Methods for stimulating proliferation of cells in the epidermis and dermis of mammalian skin are disclosed. The methods comprise the step of administering to an area of the skin a composition comprising a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative. The composition can be administered topically or by injection. The invention also provides a regimen for restoring or maintaining the strength and thickness of aging skin, comprising periodically administering a composition by topical application. The invention further provides methods for stimulating proliferation of cells in the epidermis and dermis of transplanted skin. A composition for topical application is also provided by the invention.

Description of the Invention

SUMMARY OF THE INVENTION

The experiments described herein demonstrate that administration of human placental alkaline phosphatase, but not other members of the alkaline phosphatase family, enhances proliferation of human skin fibroblasts and even human keratinocytes in vitro and in vivo. Thus, PALP may be used as an active component of skin care products designed to enhance thickness and strength of human skin.

The present invention provides a method for stimulating proliferation of cells in the epidermis and dermis of undamaged mammalian skin, by topically administering to an area of the skin a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof. In one embodiment of the method, the skin is human skin. In another embodiment of the method, a physiologically compatible carrier and human placental alkaline phosphatase or active derivative are administered to the skin.

The invention also provides a regimen for restoring or maintaining the strength and thickness of aging skin, by periodically administering by topical application to the skin a composition having an effective amount of human placental alkaline phosphatase, or an active derivative. In one embodiment of the regimen, the composition is applied once per day. In another embodiment of the regimen, the composition is applied about once per week.

Also provided by the invention is a method for stimulating proliferation of cells in the epidermis and dermis of mammalian skin, including the step of administering to the skin by injection a composition comprising a physiologically acceptable carrier, and a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof, dissolved or dispersed in the carrier. In one embodiment of the method, the carrier is a physiological saline solution. In another embodiment of the method, the mode of injection is selected from intravenous, subcutaneous, intramuscular, and intraperitoneal. In yet another embodiment, the mode of injection is intradermal.

The invention further provides a method for stimulating proliferation of cells in the epidermis and dermis of transplanted skin, including the step of topically administering to an area of the transplanted skin a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof. In one embodiment of the method, the transplanted skin is human skin. In another embodiment of the method, the transplanted skin has been transplanted onto a human host. Another embodiment of the method includes a step of topically administering a therapeutically effective amount of human placental alkaline phosphatase to an area of host skin that is adjacent to the transplanted skin.

The invention also provides a method for stimulating proliferation of cells in the epidermis and dermis of transplanted skin, including the step of administering to an area of the transplanted skin by injection a composition comprising a physiologically acceptable carrier, and a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof, dissolved or dispersed in the carrier.

A composition for topical application is also provided by the invention. In one embodiment, the composition includes either vaselinum flavum or vaselinum album and an amount of human placental alkaline phosphatase effective to stimulate proliferation of cells in the epidermis and dermis of mammalian skin. In another embodiment, the composition essentially contains vaselinum flavum or vaselinum album and human placental alkaline phosphatase. In a further embodiment, the composition essentially contains vaselinum cholesterinatum and human placental alkaline phosphatase.

DETAILED DESCRIPTION OF THE INVENTION

The experiments described herein demonstrate that topically or intradermally applied human placental alkaline phosphatase enzyme, but not other types of alkaline phosphatase enzymes, enhances the number of cells in both the epidermal and dermal layers of mouse dorsal skin, and of human skin transplanted onto mouse skin. Administration of human placental alkaline phosphatase was also found to enhance DNA synthesis and proliferation of HaCaT human keratinocytes and human skin fibroblasts in vitro. In one experiment, complete digestion of PALP by the protease bromelain failed to diminish, and instead slightly enhanced, the effect of PALP on DNA synthesis in fibroblasts. These observations imply that either human placental alkaline phosphatase or an active derivative thereof can be used as an active component of skin care products designed to thicken and strengthen aging skin and repair the epidermal and dermal layers of unhealthy skin of mammals and humans.

Another therapeutic application described herein for human placental alkaline phosphatase is to increase the likelihood of survival of transplanted skin by stimulating the proliferation of dermal and epidermal cells in the transplanted skin.

The Active Component

The methods and treatment regimens described herein include administration of a composition comprising human placental alkaline phosphatase, or an active derivative thereof. The composition generally comprises human PALP, or active derivative, in a carrier.

