USE OF MINOXIDIL FOR WOUND HEALING

Abstract: A use of minoxidil in method for the promotion or acceleration of wound healing. The minoxidil can be administered by topical application, oral administration, injection or any combination thereof. Treatment with minoxidil is effective for promoting the migration of epithelial cells in a wound or in tissues such as cornea and the like. Methods for identifying binding sites for minoxidil in cells based on their affinity for the compound in attachment or chemotactic assays are described.

Description: USE OF MINOXIDIL FOR WOUND HEALING Background of the Invention The present invention is directed toward the use of minoxidil support and to modulate the healing of wounds. Generally, t present invention is useful for the improvement or acceleration the healing of wounds. Minoxidil is a synthetic chemical with proven efficacy as hypotensive agent and as a stimulant of hair growth. Thus it acts different kinds of cells and induces different phenomena. These ha useful sequelae in the treatment of hypertension and for enhanci hair growth. The healing of wounds is a complex process. Certain reactio are initiated almost immediately after wounding including t clotting of blood and the aggregation of platelets. These steps a important as they stop the bleeding and they also initiate the fir steps in the repair process. Cellular migrations are observed with the first day or two with the arrival of phagocytic cells such neutrophils and macrophages. A day or two later, fibroblastic cel enter the wound and begin to produce the connective tissue comp nents, including collagen, necessary for restoration of the derm tissue. Endothelial cells also enter the wound and form the capi laries that restore the blood supply to the area. Following the formation of the clot, epidermal cells at t margin of the wounds, as well as epithelial cells and organelles su as hair follicles, begin to migrate out from the edge of the wou and from the remnants of hair follicles in the wounded area. Wi minor wounds and in most other wound healing situations, the migrations of epithelial cells continue until the wounded area completely covered by the epidermal cells. These cells are al known as keratinocytes. Several studies have shown that the addition of exogeno factors to the wound accelerate wound healing. These wound stimul tory factors are polypeptides and can increase the formation fibrous tissue. Also one such growth factor TGFα, Schultz, et al "Epithelial Wound Healing Enhances by Transforming Growth Facto alpha and Vaccinia Growth Factor", Science 235:350-2 (1987), has be reported to increase the epithelialization of experimental wound Clinically, wound healing is impaired or abnormal in a variety conditions including diabetes, aging, following burns and in decubi tal conditions. The importance of the proper completion of each ste in the wound healing process is illustrated in burns where epiderma coverage of the burn when delayed is associated with infection, flui loss and abnormal scaring. The sources of cells involved in the healing of skin inclu fibroblasts, smooth muscle cells and endothelial cells in the derm and associated structures. Keratinocytes from the borders of t wound as well as epithelial cells from hair follicles are the maj sources of the cells which restore the epidermis. In a small cu the scar may be formed from fibroblasts, endothelial cells a epidermal cells which migrate in from the normal tissue around t wound. In a burn, smooth muscle cells may contribute the maj portion of the dermis, while epithelial cells from hair follicles m provide much of the epidermal coverage over the tissue. Information Disclosure Minoxidil is reported to selectively depress lysyl hydroxyla activity in cultured human skin fibroblast, Murad, S., et al "Selective Depression of Lysyl Hydroxylase Activity in Human Sk Fibroblasts", J. Invest. Derm., Vol. 36, pages 4963 (1986). Th finding, by Pinnell and colleagues, suggests that minoxidil could used as an antifibrotic agent, Murad, S., et al. , "Suppression Fibroblast Proliferation and Lysyl Hydroxylase Activity by Min xidil", J. Biol. Chem. , Vol. 262, pages 11973-78, (1987) and Pinnel et al., "Effects of Minoxidil on Cultured Human Fibroblasts Dermatologica, Vol. 175, Suppl. 2, pages 12-18 (1987) and therefo have therapeutic benefit in conditions characterized by an ove proliferation of connective tissue. Because lysyl hydroxyla activity has never been shown to influence or in any way contribu to cellular chemotaxis, the publications by Pinnell and colleagues not disclose or suggest to a person skilled in the art that minoxid would stimulate directed migration of skin keratinocytes or fibr blasts, or that minoxidil would accelerate the closure of wounds. Topical compositions of minoxidil are known for treatment baldness and are disclosed in U.S. Patents 4,139,619 and 4,596,81 Also, various, salts and derivatives of minoxidil, as well as ass ciated methods, are known and disclosed in U.S. Patents 3,382,24 3,461,461, 3,644,364 and 4,287,338 and published by J.M. McCall, al., Journal of Organic Chemistry, Vol. 40, page 3304 (1975). Summary of the Invention In one aspect, the invention is a method for improving accelerating the healing of wounds comprising treatment with minoxi il. The treatment can be a topical composition of minoxidil appli directly to the wound, preferably the topical composition compris from about 0.01% to about 1% minoxidil. The treatment can al comprise an injection of minoxidil in an effective amount to a celerate the healing of the wound or an oral administration minoxidil. Generally, an