The present invention provides methods of reducing cutaneous scar formation by treating a cutaneous wound with a composition comprising a therapeutic agent that is a sodium channel blocker and/or an inhibitor of the Nax/SCN7A pathway. The present invention also provides wound cover components impregnated with such compositions, kits composed of such compositions with a wound dressing or sterile wipe, and mixtures of such compositions with a topical component (e.g., cream, ointment, or gel) suitable for application to a cutaneous wound. The present invention also provides compositions, kits, devices, and methods for treating skin conditions (e.g., dermatitis, psoriasis, or other skin conditions) with such compositions and devices. Examples of such therapeutic agents include, but are not limited to, an inhibitor of a gene or protein selected from: ENac, COX-2, PGE2, PI3K, PKB, Nax Prss8, IL-1β, IL-8, SAPK, Erk gene, p38 gene, PAR2, S100A8, S100A9, S100A12.
The repair of injured skin tissue is a fundamental biological process essential to the continuity of life, but with potential for dysregulation and overcompensation. Derangements in healing can lead to excessive (hypertrophic) scar formation, for which there are a paucity of therapeutic options (1-4). Injury to the epidermis results in loss of epithelial barrier function which is not restored until the lipid barrier (stratum corneum) becomes fully competent. Of relevance to scarring, it has been demonstrated that scars have a perturbed barrier function as compared to unwounded skin for up to one year post-injury (5).
There is an extensive body of literature implicating the importance of the epidermal barrier function in cutaneous homeostasis (6, 7). Specifically, epithelial dehydration results in compensatory changes in the injured skin including up-regulation of inflammatory cytokines and activation of fibroblasts, which are implicated in hypertrophic scarring. Furthermore, it is notable that many skin disorders such as atopic dermatitis, ichthyosis and psoriasis have an impaired barrier function, and various emollients, and moisturizers improve their symptoms and reduce dermal inflammation by improving barrier function (8).
The present invention provides methods of reducing cutaneous scar formation by treating a cutaneous wound with a composition comprising a therapeutic agent that is a sodium channel blocker and/or an inhibitor of the Nax/SCN7A pathway. The present invention also provides wound cover components impregnated with such compositions, kits composed of such compositions with a wound dressing or sterile wipe, and mixtures of such compositions with a topical component (e.g., cream, ointment, or gel) suitable for application to a cutaneous wound. The present invention also provides compositions, kits, devices, and methods for treating skin conditions (e.g., dermatitis, psoriasis, or other skin conditions) with such compositions and devices. Examples of such therapeutic agents include, but are not limited to, an inhibitor of a gene or protein selected from: ENac, COX-2, PGE2, PI3K, PKB, Nax Prss8, IL-1β, IL-8, SAPK, Erk gene, p38 gene, PAR2, S100A8, S100A9, S100A12.
In certain embodiments, the present invention provides methods of reducing cutaneous scar formation comprising: applying a composition to a cutaneous wound of a subject, wherein the composition comprises a therapeutic amount of a therapeutic agent, wherein the therapeutic agent: i) is a sodium channel blocker, and/or ii) is an inhibitor of the Nax/SCN7A pathway.
In particular embodiments, the present invention provides compositions comprising: a) a therapeutic agent that is: i) is a sodium channel blocker, and/or ii) is an inhibitor of the Nax/SCN7A pathway; and b) a topical component suitable for application to a cutaneous wound, wherein the therapeutic agent is in the topical component.
In other embodiments, the present invention provides kits comprising: a) a composition comprising a therapeutic agent that is: i) is a sodium channel blocker, and/or ii) is an inhibitor of the Nax/SCN7A pathway; and b) a wound treatment component selected from the group consisting of: a sterile wipe and a wound dressing.
In some embodiments, the present invention provides a device for treating wounds comprising: a wound cover component configured to at least partially cover a cutaneous wound, wherein the wound cover component is impregnated with a therapeutic agent that is: i) is a sodium channel blocker, and/or ii) is an inhibitor of the Nax/SCN7A pathway.
In further embodiments, the present invention provides methods of treating dermatitis or psoriasis comprising: applying a composition to a dermatitis or psoriasis affected skin surface of a subject such that the symptoms of the dermatitis or psoriasis are reduced or eliminated, wherein the composition comprises a therapeutic amount of a therapeutic agent that is: i) is a sodium channel blocker, and/or ii) is an inhibitor of the Nax/SCN7A pathway.
