WO2005007072A2 - Methods and pharmaceutical compositions for healing wounds - Google Patents
Methods and pharmaceutical compositions for healing wounds Download PDFInfo
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- WO2005007072A2 WO2005007072A2 PCT/IL2004/000640 IL2004000640W WO2005007072A2 WO 2005007072 A2 WO2005007072 A2 WO 2005007072A2 IL 2004000640 W IL2004000640 W IL 2004000640W WO 2005007072 A2 WO2005007072 A2 WO 2005007072A2
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
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- A61K38/22—Hormones
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- A61K38/30—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
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- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- the present invention relates to a method and a pharmaceutical composition for inducing and or accelerating cell proliferation and/or cell differentiation and thereby accelerating the healing process of wounds. More particularly, the present invention relates to the use of modulated expression and/or activation, e.g., as initiated by membrane translocation and activation, of serine/threonine protein kinases, also known as PKCs, for inducing and/or accelerating cell proliferation and/or cell differentiation and/or cell migration thereby accelerating the healing process of wounds.
- modulated expression and/or activation e.g., as initiated by membrane translocation and activation, of serine/threonine protein kinases, also known as PKCs
- Such modulated expression may be effected in accordance with the teachings of the present invention by (i) transformation of wound cells with a PKC expressing construct; (ii) transformation of wound cells with a cis-acting element to be inserted adjacent to, and upstream of, an endogenous PKC gene ofthe wound cells; (iii) administration of insulin for inducing expression and/or activation of PKC in wound cells; (iv) transformation of wound cells with an insulin expressing construct, when expressed and secreted the insulin produced therefrom serves as an up-regulator for PKC expression and/or activation; (v) transformation of wound cells with a cis- acting element to be inserted adjacent to, and upstream of, the endogenous insulin gene of the wound cells, when expressed and secreted the insulin serves as an up- regulator for PKC expression and/or activation; (vi) implantation of insulin secreting cells to the wound; (vii) transformation of wound cells with a trans-acting factor, e.g., PDXl, for in
- Open cutaneous wounds represent one major category of wounds and include burn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers, and diabetic ulcers. Open cutaneous wounds routinely heal by a process which comprises six major components: (i) inflammation; (ii) fibroblast proliferation; (iii) blood vessel proliferation; (iv) connective tissue synthesis; (v) epithelialization; and (vi) wound contraction. Wound healing is impaired when these components, either individually or as a whole, do not function properly.
- diabetes mellitus is characterized by impaired insulin signaling, elevated plasma glucose and a predisposition to develop chronic complications involving several distinctive tissues.
- impaired wound healing leading to foot ulceration is among the least well studied.
- skin ulceration in diabetic patients takes a staggering personal and financial cost (29, 30).
- keratinocytes In vitro, keratinocytes can be maintained as basal proliferating cells with a high growth rate. Furthermore, differentiation can be induced in vitro following the maturation pattern in the epidermis in vivo. The early events include loss of hemidesmosome components (3,5) and a selective loss of the ⁇ 6 ⁇ 4 integrin and cell attachment to matrix proteins. This suggests that changes in integrin expression are early events in keratinocyte differentiation.
- the protein kinase C (PKC) family of serine-threonine kinases plays an important regulatory role in a variety of biological phenomena (8,9).
- the PKC family is composed of at least 12 individual isoforms which belong to 3 distinct categories: (i) conventional isoforms ( ⁇ , ⁇ l, ⁇ 2, ⁇ ) activated by Ca 2+ , phorbol esters and diacylglycerol liberated intracellularly by phospholipase C; (ii) novel isoforms ( ⁇ , ⁇ , ⁇ , ⁇ ) which are also activated by phorbol esters and diacylglycerol but not by Ca 2+ ; and (iii) atypical ( ⁇ , ⁇ , t) members of the family, which are not activated by Ca 2+ , phorbol esters or diacylglycerol.
- the present invention provides novel methods and compositions for treating wounds efficiently and without adverse side effects, by providing to the wound area effective amount of insulin and/or other agents capable of modulating expression and/or activity of PKC in wound-colonizing cells and acting in synergy with insulin to accelerate the process of wound healing.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of administering to the damaged skin or skin wound a therapeutically effective amount of an agent for modulating PKC production and/or PKC activation.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a therapeutically effective amount of at least one agent for modulating PKC production and/or activity; and a pharmaceutically acceptable carrier.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of administering to the damaged skin or skin wound a therapeutically effective amount of insulin and at least one additional agent acting in synergy with the insulin, so as to induce or accelerate the healing process of the damaged skin or skin wound.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a therapeutically effective amount of insulin, at least one additional agent acting in synergy with the insulin, and a pharmaceutically acceptable carrier being designed for topical application ofthe pharmaceutical composition.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of administering to the damaged skin or skin wound a single dose of a therapeutically effective amount of insulin, thereby inducing or accelerating the healing process ofthe damaged skin or skin wound.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a single dose-unit of insulin selected capable of inducing or accelerating the healing process of the damaged skin or skin wound, and a pharmaceutically acceptable carrier being designed for topical application of the pharmaceutical composition.
- a method of inducing or accelerating a healing process of an old skin wound comprising the step of administering to the old skin wound a single dose of a therapeutically effective amount of insulin, thereby inducing or accelerating the healing process ofthe old skin wound.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of implanting into the damaged skin or skin wound a therapeutically effective amount of insulin secreting cells, so as to induce or accelerate the healing process ofthe damaged skin or skin wound.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, insulin secreting cells, and a pharmaceutically acceptable carrier being designed for topical application ofthe pharmaceutical composition.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of transforming cells of the damaged skin or skin wound to produce and secrete insulin, so as to induce or accelerate the healing process ofthe damaged skin or skin wound.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a nucleic acid construct being designed for transforming cells of the damaged skin or skin wound to produce and secrete insulin, and a pharmaceutically acceptable carrier being designed for topical application of the pharmaceutical composition.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of transforming cells ofthe damaged skin or skin wound to produce a protein kinase C, so as to induce or accelerate the healing process ofthe damaged skin or skin wound
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a nucleic acid construct being designed for transforming cells of the damaged skin or skin wound to produce a protein kinase C, and a pharmaceutically acceptable carrier being designed for topical application of the pharmaceutical composition.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of administering to the damaged skin or skin wound a therapeutically effective amount of PKC activator, so as to induce or accelerate the healing process ofthe damaged skin or skin wound.
- a pharmaceutical composition of inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a therapeutically effective amount of PKC activator, so as to induce or accelerate the healing process of the damaged skin or skin wound, and an acceptable pharmaceutical carrier.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound comprising the step of administering to the damaged skin or skin wound a therapeutically effective amount of copolymer- 1.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a therapeutically effective amount of copolymer- 1 and a pharmaceutically acceptable carrier being designed for topical application of the pharmaceutical composition.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound comprising, as an active ingredient, a therapeutically effective amount of copolymer- 1 and a pharmaceutically acceptable carrier being designed for topical application of the pharmaceutical composition.
- a method of inducing or accelerating a healing process of a damaged skin or skin wound including modulating expression and/or activity of at least one PKC isoform in dermal cells colonizing ihe damaged skin or skin wound; and administering to the dermal cells a therapeutically effective amount of at least one additional agent selected from the group consisting of a hormone, a growth factor, an adipokine, PKC ⁇ RACK and GW9662 with the modulating expression and or activity ofthe PKC isoform to thereby induce or accelerate the healing process ofthe damaged skin or skin wound.
- a pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound including, as an active ingredient, a therapeutically effective amount of a substance for modulating expression or activation of at least one PKC isoform and at least one additional agent selected from the group consisting of a hormone, a growth factor an adipokine, PKC ⁇ RACK and GW9662 and a pharmaceutically acceptable carrier.
- the wound is selected from the group consisting of an ulcer, a diabetes related wound, a burn, a sun burn, an aging skin wound, a corneal ulceration wound, an inflammatory gastrointestinal tract disease wound, a bowel inflammatory disease wound, a Crohn's disease wound, an ulcerative colitis, a hemorrhoid, an epidermolysis bulosa wound, a skin blistering wound, a psoriasis wound, saborehic dermatitis wound an animal skin wound, an animal diabetic wound, a retinopathy wound, an oral wound (mucositis), a vaginal mucositis wound, a gum disease wound, a laceration, a surgical incision wound and a post surgical adhesis wound.
- the ulcer is a diabetic ulcer, a pressure ulcer, a venous ulcer, a gastric ulcer and an HIN related ulcer.
- the insulin is recombinant.
- the insulin is of a natural source.
- the additional agent is a platelet-derived growth factor.
- the additional agent is a PKC- ⁇ inhibitor.
- administering is effected by a single application.
- the old skin wound is at least 2 days old.
- the insulin has an insulin concentration ranging from 0.1 ⁇ M to lO ⁇ M.
- the dose-unit of insulin is 0.001 to 5 nM in 0.01 - 0.2 ml ofthe pharmaceutical composition.
- the dose of insulin is ranging from 0.01 to 0.5 nM in 0.01 - 0.2 ml of the pharmaceutical composition.
- the pharmaceutical composition is selected from the group consisting of an aqueous solution, a gel, a cream, a paste, a lotion, a spray, a suspension, a powder, a dispersion, a salve and an ointment.
- the pharmaceutical composition includes a solid support.
- the cells are transformed to produce and secrete insulin.
- the cells are transformed by a recombinant PDXl gene and therefore the cells produce and secrete natural insulin.
- the cells are transformed by a cis-acting element sequence integrated upstream to an endogenous insulin gene of the cells and therefore the cells produce and secrete natural insulin.
- the insulin secreting cells are capable of forming secretory granules.
- the insulin secreting cells are endocrine cells.
- the insulin secreting cells are of a human source. According to still further features in the described preferred embodiments the insulin secreting cells are of a histocompatibility humanized animal source. According to still further features in the described preferred embodiments the insulin secreting cells secrete human insulin. According to still further features in the described preferred embodiments the insulin secreting cells are autologous cells. According to still further features in the described preferred embodiments the cells are selected from the group consisting of fibroblasts, epithelial cells and keratinocytes. According to still further features in the described preferred embodiments the cells are transformed to produce a protein kinase C transcription activator and therefore the cells produce natural protein kinase C.
- the cells are transformed by a cis-acting element sequence integrated upstream to an endogenous protein kinase C of the cells and therefore the cells produce natural protein kinase C.
- the cells are transformed by a recombinant protein kinase C gene and therefore the cells produce recombinant protein kinase C.
- the protein kinase C is selected from the group consisting of PKC- ⁇ l, PKC- ⁇ 2, PKC- ⁇ ,
- the protein kinase C is selected from the group consisting of PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ and PKC- ⁇ .
- the copolymer- 1 is contained in a pharmaceutical composition adapted for topical application. According to still further features in the described preferred embodiments the
- PKC isoform is selected from the group consisting of PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , and PKC- ⁇ .
- the hormone is insulin.
- the growth factor is selected from the group consisting of IL-6, KFG and TNF ⁇ .
- the adipokine is adipsin or adiponectin.
- FIG. 1 demonstrates effective over-expression of PKC isoforms utilizing recombinant adenovirus vectors: Left panel: four day old primary keratinocytes were infected for 1 hour utilizing ⁇ -gal adenovirus 48 hours following infection, cells were fixed and activation of ⁇ -galactosidase protein was quantified by the induction of blue color reaction in comparison to uninfected keratinocytes. Right panel: four day old primary keratinocytes were infected for 1 hour utilizing recombinant isoform specific PKC adenovirases.
- FIG. 2 shows that PKC activation by bryostatin 1 induces translocation of over-expressed PKC isoforms.
- Four day old primary keratinocytes were infected for 1 hour with isoform specific recombinant PKC adenovirases.
- Twenty four hours following infection cells were either untreated (C) or stimulated with bryostatin 1 (B) for 30 minutes, and fractionated. Protein samples were subjected to Western blotting and analyzed using isoform specific anti-PKC antibodies.
- FIG. 3 shows that over-expressed PKC isoforms are active in their native form.
