The effect of selective inhibitors on the rate of artemisinin disappearance was investigated in microsomes pooled from 10 livers (Human Biologics International, Scottsdale, Arizona, USA). The selective inhibitors 8-methoxypsoralen (500 μm), orphenadrine (500 μm), ketoconazole (4 μm) and troleandomycin (50 μm) were used to investigate the involvement of CYP2A6 , CYP2B6 [13, 14], CYP3A4 [15, 16] and CYP3A4 , respectively. The effect of orphenadrine in combination with ketoconazole or 8-methoxypsoralen was investigated in these microsomes as well.
Inhibition studies were also performed with characterized microsomes. Microsomes (Human Biologics International, Scottsdale, Arizona, USA) from one human liver (HBI 102) with high S-mephenytoin N-demethylation (CYP2B6) and dextromethorphan N-demethylation (CYP3A4) activity and microsomes from one liver (HBI 103) with low CYP2B6 and CYP3A4 activity were incubated with artemisinin in the presence of the CYP2B6 inhibitor orphenadrine (500 μm). The effect of the CYP3A4 inhibitor ketoconazole (4 μm) was also investigated in these two livers. In addition, artemisinin was coincubated with the antimalarial R,S-mefloquine (200 μm).
The inhibitors were dissolved in dimethylsulphoxide (DMSO) (final concentration <0.5
. The total incubation volume was 1 ml and the incubation was maintained at 37° C in a shaking water-bath. The reaction was initiated by addition of ice-cold NADPH after 2 min preincubation (10 min preincubation with troleandomycin) and the metabolic activity was terminated by addition of ice-cold MeOH (200 μl) to 100 μl sample at 0, 10, 20, 30 and 40 min. After centrifugation at 10 000 g for 10 min, 60 μl of the supernatant was injected directly onto the h.p.l.c. column. Control experiments were performed in parallel with artemisinin incubated without inhibitor but with 0.5% DMSO. All inhibition incubations were performed in triplicate.
In addition, the specificity of orphenadrine, ketoconazole and 8-methoxypsoralen in inhibiting artemisinin metabolism was investigated in triplicate in microsomes from human B-lymphoblastoid cell lines expressing CYP2B6, CYP3A4 and CYP2A4, respectively.
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In incubations with microsomes from human lymphoblastoid cell lines expressing different isoenzymes, artemisinin disappearance rates (nmol min−1 mg−1 protein), adjusted for native activity were calculated. In incubations with characterized microsomes and pooled microsomes, artemisinin first-order elimination rate constants were estimated by log-linear regression of drug concentration–time data. The inhibition of artemisinin metabolism by the specific inhibitors was evaluated by comparing the first-order elimination rate constants (k) in the presence and absence of inhibitors [% inhibition=100×(kabsence-kpresence)/ kabscence].
To compare artemisinin disappearance rates and specific CYP450 activities in characterized human liver microsomes, regression analysis was performed on log-transformed data for both dependent and independent variables due to skew distribution and in particular microsomes from one donor exhibiting generally very high enzyme activities and artemisinin disappearance rates. Since simple linear regressions of artemisinin metabolism rates against individual, specific enzyme activities in microsomes from different livers do not apply when enzyme activities correlate (coregulation), analysis by multiple regressions was initially considered for investigating the relative contribution of individual isoenzymes to the disappearance of artemisinin in characterized liver microsomes. After both forward and backward stepwise multiple regression, CYP2B6 activity was selected as explaining most of the variability in artemisinin disappearance rates, whereas the inclusion of other enzymes into the regression model was inconsistent (results not shown). The relationship between log artemisinin disappearance rate constants and log S-mephenytoin N-demethylation activities (CYP2B6 activity) (slope=0.97±0.04 (s.e.mean),r2=0.94) obtained from a simple regression analysis (Statistica, StatSoft, Tulsa, Okla) was by visual inspection of the plot not completely linear. Due to possible nonlinearities and subgroups with different contributions of individual isoenzymes, a tree-based model  was therefore chosen for the final regression analysis using Xpose 2.0 software . This is further considered in the discussion section. Results are presented as median (range).
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Incubations with cDNA-expressed human CYP450 isoenzymes
In incubations with microsomes from the AHH-1 TK+/−human lymphoblastoid cell line, the most rapid disappearance of artemisinin, about 6 nmol min−1 mg−1 protein, was observed in microsomes expressing CYP2B6 (Figure 2). The rate of artemisinin metabolism was 7-fold and 10-fold higher in microsomes with cDNA-expressed CYP2B6 compared to cDNA-expressed CYP2A6 and CYP3A4, respectively. Low disappearance rates were seen in incubations with recombinant CYP1A1 and oxidoreductase. No or negligible disappearance of artemisinin was found with microsomes expressing CYP1A2, 2C19, 2C8, 2C9, 2D6, 2E1 and epoxide hydrolase. There was no difference in the relative order of the velocities between the different cDNA expressed enzymes in incubations with 1.0 mg ml−1 microsomal protein compared with 2.5 mg ml−1 protein, with the exception of CYP2E1 where the rate of artemisinin disappearance (mg−1 protein) was more rapid when incubated with 2.5 mg ml−1 compared with 1.0 mg ml−1 protein.