Clozapine Metabolism Research Articles

Reconstitution of the interplay between cytochrome P450 and human glutathione S-transferases in clozapine metabolism in yeast

Clozapine, an often-prescribed antipsychotic drug, is implicated in severe adverse drug reactions (ADRs). Formation of reactive intermediates by cytochrome P450s (CYPs) has been proposed as a possible explanation for these ADRs. Moreover, a protective role for human glutathione S-transferases (hGSTs) was recently shown using purified enzymes. We investigated the interplay between CYP bioactivation and GST detoxification in a reconstituted cellular context using recombinant yeast expressing a bacterial CYP BM3 mutant (M11), mimicking the drug-metabolizing potential of human CYPs, combined with hGSTA1-1, M1-1 or P1-1. Clozapine and the N-desmethylclozapine metabolite caused comparable growth inhibition and reactive oxygen species (ROS) formation, whereas the clozapine-N-oxide metabolite was clearly less toxic. Clozapine metabolism by BM3 M11 and the hGSTs in yeast was confirmed by identification of stable clozapine metabolites and hGST isoform-specific glutathione-conjugates. Oxidative metabolism of clozapine by BM3 M11 increased ROS formation and growth inhibition. Co-expression of hGSTP1-1 protected yeast from BM3 M11 induced growth inhibition in presence of clozapine, whereas similar expression levels of hGSTA1-1 and hGSTM1-1 did not. ROS formation was not lowered by hGSTP1-1 co-expression and was unrelated to mitochondrial electron transport chain (mETC) activity. We present a novel cellular model to study the effect of CYP and GST interplay in drug toxicity.

Classics in Chemical Neuroscience: Clozapine

Clozapine was the first true breakthrough in schizophrenia treatment since the discovery of chlorpromazine in 1950, effectively treating positive, negative, and some cognitive symptoms, as well as possessing unprecedented efficacy in treatment-resistant patients. Despite over 30 years of intense study, the precise molecular underpinnings that account for clozapine's unique efficacy remain elusive. In this Viewpoint, we will showcase the history and importance of clozapine to neuroscience in general, as well as for the treatment of schizophrenia, and review the synthesis, pharmacology, drug metabolism, and adverse events of clozapine.

Increasing the clozapine: Norclozapine ratio with co-administration of fluvoxamine to enhance efficacy and minimize side effects of clozapine therapy

Although clozapine is the only antipsychotic agent to have demonstrated superior efficacy in treatment-refractory schizophrenia, one- to two-thirds of patients do not respond adequately despite acceptable dosages and plasma levels. Moreover, a significant number of patients stop the therapy for various reasons, including its side effects, many of which are thought to be related to its active metabolite, norclozapine. However, combining clozapine with the SSRI antidepressant fluvoxamine decreases norclozapine formation by inhibiting the CYP450 1A2 isoenzyme. Lowering norclozapine levels in this way while maintaining therapeutic clozapine levels increases the clozapine: norclozapine ratio; the potential benefits include both a reduction of such side effects as sedation, weight gain, metabolic disturbances, and neutropenia, and an increase in efficacy. The optimal ratio of clozapine to norclozapine has not yet been defined, but a ratio of two or more implies that saturation of clozapine metabolism has been reached. We hypothesize that co-administration of clozapine and fluvoxamine at dosages that will produce therapeutic plasma levels of clozapine and a clozapine: norclozapine ratio of two or more will increase efficacy and tolerability of clozapine therapy in treatment-resistant schizophrenic patients.

Association between CYP1A2 gene single nucleotide polymorphisms and clinical responses to clozapine in patients with treatment-resistant schizophrenia.

Despite clozapine's superior clinical efficacy in treatment-resistant schizophrenia (TRS), its adverse effects, need for periodic leukocyte monitoring, cost and variable clinical outcomes mandate a clinical need to predict its treatment response. Although cytochrome P450 1A2 (CYP1A2) is the principal determinant of metabolism of clozapine, the role of CYP1A2 gene in the clinical response to clozapine is uncertain. Hence, we investigated its association with treatment responses and adverse events of clozapine in TRS. We evaluated four single nucleotide polymorphisms (SNP) in the CYP1A2 gene, clinical responses and serum clozapine levels in 101 consecutive patients with TRS on stable doses of clozapine. We defined clozapine response a priori and investigated allelic and genotypic associations. We assessed the socio-demographic and clinical profiles, premorbid adjustment, traumatic life events, cognition and disability of the participants, using standard assessment schedules for appropriate multivariate analyses. Our results revealed that CYP1A2 gene SNP (*1C, *1D, *1E and *1F) were not associated with clozapine treatment response, adverse effects, serum clozapine levels or with disability (p values > 0.10). As CYP1A2 gene SNP do not help to predict the clinical response to clozapine, routine screening for them prior to start clozapine is currently unwarranted. We suggest future longitudinal genome-wide association studies investigating clinical and pharmacogenetic variables together.

