(T1230-03-16) Development and Evaluation of a Drug Delivery System Based on Polymeric Nanoparticles of Nitazoxanide

Purpose: Nitazoxanide (NTZ) is a broad-spectrum anti-infective thiazolide developed as an antiparasitic agent, it has shown in vitro activity against DNA viruses (e.g. Hepatitis B virus) and RNA viruses (e.g. hepatitis C virus), which that repositions it as a molecule of interest. However, its therapeutic use is limited by its low aqueous solubility (7.5 µg/mL), hindering its absorption and consequently its oral bioavailability (≤ 3%).^1,2 Among the strategies to improve the aqueous solubility of drugs are polymeric nanoparticles, a drug delivery system that due to its characteristics (size, the ability to modify its surface, high drug loading capacity and controlled release) allow improving the pharmacokinetic properties of drugs, obtaining an orientation to target cells and reduce their toxicity.³ This research aims to develop a polymeric nanosystem that allows NTZ encapsulation, and to evaluate aqueous solubility improvement, controlled release of the drug and permeability.

Methods: The polymers selected were polycaprolactone (PCL; MVW 14,000) and polyvinylalcohol (PVA; emulsion stabilizer); and, by their good biocompatibility and biodegradation properties. The organic solvent was ethyl acetate, and deionized water as aqueous system. The preparation of nanoparticles occurred by emulsion/evaporation method, and its characterization included particle size and Zeta potential measurement, powder X-ray diffraction (PXRD), Infrared spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). Design of experiments (DoE) 2² (PCL and PVA amount as factors) was analyzed by Statgraphics Centurion XVI software was used to optimize four variables: particle size, polydispersivity index (PDI), encapsulation efficiency (EE) and charge capacity (CC). Experimental drug release was carried out by dialysis method⁴, and permeability through pig intestinal tissue in Franz cell. The quantification method for NTZ was performed by HPLC reverse phase column Waters C18 (4.6 x 250 mm), mobile phase methanol: phosphate buffer (25:75); and properly validated. A standard of Tizoxanide (TZX, degradation product and active metabolite of nitazoxanide) was used to determinate the plausible deacetylation of NTZ.

Results: The quantification of NTZ was based on its UV/vis absorption maximum at 347 nm. The HPLC method was validated with r = 0.9993, VC = 1.70%, Recovery = 0.47%, detection limit (DL) = 0.048 µg/mL and quantification limit (QL) = 0.146 µg/mL. No interference due polymers nor solvents is observed in the HPLC method therefore it is specific for NTZ. As a result of our experimental design 2², we obtained a series of four formulations as shown in Table 1 (rows F1-F4). The DoE analysis pointed out to PVA as the main negative factor over CC values but without relevance for particle size, nor PDI; the particle size has an average value of 560 nm. The drug release by dialysis method showed that no formulated NTZ reached a 2.5% at 72 h; meanwhile the Formulations 1 – 4 reached values among 13 – 20% in sequence F4 <  F1  < F3 = F1. A final DoE optimization resulted in PNP-NTZ formulation (Table 1) with theoretical and experimental sustained drug release of 22% and 25%, respectively. The EE and CC do not suffer a drastic decrease, remaining in acceptable average values of 96% and 1%, respectively. For PNP-NTZ formulation, FT-IR spectroscopy and PXRD shows the presence of NTZ; and DSC shows a diminished melting point when nanoparticles are compared to NTZ. Finally, permeability studies on pig intestinal tissue showed that no formulated NTZ has a permeability of 8.38 x 10^-6 cm/s, and PNP-NTZ reached a permeability of 4.22 x 10^-5 cm/s, 5 times higher than pure NTZ. No TZX was observed in both permeability studies and drug release.

Conclusion: Nitazoxanide is a worldwide used antiparasitic agent with high antiviral potential. However, its physicochemical properties are not recommended for continuous dosing. The formulation guided by experimental design of NTZ polymeric nanoparticles with PCL and PVA allowed us to achieve an optimal formulation with a sustained release  and permeability 10 times and 5 times greater than the pure drug, allowing NTZ remain stable during all experimental stage. Particle size and PDI show that our nanoparticles are suitable for the next formulation studies before in vivo experiments.

References: 1. François-Rossignol, J. (2014). Nitazoxanide: A first-in-class broad spectrum antiviral agent. Antiviral Res.,110, 94-103.
2. Korba, B., Montero, A., & Farrar, K. (2008). Nitazoxanide, Tizoxanide and other Thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication. Antiviral Res., 77(1), 56-63.
3. Lembo, D., Donalisio, M., & Civra, A. (2018). Nanomedicine formulation for the delivery of antiviral drugs: A Promising solution for the treatment of viral infection. Expert Opin. Drug Deliv., 15(1), 93-114.
4. Bohrey, S., Chourasiya, V., & Pandey, A. (2016). Polymeric Nanoparticles Containing Diazepam: Preparation, Optimization, Characterization, in-Vitro Drug Release and Release Kinetic Study. Nano Converg, 3(1), 1-7.

Acknowledgements: The authors acknowledge the financial support of CONAHCyT-Mexico through grants 792615, 1264202, and Ciencia de Frontera 2019 project 1046617.


Table 1. Experimental response variable values obtained for NTZ nanoparticles.