Citalopram and desmethylcitalopram in vitro: human cytochromes mediating transformation, and cytochrome inhibitory effects.

BACKGROUND: Biotransformation of citalopram (CT), a newly available selective serotonin reuptake inhibitor antidepressant, to its principal metabolite, desmethycitalopram (DCT), and the capacity of CT and DCT to inhibit human cytochromes P450, were studied in vitro. METHODS: Formation of DCT from CT was evaluated using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. Cytochrome inhibition by CT and DCT in liver microsomes was studied using isoform-specific index reactions. RESULTS: Formation of DCT from CT in liver microsomes had a mean apparent K(m) of 174 mumol/L. Coincubation with 1 mumol/L ketoconazole reduced reaction velocity to 46 to 58% of control values, while omeprazole, 10 mumol/L, reduced velocity to 80% of control. Quinidine produced minimal inhibition. DCT was formed from CT by heterologously expressed human P450-2D6, -2C19, -3A4. After accounting for the relative abundance of individual cytochromes, 3A4 and 2C19 were estimated to make major contributions to net reaction velocity, with a possible contribution of 2D6 at therapeutic CT concentrations. CT and DCT themselves produced negligible inhibition of 2C9, 2E1, and 3A, and only weak inhibition of 1A2, 2C19, and 2D6. CONCLUSIONS: Formation of DCT from CT is mediated mainly by P450-3A4 and 2C19, with an additional contribution of 2D6. CT at therapeutic doses in humans may produce a small degree of inhibition of P450-1A2, -2C19, and -2D6, but negligible inhibition of P450-2C9, -2E1, and -3A.

Nefazodone, meta-chlorophenylpiperazine, and their metabolites in vitro: cytochromes mediating transformation, and P450-3A4 inhibitory actions.

RATIONALE: Understanding of the mechanisms of biotransformation of antidepressant drugs, and of their capacity to interact with other medications, is of direct relevance to rational clinical psychopharmacology. OBJECTIVES: To determine the human cytochromes P450 mediating the metabolism of nefazodone, and the inhibitory activity of nefazodone and metabolites versus human P450-3A. METHODS: Biotransformation of nefazodone to its metabolic products, and of meta-chlorophenylpiperazine (mCPP) to para-hydroxy-mCPP, was studied in vitro using human liver microsomes and heterologously expressed human cytochromes. Nefazodone and metabolites were also tested as inhibitors of alprazolam hydroxylation, reflecting activity of cytochrome P450-3A isoforms. RESULTS: mCPP and two hydroxylated derivatives were the principal metabolites formed from nefazodone by liver microsomes. Metabolite production was strongly inhibited by ketoconazole or troleandomycin (relatively specific P450-3A inhibitors), and by an anti-P450-3A antibody. Only heterologously expressed human P450-3A4 mediated formation of nefazodone metabolites from the parent compound. Nefazodone, hydroxy-nefazodone, and para-hydroxy-nefazodone were strong 3A inhibitors, being more potent than norfluoxetine and fluvoxamine, but less potent than ketoconazole. The triazoledione metabolite and mCPP had weak or negligible 3A-inhibiting activity. Formation of parahydroxy-mCPP from mCPP was mediated by heterologously expressed P450-2D6; in liver microsomes, the reaction was strongly inhibitable by quinidine, a relatively specific 2D6 inhibitor. CONCLUSION: The complex parallel biotransformation pathways of nefazodone are mediated mainly by human cytochrome P450-3A, whereas clearance of mCPP is mediated by P450-2D6. Nefazodone and two of its hydroxylated metabolites are potent 3A inhibitors, accounting for pharmacokinetic drug interactions of nefazodone with 3A substrate drugs such as triazolam and alprazolam.

Gepirone and 1-(2-pyrimidinyl)-piperazine in vitro: human cytochromes mediating transformation and cytochrome inhibitory effects.

Biotransformation of gepirone to its principal metabolite, 1-(2-pyrimidinyl)-piperazine (1-PP), was studied in human liver microsomes and in microsomes from cDNA-transfected human lymphoblastoid cells. Formation of 1-PP from gepirone in liver microsomes proceeded with a mean apparent Km ranging from 335 to 677 microM. Coincubation with 1 microM ketoconazole reduced reaction velocity to less than 5% of control values at a gepirone concentration of 250 microM. Three other metabolites, presumed to be hydroxylated products, were also formed from gepirone. Formation of all three products was reduced to approximately 20% of control values by 1 microM ketoconazole; quinidine at 1 microM produced a small reduction in formation (91-94% of control) of two of the metabolites. 1-PP was formed from gepirone exclusively by pure P450-3A4 with a Km of 849 microM; Km values for the other metabolites were 245, 240, and 415 microM. Two of the products were also formed by P450-2D6. The results indicate that 3A4 is the principal cytochrome mediating 1-PP formation, as well as formation of the other metabolites. The properties of gepirone and 1-PP themselves as cytochrome inhibitors were tested in human liver microsomes using index reactions representing activity of P450-1A2, -2C9, -2C19, -2D6, -2E1 and -3A. Gepirone and 1-PP produced negligible inhibition of all these reactions. Thus gepirone at therapeutic doses in humans has a low likelihood of inhibiting P450-mediated drug metabolism involving these cytochromes.

