Diurnal activity and pulsatility of the hypothalamus-pituitary-adrenal system in male depressed patients and healthy controls.

There is only sparse and ambiguous information about circadian and pulsatile secretion features of the hypothalamus-pituitary-adrenocortical system in depression. We studied 15 severely depressed (Hamilton Depression Scale 30.4 +/- 6.7) male patients (age 22-72 yr; mean, 47.7 +/- 14.8) and 22 age-matched male controls (age 23-85 yr; mean, 53.1 +/- 18.2). Twenty-four-hour blood sampling from 0800-0800 h with 30-min sampling intervals was performed; from 1800-2400 h, blood was drawn every 10 min. Multivariate analysis of covariance, with the covariate being age, revealed mean 24-h cortisol (315.9 +/- 58.5 vs. 188.2 +/- 27.3 nmol/L) and mean ACTH (7.82 +/- 1.94 vs. 5.79 +/- 1.28 pmol/L) to be significantly increased in depressed patients. The frequency of cortisol (2.6 +/- 0.7 vs. 1.3 +/- 1.0 pulses/6 h) and ACTH (2.6 +/- 1.6 vs. 1.6 +/- 1.4 pulses/6 h) pulses during the evening were higher in patients compared to controls. The flattened circadian cortisol variation and reduced time of quiescence of cortisol secretory activity (140 +/- 116 vs. 305 +/- 184 min) in patients suggest disturbances of circadian functions. We conclude that increased hypothalamus-pituitary-adrenocortical activity in depression is related to a greater frequency of episodic hormone release, and we hypothesize that the observed circadian changes might be partly due to altered mineralocorticoid and glucocorticoid receptor capacity and function.

Simultaneous assessment of CYP3A4 and CYP1A2 activity in vivo with alprazolam and caffeine.

Alprazolam (ALP) and caffeine (CAF) were suggested as probe drugs for the activities of CYP3A4 and CYP1A2, respectively. We investigated the disposition of oral ALP (1 mg) and CAF (100 mg) in 17 normal volunteers to establish and validate a procedure for the simultaneous assessment of CYP3A4 and CYP1A2 enzyme activity. Nine received ALP alone, ALP and CAF and CAF alone in an open three-way crossover study to test for pharmacokinetic interaction. Four received ALP after a 2-day pretreatment with ketoconazole, an inhibitor of CYP3A4, and four normal volunteers received ALP after 4 days of rifampin, an inducer of CYP3A4. AUC values of ALP and CAF administered alone were not different from AUC values when both drugs were administered combined, indicating that there is no metabolic interaction. The ratio formed of paraxanthine and CAF correlated significantly with systemic CAF clearance at 3, 4, 6, 8, 10 and 24 h. There was a strong correlation between AUC values of ALP and CAF and the plasma concentration obtained 6, 8, 10, or 24 h after ingestion of the drug. Ketoconazole and rifampin pretreatment significantly changed AUC values of ALP (mean AUC values in microg/l h: ALP = 242.2, ALP + ketoconazole = 426.2, ALP + rifampin = 28.4, ANOVA F = 17.7, P < 0.001). We conclude that ALP and CAF can be administered simultaneously for the assessment of CYP activity. Plasma concentrations 6, 8, 10, and 24 h after drug ingestion reflect AUC of ALP and CAF and therefore in-vivo CYP3A4 and CYP1A2 activity, respectively.

A double-blind comparison of lorazepam and oxazepam in psychomotor retardation and mutism.

