WO2002094256A1 - Lysine and/or analogues and/or polymers thereof for promoting wound healing and angiogenesis - Google Patents

Lysine and/or analogues and/or polymers thereof for promoting wound healing and angiogenesis Download PDF

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Publication number
WO2002094256A1
WO2002094256A1 PCT/IN2001/000105 IN0100105W WO02094256A1 WO 2002094256 A1 WO2002094256 A1 WO 2002094256A1 IN 0100105 W IN0100105 W IN 0100105W WO 02094256 A1 WO02094256 A1 WO 02094256A1
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lysine
wounds
wound
salts
analogues
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PCT/IN2001/000105
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French (fr)
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Debatosh Datta
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Debatosh Datta
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Application filed by Debatosh Datta filed Critical Debatosh Datta
Priority to PCT/IN2001/000105 priority Critical patent/WO2002094256A1/en
Publication of WO2002094256A1 publication Critical patent/WO2002094256A1/en
Priority claimed from US11/760,972 external-priority patent/US20070243135A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/01Hydrolysed proteins; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic, hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • A61K31/195Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]

Abstract

Use of cationic amino acid I-lysine and/or oligo-lysine (both I- and d- isomer) (most preferably upto molecular wieght 1000) and/or lysine (both I- and d-) analogues (R- group elongated upto C-12) either alone or in various combinations thereof as biocompatible, potent adjuvant wound healing agents for the treatment of protracted wounds (chronic) as well as acute wounds including leprotic ulcer, longstanding diabetic foot ulcer, decubitus ulcer, etc...and including other clinical conditions, e.g. ischaemic myocardium, and cerebral tissue as well as some solid tumors.

Description

LYSINE AND/OR ANALOGUES AND/OR POLYMERS THEREOF FOR PROMOTING WOUND HEALING AND ANGIOGENESIS

Wound management, deep and superficial, acute and chronic, constitute a major part of surgical and medical care. This is true both for home and institutional management. Of the different types, clean-cut protected wounds are easier to manage whereas contamination - in any way - leads to poor healing. There are still others where healing is a major problem, e.g. wounds in Diabetic subjects.

The present invention is related to effective healing of various types of wounds using healing agents that can be incorporated in various types of formulations and modes for their use including topical applications on the wounds.

Background of the nvention

Effective, speedy and scar-less wound healing is a long felt need. The search for cost- effective wound healing agents with improved rate and quality of healing coupled with the desired degree of cell/tissue compatibility continues over several decades. It is also desirable that such agents are readily available at affordable costs in developing and least developed nations and should be capable of being incorporated in a wide range of formulations and modes for easy topical and systemic applications. Most efforts thus far have been directed to the identification of molecules that are either Epidermal Growth Factor, vascular endothelial growth factor (VEGF), delivery of Platelet derived Growth Factor PDGF, various other stimulating factors etc. However most of the solutions (agents and preparations) are either complex, and in most cases leave scar on the skin after the wound healing process is completed. The present invention identifies an effective solution to this long-standing problem and describes the use of ysine and some of its derivatives in topical and parenteral routes for inducing angiogenesis leading to a qualitatively better and faster healing, probably by changing the basic tenet of adult wound repair. Another remarkable feature of the present invention is in the use of lysine to support healing process in really protracted wounds, e.g. in leprotic ulcer, longstanding diabetic foot ulcer etc including its angiogenic role(s) in some other clinical conditions, e.g. ischaemic myocardium, and cerebral tissue as well as solid tumors. Prior Art

Extensive work in the field of augmented wound healing using growth factors with known mitogenic potential is available in scientific literature. EGF (Epidermal Growth Factor) has been tried in improving wound strength and accelerating the rate of repair (Zhou J

' et. al., 1997). Delivery of PDGF (Platelet derived Growth Factor) using cultured dermal fibroblasts transduced trovirally with PDGF-B gene and adenoviral mediated gene transfer has been tried to overcome the ischaemic defect in wound healing (Breitbart AS et. al., 1999; Liechty KW et. al., 1999). Granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) also has been claimed

I 0 to show promise (Jaschke E et. al., 1999; Grzybowski J, 1999). KGF-2 (Keratinocyte Growth Factor-2) has been shown to accelerate wound healing with increase in wound strength and no obvious scarring (Junienez PA, Rampy MA, 1999). bFGF (Fibroblast Growth Factor -β) carried on a collagen matrix for inhibiting wound contracture has been tried out in diabetic and decubitus ulcers (Ono I et. al., 1999). TGF-β delivered through a

5 collagen scaffold has also been tried in enhancing wound healing (Pandit A, 1999). There is support for addition of protease inhibitors in conjunction with any treatment using growth factors (Trengove NJ et. al., 1999). Collagen and hyaluronan have been used by many as carriers for growth factors. Both per se have been used for tissue augmentation and wound healing (Ruszczak Z, Schwartz RA, 1999; Baichey G et. al.,

'0 1995; Jia C et. al., 1998). Platelet release treatment has also been tried in improving skin healing in diabetic rats through endogenous growth factor secretion (Moulin V et. al., 1998).

Accelerated wound healing has been seen in mice with a disruption of thrombospondin 2 gene (Kyriahides TR et. al., 1999). Mice lacking Smad 3 have also shown accelerated >5 healing (Ashcrott GS et. al., 1999).

Chitosan-heparin membrane is claimed to cause increased stabilization and concentration of growth factor in wound area. Immobilized heparin has also been shown to induce accelerated healing (Kratz G et. al., 1998). Water-soluble chitin has also been used as a wound-healing accelerator (Cho YW et. al., 1999). It gave high tensile 50 strength & arrangement of collagen fibres similar to normal skin. It has also been shown that positively charged dextran beads stimulate wound healing (Tawil NJ et. al., 1999). Thymosin beta 4 also has been shown to accelerate wound healing by increasing re- epithelialisation by stimulating keratinocyte migration, increasing collagen deposition and causing angiogenesis (Malinda KM et. al., 1999).

A synthetic analogue of PGE2 (Prostaglandin E2) has also been evaluated for its effect on wound healing. It was proposed it might be beneficial during early stages of inflammation rather than during later stages of remodelling (Talwar M et. al., 1996). Silver has also been shown to aid healing in sterile skin, though its mechanism of action is unknown (Lansdown AB et. al., 1997). Topical estrogen has also been shown to accelerate cutaneous wound healing especially in aged humans associated with altered inflammatory response (Ashcroft GS et. al., 1999). Amebin -1 , a natural product isolated from Arnebia mobilis, has been shown to accelerate normal and hydrocortisone induced impaired wound healing (Sidhu GS et. al., 1999 Nov). Curcumin (diferuloyl methane), a natural product from rhizomes of Curcunia procera, also showed enhanced wound healing (Sidhu GS et. al., 1999 Sep-Oct). The healing potential of Calotropis procera is also being evaluated (Rasik AM et. al., 1999).

Although many of these agents/proteins/growth factors has been shown to support wound healing and Angiogenesis processes, the profound advantage with this amino acid lies in the facts of extremely low cost, highest degree of compatibility with no known adverse/side effects even in cases of extremely high levels of systemic loading, easy availability and its unique technically superior role(s) in the process of "adult healing", where it augments the initial process of Angiogenesis (hierarchical new capillary generation/formation in ischaemic tissue) and subsequent steps of enhanced "pouring in" of inflammatory cells/platelets, other blood proteins, growth factors and other agents in the ischaemic wound bed is exactly as per the requirements (optimum level) and is not really interfered with as in the cases of all other agents (growth factors) and preparations and formulations, where one or more of the growth factors are being delivered to the wound bed. In primary closure, the amino acid and derivatives support extremely rapid cellular division and filling up of the wound void rather than much of matrix laying. On a more tissue specific Angiogenesis phenomenon, a single intrapericardial injection of FGF-2 in a porcine model of chronic myocardial ischaemia has been shown to cause functionally significant angiogenesis without any adverse outcomes (Laham RJ et. al., 2000). This has been one of the many studies being carried out for induction of angiogenic response in ischaemic myocardium.

