Effects of CYP2D6 status on harmaline metabolism, pharmacokinetics and pharmacodynamics, and a pharmacogenetics-based pharmacokinetic model

Abstract

Harmaline is a beta-carboline alkaloid showing neuroprotective and neurotoxic properties. Our recent studies have revealed an important role for cytochrome P450 2D6 (CYP2D6) in harmaline O-demethylation. This study, therefore, aimed to delineate the effects of CYP2D6 phenotype/genotype on harmaline metabolism, pharmacokinetics (PK) and pharmacodynamics (PD), and to develop a pharmacogenetics mechanism-based compartmental PK model. In vitro kinetic studies on metabolite formation in human CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) hepatocytes indicated that harmaline O-demethylase activity (V(max)/K(m)) was about 9-fold higher in EM hepatocytes. Substrate depletion showed mono-exponential decay trait, and estimated in vitro harmaline clearance (CL(int), microL/min/10(6)cells) was significantly lower in PM hepatocytes (28.5) than EM hepatocytes (71.1). In vivo studies in CYP2D6-humanized and wild-type mouse models showed that wild-type mice were subjected to higher and longer exposure to harmaline (5 and 15mg/kg; i.v. and i.p.), and more severe hypothermic responses. The PK/PD data were nicely described by our pharmacogenetics-based PK model involving the clearance of drug by CYP2D6 (CL(CYP2D6)) and other mechanisms (CL(other)), and an indirect response PD model, respectively. Wild-type mice were also more sensitive to harmaline in marble-burying tests, as manifested by significantly lower ED(50) and steeper Hill slope. These findings suggest that distinct CYP2D6 status may cause considerable variations in harmaline metabolism, PK and PD. In addition, the pharmacogenetics-based PK model may be extended to define PK difference caused by other polymorphic drug-metabolizing enzyme in different populations.

Figure 1

Schematic representation of the PK/PD model consisting of a pharmacogenetics-based compartmental PK model of harmaline following i.p. and i.v. administration, and an indirect response pharmacodynamic model with stimulation of K_out (IDRIV) for harmaline-induced hypothermia following i.p. administrations. PK model: Xa, drug amount at the absorption site; K_a, absorption rate constant; F, bioavailability. X_C and X_T represent harmaline amount in central and peripheral compartment, respectively; V_C and V_T represent volume of distribution of harmaline in central and peripheral compartment, respectively; CL_CYP2D6 and CL_other represent clearance of drug from central compartment contributed by CYP2D6-mediated metabolism and other elimination pathways, respectively; CL_D represents distribution clearance of harmaline between central and peripheral compartments. PD model, k_in, zero order rate constant of heat production; k_out, the first order rate constant of heat loss; S_max, the maximum fractional drug effect; SC₅₀, drug concentration required to produce 50% of the maximum effect. Of particular note, drug (e.g., harmaline) is eliminated by CL_CYP2D6 and CL_other in Tg-CYP2D6 mice (or EMs), i.e., CL = CL_CYP2D6 + CL_other, whereas the drug is eliminated by CL_other only in wild-type control mice (or PMs), i.e., CL = CL_other.

Figure 2

Eadie-Hofstee plots of harmalol formation from harmaline in human CYP2D6 EM (A) and PM (B) hepatocytes. Values for individual hepatocyte samples represent mean ± SEM of triplicate experiments. CYP2D6 EM hepatocytes (N = 5) exhibit monophasic kinetics, whereas PM hepatocytes (N = 4) show biphasic kinetics.

Figure 3

Harmaline depletion (A) and harmalol production (B) in human CYP2D6 EM (N = 6) and PM (N = 4) hepatocytes. Values represent mean ± SD. Significant difference in both harmaline depletion and harmalol production is shown for the variations of incubation time and CYP2D6 genotype/phenotype (P < 0.0001; two-way ANOVA).

Figure 4

Serum harmaline concentration vs. time curves in wild-type and Tg-CYP2D6 mice administered i.v. (A) or i.p. (B) with harmaline (5 and 15 mg/kg). Values represent mean ± SD (N = 3 at each time point). For each dose, significant difference in harmaline concentrations is shown for the variations of CYP2D6 genotype and time (P < 0.05; two-way ANOVA).

Figure 5

Hypothermic effects induced by harmaline (5 and 15 mg/kg, i.p.) in wild-type and Tg-CYP2D6 mice. Values represent mean ± SD (N = 8 in each group). For the high dose (15 mg/kg), significant difference is shown for the variations of CYP2D6 genotype and time (P < 0.001; two-way ANOVA). Difference (*P < 0.05, **P < 0.01) is also noted between the two genotyped mice treated with 15 mg/kg harmaline at specific time points.

Figure 6

Marble-burying behavior of wild-type and Tg-CYP2D6 mice exposed to harmaline (0–10 mg/kg, i.p.). Values represent mean ± SD (N = 14 in each group). The dose responses are significantly different between the two genotyped mice (P < 0.001; two-way ANOVA). Difference (*P < 0.05, ***P < 0.001) is also noted between the two genotyped mice at specific doses. It is notable that wild-type mice exhibit a significantly lower ED₅₀ value (5.00 ± 1.04 mg/kg) and steeper Hill slope (−5.79 ± 1.58) than Tg-CYP2D6 mice (6.75 ± 1.05 mg/kg and −4.28 ± 0.84, respectively).