Jibran Y. Khokhar

Rat brain CYP2B-enzymatic activation of chlorpyrifos to the oxon mediates cholinergic neurotoxicity.

Chlorpyrifos is a commonly used insecticide that can be metabolically activated by CYP2B to the acetylcholinesterase inhibitor chlorpyrifos-oxon causing cholinergic overstimulation and neurotoxicity. Rat brain extracts can also activate chlorpyrifos in vitro, and the lack of circulating oxon in serum suggests that metabolic activation within the brain may be responsible for chlorpyrifos neurotoxicity. Rats received intracerebroventricular (ICV) injections of CYP2B mechanism-based inhibitors (MBI), once or repeatedly, followed by chlorpyrifos (62.5-250 mg/kg sc). Rats were assessed for neurochemical (acetylcholinesterase activity), physiological (temperature), and behavioral measures (e.g., gait, righting reflex, arousal, incline angles) at 4 hours 3 days after chlorpyrifos treatment. ICV CYP2B MBIs increased brain chlorpyrifos levels, decreased brain chlorpyrifos-oxon levels, and attenuated the reduction in brain acetylcholinesterase; there was no effect on serum chlorpyrifos levels or acetylcholinesterase activity reduction. Inhibition of brain chlorpyrifos metabolism by CYP2B MBIs blocked centrally mediated hypothermia but not peripherally mediated hyperthermia. A single ICV MBI treatment significantly attenuated chlorpyrifos neurotoxicity mediated behavioral outcomes at 1 day after chlorpyrifos treatment with a gradual worsening of behavioral scores through day 3, suggesting a recovery of brain CYP2B activity and an increase in local chlorpyrifos activation. Daily ICV MBI injections attenuated neurotoxicity across all test days consistent with prolonged inhibition of brain chlorpyrifos activation. Thus, rat brain CYP2B contributes significantly to chlorpyrifoss neurotoxic effects. Variable human brain CYP2B levels, influenced by genetics and environmental exposures, may contribute to interindividual differences in neurotoxicity. Therapeutic inhibition of brain CYP2B could also be explored as a treatment for exposure to CYP2B-activated neurotoxins.

Pharmacogenetics of Drug Dependence: Role of Gene Variations in Susceptibility and Treatment

Drug dependency is a highly prevalent mental health disorder that imposes a significant burden on those directly affected, health care systems, and society in general. There is substantial heritability in the susceptibility to drug addiction, which indicates that there are genetic risk factors. Variation in the human genome is abundant and can directly affect drug dependency phenotypes, for example, by altering the function of a gene product or by altering gene expression. Pharmacogenetic studies can assess the effects of genetic variation on the risk for a particular phenotype (e.g., being an alcoholic). In addition, pharmacogenetic variability in treatment efficacy and adverse reactions can be investigated to identify particular genetic variants associated with altered responses. This review highlights examples of genetic variations that are important in the development and maintenance of specific drug dependencies as well as those that affect the response to treatment.

Drug Metabolism within the Brain Changes Drug Response: Selective Manipulation of Brain CYP2B Alters Propofol Effects

Drug-metabolizing cytochrome P450 (CYPs) enzymes are expressed in the liver, as well as in extrahepatic tissues such as the brain. Here we show for the first time that drug metabolism by a CYP within the brain, illustrated using CYP2B and the anesthetic propofol (2, 6-diisopropylphenol, Diprivan), can meaningfully alter the pharmacological response to a CNS acting drug. CYP2B is expressed in the brains of animals and humans, and this CYP isoform is able to metabolize centrally acting substrates such as propofol, ecstasy, and serotonin. Rats were given intracerebroventricularly (i.c.v.) injections of vehicle, C8-xanthate, or 8-methoxypsoralen (CYP2B mechanism-based inhibitors) and then tested for sleep time following propofol (80 mg/kg intraperitoneally). Both inhibitors significantly increased sleep-time (1.8- to 2-fold) and brain propofol levels, while having no effect on plasma propofol levels. Seven days of nicotine treatment can induce the expression of brain, but not hepatic, CYP2B, and this induction reduced propofol sleep times by 2.5-fold. This reduction was reversed in a dose-dependent manner by i.c.v. injections of inhibitor. Sleep times correlated with brain (r=0.76, P=0.0009), but not plasma (r=0.24, P=0.39) propofol concentrations. Inhibitor treatments increased brain, but not plasma, propofol levels, and had no effect on hepatic enzyme activity. These data indicate that brain CYP2B can metabolize neuroactive substrates (eg, propofol) and can alter their pharmacological response. This has wider implications for localized CYP-mediated metabolism of drugs, neurotransmitters, and neurotoxins within the brain by this highly variable enzyme family and other CYP subfamilies expressed in the brain.

