AU2004264777A1 - Topical ointment for vesicating chemical warfare agents - Google Patents

Description

WO 2005/016353 PCT/US2004/000833 TOPICAL OINTMENT FOR VESICATING CHEMICAL WARFARE AGENTS This application claims the benefit of priority from provisional application 60/439919, filed January 14, 2003. STATEMENT OF GOVERNMENT INTEREST 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 (Bis-(2-chloroethyl) sulfide) 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 1 WO 2005/016353 PCT/US2004/000833 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

LD

5 o 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 D 3 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 2 WO 2005/016353 PCT/US2004/000833 autoimmune diseases. Vitamin D 3 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 D 3 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

D

3 . (See Chen, et al.; 1991; "l1, 25-Dihydroxyvitamin D 3 : 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 D 3 . Topical treatment with vitamin D 3 to enhance keratinocyte proliferation has also been studied. (See Garach-Jehoshua, et al.; 1999; "1,25-Dihydroxyvitamin D 3 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

D

3 keratinocyte proliferation and specific growth factors, cell density and concentration of D 3 without its specific use for treatment of HD induced skin trauma or IL-6 mechanism involved in the wound healing process as induced by D 3 in the presence of HD gas. 3 WO 2005/016353 PCT/US2004/000833 The release of cytokines induced by sulfur mustard (HD) has been studied in different in vitro systems since 1993. These studies suggested that vitamin D 3 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-a, 25(OH) 2

D

3 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-a, 25(OH) 2

D

3 on cell proliferation of HD stimulated human skin fibroblasts (HSF) / human epidermal keratinocytes (HEK) cells. 4 WO 2005/016353 PCT/US2004/000833 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 D 3 , or 1-a. 25 (OH) 2

D

3 as an effective antidote for HD-induced skin trauma. The present invention also provides delivery of 1-alpha, 25-dihydroxyvitamin

D

3 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-a, 25(OH) 2

D

3 (2 x 10 9 M) on the HSF secretion of IL-8 induced by HD (104 M). Figure 1(b) shows the effect of 1-a, 25(OH) 2

D

3 (1 x 10-1 0 M) on the HSF secretion of IL-6 induced by HD (104M). Figure 2(a) shows the effect of 1-a, 25(OH) 2

D

3 (2.0 x 10- M) on the HEK secretion of IL-8 induced by HD (104M). Figure 2(b) shows the effect of 1-a, 25(OH) 2

D

3 (1.0 x 10- 9 M) on the HEK secretion of IL-6 induced by HD (1 0 4 M). Figure 3(a) shows fluorescence measurements using the CyQuant Cell Proliferation Assay on HEK (5 x 106 cell/mL) treated with 1-a, 25(OH) 2

D

3 (5 x 10~ 9 M) after exposure to HD (1 0- 4

M).

WO 2005/016353 PCT/US2004/000833 Figure 3(b) shows fluorescence measurements using the CyQuant Cell proliferation assay on HEK (1x0 cell/mL) treated with 1-a, 25(OH) 2

D

3 (1 x 10~' M) after exposed to HD (1 0 4 M). Figure 4 shows the dose response effect of 1-a, 25(OH) 2

D

3 (1 x 10-1 to 1 x 10~7 M) on HEK cell proliferation in HD stimulated and then treated with 1-a, 25(OH) 2

D

3 . 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-a, 25(OH) 2

D

3 (2 x 10-9 W. Figure 5(c) is an optical micrograph of HD-stimulated HEK. Figure 5(d) is an optical micrograph of HEK treated with 1-a, 25(OH) 2

D

3 (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 6 WO 2005/016353 PCT/US2004/000833 cytokines, tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1) and interleukin (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 D 3 is produced in the skin and is metabolized primarily in the liver to the major circulating form 25-hydroxy vitamin D 3 which is metabolized in the kidney to produce the biologically active form of the hormone, 1-a, 25-dihydroxyvitamin D 3 . The skin not only participates in the production of the vitamin D 3 but also contains receptors for 1-ac,25(OH) 2

D

3 , 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 1-a,25(OH) 2

D

3 from 25 hydroxy vitamin D 3 . The production of l-cc,25(OH) 2

D

3 varies with the degree of differentiation of keratinocytes and is regulated by exogenous 1-a,25(OH) 2

D

3 . In addition, receptors for the active metabolite of vitamin D 3 , I -a,25(OH) 2

