Genotype and co-medication dependent CYP2D6 metabolic activity: effects on serum concentrations of aripiprazole, haloperidol, risperidone, paliperidone and zuclopenthixol.

PURPOSE: Therapeutic drug monitoring (TDM) of antipsychotics can aid in therapy optimization, explaining adverse effects or non-response. One reason for therapeutic failure or adverse effects is caused by genetic variations in the cytochrome P450 drug-metabolizing genes. The aim of this study was to evaluate the impact of CYP2D6 polymorphisms on steady-state serum concentrations of antipsychotics metabolized by CYP2D6, taking into account the co-medication with CYP2D6 inhibitors. METHODS: Serum and EDTA samples were collected from 82 psychiatric patients. After a liquid-liquid extraction, serum samples were analyzed using an ultra-high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method for quantification of the antipsychotics. CYP2D6 genotyping was performed using the Luminex xTAG® CYP2D6 Kit v3 (Luminex Corporation). Patients were divided into five phenotype subgroups by calculation of the activity score (AS): poor metabolizers (PM; AS 0), intermediate metabolizers (IM; AS 0.5-1), extensive metabolizers with slow activity (EM-s; AS 1-1.5), extensive metabolizers with fast activity (EM-f; AS 2), and ultra-rapid metabolizers (UM; AS >2). The influence of the phenotypes on the concentration-to-dose and metabolite-to-parent ratios was evaluated. RESULTS: Overall, 6.1 % UM (n = 5), 25.6 % EM-f (n = 21), 46.3 % EM-s (n = 38), 1.2 % EM-s/EM-f (n = 1), 6.1 % IM (n = 5), and 14.6 % PM (n = 12) were found, taking co-administration of strong and moderate CYP2D6 inhibitors into account (phenoconversion). It was demonstrated that CYP2D6 polymorphisms affect the serum concentrations of aripiprazole (n = 18), haloperidol (n = 11), risperidone (n = 20), and zuclopenthixol (n = 6), while no influence was seen on the paliperidone serum concentrations (n = 31). CONCLUSIONS: Even with a small number of patients per antipsychotic, the importance of CYP2D6 genotyping was still clearly stated. This study illustrates the high potential of combining TDM and CYP2D6 genotyping in clinical practice.

Simultaneous determination of 25 common pharmaceuticals in whole blood using ultra-performance liquid chromatography-tandem mass spectrometry.

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of 25 common pharmaceuticals in whole blood. The selected pharmaceuticals represent the most frequently detected drugs in our forensic laboratory with basic properties such as analgesics, antidepressants, antihistamines, antihypertensives, antipsychotics and ß-blockers. Whole blood samples were extracted with butyl acetate after adjusting pH with 2M NaOH. The target analytes were separated on a 100 × 2.1 mm ACQUITY BEH 1.7 µm C18 column by a formic acid/acetonitrile gradient elution using a Waters ACQUITY Ultra-Performance Liquid Chromatography system. Quantification was performed on a Waters tandem quadrupole ACQUITY TQD using multiple reaction monitoring in positive mode. The analytes were eluted within 11 min. The limit of quantification (LOQ) ranged from 0.002 to 0.01 mg/kg depending on the analyte. A good linear behavior was achieved for all analytes in the range from LOQ to 1.0 or 2.0 mg/kg blood. The absolute recoveries were between 55-87% for all compounds except norfluoxetine (44%). The method showed acceptable precision and accuracy for almost all analytes. Only unstable compounds like levomepromazine, methylphenidate, mirtazapine, norfluoxetine and zuclopenthixol deviated more. The method was successfully applied to more than 200 authentic blood samples within a year from forensic investigations.

Characterisation of zuclopenthixol metabolism by in vitro and therapeutic drug monitoring studies.

