Winnonlin 5

Pharmacokinetic parameters were determined using non-compartmental techniques with WinNonlin® 5.0 software (Pharsight Corporation, Mountain View, CA, USA). SHAM analysis (i.e., Slope, Height, Area, and Moment) [19 ] utilized plasma concentration-time data to estimate the area under the curve (AUC), terminal elimination half-life (t1/2), and the area under the first moment of the plasma concentration-time curve (AUMC). The AUC was calculated for each animal using the piecewise log trapezoidal areas. The non-compartmental parameters were calculated for each rat before averaging dose groups.

The bio-availability kinetics of the CFM-4 NLF formulation, and CFM-4 free drug (FD) were conducted in rodents (Sprague Dawley Rats). Rats were fasted overnight before the start of the experiments and randomly divided into three experimental groups receiving CFM-4 FD and CFM-4 NLF at 40 mg/kg orally and CFM-4 solution (CFM-4 sol) at 5 mg/kg by intravenous route. After the drug administration, blood samples (250 μl) were withdrawn from tail veins at predetermined time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h).
The blood samples were collected directly into heparinized microvet blood collection tubes and plasma was obtained by centrifugation at 10,000 rpm for 10 min and then stored at −80 °C until analysis. CFM-4 was extracted from the plasma by protein precipitation method and extracted samples were dissolved in mobile phase and samples were analyzed by HPLC. Oral bioavailability of CFM-4 FD and CFM-4 NLF along with their pharmacokinetic parameters such as area under curve (AUC), Cmax, t1/2, and t max were estimated. Pharmacokinetic parameters were analyzed using non-compartmental techniques with WinNonlin^® 5.0 software (Pharsight Corporation, Mountain View, CA, USA).

Pharmacokinetics of DIM-P and Doc in BALB/c mice were determined following I.V. administration of NDi, NDo, NDDs & ENDDs (DIM-P equivalent to 5.0 mg/kg, Doc equivalent to 10.0 mg/kg). Animals were randomly distributed into experimental groups (n = 6) and fasted overnight prior to experiment. Blood samples (250 µL) were collected by heart puncture at the following time points: 0, 0.017, 0.25, 0.5, 0.75, 1, 3, 6, 8, 12 and 24 h. Samples were processed and extracted drug was analyzed by HPLC analysis (36 (link)). Briefly, DIM-P was separated from plasma by protein precipitation using acetonitrile and samples were centrifuged for 15 min at 10,000 g. Sample was run on a mobile phase consisting of acetonitrile and water (90:10% v/v) using a Waters Symmetry® C18 guard column (5 µm, 3.9×20 mm) and a Waters Symmetry® C18 column (5 µm, 4.6 × 250 mm) at a flow rate of 1.0 mL/min and DIM-P was monitored at 242 nm. Pharmacokinetic parameters were calculated using non-compartmental techniques with WinNonlin® 5.0 software (Pharsight Corporation, Mountain View, CA, USA).