The active component in the methods and compositions of the present invention is human PALP, or an active derivative thereof. As used herein, the term "PALP" and the phrase "human PALP" are used interchangeably to refer to human placental alkaline phosphatase. As used herein, the phrase "active component" means a protein, enzyme, or peptide that, when administered at an appropriate concentration or quantity, is effective to stimulate proliferation of cells in the dermis or in the epidermis of mammalian skin, or is effective to restore strength or thickness to aging skin.

Placental alkaline phosphatase is a member of the alkaline phosphatase group of enzymes that hydrolyze phosphate-containing compounds at alkaline pH. Mature PALP is a dimer of two identical glycosylated subunits. Subunits of human placental alkaline phosphatase ("PALP") have an approximate molecular weight of 66 kDa, as determined by gel electrophoresis.

As is demonstrated by the Examples herein, whole PALP enzyme in its native state is not required to achieve a beneficial effect. Derivatives of PALP are therefore suitable as an active component for the practice of the present invention. For example, digestion of PALP by a protease, such as bromelain, provides an active derivative. Likewise, one who is skilled in the art may synthesize or develop an active derivative that is a smaller fragment of a PALP amino acid sequence and demonstrates efficacy similar to that of native PALP enzyme. By way of example, modification of a PALP amino acid sequence, or a sequence of smaller PALP peptides, by exchanging amino acids at critical sites to yield an active derivative may improve the beneficial effects disclosed herein. In the practice of the present invention, it is envisioned that modified PALP, smaller PALP-derived peptides, or modified PALP-derived peptides may be similarly effective or even more effective than the native PALP enzyme, and are each considered to be active derivatives.

Human PALP in solid form is available commercially from Sigma Chemical (St. Louis, Mo.), for example (Sigma catalog number P3895; CAS Registry Number 9001-78-9). Another commercial source of human PALP is Calbiochem (San Diego, Calif.; catalog number 524604).

Human PALP, or an active derivative, may also be obtained by chemical synthesis using conventional methods. For example, solid-phase synthesis techniques may be used to obtain synthetic PALP or an active derivative.

Recombinant methods of obtaining quantities of PALP (or an active derivative) are also suitable. Since the cDNA of PALP is available, recombinant protein can be produced by one of the many existing conventional methods for recombinant protein expression. PALP has been cloned and overexpressed in several cell lines, as described by Millan, et al. [Millan, J. L. and Fishman, W. H., "Biology of human alkaline phosphatases with special reference to cancer." Critical Tev. Clin. Sci. 22, 1-39 (1995)]. Production of recombinant PALP has been difficult to date, although low yields have been obtained from bacteria using Escherichia coli [Beck, R., and Burtscher, H., "Expression of human placental alkaline phosphatase in Escherichia coli." Protein Expression and Purification 5, 192-197 (1994)] and from yeast using Pichia pastoris [Heimo, H., Palmu, K., and Suominen, I., "Human placental alkaline phosphatase: Expression in Pichia pastoris, purification and characterization of the enzyme." Protein Expression and Purification 12, 85-92 (1998)].

Recombinant PALP enzymes for the present study were prepared by a method described recently [Kozlenkov, A., Manes, T., Hoylaerts, M. F., and Millan, J. L., "Function assignment to conserved residues in mammalian alkaline phosphatases." J. Biol. Chem. 277, 22992-22999 (2002)].

A PALP preparation that is commercially available, synthesized, or produced by a recombinant method will generally contain impurities. Impure PALP preparations can be used as starting material to obtain homogeneous PALP by successive chromatographic steps, as described in detail in Example 1. Impure PALP preparations may also be used in formulating a composition for use in the practice of the present invention, so long as the composition comprises therapeutically effective amount of the active component, and any impurities are not toxic and do not interfere with the beneficial effects of the active component.

A preparation comprising human PALP may also be obtained by extraction from placental tissue. Human placenta synthesizes the enzyme during pregnancy, so that toward the end of third term the enzyme's level in the placenta tissue and maternal/fetal blood becomes very high. By way of example, a preparation may be obtained by butanol extraction of homogenized placenta. Other methods of extraction from placental tissue are also suitable.

