WO2005016353A1

WO2005016353A1 - Topical ointment for vesicating chemical warfare agents

Info

Publication number: WO2005016353A1
Application number: PCT/US2004/000833
Authority: WO
Inventors: Carmen F. Arroyo; Damon L. Burman
Original assignee: U.S. Army Medical Research And Material Command Department Of The Army
Priority date: 2003-01-14
Filing date: 2004-01-14
Publication date: 2005-02-24
Also published as: AU2011200498A1; EP1589977A1; AU2004264777A1

Abstract

The regulatory effects of the active form of vitamin D, 1-α, 25-dihydroxyvitamin D3(1-α, 25(OH)2D3) were assessed on the cytokine and chemokine secretion induced by sulfur mustard (HD) on human skin fibroblasts (HSF) and human epidermal keratinocytes (HEK). Stimulation of HSF with HD (10-4 M for 24 hours at 37° C) resulted in about a 5-fold increase in the secretion of interleukin-6 (IL-6) and over a 10- fold increase for interleukin-8 (IL-8), which was inhibited by 1-α, 25(OH)2D3, at ≤ 10-9 M. 1-α, 25(OH)2D3 also suppressed IL-8 secretion by five-fold and IL-6 by four-fold on HD-stimulated HEK at concentrations ≤ 10-9 M. The effect of 1-α, 25(OH)2D3 was dose-dependent for the suppression of IL-6 and IL-8 induced by HD on HSF/HEK, apparent at nanomolar concentrations. Results indicate that the suppression of these inflammatory mediators by 1-α, 25(OH)2D3 is dependent on the source of the primary cultures, cell densities, and kinetics of pretreatments. In addition, a composition for a topical ointment for wound healing in mammals exposed to vesicating chemical warfare agents is also disclosed. The composition includes a compound or the combination of a 1-α, 25(OH)2D3 and shark liver oil. The steroid hormone 1-α, 25(OH)2D3 (calcitriol), in addition to its crucial role in calcium homeostasis, provides benefits to the immune system by regulating cell proliferation, differentiation and maturation. These benefits are effectuated through the control of protooncogenes and the regulation of cytokine production.

Description

TOPICAL OINTMENT FOR VESICATING CHEMICAL WARFARE AGENTS This application claims the benefit of priority from provisional application 60/439919, filed January 14, 2003.

STATEMENT OFGOVERNMENTINTEREST The invention described herein may be manufactured, used and licensed by or for the U.S. Government, as represented by the Secretary of the Army.