OBJECTIVE: Zuclopenthixol pharmacokinetics is incompletely characterised. We investigated potential interactions mediated through cytochrome P450 enzymes. METHOD: In vitro, we examined the impact of CYP2D6 and CYP3A4 inhibitors on zuclopenthixol metabolism in microsomes from six human livers. Subsequently, we compared dose-corrected serum zuclopenthixol concentrations in 923 samples from a therapeutic drug monitoring database from patients prescribed oral (n = 490) or injected (n = 423) zuclopenthixol alone or with fluoxetine, paroxetine, levomepromazine or carbamazepine. RESULTS: In vitro fluoxetine, paroxetine, ketoconazole and quinidine all significantly inhibited zuclopenthixol metabolism. Ketoconazole and quinidine together abolished zuclopenthixol disappearance. Clinically, dose-corrected oral zuclopenthixol serum concentrations increased significantly, after adjustment, by 93%, 78% and 46% during co-treatment with fluoxetine, paroxetine and levomepromazine and decreased 67% with carbamazepine. Carbamazepine caused dose-dependent reductions in the oral zuclopenthixol concentration-dose ratio (P < 0.001), fluoxetine (P < 0.001) and paroxetine (P = 0.011) dose-dependent increases and levomepromazine an increase related to its serum concentration (P < 0.001). Results for injected zuclopenthixol were similar but not all reached statistical significance. CONCLUSION: The In vitro study suggests zuclopenthixol is metabolised primarily by CYP2D6 and CYP3A4. The clinical study supports this, demonstrating the impact of co-prescribed inhibitors or inducers. Guidelines should incorporate these interactions noting the potential for zuclopenthixol-related toxicity or treatment failure.

Liquid chromatographic analysis of the cis(Z)- and trans(E)-isomers of clopenthixol in human plasma using a novel solid phase extraction procedure.

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in human plasma has been developed. The chromatographic analysis was carried out isocratically on a reversed-phase column (C(8) 150 x 4.6 mm I.D., 5 microm) using a mixture of 25 mM phosphate buffer and acetonitrile (65:35 v/v, pH* 3.0) as the mobile phase, and ultraviolet detection at 230 nm. Plasma sample pretreatment was accomplished by means of an original solid-phase extraction (SPE) procedure carried out on cyanopropyl cartridges, with a high extraction yield and good selectivity. Under the optimum conditions, calibration graphs of spiked human plasma samples were obtained over the concentration ranges 1-300 ng ml(-1) for cis(Z)-clopenthixol and 1-200 ng ml(-1) for trans(E)-clopenthixol. The limit of detection (LOD) was 0.3 ng ml(-1) for both cis(Z)- and trans(E)-isomers of clopenthixol. The method was successfully applied to the determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in plasma samples of schizophrenic patients undergoing therapy with zuclopenthixol.

Maintenance therapy with zuclopenthixol decanoate: associations between plasma concentrations, neurological side effects and CYP2D6 genotype.

RATIONALE: Several antipsychotic drugs are metabolised by the polymorphic cytochrome P(450) CYP2D6. The impact of the polymorphism on the plasma levels and the occurrence of side effects have not been clearly established. OBJECTIVE: To investigate the impact of the CYP2D6 polymorphism on the steady-state plasma concentrations of zuclopenthixol and the occurrence of extrapyramidal side effects (EPS) and tardive dyskinesia (TD) during treatment with zuclopenthixol-decanoate. METHODS: Fifty-two clinically stable schizophrenic outpatients on monotherapy with zuclopenthixol-decanoate (100-400 mg/4 weeks) were genotyped for the CYP2D6 variants CYP2D6*3 and CYP2D6*4. Steady-state plasma levels of zuclopenthixol were analysed using high-performance liquid chromatography. Assessments of EPS, TD and psychopathology were performed twice with an 8-week interval using the extrapyramidal symptoms rating scale, the abnormal involuntary movement scale and the brief psychiatric rating scale. RESULTS: Thirty-five patients were homozygous extensive metabolisers (EMs), 13 were heterozygous EMs and 4 were poor metabolisers (PMs). While there were no significant genotype-related differences in the doses of zuclopenthixol decanoate, PMs as well as heterozygous EMs had significantly higher steady-state plasma levels of zuclopenthixol than homozygous EMs (median 9.5 nmol/l; 8.2 nmol/l and 5.9 nmol/l, respectively, P<0.05). The median dose-corrected plasma concentrations were 0.029, 0.038 and 0.048 nmol.l(-1).mg(-1) in homozygous EMs, heterozygous EMs and PMs, respectively, with statistically significant differences between homozygous EMs and heterozygous EMs ( P=0.014) and homozygous EMs and PMs ( P=0.03). Patients with neurological side effects were significantly older than patients without ( P=0.02 in case of parkinsonism and P=0.04 in case of TD). Mutant CYP2D6*3 and *4 alleles tended to occur more frequently in patients with neurological side effects. An odds ratio (OR) of 2.3 (95% confidence interval 0.7-6.9) for development of parkinsonism and an OR of 1.7 (95% confidence interval 0.5-4.9) for TD was calculated in an individual with at least one mutated allele. However, the ORs were not statistically significant. CONCLUSIONS: The higher zuclopenthixol steady-state plasma concentrations in heterozygous EM and PM schizophrenic patients receiving monotherapy with zuclopenthixol-decanoate than in homozygous EMs indicates a significant role of CYP2D6 in the systemic elimination of zuclopenthixol. The tendencies for patients carrying at least one mutated CYP2D6 gene to have an increased risk of parkinsonism and TD are in accordance with previous studies. Age was a significant risk factor for neurological side effects.

