clozapine-n-oxide and norclozapine

Topics: Adult; Antidepressive Agents, Second-Generation; Antipsychotic Agents; Clozapine; Drug Therapy, Combination; Fluvoxamine; Humans; Male; Schizophrenia

To study the effect of influenza vaccine on serum clozapine, N-desmethylclozapine and clozapine-N-oxide steady-state concentrations in patients with schizophrenia.. This was an open-label study in 14 schizophrenic inpatients (with 2 drop-outs) using clozapine. Serum trough concentrations of clozapine. N-desmethylclozapine and clozapine-N-oxide, as well as the concentration of c-reactive protein (CRP), were measured immediately before conventional trivalent influenza vaccination and 2, 4, 7 and 14 days after the vaccination.. Influenza vaccination had no significant effect on serum concentrations of clozapine, N-desmethylclozapine or clozapine-N-oxide. No changes in the clinical effects of clozapine were observed after vaccination. Influenza vaccination did not increase CRP. However, two drop-out patients who developed upper respiratory and abdominal symptoms had increased and elevated serum concentrations of clozapine, compared with the baseline.. Influenza vaccination using conventional trivalent influenza vaccine does not affect serum concentrations of clozapine or its main metabolites. However, an infection-related increase in CRP may be associated with increased serum concentration of clozapine.

Topics: Adult; Antipsychotic Agents; C-Reactive Protein; Clozapine; Drug Interactions; Female; Humans; Influenza Vaccines; Male; Middle Aged; Schizophrenia; Vaccination

Two separate studies were carried out to assess the effect of valproic acid on the steady-state plasma concentrations of clozapine and its major metabolites norclozapine and clozapine N-oxide in psychotic patients. In the first study, concentrations of clozapine and metabolites were compared between patients treated with clozapine in combination with sodium valproate (n = 15) and control patients treated with clozapine alone (n = 22) and matched for sex, age, body weight, and antipsychotic dosage. Patients comedicated with valproate tended to have higher clozapine levels and lower norclozapine levels, but the differences did not reach statistical significance. In a subsequent study, plasma concentrations of clozapine and its metabolites were determined in 6 patients with schizophrenia stabilized on clozapine therapy (200-400 mg/d) before and after treatment with sodium valproate (900-1200 mg/d) for 4 weeks. Mean plasma concentrations of clozapine and its metabolites did not change significantly throughout the study, but there was a trend for clozapine levels to be higher and for norclozapine levels to be lower after valproate. Overall, these findings suggest that valproic acid may have an inhibiting effect on the CYP1A2- or CYP3A4-mediated conversion of clozapine to norclozapine. However, the interaction is unlikely to be clinically significant.

Topics: Adult; Anticonvulsants; Antipsychotic Agents; Clozapine; Drug Interactions; Drug Therapy, Combination; Female; Humans; Male; Middle Aged; Mood Disorders; Schizophrenia; Valproic Acid

Results presented in this article are focused on the variability in pharmacokinetics. The purpose of this study was (1) to investigate intra- and interindividual variabilities of pharmacokinetic parameters of clozapine and its two main metabolites in plasma after multiple oral administration in 8 chronic schizophrenic patients (Study 1) and (2) to gain more information regarding plasma concentrations of these drugs after multiple doses in a group of 25 treatment-responsive patients (Study 2). Patients were treated with clozapine in fixed daily doses (given every 8-12 hours) between 200 and 900 mg. Plasma drug concentrations were determined by high-performance liquid chromatography. The mean volume of distribution and the total plasma clearance of clozapine, uncorrected for bioavailability, were 7 L/kg and 40.5 L/h, respectively. The terminal elimination half-lives averaged 10.5 hours for clozapine, 19.2 hours for norclozapine, and 8.6 hours for the N-oxide metabolite. Significant relationships were observed between clozapine and norclozapine (or clozapine N-oxide) plasma concentrations. Large inter- and intrapatient variations in pharmacokinetics were observed. Clozapine was generally well tolerated by the patients, with sedation, hypersialorrhea, and tiredness as the most common side effects encountered.

Topics: Adult; Antipsychotic Agents; Chronic Disease; Clozapine; Dose-Response Relationship, Drug; Female; Humans; Male; Metabolic Clearance Rate; Middle Aged; Schizophrenia; Time Factors

N-Desmethylclozapine and clozapine N-oxide are major metabolites of the atypical neuroleptic clozapine in humans and undergo renal excretion. The aim of this study was to investigate to what extent the elimination of these metabolites in urine contributes to the total fate of clozapine in patients and how they are handled by the kidney.. From 15 psychiatric patients on continuous clozapine monotherapy, blood and urine samples were obtained during four 2 h intervals, and clozapine and its metabolites were assayed in serum and urine by solid-phase extraction and h.p.l.c. Unbound fractions of the compounds were measured by equilibrium dialysis.. The following unbound fractions in serum were found (geometric means): clozapine 5.5%, N-desmethylclozapine 9.7%, and clozapine N-oxide 24.6%. Renal clearance values calculated from unbound concentrations in serum and quantities excreted in urine were for clozapine on average 11% of the creatinine clearance, whereas those of N-desmethylclozapine and clozapine N-oxide amounted to 300 and 640%, respectively. The clearances of unbound clozapine and N-desmethylclozapine increased with increasing urine volume and decreasing pH. All renal clearance values exhibited large interindividual variations. The sum of clozapine and its metabolites in urine represented on average 14% of the dose.. Clozapine, N-desmethylclozapine and clozapine N-oxide are highly protein-bound in serum. Clozapine is, after glomerular filtration, largely reabsorbed in the tubule, whereas the metabolites undergo net tubular secretion. Metabolic pathways alternative or subsequent to N-demethylation and N-oxidation must make major contributions to the total fate of clozapine in patients.