It would be interesting to study details of angiogenic response in ischaemic myocardium where normally angiogenic factors are available at a higher concentration than adequately perfused myocardium (Laham RJ et. al., 2000; Laham RJ et. al., 1999) Repair/healing process of the aschaemic myocardium involves formation of new blood capillaries (angiogenesis) in the ischaemic tissue. Angiogenesis process being same everywhere, ischaemic tissue angiogenesis (in chronic wound bed) has been shown amply by the amino acid and derivatives.

Patent No. 5.457.093 discloses a pharmaceutical composition, comprising a) an effective wound healing amount of platelet derived growth factor (PDGF); b) a pharmaceutically acceptable cellulose polymer; and c) a pharmaceutically acceptable positively charged chemical species selected from the group consisting of lysine, arginine, histidine, protamine, aminoguanidine, zinc and magnesium, wherein the composition is an aqueous gel having a viscosity in the range of about 50,000 to about 150,000 cps at room temperature. However this healing process suffers from shortcomings such as presence of scar in the healed wound. The primary agent here is a genetically engineered component of PDGF-β a growth factor (which augments the Keratinocytes/fibroblast division/differentiation process) in a particular concentration. This in a way is interference in the natural events in healing process, which is highly interactive process and involves more than one growth factors/agents and cells iyin the wound bed area. This delivery of single growth factor probably does not reflect the entire story of wound healing and hence, leads to scarring.

Patent no: 6203805 discloses a topical composition comprising collagen enhancing effective amounts of a whey protein, a retinoid, a vitamin E or derivatives thereof and an ascorbic acid or derivatives thereof, said vitamin E being in an amount of 500 mu.g/ml or less and said ascorbic acid being in an- amount of from about 50 mu.g/ml to about 100 .mu.g/ml and less than said amount of vitamin E and said retinoid being in an amount of from about

1. mu.g/ml to about 100 mu.g/ml. However this healing process suffers from shortcomings such as the presence of scar in the healed wound, resulting from enhanced collagen synthesis induced by the product in this patent. Also, it talks of vitamin C induced enhanced collagen transfixation which is bound to result in scarring. This healing process is also slow.

Patent no. 6201164 discloses a hydrocolloid gel composition, especially a wound gel composition, which comprises: a) a water insoluble, water swellable cross-linked cellulose derivative; b) an alginate; and c) distilled water in a sufficient amount to make up the difference between the amount of ingredients (a)+(b) and 100% and wherein the gel comprises from 0.05 to 5.00% by weight of alginate, shows better absorption capacity than known gels, accelerates the healing by serving as a slow-release system for active ingredients promoting wound healing.

Patent no. 6197751 discloses an invention related to methods for promoting tissue repair, angiogenesis and cell migration. The method of the invention utilizes thymosin a1 (T. alpha.1 ) peptide to promote tissue repair, angiogenesis and cell migration. The invention further relates to modulating T.alpha.1 activity in tissues.

Patent no. 6187743 discloses the enhanced healing of wounds by contacting the wound surfaces with a dressing of D-glucose polysaccharide obtained by hydrolysis of starch containing ascorbic acid, collagen type I and alpha-tocopherol acetate. The collagen type I, and the ascorbic acid in the dressing are the typical chemotactic agents of neutrophils, fibroblasts and/or endotheϋal cells. These combinations can be present in the dressing as a gel. The composition material can be applied to the wound as a particulate material such as a powder, or as material such as a gel, paste, dispersion, solution or syrup. Wounds, in particular those occurring in the skin as second and third degree burns, stasis ulcers, trophic lesions, such as decubitus ulcers, severe cuts and abrasions which are commonly resistant to the natural healing process, may be treated with this composition. The application of this combination to open wounds greatly accelerates the rate of. healing and reduces scarring. The invention includes an article useful in the therapy for surface wounds comprising the suspension of particles on non- adherent gauze and a bandage tape.

US Patent no. 614171 & 5618516 describe a pharmaceutical composition containing ketoprofen lysine salt having concentration between 10 to 20% p/volume of solution 5 which allows for topical treatment, the maximum absorption pf active principle. The foam is applied for the local treatment in painful and phlogistic conditions, of rheumatic or traumatic nature, of the articulations of the muscles of tendons and of the ligaments. The patent does not indicate any application towards wound healing applications.

US Patent no 5480869 describes anti-inflamatory peptide analogs and treatment to I0 inhibit vascular leakage in injured tissues. Peptide analogs of the invention have the primary sequence T()N-A()1-A()2-A()3-A()4-A()5-A()6-T()C where one o the moieties is in the D configuration. In this peptide sequence A()1 , A()2 can be D or L Lysine. This patent does not indicate the use of Lysine alone independent of the peptide analog.

The use of various types of peptides containing lysine in cosmetic skin treatment such 15 as wrinkles is described in US patent no. 5492894. Once again this does not disclose the use of lysine alone (independent of a peptide) for any wound healing application.

Similarly the use of synthetic cross-linked amino-acid containing polymers selected from a group of lysine, arginine, etc (US Patent 5505952); or tripeptide derivatives represented by the formula T!X_A()1_A()2_A()3_H having A()1 as L- or D- pyroglutamic

>0 acid residue, A()2 selected from a group consisting of glysine, L-lysine, L-proline, etc., and A()3 is an L-, D-, or DL arginal residue etc. (US Patent no. 5508385); or cosmetic or dermatological composition characterized in that it contains in a suitable vehicle, an anti oxidant system possessing a synergic effect consisting of the combination of a lauruylmethionate of lysine, histidine or arginine or of at least one polyphenol chosen 5 from specific derivatives of carboxylic acids and its salts, an ester or amide of caffeic acid and a flavonoid or an extract containing flavonoids and a rosemary extract (US patent no. 5587171 ); or medicinal compositions for enhancing the wound healing process in humans consisting essentially of a therapeutic wound healing enhancer containing protein Lumicarmine in which the 10% amino acid residue is lysine and arginine and 80% being praline, glycine, and glutamic acid (US Patent no 5384308); or use of L-lysine pyruvate ( US Patent 4734276); or use of GHL-Cu as a wound healing and anti-inflamatory agent (Patent no. 47600051 ); or compositions comprising of L- lysine, gibberellic acid and urea in an inert carrier comprising water for treatment of herpes simplex, cold sores, lesions, warts, blisters, burns, ulcers and other painful skin conditions for topical applications (US Patent no 4424232) are known approaches in wound healing methods.

Object of the Invention / Summary of the Invention:

Repair process is a combination of extremely intricate interactions between few cell types and a number of matrix components. Major concerns still existent extensively in the area f wound healing are: a) degree of neovascularization/Angiogenesis in the wound / ischaemic area b) rate of healing, c) degree of fibrosis, and d) deformation and residual scar.

