A PROCESS OF PREPARATION OF TOPICAL GEL FOR TREATING METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS INFECTION AND PRODUCTS THEREOF

Abstract

TITLE: A PROCESS OF PREPARATION OF TOPICAL GEL FOR TREATING METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS INFECTION AND PRODUCTS THEREOF APPLICANT: KARUNYA INSTITUTE OF TECHNOLOGY AND SCIENCES ABSTRACT The present invention discloses a process of preparation of topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections comprises of following steps; a. Preparation of BiVO4 nanoparticles comprises of mixing Bismuth nitrate pentahydrate dissolved in acetic acid: ethanol and water and Ammonium metavanadate dissolved in ammonia and water and maintaining pH at 7 followed by autoclaving and filtering with ethanol and distilled water and placing obtained residue in hot air oven followed by grinding to form BiVO4 nanoparticles; b. Preparation of topical formulation comprises of melting Petroleum jelly in water bath and adding the BiVO4 nanoparticles followed by mixing thoroughly until homogenous to form topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections. The present invention also discloses a topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections prepared by the process as described above.

Specification

Description:Form 2


THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)






"A PROCESS OF PREPARATION OF TOPICAL GEL FOR TREATING METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS INFECTION AND PRODUCTS THEREOF"






in the name of KARUNYA INSTITUTE OF TECHNOLOGY AND SCIENCES an Indian national having address at KARUNYA INSTITUTE OF TECHNOLOGY AND SCIENCES, KARUNYA NAGAR, COIMBATORE, COIMBATORE - 641114, TAMIL NADU, INDIA.


The following specification particularly describes the invention and the manner in which it is to be performed.
FIELD OF THE INVENTION:

The present invention generally relates to the field of medicine. Specifically, the present invention relates to a process for preparing topical formulation. More particularly, the present invention relates to a process for preparing topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections and product thereof.

BACKGROUND OF THE INVENTION:

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious pathogen that causes patient mortality. Over 50% of infections around the world are caused by MRSA. MRSA has emerged as a community-associated pathogen (CA-MRSA), usually resulting in skin infections with abscess formation and cellulites in individuals. Methicillin-resistant Staphylococcus aureus (MRSA) is a highly concerning pathogen due to its resistance to multiple antibiotics and its ability to cause severe hospital-acquired infections. The World Health Organization (WHO) has classified MRSA as a "priority pathogen" due to its potential to cause life-threatening diseases such as endocarditis, pneumonia, bacteremia etc. Over time, MRSA has developed various mechanisms of resistance to antibiotics, making it even more challenging to treat.

Staph infections are treated with a class of antibiotics called beta-lactams, which include methicillin, oxacillin, penicillin, and amoxicillin. MRSA infections, however, have become resistant to many of these common antibiotics. Accordingly, treatment of MRSA infections includes using a variety of antibiotics in which the bacteria has not developed any resistance, such as vancomycin, a glycopeptide antibiotic. It is the preferred treatment mechanism for patients suffering from serious MRSA infections, and is given orally or intravenously. Intravenous or oral administration of vancomycin is associated with a high degree of adverse reactions with the kidney and liver, and in some cases can result in lethal outcomes. These delivery mechanisms have not been useful for MRSA infections located on the skin or associated with wounds. Accordingly, a need exists for an effective MRSA composition which does not need to be given orally or intravenously.

There are reports available in the state of the art relating to formulations for treating Methicillin-resistant Staphylococcus aureus.

WO2012149116A2 discloses a formulation of liposomal vancomycin that is highly effective in the treatment of bacterial infections, and particularly MRSA infections. The present invention further teaches methods of using the formulations disclosed herein for the treatment of bacterial infections. The inventors determined that certain formulations disclosed herein are likely to result in lower toxicity than what is normally associated with vancomycin treatment.

