MXPA99000515A - Methods to alter the growth and pigmentation of the hair through apoptosis in the folicular papilas, and compositions for the my - Google Patents

Abstract

The present invention utilizes serine proteases and their ability to induce programmed cell death and apoptosis in follicular papillae to affect changes in hair growth and pigmentation in mammals, compositions are also described that have an agent with a structure portion similar to a portion of the trypsin molecule, which allows the agent to induce programmed cell death and apoptosis in the same way as trypsin

Description

METHODS TO ALTER THE GROWTH AND PIGMENTATION OF THE HAIR THROUGH APOPTOSIS IN THE FOLICULAR PAPILAS, AND COMPOSITIONS FOR THE SAME RECIPROCAL REFERENCE TO RELATED REQUEST This application claims the benefit of United States Provisional Application No. 60 / 021,829, filed July 12, 1996, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION This invention relates to methods for producing programmed cell death and apoptosis in follicular papillae, and effective compositions for the same. More specifically, the present invention is directed to methods for changing the growth rate and pigmentation of hair by topical application of a protease or a molecule that affects a receptor-mediated signal transduction pathway.

BACKGROUND OF THE INVENTION A primary function of mammalian hair is to provide environmental protection. However, this function has been largely lost in humans, in whom hair is maintained or removed essentially for cosmetic purposes. A Many procedures are used to remove undesirable hair, including shaving, electrolysis, hair removal and injection of therapeutic antiandrogens. These conventional methods are not without drawbacks. Shaving, for example, can result in scratches and cuts on the surface of the skin, may leave a feeling of increased speed of resumption of hair growth, and can also leave an undesirable incipient beard. Although the ^^ Electrolysis can maintain an undesirable hair-free area for a prolonged period, the process is often costly and painful, and can result in the formation of a scar. Hair removal can not only cause pain and Discomfort, but often results in poor removal of short hair. Several inconvenient side effects, such as effects on muscularity, B often accompany the use of antiandrogens. In addition, with the exception of electrolysis, all these methods must be repeated frequently on a daily basis, creating an inconvenience for the user. For these reasons, chemical methods are required to retard hair growth. As described in the patent of E.U.A. No. 5,455,234 by Ahlu alia and others, the hair growth of the mammals can be altered by applying an inhibitor of the cysteine enzyme pathway to the skin, which will retard or stop the metabolic pathways for the speed and character of hair growth with which cysteine is associated. However, this inhibition of cysteine also causes a deficiency of this amino acid in the treated area. Similarly, in the U.S. Patent. No. 5,095,007 by Ahluwalia, Patents of E.U.A. Nos. 5,096,911 and 5,468,476 by Ahluwalia et al., And Patents of E.U.A. Nos. 5,132,293 and 5,143,925 by Shander et al., Enzyme inhibitors are used to alter hair growth. In each of these cases, a biochemical pathway was altered to inhibit the production of a final product. It would be convenient to provide a method to chemically affect the growth and pigmentation of the hair, which does not deprive the user of the necessary amino acids or cause inconvenient side effects for the same.

BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, a method has been found to affect changes in the growth and pigmentation of hair in mammals, which comprises, consists essentially of, or consists of: topically applying to the skin of a mammal, an effective amount of a topically active composition comprising a protease within a sufficient time regarding the induction of hair growth by telogenesis.

In another embodiment of the present invention, a method has been found for inducing programmed cell death and k apoptosis within a follicular papilla comprising, consisting essentially of, or consisting of: topically applying to the skin of a mammal a topically active agent which have a first portion that is similar to a second portion of a three-dimensional structure of trypsin, whereby the first portion and the second portion are capable of signal transduction mediated by receiver. In another embodiment of the present invention, there has been found a composition comprising, consisting essentially of, or consisting of: a) a topically active agent having a first portion that is similar in shape to a second portion of a three-dimensional structure of trypsin, whereby the first portion and the second portion are capable of performing signal transduction mediated by receptor; and b) a pharmaceutically or cosmetically acceptable vehicle. In yet another embodiment of the present invention, there has been found a composition comprising, consisting essentially of, or consisting of: a) a topically active agent comprising a protease capable of inducing apoptosis in a follicular papilla; and b) a pharmaceutical or cosmetically acceptable vehicle. The compositions and methods of this invention provide unique and convenient means to delay hair growth and pigmentation, inducing apoptosis in the follicular papillae.

BRIEF DESCRIPTION OF THE DRAWINGS The file of this patent contains at least one drawing made in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office after requesting and making the payment of the necessary fees. The invention will be better understood, and other advantages will be apparent, when reference is made to the following detailed description of the invention and the accompanying drawings, in which: Figures 1 (a) and 1 (c) are representations illustrating a view in cross-section of the skin of an untreated C57BL / 6 mouse labeled with fluorescent trypsin. Figure 1 (b) is a representation illustrating a cross-sectional view of the skin of a C57BL / 6 mouse 12 days after topical treatment with fluorescent trypsin. Figure 2 are color representations of the dorsal view of the skin of C57BL / 6 mice, some of which were treated with vehicle or trypsin (1%) immediately after induction of hair growth, taken: (a) days after the induction of hair growth; (b) 11 days after the induction of hair growth; and (c) .14 days after the induction of hair growth. In Figure 2 (a), the mouse on the left was treated, while the mouse on the right was not treated. In Figures 2 (b) and 2 (c), the mouse on the left was not treated, the mouse in the center was treated with the vehicle, and the mouse on the right was treated with trypsin. Figure 3A is a representation illustrating the histology of hair follicles of the skin of untreated mice at: (a) 4 days; (b) 5 days; (c) 8 days; and (d) 12 days after the induction of hair growth. Figure 3B is a representation illustrating the histology of the hair follicles of the skin of mice treated with trypsin a: (a) 4 days; (b) 5 days; (c) 6 days; (d, e) 8 days; and (f) 12 days after the induction of hair growth. Figure 4 (A) is a representation illustrating the cross-sectional view of tissue from skin TUNNELED from an untreated mouse at: (a) 1 day; (b) 2 days; and (c) 3 days after the induction of hair growth. Figure 4 (B) is a depiction illustrating the cross-sectional view of tissue from the TUNNEL-stained skin of a trypsin-treated mouse at: (a) 1 day; (b) 2 days; (c) 3 days; (d) 4 days; (e) 5 days after induction of hair growth ((a-e) were individual applications of trypsin); and (f) 8 days after induction of hair growth (daily applications of trypsin). Figure 5 is a representation illustrating the profile of the genetic expression of the skin of a mouse treated with trypsin and the skin of an untreated mouse during a delayed growth cycle of the hair, detected by polymer chain reaction-by transcription Reverse ("RT-PCR"). Figure 5 (A) illustrates mRNA levels during days 1 to 11 -10 of the delayed hair growth cycle. Figure 5 (B) illustrates mRNA levels at day 8 of the delayed hair growth cycle. The 25ng (A) and 250ng (B) RT-PCR products of total RNA are shown.