BACKGROUND: An increasing number of case reports indicate a superior therapeutic response of catatonialike symptoms, such as severe psychomotor disturbance and mutism, associated with psychiatric disorder to the benzodiazepine lorazepam (LO). Equivocal results, however, are also reported with regard to other benzodiazepines for the treatment of this syndrome. The purpose of this study was to compare the effects of LO and oxazepam (OX), benzodiazepines with comparable pharmacokinetics, on psychomotor retardation and mutism associated with psychiatric disorder. METHODS: Twenty-one hospitalized patients with severe psychomotor retardation and mutism were treated with 2 mg LO and 60 mg OX in a double-blind crossover study design. RESULTS: Both benzodiazepines significantly reduced psychomotor symptoms. When administered for the first time, 4 of 7 patients with LO and 6 of 10 patients with OX improved at least 50% on visual analog scale (VAS) rating. Reduction in symptoms was significant with LO and OX treatment on either day of treatment. The second time, however, LO was significantly better compared with OX in alleviating the target symptoms. CONCLUSIONS: Both OX and LO are effective for the treatment of psychomotor retardation. Thus, the beneficial effect of LO on psychomotor retardation and mutism is not a unique pharmacodynamic property but more likely due to its pharmacokinetic profile. The differential effect of the two benzodiazepines on the second day of treatment warrants further clarification. Several hypotheses are evaluated.

Combined dexamethasone/corticotropin-releasing hormone test in acute and remitted manic patients, in acute depression, and in normal controls: I.

Hypothalamic-pituitary-adrenal system (HPA)-function in patients with mania (n = 11), depression (n = 11, unipolar) and in control subjects (n = 11) was studied; six of the acutely manic patients were reevaluated after a symptom-free interval of at least 6 months. The combined dexamethasone-suppression/human CRH-challenge test was used to probe HPA-system function. After CRH and dexamethasone pretreatment, ACTH and cortisol release were significantly increased in both manic and depressed patients in comparison to the control group. In the remitted patients with mania, a significant decrease in hormonal release after DEX and CRH was evident when compared to the acute manic episode, but the degree of CRH-stimulated hormone secretion in these remitted patients was still significantly larger than in normal controls. This study demonstrates that acute and remitted manic episodes are associated with a profoundly dysregulated HPA-system activity.

The combined dexamethasone/corticotropin-releasing hormone stimulation test is more closely associated with features of diurnal activity of the hypothalamo-pituitary-adrenocortical system than the dexamethasone suppression test.

BACKGROUND: The dexamethasone suppression test (DST) is a widely used endocrine test in psychiatry, but was reported to not allow reliable inferences with regard to the basal activity of the hypothalamo-pituitary-adrenocortical (HPA) system. We compared the association of the standard DST and the combined dexamethasone/corticotropin-releasing hormone (DEX/CRH) challenge with parameters of diurnal cortisol profiles. METHODS: We performed a DEX/CRH challenge and 24-hour cortisol profiles in 25 depressed patients (mean age: 47.4 +/- 16.0 years) and 33 age-matched healthy controls (mean age: 51.4 +/- 19.3 years). RESULTS: A path analysis showed cortisol area under the curve (AUC) after CRH (= DEX/CRH status) to be dependent upon minimal 24-hour cortisol and evening frequency of pulsatile cortisol release. In contrast, postdexamethasone cortisol (= DST status) was related to 24-hour mean cortisol. Simple linear regressions supported an association of cortisol AUC with several parameters of the diurnal cortisol profiles, which was not true for the standard DST. CONCLUSIONS: We conclude that the combined DEX/CRH challenge test is more closely associated with the activity of the HPA system than the standard DST in healthy and depressed subjects.

Combined dexamethasone/corticotropin-releasing hormone test in patients with schizophrenia and in normal controls: II.

Hypothalamic-pituitary-adrenal system (HPA) function was tested in 24 patients with schizophrenia and compared to 24 age-matched healthy volunteers using the combined dexamethasone-suppression (DST/CRH) corticotropin-releasing hormone stimulation test (DST/CRH). After stimulation with CRH, the dexamethasone-pretreated patients released significantly more cortisol, but a similar amount of adrenocorticotropic hormone (ACTH) in comparison to controls. No association between DST status and degree of severity of illness and/or current medication was found. However, in comparison to unmedicated patients, those patients currently receiving antipsychotics, who were also those with a lesser degree of severity of illness, showed a decreased release of CRH-stimulated cortisol and ACTH. This study demonstrates that schizophrenic patients have a dysregulation of the HPA system as assessed with the DEX/CRH test. Overall, however, the degree of HPA-system dysfunction in schizophrenic patients seems to be of a lesser magnitude than in patients with affective disorders.

