HK1156515B - Pharmaceutical compositions and methods for accelerating wound healing - Google Patents

Description

Pharmaceutical compositions and methods for accelerating wound healing

The application is a divisional application of an invention patent application with the application number of '200480029538. X' entitled 'pharmaceutical composition and method for accelerating wound healing'.

Field and background of the invention

The present invention relates to methods and pharmaceutical compositions for accelerating the wound healing process. In particular, the present invention utilizes bioactive molecules (adipokines) secreted by adipocytes, and bioactive molecules that modulate adipocyte differentiation, proliferation, and/or activity, to induce or accelerate the healing process of skin wounds.

The primary goal of wound therapy is to allow the wound to close. Open skin wounds are a major wound type, including burns, neuropathic ulcers, decubitus ulcers, venous stasis ulcers and diabetic ulcers.

Open skin wounds typically heal by a process that includes six major links: (i) inflammation; (ii) fibroblast proliferation; (iii) vascular proliferation; (iv) connective tissue generation; (v) epithelialization; (vi) the wound contracts. When these links do not function properly, individually or in their entirety, wound healing is affected. Wound healing may be affected by a number of factors, including malnutrition, infection, pharmacological agents (such as actinomycin and steroids), advanced age and diabetes [ see Current Surgical Diagnosis & Treatment of Hunt and Goodson (Way; Appleton & Lange), pp.86-98 (1988) ]. The common problem of wound healing also occurs after surgical operation on parts of the body, i.e. successful operation but non-healing of open wounds.

The skin is a stratified squamous epithelium in which cells that grow and differentiate are strictly differentiated. In physiological states, proliferation is limited to basal cells attached to the basement membrane. Differentiation is a spatial process in which basal cells lose adhesion to the basement membrane, stop DNA synthesis and undergo a series of morphological and biochemical changes. The final developmental maturation step is the production of the stratum corneum which forms the protective barrier of the skin (1, 2). The earliest changes observed when the basal cells begin to differentiate are related to the ability of the basal cells to detach and move away from the basement membrane (3). Similar changes are associated with the wound healing process, in which the ability of cells to migrate into the wound area and proliferate is enhanced. These processes must be followed in order to reconstitute the skin layer and induce proper differentiation of the epidermal layer.

With the development of culture systems for mouse and human keratinocytes (2, 4), the analysis of the regulatory mechanisms of epidermal cell growth, differentiation and migration has been greatly facilitated. In vitro, keratinocytes can be used as basal proliferating cells with a high growth rate. Furthermore, differentiation may be initiated in vitro after the maturation pattern of the epidermis in vivo. Initial phenomena included loss of the hemidesmosome moiety (3, 5) and selective loss of α 6 β 4 integrin cell attachment to the matrix protein. This suggests that changes in integrin expression are an early phenomenon in keratinocyte differentiation. Early hemidesmosome contact loss leads to upper basal migration of keratinocytes and is associated with the induction of keratin 1(K1) in cultured keratinocytes and in skin (1, 3, 6). Furthermore, differentiation of the granular layer phenotype is associated with the following factors: negative regulation of β 1 and β 4 integrin expression, loss of attachment potential to all matrix proteins and subsequent formation of the cornified envelope and cell death. The differentiated cells eventually shed from the culture dish as mature scales (2, 7). The in vitro differentiation procedure described above follows the maturation pattern of the epidermis in vivo.

Wound healing may be initiated by various bioactive agents in the body that directly or indirectly accelerate epidermal cell proliferation, differentiation and/or migration. Thus, in U.S. Pat. nos. 5,591,709 and 5,461,030 it is described to induce wound closure by using non-steroidal anabolic hormones such as insulin, growth hormone, triiodothyronine and thyroxine. U.S. Pat. No. 5,145,679 describes the induction of wound closure by the use of insulin and pancreatin. Us patent 6,541,447 describes the induction of wound closure by using a mixture of growth factors and growth hormones and international application PCT/IL01/00675 describes the induction of wound closure by using PKC modulating agents. However, there is no teaching in the prior art regarding the use of adipocytes, adipocyte modulators or molecules secreted by adipocytes to induce or accelerate processes associated with wound healing.

Accordingly, it would be highly beneficial to have a widely recognized need for a new method of promoting wound healing. The present invention provides a novel method of treating a wound, which uses adipocytes, cells capable of differentiating into adipocytes, products secreted from adipocytes, and adipocyte modulators to induce or accelerate processes associated with wound healing.

Summary of The Invention

In the course of experiments conducted in wound healing studies, the inventors of the present invention found that adipocytes are closely related to migrating keratinocytes at the wound gap (gap) during the early healing process, indicating that adipocytes, adipocyte modulators and adipokines are involved in the wound healing process and thus may be used to influence the wound healing process.

In practicing the present invention, as further described in the preferred embodiment and in the examples section below, it was found that administration of an adipokine or adipocyte modulator to a wound does substantially effectively promote healing of the wound.

Thus, according to one aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising administering a therapeutically effective amount of an adipokine to the skin wound, thereby inducing or accelerating the healing process of the skin wound.

In another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising administering to the skin wound a therapeutically effective amount of an agent capable of modulating the expression and/or secretion of adipokines, thereby inducing or accelerating the healing process of the skin wound.

In yet another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising administering to the skin wound a therapeutically effective amount of an agent capable of modulating adipocyte differentiation, thereby inducing or accelerating the healing process of the skin wound.

In yet another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising administering to the skin wound a therapeutically effective amount of an agent capable of attracting adipocytes to the skin wound, thereby inducing or accelerating the healing process of the skin wound.

In yet another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising administering to the skin wound a therapeutically effective amount of an agent capable of enhancing proliferation of adipocytes in the skin wound, thereby inducing or accelerating the healing process of the skin wound.

In yet another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising implanting a therapeutically effective amount of adipocytes in the skin wound, thereby inducing or accelerating the healing process of the skin wound.

In yet another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising implanting a therapeutically effective amount of preadipocytes in the skin wound, thereby inducing or accelerating the healing process of the skin wound.

In yet another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising implanting a therapeutically effective amount of stem cells in the skin wound, thereby inducing or accelerating the healing process of the skin wound.

In yet another aspect of the present invention, there is provided a method for inducing or accelerating a healing process of a skin wound, the method comprising transforming skin wound cells to express and secrete adipokine, thereby inducing or accelerating the healing process of the skin wound.

