Cytochrome P450 enzymes and metabolism of drugs and neurotoxins within the mammalian brain.

Cytochrome P450 enzymes (CYPs) that metabolize xenobiotics are expressed and active in the brain. These CYPs contribute to the metabolism of many centrally acting compounds, including clinically used drugs, drugs of abuse, and neurotoxins. Although CYP levels are lower in the brain than in the liver, they may influence central substrate and metabolite concentrations, which could alter resulting centrally-mediated responses to these compounds. Additionally, xenobiotic metabolizing CYPs are highly variable due to genetic polymorphisms and regulation by endogenous and xenobiotic molecules. In the brain, these CYPs are sensitive to xenobiotic induction. As a result, CYPs in the brain vary widely, including among humans, and this CYP variation may influence central metabolism and resulting response to centrally acting compounds. It has been demonstrated, using experimental manipulation of CYP activity in vivo selectively within the brain, that CYP metabolism in the brain alters central substrate and metabolite concentrations, as well as drug response and neurotoxic effects. This suggests that variability in xenobiotic metabolizing CYPs in the human brain may meaningfully contribute to individual differences in response to, and effects of, centrally acting drugs and neurotoxins. This chapter will provide an overview of CYP expression in the brain, endogenous- and xenobiotic-mediated CYP regulation, and the functional impact of CYP-mediated metabolism of drugs and neurotoxins in the brain, with a focus on experimental approaches in mice, rats, and non-human primates, and a discussion regarding the potential role of xenobiotic metabolizing CYPs in the human brain.

The role of CYP2D in rat brain in methamphetamine-induced striatal dopamine and serotonin release and behavioral sensitization.

RATIONALE: Cytochrome P450 2D (CYP2D) enzymes metabolize many addictive drugs, including methamphetamine. Variable CYP2D metabolism in the brain may alter CNS drug/metabolite concentrations, consequently affecting addiction liability and neuropsychiatric outcomes; components of these can be modeled by behavioral sensitization in rats. METHODS: To investigate the role of CYP2D in the brain in methamphetamine-induced behavioral sensitization, rats were pretreated centrally with a CYP2D irreversible inhibitor (or vehicle) 20 h prior to each of 7 daily methamphetamine (0.5 mg/kg subcutaneous) injections. In vivo brain microdialysis was used to assess brain drug and metabolite concentrations, and neurotransmitter release. RESULTS: CYP2D inhibitor (versus vehicle) pretreatment enhanced methamphetamine-induced stereotypy response sensitization. CYP2D inhibitor pretreatment increased brain methamphetamine concentrations and decreased the brain p-hydroxylation metabolic ratio. With microdialysis conducted on days 1 and 7, CYP2D inhibitor pretreatment exacerbated stereotypy sensitization and enhanced dopamine and serotonin release in the dorsal striatum. Day 1 brain methamphetamine and amphetamine concentrations correlated with dopamine and serotonin release, which in turn correlated with the stereotypy response slope across sessions (i.e., day 1 through day 7), used as a measure of sensitization. CONCLUSIONS: CYP2D-mediated methamphetamine metabolism in the brain is sufficient to alter behavioral sensitization, brain drug concentrations, and striatal dopamine and serotonin release. Moreover, day 1 methamphetamine-induced neurotransmitter release may be an important predictor of subsequent behavioral sensitization. This suggests the novel contribution of CYP2D in the brain to methamphetamine-induced behavioral sensitization and suggests that the wide variation in human brain CYP2D6 may contribute to differential methamphetamine responses and chronic effects.

Addressing the instability issue of dopamine during microdialysis: the determination of dopamine, serotonin, methamphetamine and its metabolites in rat brain.

Dopamine is a catecholamine neurotransmitter that degrades rapidly in aqueous solutions; hence, its analysis following brain microdialysis is challenging. The aim of the current study was to develop and validate a new microdialysis coupled LC-MS/MS system with improved accuracy, precision, simplicity and turnaround time for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine analysis in the brain. Dopamine degradation was studied with different stabilizing agents under different storage conditions. The modified microdialysis system was tested in vitro, and was optimized for best probe recovery, assessed by %gain. LC-MS/MS assay was developed and validated for the targeted compounds. Stabilizing agents (ascorbic acid, EDTA and acetic acid) as well as internal and cold standards were added on-line to the dialysate flow. Assay linearity range was 0.01-100 ng/mL, precision and accuracy passed criteria, and LOQ and LLOQ were 0.2 and 1.0 pg, respectively. The new microdialysis coupled LC-MS/MS system was used in Wistar rats striatum after 4 mg/kg subcutaneous methamphetamine. Methamphetamine rapidly distributed to rat striatum reaching an average ~200 ng/mL maximum, ~82.5 min post-dose. Amphetamine, followed by 4-hydroxymethamphetamine, was the most abundant metabolite. Dopamine was released following methamphetamine injection, while serotonin was not altered. In conclusion, we proposed and tested an innovative and simplified solution to improve stability, accuracy and turnover time to monitor unstable molecules, such as dopamine, by microdialysis.

Human CYP2D6 Is Functional in Brain In Vivo: Evidence from Humanized CYP2D6 Transgenic Mice.