DETAILED DESCRIPTION OF THE INVENTION As used herein, "mammal" must mean any member "of the higher vertebrate animals comprising the Mammalia class", as defined in Webster's Medical Dictionary 407 (1986), and includes, but is not limited to, humans. As used herein, "(%, w / v)" must mean grams of a given component per 100 ml of the total composition. As used herein, "receptor" must include both intracellular and intracellular receptors. extracellular, and must mean those molecules capable of receiving and transducing a signal.

Topically active agents suitable for use in the composition of the present invention include proteases, compounds having a structure substantially similar to a portion of the three-dimensional structure of trypsin, or mixtures thereof. Preferred proteases are serine proteases that include, but are not limited to, trypsin, carboxypeptidase-Y, protease IV, subtilisin, or mixtures thereof. The protease of choice is trypsin. Although it is not desired to be limited by this theory, it is thought that proteases capable of retarding the growth and pigmentation of hair do so by altering the hair growth cycle by inducing programmed cell death and apoptosis within the follicular papillae, and that this alteration is probably induced by a receptor-mediated signal transduction. Preferably, the protease is present in an amount, based on the total volume of the composition of the present invention, of from about 0% (w / v) to about 5% (w / v), and more preferably around 0.01% (w / v) to approximately 1% (w / v). As shown by example 10 herein, it has been unexpectedly found that the proteolytic activity of a protease is not the only factor contributing to the induction of programmed cell death. Although it is not desired to be limited by any theory, it is thought that a receptor-mediated signal transduction event may also contribute to the induction of programmed cell death of follicular papillae, and that this type of agent could be related to a Structural component of the topically active agent. Therefore, a second suitable topically active agent includes compounds that possess a portion that is substantially similar to the portions of a three-dimensional structure of a trypsin molecule that affects this receptor-mediated signal transduction. As used herein, "substantially similar" I means that the compounds include a sufficient amount of a structure that behaves like a ligand that can occupy a receptor, can displace a ligand from the receptor, or can activate or inactivate a receptor by breaking up proteolytic, and thus contribute to the induction of programmed cell death. Examples of such compounds that possess portions that are substantially similar to the? structure of a portion of the three-dimensional trypsin include, but are not limited to, proteinasitically trypsin It is inactive, which is a form of trypsin that is unable to digest proteins such as collagen. Preferably, these compounds are present in the composition of the present invention in an amount based on the total volume • of the composition, from about 0% (w / v) to about 5% (w / v), and more preferably from about 0.01% (w / v) to about 1% (w / v).

If the assortment parameters of the topically active pharmaceutical or cosmetic agent so require, the topically active composition of the present invention may be preferably formed in addition to a pharmaceutical or cosmetically acceptable carrier capable of functioning as an assortment system to allow agent penetration. topically active in the hair follicle. Although any vehicle commercially available to supply the protease to the appropriate appendage of the skin, which in this case is the hair follicle, is suitable for use as the pharmaceutical or cosmetically acceptable vehicle, liposomes are preferred. Liposomes are more preferably non-ionic and are formed of a) glycerol dilaurate or glycerol distearate; b) compounds having the steroid-based structure present in cholesterol; and c) fatty acid ethers having from about 12 to about 18 carbon atoms, wherein the constituent compounds of the liposomes are in a ratio of about 53:10:22 to about 63:20:32, and preferably around 55:12:24 to about 61:18:30, respectively. Liposomes formed from glycerol dilaurate / cholesterol / polyoxyethylene stearyl ether 10 (GDL), are most preferred. Preferably, the liposomes are present in an amount, based on the total volume of the composition, of from about 10 mg / ml to about 100 mg / ml, and more preferably from about 25 mg / ml to about 50 mg / ml . A ratio of approximately 58:15:27 is most preferred. Suitable liposomes can preferably be prepared in accordance with the protocol described in example 2, although other methods commonly used in the art are also acceptable. The composition described above can be prepared by combining the desired components in a suitable container, and mixing them under ambient conditions in any of the conventional high-shear mixing media well known in the art for preparations of non-ionic liposomes, such as those described in * Niemiec et al., "Influence of Nonionic Liposomal Composition On topical Delivery of Peptide Drugs Into Pilosebacious Units; An In Vivo Study Using the Hamster Ear Model ", 12 Pharm. Res. 1184-88 (1995) ("Niemiec"), which is incorporated in his whole in the present as reference. In a preferred embodiment, can the pH of the topically active pharmaceutical or cosmetic composition be adjusted? with a known pH adjuster including, but not limited to, N-2-hydroxyethylpiperazino-N'-2-ethanesulfonic acid, available from, for example, Life Technologies, under the tradename "Hepes", or (hydroxymethyl) aminomethane, available from, for example, Life Technologies under the tradename "Tris", citric acid, and mixtures of them, to produce a pH of from about 2 to about 8 in the final composition.