D

3 , have been found in dermal fibroblasts, endothelial cells, and activated T lymphocytes . 7 WO 2005/016353 PCT/US2004/000833 Test Studies Reagents: Sulfur mustard (2, 2'-dichlorodiethyl sulfide, HD, 5 RL 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%. 1-a,25-dihydroxyvitaminD 3 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 m # CCD-39Sk) was purchased from American Type Culture Collection (ATCC" ), Manassas, VA, USA. The HSF cells, ATCC m 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 100x (Sigma®, catalog # M6395; 0.7 M), 5 mL of MEM nonessential amino acid 100x (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'Lean, 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 cm 2 flask at ~ 550,000 cells per flask then incubated at 37' C in a humidified 5 % CO 2 8 WO 2005/016353 PCT/US2004/000833 atmosphere. HSF were cultivated for seven days, divided and seeded in a 150 cm 2 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 m ). The second passage of keratinocytes were subcultured in 150 cm 2 flasks at a seeding density of ~ 2.5 x 103 cells per cm 2 in KGM M for seven days. When HSF/HEK reached a desired confluence of ~ 75 - 85 % in 150 cm 2 flasks, the human skin cells were exposed to HD (104 M) for 24 hours at 370 C. The levels IL-8 and IL-6 on cell supernatants were determined by enzyme-linked immunosorbent assay (ELISA). For 1-a, 25(OH) 2

D

3 treatments, 96-well plates seeded with ~ 5 x 10 3 cells per well were incubated with 1-a, 25(OH) 2

D

3 (10~ 1 to 10~6 M) using isopropanol (0.08%) as vehicle control for 24 hours at 37' C. 1-a, 25(OH) 2

D

3 dissolved in isopropanol were added at concentrations ranging from 1011 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 cm 2 culture flasks containing fresh KGM or FGM media respectively were exposed to 10-4 M HD per flask. This concentration of HD (104 M) was estimated to be needed to produce the observed 9 WO 2005/016353 PCT/US2004/000833 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

CO

2 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 m / FGM containing HD (10-4 M) indicate that the inclusion of HD for 24 hours at 370 C does not influence or affect the ELISA kit or experiments. For experiments performed using 96-well plates, four microliters ( tL) 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 2 humidifier at 370 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 T M (R&D Systems, Inc. Minneapolis, MN, USA) were used for the determination of soluble human chemokine/cytokine in cell supernatants. Levels of IL-8 and IL-6 were determined from cell supernatants of HSF/HEK control (no HD-stimulated), treated with 1-ox,25(OH) 2

D

3 (isopropanol (0.08 %) as vehicle control), RD-stimulated and 10 WO 2005/016353 PCT/US2004/000833 HD-stimulated then treated with 1-ct, 25(OH) 2

D

3 . 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 pL) of the CyQUANT* GR dye stock solution was added and mixed thoroughly. Two hundred microliters (200 pL) 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 (Cytofluor*, 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 gL 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. 11 WO 2005/016353 PCT/US2004/000833 The standard curve under these experimental conditions was from 50 to 50,000 cells per 200 ptL sample. Clonogenic assaV 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 pim 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 -a, 25 (OH) 2

D

3 for twelve or 24 hours, after which time the cell number/viability was determined by the colorimetric MTT assay. Twenty microliters (20 gL) 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 pL/well dimethylsulfoxide (DMSO 100 %; Sigma D 8779) and 25 pL/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 staining 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 12 WO 2005/016353 PCT/US2004/000833 adjusted to 7.4 with a solution of hydrochloric acid (HCl; 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. *P-values of < 0.05 and ** P-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 supernatants from HSF and HEK treated with HD (10~4) over a period of 24 hours at 37 'C. 13 WO 2005/016353 PCTIUS2004/000833 00 0 - -H 00 00 06 0 U C ci 00 00 . 00 en 00 00 00 -4 - Z5 - 3 -- -u u4 u N u u 140 WO 2005/016353 PCT/US2004/000833 As shown in Table 1, the secretion levels of IL-6 in HD-stimulated HSF supernatants 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 supernatants 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 1-a,25(OH) 2

D

3 suppression of HD induced IL-8 and IL 6 the levels of IL-8 and IL-6 in supernatants from HD-stimulated HSF/HEK were assessed for a range of concentrations from 10-" M to 10-6M of 1-a, 25(OH) 2

D

3 . As shown in figure 1(a), vitamin D 3 inhibited IL-8 secretion by HD stimulated HSF at 80% at a concentration of 2.0 x 10- 9 M. As shown in figure 1(b), 1-a, 25(OH) 2

D

3 suppressed IL-6 secretion by 82% in HD-stimulated HSF at a concentration of 1 x 10~ 1 0 M. Analysis by Dunnet's multiple comparison test showed the inhibition of IL-8 and IL-6 by 1-a, 25(OH) 2

D

3 to be significant at every concentration tested (*P < 0.05). The effects of 1-a, 25(OI-I) 2

D

3 on HD-stimulated human epidermal keratinocytes was also tested. Figures 2(a) and 2(b) show that 1-a, 25(OH) 2

D

3 suppressed IL-8 and IL-6 levels induced by HD in HEK supernatants to a similar extent as HD in HSF. As shown in figure 2(a), 1-a, 25(OH) 2