In some embodiments, the therapeutic agent is an inhibitor of at least one of the following: a) epidermal sodium channel (ENac) mRNA or protein, b) cyclooxygenase-2 (COX-2) mRNA or protein, c) prostaglandin E2 (PGE2) mRNA or protein, d) phosphoinositide 3 kinase (PI3K) mRNA or protein, e) protein Kinase B (PKB or Akt) mRNA or protein, f) Nax (SCN7A) mRNA or protein, g) Prss8 mRNA or protein, h) interleukin-1β (IL-1β) mRNA or protein, i) interleukin 8 (IL-8) mRNA or protein, j) SAPK mRNA or protein, k) Erk mRNA or protein, 1) p38 mRNA or protein, m) PAR2 mRNA or protein, n) S100A8 mRNA or protein, o) S100A9 mRNA or protein, and p) S100A12 mRNA or protein. In particular embodiments, the agent inhibits the gene of the recited protein. In other embodiments, the recited protein (or gene encoding the protein) is human.
In some embodiments, the present invention provides methods of reducing cutaneous scar formation comprising: applying a composition to a cutaneous wound of a subject (e.g., a mammalian subject, human subject, a dog, a cat, a horse, etc.), wherein the composition comprises a therapeutic amount of a therapeutic agent selected from the group consisting of: a) an epidermal sodium channel (ENac) inhibitor; b) a cyclooxygenase-2 (COX-2) inhibitor; c) a prostaglandin E2 (PGE2) inhibitor; d) a phosphoinositide 3 kinase (PI3K) inhibitor, and e) a protein Kinase B (PKB or Akt) inhibitor.
In certain embodiments, said treating is repeated on at least 3 separate days (e.g., at least 3, 4, 5, . . . 10 . . . 15 . . . 20 . . . or 30 separate days). In some embodiments, said treating is repeated daily or bi-daily for at least a week.
In particular embodiments, the present invention provides methods of treating a skin condition (e.g., such as dermatitis (e.g., eczema, rash, seborrheic dermatitis, etc.), psoriasis, etc.) comprising: applying a composition to a dermatitis skin surface of a subject (e.g., a mammalian subject, human subject, a dog, a cat, a horse, etc.) such that the symptoms of dermatitis are reduced or eliminated, wherein the composition comprises a therapeutic amount of a therapeutic agent selected from the group consisting of: a) an epidermal sodium channel (ENac) inhibitor; b) a cyclooxygenase-2 (COX-2) inhibitor; c) a prostaglandin E2 (PGE2) inhibitor; d) a phosphoinositide 3 kinase (PI3K) inhibitor; and e) a protein Kinase B (PKB or Akt) inhibitor.
In certain embodiments, the present invention provides methods of reducing cutaneous scar formation comprising: applying a composition to a cutaneous wound of a subject such that the resulting scar formed from healing of the wound is smaller and/or less visible than would be present if the wound were un-treated, wherein the composition comprises a therapeutic agent is selected from the group consisting of: a) an epidermal sodium channel (ENac) inhibitor; b) a cyclooxygenase-2 (COX-2) inhibitor; c) a prostaglandin E2 (PGE2) inhibitor; d) a phosphoinositide 3 kinase (PI3K) inhibitor; and e) a protein Kinase B (PKB or Akt) inhibitor. In certain embodiments, the resulting scar has a lower skin elevation index score than if the wound were un-treated. In other embodiments, the therapeutic amount is about 0.1 to 10 mg per 1 cm2 of the cutaneous wound.
In particular embodiments, the present invention provides compositions comprising: a) a therapeutic agent selected from the group consisting of: i) an epidermal sodium channel (ENac) inhibitor; ii) a cyclooxygenase-2 (COX-2) inhibitor; iii) a prostaglandin E2 (PGE2) inhibitor; iv) a phosphoinositide 3 kinase (PI3K) inhibitor; and v) a protein Kinase B (PKB or Akt) inhibitor; and b) a topical component suitable for application to a cutaneous wound, wherein the therapeutic agent is in the topical component. In certain embodiments, the topical component is selected from the group consisting of: a topical cream, a topical foam, a topical gel, a topical lotion and a topical ointment. In other embodiments, the concentration of the therapeutic agent in the composition is about 0.1% to about 1%).
