated from each culture, combined as a single fluid, and stored at -20 C. Individual cultures of keratinocytes were used for generation of KDCM for up to 6 consecutive days.

Prior to use, the combined fluid KDCM was thawed; filtered sterilized; and supplemented with 2% fetal bovine serum (FBS) and 100 ug/ml of a bovine hypothalmic extract (BHE) previously demonstrated to contain a potent melanocyte mitogen. This supplemented preparation was then stored at 4 C until used. A sham conditioned medium was prepared by incubating the KPGM formulation in 60 mm dishes containing no cells whatsoever and processing identically with keratinocyte containing dishes.

Melanocyte Bioassay Melanocytes were seeded at 2 x 10⁴ cells per

35 mm dish and combined with KPGM supplemented with

2% FBS and 100 ug/ml of BHE, now designated as complete melanocyte medium. After 24 hours incubation at 37 C, the grown melanocyte cultures, in duplicate or triplicate, received one of the following: free complete melanocyte medium; or supplemented whole KDCM. Each melanocyte culture was then incubated for 6-7 days at 37 C. Subsequently, each melanocyte culture was harvested and the cell number per dish empirically determined. Each melanocyte culture was washed with a 0.4 mM EDTA in PBS; treated with 1 ml of a mixture containing 0.13% trypsin and 0.2 mM EDTA; then incubated approximately 10 minutes at 37 C; followed by addition of 1 ml of PBS. Subsequently, a 0.5 ml aliquot of each resulting suspension was diluted to 10 ml total volume using isotonic saline and processed using a particle counter (Model ZM, Colter Science). To determine melanin content, the remaining suspension was centrifuged for 5 minutes in a microcentrifuge; the supernatant discarded; and the resulting cell pellet dissolved in 0.1 ml of a 1 M NaOH which was subsequently diluted with 0.4 ml of water. Melanin concentration was calculated by determination of optical density at 475 nanometers; and values extrapolated by comparison with a standard curve of determinations for synthetic melanin, a measurement of melanogenesis which correlates extremely well with ¹⁴C-DOPA incorporation and wiih tyrosinase activity [Friedmann and Gilchrest, J . Cell. Physiol. 133:88-94 (1987)]. Melanin values were expressed as total melanin per culture; or as melanin content per cell; or as percent of untreated controls.

In certain instances, phase contrast micrographs were taken using an inverted microscope after the cultures were washed once with phosphate buffered saline.

KDCM Effects

The incubation of grown melanocytes in supplemented KDCM are demonstrated by the micrographs seen in Figs, 1a and 1b respectively. In each instance, major increases in melanocyte dendricity and substantial decreases in perinuclear cytoplasmic area for melanocytes are clearly visible. When increasing proportions of supplemented KDCM preparations were added to fresh complete melanocyte medium, a clear dose-dependent growth response was observed as is graphically illustrated by Fig. 2. At 100% concentrations, the supplemented KDCM preparation produced on average more than a 4 fold increase in melanocyte yield over than of unsupplemented paired cultures, corresponding to an increase of from approximately 2 - 4 doublings of cell population per week. This ability to induce melanocyte proliferation was empirically demonstrated to exist in each of 22 normal human melanocyte lines tested; each melanocyte line demonstrated an increased cell yield, with a range of cell proliferation response demonstrating increases from 179%-1,334% over that of controls.