Topics: Adult; Antipsychotic Agents; Blood Proteins; Clozapine; Female; Humans; Kidney; Male; Metabolic Clearance Rate; Middle Aged; Schizophrenia

The pharmacokinetic parameters of clozapine and its two main metabolites, N-desmethylclozapine (norclozapine, active metabolite) and clozapine N-oxide, were evaluated, after oral administration, in 19 patients with chronic schizophrenia. Plasma and red blood cell (RBC) drug concentrations were determined by high-performance liquid chromatography. Large interpatient variations in pharmacokinetic parameters of clozapine and its two metabolites were observed. Plasma clozapine concentration peaked, on average, at 2.3 hours. The mean volume of distribution and the total plasma clearance, uncorrected for bioavailability, were 6 L/kg and 38 L/hr, respectively. The terminal elimination half-lives averaged 7.6 hours for clozapine, 13 hours for norclozapine, and 7 hours for the N-oxide metabolite. The mean RBC/plasma concentration ratios were 23, 61, and 81% for clozapine, N-desmethylclozapine, and clozapine N-oxide, respectively. From RBC concentration data, the mean elimination half-lives were 7.6 hours for clozapine, 16 hours for N-desmethylclozapine, and 8 hours for the N-oxide metabolite. The average value for blood clearance of clozapine was 54.7 L/hr. Significant correlations were observed between dose and maximum plasma concentrations and between dose and area under the curve concentrations; these results suggested linear steady-state pharmacokinetics over the range of concentrations studied.

Topics: Adolescent; Adult; Antipsychotic Agents; Chronic Disease; Clozapine; Female; Humans; Male; Middle Aged; Schizophrenia

Clozapine is one of the most effective drugs for resistant schizophrenia, but its severe metabolic and hematological side effects limit the use of clozapine. It has been reported that clozapine blood concentrations should be maintained between 350-600 ng/mL. Our aim was to develop a determination method for clozapine and its main metabolites norclozapine and clozapine-N-oxide, to perform validation studies and to investigate the change of various biochemical parameters in patients using clozapine.. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for clozapine measurement. Thus, blood samples were collected from 38 patients with schizophrenia and 32 healthy volunteers. Biochemical and hematological parameters were measured by Beckman-Coulter AU 5800 (Beckman Coulter, Brea, USA) and Beckman Coulter LH 780 analyzer (Beckman Coulter, Miami, FL, USA), respectively. Hormone levels were analyzed using Cobas 6000 analyzer (Roche Diagnostics, Germany).. The LCMS/MS method was linear between 1.22-2500 ng/mL (r. This LC-MS/MS method was rapid, simple, cost-effective and suitable for the routine clozapine monitoring. Furthermore, norclozapine and clozapine-N-oxide were also determined. Monitoring of metabolic and hematological parameters with clozapine levels is very important. However, the limitations of the study were that the method was not validated for norclozapine and clozapine-N-oxide, so the validation parameters were not evaluated for these two metabolites.

Topics: Adult; Antipsychotic Agents; Blood Cell Count; Blood Glucose; Case-Control Studies; Cholesterol; Chromatography, Liquid; Clozapine; Drug Monitoring; Hemoglobins; Humans; Limit of Detection; Reproducibility of Results; Schizophrenia; Tandem Mass Spectrometry; Triglycerides

Epstein-Barr virus (EBV), a member of the

Topics: Antipsychotic Agents; Burkitt Lymphoma; Cell Line, Tumor; Clozapine; Dose-Response Relationship, Drug; Epstein-Barr Virus Infections; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Humans; Immediate-Early Proteins; Phosphoproteins; Trans-Activators; Virus Activation; Virus Latency

The atypical antipsychotic agent clozapine (CLZ) is effective in many patients who are resistant to conventional antipsychotic drugs. Cytochromes P450 (CYPs) 1A2 and 3A4 oxidize CLZ to norCLZ and CLZ N-oxide in human liver. Concurrent treatment with inducers and inhibitors of CYP1A2 modulates CLZ elimination that disrupts therapy. Drug-drug interactions involving CYP3A4 are also significant but less predictable. To further characterize the factors underlying these interactions, we used samples from a cohort of human livers to assess variation in CLZ oxidation pathways in relation to intrinsic CYP3A4 and CYP1A2 activities and the effects of the corresponding selective inhibitors ketoconazole (0.2 and 2 μM) and fluvoxamine (1 and 10 μM). The CYP3A4-selective inhibitor ketoconazole (2 μM) impaired CLZ N-oxide formation in all 14 of the livers used in inhibition studies (≥50% inhibition) while the CYP1A2-selective inhibitor fluvoxamine (10 μM) decreased norCLZ formation in nine. Ketoconazole effectively inhibited CLZ metabolism in five of seven livers that catalysed CYP3A4-dependent testosterone 6β-hydroxylation at or above the median rate and in four other livers with lower intrinsic CYP3A4 activity. Similarly, fluvoxamine (10 μM) readily inhibited CLZ oxidation in seven livers with high CYP1A2-mediated 7-ethoxyresorufin O-deethylation activity (at or above the median) and three livers with lower intrinsic CYP1A2 activity. In three livers, CLZ biotransformation was impaired by both ketoconazole and fluvoxamine, consistent with a major role for both CYPs. These findings suggest that the intrinsic activities of CYPs 1A2 and 3A4 are unrelated to the response to CYP-selective inhibitors and that assessment of the activities in vivo may not assist the prediction of drug-drug interactions.

Topics: Antipsychotic Agents; Biotransformation; Clozapine; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1A2 Inhibitors; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Drug Interactions; Fluvoxamine; Humans; Hydroxylation; Ketoconazole; Liver; Microsomes, Liver; Oxidation-Reduction

Studying how and where drugs are metabolized in the brain is challenging. In an entire organism, peripheral metabolism produces many of the same metabolites as those in the brain, and many of these metabolites can cross the blood-brain barrier from the periphery, thus making the relative contributions of hepatic and brain metabolism difficult to study in vivo. In addition, drugs and metabolites contained in ventricles and in the residual blood of capillaries in the brain may overestimate drugs' and metabolites' concentrations in the brain. In this study, we examine locusts and zebrafish using matrix assisted laser desorption ionization mass spectrometry imaging to study brain metabolism and distribution. These animal models are cost-effective and ethically sound for initial drug development studies.

