PLACENTAL ALKALINE PHOSPHATASE TO PROMOTE SCAR-FREE HEALING OF ACNE VULGARIS AND OTHER INFLAMMATORY SKIN LESIONS CAUSED BY MICROBIAL INFECTIONS

Abstract

This invention demonstrates that topically applied placental alkaline phosphatase dispersed in Vaselinum cholesterinatum promotes scar-free healing of acne vulgaris of bacterial origin. Other inflammatory skin conditions caused by bacterial infection and improved by placental alkaline phosphatase include nail infections as well as infections of non-healing diabetic wounds, puncture wounds, surgical wounds and incisions, burns, abrasions, lacerations, ulcers, gum disease, skin tears, or wounds caused by bites or stings. Placental alkaline phosphatase was also found to promote healing of skin lesions, with or without co-administering an antiviral drug, caused by reactivated Varicella zoster (Shingles), Herpes Simplex Virus-1 (oral herpes), or Herpes Simplex Virus 2 (vaginal/genital herpes). In all of these cases, placental alkaline phosphatase was found to significantly reduce irritation or pain associated with bacterial or viral infection and resulting skin damage.

Claims

1. A method for promoting healing of acne and other skin lesions caused by bacterial infection comprising the step of topically administering to an area of the skin lesion a composition comprising a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof, dispersed in a suitable carrier.

2. The method of claim 1, wherein the other skin lesions include nail infections and infected wounds including non-healing diabetic wounds, puncture wounds, surgical wounds and incisions, burns, abrasions, lacerations, ulcers, gum disease, skin tears, or wounds caused by bites or stings.

3. The method of claim 1, wherein the amount of placental alkaline phosphatase or an active derivative thereof in the topically administered composition is 0.1 to 0.5 mg, 0.51 to 2 mg, and 2.1 to 10 mg PLAP per one gram carrier applied to light, moderate, and severe acne lesions, respectively.

4. The method of claim 2, wherein the amount of placental alkaline phosphatase or an active derivative thereof in the topically administered composition is 1.0 to 10.0 mg per 1 gram carrier.

5. The method of claim 4, wherein the amount of placental alkaline phosphatase or an active derivative thereof in the topically administered composition is 1 mg and 5 mg per 1 gram carrier when the size of the infected lesion is 1 cm.sup.2 and 10 cm.sup.2, respectively with every two cm.sup.2 increase in the infected area resulting in the addition of 1 mg placental alkaline phosphatase per 1 g carrier.

6. The method of claim 1 wherein topical treatment of acne is applied daily as long as needed.

7. The method of claim 2, wherein in case of nail infection, on the first day the treatment is repeated hourly for 3 hours followed covering the lesion. The treatment is repeated on the second day and if still needed on the third day.

8. The method of claim 2, wherein topical treatment of infected wound applied daily as long as needed.

9. A method for promoting healing of skin lesions caused by reactivated Varicella zoster (Shingles), Herpes Simplex Virus-1 (oral herpes), or Herpes Simplex Virus 2 (mostly vaginal/genital herpes) comprising the step of topically administering to an area of the skin lesion a composition comprising a therapeutically effective amount of human placental alkaline phosphatase, or an active derivative thereof, dispersed in a suitable carrier.

10. The method of claims 9, wherein. the amount of placental alkaline phosphatase or an active derivative thereof is 2.0 mg and between 2.1 to 10.0 mg per 1 gram carrier when the lesion is 10.0 cm.sup.2 or smaller and when the lesion is larger than 10 cm.sup.2, respectively.

11. The method of claim 9, wherein the skin lesion is Shingles, which is also treated with an orally administered antiviral drug, for example acyclovir, valacyclovir or famciclovir, as prescribed, if the lesion is larger than 2.0 cm.sup.2; if the lesion is smaller than 2.0 cm.sup.2, treatment with an orally administered antiviral drug is optional.

12. The method of claim 9, wherein co-treatment of the skin lesions caused by reactivated Herpes Simplex Virus-1 or Herpes Simplex Virus 2 with an oral antiviral drug is optional regardless of the lesion’s size.