In alternative embodiments, the topically active pharmaceutical or cosmetic composition may optionally be combined with other ingredients such as humectants, foaming agents, cosmetic adjuvants, antioxidants, surfactants, conditioners, fragrances, viscosifiers, pH regulating agents, preservatives and the like. , in an amount that does not destroy the liposomal structure to produce cosmetic or pharmaceutical products such as, but not limited to, shaving creams, shaving gels, shaving powders, chemical depilatory creams, and the like. In another embodiment of the present invention, a method has been found to affect changes in the growth and pigmentation of mammalian hair, consisting of in applying the topically active pharmaceutical or cosmetic composition to the surface of the animal's skin almost at the time of induction of hair growth. M The induction of hair growth from telogen follicles can be carried out by any method well known in the art including, but not limited to, shaving, waxing, chemical hair removal, and combinations thereof. Although the time at which the topically active pharmaceutical or cosmetic composition is applied to the skin, as well as Also the amount of time during which the composition remains on the skin may vary, the skilled artisan can easily become aware without undue experimentation, that the topically active composition is preferably applied to the surface of the skin immediately before, immediately after, or simultaneously, with the induction of hair growth from a state of telogenesis. As used herein, "immediately" should be defined as being within a period of about 1 hour. More preferably, the topically active pharmaceutical or cosmetic composition is applied simultaneously with, or immediately after, the induction of hair growth, and is left on the skin for a period sufficient to retard hair growth, and which is preferably at least about 5 minutes, and more preferably is at least about 15 minutes after the time of application. The topically active pharmaceutical or cosmetic composition should be applied in an amount effective to affect changes in the growth and pigmentation of mammalian hair. As used herein, "effective amount" must mean an amount sufficient to cover the region of the skin surface where a delay in the growth and pigmentation of the hair is desired. Preferably, the composition is applied to the surface of the skin so that, based on a square centimeter of skin surface, from about 2μl / cm2 to about 8μl / cm of topically active agent, it is present when a delayed growth and pigmentation of the hair. It has been unexpectedly found that when topically active agents, such as serine proteases, are applied topically to the skin of a mammal almost at the time of performing the induction of hair growth, achieving a significant delay in growth and pigmentation thereof for at least about 9 days. It is further thought that, since the hair growth cycle in humans is often slower than in mice, it is also likely that the hair growth delay in humans is considerably greater than 9 days. The invention described illustratively herein may be practiced suitably in the absence of any component, ingredient, or step that is not specifically described herein. Several examples are given below to better illustrate the nature of the invention and the manner of carrying it out. However, it should not be considered that the invention is limited to the details thereof.

EXAMPLES EXAMPLE 1 Depilation of test subjects in the mouse system Hair growth was induced in 12 female C57BL / 6 mice obtained from Charles River (Kingston, NY), which were 6 to 10 weeks old and were in the resting phase (telogenesis) of their respective cycle of hair growth, by waxing, the fur of each respective animal, in accordance with the procedure described in Stenn et al., "Glucocorticoid Effect on Hair Growth Initiation: A Reconsideration," 6 Skin Pharmacol, 125-134 (1993). As is well known in mice, the growth phase (anagenesis) begins synchronously in all hair follicles at the time of hair removal. As illustrated in table 1, the following observations were made at the induction site: TABLE 1 Observations on the induction site As shown in table 1, hair growth was visible several days after hair removal as the animal's pink skin began to darken. This is probably due to hair pigmentation on the shaft, since the C57BL / 6 mouse contained melanocytes only in the hair follicles and not in the epidermis. Hair growth from telogenesis, in 12 similar C57BL / 6 mice, was also induced by chemical hair removal with a hair remover available from Carter-Wallace Incorporated under the trade name "Nair" in an amount sufficient to moisten the back of each mouse. In chemical hair removal, the lower portion of the follicles remained intact, and the lower portions of the hair axes of the previous cycle remained intact in the dermis until their expulsion by the newly formed hairs. The growth pattern of the new hair growth cycle was identical to that described in Table 1. Since the hair growth cycle in murine not only varies between strains, but also between each of the animals, a sample was isolated skin of 2 cm by 1 cm of each mouse with scissors, was fixed with a buffered 10% formalin solution, having a pH of about 6.9 to 7.1 at 25 ° C, available from Stephens Scientific, and was formed then in a paraffin block in accordance with well-known procedures. The block was then cut with microtome, stained with H & E stain, and examined histologically to verify the phase of the hair growth cycle using procedures well known in the art. The histological representations obtained from this example are illustrated in Figure 3A. This example shows that the hair growth cycle for C57BL / 6 mice averaged approximately 25 days, and reports a similar time regulation of the hair follicle and shaft development regardless of the method used for hair removal.

EXAMPLE 2 Preparation of topically active compositions A sufficient quantity of lyophilized trypsin available from Sigma-Aldrich Corporation was mixed in a buffered aqueous solution of N-2-hydroxyethylpiperazino-N'-2-ethanesulfonic acid at 0.05 M, available from Life Technologies, under the trade name of "Hepes", so that the pH of the resulting solution was about 7.4, and the concentration of trypsin in the solution was about 2% (w / v). A volume of the resulting trypsin solution was then mixed with a volume of glycerol dilaurate liposomes (5%) / cholesterol / polyoxyethylene stearyl ether 10 in aqueous carrier, which was prepared by the methods described in Niemiec, to produce a Trypsin concentration at 1% (w / v) in the resulting topically active composition.