According to a further aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating a wound healing process of the skin, comprising a therapeutically effective amount of adipokine as an active ingredient, and a pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition.

According to a further aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating a wound healing process of the skin, comprising a therapeutically effective amount of an agent capable of modulating the expression and/or secretion of adipokines as an active ingredient, and a pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition.

According to a further aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating a wound healing process of the skin, comprising a therapeutically effective amount of an agent capable of modulating differentiation of adipocytes, as an active ingredient, and a pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition.

According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating a healing process of a skin wound, comprising a therapeutically effective amount of an agent capable of attracting adipocytes to the skin wound, as an active ingredient, and a pharmaceutically acceptable carrier for topical application of the pharmaceutical composition.

According to a further aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating a wound healing process of the skin, comprising a therapeutically effective amount of an agent capable of enhancing proliferation of adipocytes, as an active ingredient, and a pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition.

According to a further aspect of the present invention there is provided a method of measuring the ability of an adipokine or an adipocyte modulator to induce or accelerate a wound healing process, the method comprising administering an adipokine or an adipocyte modulator to a wound and then assessing keratinocyte migration and/or epidermal closure of the wound, thereby measuring the ability of the adipokine or the adipocyte modulator to induce or accelerate wound healing.

According to still further features in preferred embodiments of the invention described below, the adipokine is selected from the group consisting of lipoprotein, adiponectin, resistin, leptin, lipoprotein lipase, angiotensinogen, angiotensin-like 4 (angiotensin-like 4), 1-butyrylglycerol, matrix metalloproteinase 2, matrix metalloproteinase 9, vascular endothelial growth factor, interleukin 6, and tumor necrosis factor alpha. Preferably, the adipokine is a lipoprotein.

According to still further features in the described preferred embodiments the adipocyte modulator is a PPAR modulator, preferably a PPAR- γ antagonist, more preferably GW 9662.

According to still further features in the described preferred embodiments the adipocytes are human adipocytes. Preferably autologous human adipocytes.

According to still further features in the described preferred embodiments the preadipocytes are human preadipocytes. Preferably autologous human preadipocytes.

According to still further features in the described preferred embodiments the stem cells are human stem cells. Autologous human stem cells are preferred.

According to still further features in the described preferred embodiments the implanting further comprises modulating expression and/or secretion of adipokines.

According to still further features in the described preferred embodiments the modulation is effected by differentiation.

According to still further features in the described preferred embodiments differentiating is effected by exposing the preadipocytes to a substance capable of enhancing differentiation of the preadipocytes into adipocytes.

According to still further features in the described preferred embodiments the enhancing differentiation is effected by exposing the stem cells to an agent capable of enhancing differentiation of the stem cells into adipocytes.

According to still further features in the described preferred embodiments the wound is selected from the group consisting of an ulcer, a burn, a laceration, and a surgical incision.

According to still further features in the described preferred embodiments the pharmaceutical composition carrier 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.

According to still further features in the described preferred embodiments the pharmaceutical composition includes a solid carrier.

According to still further features in the described preferred embodiments the adipocyte modulator is an adipocyte differentiation modulator or an adipocyte activity modulator.

According to still further features in the described preferred embodiments the wound is an incision wound made in an experimental animal.

According to still further features in the described preferred embodiments the administration of the adipokine or adipocyte modulator is done at one or more concentrations.

According to still further features in the described preferred embodiments the administration of the adipokine or adipocyte modulator is accomplished in one or more administrations.

The present invention provides novel pharmaceutical compositions and methods for treating wounds that utilize adipocytes, cells capable of differentiating into adipocytes, adipocyte modulators, and molecules secreted by adipocytes to induce or accelerate wound healing.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. If not, reference is made to the patent specification, including definitions. In addition, these materials, methods, and examples are merely illustrative.

Brief Description of Drawings

The invention is described below by way of example only with reference to the accompanying drawings. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, including the best mode for carrying out the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

figure 1 illustrates the effect of insulin on recruitment of adipocytes and migration of epidermal cells to the wound area. A wound was obtained on the back of C57BL mice by cutting. Wounds were treated with daily topical application of healing promoting insulin (1 μ M) for six days, after which mice were sacrificed and their wounds were analyzed for epidermal cell migration and adipocyte recruitment. Epidermal cell migration was determined by staining with K14 antibody and was considered positive if the wound stained positive along the entire wound seam. Recruitment of lipoproteins was determined by H & E staining and considered positive if adipocytes were detected within granulation tissue. Dark bars represent insulin treatment and light bars represent buffer treated controls. Results are expressed as percentage of closed (positive) wounds, each bar representing the mean ± standard error of six replicate experiments.

FIGS. 2A-B are histochemical micrographs illustrating the association of adipocytes with wound healing process. A wound was obtained on the back of C57BL mice by cutting. The injured mice were sacrificed seven days later, and then sectioned and stained with K14 antibody to highlight migrating epidermal cells. Micrographs demonstrate the presence of a large number of recruited adipocytes at the wound seam early in the wound healing process (FIG. 2A, x20 magnification; FIG. 2B, x10 magnification).

Figure 3 illustrates the effect of PPAR γ antagonists (GW9662) on primary keratinocyte migration in vitro. Cultured keratinocytes were either untreated (control) or treated with 2 μ MGW 9662. Migration of keratinocytes was observed under an optical microscope. The upper column shows the micrograph of the pre-treated (day 0) culture, and the lower left and table columns show the resulting control and treated cultures (day 2), respectively. The blue line marks the edge of migrating keratinocytes and the arrows indicate enhanced migration of GW9662 treated cultures relative to untreated controls.

Fig. 4 is a graph illustrating the effect of PPAR γ antagonist (GW9662) and lipoprotein lowering on wound healing in vivo. A wound was obtained on the back of C57BL mice by cutting, and then measured (day 0). The wound area was treated for six days with daily topical application of PBS (control), lipoprotein lowering or 2 μ M GW 9662. The mice were then sacrificed and their wound area was measured (day 6). The wound area fraction (% wound contraction) that contracted from the initial wound area within six days was calculated for each treatment. The graph shows that both GW9662 and the lipoprotein lowering substantially promoted wound contraction relative to the buffer treated control group.

