Drug metabolism within the brain changes drug response: selective manipulation of brain CYP2B alters propofol effects

Abstract

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.

Figure 1

Selective inhibition of rat brain CYP2B activity after intracerebroventricular (i.c.v.) injection of CYP2B mechanism-based inhibitor (MBI). (a) Radioactivity was detected following immunoprecipitation of rat brain frontal cortex tissue using antibodies specific to CYP2B, but not to CYP2A, CYP2D, CYP2E, or CYP3A, indicating a specificity of MBI activation and irreversible binding to CYP2B enzyme following i.c.v. injection of radiolabeled MBI. (b) Rat brain CYP2B activity was significantly increased by 7-day nicotine treatment in the brainstem (↔P=0.006). Both basal (↔P=0.05) and nicotine-induced CYP2B (↔P=0.03) activity in the brain were significantly reduced in animals pretreated with i.c.v. C8-xanthate (C8-X) compared with the i.c.v. artificial cerebro-spinal fluid (ACSF) pretreated animals in the brainstem. In the frontal cortex, which has low basal activity, 7-day nicotine treatment also increased CYP2B activity (↔P=0.04) and pretreatment with i.c.v. C8-X inhibited this (↔P=0.05).

Figure 2

Intracerebroventricular (i.c.v.) injection of CYP2B mechanism-based inhibitor (MBI) extends propofol-induced sleep time and increases brain propofol concentration. I.c.v. injection of the CYP2B MBIs, (a) 20 μg C8-xanthate (C8-X) (^*P=0.02) and (b) 40 μg radiolabeled MBI 8-methoxypsoralen (8-MOP) (^*P=0.04), significantly increased propofol sleep time, whereas i.c.v. injections of artificial cerebro-spinal fluid (ACSF) (vehicle control) did not alter sleep time. (c) Sleep times correlated with propofol levels in the brain (includes ACSF, C8-X, and 8-MOP animals), but not with (d) plasma propofol levels (gray: ACSF; black: C8-X; dark gray: 8-MOP). (e) Brain propofol levels were significantly higher in the C8-X (^*P=0.01)- and 8-MOP (^*P=0.04)-treated animals when compared with the ACSF controls. (f) No differences in plasma propofol concentrations were found following either MBI treatment. (g) There was no effect of either CYP2B MBI on in vitro hepatic CYP2B-mediated nicotine metabolism; there were no differences in ex vivo nicotine oxidation rates between i.c.v. ACSF and MBIs C8-X or 8-MOP (0.07±0.02, 0.07±0.04, and 0.08±0.04 nmol/min/mg, respectively), indicating no detectable effect of MBI injected i.c.v. on hepatic CYP2B metabolic activity.

Figure 3

Nicotine-induced CYP2B reduces propofol-induced sleep time, with no effect of nicotinic acetylcholine receptors (nAChR) blockade on this response. (a) Seven-day nicotine treatment significantly reduced mean sleep time by 60% compared with the animals' sleep time at baseline (n=52, ^*P<0.0001). Diamonds represent mean sleep time±standard deviation. (b) An intracerebroventricular (i.c.v.) injection of C8-xanthate (C8-X) reversed the reduction in sleep time owing to nicotine treatment and sleep time was extended beyond the baseline sleep times (P=0.02; n=4). (c) Pretreatments with the nAChR blocker chlorisondamine and (d) the nAChR antagonist mecamylamine did not alter propofol sleep at baseline or the reduction in sleep time following nicotine treatment, and i.c.v. C8-X reversed the reduction in both groups (↔P<0.05; n=6/group).

Figure 4

Rat brain CYP2B inhibition dose dependently reverses nicotine-induced sleep-time reduction. (a) A low dose of C8-xanthate (C8-X) (0.625 μg) did not reverse nicotine-induced reduction in sleep times, whereas 1.25–10 μg of C8-X given intracerebroventricularly (i.c.v.) reversed the effects of nicotine treatment returning the sleep times to those seen at baseline. Doses of C8-X between 20 and 80 μg not only reversed the reduction in sleep time after 7-day nicotine treatment, but also significantly extended it beyond baseline sleep times (n=4/group per C8-X dose, n=12/ACSF group; ↔P<0.05). (b) CYP2B inhibition dose dependently increased rat brain propofol concentration with doses between 5 and 80 μg of C8-X, resulting in significantly higher brain propofol levels compared with the animals that received 0 μg C8-X (^*P<0.05). Inset: Sleep times were correlated with brain propofol levels and not with plasma propofol levels (r=−0.03, P=0.84).

Figure 5

Rat brain CYP2B manipulation shifts the propofol sleep-time dose–response curve. A dose of 40 mg/kg was inactive at baseline—none of the animals slept (sleep time: 0 min). An intracerebroventricular (i.c.v.) injection of C8-xanthate (C8-X) significantly increased sleep time consequently rendering the 40 mg/kg dose active (sleep time: 9 min; apparent dose: 62 mg/kg; P=0.009; n=9). Upon administration of a 50 mg/kg dose of propofol, the animals slept an average of ∼5 min at baseline. Induction of brain CYP2B activity by 7-day nicotine treatment abolished the propofol response—none of the animals slept (sleep time: 0 min; apparent dose: 40 mg/kg; P=0.02; n=9). After i.c.v. C8-X, the rats displayed significantly longer sleep times (sleep time: 19 min; apparent dose: 70 mg/kg; P=0.04; n=9). Dashed arrows indicate apparent dose (graph is plotted on a log x axis, but values on x axis are actual doses).