WO1995026194A1

WO1995026194A1 - Allantoin-metal complexes for skin and hair

Info

Publication number: WO1995026194A1
Application number: PCT/US1995/003849
Authority: WO
Inventors: Loren R. Pickart
Original assignee: Skin Biology, Inc.
Priority date: 1994-03-28
Filing date: 1995-03-27
Publication date: 1995-10-05
Also published as: AU2198995A

Abstract

Methods and compositions are provided for protecting irritaded or damaged skin from further oxidative and biochimichal damage and thus permitting natural healing processes to progress, for accelerating the rate of healing of burns and surgical wounds, and for increasing the size of hair follicles and the rate of hair growth. The compositions comprise complexes of allantoin and ionic metals such as copper(II) salts and tin(II) salts.

Description

ALLANTOIN-METAL COMPLEXES FOR SKIN AND HAIR

Background of the Invention

Allantoin, a metabolic product of uric acid, is extensively used as a component of skin and hair products.

The compound, which is generally well tolerated by the skin, is anecdotally reported to possess soothing properties on the skin and is often included in preparations designed to aid skin healing. Examples of the use of allantoin include a preparation for the treatment of bedsores and skin sores as reported in JP 4208219, for burned skin as reported in JP 55104205, to improve skin elasticity as reported in JP 5310548, and as a skin soothing agent in a moisturizing cream, as reported in US 4,863,725. Scientific evidence for direct skin or hair stimulating properties of allantoin is sparse, however. Klouchek-Popova et al., Acta Phvsiol. Pharmacol. Bulg.. 8: 63-67 (1982) reported that an unguentum (ointment) containing allantoin in combination with fractions Cl and C5 isolated from the flowers of Cals°ndula officinal improved the healing of standard skin wounds in Wistar albino rats. Tauschel and Rudolph, Arzneimittelforschung 32: 1096-1100 (1982) reported that a mixture of heparin, allantoin and dexpanthenol in a specific ointment base produced a more rapid regression of erythema caused by ultraviolet radiation of guinea pig skin.

The restoration of function of damaged and wounded skin continues to be a major health problem despite the development of various medications. A major need exists for improved and low cost skin-care products and medicaments in less-developed countries, particularly in the tropics, where conditions such as inadequate health care, widespread skin fungal diseases, and the use of flammables such as kerosene for cooking and similar conditions give rise to high incidences of serious skin injuries. For such countries, there is a critical need is to produce effective medicines that can be produced at very low cost using readily available materials. Many approaches to skin healing that are currently being developed, such as the production of growth factor proteins and pharmaceutical drugs with wound healing properties, and occlusive dressings for wounds, will remain beyond the economic reach of patients in these countries for the next few decades. Furthermore, even in more developed countries increasing demands for cost-containment in medical services necessitate the development of low-cost products for skin care and pharmaceuticals for wound healing. Procedures such as hospitalization for the treatment of diabetic skin ulcers are increasingly restricted. Delayed healing or incomplete healing in humans and other animals causes additional pain and suffering for the patient and markedly increases wound complications and medical costs, and often the wound continues as a chronic sore that requires extensive attention and medical care to control infection and tissue necrosis. Even when such wounds finally heal, the wound area is frequently devoid of the ability to respond to tactile stimulation and is often filled with excessive deposits of immature collagen that produces permanent scarring. The urgent need for improved wound-healing compositions also extends to wounds generated by surgical procedures. The success of surgical procedures, especially in very ill or elderly patients, is typically a function of the adequacy and speed of post-surgical healing. Excessive inflammation can also impair the normal healing response of injured or wounded skin. While the inflammatory process and its concomitant influx of white cells into the afflicted area are an integral part of the natural healing process, in some cases the inflammatory process becomes excessive and delays healing. The wounded tissue becomes locked in an early phase of the healing process and cannot proceed to completion. In such instances, compounds with anti-inflammatory activities are used to allow the process to proceed normally. One approach to the treatment of clinical conditions associated with inflammation and impaired wound healing has been the use of metal ions complexed to organic molecules or amino acids, amino acid derivatives and peptides. Some of these complexes possess anti-inflammatory activity, while others possess both anti-inflammatory activity and healing actions. Yet other complexes reportedly possess hair growth stimulating actions in addition to anti-inflammatory and/or healing activities, as described in, for example, Applicant's U.S. Patent No. 5,382,431, which is incorporated in its entirety by reference herein.