EXAMPLE 3 Assortment of trypsin in hair follicles 12 days after depilating the skin of a 6 cm by 2 cm section of 4 female C57BL / 6 mice from 7 to 8 weeks, available from Charles River (Kingston, NY), approximately 100 μL was applied thereto / mouse of the topically active composition of example 2. The trypsin used in this example was fluorescently labeled with a FluoReporter kit for protein labeling available from Molecular Probes, Inc. in accordance with its accompanying protocol (1997), and using a fluorescent brand of fluorescein 5 isothiocyanate (FITC) also available from Molecular Probes, Inc. .. Approximately one and four hours after the application of the treatment with fluorescent trypsin, a 2 cm by 1 cm sample of the surface of the skin of each mouse .Q was isolated and formed in a paraffin block, which was then cut with microtome, and examined histologically according to the procedure described in example 1. As shown in example 1, most of the Fluorescent marking was found inside the hair follicle. Mice were examined in the 1 hour interval (FIG.1 (c)), and the 4 hour interval (FIG.1 (a)) showed identical histological staining patterns, without additional skin penetration at the last point of time. This observation is suggested against a possible digestion of the nonspecific extracellular matrix by the protease, which would probably have shown a deeper penetration of the fluorescent dye in the stratum corneum at the last point of time. This example was repeated in 4 C57BL / 6 female mice of 7 to 8 weeks, but with the exception that the mice were also treated daily for 12 hours after induction with trypsin treatment at 1% (w / v) of Example 2. Four hours after application of the treatment with fluorescent trypsin on the 12th day after induction, the skin of the mice was analyzed using similar histological methods. As illustrated in Fig. Ib, no significant change was observed in the trypsin assortment pathway in the hair follicles of the treated skins. However, minimal staining in the outer portion of the stratum corneum of skins treated with trypsin indicated some loss of barrier integrity. This example shows that the application of a topically active composition containing trypsin to the surface of the skin of anagenic animals, resulted in the assortment of trypsin mainly towards the hair follicle, after its short and long term use.

EXAMPLE 4 Delays in hair growth and pigmentation by trypsin An individual topical application of the topically active composition prepared in Example 2 was applied to 12 C57BL / 6 female mice for 7 to 8 weeks, immediately after induction of hair growth in accordance with the method described in Example 3.

As shown in Figure 2, a single application of trypsin applied after the induction of hair growth delayed the growth and pigmentation thereof. The darker skin color indicated a more progressive stage of the hair growth cycle, which appears before the hair axes are visible. The untreated controls and the controls treated with liposomes (vehicle) showed dark skin 7 to 8 days after the induction of hair growth (Figure 2a, left, treated, right, untreated), while their hair axes they were visible at 11 to 13 days (figure 2b, left, untreated, center, vehicle, right, treated). In contrast, the skin of animals treated with trypsin remained pink until day 8 (Figure 2a, left mouse); it was slightly darker pink on day 11 (Figure 2b, right mouse); and it was gray around day 14 (figure 2c, • right mouse). Although the hair axes of the animals treated with trypsin were first visible on days 15 to 17, these hair axes were of reduced quality and were not as thick as in the control; however, within about another 4 to 7 days, these hair axes were almost indistinguishable from those in the controls in all features, except in their length. This example shows that trypsin was effective in delaying hair growth and pigmentation.

EXAMPLE 5 Histology of hair follicles treated with trypsin A topically active composition as prepared in Example 2 was applied to the skin surface of 3 C57BL / 6 female mice for 7 to 8 weeks, after induction of hair growth, in accordance with the method described in the example 3. Samples of 1 cm by 2 cm were obtained from the skins of untreated, vehicle-treated mice (not shown), and treated with trypsin, stained with H & E, and then examined histologically by the procedure described in example 1 As illustrated in figure 3, the histological analysis of the stained samples showed that the development of the hair follicles in the mice treated with trypsin was delayed significantly. More specifically, the characteristic stratified structure, expanded follicular papillae and new pigmentation of the hair were first observed several days after the date of the first observation of said characteristics in the untreated and vehicle-treated controls. In addition, skin follicles from mice treated with trypsin showed a dilated infundibulum, as best illustrated in Figure 3B (ae), which was not present in the untreated and vehicle-treated mice, as shown in FIG. Figure 3A (ab). This observation supports the fact that treatment with the trypsin-containing composition also resulted in hair having a reduced quality, ie thinner and scarcer. At 7 to 8 days after treatment with trypsin, the lower part of some of the treated follicles began to form a stratified epithelial structure, as illustrated in Figure 3B (de), but the upper part exhibited an empty structure, as it is best shown in Figure 3B (d). About 11 to 12 days after treatment with trypsin, most follicles treated matured, but showed reduced pigmentation and shorter shaft length, which resembled the characteristics of untreated follicles at 4 to 5 days after induction (compare Figure 3A (d) with Figure 3B (f)). This example also showed by histological analysis that the topical application of trypsin to the skin of mice induced significantly delayed growth and pigmentation of the hair.

