MXPA98004707A - Composition that stimulates the growth of cabe - Google Patents

Abstract

The present invention relates to compositions useful in the stimulation of hair growth. These compositions comprise a conditioned medium obtained from a cell culture of human mononuclear cells or a conditioned or extracted medium obtained from a cell culture of normal or transformed mammalian cells or cancer cells.

Description

COMPOSITION THAT STIMULATES HAIR GROWTH FIELD OF THE INVENTION The present invention relates to factors that are capable of stimulating hair growth in a subject.

BACKGROUND OF THE INVENTION Physiology of Hair Growth. From the early fetal development, and throughout the life of animals including humans, hair follicles (H Fs) go through many cycles of degeneration and regrowth. In the growth of human hair, during the neonatal period and in the course of adolescence, the hair follicle on the scalp over time becomes thicker as the follicles gradually enlarge with each new cycle (DeVillez RL: In: Cu rrent Concept, A Scope Publication, by Upjohn Co., Kalamazoo, Ml, 1986; pp. 4-27; Takashima I, Kawagihi I: In: All K, and others (eds): Biology and Disease of Hair, Baltimore, University Park Press, 1976, pp. 457-471). Hair follicles are composed of many types of cells, such as epidermal and mesenchymal cells. The cells '' mesenchymes are known to play a role as 'inducing organizers' of fetal and postnatal follicles (Chase H B. Physiol Rev. 1954; 34: 1 13-126). The follicular germ cells, which are bulbar matrix cells, are responsible for the great mitotic proliferation in the hair follicle. During development, the cells in the matrix proliferate with upward migration and differentiation into internal protection cells and external protection of the hair matrix. The hair matrix group, located in the central axis of the hair follicle, also differentiates in the cells that make up the marrow, hair cortex, and hair cuticle. These cells show a continuous upward migration with keratinization of the cortical and cuticle cells that are essential for the hair-making process in a growing follicle (Chase H B. Physiol Rev. 1954; 34: 1 13-126; Hashimoto K Br J Dermatol 1971; 83: 167- 1 76). The hair cycles are divided into three stages: 1) Anagen, which is the active growth phase of the hair follicle cycle, 2) Catagen, a regressive stage, 3) Telogen, resting stage. (DeVillez RL: In: Current Concept, a Scope publication by Upjohn Co., Kalamazoo, M l, 1986; pp. 4-27; Takashima I, Kawagishi I: In: All K, and others (eds): Biology and Disease of Hair. Baltimore, Univ. Park Press, 1976: pp. 457-471; Chase H B. Physiol Rev. 1954; 34: 1 13-126; Kligman AM. J I nvest Dermatol 1959; 33: 307-316). The duration ^ Relative of these stages varies with the individual's age, hormonal factors, nutritional and health status, as well as genetics.

Growth factors responsible for the stimulation of hair growth have not yet been elucidated. Of the 100,000 to 150,000 hairs on the scalp of a human adult, approximately 90% are anagen, with the remaining 10% in the telogen phase. Approximately 50 to 100 grouped hairs are protected every day. The growth rates of human hair vary slightly depending on the body region, with 0.44 m / day at the apex of the scalp after 0.27 mm / day for the beard or body hair. (Moretti G, Rampini E, Reborah A: Int J Dermatol 15: 277-285, 1976, Orentriech N, Durr NP: Clin Plast Surg 9: 197-205, 1982, Katz M, Wheeler KE, Radowsky M. and others Med. bi Comput Comput 17: 333-336, 1979. In animal species such as rats and mice, all hairs are apparently in the same state of activity, where all cyclic changes are synchronized (DeVillez RL: In: Current Concept, a Scope publication by Upjohn Co., Kalamazoo, Ml, 1986; pp. 4-27; Takashima I, Kawagishi I: In: All K, and others (eds): Biology and Disease of Hair, Baltimore, Uviv. Park Press , 1976: pp. 457-471) The first cycle of hair growth in rats begins shortly after birth and continues through approximately the twenty-first day of life.The second cycle begins approximately after day 35. Young rat (8-12 days) has previously been used as a ^ model for alopecia induced by chemotherapy and has become a number and novel observations (H ussein, A. M., Jiménez, J .J. , McCall, C.A., and Yunis, A.A .: Science 249: 1564, 1990; Jiménez, J.J., Wong, G.H.W., Yunis A.A .: FASEB J. 5: 2456, 1991; and Jiménez, J.J., Huang, H.S., and Yunis, A.A. Cancer Invest. 10 (4): 269, 1992; Jiménez, J.J., Sawaya, M.E. and Yunis, A.A .: Cancer Res. 52: 413, 1992; Jiménez, J.J., Sawaya, M.E. and Yunis, A.A .: FASEB J. 6: 911, 1992; and Jiménez J.J., Alvarez, E., Bustamante, C.D., and Yunis, A.A. Am. J. Of the Med. Sci 210 (2): 43, 1995. Rats treated with chemotherapy during the first cycle of hair growth became totally alopecic in 10 days. These rats remain with total alopecia until the second cycle of hair growth. In this way, it takes 20 to 30 days for rats to recover from alopecia. More recently, a hair remover, NeetR, has been used in rats during their first hair growth cycle. This proposal makes the rats immediately alopecic, and the hair does not grow back until the second cycle. It is believed that this long latent period can be effectively used to test various substances for their potential ability to stimulate hair growth and thus reduce recovery time. An object of the present invention is to determine the possible existence of activity that stimulates hair growth or factors that stimulate hair growth.