A Puzzling Case of Increased Serum Clozapine Levels in a Patient With Inflammation and Infection

A 23-year old male with a history of schizophrenia treated with clozapine 900 mg/d was admitted to the hospital for a gastrointestinal infection. The trough serum concentration of clozapine at admission was 9074 nmole/L, that is, almost 4-fold the upper limit of the reference range. The patients did not report any adverse effects of clozapine. The clozapine concentration 1 month earlier had been 1919 nmole/L, which is well within the reference range. There seems to be 2 different mechanisms explaining the increase in clozapine levels in this patient. First, a downregulation of CYP enzyme activities, which primarily seems to be mediated by interleukin-6, takes place during infection and inflammation. Second, the concentration of the acute phase protein α1-acid glycoprotein (AGP; orosomucoid) increases during infection and inflammation. As approximately 95% of clozapine is bound to AGP, the concentration of clozapine will increase in parallel with the increase in AGP. A therapeutic drug monitoring analysis measures the total drug concentration (ie, the concentration of unbound plus plasma protein bound drug), whereas the concentration of free drug exerts its pharmacological effects. Thus, this second mechanism will, in contrast to the first mechanism, not affect the clinical effect of clozapine. Although the patient was also treated with ciprofloxacin, which has been reported to inhibit the metabolism of clozapine, the clozapine levels did not further increase. This case illustrates the complex interrelationship between serum levels of clozapine and an intercurrent infection treated with potentially interacting antibiotics.

Interaction of St John’s wort (Hypericum perforatum) with clozapine

St John's wort (Hypericum perforatum) is notorious for its ability to induce the enzymes of the P450 system. Especially, it induces CYP1A2 and CYP3A4, enzymes that are closely involved in the metabolism of clozapine. We present a patient with schizophrenia, who was stable on a fixed dose with stable plasma level of clozapine, and who deteriorated after she started self-medicating with St John's wort. The reduced plasma clozapine level and the psychiatric condition normalized after the withdrawal of St John's wort. It is possible that, beside the induction of P450-enzymes, the induction of P-glycoprotein by St John's wort aggravated psychiatric deterioration of the patient. Physicians should be alert to patients self-medicating with over-the-counter medicines, especially when these medicines can lower clozapine concentrations below the therapeutic range.

Case Report: The clinical implications of clozapine and cigarette smoking

INTRODUCTION Cigarette smoking is prevalent among psychiatric patients, with rates several times higher than the general population. As an example, up to 80% of schizophrenic patients smoke cigarettes. Clozapine is extensively metabolized by cytochrome P450 isoenzyme 1A2 and polycyclic aromatic hydrocarbons (PAHs) present in cigarette smoke are thought to induce CYP 1A2. Therefore concurrent smoking with clozapine can potentially lead to decreased clozapine levels and decreased efficacy, while smoking cessation can potentially lead to increased levels and toxicity. Enzyme induction has been reported to result in a 20 to 40% lower mean clozapine concentration in smokers compared to nonsmokers. We present a case of altered serum drug concentrations in association with changes in smoking status in a patient upon admission to a nonsmoking inpatient psychiatric facility.

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

In the present study, a site-saturation mutagenesis library of drug-metabolizing CYP102A1 M11H with all 20 amino acids at position 87 was applied as a biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug in which formation of reactive metabolites is considered to be responsible for several adverse drug reactions. Reactive intermediates of clozapine can be inactivated by GSH to multiple GSH conjugates by nonenzymatic and glutathione transferase (GST)-mediated conjugation reactions. The structures of several GST-dependent metabolites have not yet been elucidated unequivocally. The present study shows that the nature of the amino acid at position 87 of CYP102A1 M11H strongly determines the activity and regioselectivity of clozapine metabolism. Some mutants showed preference for N-demethylation and N-oxidation, whereas others showed high selectivity for bioactivation to reactive intermediates. The mutant containing Phe87 showed high activity and high selectivity for the bioactivation pathway and was used for the large-scale production of GST-dependent GSH conjugates by incubation in the presence of recombinant human GST P1-1. Five human-relevant GSH adducts were produced at high levels, enabling structural characterization by (1)H NMR. This work shows that drug-metabolizing CYP102A1 mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs to enable their isolation and structural elucidation.

