(T0930-03-13) Comprehensive Study on Injectable Capsaicin In Situ Forming Implant for Ultra-Long-Acting Anti-Obesity: Formulation, Characterization, Drug Release, Stability, and Cytotoxicity Studies

Purpose: Obesity, a major health concern that leads to various diseases like type II diabetes, cardiovascular diseases, and cancer, necessitates drug delivery over a prolonged period, whereas oral or transmucosal delivery is limited to 24 hours to seven days. Capsaicin, a TRPV1 agonist, has been established as an anti-obesity by enhancing metabolism, energy expenditure, and thermogenesis to counter HFD-induced obesity. However, its low solubility in an aqueous environment, the short biological half-life and bioavailability, and adverse side effects orally hampered the clinical development of capsaicin. The most effective strategy for addressing these issues is by entrapping capsaicin within polymeric carriers to generate a sustained-release delivery system, ensuring only a small amount of drug is present in the blood, reducing adverse effects, and preventing intracellular enzyme degradations, thus enhancing bioavailability. Therefore, it is necessary to develop an ultra-long-acting delivery for capsaicin as an anti-obesity, for 1 to 6 months. In-situ forming implants (ISFIs) overcome some drawbacks of other controlled delivery systems like microspheres, liposomes, and surgical implants. ISFI has a simple manufacturing and high loading efficiency, suitability for heat-sensitive drugs, generating a site-specific action and extended drug release, diminishing injection frequency, and enhancing patient compliance. ISFI is a liquid formulation that transforms into solid implant upon injection and contact with the aqueous environment in the body. Therefore, it is less complicated to administer and less invasive than a surgical implant since ISFI utilizes a biodegradable and biocompatible polymer, like Polylactide-co-glycolide (PLGA). Moreover, drug-release retarders should be incorporated within ISFI to minimize the burst effect generally exhibited by polymeric-based formulations. Therefore, this research aimed to develop capsaicin-loaded ISFI as an ultra-long-acting anti-obesity that is stable and injectable with a minimum burst effect and biocompatible (non-toxic) with the injection site.

Methods: Capsaicin-loaded ISFIs were prepared by dispersing capsaicin into PLGA-DMSO solution with three different release retarders, as shown in Figure 1, then compared their visual appearances, viscosities, solidifying times, the initial burst, and drug release profiles. Viscosity was measured using a Brookfield viscometer at 25°C, solidifying time in a thermostatically controlled water bath at 37 °C. In-vitro release study was performed for up to 5 months and analyzed using a validated HPLC method. The initial burst release was investigated for the cumulative drug release after 24 hours. The drug release kinetics were studied using zero-order, first-order, Higuchi’s, Hixon-Crowell, and Korsmeyer-Peppas models. The selected ISFI underwent surface morphological features, thermal analysis, rheological behavior, and injectability parameters, and then studied its stability. The morphological features of the solidified implant were observed using SEM. Thermal analysis of Capsaicin, PLGA, physical mixture of PLGA-Capsaicin, Maisine, liquid ISFI, and solidified ISFI were performed using DSC. The rheological behavior was determined using a Bohlin rheometer at 25°C, then calculated with Furrow's equation. Injectability parameters were predicted by a mathematical approach of the Hagen–Poiseuille law for six different syringe and needle dimensions. Stability study was done for six months at room temperature, and cytotoxicity study was performed using AlamarBlue assay and AM-PI staining in mouse fibroblast L929 cell lines.

Results: The results showed that all formulations were clear and transparent to yellowish and solidified spontaneously upon contact with the release medium. As shown in Fig. 2A, the assays of 8 ISFIs were greater than 95% of drug recovery. Increasing drug concentration after adding the release retarders increased the viscosity, where CM75 showed 134.97 ± 0.12 mPa.s of viscosity at 20 rpm. Capsaicin-loaded ISFIs showed a triphasic pattern of drug release, as shown in Fig. 2B, where CM75 was entirely released sustainably after 5 months and generated the lowest initial burst release of 7.97% ± 1.29%. As shown in Fig. 2C, the drug release kinetics described that most ISFIs, including CM75, followed a Korsmeyer-Peppes kinetics model. The SEM micrographs in Fig. 3A revealed that the implant formed a “hollow and sponge-like” structure, confirming the formation of various pore-size features on the surface morphology of the solidified implant, allowing the drug release in a sustained and prolonged manner. The DSC thermograms in Fig. 3B confirmed the crystallinity of pure Capsaicin with a single endothermic peak, a degradation temperature of the CM75 liquid implant, and performed the glass transition and degradation temperatures of the solidified implant. The Furrow constant from the plot, as shown in Fig. 3C and 3D, confirmed that CM75 followed Newtonian fluid behavior and the injection force values met the acceptance criterion of less than 40 N. The stability study in Fig. 3E showed that CM75 has remained stable with no color changes with a recovery percentage of 96.35 ± 0.58% after 6 months. The cytotoxicity results showed ± 100% viability, indicating that the system is biocompatible for a subcutaneous route.

Conclusion: Capsaicin has been successfully loaded in a PLGA-based ISFI and generated an ultra-long-acting injectable with a very minimum burst effect, stable, and biocompatible subcutaneously, where CM75 is the best formulation.

References: 1. Baskaran, P. et al., 2017. TRPV1 Activation Counters Diet-Induced Obesity Through Sirtuin-1 Activation and PRDM-16 Deacetylation in Brown Adipose Tissue. Int J Obes (Lond) 41, 739–749. https://doi.org/10.1038/ijo.2017.16
2. Ibrahim, T.M.et al., 2021. An overview of PLGA in-situ forming implants based on solvent exchange technique: effect of formulation components and characterization. Pharmaceutical Development and Technology 26, 709–728. https://doi.org/10.1080/10837450.2021.1944207
3. Justin C. M. et al., 2022, Anti-cancer activity of sustained-release capsaicin formulations, Pharmacology & Therapeutics, Vol. 238, 108-177, ISSN 0163-7258, https://doi.org/10.1016/j.pharmthera.2022.108177.


Figure 1. ISFI Composition and formulation


Figure 2. Characteristics of the prepared Capsaicin-ISFIs and the drug release profiles


Figure 3. Characterization of the selected Capsaicin-ISFI: SEM micrographs, DSC thermograms, rheology and injectability parameters, stability, and cytotoxicity/biocompatibility profiles