BACKGROUND OF THE INVENTION Sulfur mustard (R/-y-(2-chloroethyl) sulfϊde) is an alkylating agent with pathophysiological properties as a blistering agent and has been employed as a chemical warfare agent. Also known as HD, sulfur mustard gas was first used by the Germans during World War I. Due to its highly destructive effects, it was subsequently banned by the Geneva Convention. However, threats of the release of mustard gas on unsuspecting soldiers and civilians remains a dangerous reality, since no definitive drug therapy for HD induced cutaneous vesication is known to exist. Exposure to HD produces irritation, generates blisters in the human skin and causes burning sensations in the eyes and lung. HD also has genotoxic properties, and after acute exposure can exert systemic effects such as bone marrow and immune depression. In normal human skin, the first symptoms occur at approximately 6 to 8 hours after contact. Edema develops in the dermis and a separation of the epidermal- dermal junction occurs, leading to the progressive formation of blisters. Although the biochemical mechanism(s) behind the blistering action is not well understood, activation of cytokines has been suggested to contribute to this process. Wound healing proceeds via an overlapping pattern of events including coagulation, inflammation, epithelialization, formation of granulation tissue, matrix and tissue remodeling. The process of repair is mediated in large part by interacting molecular signals, primarily cytokines that motivate and orchestrate the manifold cellular activities, which underscore inflammation and healing. Continuing progress in deciphering the essential/complex role of cytokines in wound healing provides opportunities to explore pathways to inhibit/enhance appropriate cytokines to control or modulate pathologic healing of vesicating agents. A favorable degree of protection had been reported by dexamethasone in acute poisoning of rats with HD. (See Vojvodic, V., Z. Milosavljevic, B. Boskovic & N. Bojanic: The protective effect of different drugs in rats poisoned by sulfur and nitrogen mustards. Fundam. Appl. Toxicol., 1985, 5, S160-S160.) Vojvodic, et al., concluded that dexamethasone (a) prolonged the survival time in rats poisoned by 3 LD₅₀ s of HD, (b) diminished the lethality (with the protective indices ranging from 1.5 to 2.7), (c) antagonized the decrease of body weight, and (d) lessened the degree of pathological organ changes. Further studies have recently shown that dexamethasone can reduce the inflammatory effect induced by HD in primary alveoli macrophages. (See Amir, A., S. Chapman, T. Kadar, Y. Gozes, R. Sahar & N. Allon; Sulfur Mustard Toxicity In Macrophages: Effect of Dexamethasone. J. Appl. Toxicol., 2000 20, S51- S58.) Although topically applied steroids and systemically administered dexamethasone appear to be promising, they have shown unwanted side effects associated with steroids such as impaired wound healing (Barbier, et al., 1986; Babin, et al., 2000). Vitamin D₃ has recently undergone considerable study due to its effects on proliferation, differentiation and immunomodulatory activities in treating inflammatory diseases, dermatological conditions, osteoporosis, certain cancers and autoimmune diseases. Vitamin D₃ has already been approved by the Food and Drug Administration as a tropical treatment for psoriasis. (See Nagpal, et al.; 2001; "Vitamin D Analogs: Mechanism of Action and Therapeutic Applications;" Current Medicinal Chemistry, 8, 1661-1679. See also Holland, et al.; "Changes In Epidermal Keratin Levels During Treatment Of Psoriasis With Calcipotriol (MC903).) Nagpal, et al., is distinguished from the present invention because Nagpal, et al., are directed to the antiproliferation of keratinocytes, and specifically preventing the expression of IL-6. Further studies have explored the effect of a topical application of vitamin D₃ on wound healing. Lab tests on full thickness wounds in 8-week-old male rats showed dose-dependent responses with increased healing at higher concentrations of D₃. (See Chen, et al.; 1991; "lα, 25-Dihydroxyvitamin D₃: A Novel Agent For Wound Healing;" Vitamin D: Gene Regulation Structure-Function Analysis & Clinical Application.) The Chen, et al., reference is distinguished from the present invention because Chen, et al., are directed to generic wound healing using higher concentrations of vitamin D₃. Topical treatment with vitamin D₃ to enhance keratinocyte proliferation has also been studied. (See Garach-Jehoshua, et al.; 1999; "1, 25-Dihydroxyvitamin D₃ Increases the Growth-Promoting Activity of Autocrine Epidermal Growth Factor Receptor Ligands in Keratinocytes;" V. 140, No. 2 713-721.) This reference is distinguished from the present invention as it only explores the relationship between D₃ keratinocyte proliferation and specific growth factors, cell density and concentration of D₃ without its specific use for treatment of HD induced skin trauma or IL-6 mechanism involved in the wound healing process as induced by D₃ in the presence of HD gas. The release of cytokines induced by sulfur mustard (HD) has been studied in different in vitro systems since 1993. These studies suggested that vitamin D₃ may have significant impact in the treatment of HD induced skin trauma, thereby leading to the present invention as will be discussed herein below. (See Arroyo CM, Von Tersch RL, Broomfield CA, 1995, "Activation of Alpha-human tumor necrosis factor (TNF-alpha) by human monocytes (THP-1) exposed to 2-chloroethyl-ethyl sulphide (H-MG)," Human and Experimental Toxicology 14: 547-553; Arroyo CM, Carmichael AJ, 1997, "Role of cytokine and nitric oxide in the inflammatory response produced by sulfur mustard (HD)," Journal Chemical Society Perkin Transaction, 2: 2495-2499; Arroyo CM, Schafer RJ, Jurt EM, Broomfield CA, Carmichael AJ, 1999, "Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure," Human and Experimental Toxicology 18: 1-11; Arroyo CM, Schafer RJ, Kurt EM, Broomfield CA, Carmichael AJ, 2000, "Response of normal human keratinocytes to sulfur mustard: cytokine release," Journal Applied Toxicology 20: S63-S72; and Arroyo CM, Broomfield CA, Hackley BE, 2001, "The role of interleukin-6 (IL-6) in human sulfur mustard (HD) toxicology," International Journal of Toxicology., 20: 281-296.)

