br Materials and methods br Results
Materials and methods
Results Metabolic stability of compounds was studied by incubation with pooled human liver microsomes. Piperine was the metabolically most stable compound, with a t1/2 of 141min (Fig. 2, and Table 1). SCT-29 and LAU 397 showed t1/2 of approx. 45min, whereas a t1/2 of only 24.9min was found for LAU 399 (Fig. 2, and Table 1). Apparent intrinsic clearance was calculated (Table 1) and used to categorize compounds into low, medium, or high clearance . Piperine was thus a low clearance compound, SCT-29 and LAU 397 were medium clearance, and LAU 399 was a high clearance compound. However, due to non-specific binding of substrates to microsomes, the unbound fractions were determined and employed to calculate unbound intrinsic clearance for each molecule (Table 1). It appeared that all analogs had high unbound intrinsic clearance (267–411μl/min/mg protein). Human hepatic clearance and extraction ratio were predicted from in vitro microsomal stability data using the liver well-stirred model. In vitro unbound intrinsic clearance of each 86 5 was converted into in vivo by applying physiological scaling factors (Table 2). Binding of piperine and analogs in whole blood was determined since extensive binding negatively affects distribution of the compounds into deeper compartments and hence, concentrations at the target site. Unbound fractions of compounds in whole blood were calculated, and used for the prediction of human hepatic clearance (Table 2). The recovery of test compounds was checked in the whole blood binding assay. The mean% recovery±SD (n=3) was 83.4±9.27 for piperine, 85.8±13.1 for SCT-29, 82.1±11.4 for LAU 397, 95.5±21.9 for LAU 399. In addition, the hepatic extraction ratio was calculated (Table 2) to classify the compounds into low, medium, or high extraction categories . Based on these criteria, all compounds were low extraction ratio compounds. Metabolite profiles of compounds after incubation with human microsomes were analyzed by high resolution (HR)-MS, and the acquired data were further processed by metabolite identification software. The presence of a diagnostic product ion from the parent compound in the MS spectra of metabolites supported metabolite identification. The fragmentation patterns of parent compounds are summarized in Fig. S2. For piperine, extracted ion chromatograms of the blank, t0, and t120 samples are shown for m/z 201.0547 (diagnostic product ion) (Fig. S3). Extracted ion chromatograms of the t120 sample for m/z 201.0547 (diagnostic product ion), and for m/z 302.1387 and 304.1544 (metabolites) are given in Fig. 3. Microsomal incubation of piperine yielded four metabolites M1-M4 (Table 3). Metabolites M1-M3, all with m/z of 302.1387, were formed by hydroxylation of the piperidine ring (Fig. 4). M4 (m/z 304.1544) was likely formed via opening of the piperidine ring by N-dealkylation, and reduction of the aldehyde (Fig. 4). MS spectra of piperine and metabolites are shown in Fig. S4.