One of the objects of the invention is in establishing the use of the cationic amino acid lysine, its isomeric forms (L- or D- either alone or, in various combinations and salts thereof), short oligomers of lysine (both L- and D-) (most preferably upto M.W. 1000 approximately), when applied as it is, or derived in-vivo from other higher order polymers involving only Lysine or a combination of lysine and other amino acids (either I- or d- isomers or various combination of the two), to support and enhance a wound healing process resulting in a qualitatively better and much quicker wound healing with less scar and deformation in primary closures as well as in chronic wounds.

Another object of the present invention is to exploit the essential amino acid lysine, its isomeric forms (L- or D- either alone or, in various combinations amongst themselves), salts thereof and the short oligomers (most preferably upto M.W. 1000) and salts thereof, derivatives (e.g. acetyl-lysine/ oligo-lysine) as the active ingradient(s) (with or without one or more additive(s)) and their ability to induce extensive angiogenesis in some clinical conditions e.g. ischaemic myocardium (in angina cases and other related clinical conditions) and ischaemic cerebral tissue as well as in some solid tumors. It is yet another object of the present invention to utilize cationic amino acid lysine and its isomeric forms (L- and/or D-), salts thereof, including the short oligomers (both L- and D- and most preferably upto the M.W. 1000), and their salts as active ingradients, in formulations suitable for the topical and/or systemic applications such as in the forms of powder, paste, gels, foams, strips, bandage, tapes, gauze, injectables, drops and oral preparations (with or without suitable additive(s)) and their like for its induced and controlled inflammatory response in primary as well as in "passive" chronic wounds (e.g. in bed sores, venous ulcers, diabetic ulcers, first . and second degree burns, leprotic ulcers etc.) including induction of profuse angiogenic response, qualitatively I 0 much better healing (with least scar or, no scar and deformation and contracture) with rapid rate of healing.

It is yet another object of the present invention to utilize cationic amino acid lysine (both L- and D- isomers) including the short oligomers (most preferably upto the M.W. - 1000) and salts thereof etc. either alone or, in various combinations amongst 15 themselves as active ingradient(s) in combination with other additive(s) drawn from a list (mentioned above), for topical application on the scalp for the purpose of growth of hair.

Another objective of the present invention to utilize and therapeutically exploit the synthetic lysine derivatives/analogues (R groups elongated upto C12) (both L- and D-

20 forms as well as their oligomers and salts thereof as the active ingradient(s)), in the area of augmented and qualitatively better healing of wounds (both acute and chronic) as well as induction of angiogenesis and development of collateral circulation in ischaemic tissue (e.g. myocardium cerebrum), where only the R-group of the basic molecule is extended from C-5 to C-12 (and the remaining part of the molecule remains

'.5 same and hence the property of augmentation of the healing process, through extensive angiogenesis and rapid cellular division in the wound bed remains same.

In another aspect of the present invention, the medicament(s) for topical and/or systemic applications for the treatment of acute (primary closure) and chronic wounds

(including in tissues with ischaemic conditions, may or may not comprise of one or more

30 of the additive(s) (in addition to the active ingradient(s) mentioned) such as extracts of leaves from the plant Aloe vera, Alpha Hydroxy Acids (mostly glycolic and lactic acid), Antioxidant(s), Ascorbic acid, Balm mint extract (from leaves of Melissa officinalis), Boric acid, caprylic and capric triglycerides, Cetearyl alcohol, Cetyl alcohol, Chamomile extract (from flowers and leaves of Anthemic nobilis), Cyclomethicone and other emollients, exfoliants (e.g. beta hydroxy acids), Fennel extract (from the fruit of Foeniculum vulgare), Glycerol, Glycolic acid, Humectant, Hyaluronic acid, Kojic acid (from mould grown on rice or soyabean), Lactic acid, Methyl paraben, Mulberry extract, PEG-100 stearate, Polysorbates - 20,60,80, Retinyl Palmitate, Tocopheryl acetate, Propyl Paraben, Propylene glycol, other skin conditioning agents, Zinc oxide, Vitamin A and Vitamin E compounds, Cyclomethicone, Dimethicone, aqueous solution of PVP, Pyrogen free water, Parenterally acceptable oil (e.g. Arachis oil), Stabilizing Agent(s) and buffer substance(s) in presence or absence of fragrant additives (e.g. essential oil like Orange oil etc.) where the total active ingradient(s), (either any of the active ingradients mentioned above or in various combinations thereof) will be in the range of 0.1 to 100.0 % (w/w) or (w/v) (depending on the formulation and the base/carrier used), preferably in the range of 0.1 - 20 %, more preferably in the range of 1 - 10% and still more preferably in the range of about 5 -10 % and most preferably around 10%.

Detailed description of the invention:

The present invention is based on the astonishing and totally new finding that the cationic amino acid lysine (both D- and L- isomers), oligomers of the amino acid preferably in the M.W. range of about 300 to 3000 (from both D- and L- isomers), either applied as such (along with one or more additive(s)) or derived in-vivo from various higher order (higher M.W.) lysine (and other amino acid) polymeric / oligomeric species, synthetic derivative(s) / analogue(s) of lysine (where R- group is variously elongated upto a level of C-12, by introduction of -CH2 groups and/or other reactive groups, (e.g.

C=0 group), but keeping the two amino groups at the two ends of the molecule intact - one attached to the chiral carbon atom and the other, at the distal end of the R- group, elongated or otherwise) and salts thereof, either alone or in various combinations thereof may be used to treat, various types of wounds - both acute wounds (e.g. in cuts, bruises, lacerations, accidental wounds, burns, surgical wounds etc.) as well as chronic wounds (e.g. decubitus "ulcers and other pressure sores and ulcers, diabetic ulcers and sores, venous ulcers, leprotic ulcers etc.) as well as various other types of wounds and ulcers, including inducing extensive angiogenic responses in ischaemic wounds/ tissues (e.g. in ischaemic myocardium and ischaemic cerebral tissue). Based on the recent observations of researchers (Laham RJ et al., 1999 and 2000) where quantitatively more synthesis and elaboration (secretion) of βFGF and other angiogenic factors (responsible for induction of angiogenic response) in ischaemic myocardium have been shown, these molecule(s)'s (either alone or in combination) abilities to possibly bridge the angiogenic (growth) factor(s) to their respective receptor site(s) on the target cell membrane(s) (mostly the endothelial cells in chronic wound/ ischaemic tissue) on /to a quantitatively augmented degree - in a concentration / dose dependent way (thereby indicting to a concentration window of the molecule(s)), can be taken advantage of in the induction of controllable, therapeutic angiogenesis induced by an extremely biocompatible (having no known toxicity signs and symptoms even in extremely high loading doses - both in animals and human beings) and extremely low- cost natural molecule, in sharp contrast to the current global (research and clinical) approaches of employing extremely high-cost and extremely labile agents - mostly the angiogenic factors, delivered locally at the ischaemic tissue site(s). In sharp contrast, the amino acid and/or its derivatives, as mentioned, either alone or in combinations (with or without additive molecule(s)), in the form(s) of formulations) can be administered / employed through oral and/or parenteral route(s) or a combination thereof for easy and maximal therapeutic benefits, in addition to possible on-site / in-situ delivery (e.g. in the ischaemic myocardial tissue and iscahemic cerebral tissue etc.) through mechanical/ chemical means (e.g. angiography catheter, liposomal delivery).