In some embodiments, the present invention provides kits comprising: a) a composition comprising a therapeutic agent selected from the group consisting of: i) an epidermal sodium channel (ENac) inhibitor; ii) a cyclooxygenase-2 (COX-2) inhibitor; iii) a prostaglandin E2 (PGE2) inhibitor; iv) a phosphoinositide 3 kinase (PI3K) inhibitor; and v) a protein Kinase B (PKB or Akt) inhibitor; and b) a wound treatment component selected from the group consisting of: a sterile wipe and a wound dressing. In particular embodiments, the wound dressing is selected from the group consisting of: gauze, adhesive bandage, films, gels, foams, hydrocolloids, alginates, hydrogels, polysaccharide pastes, granules and beads.
In further embodiments, the present invention provides devices for treating wounds comprising: a wound cover component configured to at least partially cover a cutaneous wound, wherein the wound cover component is impregnated with a therapeutic agent selected from the group consisting of: i) an epidermal sodium channel (ENac) inhibitor; ii) a cyclooxygenase-2 (COX-2) inhibitor; iii) a prostaglandin E2 (PGE2) inhibitor; iv) a phosphoinositide 3 kinase (PI3K) inhibitor; and v) a protein Kinase B (PKB or Akt) inhibitor.
In certain embodiments, the wound cover component is impregnated with the therapeutic agent such that the therapeutic agent migrates out of the wound cover component when it is applied to a cutaneous wound. In further embodiments, the wound cover component is selected from the group consisting of: gauze, an adhesive bandage, and a film.
In some embodiments, the ENac inhibitor is selected from the group consisting of: amiloride, triamterene, benzamil, GS9411, P-365, and pyrazine derivatives (see, e.g., U.S. Pat. No. 8,372,845, which is herein incorporated by reference for compounds recited therein). Additional ENac inhibitors can be found by using the screening methods of U.S. Pat. No. 8,105,792, herein incorporated by reference. In further embodiments, the COX-2 inhibitor is selected from the group consisting of: celecoxib (Celebrex), valdecoxib (Bextra), rofecoxib (Vioxx), diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tolmetin, acetaminophen, and miRNA targeting the COX-2 gene (e.g., miR-26b, miR-199a*, miR-16, miR-101a, miR-143, miR-144, miR-145, miR-199a, miR-542-3p, and miR-543). In particular embodiments, the PGE2 inhibitor is selected from the group consisting of: curcumin, SC-560, AH6809, sulforaphane, wagonin, rifampin, and miRNA targeting the prostaglandin E2 gene. In other embodiments, the PI3K inhibitor is selected from the group consisting of: LY294002, Wortmannin, demethoxyviridin, Perifosine, CAL101, PX-866, IPI-145, BAY 80-6946, BEZ235, TGR 1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE-477, CUDC-907, AEZS-136, and miRNA targeting the phosphoinositide 3 kinase gene (e.g., miR-7). In particular embodiments, the PKB inhibitor is selected from the group consisting of: VQD-002, perifosine, miltefosine, AZD5363, and MK-2206.
The present invention provides methods of reducing cutaneous scar formation by treating a cutaneous wound with a composition comprising a therapeutic agent that is a sodium channel blocker and/or an inhibitor of the Nax/SCN7A pathway. The present invention also provides wound cover components impregnated with such compositions, kits composed of such compositions with a wound dressing or sterile wipe, and mixtures of such compositions with a topical component (e.g., cream, ointment, or gel) suitable for application to a cutaneous wound. The present invention also provides compositions, kits, devices, and methods for treating skin conditions (e.g., dermatitis, psoriasis, or other skin conditions) with such compositions and devices. Examples of such therapeutic agents include, but are not limited to, an inhibitor of a gene or protein selected from: ENac, COX-2, PGE2, PI3K, PKB, Nax Prss8, IL-1β, IL-8, SAPK, Erk gene, p38 gene, PAR2, S100A8, S100A9, S100A12. Although it is known that the inflammatory response which results from disruption epithelial barrier function after injury results in excessive scarring, the upstream signals were unknown. Epithelial disruption results in transepithelial water loss (TEWL). In developing embodiments of the present invention, it was hypothesized that epithelial cells can sense TEWL via changes in sodium homeostasis and sodium flux into keratinocytes. In developing embodiments of the present invention, it was also hypothesized that these changes in sodium flux result in activation of pathways responsible for keratinocyte-fibroblast signaling, and ultimately lead to activation of fibroblasts. In the Examples presented below, it was demonstrated