Fig. 5 is a histochemical micrograph illustrating the effect of PPAR γ antagonist (GW9662) and lipoprotein lowering on wound closure in vivo. A wound was obtained on the back of C57BL mice by cutting. The wound area was then measured (day 0). Wounds were treated for six days with daily topical application of PBS (control), 1 μ M lipoprotein lowering or 2 μ M GW 9662. The mice were then sacrificed and their wounds were fixed with paraformaldehyde and observed under a binocular microscope at x5 magnification. The micrograph shows the wound area treated with GW9662 or the lipoprotein lowering, which is substantially smaller than the wound area of the buffer control group.

FIG. 6 illustrates the effect of lipoprotein lowering on epidermal cell migration and wound closure. A wound was obtained on the back of C57BL mice by cutting. Treatment was administered topically as 1 μ M of lipoprotein per day for seven days, followed by sacrifice, sectioning and analysis of epidermal closure and migration by K14 antibody staining. Epidermal closure was considered positive if the wound along the entire wound seam was positively stained. Epidermal migration is considered positive if the wound is positively stained but not completely along the wound seam. The bar chart shows that lipoprotein lowering significantly enhances epidermal closure and epidermal migration. Each bar represents the average of six replicates.

Fig. 7 is a histochemical micrograph illustrating the effect of PPAR γ antagonist (GW9662) and lipoprotein lowering on wound closure (contraction). A wound was obtained on the back of C57BL mice by cutting. Daily treatment with lipoprotein lowering (1 μ M), GW9662(2 μ M) for 6 days, or no treatment (control). Treated mice were sacrificed six days after injury. Histochemical wound sections were stained by H & E (upper panel) or K14 antibody (lower panel) and visualized under an optical microscope at x5 magnification. Positive contraction is considered if both sides of the skin wound (marked by black lines) are visible within a single visual field. The area of open wound in the untreated control section (right) was too large to be contained in a single visual zone (thus considered negative skin contraction), while the section treated with lipoprotein (left) and the section treated with GW9662 (middle) showed positive skin contraction.

Figure 8 illustrates the effect of PPAR γ agonist (troglitazone) on primary keratinocyte migration in vitro. Cultured keratinocytes were either untreated (control) or treated with 100 μ M troglitazone and their migration was observed under an optical microscope. The upper panel shows micrographs of the cultures before treatment (time 0), and the lower left and right panels show the resulting control and treated cultures, respectively (within 48 hours). The lines mark the edges of the cultured keratinocytes, indicating that migration of the cultured keratinocytes after treatment with troglitazone was substantially inhibited relative to the untreated control.

Fig. 9 is a histochemical micrograph illustrating the effect of insulin and PPAR γ agonist (troglitazone) on wound closure in vivo. A wound was obtained by cutting on the back of C57BL mice and treated daily with PBS (control), insulin (10nM), troglitazone (100 μ M) or troglitazone (100 μ M) + insulin (10nM) for 6 days. The mice were then sacrificed and their wounds were fixed with paraformaldehyde and observed under a binocular microscope at x5 magnification. Micrographs show that the area of the wound treated with insulin is substantially smaller than the buffer control, whereas the wounds treated with troglitazone and troglitazone + insulin are much larger than the buffer control.

Figure 10 illustrates the effect of insulin and PPAR γ agonist (troglitazone) on wound closure. A wound was obtained on the back of C57BL mice by cutting. Wounds were treated daily with topical PBS (control), insulin (10nM), troglitazone (100. mu.M) or troglitazone (100. mu.M) + insulin (10nM) combinations for 6 days, then sacrificed, sectioned and analyzed for wound closure. Wound closure was measured by staining with K14 and K1 antibodies. Wound closure was considered positive if the wound was positively stained along the entire wound seam. Each bar represents the average of six replicates.

Description of the preferred embodiments

The present invention relates to methods and pharmaceutical compositions for accelerating the wound healing process. In particular, the present invention utilizes adipocytes, cells that can differentiate into adipocytes, bioactive molecules (adipokines) secreted by adipocytes, and adipocyte modulators to accelerate the healing process of skin wounds.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the details of construction and the arrangement of the parts illustrated in the description or illustrated in the following section of the embodiment are not limiting upon the application of the invention. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

Adult skin comprises two layers, the keratinized stratified epithelium and the underlying thick collagen-rich skin connective tissue that provides support and nutrition. The skin is a protective barrier against the outside. Thus, any damage or rupture on the skin must be repaired quickly and effectively. As described in the background section above, the formation of a clot that embolizes an initial wound is the first stage in achieving skin repair. Thereafter, inflammatory cells, fibroblasts and capillaries embrace the clot to form granulation tissue. The next stage involves regeneration of the epithelium at the wound, where the basal keratinocytes must lose their hemidesmosomal association and migrate into the granulation tissue to cover the wound. After migration of keratinocytes, the keratinocytes enter an increased stage of proliferation, which can replace the cells lost during wound formation. After a single layer of keratinocytes covers the wound (i.e. the epidermis closes), a new stratified epidermis is formed and the basement membrane is reconstructed (8-11).

When experimented in wound healing studies, the inventors surprisingly found that adipocytes are closely associated with migrating keratinocytes in the wounded area during the early wound healing process. Thus, example 1 of the following embodiment section illustrates that the appearance of migrating keratinocytes at the wound seam is directly related to the appearance of recruited adipocytes at the same area. In addition, the insulin-treated wounds recruited more adipocytes to the wound sutures, which subsequently healed faster than the control, untreated wounds. The above-mentioned newly discovered close relationship between adipocyte and keratinocyte migration and wound healing, and the direct correlation between recruited adipocytes and wound healing efficiency, suggests that adipocytes, adipocyte modulators and adipocyte products (adipokines) are involved in the wound healing process and thus can be used to influence the wound healing process.

Adipocytes secrete a large number of biologically active molecules, called adipokines, which play an important role in maintaining the homeostasis of energy by regulating insulin secretion, insulin activity, glucose and lipid metabolism, energy balance, inflammation, and replication.

However, there is no teaching or suggestion in the prior art regarding bioactive molecules secreted by adipocytes that may be involved in wound healing.

Based on the initial findings described above, while further practicing the present invention, the inventors of the present invention expected and subsequently discovered that a typical adipokine, a lipoprotein selected from known adipokines, significantly accelerated keratinocyte migration in vitro and effectively promoted healing of skin wounds in vivo (see example 3 of the embodiment section below).