Topics: Animals; Antipsychotic Agents; Brain; Capillaries; Clozapine; Drug Development; Grasshoppers; Molecular Imaging; Neurons; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Zebrafish

Herbal supplements are increasingly used in psychiatric practice. Our epidemiological study has identified several herbal preparations associated with adverse outcomes of antipsychotic therapy. In this study, we evaluated the in vitro effects of four herbal preparations-Radix Rehmanniae (RR), Fructus Schisandrae (FS), Radix Bupleuri (RB) and Fructus Gardeniae (FG)-on cytochrome P450s (CYPs) involved in the metabolism of clozapine in human liver microsomes (HLMs) and recombinant human cytochrome P450 enzymes (rCYPs). N-desmethylclozapine and clozapine N-oxide, two major metabolites of clozapine, were measured using high-performance liquid chromatography (HPLC). FG, RR and RB showed negligible inhibitory effects in both in vitro systems, with estimated half-maximal inhibitory concentrations (IC50) and apparent inhibitory constant values (Ki) greater than 1 mg/mL (raw material), suggesting that minimal metabolic interaction occurs when these preparations are used concomitantly with clozapine. The FS extract affected CYP activity with varying potency; its effect on CYP 3A4-catalyzed clozapine oxidation was relatively strong (Ki: 0.11 mg/mL). Overall, the weak-to-moderate inhibitory effect of FS on in vitro clozapine metabolism indicated its potential role in herb-drug interaction in practice.

Topics: Antipsychotic Agents; Chromatography, High Pressure Liquid; Clozapine; Cytochrome P-450 Enzyme System; Gene Expression Regulation; Herb-Drug Interactions; Humans; In Vitro Techniques; Microsomes, Liver; Plant Preparations

The basis of the unique clinical profile of the antipsychotic clozapine is not yet elucidated. Brain histamine receptors may play a role in schizophrenia and its treatment, but their involvement in the profile of clozapine remained unknown.. We explored the properties of clozapine and its two metabolites, N-desmethylclozapine (NDMC) and clozapine N-oxide, at the four human histaminergic receptors. We compared their active concentrations with their blood concentrations in patients treated by clozapine. We investigated the changes in receptor densities induced in rat brain by repeated administration of a therapeutic dose of clozapine.. Clozapine and NDMC behaved as very potent, and partial, H(1)-receptor inverse agonists, weak, and full, H(2)-receptor inverse agonists, moderate, and protean, H(3)-receptor agonists, and moderate, and partial, H(4)-receptor agonists. Taking into account their micromolar mean blood concentrations found in 75 treated patients, and assuming that they are enriched in human brain as they are in rat brain, a full occupation of H(1)-, H(3)-, and H(4)-receptors, and a partial occupation of H(2) receptors, is expected. In agreement, repeated administration of clozapine at a therapeutic dose (20 mg/kg/day for 20 days) induced an up-regulation of H(1)- and H(2)-receptors in rat brain.. Clozapine and its active metabolite NDMC interact with the four human histamine receptors at clinically relevant concentrations. This interaction may substantiate, at least in part, the atypical antipsychotic profile of clozapine, as well as its central and peripheral side effects such as sedation and weight gain.

Topics: Animals; Antipsychotic Agents; Brain; Clozapine; Drug Inverse Agonism; Histamine Agonists; Humans; Male; Rats; Rats, Wistar; Receptors, Histamine; Retrospective Studies; Up-Regulation

An LC-MS/MS method for the determination of the atypic neuroleptic clozapine and its two main metabolites norclozapine and clozapine-N-oxide has been developed and validated for serum and urine. After addition of d4-clozapine as deuterated internal standard a fast single-step liquid-liquid extraction under alkaline conditions and with ethyl acetate as organic solvent followed. The analytes were chromatographically separated on a Synergi Polar RP column using gradient elution with 1 mM ammonium formate and methanol. Data acquisition was performed on a QTrap 2000 tandem mass spectrometer in multiple reaction monitoring mode with positive electrospray ionization. Two transitions were monitored for each analyte in order to fulfill the established identification criteria. The validation included the determination of the limits of quantification (1.0 ng/mL for all analytes in serum and 2.0 ng/mL for all analytes in urine), assessment of matrix effects (77% to 92% in serum, 21 to 78% in urine) and the determination of extraction efficiencies (52% to 85% for serum, 59% to 88% for urine) and accuracy data. Imprecision was <10%, only the quantification of norclozapine in urine yielded higher relative standard deviations (11.2% and 15.7%). Bias values were below ±10%. Dilution of samples had no impact on the correctness for clozapine and norclozapine in both matrices and for clozapine-N-oxide in serum. For quantification of clozapine-N-oxide in urine a calibration with diluted calibrators has to be used. Calibration curves were measured from the LOQ up to 2,000 ng/mL and proved to be linear over the whole range with regression coefficients higher than 0.98. The method was finally applied to several clinical serum and urine samples and a cerebro-spinal fluid sample of an intoxicated 13-month-old girl.

Topics: Antipsychotic Agents; Chromatography, Liquid; Clozapine; Humans; Limit of Detection; Tandem Mass Spectrometry

Topics: Adult; Antipsychotic Agents; Biotransformation; Chemical and Drug Induced Liver Injury; Clozapine; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Humans; Isoxazoles; Liver Function Tests; Paliperidone Palmitate; Pyrimidines; Reference Values; Risperidone; Schizophrenia

The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant delta-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate delta-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- (K(i)=26 nM) and low-affinity (K(i)=3 microM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [(35)S]GTPgammaS binding (EC(50)=905 nM) and inhibited cyclic AMP formation (EC(50)=590 nM) as effectively as delta-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the delta-opioid antagonist naltrindole, but not by the micro-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) or the kappa-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D(2) or serotonin 5HT(1A) receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at delta-opioid receptors of human frontal cortex.

Topics: Adult; Aged; Binding, Competitive; Clozapine; Cyclic AMP; Frontal Lobe; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Middle Aged; Protein Binding; Radioligand Assay; Receptors, Opioid, delta

The atypical antipsychotic drug clozapine (CLZ) is effective in a substantial number of patients who exhibit treatment-resistance to conventional agents. CYP1A2 is generally considered to be the major enzyme involved in the biotransformation of CLZ to its N-demethylated (norCLZ) and N-oxygenated (CLZ N-oxide) metabolites in liver, but several studies have also implicated CYP3A4. The present study assessed the interplay between these cytochrome P450s (P450s) in CLZ biotransformation in a panel of hepatic microsomal fractions from 14 individuals. The relative activity of P450s 1A2 and 3A4 in microsomes was found to be a major determinant of the relative susceptibility of norCLZ formation to inhibition by the P450-selective inhibitors fluvoxamine and ketoconazole. In contrast, the activity of CYP3A4 alone was correlated with the susceptibility of CLZ N-oxide formation to inhibition by these agents. These findings suggest that both P450s may be dominant CLZ oxidases in patients and that the relative activities of these enzymes may determine clearance pathways. In vivo assessment of CYP1A2 and CYP3A4 activities, perhaps by phenotyping approaches, could assist the optimization of CLZ dosage and minimize pharmacokinetic interactions with coadministered drugs.