that perturbations in epithelial barrier function lead to increased TEWL and increased sodium flux in keratinocytes. It was identified that sodium flux in keratinocytes is mediated by epithelial sodium channels (ENaC). It was also demonstrated that activation of ENaC cause increased secretion of proinflammatory cytokines and activation of fibroblast via the COX-2/prostaglandin E2 (PGE2) pathway. Similar changes in signal transduction and sodium flux occur by increasing the sodium concentration in the media in epithelial cultures, or human ex vivo skin cultures (HESC) in vitro. Blockade of ENaC activation, prostaglandin synthesis by COX-2, or PGE2 receptors all reduce markers of fibroblast activation and collagen synthesis. In addition, as described in the Examples below, employing a validated in vivo excessive scar model in the rabbit ear, it was demonstrated that utilization of either an ENaC sensitive sodium channel blocker or a COX-2 inhibitor results in a marked reduction in scarring. Other experiments demonstrated that the activation of COX-2 in response to increase sodium flux is mediated through the PIK3/Akt pathway. While the present invention is not limited to any particular mechanism, and an understanding of the mechanism is not necessary to practice the invention, these results appear to indicate that ENaC responds to small changes in sodium concentration with inflammatory mediators, and suggests that ENaC pathway is a potential target for a novel strategy to prevent fibrosis.
Work conducted during the development of embodiments of the present invention showed successful knocked down of the expression of Nax, Prss8, and ENaC with shRNA in human keratinocytes (HK). The shRNA sequences employed were as follows:
In work conducted during the development of embodiments of the present invention, three downstream genes, Cyclooxygenase 2 (COX-2), Interleukin-1β (IL-1β), and Interleukin 8 (IL-8) were found related to this Nax-Prss8-ENaC pathway. As a consequence of the ENaC knockdown in HK, one of the important inflammatory factors, COX-2 was significantly down-regulated with the knockdown of Nax, Prss8, or ENaC in keratinocytes compared to the wild type during increased water loss. However, the expressions of the other two cytokines, IL-1β and IL-8 were only down-regulated with the knockdown of Nax or Prss8, but not ENaC. This suggests that Nax may initiate two different pathways. One is to regulate the expressions of IL-1β and IL-8. The other pathway is through activation of ENaC and further regulates the expression of COX-2. Both pathways were conducted by Prss8 following the Nax.
To further confirm the pathway of Nax-Prss8-ENaC, a Prss8 homolog, trypsin was used to activate the ENaC with the knockdown of Nax. With the knockdown of Nax, keratinocytes failed to be activated by cell differentiation medium. Additional trypsin in the differentiation medium successfully recovered the differentiation of Nax knockdown cells. Meanwhile, the additional trypsin also up-regulated the expression of COX-2, IL-1β, and IL-8 in the Nax knockdown keratinocytes under the stimulation of high concentration of sodium. The results suggested that Nax can conduct the activation of ENaC and the other pathway through Prss8.
In work conducted during the development of embodiments of the present invention, co-cultured with human dermal fibroblasts (HDF), the wild type HK dramatically activated the HDF with increased sodium concentration. However, Nax knockdown HK failed to activate most of the co-cultured HDF with the increased sodium concentration. Since the HDF is the main player of skin hypertrophic scar formation, this suggests the importance of HK Nax in determination of scar hypertrophy in wound healing.
Inhibitors of Prss8 (e.g., human Prss8) mRNA or protein include, but are not limited to, aprotinin, antipain, leupeptin, benzamidine, hepatocyte growth factor activator gene or protein inhibitor 1 (HAI-1), antibodies directed toward the Prss8 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward Prss8 mRNA.
Inhibitors of epidermal sodium channel (ENac) (e.g., human ENac) mRNA or protein include, but are not limited to, amiloride, triamterene, benzamil, GS9411, P-365, pyrazine derivatives, antibodies directed toward the ENac protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward ENac mRNA.
Inhibitors of cyclooxygenase-2 (COX-2) (e.g., human COX-2) mRNA or protein include, but are not limited to, celecoxib (Celebrex), valdecoxib (Bextra), rofecoxib (Vioxx), diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tolmetin, acetaminophen, antibodies directed toward the COX-2 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward ENac mRNA.
