The present invention provides methods and pharmaceutical compositions for use in topical delivery of genetic material and/or proteins. In addition, the present invention provides methods and compositions for enhancing and/or controlling chronic wounds by applying a wound care device comprising a cationic amphiphile, cholesterol and a genetic material, the said amphiphile with remarkable gene transfer properties. The area of medical science that is likely to benefit most from the present invention is non-viral gene therapy of chronic wounds.

Background and Prior Art Information:

Wound healing is a complex and highly integrated cascade of events involving communications and interactions between fibroblasts, endothelial cells, keratinocytes, inflammatory cells and extracellular cell matrix (Blakytny et al. Diabetic Medicine 2006;23:594-608). Among the many factors contributing to the pathogenesis of chronic or impaired wound healing (e.g. chronic non-healing diabetic wounds), deficiency of growth factors are of paramount importance. This is because wound proteases destroy the peptide growth factors at a fast rate. For instance, not only the non-healing chronic dermal ulcers have been shown to be deficient in platelet derived growth factor-B (PDGF-B), but elevated levels of PDGF-B have been detected in the serum of patients capable of healing their ulcers (Chleboun et al. Cardiovasc. Surg 1995;3:285-290, Pierce et al. J. Clin. Invest. 1995;96:1336-1350). Because of its central role in all phases of wound healing, clinical interest has been witnessed in the past for exogenous applications of PDGF-B in chronic non-healing wound. However, favorable effects in such topical applications have demanded uses of large and repeated doses of PDGF-B proteins for even modest clinical effect (Mustoe et al. J. Clin. Invest. 1991;87:694-703, Mustoe et al. Arch. Surg. 1994;129:213-219, Pierce et al. Am. J. Pathol. 1991;138:629-646, Wieman et al. Diabetes Care. 1998; 21:822-827). Clearly, there is a need for development of a more effective means of delivering PDGF-B to body cells participating in the wound healing process. Problems of frequent administrations and unstable protein formulations are being increasingly circumvented by the elegant approach of gene therapy. Gene transfer as a method for treating chronic wounds promises intracellular introduction of therapeutic genes into the wounded tissues thereby inducing the regional cells to produce and excrete therapeutic protein, such as PDGF-B. In other words, gene therapeutic approach for treating chronic wounds is, in principle, capable of solving the problems associated with using unstable protein formulations and frequent administrations.

One of the key challenges in ensuring clinical success of gene therapy is designing safe & efficacious carriers of the therapeutic genes of interest. Contemporary transfection vectors can be broadly classified into two major types: viral and non-viral. Viruses, nature’s own infecting vehicles, have evolved exquisite mechanisms through the course of evolution to deliver their genetic material into host cells. Owing to such natural ability to infect cells, viruses have been used as vectors in gene therapy by replacing the genes essential for the replication phase of their life cycles with the therapeutic genes of interest. For instance, it has been demonstrated that adenoviral-mediated over expression of PDGF-B is capable of causing enhanced neovascularization and wound healing in diabetic mice through endothelial precursor cells (EPC) recruitment to the wound bed (Keswani, S. G. et al. Wound Rep. Reg. 2004;12:497-504, Sylvester K. G. et al. Surg Forum. 1998;49:653-654, Kitano, Y. et al. Surg Forum. 1998;49:651-653, Lim F. Y. et al. Wound Rep. Reg. 2002;10:A34). However, viral vectors are potentially capable of: generating replication competent virus through various recombination events with the host genome; inducing inflammatory and adverse immunogenic responses and producing insertional mutagenesis through random integration into the host genome. All these alarming concerns associated with the use of viral vectors call for reassessing the use of viral vectors with regard to their safety in human gene therapy. Contrastingly, non-viral transfection vectors, mainly cationic polymers and cationic liposomes, possess many important safety advantages. Cationic liposomes in particular, are significantly less immunogenic. Manufacturing viral vectors in large scale are technically demanding with prohibitively high preparation cost while robust manufacture of cationic transfection lipids (used in preparing cationic liposomes) are technically quite feasible. Unlike viral vectors, they have no restrictions on the size of DNA to be delivered; cationic liposomes can deliver nucleic acids of essentially unlimited size ranging up to large mammalian artificial chromosomes. A greater degree of control can be exercised over the lipids’structure on a molecular level and the products can be highly purified. Use of cationic liposomes does not require any special expertise in handling and preparation techniques. Cationic liposomes can be covalently grafted with receptor specific ligands for accomplishing targeted gene delivery. Such multitude of distinguished favorable clinical features are increasingly making cationic liposomes as the non-viral transfection vectors of choice for delivering genes into body cells.

