(M1030-05-28) In Vitro and In Vivo Evaluation of Topical 4-phenylbutyric Acid Microsponge Formulation as a Potential Antidote Against Arsenical-Induced Skin Injury

Purpose: Lewisite is a highly toxic arsenical and chemical warfare agent reported to be present in stockpiles in several countries. Accidental or deliberate exposure to lewisite by civilians remains a threat to public health. Acute exposure to lewisite causes pain, inflammation, burning, erythema, edema, and blistering (1). The general antidote, dimercaprol, can only be administered systemically for pulmonary injury and is ineffective for cutaneous injury due to its inherent toxicity and poor water solubility. Thus, it is important to develop effective topical formulations to combat and mitigate the risks of skin injury. 4-phenylbutyric acid (4-PBA), a chemical chaperone, is identified and studied as a novel drug agent against Lewisite injury(2). The FDA has approved 4-PBA for treating urea cycle disorder, and recent studies indicate its involvement in blocking the unfolded protein response (UPR) signaling pathway, thus mitigating skin injury caused by lewisite exposure. We developed 4-PBA microsponges that incorporated the optimized formulation in gel vehicles and tested their sustained in vitro permeation across dermatomed human skin. The in vivo efficacy was tested in hairless mice after treatment with phenylarsine oxide (PAO), a chemical lewisite surrogate, to demonstrate the feasibility of topical delivery of microsponges for lewisite injury.

Methods: 4-PBA microsponges were prepared using the quasi-emulsion solvent diffusion technique using Eudragit RS100 polymer. Fig. 1 depicts the overall formulation process, wherein various parameters, such as the organic solvent drug-polymer ratio, were optimized for high drug loading, uniform particle size, and drug entrapment. Based on the results (Fig. 1), F7 was selected as the optimal batch and incorporated in different vehicles- phosphate-buffered saline, Carbopol 980NF, and carboxymethylcellulose sodium, tested for in vitro release study involving dialysis membrane (MWCO:14000kDa) on vertical Franz cells. In vitro permeation testing (IVPT) was performed using dermatomed human skin with finite (10 µL) of 4-PBA microsponges (3 mg/mL) in the aforementioned vehicles. 4-PBA microsponges in CMC gel were tested in vivo in Ptch1^+/-/SKH-1 hairless mice. Briefly, the anesthetized animals were topically treated with ethanol-diluted PAO (100 µL/mouse) across a 2 × 2 cm² area on the dorsal skin. Control mice were treated identically with the vehicle alone. Post 30-minute exposure of PAO, 4-PBA (3 mg/mouse) Eudragit RS-100 microsponges were applied to mice, and placebo microsponges were applied to the control group. After 24 h, exposed areas were measured for skin bi-fold thickness and Draize score. Post-euthanasia skin tissues were harvested to analyze the apoptosis, measured using a TUNEL assay and a commercial apoptosis detection kit. Images were captured using a Keyence fluorescence microscope. Cytokine levels were measured using Th1/Th2 Extended ProcartaPlex™ Immunoassay kit on a MAGPIX Luminex system. Skin tissue lysate was homogenized in an ice-cold RIPA lysis buffer to perform western blot analysis. Protein concentration in clear skin tissue lysates was measured using a Bio-Rad protein assay DC kit. Thirty to forty micrograms of protein from these samples were electrophoretically separated on SDS-PAGE, transferred to a PVDF membrane, and incubated with 5% nonfat dry milk for 1 hour to block nonspecific sites.

Results: Amongst F1-F10, formulation F7 showed the highest drug loading, optimal encapsulation efficiency, and uniform particle size and was thus selected for further testing (Fig.1b). Compared to the control, 4-PBA microsponges in PBS showed a sustained permeation profile (Fig.2a), 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, along with a desirable rheological profile (Fig.2b,c). PAO treatment significantly enhanced the expression of inflammation regulatory cytokines, namely IL-1β, IL6, and GM-CSF, and the number of apoptotic cells present in the epidermal and hypodermal compartments of the skin. Treatment with microsponge formulated 4-PBA showed protection in the gross tissue damage and at the molecular level by attenuating the expression of ATF-4 and CHOP in skin lysates. It also reduced skin bifold and Draize scores (combined observations of erythema, edema, and necrosis) by 11% and 41 %, respectively (Fig.3a,b,c). Similarly, the significant reduction in the levels of IL-1β, IL6, and GM-CSF was observed by 119%, 44%, and 121%, respectively (Fig.3d), and apoptosis was reduced by 45% (Fig.3e). Since PAO and other arsenicals, including lewisite, act by inducing ER stress, we assessed the expression of ER stress transcription factors ATF-4 and CHOP, where reduced expression was observed (Fig.3f).

Conclusion: 4-PBA microsponges were formulated and characterized, and an optimized formulation was incorporated into topical gels. The release and permeation study resulted in the sustained delivery of 4-PBA for up to 24 hours. In vivo evaluation suggested significant protective efficacy of 4-PBA microsponges against vesicant exposure. Thus, we demonstrated the feasibility of developing topical 4-PBA microsponges for lewisite-mediated skin toxicity.

References: 1. Agency for Toxic Substances and Disease Registry C. ToxFAQsTM for Blister Agents: Lewisite (L), Mustard-Lewisite Mixture (HL). 2011 [cited 2024 Jan 2]. Available from: https://wwwn.cdc.gov/TSP/ToxFAQs/
2. 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.

Acknowledgements: This project was funded by NIH/NIAMS U01AR078544-01 a titled “Optimization of Novel Molecular Target-based Drugs for Arsenical Skin Injury.”


(a) Quasi-emulsion solvent diffusion technique for formulation of 4PBA microsponges, (b) summary table for various batches of 4-PBA microsponges along with evaluation parameters, (c) rheological analysis of 4-PBA microsponge loaded in Carbopol 980NF gel and carboxymethylcellulose (CMC) gel, (d) Fourier-Transform Infrared Spectroscopy (FTIR) to determine potential interaction within the 4-PBA microsponge, (e) Scanning electron microscopy images of F7 microsponge magnified to depict the porous surface


(a) Results of the in vitro release study using dialysis membrane, (b) results of the in vitro permeation study on dermatomed human skin showing the permeation profile, and (c) total delivery of 4-PBA from the microsponges from various vehicles at 24 h. Statistical analysis was performed using ordinary-way ANOVA with Dunnett’s multiple comparisons tests. **** indicates p<0.001.


Results of the in vivo PAO and 4-PBA gel formulation exposure depicting (a) Morphological changes post-exposure (PAO and 4-PBA), (b) Skin Bi-fold thickness post-exposure (PAO and 4-PBA), (c) Draize Score, (d) Cytokine levels representing immune response post-exposure (PAO and 4-PBA), (e) TUNEL assay (DNA damage) (PAO and 4-PBA) and (f) Western Blots and Densitometry post-exposure (PAO and 4-PBA). Statistical analysis was performed using either one-way or two-way ANOVA.