Analysis of TDM data of clozapine: A daily consumption of less than 10 cigarettes is sufficient for maximum induction of clozapine metabolism

Analysis of TDM data of clozapine: A daily consumption of less than 10 cigarettes is sufficient for maximum induction of clozapine metabolism

Factors affecting interindividual differences in clozapine response: a review and case report

Clozapine is the most powerful new‐generation antipsychotic. Although this drug leads to great therapeutic benefits, two types of undesirable conditions frequently occur with its use: side effects and resistance to treatment. Therapeutic drug monitoring of clozapine would be very useful to avoid both these situations. The necessity of monitoring the therapy is the result of a wide interindividual variability in the metabolism of clozapine. In this review, we highlight all the conditions underlying this variability, analyzing them one by one. Relevant literature was identified through a search of MEDLINE and PubMed. In addition, the case of a treatment‐resistant patient with accelerated metabolism of clozapine is reported as representative of utility of therapeutic drug monitoring in terms of clozapine dose adjustment. Genetic polymorphisms of cytochrome P450 enzymes and of neurotransmitter receptors; drug interactions; interactions of clozapine with other substances such as food and drink; smoking; and nonmodifiable variables such as age, ethnicity, and gender have been examined in relation to the existing scientific literature. The laboratory techniques that clinicians could use to identify these variables and adequate therapies are also reviewed.

Confounding factors for variation of clozapine plasma levels: drug interactions with proton pump inhibitor or infectious etiologies?

Clozapine is an atypical antipsychotic used to treat schizophrenia in patients only after failure of response of other antipsychotic drugs because of its agranulocytosis risk. Because of frequent occurrence of gastrointestinal adverse drug reactions (ADR), proton pump inhibitors (PPIs) are often prescribed in patients exposed to clozapine [1, 2] Clozapine is metabolized mainly through the 1A2 isoenzyme, CYP3A4, only in patients with reduced CYP1A2 activity and moderate contribution of CYP2C19 [3]. Agents that induce or inhibit CYP1A2 may increase or decrease, respectively, the metabolism of clozapine [2]. It is well known that smokers need to increase twofold the dose of clozapine compared with nonsmokers because of CYP1A2 induction by cigarette smoke [2]. We report a case of change in clozapine metabolism after substitution of a racemic PPI omeprazole by its (s)-isomer, esomeprazole, associated with a possible infectious event. Case report

Schizophrenia and Obsessive-Compulsive Disorder

Back to table of contents Previous article Next article Communications and UpdatesFull AccessSchizophrenia and Obsessive-Compulsive DisorderKavi Devulapalli, B.A, M.P.H., and Henry A. Nasrallah, M.D.Kavi DevulapalliSearch for more papers by this author, B.A, M.P.H., and Henry A. NasrallahSearch for more papers by this author, M.D.Published Online:1 Nov 2010https://doi.org/10.1176/appi.ajp.2010.10081111AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: In the July 2010 issue of the Journal, Carolyn I. Rodriguez, M.D., Ph.D., et al. (1) presented an interesting case illustrating the complexities of co-occurring psychotic and obsessive-compulsive disorders (OCD). The authors should be commended for their careful review of the differential diagnosis and treatments for a patient presenting with these intertwined symptoms.In the case study, the patient was found to have manifested obsessive compulsive symptoms prior to his first psychotic episode. Such a presentation is consistent with the results of a meta-analysis we conducted regarding the temporal relationship of OCD and schizophrenia in patients suffering from both disorders (2). Our analysis showed that in patients diagnosed with both disorders, OCD tends to precede schizophrenia by 1 year. While our study did not find this result to be statistically significant, it did nearly reach significance (p=0.066), suggesting the potential for statistical significance in studies with a larger sample size. Clearly, Dr. Rodriguez et al.'s suggestions for longitudinal studies of young people at risk for psychosis or OCD would provide for much needed insight into the epidemiology and clinical phenomenology of cooccurring obsessive compulsive and psychotic symptoms.In their discussion of the potential adverse drug-drug interactions between clozapine and fluvoxamine, the authors appropriately mentioned cytochrome 3A4. We would like to also mention that cytochrome 1A2 is even more significantly involved in the metabolism of clozapine (3). As a strong inhibitor of cyto-chrome 1A2, combining fluvoxamine with clozapine in patients suffering from both OCD and treatment-resistant schizophrenia may produce serious adverse effects. Further, nicotine in tobacco is an inducer of cytochrome 1A2 and may lead to attenuation of the efficacy of clozapine unless the dose is increased.Cleveland, OhioCincinnati, Ohioaccepted for publication in August 2010.Dr. Nasrallah has received research grant support from Forest, Janssen, Otsuka, and Shire; and he has served on the advisory board or speaker's bureau of AstraZeneca, Janssen, Merck, Novartis, Pfizer, and Sepracor. Dr. Devulapalli reports no financial relationships with commercial interests.