The following references are examples of cationic liposomes and their formulations that are known in the art to be useful for enhancing the intracellular delivery of genetic materials.
Felgner et al., Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 7413-7417 reported the first use of a highly efficient cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride(DOTMA) as the non-viral DNA transfer reagent.
U.S. Pat.Nos. 4,897,355 and 4,946,787 (1990) reported the synthesis and use of N-[.omega..(.omega.-1)-dialkyloxy]-and N-[..omega..(.omega.-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium amphiphiles and their pharmaceutical formulations as efficient transfection vectors.
Leventis, R.and Silvius, J.R Biochim. Biophys. Acta. 1990; 1023: 124-132 reported the interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles.
U.S. Pat. No. 5,264,618 (1993) reported the synthesis and use of additional series of highly efficient cationic lipids for intracellular delivery of biologically active molecules.
Felgner et al. J. Biol. Chem. 1994; 269: 2550-2561 reported enhanced gene delivery and mechanistic studies with a novel series of cationic lipid formulations.
U.S. Pat. No. 5,283,185 (1994) reported the synthesis and use of 3?[N-(N1,N1-dimethylaminoethane)carbamoyl]cholesterol, termed as "DC-Chol"for delivery of a plasmid carrying a gene for chloramphenicol acetyl transferase into cultured mammalian cells.
U.S. Pat. No. 5,283,185 (1994) reported the use of N-[2-[[2,5-bis[(3-aminopropyl)amino]-1-Oxopentyl]aminoethyl]-N,N-dimethyl-2,3-bis-(9-octadecenyloxy)-1-Propanaminium tetra(trifluoroacetate), one of the most widely used cationic lipids in gene delivery. The pharmaceutical formulation containing this cationic lipid is sold commercially under the trade name "Lipofectamine".
Solodin et al. Biochemistry 1995; 34: 13537-13544 reported a novel series of amphilic imidazolinium compounds for in vitro and in vivo gene delivery.
Wheeler et al. Proc. Natl. Acad.Sci. U.S.A. 1996; 93: 11454-11459 reported a novel cationic lipid that greatly enhances plasmid DNA delivery and expression in mouse lung.
U.S.Pat No. 5,527,928 (1996) reported the synthesis and the use of N,N,N,N-tetramethyl-N,N-bis (hydroxy ethyl)-2,3-di(oleolyoxy)-1,4-butanediammonim iodide i.e pharmaceutical formulation as transfection vector.
U.S.Pat.No. 5.698,721 (1997) reported the synthesis and use of alkyl O-phosphate esters of diacylphosphate compounds such as phosphatidylcholine or posphatidylethanolamine for intracellular delivery of macromolecules.
U.S.Pat. Nos. 5,661,018; 5,686,620and 5,688,958 (1997) disclosed a novel class of cationic phospholipids containing phosphotriester derivatives of phosphoglycerides and sphingolipids efficient in the lipofection of nucleic acids.
U.S. Pat.No. 5,614,503 (1997) reported the synthesis and use of an amphiphatic transporter for delivery of nucleic acid into cells, comprising an essentially nontoxic, biodegradable cationic compound having a cationic polyamine head group capable of binding a nucleic acid and a cholesterol lipid tail capable of associating with a cellular membrane.
U.S.Pat.No. 5,705,693 (1998) disclosed the method of preparation and use of new cationic lipids and intermediates in their synthesis that are useful for transfecting nucleic acids or peptides into prokaryotic or eukaryotic cells. These lipids comprise one or two substituted arginine, lysine or ornithine residues, or derivatives thereof, linked to a lipophilic moiety.
U.S.Pat. No.5, 719,131 (1998) has reported the synthesis of a series of novel cationic amphiphiles that facilitate transport of genes into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, alkylamines or polyalkylamines.
US. Patent No. 5,527,928, (1996) reported on the synthesis and transfection biology of a novel cationic lipid namely, N, N, N’, N’-tetramethyl-N, N’-bis (2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butaneammonium iodide.
US Patent 6,541,649 (2003) disclosed novel cationic amphiphiles containing N-hydroxyalkyl head-group and its formulation for intracellular delivery of genetic materials.
US Patent 6, 503, 945 (2003) disclosed novel cationic amphiphiles containing N-hydroxyalkyl head-group and its formulation for intracellular delivery of genetic materials.
US Patent 7,157,439 (2007) disclosed methods and compositions for improving and/or controlling wound healing by applying a wound care device comprising HoxD3 and HoxA3 and/or HoxB3 novel cationic amphiphiles containing N-hydroxyalkyl head-group and its formulation for intracellular delivery of genetic materials.
OTHER PUBLICATIONS
Behr, J.P. et al. Proc. Natl. Acad. Sci. USA, 1989; 86: 124-132.
Levantis, R. et al. Biochim. Biophys. Res. Commun. 1991; 179: 280-285.
Akao, T. et al. Biochem. Mol. Biol. Int. 1994; 34: 915-920.
Felgner, J. H. et al. Proc. Natl. Acad. Sci. USA. 1996; 93: 11454-11459.
Bennett, M. J. et al. J. Med. Chem. 1997; 40: 4069-4078.
Blessing, T. et al. J. Am. Chem. Soc. 1998; 120: 8519-8520.
Wang, J. et al. J. Med. Chem. 1998; 41: 2207- 2215.
Lim, Y. et al. J. Am. Chem. Soc. 1999; 121: 5633-5639.
Lim, Y. et al. J. J. Am. Chem. Soc. 2000; 122: 6524-6525.
Zhu, J. et al. J. Am. Chem. Soc. 2000; 122: 3252-3253.
Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000; 122: 10761-10768.
Ferrari, M. E.; Rusalov, D.; Enas, J.; Wheeler, C. J.; Nuc. Acid. Res. 2001, 29, 1539-1548.
Banerjee, R.; Das, P. K.; Srilakshmi, G.V.; Chaudhuri, A.; Rao, N. M. J. Med. Chem.1999, 42, 4292-4299.
Banerjee, R.; Mahidhar, Y. V.; Chaudhuri, A.; Gopal, V.; Rao, N. M.
J. Med. Chem. 2001, 44, 4176-4185.
Singh, S. R.; Mukherjee, K.; Banerjee, R.; Chaudhuri, A.; Hait, S. K.; Moulik, S. P.; Ramadas, Y.; Vijayalakshmi, A.; Rao, N. M. Chem. Eur. J. (in press).
Floch, V.; Bolc''h, G. Le.; Gable-Guillaume, C.; Bris, N. Le.; Yaouanc, J-J.; Abbayes, H. Des.; Fe''rec, C.; Cle''ment, J-C. Eur. J. Med. Chem., 1998, 33, 923-934.
Solodin, I.; Brown, C.; Bruno, M.; Chow, C.; Jang, E-H.; Debs, R.; Heath, T. Biochemistry, 1995, 34, 13537-13544.
Karmali, P. P.; Majeti, B. K.; Bojja S.; and Chaudhuri, A. Bioconjugate Chemistry 2006, 17, 159-171.
Bharat M. K.; Karmali, P. P.; Reddy, B. S.; Chaudhuri, A. J. Med. Chem. 2005, 48, 3784-3795.
Sen, J. and Chaudhuri, A. J. Med. Chem. 2005, 48, 812-820.
Sen, J. and Chaudhuri, A. Bioconjugate Chemistry 2005, 16, 903-912.
Bharat M. K.; Karmali, P. P.; Chaudhuri, A. Bioconjugate Chemistry 2005, 16, 676-684.
Mukherjee, K. M., Sen, J. and Chaudhuri, A. FEBS Letters 2005, 579, 1291-1300.
Mahidhar, Y. V., Rajesh, M.; Madhavendra, S. S.; Chaudhuri, A. J. Med. Chem. 2004, 47, 5721-5728.
Mahidhar, Y. V., Rajesh, M.; Chaudhuri, A. J. Med. Chem. 2004, 47, 3938-3948.
Valluripalli, V. K. and Chaudhuri, A. FEBS Letters 2004, 571, 205-211.
Singh, R. S.; Gonçalves, C.; Sandrin, P.; Pichon, C.; Midoux, P.; Chaudhuri, A. Chemistry and Biology 2004, 11, 713-723.
Majeti, B. K.; Singh, R. S.; Yadav, S. K.; Reddy, S. B.; Ramkrishna, S.; Diwan, P. V.; Madhavendra, S. S.; Chaudhuri, A. Chemistry and Biology 2004, 11, 427-437.
Karmali, P. P.; Valluripalli, V. K.; Chaudhuri, A. J. Med. Chem. 2004, 47, 2123-2132.
Singh, R. S. and Chaudhuri, A. FEBS Letters 2004, 556, 86-90.
Kumar, V. V.; Pichon, C.; Refregiers, M.; Guerin, B.; Midoux, P.; Chaudhuri, A. Gene Therapy 2003, 10, 1206-1215.
Valluripalli, V. K.; Singh, R. S ; Chaudhuri, A. Curr. Med. Chem. 2003, 10, 1297-1306.

OBJECT OF INVENTION:

The object of the present invention is to provide pharmaceutical compositions and methods for improved healing of chronic wounds.

SUMMARY OF THE INVENTION:
The present invention provides pharmaceutical compositions and methods for localized (topical) delivery of genetic materials. In addition, the present invention provides methods and compositions for improving and/or controlling wound healing by topical application of formulations comprising RGD-lipopeptide A, cholesterol and a recombinant plasmid DNA encoding at least one of the therapeutic growth factors of interest, such as protein PDGF-B. Moreover, the present inventions provide methods and pharmaceutical formulations for improved wound healing in subjects with impaired wound healing efficiencies, such as diabetic rats.
Accordingly the present invention provides methods of treating a wound comprising the step of applying a wound care formulations containing an integrin receptor specific cationic RGD-lipopeptide having the structure A,

cholesterol and a therapeutic gene such as a plasmid DNA encoding the growth factor PDGF-B.

In still another embodiment of the invention, the wound having impaired healing capabilities is a diabetic wound. In yet another embodiment of the invention, the pharmaceutical composition comprising the RGD-lipopeptide, cholesterol and the genetic material is applied to a localized area of the subject having impaired wound healing capabilities. In particularly preferred embodiments, the topical applications of the wound healing formulations are carried out under conditions such that wound healing is accelerated, under conditions such that the wound closure is accelerated or under conditions such that expression of the growth factor PDGF-BB in the wound bed is enhanced.

Moreover, the present invention provides methods comprising: creating wounds in a subject; applying the pharmaceutical composition of the RGD-lipopeptide, cholesterol and the therapeutic gene of interest, such as the genetic material encoding a growth factor, to the wound bed of the subject.
In an embodiment of the invention, the wound has impaired healing capabilities, and in a subset of these, the wound having delayed healing characteristics is a diabetic wound.

In another embodiment of the invention, the formulation where the RGD lipopeptides may be used in pure form or in combination with helper lipids. In still another embodiment of the invention, the formulation where the helper lipid may be selected from the group of phosphatidylethanolamine, phosphatidylglycerol, cholesterol. In an embodiment of the invention, the formulation where the colipid can be selected from sterol group or a neutral phosphatidyl ethanolamine or a neutral phosphatidyl choline. In another embodiment of the invention, the formulation, where the colipid is preferentially selected from DOPE or cholesterol
In still another embodiment of the invention, the formulation, where the range of molar ratio of RGD lipopeptide to colipid is 3:1-1:1. In yet another embodiment of the invention, the formulation, where in the preferred molar ratio of RGD lipopeptide to colipid is 2:1. In an embodiment of the invention, the formulation where the genetic material can be selected from a group of nucleic acid that encodes for a therapeutically important growth factor, such as plate derived grow factor (PDGF-B). In yet another embodiment of the invention, the formulation where the said formulation is administered intravenously, intramuscular, intraperitonial mode. In another embodiment of the invention, the formulation where the said formulation is administered to cells at a ratio 0.1 to 0.5 microgram of DNA to 50,000 cells. In still another embodiment of the invention, the formulation where the said formulation comprises amount of amphiphile in the range of 9.0 to 0.3 microgram from a lipopeptide to DNA charge ratio ranging from 0.3:1 to 9:1

Brief description of drawings:

Figure 1 is a schematic representation of the synthetic scheme following in preparing the RGD-lipopeptide.
Figure 2 depicts In vitro transfection efficiencies of RGD-lipopeptide in Balb c mouse fibroblast cells (3T3) using cholesterol as colipid (at lipid:cholesterol mole ratio of 1:1) in absence and in presence of integrin antagonist cyclic RGDfV. Units of ?-galactosidase activity were plotted against the varying lipid to DNA (+/) charge ratios. The o-nitrophenol formation (micromoles of o-nitrophenol produced per 5 min) was converted to ?-galactosidase activity units using the standard curve obtained with pure commercial ?-galactosidase.

Figure 3 shows the relative gene transfection efficacies of RGD-lipopeptide and RGE-lipopeptide in mouse fibroblast cells.

Figure 4 is a representative Western Blot for Expression of PDGF-BB protein in mouse fibroblast (3T3) cells. Lane 1; Pure PDGF-BB protein; Lane 2; Lysate of the cells transfected with RGD lipopeptide and PDGF-B plasmid DNA; Lane 3 : Lysate of the cells transfected with RGD lipopeptide and ß-gal plasmid DNA; Lane 4: Lysate of the untransfected cells.