Various forms of chemical modification(s) of the native lysine molecule may generate the synthetic lysine analogues and their oligomers and salts which can posses the profound angiogenic response shown by the basic molecule. Accordingly the present invention concerns the uses of lysine and its various salts, synthetic analogues of lysine, and various salts thereof and the oligomers (either employed as such or derived in-vivo) in the most preferred M.W. range of - 1000 (approximately) including various salts thereof in the treatments of various types of ischaemic conditions and Wounds as mentioned (including cerebral and myocardial ischaemias). Lysine has been only one of the ingradients (along with many other amino. acids, cationic amino acids and other unrelated molecules including positively charged ions and compounds in addition to the distinctly and discreetly described active ingradient - PDGF β in the US patent number 5457093. Also, lysine has been one of the aminoacids in the tripeptide complex - L-lysyl-glycyl-L-histidine: copper (II) - which has been described to have wound healing property in US patent no. 5591711. Among various other preparations and/or formulations with claims of various degrees of wound healing properties, lysine, if at all, has always been only one of the many components in the formulations.

Accordingly only lysine, its definite oligomers (with a most preferred M.W. range) and well described and defined analogues (of various lengths and having intact terminal amino groups) either alone or in various combinations thereof - as active ingradient(s) are described for the first time ever in the treatments of ischaemic tissue reperfusion as well as in acute and chronic wounds repair and tissue regeneration, and these new biological properties of the molecule on its own or its derivatives are not at all obvious to the workers skilled in the area.

The above mentioned amino acid, analogues / derivatives and oligomers and salts thereof may be used as such because they are very soluble in water and produce pH values which are in the neutral range.

Salt formation could come into question in case salt formation should affect the aqueous solubility (salts of low soluble substances to increase the solubility and salts of very soluble substances to reduce solubility) or to modify the pH of aqueous solution.

Salts of the analogues and derivatives should be therapeutically acceptable and preferably without any significant effect on the aqueous solubility. Furthermore, the salts of the analogues should be physiologically acceptable and without effect(s) upon the stability of the analogue. The salts of lysine, derivatives/ analogues and oligomers maybe in the form of inorganic acid salts such as salts of hydrochloric acid, sulphuric acid, phosphoric acid and hydrobromic acid or a sodium salt, a potassium salt, an ammonium salt, a magnesium salt or a calcium salt or organic acid salts such as a salt of acetic acid, lactic acid, maleic acid, fumaric acid, tartaric acid, citric acid, methane sulphonic acid and p- toluene sulfonic acid or a monoethanolamine salt, a diethanolamine salt , or a triethanolamine salt, and the like.

The present invention, as mentioned, concerns with the treatment of all kinds of wounds- acute and chronic, and both of human and animal origins, including treatments of all types of infected wounds without any simultaneous antibiotic coverage (therapy) for the protection of the wounds in question. This lowers the cost of therapy very significantly and results from the antimicrobial property of the aminoacid, its derivatives / analogues and oligimers, including salts thereof. Additionaly, the quality of healing has been observed persistently to be very good with near-zero (nearly absent) degree of non-specific fibrosis, wound contracture and deformation. This much better quality of healing in terms of lack of fibrosis and contracture, results from the ability of the amino acid (and its oligomers, derivatives and analogues and salts thereof) with the mentioned characteristic chemical structure to bind the various serum-derived growth factors to their dedicated receptors on the surface of target cells (e.g: binding angiogenic factors to the dedicated receptor sites on the endothelial cells) thereby augmenting the cell division / migration process(es). The clinical conditions include all types of wounds, infected or otherwise, acute or chronic, diabetic wounds, bedsores, surgical wounds (mostly for much better quality of healing), cosmetic surgery cases, burns (first and second degree and wherever basal dermo -epidermal layer including the keratinocytes are existent in the wounds), growth of hair (in cases of hair loss of any origin where the primary hair follicular cells are existent in the dermal layer), and in cardiac muscle reperfusion in ischaemic condition (e.g. in angina) as well as in perfusion of ischaemic cerebral tissue. In veterinary area of applications, possible clinical conditions where these are applicable include: fur growth, hump sores, mastitis (in cattle), foot-mouth-disease etc. Various other internal wounds of human origin (e.g. peptic ulcer, deuodenal / gastric ulcer, gastritis, ulcerative colitis etc.) can be treated also. The advantages of topical and systemic applications with lysine and derivatives include:

1. Rate of angiogenesis and healing are much faster.

2. Quality of repurfusion and healing is much much better than any known agents in the 5 same category of extreme low cost of therapy.

3. Extremely high degree of compatibility, (no side-effects).

4. Extremely low cost of treatment.

5. Long shelf-life in sharp contrast to various growth factors employed in the area of angiogenesis and wound repair.

0 6. Extremely easy availability.

7. Oral and parenteral modes of application are extremely simple and low cost compared to the angiogenic factors being delivered to the coronary circulation through angiography catheter requiring extremely sophisticated infrastructural facility as well as very high degree of skill.

5 8. The aminoacid (and derivatives, oligomers) basically does not interfere with the normal repair process apart from preventing non-specific fibrosis on one hand and augmenting the endothelial cell division and migration resulting in much augmented angiogenesis and controlled degree of inflammation. In sharp contrast all other wound repair formulations deliver one or more (but a restricted number) of the growth factors to

!0 the wound / ischaemic bed. This results in interference in the normal process of healing which is basically an extremely intricate and complicated interaction process involving a number of cells, their products, serum derived growth factors, inter-cellular matrix materials and others. By augmenting the cell division in the ischaemic / wound bed, the aminoacid (and derivatives) basically change the fundamental tenet of adult wound

25 repair and reperfusion. Under the influence these active ingradients the adult repair process mimics the foetal /neonatal repair process where scarring and matrix laying is negligible. Although scarless healing is promoted by the mentioned active ingredients, mechanical property (e.g. load bearing capacity of the wound) of the wound is really much augmented under its influence.

30 The active ingredient(s) in the pharmaceutical compositions for skin wound repair, according to the invention, may comprise of 0.1% to 100%, preferably from 0.1 to 20%, more preferably from 1 to 10%, even more preferably from 5 to 10% and most preferably 10% (w/v) or (w/w) (depending on the preparation in question) of lysine, synthetic analogue / derivative, oiigomer(s), salt(s) thereof, either alone or in combinations. The pharmaceutical compositions may be in the form of wet dressing, tapes, bandages, lotion, solution, drop, aerosol, powder, cream, gel, ointment, fixed dressing, paste, occlusive and semi occlusive dressing, tincture, hydrocolloid, alginate or foam etc. Total active ingradient(s) in oral preparations (in the forms of tablets / capsules etc.) for uses in the healing of internal wounds (e.g. Gl tract wounds) and for induction of angiogenic response in reperfusion of ischaemic tissues may comprise of 5% to 100% (w/w) and most preferably about 75%, whereas parenteral preparations (e.g. in the form of injectables) for wound repair, angiogenesis and reperfusion may comprise of total active ingradient(s) in a range of 5 - 500 mg/ml, more preferably in a range of.20 - 200 mg/ml, still more preferably 50 - 150 mg/ml and most preferably about 100 mg/ml. The oral and parenteral dosage may be fixed on a daily (24 hrs.) basis and should be determined in such a way that sustained blood level of the active ingradient(s) should be in the range of 30 μg/ml to 175 μg/ml, more preferably in the range of 30 μg/ml to 160 μg/ml and most preferably in the range of 35 μg/ml to 155 μg/ml for maximal benefit. Wound dressings with the formulations containing the described concentrations of active ingradient(s), either alone or in various combinations amongst themselves, in addition or absence of the described additive molecules/ extracts / compounds, should take into consideration the followings: wounds should be adequately applied with the preparation, wound bed should have enough supply of blood (initial removal of sloughs may be necessary), wounds should be kept moist and the dressing should be kept in place for a period of 48 to 72 hrs. before replacement. Depending on the severity of the wound (in addition to the topical route) and ischaemia, a systemic total daily dosage may be fixed in a range of 2 to 8 gm. (administered in conveniently split amounts, 3-4 times a day).