The use of copper salts or complexes as anti- inflammatory agents for the healing of stomach ulcers in the treatment of patients suffering from acute or chronic arthritis dates back to the 1940's and 1950's (see, e.g., reviews by Sorenson, Inflammation 3:317-331 (1976); Agents and Actions 8:305-331 (1981), and Comprehensive Therapy 11:49-64 (1985)). The use of copper salts and complexes, such as copper-salicylate complex, seems to have been abandoned, apparently due to the early promise of the steroidal anti- inflammatorys, such as hydrocortisone. Other complexes of copper with amino acids (tryptophan, lysine) , with non- steroidal anti-inflammatory drugs (indomethacin, ketoprofen, acetylsalicylic acid) or with fatty acids (oleic, lauric and caprylic acids) have been studied but, despite their promise, were rarely developed beyond the preclinical phases, apparently due to problems of irritation, toxicity, and inadequate efficacy.

While many copper-complexes have been reported to possess anti-inflammatory properties, a more limited group have been reported to also possess healing actions. Heintze (U.S. Pat. No. 4,123,511) reported that a copper oleate complex had anti-inflammatory and skin healing activity. Sorenson (U.S. Pat. No. 4,440,754) described the use of complexes of copper(II) salts and amino acids, such as tryptophan or lysine, or with organic molecules such as 3,5- diisopropylsalicylic acid, acetylsalicylic acid or salicylic acid, to prevent and heal gastrointestinal ulcers. Using a wound-healing model, Townsend, Sorenson et al.. Agents and Actions 8:305-325 (1981)) found salicylate-copper accelerated the rate of healing and improved the quality of healing of surgically-induced ulcers in rats. Also, Sorenson wrote (ibid, and Inflammation 3:317-331 (1976)) that Townsend had demonstrated that copper(II)-(tryptophan)2 increased the rate of ulcer healing in a surgically-induced ulcer model. The increased healing was purportedly due to a more rapid re- epithelialization of the wound and an increase in the quantity and quality of the collagen. Fine collagen fibers in a normal orientation developed in treated animals, in contrast to non- treated animals in which the new collagen was very dense and composed of thick, wavy disoriented bundles, resembling scar tissue. Federici and Bertolotto (EP 450,398 and IT

9,019,948) reported that chondroitin sulfate-copper(II) complexes possessed anti-inflammatory activity. European Patent No. EP 66,283 discloses "eustatic" compositions which contain a non-toxic metal ion (including copper) and a glycosamino-glycan of hyaluronic acid or chondroitin sulfate useful as a cicatrizant (wound healing by closure) .

UK Patent Application GB 2 044 265 describes metal complexes (including copper) of adenosine triphosphate as aiding the recovery of bone tissue in cases of fractures as well as in osteoporosis and bone cysts.

Konishi (US Pat. No. 4,461,724) reports the tetrapeptide Gly-Ser-His-Lys and peptides of related structures possess anti-inflammatory and healing actions when complexed with metals such as ionic copper and zinc. Yu (U.S. Patent 4,053,630) discloses the use of cysteic acid and its derivatives cysteine sulfinic acid or homocysteic acid, chelated to metal ions such as ferric, cupric, zinc or aluminum, to form compositions that alleviated symptoms of diseases characterized by defects of keratinization and achieved a remission of ichthyosis, dandruff and acne. Bertelli (U.S. Patent 4,156,737) suggests that copper complexes of p-aminomethyl-benzene-sulfonamide possess healing and protective effects on skin burns. Van Scott (U.S. Patent 4,283,386) teaches that metallic (copper, zinc, or aluminum) salt forms of cysteic acid, cysteine sulfinic acid and homocysteic acid have therapeutic actions that produce remissions of dry and broken skin, keratoses, warts and palmar and plantar hyperkeratosis.