Figure 5 depicts the relative wound healing efficiency profiles in diabetic S/D rats injected with: RGD-lipopeptide:rhPDGF-B complex (¦); naked rhPDGF-B (?)and 5% aq. Glucose (?)in diabetic S/D rats.

Figure 6 shows representative photographs of the wounds of diabetic S/D rats at the time of wounding and on 5th day after treatment. Wound sizes for diabetic S/D rats at the time of wounding (A-C); wound sizes on the 5th day after treatment for diabetic S/D rats treated with: only with: 5% aq glucose (D); RGD-lipopeptide:rhPDGF B complex (E) and naked rhPDGF B (F).

Figure 7. Recombinant PDGF (vb5 PC DNA3.1 ) vector map.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides pharmaceutical compositions and methods for localized (topical) delivery of genetic material. In addition, the present invention provides methods and compositions for improving wound healing by topical application of a pharmaceutical composition comprising a RGD-lipopeptide, a co-lipid (e.g. cholesterol) and a genetic material encoding growth factor. Moreover, the present invention provides methods and compositions for improved wound healing in subjects with impaired wound healing characteristics, such as diabetic rats.

I. Overview of Wound Healing

Wound healing process comprises of four distinct but overlapping phases: hemostasis, inflammation, proliferation and remodeling (Diegelmann, R. F. and Evans, M. C. Frontiers in Bioscience, 2004;9:283-289). Hemostasis begins immediately after the tissue is injured. As the blood components spill into the site of injury, the blood platelets get exposed to collagen and other extracellular matrix components of the injured area. This contact triggers the platelets to release various clotting factors, plate-derived growth factors (PDGF) and transforming growth factor beta (TGF-ß). Following hemostasis, in the inflammation phase, body’s inflammatory cells e.g. neutrophils first migrate to the wound sites followed by migration of the various other immune cells of our body including lymphocytes, monocytes and macrophages to the wound site. Stimulated neutrophils release proteases and reactive oxygen species into the surrounding medium of the wound bed to protect possible assaults from various invading pathogenic microorganisms. Microphages take part in this inflammation stage to expedite the phagocytic removal of foreign materials & damaged tissues as well as to release more PDGF and TGF-ß. Once the wound bed is cleaned, proliferation stage begins in which fibroblasts migrate into the wound area and deposit new extracellular matrix. Formation of new granulation tissues and new blood vessels in the injured area ensue in the cellular proliferation stage. In the cellular proliferation stage, fibroblasts, endothelial cells as well as epithelial cells migrate to the wound site. The fibroblast cells produce the collagen needed for wound repair. The epithelial cells migrate from free edges of the wound tissue across the wound for re-epithelization followed by their proliferation at the periphery of the wound. In the final phage of wound remodeling, replacement of the granulation tissue with collagen fibres takes place followed by devascularization of the granulation tissue eventually leading to the formation of a scar over the wound area. The newly deposited extracellular matrix (e.g. collagen) becomes cross-linked and organized during the final remodeling stage.

All the above mentioned efficient and orderly processes are lost in cases of impaired or chronic wounds, such as diabetic wounds. The chronic ulcers are locked into a state of lasting inflammation characterized by migration of abundant neutrophils with their associated reactive oxygen species (ROS) and degradative enzymes. Excessive infiltration of the chronic ulcers by neutrophils is responsible for chronic inflammation characteristic of non-healing pressure ulcers. The connective tissue matrices are destroyed by the degradative enzyme collagenase (matrix metalloproteinase-8) secreted by the neutrophils (Nwomeh, B. C. et al. Wound Repair Regeneration. 1998;6:127, Nwomeh, B. C. et al. J. Surg Res. 1999;81:189). In addition, the neutrophils release the proteolytic enzyme elastase capable of destroying various healing factors including PDGF and TGF-ß (Yager, D. R. et al. J. Invest. Dermatol. 1996;107:743). Another important marker of chronic non-healing wounds is excessive accumulation of reactive oxygen species (ROS) in the wound bed that further damage the cells and healing tissues (Wenk, J. et al. J. Invest. Dermatol. 2001;116:833).

II. Gene therapy for chronic diabetic wounds

Chronic non-healing diabetic wounds account for 25-50 percent of total diabetic health costs annually representing billons of dollars worldwide (Keswani, S. G. et al. Wound Rep. Reg. 2004;12:497-504). Among the various factors contributing to the pathogenesis of impaired diabetic wounds, deficiency in various growth factors in general and PDGF-B in particular plays a dominant role. However, as mentioned in the above section, due to excessive accumulation of several degradative protease enzymes in the wound bed, a favorable therapeutic effects from the topical (localized) applications of PDGF-B factors on the wound bed requires large and repeated doses of PDGF-B (Mustoe et al. J. Clin. Invest. 1991;87:694-703, Mustoe et al. Arch. Surg. 1994;129:213-219, Pierce et al. Am. J. Pathol. 1991;138:629-646, Wieman et al. Diabetes Care. 1998;21:822-827). In addition, short shelf life and inefficient delivery to target cells are two major concerns associated with direct topical administrations of growth factors. To this end, delivery of genes encoding growth factors to the wound beds is an an attractive alternative strategy to direct topical applications of expensive growth factors. Such gene therapeutic approach can offer targeted local and persistent delivery of de novo synthesized growth factors to the wound bed over many days (Andree C. et al. Proc. Natl. Acad. Sci. USA.1994;91:12188-12192, Benn S.I. et al. J. Clin. Invest. 1996;98:2894-2902, Eming S.A. J. Invest. Dermatol. 1999;112:297-302, Eriksson E. et al. J. Surg. Res. 1998;78:85-91, Vogt, P. M. et al. Proc. Natl. Acad. Sci. USA.1994;91:9307-9311, Krueger G. G. et al. J. Invest. Dermatol. 1999;112:233-239, Sun L. et al. J. Invest. Dermatol. 1997;108:313-318).

However, efficient delivery and expression of genes at the physiological level into our body cells (a process biologists call "transfection") still remains a major challenge in gene therapy. Both the macromolecular genes (DNA) and biological cell surfaces are negatively charged and therefore, spontaneous entry of naked genes (DNA) inside cells is unlikely to be an efficient process. Putting it differently, the problems of developing clinically viable gene therapy methods and designing safe & efficient gene delivery reagents (popularly known as “transfection vectors”) are inseparable: shortcomings in one is going to adversely affect the success of the other. This is why there has been an upsurge of global interests in designing efficacious transfection vectors for use in gene therapy during the last decade.

Contemporary transfection vectors can be broadly classified into two major types: viral and non-viral. Viruses, nature’s own infecting vehicles, have evolved exquisite mechanisms through the course of evolution to deliver their genetic material into host cells. Thus, owing to their natural ability to infect cells, viruses have been used as vectors in gene therapy through replacement of the genes essential for the replication phase of virus life cycles with the therapeutic genes of interest. For instance, it has been demonstrated that skin wounds transfected by adenoviral vector containing PDGF-B gene is capable of providing transgene expression for up to 2 weeks and such adenoviral mediated PDGF-B gene transfer to ischemic rabbit excisional wounds enhanced wound reepithelialization (Liechty K. W. et al. Wound. Rep. Reg. 1999;7:148-153, Liechty K. W. et al. J. Invest. Dermatol. 1999;113:375-383). Eming et al. succeeded in retrovirally transducing human keratinocytes to over express PDGF-A. When seeded onto an acellular dermal matrix and transplanted into full thickness excisional wounds on mice these composite skin substitutes exhibited reduced wound contraction and enhanced re-vascularization (Eming S. A. et al. Hum. Gene. Ther. 1998;9:529-539). Promising results using adenoviral mediated PDGF-B gene therapy in animal models has provided the necessary support for the approval of a phase I clinical trial to establish the safety and efficiency of using adenoviral mediated PDGF-B gene transfer for the treatment of diabetic insensate foot ulcers (Margolis D.J. et al. Wound Rep. Reg. 2000;8:480-493). Despite of all these positive attributes of viral vectors, viral vectors are potentially capable of: generating replication competent virus through various recombination events with the host genome; inducing inflammatory and adverse immunogenic responses and producing insertional mutagenesis through random integration into the host genome. For example, the first fatality in gene therapy clinical trial involving the use of viral transfection vector was attributed to an inflammatory reaction to an adenovirus vector (Hollon, T. Nature Med 2000;6: 6). Ectopic chromosomal integration of viral DNA has been demonstrated to either disrupt expression of a tumor-suppressor gene or to activate an oncogene (Schroder, A. R. et al. Cell 2002;110: 521-529, Woods, N. B. et al. Blood 2003;101: 1284-1289). Recently, it has been reported that retrovirus vector insertion near the promoter of the proto-oncogene LMO2 in 2 human patients with X-linked severe combined immunodeficiency (SCID-XI) is capable of triggering deregulated premalignant cell proliferation with unexpected frequency (Hacein-Bey-Abina, S., et al. Science 2003;302: 415-419).