SUMMARY The present invention shows how 1-α, 25(OH)₂ D₃ regulates the induction of cytokines, including interleukin-6 (IL-6) and interleukin-8 (IL-8) involved in the cytoxicology of HD and the affect of 1-α, 25(OH)₂D₃ on cell proliferation of HD stimulated human skin fibroblasts (HSF) / human epidermal keratinocytes (HEK) cells. The present invention provides development of a topical composition with specific activity for HD, low toxicity and few side effects for treatment of dermatological disorders caused by sulfur mustard. This development has led to the use of vitamin D₃, or 1-α. 25 (OH)₂D₃ as an effective antidote for HD- induced skin trauma. The present invention also provides delivery of 1 -alpha, 25-dihydroxyvitamin D₃ alone or the combination of shark liver oil, an eicosapentaenoic acid (EPA) compound, in a topical preparation for the treatment of wounds for optimum healing of blistering caused for vesicant chemical warfare exposure, as well as for open sores, burns, incisions and wounds in mammals.

DESCRIPTION OF THE DRAWINGS Figure 1(a) shows the effect of 1-α, 25(OH)₂D₃ (2 x 10^-9 M) on the HSF secretion of IL-8 induced by HD (10^"4 M). Figure 1(b) shows the effect of 1-α, 25(OH)₂D₃ (1 x 10 ⁰ M) on the HSF secretion of IL-6 induced by HD (10 M). Figure 2(a) shows the effect of 1-α, 25(OH)₂D₃ (2.0 x 10^"9 M) on the HEK secretion of IL-8 induced by HD (10 M). Figure 2(b) shows the effect of 1-α, 25(OH)₂D₃ (1.0 x 10^"9M) on the HEK secretion of IL-6 induced by HD (10^"4M). Figure 3(a) shows fluorescence measurements using the CyQuant Cell Proliferation Assay on HEK (5 x 10° cell/mL) treated with 1-α, 25(OH)₂D₃ (5 x 10^" ⁹M) after exposure to HD (10^"4M). Figure 3(b) shows fluorescence measurements using the CyQuant Cell proliferation assay on HEK (lxl 0⁶ cell/mL) treated with 1-α, 25(OH)₂D₃ (1 x 10^"9 M) after exposed to HD (10^"4M). Figure 4 shows the dose response effect of 1-α, 25(OH)₂D₃ (1 x 10^"10 to 1 x 10^"7 M) on HEK cell proliferation in HD stimulated and then treated with 1-α, 25(OH)₂D₃. Figure 5(a) is an optical micrograph of control HEK (vehicle control used) showing typical cellular morphology. Figure 5(b) is an optical micrograph of HEK treated with 1-α, 25(OH)₂D₃ (2 x lO^-9 M). Figure 5(c) is an optical micrograph of HD-stimulated HEK. Figure 5(d) is an optical micrograph of HEK treated with 1-α, 25(OH)₂D₃ (2 x 10^"9 M).for 24 hours, after HD stimulation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Development of antidotes for sulfur mustard (HD) is particularly important due to the rapid degenerative effects of HD upon human dermal contact. Skin injuries caused by HD activate a complex phenomenon involving resident epidermal cells, fibroblasts of dermis, and endothelial cells as well as invading leukocytes interacting with each other under the control of a network of cytokines and lipid mediators. Keratinocyte as well as fibroblast skin cells play an important role in the initiation and perpetuation of skin inflammatory reactions of HD through the release of and response to cytokines/chemokine. Activation of skin cells in the context of the wound microenvironment results in enhanced release of chemokines, recruitment of reinforcements, and amplification of the response, with the further release of cytokines, tumor necrosis factor-alpha (TNF-α), inter leukin-1 (IL-1) and inter leukin (IL-6) that act as paracrine, autocrine and, potentially, endocrine mediators of host defense. Consequently, cytokines, which are central to this constellation of events, have become targets for therapeutic intervention to modulate the wound healing process. Therefore, depending on the cytokine and its role, it may be appropriate to either enhance (recombinant cytokine, gene transfer) or inhibit (cytokine or receptor antibodies, soluble receptors, signal transduction inhibitors, antisense) the cytokine to achieve the desired outcome. Vitamin D₃ is produced in the skin and is metabolized primarily in the liver to the major circulating form 25-hydroxy vitamin D₃ which is metabolized in the kidney to produce the biologically active form of the hormone, 1-α, 25-dihydroxyvitamin D₃. The skin not only participates in the production of the vitamin D₃ but also contains receptors for l-α,25(OH)₂D₃, therefore, suggesting a role of this hormone in the growth and differentiation of the skin. Cultures of neonatal human foreskin keratinocytes have demonstrated that these cells produce l-α,25(OH) D₃ from 25- hydroxy vitamin D₃. The production of l-α,25(OH)₂D₃ varies with the degree of differentiation of keratinocytes and is regulated by exogenous l-α,25(OH)₂D₃ . In addition, receptors for the active metabolite of vitamin D₃, l-α,25(OH)₂D₃, have been found in dermal fibroblasts, endothelial cells, and activated T lymphocytes .