Raw placental extracts that are not further enriched in PALP by using physical concentration methods cannot be expected to have physiological effects similar to those observed for a preparation of purified or homogeneous PALP, for at least two reasons. First, the relative concentration of PALP in an extract will be too low to expect a readily detectable effect in the skin. Second, raw placental extracts contain not only many different proteins but also other kinds of compounds, such as many lipids, proteolipids, carbohydrates, metals, vitamins, and the like.

Therefore, if placenta-derived PALP preparation is to be used in the practice of the present invention, a raw extract should be treated to enrich the concentration of PALP and obtain a purified preparation. A purified preparation will have a higher concentration of the active component than is found in a raw tissue extract, such as a raw placental extract. The term "purified" is used herein to encompass compositions that are obtained from a starting material by one or more purification steps (such as solvent extraction, column separation, chromatographic separation, etc.) that enrich the concentration of the active component, relative to the starting material. The term "purified" should not be construed to connote absolute purity.

The term "homogeneous" is used herein to indicate a composition that yields a single protein band in an electrophoretic gel separation, such as by the SDS-PAGE technique described in Example 1. The phrases "homogeneous human placental alkaline phosphatase" or "homogeneous PALP" therefore include compositions that contain predominantly PALP and yield a single band for PALP enzyme in an electrophoretic separation. Homogeneous PALP may be obtained, for example, from a raw placental extract (or from a purified preparation) by the purification procedures described in Example 1.

In the experiments described in the following Examples using placenta-derived PALP preparations, either a homogeneous PALP preparation obtained by a purification procedure described in Example 1 or a commercially available PALP preparation was employed. The commercial PALP preparation contains only a few proteins (see Example 1) as impurities, and the homogeneous PALP preparation contains no other proteins as detectable impurities. Thus, these preparations are qualitatively different from the previously used placenta extracts. Recombinant PALP preparations also contained no other proteins as detectable impurities.

As a result, the effects observed in the present experiments are qualitatively different from those that might have been observed previously by others using raw placental extracts. Based on any previously reported effects using placental extracts, the effects that were observed in the present experiments on the growth of skin cells using the PALP-containing compositions described herein could not have been predicted.

The likelihood of any toxic effects via local or intradermal application of PALP is insignificant because the protein's concentration in the circulation will be very small (probably undetectable). Even systematically applied, PALP is unlikely to be toxic, considering that the enzyme is produced by the placenta at a very high level during the second and third trimester of the pregnancy, and that the enzyme is readily detectable in the maternal blood. Furthermore, the in vivo half-life of PALP in humans is relatively short. The half-life has been measured as about 7 days [Clubb, J. S., Neale, F. C., and Posen, S., "The behavior of infused human placental alkaline phosphatase in human subjects." J. Lab. & Clin. Med. 66, 493-507 (1965)].

A further consideration in the practice of the invention is the degree of purity that is required for a PALP preparation that is to be administered to a human subject. An advantage of using a preparation comprising highly purified or homogeneous PALP in the methods and treatment regimens of the present invention is that possible side effects caused by contaminating proteins will not likely be an issue. However, impure PALP or PALP that is purified but not homogeneous (such as that comprising the commercially available human PALP from Sigma) may also be used in the compositions described herein, so long as no adverse effects are observed. Since each additional purification step results in significant loss of the enzyme, using a less pure PALP material for PALP preparations would be more cost-effective.

Compositions for Topical Administration

The present invention includes methods for stimulating proliferation of skin cells, and methods for restoring strength and thickness to aging skin, comprising a step of topically applying a composition comprising human placental alkaline phosphatase to the skin. The composition generally comprises human PALP or active derivative in a carrier. In a composition for administration to a human, the carrier should be a physiologically compatible or acceptable carrier.

For topical application, appropriate forms of the composition include, for example, creams, gels, lotions, unguents, emollients, colloidal dispersions, suspensions, emulsions, oils, sprays, foams, mousses, and the like. Compositions suitable for topical administration may also include, for example, liposomal carriers made up of lipids or special detergents.

Compositions suitable for topical application in the practice of the present invention generally include the active component as a minor ingredient, and the physiologically compatible carrier as a major ingredient. In some embodiments, the compositions may include one or more additives or enhancers, such as preservatives, buffers, moisture-control compounds, or antibiotics, for example. In other embodiments, the composition essentially contains the carrier and the active component. As used herein, the phrase "essentially contains" means that the composition has no other ingredient, other than the recited active component, that significantly stimulates proliferation of cells in the epidermis or dermis.

