EP1589977A1 - Onguent topique pour agents de guerre chimique vesicants - Google Patents

Onguent topique pour agents de guerre chimique vesicants

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Publication number
EP1589977A1
EP1589977A1 EP04775725A EP04775725A EP1589977A1 EP 1589977 A1 EP1589977 A1 EP 1589977A1 EP 04775725 A EP04775725 A EP 04775725A EP 04775725 A EP04775725 A EP 04775725A EP 1589977 A1 EP1589977 A1 EP 1589977A1
Authority
EP
European Patent Office
Prior art keywords
hek
hsf
composition
recited
secretion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04775725A
Other languages
German (de)
English (en)
Inventor
Carmen F. Arroyo
Damon L. Burman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Army Medical Research and Development Command
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US Army Medical Research and Development Command
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Filing date
Publication date
Application filed by US Army Medical Research and Development Command filed Critical US Army Medical Research and Development Command
Publication of EP1589977A1 publication Critical patent/EP1589977A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Definitions

  • Sulfur mustard (R/-y-(2-chloroethyl) sulf ⁇ de) is an alkylating agent with pathophysiological properties as a blistering agent and has been employed as a chemical warfare agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • dexamethasone (a) prolonged the survival time in rats poisoned by 3 LD 50 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.
  • 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 .
  • 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.
  • 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.
  • the present invention shows how 1- ⁇ , 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- ⁇ , 25(OH) 2 D 3 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 D 3 , or 1- ⁇ . 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.
  • EPA eicosapentaenoic acid
  • Figure 1(a) shows the effect of 1- ⁇ , 25(OH) 2 D 3 (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- ⁇ , 25(OH) 2 D 3 (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- ⁇ , 25(OH) 2 D 3 (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- ⁇ , 25(OH) 2 D 3 (1.0 x 10 "9 M) on the HEK secretion of IL-6 induced by HD (10 "4 M).
  • Figure 3(a) shows fluorescence measurements using the CyQuant Cell Proliferation Assay on HEK (5 x 10° cell/mL) treated with 1- ⁇ , 25(OH) 2 D 3 (5 x 10 " 9 M) after exposure to HD (10 "4 M).
  • Figure 3(b) shows fluorescence measurements using the CyQuant Cell proliferation assay on HEK (lxl 0 6 cell/mL) treated with 1- ⁇ , 25(OH) 2 D 3 (1 x 10 "9 M) after exposed to HD (10 "4 M).
  • Figure 4 shows the dose response effect of 1- ⁇ , 25(OH) 2 D 3 (1 x 10 "10 to 1 x 10 "7 M) on HEK cell proliferation in HD stimulated and then treated with 1- ⁇ , 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- ⁇ , 25(OH) 2 D 3 (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- ⁇ , 25(OH) 2 D 3 (2 x 10 "9 M).for 24 hours, after HD stimulation.
  • HD sulfur mustard
  • 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- ⁇ ), 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.
  • TNF- ⁇ tumor necrosis factor-alpha
  • IL-1 inter leukin-1
  • IL-6 inter leukin-6
  • 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- ⁇ , 25-dihydroxyvitamin D 3 .
  • the skin not only participates in the production of the vitamin D 3 but also contains receptors for l- ⁇ ,25(OH) 2 D 3 , therefore, suggesting a role of this hormone in the growth and differentiation of the skin.
  • l- ⁇ ,25(OH) D 3 Cultures of neonatal human foreskin keratinocytes have demonstrated that these cells produce l- ⁇ ,25(OH) D 3 from 25- hydroxy vitamin D 3 .
  • the production of l- ⁇ ,25(OH) 2 D 3 varies with the degree of differentiation of keratinocytes and is regulated by exogenous l- ⁇ ,25(OH) 2 D 3 .
  • receptors for the active metabolite of vitamin D 3 , l- ⁇ ,25(OH) 2 D 3 have been found in dermal fibroblasts, endothelial cells, and activated T lymphocytes .
  • Sulfur mustard (2, 2'-dichlorodiethyl sulfide, HD, 5 ⁇ L in 10 mL KGM TM , 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- ⁇ ,25-dihydroxyvitaminD 3 was obtained from Aldrich Chemical Company (Milwaukee, WI, USA) as 99% pure crystalline solids and were verified by NMR.
  • HSF human skin fibroblast
  • 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 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.
  • HEK cells 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 KGMTM).
  • KGM keratinocyte growth medium
  • KGM keratinocyte growth medium
  • Human IL-8 and IL-6 immunoassays produced by QuantikineTM 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- ⁇ ,25(OH) 2 D 3 (isopropanol (0.08 %) as vehicle control), HD-stimulated and HD-stimulated then treated with 1- ⁇ , 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.
  • 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 ⁇ L) of the CyQUANT ® GR dye stock solution was added and mixed thoroughly. Two hundred microliters (200 ⁇ L) 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 ® ,
  • This assay has a linear detection range extending from 50 or fewer to at least 50,000 cells in 200 ⁇ L 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 ⁇ L 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 ⁇ m 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- ⁇ , 25(OH) 2 D 3 for twelve or 24 hours, after which time the cell number/viability was determined by the colorimetric MTT assay.
  • PBS phosphate buffered saline
  • 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.
  • Table II shows the effect of HD, 1- ⁇ , 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%).
  • HD Upon exposure, HD induces stimulation of certain cytokines and chemokine such as interleukin i (IL-1), IL-6, tumor necrosis factor-alpha (TNF- ⁇ ) 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.
  • IL-1 interleukin i
  • IL-6 tumor necrosis factor-alpha
  • IL-8 tumor necrosis factor-alpha
  • the present invention shows that 1- ⁇ , 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- ⁇ , 25(OH) 2 D 3 inhibits the secretion of these cytokine/chemokine where IL-8 secretion was inhibited more than IL-6 secretion.
  • the pharmaceutically effective amount of 1- ⁇ , 25(OH) 2 D 3 is in the concentration range of 10 "9 or 10 "10 M, with a specific concentration of 2 x 10 "9 M.
  • the present invention also supports recent findings of the stimulatory effects of 1- ⁇ , 25(OH) 2 D 3 on keratinocyte proliferation.
  • the present invention also supports recent findings of the stimulatory effects of 1- ⁇ , 25(OH) 2 D 3 on keratinocyte proliferation.
  • the present invention also supports recent findings of the stimulatory effects of 1- ⁇ , 25(OH) 2 D 3 on keratinocyte proliferation.
  • the present invention also supports recent findings of the stimulatory effects of 1- ⁇ , 25(OH) 2 D 3 on keratinocyte proliferation.
  • 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.
  • the effective amount generally ranges from 5 ng/mL to 100 ug/mL and more preferably from 50 ng/mL to 2 ug/mL.
  • the human growth hormone is utilized in an effective amount of at least about 0.05 ng/mL.
  • 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.
  • 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.