Test Studies Reagents:

Sulfur mustard (2, 2'-dichlorodiethyl sulfide, HD, 5 μL in 10 mL KGM^™, 4 x 10^"3 M) was acquired from the U.S. Army Soldier and Biological Chemical Command (Aberdeen Proving Ground, MD, USA). The purity of HD was verified by NMR to be greater than 95%. l-α,25-dihydroxyvitaminD₃ was obtained from Aldrich Chemical Company (Milwaukee, WI, USA) as 99% pure crystalline solids and were verified by NMR. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma^® Chemical Corp., St. Louis, MO, USA (catalog # M2128). Cell culture and chemical treatments: The human skin fibroblast (HSF) cell line (ATCC^™ # CCD-39Sk) was purchased from American Type Culture Collection (ATCC^™), Manassas, VA, USA. The HSF cells, ATCC^™ CCD-39Sk, were cultured in 425 mL minimum essential medium (MEM Eagle, Sigma^® Chemical Corp., St. Louis, MO, USA; catalog # M5650) supplemented with 5 mL of MEM vitamins lOOx (Sigma^®, catalog # M6395; 0.7 M), 5 mL of MEM nonessential amino acid lOOx (Sigma^®, catalog # M7145), 10 mL of glutamine (Sigma^®, catalog # G7513; 0.2 M), 50 mL of fetal bovine solution (Sigma^® , catalog # F2442) and 5 mL of 50x amino acids (Flow Laboratories, M^cLean, Virginia, USA; catalog # 16-011-49). The pH of the fibroblast growth medium (FGM) was adjusted with sodium bicarbonate (0.7 M; Sigma^®, catalog # S8875) to pH = 7.4. The frozen HSF ampoules were thawed and seeded in a 150 cm² flask at ~ 550,000 cells per flask then incubated at 37° C in a humidified 5 % CO₂ atmosphere. HSF were cultivated for seven days, divided and seeded in a 150 cm² flask at -550,000 per flask for seven additional days. Cryopreserved HEK cells from Clonetics^® (BioWhittaker, Inc., Walkersville, MD, USA) identified as HEK 6207 and HEK 6717 from breast skin of adult females were cultured. These normal HEK cells were grown in keratinocyte basal medium at 15 x 10^"5 M calcium and supplemented with 5 mg/mL insulin, 0.1 ng/L recombinant epidermal growth factor, 0.4 % bovine pituitary extract, 0.5 mg/mL hydrocortisone, 50 mg/mL gentamicin and 50 ng/mL amphotericin-B (henceforth referred to as keratinocyte growth medium (KGM) or KGM™). The second passage of keratinocytes were subcultured in 150 cm² flasks at a seeding density of - 2.5 x 10³ cells per cm² in KGM^™ for seven days. When HSF/HEK reached a desired confluence of ~ 75 - 85 % in 150 cm² flasks, the human skin cells were exposed to HD (10^"4 M) for 24 hours at 37° C. The levels IL-8 and IL-6 on cell supernatants were determined by enzyme-linked immunosorbent assay (ELISA). For 1-α, 25(OH)₂D₃ treatments, 96-well plates seeded with ~ 5 x 10³ cells per well were incubated with 1-α, 25(OH)₂D₃ (10^"n to 10^"6 M) using isopropanol (0.08%) as vehicle control for 24 hours at 37° C. 1-α, 25(OH)₂D₃ dissolved in isopropanol were added at concentrations ranging from 10^"11 to 10^"6 M to control cells, no HD- stimulated cells and HD-stimulated cells when the cells reached a confluence of 75 - 85 %. Sulfur mustard (HD) exposure: For ELISA experiments: HSF/HEK in 150 cm² culture flasks containing fresh KGM^™ or FGM media respectively were exposed to 10^"4 M HD per flask. This concentration of HD (10^"4 M) was estimated to be needed to produce the observed effects in the skin of HD casualties. Cell viability experiments (trypan blue exclusion and MTT-assay) of controls (no HD-stimulated) and HD stimulated cells showed that the cell viability for controls was greater than 95 % and approximately 75 % or lower for the concentration 10^"4 M of HD under identical culture conditions (data not shown). The culture flasks were maintained in a safety chemical fume hood for one hour at room temperature to allow venting of volatile-HD and then transferred to a CO₂ incubator at 37° C for additional time of 24 hours. After the indicated incubation time, cells were centrifuged, the supernatant media were removed, and final dilutions were made. Control experiments carried out with KGM^™/ FGM containing HD (10^"4 M) indicate that the inclusion of HD for 24 hours at 37° C does not influence or affect the ELISA kit or experiments. For experiments performed using 96-well plates, four microliters (μL) of HD (diluted in cell-cultured media for a final concentration equivalent to 10^"4 M) were added to each well. The well plates were maintained at room temperature in a chemical fume hood for approximately an hour and then transferred to a CO humidifier at 37° C until a particular indicated incubation time was reached. After the specific incubation time, cell proliferation experiments were performed. ELISA and cell proliferation assay: ELISA experiments were performed as described in the manufacturer's literature. Human IL-8 and IL-6 immunoassays produced by Quantikine™ (R&D Systems, Inc. Minneapolis, MN, USA) were used for the determination of soluble human chemokine/cytokine in cell supematants. Levels of IL-8 and IL-6 were determined from cell supematants of HSF HEK control (no HD-stimulated), treated with l-α,25(OH)₂D₃ (isopropanol (0.08 %) as vehicle control), HD-stimulated and HD-stimulated then treated with 1-α, 25(OH)₂D₃. The optical density was measured using a microplate reader. The absorbance of each well was read at 450 ± 10 nm, and a standard curve was constructed to quantitate chemokine/cytokine concentrations in the cell supernatant samples. The CyQUANT^® Cell Proliferation Assay kit was obtained from Molecular Probes, Inc. (Eugene, Oregon, USA, catalog # C-7026), and it was used as indicated in the experimental protocol provided by the product printed material. The CyQUANT^® kit uses a proprietary green fluorescent dye, called CyQUANT^® GR dye, which has strong fluorescent enhancement when bound to cellular nucleic acids. This cell proliferation kit can detect much lower cell numbers than neutral red or methylene blue assays. Typical used reagent preparation in these studies, IX cell- lysis buffer stock solution was prepared by mixing 1 mL of the 20X stock with 19 mL of nuclease-free distilled water. Fifty microliters (50 μL) of the CyQUANT^® GR dye stock solution was added and mixed thoroughly. Two hundred microliters (200 μL) of the CyQUANT^® GR dye/cell-lysis buffer was added to each well, gently mixed and incubated for 3 minutes at room temperature, protected from light. Fluorescence measurements were made using a multi-well plate reader Series 4000 (Cytofiuor^®,