Niwa (Dermatologica 179 SI: 101-106 (1989)) and Bergren et al. (Am. Surg. 54:333-336 (1988)) found that the anti-inflammatory protein Cu,Zn-superoxide dismutase also acts to enhance healing processes. Pickart (see, e.g., PCT Publications WO 91/14437, WO

91/12267, WO 91/05797, WO 91/03488, WO 89/12441, WO 88/26448, WO 88/08851, EP Patents EP 190,736, EP 189,182; and U.S. Pat. No. 4,767,753) describes the synthesis and use of metal complexes of Gly-L-His-L-Lys as anti-inflammatory and healing agents.

A number of metal complexes have been used to promote hair growth. Yamashiki (Japan Pat. 70018997) used a complex of copper-pantothenate to purportedly promote growth of hair roots and promote skin functions. Morelle (U.K. Pat. GB 2097256, DE Pat. 32212448) used amino acid derivatives (N- butyryl amino acids) complexed with copper and other metals for cosmetic and therapeutic purposes, including use as hair and skin stimulants. Banfi et al. (U.S. Pat. No. 4,503,047) disclose a composition containing primarily one or more sulfur-containing amino acid(s) and copper(II) ions plus smaller amounts of allyl isothiocyanate and rhodanide ions to produce hair growth stimulating actions. Pickart (e.g., WO 91/07431, 88/08695 and EP 288,278) found a number of metal complexes of derivatives of Gly-L-His-L-Lys to increase hair follicle size and the rate of hair growth.

Despite the therapeutic promise of the above- mentioned metal complexes, toxicity and tissue irritation occur with many metal complexes (see, e.g., Johnson et al., Inorg. Chem. Acta 67:159-165 (1982); Pickart et al., Biochem. Pharm. 32:3868-3871 (1983); and Pickart et al., Lvmphokines 8:425-446 (1983)). For example, while copper-salicylate complexes and numerous copper-salicylate analogs possess anti- inflammatory activities, other salicylate analogs such as the copper(II) complex of salicy1aldehyde benzoyl hydrazone are highly toxic to tissues. Similarly, copper(II)-Gly-L-His-L- Lys supports cellular viability and possesses anti- inflammatory and healing actions, yet close synthetic aroylhydrazone analogs of its copper-binding region are extremely toxic to cells and tissues.

Another problem with the therapeutic use of copper complexes concerns the binding affinity of copper ion to the complexing molecule. While a defined copper complex can be synthesized, its therapeutic use places it in the physiological milieu of the tissues where a plethora of literally hundreds of compounds compete for binding to the copper ion, which can form electrostatic bonds to as many as six separate molecules. If the copper is removed from the complex and becomes loosely-bound, then tissue irritation occurs (see Raju et al., J. Natl. Cancer Inst. 69:1183-1188 (1982)) .

Further complications arise when such metal complexes are formulated into carrier creams or ointments. Various chemicals are added to the formulations to increase adherence to skin and wound surfaces and to enhance the penetration of the complexes into the target tissue. Yet, since many of these substances also bind to the metals, the expected therapeutic benefits may be nullified or significantly attenuated. Also, detergents such as sodium dodecyl sulfate are used to help blend oil and water phases of the emulsions and stabilize the formulations. However, such detergents are themselves tissue irritants that can delay healing. Another problem encountered with many of the metal complexes intended for therapeutic use is that they cannot be heat-sterilized; hence, to meet safety requirements, high concentrations of antimicrobial chemicals must be added during manufacture to inhibit the growth of microorganisms and the transmission of viruses. These antimicrobial agents may also inhibit the viability and function of a host's cells such as macrophages and fibroblasts that are involved in the maintenance and repair of skin and other tissue, and thus these agents may retard the healing response.

What is needed in the art are compositions useful in tissue protection, tissue healing, and/or stimulation of hair growth, which compositions could be conveniently produced and at low cost. Preferably, the compositions could be sterilized without loss of bioactivity and could be formulated for topical application without the use of detergents or other potentially irritating compounds. To speed the time and expense required for regulatory approvals, the compositions should be prepared from materials that are generally recognized as safe by regulatory agencies and thus could be used with minimal safety concerns and regulatory barriers. Quite surprisingly, the present invention fulfills these and other related needs.