All the above-mentioned alarming concerns associated with the use of viral vectors call for reassessing the use of viral vectors with regard to their safety in human gene therapy (Check, E. Nature 2003;423: 573-574). Contrastingly, non-viral transfection vectors, mainly cationic polymers and cationic liposomes, possess many important safety advantages. Cationic liposomes in particular, are significantly less immunogenic. Manufacturing viral vectors in large scale are technically demanding with prohibitively high preparation cost while robust manufacture of cationic transfection lipids (used in preparing cationic liposomes) are technically quite feasible. Unlike viruses, they have no restrictions on the size of DNA to be delivered; cationic liposomes can deliver nucleic acids of essentially unlimited size ranging up to large mammalian artificial chromosomes. A greater degree of control can be exercised over the lipids’ structure on a molecular level and the products can be highly purified. Use of cationic liposomes does not require any special expertise in handling and preparation techniques. Cationic liposomes can be covalently grafted with receptor specific ligands for accomplishing targeted gene delivery. Such multitude of distinguished favorable clinical features are increasingly making cationic liposomes as the non-viral transfection vectors of choice for delivering genes into body cells (Valluripalli V.K. Curr. Med. Chem. 2003;10:1185-1315). To this end, the present invention provides non-viral gene therapeutic methods and compositions for improving and/or controlling wound healing by topical application of formulations comprising RGD-lipopeptide A (synthesis, purification and spectral characterization of the RGD-lipopeptide A has been described in PCT Patent Application No. IB07/00826 filed on 30.03.2007), cholesterol and a recombinant plasmid DNA encoding at least one of the therapeutic growth factors of interest, such as protein PDGF-B. Moreover, the present inventions provide methods and pharmaceutical formulations for improved wound healing in subjects with impaired wound healing efficiencies, such as diabetic rats. The lipophilicity of the hydrophobic domains and the hydrophilicity of the polar RGD-head group domains are such that when the cationic lipopeptide is confronted with aqueous solutions, lipid aggregates (popularly known as “liposomes”) are formed in the presence or absence of a second compound.

III. Formulations

The invention provides novel formulation comprising optimal amounts of the cationic RGD-lipopeptide, biological macromolecules and the co-lipids. One or more additional physiologically acceptable substances may be included in the pharmaceutical formulation of the invention to stabilize the formulation for storage or to facilitate successful intracellular delivery of the biologically active molecules. Co-lipids according to the practice of the present invention are useful in mixing with the RGD-lipopeptide A. Cholesterol is an excellent co-lipid for use in combination with the RGD-lipopeptide A to facilitate successful intracellular delivery of the biologically active molecules. A preferred range of molar ratio of RGD-lipopeptide to co-lipid is 2:1. As such, it is within the art to vary the said range to a considerably wide extent. Typically, liposomes were prepared by dissolving the RGD-lipopeptide and the co-lipid (Cholesterol or DOPE) in the appropriate mole ratio in a mixture of methanol and chloroform in a glass vial. The solvent was removed with a thin flow of moisture free nitrogen gas and the dried lipid film was then kept under high vacuum for 8 h. The dried lipid film was hydrated in sterile deionized water in a total volume of 1 mL at RGD-lipopeptide concentration of 1 mM for a minimum of 12 h. Liposomes were vortexed for 1-2 minutes to remove any adhering lipid film and sonicated in a bath sonicator (ULTRAsonik 28X) for 2-3 minutes at room temperature to produce multilamellar vesicles (MLV). MLVs were then sonicated with a Ti-probe (using a Branson 450 sonifier at 100% duty cycle and 25 W output power) for 1-2 minutes to produce small unilamellar vesicles (SUVs) as indicated by the formation of a clear translucent solution. Biologically active molecules that can be administered intracellularly in therapeutic amounts using the RGD-lipopeptides of the present invention include ribosomal RNA, antisense polynucleotide of RNA or DNA, polynucleotide of genomic DNA, cDNA or mRNA that encodes for any therapeutic growth factor important for wound healing.

In a further embodiment, the RGD-lipopeptide A may be used either in pure form or in combination with other lipids or helper lipids such as cholesterol, phosphatidylethanolamine, phosphatidylglycerol, etc. The said therapeutic formulation may be stored at 0?C-4?C until complexed with the biologically active therapeutic molecules. Agents that prevent bacterial growth and increase the shelf life may be included along with reagents that stabilize the preparation, e.g., low concentrations of glycerol.

In yet another embodiment, the formulation of the RGD-lipopeptide A, co-lipids (cholesterol or DOPE) and the biologically active therapeutic molecules may be administered intravenously besides other routes such as intramuscular and intraperitonial. Further, the said formulations may be administered to diabetic rat subjects in combination with 10-200 microgram of DNA for obtaining therapeutic benefits.

IV. Integrin-receptor Specificity of the RGD-Lipopeptide A

Findings in the initial in vitro reporter gene expression assay in 3T3 cells (Balb c mouse fibroblast) using p-CMV-SPORT-ß-galactosidase plasmid DNA as the reporter gene indicated the RGD-lipopeptide A (in combination with cholesterol as co-lipid at RGD-lipopeptide:cholesterol mole ratio of 2:1) to be the most promising for in vivo use. The efficacies of the RGD-lipopeptide (Figure 2) in transfecting 3T3 cells got severely compromised when the gene transfer experiments were carried out by treating the cells with the cyclic RGDfV-peptides, the commercially available efficient integrin antagonists (Figure 2). Since RGD and not RGE peptides are ligands for integrin receptors, we also synthesized the RGE-lipopeptide B following exactly the same synthetic protocols as outlined in Figure 1 for synthesis of the RGD-lipopeptide. The RGE-lipopeptide B showed significantly reduced gene transfer efficacies in 3T3 cells (Figure 3).

Thus, the transfection results summarized in Figures 2 & 3, taken together, provide convincing support for the involvement of integrin receptors in the cellular uptake of the RGD-lipopeptide:plasmid DNA complexes in mouse fibroblast cells.

V. Expression of PDGF-BB in mouse fibroblast cells transfected with RGD-Lipopeptide A: rhPDGF-B complex and wound healing efficiencies of the RGD-Lipopeptide A formulations.

As described above, the platelet derived growth factor B (PDGF-B) is thought to play a central role in all phases of wound healing. Inspired by the impressively high integrin-receptor mediated gene transfer efficacy of the RGD-lipopeptide in mouse fibroblast cells (Figure 2 & 3), we next evaluated the expression of the gene encoding rh-PDGF-B growth factor in mouse fibroblast (3T3) cells by transfecting 3T3 cells with rhPDGF-B plasmid encoding recombinant human PDGF-B in complexation with the RGD-lipopeptide A:cholesterol liposomes. Western blot analysis revealed significant expression of rhPDGF-B in 3T3 cells transfected with RGD-lipopeptide:rhPDGF-B complex (Figure 4). The significant expression of rhPDGF-BB in 3T3 cells transfected with the RGD-lipopeptide (Figure 4) provided us impetus for conducting pre-clinical studies aimed at evaluating the systemic potential of the RGD-lipopeptide in healing wounds in diabetic rats (as a model for chronic wound healing by non-viral gene therapy). Sprague Dawley (S/D) rats were made diabetic by a single injection of streptozotocin (STZ) after overnight fasting. After the rats became diabetic i.e. after they became hyperglycemic (blood sugar > 300 mg/dl), incisional wounds were created in the back of the rats. 48 h post wound creation, rats were subcutaneously injected (for single time only) near the wound areas with: (a) RGD-lipopeptide: rhPDGF-B plasmid DNA complex in 5% aqueous glucose; (b) naked rhPDGF-B plasmid DNA complex in 5% aqueous glucose and (c) 5% glucose solution. As summarized in Figure 5, the rate of wound healing in S/D rats single time injected with RGD-lipopeptide: rhPDGF-B plasmid DNA complex in 5% glucose solution were healed significantly faster than those for rats singly injected with naked rhPDGF-B plasmid DNA complex in 5% glucose or with 5% glucose solution. A representative photograph taken on day 5 after treatment are shown in Figure 6. Clearly the findings summarized in Figures 5 & 6 demonstrate the future clinical potentials of the formulations containing RGD-lipopeptide A, cholesterol and the plasmid DNA encoding growth factors for future use in non-viral gene therapy of chronic wounds.

Applications:

The formulations described in the present invention can be exploited for efficient delivery of genetic materials encoding various growth factors in non-viral gene therapy of chronic wounds. The formulations are useful for delivering polyanions, polypeptides or nucleopolymers into cells via integrin receptors. The formulations of the cationic RGD-lipopeptide A disclosed herein can be used to deliver an expression vector into wound cells of our body towards healing of chronic wounds. In particular, towards improving wound healing in subjects with impaired healing capacities (such as diabetic patients), the presently disclosed formulations of the RGD-lipopeptide A hold potential for delivering various growth factor encoding genes to the wound cells of diabetic patients (such as macrophages, fibroblasts, keratinocytes, etc.).