PerSeptive Biosystems, Massachusetts, USA) with excitation at 485 ± 10 nm and emission detection at 530 ± 12 nm. This assay has a linear detection range extending from 50 or fewer to at least 50,000 cells in 200 μL volumes using a single dye concentration. The fluorescence enhancement is directly proportional to the binding of the green fluorescent dye to cellular nucleic acids therefore to the density of cells. CyQUANT^® Cell Proliferation assays is ideal for cell proliferation studies as well as for routine cell counts and can be used to monitor the adherence of cells to surface. The standard curve under these experimental conditions was from 50 to 50,000 cells per 200 μL sample. Clonosenic assay Human skin cells (HEK/HSF) were cultured in a 96-well plates and clonogenicity was measured spectrophotometrically in an MTT-assay. Briefly, MTT was dissolved in phosphate buffered saline (PBS, pH 7.4) filtered through a 0.22 μm filter (Millipore, Massachusetts, USA) to remove formazan crystals and stored at -20° C in the dark (5 mg/mL). Cytotoxicity assay monolayers of human skin cells were treated with increasing concentrations of 1-α, 25(OH)₂D₃ for twelve or 24 hours, after which time the cell number/viability was determined by the colorimetric MTT assay. Twenty microliters (20 μL) of MTT was added to each well and incubated for 5 hours. The supernatant was aspirated without disturbing the formazan crystals. The formazan crystals were dissolved in 150 μL/well dimethylsulfoxide (DMSO 100 %;