Summary of the Invention

The present invention is based on the discovery of methods to markedly increase the skin active properties of allantoin by complexing it with ionic metals such as copper or tin. Thus, the present invention provides allantoin-metal compositions and methods for accelerating the healing of topical wounds and skin irritation, for protecting skin from damaging effects of oxidation, and for increasing the size of hair follicles and the rate of hair growth in warm-blooded animals. The compositions useful in these methods, including pharmaceutical compositions, are prepared from complexes of allantoin and an ionic metal such as copper, indium, zinc, tin, etc.

Thus, in one aspect the invention provides methods for preparing the pharmaceutical compositions useful in accelerating the healing of topical wounds or increasing hair follicle size and hair growth in a warm-blooded animal.

Allantoin is combined with an aqueous solution of an ionic metal salt, then adjusted to a useful acidity range for skin products. The resulting aqueous mixture is composed of complexes of allantoin and the metal ions. The amount of water in the mixture is adjusted to produce a paste-like mixture. The mixture may be used directly or combined with a pharmaceutically acceptable carrier to form a cream, lotion, etc. in a concentration of from about 5% to about 50% allantoin-ionic metal complex or more. The preparation may be sterilized or pasteurized, as desired, without destroying the healing or hair-growth stimulating activity of the allantoin- ionic metal complex.

In other embodiments the invention provides methods for enhancing the recovery of skin of a warm-blooded animal from wounds, such as surgical incisions, burns, inflammation or minor irritation due to oxidative damage, etc. The methods comprise administering to the skin wound or irritation a therapeutically or, in some cases a prophylactically effective amount of a composition which comprises the allantoin-ionic metal complex.

Yet other embodiments relate to compositions and methods for increasing hair follicle size and the rate of hair growth in warm-blooded animals, such as humans. The methods comprise administering to the skin in the area in which hair growth is desired an amount of allantoin-ionic metal complex sufficient to increase hair follicle size and the rate of hair growth in the animal. Typically, the composition will be administered topically as a cream, and will be applied on a daily basis until hair growth is observed and for a time thereafter sufficient to maintain the desired amount of hair growth.

Description of the Specific Embodiments

Compositions and methods are provided by the present invention for topical skin treatments to protect damaged skin and thereby allow natural healing processes to proceed, to enhance tissue regenerative processes in the skin of warm blooded animals, and to stimulate hair growth in warm blooded animals. The compositions are formed by complexing allantoin with ionic metals such as copper, zinc, tin or the like. Methods are provided for improving the recovery of wounds and surgical incisions, and stimulating hair growth in warm¬ blooded mammals. By allantoin is commonly meant glyoxylic diureide or glyoxyuric acid diuride, which is produced by the oxidation of uric acid and is widely available in nature. Allantoin can be prepared synthetically, as described in, e.g., U.S. Patent No. 2,158,098, incorporated herein by reference, or can be obtained from a variety of commercial suppliers.

To produce the allantoin-ionic metal complexes useful in the present invention, the allantoin is complexed via admixture or other suitable procedure with one or more ionic metals, such as copper, indium, tin, zinc, or the salts thereof, such as sulfate, acetate, phosphate, chloride, citrate, succinate, oxalate, cinnamate, tartrate, fumarate, maleate, glutarate, etc. By complexed is meant that the allantoin and the metal ions form electrostatic bonds, although this mechanism is offered by way of explanation only and not by way of limitation. In one method for preparing the allantoin-ionic metal complex, allantoin, at a concentration of about 5 to 50% (weight/volume) , is mixed with a aqueous solution of the metal salt (copper(II) chloride, tin(II) chloride, tin(IV) chloride, indium(III) chloride, or zinc(II) chloride) at a salt concentration of about 10 to 50% (w/v) , more preferably about 20% (w/v) .

The allantoin-ionic metal complexes of the invention may be administered for a variety of therapeutic, prophylactic or cosmetic uses to humans or in veterinary applications to other warm-blooded animals. Among veterinary animals particularly well suited for treatment with the present compositions are species of equine, bovine, porcine, ovine, caprine, canine, avian, feline, etc.

