(T1430-03-16) Development of Microemulsion Formulations with D-Limonene and Terbinafine Hydrochloride for Treatment of Cutaneous Fungal Infections

Purpose: D-limonene is the main product obtained from the purification of citrus essential oils [1]. It's recognized as GRAS material, with low toxicity, no mutagenicity, carcinogenicity, or nephrotoxic risks for humans [2]. Belonging to the terpene family, it's widely known for its therapeutic effects, with its antifungal activity [3]. Terbinafine, on the other hand, is an antifungal agent indicated for onychomycosis treatment caused by dermatophytes, as well as athlete's foot. However, treating fungal infections often takes a long time, which can affect patient adherence to the treatment, highlighting the need for new formulations [4]. The objective of this study is to formulate microemulsion formulations incorporating D-limonene and Terbinafine hydrochloride, aiming to create a microemulsion formulation for the treatment of cutaneous fungal infections such as athlete's foot and nail mycoses.

Methods: Formulations were developed using a titration method with Labrasol^® and D-limonene mixtures, varying from 1:9 to 9:1 ratio to generate a pseudo ternary phase diagram. Surfactant selection was based on its HLB value proximity to the oily phase to enhance microemulsion physical stability. Microemulsions were produced by mixing water, Labrasol^®, and D-limonene in predetermined proportions. Terbinafine hydrochloride was accurately weighed and then incorporated by mixing into the microemulsion at a concentration of 1% (w/v). Microemulsion formation was visually assessed, via turbidity/translucent transitions and classified as microemulsions. Optical clarity was evaluated using a Genesys Bio 10 spectrophotometer (Thermo Scientific), and physical stability was assessed using a Fisher Scientific AccuSpin Micro 17 centrifuge at 10,000 min^-1xg for 10 minutes. The drug solubilization capacity was analysed using the equilibrium method, where the microemulsion, water, 0.1 M HCl, and pH 4.5 and pH 6.8 buffer solutions were saturated with terbinafine hydrochloride, agitated for 48 hours at 37°C ± 0.5°C, and quantified using a spectrophotometer. Drug release of the formulation was evaluated using a Franz diffusion cell, with the donor compartment filled with 1 mL of the microemulsion. A synthetic 0.22 µm Polyvinylidene Fluoride (PVDF) membrane impregnated with octanol was placed between compartments. The receptor compartment was filled with 10 mL of acetate buffer solution pH 4.5 mixed with 10% isopropyl alcohol to ensure sink conditions. The medium was maintained at 37°C ± 0.5°C and stirred with magnetic stirring at 600 rpm. At different time intervals, 0.5 mL samples were withdrawn and analysed for drug content using a Genesys Bio 10 spectrophotometer (Thermo Scientific), totalling 4 hours of assay.

Results: Microemulsion formulations obtained from the pseudoternary phase diagram were found being homogeneous, transparent, low-viscosity, and stable after centrifugation. Among these formulations, all showed transmittance ranging from 74.2 to 99.8%, indicating good optical clarity consistent with microemulsion characteristics. These microemulsions also effectively increased terbinafine hydrochloride solubility, with 30.54 mg/mL solubilized, compared to 6.08, 16.51, 2.37, and 0.01 mg/mL solubility in water, 0.1 M HCl, pH 4.5, and pH 6.8, respectively (Figure 1). Finally, diffusion studies showed that initially, a simple aqueous solution of 1% terbinafine hydrochloride appeared to have greater flux in the first 3 hours (Figure 2). However, after this period, there was an increase in release by the microemulsion, penetrating the membrane. However, further studies are needed to optimize the D-limonene/terbenafine-containing microemulsion formulation.

Conclusion: Based on the presented results, it can be concluded that obtaining microemulsions of D-limonene and terbinafine hydrochloride is feasible, resulting in products with good characteristics and physical stability. Additionally, the microemulsion can increase the solubility of terbinafine hydrochloride, allowing its incorporation into the formulation by simple addition of the drug. After 3 hours, the microemulsion also demonstrated increased drug release through the membrane compared to the individual components. However, further studies are needed to confirm the formulation's efficacy and optimize its performance.

References: [1] Bizzo HR, Hovell AMC, Rezende CM. Essential oils in Brazil: general aspects, development, and perspectives. Química Nova. 2009;32(3):588-594. FapUNIFESP (SciELO).
[2] Sun J. D-Limonene: Safety and Clinical Applications. Altern Med Rev. 2007;12(3):259-264.
[3] Leite-Andrade MC, de Araújo Neto LN, Buonafina-Paz MDS, de Assis Graciano dos Santos F, da Silva Alves AI, de Castro MCAB, Mori E, de Lacerda BCGV, Araújo IM, Coutinho HDM, et al. Antifungal Effect and Inhibition of the Virulence Mechanism of D-Limonene against Candida parapsilosis. Molecules. 2022; 27:8884.
[4] Baheti A, Srivastava S, Sahoo D, Lowalekar R, Panda BP, Padhi BK, Raghuvanshi R. Development and Pharmacokinetic Evaluation of Industrially Viable Self-microemulsifying Drug Delivery Systems (SMEDDS) for Terbinafine. Curr Drug Deliv. 2016 Mar 14;13(1):65-75. doi: 10.2174/1567201812666150120153357.

Acknowledgements: The authors would like to thank the School of Pharmaceutical Sciences (Faculdade de Ciências Farmacêuticas) of the University of São Paulo (FCF-USP) - Process Number 22.1.00142.09.7 - for the Bruna Belem’s scholarship. The authors would also like to acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship awarded to Bruna Belem under the Institutional Internationalization Program (PRINT) - Process number 88887.884749/2023-00.


Fig.1 – Solubility of terbinafine hydrochloride in different media.


Fig. 2 – Results obtained in the Franz diffusion cell, comparing the microemulsion with the respective isolated excipients.