13. The method of claims 9 to 12, wherein the treatments with topical placental alkaline phosphatase or an active derivative are administered once daily until the lesion is healed.

14. The method of claim 1, wherein the suitable carrier is Vaselinum cholesterinatum.

15. The method of claims 1 and 9, wherein the suitable carrier is Vaselinum, flavum, Vaselinum albaum, a gel, a lotion, an unguent, an emollient, a colloidal dispersion, a suspension, an emulsion, an oil, a spray, a liposomal preparation, a non-ionic detergent, foams, mousses, and the like as used in the skin care industry that are safe to use on the skin.

16. The method of claims 1 and 9, wherein placental alkaline phosphatase or an active derivative thereof effectively reduces pain and irritation caused by the lesion.

17. The method of claims 1 and 9, wherein the active derivative is a recombinant form of the full length placental alkaline phosphatase.

18. The method of claim 17, wherein the active derivative has catalytic alkaline phosphatase activity and contains 80 to 100% of the sequence of the full length placental alkaline phosphatase.

19. The method of claim 2, wherein placental alkaline phosphatase or an active derivative thereof dispersed in a suitable carrier is topically administered onto a wound to reduce inflammation and associated pain caused by bacterial infection along with orally administered antibiotics as prescribed.

20. The method of claim 2, wherein placental alkaline phosphatase or an active derivative thereof dispersed in a suitable carrier is topically administered onto a wound not responding to antibiotics to reduce inflammation and associated pain caused by drug resistant bacterial infection.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows a section of right side of face of a 16-year-old female subject before treatment (A) and after daily treatment with PLAP cream for 3 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 2 months with only small visible positive effects seen.

[0033] FIG. 2 shows a section of left side of face of the same female subject shown in FIG. 1 before treatment (A) and after daily treatment with PLAP cream for 3 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 2 months with only small visible positive effects seen.

[0034] FIG. 3 shows a section of right side of face of a 17-year-old male subject before treatment (A) and after daily treatment with PLAP cream for 3 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 3 months with only small visible positive effects seen.

[0035] FIG. 4 shows a section of left side of face of the same male seen shown in FIG. 3 before treatment (A) and after daily treatment with PLAP cream for 3 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 3 months with only small visible positive effects seen.

[0036] FIG. 5 demonstrates that combined daily treatment of a 72-year-old female subject with PLAP containing cream and acyclovir (four times daily as prescribed) for eight days helped to greatly reduce her frequently recurring shingles on her back.

DETAILED DESCRIPTION

A. The Active Agent

[0037] The active agent is full length placental alkaline phosphatase (PLAP) or an active derivative of PLAP. The alkaline phosphatase group of enzymes hydrolyze phosphate-containing compounds at alkaline pH. Mature PLAP is a dimer of two identical glycosylated subunits. Each subunit has an approximate molecular weight of 66 kDa, as determined by gel electrophoresis. As used herein, the phrase “active derivative of PLAP” means a sequence specifically derived from PLAP produced by a recombinant, enzymatic, or chemical method that can promote healing of acne. The recombinant active derivative (rPLAP) has catalytic alkaline phosphatase activity and contains at least 80% of the sequence of the full length PLAP.

[0038] Both PLAP and rPLAP; stimulate proliferation of skin fibroblasts and epidermal cells [Kiss, Z. U.S. Pat. 7,374,754, titled: Use of placental alkaline phosphatase to promote skin cell proliferation] which is important with respect to the healing phase of acne.

[0039] In the present application, for human use PLAP was highly purified from commercial PLAP (Sigma-Aldrich) as described in detail earlier [Kiss, Z. U.S. Pat. 7,374,754, titled: Use of placental alkaline phosphatase to promote skin cell proliferation]. The rPLAP used for in vitro studies with skin cells were prepared using a previously described method [Kozlenkow, A., Manes, T., Hoylaerts, M.F. and Millan, J.L. Function assignment to conserved residues in mammalian alkaline phosphatase. J. Biol. Chem. 277, 22992-22999, 2002].