Inhibitors of prostaglandin E2 (PGE2) (e.g., human PGE2) mRNA or protein include, but are not limited to, curcumin, SC-560, AH6809, sulforaphane, wagonin, rifampin, antibodies directed toward the PGE2 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward PGE2 mRNA.
Inhibitors of phosphoinositide 3 kinase (PI3K) (e.g., human PI3K) mRNA or protein include, but are not limited to, LY294002, Wortmannin, demethoxyviridin, Perifosine, CAL101, PX-866, IPI-145, BAY 80-6946,BEZ235, TGR 1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE-477, CUDC-907, AEZS-136, antibodies directed toward the PI3K protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward PI3K mRNA.
Inhibitors of protein Kinase B (PKB or Akt) (e.g., human PKB) mRNA or protein include, but are not limited to, VQD-002, perifosine, miltefosine, AZD5363, and MK-2206, antibodies directed toward the PKB protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward PKB mRNA.
Inhibitors of Nax (SCN7A) (e.g., human Nax) mRNA or protein include, but are not limited to, antibodies directed toward the Nax protein (e.g., a monoclonal antibody directed towards the human protein), and siRNA or miRNA directed toward Nax mRNA (see SEQ ID NOs: 1 and 2, and those described in Ke et al., Neuroscience, 2012, Dec. 27:227:80-9, herein incorporated by reference).
Inhibitors of interleukin-1β (IL-1β) (e.g., human IL-1beta) mRNA or protein include, but are not limited to, Canakinumab (Ilaris) or other antibodies directed toward the IL-1β protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward IL-1β mRNA (e.g., as described in Peng et al., Glia. 2006 Nov. 1; 54(6):619-29, herein incorporated by reference).
Inhibitors of interleukin 8 (IL-8) (e.g., human IL-8) mRNA or protein include, but are not limited to, Reparixin, molecules disclosed in U.S. Pat. No. 6,448,379 (herein incorporated by reference), the peptide Ac-Arg-Arg-Trp-Trp-Cys-Arg-NH2 (SEQ ID NO: 7), antibodies directed toward the IL-8 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward IL-8 mRNA (see, e.g., Merritt et al., JNCI J Natl Cancer Inst Volume 100, Issue 5, Pp. 359-372, herein incorporated by reference).
Inhibitors of SAPK (e.g., human SAPK) mRNA or protein include, but are not limited to, SP600125, DJNK11, antibodies directed toward the SAPK protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward SAPK mRNA (see e.g., Shen et al., Cell Biochem Biophys. 2012 September; 64(1):17-27, herein incorporated by reference).
Inhibitors of Erk (e.g., human Erk) mRNA or protein include, but are not limited to, SCH772984, FR180204, AEZS-131, PD98059, antibodies directed toward the Erk protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward Erk mRNA.
Inhibitors of p38 (e.g., human p38) mRNA or protein include, but are not limited to, SB203580, LY2228820, SC-68376, VX-745, antibodies directed toward the p38 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward p38 mRNA (see e.g., Chen et al., Cancer Res Dec. 1, 2009 69; 8853; herein incorporated by reference).
Inhibitors of PAR2 (e.g., human PAR2) mRNA or protein include, but are not limited to, K-12940, K-14585, P2pal-18S, antibodies directed toward the PAR2 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward PAR2 mRNA (see, e.g., Lin et al., Int J Biochem Cell Biol. 2008; 40(6-7): 1379-1388).
Inhibitors of S100A8 (e.g., human S100A8) mRNA or protein include, but are not limited to, arachidonic acid, antibodies directed toward the S100A8 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward S100A8 mRNA.
Inhibitors of S100A9 (e.g., human S100A9) mRNA or protein include, but are not limited to, arachidonic acid, quinoline-3-carboxamide compounds, antibodies directed toward the S1009 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward S100A9 mRNA. Inhibitors of S100A12 (e.g., human S100A12) mRNA or protein include, but are not limited to, epigallocatechin-3-gallate (EGCG), antibody ab37657, or other antibodies directed toward the S10012 protein (e.g., a monoclonal antibody directed toward the human protein), and siRNA or miRNA directed toward S100A12 mRNA.