Age-associated changes of pituitary-adrenocortical hormone regulation in humans: importance of gender.

In the present study the hypothesis was tested that in normal human aging an insensitivity of the glucocorticoid feedback signals is acquired. Thus, 40 healthy elderly (mean age: 69 +/- 5 years) and 20 younger (mean age: 34 +/- 8 years) individuals underwent a combined dexamethasone suppression/CRH-stimulation test. Cortisol secretion after dexamethasone (DEX) pretreatment and before CRH was increased in the older age group, but none of the subjects escaped DEX-induced suppression of cortisol. However, after additional CRH administration to the DEX-pretreated volunteers, the older group released significantly more cortisol than their young counterparts. Within the group of the elderly only, a positive correlation between BASAL, DEX-pretreated cortisol concentration and post-CRH steroid responses was found. Gender profoundly affected DEX/CRH-test outcome: females, regardless of age, had an increased hormonal secretion in comparison to males. It is concluded that, during human aging, adaptive changes in glucocorticoid receptors take place, allowing for the system to maintain "peripheral" glucocorticoid homeostasis, but that more sophisticated challenge procedures such as the DEX/CRH test reveal an age-related increase in HPA system activity.

Pituitary-adrenal-system regulation and psychopathology during amitriptyline treatment in elderly depressed patients and normal comparison subjects.

OBJECTIVE: This study was done to compare the effects of 6-week treatment with amitriptyline on hypothalamic-pituitary-adrenocortical (HPA) regulation in elderly depressed patients and age-matched comparison subjects. METHOD: A combined dexamethasone-suppression/CRH-stimulation (dexamethasone/CRH) test was administered before initiation of amitriptyline treatment and at the end of weeks 1, 3, and 6 of treatment. Thirty-nine depressed inpatients, mean age = 69 years, completed the study. Fourteen normal volunteers, mean age = 67 years, served as comparison subjects. RESULTS: In relation to the comparison subjects, the depressed patients had a profoundly abnormal HPA response, in particular an exaggerated cortisol release in the dexamethasone/CRH test. This abnormality began to disappear after 1 week of treatment with amitriptyline. In contrast, amitriptyline did not affect neuroendocrine regulation in the comparison subjects at any time during the test period. CONCLUSIONS: The data suggest that amitriptyline affects HPA regulation in hypercortisolemic depression only, and they raise the possibility that normalization of its feedback control is related to the antidepressive effect of amitriptyline.

Low lumbar bone mineral density in patients with major depression.

Major depression is associated with hypercorticoidism, a risk factor for osteoporosis. However, it is unknown whether depressive disorders are associated with alterations in bone mineral density. The authors measured the density of trabecular bone from the first to the third lumbar vertebra by quantitative computerized tomography in 80 depressed inpatients older than 40 years and in 57 healthy comparison subjects. An analysis of covariance model with age as a covariate showed a significant effect of diagnosis on the dependent variable spinal bone mineral density: depressed patients had lower values. Other factors could not explain the finding. The authors conclude that major depression is a significant risk factor for osteoporosis.

O- and N-demethylation of venlafaxine in vitro by human liver microsomes and by microsomes from cDNA-transfected cells: effect of metabolic inhibitors and SSRI antidepressants.