EXAMPLE 6 Induction of apoptosis by trypsin in the follicular papillae A topically active composition, as prepared in Example 2, was applied to the skin surface of 3 C57BL / 6 female mice for 7 to 8 weeks, after induction of hair growth in accordance with the method described in example 3 Samples of 1 cm x 2 cm were obtained from the skins of untreated mice and treated with trypsin by the | procedure described in example 1, and then they were analyzed by an end marking procedure ("TUNNEL") with cut by dUTP-biotin mediated by TdT, as described in Gavrieli et al., "Identification of Programmed Cell Death in situ Via Specific Labelling of Nuclear DNA Fragmentation ", 119 Jl. Cell Biology 493-501 (1992) ("Gavrieli"). During this procedure, sections of skin LO prepared with paraffin were stained using an "Apop TagTM Plus in situ apoptosis detection kit" available from Oncor Inc., as specified in the protocol of the apoptosis detection kit. Apop Tag TM Plus in situ "by Oncor, Inc. (Feb. 1995), which is based on the labeling of 15 fragmented DNA ends, as described in Gavrieli. Figure 4 shows a histological analysis, where the dye has a peroxidase end point (coffee) and a > counter-coloration of methyl green. In the same way, paraffin sections 20 suitable for TUNNEL staining were prepared from the untreated skins of mice. These sections were stained and analyzed histologically, and their resulting representations are provided in Figure 4A (a-c). As illustrated in FIG. 4, samples stained with TUNNEL defined apoptotic cells by morphology (condensed or fragmented nuclei and cytoplasm, or apoptotic bodies) and by the color of their dye (DNA fragmented within condensed nuclei). it was dyed brown). More specifically, Figure 4B (a-e) illustrated 5 that apoptotic bodies were detected within the follicular papillae treated during the first week after the induction of hair growth. However, no other portion of the follicle, epidermis or dermis was affected by trypsin treatment. In contrast, a minimal level of apoptosis was occasionally detected in anagen follicles • untreated early, as illustrated in the untreated sample stained with TUNNEL of Figure 4A (a-c); however, apoptosis always occurred in the isthmus of the follicle, and thus well above the follicular papilla. Most follicles without treated did not show any cell death. This example showed that the application of trypsin to the depilated skin resulted in an increase in the apoptotic figures within the follicular papillae of the follicles treated with trypsin, with respect to that of untreated controls. or treated with vehicle.

EXAMPLE 7 Changes in gene expression induced by trypsin during the hair growth cycle A topically active composition as prepared in Example 2 was applied to the surface of the skin of six female C57BL / 6 mice for 7 to 8 weeks, after induction of hair growth in accordance with the method described in the example 3. At the time intervals indicated in Figure 5, the skins of untreated mice and mice treated with trypsin were obtained as described in example 1, and then their RNA molecules were extracted using reagent "RNA Stat-60"available from Tel-Test" B "Incorporated, as described in Chomczymski," Single Step Method of RNA Isolation By Acid Guanidinium Thiocyanate-phenol-chloroform extraction ", 162 Anal. Biochem. 156-59 (1987) .A sufficient quantity of RNAse-free DNase available from Promega Corporation under the trade name "RNase-free DNase RQ1" was then added to the RNA extracted from each mouse, so that each respective product contained 200 ng of DNase-treated RNA using the procedure described in "RNase-free DNase", protocol published by Promega Corporation (May 1995). The resulting 200 ng of RNAse treated with DNase were transcribed in inverted form ("RT") by the procedure described in "Superscript II Reverse Transcriptase", a protocol published by Gibco-BRL (now Life Techonologies Incorporated) (April 1992), using | random hexamers such as random primers that are commercially available from Life Technologies Incorporated. The resulting RT products were then amplified by polymerase chain reaction ("PCR") using approximately 0.5 units (per 100 μl reaction) of a thermostable DNA polymerase that is available commercially from Perkin-Elmer-Cetus Corporation under the trade name "Taq polymerase", and approximately 0.1 μmoles / reaction of gene-specific primers, available from Clontech Laboratories, Inc. ("Clontech"), or designed as described in table 2 of according to the procedures described in the protocol accompanying the Clontech primers for glyceraldehyde-3-phosphtho-dehydrogenase (G3PDH), transcription factors and , mouse cytokines, or as described in table 2. Table 2 illustrates some of the DNA primers used, the amount of MgCl2 that is required for the PCR reaction, and the length of the PCR cycle.

TABLE 2 DNA primers used in the RT-PCR test *** TABLE 2 (CONTINUED) The PCR products were then analyzed by 2% agarose gels / ethidium bromide, and analyzed according to methods well known in the art for comparing the expression level of certain genes during the hair growth cycle in untreated mice and treated with trypsin. When necessary for better visualization, the resulting PCR products were precipitated with ethanol in accordance with well-known procedures. When initiators were used for G3PDH, only 10% of the PCR reaction products were used. A sample of RNA 5 from the skin of a C57BL / 6 female mouse of 7 or 8 weeks, and which was not transcribed in reverse, was used as a negative control for each PCR amplification. A sample of RNA from the skin of a six-month-old female C57B1 / 6 mouse that had an unsynchronized hair growth cycle was used as positive control when there were no commercial positive controls. The migration of the RT-PCR products on the gels was always identical to that of the positive controls, and to that of the reported amplifier sizes. 15 The relative quality of each reaction product of The respective RT-PCR was then compared by analyzing the level of G3PDH mRNA, a "domestic" gene, in each respective product.

W. As illustrated in figure 5, it was found that the expression of the G3PDH gene is similar at all time points examined, which thus allowed the comparison of the relative levels of gene expression for the desired genes. To verify that the delay in follicular development was not the result of irritation or nonspecific inflammatory response, mRNA levels were analyzed. genes that are typically overregulated during such situations. After examining the gene mRNA levels of the RT-PCR products, it was found that there was no change in mRNA levels of IL-6, IL-10 and GM-CSF, a slight upregulation of FNTalpha, a down-regulation of FNTß and FNT-RI, and moderate down regulation in macrophage-inducible protein (MIP), as illustrated in Table 3.

TABLE 3 Patterns of genetic expression TABLE 3 (CONTINUED) * Note that the RT-PCR test is only semiquantitative. The comparisons are valid only for each amplified sequence within the cycles, of different hair growth, and not between different genes.

No detectable expression + A strong band + / - A very weak band ++ A stronger band +/- A weak band +++ A very strong band.