The therapeutic agents of the present invention may be formulated in compositions for topical administration, such as in ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In certain embodiments, topical formulations comprise patches or dressings such as a bandage or adhesive plasters impregnated with the therapeutic agent, and optionally one or more excipients or diluents. In some embodiments, the topical formulations include a compound(s) that enhances absorption or penetration of the therapeutic agent(s) through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide (DMSO) and related analogues.
If desired, the aqueous phase of a cream base includes, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. In some embodiments, oily phase emulsions of this invention are constituted from known ingredients in an known manner. This phase typically comprises a lone emulsifier (otherwise known as an emulgent), it is also desirable in some embodiments for this phase to further comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
In certain embodiments, a hydrophilic emulsifier is included together with a lipophilic emulsifier so as to act as a stabilizer. It some embodiments it is also preferable to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include, for example, Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
The choice of suitable oils or fats for the formulation is based on achieving the desired properties (e.g., cosmetic properties), since the solubility of the active compound/agent in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus creams should preferably be a non-greasy, non-staining and washable products with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
This Example describes experiments conducted to evaluate the mechanism by which TEWL (trans-epidermal water loss) leads to pro-inflammatory cytokine expression and increased scarring. It was hypothesized that changes in epithelial hydration and sodium homeostasis are monitored through ENaC (epithelial sodium channel) and that this protein regulates downstream inflammatory pathways leading to fibroblast activation. Remarkably, blocking ENaC or ENaC mediated signal transduction with a commercially available sodium channel blocker (amiloride) or a COX-2 inhibitor lead to significant improvement in scarring. Given that compromised barrier function with increased TEWL is a major factor in many types of inflammatory dermatitis, these targets are useful for many skin diseases.
Increased trans-epidermal water loss results in higher sodium flux. To estimate the change of sodium flux of skin, the sodium flux was measured in human ex vivo skin culture (HESC), which retains important elements of in vivo skin and recapitulates the features of human wound repair (25, 26). The HESCs were cultured in an air/liquid interface after tape-stripping the epithelium to remove the stratum corneum and impair the barrier function of skin. Conditions to increase TEWL were created by placing the cultured HESCs in an air-flow chamber with reduced humidity. Control HESCs were cultured in closed chamber which maintains a humid environment. To validate increased TEWL, expression levels of aquaporin 3 (AQP3) were measured, which has been reported as a molecular marker to indicate the hydration conditions of skin (27, 28). Western blot analysis showed that expression of AQP3 protein was increased by 80% in cultured HESC cells placed in the dry air-flow chamber over controls. This study was replicated in stratified keratinocyte cultures (HaCaT) to validate results. Conditions to promote water loss from keratinocytes were created by exposing the surface of cultured cells to air-flow. Control stratified keratinocyte cultures were kept in a liquid environment of standard media. Both mRNA and protein expression analysis showed enhanced expression of AQP3 by increased TEWL in stratified keratinocytes culture.
Changes in sodium flux were evaluated using a scanning ion-selective electrode technique (SIET). HESCs placed in air-flow chambers demonstrated increased sodium flux compared to control HESCs (
Water loss may also be modeled by increasing the concentration of sodium in the culture medium and such a study was conducted to confirm the results above. Sodium concentration was increased in the culture medium, which contains 110 mM sodium, by 10% (11 mM), to 121 mM sodium. Increasing sodium concentration in the culture medium enhanced the sodium flux in HESC and HaCaT cells by five and two fold, respectively, compared to controls (
(
The amiloride-sensitive ENaC facilitates sodium absorption in many epithelia tissues, such as kidney (29) and lung (30). It was investigated whether ENaC mediates the changes in sodium sodium flux caused by increased water loss or increased sodium concentration. This was accomplished by evaluating changes in sodium flux after pharmacological blockade and gene knockdown of ENaC. Treatment of stratified keratinocytes with amiloride (10 μg/ml) completely abolished the enhanced sodium flux due to increased water loss (