The biotransformation of venlafaxine (VF) into its two major metabolites, O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) was studied in vitro with human liver microsomes and with microsomes containing individual human cytochromes from cDNA-transfected human lymphoblastoid cells. VF was coincubated with selective cytochrome P450 (CYP) inhibitors and several selective serotonin reuptake inhibitors (SSRIs) to assess their inhibitory effect on VF metabolism. Formation rates for ODV incubated with human microsomes were consistent with Michaelis-Menten kinetics for a single-enzyme mediated reaction with substrate inhibition. Mean parameters determined by non-linear regression were: Vmax = 0.36 nmol/min/mg protein, K(m) = 41 microM, and Ks 22901 microM (Ks represents a constant which reflects the degree of substrate inhibition). Quinidine (QUI) was a potent inhibitor of ODV formation with a Ki of 0.04 microM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 microM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, -2C19, and -2D6. CYP2D6 was dominant with the lowest K(m), 23.2 microM, and highest intrinsic clearance (Vmax/K(m) ratio). No unique model was applicable to the formation of NDV for all four livers tested. Parameters determined by applying a single-enzyme model were Vmax = 2.14 nmol/min/mg protein, and K(m) = 2504 microM. Ketoconazole was a potent inhibitor of NDV production, although its inhibitory activity was not as great as observed with pure 3A substrates. NDV formation was also reduced by 42% by a polyclonal rabbit antibody against rat liver CYP3A1. Studies using expressed cytochromes showed that NDV was formed by CYP2C9, -2C19, and -3A4. The highest intrinsic clearance was attributable to CYP2C19 and the lowest to CYP3A4. However the high in vivo abundance of 3A isoforms will magnify the importance of this cytochrome. Fluvoxamine (FX), at a concentration of 20 microM, decreased NDV production by 46% consistent with the capacity of FX to inhibit CYP3A, 2C9, and 2C19. These results are consistent with previous studies that show CYP2D6 and -3A4 play important roles in the formation of ODV and NDV, respectively. In addition we have shown that several other CYPs have important roles in the biotransformation of VF.

Metabolism of drugs by cytochrome P450 3A isoforms. Implications for drug interactions in psychopharmacology.

Members of the P450 3A subfamily are the most abundant of the human hepatic cytochromes. CYP3A isoforms mediate the biotransformation of many drugs, including a number of psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents. Activity of CYP3A in humans is variable among individuals, but there is no evidence of genetic polymorphism. Significant amounts of CYP3A are present in the gastrointestinal tract, and may contribute to presystemic extraction of drugs such as cyclosporin. The azole antifungal agents ketoconazole and itraconazole are potent inhibitors of human CYP3A isoforms. Selective serotonin reuptake inhibitor (SSRI) antidepressants are also CYP3A inhibitors, but much less potent than ketoconazole or itraconazole. In vitro models can provide important information on the qualitative and quantitative activity of potential inhibitors of human cytochromes. However, in vitro inhibition constant (Ki) values alone do not predict the magnitude of an in vivo interaction, nor whether an interaction will be of clinical importance. For example, SSRIs are predicted to impair clearance of the antihistamine terfenadine in humans. However, the magnitude of this effect is much less than would be associated with a pharmacokinetic interaction of clinical importance.

In vitro approaches to predicting drug interactions in vivo.

In vitro metabolic models using human liver microsomes can be applied to quantitative prediction of in vivo drug interactions caused by reversible inhibition of metabolism. One approach utilizes in vitro Ki, values together with in vivo values of inhibitor concentration to forecast in vivo decrements of clearance caused by coadministration of inhibitor. A critical limitation is the lack of a general scheme for assigning intrahepatic exposure of enzyme to inhibitor or substrate based only on plasma concentration; however, the assumption that plasma protein binding necessarily restricts hepatic uptake is not tenable. Other potential limitations include: flow-dependent hepatic clearance, "mechanism-based" chemical inhibition, concurrent induction, or a major contribution of gastrointestinal P450-3A isoforms to presystemic extraction. Nonetheless, the model to date has provided reasonably accurate forecasts of in vivo inhibition of clearance of several substrates (desipramine, terfenadine, triazolam, alprazolam, midazolam) by coadministration of selective serotonin reuptake-inhibitor antidepressants and azole antifungal agents. Such predictive models deserve further evaluation, since they may ultimately yield more cost-effective and expeditious screening for drug interactions, with reduced human drug exposure and risk.

Insulin-like growth factor-I (IGF-I) plasma concentrations are increased in depressed patients.

