Contributions of five human cytochrome P450 isoforms to the N-demethylation of clozapine in vitro at low and high concentrations.

The authors assessed the in vitro contribution of cytochrome P450 (CYP) isoforms 1A2, 3A4, 2C9, 2C19, and 2D6 to the N-demethylation of clozapine mediated by human liver microsomal preparations (HLM). In contrast to previous studies, the authors focused on a relatively low hepatic concentration level, 5 microM, to assess the conditions at a therapeutically relevant hepatic concentration level of clozapine. The optimal concentrations of specific inhibitors were initially established using cDNA-expressed CYP isoforms. The mean contributions of CYPs 1A2, 2C19, 3A4, 2C9, and 2D6 amounted to 30%, 24%, 22%, 12%, and 6%, respectively, with regard to the total HLM-mediated N-demethylation. Thus, the present in vitro study on clozapine N-demethylation suggests that CYP1A2 is the most important form at low concentrations, which is in agreement with clinical findings. CYP2C19 is also of considerable importance, while the roles of CYP2C9 and 2D6 are more modest. CYP3A4 attained a dominating role with an average contribution of 37% at a high clozapine concentration (50 microM). The rate of other metabolic routes mediated by CYP2D6 only corresponded to about one fifth of the CYP2D6 catalyzed N-demethylation rate.

Fully automated on-line determination of olanzapine in serum for routine therapeutic drug monitoring.

A method is presented for unattended fully automated extraction and on-line determination of the atypical antipsychotic drug olanzapine in serum. An ASPEC automatic sample-preparing apparatus with Isolute cyanopropyl-bonded silicagel cartridges was used for solid-phase extraction of the drugs from serum. The adsorbed drugs were eluted with methanol and an aliquot injected into a high-performance liquid chromatograph (HPLC) apparatus. Trifluoperazine was used as internal standard, and the analytes were separated on an unmodified silicagel column using methanol-ammonium acetate buffer pH 9.9 (85:15) as mobile phase. Ultraviolet detection at 257 nm was used for quantitation. Within the therapeutic range for the serum concentration of olanzapine, the interday variations for the quantitative determinations were <8%. Comparisons between concentrations measured using liquid-liquid extraction and the present on-line extraction method showed good agreement. Other drugs often used in combination with olanzapine did not interfere with the quantitative determinations. The method has been in routine use for more than 1 year for therapeutic drug monitoring.

Determination of nortriptyline in human serum by fully automated solid-phase extraction and on-line high-performance liquid chromatography in the presence of antipsychotic drugs.

A fully automated on-line method for determination of nortriptyline in human serum was developed using an ASPEC XL (Gilson) solid-phase extraction apparatus in combination with high-performance liquid chromatography. Solid phase extraction was performed on cyanopropyl cartridges. HPLC was carried out using a C18 column with a mobile phase of acetonitrile-0.01 M triethylamine (34:66 v/v) buffer, pH 3.0. UV detection was at 242 nm. The Inter-day CV% was <5%. Comparison with liquid-liquid extraction of serum from patients treated with nortriptyline showed good agreement. Studies of analytical interference from coadministered psychoactive drugs revealed that only imipramine and a methotrimeprazine metabolite interfered.

[Monitoring of serum olanzapine during antipsychotic treatment]. / Serummonitorering af olanzapin under antipsykotisk behandling.

Olanzapine steady-state serum concentrations of 334 patients were measured by high pressure liquid chromatography. The patients were subjected to routine therapeutic drug monitoring of antipsychotic treatment. In cases with more than one serum determination per patient, only the first measurement was included. Eighty-four percent of the patients received daily doses in the recommended range from 5 to 20 mg. The interval 25-140 nmol/l covered about 80% of the concentrations observed for the daily dose range 5-20 mg. This interval may be used as a reference for therapeutic drug monitoring of olanzapine, indicating whether a patient has a typical or an atypical concentration level at a given dose. Such a judgment may be helpful in situations characterized by side effects, insufficient effect, doubts about compliance or possible pharmacokinetic interactions.

Citalopram and breast-feeding: serum concentration and side effects in the infant.

