Methods and pharmaceutical compositions for healing wounds

This is a continuation of U.S. patent application Ser. No. 10/644,775 filed Aug. 21, 2003, pending, which is a continuation-in-part of U.S. patent application Ser. No. 10/169,801, filed Jul. 9, 2002, which is a National Phase of PCT/IL01/00675, filed Jul. 23, 2001, which claims priority of U.S. patent application Ser. No. 09/629,970, filed Jul. 31, 2000, now abandoned. This application also claims the benefit of priority of U.S. provisional patent application No. 60/486,906, filed Jul. 15, 2003.

The present invention relates to a method and a pharmaceutical composition for inducing and/or accelerating cell proliferation, and/or cell differentiation and thereby accelerating the healing process of wounds. More particularly, the present invention relates to the use of modulated expression and/or activation, e.g., as initiated by membrane translocation, of serine/threonine protein kinases, also known as PKCs, for inducing and/or accelerating cell proliferation and/or cell differentiation and thereby accelerating the healing process of wounds. Such modulated expression may be effected in accordance with the teachings of the present invention by (i) transformation of wound cells with a PKC expressing construct; (ii) transformation of wound cells with a cis-acting element to be inserted adjacent to, and upstream of, an endogenous PKC gene of the wound cells; (iii) administration of insulin for inducing expression and/or activation of PKC in wound cells; (iv) transformation of wound cells with an insulin expressing construct, when expressed and secreted the insulin produced therefrom serves as an up-regulator for PKC expression and/or activation; (v) transformation of wound cells with a cis-acting element to be inserted adjacent to, and upstream of, the endogenous insulin gene of the wound cells, when expressed and secreted the insulin serves as an up-regulator for PKC expression and/or activation; (vi) implantation of insulin secreting cells to the wound; (vii) transformation of wound cells with a trans-acting factor, e.g., PDX1, for induction of endogenous insulin production and secretion, the insulin serves as an up-regulator for PKC expression and/or activation; and (viii) administration to the wound of a PKC modulator.

The present invention, as is realized by any of the above methods, can also be practiced ex-vivo for generation of skin grafts.

The primary goal in the treatment of wounds is to achieve wound closure. Open cutaneous wounds represent one major category of wounds and include burn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers, and diabetic ulcers.

Open cutaneous wounds routinely heal by a process which comprises six major components: (i) inflammation; (ii) fibroblast proliferation; (iii) blood vessel proliferation; (iv) connective tissue synthesis; (v) epithelialization; and (vi) wound contraction. Wound healing is impaired when these components, either individually or as a whole, do not function properly. Numerous factors can affect wound healing, including malnutrition, infection, pharmacological agents (e.g., actinomycin and steroids), advanced age and diabetes [see Hunt and Goodson in Current Surgical Diagnosis & Treatment (Way; Appleton & Lange, pp. 86-98 (1988)].

With respect to diabetes, diabetes mellitus is characterized by impaired insulin signaling, elevated plasma glucose and a predisposition to develop chronic complications involving several distinctive tissues. Among all the chronic complications of diabetes mellitus, impaired wound healing leading to foot ulceration is among the least well studied. Yet skin ulceration in diabetic patients takes a staggering personal and financial cost (29, 30). Moreover, foot ulcers and the subsequent amputation of a lower extremity are the most common causes of hospitalization among diabetic patients (30-33). In diabetes, the wound healing process is impaired and healed wounds are characterized by diminished wound strength. The defect in tissue repair has been related to several factors including neuropathy, vascular disease and infection. However, other mechanisms whereby the diabetic state associated with abnormal insulin signaling impairs wound healing and alter the physiology of skin has not been elucidated.

There is also a common problem of wound healing following surgical procedures in various parts of the body, the surgery succeeds but the opening wound does not heal.

Skin is a stratified squamous epithelium in which cells undergoing growth and differentiation are strictly compartmentalized. In the physiologic state, proliferation is confined to the basal cells that adhere to the basement membrane. Differentiation is a spatial process where basal cells lose their adhesion to the basement membrane, cease DNA synthesis and undergo a series of morphological and biochemical changes. The ultimate maturation step is the production of the cornified layer forming the protective barrier of the skin (1, 2). The earliest changes observed when basal cells commit to differentiate is associated with the ability of the basal cells to detach and migrate away from the basement membrane (3). Similar changes are associated with the wound healing process where cells both migrate into the wound area and proliferative capacity is enhanced. These processes are mandatory for the restructuring of the skin layers and induction of proper differentiation of the epidermal layers.