The profile of gene expression in Table 3 is suggested against an inflammatory reaction or a response to skin irritation. Moderate upregulation was observed at the level of IL-lalfa mRNA (Figure 5b and Table 3), a gene associated with the inhibition of hair growth in culture. Since the induction of IL-lalfa could have also resulted from the loss of epidermal barrier function, the barrier integrity was analyzed by measuring transepidermal water loss (TEWL). The TEWL was measured with an evaporimeter "Evaporimeter EPI", available from Servomed AB, first normalizing the evaporimeter to ambient humidity, and then placing the probe on the dorsal skin of the test subject. The results showed only a moderate increase in the TEWL, which did not correlate with the trypsin concentration, as shown in Table 4.

TABLE 4 TEWL in skins treated with trypsin Therefore, it is likely that any upregulation of IL-lalfa in the RT-PCR product, as illustrated in Table 3 in Figure 5, is associated with the inhibition of hair growth. As illustrated in Figures 5a-b and Table 3, a stronger increase in mRNA levels demonstrated for 1L-Iß and IFNgamma was demonstrated, which are known to be up-regulated in Alopecia Areata, and which are associated with inhibition. of hair growth. This further suggested that the only genes upregulated by trypsin and associated with the inflammatory process were those associated with the | inhibition of hair growth. As illustrated in Table 3, a slight reduction in mRNA levels of c-myb, s-myc and c-fos was detected, while the level of c-jun was slightly increased over the period of delay . Collagenase, an enzyme regulated by an AP-1 response element, was also slightly reduced. This observation of a ^ The general light reduction in mRNA levels of several genes also reflected the general delay in the growth of skin hair treated with trypsin. As illustrated in Figure 5A of Table 3, tyrosinase, a key enzyme for hair pigmentation, was subtracted during the delay in the hair growth period, while its mRNA level increased as the follicles began to overcome the delay. POMC, the forerunner W of melanocyte-stimulating hormone of melanogenous peptides (MSH), was moderately subdued throughout the period of the delayed, as shown in Figure 5A and Table 3. The down-regulation of these two genes indicated that trypsin also affected the regulation of hair pigmentation. This example showed that perturbation of the hair growth cycle by trypsin can be understood by examining the expression pattern of a series of genes throughout the delay period. The changes induced by trypsin in mRNA levels throughout the hair growth cycle were clearly demonstrated, indicating a regulatory function for trypsin in hair growth and pigmentation. In addition, this example also reflected the specific nature of the topical application of trypsin on the genes for hair growth and pigmentation.

EXAMPLE 8 Other serine proteases that affect hair growth A topically active composition as prepared in Example 2 was applied to the skin surface of C57BL / 6 female mice for 7 to 8 weeks, after induction of hair growth in accordance with the method described in Example 3. The procedure of Example 3 was repeated, but replacing the trypsin with carboxypeptidase-Y, protease IV and subtilisin, respectively. After 8 days after the treatment, the skin of each respective mouse was examined using a Minolta model CR300 chromometer to compare their respective skin colors. It was observed that trypsin, a member of the endopeptidase family that cuts a protein on the C side of arginine and lysine, induced the longest delay in hair growth. In contrast, carboxypeptidase-Y, which hydrolyzes amino acids L at the C-terminus of proteins, and protease IV, a member of the nonspecific endopeptidase family that cuts 56% of peptide bonds at neutral pH, had only one effect of minimal delay, whereas subtilísin, a member of the nonspecific peptidase family that cleaves the peptide bonds at alkaline pH, only slightly increased the hair growth rate. The results of this example are illustrated in Table 5.

TABLE 5 Color measurements of skins treated with serine protease (The scale L: 0 = black and 100 = white, is a measure of the pigmentation of the hair observed from the surface of the skin).

As illustrated in Table 5, the hair follicles treated with trypsin were the least developed, and seemed to impart the lightest color to the skin. This example suggests that the appearance of skin color is related to hair development in the hair follicle. Since the results obtained from the skins treated with trypsin showed that the trypsin is relatively superior to delay the growth and pigmentation of the hair, this example supports the opinion that the example of trypsin on hair growth and pigmentation it may not be exclusively the result of a non-specific proteolytic digestion, but rather may be the result of a specific activity induced by trypsin, which causes the activation of the programmed cell death pathway.

EXAMPLE 9 Effect of trypsin on an early step in the induction of hair growth A topically active composition as prepared in Example 2 was applied to the skin surface of 3 C57BL / 6 female mice for 7 to 8 weeks, after induction of hair growth, in accordance with the method described in FIG. example 3. This procedure was repeated in nine other mice, but the treatment was applied 2, 3 or 7 times per week, respectively.

After observing the skin color of the mice for 8 days, the skin of the treated mice was clearer than that of the untreated controls. This suggested »Furthermore, the hair follicles treated with trypsin were the least developed, and they seemed to impart the lightest color to the skin. Samples of 1 cm x 2 cm were obtained from the skins of the treated mice respectively, stained with H &E, and examined histologically by the procedure described in example 1. The histological analysis of the stained samples supports the fact that that additional daily treatments of animals with trypsin did not prolong the delay in hair growth. This indicated that the induction of cell death by a serine protease resulted from a specific and timely event. To determine the time regulation of this specific event, a topically active composition, such as W- described in Example 2, was applied to the skin surface of C57BL / 6 female mice for 7 to 8 weeks, after 20 days. induction of hair growth in accordance with the method described in example 3, except that the treatment was applied in several time intervals, as indicated in Table 6. As illustrated in Table 6, the color chromometric measurements of the skin obtained in accordance with The method described in Example 8, showed an increase in darkness (more pigment, less delay) that correlated with the prolonged time between the induction of hair growth and the application of trypsin. Mice treated immediately after induction of hair growth showed the longest delay in hair growth and pigmentation. Mice treated 2 and 4 hours after the induction of hair growth, also exhibited a delay, but a progressively shorter hair growth cycle. Mice treated 6 or more hours after depilation showed no delay in hair growth and were indistinguishable from the untreated control.