D

3 inhibited IL-8 at a concentration of 2 x 10-9 M (*P<0.05) by 80%. As shown in figure 2(b), 1-a, 25(OH) 2

D

3 inhibited IL-6 at a concentration of 1 x 109 M (*P< 0.05) by 73%. These findings are consistent with the fact that the drug-receptor dissociation constant (KD) of 1-a, 25(OH) 2

D

3 for its receptor is on the order of 1010 M. Thus, as per the present 15 WO 2005/016353 PCT/US2004/000833 invention, the most relevant effects of 1-a, 25(OH) 2

D

3 on HD induced skin trauma occur in the nanomolar range . As shown in figure 3(a), cell viability decreased as the dosage of 1-a, 25(OH) 2

D

3 increased (1 x 1010 to 1 x 10-6 M). Stimulation with HD ( 10 - M) decreased cell number by 56 % as shown in figure 3(b). However, the treatment with 1-a, 25(OH) 2

D

3 had a synergistic effect on cell proliferation and cell proliferation enhanced 1.7 fold in the presence of 1-cc, 25(OH) 2

D

3 . Higher cell numbers were seen at 1 x 10- M concentrations of 1-a, 25(OH) 2

D

3 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-a, 25(OH) 2

D

3 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-a, 25(OH) 2

D

3 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%). 16 WO 2005/016353 PCTIUS2004/000833 01 00 CN r-- 00 9- oq 6 6 o'-' C' 0 0 0 o o kn All a)~H-H N O ~ e 00 0 to A 6 6l 'A0 CY)I C0 -H -H -H 9-H -H -H C 2 VI 00000 0 0 01 '6d +1 - - - H 0~C 0 rx N 41 - -H 4 Ql 0 0 0 A0 o~P p 'O0 ;-.1 6 0 0 o. u~ 0 u~ 0 1700 WO 2005/016353 PCT/US2004/000833 As shown in figure 4, in the presence of HD (10~ 4 M), proliferation decreased by 50%. Proliferation increased 1.5 fold when HEK cells were treated with 1-a, 25(OH) 2

D

3 after HD-stimulation. The higher proliferation effect was seen with 1-a, 25(OH) 2

D

3 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 9 M for 1-a, 25(OH) 2

D

3 . The effect of 1-a, 25(OH) 2

D

3 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-a, 25(OH) 2

D

3 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-c, 25(OH) 2

D

3 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-a, 25(OH) 2

D

3 ( 2 x 109 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, ic, molecular weight, polarity, state of ionization and vehicle, influence skin absorption of HD 18 WO 2005/016353 PCT/US2004/000833 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-a) 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-a, 25(OH) 2

D

3 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-cc, 25(OH) 2

D

3 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 -a, 25(OH) 2

D

3 decreases DNA binding of nuclear factor -kappaB (NF-kappaB) in human fibroblasts due to de novo protein synthesis suggests that 1-cc, 25(OH) 2

D

3 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-a, 25(OH) 2

D

3 , 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-a, 25(OH) 2

D

3 is in the concentration range of 10 9 or 1010 M, with a specific concentration of 2 x 10~ 9 M. The present invention also supports recent findings of the stimulatory effects of 1-a, 25(OH) 2

D

3 on keratinocyte proliferation. Thus, the present invention also shows that 1-cc, 25(OH) 2

D

3 increased cell proliferation of ID stimulation in HEK by 1.7-fold, as shown in table U1, above. 19 WO 2005/016353 PCT/US2004/000833 The present invention, based on the histochemical staining studies shown in figures 5(a) through 5(d), also provide that 1x-a, 25(OH) 2

D

3 lessens the HD-induced abnormal signs of cellular degeneration. In an additional embodiment, the present invention also provides delivery of 1-alpha, 25-dihydroxyvitamin D 3 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 D 3 , 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 D 3 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 20 WO 2005/016353 PCT/US2004/000833 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. 21

Claims (11)

1. A biologically active topical composition for treating sulfur mustard gas skin injuries comprising a pharmaceutically effective amount of 1 -a, 25(OH) 2 D 3 .

2. A biologically active topical composition as recited in claim 1, wherein said pharmaceutically effective amount of said 1-a, 25(OH) 2 D 3 is in the range of 10~9 and 10- 0 M.

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

4. A method for treating sulfur mustard gas skin injuries comprising: (a) administering a biologically active topical composition of 1-a, 25(OH) 2 D 3 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-a, 25(OH) 2 D 3 in the concentration range of 10 and 1040 M.

6. A method for treating sulfur mustard gas skin injuries as recited in claim 4, wherein said treatment further comprises administering said 1-a, 25(OH) 2 D 3 in a concentration of 2 x 1 0 9 M. 22 WO 2005/016353 PCTIUS2004/000833

7. A composition for topically treating burns, open sores, incisions and wounds in mammals comprising an effective amount of 1-alpha, 25 dihydroxyvitamin D 3 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 D 3 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 I ng/mL. 23