WO2010002415A1 discloses a method of treating infection by methicillin-nonsusceptible bacteria, vancomycin-nonsusceptible bacteria, penicillin-nonsusceptibale bacteria, clarithromycin-nonsusceptible bacteria, or metronidazole-nonsusceptible bacteria by administering to a subject in need thereof an effective amount of a quinolone compound of the formula shown in the specification.

WO2013134225A1 discloses a vaccine formulation effective against Staphylococcus aureus, including Methicillin-resistant Staphylococcus aureus (MRSA) as well as methods of using the vaccine formulations in the treatment and prevention of S. aureus infections in a subject. A vaccine formulation comprising: (a) five S. aureus polypeptides, or portions thereof, or variants thereof, or combinations thereof, wherein the S. aureus polypeptides are (i) S. aureus polypeptide SA0037 set forth in SEQ ID NO: 13, (ii) 5". aureus polypeptide SA0119 set forth in SEQ ID NO: 14, (iii) 5". aureus polypeptide SA0486 set forth in SEQ ID NO: 15, (iv) 5". aureus polypeptide SA0688 set forth in SEQ ID NO: 16, and (v) S. aureus glucosaminidase set forth in SEQ ID NO: 17, and(b) a pharmaceutically acceptable carrier or diluent.
Though conventional treatments for MRSA infections exhibit advantages in the prior arts, they do have disadvantages such as ineffective and causing undesirable side effects.

Thus, there exists a need in the state of the art for an alternative composition with enhanced efficiency for treating Methicillin-resistant S. aureus (MRSA) infections.

Hence an attempt has been made to develop a topical gel formulation for providing an effective therapeutic intervention against MRSA infections overcoming the above said drawbacks.

OBJECTIVE OF THE INVENTION:

The main object of the present invention is to develop a process for preparing topical formulation.

Another object of the present invention is to develop a process for preparing topical gel formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.

Another object of the present invention is to prepare a topical gel formulation employing BiVO4 nanoparticles and Petroleum jelly.

Yet another object of the present invention is to prepare a topical gel formulation exhibiting enhanced antibacterial and anti-inflammatory activities.

Further object of the present invention is to utilize the developed topical gel formulation for providing an effective therapeutic intervention against MRSA infections.

SUMMARY OF THE INVENTION:

The present invention discloses a process of preparation of topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections comprises of following steps;

a. Preparation of BiVO4 nanoparticles comprises of mixing Bismuth nitrate pentahydrate dissolved in acetic acid: ethanol and water and Ammonium metavanadate dissolved in ammonia and water and maintaining pH at 7 followed by autoclaving and filtering with ethanol and distilled water and placing obtained residue in hot air oven followed by grinding to form BiVO4 nanoparticles;

b. Preparation of topical formulation comprises of melting Petroleum jelly in water bath and adding the BiVO4 nanoparticles followed by mixing thoroughly until homogenous to form topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.

The present invention also discloses a topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections prepared by the process as described above.
DETAILED DESCRIPTION OF THE INVENTION:

The present invention discloses a process of preparation of topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections and product thereof.

The BiVO4 nanoparticles used in this research were synthesized using solvo-thermal technique. The precursor materials used were Bismuth nitrate pentahydrate (Bi(NO3)3•5H2O) and ammonium metavanadate NH4VO3.

The present invention comprises of mixing (Bismuth nitrate pentahydrate (Bi (NO3)3•5H2O) dissolved in acetic acid: ethanol and water) and (Ammonium metavanadate (NH4VO3 dissolved in ammonia and water) and pH was maintained at 7 using ammonia solution and acetic acid and processed under specific conditions such as autoclaving, cooling, filtration etc. The obtained solution was filtered with ethanol and distilled water thrice and the filtered residue was kept in hot air oven. Then the obtained sample was grinded thoroughly to form BiVO4 nanoparticles.

Mixing the pure petroleum jelly melted in water bath with the BiVO4 nanoparticles until it becomes homogenous to form topical gel formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.