Protease inhibitors as inhibitors of human cytochromes P450: high risk associated with ritonavir.

Four protease inhibitor antiviral agents (ritonavir, indinavir, nelfinavir, saquinavir) were evaluated as in vitro inhibitors of the activity of six human cytochromes using an in vitro model based on human liver microsomes. Ritonavir was a highly potent inhibitor of P450-3A activity (triazolam hydroxylation), having inhibitory potency slightly less than ketoconazole. Indinavir was also a potent 3A inhibitor, while nelfinavir and saquinavir were less potent. Ritonavir had high inhibition potency against cytochrome P450-2C9 (tolbutamide hydroxylation), -2C19 (S-mephenytoin hydroxylation), and -2D6 (dextromethorphan O-demethylation and desipramine hydroxylation), while the other protease inhibitors had one or more orders of magnitude lower inhibitory activity against these reactions. None of the protease inhibitors had important inhibitory potency against P450-1A2 (phenacetin O-deethylation) or -2E1 (chlorzoxazone hydroxylation). Thus, among available protease inhibitors, ritonavir carries the highest risk of incurring drug interactions due to inhibition of cytochrome P450 activity.

In situ hybridization histochemistry as a method to assess GABA(A) receptor subunit mRNA expression following chronic alprazolam administration.

Previous work in our laboratory has demonstrated region-specific effects for chronic alprazolam on binding and function at the GABA(A) receptor. The present study evaluated regional changes in mRNA expression of several subunits of the GABA(A) receptor following chronic alprazolam administration that might underlie these effects. Mice received alprazolam (2 mg/kg/day) or vehicle via subcutaneously implanted osmotic pumps for 1, 7, 14 or 28 days. In situ hybridization histochemistry was performed on tissue sections using [35S]dATP oligonucleotide probes corresponding to the alpha1 and gamma2 subunits of the GABA(A) receptor. Specific hybridization was clearly demonstrated and alpha1 subunit mRNA expression in frontoparietal cortex (layers II-IV) on day 1 of infusion was reduced in animals receiving alprazolam compared to vehicle. On subsequent days, there were no alterations in the levels of alpha1 subunit mRNA in the frontoparietal cortex, hippocampus or dentate gyrus. Expression of gamma2 subunit mRNA was increased on day 1 in the frontoparietal cortex (layer VI), hippocampus and dentate gyrus. mRNA expression was also increased in the dentate gyrus on day 28 of infusion. Comparison of the present study with the results of chronic treatment with other benzodiazepines clearly demonstrates that the pattern of mRNA subunit alterations obtained is both treatment- and region-specific. This makes a definitive conclusion regarding benzodiazepines and their interactions with GABA(A) receptors difficult at best.

Pharmacodynamic and receptor binding changes during chronic lorazepam administration.

To assess pharmacodynamic and neurochemical aspects of tolerance, lorazepam (2 mg/kg/day), or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice via implantable osmotic pump. Open-field behavior, benzodiazepine receptor binding in vitro, receptor autoradiography, and muscimol-stimulated chloride uptake were examined at both 1 and 14 days. Open-field activity was depressed in lorazepam-treated animals on Day 1. On Day 14, open-field parameters were indistinguishable from those of vehicle-treated animals, indicating behavioral tolerance. Benzodiazepine binding, as determined by the specific binding of [125I]diazepam, was also decreased in cortex on Day 14. Hippocampal binding was unchanged following chronic lorazepam exposure. Apparent affinity in cortical membrane preparations was unchanged, indicating that altered ligand uptake was due to decreased receptor number. Muscimol-stimulated chloride uptake into cortical synaptoneurosomes from lorazepam-treated animals was not significantly different on Day 1 or Day 14 compared to vehicle-treated animals. These results confirm that down-regulation of benzodiazepine receptor binding is closely associated with behavioral tolerance to benzodiazepines. These observed changes in binding are not necessarily associated with robust changes in receptor function.

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro: role of CYP2C9, CYP2C19, CYP2D6, and CYP3A.