oral administration comprises from about to about a 100 mg dose per day of minoxidil. Furthermore, the treatment can be a combination of topica injection or oral administration of minoxidil. In yet another aspect, the present invention is a method f promoting the migration of epithelial cell comprising the treatme of such epithelial cells with minoxidil. The method can comprise topical composition of minoxidil applied directly to the epitheli cells or the vicinity of the epithelial cells to be effecte Generally, the topical composition comprises from about 0.01% about 1% minoxidil. The method can also comprise an injection minoxidil in an effective amount to promote the migration of t epithelial cells or an oral administration of minoxidil. Generall an oral administration comprises from about 5 to about a 100 mg do per day of minoxidil. Furthermore, the treatment can comprise a combination of topical, injection or oral administration of minoxi il. The present invention has found minoxidil to be chemotactic f keratinocytes and for fibroblasts, additionally minoxidil is able stimulate the general mobility of these cells. It has also be demonstrated that these cells will adhere to minoxidil demonstrati that the cells have minoxidil receptors or binding groups on the surfaces. Figure 1 shows the migration of human keratinocyte (epiderma cells in chemotaxis assays as they respond to BSA (bovine ser albumin), EGF (epidermal growth factor lOng/ml) , TGFα (transformi growth factor alpha lOng/ml) , TGFβ (transforming growth factor be lOng/ml) , PDFG (platelet derived growth factor lOng/ml) , EC (endothelial cell growth factor lOng/ml) , FGF (fibroblast grow fac or lOng/ml) . Figure 2 shows the chemotactic response of human keratinocyte (epidermal cells) to minoxidil, minoxidil sulfate and a minoxidi analogue. BSA indicates the migration of the cells in response t this protein which was added to all samples. EGF at lOOng/ml wa added as a known positive chemoattractant. Figure 3 shows the response of human keratinocytes (epiderma cells) in chemotaxis assays in Boyden chambers. Minoxidil was place in some experiments in the bottom compartment, in others in the to and in others in both top and bottom compartments. Figure 4 shows studies which relate to the mechanism of actio of minoxidil in promoting chemotaxis. In addition to minoxidil, EG and BSA, cyclic AMP plus IBMX were added to some samples, whil verapamil (a calcium channel blocker) , BAY K8644 (a calcium agonist were added to the samples in other studies. Figure 5 shows that human keratinocytes have the ability to bin to minoxidil and to a lesser extent to an analogue of minoxidil. I these studies, the compounds were dried onto a plastic petri dish an the ability of these substrates to allow cells to bind to immobilize minoxidil was tested. Figure 6 shows that increasing amounts of calcium ion preven the keratinocytes from migrating toward attractants. This occur regardless of the chamber in which the calcium is placed. Detailed Description of the Invention The present invention is directed toward the use of minoxidil t induce the migration of epidermal and other cells and thus to improv the healing and restoration of skin and other tissues damaged b trauma, burns, aging, environmental factors etc. The epidermal cell referred to here include keratinocytes and epithelial cells in hai follicles which serve as a source of cells for the coverage o lesions of skin and other organs such as the cornea. The active component of the subject compositions is minoxidi which chemically is 6-amino-l,2-dihydro-hydroxy-2-imino-4-piperidino pyrimidine and analogs thereof. The preparation of these compoun are described in U.S. Patents 3,382,247, 3,461,461 and 3,644,364 a J.M. McCall, et al. , Journal of Organic Chemistry, Vol. 40, page 33 (1975) all of which are hereby incorporated by reference. Relat compounds are sulfoxypyrimidinium, -pyridinium, and -triazinium whi are described in U.S. Patent 4,287,338 herein incorporated by refe ence. Hereinafter, the term "minoxidil" means any of the vario forms of 6-amino-1,2-dihydro-hydroxy-2-imino-4-piperidinopyrimidin derivatives and analogs thereof. In one preferred composition, the various minoxidil compoun were synthesized by The Upjohn Company, Kalamazoo, MI, and we dissolved in acetone plus physiological saline 0.15M NaCl, plus 0.0 phosphate buffer, pH 7.4. Samples dissolved in acetone were dilut with media to a maximum of 1% acetone. Human keratinocytes were grown in an incubator maintained 37°C in 95% air - 5% CO2. Keratinocytes cultures at near confluen were rinsed with phosphate buffered saline PBS and removed from t dish with trypsin (0.025%) in PBS. To measure cell migration, chemotaxis assays were carried ou Polycarbonate filters (12 micron pore size) were coated with type collagen (10 micrograms/filters) and placed in a modified Boyd chamber. Cells (human keratinocytes) were harvested and prepared described above, and 3.0 x 10 5 cells in 0.8 ml were placed in t upper compartment of the Boyden chamber. The lower compartment the Boyden chamber contained the chemoattractants including vario growth factors, minoxidil or related compounds dissolved in seru free Eagles #2 Medium with 0.1% BSA (0.2 ml). The chambers were th incubated for six hours at 37° C, 5% CO2 and 95% air. The cel which had attached to the upper side of the filter were mechanical removed. The cells which had migrated to the lower side of t filter were fixed in methanol r and then stained with hematoxylin a eosin. Each sample was assayed in quadruplicate, and the cells in least 10 microscopic fields