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLE 1:

Synthesis of the RGD-lipopeptide A (Figure 1).
Step (a): Solid HOSu (0.81 g, 7.03 mmol) and DCC (1.45 g, 7.03 mmol) were added sequentially to an ice cold and stirred solution of N?BOC-N?-Z-L-Lysine (2.7 g, 7.03 mmol) in dry DCM (10 mL). After half an hour, N-2 aminoethyl-N,N-di-n-hexadecylamine (I, 3.6 g, 7.03 mmol, prepared as described earlier (Majeti, B.K et al, Bioconjug Chem. 2005;16:676-684) and DMAP (catalytic) dissolved in dry DCM (10 mL) were added to the reaction mixture. The resulting solution was left stirred at room temperature for 16 hours, solid DCU was filtered and the solvent from the filtrate was evaporated. The residue was taken in ethyl acetate (100 mL) and washed sequentially with ice-cooled 1N HCl (1 x 100 mL), saturated sodium bicarbonate (1 x 100 mL) and water (2 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation. The residue upon column chromatographic purification with 60-120 mesh silica gel using 12% acetone-hexane (v/v) as eluent afforded 3.7 g (60%) of the pure intermediate (II). (Rf = 0.5 using 30% Acetone-Hexane v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.2-1.3 [bs, 52H, -(CH2)13-]; 1.3-1.65 [m, 4H, LysC?H2+ LysC?H2; 9H, CO-O-C(CH3)3; 4H, -N(-CH2-CH2-)2]; 1.7-1.8 [m, 2H, LysC?H2]; 2.4 [t, 4H, -N(-CH2-CH2-)2]; 2.55 [t, 2H, -N-CH2-CH2- NH-CO]; 3.1-3.4[m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-]; 4.0 [m, 1H, LysC?H]; 4.9-5.1 [m, 1H, NH-CO-O-CH2-C6H5; 2H, COO-CH2-C6H5]; 5.2 [m,1H, LysC?H-NH-CO-]; 6.7 [m, 1H, -CH2-CH2-NH-CO-]; 7.1-7.3 [m, 5H, COO-CH2-C6H5].
LSIMS : m/z= 871 [M+1]+ for C53H98O5N4

Step (b): The intermediate II obtained in step (a) (3.4 g, 3.90 mmol) was dissolved in dry DCM (10 mL) and TFA (4 mL) was added at 0oC. The resulting solution was left stirred at room temperature for 5 h. to ensure complete deprotection. Excess TFA was removed by nitrogen flushing. The resulting compound was dissolved in DCM (100 mL) and triethyl amine (10 mL) was added and stirred at room temperature for 15 minutes. The solvent was completely removed by rotary evaporation. The residue was taken in DCM (100 mL) and washed with water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation afforded 2.76 g (91% yield). (Rf = 0.2 using 10% Methanol-chloroform v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.1-1.6 [m, 52H, -(CH2)13-, 4H, LysC?H2+ LysC?H2; 4H, -N(-CH2-CH2-)2]; 1.7-1.9 [m, 2H, LysC?H2]; 2.3-2.6 [m, 4H, -N(-CH2-CH2-)2, 2H, -N-CH2-CH2- NH-CO]; 3.1-3.40 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-, 1H, LysC?H]; 4.8-5.0 [m, 1H, NH-CO-O-CH2-C6H5]; 5.0-5.1 [s, 2H, COO-CH2-C6H5]; 7.2-7.4 [m, 5H, COO-CH2-C6H5]; 7.6 [m, 1H, LysC?H-CO-NH-].
Step (c): Solid HOSu (0.41 g, 3.57 mmol) and DCC (0.74 g, 3.57 mmol) were added sequentially to an ice cold and stirred solution of N-t-butyloxycarbonyl-L-Aspartic acid-?-benzylester prepared conventionally from L-Aspartic acid-?-benzyl ester (Bodanszky, M et al, the presence of peptide synthesis springer-Verlag, Berlin Heidelberg,1984 page no:20) (1.15 g, 3.57 mmol) in dry DCM (15 mL). After half an hour, the intermediate obtained in step b (2.75 g, 3.57 mmol) and DMAP (catalytic) dissolved in dry DCM (15 mL) were added to the reaction mixture. The resulting solution was left stirred at room temperature for 16 hours, solid DCU was filtered and the solvent from the filtrate was evaporated. The residue was taken in DCM (100 mL) and washed with water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation. The residue upon column chromatographic purification with 60-120 mesh silica gel using 15 % acetone-hexane (v/v) as eluent afforded 2.85 g (74.2%) of the pure intermediate (III). (Rf = 0.4 using 30% Acetone-Hexane v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.2-1.3 [bs, 52H, -(CH2)13-]; 1.3-1.5 [m, 4H, LysC?H2+ LysC?H2; 9H, CO-O-C(CH3)3; 4H, -N(-CH2-CH2-)2 ]; 1.7-1.8 [m, 2H, LysC?H2]; 2.4 [t, 4H, -N(-CH2-CH2-)2]; 2.5 [t, 2H, -N-CH2-CH2- NH-CO]; 2.6-2.8 [m, 1H, Asp C?H]; 2.95-3.05 [m, 1H, Asp C?H]; 3.1-3.3 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-]; 4.3 [m, 1H, LysC?H]; 4.45 [m, 1H, AspC?H]; 4.8-5.1 [m, 1H, NH-CO-O-CH2-C6H5; 4H, COO-CH2-C6H5]; 5.6 [m,1H, BOC-NH]; 6.7 [m, 1H, -CH2-CH2-NH-CO-]; 7.1-7.3 [m, 10H, COO-CH2-C6H5]; 7.5 [m, 1H, LysC?H-NH-CO].
LSIMS : m/z= 1077 [M+1]+ for C64H109O8N5
Step (d): The intermediate III obtained in step (c) (2.85 g, 2.65 mmol) was dissolved in dry DCM (6 mL) and TFA (3 mL) was added at 0oC. The resulting solution was left stirred at room temperature for 5 h. to ensure complete deprotection. Excess TFA was removed by nitrogen flushing. The resulting compound was dissolved in DCM (50 mL) and triethyl amine (5 mL) was added and stirred at room temperature for 15 minutes. The solvent was completely removed by rotary evaporation. The residue was taken in DCM (100 mL) and washed with water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation afforded 2.58 g (92% yield). (Rf = 0.3 using 10% Methanol-chloroform v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.1-1.3 [bs, 52H, -(CH2)13-]; 1.3-1.6 [m, 4H, LysC?H2+ LysC?H2; 4H, -N(-CH2-CH2-)2]; 1.8-2.0 [m, 2H, LysC?H2]; 2.3-2.6 [m, 4H, -N(-CH2-CH2-)2, 2H, -N-CH2-CH2- NH-CO]; 2.7-2.9 [m, 1H, Asp C?H; 1H, Asp C?H]; 3.1-3.35 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-]; 3.65 [t, 1H, AspC?H]; 4.3 [m, 1H, LysC?H]; 4.45 [m, 1H, AspC?H]; 5.0-5.2 [m, 1H, NH-CO-O-CH2-C6H5, 4H, COO-CH2-C6H5]; 6.75 [m, 1H, -CH2-CH2-NH-CO-]; 7.2-7.4 [m, 10H, COO-CH2-C6H5]; 7.9 [m, 1H, LysC?H-NH-CO].

Step (e). N-t-butyloxycarbonyl-L- Glycine (0.43 g, 2.46 mmol) was coupled with the intermediate obtained in step (d) (2.4 g, 2.46 mmol) in presence of solid HOSu (0.51 g, 2.46 mmol), DCC (0.28 g, 2.46 mmol) and DMAP (catalytic) following essentially the same protocol as described above in step (c). The resulting crude product upon column chromatographic purification with 60-120 mesh silica gel using 30-35% acetone in petroleum ether (v/v) as eluent afforded 1.5 g (54% yield) of intermediate (IV) as a gummy solid. (Rf = 0.45 using 35% Acetone-Hexane v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.1-1.3 [m, 52H, -(CH2)13-]; 1.3-1.6 [m, 4H, LysC?H2+ LysC?H2; 9H,CO-O-C(CH3) 3; 4H, -N(-CH2-CH2-)2 ]; 1.8-1.9 [m, 2H, LysC?H2]; 2.4 [t, 4H, -N(-CH2-CH2-)2]; 2.5 [t, 2H, -N-CH2-CH2- NH-CO]; 2.6-2.9[m, 2H, Asp C?H]; 3.1-3.3 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-]; 3.5-3.8 [m, 2H, Gly C?H2]; 4.2-4.4 [m, 1H, LysC?H]; 4.7-4.9 [m, 1H, AspC?H]; 4.95-5.1 [m, 4H, COO-CH2-C6H5]; 5.5-5.7 [m,1H, BOC-NH]; 6.85 [m, 1H, -CH2-CH2-NH-CO-]; 7.2-7.3 [m, 10H, COO-CH2-C6H5]; 7.4-7.6 [m, 1H, LysC?H-NH-CO; m, 1H, Gly C?H2-NH-CO].
LSIMS : m/z= 1133 [M+1]+ for C66H112O8N6