In addition, each melanocyte cell line tested demonstrated increases of melanin per cell culture in total as is graphically illustrated by Figs. 2 and 3 respectively. A significant increase In melanin content on a per cell basis was also observed (157±29% of controls; a range of from 49-540%) in contrast to the usual inverse relationship between melanin content and growth rates in cultured melanocytes in each systems. Incubation with sham conditioned medium has no effect on melanocytes in three different experiments producing an average 94±4% of the yield from control cells and 116±5% of the melanin content in comparison to control cells. In addition, there was no change in dendricity compared to control cultures of melanocytes incubated in fresh complete melanocyte medium. The increased dendricity of melanocytes cultured with supplemented KDCM was visible within 24 hours incubation time as is seen by the micrographs of Figs. 4a and 4b; and became increasingly prominent after one week's incubation time as demonstrated by the micrographs of Figs. 4c and 4d. The reduction in apparent cell size of melanocytes was not observable after one day's incubation with supplemented KDCM, but was readily visible by the seventh day. Under phase contrast microscopy, melanocytes cultured in supplemented KDCM contained more golden intracellular inclusions which were predominantly perinuclear and at the tip of the dendrites - a cellular appearance which indicates an increased melanosome number and degree of melanization. The growth promoting activity of the KDCM preparation exists in its own right and was demonstrated in the absence of supplements - that is, in the absence of either a blood serum supplement or a fibroblast growth factor supplement. In four different human melanocyte lines, the KDCM preparation containing supplements of 2% fetal bovine serum and BHE produced a 4 fold increase in cell yield in comparison to control cells (396±93% of control). In comparison, the KDCM preparation containing the BHE but without the FBS produced 102±38% of the cell yield obtained from complete, non-conditioned serum-containing melanocyte medium; similarly, the KDCM preparation containing FBS but without BHE produced 72±29% of the cell yield observed using cells grown in complete, non-conditioned medium. These empirical data demonstrate unequivocally that the KDCM preparation provides growth promoting activity independently of either the FBS and BHE supplement.

Experiment 2: Effects of KDCM Fractions

Biological Activity Of KDCM Fractions

Initially, whole KDCM was obtained following the procedure described within Experiment 1 above. Subsequently, whole KDCM was fractionated by ultrafiltration using conventionally known techniques and exclusion filters of 500, 2,000, or 10,000 daltons. Each KDCM fraction was then combined with grown melanocytes in culture and evaluated by the bioassay procedure previously described herein. The results are summarized by Figs. 5 and 6 respectively and by the data of Table III below.

Table III

PERCENT NEGATIVE CONTROLS PERCENT POSITIVE CONTROLS

CULTURE MELANOCYTE MELANIN MELANIN MELANOCYTE MELANIN MELANIN ADDITIVE YIELD PER CELL PER CULTURE YIELD PER CELL PER CULTURE

Rententate 210±42^b 132±20 228±47^b* 60+7^a 86+9 48±6^a (MW > 10 kDa) n = 12

Filtrate 220+32^a 206±34^b** 427±123^b 63±10^a 167±31^b 92±11

(MW < 0.5 kDa) n = 7 Negative controls are values obtained in complete melanocyte medium. Positive controls are values obtained in homologous unfractionated KDCM. The retentate values represent pooled results from experiments using 12 different melanocyte cell lines, and 7 lots of KDCM. The ultrafiltrate values are pooled results from experiments using 7 lines, and 4 different lots of KDCM. * n=11 ** n=6 a : p<0.01, b:p<0.05

Empirically, the retentates obtained after ultrafiltration with exclusion filters of 10, 2, or 0.5 kDa each contained approximately one-half of the growth promoting activity of whole KDCM. Because the results with each retentate from each fractionated size range above 10,000 daltons were similar, the retentate data was pooled.

Overall, as is illustrated by Figs. 5 and 6 respectively, when the retentates were concentrated 10 times and reconstituted to the original volume using basal melanocyte culture medium, more than a doubling in both cell yield and melanin production per culture was observed after 7 days incubation over that for controls even though there was no significant increase in melanin production per melanocyte. The amount of cell proliferation and the total melanin content per culture of the pooled retentates were found to be statistically less than the proliferation and melanin production yielded by whole KDCM, by approximately 50%. In addition, melanocytes cultured in pooled retentate exhibited a slightly more spread cellular morphology but did not demonstrate any increase in dendricity in comparison to controls. A subsequent experiment in which whole KDCM was first dialysed against PBS using exclusion membranes retaining products greater than 12,000-14,000 daltons, followed by dialysis against melanocyte medium, provided substantially similar results: the retentate provided 180% increases in cell proliferation in comparison to controls; while whole homologous KDCM demonstrated 340% cell proliferation in comparison to controls. In addition, the 12-14 kDa retentates did not increase melanocyte dendricity or melanin content (either per cell or per culture) in any meaningful manner.

