4. DESCRIPTION:

Introduction

Infectious diseases including wound infections are a major concern worldwide and the bacterial infection predispose wounds to prolonged inflammatory conditions which contribute to delay the wound healing. Henee topical applications of effective antimicrobial agents are needed to reduce both the risk of infection and overall time required for wound healing. Despite antimicrobial chemotherapy, management of wound infections caused by Gram-negative pathogenic bacteria of P. aeruginosa is difficult due to the emergence of resistance to the currently available antibiotics and some conventional antibiotics reléase virulence factors which stimulate the overproduction of local pro-inflammatory cytokine such as TNF-a which may hamper wound healing.
TNF-a can act beneficial or deleterious effect in a dose dependent manner showing the importance of accurate cytokine regulation in wound healing.

So, prolonged expression of TNF-a inhibits angiogenesis (VEGF-A) and it further promotes tissue degradation or decreases collagen deposition (COL-1) and its crosslinking resulting in delayed wound healing through higher production of matrix metalloproteinase (MMP) especially MMP-9. Therefore, alternative antimicrobial agents are needed for the treatment of skin infection caused by P. aeruginosa and this has prompted us to an intensive search for suitable topical anti-infective agent and to focus on preparations of such an agent that can kill microbes and promote tissue regeneration whüe limiting drug resistance and tissue damage.

Concern about drug resistance and toxicity of the available topical anti-infective agent to make hybrid molecule strategy has drawn increased attention in drug design and development. Conceptually, this strategy incorporates structural features that are essential to the biological activities of different drug structures into a single molecule. Compared to the combination of antibiotics, hybrid molecules may provide certain advantages: (1) enhanced poteney by self-synergy within one molecule; (2) reduced risk of developing drug resistance; and (3) reduced toxic side effeets compared to the administration of múltiple agents. Herein, we report the preparation of a new antimicrobial complex tyrocidine hydrochloride+Ag complex using tyrocidine hydrochloride and silver nanoparticles as potential treatment agent for chronic wound infection.

Summary of the Invention

The present invention involves the preparation of topical tyrocidine hydrochloride+Ag complex to be used in the treatment of wound infections composed of tyrocidine hydrochloride and silver nanoparticles. Tyrocidine hydrochloride+Ag complex effectively suppressed the growth of drug resistant P. aeruginosa in in vitro and in vivo more over it down-regulates the pro-infiammatory cytokine such as TNF-a as a results the dysregulated MMP-9 and its inhibitor TIMP-1 expressions were restored in normal level leads to prober collagen deposition and its crosslinking resulting re-epithelization in infected wounds not found otherwise in its individual constituents namely tyrocidine hydrochloride and silver nanoparticles. The present study demonstrated that topical application of tyrocidine hydrochloridei|~Ag complex enhanced re-epithelization in infected wounds to reduce the wound healing time of acute wounds.

Background of the Invention

Wound infection is the major difficulty in the field of wound care management, because such infections can retard wound healing by causing a wound environment not conducive to healing. The remedy for the above mentioned harms would be the use of topical antimicrobial compounds. Antimicrobial ointments such as silver sulfadiazine, bacitracin, fusidic acid, mupirocin and neomycin sulfate are used to reduce the risk of infection in the wounds. However these antimicrobials have some adverse effects, interference with wound healing or induction of drug resistance. An acceptable topical. anti-infective agent would need to demónstrate activity against the spectrum of bacteria that are known to cause and aggravate wound infection leading to serious adverse effects. So, the development of alternative therapeutic approaches has received special attention including the formulations/combination of antibiotics with different modes of action for topical treatment of wound infections.

In literature, the antimicrobial peptide such as tyrocidine hydrochloride isolated from Brevibácillus brevis and the inorganic antimicrobial agent silver nanoparticles are well known for treatment of wound infection, but, there is no report on synergistic effect between tyrocidine hydrochloride and silver nanoparticles on microbial pathogens. In this study, we demónstrate a synergistic effect between tyrocidine hydrochloride and silver nanoparticles towards clinical P. aeruginosa isolates.

The novelty of this invention relates tp the development of a new antimicrobial complex tyrocidine hydrochloride+Ag complex for treatment of wound infection to aid wound healing through down regulation of pro-inflammatory cytokine such as TNF-a leading to the restoration of dysregulated MMP-9 expression. The topical antimicrobial agent described herein could lead to a new spectrum of antimicrobials useful in the treatment of chronic infected wounds.

Objectives of the Invention

♦ The main objective of this present invention is to prepare a topical antimicrobial agent useful for treatment of wound infection.

♦ The second objective is to provide a process of functionalization- of tyrocidine hydrochloride with silver nanoparticles to develop a new topical antimicrobial agent such as tyrocidine hydrochloride+Ag complex.

