Category: Formulation and Quality
Purpose: Currently, drug resistance is the main problem for the treatment of malignant melanoma. Alternative therapeutic approaches are needed for the treatment. Angiogenesis is another issue along with the development of resistance in melanoma and protein kinase C inhibitor was found to be able to inhibit angiogenesis. Proteolysis targeting chimera (PROTAC) is capable to degrade targeted protein by hijacking the ubiquitin-proteasome system (UPS). The purpose of the study is to address the limitations of resistance by Protein kinase C inhibitor anchored BRD4 PROTAC(ARV) PEGylated nanoliposomes (Lpc). Moreover, long circulation of nanoliposomes facilitate accumulation in melanoma tumor matrix and minimize off-target side effects.
Methods: Lpc was prepared by modified hydration method. Particle size and zeta potential were measured using dynamic light scattering (DLS). In vitro cytotoxicity of Palmitoyl carnitine, BRD4 inhibitor and Lpc were evaluated in A375 vemurafenib-resistant (A375R) and SK-mel-28 vemurafenib-resistant (Sk-mel-28R) cell lines by MTT assay. Effect of drug combination was tested by Combenefit software and combination Index was calculated. order to study the effect of BRD4 inhibitor, protein kinase C inhibitor and Lpc on angiogenesis, vascular mimicry study and HUVEC tube formation assay using a Matrigel basement membrane model were carried out. Apoptosis study was carried out by Annexin V apoptosis kit. In Scratch assay was used to study cell migration in vitro. Clonogenic assay was carried out to determine cell reproductive death after treatment. Lpc was used for in vitro release study and stability study, samples at different time intervals were withdrawn and analyzed by HPLC.
Results: Particle size of Lpc is in the range of 105.25 ±2.76 nm with poly dispersity index 0.224, zeta potential showed positive value of 26.6 ± 6.25 mV due to palmitoyl carnitine. IC50 of Palmitoyl carnitine, BRD4 inhibitor and Lpc in resistant cell lines are 22.27-25.88 μM, 0.21 μM and 0.14-0.2 μM respectively. Specific combination of protein kinase C inhibitor and BRD4 inhibitor showed synergistic effect with combination index less than 1. Moreover, we surprisingly found that palmitoyl carnitine exhibites the function of stabilizing BRD4 liposome as a cationic molecule. Vascular mimicry study showed melanoma cells are significantly inhibited after treatment withBRD4 inhibitor, protein kinase C inhibitor and Lpc (Figure 1). HUVEC angiogenesis assay showed HUVEC cells form more tubular vascular networks compared to treatment groups after incubation for 10 hours. Lpc showed 26.07% apoptosis in A375R (Figure 2). Lpc and ARV treated group showed inhibition of migration and proliferation in vemurafenib-resistant cell lines. The number of colonies was dramatically affected by the exposure to the drug. Untreated control with 119 clones and 50nM ARV treated group showed 14 colonies in A375R after 5 days incubation. In vitro release showed no burst release of Lpc within 24h. Lpc was found to be stable within one month at room temperature.
Conclusion: Protein kinase C inhibitor anchored BRD4 PROTAC PEGylated nanoliposomes was successfully developed which is very promising for the treatment of BRAFV600E-mutated vemurafenib- resistant melanoma.