HPLC-ESI-MS/MS determination of zuclopenthixol in a fatal intoxication during psychiatric therapy.

The first non-suicidal fatality due to intramuscular administration of Cisordinol (zuclopenthixol, ZPT) is described. A new, rapid, and sensitive method for the determination of ZPT in postmortem specimens has been developed. High performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was employed for drug confirmation and quantitation. Sample clean up was performed using a simple liquid-liquid extraction procedure. The postmortem concentration of ZPT in heart blood was 0.68 microg/ml. Furthermore, zotepine, carbamazepine, and chlorprotixene were detected in body fluids. The proposed method enables the unambiguous identification and quantitation of ZPT and other neuroleptic drugs in clinical and forensic specimens.

Concentrations of cis(Z)-clopenthixol and trans(E)-clopenthixol in a lethal case involving zuclopenthixol, diazepam, and cyamemazine.

cis(Z)-Clopenthixol and trans(E)-clopenthixol were determined by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode-array detection in necropic samples from a postmortem case. The peripheral blood concentrations of cis(Z)-clopenthixol and trans(E)-clopenthixol were 278 and 177 ng/mL, respectively. The level of the active cis(Z)-isomer is within the toxic range. Other associated drugs' concentrations were within their therapeutic ranges. Postmortem redistribution of the drug and instability of the drug due to trans-isomerization were discussed.

Therapeutic drug monitoring of haloperidol, perphenazine, and zuclopenthixol in serum by a fully automated sequential solid phase extraction followed by high-performance liquid chromatography.

In Denmark, haloperidol, perphenazine, and zuclopenthixol are among the most frequently requested antipsychotics for therapeutic drug monitoring. With the number of requests made at the authors' laboratory, the only rational analysis is one that can measure all three drugs simultaneously. The authors therefore decided to develop an automated high-performance liquid chromatography (HPLC) method. Two milliliters serum, 2.0 mL 10 mmol/L sodium phosphate buffer (pH 5.5), and 150 microL internal standard (trifluoperazine) solution were pipetted into HPLC vials and extracted on an ASPEC XL equipped with 1 mL (50 mg) Isolute C2 (EC) extraction columns and acetonitrile-methanol-ammonium acetate buffer (60:34:6) as extracting solution. Three hundred fifty microliters was analyzed by HPLC; a 150 x 4.6-mm S5CN Spherisorb column with a mobile phase of 10 mmol/L ammonium acetate buffer-methanol (1:9), a flow rate of 0.6-1.7 mL/min, and ultraviolet detection at 256 and 245 nm were used. Reproducibility was 5-12% and the lower limit of quantitation was 10, 1, and 5 nmol/L (4, 0.4, and 2 ng/mL) for haloperidol, perphenazine, and zuclopenthixol, respectively. The method was found to be sufficiently selective and robust for routine analysis.

Flow-injection chemiluminometric determination of some thioxanthene derivatives in pharmaceutical formulations and biological fluids using the [Ru(dipy)3(2+)]-Ce(IV) system.

