Elsevier

Biochemical Pharmacology

Volume 78, Issue 6, 15 September 2009, Pages 617-624
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2009.05.011Get rights and content

Abstract

Harmaline is a β-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 (Vmax/Km) was about 9-fold higher in EM hepatocytes. Substrate depletion showed mono-exponential decay trait, and estimated in vitro harmaline clearance (CLint, μL/min/106 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 15 mg/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 (CLCYP2D6) and other mechanisms (CLother), 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 ED50 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.

Introduction

Cytochrome P450 2D6 (CYP2D6) is one of the most important polymorphic phase I drug-metabolizing enzymes that is involved in the biotransformation of 20–30% of marketed drugs and some endogenous substrates [1], [2]. Individuals with impaired or deficient CYP2D6 activity could be more sensitive to CYP2D6-metabolized drugs, which is likely dependent on the fraction of CYP2D6-mediated metabolism in total drug clearance and the range between therapeutic and toxic doses of the drug. CYP2D6 also metabolizes a variety of drugs of abuse such as amphetamines [3], [4], [5], [6], indoleamines [7], [8], [9] and many other designer drugs [10], [11]. Delineation of the impact of CYP2D6 on the metabolism, pharmacokinetics (PK) and pharmacodynamics (PD) of these substances would provide increased understanding of individual vulnerability to and/or protection from illicit drugs of abuse [12].

Harmaline, 3,4-dihydro-7-methoxy-1-methyl-β-carboline, is a major form of alkaloid in the seeds of Peganum harmala (Syria rue) that has been used in traditional medicine and for recreational purpose [13]. Harmaline is also a typical psychotropic ingredient in the recreational beverage Ayahuasca, which shows potential in treating many psychiatric disorders [14], whereas the therapeutic potential and medical uses are hampered by many safety issues including abuse liability. Harmaline readily modulates the levels of amine neurotransmitters via inhibition of monoamine oxidase. It may directly bind to specific receptors including 5-HT, benzodiazepine and imidazoline receptors. With diverse biochemical properties, harmaline has shown neuroprotective, anxiolytic, antinociceptive, antioxidant and vasorelaxant properties in animal models [15], [16], [17], [18], [19]. On the other hand, harmaline alone or along with other tryptamine substances may result in severe or even fatal toxicities in animals [20], [21], [22] and in humans [23], [24], [25]. Of particular note, harmaline acts on the olivo-cerebellar system, causes rhythmic activity, and leads to postural tremors in animal models. Harmaline-induced tremor has many similar characteristics as essential tremor, a common movement disorders in humans. Therefore, harmaline tremor has been widely used as a model to understand the pathogenesis of essential tremor and to develop therapeutic agents for generalized tremor [21], [22], [26].

Following an acute administration to rats, harmaline is rapidly distributed into different organs and mainly metabolized via O-demethylation [27]. Recently we have shown that harmaline O-demethylation is primarily catalyzed by cytochrome P450 2D6 (CYP2D6) and CYP1A2 in human liver microsomes [28]. This study, therefore, aimed to (1) define the effects of CYP2D6 status on harmaline metabolism, pharmacokinetics and pharmacodynamics, and (2) use harmaline as a model drug to develop pharmacogenetics-based PK compartmental model to provide increased understanding of the PK difference caused by CYP2D6 metabolism.

Section snippets

Chemicals and materials

Harmaline hydrochloride dihydrate, harmalol hydrochloride dihydrate and β-glucuronidase were purchased from Sigma–Aldrich (St. Louis, MO). Cryopreserved human hepatocytes, InVitroGRO HT Medium, KHB buffer and Torpedo Antibiotic Mix were purchased from Celsis (Chicago, IL) or BD Biosciences (San Jose, CA). The lot numbers of CYP2D6 PM hepatocyte samples were VTA (dextromethorphan O-demethylase activity, 1 pmol/min/106 cells; CYP2D6 genotype, *3/*4), ETR (1 pmol/min/106 cells; *4/*4), PFM (1 

Harmaline O-demethylase activity is much lower in human CYP2D6 PM hepatocytes

To delineate the effects of CYP2D6 status on harmaline metabolism, we first compared harmalol production between CYP2D6 EM and PM hepatocytes in vitro. Interestingly, all EM hepatocytes (N = 5) showed monophasic Michaelis–Menten enzyme kinetics, whereas PM hepatocytes (N = 4) exhibited biphasic kinetics (Fig. 2). Estimated enzyme efficiency (Vmax/Km, μL/min/106 cells) was about 9-fold lower in CYP2D6 PM hepatocytes (0.71 ± 0.44) than that in CYP2D6 EM hepatocytes (6.32 ± 2.35) (Table 1). The results

Discussion

Humans are exposed to endogenously synthesized, dietary or recreational β-carbolines, an important group of indoleamines that have a wide spectrum of biochemical, pharmacological and toxicological properties. Harmaline represents a β-carboline alkaloid that shows both neurotoxic and neuroprotective effects, and cases of harmaline-related intoxication have been documented in recent years [23], [24], [25]. Our previous study has revealed an important contribution of polymorphic CYP2D6 to

Acknowledgements

The project was supported by Award Number R01DA021172 from the National Institute On Drug Abuse (NIDA), National Institutes of Health (NIH). The authors also thank the Pharmaceutical Sciences Instrumentation Facility at University at Buffalo for use of LC-MS system that was obtained with Shared Instrumentation Grants S10RR014592 from the National Center for Research Resources, NIH.

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