BRIEF DESCRIPTION OF THE INVENTION It has been found that the medium is conditioned or extracted from normal mammalian cells, transformed or with cancer, and preferably conditioned or extracted from human mononuclear cells obtained from bulky coatings and MIA PaCa conditioned medium from human pancreatic carcinoma cell line (Yunis, AA, Arimura, GK, and Russin, DJ., Int. J. Of Cancer, 19: 128, 1977) possesses activity that stimulates hair growth. This activity was determined by the use of a sieving process where several compounds and / or cytokines were tested.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photograph of two groups of rats that were tested with CTX. The upper group of rats was tested with a composition that stimulates hair growth in accordance with the present invention and the lower group was tested with control medium. Figure 2a is a photograph of a group of rats that were tested with CTX and then with a composition that stimulates hair growth in accordance with the present invention.

Figure 2b shows a second group of rats treated with CTX and then with control medium. Figure 3 shows a group of rats treated with CTX. The groups on the right and left were tested with compositions according to the invention while the central group was treated with control medium.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates, in general, to compositions for stimulating hair growth and methods for stimulating hair growth. Hair growth stimulation may be desirable, for example, under conditions of alopecia induced by chemotherapeutic treatment or in subsequent hair graft treatment. Additionally, hair growth stimulation may be a goal to simply improve normal hair growth in a subject or to alleviate the effects of thin hair. It should be noted that the stimulation of hair growth as used herein is intended to encompass the stimulation of existing hair growth or the stimulation of hair growth from the hair follicle. The stimulation of hair growth is a commonly sought goal • in various situations. Alopecia is a common side effect and alteration of several chemotherapeutic agents. There is an urgent need in such situations to improve hair growth to restore the original state of a subject suffering from such alopecia. Similarly, the stimulation of hair growth is a requirement after the hair grafting procedures so that the grafted hair can grow rapidly and provide more complete hair conditions than would be present before the grafting procedure. hair. Some normal subjects may also wish to have a more complete hair condition and, thus, a composition that stimulates hair growth would also be desirable for such subjects. Compositions suitable for use in the present invention comprise formulations containing the active materials according to the present invention either by themselves or in association with a pharmaceutically acceptable carrier thereof and optionally other therapeutic ingredient (s). (s) The vehicle (Hashioto K. Br J. Dermatol 1 970; 83: 167-176) must be "acceptable" in the sense of being compatible with the other ingredients of the preparation and not harmful to the container thereof. The present active materials are usually administered in topical form such as a liquid ointment, a lotion, a cream or a gel. Additionally, the present active materials may ^ administered intracutaneously (intradermally). Other therapeutic ingredients that may be present include, for example, vitamin D3 and its analogs, derivatives and active metabolites or a potassium channel opener such as minoxidil, chromocalin or pinacidil. The concentration of the active ingredients will generally be between about 1 and 100 μg / g. The present formulations can be applied one to several times a day for prolonged periods if so required. Suitable preparations for topical administration include liquid and semi-liquid preparations such as liquid ointments, lotions, applicators, oil-in-water or water-in-oil emulsions such as creams, ointments, pastes or gels, or solutions or suspensions. The intracutaneous preparations contain the present active material and the generally known excipients, carriers and additives, intracutaneously accepted. In addition to the ingredients mentioned above, the preparations of this invention may include one or more additional ingredients such as diluents, regulators, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives, for example, methyl hydroxybenzoate (including anti -oxidants), emulsifying agents and the like. The preparations as mentioned above, may also contain therapeutically active compounds usually applied in the treatment mentioned above. In the topical treatment, ointments, creams, ^ gels or lotions containing 1-100 μg / g of the present active ingredients are administered.

