WO2000057902A1

WO2000057902A1 - Method and pharmaceutical composition for wound healing

Info

Publication number: WO2000057902A1
Application number: PCT/IL2000/000173
Authority: WO
Inventors: Israel Blumenfeld; Yehuda Ullmann; Dov Laufer; Erela Livne
Original assignee: Technion Research And Development Foundation Ltd.
Priority date: 1999-03-25
Filing date: 2000-03-16
Publication date: 2000-10-05
Also published as: AU3321100A; IL129180A0

Abstract

A method of treating a wound is provided. The method is effected by topically applying a chemokine to the wound. A pharmaceutical composition for topically treating a wound is further provided. The composition contains an effective concentration of a chemokine and a pharmaceutically acceptable carrier. Figure 4 is a histogram showing percent contraction in control, TGF-beta 1+bFGF and IL-8 treated animals.

Description

METHOD AND PHARMACEUTICAL COMPOSITION FOR WOUND

HEALING

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a method and pharmaceutical composition effective in wound healing and, more particularly, to a method and pharmaceutical composition in which chemokines, interleukin-8 and interleukin-8 analogs in particular, are used to facilitate and enhance wound healing. Wound healing involves a complex programmed sequence of cellular and molecular processes including inflammation, cell migration, angiogenesis, provisional matrix synthesis, collagen deposition and reepithelization (1). Until recently, no pharmacological agents that could reproducibly accelerate wound healing, have been identified. The identification of cytokines and growth factors as mediators of many of the processes integral to tissue repair has sparked renewed interest in the development of potential therapeutics to enhance deficient tissue repair (2). Growth factors and cytokines are considered candidate therapeutics because they are synthesized by and stimulate cells required for tissue repair (e.g., platelets, macrophages, endothelial cells, keratinocytes and fϊbroblasts). However, such factors and cytokines are deficient in chronic wounds in- vivo, and in pharmacological composition for wound repair enhancement (3).

Normal wound healing is thought to occur in three stages: (i) directed and sequential migration of neutrophils, monocytes, keratinocytes and fibroblasts into the wound over the first several days; (ii) activation of wound macrophages and fibroblasts resulting in de-novo synthesis of growth factors and cytokines, extracellular matrix (ECM) proteins and in proliferation of fibroblasts which take place during the next 2-3 weeks; and (iii) remodeling which is associated with active collagen turnover and cross linking, which takes place from two weeks to one year post wounding.

Cytokines are usually grouped into "families" based on structural and functional similarities. The accumulation and activation of leukocytes in the wounded tissues is the most characteristic expression of inflammation. The mechanism of leukocyte recruitment has been studied for decades and the breakthrough came about few years ago with the discovery of interleukin-8 (IL-8) and several related chemotactic cytokines. IL-8 and related chemotactic cytokines are now collectively called chemokines. They are small proteins of 70-80 amino acids with four conserved cysteins which form two disulfide bonds, a short amino terminal and a longer carboxyl terminal sequence (4).

Certain cytokines remain attached to the cell membrane and the interaction mitigated thereby involves direct cell contact which is known as juxtacrine interaction (3).

Cytokines have the ability to affect all aspects of the cellular phenotype. Recently, a great deal has been learned about the intracellular mechanism of how cytokines cause phenotypic changes in cells.

The binding of cytokines to their respective surface receptors leads to the initiation of specific cell transduction pathways. In addition to binding growth factors, many receptors must form dimers as a requirement for cell transduction. Most cytokines activate tyrosine kinases that are attached to the receptor. The receptor-associated tyrosine kinase then activates a series of other kinases that initiate a specific signal transduction pathways.

Cytokines and growth factors have a potential to improve wound healing through several mechanisms (i) they have chemotactic activities that attract inflammatory cells, fibroblasts and keratinocytes into the wound; (b) they act as mitogens to stimulate cellular proliferation; (iii) cytokines and growth factors can stimulate angiogenesis, the ingrowth of new blood vessels into the wound; (iv) they have a profound effect on the production and degradation of extracellular matrix (ECM); finally, (v) they influence the synthesis of other cytokines and growth factors by neighboring cells.