- Four day old primary keratinocytes were infected for 1 hour with isoform specific recombinant PKC adenovirases. Eighteen hours following infection, cell lysates from uninfected control cells (C) and PKC isoforms over-expressing cells (OE) were immunoprecipitated using isoform specific anti-PKC antibodies. Immunoprecipitates were subjected to PKC activity assay as described in the Examples section that follows.
- FIG. 4 demonstrates that over-expression of specific PKC isoforms induces distinct morphologic changes in primary keratinocytes.
- FIG. 5 shows distinct localization of over-expressed PKC isoforms in infected primary keratinocytes.
- Primary keratinocytes were plated on laminin 5-coated glass slides. Cultures were either untreated or infected with different recombinant PKC adenovirases. Twenty four hours following infection, cells were fixed, washed and air-dried. Cultures were analyzed by immunofluorescence using isoform specific anti- PKC antibodies, followed by FITC conjugated secondary antibodies.
- FIG. 6 demonstrates that PKC isoforms specifically regulate ⁇ 6 ⁇ 4 integrin expression.
- Five day old primary mouse skin keratinocytes were untreated or infected with PKC ⁇ , PKC ⁇ , PKC ⁇ or PKC ⁇ recombinant adenovirases. Forty eight hours post infection, membranal cell fractions were subjected to SDS-PAGE electrophoresis, transferred to nitrocellulose filters, immunoblotted with anti ⁇ 6 and anti- ⁇ 4 antibodies and analyzed by ECL.
- FIG. 7 shows that over-expression of PKC ⁇ and PKC ⁇ induces keratinocyte proliferation.
- FIG. 8 demonstrates the PKC isoforms over-expression effects on hemidesmosomal localization ofthe ⁇ 6 ⁇ 4 integrin.
- Primary keratinocytes were plated on laminin 5 coated glass slides and keratinocyte cultures were maintained in low Ca MEM for 48 hours. Following that period of time, cultures were left untreated (A), or infected with PKC ⁇ , PKC ⁇ , PKC ⁇ or PKC ⁇ recombinant adenovirases (B-E, respectively).
- FIGs. 9A-B shows that over-expressed PKC ⁇ and PKC ⁇ induce keratinocyte detachment in vitro.
- A -Primary keratinocytes were either untreated (C) or infected with recombinant PKC ⁇ , ⁇ , ⁇ or ⁇ adenovirases.
- FIG. 10 demonstrates that PKC ⁇ is expressed in actively proliferating keratinocytes. Primary keratinocytes were plated on laminin 5-coated glass slides.
- FIG. 11 demonstrates that PKC ⁇ induces, while PKC ⁇ mutant reduces, keratinocyte proliferation.
- Primary skin keratinocytes were infected for 1 hour with recombinant PKC ⁇ or a dominant negative mutant of PKC ⁇ (DNPKC ⁇ or PKC DN ⁇ ) adenovirases.
- DNPKC ⁇ or PKC DN ⁇ a dominant negative mutant of PKC ⁇
- FIGs. 12A-B demonstrate that PKC ⁇ and DNPKC ⁇ over-expressions specifically regulate PKC localization and cellular morphology.
- Primary skin keratinocytes were infected for 1 hour with recombinant PKC ⁇ or a dominant negative mutant of PKC ⁇ (PKC DN ⁇ ) adenovirases.
- PKC DN ⁇ a dominant negative mutant of PKC ⁇
- Forty eight hours post infection keratinocytes were fixed and subjected to (A) bright field photography (x 20) and (B) immunofluorescence analysis utilizing PKC ⁇ specific antibodies followed by FITC conjugated secondary antibodies as described in experimental procedures. Control- uninfected cells.
- 13A-B show that inhibition of PKC ⁇ expression induces keratinocyte differentiation in proliferating keratinocytes.
- Primary skin keratinocytes were either maintained proliferating in low Ca 2+ medium or differentiated in 0.12 mM Ca 2+ for 24 hours. Thereafter, keratinocytes were infected for 1 hour with recombinant PKC ⁇ or a dominant negative mutant of PKC ⁇ (PKC DN ⁇ ) adenovirases. Twenty four hours after infection, keratinocytes were either maintained in low Ca medium or transferred to differentiating medium containing 0.12 mM Ca 2+ for an additional 24 hours. Forty eight hours after infection, keratinocytes were extracted and subjected to SDS-PAGE gels.
- FIG. 14 demonstrates that topical in vivo expression of PKC ⁇ enhances the formation of granulation tissue and accelerates wound healing in mice incisional wounds.
- Whole skin 7 mm incisions were created on the back of nude mice.
- Topical application of control ⁇ -gal, PKC ⁇ and PKC ⁇ adenovirus suspension was applied at Id and 4d following wounding.
- Whole skin wounds were fixed in 4 % paraformaldehyde and skin sections were analyzed histologically by H&E staining and bright field microscopy. E - epidermis, D - dermis.
- FIG. 14 demonstrates that topical in vivo expression of PKC ⁇ enhances the formation of granulation tissue and accelerates wound healing in mice incisional wounds.
- Whole skin 7 mm incisions were created on the back of nude mice.
- Topical application of control ⁇ -gal, PKC ⁇ and PKC ⁇ adenovirus suspension was applied at Id and 4d following wounding.
- FIG. 16 shows that insulin but not IGFl induces PKC ⁇ activity.
- To determine _7 PKC ⁇ activity five-day keratinocyte cultures were stimulated with 10 M insulin
- FIGs. 17A-B show that insulin and IGFl have an additive effect on keratinocyte proliferation.
- Proliferating keratinocytes were maintained for five days in low Ca 2+ medium (0.05 mM) until they reached 80 % confluence.
- A Five-day keratinocyte cultures were stimulated for 24 hours with insulin or IGFl at the designated concentrations.
- B In parallel, keratinocytes were stimulated with 10" ' M insulin (Ins) and increasing doses of IGFl (IGF). At each concentration the right column (striped bar) represents proliferation observed when both hormones were added together. The left bar demonstrates the separate effect of 10"7 M insulin (red bars) and increasing concentrations of IGFl (gray bars). Thymidine incorporation was measured as described in experimental procedures. The results shown are representative of 6 experiments.
- FIGs. 18A-B demonstrate the over-expression of recombinant PKC adenovirus constructs. Keratinocyte cultures were infected utilizing recombinant adenovirus constructs containing wild type PKC ⁇ (WTPKC ⁇ ), wild type PKC ⁇ (WTPKC ⁇ ), or a dominant negative PKC ⁇ mutant (DNPKC ⁇ ).
- WTPKC ⁇ wild type PKC ⁇
- WTPKC ⁇ wild type PKC ⁇
- DNS ⁇ dominant negative PKC ⁇ mutant
- FIG. 19 shows the effects of PKC over-expression on insulin or IGFl -induced proliferation.
- Non-infected (light blue bars), or cells over-expressing WTPKC ⁇ (dark -7 blue bars) or DNPKC ⁇ (slashed blue bars) were treated for 24 hours with 10 M insulin (Ins), 10 " M IGFl (IGF) or both (Ins+IGF).
- Thymidine incorporation was measured as described in experimental procedures.
- Each bar represents the mean ⁇ SE of 3 determinations in 3 experiments done on separate cultures. Values are expressed as percent of control, unstimulated cells from the same culture in each experiment.
- PKC ⁇ activity specifically abrogates insulin induced keratinocyte proliferation.
- Primary keratinocytes were cultured as described in the Examples section that follows. Non-infected cells or keratinocytes infected with DNPKC ⁇ were stimulated for 24 hours with the following growth factor concentrations: 10 -7 M insulin (Ins), 10 "8 M IGFl (IGF), 10 ng ml EGF, 10 ng/ml
- PDGF PDGF
- 1 ng/ml KGF PDGF
- 5 ng/ml ECGF Thymidine incorporation was measured as described in the Examples section that follows. Each bar represents the mean ⁇ SE of
- FIG. 21 shows that over-expression of PKC ⁇ mediates specifically insulin induced keratinocyte proliferation.
- Primary keratinocytes were cultured as described under Figure 1.
- Non-infected cells or keratinocytes infected with over-expressed WTPKC ⁇ were stimulated for 24 hours with the following growth factor concentrations: 10 "7 M insulin (Ins), 10 "8 M IGFl (IGF), 10 ng/ml EGF, 10 ng/ml PDGF, 1 ng/ml KGF or 5 ng/ml ECGF.
- Thymidine incorporation was measured as described in the Examples section that follows.
- FIGs. 22A-B substantiate the significance of PKC ⁇ and PKC ⁇ in the wound healing process of skin in vivo. Utilizing in vivo mouse model of newly developed isoform specific PKC null mice, PKC ⁇ , PKC ⁇ and PKC ⁇ null mice and their wild type littermates were subjected to a wound healing study. Mice were anesthetized and a skin through punch biopsies of 4 mm in diameter were created on the mice back.
- FIG. 23 identifies a specific interaction between STAT3 and PKC ⁇ in primary skin keratinocytes.
- Primary keratinocytes were either untreated (upper panel) or infected for 1 hour with isoform specific, recombinant PKC adenovirases (lower panel). Cells were extracted and immunoprecipitated (IP) with isoform specific PKC antibodies.
- FIG. 24 demonstrates the importance of PKC ⁇ activation to insulin induced transcriptional activation of STAT3.
- Primary keratinocytes were plated on glass slides and maintained for 5 days in low Ca ++ medium (0.05 mmol/1) until they reached
- FIG. 25 demonstrates that overexpression of DN PKC ⁇ inhibits keratinocyte proliferation induced by overexpression of PKC ⁇ and STAT3.
- FIG. 26 demonstrates the importance of insulin concentrations and frequency of applications on wound healing in vivo.
- FIG. 27 demonstrates histological effects of insulin concentrations and frequency of applications on wound healing in vivo. Wound incisions were performed on the back of 8-10 week old C57BL mice and were treated with different concentrations of insulin and frequencies of applications (i.e., seven daily repeat applications vs. a single application).
- FIG. 28 demonstrates a synergistic effect of combining insulin and platelet- derived growth factor (PDGF-BB) on wound healing in vivo.
- PDGF-BB platelet- derived growth factor
- Wound incisions were performed on the back of 8-10 week old C57BL mice and were treated with a single application of insulin, PDGF-BB, or with insulin and PDGF-BB combined.
- the treated mice were sacrificed seven days after wounding and biopsies were taken for histological analyses of epidermal and dermal closure (wound contraction).
- Epidermal closure was assessed by Keratin 14 (K14) antibody staining (left panel) and was considered positive if the wound was stained positive across the entire gap.
- the dermal closure was considered positive if both dermal wound sides could be observed under a light microscope in a single field at xlOO magnification (right panel).
- FIGs. 29A-D are photographs illustrating the morphological effect of combining insulin and a PKC ⁇ inhibitor on wound healing in vivo. Wound incisions were performed on the back of 8-10 week old C57BL mice and were either untreated (control) or treated with insulin (HO/01) combined with a PKC ⁇ inhibitor (HO/02). Skin biopsies were removed 7 days after wounding for morphological observations. Figures 29A-B show control wounds while Figures 29C-D show treated wounds.
- FIG. 30 is a histo-micrograph illustrating the combined effect of insulin and a PKC ⁇ inhibitor on dermal closure (wound contraction).
- FIG. 31 is a histo-micrograph illustrating the combined effect of insulin and a
- PKC ⁇ inhibitor on epidermal closure were performed on the back of 8-10 week old C57BL mice and were either untreated or treated daily with insulin
- FIG. 32 is a histo-micrograph illustrating the combined effect of insulin and a
- FIG. 33 demonstrates the quantitative effect of insulin combined with a PKC ⁇ inhibitor on wound healing in vivo.
- FIGs. 34A-G are photographs illustrating the combined effect of inhibiting expression and or activity of PKC ⁇ and modulating expression and/or activity of another PKC isoform in dermal cells, or administering a hormone to the dermal cells, on the closure of in vitro skin wounds.
- FIGs. 35A-H are photographs illustrating the combined effect of inhibiting expression and/or activity of PKC ⁇ and modulating expression and/or activity of another PKC isoform in dermal cells, or administration of a growth factor to the dermal cells, on the closure of in vitro skin wounds.