Examples:

Few representative formulations can be as follows: Example 1

For application directly or by a mechanical spray:

Lysine 10 g

Methyl Paraben 70 mg

Propyl Paraben 30 mg

Purified Water 89.9 g

100.0 g

Example 2

Solution for direct application / or in the form of impregnated dressing:

Lysine 10.0 g

Methyl Paraben 0.2 g

Propylene Glycol 9.8 g

Purified water 80.0 g

100.0 g

Example 3

Viscous solution for direct application (e.g. in the form of a lotion):

Lysine 10.0 g

Methyl Paraben 0.1 g

Propylene Glycol 8.0 g

Methyl Cellulose (1500) 4.0 g

Purified Water 77.9 g

100.0 g Example 4

Application as water-miscible gel or hydrophilic gel :

Lysine 10.0 g

Carboxymethyl Cellulose 4.0 g

(sodium)

Methyl Paraben 0.1 g

Propylene Glycol 22.0 g

Purified Water 63.9 g

100.0 g

Example 5

Water miscible water-free gel form of application:

Lysine 10.0 g

Macrogol (3000) 40.0 g

Macrogol (400) 50.0 g

100.0 g

Example 6

Hydrophobic ointment form of application :

Lysine 10.0 g

Polyethylene 8.5 g

Liquid Paraffin 81.5 g

100.0 g

Example 7 Water absorbing ointment form of application:

Lysine 10.0 g

Cetostearyl alcohol 27.5 g

Polysorbate 80 2.5 g

Petroleum jelly 50.0 g

Liquid paraffin 10.0 g

100.0 g

Example 8

Cream form for application:

(A) Lysine 10.0 g

(B) Polysorbate 80 0.45 g

Cetyl alcohol 4.5 g

Liquid paraffin 4.5 g

Glycerol monostearate 5.4 g

40-50

(C) Glycerol 85% 4.0 g

Sorbitol 6.0 g

Methyl Paraben 0.1 g

Purified water 65.15 g

(B) is mixed and melted together at around 65 - 70 ° C. (C) is heated to boiling point and cooled to 70 ° C approximately and mixed with (B). 10 grams of Lysine (A) of a particular particle size ( < 300 μm), put in 90.0 gram of the mixture of (B) and (C). Enhancement of active ingradient concentration to more than 15% might result in appearance of particulate matters in the bulk. This can be overcome by using hydrophilic polymer e.g. PVP. ~ Example 9

Hydrophobic gel form (water immiscible):

Lysine 10.0 g

White soft paraffin 75.0 g

Liquid paraffin 15.0 g

100.0 g

Example 10

Dry powder form for direct application:

Lysine 10.0 g

Boric acid 90.0 g

100.0 g Example 11

In the form of tablets for rapid dissolution in intestinal tract for wound repair and/or ischaemic tissue angiogenesis:

Lysine 500 mg

Cellulose (microcrystalline)IOO mg

Povidone 18 mg ' Magnesium Stearate 5 mg

Talc 27 mg

650 mg

Example 12 Tablet formulation for oral application for wound healing and/or angiogenesis: Lysine 500 mg Calcium monohydrogen

Phosphate 82 mg

Croscaramellose Sodium 25 mg

Povidone 18 mg

Magnesium Stearate 5 mg

Talc 27 mg

657 mg Example 13

Topical application forms/ointment/gels/cream forms for wound healing:

Representative formulation in example 1 - 9 are repeated using either L- and/or D- isomers of monomeric Lysine and/or their salts, in isolation or in varied combinations thereof, or any or both of the mentioned isomers in varied combinations with oligo-lysine (either L- and/or D-) (upto a M.W. of 2500, preferably upto a M.W of 2000, more preferably upto a M.W. of 1500 and most preferably upto a M.W. of 1000), Lysine analogues/derivatives (R-groups extended upto C-12) and/or their salts, or lysine analogues/derivatives (mentioned above) in various combinations with oligolysine(s) (mentioned above) as active ingradients. All formulations containing one or more of the above mentioned active ingradients can be given various forms of formulations (physically) where the additive(s) (one or more) in various combinations and concentrations is/are drawn/selected from a list containing extract of leaves from the plant Aloe vera, Alpha Hydroxy acids (mostly glycolic and lactic acids), Antioxidants, Ascorbic acid, Balm mint extract (from leaves of Melissa officinalis), Caprylic and Capric triglycerides, Cetearyl alcohol, Cetyl alcohol, Chamomile extract (from flowers and leaves of Anthemis nobilis), Cyclomethicone and other emollients, Exfolients (e.g. beta hydroxy acids), fennel extract (from the fruit of Foeniculum vulgare), Glycerol, Glycolic acid, Humectant, Hyaluronic acid, Boric acid, Kojic acid (from mould grown on rice or soyabean), Lactic acid, Methyl paraben, Mulberry extract, PEG-100 stearate, Polysorbates - (20,60,80), Propyl Paraben, Propylene glycol, Retinyl Palmitate, other skin conditioning agents, Tocopheryl acetate, Zinc oxide, Cyclomethicone and Dimethicone in presence or absence of fragrant additives (e.g. essential oil like Orange oil etc.), where total concentration (w/w) or (w/v) of the active ingradient(s), (lysine, its oligomers, derivatives/analogues and their salts) will be in the range of 0.1 to 100.0 %, preferably in the range of 0.1-20 %, more preferably in the range of 1 - 10% and most preferably in the range of about 10 %. Example 1 :

Other tablet forms for application:

Representative formulation of example 10 is repeated using either L- and/or D- isomer(s) of monomeric lysine and/or their salts, in isolation or in varied combinations thereof, or any or both of the mentioned isomers in varied combinations with oligolysine(s) (mentioned in example 13), lysine analogues/ derivatives (R - groups extended upto C-12) and/or their salts (mentioned in example13), where total concentration(s) of the active ingradient(s) (one or more in various combinations) will be between 5-100% (w/w), and preferably between 10-80% and more preferably between 50-80% and most preferably about 75%.

Example 15:

Injectable form for ischaemic tissue angiogenesis and / or wound repair :

Lysine (and/or analogues/ derivatives/ oligomer(s) 10Omg/ml and / or salts thereof, as in example 13 &14 above) (in purified deionized water of injection grade)

[The total concentration of the active ingradient(s) in examplelδ should be between 5- 500mg/ml, preferably between 20-200mg/ml, more preferably between 50-100mg/ml and most preferably about 100mg/ml] Experimental documents and clinical studies:

To illustrate the remarkable beneficial effect of the present invention, selected cases of the present invention, selected cases of acute (surgical wound) arid chronic wounds treated with the amino acid is presented unless otherwise stated the topical 100% (w/w) powder form application is to be assumed. The invention will now be explained using non-limiting examples:

Example 16

To investigate the wound healing properties of lysine, the following experiment was performed: Wound was made in the dorsal skin (3.0 mm deep) in a rabbit following shaving and antiseptic dressing under local anaesthesia. L-lysine HCI in dry form was incorporated in the wound (dry powder form applied topically in most preferred optimum concentration of ~ 0.25-100 mg/sq.in wound (within total range of 1 - 100 mg/sq.in of the wound)) while closing with silk suture. The wound was not protected with antibiotics. Similar wound was made in a matched control rabbit except nothing was applied before suturing. Scanning electron micrograph of incised wound in the experimental rabbit showed extensive granulation tissue formation. (Age of the wound is 72 hrs). The control showed distinct lack of granulation tissue response. Scanning electron micrograph of experimental wound with L-lysine HCI injected intraperitoneally (10% w/v) also showed extensive cellular expansion compared to matched control (figs.1 ,2, 3 in page 1/7).