The present invention further relates to a method for treating subjects suffering from or at risk of having alopecia, said method consisting of topically administering to a subject in need of treatment to stimulate hair growth an effective amount of the present materials active, alone or in combination with one or more therapeutically active compounds usually applied in said treatment. The treatment with the present compounds concomitantly with other therapeutically active compounds can be simultaneous or with intervals. In the present invention, the unknown existence of an activity or factors that stimulate hair growth has been discovered. The activity or factors that stimulate hair growth are produced by normal or transformed mammalian cells including, for example, normal or transformed human cells as well as other tumor cells or cell lines. Compositions that stimulate hair growth condition media obtained from cultures of said cells and preferably from human mononuclear cells or pancreatic cancer cells of a human. A sieving process was carried out where a number of compositions and / or cytokines were tested. In order to test two models were used: 1. Rats that were made bald with chemotherapy / (Cytoxan). 2. Rats that were made alopecia by epilation using the NeetR hair remover. From the sieving process, it was unexpectedly discovered that the conditioned medium of human mononuclear cells (obtained from bulky coatings) and conditioned medium of the MIA PaCa of human pancreatic carcinoma cell line (Yunis, AA, Arimura, GK, and Russin, DJ., I nt.

J. Of Cancer, 19: 128, 1977) contained activity that stimulates hair growth (HGSA). In rats that were made bald with Cytoxan (CTX) or NeetR, the treatment with Cell Conditioner Human Mononuclear, H MCC, resulted in faster recovery of hair regrowth than controls, ie, H MCC contains activity or factor that stimulates hair growth. In this way, it can be concluded that human mononuclear cells produce activity or factor that stimulates hair growth. In rats that were made bald with NeetR (model of Cytoxan not used), treatment with Human Pancreatic Cancer Cell Conditioner, MPCM, also resulted in faster hair regrowth than in controls. In this way, certain cancer cells can also produce activity or factor that stimulates hair growth. Although the above invention has been described in detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope thereof as described in the specification and as defined in the appended claims.

EXPERIM ENTALES PROCEDURES Sprague Dawley rats were used. The rats were fed and housed in accordance with the N I H guidelines. Cyclophosphamide or Cytoxan (CTX) was from Adria Laboratories (Columbus, OH). CTX (35 mg / kg) was given only for one day on day 1 1.

PREPARATION OF CONDITIONED MEDIUM FROM HUMAN MONONUCLEAR CELL PREPARATIONS Bulky coatings were purchased from American Red Cross, North American Red Cross, (Miami, FL). Bulky coatings were diluted 1: 4 with Dulbecco's Modified Eagle Media, (GIBCO) with 10% fetal bovine serum (Media Facility Cancer Center, Miami, FL). The cell suspension was placed on a gradient (Histopaque-1077, Sigma Diagnostics) and centrifuged for 40 minutes at 1400 rpm. The interface was collected, diluted 1: 5 with 10% fetal bovine serum (FCS) and centrifuged for 10 minutes at 800 rpm. This procedure was repeated to wash the mononuclear cells. The cells were resuspended in DMEM with 1% FCS, were counted and verified for morphology. This procedure produced an average of 35% monocytes and 65% lymphocytes. The cells were then plated at 3x106 / ml in DMEM with 1% FCS in tissue culture dishes (Sarsteadt) and incubated for 24 hours in a 37 ° humidified incubator with 5% CO2. At the end of the incubation period, the supernatant was collected and centrifuged at 1000 rpm at 5 ° C for 10 minutes. The conditioned medium, which contains the activity that stimulates hair growth (conditioned medium of human mononuclear cell or HMCCM), was collected and filtered with 0.2 μ filter. Samples were aliquoted and those not used immediately were stored at -70 °.

PREPARATION OF M EDIO CONDITIONING MIA PaCa MIA PaCa cells were grown for confluence in DMEM with 10% FCS and 2.5% horse serum (HS). Plates were washed for 2 and a fresh medium was added. The cells were then incubated for 24 hours in a humidified incubator a 37 ° with 5% CO2. At the end of the incubation period, it was collected and ^ centrifuged the supernatant at 1000 rpm at 5 ° for 10 minutes. The conditioned medium (MIA PaCa conditioned medium or MPCM), which contains the activity that stimulates hair growth, was collected and filtered with 0.2 μ filter. Sas were aliquoted and those not used immediately were stored at -70 °. The control medium was prepared in a similar manner but without cells. Serum-free MPCM (S.M. MPCM) was prepared as follows: MIA PaCa cells were grown for confluence in DMEM with 10% FCS and 2.5% H.S. the plates were washed by two with S.F.'s medium and fresh S.F.'s medium was added. After 48 hours of incubation, the supernatant was collected, centrifuged at 1000 rpm at 5 ° C for 10 minutes, filtered with 0.2 μ filter and used for ultrafiltration.