Step (f): The intermediate (IV) obtained in step (e) (0.7 g, 0.62 mmol) was deprotected following essentially the same protocol as described above in step (b). The resulting product upon rotary evaporation afforded 0.6 g (93% yield) of intermediate (Rf = 0.2 using 5% Methanol-chloroform v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.1-1.3 [m, 52H, -(CH2)13-]; 1.3-1.6 [m, 4H, LysC?H2+ LysC?H2; 4H, -N(-CH2-CH2-)2]; 1.8-1.9 [m, 2H, LysC?H2]; 2.6-2.9 [m, 4H, -N(-CH2-CH2-)2; 2H, -N-CH2-CH2- NH-CO; 2H, Asp C?H]; 3.1-3.5 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-; Gly C?H2-NH2; Gly C?H2-NH2]; 4.2-4.45 [m, 1H, LysC?H]; 4.7-4.9 [m, 1H, AspC?H]; 5.0-5.1 [m, 4H, COO-CH2-C6H5]; 5.85 [m, 1H, -CH2-CH2-NH-CO-]; 7.2-7.3 [m, 10H, COO-CH2-C6H5]; 7.6-7.7 [m, 1H, LysC?H-NH-CO; m, 1H, Gly C?H2-NH-CO].

Step (g). N?-t-butyloxycarbonyl-N?-nitro- L-Arginine (0.18 g, 0.57 mmol) was coupled with the intermediate obtained in step (f) (0.59 g, 0.57 mmol) in presence of solid HOSu (0.066 g, 0.57 mmol), DCC (0.12 g, 0.57 mmol) and DMAP (catalytic) following essentially the same protocol as described above in step (c). The resulting crude product upon column chromatographic purification with 60-120 mesh silica gel using 5% methanol in DCM (v/v) as eluent afforded 0.35 g (46% yield) of intermediate V. (Rf = 0.2 using 5% Methanol-chloroform v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.0-1.9 [m, 52H, -(CH2)13-; 4H, LysC?H2+ LysC?H2; 9H,CO-O-C(CH3)3; 4H, -N(-CH2-CH2-)2; Arg C? H2+ Arg C?H2; m, 2H, LysC?H2]; 2.5-2.6 [m, 1H, Asp C?H1]; 2.8-3.3 [m, 4H, -N(-CH2-CH2-)2; 2H, -N-CH2-CH2- NH-CO; 2H, Lys?CH2; 1H, Asp C?H2; Arg C?H2]; 3.4-3.7 [m, 2H, -N-CH2-CH2-NH-CO-]; 3.7-4.2 [m, 2H, Gly C?H2; 1H, LysC?H; 1H, Arg C?H; 4.6-4.8 [m, 1H, AspC?H; 4.9-5.1 [m, 4H, COO-CH2-C6H5]; 5.6-5.7 [m,1H, BOC-NH]; 6.1-6.3 [m, 2H, -CH2-CH2-NH-CO-; NH-COO-CH2-C6H5]; 7.1-7.3 [m, 10H, COO-CH2-C6H5]; 7.6-7.9 [m, 1H, LysC?H-NH-CO; 1H, Gly C?H2-NH-CO; 1H, Asp C?H-NH-CO]; 8.0-8.3 [m, 3H, Guanidine NH-CN-NH2].
LSIMS : m/z= 1335 [M+1]+ for C72H123O12N11.

Steps (h,i,j): The intermediate obtained in step (g) (0.1 g, 0.075 mmol) was dissolved in dry DCM (2 mL) and TFA (0.5 mL) was added at 0oC. The resulting solution was left stirred at room temperature for 5 h. to ensure complete deprotection. Excess TFA was removed by nitrogen flushing. The resulting compound was dissolved in methanol (3 mL) and 10% Pd/C was added to it. The reaction mixture was stirred at room temperature for 20 h in presence of hydrogen gas. The reaction mixture was then diluted with methanol (50 mL) and the catalyst was filtered through celite. Solvent was rotary evaporated followed by chloride ion exchange chromatography (using Amberlyst A-26 chloride ion exchange resin) and crystallization in acetone afforded 0.037 g (50% yield) of the pure target lipid 1 (Rf = 0.10 using 35% Methanol-chloroform v/v, as the TLC developing solvent).

1H NMR (400 MHz, CDCl3 +CD3OD):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.0-2.2 [m, 52H, -(CH2)13-; 4H, LysC?H2+ LysC?H2; 4H, -N(-CH2-CH2-)2; Arg C? H2+ Arg C?H2; m, 2H, LysC?H2]; 2.6-3.3 [m, 1H, Asp C?H1; 4H, -N(-CH2-CH2-)2; 2H, -N-CH2-CH2- NH-CO; 2H, Lys?CH2; 1H, Asp C?H2]; 3.4-4.8 [m, 2H, -N-CH2-CH2-NH-CO-; 2H, Gly C?H2; Arg C?H2; 1H, LysC?H; 1H, Arg C?H; 1H, AspC?H + CD3OD].
LSIMS : m/z= 966 [M+1]+ for C51H105O6N10

EXAMPLE 2:
Synthesis of the RGE-lipopeptide B:
Step (a): Solid HOBt (0.488 g, 3.62 mmol) and EDCI (0.693 g,3.62 mmol) were added sequentially to an ice cold and stirred solution of N?BOC-N?-Z-L-Lysine (1.15 g, 3.62 mmol) in dry DCM (10 mL). After half an hour, N-2 aminoethyl-N,N-di-n-hexadecylamine (I, 1.67 g, 3.28 mmol, prepared as described earlier (Majeti, B.K et al, Bioconjug Chem. 2005; 16; 676-684) dissolved in dry DCM (10 mL) were added to the reaction mixture. The resulting solution was left stirred at room temperature for 16 hours. The solution was taken in chloroform (50 mL) and washed sequentially with ice-cooled 1N HCl (2 x 100 mL), saturated sodium bicarbonate (2 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation. The residue upon column chromatographic purification with 60-120 mesh silica gel using 12% acetone-hexane (v/v) as eluent afforded 1.72 g (60%) of the pure intermediate (II). (Rf = 0.5 using 30% Acetone-Hexane v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.2-1.3 [bs, 52H, -(CH2)13-]; 1.3-1.65 [m, 4H, LysC?H2+ LysC?H2; 9H, CO-O-C(CH3)3; 4H, -N(-CH2-CH2-)2]; 1.7-1.8 [m, 2H, LysC?H2]; 2.4 [t, 4H, -N(-CH2-CH2-)2]; 2.55 [t, 2H, -N-CH2-CH2- NH-CO]; 3.1-3.4[m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-]; 4.0 [m, 1H, LysC?H]; 4.9-5.2 [m, 1H, NH-CO-O-CH2-C6H5; 2H, COO-CH2-C6H5 ; m,1H, LysC?H-NH-CO-]; 6.7 [m, 1H, -CH2-CH2-NH-CO-]; 7.1-7.3 [m, 5H, COO-CH2-C6H5].
LSIMS : m/z= 871 [M+1]+ for C53H98O5N4

Step (b): The intermediate II obtained in step (a) (0.85 g, 0.98 mmol) was dissolved in dry DCM (10 mL) and TFA (4 mL) was added at 0oC. The resulting solution was left stirred at room temperature for 5 h. to ensure complete deprotection. Excess TFA was removed by nitrogen flushing. The resulting compound was dissolved in DCM (100 mL) and triethyl amine (10 mL) was added and stirred at room temperature for 15 minutes. The solvent was completely removed by rotary evaporation. The residue was taken in DCM (100 mL) and washed with water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation afforded 0.708 g (94% yield). (Rf = 0.2 using 10% Methanol-chloroform v/v, as the TLC developing solvent).

Step (c): Solid HoBt (0.188 g, 1.38 mmol) and EDCI (0.264g, 1.38 mmol) were added sequentially to an ice cold and stirred solution of N-t-butyloxycarbonyl-L-Glutamic acid-?-benzylester prepared conventionally from L-Glutamic acid-?-benzyl ester ((0.464 g, 1.38 mmol) in dry DCM (15 mL). After half an hour, the intermediate obtained in step b (0.708 g, 0.92 mmol) dissolved in dry DCM (15 mL) were added to the reaction mixture. The resulting solution was left stirred at room temperature for 16 hours,. The solution was taken in chloroform (50 mL) and washed sequentially with ice-cooled 1N HCl (2 x 100 mL), saturated sodium bicarbonate (2 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation. The residue upon column chromatographic purification with 60-120 mesh silica gel using 15 % acetone-hexane (v/v) as eluent afforded 0.7 g (70%) of the pure intermediate (III). (Rf = 0.4 using 30% Acetone-Hexane v/v, as the TLC developing solvent).