♦ The third objective is to analyze the therapeutic valué of tyrocidine hydrochloride +Ag complex in in vivo using excision-wound model in Swiss albino mice.

♦ The fourth objective is to analyze the gene expressions profile of infected and treated wounded skin.

♦ The fifth objective is to demónstrate speedy recovery with minimal scarring to rapidly restore the structural and functional properties to the levéis of normal tissue, involving re- epithelization and orchestrated regeneration of the skin appendages.
Methodology

Preparation and Characterization of Tyrocidine Hydrochloride+Ag Complex
For preparation of new antimicrobial complex such as tyrocidine hydrochloride +Ag complex, the raw materials such as tyrocidine hydrochloride and silver nanoparticles are extracted and synthesized from the bacterial strain Brevibacillus brevis KN8(2).

Tyrocidine hydrochloride (1 mg/ml) and silver nanoparticles (10 ug/ml) are mixed at the desired ratio (1:1) without functionalizing agents to avoid its negative effects, and then the mixture is gently stirred for 2 h and incubates at room temperature to self-assemble the tyrocidine hydrochloride with silver nanoparticles for 24 h. After the incubation period the colour of the reaction mixture turned from • yellow to white (Fig.l) indicates the surface functionalization between tyrocidine hydrochloride and silver nanoparticles.
Preliminary to confirm the surface functionalization between tyrocidine hydrochloride and silver nanoparticles, the reaction mixture is subjected to UV-Visible spectroscopy, and this shows that there are significant changes in the UV-Visible absorption spectrum of tyrocidine hydrochloride i.e., the second peak 381 did not shift whereas the first one slightly shifted from 212 to 211 nm with a new peak at 377 nm corresponding to silver nanoparticles was also obtained (Fig.2). The appearance of new peak at 377 nm indicates successful surface functionalization occurring between tyrocidine hydrochloride and silver nanoparticles.

FT-IR spectrum of the reaction mixture shows there are a slight changes in the functional groups of tyrocidine hydrochloride in the finger print región such as ester group 474.81 región which is broadened and shifted to lower wavelength 466.29 (Fig. 3).
After tyrocidine hydrochloride functionalization with silver nanoparticles a considerable modification occurs on the shape and size of silver nanoparticles which is clearly evinced from FESEM. FESEM image of nanosilver shows cube shaped particles whereas FESEM of tyrocidine hydrochloride functionalization silver shows rectangular rod shaped particles (Fig. 4).

For confirmation of surface functionalization between tyrocidine hydrochloride and silver nanoparticles, EDAX spectroscopy analysis is performed. The EDAX spectrum of nanosilver shows only silver signature peaks, whereas the EDAX spectrum of tyrocidine hydrochloride functionalized nanosilver shows the presence of both silver and chlorine.

These two elements are indicative of silver particles and the tyrocidine hydrochloride in the reaction mixture further confirms the functionalization (Fig.5).

On the basis of spectral data including UV-Visible absorption spectrum, FTIR, EDAX, spectra the structure of the conjugated antimicrobial compound developed from the raw materials tyrocidine hydrochloride and silver nanoparticles is proposed as tyrocidine hydrochloride+A8 complex (Fig. 6).

The antibacterial activity of tyrocidine hydrochloride is increased in the presence of silver nanoparticles against P. aeruginosa (Fig. 7). The increase in fold área of silver nanoparticles fiínctionalized tyrocidine hydrochloride is 12.00% indicating that silver nanoparticles fiínctionalized tyrocidine hydrochloride has additive/synergistic antibacterial activity compared to its individual constituents such as tyrocidine hydrochloride and silver nanoparticles.

Skin Wound Preparation, Histological Observation and Reverse Transcriptase-Polymerase Chain Reaction Analysis
Swiss albino mice were anesthetized by i.p. injection of ketamine (24 mg/kg of body weight) and the dorsum was shaved and excisión wonnds (1.5 X 1.5 cm) were created on the upper skin of each mouse using sharp scissors and scalpel. Pseudomonas aeruginosa strain was grown in Luria-Bertani (LB) médium. Aliquots (50 ul) of overnight cultures were sub cultured in fresh LB broth and grown at 37°C for 4 h to an optical density of approximately 0.9 at 600 nm. A 100 ul aliquot of each culture was then pelleted, washed in phosphate-buffered saline (PBS) and serially diluted (tenfold serial dilutions) in PBS.