Two subfamilies of chemokines are distinguished according to the arrangement of the first two cysteins which are either separated by one amino acid (CXC chemokines) or, adjacent (CC chemokines). CXC chemokines act mainly on neutrophil leukocytes while CC chemokines are inactive on these cells (5).

IL-8 is generated as a 99 amino acid precursor and is secreted after cleavage of a leader sequence of 20 residues. The mature protein is processed extracellularly at the amino terminus leading to a marked increase of the specific activity (6).

Nuclear magnetic resonance spectroscopy shows that IL-8 exists as a dimer in solution (7). Recently an analog of IL-8 was synthesized which was as effective as natural IL-8, so the conclusion is that the biologically relevant form of IL-8 is the monomer. The gene of IL-8 has been cloned and sequenced (8) and has been mapped to human chromosome 4ql2-21. Fibroblasts, endothelial cells, monocytes and keratinocytes are major producers of IL-8 when stimulated by agents such as IL-1 and TNF-α.

In rapid succession several analogs of IL-8 were reported: neutrophil activating protein 2(NAP-2), three GRO proteins (GRO α, β, γ) and epithelial cell-derived neutrophil activating protein (ENA78) (9).

They are all CXC chemokines and share with IL-8 the properties of neutrophil chemoattractant, inducing a shape change chemotaxis, a transient rise of the intracellular free Ca²⁺ concentration, release of granules content, upregulation of adhesion proteins, formation of bioactive lipids and respiratory burst (9).

Human neutrophils possess on the average 64,500 ± 14,000 receptors to IL-8 and its analogs. There is evidence of existence of two receptors on neutrophils, one with high affinity for all three ligands and the other with high affinity for IL-8 (8, 9). The receptor which is selective for IL-8 is termed IL-8 Rl or IL-8 Ra and the other, IL-8 R2 or IL-8 Rb (4).

IL-8 Rl was detectable in a variety of cell types, such as leukocytes, lymphocytes, as well as in fibroblasts. IL-8 was found to be produced by many cells, including, for example, endothelial cells, fibroblasts, keratinocytes, synovial cells and chondrocytes. IL-8 may have an important function in wound healing as it is expressed in the stratum granulosum attracting polymorphonuclear cells stimulating angiogenesis and keratinocytes mitogenesis. It promotes epidermal proliferation (10) associated with rapid and transient mobilization of Ca²⁺ in vitro. IL-8 promotes angiogenesis and induces HLA-DR on keratinocytes (11). IL-8 appears, therefore, to be a pivotal factor by which fibroblasts influence epidermal growth in epidermal regeneration and wound healing. Probably there is paracrine loop between dermal and epidermal IL-8 production.

IL-8 may play an important role in the homeostasis of normal epidermis as well as in wound healing in which dermal-epidermal interaction and keratinocytes migration are critical to re-establishment of the barrier function (12).

Some overlap between the different stages of wound repair can occur. A number of growth factors are secreted from α granules of platelets which are strong chemoattractants to inflammatory cells (13). TGF-βl isolated from blood platelets has major effect on tissues from mesemchymal origin. It was shown to induce wound repair following application in rat (14) and in human (15). Addition of TGF-β prior to experimentally induced excision wound enhanced wound repair (16).

Fibroblast growth factor (FGF) was also found to induce angiogenesis and stimulate endothelial and smooth muscle cells (17). It was reported that application of bFGF on pressure wound in paraplegs resulted in dose response healing of the wound (18).

Growth factors are good candidates for treatment of wound healing because they are naturally produced by the cells and enhance tissue repair

(19). Their use in chronic wounds was found to enhance repair. Clinical studies have shown that FGF applied to chronic and diabetic ulcers enhanced their repair significantly (20, 21, 22).