BACKGROUND: During treatment of postpartum depression with antidepressant drugs, the mothers often strongly wish to continue breast-feeding although the long-term safety of exposing infants to low doses of antidepressants has not been established. METHODS: Citalopram in breast milk and in the serum of a nursing mother and her infant was determined by high-performance liquid chromatography. RESULTS: During treatment with 40 mg/day of citalopram, the concentration of the drug in milk and serum was 205 ng/mL and 98.9 ng/mL, respectively. Her infant obtained 12.7 ng/mL of citalopram in serum and uneasy sleep was observed. Sleep was normalized when the dose was halved and two breast-feedings were replaced with artificial nutrition. CONCLUSION: The amount of citalopram and other selective serotonin inhibitors (SSRIs) passed to breast milk and delivered to the child correlates to the serum concentration of the mother. The lowest possible effective serum concentration should be used and breast-feeding during the drug absorption phase may be avoided.

Fluvoxamine-Clozapine drug interaction: inhibition in vitro of five cytochrome P450 isoforms involved in clozapine metabolism.

Administration of fluvoxamine to patients receiving clozapine therapy may increase the steady-state serum concentrations of clozapine by a factor of 5 to 10. The authors undertook in vitro studies to disclose the mechanism behind this clinically important interaction. In a human liver microsome preparation, fluvoxamine showed a concentration-dependent inhibition of clozapine N-demethylation. Fluvoxamine was much less effective as an inhibitor of clozapine N-oxidation. Fluvoxamine also inhibited in a concentration-dependent manner the activity of all five cytochrome P450 (CYP) isoforms previously determined to be capable of catalyzing the demethylation of clozapine. Fluvoxamine inhibited CYP1A2 and 2C19 with the highest affinities (Ki values of 0.041 and 0.087 microM, respectively). The Ki values for CYP2C9 and 2D6 were 2.2 and 4.9 microM, respectively, whereas the Ki for CY3A4 was 24 microM. Assuming a hepatic tissue concentration of fluvoxamine in the range of 4 to 7 microM under therapeutic conditions, a clinically significant inhibition of all but CYP3A4 is expected in relation to clozapine N-demethylation. No significant effect of fluvoxamine on clozapine N-oxidation is to be expected under therapeutic conditions. Because of the large interindividual variability of the quantity of the various CYP isoforms in liver tissue, it is not possible to predict the fluvoxamine-induced increase in the plasma concentration of clozapine of an individual patient.

Identification of the human cytochrome P450 isoforms mediating in vitro N-dealkylation of perphenazine.

AIMS: To identify the human cytochrome P450 (CYP) isoforms mediating the N-dealkylation of the antipsychotic drug perphenazine in vitro and estimate the relative contributions of the CYP isoforms involved. METHODS: cDNA-expressed CYP isoforms were used to identify the isoforms that are able to mediate the N-dealkylation of perphenazine, which is considered a major metabolic pathway for the drug. Using human liver microsomal preparations (HLM), inhibition studies were carried out to establish the relative contributions of the CYP isoforms involved in the N-dealkylation reaction. RESULTS: CYP isoforms 1A2, 3A4, 2C8, 2C9, 2C18, 2C19 and 2D6 were able to mediate the N-dealkylation of perphenazine. Reaction velocities and their relative abundance in HLM suggested that CYP1A2, 3A4, 2C19 and 2D6 were the most important contributors to N-dealkylation. Apparent Km values of CYP1A2 and CYP2D6 were in the range 1-2 microM, and Km values of CYP2C19 and CYP3A4 were 14 microM and 7.9 microM, respectively. Ketoconazole inhibition of N-dealkylation mediated by a mixed HLM indicated that CYP3A4 accounted for about 40% of perphenazine N-dealkylation at therapeutically relevant concentrations. The contribution of the CYP isoforms 1A2, 2C19 and 2D6 amounted to 20-25% each as measured by the percentage inhibition obtained by addition of furafylline, fluvoxamine or quinidine, respectively. HLM-mediated N-dealkylation of perphenazine accounted for 57% of the total amount of substrate consumed during incubation. CONCLUSIONS: The present in vitro study suggests that CYP isoforms 1A2, 3A4, 2C19 and 2CD6 are primarily involved in the N-dealkylation of perphenazine. The relatively modest role of CYP2D6 is at variance with in vivo studies, which indicate a greater contribution of this isoform. Alternative metabolic pathways, corresponding to 43% of the HLM-mediated metabolism of the drug, may depend more strongly on CYP2D6.

Studies on the stereoselective metabolism of citalopram by human liver microsomes and cDNA-expressed cytochrome P450 enzymes.