The compositions and pharmaceutical preparations thereof are intended for local, topical, oral or parenteral

(e.g., subcutaneous injection) administration for prophylactic and/or therapeutic or cosmetic treatment. Preferably, the pharmaceutical compositions are administered locally, e.g., topically, as a paste, cream, salve or ointment.

For administration to warm-blooded animals, the allantoin-ionic metal compositions will typically be sterilized or pasteurized and incorporated into pharmaceutical or veterinary formulations. Compositions which comprise the allantoin-ionic metal complexes can be sterilized by conventional, well known sterilization techniques, e.g., boiling or pasteurization, without substantially adversely affecting the biological activity of the allantoin-ionic metal complexes. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions and as necessary to prepare compositions for convenient administration, such an pH adjusting and buffering agents, and delivery vehicles. Actual methods for preparing pharmaceutically administrable compounds will be known or apparent to those skilled in the art and are described in detail in, for example, Remington's Pharmaceutical Science. Mack Publishing Co., Easton, PA pp. 428-429 (1975) , which is incorporated herein by reference, infra.

Depending on the intended mode of administration and the intended use, the compositions may converted to solid, semi-solid, or liquid dosage forms, such, for example, as powders, granules, crystals, liquids, suspensions, liposomes, pastes, cremes, salves, etc., and may be in unit-dosage forms suitable for administration of relatively precise dosages. The compositions may include a conventional pharmaceutical carrier or excipient and, in addition, may include other medicinal agents, growth factors, wound sealants, carriers, etc., as further described below.

For semi-solid compositions, as would be appropriate for pastes and creams intended for topical administration, the allantoin-ionic metal complexes can be provided separately or may be compounded with conventional nontoxic carriers such as, for example, aloe vera gel, squalane, glycerol sterate, polyethylene glycol, cetyl alcohol, stearic acid, and propylene glycol, among others. Such compositions may contain about 5-100% active ingredient, more preferably about 20- 40%. The concentration of the allantoin-ionic metal complexes in these formulations can vary widely, and will be selected primarily by intended use, viscosities, etc., in accordance with the particular mode of administration selected. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Science. 17th ed. , Mack Publishing Company, Easton, PA (1985) , which in incorporated herein by reference. The composition or formulation to be administered will, in any event, contain a quantity of the allantoin-ionic metal complexes sufficient to achieve the desired therapeutic or prophylactic effect in the subject being treated.

The tissue healing compositions of the invention are administered to a warm-blooded animal, such as humans, already suffering from a wound, oxidative skin damage, inflammatory skin lesions, other the like as described above, in an amount sufficient to allow the healing process to proceed more quickly than if the host were not treated. In the case of an animal suffering from decreased hair follicle size and impaired hair growth, the compositions of the invention are administered in an amount sufficient to increase hair follicle size and the rate of hair growth. Amounts adequate to accomplish these effects are defined as a "therapeutically effective doses." Amounts effective for this use will depend on the severity of the wound, sore, etc, in the case of wound healing, and the extent of decreased follicle size in the case of impaired hair growth has, and the general state of health of the patient being treated, but generally range from about 1 mg to about 25 mg per day of allantoin-ionic metal complex per day per square centimeter of wound site, with dosages of from about 5 mg to about 10 mg per day per square centimeter of wound site being more commonly used. Maintenance dosages over a prolonged period of time may be adjusted as necessary. For veterinary uses higher levels may be administered as necessary. Determining actual amounts of the allantoin-ionic metal complexes necessary to treat a particular wound or condition as described above will be through standard empirical methods well known in the art.

In prophylactic applications compositions containing the allantoin-ionic metal complexes are administered to a host susceptible to or otherwise at risk of skin lesions or similar damage, to enhance the host's own wound healing or anti- oxidative capabilities. Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts again depend on the host's condition and general state of health, but generally range from about 0.1 mg to about 10 mg per day per square centimeter of skin, more commonly from about 1 mg to about 3 mg per cm² of skin per day. Single or multiple administrations of the compositions can be carried out. The allantoin-ionic metal complexes of the invention may be administered in relatively large amounts without serious side effects, although indiscriminate use may produce discoloration of the skin. In instances where the compositions are administered to inhibit oxidative or biochemical damage to the skin or to those suffering from only mild or irritation or inflammation of the skin, the dose may be adjusted accordingly to lower maintenance levels.