per filter were counted. As shown in Figure 1, human keratinocytes are able to under directed movement in response to certain growth f ctors, particular TGF alpha, PDGF and FGF. Other growth factors EGF, ECGF and TGF be show lesser activity to mobilize the cells. At higher levels of EG i.e., 100 ng/ml, comparable positive activity was observed (Figu 2). When minoxidil was tested for chemotactic activity, it caused dose dependent migration of the keratinocytes over the range of 10 1000 μg/ml. An analogue of minoxidil, 6-amino-l,2-dihydro-l-hydrox 2-imino-pyrimidine, showed activity at 10 and 100 /xg/ml. The migration of the keratinocytes in response to minoxidil wa assessed to determine chemoattractant activity (Figure 3) . To make this assessment, the response of the cells to minoxidil when it was incorporated into the top, the bottom or both chambers was tested. In all cases, the keratinocytes were placed in the top chamber. Minoxidil when placed in the lower chamber induced the expected migration of the cells as shown in Figure 2. When minoxidil was placed in equal amounts in both chambers, little or no increase in migration was observed indicating that in the absence of a gradient migration, was not occurring. Some response was induced by minoxidil when it was placed with the cells in the top compartment. Such responses are typical of chemotactic factors which are able to stimulate random motion in cells. These results are consistent with minoxidil being both chemotactic and a stimulator of random cellular migrations. As shown in Figure 5, the attachment of keratinocytes is in¬ creased by coating the plastic dish with various concentrations of minoxidil. The minoxidil analogue had a similar but somewhat lesser activity. The fact that the keratinocytes bind to minoxidil indi¬ cates that they have binding groups on their surface for minoxidil. Since chemotaxis is often mediated via cell surface receptors which bind the attractant, attachment and chemotaxis could be mediated by the same cellular receptors. As shown in Figure 6, calcium strongly inhibits the migrating response of cells. These effects were rapid and the cells lost their migratory ability at relatively low levels of calcium. However, as shown in Figure 5 verapamil, a calcium channel blocker, had no effec on the response of the cells to minoxidil, while a calcium agonist (BAY K8644) did reduce the response of the cells. Therefore, such combinations of drugs may modulate or potentiate the response of the migrating epidermal cells. Cell adhesion was assayed by adding various amounts of minoxidi to 35 mm tissue culture dishes and 1 ml of serum-free Eagles # Medium containing 0.5% BSA. Human keratinocytes were prepared a described above and added to each dish, followed by a two hou incubation at 37° C in 5% CO2, 95% air. At the end of this period, plates were gently washed three times with PBS to remove unattache cells. Attached cells were trypsinized with 0.025% trypsin, 0.025 EDTA, and electronically counted. Medical devices can be designed to deliver minoxidil at variou concentrations directly to wounds. These might include pastes, gel or emulsions applied directly to the surface of the wound or mater ials incorporated in a surface covering material or by in ection. I general, minoxidil would be supplied in concentrations of 0.01% to 1 in topical application. Methods of preparation and formulations fo topical minoxidil are described in U.S. Patent 4,139,619 and 4,596,812 herein incorporated by reference. Under other situations oral administration at levels of 5-100 mg/day could be used alone o in combination with topical treatment. Minoxidil in the present invention may be used in the form of liquid, such as eye drops or lotions, or as a salve or gel which ma be applied to promote cell migration or attachment, or in any othe convenient form. Accordingly, the compound may be contained in an pharmaceutically acceptable carrier which is appropriate for th delivery means intended.

Claims: CLAIMS 1. Use of minoxidil for accelerating the healing of wounds by oral, injection or topical administration of an effective amount o minoxidil. 2. The use of Claim 1 wherein said treatment is a topical composi¬ tion of minoxidil applied directly to the wound. 3. The use of Claim 2 wherein said topical composition comprise from about 0.01% to about 1% minoxidil. 4. The use of Claim 1 wherein said oral administration comprise from about 5 to about a 100 mg dose per day of minoxidil. 5. The use of Claim 1 wherein said treatment comprises a combina tion of topical, injection or oral administration of minoxidil. 6. A use of minoxidil to prepare a medicament for promoting th migration of epithelial cells by treating epithelial cells wit minoxidil. 7. The use of Claim 6 wherein said treatment is a topical composit ion of minoxidil applied directly to the epithelial cells or th vicinity of the epithelial cells to be effected, an injection o minoxidil or an oral administration of minoxidil in an effectiv amount. 8. The use of Claim 7 wherein said topical composition comprise from about 0.01% to about 1% minoxidil. 9. The use of Claim 7 wherein said oral administration comprise from about 5 to about a 100 mg dose per day of minoxidil. 10. The use of Claim 6 wherein said treatment comprises a combinati on of topical, injection or oral administration of minoxidil.

Inventors: LEDBETTER STEVEN R, MARTIN GEORGE R, SANK ANTHONY C
Applicants: LEDBETTER STEVEN R, MARTIN GEORGE R, SANK ANTHONY C, UPJOHN CO, US HEALTH, HEALTH & HUMAN SERVIC DEPART
Application year: 1989
Main classification: A61K31
IPC codes: A61K31, A61P17, C07D239