Table 1: Composition of topical gel formulation:

S.NO INGREDIENTS AMOUNT
1 Petroleum jelly 1gm
2 BiVO4 nanoparticles 0.01-0.02g

The prepared topical formulation of the present invention was then subjected to experimental studies to determine the antibacterial and anti-inflammatory activities.

The study utilized nulliparous, non-pregnant female BALB/c mice, aged 8 to 12 weeks, weighing between 25 and 35 grams. There were a total number of 30 animals, divided into five groups (Control groups - Group 1, Group 2 and Experimental groups - Group 3, Group 4, Group 5), consisting of six animals each as outlined in Table 2. Group 1 received solely petroleum jelly as the carrier, while Group 2 was administered linezolid, a standard drug, orally at a dose of 25mg/kg body weight. Group 3, Group 4, and Group 5 received 0.5%, 1%, and 2% topical formulation of the present invention (BiVO4 nanoparticles in petroleum jelly),respectively.

Table 2: Grouping of animals and the specific drugs administered to each group.

S. No Drug administered No. of animals
1. Carrier (Petroleum jelly) 6
2. Standard drug (Linezolid) 6
3. 0.5% Topical formulation 6
4. 1% Topical formulation 6
5. 2% Topical formulation 6
6. Total 30

Low dose: To achieve low dose (0.5% concentration), 0.01g of BiVO4 nanoparticles were mixed with 1.00g of petroleum jelly.

Medium dose: In this case (1% concentration), 0.01g of BiVO4 nanoparticles were mixed with 1.00g of petroleum jelly.

High dose: Here, (2% concentration) 0.02g of BiVO4 nanoparticles were mixed with 1g of petroleum jelly.

The experimental animals were anesthetized using isoflurane 24 hours prior to induction of MRSA infection. Hair on the dorsal lumbar side was removed with a trimmer to optimize injection site visualization and topical application. On Day 0, animals were anesthetized with isoflurane again, and the injection site was lightly swabbed with 70% ethanol before administering a 40 μl intradermal MRSA injection. The BiVO4 test doses were prepared by mixing the measured amount of BiVO4 nanoparticles with the necessary amount of pure petroleum jelly to achieve concentrations of 0.5%, 1% and 2%. These doses were applied topically to the animals over 5-day duration to treat MRSA infection.

The wound site underwent a gentle swabbing with 70% ethanol, and tissue samples were collected from the animals 24 hours later to quantify bacterial load. Subsequently, the wound, measuring approximately 1cm2, was excised for bacterial enumeration after homogenization in 1 mL of tryptic soy broth. Following tissue homogenization, the levels of cytokines (IL-6, TNF α, IL-6, and MCP 1) produced by MRSA were assessed, and the results of the experimental groups were compared with those of the control group to determine the effectiveness of topical formulation of the present invention (BiVO4 nanoparticles in petroleum jelly).

Observations of the animals were conducted at 24 hours and 48 hours post treatment. Skin samples collected from mice infected with the pathogen underwent centrifugation at 4000 rpm for 10 minutes, and the resulting supernatants were used for analyzing cytokine levels. ELISA kits were employed to detect specific cytokines including Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-1beta (IL-1β), and Monocyte Chemoattractant Protein-1 (MCP-1), following the manufacturer's protocol. Statistical analyses were conducted using GraphPad Prism 6.0 software. Two-tailed Student's t-test was employed to calculate p-values, with a significance level set at p < 0.05 to determine statistical significance of the results.

It was evident from the results that there was considerable improvement in wound healing in experimental groups compared to the control group that received petroleum jelly alone. The main objective of the study was to evaluate the levels of four cytokines across the study groups. In the MRSA control group, which received only carrier petroleum jelly, the levels of all four tested cytokines - IL-6, TNF-α, IL-1β, and MCP-1 appeared to increase. Conversely, in experimental groups, including those receiving different concentrations of topical formulation, the levels of these cytokines were comparatively lower when compared to the MRSA control group. Notably, Group 5, which received treatment with 2% topical formulation, exhibited results very similar to the group treated with the standard drug (linezolid), followed by the groups receiving 1% and 0.5% topical formulation.