The compositions of the invention, including pharmaceutical compositions, may be administered alone or as adjunct therapy or prophylaxis. The allantoin-ionic metal compositions can be used in combination with other factors found to improve other aspects of healing. In this manner, a synergistic effect may be attained that yields a clinical efficacy greater than that realized with any single factor. Further, while the compositions described herein stimulate a spectrum of healing processes, clinical wounds may differ considerably in their properties and healing patterns, leading one to utilize a combination of a composition described herein and a growth factor or a preparation such as described in commonly owned copending U.S. Patent 5,382,431 and patent applications USSN 08/219,047 and 08/219,681, each of which is incorporated herein by reference in its entirety. For example, nerve regeneration is defective in many burns and thus one can add a specific nerve growth factor to supplement the composition to enhance nerve regrowth into the burn area. Examples of factors with other reported healing properties include epidermal growth factor, fibroblast growth factor, nerve growth factor, transforming growth factors, angiogenic growth factors, heparin, fibronectin, fibrin, platelet-derived growth factor, enzymatic superoxide dismutase, extracts of blood or factors from the blood, and other similar factors. The following examples are offered by way of illustration, not by way of limitation.

EXAMPLE I Preparation of Active Allantoin-ionic Metal Complexes

This Example describes the preparation of allantoin- ionic metal complexes having biological activities described herein. Allantoin was purchased from Sigma Chemical Company, St. Louis, MO, as was cupric chloride hydrate (no. C 6641) . Tin (II) chloride, 99% pure, was purchased from Aldrich Chemical Company, Milwaukee, WI.

In one method for preparing the allantoin-metal complex, the allantoin was mixed with sufficient room temperature water (about 23°C) to form a thick paste. To obtain this paste, allantoin (1 gram) was mixed with 2 milliliters of water. The paste was then mixed with a aqueous solution of cupric chloride hydrate or tin (II) chloride, at a concentration of about 20% (w/v) . The volume of metal salt solution added was that amount needed to obtain a final metal concentration in the paste of 0.3 to 1.2% (weight/weight).

The addition of the metal salt reduced the pH of the paste to about 3.0. The pH of the paste was then raised to 6.5 by careful addition of 1 N sodium hydroxide. This pH is useful for skin preparations but other pH levels near neutrality or in the slightly acidic range are also efficacious.

The paste can be applied directly to the skin or is formulated into skin creams and lotions at concentrations of usually 5 to 20% (w/w) although higher concentration are also effective.

The allantoin-copper complexes are used to treat damaged or irritated skin. Damaged or irritated skin healed faster after treatment with such a composition. As described further below, the allantoin-copper and allantoin-tin were used to promote hair growth and the enlargement of hair follicles. In hair growth models in mice, application of these complexes to the skin produced a marked stimulation of hair growth after 15 to 20 days.

EXAMPLE II Healing of Surgical Wounds with Allantoin-Copper Complexes

This Examples demonstrates the use of a paste prepared with the allantoin-copper complexes to hasten the healing of surgical incision wounds in animals.

Surgical incisions (1.25 cm) were made on the backs of anesthetized, 35 gram, Swiss-Webster mice. Immediately after surgery and 24 hours later, the wounds were covered with a thin film of the paste containing the active allantoin- copper complex prepared in accordance with Example I above. Control wounds were untreated or contained allantoin without copper. As seen in Table 1, wounds treated with the active allantoin-copper complex healed faster than control wounds. Since rapidly healing wounds tend to contract and become more rounded, the healing activity can be related to the length of the wound after 15 days. Each group consisted of eight mice.

Table 1: Effect of allantoin-copper complex on incision length.