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  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'on a évalué les effets régulateurs de la forme active de la vitamine D, 1-α, 25-dihydroxyvitamine D3 (1-α, 25(OH)2D3) sur la sécrétion de cytokines et de chimiokines induite par l'ypérite (HD) sur des fibroblastes cutanés humains (HSF) et sur des kératinocytes épidermiques humains (HEK). La stimulation de HSF par HD (10-4 M pendant 24 heures à 37° C) a eu pour effet de multiplier par cinq environ la quantité d'interleukine-6 (IL-6) sécrétée et par plus de dix la quantité d'interleukine-8 (IL-8) sécrétée, lesquelles sécrétions ont pu être inhibées par 1-α, 25(OH)2D3, à = 10-9 M. De plus, 1-α, 25(OH)2D3 a également divisé par cinq la quantité d'IL-8 sécrétée et par quatre celle d'IL-6 sur des HEK stimulés par HD à des concentrations = 10-9 M. L'effet de 1-α, 25(OH)2D3 était lié à la dose pour la diminution de la sécrétion de IL-6 et de IL-8 induite par HD sur HSF/HEK, et visible à des concentrations nanomolaires. Les résultats révèlent que la réduction de ces médiateurs inflammatoires par 1-α, 25(OH)2D3 est subordonnée à la source des cultures primaires, aux densités cellulaires et à la cinétique des prétraitements. En outre, l'invention a trait à une composition permettant de préparer un onguent topique pour le traitement de plaies chez des mammifères exposés à des agents de guerre chimique vésicants. La composition selon l'invention contient un composé ou une combinaison de 1-α, 25(OH)2D3 et d'huile de foie de requin. L'hormone stéroïde 1-α, 25(OH)2D3 (calcitriol) joue non seulement un rôle primordial dans l'homéostase du calcium, mais a également un effet bénéfique sur le système immunitaire, car elle régule la prolifération, la différenciation et la maturation cellulaires, et ce grâce à la limitation des protoocongènes et à la régulation de la production de cytokines.
EP04775725A 2003-01-14 2004-01-14 Onguent topique pour agents de guerre chimique vesicants Withdrawn EP1589977A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US43991903P 2003-01-14 2003-01-14
US439919P 2003-01-14
PCT/US2004/000833 WO2005016353A1 (fr) 2003-01-14 2004-01-14 Onguent topique pour agents de guerre chimique vesicants

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EP1589977A1 true EP1589977A1 (fr) 2005-11-02

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AU (2) AU2004264777A1 (fr)
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US20090187060A1 (en) 2008-01-22 2009-07-23 E-Z-Em, Inc. Method and Formulation for Neutralizing Toxic Chemicals and Materials
RU2397770C1 (ru) * 2008-12-30 2010-08-27 Закрытое акционерное общество Фармацевтическое научно-производственное предприятие "Ретиноиды" Поливитаминная мазь для смягчения, питания и заживления кожи и способ ее получения
RU2642603C2 (ru) * 2016-06-14 2018-01-25 Федеральное государственное бюджетное военное образовательное учреждение высшего образования Военно-медицинская академия им. С.М. Кирова Министерства обороны Российской Федерации (ВМедА) Способ профессионального отбора лиц для работ по уничтожению боевых отравляющих веществ

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PT892638E (pt) * 1996-04-04 2003-03-31 Cilag Ag Formulacao topica de vitamina d a base de lipossomas

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WO2005016353A1 (fr) 2005-02-24
AU2011200498A1 (en) 2011-03-03

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