First demonstration that brain CYP2D-mediated opiate metabolic activation alters analgesia in vivo.

The response to centrally acting drugs is highly variable between individuals and does not always correlate with plasma drug levels. Drug-metabolizing CYP enzymes in the brain may contribute to this variability by affecting local drug and metabolite concentrations. CYP2D metabolizes codeine to the active morphine metabolite. We investigated the effect of inhibiting brain, and not liver, CYP2D activity on codeine-induced analgesia. Rats received intracerebroventricular injections of CYP2D inhibitors (20 μg propranolol or 40 μg propafenone) or vehicle controls. Compared to vehicle-pretreated rats, inhibitor-pretreated rats had: (a) lower analgesia in the tail-flick test (p<0.05) and lower areas under the analgesia-time curve (p<0.02) within the first hour after 30 mg/kg subcutaneous codeine, (b) lower morphine concentrations and morphine to codeine ratios in the brain (p<0.02 and p<0.05, respectively), but not in plasma (p>0.6 and p>0.7, respectively), tested at 30 min after 30 mg/kg subcutaneous codeine, and (c) lower morphine formation from codeine ex vivo by brain membranes (p<0.04), but not by liver microsomes (p>0.9). Analgesia trended toward a correlation with brain morphine concentrations (p=0.07) and correlated with brain morphine to codeine ratios (p<0.005), but not with plasma morphine concentrations (p>0.8) or plasma morphine to codeine ratios (p>0.8). Our findings suggest that brain CYP2D affects brain morphine levels after peripheral codeine administration, and may thereby alter codeines therapeutic efficacy, side-effect profile and abuse liability. Brain CYPs are highly variable due to genetics, environmental factors and age, and may therefore contribute to interindividual variation in the response to centrally acting drugs.

Differential induction of ethanol-metabolizing CYP2E1 and nicotine-metabolizing CYP2B1/2 in rat liver by chronic nicotine treatment and voluntary ethanol intake.

Alcohol and nicotine are frequently co-used and co-abused, and use of both drugs alone can affect hepatic drug metabolism. We investigated the influences of chronic nicotine treatment and voluntary ethanol intake on the induction of rat hepatic cytochrome P450 (CYP) enzymes that metabolize ethanol and nicotine. Rats were trained to voluntarily drink ethanol (6% v/v, 1 h) with nicotine pretreatment for 10 days. Another group of rats were treated with the same nicotine doses alone. Hepatic CYP2E1, CYP2B1/2 and CYP2D1 proteins were assessed by immunoblotting. Nicotine pretreatment (0.4, 0.8 and 1.2 mg/kg) increased voluntary ethanol intake on day 10 by 1.8, 2.0, and 1.4 fold respectively compared to saline pretreatment (P<0.01-0.3). CYP2E1 was increased 1.7, 1.8, and 1.4 fold by the three doses of nicotine alone (P<0.02-0.21); CYP2E1 levels were increased by voluntary ethanol intake alone and a further 2.4, 2.2, and 1.8 fold by 0.4, 0.8, and 1.2 mg/kg nicotine respectively versus saline pretreatment (P<0.002-0.06). CYP2B1/2 proteins were not induced by nicotine alone, but were increased by 2.2-2.5 fold by ethanol drinking (P<0.05). CYP2E1 (r=0.67, P<0.001) and CYP2B1/2 levels (r=0.49, P=0.007) correlated with alcohol consumption on day 10. There was no change in CYP2D1. Chronic nicotine increased voluntary ethanol intake thereby enhancing CYP2E1 and CYP2B1/2 levels. Thus CYPs are regulated not only directly by nicotine and ethanol, but also indirectly via an increase in the ethanol consumption in the presence of nicotine pretreatment. Together this may contribute to the co-abuse of these drugs and alter the metabolism of clinical drugs and endogenous substrates.