The carrier may be in any form appropriate for topical application to the skin. Any physiologically compatible carrier in which the active component is at least minimally soluble is suitable for topical compositions of the present invention. A physiologically acceptable carrier is one that is non-toxic, does not cause an adverse physical reaction upon administration, and in which the active component is sufficiently soluble or compatible so that the composition can provide an effective amount of the active component. The carrier should also provide a composition of appropriate consistency for topical administration and should be capable of achieving proper distribution of the active component to the treated tissue.

Suitable carriers generally include, for example, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, mixtures thereof and the like. Buffered solutions can also serve as carriers.

In some embodiments, the topical composition is a gel. The gel may include as a carrier methyl cellulose, agar, vaseline or petrolatum, agarose, gelatin, calcium alginate or combinations thereof. By way of example, the active component can be incorporated into sterile 3% by weight methyl cellulose gel, 1% by weight agarose gel, 4% by weight gelatin gel, or 1 to 3% by weight calcium alginate.

Gels of more complex composition can also be formulated. In some embodiments, the carrier includes vaselinum flavum (yellow petrolatum), vaselinum album (white petrolatum), or vaselinum cholesterinatum. Commercially available vaselinum cholesterinatum consists of about 1.5 wt.-% cholesterol, about 5.0 wt.-% cerae lanae, and about 93.5 wt.-% vaselinum flavum.

Additives or enhancers may optionally be included in the topical compositions. The criterion for using an additive is that it increases, or at least does not significantly impair, the effectiveness of the active component in achieving the desired beneficial effect. Additives or enhancers in compositions for topical application may include various ingredients, for example, preservatives (such as parabens, quaternary ammonium compounds, alcohols, phenols, essential oils and the like), buffers, antioxidants (such as vitamin E), antimicrobials, vitamins, and moisture-control agents (such as glycerine, propylene glycol, and the like). Other potential additives include, for example, analgesics, anesthetics, anti-acne agents, anti-dermatitis agents, anti-pruritic agents, anti-inflammatory agents, anti-hyperkeratolytic agents, antiperspirants, anti-psoriatic agents, anti-seborrheic agents, anti-aging agents (such as retinoids), anti-wrinkle agents, skin-lightening agents, depigmenting agents, corticosteroids, additional tanning agents or hormones. Other additives may include, for example, colorants, sunscreens, emulsion stabilizers, preservatives, fragrances, humectants, waterproofing agents, viscosity modifying agents, and the like.

In one embodiment, the composition includes a penetration-enhancing additive that enhances penetration of the human placental alkaline phosphatase into the skin. Many conventional penetration enhancers are suitable in the practice of the invention. Non-limiting examples of suitable penetration enhancers include: sulfoxides such as dimethyl sulfoxide (DMSO); alcohols such as ethanol; polyols such as propylene glycol; surfactants such as sodium lauryl sulfate, lecithin, docusate sodium, and polysorbates; fatty acids such as lauric acid, myristic acid, palmitic acid, and stearic acid; esters such as isopropyl palmitate and isopropyl myristate; and amides such as urea.

Compositions for topical administration can be made using any number of suitable techniques. For example, a carrier, a preparation comprising an active component, and any optional additives can be mixed together using a commercial mixer to form a suspension, gel, solution or the like. Conventional methods known in the art are suitable. The compositions can be additionally processed after formulation. Sterilization, for example, can be conducted by filter sterilization, irradiation or the like. Methods for conducting these steps are also conventional in the art.

Both purified PALP preparations and homogeneous PALP preparations obtained from placental extracts can be used as the active component in the compositions described herein. The less pure the PALP preparation is, proportionally more of the PALP preparation must be used for the topical composition in order to provide an effective amount of the active component. Alternatively, a preparation comprising synthetic PALP or active derivative, or recombinant PALP or active derivative, can be employed as the active component. A preparation comprising an active component can also be derived from any of these PALP sources, such as by digestion of a PALP preparation with bromelain, for example.