CYP2D metabolizes many drugs that act within the brain, and variable expression of CYP2D in the brain may alter local drug and metabolite levels sufficiently to affect behavioral responses. Transgenic mice that express human CYP2D6 (TG) were compared to wild type mice (WT). Following selective inhibition of human CYP2D6 in TG brain, we demonstrated in vivo that human CYP2D6 in the brain was sufficient to alter a drug-induced behavioral response. After a 4-h pre-treatment with intracerebroventricular (i.c.v.) propranolol, CYP2D activity in vivo and in vitro was reduced in TG brain, whereas CYP2D activity in vivo, but not in vitro, was reduced in WT brain. After a 24-h pre-treatment with i.c.v. propranolol, CYP2D activity in vivo and in vitro was reduced in TG brain, whereas CYP2D activity in vivo and in vitro was not changed in WT brain. These results indicate that i.c.v. propranolol irreversibly inhibited human CYP2D6 in TG brain but not mouse CYP2D in TG and WT brain. Pre-treatments with propranolol did not change liver CYP2D activity in vivo or in vitro. Furthermore, 24-h pre-treatment with i.c.v. propranolol resulted in a significant decrease of the haloperidol-induced catalepsy response in TG, but not in WT, without changing serum haloperidol levels in either mouse line. These studies reveal a new tool to selectively and irreversibly inhibit human CYP2D6 in TG brain and indicate that human CYP2D6 has a functional role within the brain sufficient to impact the central nervous system response from peripherally administered drugs.

Human CYP2D6 in the Brain Is Protective Against Harmine-Induced Neurotoxicity: Evidence from Humanized CYP2D6 Transgenic Mice.

CYP2D6 metabolically inactivates several neurotoxins, including beta-carbolines, which are implicated in neurodegenerative diseases. Variation in CYP2D6 within the brain may alter local inactivation of neurotoxic beta-carbolines, thereby influencing neurotoxicity. The beta-carboline harmine, which induces hypothermia and tremor, is metabolized by CYP2D6 to the non-hypothermic/non-tremorgenic harmol. Transgenic mice (TG), expressing human CYP2D6 in addition to their endogenous mouse CYP2D, experience less harmine-induced hypothermia and tremor compared with wild-type mice (WT). We first sought to elucidate the role of CYP2D in general within the brain in harmine-induced hypothermia and tremor severity. A 4-h intracerebroventricular (ICV) pretreatment with the CYP2D inhibitor propranolol increased harmine-induced hypothermia and tremor in TG and increased harmine-induced hypothermia in WT. We next sought to specifically demonstrate that human CYP2D6 expressed in TG brain altered harmine response severity. A 24-h ICV propranolol pretreatment, which selectively and irreversibly inhibits human CYP2D6 in TG brain, increased harmine-induced hypothermia. This 24-h pretreatment had no impact on harmine response in WT, as propranolol is not an irreversible inhibitor of mouse CYP2D in the brain, thus confirming no off-target effects of ICV propranolol pretreatment. Human CYP2D6 activity in TG brain was sufficient in vivo to mitigate harmine-induced neurotoxicity. These findings suggest that human CYP2D6 in the brain is protective against beta-carboline-induced neurotoxicity and that the extensive interindividual variability in CYP2D6 expression in human brain may contribute to variation in susceptibility to certain neurotoxin-associated neurodegenerative disorders.

Attenuation of MK-801-induced behavioral perseveration by typical and atypical antipsychotic pretreatment in rats.

The noncompetitive NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5-10-imine maleate (MK-801) has been shown to increase the probability of operant responding during extinction and reduce infralimbic prefrontal cortical activation, possibly modeling the cognitive dysfunction symptomology, and underlying cause, in patients with schizophrenia. The present study sought to determine if typical and/or atypical antipsychotics would attenuate the MK-801-induced behavioral perseveration and whether this would be associated with concomitant changes in phosphorylated ERK1/2 (pERK1/2) labeling in the infralimbic cortex (IL). Male, Long Evans rats were pretreated with the typical antipsychotic, Flupenthixol (0, 0.125, 0.25 or 0.5 mg/kg) or the atypical antipsychotic, aripiprazole (0, 0.3, 1.0, 3.0 mg/kg), then given 0.1 mg/kg MK-801 followed by a 60-min appetitive operant extinction session. Flupenthixol produced a dose-dependent decrease in MK-801-induced bar pressing behavior and locomotor activity and a dose-dependent increase in IL pERK1/2 labeling. Aripiprazole produced a U-shaped dose-response curve on MK-801-induced bar pressing behavior, a dose-dependent decrease in locomotor activity but no changes in IL pERK1/2 labeling. The attenuation of the MK-801-induced behavioral (bar pressing, locomotion) profile by Flupenthixol indicates a clear dopaminergic contribution to this behavior. The behavioral effect of aripiprazole may be due to its a) binding to presynaptic dopamine receptors at the midrange dose decreasing dopamine output and b) binding to postsynaptic dopamine receptors at the higher dose increasing dopamine tone. While both classes of antipsychotics can normalize perseverative behavioral symptoms, the underlying prefrontal cortical dysregulation seems to persist.