Development and validation of a quantification method for cucurbitacins E and I

34 Cucurbitacin E is a potential drug candidate due to its anticancer activity, recognition of 35 its molecular targets, and synergism with other drugs used for cancer treatment. 36 However, the use of cucurbitacin E in clinical practice is not possible because of 37 important knowledge gaps in its preclinical and clinical pharmacokinetic characteristics. 38 Cucurbitacin E is hydrolyzed to cucurbitacin I in plasma and in human liver 39 microsomes. The aim of this study was to evaluate the population pharmacokinetics of 40 cucurbitacin E and of its metabolite cucurbitacin I in rats. The method for the sequential 41 analysis of cucurbitacins E and I in rat plasma was developed using LC-MS/MS. 42 Plasma aliquots of 50 μL were deproteinized with acetonitrile and clobazam was added 43 as internal standard. The extracts were injected into an RP-18 column and eluted with a 44 mobile phase consisting of a mixture of acetonitrile:water:methanol (32:35:33, v/v/v). 45 The method was precise and accurate, showing linearity in the range of 1-100 ng 46 cucurbitacin E/mL plasma and of 0.4-200 ng cucurbitacin I/mL plasma. The method 47 was applied to the pharmacokinetic evaluation of cucurbitacin E administered 48 intravenously to male Wistar rats (1 mg/kg). Serial blood samples were collected up to 49 24 h after administration. The plasma concentrations of cucurbitacin E were quantified 50 up to 16 h, while the plasma concentrations of cucurbitacin I remained below the limit 51 of quantification. A population pharmacokinetic model was developed for cucurbitacin 52 E using the NONMEM program, with adequate goodness of fit and predictive 53 performance. The following pharmacokinetic parameters were obtained: release time of 54 0.45 h, volume of distribution of 27.22 L, clearance of 4.13 L/h, and elimination half55 life of 4.57 h. 56 57