TABLE 6 Measurements of skin color treated with trypsin ** TABLE 6 (CONTINUED) ** The skin samples were tested 8 days after waxing. This example shows that the application of trypsin to the skin is effective in delaying the growth and pigmentation of the hair when applied within about 4 hours, and preferably immediately after the induction of hair growth.

EXAMPLE 10 The effect of trypsin may involve a receptor-mediated pathway A topically active composition, as prepared in Example 2, was applied to the surface of the skin of female C57BL / 6 mice for 7 to 8 weeks, after induction of hair growth in accordance with the method described in the example 3, with the exception that the trypsin concentration was varied between 1% (w / v) and 0.01% (w / v) (4xl0 ~ 4M -4xlO_6M). At 9 days after depilation, the mice were analyzed colorimetrically by the procedure described in example 9. As illustrated in table 7, the results of the colorimetric analysis showed an increase in brightness (L, more white, less pigment). ) that correlated with a decrease in trypsin concentration and with an increase in the delay of the hair growth cycle.

TABLE 7 Brilliance of the skin, in skins treated with trypsin ** L scale: O = black, 100 = white. ** Samples were analyzed 9 days after depilation.

This example suggests the presence of a receptor mediated mechanism that includes desensitization with higher doses.

EXAMPLE 11 The proteolytic activity of trypsin is unnecessary to retard hair growth After the composition of Example 2 was incubated at room temperature for 48 hours, its proteolytic activity was analyzed by serine protease activity detection equipment available from PanVera Corporation, which showed that the preparation had no proteolytic activity detectable The inactive composition was applied to the skin surface of 3 C57BL / 6 female mice for 7 to 8 weeks, after induction of hair growth in accordance with the method described in example 10. At 9 days after After depilation, the mice were analyzed colorimetrically by the procedure described in Example 8. As illustrated in Table 7, it was evident that the lack of proteolytic activity of trypsin did not reduce the activity of this preparation to retard hair growth. This example was repeated, except that the incubated trypsin was replaced with boiled trypsin for 10 minutes at a temperature of 100 ° C. The results showed that the use of boiled trypsin, which was also proteolytically inactive, had no effect of any delay on hair growth. The boiling, which denatures the three-dimensional structure of trypsin, suggested that the three-dimensional structure of trypsin, and not only its proteolytic activity, contributed to the effect of retardation on the hair growth cycle. Regardless of whether trypsin blocked a survival signal or activated a death receptor to induce this process, this example suggests that the configuration of the trypsin molecule might also have been responsible for the induction of programmed cell death and apoptosis in the follicular papillae EXAMPLE 12 Use of a composition containing trypsin Liposomes of glycerol dilaurate / cholesterol / polyoxyethylene stearyl ether 10 were prepared, according to the procedure described in Niemiec, where the constituent compounds of the liposomes are in a ratio of about 58:15:27, respectively. After cooling the resulting liposomes to a temperature of about 40 ° C, the liposomes were added with normal mixing to an amount of a thickening agent formed of polyacrylamide (e) C13-C14 isoparaffin (and) laureth-7, available from from Seppic, Inc., under the trade name of "SEPIGEL 305", so that the amount of the thickening agent in the final composition is about 1-3% (w / v).

A sufficient amount of trypsin was then added to produce a 1% (w / v) trypsin composition. This composition is suitable for immediate topical application. The resulting product possesses characteristics and properties of an effective shaving gel.