Empirical testing of the 0.5, 2, and 10 kDa ultrafiltrates revealed a very different effect upon living human melanocytes. Initially, the fractions less than 10,000 daltons were able to induce melanocyte proliferation to approximately double its paired control under identical test conditions; but, unlike the retentate fractions, the ultrafiltrate fractions less than about 10,000 daltons were able to significantly increase melanocyte dendricity and significantly increase both the melanin content per cell and the total melanin content per culture. The increased growth produced by the ultrafiltrate (like that of the retentate) was approximately one-half of that induced by incubation with whole KDCM; on the other hand, the melanin content increase per cell was slightly greater than that produced by whole KDCM. Melanocytes cultured in ultrafiltrate demonstrated a distinct increase in dendricity within 24 hours incubation time, but the degree of dendricity was usually less than that provided by the use of whole KDCM.

Experiment 3: Comparison of Effects Among KDCM And Known Cellular Mediators

This experimental series evaluated whole KDCM in comparison to a variety of cellular mediators which are either known to be produced by keratinocytes in culture or have recently been reported to be able to stimulate melanocytes in-vivo or in-vitro. The test procedure followed that previously described within Experiment 1. Briefly, melanocytes in complete melanocyte medium were combined individually with each mediator and incubated for identical periods of time at 37 C. Subsequently, each chemical mediator was evaluated for its effects upon living melanocytes. The results are provided by Table IV below.

Table IV

MEDIATOR EFFECT IN COMPLETE MELANOCYTE MEDIUM

MELANIN

GROWTH PER CELL

CONCENTRATION (% Control) DENDRICITY (% Control)

Basic Fibroblast Growth Factor 1 ng/ml 62 no effect 81 Interleukin 1² 1 ng/ml (alpha) 92 no effect 141

(beta) 97 no effect 103

Prostaglandin E2³ 1 ng/ml 108±7 no effect 116±12

1,2-hydroxyelcosa-tetraenoic acid 0.1 uM 102±13 no effect

Leukotriene B4 0.1 nM 98± 1 1 no effect 1 17 ±5

100 nM 98±11 no effect 121±6

Adenosine 3', 5'-cyclic monophosphate analogues

8-bromo-cAMP 100 uM 89 no effect 75 dibutrylcyclic-cAMP 100 uM 140 no effect 94

3-isobutyl-1-methylxanthine 100 uM 96 slight increase 194

Forskolin 10 uM 140 197

Phorbol-1,2-tetradecanoate-1,3-acetate 100 nM 69 large increase 115

1. Basic FGF largely substitutes for BHE in the culture system.

2. Interleukin 1 alpha and beta had no effect on any property either in the presence or absence of indomethacin, a factor known for potential responsiveness of interleukin in some cell types [Libby et al., J. Clin. Invest. 81:487-494 (1988)].

3. Prostaglandin E2 is the major prostanoid produced by keratinocytes. The addition of indomethacin directly to melanocytes did not effect melanocyte growth of melanization. Furthermore, indomethacin did not inhibit the ability of keratinocytes to release the active species, suggesting that other prostanoids are not involved.

4. In cells cultured in the absence of choleragen.

Experiment 4: Discrimination Ability Of Human Melanocytes

This experimental series demonstrates the selectivity and discrimination ability of human melanocytes with respect to specific mitogens and cell mediators previously reported to be active with melanocytes from animal origins, in particular, the

S91 mouse melanoma cell line. Two individual experimental series were performed to empirically demonstrate the biological activity and effects of specific agents. The agents were melanocyte stimulating hormone (hereinafter "MSH") reported to be active in the literature; and melanocyte growth factor (hereinafter "MGF") also reported to be biologically active in the scientific literature. Each of these agents, MSH and MGF we r e in d i vi d ua l l y a d d e d t o comp l e t e me lan o c y t e medium formulated as previously described herein at varying concentrations; and this mixture combined with human melanocytes obtained and cultured as previously described within Experiment 1. The results are graphically illustrated by Figs. 7 and 8 respectively.