Results:

The result of these experiments is illustrated in figures 1 to 3. In primary closure of wounds, under the influence of this amino acid and its derivatives/oligomers/analogues the repair process is characterized by more of cellular division process (e.g. in neonatal repair process) rather than by the predominant matrix laying (as is the feature of adult repair process). Topical as well as systemic applications support faster and better scarless healing. Example 17

To prove the long time cosmetic effect of Lysine induced quality of healing, SEM of 4- month-old experimental wound with L-lysine HCI preparation applied topically was compared with 4-month-old control. Extensive deformation of the control wound compared to the experimental healing was observed. SEM analysis of tissue just below the surface wound (4 month old) in experimental wound and tissue below the control (deformed) wound showed substantial non-specific fibrosis in tissues below the control wounds, resulting in scar retraction whereas experimental wound showed a high degree of absence of non-specific fibrosis. This non-specific fibrosis in control wound causes downward pulling of surface tissue resulting in contracture and deformation. The wound-healing agent in its topically applicable forms is therefore of value in the prevention of scarring in primary and chronic wounds, (figs. 4-11 in pages 2/7,3/7,4/7). Most preferred optimum amounts incorporated in the experimental wounds, at the time of surgery (4 months back) ranged between 0.25 to 100 mg/sq.in of the wound.

Results:

Degree of non-specific fibrosis in the wound area was much more in control wounds compared to lysine mediated healing. Deformation and scarring of healed primary wounds, in_absence of lysine, was substantial, compared to lysine augmented healing. This scarring and deformation in the control wound was the result of non-specific (non- orderly) fibrous tissue net formation below the wound area. This was totally absent in the lysine treated wounds.

Example 18

As glycine is present at every third position in the primary sequence of Collagen, 14C- Glycine incorporation in the wound tissue was supposed to give an idea of differential collagen synthesis in experimental and control wounds. Free skin glycine incorporation from control and experimental animals indicated a basal incorporation and collagen synthesis while in control and experimental wounds, glycine incorporation showed enhancement compared to non-wounded skins respectively. Comparatively low incorporation in experimental wound in presence of L-lysine HCI 100% (w/w) applied topically (in the most preferred optimum amount of 0.25 to 100 mg/sq.in) was noted (illustrated in fig. 12 page 4/7).

Result:

14C-Glycine incorporation was a distinct and definite way of showing the degree of collagen formation in the wound areas versus non-wound areas in the two groups. Clearly, 14C-Glycine incorporation pattern (data) corroborated well with lysine induced less non-specific fibrosis resulting in less scarring.

Example 19

Non-specific protein synthesis pattern following topical application of 100% (w/w) powder formulation to acute clean-cut surgical wound was exhibited by 35S-Methionine incorporation profile 72 hrs post surgery in both experimental and control wounds (no application). Significant difference in the 35S-Methionine incorporation in the experimental wound tissue was noted indicating a probable surge in overall enhancement in protein synthesis, required for rapid cellular division in situ (fig.13 in page 5/7). Results:

Significantly more 35S-Methionine incorporation in amino acid treated (0.25 to 100 mg/sq.in) wound area indicated enhanced synthesis of protein overall. This is a basic requirement of rapid cell division and expansion in the wound bed which is one of the bench marks of the present invention. Example 20

Histopathology of experimental and control animal wounds (X 200) were performed in experimental (following topical application) and control animals. Significant dermo- epidermal thickening in the experimental group with topically applied lysine HCI was noted. Non-uniform thickening was possibly due to non-uniform application of the dry powder. (0.25 to 100 mg/sq.in) (figs.14, 15 in page 5/7).

Results: The amino acid formulation applied topically in acute model animal wound (primary closure) resulted in significant and remarkable thickening of dermo-epidermal layers showing cellular proliferation primarily in the wound bed under the influence of amino acid followed by wound remodeling which mimics fetal/neonatal repair processes. Example 21

Topically applied L-lysine HCI alone induced extensive angiogenesis and optimum inflammatory cell migration in the chronic wound bed following 48-72 hourly interval free topical application and dressing (10 days post institution of therapy) (X200). Wound bed showed very "passive" capillary structures and near total absence of inflammatory cells before starting the L-lysine HCI topical treatment (7 weeks old bedsore). (Optimum amount of 0.25 to 100 mg/sq.in of the wound bed) (figs.16, 17, 18 in page 6/7).

Results:

Topical application of the amino acid alone resulted in extensive angiogenic response within the first week thereby promoting formation of healthy granulation tissue and faster healing. The wound was not at all covered with any form of antibiotic preparation during the amino acid therapy.

Example 22

Topically applied monomeric lysine preparation (alone) induced extensive angiogenic response and formation of healthy granulation tissue within 7 to 10 days of institution of therapy in cases of chronic and protracted human wounds. (0.25 to 100 mg/sq.in)(fιgs.19,20 in page 7/7). Results:

The amino acid formulation was sprinkled freely as shown in left-hand photograph and the wound was dressed every 48-72 hours. The shown wound here was not covered with any antibiotic during the therapy period with the amino acid. Example 23

Topical lysine formulation (alone) therapy in case of infected amputation wound. The healing agent was applied topically at 48-72 hourly intervals with the wound being kept moist, (figs.21 ,22 in page 7/7).

Results:

Healthy granulation tissue and angiogenesis within 10 days. The wound was not protected with any antibiotic during the period.

Example 24

A 25 year old male, apparently otherwise healthy approached with a complaint of rapid loss of hair and frontal alopecia and baldness. He had a family history of baldness. The patient used the dry powder form of monomeric L-lysine HCL (each time 5 gm approximately, once a day before going to bed) made into a thin paste with water just before hand application of the preparation to the scalp with gentle rubbing and massaging for 15 min. Immediately on starting the topical application therapy the nonspecific regular hairloss was reversed and on 4 weeks (once a day) application, significant growth of visible hair in all the areas of scalp was evident. During this period, the subject did not use any other medication -oral or locally applicable - for hair growth.

Example 25

Parenteral (sub-cutaneous injection, of 1 ml on alternate days for a total period of 21 days of L-lysine HCL monomer at concentration of 5 mg/ml) coupled with antigen (dead M.TB, BSA) (1 μg/ kg of the body weight in animals on 0 day and 7 th day) enhanced the antibody titre (against the antigen employed for inoculation). The enhanced Ab (polyclonal antibody) response was obvious from 12th day onwards and antibody titre was very significantly different (higher) than the matched controls. Antibody estimation was carried out utilizing Radioimmunoassay technique (RIA). Also, CD4+ and CD8+ cell population in peripheral circulation was augmented significantly following the application protocol of antigen and L-lysine HCL. L-lysine HCL augments both the antibody response (titre) as well as the cell-mediated immunity, as was evident from augmentation of CD4+ cell counts in peripheral circulation, in both MHC-I and MHC-II loaded antigen (M.TB as well as BSA respectively). Results:

Lysine acts as a biocompatible low molecular weight adjuvant with significant role in the formulation of new vaccines.