The involvement of CYP enzymes in the metabolism of citalopram was studied, inclusive the conversion of demethylcitalopram to didemethylcitalopram and the formation of citalopram N-oxide, which both have not been considered previously. Using human mixed liver microsomes and cDNA-expressed CYP enzymes, we confirmed that CYP3A4, 2C19 and 2D6 are involved in the first demethylation step of citalopram, all favouring conversion of the biologically active S-enantiomer. Inhibitor studies indicated that at therapeutic citalopram concentrations CYP3A4 was responsible for 40-50% of demethylcitalopram formation, while the contribution of CYP2C19 increased and that of CYP2D6 tended to decrease with increasing drug concentration. CYP2D6 exclusively mediated the second demethylation step, and citalopram N-oxide was also exclusively formed by CYP2D6. None of the studied CYP enzymes mediated deamination to the propionic acid derivative.

Olanzapine serum concentrations in psychiatric patients given standard doses: the influence of comedication.

We recorded steady state serum concentrations of olanzapine in 56 psychiatric patients under routine conditions. Twenty-two patients (39%) underwent monotherapy; the rest received additional psychotropic drugs. Doses were given once daily in the evening, and serum olanzapine levels were measured 12 hours later. For the whole group, the concentration-to-dose ratio (C/D) varied 26-fold, with a median value of 4.8 (nmol/L)/(mg/24 hours), but 80% of the patients had C/D values within the range 2 to 10 (nmol/L)/(mg/24 hours). All but three patients received standard doses (5-20 mg/24 hours), of whom 80% had serum concentrations of olanzapine within the range 22 to 146 nmol/L. Patients comedicated with potential inhibitors of CYP2D6 and other drugs displayed a median C/D approximately 40% higher than the group undergoing monotherapy. Patients comedicated with carbamazepine had a median C/D 36% lower than that of the monotherapy group. Therefore, the serum concentration range (12-hour values) of 25 to 150 nmol/L can be expected for patients receiving a standard daily dose.

Determination of olanzapine in serum by high-performance liquid chromatography using ultraviolet detection considering the easy oxidability of the compound and the presence of other psychotropic drugs.

A method for determination of the atypical neuroleptic drug olanzapine in serum was developed. After a single-step liquid-liquid extraction, the compound was separated from other constituents on a normal-phase silica gel column using a buffer-methanol mobile phase and measured by UV absorption at 270 nm. Addition of 0.25% ascorbic acid to serum protects olanzapine against oxidation during extraction and stabilizes the easily oxidised compound during storage. Inter-day variation was <8% at serum levels found in olanzapine treated patients. Analytical interference from coadministered psychoactive drugs and their metabolites were studied. Only risperidone, also a relatively newly developed antipsychotic drug, interfered, but the most commonly used antidepressants and traditional antipsychotics and their metabolites did not interfere.

Serum concentrations and side effects in psychiatric patients during risperidone therapy.

Steady state serum concentrations of risperidone and 9-hydroxyrisperidone (9-OH-risperidone), the active moiety, were measured in 42 patients. The concentration-to-dose ratios (C/D) varied by a factor of 20, from 1.8 to 36.8 (nmol/l)/(mg/24 hours), and 90% of the active moiety was constituted of 9-OH-risperidone. No correlation between the serum concentration of the active moiety and the side effects evaluated by the UKU Side Effect Scale was found. The absence of CYP2D6 (poor metabolizers) or the coadministration of drugs other than benzodiazepines increased the ratio between parent compound and metabolite but did not significantly influence the C/D of the total active moiety. A therapeutic range for serum risperidone has not been established, but 6 mg/day is considered the optimum dose for most patients. The authors found that in 90% of 22 patients administered 6 mg/day risperidone, the serum levels were within 50 to 150 nmol/l.

Therapeutic drug monitoring of clozapine treatment. Therapeutic threshold value for serum clozapine concentrations.