Fig. 7 illustrates the MSH effect on human melanocytes at concentrations ranging from 10^-8-10^-14 M and reveals MSH to be effectively inactive for cultured human melanocytes. Clearly, the empirical data reveal a cellular growth and a melanin production which is substantially equivalent to that for control cells. Similarly, Fig. 8 demonstrates the effect of MGF on proliferating human melanocytes at concentration ranges from 0-100 ug/ml. Clearly, increase in concentrations of MGF cause a substantial increase in human melanocyte concentration; this increase in melanocyte growth, however, Is directly coupled with a substantial decrease in melanin content per cell with increasing concentration of MGF. Accordingly, MGF is able to induce melanocyte proliferation but is clearly unable to cause meaningful increases in melanin content on a per cell basis.

Experiment 5: Effects Of Diacylglycerols

This experimental series investigates the biological effects of diacylglycerols (DAG) upon human melanocytes and reveals the effects of pretreatment of human melanocytes with specific agents prior to incubation with a DAG. A first experimental series was performed which utilized human melanocytes cultured in the manner previously described and combined these cells with the preferred DAG, 1-oleoyl-2-acetyl-glycerol, selected for its efficiency of intercalating into melanocyte membranes. The preferred DAG was combined with human melanocytes in complete melanocyte medium and incubated together for a period of 6 days at 37°C. The results are graphically illustrated by Figs. 9a and 9b which evidence a clear dose dependent response to increased melanin content at concentration levels ranging from 25 - 200 uM with no meaningful effects on melanocyte proliferation or growth. At an optimal concentration of 100 uM, this DAG produced an average of a 4 fold Increase in cellular melanin content per cell over the untreated control. It will also be noted that this DAG did not increase the dendricity of human melanocytes and did not meaningful effect human melanocyte growth.

A second experimental series was conducted in which the cultured human melanocytes were first pretreated with tetradecanoyl phorbol-13-acetate (hereinafter "TPA") at a concentration of 100 nM. Initially, the human melanocytes were obtained and grown as previously described within Experiment 1. Subsequently, the cultured human melanocytes were combined with 100 nM of TPA in melanocyte medium and incubated at 37 C for 24 hours. The DAG was then added and the culture incubated again for 6 days. The melanin content of the cells was then evaluated in accordance with the procedure as previously described herein. The results are graphically illustrated by Figs. 10 and 11 respectively.

TPA alone induced extreme dendricity and diacylgylcerol induced increased melanization. The combination was equally effective at melanization and the induction of dendricity. Such combinations would be able to increase both synthesis and pigment transfer.

Fig. 10 empirically demonstrates that neither the DAG analogue, nor the pretreatment with TPA, nor their combination has any substantial effect on human melanocyte proliferation. In comparison, Fig. 11 empirically demonstrates major and significant increases in melanin content per human melanocyte as a function of TPA pretreatment in amounts meaningfully greater than that of the chosen DAG analogue alone. Clearly, for maximum melanin production, it is most desirable to pretreat human melanocytes prior to administration of the chosen DAG analogue.

Experiment 6: Effects Of Diacylglycerol Analogues On Non-Human Melanocytes

This experimental series was performed to evaluate the effect of a DAG on melanocytes derived from animal sources, rather than humans. In this experimental series, murine S91 melanoma cells were grown and maintained in culture using conventional techniques [Friedmann and Gilchrest, J. Cell. Physiol. 133:88-94 (1987)]. The grown S91 melanoma cells were then combined with 100 uM of the preferred DAG, 1-oleoyl-2-acetyl-glycerol; or with 100 uM of 3-isobutyl-1-methylxanthine (IBMX). Each chemical agent was combined with the S91 melanoma cells in Dulbecco's Minimal Eagle's Medium containing 2% FBS at 37 C for 7 days. The results are graphically illustrated by Figs. 12 and 13 respectively.

