(W1230-02-09) Development of Rapidly Soluble Mebendazole Nanosuspension for Colorectal Cancer

Purpose: Mebendazole (MBZ) has been proven for the treatment of various cancers. Unfortunately, poor water solubility and bioavailability limit its application as a potential molecule. As MBZ has proven as a trailblazer repurposing molecule for the treatment of colorectal cancer, its improved bioavailability may also offer some advantages to anti-cancer activity. Therefore, colon-targeted MBZ must be reformulated using the benefits of nanotechnology that can deliver drugs specifically at the desired site of action, improve drug effectiveness, and enhance drug bioavailability. The purpose of the study was to enhance dissolution rate and anticancer efficacy by formulating MBZ-based nanosuspension (NS). Nanosuspension technology has been useful for brick dust insoluble compounds with the benefits of improved efficiency in terms of saturated solubility, fast dissolution, absence of Ostwald ripening, and large-scale versatility for the production of BCS class II and IV drugs. Therefore, the objectives of the study are: (a) To formulate and characterize MBZ Nanosuspension (MBZ NS). (b) To investigate MBZ NS anticancer activity in various colorectal cancer cell lines.

Methods: MBZ NS was formulated using a NETZSCH DeltaVita® 1 (NETZSCH Premier Technologies, LLC. PA, USA) dual centrifugation (DC) nano-milling method utilizing Hydroxy Propyl Methyl Cellulose E5 (HPMC E5) and Sodium Lauryl Sulfate (SLS) as stabilizers (Fig. 1). Screening of suitable stabilizers was done with keeping certain parameters constant such as drug concentration (4% w/v), polymer as stabilizer (1% w/v), surfactant stabilizers (0.1% w/v), 10 gm milling beads (0.3 mm), milling time (2 h), and stirring rate (1500 rpm). The optimized MBZ NS was characterized using dynamic light scattering particle size analyzer for particle size, polydispersity index (PDI), zeta potential (ZP), and in vitro drug release was investigated. Solid-state characterization of pure MBZ and freeze-dried MBZ NS were evaluated using differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FT-IR). In vitro cytotoxicity of MBZ, MBZ physical mixture (MBZ-PM), and MBZ NS was assessed on HT29 and HCT116 using an MTT assay. Furthermore, In vitro cytotoxicity of the HPMC and SLS was evaluated in both cell lines. Also, In Vitro Live/Dead Cell Assay was performed to see the effect of MBZ and MBZ NS on cell viability within 3D multicellular tumor spheroids of HCT 116.

Results: After screening the stabilizer, it was found that excellent stability was noticed with the combination of HPMC and SLS (Fig. 2A), which was reflected by the lowest particle size and PDI and MBZ NS formulation remained stable for 1 month of storage at room temperature. The optimized MBZ NS resulted in particle size of 362.6 ± 12.3 nm, PDI of 0.167 ± 0.01, and ZP of -14.6 ± 1.208 mV (Fig. 2B). Furthermore, MBZ NS exhibited 20 folds with higher dissolution than MBZ in colonic pH (Fig. 2D). DSC data revealed the crystallinity of the MBZ NS (Fig. 2C). IC₅₀ of MBZ NS was found to be significantly lower than the MBZ-PM in HT29 and HCT116. (Fig 3A). Furthermore, both HPMC and SLS at higher concentrations portrayed similar cellular morphology as control, implying their safety (Fig 3B). Additionally, 1.5 folds and 1.8 folds reduction in the area of 3D tumor spheroids after treatment with MBZ NS was observed compared to control and MBZ-PM. From the In Vitro Live/Dead Cell Assay higher amount of red fluorescence in treatment groups confirmed a large number of dead cells compared to the control (Fig 3C).

Conclusion: In conclusion, MBZ NS was successfully designed with HPMC-SLS stabilizers through the dual centrifugation method, and its colorectal cancer activity was assessed in HT 29 and HCT 116 cell lines. The optimized MBZ NS increased the dissolution rate and improved anticancer activity in colorectal cancer through 2D assay and 3D tumor spheroid assay. Therefore, the developed MBZ NS might be a suitable oral formulation for colorectal cancer treatment.