5 Test group Length of wound after 15 days (cm.)

Control 0.76 ± 0.10

J Allantoin 0.78 ± 0.12

10 no copper

Allantoin-

1.0% copper complex 0.32 ± 0.06

15 Allantoin-

0.3% copper complex 0.39 ± 0.13

20

EXAMPLE III Reduction in Post-Burn Inflammation of Skin

This Example demonstrates the ability of allantoin-

25 copper complex to reduce inflammation associated with mild skin burns.

Very mild thermal burns were induced on the shaved backs of anesthetized mice (8 mice in each group) by placing a circular (1.25 cm diameter, irritated area = 1.22 cm²) brass

30 rod (60°C) in contact the skin for 5 seconds. This produced a mild skin irritation characterized by redness and swelling, but rarely a loss of skin tissue. Immediately after inducing the thermal injury, the irritated area was covered with a thin film of the paste containing allantoin with no copper or

35 allantoin with 1.0% copper ion. Control wounds were untreated. Wounds were observed at daily intervals. At day 3, the untreated thermal injuries were still reddish and swollen while the skin treated with the allantoin-copper complex had minimal reddishness and swelling. Treatment with

40 allantoin without copper had much less of an effect on reducing post-burn wound inflammation. EXAMPLE IV Pasteurization of Active Allantoin-Copper Composition

Pasteurization consists of heating a solution to 160°F for 30 minutes which process kills all but the hardiest microorganisms. For this Example, allantoin was complexed with copper chloride by the methods described above in Example I. The resultant paste was thoroughly mixed, then heated to 160°F for 30 minutes. After cooling to room temperature, the pastes were applied to surgical incision wounds in mice in the manner described in Example II. Healing activity was similar to that observed with unpasteurized active composition, as shown in Table 2. There were six mice in each group.

Table 2: Effect of pasteurization on active complex.

Test group Length of wound after 15 days (cm)

Control 0.78 ± 0.15

Allantoin-

1.0% copper complex 0.29 ± 0.13 (unpasteurized)

Allantoin-

1.0% copper complex 0.30 ± 0.04

(pasteurized)

EXAMPLE V

Sterilization of Active Composition by Boiling

This Example demonstrates that the allantoin-copper complex can be sterilized by boiling and yet retains substantially all of the activity of the unsterilized formulation. This presents considerable advantage by avoiding the necessity of including potentially sensitizing antimicrobial agents in the compositions. Sterilization by boiling kills virtually all microorganisms. For this test, allantoin was complexed with copper chloride by the procedure described above in Example I. The mixture was thoroughly mixed, then heated in a boiling water bath for 10 minutes. After cooling to room temperature, the active composition was applied to surgical wounds in mice in the manner described in Example II. The healing activity observed with the sterilized allantoin-copper complexes was similar to results obtained with unsterilized active compositions, as shown in Table 4. Each group had six mice.

Table 4: Effect of sterilization on active complex.

Test group Length of wound after 15 days (cm)

Control 0.79 ± 0.12

Allantoin- 1.0% copper complex 0.34 ± 0.13 (no boiling)

Allantoin-

1.0% copper complex 0.35 ± 0.17 (boiled)

EXAMPLE VI Stimulation of Hair Growth by Injection

This Example describes the use of compositions containing allantoin-copper and allantoin-tin complexes to stimulate the growth of hair follicles in warm blooded animals.

The model used in this test was a mouse model that has been found to successfully predict the therapeutic response in humans (see, e.g., U.S. Pat. No. 5,118,665, which is incorporated herein by reference) . Hair growth in mammals proceeds through actively growing stages (anagen) followed by dormant stages (telogen) . The test method generally involves applying the hair-growth stimulant to the skin of mice in telogen phase. Female, Swiss-Webster mice begin a telogen phase at about 45 days of age that lasts until about 90 days of age. After application of the active substance, enhanced hair growth is noted within 14 days. For this test, mice 50 days of age were used.

Allantoin complexes containing copper(II) and tin(II) were tested. For testing, the allantoin-metal pastes were mixed with saline (25% allantoin complex and 75% physiological saline by weight). Mice were shaved, then 0.05 ml of the mixture was infiltrated immediately below the skin by injection. Control mice were injected with an equal volume of saline or with allantoin not complexed with the metal. Each group contained 10 mice. After 20 days, the groups were compared. The percentage of mice with hair growth at the injection site and the relative strength of the hair growth response (on a scale of 1 to 5 where 1 is barely noticeable growth and 5 is very strong hair growth) were determined. The results, shown in Table 5, indicate that all complexes stimulated hair growth.