Fig. 12 empirically demonstrates that the chosen DAG at concentrations ranging from 1-200 uM effectively failed to induce proliferation of S91 melanoma cells; the IBMX was similar in effect and also failed to produce any cellular growth. In comparison, Fig. 13 reveals major differences between the chosen DAG and IBMX to induce increases in melanin production. Clearly, IBMX at a concentration of 100 uM caused a major increase in melanin production in. S91 melanoma cells; in comparison, the chosen DAG consistently failed to induce any substantial increases in melanin production in comparison to controls. It is thus unequivocally demonstrated that the biological effects of DAG are selective and discriminatory in accordance with the source of origin for the melanocytes. It is incontrovertible that this DAG has no meaningful effect on S91 melanoma cells, cells derived from mice; on the other hand, administration of this DAG to human melanocytes clearly and unequivocally causes major substantial increases in melanin content without concommitant cell proliferation of human melanocytes.

The present invention is not to be restricted in form nor limited in scope except by the claims appended hereto.

Claims

What we claim is:

1. A method for inducing melanocyte proliferation, and method comprising the steps of: obtaining a preparation containing at least a fraction of the products generated and released by keratinocytes into the surrounding medium during in-vitro culture using a medium comprising, at least an isotonic salt solution of about neutral, pH value for minimal maintenance of grown keratinocytes in culture,

0 - 20 mg/ml albumin, 0 - 100 ug/ml inositol, and 0 - 50 ug/ml choline chloride; adding to said preparation at least one supplemental selected from the group consisting of a fibroblast growth factor, a blood serum, and a cyclic adenosine monophosphate mediation to form a supplemented preparation; and administering said supplemented preparation to living melanocytes.

2. A method for inducing melanocyte proliferation, said method comprising the steps of: obtaining a preparation containing at least a fraction of the products generated and released by keratinocytes into the surrounding medium during in-vitro culture using a medium comprising, essential amino acids, vitamins, nutrients, and salts for maintenance of keratinocyts in culture, 0 - 100 ug/ml insulin, 0 - 10 nM of an iodothyronine,

0 - 100 ug/ml transferrin, 0 - 15 M of a cortisone, and 0 - 100 ng/ml epidermal growth factor; adding to said preparation at least one supplement selected from the group consisting of a fibroblast growth factor, a blood serum, and a cyclic adenosine monophosphate mediator to form a supplemented preparation; and administering said supplemented preparation to living melanocytes.

3. The method as recited in claim 1 or 2 wherein said melanocytes are maintained in culture.

4. The method as recited in claim 1 or 2 wherein said melanocytes are within the skin of a living subject.

5. A method for increasing the melanin content of skin in-vivo, said method comprising the steps of: obtaining a preparation containing at least a fraction of the products generated and release by keratinocytes into the surrounding medium during in-vitro culture using a medium comprising, at least an isotonic salt solution of about neutral pH value for minimal maintenance of grown keratinocytes in culture,

0 - 20 mg/ml albumin, 0 - 100 ug/ml inositol, and

0 - 50 ug/ml choline chloride; adding to said preparation at least one supplement selected from the group consisting of a fibroblast growth factor, a blood serum, and a cyclic adenosine monophosphate mediator to form a supplemented preparation; and administering said supplemented preparation to living melanocytes.

6. A method for increasing the melanin content of skin in-vivo, said method comprising the steps of: obtaining a preparation containing at least a fraction of the products generated and released by keratinocytes into the surrounding medium during in-vitro culture using a medium comprising, essential amino acids, vitamins, nutrients, and salts for maintenance of keratinocytes in culture, 0 - 100 ug/ml insulin,

0 - 10 nM of an iodothyronine, 0 - 100 ug/ml transferrin, 0 - 15 M of a cortisone, and 0 - 100 ng/ml epidermal growth factor; adding to said preparation at least one supplement selected from the group consisting of a hypothalmic extract and a blood serum to form a supplemented preparation; and administering said supplemented preparation to the skin of a living subject.