The analysis of mechanisms regulating growth and differentiation of epidermal cells has been greatly facilitated by the development of culture systems for mouse and human keratinocytes (2,4). In vitro, keratinocytes can be maintained as basal proliferating cells with a high growth rate. Furthermore, differentiation can be induced in vitro following the maturation pattern in the epidermis in vivo. The early events include loss of hemidesmosome components (3,5) and a selective loss of the α6β4 integrin and cell attachment to matrix proteins. This suggests that changes in integrin expression are early events in keratinocyte differentiation. The early loss of hemidesmosomal contact leads to suprabasal migration of keratinocytes and is linked to induction of Keratin 1 (K1) in cultured keratinocytes and in skin (1, 3, 6). Further differentiation to the granular layer phenotype is associated with down regulation of both β1 and β4 integrin expression, loss of adhesion potential to all matrix proteins and is followed by cornified envelope formation and cell death. Differentiating cells ultimately sloughs from the culture dish as mature squames (2, 7). This program of differentiation in vitro closely follows the maturation pattern of epidermis in vivo.

Recent studies in keratinocytes biology highlights the contribution of Protein Kinase C pathways, which regulate skin proliferation and differentiation. The protein kinase C (PKC) family of serine-threonine kinases plays an important regulatory role in a variety of biological phenomena (8,9). The PKC family is composed of at least 12 individual isoforms which belong to 3 distinct categories: (i) conventional isoforms (α, β1, β2, γ) activated by Ca²⁺, phorbol esters and diacylglycerol liberated intracellularly by phospholipase C; (ii) novel isofomis (δ, ε, η, θ) which are also activated by phorbol esters and diacylglycerol but not by Ca²⁺; and (iii) atypical (ζ, λ, τ) members of the family, which are not activated by Ca²⁺, phorbol esters or diacylglycerol.

On activation, most but not all isoforms are thought to be translocated to the plasma membrane from the cytoplasm. The type of isoform and pattern of distribution vary among different tissues and may also change as a function of phenotype. Numerous studies have characterized the structure and function of PKC because of its importance in a wide variety of cellular endpoints of hormone action. Five PKC isoforms—α, δ, ε, η and ζ—have been identified in skin in vivo and in culture. Recent studies have shown that the PKC signal transduction pathway is a major intracellular mediator of the differentiation response (10,11). Furthermore, pharmacological activators of PKC are powerful inducers of keratinocyte differentiation in vivo and in vitro (4, 12), and PKC inhibitors prevent expression of differentiation markers (10).

While conceiving the present invention, it was hypothesized that PKC isoforms over-expression and/or activation may be beneficial for accelerating wound healing processes. The limitations for investigating the role of distinct PKC isoforms in skin cells proliferation and/or differentiation has been hampered as result of the difficulty in introducing foreign genes efficiently into primary cells, by conventional methods. The short life span, differentiation potential and the inability to isolate stable transformants do not allow efficient transduction of foreign genes into primary skin cells.

Prior art describes the potential use of insulin as a therapeutic agent for healing wounds. Thus, U.S. Pat. Nos. 5,591,709, 5,461,030 and 5,145,679 describe the topical application of insulin to a wound to promote wound healing. However, these patents describe the use of insulin in combination with glucose since the function of the insulin is to enhance glucose uptake and to thus promote wound healing.

U.S. patent application Ser. No. 09/748,466 and International Patent Application No. PCT/US98/21794 describe compositions containing insulin for topical application to skin for the purpose of improving skin health or treating shallow skin injuries. However, none of these patent applications teaches the use of insulin for treating chronic, Grade II or deep wounds.

International Patent Application No. PCT/US01/10245 describes the use of cyanoacrylate polymer sealant in combination with insulin or silver for wound healing. However, the use of insulin in combination with another biologically active agent capable of modulating the expression and/or activation of PKC is not taught nor suggested in this application.

International Patent Application No. PCT/US85/00695 describes topical application of insulin for treating diabetes. However, this patent application fails to teach the use of insulin for the purpose of treating diabetes non-related wounds.

International Patent Application No. PCT/US92/03086 describes therapeutic microemulsion formulations which may contain insulin. However the use of the formulated insulin for the purpose of wound healing is not taught in this disclosure.

U.S. Pat. Nos. 4,673,649 and 4,940,660 describe compositions for clonal growth of human keratinocytes and epidermal cells in vitro which include epidermal growth factor and insulin. Both of these patents teach the use of insulin for the development of cultured skin cells which may be used for grafting. However, the application of insulin on wounds in vivo is not taught by these patents.

None of the above cited prior art references teach or suggest the use insulin for modulating the expression and/or activation of PKC, so as to accelerate the healing process of wounds. Furthermore, the prior art fails to teach or suggest utilizing nucleic acid constructs or genetic transformation techniques for providing insulin to wounds, so as to accelerate the healing process of the wounds.

There is a widely recognized need for, and it would be highly advantageous to have, new approaches for accelerating the processes associated with wound healing. In addition, there is a widely recognized need for, and it would be highly advantageous to have, an efficient method to insert recombinant genes into skin cells which will accelerate cell proliferation and/or differentiation processes and wound healing.