[0040] Other recombinant methods to obtain quantities of PLAP (and active derivative) are also suitable. Since cDNA of PLAP is available, recombinant protein can be produced by one of the existing conventional methods for recombinant protein expression as reported by others [Beck, R. and Burtscher, H. Expression of human placental alkaline phosphatase in Escherichia coli. Protein Expression and Purification 5, 192-197, 1994; Heimo, H., Palmu, K. and Suominen, I. Human placenta alkaline phosphatase: Expression in Pichia pastoris, purification and characterization of the enzyme. Protein Expression and Purification 12, 85-92, 1998].

[0041] Bacterial expression yields non-glycosylated PLAP. So far there is no reported evidence that the biological effects of native glycosylated PLAP and bacteria produced PLAP would be significantly different. Thus, in the methods of the present invention native glycosylated PLAP and its active derivatives as well as non-glycosylated PLAP and its active derivatives may be used interchangeably.

[0042] If placenta derived PLAP preparation is to be used in the practice of the present invention, a raw extract should be treated to enrich the concentration of PLAP and obtain a highly purified preparation. A highly purified preparation will have a much higher concentration of the active component than found in a raw tissue extract. A highly purified PLAP preparation does not contain detectable amounts of other proteins or other impurities, or it contains such minimum amounts of reliably identified contaminants that the benefits of using such preparation far out-weight the accompanying potential risks. A highly purified PLAP preparation obtained from a starting placenta derived material by several purification steps (such as solvent extraction, column separation, chromatographic separation, etc.) that enrich the concentration of PLAP, relative to the starting material, to an extent that PLAP is the dominating component, and the remaining components do not pose any significant health risk and do not reduce the beneficial effects of PLAP. The term “highly purified” should not be construed to connote absolute (100%) purity.

B Methods of Use

Local Treatment of Acne With PLAP Cream

[0043] Highly purified placenta derived PLAP, or essentially pure recombinant PLAP, or an essentially pure active derivative thereof, is dispersed in a suitable carrier and gently massaged onto the acne lesion. The carrier used in the invention is Vaselinum cholesterinatum, but it may be replaced by Vaselinum flavum or Vaselinum album. Vaselinum cholesterinatum consists of about 1.5 wt.-% cholesterol, about 5.0 wt.-% cerae lanae, and about 93.5 wt.-% Vaselinum flavum. In this invention, for a preparation containing PLAP or rPLAP in a carrier the term “PLAP cream” is used.

[0044] Other appropriate forms of PLAP containing compositions may contain other suitable carriers in the forms of gels, lotions, unguents, emollients, colloidal dispersions, suspensions, emulsions, oils, sprays, liposomes, non-ionic detergents, foams, mousses, and the like as used in the skin care industry with no damaging effects on the skin. Preferably, the chosen carrier would enable PLAP to exert its antiacne effect or at least would not reduce it. In addition, the carrier should not be comedogenic and should not inhibit interaction of PLAP with the acne lesion. In the preparation of the carrier-PLAP mixture, PLAP is first dissolved in distilled water or a suitable buffer and then mixed with the carrier. The PLAP cream contains highly purified PLAP or rPLAP, in the amount of 0.1 to 10 mg per one gram carrier gently massaged onto the area of acne lesion. The amount of PLAP depends on the severity of acne. In cases of light, moderate and severe acnes the recommended amount of PLAP per 1 gram carrier is 0.1 to 0.5 mg, 0.6 to 2 mg, and 2.1 to 10 mg PLAP, respectively. Before each daily treatment the acne lesion needs to be cleaned with a facial cleanser as recommended by the manufacturer. The length of the daily treatments is typically for about 3 weeks, but it may be extended until the lesion is healed.