Thus, according to an aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising administering a therapeutically effective amount of an adipokine to the skin wound, thereby inducing or accelerating the healing process of the skin wound.

The term "wound" as used herein broadly includes injuries to the skin and subcutaneous tissue that are induced in different ways (e.g., bed sores caused by prolonged bed rest, wounds caused by trauma (trauma), cuts, ulcers, burns, surgical incisions, etc.) and have varying characteristics.

Wounds are generally classified into the following four categories according to the depth of the wound: (i) stage I: wounds limited to the epidermis; (ii) II stage: wounds that develop into the dermis; (iii) grade III: wounds that progress to the subcutaneous tissue; (iv) class IV (or full-thickness wound)): the wound exposes bone (e.g., a compression point where bone is exposed such as a larger trochanter or sacrum).

The term "partial cutaneous wound" as used herein includes class I-III wounds; examples of partial cortical wounds include burns, pressure sores, venous stasis ulcers and diabetic ulcers.

The term "deep wound" as used herein is meant to include both class III and IV wounds.

The term "chronic wound" as used herein refers to a wound that does not heal within thirty days.

The term "healing" in reference to a wound refers to the process of repairing a wound via scarring.

It is contemplated that the present invention can treat all wound types including deep wounds and chronic wounds.

The term "adipokine" as used herein refers to any biologically active molecule secreted by adipocytes in vivo or in vitro, including, but not limited to, enzymes, growth factors, cytokines, and hormones secreted by adipocytes. Preferably, the adipokines of the invention are selected from complement D (lipoprotein lowering), C3, and B; vascular endothelial cell growth factor (VGEF), adiponectin (Acrp 30); resistin; a leptin; lipoprotein lipase (LPL); an angiotensinogen; angiotonin-like substance 4; 1-butyryl glycerol (glycerol monobutyrate); matrix metalloproteinases 2 and 9; tumor necrosis factor alpha (TNF α), and interleukin 6. The preferred adipocyte is a lipoprotein.

In addition to administering adipokines to wounds, according to certain embodiments of the invention, adipocyte modulators may induce or accelerate healing of wounds.

The term "adipocyte modulator" as used herein refers to any molecule capable of modulating the expression and/or secretion of adipokines in adipocytes, differentiation of adipocytes, proliferation of adipocytes, migration of adipocytes, or attraction of adipocytes to wounds.

Adipocytes are differentiated from preadipocytes in a process known as adipogenesis. In culture, adipogenesis is completely dependent on insulin, dexamethasone and isobutylmethylxanthine, emphasizing the involvement of the insulin, glucocorticoid and cyclic adenosine monophosphate pathways.

While many signals and biochemical pathways play a necessary role in this process, most of the known changes during adipogenesis are at the level of gene transcription. The key transcription factors involved in the adipogenesis process include proteins belonging to the CCAAT/enhancer binding protein family, adipocyte determinant and differentiation dependent factor 1 (also known as sterol regulatory factor binding protein 1), and the peroxisome proliferator activator receptor gamma (Rangwala and Lazar, Ann. Rev. Nutt.20: 535-539, 2000).

Peroxisome proliferator-activated receptors (PPARs) include three types: PPAR α, PPAR β and PPAR γ. They are ligand-inducible nuclear receptors that directly modulate the activity of a gene by binding to a nucleotide sequence specified in the promoter region of the target gene. PPAR γ plays a crucial role in terminal differentiation by transactivating adipocyte-specific genes. Recent findings indicate that there is interference between PPAR and cholesterol metabolic pathways in the epidermis. All PPAR isoforms are expressed in immature and mature skin. PPAR γ expression increases significantly in late fetal maturation. After birth, and in adult skin, PPAR γ expression is reduced. During epidermal formation, PPAR β and PPAR α in keratinocyte differentiation show important roles (Wabli W., Swiss Med. Wkly.132: 83-91, 2002). It was also demonstrated that PPAR β and PPAR α were upregulated at the margins of wounded skin and that wound healing was affected in naive mice of these isoforms (Michalnik et al, J.cell biol. 154: 799-. The prior art also does not describe or suggest the involvement of PPAR γ in wound healing.

Us patent 6,403,656 describes the use of PPAR γ activators to treat skin disorders associated with abnormal differentiation of epidermal cells. In addition, International application PCT/US99/28101 describes the use of PPAR γ activators such as prostaglandins J2 or D2 to treat obesity and diabetes. However, these publications do not teach or suggest the use of PPAR γ activators or inhibitors to heal wounds.

While further practicing the present invention, the inventors of the present invention found that PPAR γ activity is inversely correlated to the wound healing process. Thus, example 2 of the embodiments section below illustrates the inhibition of wound contraction by the administration of troglitazone, a PPAR γ agonist. On the other hand, administration of the PPAR γ antagonist GW9662 promotes wound contraction.

Thus, according to another aspect of the present invention, there is provided a method of inducing or accelerating a healing process of a skin wound, the method comprising administering to the skin wound a therapeutically effective amount of an agent capable of modulating adipocyte differentiation, thereby inducing or accelerating the healing process of the skin wound. According to this aspect of the invention, the agent may be an agonist or antagonist of any factor involved in adipocyte differentiation, such as transcription factors including, but not limited to, proteins belonging to the CCAAT/enhancer binding protein family, adipocyte determinant and differentiation dependent factors, and PPAR γ. The agent is preferably a PPAR γ antagonist, more preferably GW 9662.

In addition to adipocyte differentiation modulators, the use of other adipocyte modulators may also promote the process of wound healing. Thus, according to the teachings of the present invention, administration of a therapeutically effective amount of an agent to a skin wound is capable of inducing or accelerating the healing process of the skin wound, the agent being capable of: (i) modulating expression and/or secretion of adipokines in adipocytes, (ii) enhancing proliferation of adipocytes, (ii) enhancing migration of adipocytes, (iii) attracting adipocytes to the wound region.

One of ordinary skill in the relevant art can readily determine, in accordance with the assays provided in the context of the present invention, a particular agent, such as an adipokine or adipocyte modulator, to perform a test as to whether any of the above particular agents is indeed an inducer or promoter of the wound healing process.