In conclusion, the oral administration of Linezolid at a dose of 25mg/kg body weight for five days, initiated 48 hours post MRSA infection induction, resulted in a significant (P≤0.01) reduction in the levels of all measured inflammatory cytokines (IL-6, TNF α, IL1β, and MCP1). Similarly, topical treatment with 1% BiVO4 significantly (P≤0.05) reduced the levels of IL-6, IL1β, and MCP 1, while treatment with 2% BiVO4 significantly (P≤0.01) reduced the levels of IL-6, TNF α, IL1β, and MCP 1 compared to the MRSA control group.

The 2% topical formulation for the treatment of MRSA infections, wherein the composition demonstrate a significant reduction in inflammatory markers IL-6, TNF α, IL1β, and MCP 1, thereby indicating its efficacy in mitigating MRSA-induced inflammation.
The topical formulation results in a substantial decrease in MRSA bacterial load and a notable improvement in wound healing, thereby providing an effective therapeutic intervention against MRSA infections.

Thus, 2% topical formulation demonstrated enhanced antibacterial activity, anti-inflammatory activity and wound healing activity against MRSA-infected mice comparable to the control group and holds the potential to be used as an alternative wound healing therapy, and can be incorporated in wound healing topical ointments and gels.

Advantages:

• The topical gel formulation of the present invention is used for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.
• The process for preparing the topical gel formulation is simple and cost-effective.
• The topical gel formulation of the present invention exhibits significant reduction in MRSA-induced inflammation and bacterial load.
• The topical gel formulation of the present invention is helpful in improving wound healing.

In one of the preferred embodiments, the present invention shall disclose a process of preparation of topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections comprises of following steps;

a. preparation of BiVO4 nanoparticles comprises of mixing Bismuth nitrate pentahydrate dissolved in acetic acid: ethanol and water and Ammonium metavanadate dissolved in ammonia and water and maintaining pH at 7 followed by autoclaving and filtering with ethanol and distilled water and placing obtained residue in hot air oven followed by grinding to form BiVO4 nanoparticles;
b. preparation of topical formulation comprises of melting Petroleum jelly in water bath and adding the BiVO4 nanoparticles followed by mixing thoroughly until homogenous to form topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.
In another preferred embodiment, the present invention shall disclose a topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections prepared by the process as described above.

In yet another preferred embodiment, the present invention shall disclose a process of preparation of topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections comprises of following steps;

a. preparation of BiVO4 nanoparticles comprises of mixing 0.97gms of Bismuth nitrate pentahydrate dissolved in 50mL of 1:1:3 ratio of acetic acid: ethanol and water and 0.023gms of Ammonium metavanadate dissolved in 20mL of 3:1 ratio of ammonia and water and maintaining pH at 7 using ammonia solution and acetic acid followed by autoclaving at 180oC for 3 hour and filtering with ethanol and distilled water for three times and placing obtained residue in hot air oven at 80oC for overnight or 6 to 7 hour followed by grinding to form BiVO4 nanoparticles;
b. preparation of topical formulation comprises of melting 1gm of petroleum jelly in water bath and adding 0.01-0.02g of the BiVO4 nanoparticles followed by mixing thoroughly until homogenous to form topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.

In further preferred embodiment, the present invention shall disclose a topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections prepared by the process as described above.

Working example 1:

Process of preparation of topical formulation

0.97gms of Bismuth nitrate pentahydrate dissolved in 50mL of 1:1:3 ratio of acetic acid: ethanol and water and 0.023gms of Ammonium metavanadate dissolved in 20mL of 3:1 ratio of ammonia and water were mixed and pH was maintained at 7 using ammonia solution and acetic acid further autoclaved at 180oC for 3 hour and filtered with ethanol and distilled water for three times and obtained residue was placed in hot air oven at 80oC for overnight or 6 to 7 hour and ground to form BiVO4 nanoparticles. 1gm of Petroleum jelly was melted in water bath and 0.01-0.02g of the BiVO4 nanoparticles was added and mixed thoroughly until homogenous to form topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.