- Cultured primary skin keratinocytes were infected with dominant negative (DN) kinase inactive PKC ⁇ . Twenty four hours later scratches were performed and the cultures and were either left untreated (Figure 35A) or infected with wild type (WT) PKC ⁇ (Figure 35D), WT PKC ⁇ ( Figure 35E) or WT PKC ⁇ ( Figure 35F).
- DN dominant negative
- FIGs. 36A-B are photographs illustrating the combined effect of inhibiting expression and/or activity of PKC ⁇ in dermal cells and administering a growth factor to the dermal cells, on the closure of in vitro skin wounds.
- Cultured primary skin fibroblasts were infected with dominant negative (DN) kinase inactive form of PKC ⁇ (DN ⁇ ).
- DN dominant negative
- FIGs. 37A-D are photographs illustrating the combined effect of inhibiting expression and/or activity of PKC ⁇ in dermal cells and administering a growth factor or a hormone to the dermal cells, on the closure of in vitro skin wounds.
- Cultured primary skin keratinocytes were infected with dominant negative (DN) kinase inactive form of PKC ⁇ (DN ⁇ ).
- DN dominant negative
- FIGs. 38A-E are photographs illustrating the combined effect of inhibiting expression and or activity of PKC ⁇ in dermal cells and administering a growth factor, insulin or GW9662 to the dermal cells, on the closure of in vitro skin wounds.
- FIGS. 39A-E are photographs illustrating the combined effect of promoting expression and/or activity of PKC ⁇ and modulation expression and/or activity of another PKC isoform in dermal cells, or administering a hormone to the dermal cells, on the closure of in vitro skin wounds.
- Cultured primary skin keratinocytes were infected with wild type (WT) kinase form of PKC ⁇ (DN ⁇ ). Twenty four hours later scratches were performed and the cultures were either left untreated (Figure 39 A) or infected with WT PKC ⁇ (PKC ⁇ ; Figure 39B), WT PKC ⁇ (PKC ⁇ ; Figure 39C) or DN PKC ⁇ (PKC ⁇ ; Figure 39D).
- FIGs. 40A-F are photographs illustrating the effect of administering copolymer- 1, insulin, PKC ⁇ pseudosubstrate, or combinations thereof, on the closure of in vitro skin wounds.
- Cultured primary skin keratinocytes were either left untreated (Figure 40A), or treated with insulin only (6.7 x 10 "7 M; Figure 40B), copolymer-1 only (55 ⁇ g/dish; Figure 40C), a mixture of insulin and PKC ⁇ pseudosubstrate (6.7 x 10 "7 M and 10 7 M, respectively; Figure 40D), a mixture of copolymer-1 and insulin (55 ⁇ g/dish and 6.7x10 "7 M, respectively; Figure 40E) or a mixture of copolymer-1, insulin and PKC ⁇ pseudosubstrate (55 ⁇ g/dish, 6.7xlO "7 M and 10 7 M, respectively; Figure 40F). Photographs were taken 48 hours following treatment. FIGs.
- FIGs. 41A-D are photographs illustrating the effect of copolymer-1, insulin, PKC ⁇ pseudosubstrate, or combinations thereof, on wound healing in vivo.
- Wounded mice were either left untreated (Figure 41 A) or treated daily for 4 days with topical applications of copolymer-1 (55 ⁇ g/ml: Figure 41B), a mixture of copolymer-1 and insulin (55 ⁇ g/ml and l ⁇ M, respectively; Figure 41 C), or a mixture of copolymer-1, insulin and PKC ⁇ pseudosubstrate (55 ⁇ g/ml, l ⁇ M and I ⁇ M , respectively; Figure 4 ID). Photographs were taken 4 days post wounding. FIGs.
- FIGS 42A-H are histo-micrographs illustrating the effect of thymus proximity to the wound gap on the wound healing process.
- Figures 42A-B show normal adult rodent thymus at x200 magnification.
- Figures 42C shows a 7 day old wound magnified at x40, thymus is observed in close proximity of the wound gap (in red square; magnified at x200 in Figure 42D). The wound is re-epithelized, granulation tissue is formed and dermal contraction is in progress.
- FIG. 42E-F show a 9 day old wound of a STZ diabetes mouse magnified at x40 ( Figure 42E) and x200 ( Figure 42F), no thymus is observed in close proximity of the wound gap and no re- epithelization, tissue granulation, or dermal contraction is observed.
- Figure 42G shows a 9 day old wound of a STZ diabetic mouse magnified at x40. The wound was treated with a mixture of insulin and PKC ⁇ pseudosubstrate. Thymus is observed in close proximity of the wound gap (in red square; magnified x20 in Figure 42H). The wound is re-epithelized, granulation tissue is formed and dermal contraction is in progress.
- FIG. 43 is a photograph illustrating the effect of insulin combined with a
- PKC ⁇ inhibitor on the healing of wounds and damaged skin.
- Longitudinal wound incisions were effected on the back of Large Whites& Landrace domestic pigs and treated daily for 15 days with either PBS (control) or a mixture of 1 ⁇ M insulin and 1 ⁇ M PKC ⁇ pseudosubstrate (HO/03/03).
- the wounds were photographed 30 days post wounding.
- the HO/03/03 treated wounds are completely healed with no scar formation and exhibit markedly improved skin aesthetics as compared with the buffer control.
- the present invention is of methods and pharmaceutical compositions designed for modulating the expression and/or activation of serine/threonine protein kinases, also known as PKCs, for inducing and/or accelerating cell proliferation and/or cell differentiation, and thereby accelerate the healing process of wounds.
- serine/threonine protein kinases also known as PKCs
- Such modulated expression may be effected in accordance with the teachings of the present invention by, for example, (i) fransformation of wound cells with a PKC expressing construct; (ii) transformation of wound cells with a cis-acting element to be inserted adjacent to, and upstream of, an endogenous PKC gene ofthe wound cells; (iii) administration of insulin and other agents acting in synergy with insulin for modulating the expression and/or activation of PKC in wound cells; (iv) transformation of wound cells with an insulin expressing construct, when expressed and secreted the insulin produced therefrom serves as an up-regulator for PKC expression and/or activation; (v) transformation of wound cells with a cis-acting element to be inserted adjacent to, and upstream of, the endogenous insulin gene of the wound cells, when expressed and secreted the insulin serves as an up-regulator for PKC expression and or activation; (vi) implantation of insulin secreting cells to the wound; (vii) transformation of wound cells with
- the first stage of the repair is achieved by formation of the clot that plugs the initial wound. Thereafter, inflammatory cells, fibroblasts and capillaries invade the clot to form the granulation tissue. The following stages involve re-epithelization of the wound where basal keratinocytes have to lose their hemidesmosomal contacts, keratinocytes migrate upon the granulation tissue to cover the wound. Following keratinocyte migration, keratinocytes enter a proliferative boost, which allows replacement of cells lost during the injury.
- EGF EGF family of growth factors
- PDGF PDGF
- TGF ⁇ l growth factor family of growth factors
- EGF and KGF are thought to be intimately involved in the regulation of proliferation and migration of epidermal keratinocytes (25,26).
- Fundamental to the understanding of wound healing biology is a knowledge of the signals that trigger the cells in the wound to migrate, proliferate, and lay down new matrix in the wound gap.
- wound refers broadly to injuries to the skin and subcutaneous tissue initiated in any one of a variety of ways (e.g., pressure sores from extended bed rest, wounds induced by trauma, cuts, ulcers, burns and the like) and with varying characteristics. Wounds are typically classified into one of four grades depending on
- Grade I wounds limited to the epithelium
- Grade II wounds extending into the dermis
- Grade III wounds extending into the subcutaneous tissue
- Grade IN or full-thickness wounds: wounds wherein bones are exposed (e.g., a bony pressure point such as the greater trochanter or the sacrum).
- partial thickness wound refers to wounds that encompass Grades I- III; examples of partial thickness wounds include burn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers.
- deep wound is meant to include both Grade III and Grade IN wounds.
- wound in respect to a wound refers to a process to repair a wound as by scar formation.
- inducing or accelerating a healing process of a skin wound refers to either the induction of the formation of granulation tissue of wound contraction and/or the induction of epithelialization (i.e., the generation of new cells in the
- Wound healing is conveniently measured by decreasing wound area.
- the present invention contemplates treating all wound types, including deep wounds and chronic wounds.
- chronic wound refers to a wound that has not healed within thirty days.
- transforming cells refers to a transient or permanent alteration of a cell's nucleic acid content by the incorporation of exogenous nucleic acid which either integrates into the cell genome and genetically modifies the cell or remains unintegrated.
- cis-acting element is used herein to describe a genetic region that serves as an attachment site for DNA-binding proteins (e.g., enhancers, operators and promoters) thereby affecting the activity of one or more genes on the same chromosome.
- trans-acting factor is used herein to describe a factor that binds to a cis-acting element and modulates its activity with respect to gene expression therefrom.
- PDXl is a trans-acting factor which binds to the insulin gene promoter and modulates its activity.
- transcription activator is used herein to describe a factor that increases gene expression.
- a trans-acting factor is an example of a direct transcription activator.
- activator is used herein to describe a molecule that enhances an activity.
- modulated expression and/or activation used herein refers to enhanced or inhibited expression and/or activation.
- PKC is a major signaling pathway, which mediates keratinocyte proliferation and differentiation. PKC isoforms ⁇ , ⁇ , ⁇ , ⁇ and ⁇ are expressed in the skin (4, 10).
- PKC PKC
- insulin may serve as a therapeutic agent for modulating the expression and/or activation of PKC so as to accelerate the healing process of wounds.
- the characteristics of distinct PKC isoforms and their specific effects on cell proliferation and/or differentiation are of great importance to the biology of skin wound healing.
- Utilizing PKC adenovirus constructs enabled to identify the specific roles of a variety of PKC isoforms in the wound healing process in vitro and in vivo. All isoforms were able to specifically affect different aspects of keratinocyte growth and differentiation.
- Two isoforms, PKC ⁇ and PKC ⁇ could specifically regulate integrin regulation (see Example 6 below), adherence to the basement membrane (see Example 9 below) and hemidesmosome formation (see Example 8 below).
- PKC ⁇ and PKC ⁇ Two isoforms, PKC ⁇ and PKC ⁇ , were found to regulate the proliferation potential of epidermal keratinocytes (see Examples 7 and 11 below).
- a dominant negative isoform of PKC ⁇ (DNPKC ⁇ ) was able to specifically induce differentiation in actively proliferating keratinocytes (see Example 12 below).
- DPKC ⁇ a dominant negative isoform of PKC ⁇
- Example 12 the importance of distinct PKC isoforms to the wound healing process in skin was also verified in an in vivo system. Utilizing PKC null mice where expression of distinct PKC isoforms was abolished it is shown herein that PKC ⁇ and PKC ⁇ which were found to be required for both adhesion and motility processes in skin keratinocytes are also important in the in vivo wound healing process in an animal model (see Example 19).
- PKC ⁇ has a unique tissue distribution. It is predominantly expressed in epithelial tissues (27,28). In situ hybridization studies as well as immunohistochemical studies have demonstrated PKC ⁇ is highly expressed in the differentiating and differentiative layers (27). The results presented herein suggest the role of PKC ⁇ as a functional regulator of both proliferation and differentiation of skin depending on the cellular physiology.
- PKC ⁇ When keratinocytes are maintained in a proliferative state under low Ca 2+ conditions, PKC ⁇ induced the proliferation rate five to seven times above control keratinocytes. However, when cells were induced to differentiate by elevating the Ca 2+ concentration, differentiation was induced in a faster and higher rate in comparison to control cells (see Example 12). This could explain the ability of PKC ⁇ to dramatically induce wound healing and formation of granulation tissue as both proliferative capacity and formation of differentiation layers were achieved. Interestingly, the wound healing results in vivo and the expression of PKC ⁇ in embryonic tissue, which normally does not express PKC ⁇ at high levels in adulthood, would suggest a possible role for PKC ⁇ in the proliferation and tissue organization of other tissues as well.