In contrast to mucosal wounds which heal in a liquid environment, cutaneous wounds have increased water loss. It was hypothesized that increased sodium flux associated with TEWL upon cutaneous injury results in up-regulation of inflammatory genes including COX-2 and the downstream end product prostaglandin E2 (PGE2). The expression of COX-2 mRNA in epidermis of HESC was increased by 3.3±1.7 (mean±s.e.m.) fold within 4 hours of increased water loss treatment (
ELISA was performed to quantify the amount of PGE2 secreted by cells into the culture medium (
Previous reports have identified phosphorylation cascades that can activate COX-2 expression (32-34). In this Example, it was tested whether phosphorylation of Akt mediates the up-regulation of COX-2 expression and activity caused by increased sodium flux. Phosphorylation of these pathways was evaluated 10, 30, 60 and 240 minutes after HaCaT cells were treated with increased water loss or increased sodium concentrations. Phophorylation of Akt was rapidly increased at 10 min with increased water loss or 10% higher sodium concentration in culture, and the increase lasted until 60 minutes post treatment (
It is known that phosphatidylinositol 3-kinases (PI3K) activation activates phosphorylation of Akt. This Example addressed whether PI3K/Akt signaling is involved in activation of the COX-2 pathway caused by increases in sodium flux. Treatment of PI3K inhibitor, LY294002, reduced the basal level COX-2 mRNA expression in human foreskin primary keratinocytes (HK) and PGE2 protein in culture medium (
It was investigated whether ENaC is involved in this epidermal-dermal cross talk. Human dermal fibroblasts were co-cultured with stratified HaCaT cells on 6 well plates. Increased water loss was generated by placing stratified HaCaT air/liquid interface in a dry air flow chamber. In controls, both fibroblast and stratified HaCaT were submerged in culture medium. It is known that dermal fibroblasts show increased expression of alpha smooth muscle actin (α-SMA) and pro-collagen I (pro-Col I) upon activation (35, 36). Expression of α-SMA and pro-Col I in dermal fibroblast was significantly increased by water loss when compared to controls (
These results also strongly suggest that the ENaC pathway is important for the COX-2 expression in response to sodium flux in keratinocytes (
Since the PGE2 is a downstream product of COX-2 and found to be involved in regulation of fibroblast cytoskeletal dynamics during airway epithelium injury (37), it was hypothesized that PGE2 signaling is important for keratinocyte-fibroblast signaling. Prostaglandins EP2 and EP4, after secretion from keratinocytes, and since they can activate target cells via PGE2 receptors, making them suitable for cellular interactions. Compared to the wild type HaCaT culture condition medium, the ENaC knockdown HaCaT culture medium failed to activate the dermal fibroblasts (
Sustained activation of dermal fibroblasts with accumulation of collagen is the main cause of hypertrophic scars (38, 39). The in vitro data above demonstrated that the ENaC-COX2 pathway is critical for the regulation of dermal fibroblast activation by epidermal keratinocytes. The role of the ENaC-COX-2 pathway was investigated in vivo utilizing a well validated hypertrophic scar model in the rabbit ear (31, 40-43). Amiloride, an ENaC antagonist, was topically applied to the rabbit ear wounds after re-epithelialization was complete. Control scars, located on the contralateral ear, were treated with vehicle alone. In this model, absence of scar elevation gives a scar elevation index (SEI) of 1.0, and control wounds heal with an elevated scar (SEI>1). Remarkably, rabbit ear wounds treated with 0.5 mg/wound of amiloride dramatically decreased the SEI from 1.42±0.08 to 1.06±0.05 (
The epidermis plays a critical barrier function which maintains the hydration level of the deeper layers of skin. Increased TEWL occurs immediately upon injury to the lipid containing stratum corneum. Water loss of the skin causes alteration of expression levels of many genes involved in skin barrier lipid synthesis (46), keratinocyte differentiation and desquamation (47). However, the mechanism of gene regulation in response to TEWL remains largely unknown due to the lack of methods to detect the change of the microenvironment caused by TEWL in situ. It was hypothesized that increased TEWL resulted in increased sodium flux, which was confirmed with the use the STET which demonstrated a high sensitivity to detect the ion flux under 0.01 pmol·cm-2·s-1. To reduce background to acceptable levels, cells were first treated with conditions of increased TEWL or sodium concentration, and then transferred to buffer with low sodium concentration and then measuring sodium flux compared to controls. Both keratinocytes and HESC showed increased similar sodium fluxes to increased TEWL (exposure to air with water evaporation) or 10% increased sodium, a level reached in the extracellular fluid in a water deprived thirsty animal (48).