Working example 2:

Synergism studies:

The topical gel formulation of the present invention is subjected to synergism studies, to study the effect of the topical gel formulation of the present invention viz-a-viz its components when individually applied.
Table 3: Synergism studies:

S.NO Ingredients Inhibition of Staphylococcus aureus after topical gel formulation treatment Reduction in inflammatory markers after topical gel formulation treatment
1. BiVO4 nanoparticles + +
2. Petroleum jelly - -
3. Topical gel formulation of the present invention +++ +++


S.NO Ingredients Inhibition of Staphylococcus aureus / Reduction in inflammatory markers
1. BiVO4 nanoparticles In in-vitro studies, inhibition zones were observed, with reduced biofilm formation and bacterial adherence indicating a slight suppression of S. aureus growth.
2. Petroleum jelly No effect (used as carrier for BiVO4 nanoparticles). The levels of wound healing and inflammatory cytokines were similar to control group which received no treatment.
3. Topical gel formulation of the present invention The levels of inflammatory cytokines (IL-6, TNF-α, IL-1β, and MCP-1) decreased; weight fluctuations reduced and wound healing improved. These results were comparable to those observed in the group treated with the standard drug, linezolid.

From the results it is inferred that individual ingredients BiVO4 nanoparticles exhibit anti-bacterial and anti-inflammatory activities to a certain extent whereas the topical gel formulation of the present invention using Petroleum jelly as carrier exhibits significant anti-bacterial and anti-inflammatory activities and is suitable to be applied for treating MRSA infections.

Although the invention has now been described in terms of certain preferred embodiments and exemplified with respect thereto, one skilled in art can readily appreciate that various modifications, changes, omissions, and substitutions may be made without departing from the scope of the following claims.
, Claims:WE CLAIM:

1. A process of preparation of topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections comprises of following steps

a. preparation of BiVO4 nanoparticles comprises of mixing Bismuth nitrate pentahydrate dissolved in acetic acid: ethanol and water and Ammonium metavanadate dissolved in ammonia and water and maintaining pH at 7 followed by autoclaving and filtering with ethanol and distilled water and placing obtained residue in hot air oven followed by grinding to form BiVO4 nanoparticles;

b. preparation of topical formulation comprises of melting petroleum jelly in water bath and adding the said BiVO4 nanoparticles followed by mixing thoroughly until homogenous to form topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.

2. A topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections prepared by the process as claimed in claim 1.

3. A process of preparation of topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections comprises of following steps

a. preparation of BiVO4 nanoparticles comprises of mixing 0.97gms of Bismuth nitrate pentahydrate dissolved in 50mL of 1:1:3 ratio of acetic acid: ethanol and water and 0.023gms of Ammonium metavanadate dissolved in 20mL of 3:1 ratio of ammonia and water and maintaining pH at 7 using ammonia solution and acetic acid followed by autoclaving at 180oC for 3 hour and filtering with ethanol and distilled water for three times and placing obtained residue in hot air oven at 80oC for overnight or 6 to 7 hour followed by grinding to form BiVO4 nanoparticles;

b. preparation of topical formulation comprises of melting 1gm of petroleum jelly in water bath and adding 0.01-0.02g of the said BiVO4 nanoparticles followed by mixing thoroughly until homogenous to form topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections.

4. A topical formulation for treating Methicillin-resistant Staphylococcus aureus (MRSA) infections prepared by the process as claimed in claim 3.


Dated this 24th day of OCT 2024



For KARUNYA INSTITUTE OF TECHNOLOGY AND SCIENCES
By its Patent Agent

Dr.B.Deepa
IN/PA 1477

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