- the results presented herein demonstrate that modulating expression and/or activation (membrane mobilization) of distinct PKC isoforms is an effective tool to combat wounds. Accordingly, wound healing may be promoted by enhancing the expression and/or activity of isoforms PKC ⁇ , PKC ⁇ and PKC ⁇ , or by inhibiting the expression and/or activity of isoform PKC ⁇ .
- a method of inducing or accelerating a healing process of a skin wound or damage the method is effected by administering to the skin wound a therapeutically effective amount of at least one agent for modulating PKC expression and/or activation.
- a pharmaceutical composition for effecting the method according to this aspect of the present invention therefore includes, as an active ingredient, a therapeutically effective amount of at least one agent for modulating PKC expression and/or activation; and a pharmaceutically acceptable carrier.
- skin wound refers to any type of epithelial wound including, but not limited to an ulcer such as a diabetic ulcer, a pressure ulcer, a venous ulcer, a gastric ulcer and an HIN related ulcer, a diabetes related wound, a burn, a sun burn, an aging skin wound, a corneal ulceration wound, an inflammatory gastrointestinal tract disease wound, a bowel inflammatory disease wound, a Crohn's disease wound, an ulcerative colitis, a hemorrhoid, an epidermolysis bulosa wound, a skin blistering wound, a psoriasis wound, an animal skin wound, an animal diabetic wound, a retinopathy wound, an oral wound (mucositis), a vaginal muco
- skin damage refers to any type of skin damage or condition such as, for example, wrinkles (e.g., ultraviolet irradiation-induced wrinkles), skin lines, crevices, bumps, large pores (e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands, or hair follicles), or unevenness or roughness, loss of skin elasticity (loss and/or inactivation of functional skin elastin), sagging (including puffiness in the eye area and jowls), loss of skin firmness, loss of skin tightness, loss of skin recoil from deformation, discoloration (including undereye circles), blotching, sallowness, hyperpigmented skin regions such as age spots and freckles, keratoses, abnormal differentiation, hyperkeratinization, elastosis, collagen breakdown, and other histological changes in the sfratum corneum, dermis, epidermis, the skin vascular system
- wrinkles e.g
- PKC ⁇ is a unique isoform among the PKC family of proteins involved specifically in growth and maturation of various cell types (35).
- PKC ⁇ was shown to be specifically regulated by stimulation of several growth factors including EGF, Platelet derived growth factor and neurotransmitters, its physiological effects were shown to participate in growth factor inhibition of cell growth including apoptosis, differentiation, and cell cycle retardation or arrest (36-41).
- EGF EGF
- Platelet derived growth factor and neurotransmitters its physiological effects were shown to participate in growth factor inhibition of cell growth including apoptosis, differentiation, and cell cycle retardation or arrest (36-41).
- a selective loss of the ⁇ 6 ⁇ 4 integrin complex is linked to induction of the KI in cultured mouse keratinocytes (6).
- the loss of ⁇ 6 ⁇ 4 protein expression is a consequence of transcriptional and post-translational events including enhanced processing of the ⁇ 6 and ⁇ 4 chains.
- a link was established between the activation of PKC and the processing and regulation ofthe ⁇ 6 ⁇ 4 integrin.
- the examples below show that only insulin stimulation, but not a variety of growth factors, including, but not limited to, EGF, KGF, PDGF, ECGF and IGFl, can translocate and activate PKC ⁇ , but not any of the other PKC isoforms expressed in skin.
- the importance of PKC ⁇ to insulin stimulation was further confirmed when the mitogenic stimulation by EGF, KGF, PDGF, ECGF and IGFl were not abrogated by the dominant negative mutant of PKC ⁇ and insulin appeared to be the primary activator of this PKC isoform in the regulation of keratinocyte proliferation (see Example 17). However, when keratinocytes were infected with WT PKC ⁇ keratinocytes mitogenic stimulation by EGF and KGF was enhanced.
- STAT Signal Transducers and Activators of Transcription proteins are a family of transcription factors recraited by a variety of cytokines and growth factors. Among the seven known STAT family members STAT3 is unique. Targeted disruption of STAT3 but not other STAT family members leads to early embryonic lethality. Specifically, when STAT3 was conditionally ablated in skin, skin remodeling was severely disrupted.
- STAT proteins Upon activation, STAT proteins form homo or heterodimers, translocate to the nucleus and bind to DNA response elements of target genes to induce transcription. It was found that in keratinocytes, PKC ⁇ but not other PKC isoforms expressed in skin (PKCs ⁇ , ⁇ , ⁇ and ⁇ ) is constitutively associated with STAT3 (see, Example 18). Furthermore, insulin regulates phosphorylation, activation and nuclear translocation of STAT3 via specific activation of PKC ⁇ . Inhibition of PKC ⁇ activity by a pharmacological inhibitor, rottlerin or by overexpressing a dominant negative PKC ⁇ mutant abrogated insulin induced STAT3 activation and nuclear translocation.
- modulating PKC expression and/or activation is effected by subjecting wound cells to insulin.
- This can be executed by one of a plurality of alternative ways as is further exemplified hereinunder.
- One way is the direct administration of insulin to the wound.
- a topical application of insulin on wounds at a concentration ranging from 0.1 - 10 ⁇ M effectively promoted epidermal and dermal closure and subsequently wound healing.
- the application of insulin combined with PDGF-BB growth factor, or with a PKC ⁇ inhibitor resulted in a substantial and synergetic improvement of the wound healing process over the insulin alone.
- a method of inducing or accelerating a healing process of a skin wound or damage is effected by administering to the skin wound a therapeutically effective amount of insulin and at least one additional agent acting in synergy with the insulin, so as to induce or accelerate the healing process of the skin wound or damage.
- the agent is a PKC ⁇ inhibitor.
- the agent is a growth factor such as PDGF, EGF, TGF ⁇ , KGF, ECGF or IGFl, and most preferably the agent is PDGF-BB.
- the direct administration of insulin may be effected by a single or by repeat applications. While reducing the present invention to practice, the inventors surprisingly discovered that a treatment with a single application of insulin at a concentration of l ⁇ M was substantially more effective in healing wounds than with seven repeat daily applications of insulin at a similar concentration (see Example 20 below).
- the single dose-unit comprises 0.001 to 5 nM, preferably 0.01 to 0.5 nM of insulin in, for example, an aqueous solution, gel, cream, paste, lotion, spray, suspension, powder, dispersion, salve or ointment formulation in an amount sufficient to cover a 1 cm area ofthe skin wound, e.g., 0.01-0.2 ml.
- the timing of administering insulin onto wounds may be critical, as illustrated in Example 20 in the Examples section that follows.
- a single application of insulin to a 4 day-old wound resulted in effective wound healing.
- a method of inducing or accelerating a healing process of an old skin wound by administering to the wound a single dose of a therapeutically effective amount of insulin.
- old skin wound used herein refers to a skin wound that is at least one day old, at least two days old, at least three days old, preferably, at least four days old.
- a pharmaceutical composition for inducing or accelerating a healing process of a skin wound or damage includes, as an active ingredient, a therapeutically effective amount of insulin, at least one additional agent acting in synergy with the insulin, and a pharmaceutically acceptable carrier designed for topical application of the pharmaceutical composition.
- the agent is a PKC ⁇ inhibitor or a growth factor such as PDGF, EGF, TGF ⁇ , KGF, ECGF or IGFl, and most preferably PDGF-BB.
- the pharmaceutically acceptable carrier can be, but not limited to, a gel, a cream, a paste, a lotion, a spray, a suspension, a powder, a dispersion, a salve and an ointment, as is further detailed hereinunder.
- Solid supports can also be used for prolonged release of insulin into the wound. It will be appreciated that the insulin can be native or preferably recombinant, of a human or any other suitable source.
- a pharmaceutical composition for inducing or accelerating a healing process of a skin wound or damage may include a single dose-unit of insulin selected capable of inducing or accelerating a healing process of the skin wound or damage, and a pharmaceutically acceptable carrier being designed for topical application of the pharmaceutical composition.
- the single dose-unit of insulin is ranging from 0.001 to 5 nM, preferably 0.01 to 0.5 nM, in a 0.01-0.2 ml formulation dose-unit.
- cells expressing and secreting insulin are implanted into the wound, so as to induce or accelerate the healing process of the skin wound or damage.
- Such insulin producing cells may be cells naturally producing insulin, or alternatively, such cells are transformed to produce and secrete insulin.
- the cells can be transformed by, for example, a recombinant PDXl gene (see GeneBank Accession Nos. AH005712, AF035260, AF035259) which is a trans-acting factor for the production and secretion of insulin.
- the cells can be transformed by a cis-acting element sequence, such as a strong and constitutive or inducible promoter integrated upstream to an endogenous insulin gene of the cells, by way of gene knock-in, so as to transform the cells to overproduce and secrete natural insulin.
- a cis-acting element sequence such as a strong and constitutive or inducible promoter integrated upstream to an endogenous insulin gene of the cells, by way of gene knock-in, so as to transform the cells to overproduce and secrete natural insulin.
- a cis-acting element sequence such as a strong and constitutive or inducible promoter integrated upstream to an endogenous insulin gene of the cells, by way of gene knock-in, so as to transform the cells to overproduce and secrete natural insulin.
- a pharmaceutical composition for inducing or accelerating a healing process of a skin wound or damage therefore includes, as an active ingredient, insulin secreting cells, and a pharmaceutically acceptable carrier which is designed for topical application of the pharmaceutical composition.
- the insulin secreting cells administered to a wound are capable of forming secretory granules, so as to secrete insulin produced thereby.
- the insulin secreting cells can be endocrine cells. They can be of a human source or of a histocompatibility humanized animal source. Most preferably, the insulin secreting cells, either transformed or not, are of an autologous source.
- the insulin secreted by the insulin secreting cells is preferably human insulin or has the amino acid sequence of human insulin.
- the insulin secreting cells can be fibroblasts, epithelial cells or keratinocytes, provided that a transformation as described above is employed so as to render such cells to produce and secrete insulin.
- cells of the skin wound are transformed to produce and secrete insulin, so as to induce or accelerate the healing process of the skin wound.
- a pharmaceutical composition for inducing or accelerating a healing process of a skin wound according to this aspect of the present invention therefore includes, as an active ingredient, a nucleic acid construct designed for transforming cells of the skin wound to produce and secrete insulin, and a pharmaceutically acceptable carrier designed for topical application ofthe pharmaceutical composition.
- any one of the transformation methods described above e.g., transformation with a construct encoding insulin, transformation with a construct harboring a cis- acting element for insertion downstream of an endogenous insulin gene by way of gene knock-in, and transformation with a construct encoding a trans-acting factor for activation of endogenous insulin production and secretion, can be employed in context of this embodiment ofthe present invention.
- Previous studies on the effects of distinct PKC isoforms in skin have been hampered as a result of the difficulty in introducing foreign genes efficiently into primary cells by conventional methods due to the short life span, differentiation potential and the inability to isolate stable transformants. To overcome these obstacles, viral vectors are being used to introduce genes of interest.
- Viral vectors are developed by modification of the viral genome in the form of replicative defective viruses.
- the most widely used viral vectors are the retrovirases and adenovirases, which are used for experimental as well as gene therapy purposes (13).
- the high efficiency of adenovirus infection in non replicating cells, the high titer of virus and the high expression of the transduced protein makes this system highly advantageous to primary cultures compared to retro viral vectors.
- adenovirases do not integrate into the host genome and the stable viral titers can be rendered replication deficient, these viral constructs are associated with minimal risk for malignancies in human as well as animal models (14).
- adenovirus constructs have also been used successfully with high efficiency of infection with ex vivo and in vivo approaches (15, 16).
- An adenovirus vector which was developed by I. Saito and his associates (17) was used in the present study.
- the cosmid cassette (pAxCAwt) has nearly a full length adenoviras 5 genome but lacks El A, El B and E3 regions, rendering the virus replication defective. It contains a composite CAG promoter, consisting of the cytomegalo virus immediate-early enhancer, chicken ⁇ - actin promoter, and a rabbit ⁇ -globin polyadenylation signal, which strongly induces expression of inserted DNAs (13, 17).
