In vitro inhibition of human cytochrome P450 enzymes by the novel atypical antipsychotic drug asenapine: a prediction of possible drug-drug interactions

Abstract

Background: Inhibition of cytochrome P450 (CYP) enzymes is the most common cause of harmful drug-drug interactions. The present study aimed at examining the inhibitory effect of the novel antipsychotic drug asenapine on the main CYP enzymes in human liver.

Methods: The experiments were performed in vitro using pooled human liver microsomes and the human cDNA-expressed CYP enzymes: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (Supersomes). Activities of CYP enzymes were determined using the CYP-specific reactions: caffeine 3-N-demethylation (CYP1A2), diclofenac 4'-hydroxylation (CYP2C9), perazine N-demethylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), and testosterone 6β-hydroxylation (CYP3A4). The rates of the CYP-specific reactions were assessed in the absence and presence of asenapine using HPLC.

Results: The obtained results showed that both in human liver microsomes and Supersomes asenapine potently and to a similar degree inhibited the activity of CYP1A2 via a mixed mechanism (K_i = 3.2 μM in liver microsomes and Supersomes) and CYP2D6 via a competitive mechanism (K_i = 1.75 and 1.89 μM in microsomes and Supersomes, respectively). Moreover, asenapine attenuated the CYP3A4 activity via a non-competitive mechanism (K_i = 31.3 and 27.3 μM in microsomes and Supersomes, respectively). In contrast, asenapine did not affect the activity of CYP2C9 or CYP2C19.

Conclusion: The potent inhibition of CYP1A2 and CYP2D6 by asenapine, demonstrated in vitro, will most probably be observed also in vivo, since the calculated K_i values are close to the presumed concentration range for asenapine in the liver in vivo. Therefore, pharmacokinetic interactions involving asenapine and CYP2D6 or CYP1A2 substrates are likely to occur during their co-administration to patients.

Keywords: Asenapine; Cytochrome P450; Human liver microsomes; Inhibition; cDNA-expressed CYP enzymes.

Fig. 1

The influence of asenapine on the activity of CYP1A2 measured as a rate of caffeine 3-N-demethylation. a Human liver microsomes (K_m = 709 µM, V_max = 9.67 pmol/mg protein/min). b Human cDNA-expressed CYP1A2 (Supersomes CYP1A2) (K_m = 705 µM, V_max = 1.92 pmol/pmol CYP/min). Each point represents the mean value of two independent analyses. V velocity of the reaction, I the concentration of the inhibitor (asenapine), S the concentration of the substrate (caffeine). The K_i values and mechanisms of inhibition are shown in Table 1. Dixon’s plots (the main plots): the caffeine concentration of 200 µM (■), 400 µM (▲), and 800 µM (▼). Lineweaver–Burk’s plots (inserts): control—no asenapine (✱); the asenapine concentration of 0.5 µM (x), 1 µM (○), 5 µM (□), and 10 µM (△)

Fig. 2

The influence of asenapine on the activity of CYP2D6 measured as a rate of bufuralol 1′-hydroxylation. a Human liver microsomes (K_m = 5.1 µM, V_max = 5.59 pmol/mg protein/min). B Human cDNA-expressed CYP2D6 (Supersomes CYP2D6) (K_m = 6.6 µM, V_max = 4.1 pmol/pmol CYP/min). Each point represents the mean value of two independent analyses. V velocity of the reaction, I the concentration of the inhibitor (asenapine), S the concentration of the substrate (bufuralol). The K_i values and mechanisms of inhibition are shown in Table 1. Dixon’s plots (the main plots): the bufuralol concentration of 10 µM (■), 25 µM (▲), and 50 µM (▼). Lineweaver–Burk’s plots (inserts): control—no asenapine (✱); the asenapine concentration of 0.5 µM (x), 1 µM (○), 5 µM (□), and 10 µM (△)

Fig. 3

The influence of asenapine on the activity of CYP3A4 measured as a rate of testosterone 6β-hydroxylation. a Human liver microsomes (K_m = 283 µM, V_max = 72.9 pmol/mg protein/min). b Human cDNA-expressed CYP3A4 (Supersomes CYP3A4) (K_m = 428 µM, V_max = 42.9 pmol/pmol CYP/min). Each point represents the mean value of two independent analyses. V velocity of the reaction, I the concentration of the inhibitor (asenapine), S the concentration of the substrate (testosterone). The K_i values and mechanisms of inhibition are shown in Table 1. Dixon’s plots (the main plots): the testosterone concentration of 50 µM (■), 100 µM (▲), and 200 µM (▼). Lineweaver–Burk’s plots (inserts): control—no asenapine (✱); the asenapine concentration of 0.5 µM (x), 1 µM (○), 5 µM (□), and 10 µM (△)

Fig. 4

The influence of asenapine on the activity of CYP2C9 (a) measured as a rate of diclofenac 4′-hydroxylation and CYP2C19 (b) measured as a rate of perazine N-demethylation. The enzyme activity was estimated in human liver microsomes (K_m = 136.8 µM, V_max = 21.02 nmol/mg protein/min for diclofenac 4′-hydroxylation; K_m = 199.9 µM; V_max = 1.58 nmol/mg protein/min for perazine N-demethylation) and Supersomes CYP2C9 and CYP2C19 (K_m = 131.9 µM, V_max = 213.6 pmol/pmol CYP/min for diclofenac 4′-hydroxylation; K_m = 162.3 µM, V_max = 39.4 pmol/pmol CYP/min for perazine N-demethylation). Each point represents the mean value of two independent analyses. V velocity of the reaction, I the concentration of the inhibitor (asenapine), S the concentration of the substrate (diclofenac or perazine). Dixon’s plots: a the diclofenac concentration of 5 µM (■), 10 µM (▲), and 25 µM (▼); b the perazine concentration of 50 µM (■), 100 µM (▲), and 200 µM (▼)