There is some evidence that the somatotrophic system in depression, as assessed by basal growth hormone (GH) concentrations and by GH releasing hormone (GHRH) challenge, might be dysfunctional. However, the rather limited data have been inconclusive so far and plasma concentrations of both insulin-like growth factor-1 (IGF-I) and binding proteins (IGFBP 1 to IGFBP-6) have not been measured simultaneously in depressed patients. We studied 24 severely depressed patients and 33 healthy controls and estimated 24-hour mean plasma cortisol, six-hour evening mean plasma growth hormone (GH), morning plasma IGF-I, IGFBP 2 and 3 and GH-binding protein (GH-BP). Twenty-four-hour mean cortisol (306 +/- 69 vs. 196 +/- 30 nmol/l, p < .001) and IGF-I (157 +/- 40 vs. 120 +/- 33 micrograms/l, p < .01) plasma concentrations were found to be significantly increased in depressed patients, while there was no difference in GH or binding proteins between both groups. MANOVA analysis revealed age and diagnosis to have main effects upon plasma IGF-I. Especially young age and a diagnosis of major depression are associated with higher plasma IGF-I. After treatment only patients in remission had attenuated IGF-I plasma concentrations. We conclude that plasma IGF-I is increased in acutely depressed patients similar to other states of hypercortisolemia.

Cytochrome P-450 enzymes and FMO3 contribute to the disposition of the antipsychotic drug perazine in vitro.

RATIONALE: Perazine (PER) is a phenothiazine antipsychotic drug frequently used in Germany that undergoes extensive metabolism. OBJECTIVES AND METHODS: To anticipate metabolic drug interactions and to explore the relevance of polymorphisms of metabolic enzymes, perazine-N-demethylation and perazine-N-oxidation were investigated in vitro using human liver microsomes and cDNA expressed enzymes. RESULTS: CYP3A4 and CYP2C9 were identified as the major enzymes mediating PER-N-demethylation. At 10 microM PER, a concentration consistent with anticipated in vivo liver concentrations, CYP3A4 and CYP2C9 contributed 50% and 35%, respectively, to PER-N-demethylation. With increasing PER concentrations, contribution of CYP2C9 decreased and CYP3A4 became more important. In human liver microsomes, PER-N-demethylation was inhibited by ketoconazole (>40%) and sulfaphenazole (16%). Allelic variants of recombinant CYP2C9 showed differences in PER-N-demethylase activity. The wild type allele CYP2C9*1 was the most active variant. Maximal activities of CYP2C9*2 and CYP2C9*3 were 88% and 18%, respectively, compared to the wild type activity. Perazine-N-oxidation was mainly mediated by FMO3. In the absence of NADPH, heat treatment of microsomes abolished PER-N-oxidase activity. Methimazole inhibited PER-N-oxidation, while CYP specific inhibitors had no inhibitory effect. Perazine is a potent inhibitor of dextromethorphan-O-demethylase, S-mephenytoin-hydroxylase, alprazolam-4-hydroxylase, phenacetin-O-deethylase and tolbutamide-hydroxylase activity in human liver microsomes. CONCLUSIONS: Alterations in the activity of CYP3A4, CYP2C9 and FMO3 through genetic polymorphisms, enzyme induction or inhibition bear the potential to cause clinically significant changes in perazine clearance. PER may alter the clearance of coadministered compounds metabolized by CYP2D6, CYP2C19, CYP2C9, CYP3A4 and CYP1A2.

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.

Phenacetin O-deethylation by human liver microsomes in vitro: inhibition by chemical probes, SSRI antidepressants, nefazodone and venlafaxine.