[0045] Therapeutically effective amounts of recombinant PLAP or other active derivatives of PLAP, may also be employed as the active components for the treatment of acne with the same recommended amounts as indicated for PLAP. The term “active” means that the given PLAP derivative exerts antiacne effects comparable to that of the highly purified PLAP. The term “therapeutically effective amount” in this specification indicates a dosage of PLAP or an active derivative that is effective in healing the acne lesion.

[0046] In some embodiments, the PLAP cream may include one or more well tested additives or enhancers from a list offered by pharmaceutical companies including preservatives, biologically active compounds with positive effects on the recovery of normal skin texture, buffers, moisture-control compounds, or antibiotics, for example. In other embodiments, the composition essentially contains the carrier and PLAP. As used here, the phrase “essentially contains” means that the given composition has no other ingredient in it in addition to PLAP or an active derivative and a carrier.

[0047] The PLAP cream can be made using several suitable techniques. In some embodiments, PLAP, optional additives, preservatives, antibiotics, and enhancers as well as a carrier are mixed within a commercial mixer to form a gel or the like. All conventional methods known in the art for mixing may be suitable. Various equipment is also available to manufacture liposomal preparations (which provides for controlled, sustained release of the components). In pharmaceutical composition embodiments, methodologies for the formulation are well known, and can be found, for example, in Remington’s Pharmaceutical Sciences, Eighteenth Edition, A.R. Gennaro, Ed., Mack Publishing Co. Easton, PA 1990, incorporated hereby by reference. Since PLAP activity remains stable by heating the PLAP cream for 30 min at 65 Celsius, such heating step may be included in the preparation of the final formulation for sterilization purpose.

Local Treatment of Bacterial Skin Infections Other Than Acne With PLAP Cream

[0048] PLAP cream can also be used for the local treatment of (a) nail infections, (b) already infected wounds along with antibiotics, and (c) wounds infected with drug resistant bacteria. The same carriers and additives may be used as described above for the acne treatment, The recommended amount of PLAP per 1 gram carrier is 1.0 to 10 mg. Generally, the larger the lesion or wound the more PLAP is needed. As examples, if the area of the lesion is 1 cm.sup.2 and the wound area is 10 cm.sup.2, then the recommended concentration of PLAP is 1 mg and 5 mg per 1 gram carrier, respectively. In case of nail infection, on the first day the treatment is repeated hourly for 3 hours followed covering the lesion. The treatment is repeated on the second day and if still needed on the third day. Treatment of nail infections with PLAP cream does not require co-treatment with antibiotics. The infected wounds are treated with PLAP cream as nail infections except that it is combined with antibiotics as prescribed. In cases when infected wounds do not respond to antibiotics, meaning they were infected by drug resistant bacteria, they are treated with PLAP cream daily without treatment with antibiotics.

PLAP Cream for Local Treatment of Skin Infected With Reactivated Varicella Zoster, Herpes Simplex Virus-1 (HSV-1) or Herpes Simplex Virus 2 (HSV-2)

[0049] The PLAP cream can be used for the local treatment of Shingles or other skin lesions caused by reactivated HSV-1 or HSV-2 generally, but not always, along with treatment with a prescribed antiviral drug. The same carriers and additives may be used as described for the acne treatment, The recommended amount of PLAP per 1 gram carrier is 2.0 to 10.0 mg. Generally, the larger the lesion the more PLAP per 1 gram carrier is needed. As examples, if the area of the lesion is 10 cm.sup.2 or smaller, 2 mg per 1 gram carrier is suitable. If the area of the lesion is larger than 10 cm.sup.2 then, depending on the lesion’s size, the recommended concentration of PLAP is between 2.1 to 10.0 mg per 1 gram carrier. Local treatment of the lesion with PLAP cream is performed once daily along with oral administration of the prescribed antiviral drug (usually four-times a day). Local administration of the antiviral drug is not recommended. In case of shingles, when the lesion treated with placental alkaline phosphatase or an active derivative is smaller than 2.0 cm.sup.2, the use of an antiviral drug is optional. In cases of skin lesions caused by HSV-1 or HSV-2, the use of an oral antiviral drug is optional regardless of the lesion’s size.