A flow-injection (FI) methodology using tris(2,2'-dipyridyl)ruthenium(II), [Ru(dipy)3(2+)], chemiluminescence (CL) was developed for the rapid and sensitive determination of three thioxanthene derivatives, namely zuclopenthixol hydrochloride, flupentixol hydrochloride and thiothixene. The method is based on the CL reaction of the studied thioxanthenes with [Ru(dipy)3(2+)] and Ce(IV) in a sulfuric acid medium. Under the optimum conditions, calibration graphs were obtained over the concentration ranges 0.002-6 migrograms/ml for zuclopenthixol hydrochloride, 0.5-15 micrograms/ml for flupentixol hydrochloride and 0.05-7.5 micrograms/ml for thiothixene. The limits of detection (s/n = 3) were 4.2 x 10(-9) mol/l zuclopenthixol hydrochloride, 2 x 10(-8) mol/l flupentixol hydrochloride and 4.5 x 10(-8) mol/l thiothixene. The method was successfully applied to the determination of these compounds in dosage forms and biological fluids.

Influence of Cyp2D6 genetic polymorphism on ratios of steady-state serum concentration to dose of the neuroleptic zuclopenthixol.

One hundred and nineteen psychiatric patients undergoing therapeutic drug monitoring (TDM) of the neuroleptic zuclopenthixol were genotyped with regard to Cyp2D6. Twelve patients (10.1%) were of the poor metabolizer genotype. The extensive metabolizers comprised 58 patients receiving no potentially interacting drugs and 38 patients concomitantly treated with other drugs competing for metabolism by Cyp2D6. Information on the rest (11 patients) was missing. The median steady-state serum concentration-to-dose ratio (C/D) of the PM group (2.00 nmol/L/mg) was close to that of the EM group receiving potentially interacting drugs (1.80) and approximately 60% higher than that of the remaining EM group (1.25) (p < 0.01). When judging the clinical importance of this difference, the total group variability in C/D of nearly 10-fold should be kept in mind (0.5-4.2 nmol/L/mg). In terms of serum concentrations not corrected for dose, the three groups had about similar levels, with median values from 16 to 21 nmol/L. We consider that TDM adequately takes into account dose adjustments for both EM and PM subjects in the context of this neuroleptic.

Central D2 receptor occupancy and effects of zuclopenthixol acetate in humans.

Repeated positron emission tomography (PET) measurements of D2 receptor occupancy, plasma concentrations of zuclopenthixol and reaction time were performed in three healthy subjects after injection of 12.5 mg zuclopenthixol acetate (ZPTA) in an open study design. Five control subjects were examined for reaction time only. D2 receptor occupancy was 51%, 71% and 75% after 7 h and 75%, 83% and 87% after 31 h in the three subjects. The subjects reported sedation, but reaction time was not prolonged. After the low dose of 12.5 mg ZPTA, D2 receptor occupancy exceeded the 70% assumed to be required to induce antipsychotic effect. Extrapolation of data to a clinical dose interval indicates that 50-150 mg ZPTA should induce very high D2 receptor occupancy lasting several days after injection. Such high doses may be required to induce sedation and to avoid frequent intramuscular injections in acutely psychotic patients. However, the simultaneously induced very high D2 receptor occupancy calls for careful assessment of acute extrapyramidal symptoms.

Search for a therapeutic range for serum zuclopenthixol concentrations in schizophrenic patients.

The aim of our study was to find a therapeutic range for the serum concentration of zuclopenthixol (S-Zu) in chronic schizophrenic patients. S-Zu was measured in 17 patients and dosage reduction was suggested by the laboratory if S-Zu exceeded 15 nmol/L. The clinical symptoms and side effects were evaluated blindly using the Brief Psychiatric Rating Scale (BPRS) and the UKU rating scale, respectively. S-Zu and ratings were repeated 6, 12, and 24 weeks after the final dosage adjustments. In 7 of 10 patients with S-Zu > 15 nmol/L the dosage was reduced by 20-67%. After dosage reduction the S-Zu was below 15 nmol/L in 10 of the 17 patients. The mean BPRS score and the side effects, evaluated by the UKU scale, were reduced in patients in whom the dosage was reduced. It is suggested that S-Zu in the range 5-15 nmol/L may serve as a preliminary therapeutic range for S-Zu.