Use of cytokines to enhance wound healing is crucial in the cases of burns, chronic pressure wounds, diabetes wounds and in chronic ulcers. The possibility to enhance wound healing by cytokines is beneficial during long-term hospitalization which leads to pressure wounds formation, in elderly individuals which suffer chronic wounds, in accident and battlefield inflicted wounds, and in burn wounds.

Previous studies of wound repair were conducted with growth factors. However, the possible effect of IL-8 and its analogs on wound repair has not yet been studied or attempted.

There is thus a widely recognized need for, and it would be highly advantageous to have, a method and pharmaceutical composition in which chemokines, interleukin-8 and interleukin-8 analogs in particular, are used to facilitate and enhance wound healing.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided method of treating a wound comprising the step of topically applying a chemokine to the wound. According to another aspect of the present invention there is provided a pharmaceutical composition for topically treating a wound comprising an effective concentration of a chemokine and a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the invention described below, the chemokine is capable of binding an interleukin-8 receptor. According to still further features in the described preferred embodiments the interleukin-8 receptor is selected from the group consisting of interleukin-8 receptor 1 and interleukin-8 receptor 2.

According to still further features in the described preferred embodiments the chemokine is a CXC chemokine.

According to still further features in the described preferred embodiments the chemokine is interleukin-8.

According to still further features in the described preferred embodiments the chemokine is an interleukin-8 analog. According to still further features in the described preferred embodiments the interleukin-8 analog is selected from the group consisting of neutrophil activating protein 2 (NAP-2), GRO α, GRO β, GRO γ and epithelial cell-derived neutrophil activating protein (ENA78).

According to still further features in the described preferred embodiments the chemokine is native.

According to still further features in the described preferred embodiments the chemokine is a product of genetic engineering.

According to still further features in the described preferred embodiments the chemokine induces in-vivo interleukin-8 secretion. According to still further features in the described preferred embodiments the chemokine is selected from the group consisting of interleukin-1 and TNF-α.

According to still further features in the described preferred embodiments the chemokine is applied as a pharmaceutical composition including an effective concentration of the chemokine and a pharmaceutically acceptable carrier.

According to still further features in the described preferred embodiments the pharmaceutically acceptable carrier is a lotion, an ointment, a gel, a cream, a liquid, a spray and a powder. According to still further features in the described preferred embodiments the wound is selected from the group consisting of burn wound, chronic wound, infection wound, pressure wound and cut wound.

According to still further features in the described preferred embodiments the method further comprising the step of bandaging the wound.

The present invention successfully addresses the shortcomings of the presently known configurations by providing new and effective tool for wound healing, chronic and complicated wounds, in particular. BRIEF DESCRIPTION OF THE DRAWINGS

The invention herein described, by way of example only, with reference to the accompanying drawings, wherein:

2 FIG. 1 is a histogram showing wound area (cm ) in non-treated control animals, **p<0.01.

2 FIG. 2 is a histogram showing wound area (cm ) in animals treated with TGF-βl + bFGF, ***pO.001.

2 FIG. 3 is a histogram showing wound area (cm ) in animals treated with IL-8, ***p<0.001. FIG. 4 is a histogram showing percent contraction in control, TGF-β

1 + bFGF and IL-8 treated animals.

FIG. 5 is a histogram showing percent open area in control, TGF-βl

+ bFGF and IL-8 treated animals.

FIG. 6 is a histogram showing percent epithelization in control, TGF- βl + bFGF and IL-8 treated animals.

FIG. 7 is a histogram showing epithelization and contraction area in

TGF-βl + bFGF and IL-8 treated animals.

FIG. 8 is a histogram showing percent of repair in TGF-βl + bFGF and IL-8 treated animals. FIG. 9 demonstrates the clinical appearance of burn injury on day 9 in a control animal.

FIG. 10 demonstrates the clinical appearance of burn injury following 9 days of treatment with TGF-βl + bFGF.