Developed by Shipley and Feijo in 1999 (49), the SIET is an excellent tool to measure ion fluxes in vivo since the microelectrode can be non-invasively manipulated very close around to live cells or tissues. The measurement of SIET on HESCs and keratincoytes reliably reflected the sodium consumption during the water loss. Interestingly, the sodium flux level in HESCs was measured to much higher than the differentiated keratinocyte cultures. This may reflect the difference between this two skin substitutes. The HESC model is human skin grown in vitro. It contains all the layers of differentiated keratinocytes, including stratum basale, stratum spinosum, stratum granulosum, and stratum lucidum (the stratum corneum was removed before use by tape stripping). Previous studies have demonstrated that ENaC is preferentially expressed in differentiated keratinocytes (50). Since the cultured keratinocytes in this Example were only grown into 3-4 layers, they may have not been as differentiated as HESCs and may have expressed lower levels of ENaC. This may account for the stronger sodium fluxes detected on HESCs compared to keratinocytes. In addition the HESCs have a complex organization which may have other effects on sodium flux. Nevertheless, both models showed same trend of sodium flux in response to the TEWL and high sodium concentration.
Prior to this invention, the primary signal cascade, which is activated in response to TEWL, was believed to be unknown. Based on the results obtained from this Example, increased epithelial water loss raises extracellular sodium concentration which activates ENaC resulting in an influx of sodium into keratinocytes. Recently, ENaC has been demonstrated to play a critical role in embryo implantation in the uterus through increased COX-2 expression by endometrial epithelial cells. In this system, calcium was found to be the secondary messenger to initiate the phosphorylation of cAMP response element-binding protein (CREB) in response to sodium influx (51). In this Example, SIET did not detect the appearance of calcium influx upon the stress of increased water loss on keratinocytes. To further confirm this, the calcium channel was blocked by using benidipine hydrochloride (52).
The qPCR with the calcium blocked keratinocytes suggested that the increased expression levels of COX-2 in HKs during increased water loss or sodium concentration were not abolished by blocking the calcium channel, although there was slight decrease in mRNA. Since the calcium dependent pathway is not the only way to activate CREB as a transcription factor of COX-2, the ENaC may follow another pathway to phosphorylate the CREB in skin keratinocytes. Previous studies suggested that CREB phosphorylation can be performed through several routes, such as Grb2/ERK1 in retina (53), PI3k/Akt in kidney (54) and pancreatic ductal epithelial cells (55), and PKA/p38/MSK1 in fibroblasts (56). The western results generated during this Example suggested that the phosphorylation of Akt were rapidly increased as early as 10 min post stimulation (
Taken that the ENaC activation in keratinocytes plays an important role in activation of dermal fibroblasts, antagonists targeting ENaC are useful for preventing or reducing scarring, as well as other skin conditions with increased TEWL. The ability to treat locally would limit toxicity concerns. The remarkable success of the antagonist used in this Example, amiloride, is a commercially available component that has been used in the treatment of hypertension (57) and congestive heart failure (58). The study of amiloride in rabbit ear scar treatment clearly showed the improvement of the scar formation at 28 days post wounding. Similarly, the inhibition of COX-2 activity with Celecoxib also contributed to the reduction of hypertrophy of rabbit ear scar.
While the present invention is not limited to any mechanism, it is believed that the increased water loss upon disruption of skin barrier results in a change in sodium concentration, which is sensed by ENaC. The intake of sodium in response to the activation of ENaC results in the phosphorylation of Akt through PI3K, which leads to the increase of COX-2 synthesis. The regulation of COX-2 at the transcription level is not clear although recent studies on the critical steps for embryo implantation in the uterus suggests that the CREB is the factor that initiates the production of COX-2 in epithelial cells of uterus (51). The up-regulation of COX-2 promotes the production and release of PGE2 to the matrix, which dramatically stimulates the dermal fibroblasts through receptor EP2 and EP4 to contribute to the hypertrophic scar formation (
Journal of investigative dermatology 125(2):183-200.
All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
The present Application is a continuation of U.S. patent application Ser. No. 15/241,841, filed Aug. 19, 2016, which is a continuation of abandoned U.S. patent application Ser. No. 14/469,823, filed Aug. 27, 2014, which claims priority to U.S. Provisional Application Ser. No. 61/870,607, filed Aug. 27, 2013, each of which is herein incorporated by reference in its entirety.
Number | Date | Country | |
---|---|---|---|
61870607 | Aug 2013 | US |
Number | Date | Country | |
---|---|---|---|
Parent | 15241841 | Aug 2016 | US |
Child | 16253735 | US | |
Parent | 14469823 | Aug 2014 | US |
Child | 15241841 | US |