Influence of Poloxamer 188 on Anti-Inflammatory and Analgesic Effects of Diclofenac-loaded Nanoemulsion: Formulation, Optimization and In Vitro/In Vivo Evaluation

Abstract

In this study, a polymer-stabilized nanoemulsion (PNE) was developed to improve the inflammatory and analgesic activities of diclofenac (DA). DA-PNEs were prepared from sesame oil and poloxamer 188 (P188), polysorbate 80, and span 80 as emulsifiers and optimized by a systematic multi-objective optimization method. The developed DA-PNEs exhibited thermodynamical stability with low viscosity. The mean diameter, PDI, surface charge, and entrapment efficiency of DA-PNEs were 122.49±3.42 nm, 0.226±0.08, -47.3±3.6 mV, and 93.57±3.4%, respectively. The cumulative in vitro release profile of DA-PNEs was significantly higher than the neat drug in simulated gastrointestinal fluids. The anti-inflammatory activities of DA-PNEs were evaluated in the λ-carrageenan-induced paw edema model. To investigate the effect of P188 on analgesic and anti-inflammatory activities, a formulation without P188 was also prepared and named DA-NEs. Following oral administration, DA-PNEs showed a significantly higher (p<0.05) effect in reducing pain and inflammation symptoms as compared to free diclofenac and DA-NEs. Moreover, histopathological examination confirmed that DA-PNEs meaningfully reduced the extent of paw edema, comparable to that of DA. Taken together, the findings of the in vitro and in vivo studies suggest that diclofenac-loaded P188-stabilized nanoemulsion can be considered a potential drug delivery system for treating and controlling inflammatory disorders and alleviating pains.

Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) control inflammation and pain by blocking the pro-inflammatory enzyme cyclooxygenase (COX) and thus inhibiting prostaglandin synthesis ^1,2. Although NSAIDs inhibit cyclooxygenase-2 in inflammatory tissue, most of them also affect gastric mucosal cyclooxygenase-1, which ultimately leads to gastric damage. Diclofenac (DA) is frequently implicated in decreasing inflammation and the associated pain in postoperative and dental procedures as well as other disorders including arthritis, muscle strains, bone injuries, gout, dysmenorrhea, and migraine ^3,4. Similar to other NSAIDs, DA has limited solubility in biological fluids and can cause various side effects especially, gastrointestinal (GI) ulcers and renal damage. Besides, numerous side effects have been reported in the cardiovascular system, hematological system, liver, and kidneys following receiving NSAIDs ^5,6. When administered orally, only about 50% of the absorbed dose of DA becomes systemically available, as a result of the first-pass effect ⁷. Therefore, to develop a proper formulation of DA at least three features should be considered: first, it must provide adequate drug release in the intestine, improve the extent of drug absorption, and avoid the first-pass metabolism ^8,9. To fulfill this goal, different types of drug delivery systems for DA have already been reported 10, 11, 12.

One of the best approaches to reduce the side effects and enhance the efficacy of therapeutic agents is the administration of them via liquid nanoformulations, namely nanoemulsions (NEs). NE is a promising drug delivery system considering simple production methods, improved bioavailability, and decreased dosing frequency ^13,14. NEs are a combination of two immiscible liquids stabilized by surfactants and cosurfactants with nano-sized droplets. The solubility of hydrophobic therapeutics could be enhanced by incorporating them in the inner oily part, promoting intestinal absorption and bioavailability 15, 16, 17. Moreover, oral administration of NEs can effectively improve the bioavailability and stabilize the concentration of the therapeutic agent in blood for a longer time ^18,19. Sesame oil is a commonly used vegetable oil obtained from Sesamum indicum and has been reported to have antioxidant, anti-inflammatory, immunoregulatory, antibacterial, and antiulcer potential in various preclinical studies ^20,21. Moreover, oral administration of drugs together with sesame oil improves their bioavailability and lymphatic transport ²². Sesame oil mostly contains linoleic and oleic acids, which these long-chain fatty acids that can facilitate the delivery of the lipophilic drug through the intestinal lymphatic system ²³.

For rational nanoemulsion design, excipient selection is an important factor to consider. Several surfactants such as polysorbates and poloxamers are reported to affect the absorption of P-glycoprotein (P-gp) substrates by inhibiting secretion-directed transport in Caco-2 cells, and enhancing the intestinal absorption in a rat model ^24,25. The right blend of different surfactants leads to the formation of a stable emulsion. In this study, the selected emulsifiers were span 80, tween 80, and poloxamer 188 with HLB values of 4.3, 15, and 24, respectively. Poloxamer 188 (P188), a linear nonionic triblock copolymer, is composed of hydrophilic segments of polyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO) chain. Owing to its safety, versatility, and commercial availability, P188 has been frequently used in increasing the solubility, circulation time, and cellular uptake of therapeutic agents. Due to the P-glycoprotein and a cytochrome P3A4 inhibitory activity, the P188 increases intestinal absorption and changes the pharmacokinetic parameters of P-glycoprotein (P-gp) substrate drugs ^26,27. Moreover, the PPO part of P188 adsorbs on the oily part of NE while PEO chains extend towards the aqueous medium, causing a steric shield to limit Ostwald ripening. Tween 80 (polysorbate 80, polyoxyethylene sorbitan monooleate) is a hydrophilic nonionic surfactant mainly used in the emulsification and dispersion of pharmaceutical agents. Span 80 (sorbitan monooleate), as a lipophilic liquid emulsifying agent, tends to form water in oil (W/O) emulsions.

In this study, we combined the advantages of sesame oil, P188, S80, and T80 to develop P188-stabilized nanoemulsions (DA-PNEs) for the improvement of the anti-inflammatory and analgesic activities of DA. By a systematic multi-objective optimization method, DA-PNEs were prepared. We explored the physicochemical features of DA-PNEs, such as mean droplet size, surface charge, morphology, stability, drug loading, drug release, and cell viability. Finally, the biological activity of developed nanoemulsions in the λ-carrageenan-induced paw edema model was investigated.

Read more here

Materials

Tween 80 (T80), span 80 (S80), and poloxamer 188 (P188) were purchased from Merck (Darmstadt, Germany). Sesame Oil was supplied by Etka (Rudbar, Iran). Diclofenac was obtained from the Caspian vitamin (Gilan, Iran). α-Tocopherol was acquired from Sigma-Aldrich (MO, USA). Oleic acid was purchased from Carlo Erba (Val de Reuil, France).

Zahra Karami, Mohammadreza Saghatchi Zanjani, Sina Andalib, Hossein Babaie, Paria Aminoroaia, Influence of Poloxamer 188 on Anti-Inflammatory and Analgesic Effects of Diclofenac-loaded Nanoemulsion: Formulation, Optimization and In Vitro/In Vivo Evaluation, Journal of Pharmaceutical Sciences, 2023, ISSN 0022-3549, https://doi.org/10.1016/j.xphs.2023.09.022.