FIG. 11 demonstrates the clinical appearance of burn injury following 9 days of treatment with IL-8 according to the present invention.

FIG. 12 demonstrates the microscopy of tissue morphology of wound biopsy from a control animal on day 13. Thin epithelial layer containing few rows of epithelial cells is observed.

FIG. 13 demonstrates the microscopy of tissue morphology of wound biopsy on day 13 from an animal treated with TGF-βl + bFGF. Similar appearance to the control group is observed.

FIG. 14 demonstrates the microscopy of tissue morphology of wound biopsy on day 13 from an animal treated with IL-8 according to the present invention. In comparison to the previous groups, a thick epithelial layer containing numerous rows of epithelial cells including skin appendages are observed. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a method and pharmaceutical composition which can be used for enhancement of wound healing.

Specifically, the present invention can be used to facilitate healing of wounds such as, but not limited to, pressure wounds, chronic wounds, cut wounds and burn wounds.

The principles and operation of a method and pharmaceutical composition according to the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. 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 employed herein is for the purpose of description and should not be regarded as limiting.

Large burns are followed by significant trauma induced immunomodulation. The mean plasma concentration of IL-8 in burned patients is about 60 times higher than that of healthy individuals.

Furthermore, patients with total body surface area burns has significant higher IL-8 concentrations in plasma than patients with smaller burns.

IL-8 is localized in the basal germinative cell layer or at the focal sites of ongoing neutrophil inflammation in the suprabasal cell layer. IL-8 have been implicated as a regulator of proliferation and differentiation of normal keratinocytes and as a mediator of keratinocytes maturation and migration in inflammatory processes that involve the skin.

IL-8 may thus play an important role in the homeostasis of normal epidermis as well as in the process of wound healing, in which dermal- epidermal interactions and keratinocytes migration are critical.

Dermal factors influence epidermal differentiation and appendage formation, but the rules governing these interactions and the factors that affect them are poorly understood.

Progressive skin necrosis after trauma such as burn wounds and local trauma is a frequent occurrence. During this process tissue initially appears viable with clinical evidence of perfusion. Subsequently this tissue dies, a fact that has profound clinical significance because ultimate tissue loss is much greater than that assessed at the time of initial injury. The injured skin directly initiates an inflammatory response that includes the release of neutrophil chemoattractants. In-vivo IL-8 is a specific cytokine initiator of post traumatic cascade wound healing. IL-8 is synthesized in-vivo by resident skin cell like keratinocytes (24) fibroblasts, (25) macrophages and endothelial cells.

Rot et al. (1996) demonstrated the presence of binding sites for IL-8 on the endothelium of post capillary venules and small veins in human skin (26). Another aspect of IL-8 is its ability to trigger angiogenesis in-vivo by an indirect action probably through recruitment of leukocytes (27). Besides in human skin, IL-8 induces increased micro vascular permeability (28). IL-8 stimulates neutrophils to degranulate, thereby exposing surface receptors which may promote integrin-mediated adhesion between the neutrophils and the endothelial cells.

However, the ability of IL-8 to enhance wound healing by topical application is shown herein for the first time. In the past various topical agents and bandages were used for wound healing, but topical treatment with chemokines, IL-8 and its analogs in particular, has not been studied or attempted previously.

While reducing the present invention to practice, the ability of interleukin-8 (IL-8) to enhance wound healing as compared to the effect of transforming growth factor-βl (TGF-βl) and basic fibroblast growth factor

(bFGF) applied in combination, was evaluated. It was demonstrated that topical administration of IL-8 enhanced wound healing.

Inflicted burn injury was used as a model for wound healing. Deep partial thickness burns were performed in experimental animals using aluminum templates according to the method of Kaufman et al. (23).

IL-8 and the above growth factors were applied topically using measured volumes and the treated wounds were covered by non-adherent absorbent dressing and bandaged. Treatment was repeated every two days for up to 13 days. Wound areas were recorded, photographed, and tissue biopsies were obtained on the last day and processed for general morphology.