EXAMPLES

Example 1 Production, Purification, and Spectrophotometric Assay of PLAP and rPLAP

[0050] A purification procedure consisting of several steps was performed to further purify the commercially obtained PLAP and to yield a homogeneous band in electrophoretic separation. The same purification procedure and assay was followed that had been described in detail elsewhere [She, Q.-B., Mukherjee, J.J., Huang, J.-S., Crilly, K.S. and Kiss, Z. Growth factor-like effects of placental alkaline phosphatase in human and mouse embryo fibroblasts. FEBS Lett., 469, 163-167, 2000].

[0051] Recombinant wild-type PLAP (rPLAP) was produced exactly as described by Kozlenkov et al. [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].To simplify the recovery and purification of rPLAP, the glycosylphosphatidylinositol anchoring sequence of PLAP was replaced by the FLAG octapeptide, and rPLAP was expressed as secreted, epitope-tagged, enzyme. rPLAP is an example of an active derivative of full length PLAP. It demonstrates that active derivatives of full length PLAP with altered and shorter sequences can be made using recombinant methods.

Example 2 Treatment of Acne Lesions on the Right Side of Face of a 16-Year-Old Female With PLAP Cream

[0052] FIG. 1 shows a section of right side of the face of a 16-year-old female subject with moderate acnes at multiple spots before treatment (A) and after daily treatment with PLAP cream (3-mg PLAP in 1-gram Vaselinum cholesterinatum) for 3 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 2 months that showed only small visible positive effects. In contrast, treatment with PLAP for 3 weeks was sufficient to heal the acne lesion by about 90% without visible scar tissue. Complete recovery of the original skin texture required about 5 weeks with no additional treatments needed between 3 to 5 weeks. The sites of the healed acne lesions have remained free of visible scar tissue since then (15 years).

Example 3 Treatment of Acne Lesions on the Left Side of Face of a 16-Year-Old Female With PLAP Cream

[0053] FIG. 2 shows a section of left side of the face of the same female subject shown in FIG. 1 before treatment of moderate acnes at multiple spots (A) and after daily treatment with PLAP cream (3-mg PLAP in 1-gram Vaselinum cholesterinatum) for 3 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 2 months that showed only small visible positive effects. In contrast, treatment with PLAP for 3 weeks was sufficient to heal the acne lesion by about 80-85% without visible scar tissue. Complete recovery of the original skin texture required about 6 weeks with no additional treatments needed between 3 to 6 weeks. The sites of the healed acne lesions have remained free of visible scar tissue since then (15 years).

Example 4 Treatment of Acne Lesions on the Right Side of Face of a 17-Year-Old Male With PLAP Cream

[0054] FIG. 3 shows a section of right side of face of a 17-year-old male with multiple severe acnes before treatment (A) and after daily treatment with PLAP cream (5-mg PLAP in 1-gram Vaselinum cholesterinatum) for 4 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 3 months with only small visible positive effects. In contrast, treatment with PLAP for 4 weeks was sufficient to heal the acne lesion by about 75-80% without visible scar tissue. Complete recovery of the original skin texture required about 8 weeks with no additional treatments needed between 4 to 8 weeks. The sites of the healed acne lesions have remained free of visible scar tissue since then (16 years).

Example 5 Treatment of Acne Lesions on the Left Side of Face of a 17-Year-Old Male With PLAP Cream

[0055] FIG. 4 shows a section of left side of face of the same male shown in FIG. 3 before treating his multiple severe acnes (A) and after daily treatment with PLAP cream (5-mg PLAP in 1-gram Vaselinum cholesterinatum) for 3 weeks (B). Before PLAP treatment, the acne covered area was treated with benzoyl peroxide as prescribed for 3 months with only small visible positive effects. In contrast, treatment with PLAP for 3 weeks was sufficient to heal the acne lesion by about 75-85% without visible scar tissue. Complete recovery of the original skin texture required about 6 weeks with no additional treatments needed between 3 to 6 weeks. The sites of the healed acne lesions have remained free of visible scar tissue since then (16 years).