CONCENTRATION OF MPCM This was achieved by ultrafiltration at 5 ° C. The MPCM was ultrafiltered in 400 ml loads through a 10 Kd cut molecular weight (MW) filter at an operating pressure of 1.75 kg / cm2. The concentrated MPCM was collected from two bends and the tributary was discarded. S was similarly concentrated. F. MPCM of 10 doublings. For control rats, serum-free DMEM of 10 foldings was similarly concentrated. Aliquots were formed and frozen at -70 ° until used.

EXA I Six eleven-day-old rats received CTX 35 mg / kg i. p. Ten days later, when the rats were totally rapacious, they were randomly divided into two groups of 3 rats each. The group no. 1 received 0.3 ml of HMCCM s.c. in the head area daily for 10 days. The group no. 2 received 0.3 ml of control medium s.c. in the head area daily for 10 days and served as control. The rats in the group no. 1 showed hair growth increased five days before the rats of the group did not. 2. A photograph was taken on day 35. (Figure 1).

EXA II Nineteen 1 1 day old rats received CTX 35 mg / kg i. p. Ten days later, when the rats were cotely rapacious, they were randomly divided into two groups of 9 and 10 rats, respectively. The group no. 1 (9 rats) received 0.3 μl of HMCCM s.c. in the area of the back daily for 10 days. The group no. 2 (10 rats) received 0.3 ml of control medium s. c. in the area of the back daily for 10 days and served as control. The rats of the group no. 1 showed hair growth increased five to six days earlier than the rats in the group did not. 2 - (A photograph was taken on day 35). (Figures 2a and 2b).

EXA III When using NeetR, all hair was removed from nine rats with 26 days of birth. Three groups of 3 rats were randomly selected. The group no. 1 received 0.5 ml of HMCCM s.c. in the area of the back daily for 6 days. The groups no. 2 received 0.5 ml of MPCM in a similar manner. The group no. 3 received control medium. Six days after the first injection, three independent observers, not involved in the experiments, had to select rats with increased hair growth.

STIMULATION OF HAIR GROWTH HMCCM MPCMControl M Group # 1 Group # 2 Group # 3 Observer no. 1 + + Observer no. 2 + + - Observer no. 3 + + - EXA IV When using NeetR, all hair was removed from nine rats with 23 days of birth. Three groups of 3 rats were randomly selected. The group no. 1 received 0.5 ml of 10X S. F. M PCM in the area of the back daily for 7 days. The group no. 2 received 0.5 ml of 2X MPCM (with serum). The group no. 3 received medium of 10X control S. F. and served as control. Seven days later, the first injection photographs were taken (Figure 3). The photographs clearly show that the rats that received 10X S. F. MPCM or 2X MPCM had improved hair regrowth.

Claims (7)

1. CLAIMS 1 .- A composition useful in the stimulation of hair growth in a subject comprising a conditioned medium obtained from a cell culture of human mononuclear cells.
2. 2. The composition according to claim 1, wherein the cultured cells are derived from bulky coatings.
3. 3. The composition according to claim 2, wherein the cells are plated in tissue culture dishes and incubated and harvested and filtered supernatant of the cell culture comprising the conditioned medium.
4. 4. A composition useful in stimulating hair growth in a subject comprising a conditioned or extracted medium obtained from a cell culture of normal or transformed mammalian cells or cancer cells.
5. 5. The composition according to claim 4, wherein the cells are human pancreatic carcinoma cells.
6. 6. The composition according to claim 5, wherein the cells are MIA PaCa human pancreatic carcinoma cell line.
7. 7. The composition according to claim 4, wherein the cells are plated in tissue culture dishes and incubated and harvested and filtered supernatant of the cell culture comprising the conditioned medium. 8. - A method for stimulating hair growth in a subject comprising treating said subject with an effective amount of a composition according to claim 1. 9. A method for stimulating hair growth in a subject comprising treating said subject with an effective amount of a composition according to claim 4. 10. A pharmaceutical composition useful for stimulating hair growth comprising the composition according to claim 1 and a pharmaceutically acceptable carrier. 1 .- The pharmaceutical composition according to claim 10, wherein the vehicle is suitable for topical or intracutaneous application. 12. A pharmaceutical composition useful for stimulating hair growth comprising the composition according to claim 4 and a pharmaceutically acceptable carrier. 13. The pharmaceutical composition according to claim 12, wherein the vehicle is suitable for topical or intracutaneous application. 14. The method according to claim 8, wherein the treatment of said subject is a topical or intracutaneous treatment. 15. - The method according to claim 9, wherein the treatment of said subject is a topical or intracutaneous treatment. SUMMARY The present invention relates to compositions useful in the stimulation of hair growth. These compositions comprise a conditioned medium obtained from a cell culture of human mononuclear cells or a conditioned or extracted medium obtained from a culture. cellular of normal or transformed mammalian cells or cancer cells.