Thus, the ability of an adipokine or adipokine modulator to induce or accelerate the healing process of a skin wound can be measured by administering the adipokine or adipokine modulator under consideration to the skin wound and then evaluating the keratinocyte migration and/or epidermal closure of the treated wound.

Skin wounds are preferably obtained on the backs of C57BL mice by cutting, and then treated in one or more applications, each with one or more concentrations of adipokine.

At the desired time after the wound is created, preferably about 6 days, the mice are sacrificed and wound biopsies (wound biopsies) are sampled. Wound biopsies are then analyzed for keratinocyte migration to the wound seam and/or epidermal closure of the wound seam according to methods known in the art, preferably using the procedures described in the examples section.

If the chance of keratinocyte migration and/or epidermal closure is significantly increased relative to untreated controls, it is indicative that the tested adipokine or adipocyte modulator is capable of inducing or accelerating the healing process of a skin wound.

According to another aspect of the invention, adipocytes, preferably autologous adipocytes, are implanted into a wound, thereby inducing or accelerating the healing process of skin wounds.

Adipocytes can be obtained from adipose tissue of any animal origin, preferably from human donors, most preferably from autologous human sources. Adipose tissue may be sampled from subcutaneous or perirenal locations, preferably subcutaneous, according to well-known procedures, such as surgery, suction, liposuction, abdominal wall surgery (transluminal) or transection. It is preferred to separate adipocytes from adipose tissue samples using enzymes that disrupt the physical association of the cells (e.g., collagenase) or using mechanical agitation, sonic or ultrasonic energy, and the like. Isolation of adipocytes can be performed using appropriate tissue culture techniques known in the art, for example, as detailed in International application PCT/US 00/30623. Cultured adipocytes can be grown to near confluence, then gently scraped from the growth medium and implanted on a wound.

Adipocytes can also be produced from cultured preadipocytes. The term "preadipocytes" as used herein refers to any cell capable of differentiating into adipocytes. The preadipocytes are preferably human adipocytes, more preferably autologous adipocytes isolated from the patient's own fat or other tissue. Adipose tissue may be sampled from subcutaneous or perirenal locations using well-known procedures, such as surgery, aspiration, liposuction aspiration, abdominoplasty, or transection. Preadipocytes can be isolated from sample tissue using methods such as those described by Rodbell et al (meth. enzymol.31: 103-114, 1974). The preadipocytes isolated can be cultured, expanded and differentiated in vitro into adipocytes using the methods and procedures described in Hauner et al (Journal Clin. invest., 34: 1663-. After at least 1 day, the graft compartment is removed from the wound, preferably at least 1 week after the adipose cell transplantation. The implanted adipocytes are optionally exposed to an adipocyte modulator, such as a non-limiting PPAR γ antagonist, preferably GW 9662.

According to another aspect of the invention, implanting preadipocytes into a wound induces or accelerates the healing process of the skin wound. Using the above-described methods and procedures for adipocytes but omitting the differentiation step, preadipocytes can be isolated, cultured, and expanded in vitro. Undifferentiated preadipocytes were harvested from the culture medium and implanted into the wound using the procedure described above for adipocytes. The implanted preadipocytes are optionally exposed to an adipocyte modulator, such as a non-limiting PPAR γ antagonist, preferably GW 9662.

Adipocytes and/or preadipocytes can be produced from cultured stem cells. The phrase "stem cell" as used herein refers to an immature or mature cell that is not terminally differentiated, can divide without limitation, into cells that are stem cells, or can irreversibly differentiate to produce a new type of cell, such as preadipocytes or adipocytes.

Isolation and in vitro expansion of stem cells can be accomplished using methods well known in the art. For example, Van Epps et al (Blood Cells 20: 411, 1994) and Emerson S.G (Blood 87: 3082, 1996) describe the procedure of isolated culture and human hematopoietic stem Cells from bone marrow, peripheral Blood or neonatal umbilical cord Blood, and their expansion in culture. Human embryonic stem cells (hESCs) can be prepared from human blastocyst cells obtained from human pre-embryo prior to implantation in vivo or from embryos propagated in vitro, using methods such as those described in U.S. Pat. No. 5,843,780 and Reubinoff et al (Nature Biotech.18: 399, 2000). Human mesenchymal stem cells (hmscs) can be isolated and expanded using the methods described in U.S. Pat. nos. 5,197,985, 5,486,359, and 6,214,369. Hmscs found in bone marrow, blood, dermis, and periosteum can differentiate into any particular type of interstitial tissue, such as adipose tissue.

The stem cells may be administered directly to the skin wound, differentiated in vivo into adipocytes, which may or may not include a combination of factors that promote such differentiation. In other words, stem cells may be differentiated ex vivo into preadipocytes or adipocytes, which are then implanted into a wound.

Cultured hmscs can be induced to undergo adipogenic differentiation using the method described in us patent 6,322,784. Thus, adipocytes can be generated from primary hmscs by exposing these cells to glucocorticoids and compounds that upregulate cAMP production, or by inhibiting cAMP degradation, such as phosphodiesterase inhibitors. The adipocytes generated from the stem cells are then harvested and implanted into the wound using the procedures described above, thereby inducing or accelerating the healing process of the skin wound.

An alternative embodiment of the invention is to transform wound cells to express and secrete adipokines, thereby inducing or accelerating the healing process of skin wounds.

The wound cells may be any cell type involved in the wound healing process, such as keratinocytes, adipocytes or preadipocytes. Cells may be transformed with a polynucleotide encoding an adipokine, such as, for example, lipoprotein, adiponectin, resistin, leptin, lipoprotein lipase, angiotensinogen-like 4, 1-butyrylglycerol, matrix metalloproteinase 2, matrix metalloproteinase 9, and tumor necrosis factor alpha. Alternatively, cells can be transformed with a polynucleotide encoding a polypeptide having adipokine activity, such as the polynucleotide encoding a lipoprotein/complement D activity described in U.S. patent 5,223,425.

The appropriate polynucleotide may be introduced into the cell by any method known in the art. Such methods can be found in the methods described extensively in Sambrook et al [ Molecular Cloning: ALaborator Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al [ Current Protocols in Molecular Biology, John Wileyand Sons, Baltimore, Maryland (1989) ], Chang et al [ viral Gene therapy, CRC Press, Ann Arbor, MI (1995) ], Vega et al [ Gene Targeting, CRC Press, Ann Arbor MI (1995) ], Vectors [ A Survey of Molecular cloning Vectors and the Uses, Butterworks, Boston MA (1988) ] and Gilboa et al [ Biotechnologies 4(6) ]: 504-512(1986), and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, see U.S. Pat. No. 4,866,042 for vectors involving the central nervous system, and U.S. Pat. Nos. 5,464,764 and 5,487,992 for inducing homologous recombination using a positive and negative selection.