Topics: Alleles; Aryl Hydrocarbon Hydroxylases; Biotransformation; Catalysis; Clozapine; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1A2 Inhibitors; Cytochrome P-450 CYP2B6; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dextromethorphan; Enzyme Inhibitors; Fluvoxamine; Humans; Isoenzymes; Ketoconazole; Kinetics; Microsomes, Liver; Oxidation-Reduction; Oxidoreductases, N-Demethylating; Oxygenases; Recombinant Proteins; Testosterone; Tolbutamide

Hepatic lipid infiltration (steatosis) is a complication of the metabolic syndrome and can progress to nonalcoholic steatohepatitis and severe liver injury. Microsomal cytochrome P450 (P450) drug oxidases are down-regulated in experimental steatosis. In this study we evaluated the separate and combined effects of lipid accumulation and P450 down-regulation on the microsomal oxidation of the antipsychotic agent clozapine (CLZ), the use of which is associated with an increased incidence of the metabolic syndrome. Several important drug oxidizing P450s were down-regulated, and the formation of N-desmethyl-CLZ (norCLZ) and CLZ N-oxide was decreased in microsomal fractions from orotic acid-induced early steatotic rat liver. Inclusion of lipids extracted from steatotic, but not control, liver decreased the free concentration of CLZ in microsomes and suppressed norCLZ formation; CLZ N-oxidation was unchanged. Triglycerides increased in steatotic liver to 15-fold of control, whereas increases in the monounsaturated oleic acid to 10-fold of control and total polyunsaturated and saturated fatty acids to 4- and 5-fold of control also occurred. Addition of triglycerides containing esterified omega-6 and omega-3 fatty acids inhibited the microsomal formation of norCLZ but not that of CLZ N-oxide; triglycerides esterified with unsaturated and monounsaturated fatty acids were inactive. Thus, drug oxidation may be suppressed in steatosis by P450 down-regulation and the accumulation of polyunsaturated fatty esters. In contrast, the activity of the flavin-containing monooxygenase that mediates CLZ N-oxidation was unimpaired. Lipid deposition in livers of patients with the metabolic syndrome may necessitate dosage adjustments for toxic drugs, including CLZ.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Clozapine; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Cytochromes; Fatty Acids; Fatty Acids, Unsaturated; Fatty Liver; Kinetics; Lipids; Liver; Male; Microsomes, Liver; Orotic Acid; Oxazines; Oxidation-Reduction; Rats; Rats, Wistar; Steroid 16-alpha-Hydroxylase; Triglycerides

Clozapine-induced obsessive/compulsive symptoms (OCS) have been reported by many authors. This study investigated the incidence of these side effects, together with the relation between these side effects and the plasma concentration (Cps) of clozapine and its metabolites norclozapine and clozapine-N-oxide in schizophrenic patients. One hundred and two schizophrenic patients treated with clozapine were interviewed and screened with questionnaires testing for OCS during a 1-year study period. Cps of clozapine and the metabolites were monitored using reversed-phase high-performance liquid chromatography with ultraviolet detection. Thirty-nine patients (38.2%) presented with OCS, and, of these, 29 patients (28.4%) were classified as clozapine-induced, with an average latent period of 39.8+/-22.5 months. The Cps of clozapine and norclozapine were significantly higher in patients with OCS than in those without (595.1+/-364.9 vs. 433.5+/-252.8 ng/mL, P=0.001 and 266.4+/-144.4 vs. 203.1+/-119.8 ng/mL) OCS. Clozapine-induced OCS were not uncommon side effects. The authors suggest that the emergence of these side effects may be related to higher Cps of clozapine and clinicians should routinely check for and manage these side effects.

Topics: Adult; Antipsychotic Agents; Clozapine; Dose-Response Relationship, Drug; Drug Monitoring; Female; Humans; Male; Obsessive-Compulsive Disorder; Schizophrenia

The goal of the present study was to determine the effects of clozapine (Cloz) and its metabolites norclozapine (Norcloz) and clozapine-N-oxide (Cloz-N-oxide) on the 5-HT(2) receptor system on the levels of protein and gene expression in in vitro systems of primary cortical cells of the rat and human hippocampal SHS5Y5 neuroblastoma cells.. Clinically relevant concentrations of Cloz (200/400 ng/ml) and its metabolites (200 ng/ml) were used for the examination of the effects of Cloz and its metabolites on serotoninergic 5-HT(2) receptor parameters (density, affinity and mRNA levels) as well as on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in primary cortical cells of the rat after treatment for 24 h under in vitro conditions. To compare the results to human cells, we also measured treatment-induced changes in 5-HT(2) and GAPDH mRNA levels in human hippocampal SHS5Y5 cells.. A significant decrease was found in primary cortical cells for 5-HT(2) receptor density (Cloz 200/Cloz 400/Norcloz 200 and Cloz-N-oxide 200 vs. control) and 5-HT(2A) receptor mRNA levels (Cloz 200 vs. control). 5-HT(2A) receptor mRNA levels were also significantly reduced (Norcloz 200 vs. control) in SHS5Y5 cells. GAPDH mRNA levels were not affected.. The results of the present study show that Cloz and Norcloz induce significant alterations on the 5-HT(2) receptor system in primary cortical cells of the rat and in human hippocampal cells.