Example 6 Additional Observations on Acne Treatment

[0056] The examples shown in FIGS. 1-4 and additional similar observations with one female and two male adolescents, each with severe form of acne, strongly suggested that early treatment of acnes with PLAP would result in faster healing. This idea was tested with 16-17-year-old males who started to develop acne during a 2 to 4-day period. Treatments (2-mg PLAP in 1-gram Vaselinum cholesterinatum) were applied at bedtime only once. In each case the acne was completely healed within 2-5 days without any scar tissue formation.

Example 7 Nail and Wound Infections

[0057] Six subjects with painful fingernail infection, each rapidly developed within 24 hours of the initial skin damage, were treated with PLAP cream once within hours of detecting the signs of infection. In each case, the pain subsided within about 30 minutes but at a lower level it soon returned. Then, an additional treatment was applied after about one hour of the first treatment which completely abolished the pain. The infection was resolved within 24-48 hours without further treatment. Although no controls could be included, according to medical records nail infections rarely, if ever, resolve within such a short time.

[0058] On the basis of PLAP’s effects on nail infection, PLAP is fully expected to exert beneficial anti-inflammatory effects on any kind of other wounds infected with bacteria. Such use of PLAP would be especially beneficial if the wound is infected with drug resistant bacteria. Since PLAP strongly reduces formation of adenosine from extracellular ATP that would otherwise support bacterial growth, PLAP will likely curb the growth of drug resistant bacteria. This is an extremely important aspect of PLAP effect considering that in the U.S. every year nearly 100,000 people die because of wound infection including deaths due to diabetic non-healing infected wounds.

Example 8 PLAP Promotes Healing of Shingles

Shingles With Serious Symptoms

[0059] Two individuals (one female and one male) had recurrent painful shingles at 10-16 months intervals, usually lasting for 4-6 weeks without treatment, associated with pain lasting beyond 6 weeks that prevented them to get a good night sleep. When acyclovir became available, both subjects received daily (4-times a day) oral acyclovir treatment during the last 3 cycles of recurrence which resulted in about 40-50% remission in 10-12 days and full healing by the end of third week. Acyclovir had no significant effect on pain.

[0060] When a new recurrence of shingles occurred again, the same two subjects were co-treated with local PLAP cream (5 mg PLAP in 1-gram Vaselinum cholesterinatum) and oral acyclovir. The two subjects responded to this combination treatment similarly.

[0061] FIG. 5 demonstrates the combined effects of PLAP cream and acyclovir on the female subject (72 years old). She started the treatment with acyclovir and PLAP cream on day 0. As shown, the combined treatment reduced the size of the lesion below 50% after 4 days of treatments. By day 8, the size of the lesion was only about 10-15% of the original size, and this was accompanied by significant reduction in pain which allowed her to sleep, from day 1 during each consecutive night, at least 6 hours without waking up. Complete healing of both subjects occurred by day 12 (the female subject) and day 14 (the male subject). Thus, combination treatments with PLAP and acyclovir (and most probably with other antiviral agents as well) yield significantly better results on shingles than treatments with acyclovir alone.

Shingles With Moderate Symptoms

[0062] Two female individuals, 32- and 61- year-old, respectively, had recurrent (practically yearly) moderate shingles (one had under the breast, the other had on the arm, both approximately the size of 1-2 cm.sup.2) previously lasting for about 2-3 weeks. In both cases, the shingles were associated with well noticeable irritation. After the first treatment with PLAP cream ((2.0 mg PLAP in 1-gram Vaselinum cholesterinatum) without acyclovir the irritation became unnoticeable within 1 hour and the shingles disappeared by around the 3rd and 4th day in the cases of the 32=year-old and 61-year-old woman, respectively. This indicates that shingles of moderate size can be effectively treated with PLAP without using an antiviral medication.

