(T1330-01-02) Topical and Transdermal Delivery of 4-phenyl-butyric Acid Through Chitosan Nanoparticles Dispersed in a Foam Formulation: A Potential Antidote for Skin Injury Due to Chemical Warfare Agents

Purpose: Lewisite, an arsenic-based chemical warfare agent, poses a significant threat to public health due to its potential for accidental civilian exposure. Acute exposure to lewisite causes immediate burning sensation and erythema, followed by edema and blistering. The nociceptive response of lewisite lasts for several weeks. Therefore, it is crucial to mitigate skin injury caused by lewisite and similar arsenicals to reduce pain, inflammation, and blistering. To mitigate the skin injury caused by lewisite exposure and reduce pain, inflammation, and blistering, an antidote 4-phenyl butyric acid (4-PBA) has been studied. The FDA has approved 4-PBA for treating urea cycle disorder. Our recent studies indicate its potential in blocking the unfolded protein response (UPR) signaling pathway, thus reducing skin injury. The present study aimed to develop a 4-PBA-loaded chitosan nanoparticle incorporated in a topical foam (F39) and compare low molecular weight and medium molecular weight chitosan for sustained 4-PBA delivery at the site of action. A topical foam would provide an easily spreadable formulation with no greasy residue, which is important for sensitive or inflamed skin in cases of arsenical exposure.

Methods: Various chitosan nanoparticle-based formulations (NF1-NF38) were synthesized using the ionic gelation method, using both low (LW) and medium (MW) molecular weights of chitosan. Formulation parameters, including drug-polymer ratios, amounts of TPP, TPP injection rate, and stirring speed, were optimized as described in Table 1. Initially, chitosan was dissolved in acetic acid adjusted to pH 4.5, followed by the injection of a 50 mg/ml 4-PBA solution in ethanol using a syringe pump. Pentasodium tripolyphosphate (TPP) solution (5ml) was then added dropwise to an equal volume of chitosan solution using the syringe pump under magnetic stirring at 650 rpm. Chitosan nanoparticles formed immediately and were subsequently collected, washed, lyophilized, and assessed for appearance, drug loading, particle size, and entrapment efficiency. Batch N31 (LW) and N35 (MW) were chosen for further studies. These N31 and N35 nanoparticle batches were incorporated in a topical foam (F39). F39 was composed of 15 % Tween 20 solution, 5% v/v oleic acid, 10% v/v ethanol, and 70 % v/v propylene glycol. N31 and N35 loaded foams were further tested for in vitro permeation using vertical Franz diffusion cells and a dermatomed porcine skin model (n=4). An infinite dose of 200µL (1.5mg/1ml) of nanoparticles in F39 and nanoparticle suspension in PBS was applied on the skin and compared to the unencapsulated drug in PBS vehicle as a 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 using a validated HPLC method.

Results: Formulations N1-N38 were prepared according to the process outlined in Figure 1 and were subsequently characterized. Among these formulations, N31 (LW) and N35 (MW) exhibited promising characteristics including good yield, zeta potential (~±20-30mV), drug loading (3% and 5.5% respectively), and particle size (mean diameter: 141 ± 26 nm and 176 ± 36 nm, n=10 respectively), leading to their selection for further testing. The total delivery of 4-PBA across the skin from the PBS control was observed to be the highest (1019.8 ± 65 µg/cm2), suggesting that 4-PBA possesses inherent high transdermal permeability attributed to its moderate lipophilicity and low molecular weight (164.20 g/mol), thus serving as the control. Our objective was to sustain the delivery of 4-PBA by encapsulating it in nanoparticle formulations. Compared to the control, N31 and N35 in PBS exhibited a sustained permeation profile, as depicted in Figure 2C. N31 in PBS demonstrated a significantly lower (p=0.0012) total delivery of 284.4 ± 13.31 µg/cm2, while N35 in PBS (570.61 ± 56.13 µg/cm2) showed an overall lower delivery compared to the control, although the difference was not statistically significant. Furthermore, foam-containing nanoparticles (N31 and N35) exhibited a significantly lower (p < 0.05) total delivery (101.23 ± 36.42 µg/cm2 and 245.58 ± 45.91 µg/cm2 respectively) compared to the control. However, foam (F39) with 4-PBA did not demonstrate a significant lower total delivery than the control (354.65 ± 51.03 µg/cm2). This suggests that the foam did not alter the permeation characteristics of 4-PBA, and sustained delivery was primarily achieved by the nanoparticles. The medium molecular weight of chitosan delivered more 4-PBA across the skin compared to the low molecular weight, but this higher delivery was not significant across all groups. The foam proved to be an easy-to-apply formulation that did not require rubbing and did not significantly reduce the delivery of 4-PBA any further.

Conclusion: We successfully prepared and characterized chitosan nanoparticles loaded with 4-PBA and incorporated them into a topical foam (F39). These nanoparticle formulations achieved sustained delivery of 4-PBA across dermatomed porcine skin for up to 24 hours. Thus, our study demonstrates the feasibility of developing a topical foam formulation for the sustained delivery of 4-PBA to mitigate 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. Kshirsagar S, Dandekar A, Srivastava RK, Khan J, Muzaffar S, Athar M, et al. Microneedle-mediated transdermal delivery of N-acetyl cysteine as a potential antidote for lewisite injury. Int J Pharm. 2023 Nov 25;647.
3. 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;
4. 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 1U01AR078544-01 titled “Optimization of Novel Molecular Target-based Drugs for Arsenical Skin Injury”.


Figure 1. Schematic illustration of the methods used in this study


Table 1: Formulation matrix for 4-PBA loaded chitosan nanoparticles


Figure 2: (A) Total delivery and lag time of 4-PBA nanoparticle formulations #2, and #4 (donor concentration 1.5 mg/mL infinite dosing) in dermatomed porcine skin; (B) Total delivery and lag time of 4-PBA nanoparticle formulations #1, and #3 (donor concentration 1.5 mg/mL infinite dosing) in dermatomed porcine skin; (C) Permeation profile of 4-PBA across dermatomed porcine skin from formulations #2, and #4; (D) Permeation profile of 4-PBA across dermatomed porcine skin from formulations#1, and #3.