Factors influencing the metabolism of Diazepam

Conflicting evidence suggest that perturbations of GABAergic neurotransmission play crucial roles in disrupting cortical neuronal network oscillations, memory, and cognitive deficits in Alzheimer's disease (AD). However, the role and impact of sex differences on GABAergic transmission in AD are not well understood. Using an APP knock-in mouse model of AD, APP^NLGF mice, we studied the effects of acute diazepam administration on memory and anxiety-like behavior to unveil sex-dependent dysregulation of GABAergic neurotransmission. We also examined sex differences in GABA_A receptor subunit mRNA and protein expression and the role of epigenetic regulation in hippocampus of APP^NLGF mice. We found that diazepam elicited dose-dependent suppression of locomotion in wildtype and APP^NLGF mice. However, a low dose, which had no significant effect in both male and female wildtype as well as female APP^NLGF mice, significantly suppressed locomotion in male APP^NLGF mice. Furthermore, this low dose of diazepam was more efficacious at eliciting anxiolytic-like effects in male than female APP^NLGF mice. The same low dose of diazepam disrupted recognition memory exclusively in male APP^NLGF mice. Biochemical analyses revealed that hippocampal α1 and α5 GABA_A receptor subunits mRNA and protein expression were significantly higher in male than female APP^NLGF mice and were regulated by histone H3 tri-methylation (H3K4me3) but not histone H3 acetylation. The higher sensitivity of APP^NLGF males to diazepam-induced behavioral effects may potentially be due to epigenetic-dependent upregulation of hippocampal α1 and α5 GABA_A receptor subunits expression compared to female APP^NLGF mice. These findings suggest that dysregulation of GABAergic neurotransmission plays a significant role in memory and affective behavior, particularly in male APP^NLGF mice.

A zero-concentration limit for controlled scheduled drugs in the blood of motorists was introduced in Sweden in 1999 and the annual number of arrests for driving under the influence of drugs (DUID) has since increased eight-fold. However, for prescription drugs that might cause impairment (e.g. benzodiazepines) additional proof is needed to justify prosecution, such as whether the medication was being misused. Over a 2-year period, we found 94 cases of DUID in which the concentrations of diazepam in blood was 1.1 μg/g or more. Diazepam (D) and nordiazepam (ND) were determined in whole blood by capillary gas chromatography with a limit of quantitation of 0.05 μg/g for each compound. The mean (median) and maximum concentrations of D were 2.0 μg/g (1.7 μg/g) and 7.8 μg/g and the corresponding ND concentrations were 1.5 μg/g (1.0 μg/g) and 7.6 μg/g, respectively. The concentration of D in blood exceeded 2 μg/g in 21% of cases and was over 3.0 μg/g in 11% of cases. D and ND were the only drugs present in eight cases (seven men and one women) and in another five cases ethanol was present at concentrations ranging from 0.81 to 1.98 mg/g. Polydrug use was very common in these DUID suspects and D and ND coexisted with amphetamine in 20% of cases, tetrahydrocannabinol in 18% of cases and with both these illicit drugs in 12% of cases. The next most prevalent drug combination was D, ND and morphine (mostly derived from heroin), seen in 13% of cases. Other psychoactive prescription drugs were identified in blood including alprazolam, flunitrazepam, oxazepam, zolpidem and zopiclone. This case series of DUID suspects demonstrate the high frequency of polydrug use showing preference for illicit drugs like amphetamine, cannabis and heroin, in that order. Furthermore, Swedish traffic delinquents frequently overdose with prescription drugs as exemplified here by unusually high concentrations of D and its active metabolite ND.

Phenobarbitone-pretreated male Sprague-Dawley rat liver microsomes were used to examine C₃-hydroxylation and N-dealkylation of four clinically important benzodiazepines: diazepam (DZP), prazepam (PZP), pinazepam (PIN) and halazepam (HZP). These substrates differ only in the nature of the N-substituent of the B ring and N-desmethyldiazepam (DMD) is the N-dealkylation product in each case. C₃-Hydroxylation was accordingly also studied with DMD as substrate. All monooxygenations were studied with substrates at a concentration of 10 μM, in the absence of solubilizing agents, and under conditions where the production of secondary metabolites was minimized. A 20-fold variation in the rate of C₃-hydroxylation was recorded across the five substrates with HZP showing the highest rate and DMD showing the lowest rate. An almost equally large range of variation was shown for the N-dealkylation reaction, with PZP undergoing this biotransformation more than 17 times faster than DZP. Log P values (a measure of lipophilicity) for the five substrates were determined using an HPLC method and a remarkable lack of correspondence between this substrate parameter and either of the monooxygenations was noted. This suggests that multiple substrate determinants govern the relative rates of these monooxygenations. It was, however, notable that the additive rate of metabolism of these substrates by both monooxygenase routes did show an excellent correlation with substrate lipophilicity.

A number of different progestogens, levonorgestrel (LNG), norethisterone (NET), gestodene (GSD), desogestrel (DG) and norgestimate (NORG) are used in combination with the oestrogen ethinyloestradiol (EE₂) in oral contraceptive steroid preparations. All the progestogens are acetylenic steroids and previous studies have indicated the potential of acetylenic steroids to cause mechanism-based or “suicide” inactivation of cytochrome P-450.

We have compared the effects of the different progestogens on EE₂ 2-hydroxylation (a reaction catalyzed by enzymes from the P-450IIC, P-450IIIA and P-450IIE gene families) and also the oxidative metabolism of other drug substrates (cyclosporin, diazepam, tolbutamide) by human liver microsomes. On coincubation with EE₂ as substrate, GSD, 3-keto desogestrel (3-KD, the active metabolite of desogestrel) and LNG produced some concentration-dependent inhibition of EE₂ 2-hydroxylation (maximum 32% inhibition at 100 μM 3-keto desogestrel). K_i values determined for GSD and 3-KD were 98.5 ± 12.3 and 93.2 ± 10.3 μM (mean ± SD; n = 4), respectively. Preincubation of progestogens in a small volume (50 μl) incubation for 30 min in the presence of an NADPH-generating system enhanced the inhibitory potential of all the steroids (at 100 μM, inhibition was for GSD 39%, 3-KD 46%, LNG 46%, NET 51% and NORG 43%). Inhibitory effects were therefore comparable and also similar to the macrolide antibiotic troleandomycin. The most marked inhibition seen was of diazepam N-demethylation and hydroxylation by GSD (71 and 57%, respectively) and 3-KD (62 and 50%, respectively).

In preincubation studies involving cyclosporin as the substrate, the order of inhibitory potency was GSD > 3-KD > NET > LNG for production of both metabolite M17 and M21.

The results of the study indicate that all the progestogens in common use have the propensity to inhibit a number of oxidative pathways but there is little evidence for one progestogen being more markedly inhibitory than others.