Topics: Analysis of Variance; Animals; Cells, Cultured; Cerebral Cortex; Clozapine; Dose-Response Relationship, Drug; Gene Expression Regulation; Hippocampus; Humans; In Vitro Techniques; Neuroblastoma; Neurons; Protein Binding; Radioligand Assay; Rats; Receptors, Serotonin, 5-HT2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serotonin Antagonists

Clozapine and its two major metabolites, N-desmethylclozapine and clozapine N-oxide were quantified using a high-performance liquid chromatographic method with UV detection in dog plasma following a single dose of clozapine. The analysis was performed on a 5-micrometer Hypersil CN (CPS-1; 250x4.6 mm) column. The mobile phase consisted of acetonitrile-water-1 M ammonium acetate (50:49:1, v/v/v), which was adjusted to pH 5.0 with acetic acid. The detection wavelength was 254 nm. A liquid-liquid extraction technique was used to extract clozapine and its metabolites from dog plasma. The recovery rates for clozapine, N-desmethylclozapine, and the internal standard (I.S.) were close to 100% using this method. The recovery rate for clozapine N-oxide (62-66%) was lower as expected because it is more polar. The quantitation limits for clozapine, clozapine N-oxide, and N-desmethylclozapine were 0.11, 0.05 and 0.05 microM, respectively. Intra-day reproducibility for concentrations of 0.1, 1.0 and 5.0 microM were 10.0, 4.4 and 4.2%, respectively, for N-oxide; 11.2, 4.3 and 4.9%, respectively, for N-desmethylclozapine; and 10.8, 2.2 and 4.9%, respectively, for clozapine. Inter-day reproducibility was <15% for clozapine N-oxide, <8% for N-desmethylclozapine and <19% for clozapine. This simple method was applied to determine the plasma concentration profiles of clozapine, N-desmethylclozapine and clozapine N-oxide in dog following administration of a 10 mg/kg oral dose of clozapine.

Topics: Animals; Chromatography, High Pressure Liquid; Clozapine; Dogs; Reference Standards; Reproducibility of Results; Sensitivity and Specificity

Up to now, it is not yet clear whether and how clozapine and its metabolites are metabolized in neuronal cells. The interconversion of clozapine and its metabolites, clozapine-N-oxide and norclozapine, was studied in the hippocampal neuronal in vitro system of HT22 cells. Clinically relevant concentrations of clozapine (200+400 ng/ml) and its metabolites (100+200 ng/ml) were used for the examination of the metabolizing effects after short- (4 h) and long- (24 h) term incubation. Two-way analysis of variance revealed a significant decrease of clozapine (P<0.01) and norclozapine (P<0.01) levels in the supernatants of HT22 cells after the treatment procedures. Student-Newman-Keuls tests showed a significant decrease of clozapine 400 after 24 h of incubation (P=0.01) as well as of all concentrations of norclozapine. No significant treatment effects were found for the clozapine-N-oxide degradation. Using semi-quantification by reverse transcriptase-polymerase chain reaction methods, we could show a significant increase of cytochrome P450 (CYP) 1A2 mRNA levels (P<0.05) after clozapine treatment with 200 ng/ml. The results of the present study strongly suggest that clozapine and norclozapine are metabolized in hippocampal neuronal HT22 cells by CYP1A2, whereas the levels of clozapine-N-oxide were not affected. Moreover, CYP1A2 mRNA levels were significantly changed by incubation with clozapine 200.

Topics: Analysis of Variance; Biological Transport; Cells, Cultured; Clozapine; Cytochrome P-450 CYP1A2; Hippocampus; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

In the hippocampal neuronal in vitro system of HT22 cells, we studied the effects of clozapine (Cloz) and its metabolites clozapine-N-oxide (Cloz-N-oxide) and norclozapine (Norcloz) on 5-HT transporter affinity (K(M)) and uptake (V(max)), MAO-B affinity (K(M)) and maximal velocity (V(max)), as well as on 5-HT(2) receptor affinity and density. Clinically relevant concentrations of Cloz (200 and 400 ng/ml) and its metabolites (100 and 200 ng/ml) were used for the examination of the effects after short-term (4 h) and long-term (24 h) incubation. Statistical evaluation revealed that a significantly lowered 5-HT transporter affinity (higher K(M)) was related to higher concentrations of Cloz and its metabolites. A significantly higher 5-HT transporter uptake was dependent on both high concentrations of drugs and an increased time of incubation. No significant influence of the investigated independent variables on MAO-B affinity could be demonstrated, whereas a significant drug-related increase of MAO-B velocity was detectable. Additionally, low and high concentrations of Cloz and its metabolites induced a higher 5-HT(2) receptor affinity (lower K(D)). No significant influences of the investigated independent variables on 5-HT(2) receptor density were detectable. The results of the present study show that Cloz and its metabolites induce significant alterations in serotoninergic parameters of hippocampal HT22 cells, validating the system of hippocampal HT22 cells for further examinations of the mechanisms of action of atypical neuroleptics.

Topics: Antipsychotic Agents; Carrier Proteins; Cells, Cultured; Clozapine; Dose-Response Relationship, Drug; Hippocampus; Humans; Membrane Glycoproteins; Membrane Transport Proteins; Monoamine Oxidase; Nerve Tissue Proteins; Neurons; Receptors, Serotonin; Serotonin; Serotonin Plasma Membrane Transport Proteins

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine-N-oxide in human plasma has been developed and validated. The isocratic HPLC assay uses a mobile phase consisting of an acetonitril-buffered aqueous solution containing 146 microL of triethylamine and 200 microL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydroxide solution (400:600, v/v) at a flow-rate of 0.8 ml/min and a Lichrospher 100 RP-18 reversed-phase column and UV detection at 215 nm. Doxepine was used as the internal standard. Mean recoveries for clozapine, norclozapine, clozapine-N-oxide and doxepine were 95%, 98%, 96% and 94%, respectively, whereas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1.8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concentration range of 100-1600 ng/mL was 0.9998 and 0.9997, respectively; for clozapine-N-oxide (20-200 ng/mL) it was found to be 0.9986. The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, norclozapine and clozapine-N-oxide, respectively.