- a gene of interest is inserted into the cosmid cassette, which is then co-transfected into human embryonic kidney 293 cells together with adenovirus DNA terminal protein complex (TPC).
- TPC adenovirus DNA terminal protein complex
- recombination occurs between the cosmid cassette and adenoviras DNA-TPC, yielding the desired recombinant virus at an efficiency one hundred fold that of conventional methods.
- Such high efficiency is mainly due to the use of the adenovirus DNA-TPC instead of proteinesed DNA.
- the presence of longer homologous regions increases the efficiency ofthe homologous recombination. Regeneration of replication competent viruses is avoided due to the presence of multiple EcoT221 sites.
- keratinocytes were infected with distinct PKC recombinant adenovirases, demonstrated 24 hours later effective over-expression of PKC isoforms (see example 1).
- another way by which modulating PKC expression and/or activation is effected according to the present invention is by inducing over-expression of a PKC in the skin wound cells. This can be achieved by transforming the cells with a cis-acting element sequence integrated, by way of homologous recombination, upstream to an endogenous protein kinase C of the cells and thereby causing the cells to produce natural protein kinase C.
- this can be achieved by transforming the cells with a recombinant protein kinase C gene, such as, but not limited to, PKC- ⁇ 1 gene (Accession Nos. X06318, NM002738), PKC- ⁇ 2 gene (Accession No. X07109), PKC- ⁇ gene (Accession No. L28035), PKC- ⁇ gene (Accession No. L07032), PKC- ⁇ gene (Accession No. D28577), PKC-i gene (Accession No. LI 8964), PKC- ⁇ gene (Accession No. X52479), PKC- ⁇ gene (Accession Nos. L07860, L07861), PKC- ⁇ gene (Accession No.
- a recombinant protein kinase C gene such as, but not limited to, PKC- ⁇ 1 gene (Accession Nos. X06318, NM002738), PKC- ⁇ 2 gene (Accession No. X07109),
- a pharmaceutical composition for inducing or accelerating a healing process of a skin wound therefore includes, as an active ingredient, a nucleic acid construct designed for transforming cells of the skin wound to produce a protein kinase C, and a pharmaceutically acceptable carrier designed for topical application ofthe pharmaceutical composition.
- a pharmaceutical composition of inducing or accelerating a healing process of a skin wound or damage according to this aspect of the present invention therefore includes, as an active ingredient, a therapeutically effective amount of a PKC activator, so as to induce or accelerate the healing process of the skin wound or damage, and an acceptable pharmaceutical carrier. Still yet another way by which modulating PKC expression and/or activation is effected according to the present invention by downregulating expression and/or activity of a PKC isoform.
- Downregulating activity of PKC isoform may be effected by a PKC pseudosubstrate isoform inhibitor such as, for example PKC ⁇ , PKC ⁇ or PKC?? pseudosubstrate inhibitors (CalbioChem, California USA), or another PKC isoform inhibitor such as, for example, the pharmaceutical peptide LY379196 (Elly Lilly,
- RNA interference is a two step process. The first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes
- dsRNA Introduced directly or via a transgene or a virus
- ATP-dependent manner Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore Curr.
- the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC).
- RISC RNA-induced silencing complex
- An ATP-dependent unwinding of the siRNA duplex is required for activation ofthe RISC.
- the active RISC targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus ofthe siRNA [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)].
- RNAi molecules suitable for use with the present invention can be effected as follows. First, the PKC isoform mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites.
- siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR- binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html). Second, potential target sites are compared to an appropriate genomic database
- sequence alignment software e.g., human, mouse, rat etc.
- Putative target sites which exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
- a suitable siRNA according to the present invention can be, for example, an siRNA capable of inhibiting PKC ⁇ expression such as any ofthe nucleic acid sequences set forth in SEQ ID NOs: 1-16.
- Another agent capable of downregulating a PKC isoform is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the PKC isoform.
- DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci.
- DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit
- Urokinase receptor expression and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al , 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org).
- DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and
- Downregulation of a PKC isoform can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the PKC isoform.
- Design of antisense molecules which can be used to efficiently downregulate a
- PKC isoform must be effected while considering two aspects important to the antisense approach.
- the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
- the prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example,
- antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al, Curr Opin Mol Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin Mol Ther 1 :297-306 (1999)].
- ribozyme molecule capable of specifically cleaving an mRNA transcript encoding a PKC isoform.
- Ribozymes are being increasingly used for the sequence-specific inhibition- of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al, Curr Opin Biotechnol. 9:486-96 (1998)].
- the possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
- ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al, Clin Diagn Virol.
- ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials.
- ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
- the pharmaceutical composition of inducing or accelerating a healing process of a skin wound or damage further includes at least one additional agent selected from the group consisting of a hormone, a growth factor, an adipokine, PKC ⁇ RACK and GW9662.
- a suitable hormone can be, but not limited to, insulin.
- a suitable growth factor can be, but not limited to, Interleukin-6 (IL-6), Kerotinocyte Growth Factor (KFG) or Tumor Necrosis Factor alpha (TNF ⁇ ).
- a suitable adipokine can be, but not limited to, adipsin or adiponectin. While reducing the present invention to practice, the present inventors surprisingly and unexpectedly uncovered that copolymer-1 (glatiremar acetate) is capable of substantially promoting wound healing in vitro and in vivo (see Example 26 in the Examples section which follows). While copolymer-1 has been previously known as an immunomodulating agent used for treating multiple sclerosis and central nerve system disorders (U.S. Pat. Nos.
- a pharmaceutical composition for effecting the method according to this aspect of the present invention therefore includes, as an active ingredient, a therapeutically effective amount of copolymer-1 and a pharmaceutically acceptable carrier.
- the therapeutically/pharmaceutically active ingredients of the present invention can be administered to a wound per se, or in a pharmaceutical composition mixed with suitable carriers and/or excipients.
- Pharmaceutical compositions suitable for use in context of the present invention include those compositions in which the active ingredients are contained in an amount effective to achieve an intended therapeutic effect.
- a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein, either protein, chemicals, nucleic acids or cells, or physiologically acceptable salts or prodrags thereof, with other chemical components such as traditional drags, physiologically suitable carriers and excipients.
- the purpose of a pharmaceutical composition is to facilitate administration of a compound or cell to an organism.
- Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
- physiologically suitable carrier and “pharmaceutically acceptable carrier” are interchangeably used and refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties ofthe administered conjugate.
- excipient refers to an inert substance added to a pharmaceutical composition to further facilitate processes and administration of the active ingredients. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
- topical route is preferred, and is assisted by a topical carrier.
- the topical carrier is one, which is generally suited for topical active ingredients administration and includes any such materials known in the art.
- the topical carrier is selected so as to provide the composition in the desired form, e.g., as a liquid or non-liquid carrier, lotion, cream, paste, gel, powder, ointment, solvent, liquid diluent, drops and the like, and may be comprised of a material of either naturally occurring or synthetic origin. It is essential, clearly, that the selected carrier does not adversely affect the active agent or other components of the topical formulation, and which is stable with respect to all components of the topical formulation.
- suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like.
- Ointments are colorless, odorless ointments, liquids, lotions, creams and gels.
- Ointments are semisolid preparations, which are typically based on petrolatum or other petroleum derivatives.
- the specific ointment base to be used is one that will provide for optimum active ingredients delivery, and, preferably, will provide for other desired characteristics as well, e.g., emoUiency or the like.
- an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed.
- ointment bases maybe grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases.
- Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum.
- Emulsifiable ointment bases also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.
- Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.
- W/O water-in-oil
- O/W oil-in-water
- Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight; again, reference may be made to
- Lotions are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations, in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations herein for freating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethylcellulose, or the like.
- Creams containing the selected active ingredients are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil.
- Cream bases are water- washable, and contain an oil phase, an emulsifier and an aqueous phase.
- the oil phase also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
- the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.
- Gel formulations are preferred for application to the scalp.
- gels are semisolid, suspension-type systems.
- Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contains an alcohol and, optionally, an oil.
- Carriers for nucleic acids include, but are not limited to, liposomes including targeted liposomes, nucleic acid complexing agents, viral coats and the like. However, transformation with naked nucleic acids may also be used.
- Various additives known to those skilled in the art, may be included in the topical formulations ofthe invention. For example, solvents may be used to solubilize certain active ingredients substances.
- topical preparations for the treatment of wounds according to the present invention may contain other pharmaceutically active agents or ingredients, those traditionally used for the treatment of such wounds.
- immunosuppressants such as cyclosporine, antimetabolites, such as methotrexate, corticosteroids, vitamin D and vitamin D analogs, vitamin A or its analogs, such etretinate, tar, coal tar, anti pruritic and keratoplastic agents, such as cade oil, keratolytic agents, such as salicylic acid, emollients, lubricants, antiseptics and disinfectants, such as the germicide dithranol (also known as anthralin) photosensitizers, such as psoralen and methoxsalen and UV irradiation.
- Other agents may also be added, such as antimicrobial agents, antifungal agents, antibiotics and anti-inflammatory agents.
- the topical compositions ofthe present invention may also be delivered to the skin using conventional dermal-type patches or articles, wherein the active ingredients composition is contained within a laminated structure, that serves as a drag delivery device to be affixed to the skin.
- the active ingredients composition is contained in a layer, or "reservoir", underlying an upper backing layer.
- the laminated stracture may contain a single reservoir, or it may contain multiple reservoirs.
- the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during active ingredients delivery.
- suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
- the particular polymeric adhesive selected will depend on the particular active ingredients, vehicle, etc., i.e., the adhesive must be compatible with all components of the active ingredients- containing composition.
- the active ingredients-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
- the backing layer in these laminates which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility.
- the material selected for the backing material should be selected so that it is substantially impermeable to the active ingredients and to any other components of the active ingredients-containing composition, thus preventing loss of any components through the upper surface ofthe device.
- the backing layer may be either occlusive or nonocclusive, depending on whether it is desired that the skin become hydrated during active ingredients delivery.
- the backing is preferably made of a sheet or film of a preferably flexible elastomeric material. Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, and polyesters.
- the laminated stracture includes a release liner.
- this layer is removed from the device to expose the basal surface thereof, either the active ingredients reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin.
- the release liner should be made from an active ingredients/vehicle impermeable material.
- Such devices may be fabricated using conventional techniques, known in the art, for example by casting a fluid admixture of adhesive, active ingredients and vehicle onto the backing layer, followed by lamination of the release liner. Similarly, the adhesive mixture may be cast onto the release liner, followed by lamination of the backing layer.
- the active ingredients reservoir may be prepared in the absence of active ingredients or excipient, and then loaded by "soaking" in an active ingredients/vehicle mixture.
- the active ingredients composition contained within the active ingredients reservoirs of these laminated systems may contain a number of components. In some cases, the active ingredients may be delivered "neat," i.e., in the absence of additional liquid. In most cases, however, the active ingredients will be dissolved, dispersed or suspended in a suitable pharmaceutically acceptable vehicle, typically a solvent or gel. Other components, which may be present, include preservatives, stabilizers, surfactants, and the like.
- the pharmaceutical compositions herein described may also comprise suitable solid or gel phase carriers or excipients.
- Such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols. Dosing is dependent on the type, the severity and manifestation of the affliction and on the responsiveness of the subject to the active ingredients, as well as the dosage form employed, the potency of the particular conjugate and the route of administration utilized. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch.
- dosing can be a single or repetitive administration, with course of treatment lasting from several days to several weeks or until cure is effected or diminution ofthe skin lesion is achieved.
- the present invention utilizes in vivo and ex vivo (cellular) gene therapy techniques which involve cell transformation and gene knock-in type transformation.
- Gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition or phenotype.
- the genetic material of interest encodes a product (e.g., a protein, polypeptide, peptide, functional RNA, antisense RNA) whose production in vivo is desired.
- a product e.g., a protein, polypeptide, peptide, functional RNA, antisense RNA
- the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value.