Determination of zuclopenthixol and its main N-dealkylated metabolite in biological fluids using high-performance liquid chromatography with post-column photochemical derivatization and fluorescence detection.

A highly sensitive high-performance liquid chromatographic (HPLC) method for the assay of cis-(Z)-clopenthixol (zuclopenthixol) in urine and plasma has been developed. Following solid-phase extraction, the samples are chromatographed using reversed-phase ion-pairing HPLC. After separation, the solutes, having a thioxanthene structure, are transformed on-line into thioxanthones in a photochemical reactor. The thioxanthones are highly fluorescent compounds, and therefore, low detection limits are obtained when using fluorescence detection. Detection limits for zuclopenthixol and its N-dealkylated metabolite, in plasma as well as in urine, using fluorescence detection with excitation at 260 nm and emission at 435 nm, were found to be 0.05 ng/ml and 0.2 ng/ml, respectively. The chromatographic system separates the cis-(Z)- and trans-(E)-isomers of clopenthixol from its main dealkylated metabolite. Furthermore, the chromatographic system is very suitable for study of the photochemical reaction, since the chloro-thioxanthone and thioxanthone are well separated from the isomers of clopenthixol.

Zuclopenthixol decanoate in maintenance treatment of schizophrenic outpatients. Minimum effective dose and corresponding serum levels.

23 schizophrenic outpatients in maintenance treatment with zuclopenthixol decanoate were included in a study aimed at finding the minimum effective dose and corresponding serum concentration of zuclopenthixol. Every three months the dose was gradually reduced until prodromal symptoms appeared, indicative of an incipient relapse. A slightly higher dose was then promptly reinstituted (the minimum effective dose). At each dose level, two blood samples were drawn to determine the serum concentration. This dose reduction principles proved feasible. Only one patient had a clear-cut relapse. The condition of the remaining patients was acceptably maintained by a dose increase. The minimum effective dose of zuclopenthixol was 200 mg/2 weeks (range 60-400), with a serum concentration of 22 nmol/l (7.1-69.7). There was a significant correlation between the administered dose and the corresponding serum level of the drug (r = 0.66, P < 0.01). A trend towards a positive correlation was found between the serum level at the minimum effective dose and the BPRS score (r = 0.42, P < 0.1). No correlation was found between the serum level and the side-effects or length of neuroleptic treatment. It is concluded that routine serum drug monitoring is hardly indicated in the long-term depot-neuroleptic treatment of schizophrenic patients. A strategy aiming at continually seeking the lowest effective dose on the basis of clinical parameters appears more appropriate, especially in case of depot-neuroleptic treatment.

Fluctuation of serum zuclopenthixol concentrations in patients treated with zuclopenthixol decanoate in viscoleo.

Zuclopenthixol serum concentrations were measured in 58 psychiatric patients referred for routine therapeutic drug monitoring (TDM). Patients were treated for prolonged time with zuclopenthixol decanoate in viscoleo in doses of 50-500 mg, administered intramuscularly at 14-day intervals. The serum concentration was determined at days 7 (C7) and 14 (C14) following injection. The mean ratio C7/C14 was 2.0 and was independent of the dosage given. In 14 patients, additional blood samples were drawn at day 3 (C3) following injection. The mean ratio C3/C14 of this group was 3.2. An almost log-linear decline of the serum concentration from day 3 to 14 appeared, which corresponds to an apparent half-life of zuclopenthixol in this dosage form of 7.4 days. The marked fluctuations of serum concentrations of zuclopenthixol from peak to trough levels in patients given fortnightly injections of the depot preparation indicate that shorter intervals between injections should be considered in many cases in order to diminish side effects.

[The effect of serum monitoring on concentration of perphenazine and zuclopenthixole]. / Effekten af serumkontrol på koncentrationen af perfenazin og zuklopentixol.