Results indicated that treatment with IL-8 enhanced epithelization, reduced contraction and open area values of inflicted wounds. IL-8 appeared to be the most significant in these respects as is compared with TGF-βl + bFGF or control groups. The criteria used for evaluating wound healing were measurements of (i) the total area of wound, the values of (ii) contraction and (iii) open area of the wound, the percent of (iv) open area from total area, (v) the percent of contraction, values of (vi) epithelization and (vii) percent repair. The results, which are demonstrated in Figures 1- 14 and described in more detail in the Examples section that follows, indicate that the effect of IL-8 is the most beneficial in all tested criteria. Furthermore, morphology obtained from tissue biopsies on the last day of the experiment revealed also that reepithelization was the most significant in IL-8 treated wounds.

Thus, IL-8 can contribute to the improvement of unhealed wounds resulting from, for example, bums, infections, cut, pressure and chronic wounds associated with systemic diseases, such as diabetes and in the case of insufficient blood supply, e.g., in altered angiogenesis.

According to one aspect of the present invention there is provided method of treating a wound. The method is effected by topically applying a chemokine to the wound. According to another aspect of the present invention there is provided a pharmaceutical composition for topically treating a wound. The composition contains an effective concentration of a chemokine and a pharmaceutically acceptable carrier. Effective concentrations are expected to range from e.g., about 1 or less to about 1000 or more ng per μl, depending on wound severity. Further dosing considerations are provided hereinunder.

As used herein in the specification and in the claims section below, the term "wound" refers to any injury to any portion of the body of a subject including, but not limited to, acute conditions such as thermal bums, chemical bums, radiation bums, bums caused by excess exposure to ultraviolet radiation such as sunburn, damage to bodily tissues, such as the perineum, as a result of labor and childbirth, including injuries sustained during medical procedures such as episiotomies, trauma-induced injuries including cuts, those injuries sustained in automobile and other mechanical accidents, and those caused by bullets, knives and other weapons, and post- surgical injuries, as well as chronic conditions, such as pressure sores, bedsores, conditions related to diabetes and poor circulation, and all types of acne and skin infection.

As used herein in the specification and in the claims section below, the term "treating" refers to substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or substantially preventing the appearance of clinical symptoms of a disease. It also refers to healing and repairing. According to preferred embodiments of the present invention the chemokine is capable of binding an interleukin-8 receptor, such as the interleukin-8 receptor 1 and the interleukin-8 receptor 2. The chemokine is preferably a CXC chemokine that binds the above receptors. Preferably, the chemokine is interleukin-8 or an interleukin-8 analog, such as, but not limited to neutrophil activating protein 2 (NAP-2), GRO α, GRO β, GRO γ and/or epithelial cell-derived neutrophil activating protein (ENA78). The chemokine according to the present invention can be either native, i.e., purified from tissues, cells or plasma of mammals, however, it can also be a product of genetic engineering. The genes of many of the chemokines have been isolated and their encoded proteins expressed in heterologous expression systems (e.g., animal cells, yeast cells, bacteria). These genes can be used to produce transgenic animals which produce and secrete any desired chemokine in, for example, their milk. According to a preferred embodiment of the present invention the chemokine used induces in-vivo interleukin-8 secretion. For example, interleukin- 1 and TNF-α are known to induce interleukin-8 secretion in vivo, yet these chemokines, like IL-8, were never administered topically.

The chemokine is preferably applied as a pharmaceutical composition including an effective concentration of the chemokine and a pharmaceutically acceptable carrier, such as, but not limited to, a lotion, an ointment, a gel, a cream, a liquid, a spray and a powder.

The method and composition according to the present invention are particularly useful and advantageous in treating bum wound, chronic wound, infection wound, pressure wound (bed sores) and cut wound.