Herpes (Oral)

[0063] Three female subjects having recurrent oral herpes with 6-12 months frequency during the last 3 years usually lasting for 4-6 days and accompanied by irritation or mild pain were treated once with PLAP cream (2.0 mg PLAP in 1-gram Vaselinum cholesterinatum) without an antiviral drug. In each case, the irritation was greatly reduced within one hour and the skin healed within 2 days.

Herpes (Genital)

[0064] Two male subjects having recurrent relatively small size (0.4-0.6 cm.sup.2) but moderately painful genital herpes, lasting for 10-16 days, were treated with PLAP cream (2.0 mg PLAP in 1-gram Vaselinum cholesterinatum) without an antiviral drug on two consecutive days. Pain was significantly reduced in a lasting manner within one hour of the first treatment, and the lesions resolved/healed in 3 days (0.4 cm.sup.2 lesion) and 5 days 0.6 cm.sup.2 lesion.

Example 9 Effects of PLAP, rPLAP, and ATP on the Proliferation of Human Epidermal HaCaT Cells and Human Skin Fibroblast Cell Line CCD 966 SK

[0065] The immortalized human HaCaT keratinocyte cell line, isolated in 1988 [Boukamp, P., Petrussevska, R.T., Breitkreutz, D., Hornung, J., and Markham, A., “Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line,” J. Cell. Biol., 106, 761-771, 1988], was provided for these studies by the Institute of Dermatology, Szeged University, , Szeged, Hungary). HaCaT cells were maintained in 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin -containing Dulbecco’s modified Eagle’s medium (DMEM) up to 80% confluency in an incubator (5% CO.sub.2, 37° C.). Cells were plated into 96 well plates at 20% confluency and incubated for 72 hours in an incubator (5% CO.sub.2, 37° C.) with 200 nM purified PLAP or rPLAP in the absence or presence of 100 .Math.M ATP.

[0066] CCD 966 SK fibroblasts, originally derived from a 52-year-old subject and purchased from American Type Culture Collection (Alexandria, Virginia), were at passage 5 when used. The fibroblasts were maintained and treated as described above for the HaCaT cells.

[0067] The relative changes in the number of viable cells were determined by the MTT assay. This colorimetric assay is based on the ability of living healthy cells (mostly the mitochondrial compartment), but not dead cells, to reduce 3-(4,5-dimethyl thizaol-2-yl)-2,5-tetrazolium bromide to a blue formazan product [Carmichael, J., De Graft, W.G., Gazdar, A.F., Minna, J.D. and Mitchell, J.B. Evaluation of tetrazolium-based semi-automated colorimetric assay: Assessment of chemosensitivity resting. Cancer Res. 47, 936-942, 1987] This technique is a widely used and accepted method to accurately determine the relative numbers of viable cells. For example, this is the official method used by the National Cancer Institute to screen anticancer drugs. In most cases, when the test agent does not strongly influence the oxidation-reduction balance of cells, the MTT assay is essentially a proliferation assay.

[0068] A MULTISKAN MS microplate reader purchased from Labsystems (Franklin, Massachusetts) was used to measure the formation of formazan as an increase in absorbance at a test wavelength of 540 nm and a reference wavelength of 690 nm. In the data analysis, data were expressed as mean values ± standard deviation (S.D.) of eight incubations (from eight wells) in one experiment. In each case, incubations were performed for 72 hours.

[0069] As data in TABLE 1 shows, ATP alone had no effects on the proliferation of either cell line, while both PLAP and rPLAP clearly stimulated proliferation of both cell lines. It is noteworthy that in the absence of ATP the stimulatory effects of PLAP and rPLAP were of similar magnitude. In contrast, in the presence of ATP both PLAP nor rPLAP inhibited the proliferation of epidermal cells and fibroblasts.

[0070] These results strongly indicate that depending on the concentration of extracellular ATP, PLAP and rPLAP will exert two different effects on skin cell proliferation. In the presence of ATP in the extracellular space (like in inflamed tissues), PLAP and rPLAP will inhibit proliferation of skin cells, while in the absence of ATP in the extracellular space (in the regenerative phase) PLAP and rPLAP will stimulate proliferation of skin cells thereby helping to restore the damaged skin.

