Preclinical Examination of Bwell; a 3% Tetracycline Hydrochloride Formulation Safety and Skin Penetration Studies

Abstract

Tetracycline is a broad-spectrum antibiotic, active against gram-positive and gram-negative bacteria, as well as organisms such as mycoplasma and chlamydia. The main goal of the project was to study the safety and skin penetration of a proprietary topical tetracycline formulation developed by Inventus, LLC. Overall the formulation sustained the release of tetracycline compared to the solution formulation. The formulation also took up moisture up to 42% when exposed to 65%RH.

In-vitro skin irritation studies were carried out using reconstructed human skin tissue. In-vivo irritation studies and topical bioavailability studies were conducted using SKH-1 hairless mice. The skin penetration studies of the control tetracycline solution and topical tetracycline formulation were carried out in excised porcine and human skin. To mimic the skin penetration of tetracycline in different skin wound and injury, the permeation studies were carried out after partial (50%) and complete removal of the stratum corneum (100%) by tape stripping. The results from the study demonstrate that the tetracycline formulation produced mild skin irritation comparable to baby shampoo.

The in-vitro skin penetration studies showed comparable skin penetration trend in porcine and human skin. Around 0.5-1% of the applied dose was found in the skin after 6hrs of application which increased to 3% with 48hrs treatment. In vivo mice studies showed that tetracycline was mainly retained in the skin with no measurable systemic absorption after 6hrs of treatment. However the skin damage was found to significantly increase the skin penetration of tetracycline both in-vitro and in- vivo. Overall the results from the study show that the tetracycline formulation is generally safe and can achieve therapeutic concentrations in the skin. Changes in skin barrier as in case of wound and skin injuries, can result in higher drug absorption.

Introduction

The purpose of these studies was to determine the general safety and penetration abilities of Inventus’ proprietary over the counter (OTC) First Aid antibiotic ointment with 3% Tetracycline Hydrochloride delivered by Dual Carrier Technology (DCT). The DCT is a tissue penetration technology, which forms a releasable bond to the active pharmaceutical ingredient, as the carrier transports the drug in a high penetration format through the pathogen cell wall to effectively kill the bacteria and reduce the water activity level (e.g. dehydrate) in the bacteria for a pharmaceutical and mechanical kill.

Materials & Methods

Materials & MethodsMaterials: Tetracycline Hydrochloride (Sigma Aldrich, USA), 3H Tetracycline Hydrochloride (Moravek Biochemicals and Radiochemicals, United States), Tetracycline topical ointment (3% w/v) (pharmaCline, United States), Dialysis Membrane (Spectrum Labs, United States), Sodium Hydroxide, Ethanol, Sodium Bromide, Sodium Chloride, Potassium Chloride, Sodium Phosphate Dibasic Anhydrous (Fisher Scientific, United States), Potassium Phosphate Monobasic (Sigma Aldrich, USA), Scintillation Cocktail (Ecoscint H; National Diagnostics, United States), Scotch Tape (3M, Scotch and the Plaid Design, United States), EPI-200 (Mattek Corporation, United States). SKH-1 Mice (Charles River Laboratories, United States), Porcine Skin (Procured from the Slaughter house in the Department of Animal and Range Sciences, SDSU), Human Skin (Purchased from NDRI, Philadelhpia, PA).

Study 1

Physicochemical Characterization of the Formulation- Release Studies & Moisture Uptake Studies

Method

1. Physicochemical Characterization of the Formulation
1.1. In-vitro release studies
To test the release of tetracycline from the formulation, in-vitro release studies were carried out using dialysis membrane (Cellulose ester membrane, MWCO: 3.5-5 kDa) in phosphate buffered saline (pH 7.4). About 100μl of the tetracycline solution and formulation (equivalent to 3mg of tetracycline) was placed in the dialysis cassette. At predetermined time intervals (1-24hrs) 1 ml of the samples was withdrawn from the receptor compartment and was replaced with 1ml of fresh medium. Tetracycline content was analyzed by UV visible spectrophotometery at 355nm.

Results

1. Physicochemical Characterization of the Formulation

1.1. In-vitro release studies

In vitro release study showed that compared to a simple solution the formulation sustained the release of tetracycline. However all the tetracycline was released in 24 hours from the formulation.

Figure 1: In-vitro release profile of the tetracycline solution and formulation. Values are mean ± SD (n=3)

Method
1. Physicochemical Characterization of the Formulation
1.2. Moisture uptake studies
To test the hygroscopicity of the formulation, the tetracycline formulation was exposed to various humidity conditions. Saturated solutions of sodium bromide (RH: 65%), and keep it in a desiccator at room temperature and allowed for 2 hrs to attain the desired humidity. After 2 hrs, accurately weighed amount of tetracycline formulation (about 0.5 g in triplicate) was placed in a watch glass and kept in the desiccator. At predetermined time intervals (1-48hrs) samples were removed and weighed. The increase in weight of tetracycline formulation was used to calculate the % moisture uptake.

Results

1. Physicochemical Characterization of the Formulation

1.2. Moisture uptake studies

Moisture uptake studies were performed in presence of sodium bromide in a desiccator. As can be seen from Figure 2, the moisture uptake increased with exposure time but reached saturation within 24 hrs. At the end of 48 hrs moisture uptake was 41.65±0.59%. The results show that the formulation tends to take up moisture and should be stored in an appropriate container to minimize the exposure to moisture.

Figure 2.

Moisture uptake of tetracycline formulation as a function of exposure time. Values are mean ± SD (n=3).

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