When treating a wound using the method and composition according to the present invention, the wound is preferably bandaged between successive application of chemokines, so as to reduce the risk of infection, as well known in the art. Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day or several days, with course of treatment lasting from several days to several weeks or months or until a cure is effected or a diminution of disease state is achieved. Persons ordinarily skilled in the art can easily determine optimum dosages, dosing methodologies and repetition rates.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

MATERIALS, DEVICES AND EXPERIMENTAL METHODS

Deep partial skin thickness bums were performed using guinea-pigs (250-300 grams). Twenty-four hours prior to the experiment, animals were anaesthetized (Ketamine HC1, 150 mg/kg injected intramuscularly). Back and abdomen were clipped using electrical clipper (Oster-Golden A-S, model 5-55E, 35 Watts, Head No. 80, Blade size 40). Then, the back and right flank of each animal were depilated using a regular depilatory cream. This approach ensured a thorough and uniform removal of the animal's fur. Since the clipping and the depilation procedures produce edema of the skin, bum were inflicted only 24 hours thereafter.

The thermal source for inflicting bum injuries in clipped and depilated mice was a cylindrical aluminum templates (Diameter, 3.76 cm; length of handle, 24 cm; total weight, 500 grams, 23). The templates were heated in a water bath for 2 hours prior to the injury at a constant temperature of 75 °C. Six templates were heated concurrently and were used alternatively, one for each injury, and then were returned to the heated water to ensure maintenance of the desired temperature of their surface. At least 5 minutes elapsed between reuse of any of the templates. In order to reduce significantly the biological variables among the various treated groups of bums in the experiment, the animals were anaesthetized again on the following day. Prior to inflicting the bum wound, animals were restrained and stretched flat on a wire mesh and the midline corresponding to the spine was marked. At the level of the mid distance between the twelfth rib and the horizontal line between both sacroilliac joints on the right hand side was determined as the exact location of the injury for all animals. The heated and moistened template was applied at right angle to the skin of the animal's back according to the pre-marked location, while monitoring aloud exactly 5 seconds using an analog stopwatch (Fisher

Houer, Switzerland). Only minimal pressure was required in order to ensure a perfect contact between the template surface and the underlying dorsal skin.

Animals were divided into three groups (a control group and 2 experimental groups).

Experimental animals were treated topically with measured volumes (40 μl) of IL-8 (25 ng/μl in saline), or with 20 μl of TGF-βl (10 ng/μl, dissolved in 4 mM HC1 containing 0.1 % BSA) + 20 μl bFGF (25 ng/μl in saline), all from Sigma, St. Louis, MO, USA. Control groups were treated similarly using 40 μl of saline.

Following application of cytokine, growth factors or saline, wounds were covered by a non-adherent absorbent dressing (Melolin, Smith and

Nephew Ltd., Hull, UK) and bandaged using elastic adhesive bandage

(Salvaplast Ind. Ltd., Petach-Tiqua, Israel). Treatment was repeated every 2 days for up to 13 days. On days 0, 2, 9 and 13 the diameter of each bum injury was recorded using overhead viewgraph clear transparencies, one for each animal. Bum injuries were also photographed under fixed lens distance conditions. Upon termination of the experiment, animals were anaesthetized as described above and tissue biopsies for light microscopy were obtained from a predetermined area. Tissue samples designated for light microscopy were fixed in 4 % phosphate neutral buffered formalin, pH 7.4, 48 hours, dehydrated in graded alcohols and embedded in paraplast.

Six μm-thick sections were stained with hematoxylin and eosin for general morphology and photographed using Olympus CH-2 microscope.

