(T1330-02-09) Development of 4-phenylbutyric Acid Microsponge Formulations as a Potential Antidote for Skin Injury Due to Chemical Warfare Agents

Purpose: Lewisite is an arsenic-based chemical warfare agent previously intended for use during the World Wars but is reported to be present in stockpiles in several countries. Accidental exposure of lewisite to civilians remains a threat to public health. Studies report that acute exposure to lewisite causes immediate burning sensation and erythema within 30 minutes, followed by edema and blistering. The overall skin recovery time from the lewisite exposure without any treatment is around four weeks(1). Dimercaprol is a parenterally administered antidote, however, the drawbacks include inherent toxicity, painful intramuscular administration, and low aqueous solubility. Thus, it is essential to mitigate the skin injury caused by arsenicals to reduce pain, inflammation, and blistering using novel therapeutic agents 4-phenyl butyric acid (4-PBA) is a novel drug agent for treating injury from lewisite or other arsenic-based chemicals. 4-PBA, currently used to treat urea cycle disorder, can improve protein folding and decrease the endoplasmic reticulum stress caused by lewisite in the skin and other organs(1). Our previous research has demonstrated feasibility of delivering 4-PBA through the transdermal route (2,3). The present study aimed to develop a topical formulation for sustained 4-PBA delivery at the site of action. Encapsulation of 4-PBA in microsponges would provide sustained delivery of 4-PBA at the site of skin injury, and a topical gel would prolong drug contact at the site of action.

Methods: 4-PBA microsponges were prepared (Figure 1) using the oil-in-water emulsion solvent evaporation method. The organic phase consisted of varying ratios of 4-PBA and Eudragit RS-100 (10% w/w) dissolved in dichloromethane. The organic phase was slowly injected into the aqueous phase consisting of 1% w/w polyvinyl alcohol and deionized water. The resulting emulsion was stirred until the dichloromethane evaporated. The microsponges were dried using vacuum centrifugation. Several batches of microsponges with varying formulation and process variables were characterized for particle size and uniformity by optical microscopy, drug loading, and encapsulation efficiency. Quantitative analysis of 4-PBA was performed using a validated high-performance liquid chromatography (HPLC) method. The optimized batch F7 was further used to develop a topical gel formulation. Carboxymethylcellulose sodium (CMC) and Carbopol 980 NF were gelling agents. Microsponge-loaded gels were further tested for in vitro permeation using Franz diffusion cells and dermatomed human skin model (n=4). A finite dose of 15 mg/cm² of gel formulations and microsponge suspension in PBS was applied on the skin and compared to the unencapsulated drug in PBS vehicle as control. Receptor samples were withdrawn at predetermined time points up to 24 h. The skin and receptor samples were further analyzed for 4-PBA concentrations.

Results: Formulations F1-F10 were prepared as seen in Figure 2 and characterized. Formulation F7 showed the highest drug loading, encapsulation efficiency, and particle size (mean diameter: 50.34 ± 4.32 µm, n=10) and was thus selected for further testing. Total delivery of 4-PBA into and across the skin from PBS control was found to be the highest (41.52 ± 2.54 µg/cm²), indicating that 4-PBA inherently has high transdermal permeability due to its moderate lipophilicity and low molecular weight (164.20 g/mol), thereby serving as control. Our objective was to sustain the delivery of 4-PBA by encapsulating it in microsponge formulations. Compared to the control, 4-PBA microsponges in PBS showed a sustained permeation profile, as seen in Figure 3, as well as significantly lower (p=0.0056) total delivery (14.16 ± 1.23 µg/cm²). Carbopol 980 gel (0.75% w/w) with 4-PBA microsponges showed a significantly lower (p=0.0048) total delivery than control (12.55 ± 1.41 µg/cm²). Furthermore, CMC gel (10% w/w) with 4-PBA microsponges showed a significantly lower (p=0.0019) total delivery (10.09 ± 1.23 µg/cm²) compared to the control. For both Carbopol and CMC gels, the total delivery was not significantly lower than microsponge in PBS vehicle (p=0.9204 and 0.3606, respectively), indicating that microsponge encapsulation resulted in the sustained release of 4-PBA. The gel helped to develop a formulation that was easy to apply, while not further reducing the delivery in any significant manner.

Conclusion: 4-PBA microsponges were successfully prepared and characterized and incorporated into topical gels. Microsponge formulations successfully resulted in sustained delivery of 4-PBA into and across dermatomed human skin for up to 24 h. Thus, we demonstrated the feasibility of developing a topical formulation for sustained delivery of 4-PBA for lewisite-mediated skin toxicity.

References: 1. Li C, Srivastava RK, Weng Z, Croutch CR, Agarwal A, Elmets CA, et al. Molecular Mechanism Underlying Pathogenesis of Lewisite-Induced Cutaneous Blistering and Inflammation: Chemical Chaperones as Potential Novel Antidotes. American Journal of Pathology. 2016 Oct 1;186(10):2637–49.
2. Dandekar A, Vora D, Yeh JS, Srivastava RK, Athar M, Banga AK. Enhanced Transdermal Delivery of N-acetylcysteine and 4-phenylbutyric acid for Potential Use as Antidotes to Lewisite AAPS PharmSciTech. AAPS PharmSci Tech. 2023 Jan 31;
3. Vora D, Dandekar AA, Srivastava RK, Athar M, Banga AK. Development and Evaluation of a Topical Foam Formulation for Decontamination of Warfare Agents. Mol Pharm. 2022 Dec 5;

Acknowledgements: This project was funded by NIH/NIAMS grant 1U01AR078544

An overview of the objective, experimentation, and significant results of the study

Formulation matrix and observed results for 4-PBA microsponges via solvent evaporation method

Permeation profile of 4-PBA across dermatomed human skin, (B) Total delivery of 4-PBA into and across dermatomed human skin. Statistical significance calculated using Ordinary One-Way ANOVA test with Tukey's Multiple Comparisons test. ** indicates p < 0.01; ns indicates no significant difference