It has been suggested that the minimum effective serum clozapine concentration for an acceptable clinical response (threshold value) is about 400 micrograms/L. This article argues against the use of therapeutic drug monitoring (TDM) as a tool to obtain clozapine concentrations of > or = 400 micrograms/L in the individual patient from the start of clozapine treatment. The following arguments are presented: (i) because of a great interindividual variability of the clozapine concentration to dose ratio (C/D) it can be calculated that extremely high daily doses (900 to 1800 mg/day) would be necessary in 15% of patients to obtain a clozapine concentration of 400 micrograms/L; (ii) doses of 200 to 300 mg/day are commonly used in Central Europe. although about 80% of the patients can be expected to have clozapine concentration < 400 micrograms/L; (iii) in a double-blind study, no difference in clinical response was found between patients treated with clozapine in the concentration range of 200 to 300 micrograms/L, and a group with higher clozapine concentrations; (iv) positron emission topography (PET) studies indicate that maximum receptor occupancy is obtained at clozapine concentrations of about 200 micrograms/L and no further receptor occupancy is obtained by increasing clozapine concentrations to > or = 400 micrograms/L; (v) the frequency of both severe clozapine-induced adverse effects (seizure and confusion) and more common adverse effects increases with increasing doses/clozapine concentrations. It is concluded that the antipsychotic effects and adverse effects of clozapine occur over a very broad range of serum concentrations. In most countries the majority of patients are treated with clozapine concentrations well below the 400 micrograms/L suggested as threshold concentration for optimum response. Therefore clinical judgement should always be primarily used for dose adjustments. TDM is useful to follow compliance and to adjust for extreme serum concentrations and revealing drug interactions.

Metabolism of the tricyclic antidepressant amitriptyline by cDNA-expressed human cytochrome P450 enzymes.

The metabolism of amitriptyline was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6 and 2E1. CYP 2C19 was the most important enzyme with regard to the demethylation of amitriptyline, the quantitatively most important metabolic pathway. CYP 1A2, 3A4, 2C9 and CYP 2D6 also participated in the demethylation of amitriptyline. CYP 2D6 was the sole enzyme mediating the hydroxylation of amitriptyline, and (E)-10-OH-amitriptyline was exclusively produced. CYP 2E1 did not metabolize amitriptyline. Concerning the quantitative relations, CYP 2C19 and 2D6 exhibited high affinities with Km values in the range of 5-13 mumol/l, whereas the affinities of 1A2, 3A4 and 2C9 were somewhat lower with Km values ranging from 74 to 92 mumol/l. CYP 2C19 displayed the highest reaction capacity per mole with Vmax equal to 475 mol h-1 (mol CYP)-1. The other enzymes had Vmax values in the range of 90-145 mol h-1 (mol CYP)-1. Allowing for the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that, at therapeutic doses, on average about 60% of the metabolism depended on CYP 2C19. At toxic doses, CYP 2C19 is expected to be saturated, and CYP 3A4 may now play a dominant role in the metabolism.

Simplified high-performance liquid chromatographic method for determination of risperidone and 9-hydroxyrisperidone in serum from patients comedicated with other psychotropic drugs.

A HPLC method was developed for determination of risperidone and its therapeutically active main metabolite 9-hydroxyrisperidone in serum. After a single-step liquid-liquid extraction the analytes were separated on a C18 column and measured by UV detection at 280 nm. Inter-day coefficient of variation was <7% for both compounds at serum levels occurring in patients treated with ordinary doses. Studies of analytical interference showed that the most commonly coadministered antidepressants and benzodiazepines did not interfere. Some conventional low dose neuroleptics and clozapine did interfere, but this is of minor importance, because risperidone is intended as an alternative to these drugs.

Single dose imipramine reduces nocturnal urine output in patients with nocturnal enuresis and nocturnal polyuria.

PURPOSE: We investigated the effect of imipramine on nocturnal urine output in patients with nocturnal enuresis. MATERIALS AND METHODS: There were 15 monosymptomatic enuretic patients 15 to 37 years and 8 control subjects 25 to 32 years old. We measured nocturnal urine output, urine osmolality, creatinine clearance, osmolal clearance, free water clearance, excretion of solutes, fractional excretion of sodium, fractional excretion of potassium and plasma vasopressin with and without a single oral dose of imipramine (1 mg./kg. of body weight) taken at 8 p.m. RESULTS: Baseline studies showed significantly larger and less concentrated nocturnal urine among enuretics compared with controls. We observed a marked antidiuretic effect of imipramine in 6 enuretics with severe nocturnal polyuria. The imipramine induced decrease in urine output was accompanied by reduced osmolal clearance. Approximately a third of the observed decrease in solute excretion was attributed to lower excretion of sodium and potassium. The remaining two-thirds were most likely caused by an increased tubular reabsorption of urea, which may be secondary to a sympathomimetic effect of imipramine tubules, possibly because of altered adrenal medullary function with an increase in proximal tubular sodium and water reabsorption. The resultant lower tubular flow rate facilitates tubular reabsorption of urea in the distal part of the nephron. CONCLUSIONS: Imipramine has a vasopressin independent antidiuretic effect if nocturnal polyuria is present. The antidiuretic effect of imipramine can be attributed primarily to increased alpha-adrenergic stimulation in the proximal tubules with a secondary increased urea and water reabsorption more distally in the nephron.