Nucleus accumbens deep brain stimulation in a rat model of binge eating.

Binge eating (BE) is a difficult-to-treat behavior with high relapse rates, thus complicating several disorders including obesity. In this study, we tested the effects of high-frequency deep brain stimulation (DBS) in a rodent model of BE. We hypothesized that BE rats receiving high-frequency DBS in the nucleus accumbens (NAc) core would have reduced binge sizes compared with sham stimulation in both a ‘chronic BE’ model as well as in a ‘relapse to chronic BE’ model. Male Sprague–Dawley rats (N=18) were implanted with stimulating electrodes in bilateral NAc core, and they received either active stimulation (N=12) or sham stimulation (N=6) for the initial chronic BE experiments. After testing in the chronic BE state, rats did not engage in binge sessions for 1 month, and then resumed binge sessions (relapse to chronic BE) with active or sham stimulation (N=5–7 per group). A significant effect of intervention group was observed on binge size in the chronic BE state, but no significant difference between intervention groups was observed in the relapse to chronic BE experiments. This research, making use of both a chronic BE model as well as a relapse to chronic BE model, provides data supporting the hypothesis that DBS of the NAc core can decrease BE. Further research will be needed to learn how to increase the effect size and decrease deep brain stimulation-treatment outcome variability across the continuum of BE behavior.

Intracerebroventricularly and Systemically Delivered Inhibitor of Brain Cyp2b (C8-Xanthate), Even Following Chlorpyrifos Exposure, Reduces Chlorpyrifos Activation and Toxicity in Male Rats

Chlorpyrifos is a pesticide that is metabolically activated to chlorpyrifos oxon (acetylcholinesterase inhibitor) primarily by the cytochrome P450 2B (CYP2B) enzyme subfamily in the liver and brain. We have previously shown that intracerebroventricular pretreatment with a CYP2B inhibitor, C8-Xanthate, can block chlorpyrifos toxicity. Here, we assessed whether delayed introduction of C8-Xanthate would still reduce toxicity and whether peripheral administration of C8-Xanthate could also inhibit chlorpyrifos activation in the brain and block toxicity. Male rats (N = 4-5/group) were either pretreated with C8-Xanthate (40 μg intracerebroventricular or 5 mg/kg intraperitoneal), or vehicle (ACSF or saline, respectively), 24 h before chlorpyrifos treatment (125 mg/kg subcutaneous) and then treated daily with inhibitor or vehicle until 7 days post-chlorpyrifos treatment. Additional groups received vehicle pretreatment, switching to C8-Xanthate 1, 2, 3, or 4 days after chlorpyrifos and then continuing with daily C8-Xanthate treatment until 7 days post-chlorpyrifos treatment. Neurotoxicity was assessed at baseline (before chlorpyrifos) and then daily after chlorpyrifos, using behavioral assessments (e.g., gait score). Neurochemical assays (e.g., serum and brain chlorpyrifos) were performed at the end of study. Pretreatment with C8-Xanthate completely prevented chlorpyrifos toxicity, and delayed introduction of C8-Xanthate reduced toxicity, even when started up to 4 days after chlorpyrifos treatment. Discontinuation of C8-Xanthate treatment 7 days post-chlorpyrifos treatment did not result in the reappearance of toxicity, tested through 10 days after chlorpyrifos treatment. These findings suggest that CYP2B inhibitor treatment, even days after chlorpyrifos exposure, and using a peripheral delivery route, may be useful as a therapeutic approach to reduce chlorpyrifos toxicity.