Topics: Chromatography, High Pressure Liquid; Clozapine; Humans; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

A liquid chromatography tandem mass spectrometry (LC-MS-MS) assay method for the simultaneous determination of clozapine and its N-desmethyl (norclozapine) and N-oxide metabolites in human plasma is described. The compounds were extracted from plasma by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using positive ion atmospheric pressure electrospray ionization method by a TurboIonspray source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 327 --> m/z 270 for clozapine, m/z 313 --> m/z 192 for norclozapine, m/z 343 --> m/z 256 for clozapine-N-oxide and m/z 421--> m/z 201 for internal standard. The standard curves of clozapine, norclozapine and clozapine-N-oxide were linear over the range of 1 ng/ml to 1000 ng/ml when 0.5 ml of plasma was used for the analysis (r(2) >0.998). Three pooled plasma samples collected from patients who were treated with clozapine were used as long-term quality control samples to check the validity of spiked standard curve samples made at various times. The intra- and inter-assay variations for the spiked standard curve and quality control samples were less than 14%. These variations for the long-term patient quality control samples were less than 11%. The LC-MS-MS assay for simultaneous determination of clozapine, norclozapine and clozapine-N-oxide reported here is highly specific, sensitive, accurate and rapid. This method is currently being used for the plasma level monitoring of clozapine and its N-desmethyl and N-oxide metabolites in patients treated with clozapine. The plasma levels of clozapine, norclozapine and clozapine-N-oxide varied widely within and among patients. The data revealed that the norclozapine and clozapine N-oxide metabolites were present at about 58%+/-14% and 17%+/-6% of clozapine concentrations in plasma, respectively.

Topics: Antipsychotic Agents; Chromatography, Liquid; Clozapine; Drug Monitoring; Humans; Reproducibility of Results; Schizophrenia; Spectrometry, Mass, Electrospray Ionization

Grapefruit juice can inhibit the gastrointestinal activity of cytochrome P450 (CYP) 3A4, while its effect on CYP1A2 remains controversial. Several grapefruit juice bioflavonoids also modulate the activity of the drug transporter P-glycoprotein in the gut and in the blood-brain barrier. Both CYP1A2 and CYP3A4 are involved in clozapine metabolism. This study investigated the effects of repeated ingestion of grapefruit juice on multiple-dose pharmacokinetics and pharmacodynamics of clozapine in schizophrenic patients.. Clozapine therapy was initiated for fifteen treatment-resistant schizophrenic inpatients (DSM-IV criteria). The doses were individually titrated from day -35 to day -15 and then kept unchanged from day -14 to day 49. Regular-strength grapefruit juice (250 mL) was coadministered b.i.d. with each clozapine dose from day 15 to day 28. Plasma levels of clozapine and its main metabolites (norclozapine and clozapine N-oxide) were obtained, and clinical efficacy and safety assessments were completed prior to juice administration (days 0, 7, and 14), during the coadministration (days 17, 21, and 28), and after cessation of the juice (days 35, 42, and 49).. After reaching steady states, plasma concentrations of clozapine and its metabolites and Positive and Negative Syndrome Scale scores were not significantly altered by the effect of grapefruit juice ingestion. The Clinical Global Impressions scale scores, Calgary Depression Scale scores, and side effect profiles (by the Extrapyramidal Symptom Rating Scale, the UKU Side Effect Rating Scale, and thorough examinations including electrocardiography and electroencephalography) also remained constant during the study.. Consumption of regular-strength grapefruit juice, 250 mL b.i.d., for 14 days did not significantly impact clozapine metabolism, clinical efficacy, or tolerability. One reason is that enzymes other than CYP3A4 also mediate clozapine disposition. Also, grapefruit juice inhibits CYP3A4 in the gut, but not in the liver. The preliminary results also suggest that clozapine is unlikely to be a P-glycoprotein substrate. Further rigorous studies are necessary to reconfirm these findings.

Topics: Adult; Beverages; Citrus; Clozapine; Female; Food-Drug Interactions; Humans; Male; Middle Aged; Prospective Studies; Psychiatric Status Rating Scales; Schizophrenia

It was aimed to identify the cytochrome(s) P450 (CYPs) involved in the N-demethylation and N-oxidation of clozapine (CLZ) by various approaches using human liver microsomes or microsomes from human B-lymphoblastoid cell lines. The maximum rates of formation were measured in the microsomal fraction of human livers and the Michaelis-Menten kinetics one enzyme model was found to best fit the data with mean K(M) for CLZ N-oxide and N-desmethyl-CLZ of 336 and 120 microM, respectively. Significant correlations were observed between the maximum rates of formation (Vmax) for CLZ N-oxide and N-desmethyl-CLZ with the microsomal immunoreactive contents of CYP1A2 (r = 0.92, P < 0.009 and r = 0.77, P < 0.077; respectively) and CYP3A (r = 0.89, P < 0.02 and r = 0.82, P < 0.05; respectively). Antibodies directed against CYP1A2 and CYP3A inhibited formation of CLZ N-oxide in human liver microsomes by 10.7+/-6.1%) and 37.2+/-6.9% of control, respectively, whereas CLZ N-demethylation was inhibited by 32.2+/-15.4% and 33.6+/-7.4%, respectively. Troleandomycin (CYP3A inhibitor) and furafylline (CYP1A2 inhibitor) inhibited CLZ N-oxidation in human liver microsomes by 23.2+/-12.1% and 7.8+4.3%, respectively, whereas CLZ N-demethylation was inhibited by 17.5+/-13.9% and 25.6+/-16.5%, respectively. While ketoconazole did not inhibit N-oxidation of CLZ, the N-demethylation pathway was inhibited by 34.1+/-10.0%. Formation in stable expressed enzymes indicated involvement of CYP3A and CYP1A2 in CLZ N-oxide formation and CYP2D6, CYP1A2 and CYP3A4 in CLZ N-demethylation. This apparent involvement of CYP2D6 in the N-demethylation of CLZ did not corroborate with the findings of other experiments. In conclusion, these data indicate that while both CYP isoforms readily catalyze both metabolic routes in vitro, CYP1A2 and CYP3A4 are more important in N-demethylation and N-oxidation, respectively.