Claims (3)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A method for affecting changes in hair growth and pigmentation in mammals, characterized in that it comprises: topically applying to the skin of a mammal, an effective amount of a topically active composition comprising a protease within a sufficient time with respect to induction of hair growth by telogenesis.
2. 2. The method according to claim 1, further characterized in that the protease is a serine protease.
3. 3. The method according to claim 2, further characterized in that the protease is selected from trypsin, carboxypeptidase-Y, protease IV, subtilisin, or mixtures thereof. . - The method according to claim 3, further characterized in that the protease is trypsin. 5. - The method according to claim 4, further characterized in that the protease is present in an amount, based on the total volume of the topically active composition, from about 0% (w / v) to 5% (p / v). 6. The method according to claim 5, further characterized in that the protease is present in an amount, based on the total volume of the topically active composition, from about 0.01% (w / v) to about 1% (p. / v). 7. The method according to claim 1, further characterized in that one of said changes is a delay in the growth and pigmentation of the hair. 8. The method according to claim 1, further characterized in that said topically active composition further comprises a pharmaceutical or cosmetically acceptable vehicle. 9. The method according to claim 8, ^^ further characterized in that said pharmaceutical or cosmetically acceptable carrier is a liposome, or mixture thereof. 10.- The method according to the claim 9, further characterized in that said liposome is non-ionic. 15 11.- The method according to the claim 10, further characterized in that said liposome is formed of: a) glycerol dilaurate, glycerol distearate, or a mixture thereof; b) a compound having a steroid-based structure as found in cholesterol, or a mixture thereof; and c) a fatty acid ether having from about 12 to about 18 carbon atoms, or a mixture thereof. 12. - The method according to claim 10, further characterized in that said liposome is formed of: a) glycerol dilaurate; b) cholesterol; and c) polyoxyethylene 10 stearyl ether. 13. - The method according to claim 11, further characterized in that the components of said liposome are present in a ratio of about 53:10:22 to about 63:20:32, respectively. 5 1. - The method of compliance with the claim 8, further characterized in that said pharmaceutical or cosmetically acceptable carrier is present in an amount, based on the total volume of said topically active composition, of from about 0 mg / ml to about 100 mg / ml. 15. The method according to claim iw 1, further characterized in that the induction of hair growth by telogenesis is carried out by a method comprising shaving, electrolysis, waxing, chemical depilation, or combinations thereof. . 15 16.- The method according to the claim 1, further characterized in that said time is immediately before, immediately after or simultaneously, with said induction of hair growth from telogenic hair follicles. 20 17.- The method according to the claim 16, further characterized in that said time is simultaneously with, or immediately following, the induction of hair growth. 18.- The method of compliance with the claim 25 1, further characterized in that the composition further comprises surfactants, humectants, conditioners, fragrances, colorants, or mixtures thereof. 19. The method according to claim 18, further characterized in that said composition is in the form of shaving creams, shaving gels, shaving powders, chemical depilatory creams, or other products whose purpose is to facilitate hair removal or really remove the same, or combinations thereof. 20. The method according to claim 1, further comprising leaving said composition on said skin for a period sufficient to affect said changes. 21. The method according to claim 20, further characterized in that said period is at least about 15 minutes. 22. The method according to claim 1, further comprising mixing said protease with a pH regulator comprising N-2-hydroxyethylpiperazino-N'-2-ethanesulfonic acid, (hydroxymethyl) aminomethane, or mixtures thereof. 23. - A method for inducing programmed cell death and apoptosis within a follicular papilla of a mammal, comprising: topically applying to the skin of the mammal, a topically active agent having a first portion that is similar to a second portion of a three-dimensional structure of trypsin, whereby said first portion and said second portion are capable of performing signal transduction mediated by receptor. 24. - The method according to claim 23, further characterized in that said agent is trypsin, proteolytically inactive trypsin, or combinations thereof. 25. A composition, characterized in that it comprises: a) a topically active agent having a first portion that is similar to a second portion of a three-dimensional structure of trypsin, whereby said first portion and said second portion are capable of Perform receptor-mediated signal transduction; and b) a pharmaceutical or cosmetically acceptable vehicle. 26. The composition according to claim 25, further characterized in that the topically active agent is trypsin, proteolytically inactive trypsin, or combinations thereof. 27. The composition according to claim 25, further characterized in that the topically active agent is present in an amount, based on the total volume of the composition, of about 0% (w / v) to about 5% ( p / v). 28. The composition according to claim 25, further characterized in that the topically active agent is present in an amount, based on the total volume of the composition, of about 0.01% (w / v) to about 1% ( p / v). 29. The composition according to claim 25, further characterized in that the pharmaceutical or cosmetically acceptable carrier is a liposome 30.- The composition according to claim 29, further characterized in that said liposome is nonionic. 31. The composition according to claim 30, further characterized in that said liposome is formed of: a) glycerol dilaurate, glycerol distearate, or a mixture thereof, - b) a compound having a steroid-based structure as found in cholesterol, or a mixture thereof; and c) a fatty acid ether having from about 12 to about 18 carbon atoms, or a mixture thereof. 32. The composition according to claim 30, further characterized in that said liposome is formed of: a) glycerol dilaurate; b) cholesterol; and c) polyoxyethylene stearyl ether 10. The composition according to claim 31, further characterized in that the components of said liposome are present in a ratio of about 53:10:22 to about 63:20:32, respectively . 34. The composition according to claim 25, further characterized in that said pharmaceutical or cosmetically acceptable carrier is present in an amount, based on the total volume of said topically active composition, of from about 0 mg / ml to about 100 mg / ml. 35. The composition according to claim 25, further characterized in that it comprises surfactants, humectants, foaming agents, cosmetic adjuvants, pH regulating agents, preservatives, antioxidants, conditioners, fragrances, dyes, or mixtures thereof. 36.- The composition according to claim 35, further characterized in that said composition is in the form of shaving creams, shaving gels, shaving powders, chemical depilatory creams, other products whose purpose is to facilitate hair removal or actually remove the same, or combinations thereof. 37.- A composition, further characterized in that it comprises: a) a topically active agent having a first portion that is similar to a second portion of a three-dimensional structure of trypsin, whereby said first portion and said second portion are capable to perform receptor-mediated signal transduction; and b) a pharmaceutical or cosmetically acceptable vehicle. 38.- The composition according to claim 37, further characterized in that it comprises surfactants, humectants, foaming agents, cosmetic adjuvants, pH regulating agents, preservatives, antioxidants, conditioners, fragrances, dyes, or mixtures thereof. 39. The composition according to claim 37, further characterized in that said protease is a serine protease. 40. The composition according to claim 39, further characterized in that said protease is selected from trypsin, carboxypeptidase-Y, protease IV, subtilisin, or mixtures thereof. 41. The composition according to claim 37, further characterized in that the topically active agent is present in an amount, based on the total volume of the composition, of about 0% (w / v) to about 5% ( p / v). 42. The composition according to claim 37, further characterized in that the topically active agent is present in an amount, based on the total volume of the composition, of about 0.01% (w / v) to about 1% ( p / v). 43. The composition according to claim 37, further characterized in that the pharmaceutical or cosmetically acceptable carrier is a liposome 44. The composition according to claim 43, further characterized in that said liposome is nonionic. 45. The composition according to claim 43, further characterized in that said liposome is formed of: a) glycerol dilaurate, glycerol distearate, or a mixture thereof; b) a compound having a steroid-based structure such as is present in cholesterol, or a mixture thereof; and c) a fatty acid ether having from about 12 to about 18 carbon atoms, or a mixture thereof. 46. The composition according to claim 43, further characterized in that said liposome is formed of: a) glycerol dilaurate; b) cholesterol; and c) polyoxyethylene stearyl ether 10. The composition according to claim 45, further characterized in that the components of said liposome are present in a ratio of about 53:10:22 to about 63:20:32, respectively . 48. The composition according to claim 37, further characterized in that said pharmaceutical or cosmetically acceptable carrier is present in an amount, based on the total volume of said topically active composition, of from about 0 mg / ml to about 100 mg / ml. 49. - The composition according to claim 37, further characterized in that said composition is in the form of shaving creams, shaving gels, shaving powders, chemical depilatory creams, other products whose purpose is to facilitate hair removal or actually remove the same , or combinations thereof.