- Gene Therapy Advanced in Pharmacology 40, Academic Press, 1997.
- Two basic approaches to gene therapy have evolved (1) ex vivo; and (ii) in vivo gene therapy.
- ex vivo gene therapy cells are removed from a patient or are derived from another source, and while being cultured are treated in vitro.
- a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.
- These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
- target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient.
- the host gene is defective, the gene is repaired in situ (Culver, 1998. (Abstract) Antisense DNA & RNA based therapeutics, February 1998, Coronado, CA).
- the gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell.
- the expression vehicle may include elements to control targeting, expression and transcription ofthe nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR ofthe gene may be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore, as used herein the expression vehicle may, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
- the expression vehicle can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription. Enhancers that may be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any nontranslated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
- the expression vehicle can also include a selection gene as described herein below. Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art.
- Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
- a specific example of DNA viral vector introducing and expressing recombination sequences is the adenoviras-derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
- TK herpes virus thymidine kinase
- This vector can be used to infect cells that have an adenoviras receptor which includes most tissues of epithelial origin as well as others.
- This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, in vitro or ex vivo culture of cells, a tissue or a human subject.
- Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.
- recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retroviruses, and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.
- Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
- virases are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types.
- the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
- Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection.
- the vector's genome is also engineered to encode and express the desired recombinant gene.
- the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection.
- the specific type of vector will depend upon the intended application.
- the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
- the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment.
- Patent Applications WO 94/23049, WO93/14200, WO 94/06908, WO 94/28123 also provide information.
- Tissue culture media and serum were purchased from Biological Industries (Beit HaEmek, Israel). Enhanced Chemical Luminescence (ECL) was performed with a kit purchased from BioRad (Israel). Monoclonal anti p-tyr antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Polyclonal and monoclonal antibodies to PKC isoforms were purchased from Santa Cruz (California, USA) and Transduction Laboratories (Lexington, KY). The ⁇ 6 rat antimouse mAb (GoH3) was purchased from Pharmingen (San Diego, CA).
- the antibody 6844 for the ⁇ 6A cytoplasmic domain was a gift from Dr. V. Quaranta (Scripps Research Institute, La Jolla, CA).
- the rat mAb directed against the extracellular domain of mouse ⁇ 4 (346-11 A) was a gift from Dr. S. J. Kennel (Oak Ridge National Laboratory, Oak Ridge, TN).
- Rat mAB to phosphotyrosine was purchased from Sigma (St. Louis, MO) and rabbit anti phosphoserine was purchased from Zymed (San Francisco, CA).
- Horseradish peroxidase-anti-rabbit and anti-mouse IgG were obtained from Bio-Rad (Israel).
- Leupeptin, aprotinin, PMSF, DTT, Na- orthovanadate, and pepstatin were purchased from Sigma Chemicals (St. Louis, MO). Insulin (humulinR-recombinant human insulin) was purchased from Eli Lilly France SA (Fergersheim, France). IGFl was a gift from Cytolab (Israel). Keratin 14 antibody was purchased from Babco-Convance (Richmond, CA) BDGF-BB was purchased from R&D systems (Minneapolis) and PKC ⁇ pseudosubstrate myristolated was purchased from Calbinochem (San Diego, CA). Isolation and culture of murine keratinocytes: Primary keratinocytes were isolated from newborn skin as previously described (18).
- Keratinocytes were cultured in Eagle's Minimal Essential Medium (EMEM) containing 8 % Chelex (Chelex-100, BioRad) treated fetal bovine serum. To maintain a proliferative basal cell phenotype, the final Ca 2+ concentration was adjusted to 0.05 mM. Experiments were performed five to seven days after plating.
- EMEM Eagle's Minimal Essential Medium
- Chelex Cholex-100, BioRad
- the pellet was resuspended in 250 ⁇ l PBS containing 1 % Triton X-100 with protease and phosphatase inhibitors, incubated for 30 minutes at 4 °C and spun down in a microcentrifuge at maximal speed at 4 °C. The supernatant contains the membrane fraction. Protein concentrations were measured using a modified Lowery assay (Bio-Rad DC Protein Assay Kit). Western blot analysis of cellular protein fractions was carried out as described (6). Preparation of cell lysates for immunoprecipitation: Culture dishes containing keratinocytes were washed with Ca 2+ /Mg 2+ -free PBS.
- the lysate was precleared by mixing 300 ⁇ g of cell lysate with 25 ⁇ l of Protein A/G Sepharose (Santa Cruz, CA, USA), and the suspension was rotated continuously for 30 minutes at 4 °C. The preparation was then centrifuged at maximal speed at 4 °C for 10 minutes, and 30 ⁇ l of A G Sepharose was added to the supernatant along with specific polyclonal or monoclonal antibodies to the individual antigens (dilution 1:100). The samples were rotated overnight at 4 ° C.
- Immunofluorescence Primary keratinocytes were plated on laminin 5 coated glass slides. Two days old keratinocytes were infected with PKC adenoviras for one hour, washed twice with PBS and maintained in culture in low Ca + MEM. Twenty four hours post infection; cells were fixed in 4 % paraformaldehyde for 30 minutes followed by permeabilization with 0.2 % Triton for 5 minutes. For analysis, control and PKC infected keratinocytes were rinsed with PBS and incubated overnight at 4 °C with PKC antibodies (Santa Cruz) diluted in 1 % BSA in PBS.
- Adenovirus constructs The recombinant adenovirus vectors were constructed as previously described (19). The dominant negative mutants of mouse PKCs were generated by substitution of the lysine residue at the ATP binding site with alanine. The mutant cDNA was cut from SRD expression vector with EcoR I and ligated into the pAxCAlw cosmid cassette to construct the Ax vector. The dominant negative activity of the genes was demonstrated by the abrogation of its autophosphorylation activity.
- Transduction of keratinocytes with PKC isoform genes The culture medium was aspirated and keratinocyte cultures were infected with the viral supernatant containing PKC recombinant adenovirases for one hour. The cultures were then washed twice with MEM and re-fed. Ten hours post-infection cells were transferred to serum-free low Ca 2+ -containing MEM for 24 hours. Keratinocytes from control 86 and insulin-treated or IGFl -treated cultures were used for proliferation assays, Rb uptake, or extracted and fractionated into cytosol and membrane fractions for immunoprecipitation, immunofluorescence and Western blotting as described.
- PKC activity Specific PKC activity was determined in freshly prepared immunoprecipitates from keratinocyte cultures following appropriate treatments. These lysates were prepared in RJPA buffer without NaF. Activity was measured with the use of the SignaTECT Protein Kinase C Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. PKC ⁇ pseudosubstrate was used as the substrate in these studies. 3 Cell proliferation: Cell proliferation was measured by [ H]thymidine 3 incorporation in 24 well plates. Cells were pulsed with [ H]thymidine (1 ⁇ Ci/ml) overnight. After incubation, cells were washed five times with PBS and 5 % TCA was added into each well for 30 minutes.
- Triton Lysis Buffer (1 % Triton-X 100, 10 ⁇ g/ml aprotinin and leupeptin, 2 ⁇ g/ml pepstatin, 1 mM PMSF, 1 mM EDTA, 200 ⁇ M Na2 O4, 10 mM NaF in 1 x PBS). Lysates were incubated at 4 °C for 30 minutes, and spun at 16,000 x g for 30 minutes at 4 °C. Supematants were transferred to a fresh tube.
- Immunoprecipitation of cell lysates was carried out overnight at 4 °C with 5 ⁇ g/sam ⁇ le anti- ⁇ 6/GoH3 (PharMingen) and 30 ⁇ l/sample of protein A/G-Plus agarose slurry (Santa Cruz). Beads were washed once with RIPA buffer and twice with 50 mM Tris/HCl pH 7.5. 35 ⁇ l of reaction buffer (1 mM CaCl2, 20 mM MgCl2, 50 mM Tris ⁇ Cl pH 7.5) was added to each assay.
- Proteins were separated by SDS-PAGE on a 8.5 % gel, transferred onto Profran membranes (Schleicher & Schuell) and visualized by autoradiography. Phosphorylation of histones and phosphorylation of PKC substrate peptide were used as controls for PKC activity.
- EXPERIMENTAL RESULTS EXAMPLE 1 Effective Over-expression of PKC isoforms utilizing recombinant adenovirus vectors By utilizing a recombinant ⁇ -galactosidase adenoviras a high infection rate was achieved with more then 90 % ofthe cultured keratinocyte population expressing the recombinant protein. The recombinant ⁇ -galactosidase adenoviras infection did not affect cell viability or cell growth. Furthermore, ⁇ -galactosidase expression was sustained for up to two weeks of culture and was used as a control infection in following experiments.
- EXAMPLE 3 Over-expressed PKC isoforms are active in their native form As early as 18 hours following infection, PKC kinase assays revealed that immunoprecipitates of distinct PKC isoforms were enzymatically active without further need of stimulation by PKC activators ( Figure 3). EXAMPLE 4 Over-expression of specific PKC isoforms induces distinct morphological changes in primary keratinocytes
- Each of the PKC adenovirus constructs employed induced a specific morphological change in primary keratinocytes ( Figure 4).
- Uninfected primary mouse keratinocyte cultures and ⁇ -galactosidase infected cells presented a cubidal morphology typical to the proliferative basal cell characteristics in culture.
- All PKC over-expressing keratinocytes showed morphological changes typical to PKC activation including cell elongation and the appearance of neuronal like projections.
- each one of the PKC isoforms had a characteristic effect on keratinocyte morphology.
- PKC ⁇ infection induced stratification of keratinocytes, with a typical flattened morphology.
- PKC ⁇ appeared as condensed clones of cells, presenting morphological characteristics of basal cells proliferating at prompt rate (Figure 4). Two of the isoforms appeared to effect cell matrix as well as cell-cell associations. 18-48 hours following PKC ⁇ infection, cells appeared elongated and extended with neuronal like projections. This was followed by gradual cell loss off the culture dish which occurred progressively in the course of the culture period. Over-expressing PKC ⁇ keratinocytes appeared as rounded keratinocyte clusters, which were attached loosely to the culture dish and were gradually lost several days following infection.
- EXAMPLE 5 Distinct localization of over-expressed PKC isoforms in infected primary keratinocytes The distinct morphological changes were associated with distinct cellular localization as characterized by immunofluorescence analysis. In proliferating keratinocytes, PKC ⁇ , PKC ⁇ and PKC ⁇ were expressed in the cytoplasm as well as in the plasma membrane. Similarly to endogenous protein expression, PKC ⁇ isoform was localized to the keratinocytes' perinuclear region ( Figure 5). A dynamic change in distribution was associated with PKC ⁇ and PKC ⁇ , where succeeding cell detachment PKC isoform expression was predominantly localized to the cell membrane ( Figure 5).
- EXAMPLE 6 Regulation of a ⁇ f expression by PKC isoforms experimental results The ability of specific PKC isoforms to regulate proteins which are characteristic ofthe basal phenotype ofthe proliferative basal layer was examined. As down regulation of ⁇ 6 ⁇ 4 integrin is one of the early events taking place during keratinocyte differentiation, the ability of the various PKC isoforms to regulate expression of the ⁇ 6 ⁇ 4 integrin, an integrin which is specifically localized to the hemidesmosomes of the basal layer was assessed.
- EXAMPLE 7 Over-expressed PKC ⁇ and PKC ⁇ induce keratinocytes proliferation in vitro Over-expression of PKC ⁇ and PKC ⁇ significantly induced keratinocyte proliferation five and two fold above control levels respectively (Figure 7). PKC ⁇ and PKC ⁇ did not affect cell proliferation.
- EXAMPLE 8 Over-expressed PKC Sand ⁇ induce keratinocytes detachment in vitro The adhesion properties of PKC ⁇ and ⁇ over-expressing keratinocytes was studied. In comparison to control keratinocytes no change in adhesion potential to specific matrix proteins including lamininl, laminin 5, fibronectin and collagen, was observed (data not presented).