Sigma D 8779) and 25 μL/well glycine (0.1 M; Sigma G 7126, pH 10.5) buffer. Complete solubilization was achieved by vigorously shaking on a microplate shaker for 15 minutes. Optical density was read on a microplate reader (Molecular Devices, Sunnyvale, CA, USA) attached to IBM PC/XT for data manipulation at a wavelength of 570 ± 10 nm and a reference wavelength of 630 nm. Background absorbance values were measured in wells containing similar media without cells. Surviving cell number was then indirectly determined by MTT dye reduction. Each experiment was performed with eight independent replicates and repeated four times. Histochemical staininε Human skin cells were washed in sodium cacodylate buffer (0.1 M) (sodium cacodylate, 21.40 g, Polysciences, Inc., Warrington, PA, USA, catalog # 01131), 900 mL deionized water, to a final volume of one liter using deionized water. The pH was adjusted to 7.4 with a solution of hydrochloric acid (HC1; 1.0 N). Fixation procedure using the Karnovsky's fixative mixture consisting of 2.5 % glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA, catalog # 16220) and 1.6 % paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA, catalog. # 15710) in sodium cacodylate (0.1 M) was performed as described by Petrali, et al. Cells were rinsed twice for 10 minutes. The mixed solution was replaced by distilled water and stained with hematoxylin and eosin-phloxine for light microscopic examination and photomicroscopy as previously described by Petrali et al. Data analysis: Data are expressed as means + standard deviation (SD). All immune assays and cell proliferation data points were measured at a minimum of five independent preparations and means ± SD are illustrated. Statistically significant differences were determined by Dunnett's multiple comparison tests. Dunnett's test is a specialized multiple comparison test that allows the comparison of a single control group with all other groups. ^*R-values of < 0.05 and ^** R-values of < 0.01 were considered significant. The intervention studies are presented as means + standard deviations for the obtained data. To analyze two-way treatment structures with unequal subclass numbers, we utilized the approach of analysis of messy data. Table 1, below, shows the secretion levels of IL-8 and IL-6 on supematants from HSF and HEK treated with HD (10^"4) over a period of 24 hours at 37 °C. Table I

*Cells were cultured and stimulated with HD as described in Materials and Methods section. At the end of the culture period, ELISAs were determined. Data are means ± SD (n=5).

As shown in Table 1, the secretion levels of IL-6 in HD-stimulated HSF supematants resulted in a 5-fold increase from HSF control (non-stimulated HSF). The secretion of IL-8 was significantly more pronounced than IL-6 in supematants from HSF/ HEK stimulated with HD. The secretion levels of IL-6 and IL-8 vary with the cell density, the primary culture passages and the differences between the donors as shown. In order to determine l-α,25(OH)₂D₃ suppression of HD induced IL-8 and IL- 6 the levels of IL-8 and IL-6 in supematants from HD-stimulated HSF/HEK were assessed for a range of concentrations from 10^"10 M to 10^"6M of 1-α, 25(OH)₂D₃. As shown in -figure 1(a), vitamin D₃ inhibited IL-8 secretion by HD stimulated HSF at 80% at a concentration of 2.0 x 10^"9M. As shown in figure 1(b), 1-α, 25(OH)₂D₃ suppressed IL-6 secretion by 82% in HD-stimulated HSF at a concentration of 1 x 10^" ¹⁰M. Analysis by Dunnet's multiple comparison test showed the inhibition of IL-8 and IL-6 by 1-α, 25(OH)₂D₃ to be significant at every concentration tested (*P < 0.05). The effects of 1-α, 25(OH)₂D₃ on HD-stimulated human epidermal keratinocytes was also tested. Figures 2(a) and 2(b) show that 1-α, 25(OH)₂D₃ suppressed IL-8 and IL-6 levels induced by HD in HEK supematants to a similar extent as HD in HSF. As shown in figure 2(a), 1-α, 25(OH)₂D₃ inhibited IL-8 at a concentration of 2 x 10^"9 M (*P<0.05) by 80%. As shown in figure 2(b), 1-α, 25(OH)₂D₃ inhibited IL-6 at a concentration of 1 x 10^"9 M (*P< 0.05) by 73%. These findings are consistent with the fact that the drug-receptor dissociation constant (K_D) of 1-α, 25(OH)₂D₃ for its receptor is on the order of 10^"10 M. Thus, as per the present invention, the most relevant effects of 1-α, 25(OH)₂D₃ on HD induced skin trauma occur in the nanomolar range . As shown in figure 3(a), cell viability decreased as the dosage of 1-α, 25(OH)₂D₃ increased (1 x 10^"10 to 1 x 10^"6 M). Stimulation with HD (10^"4 M) decreased cell number by 56 % as shown in figure 3(b). However, the treatment with 1-α, 25(OH)₂D₃ had a synergistic effect on cell proliferation and cell proliferation enhanced 1.7 fold in the presence of 1-α, 25(OH)₂D₃. Higher cell numbers were seen at 1 x 10^"9 M concentrations of 1-α, 25(OH)₂D₃ as shown in figure 3(b). These results were further confirmed by cell counting using the trypan blue exclusion method (data not shown). HEK cells responded to 1-α, 25(OH)₂D₃ by increased proliferation in a very similar portion as shown in figure 4. Proliferation evaluated on day seven as fraction of percentage obtained by control (100%) are presented in Table II below.