1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.2-1.3 [bs, 52H, -(CH2)13-]; 1.3-1.5 [m, 4H, LysC?H2+ LysC?H2; 9H, CO-O-C(CH3)3; 4H, -N(-CH2-CH2-)2 ]; 1.7-2.2 [m, 2H, LysC?H2; 2H,Glu C?H2]; 2.5 [m, 4H, -N(-CH2-CH2-)2; 2H, Glu C?H2]; 2.6 [t, 2H, -N-CH2-CH2- NH-CO]; 3.2-3.4 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-]; 4.15 [m, 1H, LysC?H]; 4.35 [m, 1H, GluC?H]; 5.0-5.2 [m, 1H, NH-CO-O-CH2-C6H5; 4H, COO-CH2-C6H5]; 5.4 [m,1H, BOC-NH]; 6.8-7.2 [m, 1H, -CH2-CH2-NH-CO- ; m, 1H, LysC?H-NH-CO]; 7.3 [m, 10H, COO-CH2-C6H5]
LSIMS : m/z= 1091 [M+1]+ for C65H111O8N5

Step (d): The intermediate III obtained in step (c) (0.7 g, 0.64 mmol) was dissolved in dry DCM (6 mL) and TFA (3 mL) was added at 0oC. The resulting solution was left stirred at room temperature for 5 h. to ensure complete deprotection. Excess TFA was removed by nitrogen flushing. The resulting compound was dissolved in DCM (50 mL) and triethyl amine (5 mL) was added and stirred at room temperature for 15 minutes. The solvent was completely removed by rotary evaporation. The residue was taken in DCM (100 mL) and washed with water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation afforded 0.6 g (94% yield). (Rf = 0.3 using 10% Methanol-chloroform v/v, as the TLC developing solvent).

Step (e). N-t-butyloxycarbonyl-L- Glycine (0.318 g, 1.82 mmol) was coupled with the intermediate obtained in step (d) (0.6 g, 0.61 mmol) in presence of solid HOBt (0.246 g, 1.82 mmol), EDCI (0.348 g, 1.82 mmol)) following essentially the same protocol as described above in step (c). The resulting crude product upon column chromatographic purification with 60-120 mesh silica gel using 30-35% acetone in petroleum ether (v/v) as eluent afforded 0.382 g (55% yield) of intermediate (IV) as a gummy solid. (Rf = 0.45 using 35% Acetone-Hexane v/v, as the TLC developing solvent).
1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.2-1.3 [bs, 52H, -(CH2)13-]; 1.3-1.5 [m, 4H, LysC?H2+ LysC?H2; 9H, CO-O-C(CH3)3; 4H, -N(-CH2-CH2-)2 ]; 1.7-2.2 [m, 2H, LysC?H2; 2H,Glu C?H2]; 2.5 [m, 4H, -N(-CH2-CH2-)2; 2H, Glu C?H2]; 2.6 [t, 2H, -N-CH2-CH2- NH-CO]; 3.2-3.4 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO-]; 4.15 [m, 1H, LysC?H]; 4.35 [m, 1H, GluC?H; 2H,GlyC? H2]; 5.0-5.2 [m, 1H, NH-CO-O-CH2-C6H5; 4H, COO-CH2-C6H5]; 5.8 [m,1H, BOC-NH]; 7.3 [m, 10H, COO-CH2-C6H5] 7.4-7.9 [m, 1H, -CH2-CH2-NH-CO- ; m, 1H, LysC?H-NH-CO ; m, 1H, GluC?H-NH-CO- ];
LSIMS : m/z= 1147 [M+1]+ for C67H114O8N6
Step (f): The intermediate (IV) obtained in step (e) (0.382 g, 0.33 mmol) was deprotected following essentially the same protocol as described above in step (b). The resulting product upon rotary evaporation afforded 0.32 g (92% yield) of intermediate (Rf = 0.2 using 5% Methanol-chloroform v/v, as the TLC developing solvent).

Step (g). N?-t-butyloxycarbonyl-N?-nitro- L-Arginine (0.093 g, 0.29 mmol) was coupled with the intermediate obtained in step (f) (0.24 g, 0.25 mmol) in presence of solid HoBt (0.039 g, 0.29 mmol), EDCI (0.056 g, 0.29 mmol) following essentially the same protocol as described above in step (c). The resulting crude product upon column chromatographic purification with 60-120 mesh silica gel using 5% methanol in DCM (v/v) as eluent afforded 0.16 g (50% yield) of intermediate V. (Rf = 0.2 using 5% Methanol-chloroform v/v, as the TLC developing solvent).
1H NMR (300 MHz, CDCl3):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.2-2.0 [bs, 52H, -(CH2)13- ; m, 4H, LysC?H2+ LysC?H2; 9H, CO-O-C(CH3)3; 4H, -N(-CH2-CH2-)2 ; 2H,Glu C?H2 ; 2H, LysC?H2]; 2.1-2.6[m, 4H, -N(-CH2-CH2-)2 ; m, 2H, -N-CH2-CH2- NH-CO ; 2H, LysC?H2; ;2H Arg C? H2 ; 2H, Glu C?H2]; 3.0-3.4 [m, 2H, Lys?CH2, 2H, -N-CH2-CH2-NH-CO- ; 2H, Arg C?H2]; 3.9-4.3 [m, 1H, LysC?H ; m, 1H, GluC?H; 2H,GlyC? H2]; 5.0-5.2 [m, 1H, NH-CO-O-CH2-C6H5; 4H, COO-CH2-C6H5]; 5.8 [m,1H, BOC-NH]; 7.3 [m, 10H, CO- CH2-C6H5] 7.4-7.9 [m, 1H, -CH2-CH2-NH-CO- ; m, 1H, LysC?H-NH-CO ; m, 1H, GluC?H-NH-CO- ; 1H, Gly C?H2-NH-CO];
LSIMS : m/z= 1349 [M+1]+ for C72H123O12N11.
Steps (h,i,j): The intermediate obtained in step (g) (0.1 g, 0.075 mmol) was dissolved in dry DCM (2 mL) and TFA (0.5 mL) was added at 0oC. The resulting solution was left stirred at room temperature for 5 h. to ensure complete deprotection. Excess TFA was removed by nitrogen flushing. The resulting compound was dissolved in methanol (3 mL) and 10% Pd/C was added to it. The reaction mixture was stirred at room temperature for 20 h in presence of hydrogen gas. The reaction mixture was then diluted with methanol (50 mL) and the catalyst was filtered through celite. Solvent was rotary evaporated followed by chloride ion exchange chromatography (using Amberlyst A-26 chloride ion exchange resin) and crystallization in acetone afforded 0.037 g (50% yield) of the pure target lipid 2 (Rf = 0.10 using 35% Methanol-chloroform v/v, as the TLC developing solvent).

1H NMR (400 MHz, CDCl3 +CD3OD):?/ppm = 0.9 [t, 6H, CH3-(CH2)15-]; 1.0-2.2 [m, 52H, -(CH2)13-; 4H, LysC?H2+ LysC?H2; 4H, -N(-CH2-CH2-)2; Arg C? H2+ Arg C?H2; m, 2H, LysC?H2]; 2.6-3.3 [m, 2H, Glu C?H2; 4H, -N(-CH2-CH2-)2; 2H, -N-CH2-CH2- NH-CO; 2H, Lys?CH2; m, 2H, Glu C?H2]; 3.4-4.8 [m, 2H, -N-CH2-CH2-NH-CO-; 2H, Gly C?H2; Arg C?H2; 1H, LysC?H; 1H, Arg C?H; 1H, GluC?H + CD3OD].
LSIMS : m/z= 980 [M+1]+ for C51H105O6N10

EXAMPLE 3
Evaluation of gene transfer efficacies of the RGD-lipopeptide in mouse fibroblast cells.
3T3 (Balb c mouse fibroblast) cells were seeded at a density of 12,000 cells/ well in a 96-well plate usually 18–24 h before transfection. 0.30 µg of pCMV-SPORT-gal DNA (diluted to 50 µL with DMEM) was complexed with varying amount of cationic liposomes (diluted to 50 µL with plain DMEM) for 30 min. The molar ratios (lipid/DNA) were 9:1, 3:1, 1:1 and 0.3:1. After complexation was completed, 150 µL of DMEM containing 10% FBS (CM1X) were added to the resulting lipoplexes for triplicate experiments. Cells were washed with phosphate-buffered saline (PBS), pH 7.4 (1x 200 µL) and then treated with lipoplex (100 µL). After incubation of the cell plates in a humidified atmosphere containing 5% CO2 at 37 °C for 4 h, 200 µL of DMEM containing 10% FBS (CM1X) were added to cells. The reporter gene activity was assayed after 48 h. The medium was removed completely from the wells, and cells were lysed with 50 µL of 1X reporter lysis buffer (Promega) for 30 min. The ß-galactosidase activity per well was estimated by adding 50 µL of 2X substrate (1.33 mg/ml of ONPG, 0.2M sodium phosphate, pH 7.3, and 2mM magnesium chloride) to the cell lysate in the 96-well plate. Absorption of the product ortho nitrophenol at 405 nm was converted to absolute ß -galactosidase units using a calibration curve constructed with commercial ß -galactosidase enzyme. The values of ß-galactosidase units in triplicate experiments assayed on the same day varied by less than 20%. Each transfection experiment was repeated two times and the day to day variation in average transfection efficiency was found to be within 2-fold. The transfection profiles obtained on different days were identical.