In some embodiments, the composition comprises a therapeutically effective amount of human placental alkaline phosphatase, or active derivative thereof. The term "therapeutically effective amount" in this specification and in the claims indicates a dosage that is effective in, or is targeted to, either enhancing proliferation of fibroblasts or keratinocytes, or restoring or maintaining strength or thickness of aging skin. A therapeutically effective amount of an active component may vary based on the needs or tolerance of the individual subject, the degree to which the ability of fibroblasts or keratinocytes to proliferate has degenerated, the degree to which strength or thickness of the subject's skin has deteriorated, or other criteria evident to one of ordinary skill in the art.

In one embodiment of the invention, a composition for topical application includes a sufficient quantity of an active component to enhance proliferation of fibroblasts in the dermis. In another embodiment, a composition for topical application includes a sufficient quantity of an active component to enhance proliferation of keratinocytes in the epidermis. In yet another embodiment, topical administration of the composition enhances both proliferation of fibroblasts in the dermis and proliferation of keratinocytes in the epidermis.

Generally, the concentration of active component in a composition for topical application will be at least about 0.01 wt.-% and more suitably, between about 0.1 and about 1 wt.-%. In one embodiment, the composition comprises about 0.1 to about 0.5 wt.-% of the active component.

As one suitable composition for topical application, the invention provides a composition for topical application comprising either vaselinum flavum or vaselinum album, and an amount of human placental alkaline phosphatase effective to stimulate proliferation of cells in the epidermis and dermis of mammalian skin. The composition may include an additive that enhances penetration of the human placental alkaline phosphatase into the skin. In one embodiment, the composition contains about 0.1 to about 1 wt.-% human placental alkaline phosphatase. In another embodiment, the composition essentially contains vaselinum flavum and human placental alkaline phosphatase. In another embodiment, the composition essentially contains vaselinum album and human placental alkaline phosphatase. In yet another embodiment, the composition essentially contains vaselinum cholesterinatum and human placental alkaline phosphatase.

The invention further includes the use of human placental alkaline phosphatase in the manufacture of a topical composition effective to stimulate proliferation of cells in the epidermis and dermis of undamaged mammalian skin.

Topical Administration

The present invention provides a method for stimulating proliferation of cells in the epidermis and dermis of undamaged mammalian skin, comprising the step of topically administering to an area of the skin a composition comprising a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof.

The methods described herein are suitable for mammalian skin. The subject can be canine, porcine, or bovine, for example. Most suitably, the subject is human.

In the practice of the method, topical application of the composition is effective to stimulate proliferation of cells in the epidermis and dermis of the treated skin. Proliferation is stimulated when the rate of cell replication is increased (such as by an increase in the frequency of mitosis), relative to an untreated condition. In particular, proliferation of fibroblasts in the dermis may be enhanced. The methods also may be effective to stimulate proliferation of keratinocytes in the epidermis. The methods of the invention may also be effective to increase the viability of cells in the dermis or epidermis, or both.

The composition is applied to an undamaged area of skin. The term "undamaged" as used herein with reference to an area of skin indicates that the area of skin is free from wounds including cuts or punctures, abrasions, sores, scars, bruises, or burns of moderate- to high-degree that would require medical treatment. The methods of the invention are not intended for healing wounded or significantly damaged skin.

Topical administration can be accomplished via manual application of a composition such as a cream, a lotion, a gel and the like that includes the active component. A composition may also be applied by other means, such as by spraying, applying with a pad or towelette, etc. In some embodiments, a composition can be delivered by means of a dressing, bandage, patch, or other similar covering capable of releasing a therapeutically effective amount of the active component. Other methods of delivering the PALP compositions are also within the scope of this invention.

The method may suitably be practiced using any of the compositions described above, comprising a physiologically compatible carrier and human placental alkaline phosphatase, or an active derivative thereof.

In some experiments described herein, topical administration of PALP led to the stimulation of cells in the epidermis in the hair follicles. Although not studied in detail, this observation suggests that PALP may exert a positive effect on hair strength or even on hair growth.

Regimen for Restoring or Maintaining Strength and Thickness in Aging Skin

The invention also provides a regimen for restoring or maintaining the strength and thickness of aging skin, comprising periodically administering by topical application to the skin a composition comprising an effective amount of human placental alkaline phosphatase, or an active derivative.