Hydroxylation and demethylation of the tricyclic antidepressant nortriptyline by cDNA-expressed human cytochrome P-450 isozymes.

The metabolism of nortriptyline was studied in vitro using cDNA-expressed human cytochrome P450 isozymes 1A2, 3A4, 2C19, and 2D6, CYP2D6 was the sole isozyme mediating hydroxylation of nortriptyline, the quantitatively most important metabolic pathway, and only (E)-10-OH-nortriptyline was formed. CYP2D6, 2C19, and 1A2, mentioned in decreasing order of significance, mediated the demethylation reaction of nortriptyline, whereas 3A4 did not participate in the metabolism of nortriptyline. Concerning the quantitative relations, CYP2D6 exhibited a high affinity with respect to hydroxylation and demethylation (K(m) 0.48-0.74 mumol/l), a high hydroxylation capacity (Vmax 130 mol/hr/mol CYP) and a somewhat lower demethylation capacity (Vmax 19 mol/ hr/mol CYP). The affinities of 1A2 and 2C19 were 100-fold lower (K(m) 54-118 mumol/l). The capacity of 1A2 was low (Vmax 6.8 mol/hr/ mol CYP), whereas 2C19 had the highest demethylation capacity (Vmax 93 mol/hr/mol CYP). Taking into account the relative amounts of CYP isozymes present in the liver, about 90% of the metabolism was estimated to depend on CYP2D6, with CYP2C19 and 1A2 mediating the remaining 10%. In subjects lacking the 2D6 isozyme, CYP2C19 and 1A2 are expected to be of major importance for elimination of nortriptyline.

Citalopram and desmethylcitalopram concentrations in breast milk and in serum of mother and infant.

A case is presented with measurements of the selective serotonin reuptake inhibitor (SSRI)-antidepressant citalopram in serum of mother and breast-fed infant and in breast milk. We found a milk-to-serum concentration ratio of approximately 3 for both citalopram and the main metabolite desmethylcitalopram. Peak milk concentrations of citalopram occurred 3-9 h after drug intake by the mother. The infant received approximately 5% of the mother's dose, adjusted for weight. Accordingly, the serum level in the infant of approximately 7 nM corresponded to approximately 1/15 of the trough serum concentration of the mother (104 nM). No signs of drug effects in the infant were observed.

Metabolism of clozapine by cDNA-expressed human cytochrome P450 enzymes.

The metabolism of clozapine was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6, and 2E1. CYP1A2, 3A4, 2C9, 2C19, and 2D6 were able to N-demethylate clozapine. N-Oxide formation was exclusively catalyzed by CYP3A4. CYP2E1 did not metabolize clozapine. With regard to quantitative relationships, CYP1A2, 2C9, 2C19, and 2D6 displayed KM values ranging from 13 to 25 microM, whereas CYP3A4 had a 5-10 times higher KM value. CYP2C19 and 2D6 had the highest Vmax values (149-366 mol/hr/mol CYP). Taking into account the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that at therapeutic concentrations CYP2C19 and CYP3A4 each accounted for about 35% of the metabolism. At toxic concentrations, the relative importance of CYP3A4 increased.

[Serious drug interaction between clozapine-Leponex and fluvoxamine-Fevarin]. / Alvorlig laegemiddelinteraktion mellem clozapin-Leponex og fluvoxamin-Fevarin.

A 67 year-old woman in steady-state treatment with clozapine 150 mg/24 h was co-medicated with 100 mg/24 h of fluvoxamine. During the next months the patient suffered from nausea and occasionally vomited, but these symptoms were ascribed to fluvoxamine, and as she mentally improved, both treatments were continued. Two months after the start of fluvoxamine her serum clozapine concentration was 7570 nmol/l or 7.5 fold higher than before fluvoxamine was added. The woman was admitted to hospital, suffering from abdominal pain, dehydration and fever (38.5 degrees C). Serum creatinine concentration was increased, but normalized during hydration. After 18 days care the woman felt well and was discharged from hospital. The case report shows that certain combinations of selective serotonin reuptake inhibitors and neuroleptic drugs should either be avoided or the serum concentrations of the drugs closely followed.