- PKC ⁇ differentially regulate keratinocyte proliferation and differentiation under physiological settings
- cells over-expressing PKC ⁇ isoform proliferate at an accelerated rate, five to seven times above control uninfected cells, and consistently higher then keratinocyte cultures over-expressing other PKC isoforms.
- the induction of proliferation was dependent on the differentiation state of the keratinocytes as determined by regulating the Ca 2+ concentrations in the medium.
- endogenous PKC ⁇ was localized to the perinuclear region of majority of the proliferating cells (Figure 10).
- PKC ⁇ and DNPKC ⁇ over-expression specifically regulates PKC localization and cellular morphology
- the effects of a kinase inactive dominant negative adenovirus PKC ⁇ construct were analyzed by studying the effect of infection in proliferating and differentiating keratinocytes.
- adenoviral infection of both PKC ⁇ and DNPKC ⁇ were efficient in both the proliferation and differentiation states.
- EXAMPLE 12 In vivo experiments In order to test the ability of PKC ⁇ to differentially regulate cell proliferation and differentiation in vivo, the ability of PKC ⁇ to induce healing of full incisional wounds created on the back of nude mice was assessed. The ability of the keratinocytes to express the exogenous recombinant protein was verified by utilizing a control ⁇ -gal adenoviras. As can be seen in Figure 14, two weeks after infection, ⁇ - gal expression is maintained in vitro keratinocytes as well as in vivo skin.
- PKC ⁇ can be considered as a primary candidate in regulating proliferation and differentiation of skin in the induction of wound healing processes.
- PKC ⁇ and PKC ⁇ Two PKC isoforms expressed in skin were found to affect keratinocyte proliferation: PKC ⁇ and PKC ⁇ .
- PKC isoforms ⁇ , ⁇ , ⁇ , ⁇ and ⁇ are expressed in the skin.
- PKC isoforms As activation of PKC isoforms is associated with their translocation to membrane fractions, the effects of these growth factors on the translocation of the various PKC isoforms from cytosol to the membrane were examined. As seen in Figure 15, as early as 5 minutes following stimulation, insulin specifically induced translocation of PKC ⁇ from the cytoplasm to the membrane fractions. Membrane expression of PKC ⁇ was maintained for several hours following insulin stimulation. In contrast, IGFl reduced PKC ⁇ expression in the membrane and increased its relative level of expression in the cytoplasm fraction. No other growth factor significantly affected PKC ⁇ translocation and localization. No change in distribution of the other PKC isoforms was seen following stimulation by any of the growth factors including IGFl and insulin.
- EXAMPLE 14 Insulin specifically induces activation of PKC ⁇ in proliferating keratinocytes
- kinase activity of PKC immunoprecipitates from the cytoplasm and membrane fractions of insulin and IGFl treated keratinocytes was measured.
- insulin but not IGFl increased activity of PKC ⁇ in the membrane fraction.
- No elevation in PKC ⁇ activity was observed in the cytoplasm fraction.
- the insulin-induced activation was specific for PKC ⁇ and no activation of PKCs ⁇ , ⁇ , ⁇ or ⁇ was observed for up to 30 minutes following insulin stimulation. Altogether, these results suggest selective stimulation by insulin but not IGFl of PKC ⁇ activation.
- IGFl stimulated thymidine incorporation in a dose dependent manner with maximal -7 -8 induction achieved at 10 " and 10 " M, respectively.
- the maximal stimulation by IGFl was greater than that by insulin.
- the mitogenic effects were additive (Figure 17B).
- EXAMPLE 16 The association between insulin-induced PKC ⁇ activation and insulin-induced keratinocyte proliferation
- recombinant PKC adenoviras constracts were used to over-express both wild type PKC ⁇ (WTPKC ⁇ ) as well as a kinase-inactive dominant negative mutant of PKC, which abrogates the endogenous PKC ⁇ activity (DNPKC ⁇ ).
- WTPKC ⁇ wild type PKC ⁇
- DNPKC ⁇ kinase-inactive dominant negative mutant of PKC
- EXAMPLE 17 Specificity of PKC ⁇ activation to the insulin-mediated pathway
- the specificity of PKC ⁇ activation to the insulin-mediated pathway was analyzed by investigating the effects of PKC ⁇ and DNPKC ⁇ on the mitogenic response to a variety of growth factors including: IGFl, EGF, KGF, ECGF and PDGF.
- IGFl IGFl
- EGF EGF
- KGF EGF
- ECGF ECGF
- Insulin induced PKC ⁇ activity and keratinocyte proliferation is mediated by STAT3 transcriptional activation
- the role of PKC ⁇ in insulin signaling was further characterized and found to involve induction of transcriptional activation mediated by STAT3. As seen in Figure
- PKC ⁇ and PKC ⁇ are essential to the wound healing process in vivo
- the importance of PKC isoforms in the wound healing process in vivo was established utilizing isoform specific PKC null mice.
- FIGs 22A-B when full thickness wounds were created on the back of PKC ⁇ , PKC ⁇ , PKC ⁇ null mice (knock-out, KO) and their wild type littermates, delayed wound healing was observed in PKC ⁇ and PKC ⁇ but not PKC ⁇ null mice. This data indicates that even in the absence of diabetic background, specific PKC isoforms are essential for the wound healing process in skin.
- EXAMPLE 20 Single vs. multiple applications of insulin for wound healing in vivo Wounds were effected on the back of 8-10 week old C57BL mice by incision and were treated as follows: (i) insulin 0.1 ⁇ M applied daily for 7 days; (ii) insulin l ⁇ M applied daily for 7 days (iii) insulin 10 ⁇ M applied daily for 7 days; (iv) insulin l ⁇ M applied once 4 days after wounding; and (v) vehicle (PBS) control applied daily for 7 days. All mice were sacrificed seven days after wounding and their open wound areas were measured. As seen in Figure 26, a daily treatment of insulin at 1 ⁇ M concentration was significantly more effective than daily treatments of insulin at a lower (0.1 ⁇ M) or a higher (10 ⁇ M) concentration.
- the treatment of a single application of insulin at 1 ⁇ M concentration was substantially more effective than the treatment of seven repeat daily applications of insulin at the same concentration. Since the observed wounds were covered with a scar tissue it was difficult to correctly assess the actual closure of the wound and the formation of reconstructed epidermis. Therefore the effects of insulin on epidermal and dermal closure of wounds tissue were determined by histological parameters. Epidermal closure of wounds was determined by staining wound sections with Keratin 14 antibody (K14, Babco- Convance, Richmond, CA, USA) which highlighted the formation of basal cells at the wound gap.
- Keratin 14 antibody Keratin 14 antibody
- Dermal closure of wounds was considered positive if at both wound sides the dermis could be observed in a single field observed under a light microscope at xlOO magnification. As seen in Figure 27, all insulin treatments effectively promoted epidermal and dermal closure. Similarly to the results shown in Figure 26, a daily treatment of insulin at 1 ⁇ M concentration was significantly more effective than a daily treatment of insulin at O.l ⁇ M, or lO ⁇ M concentrations. In addition, a single application of insulin at l ⁇ M concentration was substantially more effective than of seven repeat daily applications of insulin at the same concentration. Hence, these results clearly substantiate the therapeutic efficacy of insulin on wound healing in vivo as determined by morphological as well as histological parameters.
- EXAMPLE 21 Combining insulin and platelet-derived growth factor (PDGF-BB) for wound healing in vivo Wounds were effected on the back of 8-10 week old C57BL mice by incision and were treated 4 days after wounding as follows: (i) vehicle (PBS) control; (ii) insulin l ⁇ M (iii) PDGF-BB lO ⁇ M (R&D Systems, Minneapolis, USA); and (iv) insulin l ⁇ M + PDGF-BB lO ⁇ M.
- PBS vehicle
- insulin l ⁇ M insulin l ⁇ M
- PDGF-BB lO ⁇ M R&D Systems, Minneapolis, USA
- insulin l ⁇ M + PDGF-BB lO ⁇ M insulin l ⁇ M + PDGF-BB lO ⁇ M.
- EXAMPLE 22 Combining insulin and PKCa inhibitor for wound healing in vivo Wounds were effected on the back of 8-10 week old C57BL mice by incision and were treated daily for 7 days with either vehicle (PBS) control or with 0.67 ⁇ M insulin (HO/01; Humulin, Eli Lilly, USA) combined with a PKC ⁇ inhibitor (HO/02; PKC ⁇ pseudosubstrate myristolated; Calibiochem, San Diego, CA, USA).
- PBS vehicle
- PKC ⁇ inhibitor HO/02
- PKC ⁇ pseudosubstrate myristolated Calibiochem, San Diego, CA, USA
- wound closure was determined by measuring the open wound area. Dermal closure of wounds was considered positive if at both wound sides the dermis could be observed in a single field observed under a light microscope at xlOO magnification.
- Epidermal closure of wounds was determined by staining wound sections with K14 antibody which highlighted the formation of basal cells at the wound gap. Spatial differentiation of epidermal cells was determined by staining wound sections with KI antibody which highlighted newly formed epidermal cells.
- mice Seven days after wounding all mice were sacrificed and treated wounds were histologically analyzed for proliferative capacity of the epidermis (PCNA), angiogenesis, inflammation, epidermal cells and the remodeling processes at the wound gap.
- PCNA proliferative capacity of the epidermis
- angiogenesis inflammation
- epidermal cells the remodeling processes at the wound gap.
- the insulin-only treatment caused a substantial increase in the incidence of abnormal angiogenesis in the wound area, as compared with the buffer control (60% and 25%, respectively). Since the wound healing process involves rapidly proliferating epidermal cells, such increased angiogenesis may also increase the risk of initiating cancerous development.
- PKC ⁇ inhibitor no angiogenesis was observed in the treated wound area.
- Table 1 The effect of insulin only and insulin combined with PKCa inhibitor on the severit o an io enesis at the wound area
- the insulin-only treatment resulted in increased inflammation, hyperplasia of epidermal cells, delayed differentiation of the spinous layer of epidermal cells and increased scarrin. None ofthe adverse side effects which resulted from the insulin-only treatment were observed when the PKC ⁇ inhibitor was combined with insulin
- PKCa inhibitor reduces wounds inflammation Late and severe inflammatory response in wounds may suppress the process of healing, thus preventing such inflammation from development may promote the wound healing process. Accordingly, the effect of PKC ⁇ inhibitor and insulin on wound inflammation was tested in the following experiment. Wounds were effected on the back of C57BL mice by incision and were treated daily for 7 days with: (i) PBS, control; (ii) 1 ⁇ M of a PKC ⁇ inhibitor (pseudosubstrate myristolated; Calibiochem, USA); (iii) 1 ⁇ M insulin (Eli Lilly, USA); or a mixture of 1 ⁇ M PKC ⁇ inhibitor and 1 ⁇ M insulin. Seven days after wounding all mice were sacrificed and the treated wounds were observed for inflammation under a microscope. The resulting incidences of severe inflammation observed in the wound area are summarized in Table la that follows.
- EXAMPLE 25 The combined effects of modulating expression and/or activity of specific PKC isoforms in dermal cells and administering various agents to the cells on accelerating wound closure in vitro Materials and Methods: Reagents: Factor D (Adipsin) Human, CalbioChem, California USA; Recombinant TNF ⁇ Mouse, R&D Systems, Minneapolis USA; GW 9662, Cayman chemical, USA; Protein Kinase C ⁇ Pseudosubstrate Inhibitor, CalbioChem, California USA; Protein Kinase C ⁇ Pseudosubstrate Inhibitor, CalbioChem, California USA; Protein Kinase C ⁇ Pseudosubstrate Inhibitor, CalbioChem, California USA; PDGF- BB, Cytolab, Israel; IL-6, Cytolab, Israel; KGF/FGF-7, Cytolab, Israel; IGF-1, Cytolab, Israel; TGF ⁇ 2, Cytolab, Israel; Epidermal Growth Fact
- the cultured cells were further provided with one ofthe following agents: insulin (6.7xl0 “7 M), adiponectin (1 ⁇ g per dish), adipsin (2 ⁇ g/ml), IL-6 (1 ⁇ g per dish), GW9662 (l ⁇ g per dish), KGF (1 ⁇ g per dish), TNF ⁇ (12 ⁇ g/ml), TGF ⁇ , rosiglitazone, SRC inhibitors, PKC ⁇ RACK (10 "7 M) and a PKC ⁇ pseudosubstrate inhibiting peptide (10 7 M).