Table II shows the effect of HD, 1-α, 25(OH)₂D₃ and both on the proliferation of human skin cells.. Proliferation was evaluated on day seven as percentage of the value of the proliferation obtained by control (100%).

Table II

^£ Isopropanol final concentration of 0.1 %. Cells were cultured with the 1-α, 25(OH)₂. At the end of the culture period, the fluorescence measurements were determined. The linear range of the assay under these conditions was from 50 to 50,000 cells per 200 μL sample. ⁱ Significant (P <0.05) increase of the effects of 1-α, 25(OH)₂ D₃ compared to the HD group. Data are means ± SD (n=5).

As shown in figure 4, in the presence of HD (10^"4M), proliferation decreased by 50%. Proliferation increased 1.5 fold when HEK cells were treated with 1-α, 25(OH) D₃ after HD-stimulation. The higher proliferation effect was seen with 1-α, 25(OH)₂D₃ at a concentration of 1 x 10^"9 M. The observed increase depends on the primary cultures, cell densities and kinetics of treatments with a maximal effect to be 10^"9M for 1-α, 25(OH)₂D₃. The effect of 1-α, 25(OH)₂D₃ was dose-dependent, already apparent at nanomolar concentrations, and time-dependent, maximal after 24- hours incubation. Cell proliferation studies were performed using two different assays, the MTT-assay and the CyQuant® proliferation assay. Histological staining studies of human skin cells from control, ie no HD stimulated cells (vehicle controls used) to HD-stimulated and 1-α, 25(OH)₂D₃ treated cells ( at 2 x 10^"9 M) , were carried out for twenty-four hours. Photomicrograph show typical cellular morphology for control HEK, as shown in figures 5(a) through 5(d). Figure 5(a) shows photomicrograph studies of control HEK showing typical cellular morphology. As shown in figure 5(b), treated HEK with 1-α, 25(OH)₂D₃ for 24 hours revealed normal morphology similar to control. As shown in figure 5(c), HD-stimulated HEK (10^"4 M for 24 hours) showed chromatin condensation. Classical signs of cytotoxicity such as chromatin condensation induced by HD were lessened when HEK were treated with 1-α, 25(OH)₂D₃ ( 2 x 10^'9 M) for 24-hours after HD- stimulation, as shown in figure 5(d). The effect of HD on human skin is dependent upon a variety of factors. Intrinsic factors include skin age, genetic background, race, gender and the site of contact. These factors, in addition to the chemical properties of HD, ie, molecular weight, polarity, state of ionization and vehicle, influence skin absorption of HD along with the actual concentration of HD exposed to and duration of exposure. Upon exposure, HD induces stimulation of certain cytokines and chemokine such as interleukin i (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α) and IL-8 in HEK, dependent upon the factors cited above. While IL-6 secretion by human keratinocytes and fibroblasts is weak to moderate under normal conditions, HD, induces HEKs/HSFs to release growth-promoting cytokines, IL-6 and IL-8. The present invention shows that 1-α, 25(OH)₂D₃ inhibits HD induced IL-6 and IL-8 secretion human skin cells, thereby supporting previous findings that stimulated HSF/HEK increase IL-6 and IL-8 production and that 1-α, 25(OH)₂D₃ inhibits the secretion of these cytokine/chemokine where IL-8 secretion was inhibited more than IL-6 secretion. This finding in addition to other findings showing that 1-α, 25(OH) D₃' decreases DNA binding of nuclear factor -kappaB (NF-kappaB) in human fibroblasts due to de novo protein synthesis suggests that 1-α, 25(OH)₂D₃ is capable of regulating expression of cellular factors that contribute to reduced DNA binding of NF-kappaB. These findings have also provided for the present invention, that is, utilizing 1-α, 25(OH)₂D₃, in a topical composition in a pharmaceutically effective amount, as an anti-inflammatory alone or in combination with traditional inhibitors of inflammation against HD. In a preferred embodiment, the pharmaceutically effective amount of 1-α, 25(OH)₂D₃ is in the concentration range of 10^"9 or 10^"10 M, with a specific concentration of 2 x 10^"9M. The present invention also supports recent findings of the stimulatory effects of 1-α, 25(OH)₂D₃ on keratinocyte proliferation. Thus, the present invention also

shows that 1-α, 25(OH)₂D₃ increased cell proliferation of HD stimulation in HEK by 1.7-fold, as shown in table II, above. The present invention, based on the histochemical staining studies shown in figures 5(a) through 5(d), also provide that 1-α, 25(OH)₂D₃ lessens the HD-induced abnormal signs of cellular degeneration.