A preferred method of introducing the adipokine-encoding polynucleotide into the cells of a wound is by the use of a viral vector. Viral vectors have several advantages, including higher efficiency of transformation, targeting, and propagation in particular cell types. Viral vectors may also be modified for specific receptors or ligands to alter target specificity through specific cellular receptors, such as cancer cell receptors.

Retroviral vectors represent a class of vectors suitable for the present invention. Defective retroviruses are commonly used for gene transfer into mammalian cells [ see review Miller, a.d., Blood 76: 271(1990). Recombinant retroviruses, which include adipokine-encoding polynucleotides, may be constructed using well-known molecular techniques. To achieve a retroviral replication-defective, a portion of the retroviral genome can be removed, followed by packaging of the replication-defective retrovirus into viral particles, which can be used to infect target cells using a helper virus and using standard techniques. Procedures for preparing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses are described, for example, in Ausubel et al (eds., Current Protocols in molecular biology, Greene Publishing Associates (1989)). Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes and bone marrow cells.

Other suitable expression vectors may be adenoviral vectors. Adenovirus has been extensively studied and has been conventionally used as a gene transfer vector. Key advantages of adenoviral vectors include relatively high transduction efficiency of dividing and quiescent cells, natural targeting and easy-to-generate high titers to various epithelial tissues [ Russel, W.C. [ j.gen.virol.81: 57-63(2000)]. Adenovirus DNA is transported to the nucleus, but does not integrate with it. Thus, the risk of mutagenesis using adenoviral vectors is minimized, and short-term expression is particularly suitable for treating cancer cells, such as multidrug resistant cancer cells. Seth et al describe Adenoviral vectors for therapeutic use in experimental cancer treatment [ Adenoviral vectors for cancer gene therapy in: p. seth (ed.) adonvires: basic biology to Gene Therapy, Landes, Austin TX, (1999) pp. 103-120 ].

Features that limit expression to specific cell types may also be included. Such features include, for example, promoters and regulatory elements specific for the desired cell type. The viral vector may also include a nucleotide sequence encoding a signal for secretion of the antibody fragment to the outside of the cell. The secretion signal typically comprises a short sequence of hydrophobic amino acids (7-20 residues). Suitable secretion signals in the present invention are generally available and well known in the art, see for example von Heijne [ j.mol.biol.184: 99-105(1985) and Lej et al [ j.bacteriol.169: 4379(1987)].

Recombinant vectors can be administered in a variety of ways. If viral vectors are used, their targeting specificity can be exploited so that such vectors do not have to be administered locally at the tumor site. However, topical administration can provide a more rapid and effective treatment. Administration of the viral vector can also be accomplished by, for example, intravenous or subcutaneous injection into the subject. After injection, the viral vector can circulate to host cells where it recognizes appropriate target specificity for infection.

The adipokines or adipocyte modulators according to the invention may be used therapeutically either by themselves or as active ingredients in pharmaceutical compositions.

The phrase "pharmaceutical composition" as used herein refers to a preparation described herein containing one or more active ingredients and other chemical ingredients such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compound to the organism.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably and refer to a carrier or diluent that does not cause significant irritation to an organism and does not affect the biological activity and properties of the administered active ingredient. Adjuvants are included in this phrase.

The term "excipient" as used herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

For pharmaceutical formulations and methods of administration, reference may be made to the latest edition of "Remington's pharmaceutical Sciences" Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

The pharmaceutical compositions of the present invention may be prepared by methods well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and/or lyophilizing processes.

The pharmaceutical compositions of the present invention may be prepared in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which improve the processing of the active ingredients into pharmaceutical preparations.

According to the present invention, the pharmaceutically acceptable carrier is suitable for topical administration, such as, but not limited to, gels, creams, pastes, lotions, sprays, suspensions, powders, dispersions, salves and ointments, as further described below. Solid carriers may also be used to delay the release of the active ingredient within the wound.

Pharmaceutical compositions suitable for use in the context of the present invention include compositions comprising an effective amount of the active ingredient to achieve the objects of the invention. More specifically, a therapeutically effective amount means an amount of active ingredient effective to induce or accelerate wound healing.

Determination of a therapeutically effective amount is well within the ability of those skilled in the art, particularly in light of the detailed description of the prior art provided herein, the assays disclosed herein, and the examples in the following sections.

The therapeutically effective amount or dose of any of the formulations used in the methods of the invention can be initially assessed from a wound in the skin using the experimental animals described above. Such information can be used to more accurately determine effective dosages in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell culture, or in experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a range of dosage for human use. The dosage may vary with the dosage form employed and the route of administration employed. The precise composition, route of administration and dosage can be selected by the individual physician according to the patient (see, e.g., Fingl et al, 1975, "pharmaceutical Basis of Therapeutics", Chapter 1, page 1).

The dosage and interval, respectively, can be adjusted to a level of active ingredient sufficient to, for example, induce wound healing (minimum effective concentration, MEC). The MEC should vary with a variety of different formulations, but it can be estimated from in vitro data. The amount of drug necessary to achieve an MEC depends on the individual characteristics and the route of administration.

The dosage may be administered singly or in multiple doses, with the course of treatment lasting from days to weeks or until a wound reduction is achieved, depending on the severity and responsiveness of the wound to be treated.

The amount of the composition to be administered will, of course, depend on the subject being treated, the severity of the wound, the mode of administration, the judgment of the prescribing physician, and the like.

If desired, the compositions of the present invention may be stored in the form of a package or dispensing device, such as an FDA approved kit, containing one or more unit dosage forms containing the active ingredient. For example, the package may comprise a metal or plastic film, forming a hose for dispensing the topical formulation. The package or dispensing device may contain instructions for administration. The package or dispensing device may also be accompanied by a notice regarding the use or sale of the container in a form prescribed by a governmental agency regulating the manufacture of pharmaceuticals, wherein the notice is reflective of approval by the agency of the human or veterinary composition. For example, such notice may be a label related to a drug approved by the U.S. food and drug administration to be purchased with a physician's prescription or related to an approved product attachment. Compositions comprising the preparations of the invention formulated in a suitable pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for indication of treatment of a condition, as described in further detail above.