7. A method for increasing the melanin content of melanocytes, said method comprising the steps of: obtaining a diacylglycerol; and adding said diacylglycerol to the melanocytes.

8. A method for increasing the melanin content of skin in-vivo, said method comprising the steps of: obtaining a diacylglycerol; and administering said diacylglycerol to the skin of a living subject.

9. The method as recited in claim 1, 2, 5, or 6 wherein said fibroblast growth factor supplement is basic fibroblast growth factor.

10. The method as recited in claim 1, 2, 5, or 6 wherein said fibroblast growth factor supplement is bovine hypothalmic extract.

11. The method as recited in claim 1, 2, 5, or 6 wherein said fibroblast growth factor supplement includes brain extracts, brain portion extracts, and acid fibroblast growth factor.

12. The method as recited in claim 1, 2, 5, or 6 wherein said blood serum supplement is fetal bovine serum.

13. The method as recited in claim 1, 2, 5, or 6 wherein said blood serum supplement is any serum preparation derived from the blood of an animal.

14. The method as recited in claim 1, 2, or 7 wherein said melanocytes are maintained in culture for laboratory use.

15. The method as recited in claim 1, 2, 5, 6, or 7 wherein said melanocytes are found in the skin and hair bulbs of living human patients.

16. The method as recited in claim 1, 2, 5, 6, or 7 wherein said melanocytes are found in the skin and hair bulbs of living animal subjects.

17. The method as recited In claim 1, 2, 5, 6, or 7 wherein said melanocytes are within skin tissue to be used as a skin graft.

18. A topical formulation for increasing the melanin content of skin in-vivo, said formulation comprising: a fluid carrier compatible with the skin of a living subject; and a supplemented preparation in admixture with said fluid carrier, said supplemented preparation containing at least one fraction of the products generated and released by keratinocytes into the surrounding medium during in-vitro culture using a culture medium comprising, at least an isotonic salt solution of about neutral pH value for minimal maintenance of grown keratinocytes in culture,

0 - 20 mg/ml albumin, 0 - 100 ug/ml isositol, 0 - 50 ug/ml choline chloride, said products being supplemented with at least one supplement selected from the group consisting of a fibroblast growth factor, a blood serum, and a cyclic adenosine monophosphate mediator.

19. A topical formulation for increasing the melanin content of skin in-vivo, said formulation comprising: a fluid carrier compatible with the skin of a living subject; and a supplemented preparation in admixture with said fluid carrier, said supplemented preparation containing at least one fraction of the products generated and released by keratinocytes into the surrounding medium during in-vitro culture using a medium comprising: essential amino acids, vitamins, nutrients, and salts for maintenance of keratinocytes in culture,

0 - 100 ug/ml insulin, 0 - 10 nM of an indothyronine, 0 - 100 ug/ml transferrin, 0 - 15 M of a cortisone, 0 - 100 ng/ml epidermal growth factor, said products being supplemented with at least one supplement selected from the group consisting of a fibroblast growth factor, a blood serum, and a cyclic adenosine monophosphate mediator.

20. A topical formulation for increasing the melanin content of the skin in-vivo, said formulation comprising: a fluid carrier compatible with the skin of a living subject; and a diacylglycerol.

21. The topical formulation as recited In claim 18, 19, or 20 wherein said formulation is used for tanning of the skin.

22. The topical formulation as recited in claim 18, 19, or 20 wherein said formulation is used for darkening of hair.

23. The topical formulation as recited in claim 18, 19, or 20 wherein said formulation is a therapeutic treatment for vitiligo.

24. The topical formulation as recited in claim 18, 19, or 20 wherein said formulation is used to prevent discoloration of animal hair counterparts.

25. The topical formulation as recited in claim 18, 19, or 20 wherein said formulation is used to darken skin grafted tissue.