- the resulting wound closure levels were determined 24-48 hours following treatment using index values ranging from 0 (no closure) to 10 (complete closure).
- wound closure was promoted by inhibition of PKC ⁇ in fibroblasts combined with administration of KFG to the cells (wound closure index values of 7; Figure 36). Further in addition, wound closure was accelerated by the inhibition of PKC ⁇ in fibroblasts combined with administration of insulin, IL-6, KGF or GW9662 (wound closure index values of 8, 7, 9 and 8, respectively; Figures 38A-E).
- Table 2a The e ect o treatment combinations on the closure o broblasts in vitro wounds 1
- wound closure was promoted by the inhibition of PKC ⁇ in keratinocytes combined with administration of IL-6, TNF ⁇ or adiponectin to the cells (wound closure index values of 9, 9 and 7, respectively; Figures 37A-D). Further in addition, wound closure was accelerated by the promotion of PKC ⁇ activity and/or expression in keratinocytes combined with activating PKC ⁇ , activating PKC ⁇ , or inhibiting
- Tables 4a The e ect o treatment combinations on the closure o keratinocytes in vitro wounds 1
- the results indicate that wound closure can be substantially accelerated by modulating expression and/or activity of specific PKC isoforms in the dermal and epidermal cells colonizing the wound area, when combined with administering to the cells a growth factor such as IL-6, KGF, TNF ⁇ , a hormone such as insulin, an adipokine such as adipsin or adiponectin, PKC ⁇ RACK and/or GW9662.
- a growth factor such as IL-6, KGF, TNF ⁇
- a hormone such as insulin
- an adipokine such as adipsin or adiponectin
- PKC ⁇ RACK adiponectin
- Copolymer-1 is a synthetic polypeptide analog of myelin basic protein (MBP), which is a natural component of the myelin sheath. Chemically, copolymer-1 is designated L-glutamic acid polymer with L-alanine, L-lysine and L-tyrosine, acetate (salt). Its structural formula is: (Glu, Ala, Lys, Tyr) x.X CH3COOH (C5H9NO4* C3H7NO2 ® C6H14N2O2 ' C9H11NO3) x «x C2H4O2.
- the average molecular weight of glatiramer acetate is 4,700-11,000 daltons.
- copolymer-1 (55 ⁇ g/ml); (iii) a mixture of copolymer-1 (55 ⁇ g/ml) and insulin (1 ⁇ M); (iv) a mixture of PKC ⁇ pseudosubstrate inhibiting peptide (1 ⁇ M) and insulin (1 ⁇ M); and (v) a mixture of copolymer-1 (55 ⁇ g/ml), PKC ⁇ pseudosubstrate inhibiting peptide (1 ⁇ M) and insulin (1 ⁇ M).
- Wounds were morphologically assessed for (i) wound closure, (ii) scab formation and (iii) bleeding/oozing of wounds.
- results In vitro assays: Administering copolymer-1 to cultured keratinocytes promoted closure of in vitro wounds to an index value of 8 on a scale of 0 (no closure) to 10 (complete closure). The combination of copolymer-1 with PKC ⁇ pseudosubstrate inhibiting peptide, or with a mixture of PKC ⁇ pseudosubstrate and insulin, resulted in similar effects (wound closure index values of 8 and 9, respectively; Figures 40A-F). Thus, the results indicate that copolymer-1 per se is capable of substantially accelerating wound closure in vitro.
- EXAMPLE 27 Influence ⁇ f thymus secreted substances on the wound healing process
- Materials and Methods Wounds incisions were performed on the upper back (near the neck) of normal adult rodents or STZ diabetes mice. The animals were sacrificed 7 or 9 days after treatment and the wounds were histologically analyzed for the presence of thymus in the proximity of the wound area and for epidermal and dermal closure of the wounds using the staining procedure as described in Example 20 above. Results: As can be seen in Figures 42A-H, the presence of thymus in close proximity to the wound gap correlated with accelerated epithalization, granulation of tissue and dermal contraction in wounds.
- thymus secreted substances may effectively contribute to the healing process of wounds.
- thymus derived substances such as thymosin, beta thymosins (e.g., thymosin beta 4, thymosin beta 10, thymosin beta 9; thymosin beta 12, thymosin beta 14), alpha thimosins (e.g., thymosin alpha, 1/ zadaxin, prothymosin alpha, parathymosin alpha), fhymulin, IGFl, IGFII, NGF, somatostatin, thyroglobulin, parathyroid hormone and/or thymic hormonal peptides (THPs) may be utilized in treatment for accelerating the healing process of wounds.
- beta thymosins e.g., thymosin beta 4, thymosin beta 10, thymosin beta 9; thymosin beta 12, thymosin beta 14
- nPKC n a new member of the protein kinase C family predominantly expressed in lung and skin. J.Biol.Chem., 265: 22434-22440, 1990.
Abstract
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NZ545318A NZ545318A (en) | 2003-07-15 | 2004-07-15 | Pharmaceutical compositions for healing wounds comprising insulin and PKC alpha inhibitor |
JP2006520104A JP4790609B2 (en) | 2003-07-15 | 2004-07-15 | Methods and pharmaceutical compositions for wound healing |
CN2004800265295A CN1852722B (en) | 2003-07-15 | 2004-07-15 | Use of insulin and myristoylation PKC alpha Pseudo substrate in preparing medicine for healing skin wound |
EP04744980.6A EP1648474B1 (en) | 2003-07-15 | 2004-07-15 | Pharmaceutical compositions for healing wounds |
CA002532352A CA2532352A1 (en) | 2003-07-15 | 2004-07-15 | Methods and pharmaceutical compositions for healing wounds |
AU2004257485A AU2004257485B2 (en) | 2003-07-15 | 2004-07-15 | Methods and pharmaceutical compositions for healing wounds |
IL173148A IL173148A (en) | 2003-07-15 | 2006-01-15 | Use of insulin for the preparation of pharmaceutical compositions for healing wounds |
US11/332,774 US20060258562A1 (en) | 2000-07-31 | 2006-01-17 | Methods and pharmaceutical compositions for healing wounds |
HK06111616.5A HK1090843A1 (en) | 2003-07-15 | 2006-10-20 | Pharmaceutical compositions for healing wounds |
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- 2003-08-21 US US10/644,775 patent/US20040037828A1/en not_active Abandoned
-
2004
- 2004-07-15 RU RU2008138474/14A patent/RU2392959C1/en not_active IP Right Cessation
- 2004-07-15 CN CN201310452225.6A patent/CN103751211A/en active Pending
- 2004-07-15 CA CA002532352A patent/CA2532352A1/en not_active Abandoned
- 2004-07-15 EP EP12004248A patent/EP2540302A1/en not_active Withdrawn
- 2004-07-15 EP EP12004250A patent/EP2526953A1/en not_active Withdrawn
- 2004-07-15 EP EP12004247A patent/EP2526952A1/en not_active Withdrawn
- 2004-07-15 AU AU2004257485A patent/AU2004257485B2/en not_active Ceased
- 2004-07-15 EP EP04744980.6A patent/EP1648474B1/en not_active Not-in-force
- 2004-07-15 RU RU2006104617/14A patent/RU2359694C2/en not_active IP Right Cessation
- 2004-07-15 EP EP12004249.4A patent/EP2540301A3/en not_active Withdrawn
- 2004-07-15 WO PCT/IL2004/000640 patent/WO2005007072A2/en active Application Filing
-
2006
- 2006-10-20 HK HK06111616.5A patent/HK1090843A1/en not_active IP Right Cessation
-
2008
- 2008-05-15 US US12/120,954 patent/US20080280816A1/en not_active Abandoned
-
2009
- 2009-03-16 US US12/404,622 patent/US8093211B2/en not_active Expired - Lifetime
- 2009-10-14 AU AU2009225335A patent/AU2009225335B2/en not_active Ceased
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2011
- 2011-12-06 US US13/311,675 patent/US20120076764A1/en not_active Abandoned
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2013
- 2013-05-30 JP JP2013113603A patent/JP5852051B2/en not_active Expired - Fee Related
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Cited By (19)
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US7402571B2 (en) | 2000-07-31 | 2008-07-22 | Bar-Ilan University | Methods and pharmaceutical compositions for healing wounds |
US8093211B2 (en) | 2000-07-31 | 2012-01-10 | Bar-Ilan University | Methods and pharmaceutical compositions for healing wounds |
US7638484B2 (en) | 2003-08-07 | 2009-12-29 | Healor Ltd. | Methods for accelerating wound healing by administration of adipokines |
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EP2462944A2 (en) | 2005-08-29 | 2012-06-13 | HealOr Ltd. | Methods and compositions for prevention and treatment of diabetic and aged skin |
JP2009506107A (en) * | 2005-08-29 | 2009-02-12 | ヒールオア・リミテッド | Methods and compositions for preventing and treating diabetes and skin aging |
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EP2452691A2 (en) | 2005-08-29 | 2012-05-16 | HealOr Ltd. | Methods and compositions for prevention of diabetic and aged skin |
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AU2006286152B2 (en) * | 2005-08-29 | 2012-05-31 | Healor Ltd. | Methods and compositions for prevention and treatment of diabetic and aged skin |
EP1940440A4 (en) * | 2005-08-29 | 2009-11-11 | Healor Ltd | Methods and compositions for prevention and treatment of diabetic and aged skin |
EP2452687A3 (en) * | 2005-08-29 | 2012-09-05 | HealOr Ltd. | Methods and compositions for prevention and treatment of diabetic and aged skin |
EP2452686A3 (en) * | 2005-08-29 | 2012-09-05 | HealOr Ltd. | Methods and compositions for prevention and treatment of diabetic and aged skin |
EP2452691A3 (en) * | 2005-08-29 | 2012-09-05 | HealOr Ltd. | Methods and compositions for prevention of diabetic and aged skin |
EP2462944A3 (en) * | 2005-08-29 | 2012-09-12 | HealOr Ltd. | Methods and compositions for prevention and treatment of diabetic and aged skin |
US8349793B2 (en) | 2010-01-11 | 2013-01-08 | Heal0r, Ltd. | Method for treatment of inflammatory disease and disorder |
US11400137B2 (en) | 2016-11-14 | 2022-08-02 | University Of Copenhagen | Rectal insulin for treatment of inflammatory bowel diseases |
Also Published As
Publication number | Publication date |
---|---|
US20080280816A1 (en) | 2008-11-13 |
HK1090843A1 (en) | 2007-01-05 |
EP2526952A1 (en) | 2012-11-28 |
EP2540302A1 (en) | 2013-01-02 |
RU2359694C2 (en) | 2009-06-27 |
JP5852051B2 (en) | 2016-02-03 |
CN103751211A (en) | 2014-04-30 |
WO2005007072A3 (en) | 2005-05-12 |
RU2392959C1 (en) | 2010-06-27 |
US20040037828A1 (en) | 2004-02-26 |
EP2540301A3 (en) | 2013-04-10 |
AU2009225335A1 (en) | 2009-11-05 |
EP2540301A2 (en) | 2013-01-02 |
US20090192074A1 (en) | 2009-07-30 |
EP1648474A2 (en) | 2006-04-26 |
JP2013177436A (en) | 2013-09-09 |
CA2532352A1 (en) | 2005-01-27 |
RU2006104617A (en) | 2007-09-10 |
RU2008138474A (en) | 2010-04-10 |
AU2004257485A1 (en) | 2005-01-27 |
US20120076764A1 (en) | 2012-03-29 |
EP1648474B1 (en) | 2013-09-04 |
AU2004257485B2 (en) | 2010-07-01 |
US20100167987A9 (en) | 2010-07-01 |
AU2009225335B2 (en) | 2011-08-25 |
EP1648474A4 (en) | 2009-03-25 |
US8093211B2 (en) | 2012-01-10 |
EP2526953A1 (en) | 2012-11-28 |
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