100 ul aliquot of the 10"3 dilution (equivalent to approximately 104 CFU) was applied topically to the wound of each mouse. Then mice those that showed signs of infection at wounds were treated topically with different concentration of 75 \ú of the various antimicrobial agents such as tyrocidine hydrochloride+Ae complex (500+10 ug/ml) and its individual constituents such as tyrocidine hydrochloride (500 ug/ml), and silver nanoparticles (10 ug/ml) once daily for a period of 6 days. At the end of the experiment all mice in each group were euthanized by i.p. administration of 90-120 mg/kg of ketamine. Skin tissues were collected from all groups of animáis for histopathological and gene expression analysis. The ultímate aim for the treatment of skin wounds is to rapidly restore the structural and functional properties to the levéis of normal tissue, involving re-epithelization, and orchestrated regeneration of all the skin appendages. The histological sections of the tyrocidine hydrochloride+Ag complex treated group exhibited complete re-epithelialization at 16 days indicating a similar epithelial structure with the normal skin in contrast the epidermis in tyrocidine hydrochloride, silver nanoparticles treated groups and control groups was still partially unclosed (Fig. 8).

The pro-inflammatory cytokine such as TNF-a and MMP-9 play an important role in wound healing; the gene expression pattem of TNF-a and MMP-9 were analyzed by reverse transcriptase-polymerase chain reaction. When wounds were cióse to re-epithelization by day 16, the expression pattem of TNF-a and MMP-9 in tyrocidine hydrochloride+Ag complex treated skin was restored to the normal level comparable with that obsérved normal skin in contrast expression pattern of TNF-a and MMP-9 remains high (Fig. 9) in tyrocidine hydrochloride and silver nanoparticles treated groups.

Merits of the Invention

• Preparation of raw materials for development of the hybrid molecule is very easy and cheaper.

• The chemical reaction process for preparation of hybrid molecule is very simple.

• Hybrid molecule has synergistic effect within one molecule that may not be achievable by a traditional combination of múltiple antibiotics.

• Topical antibiotics provide high local concentration at the site of infection.

• Hybrid molecules may provide the advantages of cost-benefit ratio.

• Hybrid moletule reduced risk of developing drug resistance and toxic side effects.

• Hybrid molecule can promote re-epithelialization in wounded skin to shorten the wound healing time.

• Hybrid molecule heals the wound with minimal scarring and maximal function.

• Hybrid molecule alleviates TNF-a and MMP-9 activities in the wounded skin.

Demerits of the Existíng Methods

• Tyrocidine hydrochloride in alcoholic solutions causes less skin irritation temporarily.

• The significant percutaneous absorption of silver may lead to argyria conditions.

5. CLAIMS:

What claimed is:

1. The process for preparation of pharmaceutical compositions for development of hybrid molecule using the raw material tyrocidine hydrochloríde and silver nanoparticles to control Pseudomonas aeruginosa infected skin wound.

2. A pharmaceutical composition as claimed in claim 1, wherein the said composition comprising the therapeutically effective amount of tyrocidine hydrochloríde and silver nanoparticles.

3. A pharmaceutical composition as claimed in claim 2, wherein the dosage of the said composition is administered at a unit dose of at least 500+10 ug/kg body weight.

4. A pharmaceutical composition as claimed in claim 1, wherein the said composition comprising the therapeutically effective amount of tyrocidine hydrochloríde and silver nanoparticles for synergistic effect to control Pseudomonas aeruginosa infected skin wound.

5. A pharmaceutical composition as claimed in claim 3, wherein the said tyrocidine hydrochloríde is present in the amount of 500 units per gram.

6. A pharmaceutical composition as claimed in claim 3, wherein the said silver nanoparticles is present in the amount of 10 units per gram.

7. A pharmaceutical composition as claimed in claim 1, wherein the dosage of the said composition is administered in soluble form preferably in suspensión form.

8. A pharmaceutical composition as claimed in claim 1, wherein the carrier used is selected from the group consisting of ethanol and water.

9. A pharmaceutical composition as claimed in claim 1, wherein the administration route is selected from the group consisting oftopical.

10. A pharmaceutical composition as claimed in claim 1 wherein the said composition
comprising the therapeutically effective amount of tyrocidine hydrochloríde and silver
nanoparticles down-regulates the pro-inflammatory cytokine such as TNF-a.

11. A pharmaceutical composition as claimed in claim 1 wherein the said composition comprising the therapeutically effective amount of tyrocidine hydrochloride and silver nanoparticles can promote re-epithelialization in wounded skin to reduce the wound healing time with minimal scarring and maximal function of skin.

12: A pharmaceutical composition as claimed in claim 1 wherein the said composition comprising the therapeutically effective amount of tyrocidine hydrochloride and silver nanoparticles can alleviate dysregulated MMP-9 activities in the wounded skin to aid in wound healing.