First demonstration that brain CYP2D-mediated opiate metabolic activation alters analgesia in vivo
Graphical abstract
Introduction
Cytochromes P450 (CYPs) are drug-metabolizing enzymes which are primarily expressed in the liver but are also expressed in other tissues such as the brain. CYPs in the brain are active in vivo [1], and in some cell types, brain CYPs are expressed at levels as high as those in the liver [2]. There is large interindividual variation in the response to centrally acting drugs, which does not always correlate with plasma drug levels [3]. This may be caused, in some circumstances, by variation in the degree of metabolism by brain CYPs, which may affect local drug and metabolite levels in the brain, and in turn influence drug response.
Cytochrome P450 2D6 (CYP2D6) metabolizes many centrally acting drugs, including clinically prescribed drugs (e.g. risperidone, fluoxetine and codeine) as well as drugs of abuse (e.g. amphetamine, 3,4-methylenedioxymethamphetamine) [4]. Brain CYP2D6 levels are influenced by multiple factors. CYP2D6 is genetically polymorphic, which results in different phenotypes ranging from poor metabolizers, who lack CYP2D6 function, to ultrarapid metabolizers, who have multiple functional copies of CYP2D6 [5]. Levels of brain CYP2D6, unlike hepatic CYP2D6, are induced by drugs; for example, they are higher in smokers and alcoholics [6], [7], [8] and in animals exposed to nicotine and ethanol [7], [9], [10]. Brain CYP2D6 levels also increase with age while hepatic CYP2D6 levels remain the same [11], [12]. Therefore, genetics, environmental inducers and age may contribute to variation in brain CYP2D6 expression and activity, and thereby alter the metabolism of, and response to, centrally acting drugs.
CYP2D6 metabolizes codeine to the active morphine metabolite [13]. Since morphine has a 3000-fold greater affinity for the mu-opioid receptor than does codeine [14], analgesia from codeine depends on its conversion to morphine. Thus, CYP2D6 poor metabolizers, and individuals pretreated with the CYP2D6 inhibitor quinidine, produce no morphine from codeine and experience no analgesia [15], [16], [17]. Codeine is metabolized to morphine mainly in the liver; morphine then crosses into the brain where it can interact with mu-opioid receptors to elicit analgesia. However, morphine is less permeable across the blood–brain barrier (BBB) than codeine, and is also actively transported out of the brain by efflux transporters [18]; therefore, there is a delay in morphine's entry into the brain compared to codeine's entry [19]. In rats given peripheral injections of either codeine or morphine, where the doses of the two drugs were adjusted to give the same plasma morphine levels at 30 min after injection, brain morphine was only found in codeine- and not in morphine-treated rats, suggesting that at 30 min, morphine had not yet crossed into the brain from the periphery [20]. Therefore, the morphine found in the brain during the first 30 min after codeine injection may be due to local codeine metabolism in the brain.
Our objective was to examine the role of rat brain CYP2D in the metabolic activation of codeine and subsequent analgesia. The rat is a useful model of human CYP2D6-mediated drug metabolism since rat CYP2D has similar substrate specificity and enzymatic activity to human CYP2D6 [21]. The role of brain, as opposed to hepatic, CYP2D was investigated using intracerebroventricular (i.c.v.) injection of CYP2D inhibitors in rats, which inhibited brain CYP2D activity while leaving hepatic CYP2D activity unchanged. We hypothesized that compared to vehicle pretreatment, CYP2D inhibitor pretreatment would result in lower brain morphine levels and therefore lower analgesia after subcutaneous codeine injection. Clarifying the impact of brain CYP2D-mediated metabolism on codeine response will help us understand the potential role brain CYPs have in interindividual variation in the response to centrally acting drugs.
Section snippets
Animals
Male adult Wistar rats (250–300 g; Charles River, St. Constant, QC, Canada) were kept in pairs or triplets under a 12 h artificial light/dark cycle. Rats were handled daily to acclimate them to testing procedures. All procedures were approved by the Animal Care Committee at the University of Toronto.
Drugs
Propranolol hydrochloride (Sigma–Aldrich, Oakville, ON, Canada), a CYP2D mechanism-based inhibitor [22], was dissolved in artificial cerebrospinal fluid (ACSF; 126 mM NaCl, 2.68 mM KCl, 1 mM Na2HPO4, 0.88
Inhibition of brain CYP2D reduced codeine-induced analgesia
We examined whether inhibiting brain CYP2D would reduce analgesia following codeine administration. Compared to vehicle pretreatment, 20 μg i.c.v. propranolol pretreatment resulted in significantly lower %MPE at 15 (p < 0.04), 20 (p < 0.02), 30 (p < 0.006) and 40 (p < 0.005) min after 30 mg/kg s.c. codeine injection (Fig. 1A). Compared to vehicle pretreatment, 40 μg i.c.v. propafenone pretreatment resulted in significantly lower %MPE at 20 (p < 0.03), 30 (p < 0.03) and 40 (p < 0.05) min after 30 mg/kg s.c. codeine
Discussion
This is the first study to show that brain CYP2D-mediated metabolism can alter the effect of a centrally acting drug. Inhibiting brain CYP2D decreased the first 60 min of codeine-induced analgesia, but did not alter baseline nociception or morphine-induced analgesia. This confirms that the inhibitors did not lower codeine-induced analgesia by having their own effects on nociception or by interfering with morphine's mechanisms of action. Furthermore, showing that analgesia from codeine was
Conflict of interest statement
Dr. Rachel Tyndale has participated in one-day advisory meetings for Novartis and McNeil.
Acknowledgements
The authors would like to thank Dr. Sharon Miksys for her technical assistance and sharing of knowledge and advice, and Steven Lo for his technical assistance. This work was supported by a Canadian Research Chair (RFT), the Endowed Chair in Addiction for the Department of Psychiatry (RFT), Canadian Institutes of Health Research (grant number MOP97751), National Institutes of Health (grant number DA 02083), Centre for Addiction and Mental Health and the Centre for Addiction and Mental Health
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Department of Psychiatry, Dartmouth College, One Medical Center Drive, Lebanon, NH 03756, United States.