TABLE-US-00001 Combined effects of PLAP and rPLAP on the Proliferation of HaCaT and 966 SK Fibroblasts Treatment Viability of Cells (Absorption: A.sub.540) HaCaT cells CCD 966 SK None, 0 hour 0.512±0.033 0.411±0.023 None, 72 hours 0.859±0.052 0.705±0.035 ATP, 72 hours 0.841±0.037 0.686±0.029 PLAP, 72 hours 1.086± 0.047 0.853±0.040 PLAP + ATP, 72 hours 0.736±0.038 0.590±0.017 rPLAP, 72 hours 1.133±0.049 0.818±0.032 rPLAP + ATP, 72 hours 0.715±0.020 0.597±0.039

Example 10. Determination of ATP Hydrolysis in Intact HaCaT Human Epidermal Cells and CCD 966 SK Human Fibroblasts

[0071] In these experiments, PLAP was incubated with human HaCaT cells and human 966 SK fibroblasts. The plasma membranes of both cell lines contain ectonucleotidases that hydrolyze ATP to result in the formation of adenosine. If PLAP binds ATP as reported earlier, it will compete with the ectonucleotidases for ATP. In this case, in the presence of PLAP the formation of adenosine, the final product of ATP hydrolysis, should be reduced. The experiment performed under this Example served to examine this possibility.

[0072] HaCaT and 966 SK cells were grown in 10% serum containing DMEM medium (0.1 ml) in 96-well plates to confluence. First, the medium was changed for serum-free medium. Then, selected wells received 200 nM purified PLAP 5 min prior to the addition of 2,000,000 c.p.m. (0.1 mM) of [U-.sup.14C] ATP (purchased from Amersham). Incubations in an incubator were performed for 2 hours. Fifty ml aliquots were withdrawn into small centrifuge tubes followed by the addition of 0.15 ml of chloroform methanol 2:1 mixture containing 1 mM adenosine (to help UV visualization and reduce cellular uptake of [U-.sup.14C] Adenosine) to precipitate the proteins and eliminate lipids. After centrifugation, 5 .Math.l aliquots of the lower phase were transferred to fluorescent PEI-cellulose (polyethyleneimine-impregnated cellulose) plates, and after drying the plates they were developed with water in a flat bottom chamber. After drying and visualizing the adenosine and nucleotide spots under UV (all nucleotides remained in the application spots), the respective areas were cut out, placed in scintillation vials, and radioactivity was measured in a liquid scintillation spectrometer. Data is the mean of three independent determinations (from three separate wells) ± S.D. in the same experiment.

[0073] The data, shown in TABLE 2, demonstrates that in both cell lines PLAP greatly reduced the hydrolysis of ATP and related nucleotides (i.e., more radiolabeled ATP and related nucleotides remained in the medium) which coincided with dramatic reduction in the formation of radiolabeled adenosine. The difference between the ATP lost and adenosine present in the medium most probably reflects uptake of a portion of newly formed adenosine into cells which was not measured in this experiment.

[0074] The best interpretation of data is that PLAP, by binding ATP, competed with ectonucleotidases to gain access to this substrate. One major consequence of this event is that less adenosine is formed, as shown in TABLE 2.

TABLE-US-00002 PLAP reduces ATP hydrolysis and adenosine formation in HaCaT and CCD 966 SK cell cultures Treatment Total [U-.sup.14C] nucleotide [U-.sup.14C] Adenosine HaCaT cells; c.p.m./well 0 minute 1,711,445 ± 50,034 953 ± 151 2 hours; None 1,009,901 ± 44,604 548,050 ± 23,477 2 hours; PLAP, 500 nM 1,572,853 ± 33,737 71,048 ± 2,220 CCD 966 SK cells; c.p.m./well 0 minute 1,771,762 ± 34,405 1,116-963-875 2 hours; None 1,295,488 ± 48,371 372,989 ± 25,283 2 hours; PLAP, 500 nM 1,653,949 ± 42,791 34,019 ± 4,892