Topics: Antibodies; Antipsychotic Agents; Cell Line; Clozapine; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Gene Expression; Humans; Kinetics; Microsomes, Liver; Mixed Function Oxygenases; Molecular Structure

Cytochrome P450 expression in liver is influenced by several factors, including species, sex and strain. We compared metabolism formation of clozapine in different species (rat, mouse, guinea-pig, dog, monkey and man) so as to choose between species to further validate interaction studies. Liver microsomes of male and female Sprague-Dawley rats, hairless rats, OF1 mice, Balb C mice and Dunkin-Hartley albino guinea-pigs, male beagle dogs, male cynomolgus monkeys and man were used to investigate in vitro metabolism of clozapine. This process was dependent on the presence of NADPH and on the presence of microsome protein. In addition, we observed the formation of desmethyl- and N-oxide metabolites, with the rate of formation of each of these compounds varying with species, sex and strain of microsomes incubated. The desmethyl- and N-oxide metabolites formed were statistically greater in male than in female rats, mice in the two strains studied, as well as for the guinea-pigs. Levels of desmethyl clozapine formed were high for the rats and no significant difference in clozapine biotransformation was observed between Sprague-Dawley and hairless rats. For man, the formation of metabolites of clozapine was comparable with guinea-pig, dog and monkey. In addition, we screened the effect of 52 molecules, representative of 11 different therapeutic classes, on the metabolism of clozapine by rat liver microsomes. We found that most of the calcium channel blockers (diltiazem, felodipine, isradipine, lacidipine, nicardipine and nitrendipine), antifungals (ketoconazole, miconazole) and two anticancer drugs (paclitaxel, teniposide) caused more than 50% inhibition of clozapine metabolism in vitro. The extent of inhibition was increased in a concentration-dependant manner. Complementary clinical and pharmacokinetic studies should be performed to confirm these results.

Topics: Animals; Clozapine; Dogs; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Interactions; Drug-Related Side Effects and Adverse Reactions; Female; Guinea Pigs; Humans; Macaca fascicularis; Male; Mice; Mice, Inbred BALB C; Microsomes, Liver; NADP; Rats; Rats, Nude; Rats, Sprague-Dawley; Sex Factors; Species Specificity

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid-liquid extraction with n-hexane-isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 microl of 0.1 M dibasic phosphate (pH 2.2 with 25% H3PO4). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile-0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250 x 4.60 mm I.D. analytical column packed with 5 microm C6 silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were < or = 13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from -19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.

Topics: Antipsychotic Agents; Chromatography, High Pressure Liquid; Clozapine; Humans; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

The steady state plasma concentrations of clozapine and its two major metabolites, norclozapine and clozapine N-oxide, were compared in patients with schizophrenia treated with clozapine in combination with phenobarbital (n=7), and in control patients treated with clozapine alone (n=15). Patients were matched for sex, age, body weight, and antipsychotic dosage. Patients comedicated with phenobarbital had significantly lower plasma clozapine levels than those of the controls (232+/-104 versus 356+/-138 ng/ml; mean, SD, p < 0.05). Plasma norclozapine levels did not differ between the two groups (195+/-91 versus 172+/-61 ng/ml, NS), whereas clozapine N-oxide levels were significantly higher in the phenobarbital group (115+/-49 versus 53+/-31 ng/ml, p < 0.01). Norclozapine/clozapine and clozapine N-oxide/ clozapine ratios were also significantly higher (p < 0.001) in patients comedicated with phenobarbital. These findings suggest that phenobarbital stimulates the metabolism of clozapine, probably by inducing its N-oxidation and demethylation pathways.

Topics: Adult; Clozapine; Cytochrome P-450 Enzyme System; Drug Interactions; Enzyme Induction; Female; Humans; Male; Middle Aged; Phenobarbital; Schizophrenia

An isocratic high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of clozapine (8-chloro-11-(4'-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two major metabolites in plasma and red blood cells (RBCs). The method involves sample clean-up by liquid-liquid extraction with ethyl acetate. The organic phase was back-extracted with 0.1 M hydrochloric acid. Loxapine served as the internal standard. The analytes were separated by HPLC on a Kromasil Ultrabase C18 analytical column (5 microns particle size; 250 x 4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm. The limits of quantiation were 20 ng/ml for clozapine and N-desmethylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma or RBCs proved to be higher than 62%. Precision, expressed as % C.V., was in the range 0.6-15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine and two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeutic drug monitoring.

Topics: Antipsychotic Agents; Chromatography, High Pressure Liquid; Clozapine; Drug Stability; Erythrocytes; Humans; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites.. Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used.. Km and Vmax values determined in human liver microsomes were lower for the demethylation (61 +/- 21 microM, 159 +/- 42 pmol min(-1) mg protein(-1) mean +/- s.d.; n = 4), than for the N-oxidation of CLZ (308 +/- 1.5 microM, 456 +/- 167 pmol min(-1) mg protein(-1); n = 3). Formation of DCLZ was inhibited by fluvoxamine (53 +/- 28% at 10 microM), triacetyloleandomycin (33 +/- 15% at 10 microM), and ketoconazole (51 +/- 28% at 2 microM) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34 +/- 16% at 10 microM) and ketoconazole (51 +/- 13% at 2 microM), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. Km and Vmax values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions.. It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.

Topics: Antibodies; Antibody Specificity; Antipsychotic Agents; Biotransformation; Clozapine; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1A2 Inhibitors; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Humans; Isoenzymes; Kinetics; Microsomes, Liver; Mixed Function Oxygenases

Clozapine (0.625-10.0 mg kg-1 s.c.), but not the two major clozapine metabolites, N-desmethylclozapine (0.625-10.0 mg kg-1 s.c.) or clozapine-N-oxide (0.625-10.0 mg kg-1 s.c.), caused a dose-dependent decrease in core temperature in the rat. Furthermore, the clozapine-induced hypothermia (2.5 mg kg-1 s.c.) was fully antagonised by pretreatment with the selective dopamine D1 receptor antagonist (+)-5-(2,3-dihydrobenzofuran-7-yl)-3-methyl-8-nitro-2,3,4, 5-tetrahydro-1 H-3-benzazepine-7-ol, maleate (NNC 687) (4.0 mg kg-1 s.c.). NNC 687 by itself (2.0-8.0 mg kg-1 s.c.) did not affect core temperature. The present results provide further evidence for the dopamine D1 receptor agonist properties of clozapine.