The recorded values of bu injury in each time interval were used to

2 calculate the actual area (cm ) of "Open" and "Total" areas of each bum injury on days 0, 2, 9 and 13, using a morphomat system (Moφhomat 30 and Epson printer, C. Zeiss, Oberkochen, Germany). Also the values of "Total" area in each interval, the values of "Open" area on day 13, the "Percent of open area" from total area on day 0, the "Percent of contraction" (total area on day 0 minus total area on day 13), values of "Epithelization" (total area on day 13 minus open area on day 13), "Epithelization + Contraction" and % "Repair", were calculated statistically using the Instat2 software. Student's t-test and analysis of variance (ANOVA) were performed and p<0.05 was accepted as significance level. EXPERIMENTAL RESULTS

The "Total" area of all injuries appeared to decrease gradually from day 0 and up to day 13, as shown for the control group (Figure 1), in bums treated with TGF-β+ bFGF (Figure 2), and in bums treated with IL-8 (pθ.001, Figure 3). This reduction in "Total" area represented a

"Contraction" process calculated as "Percent" from the area on day 0 (Figure 4). It appeared that "Contraction" values in controls were higher than the values following treatment with IL-8 as well as following treatment with TGF-β + FGF.

Additionally, the percent values of "Open" area of wounds on day 13 were reduced significantly (p<0.05) in both IL-8 and TGF-βl + bFGF treated injuries (Figure 5) as is compared to controls.

"Percent" values of "Closed" ("Epithelization") area appeared to be similar in both treated groups as is compared to control (Figure 6). The values of "Contraction + Epithelization" appeared to be higher in IL-8 as well as in TGF-βl + bFGF treated groups as is compare to control (Figure 7). Percent "Repair" was higher in the IL-8 treated group then in the TGF-β 1 + bFGF or control groups (Figure 8). The general appearance of bum injury revealed that control injuries

(Figure 9) were open and bleedy and macroscopically appeared to be less epithelized as is compared to animals treated with TGF-βl + bFGF (Figure 10). However, in animals treated with IL-8 the wound appeared to be smooth and closed (Figure 11). Tissue moφhology revealed that on day 13 a very thin layer of epidermis was observed in control group (Figure 12). Similar result was obtained in TGF-βl + bFGF treated animals (Figure 13). In animals treated with IL-8, a thick layer of epidermis as well as well developed skin appendages were observed (Figure 14).

Although the invention has been described in conjunction with specific embodiments thereof, 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 broad scope of the appended claims. REFERENCES:

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2. D.G. Greenhanlgh. 1996. The role of growth factors in wound healing. J. Trauma. 41: 159-167.

3. Clark R.A. 1993. Basics of cutaneous wound repair. J. Dermatol. Surg. Oncol. 19:693-706.

4. Clark-Lewis et al. 1991. Stmcture-activity relationships of IL-8. J.Biol.Chem. 266:28-134.

5. M. Bagglioni Dewald B. and Moser B. 1994. Interleukin-S and related chemotactic cytokines-CXC and CC chemokines. Adv. Immun. 55: 97- 179.

6. Baggiolini M. Imbiden P. and Demers P. 1992. Neutrophilic activities and the effect of IL-8. In: Cytokines. IL-8 and related chemotactic cytokines. Eds. Baggiolini M. and Sorg C, Karger-Basel. Vol 4, pp. 1- 17.

7. G.M. Clark. 1992. NMR and x-ray analysis of the three dimensional structure of IL-8. In: Cytokines. interleukin-8 (NAP-1). Eds. Baggiolini and Sorg. C. Karger-Basel, Vol 4, pp.18-40.

8. Moser B. Schumacher C. von Tscahamer V. Clark-Lewis I and Baggiolini M. 1993. Neutrophil- activating peptides 2 and Gro. melanoma growth- stimulatory activity interact with IL-8 receptors in human neutrophils. J.Biol. Chem. 266: 10666-10671.

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10. Toschil A. et al. 1992. IL-8 stimulates calcium transients and promotes epidermal cell proliferation. J. Invest. Dermatol. 99: 294-298.

11. Kemey L. et al. 1995. IL-8 induces HLA-Dr expression on cellular human keratinocytes via specific receptor. Int. Arch. Allergy Immunol. 106:351-356.

12. Konstantinova V. Douer N. 1996. IL-8 is induced in skin equivalents and is highest in those derived from psoriatic fibroblast. J. Invest. Dermatol. 107:615-621.