The effect of therapeutic drug monitoring (TDM), including pharmacokinetic guidance, was examined in 994 psychiatric patients treated with perphenazine (Trilafon) or zuclopentixol (Cisordinol). Before monitoring of the serum level, half of the serum concentrations from patients given perphenazine tablets (466 patients) was below the therapeutic level, and about one third was above. For perphenazine depot (208 patients), almost no patients had a serum concentration below the therapeutic level whereas 40% had concentrations above. For zuclopentixol tablets (231 patienter) and zuclopentixol depot (163 patients), about 60% of the patients had concentrations above the therapeutic level. For all four groups, it was found that slightly more than half of the patients with serum concentrations outside the therapeutic level was reexamined. The dosage was changed in most of these patients (80-90%) in order to bring the concentration within the therapeutic level. Half of those in whom the dose was changed obtained concentrations within the therapeutic level. It is concluded that since the recommendations for changes of the dosage is followed to a wide extent, the therapeutic drug monitoring service does influence the treatment significantly.

Clinical evaluation and serum concentration of zuclopenthixol acetate in psychotic Asian patients: a single-dose preliminary study.

Nineteen acutely disturbed psychotic Asian patients were treated with a single intramuscular injection of 50 mg of zuclopenthixol acetate in Viscoleo. Patients were assessed clinically before and after treatment using the Brief Psychiatric Rating Scale (BPRS). Serum zuclopenthixol and the inactive geometric isomer trans(E)-clopenthixol were determined by high-performance liquid chromatography after intramuscular injection. All patients improved, with the BPRS being significantly reduced (p < 0.001) at 72 h after injection. Adverse effects were generally few. The mean +/- SEM serum zuclopenthixol concentrations at 24, 48, and 72 h were 19.9 +/- 2.8, 31.5 +/- 4.5, and 17.8 +/- 2.9 micrograms/L, respectively. trans(E)-Clopenthixol concentrations ranged from negligible to 39.5 micrograms/L. This study confirms that a single intramuscular injection of 50 mg is adequate for managing severely disturbed psychotic patients for the first 3 days. The serum zuclopenthixol concentrations attained in the Asian patients were higher than those reported in Caucasian psychiatric patients. In some patients, a considerable amount of zuclopenthixol had been transformed to trans(E)-clopenthixol.

Disposition of the neuroleptic zuclopenthixol cosegregates with the polymorphic hydroxylation of debrisoquine in humans.

The pharmacokinetics of a single oral dose of the neuroleptic drug zuclopenthixol (10 or 6 mg) was studied in 6 extensive and 6 poor metabolizers of debrisoquine. The peak plasma concentrations of zuclopenthixol did not differ between the phenotypes, whereas the plasma elimination half-life was significantly longer in poor than in extensive metabolizers (29.9 +/- 6.6 vs 17.6 +/- 6.9 h). Accordingly, the total oral plasma clearance was lower in poor than in extensive metabolizers (0.78 +/- 0.27 vs 2.12 +/- 0.65 1/h/kg). Ten of the volunteers had previously participated in a similar study in which the kinetics of perphenazine, another neuroleptic drug, were studied in poor and in extensive metabolizers of debrisoquine. There was a significant correlation between the oral clearance of perphenazine and that of zuclopenthixol among these 10 subjects. The study indicates that the disposition of zuclopenthixol, as well as that of perphenazine, is related to the genetically determined capacity to hydroxylate debrisoquine. The significance of this polymorphism for the clinical use of neuroleptics is discussed.

Milk concentrations of flupenthixol, nortriptyline and zuclopenthixol and between-breast differences in two patients.

Flupenthixol (FP), nortriptyline (NT) and zuclopenthixol, (ZCP) were determined in breast milk and plasma from 2 puerperal, lactating women with psychiatric disorders. The milk concentrations were equal to, higher and lower than those in plasma for FP, NT and ZCP, respectively. Variation in milk triglyceride concentration, but not milk pH, could partly explain between-breast differences in the milk concentrations. The study demonstrates the need for appropriate and representative milk sampling procedures. The estimated daily infant exposure averaged 0.5, 2.3 and 0.3% of the corresponding maternal weight related doses of FP, NT and ZCP. FP was also detectable in infant plasma. These drugs are not known to be harmful in small doses to breast-fed infants. However, concern about the effect of dopamine blocking agents on neurobehavioral mechanisms in animals warrants caution. If neuroleptics are required for a long period this risk must be weighed against the benefits of breast-feeding, also considering the psychological effects of the latter.