It will be appreciated that the preferred mode of administration of the active ingredients of the invention is local-regional administration, although systemic administration via acceptable routes of administration is not excluded, as is well known in the art, oral, intramuscular, intravenous, subcutaneous, transdermal, peritoneal administration using appropriate ingredients, etc.

Thus, the present invention provides a novel method and pharmaceutical composition for treating wounds, which uses adipocytes, cells capable of differentiating into adipocytes, adipocyte modulators and molecules secreted from adipocytes, to induce or accelerate safe and effective healing of wounds.

Other objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon examination of the following non-limiting examples. In addition, each of the embodiments and aspects of the invention described hereinabove, as well as those claimed below in the claims section, is supported experimentally in the following embodiments.

Examples

The invention is now described, without limitation, with reference to the following examples in connection with the above description.

Generally, the nomenclature used herein and the laboratory procedures of the invention include molecular, biochemical, microbial, and recombinant DNA techniques. The following documents fully explain the above-mentioned techniques. See, for example, "Molecular Cloning: a Laboratory Manual "Sambrook et al, (1989); "Current Protocols in Molecular Biology" volume 1-3 Ausubel, R.M., ed. (1994); ausubel et al, "Current Protocols in molecular biology", John Wiley and Sons, Baltimore, Maryland (1989); perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); watson et al, "Recombinant DNA", Scientific American Books, New York; birren et al (eds) "Genome Analysis: a Laboratory Manual series ", volume 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodology is set forth in the following U.S. patents: 4,666,828, respectively; 4,683,202; 4,801,531, respectively; 5,192,659 and 5,272,057; "Cell Biology: a Laboratory Handbook ", Cellis, volume 1-3, J.E., ed. (1994); "Culture of Animal Cells-A Manual of basic technique", Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current protocols in Immunology" 1-3 Coligan J.E., ed. (1994); stits et al, (eds), "Basic and Clinical Immunology" (eighth edition), apple & Lange, Norwalk, CT (1994); mishell and Shiigi (eds), "Selected Methods in cellular Immunology", W.H.Freeman and Co., New York (1980); useful immunoassays are widely described in the patent and scientific literature, see for example U.S. patents: 3,791,932; 3,839,153, respectively; 3,850,752, respectively; 3,850,578, respectively; 3,853,987, respectively; 3,867,517; 3,879,262, respectively; 3,901,654, respectively; 3,935,074, respectively; 3,984,533, respectively; 3,996,345; 4,034,074, respectively; 4,098,876, respectively; 4,879,219, respectively; 5,011,771 and 5,281,521; "Oligonucletides Esynthesis" Gait, M.J., ed. (1984); "Nucleic Acid Hybridization" Hames, B.D., and Higgins S.J., eds. (1985); "transformation and translation" Hames, b.d., and Higgins s.j., eds. (1984); "Animal cell culture" Freshney, r.i., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" volumes 1-317, Academic Press; "PCR Protocols: a Guide To Methods and Application ", Academic Press; san Diego, CA (1990); marshak et al, "Strategies for Protein purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all references are incorporated herein in their entirety. Other commonly used documents are provided herein. The steps described therein are provided for ease of reading herein and are well known in the art. All information contained therein is incorporated by reference in its entirety.

Test materials and methods

Materials: all standard chemicals were from Sigma-Aldrich, st. The accessory Embedding vehicle (Paraplast Embedding Me dium) was also from Sigma, and the anti-keratin 14 and anti-keratin 1 polyclonal antibodies were from Bacto-Covance (Richmond, CAUSA). Biotinylated goat anti-rabbit antibody and streptavidin-horseradish peroxidase (HRP) were from ZYMED Experimental Co., Ltd (San Francisco, CA USA). GW9662 is available from Cayman Chemicals (Ann Arbor Michigan USA). The lipoprotein for lowering blood lipid (complement factor D) is from Calibiochem (san Diego CA USA). Hematoxylin was obtained from DAKO (Carpinteria CA USA). Eosin is from ICN biomedical limited (Aurora ohario USA) and entellan is from merck (darmstadt germany).

Isolation and culture of murine keratinocytes: primary keratinocytes were isolated from fresh skin and cultured in Eagle's Minimal Essential Medium (EMEM) containing 8% Chelex (Chelex-100, BioRad) treated fetal bovine serum as described in reference 18. To maintain a proliferative basal cell phenotype, the last Ca²⁺The concentration is adjusted to 0.05 mM. The experiment was performed 5-7 days after plating.

Keratinocyte migration assay: primary mouse keratinocytes were treated or not with PPAR γ antagonist GW9622(μ M) or PPAR γ agonist troglitazone (μ M). Wound streak analysis was performed 24 hours after treatment, and representative visual zones were photographed immediately after injury (day 0) and 48 hours (day 2). The mean number of wound closures is expressed as a percentage compared to the initial wound score width.

Wound healing analysis: wounds were created by a 20mm incision in the back of C57BL mice and treated with various substances daily for 6 days. Mice were sacrificed six days after injury. Wound biopsy sampling, treatment, analysis of various wound healing parameters, i.e., wound contraction, adipocyte migration and differentiation, epidermal migration and closure, morphologically and histochemically.

Wound contraction analysis: the wound area was measured before and after treatment and the percent reduction of the wound area was calculated.

Preparation of paraffin-embedded wound sections: wound biopsies were fixed in 4% paraformaldehyde and then dehydrated to increase their ethanol concentration (50-100%). The dehydrated product was first immersed in a solution of paraffin (50%) and xylene (50%) and then in pure paraffin. The paraffin blocks were cut by a microtome and sections were loaded on a Super Frost^+TMOn a slide.

H & E staining: slides loaded with paraffin-embedded wound sections were incubated at 60 ℃ for 60 minutes, then the slides were rinsed 2 times with toluene (100%) for 10 minutes, once with ethanol (100%) for 15 minutes, and once with ethanol (100%) for 10 minutes for dewaxing. These dewaxed slides were stained with hematoxylin (stock solution) for 10 minutes, rinsed with water, stained with eosin (0.5% in DDW) for 5 minutes, and then washed with 70% ethanol solution for 1 minute. Thereafter, the slide glass was dehydrated by washing it once with a 95% ethanol solution for 5 minutes, twice with 100% ethanol for 5 minutes, twice with 100% toluene for 10 minutes, and finally sealed with entellan (MERCK DarmstadtGermany).