Topics: Animals; Benzazepines; Benzofurans; Body Temperature; Clozapine; Dopamine Agonists; Dopamine Antagonists; Male; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Sensitivity and Specificity

The metabolism of clozapine N-oxide was investigated in the rat (n = 6) after a single oral dose of 20 mg kg-1. The organic extracts of rat urine were separated by conventional high performance liquid chromatography (HPLC) and individual collected fractions were analyzed by micro-column electrospray HPLC/mass spectroscopy. The compounds identified in rat urine were clozapine N-oxide, clozapine, N-desmethylclozapine, 8-deschloro-8-hydroxyclozapine, 8-deschloro-8-thiomethylclozapine, N-desmethylclozapine, 8-deschloro-8-hydroxyclozapine, 8-deschloro-8-thiomethylclozapine, N-desmethyl-8-deschloro-8-thiomethylclozapine and 8-deschloro-8-methylsulfinylclozapine. With the exception of the unchanged clozapine N-oxide, no other metabolite containing a N-oxide functional group could be found, the concentrations of clozapine N-oxide, clozapine and N-desmethylclozapine excreted from rat urine were determined utilizing a conventional HPLC procedure with UV detection. The recoveries of these three analytes reported as the percentage of the dosage from the 0.24 h urine are 0.93 +/- 0.54%, 0.06 +/- 0.03% and 0.01 +/- 0.006% respectively.

Topics: Animals; Chromatography, High Pressure Liquid; Clozapine; Female; Male; Mass Spectrometry; Rats; Rats, Inbred Lew

Similarly to clozapine, a clozapine metabolite, N-desmethylclozapine, but not clozapine N-oxide, antagonized brain gamma-aminobutyric acid type A (GABAA) receptors at high micromolar concentrations. However, daily subcutaneous injections of clozapine (10 and 25 mg/kg) and haloperidol (0.5 mg/kg) for 14 days failed to alter the modulation by GABA of rat cerebrocortical and cerebellar benzodiazepine ([3H]flunitrazepam) or convulsant (t-[35S]bicyclophosphorothionate) binding sites of the GABAA receptor. The results thus suggest that the GABAA receptor antagonism exerted by chronic in vivo clozapine treatment is weak as compared to this treatment's actions on certain monoamine receptors and is unlikely to be involved in the therapeutic actions of clozapine.

Topics: Animals; Antipsychotic Agents; Brain; Clozapine; Drug Evaluation, Preclinical; GABA Antagonists; GABA-A Receptor Antagonists; Haloperidol; In Vitro Techniques; Logistic Models; Male; Rats; Rats, Sprague-Dawley; Time Factors

Clozapine is an antipsychotic drug with few extra-pyramidal motor side-effects, used to treat schizophrenia which is resistant to classical neuroleptic therapy. This report shows that norclozapine but not clozapine-N-oxide has the same D2 receptor affinity as clozapine. Assay results suggest a bimodal distribution which may be explained by CYP1A2 polymorphism. Extensive metabolizers could produce other active metabolites, probably other hydroxy-clozapine derivatives.

Topics: Animals; Antipsychotic Agents; Chromatography, High Pressure Liquid; Clozapine; Corpus Striatum; Female; Humans; Hydroxylation; Male; Radioligand Assay; Rats; Receptors, Dopamine D2; Schizophrenia

This study was done to test the hypothesis that serum concentration of norclozapine is a risk factor for leukopenia during treatment with clozapine.. Maximum decreases in leukocyte counts in 44 unselected patients treated with clozapine were determined and then correlated with drug doses and serum concentrations of clozapine, norclozapine, and clozapine-N-oxide.. White cell and granulocyte counts decreased by up to 60%-73%, but there were no positive correlations between these decrements and drug dose, drug level, ratio of drug level to drug dose, or ratio of norclozapine level to clozapine level, nor were the decreases related to age or gender.. While these results do not suggest in vivo hemotoxicity of norclozapine, further study of patients with clinically significant leukopenia is required.

Topics: Clozapine; Female; Follow-Up Studies; Humans; Leukocyte Count; Leukopenia; Male; Middle Aged; Psychotic Disorders; Risk Factors

Topics: Animals; Chemistry Techniques, Analytical; Chromatography, Liquid; Clozapine; Humans; Rats; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

We report a new assay to measure the serum concentrations of the atypical antipsychotic drug clozapine and two major metabolites, norclozapine and clozapine-N-oxide. The analytes and an internal standard (triprolidine) were extracted from alkalinized samples into ethyl acetate and back-extracted into 0.1 mol/L HCl. The acid extracts were chromatographed on a reversed-phase liquid chromatographic column with photodiode array detection (210-340 nm). With the 254-nm signal, between-run imprecision (CV) was < 2% for clozapine and norclozapine at 400 micrograms/L, and 4.1% for clozapine-N-oxide at 100 micrograms/L. Absolute recovery exceeded 65%, and the detection limit was approximately 3-4 micrograms/L. In 25 patients at steady state at a mean daily clozapine dosage of 269 mg (3.09 mg/kg), clozapine averaged 231 +/- 144 micrograms/L (mean +/- SD); norclozapine and clozapine-N-oxide concentrations averaged 84% and 23% that of clozapine. Analyte concentrations were significantly correlated with daily dose. The method's ability to quantify clozapine and two major metabolites simultaneously with precision and sensitivity makes it useful in pharmacokinetic studies and therapeutic monitoring.

Topics: Adult; Chromatography, Liquid; Clozapine; Female; Humans; Male; Middle Aged; Quality Control; Reference Values; Spectrophotometry, Ultraviolet

The effects of the atypical antipsychotic drug, clozapine, and its two major metabolites in man, N-desmethylclozapine and clozapine N-oxide, on 5-HT1C receptor mediated phosphoinositide hydrolysis were studied in rat choroid plexus. Clozapine and N-desmethylclozapine antagonized 5-HT-stimulated phosphoinositide hydrolysis with IC50 values of 110 and 29.4 nM, respectively. Clozapine N-oxide was less potent. None of the compounds stimulated phosphoinositide hydrolysis per se. The Ki values for [3H]mesulergine displacement in choroid plexus were in accordance with phosphoinositide hydrolysis data. In conclusion, this study demonstrates that clozapine and one of its major metabolites in man, N-desmethylclozapine, are potent 5-HT1C receptor antagonists. These properties of clozapine and N-desmethylclozapine should be considered when the atypical effects of clozapine are evaluated in vivo.

Topics: Animals; Choroid Plexus; Clozapine; In Vitro Techniques; Male; Methysergide; Phosphatidylinositols; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; Receptors, Dopamine D2; Serotonin Antagonists

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C18 reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C18 ODS Hypersil analytical column (5 microns particle size; 250 mm x 4.6 mm I.D.) using an acetonitrile-water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N',N'-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75-400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.

Topics: Chromatography, High Pressure Liquid; Clozapine; Humans; Reproducibility of Results; Spectrophotometry, Ultraviolet