Also, the antibody titre as well as CD4+ and CD8+ cell population in peripheral circulation was augmented significantly in experimental animals (mice) compared to controls, following putting back in circulation (of approximately 25 % of total WBC population in one cycle) of lysine mediated antigen (M.TB) exposed (approximately for a period of 48 hours in culture) buffy coat cells (total WBC population) in presence of subsequent antigen challenge.

L-lysine HCL significantly augments the humoral and cell-mediated immunity in adoptive immune therapy, which is becoming significantly and singularly important in cancer vaccinology. In this invention, significant role(s) of lysine in adoptive immune therapy, directed towards infective disease vaccine (in this case, M.Tuberculosis) development is described for the first time which has not been reported anywhere till date.

Prior Art Citations from Scientific Literature

1) Zhou J, Song Y, Wang Y (1997 Sep) The study of epidermal growth factor in acceleration of skin wound healing. Chung Kuo HsinFu Chung Chien Wai Ko Tsa Chin; 11 (5): 267-8.

2) Breitbart AS, Mason JM, Urmacher C, Barcia M, Grant RT, Pergolizzi RG, Grande PA (1999 Dec) Gene enhanced tissue engineering : applications for wound healing using cultured dermal fibroblasts transduced retrovirally with PDGF-B gene. Ann Plast Surg; 43(6): 632-9.

3) Liechty KW, Nesbit M, Herlyn M, Radu A, Adzick NS, Crombleholme TM (1999 Sep) Adenoviral- mediated overexpression of PDGF-B corrects ischemic impaired wound healing. J. Invest Dermatol : 1 13(3): 375-83.

4) Jaschke E, Zabernigg A, Gattringer C (1999 May) Recombinant human Granulocyte- macrophage colony stimulating factor applied locally in low doses enhances healing and prevents recurrence of chronic venous ulcers. Int J Dermatol; 38(5): 380-6.

5) Grzybowski J, Oldak E, Janiak MK (1999)- Local application of Granulocyte-colony stimulating factor, Granulocyte-macrophage colony stimulating factor and Epidermal growth factor in treatment of wounds. Postepy Hig Med Dosw; 53(1 ): 75-86.

6) Junienez PA, Rampy MA (1999 Feb) Keratinocyte growth factor - 2 accelerates wound healing in incisional wounds. J Surg Res; 81(2): 238-42..

7) Ono I, Pateshita T, Inoue M (1999) Effects of a collagen matrix containing basic fibroblast growth factor on wound contraction. J Biomed Mater Res; 48(5): 621-30.

8) Pandit A, Ashar R, Feldman D (1999 Mar-Apr) The effect of TGF-β delivered through a collagen scaffold on wound healing. J Invest Surg; 12(2): 89-100.

9) Trengove NJ, Stacey MC, Mac Auley S, Bennet N, Gibson J, Burslem F, Murphy G, Schultz G (1999 Nov- Dec) Analysis of acute and chronic wound environments : the role of proteases and their inhibitors. Wound Repair Regen; 7(6):.442-52.

10) Ruszczak Z, Schwartz RA (1999) Collagen uses in dermatology - An update. J Dermatol; 199(4): 285-9.

11) Baichey G, Penkoya R, Deliiski T (1995) The treatment of slowly healing wounds with collagen and growth factors. Khururgiia (Sofiia); 48(3): 17-19.

12) Jia C, Chen B, Arnold F (1998 Jul) The effect of ultrapure hyaluronic acid with different molecular weights on the healing of porcine full thickness skin wound. Chung Kuo Hsiu Fu Chung Chien Wai Ko Tsa Chih; 12(4): 197-200.

13) Moulin V, Lawny F, Barritault D, Caruelle JT (1998 Sep) Platelet release treatment improves skin healing in diabetic rats through endogenous growth factor secretion. Cell Mol Biol (Noisy-le-grand); 44(6): 961 -71.

14) Kyriahides TR, Tain JW, Bomstein P (1999 Nov) Accelerated wound healing in mice with a disruption of thrombospondin 2 gene. J. Invest Dermatol; 113(5): 782-7.

15) Ashcrott GS, Yang X, Glick AB, weinstein M, Lelterio JL, Mizel DE, Anzan OM, Greenwell- Wild T, Wahl SM, Deng C, Roberts AB (1999 Sep) Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol; 1(5) : 260-6.

16) Kratz G, Back M, Amander C, Larun O (1998 Dec) Immobilised heparin accelerates the healing of human wounds in vivo. Scand J Plast Reconstr Surg hand Surg 1998 Dec; 32(4): 381-5.

17) Cho YW, Cho YN, Chung SH, Yoo G, Ko SW (1999 Nov) Water soluble chitin as wound healing accelerator. Biomaterials; 20(22): 2139-45.

18) Tawil NJ, Connors D, Gies D, Bennet S, Gruskin E, Mustoe T (1999 Sep-Oct) Stimulation of woun healing by positively charge dextran beads depends upon clustering of beads and cells in close proximity to the wound. Wound Repair Regen; 7(5): 389-99.

19) Malinda KM, Sidhu GS, Mani H, Banaudha K, Maheshwari RK, Goldstein AL, Kleinman HK (1999 Sep) Thymosin beta 4 accelerates wound healing. J. Invest Dermatol; 113(3): 364-8.

2Q) Talwar M,_ Mόyana TN; Bharadwaj B7 Tan LK' (1996 Sept-Oct) The effect of synthetic analogue of PGE2 on wound healing in rats. Ann Clin Lab Sci; 26(5): 451-7.

21) Lansdown AB, Sampson B, Laupattarakasam P, Vultivirojana A (1997 Nov) Silver aids healing in the sterile skin wounds : experimental studies in Laboratory rat. Br. J Dermatol; 137(5): 728-35. 22) Ashcroft GS, Greenwell-Wild T, Horan MA, Wahl SM, Ferguson MW (1999 Oct) Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response. Am J Pathol; 155(4): 1137-46.

23) Sidhu GS, Singh AK, Banaudha KK, Gaddipati JP, Patuaik GK, Maheshwari RK (1999 Nov) Arnebin-1 accelerates normal and hydrocortisone induced impaired wound healing. J Invest dermatol; 113(5): 773-81.

24) Sidhu GS, Mani H, Gaddipati JP, Singh AK, Seth P, Banaudha KK, Patnaik GK, Maheshwari RK (1999 Sep-Oct) Curcumin enhances wound healing in streptozotocin induced diabetic rats and genetically diabetic mice. Wound Repair Regen; 7(5): 362-74.

25) Rasik AM, Raghubir R, Gupta A, Shukla A, Dubey MP, Srivastava S, Jain HK, Kulshrutha DK (1999 Dec 15) Healing potential of Calotropis procera on dermal wounds in Guinea pigs. J Ethnopharmacol; 68(1-3): 261-6.

26) Laham RJ et. al. (1999 Nov 2) Local perivascular delivery of basic fibrobiast growth factor in patients undergoing coronary bypass surgery; results of a phase I randomized, double-blind, placebo-controlled trial. Circulatiokn; 100(18): 1865-71.

27) Laham RJet. al.(2000)Jntrapericardial delivery of FGF2 induces neo-vascularisation in a porcine model of chronic myocardial ischemia. J Pharmacol Exp Ther Feb 292(2); 795-802.