Keratin 4 and keratin 14 staining: paraffin removal Process of slides loaded with Paraffin-Embedded wound sections As described above H&E staining method, and in blocking solution (5% BSA and 5% Tween 20)^TMPBS solution) for 1 hour. Then, the solution (5% BSA and 5% Tween 20) was blocked (1: 1000) with an anti-keratin 1 antibody or with an anti-keratin 14 antibody (Babco-Coonce)^TMPBS solution) was incubated at 4 ℃ overnight. Thereafter, the slides were washed with wash buffer (5% Tween 20)^TMPBS solution) was washed 5 times, followed by suspension (1: 200) in blocking solution (5% BSA and 5% Tween 20)^TMPBS solution) was incubated for 1 hour with biotinylated goat anti-rabbit antibody (ZYMED laboratories inc). Then, the slide was washed 3 times with washing buffer, and immediately thereafter, streptavidin antibody was acylated with secondary biotin in a blocking solution (1: 300) at room temperature for 1 hour. Thereafter, the slides were washed 2 times with washing buffer, 5 minutes at time, once with PBS, 5 minutes at time, once with TRIS buffer solution (0.05M PBS solution), and then incubated in DAB reagent (2 tablets dissolved in DDW, gold and silver each) for color development. The reaction was stopped by immersing the slides in water, followed by counterstaining with eosin (ICN, 0.5% DDW solution).

Results of the experiment

Example 1

Association of adipocytes with migrating keratinocytes during wound healing

Migrating epidermal cells (keratinocytes) and recruited adipocytes were observed on seven-day wound tissue (FIGS. 2A-B). The recruited adipocytes observed were essentially free of stored fat (i.e., early adipocytes). As seen in fig. 1, migrating keratinocytes were observed along the entire wound seam in about 60% of the untreated wound groups, while recruited adipocytes were also present in about 60% of the untreated wound groups. Figure 1 also shows that migrating keratinocytes and recruited adipocytes were observed in about 90% and 80% of the insulin treated wound groups, respectively.

These results show that in the early stages of wound healing, keratinocytes migrating to the wound gap area are closely related to the recruitment of adipocytes to the same area. Thus, the results confirmed that migrating adipocytes that did not differentiate completely into fat-accumulating cells are involved in the wound healing process.

Example 2

Effect of PPAR γ modulators on keratinocyte migration and wound contraction

The effect of inhibiting or enhancing the activity of peroxisome proliferator-activated receptor gamma (PPAR γ) on keratinocyte migration was evaluated in vitro. As shown in fig. 3, treatment of cultured primary mouse keratinocytes with PPAR γ antagonist GW9662 promoted migration of keratinocytes. On the other hand, treatment of cultured keratinocytes with the PPAR γ agonist troglitazone inhibited the migration of keratinocytes (fig. 8).

The effect of inhibiting or enhancing the activity of PPAR γ with PPAR γ antagonists and agonists, respectively, on wound healing was also evaluated in vivo. Accordingly, incision wounds were made on the backs of C57BL mice, treated daily with either PBs buffer (control) or different agents for 6 days. Mice were sacrificed 6 days after injury, after which the wounds were analyzed. As seen in fig. 4,5 and 6, treatment with GW9662, a PPAR γ antagonist, promoted wound contraction compared to the control group. On the other hand, similar treatment with troglitazone (PPAR γ agonist) inhibited wound contraction (fig. 9). In addition, troglitazone impaired insulin-induced wound healing (fig. 9 and 10).

Thus, the above results clearly demonstrate that PPAR γ activity hinders wound healing and that PPAR γ inhibition is effective in promoting the wound healing process. Thus, the results indicate that PPAR γ antagonists such as GW9662 can be used to effectively accelerate wound healing.

Example 3

Effect of lipoprotein lowering on keratinocyte migration and wound contraction

The effect of lipoprotein (complement factor D, secreted by adipocytes) on wound healing was also evaluated in vivo. Accordingly, incision wounds were made on the back of C57BL mice, treated daily with PBS buffer (control) or 1 μ M of lipoprotein lowering for 6 days. Mice were sacrificed 6 days after injury, and wounds were analyzed. As seen in figures 4,5 and 7, the lipoprotein lowering substantially promoted wound contraction (figures 4,5 and 7). In addition, the lipoprotein lowering increased epidermal closure from about 15% to about 30% and increased keratinocyte migration from about 30% to about 65% compared to the buffer control group (fig. 6).

These results demonstrate that adipokines, such as lipoproteins, are effective in inducing or accelerating wound healing.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

While the invention has been described with respect to various specific embodiments, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and scope of the appended claims. All publications, patents, patent applications, and sequences identified by their names and/or database listing numbers mentioned in the specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent application, or sequence was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

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Claims (11)

1. Use of a PPAR γ antagonist in the preparation of a pharmaceutical composition suitable for topical administration to induce or accelerate the healing process of skin wounds.
2. 2. The use of claim 1 wherein the PPAR γ antagonist is GW 9662.
3. 3. The use of claim 1 or 2, wherein the skin wound is a wound or ulcer caused by trauma.
4. 4. The use of claim 1 or 2, wherein the skin wound is a cut, a burn, or a surgical incision.
5. 5. Use according to claim 1 or 2, wherein the skin wound is a decubitus ulcer due to prolonged bedridden rest.
6. 6. The use of claim 1 or 2, wherein the pharmaceutical composition is selected from the group consisting of an aqueous solution, a spray, a suspension, a powder and an ointment.
7. 7. Use according to claim 1 or 2, wherein the pharmaceutical composition is selected from the group consisting of lotions, gels, creams, pastes and ointments.
8. 8. The use of claim 1 or 2, wherein the pharmaceutical composition is a dispersion.
9. 9. The use of claim 6, wherein the pharmaceutical composition comprises a solid carrier.
10. 10. The use of claim 7, wherein the pharmaceutical composition comprises a solid carrier.
11. 11. The use of claim 8, wherein the pharmaceutical composition comprises a solid carrier.