Table 5: Stimulation of Hair Growth by Allantoin-Metal Complexes.

Percent with Average hair growth at intensity of injection site hair growth

Control mice 0 0 Allantoin-no metal ion 0 0

Allantoin-1% copper(II) 100 3.5

Allantoin-1% tin(II) 90 3.0

It is evident from the above results that the subject invention provides compositions of allantoin-ionic metal complexes for topical skin, wound and surgical treatments to protect damaged skin and facilitate natural healing processes, to enhance tissue regenerative processes in the epidermis, and to stimulate hair growth in warm blooded animals. The invention also provides economical methods for preparing and formulating the compositions for topical administration. All publications and patents mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for accelerating the healing of topical wounds in a warm-blooded animal which comprises: administering to the wound a therapeutically effective amount of a composition which comprises allantoin complexed with an ionic metal.

2. The method of claim 1, wherein the ionic metal is copper(II) , and therapeutically acceptable salts thereof.

3. A method for enhancing the recovery of skin of a warm-blooded animal from irritation, which comprises: administering to the skin irritation a therapeutically effective amount of a composition which comprises allantoin complexed with an ionic metal.

4. The method of claim 3 where the ionic metal is copper(II) .

5. A method for protecting the skin from oxidative damage and aiding the recovery of skin wounds in a warm blooded animal, which comprises: administering to the skin or wound site a prophylactically or therapeutically effective amount of a composition which comprises allantoin complexed with an ionic metal.

6. The method of claim 5, wherein the ionic metal is copper(II) , and therapeutically acceptable salts thereof.

7. A method for increasing hair follicle size and the rate of hair growth in a warm-blooded animal, comprising: administering to the skin of said animal a composition which comprises allantoin complexed with an ionic metal in an amount sufficient to increase hair follicle size and the rate of hair growth in said animal. 1 8. The method of claim 7, wherein the composition

2 is administered topically or by injection into the skin.

1 9. The method of claim 7, wherein the composition

2 is administered topically on a daily basis to the skin area in

3 which enhanced hair growth is desired.

1 10. The method of claim 7, wherein the ionic metal

2 is copper(II) or tin(II) , and therapeutically acceptable salts

3 thereof.

1 11. The method of claim 7, wherein the ionic metal

2 is copper(II) .

1 12. The method of claim 7, wherein the ionic metal

2. is tin(II) .

1 13. A method for preparing a pharmaceutical

2 composition useful for accelerating the healing of topical

3 wounds or increasing hair follicle size and rate of hair

4 growth in a warm-blooded animal, which comprises combining

5 allantoin with an amount of an aqueous solution of ionic metal

6 salt sufficient to form an active composition, comprised of

7 the allantoin complexed with the ionic metal.

1 14. The method of claim 13, further comprising the

2 step of sterilizing or pasteurizing the allantoin-ionic metal

3 complex.

1 15. The method of claim 14, further comprising the

2 step of combining the isolated allantoin-ionic metal complex

3 with a pharmaceutically acceptable carrier.

1 16. The method of claim 15, wherein the isolated

2 allantoin-ionic metal complex is combined with a

3 pharmaceutically acceptable carrier to form a cream or lotion.

17. The method of claim 13, wherein the concentration of the allantoin-ionic metal complex in the pharmaceutical composition is about 10% to 50%.

18. The method of claim 13, wherein the ionic metal is copper(II) or tin(II) .

19. A pharmaceutical composition which comprises a therapeutically effective amount of allantoin complexed with an ionic metal and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, wherein the ionic metal is copper(II) , tin(II) , or a therapeutically acceptable salt thereof.

21. The pharmaceutical composition of claim 20, wherein the allantoin-ionic metal complex is present in the composition at a concentration of 5% to 50%.

22. The pharmaceutical composition of claim 21, wherein the allantoin-ionic metal complex is present in the composition at a concentration of 5% to 25%.