Desipramine enhances the ability of risperidone to decrease alcohol intake in the Syrian golden hamster.

The atypical antipsychotic clozapine reduces alcohol drinking in patients with schizophrenia. We have proposed that clozapine׳s ability to decrease alcohol drinking relates to its weak blockade of the dopamine D2 receptor and potent blockade of the norepinephrine α-2 receptor, as well as its ability to elevate plasma and brain norepinephrine. Another atypical antipsychotic, risperidone, which is a potent blocker of both the dopamine D2 receptor and norepinephrine α-2 receptor, does not decrease alcohol drinking. In this study, we used the Syrian golden hamster to test whether the ability of risperidone to reduce alcohol drinking would be enhanced if it was used in combination with the norepinephrine reuptake inhibitor desipramine. Hamsters were given free access to water and alcohol (15% v/v) until they reached a steady drinking baseline. They were then treated daily with each drug or drug combination for 20 days. Risperidone (0.2mg/kg) only transiently decreased alcohol drinking. However, 5.0mg/kg, and possibly 1.0mg/kg, desipramine added to 0.2mg/kg risperidone appeared to produce a more substantial and relatively sustained effect than risperidone alone. Data from this study provide leads toward the development of new treatments for patients with schizophrenia and alcoholism, and also for those with alcoholism alone.

The link between schizophrenia and substance use disorder: A unifying hypothesis

Substance use disorders occur commonly in patients with schizophrenia and dramatically worsen their overall clinical course. While the exact mechanisms contributing to substance use in schizophrenia are not known, a number of theories have been put forward to explain the basis of the co-occurrence of these disorders. We propose here a unifying hypothesis that combines recent evidence from epidemiological and genetic association studies with brain imaging and pre-clinical studies to provide an updated formulation regarding the basis of substance use in patients with schizophrenia. We suggest that the genetic determinants of risk for schizophrenia (especially within neural systems that contribute to the risk for both psychosis and addiction) make patients vulnerable to substance use. Since this vulnerability may arise prior to the appearance of psychotic symptoms, an increased use of substances in adolescence may both enhance the risk for developing a later substance use disorder, and also serve as an additional risk factor for the appearance of psychotic symptoms. Future studies that assess brain circuitry in a prospective longitudinal manner during adolescence prior to the appearance of psychotic symptoms could shed further light on the mechanistic underpinnings of these co-occurring disorders while identifying potential points of intervention for these difficult-to-treat co-occurring disorders.

The comparative effects of clozapine versus haloperidol on initiation and maintenance of alcohol drinking in male alcohol-preferring P rat.

Alcohol use disorder, characterized by modest levels of alcohol use, commonly occurs in patients with schizophrenia and dramatically worsens their course. Recent data indicate that the atypical antipsychotic clozapine, but not the typical antipsychotic haloperidol, decreases alcohol drinking both in patients with schizophrenia and also in the Syrian golden hamster, an animal model of moderate alcohol drinking. The present study was designed to assess the comparative effects of clozapine and haloperidol in the alcohol-preferring (P) rat, an animal model of alcoholism. First, the study investigated the comparative effects of clozapine and haloperidol on initiation of alcohol consumption in P rats, which models the early stage of alcoholism. Second, the study assessed the comparative effects of clozapine and haloperidol on maintenance of chronic alcohol consumption in P rats to provide a clue as to whether either drug may also limit alcohol consumption in alcohol-dependent patients. Clozapine attenuated the initiation of alcohol drinking and development of alcohol preference while haloperidol did not. However, neither clozapine nor haloperidol attenuated maintenance of chronic alcohol drinking. Taken together, the current data suggest that clozapine, but not haloperidol, may be effective at reducing alcohol abuse or non-dependent drinking and the P rat, used within an alcohol initiation paradigm, and may differentiate the effects of clozapine and haloperidol on alcohol drinking.