Topical application of diclofenac nanoemulsion reduces postoperative pain in rats

LM Guethe1, FL Primo1, RS Fais0,2, MP Siqueira-Moura1, WA Prado0,2, AC Tedesco1. 1University of São Paulo, Department of Chemistry FFCLRP 14040901, Brazil, 2University of São Paulo, Department of Pharmacology FMRP 14049-900, Brazil

BACKGROUND AND AIMS: Topical diclofenac is widely used in treatment of pain and inflammation. Studies demonstrate that the drug preferentially distributes to the target tissues in sufficient concentrations to produce a therapeutic effect. Topical diclofenac is well tolerated and is associated with fewer side-effects than other topical non-steroidal anti-inflammatory drugs (NSAIDs) and a lower rate of gastrointestinal complications than oral NSAIDs. Besides that, a topical formulation based on nanoemulsion is well-known due to their advantages such as biocompatibility, enhance skin permeation, and so on. In this context, the aim of the present study was to prepare nanoemulsions containing diclofenac (diclofenac-NE ) and to assess their topical effect in a model of postoperative pain in rats. METHODS: Diclofenac-loaded nanoemulsions (3, 5, and 10 mg.mL-1) were obtained by spontaneous emulsification method. Colloidal formulations were characterized by mean diameter, size distribution (PdI), and zeta potential. Aqueous solution containing free diclofenac (3, 5, and 10 mg.mL-1) was used as a control. The in vivo experiments were conducted in accordance to the Ethical Committee for Animal Experimentation from Faculty of Medicine of Ribeirao Preto/USP (No. 108/2010). Male Wistar rats (160g-180g; n=42) had their baseline paw withdrawal thresholds (PWT) to mechanical stimulation measured using an electronic von Frey Test. The animals were then submitted to paw incision and the PWT for both hind paws were measured again 2 h after the incision. Diclofenac-NE or aqueous solution of diclofenac was then applied into the incised paw and the PWT for both hind paws of each animal was measured at 10-min intervals for up to 70 min. All the results were reported as mean ± SEM and the comparisons between groups was made by analysis of variance (ANOVA) followed by Bonferroni\'s post-hoc test, or multivariate analysis of variance (MANOVA) with repeated measures to compare the groups over all times. The factors analyzed in MANOVA were treatments, time and treatment×time interaction. In the case of treatment×time interaction, one-way analysis of variance also followed by Bonferroni\'s test was performed for each time. RESULTS: Nanoemulsion formulations had mean diameters of 443 nm (10 mg), 227 nm (5 mg), and 221 nm (3 mg) with PdI about 0.4 (10 mg) and 0.3 (5 and 3 mg). Zeta potential was significantly negative ranging from – 61 to – 64 mV. Topical diclofenac-NE (10 and 5 mg) increased the PWT to mechanical stimulation of the incised paw for at least 30 min as compared to control while the diclofenac-NE application (3 mg) did not alter PWT. The curves in diclofenac-NE (10 mg) group were significantly different regarding treatment (F5,18=1603; P<0.0001) and time (F8,144=120.3; P<0.0001), and had significant treatment×time interaction (F40,144=35.32; P<0.0001). Diclofenac-NE (5 mg) group also showed curves significantly different regarding treatment (F5,18=1184; P<0.0001) and time (F8,144=174.9; P<0.0001), and had significant treatment×time interaction (F40,144=54.73; P<0.0001). CONCLUSIONS: Spontaneous emulsification method is able to produce droplets in the nanometer range and a homogeneous formulation having zeta potential values higher than – 30 mV. Moreover, we suggest that diclofenac-NE has an anti-hyperalgesic effect on postoperative pain state. FINANCIAL SUPPORT: CAPES.