13. Assoian R.K., Grontendorst G.R. Miller D.N. Spom M.B. 1984. Cellular transformation by coordinated action of three growth factors from human platelates. Nature 309:804-806. 14. Mustoe T.A. Pierce G.F. Thomson et al. 1987. Accelerated healing of incisional wound in rats induced by transforming growth factor-b. Science 347:1333-1336.

15. Cromack D.T. Poras-Reyes B. Pordy J. et al. 1993. Acceleration of tissue repair by transforming growth factor-b 1 : Identification of in-vivo mecahnism of action with radiotherapy-induced specific healing deficits. Surgery 113:36-42.

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17. Maciaf T. Zahn X. Garfinkel S et al. 1994. Novel mecahnism of fibroblast growth factor function. Rec. Prog. Horm. Res. 49:105-123.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating a wound comprising the step of topically applying a chemokine to the wound.

2. The method of claim 1, wherein said chemokine is capable of binding an interleukin-8 receptor.

3. The method of claim 2, wherein said interleukin-8 receptor is selected from the group consisting of interleukin-8 receptor 1 and interleukin-8 receptor 2.

4. The method of claim 1, wherein said chemokine is a CXC chemokine.

5. The method of claim 1, wherein said chemokine is interleukin-8.

6. The method of claim 1, wherein said chemokine is an interleukin-8 analog.

7. The method of claim 6, wherein said interleukin-8 analog is selected from the group consisting of neutrophil activating protein 2 (NAP- 2), GRO α, GRO β, GRO γ and epithelial cell-derived neutrophil activating protein (ENA78).

8. The method of claim 1 , wherein said chemokine is native.

9. The method of claim 1, wherein said chemokine is a product of genetic engineering.

10. The method of claim 1, wherein said chemokine induces in- vivo interleukin-8 secretion.

11. The method of claim 10, wherein said chemokine is selected from the group consisting of interleukin-1 and TNF-α.

12. The method of claim 1, wherein said chemokine is applied as a pharmaceutical composition including an effective concentration of said chemokine and a pharmaceutically acceptable carrier.

13. The method of claim 12, wherein said pharmaceutically acceptable carrier is a lotion, an ointment, a gel, a cream, a liquid, a spray and a powder.

14. The method of claim 1 , wherein said wound is selected from the group consisting of bum wound, chronic wound, infection wound, pressure wound and cut wound.

15. The method of claim 1, further comprising the step of bandaging the wound.

16. A pharmaceutical composition for topically treating a wound comprising an effective concentration of a chemokine and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition for claim 16, wherein said chemokine is capable of binding an interleukin-8 receptor.

18. The pharmaceutical composition for claim 17, wherein said interleukin-8 receptor is selected from the group consisting of interleukin-8 receptor 1 and interleukin-8 receptor 2.

19. The pharmaceutical composition for claim 16, wherein said chemokine is a CXC chemokine.

20. The pharmaceutical composition for claim 16, wherein said chemokine is interleukin-8.

21. The pharmaceutical composition for claim 16, wherein said chemokine is an interleukin-8 analog.

22. The pharmaceutical composition for claim 21, wherein said interleukin-8 analog is selected from the group consisting of neutrophil activating protein 2 (NAP-2), GRO α, GRO β, GRO γ and epithelial cell- derived neutrophil activating protein (ENA78).

23. The pharmaceutical composition for claim 16, wherein said chemokine is native.

24. The pharmaceutical composition for claim 16, wherein said chemokine is a product of genetic engineering.

25. The pharmaceutical composition for claim 16, wherein said chemokine induces in-vivo interleukin-8 secretion.

26. The pharmaceutical composition for claim 25, wherein said chemokine is selected from the group consisting of interleukin- 1 and TNF-α

27. The pharmaceutical composition for claim 16, wherein said pharmaceutically acceptable carrier is a lotion, an ointment, a gel, a cream, a liquid, a spray and a powder.