Biotransformation of phenacetin via O-deethylation to acetaminophen, an index reaction reflecting activity of Cytochrome P450-1A2, was studied in microsomal preparations from a series of human livers. Acetaminophen formation was consistent with a double Michaelis-Menten system, with low-Km (mean Km1 = 68 microM) and high-Km (mean Km2 = 7691 microM) components. The low-K(m) enzyme accounted for an average of 96% of estimated intrinsic clearance, and was predicted to contribute more than 50% of net reaction velocity at phenacetin concentrations less than 2000 microM. Among index inhibitor probes, alpha-naphthoflavone was a highly potent inhibitor of the low-Km enzyme (Ki1 = 0.013 microM); furafylline also was a moderately active inhibitor (Ki1 = 4.4 microM), but its inhibiting potency was increased by preincubation with microsomes. Ketoconazole was a relatively weak inhibitor (Ki1 = 32 microM); quinidine and cimetidine showed minimal inhibiting activity. Among six selective serotonin reuptake inhibitor (SSRI) antidepressants, fluvoxamine was a potent inhibitor of 1A2 (mean Ki1 = 0.24 microM). The other SSRIs were more than tenfold less potent. Mean Ki1 values were: fluoxetine, 4.4 microM; norfluoxetine, 15.9 microM; sertraline, 8.8 microM; desmethylsertraline, 9.5 microM; paroxetine, 5.5 microM. The antidepressant nefazodone and four of its metabolites (meta-chloro-phenylpiperazine, two hydroxylated derivatives, and a triazoledione) were very weak inhibitors of P450-1A2. Venlafaxine and its O- and N-desmethyl metabolites showed minimal inhibitory activity.

Human cytochromes mediating N-demethylation of fluoxetine in vitro.

Biotransformation of the selective serotonin reuptake inhibitor antidepressant, fluoxetine, to its principal metabolite, norfluoxetine, was evaluated in human liver microsomes and in microsomes from transfected cell lines expressing pure human cytochromes. In human liver microsomes, formation of norfluoxetine from R,S-fluoxetine was consistent with Michaelis-Menten kinetics (mean K(m) = 33 microM), with evidence of substrate inhibition at high substrate concentrations in a number of cases. The reaction was minimally inhibited by coincubation with chemical probes inhibitory for P450-2D6 (quinidine), -1A2 (furafylline, alpha-naphthoflavone), and -2E1 (diethyldithiocarbamate). Substantial inhibition was produced by coincubation with sulfaphenazole (Ki = 2.8 microM), an inhibitory probe for P450-2C9, and by ketoconazole (Ki = 2.5 microM) and fluvoxamine (Ki = 5.2 microM). However, ketoconazole, relatively specific for P450-3A isoforms only at low concentrations, reduced norfluoxetine formation by only 20% at 1 microM, and triacetyloleandomycin (> or = 5 microM) reduced the velocity by only 20-25%. Microsomes from cDNA-transfected human lymphoblastoid cells containing human P450-2C9 produced substantial quantities of norfluoxetine when incubated with 100 microM fluoxetine. Smaller amounts of product were produced by P450-2C19 and -2D6, but no product was produced by P450-1A2, -2E1, or 3A4. Cytochrome P450-2C9 appears to be the principal human cytochrome mediating fluoxetine N-demethylation. P450-2C19 and -3A may make a further small contribution, but P450-2D6 is unlikely to make an important contribution.

Long-term therapeutic drug monitoring of clozapine and metabolites in psychiatric in- and outpatients.

RATIONALE: Clozapine is a unique antipsychotic drug, outstanding for its lack of extrapyramidal side-effects and its superior efficacy in refractory schizophrenia. However, an unambiguous concentration-response relationship has not yet been established. OBJECTIVE: We investigated serum concentrations of clozapine, norclozapine and clozapine-N-oxide in psychiatric in- and outpatients to identify particular metabolic patterns in clozapine responders and non-responders and putative threshold levels for clozapine response. METHODS: Psychiatric assessments, CYP2D6 genotype, and weekly serum concentrations of clozapine, norclozapine and clozapine-N-oxide were obtained in 34 adult schizophrenic in-and outpatients (18 men, 16 women) during 10 weeks of clozapine treatment with a naturalistic dose design. RESULTS: Responders (n=21) displayed significantly lower serum concentrations of clozapine corrected for dose compared to non-responders (n=13; P<0.05), while none of the other parameters (absolute clozapine concentration, metabolite ratios, gender) were different. Smokers had significantly lower dose-corrected clozapine concentrations. A positive correlation was observed between age and average steady state clozapine concentrations. CONCLUSIONS: These findings indicate a possible link between CYP activity and response to clozapine that is not mediated through differences in serum concentrations. No clinically meaningful pattern in serum parameters could be identified that differentiates responders from non-responders. Thus, clozapine TDM seems ineffective for predicting clinical response. Smoking behavior is a major determinant of clozapine clearance while CYP2D6 genotype does not impact clozapine disposition.