Claims

Claims Use of the cationic amino acid l-lysine and/or d-lysine and/or oligo-lysine (both I- and d- isomer) (most preferably upto molecular weight 1000) and/or lysine (both I- and d-) analogues (R- group elongated upto C-12) either alone or in various combinations thereof as biocompatible, potent adjuvant wound healing agents administered topically either alone or with one or more of the compounds as in example 13 (topical dosage / applied amounts ranges between 0.25 to 100 mg of the active ingradient(s) /sq.in of the wound area) or through i.v. route or orally — singly or through formulations (oral and parenteral dosages to be adjusted in such a way that most desired sustained bioavailability/blood level of the amino acid / analogues/ oligomers ranges between (approximately) 35 μg/ml to 155 μg/ml to support and enhance wound healing and angiogenesis (collateral formation) through a process of cell division and endothelial cell migration/division resulting in an augmented angiogenic response and optimum level of cell(s) and serum derived growth factor(s)' infiltration in the wound/ ischaemic area, resulting in qualitatively better and much quicker wound healing (and angiogenesis), with less scar and deformation in protracted wounds (chronic) as well as acute wounds, (including leprotic ulcer, longstanding diabetic foot ulcer, decubitus ulcer, etc. and including other clinical conditions, e.g. ischaemic myocardium, and cerebral tissue as well as some solid tumors.)
The application modes in claim 1 include treatment using the angiogenic/healing agent(s) as such or in formulations in the forms of powder, paste, gels, foams, drops, incorporated in strips, bandage, tapes, gauze, injectables, oral preparations and their like for its/their optimal angiogenic and controlled inflammatory response leading to augmented healing and collateral circulation development in ischaemic tissue (Myocardium/ cerebral tissue / or chronic wound bed etc.)
The total amount of active ingredient(s) in these above formulations as in (2) above and modes of application(s) in the form(s) of lysine, monomeric (both I- and d- configurations) and oligomers (upto molecular weight 1000), analogues (upto R group length of C-12), analogue oligomers (dp- upto 5 to 6) and salts thereof, either alone or in various combination(s) either in presence or in absence of additive molecule(s) / extracts / compounds in the form of a formulation, would range from 0.1 to 100% (either one component or in combinations) per unit volume / per unit weight of the formulation or per unit area of the wound or preparation (strip/bandage/tapes/gauze etc) or per unit piece (e.g. tablets/capsule etc.) depending on the formulation and application in question.
4. Use as in claims 1 & 2, were monomeric lysine and various salts thereof, lysine analogues (as mentioned in claim 3) and salts thereof, short oligomers and salts thereof, either in various combinations or alone, in presence and/ or in the absence of one or more of the active molecules/ compounds/ extracts, as mentioned in example 13, helps in the growth of new hair and prevents loss of hair.
5. Use according to claims 1- 4, of lysine, oligomers, analogues and various salts thereof, either alone or in combinations, in presence and / or absence of one or more of the molecules/ compounds / extracts from the list as in example 13, causes healing of animal wounds (e.g. hump sores, foot mouth ulcers and others etc.) as well as animal clinical conditions (e.g. Mastitis etc.).
6. Use according to claims 1-5, where lysine, analogues/ derivatives, oligomers and various salts thereof either alone or various combinations augment the humoral and cell - mediated immunity following Mycobacterium Tuberculosis (and other related organisms) innoculation, thereby acting as a low molecular weight biocompatible adjuvant molecule for the development of new vaccine(s) against M. Tuberculosis infection.
7. Use according to claims 1-6, where lysine analogues/ derivatives, oligomers and various salts thereof either alone or in various combinations augments the process of adoptive immune therapy against M.Tuberculosis infection. These active ingradient(s), either alone or various combinations, also augment the adaptive immune- therapy process in other infective disease conditions. This constitutes a new mode of vaccination against various infective agents (which give rise to various infective diseases).
8. Use according to claims 1-7, wherein lysine and its various salts, oligomers and salts thereof as well as analogues/ derivatives and various salts thereof, either alone or in various combinations, when applied systemically through oral or parenteral routes or a combination of the two, giving rise to a sustained blood level of 30 μg/ml to 180 μg/ml, preferably 30 μg/ml to 170 μg/ml, more preferably 30 μg/ml to 160 μg/ml and most preferably 35 μg/ml to 155 μg/ml, result in an augmentation of angiogenic / neovascularization response in ischemic tissue (e.g. ischemic myocardium, ischemic cerebral tissue etc), resulting in a new mode of therapy involving controllable therapeutic angiogenesis induced by an extremely biocompatible and extremely low cost molecule / its derivatives / oligomers/ analogues in sharp contrast to the existing extremely complicated and high cost therapy ( in angina cases).
9. Use according to claim 1-3 wherein the active ingredient(s) mentioned can be , applied to clinical conditions which include all types acute wounds (infected / uninfected)- e.g. all surgical wounds, cosmetic surgery wounds, burns (1st and 2nd degree mostly), cuts, bruises, lacerations etc., and other acute infected wounds, as well as ail chronic wounds- infected or otherwise ( e.g. diabetic ulcers/ wounds, decubitus ulcers and other pressure sores, leprotic ulcers, venous ulcers etc.), and various internal wounds with systemic formulations ( e.g. peptic ulcer, gastritis, deuodenai and gastric ulcers etc.).
10. Use of lysine and various salts, analogues / derivatives and salts thereof, oligomers and salts thereof as in claims 1-9, either alone or in various combination augment the production of polyclonal antibody in inoculated animal against any given antigen, as in claims 6 and 7.
11. Use of active ingredients as in claims 1-3 and 9 where in lysine and salts thereof, analogues / derivatives and salts thereof and oligomers and salts thereof of maybe employed alone or in combinations either in presence or absence of one or more of the additive molecules /compounds / extracts as in e.g. 13, for topical application therapy of various kinds of wounds.
12. Forms of formulations in skin wounds repair involving the said active ingradients (one or more) as in claims 1-11 either in presence or absence of one or more additive molecules/ compounds/ extracts maybe from a list including wet dressings, lotion, solutions, drops, tincture, aerosols, soap, bath, shampoo, powder, cream, gel, ointment, fixed dressing, paste, occlusive dressing and semi occlusive dressing, hydrocolloid, alginate, foam or a combination thereof. 13. Use according to claims 1-12 wherein total content of the active ingredient(s), one or more, comprises from 0.1 to 100%, preferably from 0.1 to 20.0%, more preferably from 1.0 to 10.00%, even more preferably from 5.00 to 10.00% and most preferably 10.00% (approximate) (w/w or w/v depending on the formulation in question), in presence and / or absence of one or more of the additive compounds/ molecules/ extracts as in example 13.
PCT/IN2001/000105 2001-05-23 2001-05-23 Lysine and/or analogues and/or polymers thereof for promoting wound healing and angiogenesis WO2002094256A1 (en)

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US11/760,972 US20070243135A1 (en) 2001-05-23 2007-06-11 Method of treating fetal growth retardation and placental ischaemia and insufficiency
US12/045,814 US20080153910A1 (en) 2001-05-23 2008-03-11 Lysine in therapeutic angiogenesis for cardiac and cerebral/neural reperfusion

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WO2009078040A2 (en) * 2007-12-17 2009-06-25 Debatosh Datta Compounds inducing reperfusion in ischaemic tissues
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US10058642B2 (en) 2004-04-05 2018-08-28 Bluesky Medical Group Incorporated Reduced pressure treatment system
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WO2009078039A3 (en) * 2007-12-17 2010-11-25 Debatosh Datta Compound inducing angiogenesis response in ischaemic tissues
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