(M1530-06-35) Formulation and Evaluation of Antibacterial Activity of K21 Topical Liquid and Gel Formulations

Purpose: K21 is a potent antimicrobial agent that is already approved for use in preventing dental infections. It is insoluble in water but soluble in alcohol. This project aimed to develop a non-alcoholic liquid and gel formulation of K21 for potential use as a presurgical rub. The liquid formulation was tested for antibacterial activity against Staphylococcus aureus using the ASTME2315 method, while the gel formulation was tested using the Disc-diffusion method. Additionally, a human volunteer study assessed the microbial load on hands after treatment with a 0.5% K21 liquid formulation.

Methods: Solubility studies were performed in different solvent systems, with K21 incorporated incrementally until saturation. Subsequent solubility studies were conducted in binary and ternary solvent systems. The antibacterial activity of 0.5% K21 formulation developed in the ternary solvent system was assessed as per ASTM E2315 method by directly inoculating 1 gram formulation with 50µL (10ᵔ6 CFU/mL) of S. aureus bacterial inoculum at contact times of 30, 60, and 120 seconds. At the end of each contact time, the formulation was deactivated by adding 9mL of neutralizer, Leethen broth. After 10 minutes, the resulting test solution was serially diluted with sterile Phosphate Buffer Saline (PBS), plated on Tryptic Soy agar, and incubated at 37ᵒC for 24 hours. PBS was used instead of formulation in the control group. Further neutralizer effectiveness on the formulation was tested by inoculating 0.1mL (300-1000CFU) bacteria in Leethen broth, followed by vortexing, and adding formulation after 10 mins. The test suspension was then plated in Tryptic Soy agar and incubated at 37 ᵒC for 24 h. Neutralizer toxicity on bacterial inoculum was tested by following the above procedure and replacing the formulation with PBS. In inoculum control, Leethen broth and formulation were replaced by PBS. The antibacterial activity of gel formulation was evaluated by the Disc-diffusion method against S. aureus. In the human subject studies, one hand of the volunteers was treated with K21 topical liquid formulation (250µL) while the other was left untreated. Both hands were covered with a powder-free sterile glove for five hours. Later, 1mL sterile PBS was added to the glove, rinsed thoroughly, and 100µL was inoculated on a nutrient agar plate and incubated at 37°C for 24 hr.

Results: The K21 was soluble only in Kollicream® OA, Transcutol, and Hexylene glycol. The solubility was enhanced in the binary mixture of Kollicream® OA and Hexylene glycol up to 2%. The final solvent composition in which the K21 0.5% formulation was prepared was 41.16% Kollicream® OA, 41.16% hexylene glycol, and 17.09% transcutol. Transcutol was incorporated to eliminate the turbidity. In the gel formulation, 3% w/v hydroxypropyl cellulose was incorporated. When tested using the ASTM E2315 method, the liquid formulation resulted in a 99.98% reduction (60 sec contact time) in bacterial count. Further, it was confirmed that the neutralizer effectively deactivated the formulation and was nontoxic to bacteria. The gel (dose-loaded 10 mg formulation) resulted in a zone of inhibition significantly higher than the control in the Disc-diffusion method. In the human volunteer study, sweat (PBS) recovered from individuals with K21 topical liquid formulation had no viable organisms on the plate, whereas the untreated hand sweat resulted in several colonies of microorganisms.

Conclusion: The project aimed to investigate the feasibility of developing a K21 topical solution and topical gel. The formulation developed was stable for one year. The liquid formulation and human subject study passed the ASTM E2315 antimicrobial test. K21 gel formulation showed significant antibacterial activity in the Disc-diffusion method plate compared to the control. Formulation excipients did not affect the antimicrobial activity of K21. The ternary solvent system formulation could be an excellent presurgical rub. The gel formulation can be a good antibacterial application to treat local infections on the skin.

Acknowledgements: Institute for Drug Delivery and Biomedical Research, Bengaluru, Karnataka 560086, India
Topical Products Testing, Batesville, MS 38606, USA
Fitebac Pharma LLC, Marietta, GA 30064, USA