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (Km (substrate concentration producing maximum reaction velocity) 3-13 microM). Formation of dextrorphan from 25 microM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 microM); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, and -2D6 but not by those expressing CYP1A2, -2E1 or -3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 microM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean Km of 259 microM. For formation of 3-methoxymorphinan from 25 microM dextromethorphan the IC50 for ketoconazole was 1.15 microM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, -2D6, and -3A4, but not by those expressing CYP1A2 or -2E1. CYP2C19 had the highest affinity (Km = 49 microM) whereas CYP3A4 had the lowest (Km = 1155 microM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and -2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.

Zolpidem metabolism in vitro: responsible cytochromes, chemical inhibitors, and in vivo correlations.

AIMS: To determine the human cytochromes mediating biotransformation of the imidazopyridine hypnotic, zolpidem, and the clinical correlates of the findings. METHODS: Kinetic properties of zolpidem biotransformation to its three hydroxylated metabolites were studied in vitro using human liver microsomes and heterologously expressed individual human cytochromes. RESULTS: The metabolic product termed M-3 accounted for more than 80% of net intrinsic clearance by liver microsomes in vitro. Microsomes containing human cytochromes CYP1A2, 2C9, 2C19, 2D6, and 3 A4 expressed by cDNA-transfected human lymphoblastoid cells mediated zolpidem metabolism in vitro. The kinetic profile for zolpidem metabolite formation by each individual cytochrome was combined with estimated relative abundances based on immunological quantification, yielding projected contributions to net intrinsic clearance of: 61% for 3 A4, 22% for 2C9, 14% for 1A2, and less than 3% for 2D6 and 2C19. These values were consistent with inhibitory effects of ketoconazole and sulfaphenazole on zolpidem biotransformation by liver microsomes. Ketoconazole had a 50% inhibitory concentration (IC50 ) of 0.61 microm vs formation of the M-3 metabolite of zolpidem in vitro; in a clinical study, ketoconazole coadministration reduced zolpidem oral clearance by approximately 40%, somewhat less than anticipated based on the IC50 value and total plasma ketoconazole levels, but much more than predicted based on unbound plasma ketoconazole levels. CONCLUSIONS: The incomplete dependence of zolpidem clearance on CYP3A activity has clinical implications for susceptibility to metabolic inhibition.

Effects of ketoconazole on triazolam pharmacokinetics, pharmacodynamics and benzodiazepine receptor binding in mice.

We previously demonstrated that ketoconazole is a potent inhibitor of triazolam biotransformation in vitro and in vivo. Despite significant elevations in triazolam plasma levels with coadministration of ketoconazole, the pharmacodynamic enhancement was lower than predicted based on plasma levels of triazolam. The present study examines the effects of ketoconazole on benzodiazepine receptor binding in vitro as well as on open-field behavior in male CD-1 mice. Triazolam alone inhibited [3H]flunitrazepam binding with an IC50 value of 0.85 nM and a Ki value of 0.50 nM. Ketoconazole alone also competitively antagonized [3H]flunitrazepam binding in a concentration-dependent manner with an IC50 value of 1.56 microM and a Ki value of 1.17 microM. In the presence of 1, 3 or 9 microM ketoconazole, the IC50 value of triazolam was increased to 1.11, 1. 58 and 5.73 nM, respectively, whereas maximal binding was reduced by 36%, 69% and 89%. Coadministration of 50 mg/kg ketoconazole and triazolam (0.1-0.3 mg/kg) to intact animals significantly elevated plasma and brain triazolam levels. Ketoconazole could be measured in mouse brain at levels averaging 31% of those in plasma. Ketoconazole alone had minimal or no effect on open field activity, but it significantly potentiated the decreased activity seen with triazolam administration. The ability of ketoconazole to inhibit triazolam displacement of [3H]flunitrazepam binding may explain the muted pharmacodynamic effect of this benzodiazepine in the presence of ketoconazole. Based on these results, it is likely that ketoconazole acts as a neutral ligand at the benzodiazepine receptor.

Appetite suppressant drugs as inhibitors of human cytochromes P450: in vitro inhibition of P450-2D6 by D- and L-fenfluramine, but not phentermine.

The activity of D-fenfluramine, L-fenfluramine, and phentermine as inhibitors of five human cytochromes P450 was evaluated using human liver microsomes in vitro. All three compounds produced negligible inhibition of P450-1A2, -2C9, -2E1, and -3A. Phentermine also did not inhibit P450-2D6. However, D- and L-fenfluramine significantly inhibited P450-2D6 activity as measured by dextromethorphan O-demethylation, with mean 50% inhibitory concentrations (15.1 microM) within one order of magnitude of that for fluoxetine (2.7 microM). Findings from the in vitro assay are consistent with clinical studies showing significant inhibition of desipramine clearance by coadministration of fenfluramine.