In an additional embodiment, the present invention also provides delivery of 1 -alpha, 25-dihydroxyvitamin D₃ alone or the combination of shark liver oil, an eicosapentaenoic acid (EPA) compound, in a topical preparation for the treatment of wounds for optimum healing of blistering caused for vesicant chemical warfare exposure, as well as for open sores, burns, incisions and wounds in mammals. In the case of 1 -alpha, 25-dihydroxyvitamin D₃, the effective amount generally ranges from 5 ng/mL to 100 ug/mL and more preferably from 50 ng/mL to 2 ug/mL. Also according to the present invention the human growth hormone is utilized in an effective amount of at least about 0.05 ng/mL. Most preferably, the cell growth- stimulating compound includes a mixture of shark liver oil, 1-alpha, 25- dihydroxyvitamin D₃ and human growth hormone, each compound being present in an amount ranging from at least about 0.05 ng/mL, preferably at least about 0.5 ng/mL, and more preferably at least 1 ng/mL.

The of 1-alpha, 25-dihydroxyvitamin D3, shark liver oil formulation according to the present invention may also include an effective amount of an antimicrobial agent, for example, antibiotics and antifungal agents, such as nystatin and antiviral agents. Nystatin is a common topical treatment for thrush and an antimycotic polyene antibiotic obtained from Streptomyces noursei. The antimicrobial agent may be added for its ability to treat an infection, or alternatively, for its prophylactic effect in avoiding an infection. Excipients: include benzyl alcohol, cetostearyl alcohol, polysorbate 60; Dose: apply 2-3 times daily continuing for 7 days after lesions have healed. While many carries may be effectively utilizedfor the sharl liver oil based formulation, liposome is preferred, due to its rapid infusion into the skin. Novasome® microvesicles - a liposome based technology created from surfactant systems rather than phospholipids - to encapsulate moisture within skin treatments, shampoos and sprays to achieve a hydrating effect may be utilized. These Novasome® microvesicles carry both water-based as well as lipid-based compounds and are proven to be a very effective hydrator. Novasome® microvesicles (nonphospholipid liposomes) help provide an elegant texture, a rich creamy feel that leaves skin smooth and soft. The time-release system means the effects of moisturizers and glycolic acid last longer on the skin, with less potential for irritation.

Claims

What is claimed is:

1. A biologically active topical composition for treating sulfur mustard gas skin injuries comprising a pharmaceutically effective amount of 1-α, 25(OH)₂D₃.

2. A biologically active topical composition as recited in claim 1, wherein said pharmaceutically effective amount of said 1-α, 25(OH)₂D₃ is in the range of 10^"9 and 10^"10M.

3. A biologically active topical composition as recited in claim 1, wherein said pharmaceutically effective amount of said 1-α, 25(OH)₂D₃ is 2 x 10^"9 M.

4. A method for treating sulfur mustard gas skin injuries comprising: (a) administering a biologically active topical composition of 1-α, 25(OH)₂D₃ in a pharmaceutically effective amount; (b) inhibiting sulfur mustard induced secretion of IL-6 and IL-8; and (b) increasing cell proliferation of human epidermal keratinocytes.

5. A method for treating sulfur mustard gas skin injuries as recited in claim 4, wherein said treatment further comprises administering said 1-α, 25(OH)₂D₃ in the concentration range of 10^"9 and 10^"10 M.

6. A method for treating sulfur mustard gas skin injuries as recited in claim 4, wherein said treatment further comprises administering said 1-α, 25(OH)₂D₃ in a concentration of 2 x 10^"9M.

7. A composition for topically treating burns, open sores, incisions and wounds in mammals comprising an effective amount of 1-alpha, 25- dihydroxyvitamin D₃ in combination with an effective amount of eicosapentaenoic acid in a liposome carrier.

8. A composition as recited in claim 7 and further comprising said of 1- alpha, 25-dihydroxyvitamin D₃ being present in a range between 5 ng/mL and 100 ug/mL.

9. A composition as recited in claim 8 and further comprising an antimicrobial agent.

10. A composition as recited in claim 9, and further comprising said combination present in a concentration of at least 0.5 ng/mL.

11. A composition as recited in claim 9, and further comprising said combination present in a concentration of at least 1 ng/mL.