A characteristic of aging skin is that skin cells, particularly fibroblasts, progressively lose their ability to proliferate. Aging skin is further characterized by a decrease in flexibility and tensile strength. Induction of relatively long-lasting changes in the skin requires agents and/or procedures that enhance the number of viable keratinocytes in the epidermis and fibroblasts in the dermis. Long-lasting changes can be achieved by either enhancing the lifetime of the cells or stimulating their proliferation, or both. Agents that enhance proliferation of epidermal and dermal cells in the skin, including fibroblasts and keratinocytes, are likely to be effective in restoring or maintaining the strength and thickness of aging skin.

In the practice of the regimen, any of the compositions described above may be used. The composition is applied topically to the skin, as described above. The composition is applied periodically over a period of time. As used with respect to the regimens described herein, the term "periodically" refers to repeated administration targeted to restoring or maintaining the strength and thickness of the skin, over the time of treatment. The term "periodically" includes repeated administration at fixed intervals, but also includes repeated administration over irregular intervals as is required by the subject's condition. The composition can be administered as needed. Alternatively, the composition can be administered two or more times a day. The frequency of administration of the composition can vary and depend on the type of skin, the location of the treated skin, the concentration of the active component in the composition, and the method used to administer the composition.

Generally, a therapeutically effective amount of the active component is administered. In the regimens of the invention, however, the effective amount of the active component that is administered does not need to be identical for each separate administration. More or less of the active component may be administered in separate administrations, as the subject's needs dictate. A medical professional supervising treatment can adjust administered doses to obtain desired results.

The frequency of application and the duration of the regimen will depend on the physiological state of the skin and, on the magnitude of response, and the level of satisfaction by the treated subject. In one embodiment of the regimen, the composition is applied once per day. In another embodiment of the regimen, the composition is applied about once per week.

For topical application, a recommended treatment schedule includes once-a-day treatment for the first week, three-times-a-week treatment for the second week, twice-a-week treatment for the third week, and once-a-week treatment for the following weeks, for a time period that is determined by the level of success.

While the important structural changes in the skin are expected to take place during the first three weeks after the regimen is commenced, a once-a-week treatment schedule may be desired after the first three weeks to maintain the improved texture of the skin. In other embodiments involving skin that is resistant to treatment or is badly deteriorated, the composition may be administered periodically for several months or more.

Administration by Injection

Also provided by the invention is a method for stimulating proliferation of cells in the epidermis and dermis of mammalian skin, comprising the step of administering to the skin by injection a composition comprising a physiologically acceptable carrier, and a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof, dissolved or dispersed in the carrier.

As a complement to the topical methods described above, other application methods, including various forms of injections, will also elicit the desired effects in the skin. In the case of injection, the active component will be transported to the skin either directly (such as for intradermal application or partly subcutaneous application) or via the blood supply (such as for intravenous, intraperitoneal, or subcutaneous applications). A composition comprising an active component may be administered via intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal injection, intramuscular injection, or any other mode of delivery that ensures appropriate distribution and relative stability of the enzyme in the body.

For injection of a composition comprising an active component, the carrier can be any physiologically acceptable carrier that does not cause an undesirable physiological effect and is capable of ensuring proper distribution of the active component in the treated tissue. The active component is dissolved or dispersed in the physiologically acceptable carrier. Examples of carriers include physiological saline and phosphate-buffered saline. Alternatively, the active component may be enclosed in liposomes such as immunoliposomes, or other delivery systems or formulations as are known to the art may be employed. By way of example, the active component can be readily dissolved in physiological saline (0.9 M NaCl), or in any other physiologically competent carrier, to yield a solution for injection.

An injectable composition can be prepared by dissolving or dispersing a suitable PALP preparation in the carrier using conventional methods. As examples only, one suitable composition for the practice of the method comprises human PALP dissolved in a 0.9 N physiological salt solution to yield a PALP concentration of 10 mg/mL. Another suitable composition comprises human PALP dissolved in a 0.9 N physiological salt solution to yield a PALP concentration of 30 mg/mL.

The injectable composition can be modified by any number of additives, as listed above for the topical application, that can be dissolved or suspended in the composition and that are expected to enhance the effects of the active component or diminish any potential side effect.

In one embodiment of the method, the mode of injection is selected from intravenous, subcutaneous, intramuscular, and intraperitoneal. The mode of injection is selected to provide either local delivery (such as by intradermal application or partly subcutaneous application) or systemic delivery via the blood supply (such as for intravenous, intraperitoneal, or subcutaneous applications).