EXAMPLE 4
Evaluation of rhPDGF-B Gene Expression in Mouse Fibroblast Cells by Western Blot.
Mouse fibroblast cell lines were cultured on a 25 cm2 tissue culture flask till they reached a confluency of about 70%. 3 µg of pDNA was complexed with required volume of 1 mM liposome solution in sterile water to give +/- charge ratios of 9:1 in plain DMEM (total volume made up to 1 mL) for 10–15 min. The complexes were then added to the cells. After 1 hr of incubation, medium was changed to complete medium containing 10% FBS. After 48 h the cells were detached from the flask using a cell scrapper. Whole cell lysates were prepared by lysing the cells as mentioned elsewhere (Caruccio, L. Banerjee, R. J. Immunol. Metthods. 1999;230:1-10). Total protein content in each sample was determined by BCA method (Smith, P. K. et al. Anal. Biochem. 1985;150:76-85). 15 µg of cell lysate and 100 ng of pure PDGF protein was loaded and separated on a 7.5% polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membrane (Hybond-C extra, Amersham Biosciences, NJ) using wet blotting. Membrane was blocked for 1.5 h with 3% BSA solution in PBS-T (phosphate buffer saline containing 0.05% Tween-20). Blot was then incubated with polyclonal antibody raised against PDGF-BB of human origin in rabbit (Chemicon International) at 1:500 dilution for overnight at 4 0C. After washing three times with PBS-T, the membrane was incubated with goat anti rabbit secondary antibody conjugated to horseradish peroxidase (Bangalore Genei, India) at 1:1000 dilution for 60 min. After washing for three times with PBS-T protein bands were visualized using TMB-Blotting methods with TMB (Pierec Biotechnology Inc, Pittsburgh, PA) according to the manufacturer’s protocol.

EXAMPLE 5
Healing of Wounds in Streptozotocin induced Diabetic Sprague-Dawley (S/D) Rats.
Adult Sprague Dawley (S/D) male rats (230-260 g) were obtained from National Institute of Nutrition, Hyderabad, India and were allowed to acclimatize to IICT’s animal house conditions for 5 days. All of the in vivo experiments were performed in accordance with the Institutional Bio-Safety and Ethical Committee guidelines using an approved animal protocol. S/D rats were made diabetic by a single injection of streptozotocin (STZ, Sigma, St Louis, MO, USA ) in citrate buffer, pH 4.5 (45 mg/kg, i.p.) after overnight fasting. Daily blood glucose measurements were performed by blood analyzer (BAYER CORP., USA, Express plus, Model No. 15065) for all animals using retroorbital blood samples. Diabetic status was defined as blood glucose levels (non-fasting) higher than 300 mg/dL. Fourteen days after STZ treatment, the backs of the diabetic rats were shaved and the rats were anesthetized with ether solution. A 1.7-cm (radii) circular dorsal skin incision was produced with a scalpel down to the level of the loose subcutaneous tissues.

For the first 48 h post wounding the animals was left undisturbed and the treatments were as follows

Drug Dose Route Vehicle n
i) Lipid-DNA Complexe made with
RGD lipopeptide and plasmid once subcutaneously 5% glucose 4
DNA encoding PDGF-B
Protein

ii)Naked plasmid DNA encoding once subcutaneously 5% glucose 3
PDGF-B protein

iii)Control once subcutaneously 5% glucose 3

Measurement of wound area was started on the 3rd day after treatment and area measurements were taken on every alternative day. The outline of the wound was traced as accurately as possible on a superimposed sheet of sterile cellophane. This outline was then transferred directly to a sheet of tracing paper and the area within it was determined by counting the number of the smallest squares on the tracing paper. The wound healing process was monitored by calculating the percent reduction in the wound area calculated with reference to wound area on day zero. Measurement of wound area was started on 3rd day after treatment and area was taken on every alternative days. Tracings of wound edges using transparent sheets have been used.

EXAMPLE 6
Cloning of recombinant human PDGF into eukaryotic expression vector.

mRNA obtained, from human umbilical endothelial cells were amplified by RT-PCR using PDGF encoding gene specific primers for 25 cycles. The vector DNA and PDGF-B specific primer amplified product was digested with restriction enzymes at 37ºC for 1 hour. Gel purified vector DNA (Ppic 3.5kb) and PDGF-B insert was ligated using T4-DNA ligase, using the directional cloning approach. Following ligation, DNA was transformed into E.coli competent cells, by heat shock method. Subsequently, LB medium (1 ml) was added to the competent cells, incubated at 37ºC for 40 minutes and then plated onto LB agar plates supplemented with 100ug/ml ampilcillin. Plasmid preparation was made from the single isolated colonies and the recombinant clone was identified by restriction digestion. Recombinant pdgf was further sub-cloned into eukaryotic expression vector. The pdgf containing plasmid DNA was digested with restriction enzymes (EcoR1 and Bam H1) at 37ºC for 1 hour and insert was ligated using T4-DNA ligase into PC DNA 3.1 cut with BamH1 and EcoR1 DNA was transformed into E.coli competent cells, by heat shock method. Plasmid preparation was made from the single isolated colonies and the recombinant clones were identified by restriction digestion and conformed by DNA sequencing. The recombinant PDGF (vb5 PC DNA3.1, the vector map depicted in Figure ) was transfected into Eukaryotic cell lines.

We Claim:

1. A method comprising: a) providing ; i) a subject with a diabetic skin wound, and ii) a pharmaceutical composition comprising of a gene transfer vehicle and an expression vector encoding cellular growth factor; and b) applying the said pharmaceutical composition to said wound under conditions suitable for transfecting at least one of the various cells involved in the wound healing process.
2. The method of claim 1, wherein the said applying is under conditions in which wound closure is accelerated.
3. The method of claim 1, wherein the said pharmaceutical composition further comprises a RGD-lipopeptide material.
4. The method of claim 1, wherein the said expression vector encodes the growth factor PDGF-BB.
5. The method of claim 1, wherein at least one of the cells involved in wound healing shows enhanced level of expression of PDGF-B gene.
6. The method of claim 1, wherein the said composition is topically applied to the wound bed.
7. The method of claim 1, wherein said wound is chronic diabetic wound.
8. The method of claim 1, wherein said gene transfer vehicle is selected from the group consisting of liposomes, cationic lipopeptide and cholesterol.
9. The method of claim 1, wherein the pharmaceutical composition comprising a RGD-lipopeptide, a co-lipid, and a genetic material encoding growth factor.
10. The formulation as claimed in claim 9 wherein the RGD lipopeptides may be used in pure form or in combination with helper lipids.
11. The formulation as claimed in claim 9 wherein the colipid is selected from sterol group or a neutral phosphatidyl ethanolamine or neutral phosphatidyl choline.
12. The formulation as claimed in claim 9, where in the co lipid is preferentially selected from DOPE or cholesterol.
13. The formulation as claimed in claim 9, where in the molar ratio of RGD lipopeptide to colipid used is in the range of 1:1 to 3:1.
14. The formulation as claimed in claim 9, where in the preferred molar ratio of RGD lipopeptide to colipid is2:1.
15. The formulation as claimed in claim 9 wherein the nucleic acid is selected from the group of a circular or linear plasmid or is a ribonucleic acid, polynucleotide of genomic DNA, cDNA or mRNA encoding growth factors.
16. The formulation as claimed in claim 12 wherein the said formulation is administered intravenous, intramuscular, subcutaneous or intraperitonial mode.
17. The formulation as claimed in claim 9 wherein the said formulation comprises amount of amphiphile in the range of 9.0 to 0.3 microgram from a lipopeptide to DNA charge ratio ranging from 0.3:1 to 9:1.
18. A RGD-lipopeptide complex, formulation comprising it, and a transfection complex for curing chronic wounds as herein substantially described with reference to examples and drawings accompanying this specification.

Dated 4th June of 2007

Scientist

Figure 1.

Figure 2.

Figure 3.

Figure 4.


Figure 5.

Figure 6.

Figure 7.
PDGF-BB gene

ABSTRACT

PHARMACEUTICAL COMPOSITIONS AND METHODS FOR IMPROVED HEALING OF CHRONIC WOUNDS

The present invention provides methods and pharmaceutical compositions for use in topical delivery of genetic material and/or proteins. In addition, the present invention provides methods and compositions for enhancing and/or controlling chronic wounds by applying a wound care device comprising a cationic amphiphile, cholesterol and a genetic material, the said amphiphile with remarkable gene transfer properties. The area of medical science that is likely to benefit most from the present invention is non-viral gene therapy of chronic wounds.