Midazolam hydroxylation by human liver microsomes in vitro: inhibition by fluoxetine, norfluoxetine, and by azole antifungal agents.

Biotransformation of the imidazobenzodiazepine midazolam to its alpha-hydroxy and 4-hydroxy metabolites was studied in vitro using human liver microsomal preparations. Formation of alpha-hydroxy-midazolam was a high-affinity (Km = 3.3 mumol/L) Michaelis-Menten process coupled with substrate inhibition at high concentrations of midazolam. Formation of 4-hydroxy-midazolam had much lower apparent affinity (57 mumol/L), with minimal evidence of substrate inhibition. Based on comparison of Vmax/Km ratios for the two pathways, alpha-hydroxy-midazolam formation was estimated to account for 95% of net intrinsic clearance. Three azole antifungal agents were inhibitors of midazolam metabolism in vitro, with inhibition being largely consistent with a competitive mechanism. Mean competitive inhibition constants (Ki) versus alpha-hydroxy-midazolam formation were 0.0037 mumol/L for ketoconazole, 0.27 mumol/L for itraconazole, and 1.27 mumol/L for fluconazole. An in vitro-in vivo scaling model predicted inhibition of oral midazolam clearance due to coadministration of ketoconazole or itraconazole; the predicted inhibition was consistent with observed interactions in clinical pharmacokinetic studies. The selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine and its principal metabolite, norfluoxetine, also were inhibitors of both pathways of midazolam biotransformation, with norfluoxetine being a much more potent inhibitor than was fluoxetine itself. This finding is consistent with results of other in vitro studies and of clinical studies, indicating that fluoxetine, largely via its metabolite norfluoxetine, may impair clearance of P450-3A substrates.

Biotransformation of mestranol to ethinyl estradiol in vitro: the role of cytochrome P-450 2C9 and metabolic inhibitors.

Mestranol, the estrogen component of some oral contraceptive formulations, must be demethylated to its active metabolite, 17 alpha-ethinyl estradiol, to produce estrogenic activity. To investigate the transformation of mestranol to ethinyl estradiol, an in vitro assay was used with human liver microsomes from four different donors. Incubation of a fixed concentration of mestranol (3 mumol/L) with varying concentrations of CYP inhibitors revealed strong inhibition of ethinyl estradiol formation by sulfaphenazole, a specific CYP2C9 inhibitor, with an average inhibitor concentration at one half of Emax (IC50) of 3.6 mumol/L (range, 1.8-8.3 mumol/L) and an average maximal inhibitory capacity (Emax) of 75% (range, 60-91%). Troleandomycin (a CYP3A3/4 inhibitor) and quinidine (a CYP2D6 inhibitor), however, produced no substantial inhibitory activity. alpha-Naphthoflavone (a CYP1A1/2 inhibitor only at concentrations < 2 mumol/L and a CYP2C9 inhibitor at higher concentrations) had a weak inhibitory effect on ethinyl estradiol formation (< 20% decrease in mestranol demethylation activity). Of the three antifungal azoles tested, miconazole strongly inhibited mestranol demethylation, with an average IC50 of 1.5 mumol/L (range, 0.7-3.2 mumol/L) and an average Emax of 90% (range, 77-100%), whereas fluconazole displayed relatively weak inhibition only at the highest concentration of 50 mumol/L (mean reduction in demethylation activity was 29%). Itraconazole produced no meaningful inhibition. Strong inhibition of ethinyl estradiol formation by sulfaphenazole suggests a major contribution of CYP2C9 to this reaction.