Since human PALP is a relatively large protein, and its actions involve multiple layers of skin, systemic administration is an appropriate mode of administration. Systemic administration of PALP is reported to be effective to modulate blood glucose levels, although the effect is not expected to be observed unless the subject already has an elevated blood glucose level (such as for a diabetic). In such cases, it may be desirable to avoid systemic administration in the practice of the methods of the present invention.

A common way to express a suitable dosage for systemic administration is grams of active agent per square meter of body surface area for the subject. Several formulas are known for estimating a human subject's body surface area, based on the human's height (in cm) and mass (in kg). Table 1 (see Original Patent) lists a variety of known formulas for estimating body surface area (BSA) proposed by researchers. Other suitable formulas may likewise be employed.

In case of intravenous, intramuscular, intraperitoneal, or subcutaneous application, the subject may be administered about 1 to about 5 g/m.sup.2 once daily. Alternatively, the subject may be administered about 0.2 to about 1.0 g/m.sup.2 once daily.

In another embodiment, a subject may be administered by intravenous, intramuscular, intraperitoneal, or subcutaneous application about 0.2 to about 3.0 g/m.sup.2 once or twice weekly. Alternatively, the subject may be administered about 0.2 to about 3.0 g/m.sup.2 once biweekly by intravenous, intraperitoneal, or subcutaneous application.

In another embodiment, the subject may be administered about 1 to about 5 g/m.sup.2 by intravenous, intramuscular, intraperitoneal, or subcutaneous application once daily for several days, with treatment then continued by less frequent applications of smaller doses.

If the PALP solution is injected locally, such as when the mode of injection is intradermal, aliquots of about 10 to about 100 .mu.L per injection site may be administered. The concentration of the active component in the injectable composition may be in the range of about 1 to about 30 mg/mL. Alternatively, the concentration of the active component may be in the range of about 2 to about 10 mg/mL. In one embodiment, a plurality of injection sites is treated for one administration.

Intradermal application may be an especially effective mode of application in the practice of the present invention. Intradermal application may require use of less of the active component, as compared to other modes of injection. Also, for localized application the active component may be more effectively delivered or transported to the epidermal and dermal layers of the treated skin.

The invention further includes the use of human placental alkaline phosphatase in the manufacture of an injectable composition effective to stimulate proliferation of cells in the epidermis and dermis of undamaged mammalian skin.

Application to Transplanted Skin

The invention further provides a method for stimulating proliferation of cells in the epidermis and dermis of transplanted skin, comprising the step of topically administering to an area of the transplanted skin a composition comprising a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof. Any of the topical compositions described above are suitable in the practice of this method. The method may be effective to enhance the chance of survival of the transplanted skin (i.e., to increase the likelihood of a successful transplant).

In one embodiment of the method, the transplanted skin is human skin. In another embodiment of the method, the transplanted skin has been transplanted onto a human host. Another embodiment of the method includes a step of topically administering the composition to an area of host skin that is adjacent to the transplanted skin.

The invention further includes the use of human placental alkaline phosphatase in the manufacture of an topical composition effective to stimulate proliferation of cells in the epidermis and dermis of transplanted mammalian skin.

The invention also provides a method for stimulating proliferation of cells in the epidermis and dermis of transplanted skin, comprising the step of administering to an area of the transplanted skin by injection a composition comprising a physiologically acceptable carrier, and a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof, dissolved or dispersed in the carrier. The method may be effective to enhance the chance of survival of the transplanted skin (i.e., to increase the likelihood of a successful transplant).

In one embodiment of the method, the transplanted skin is human skin. In another embodiment of the method, the transplanted skin has been transplanted onto a human host. Another embodiment of the method includes a step of administering the composition by injection to an area of host skin that is adjacent to the transplanted skin. In another embodiment of the method, the mode of injection is intradermal.

The invention further includes the use of human placental alkaline phosphatase in the manufacture of an injectable composition effective to stimulate proliferation of cells in the epidermis and dermis of transplanted mammalian skin.
 

Claim 1 of 29 Claims

1. A method for stimulating proliferation of cells in an epidermis and a dermis of undamaged mammalian skin, comprising the step of topically administering to an area of the skin a composition comprising Vaselinum flavum, Vaselinum album or Vaselinum cholesterinatum, and human placental alkaline phosphatase.
 

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