ABCB1 and Cytochrome P450 Polymorphisms

To examine the genetic factors influencing clozapine kinetics in vivo, 75 patients treated with clozapine were genotyped for CYPs and ABCB1 polymorphisms and phenotyped for CYP1A2 and CYP3A activity. CYP1A2 activity and dose-corrected trough steady-state plasma concentrations of clozapine correlated significantly (r = -0.61; P = 1 x 10), with no influence of the CYP1A2*1F genotype (P = 0.38). CYP2C19 poor metabolizers (*2/*2 genotype) had 2.3-fold higher (P = 0.036) clozapine concentrations than the extensive metabolizers (non-*2/*2). In patients comedicated with fluvoxamine, a strong CYP1A2 inhibitor, clozapine and norclozapine concentrations correlate with CYP3A activity (r = 0.44, P = 0.075; r = 0.63, P = 0.007, respectively). Carriers of the ABCB1 3435TT genotype had a 1.6-fold higher clozapine plasma concentrations than noncarriers (P = 0.046). In conclusion, this study has shown for the first time a significant in vivo role of CYP2C19 and the P-gp transporter in the pharmacokinetics of clozapine. CYP1A2 is the main CYP isoform involved in clozapine metabolism, with CYP2C19 contributing moderately, and CYP3A4 contributing only in patients with reduced CYP1A2 activity. In addition, ABCB1, but not CYP2B6, CYP2C9, CYP2D6, CYP3A5, nor CYP3A7 polymorphisms, influence clozapine pharmacokinetics.

Ciprofloxacin Strongly Inhibits Clozapine Metabolism

We report on two cases of drug-drug interactions between ciprofloxacin and clozapine. The first case was a 46-year-old male patient receiving a daily dose of clozapine 900 mg. He was admitted to hospital with urosepsis and was treated with a 5-day course of ciprofloxacin and amoxicillin. Two days after completion of antibacterial therapy, the patient developed symptoms of rhabdomyolysis. Clozapine therapy was discontinued and measurement of the patient's clozapine plasma concentration 1 day after cessation of clozapine therapy and 3 days after cessation of ciprofloxacin treatment showed that it was in excess of recommended therapeutic levels. The second patient was a 58-year-old male patient treated with a daily dose of clozapine 300 mg. He was admitted to hospital because of delirium and suspected urinary tract infection or pneumonia. Treatment with ciprofloxacin was initiated. Measurement of clozapine plasma concentrations prior to and 3 days after commencement of ciprofloxacin showed that clozapine concentrations doubled over that time period. We suggest that inhibition of cytochrome P450 (CYP) enzymes 1A2 and 3A4 by ciprofloxacin resulted in delayed clozapine metabolism and elevated clozapine plasma concentrations. This might cause severe adverse effects. We advise using another antibacterial agent or reducing the clozapine dose and monitoring clozapine levels when this antipsychotic agent is used in combination with ciprofloxacin.

Variability of the In Vivo Metabolism of Clozapine

Clozapine, the gold standard of antipsychotic treatment in treatment-refractory patients with schizophrenia, is metabolized in vivo to clozapine-N-oxide and N-desmethylclozapine (NDMC = norclozapine). N-desmethylclozapine is an active metabolite of clozapine and combines unique pharmacological properties. Because little is known about the rate of metabolic conversion of clozapine in vivo, we assessed the association between clozapine dose and plasma levels for clozapine and NDMC. Plasma levels of clozapine and NDMC were measured in 485 blood samples from 108 patients with schizophrenia treated with clozapine. %NDMC, the ratio of NDMC to total clozapine (NDMC + clozapine), was used as a measure of the in vivo metabolism of clozapine. Daily clozapine doses correlated significantly with clozapine levels and NDMC levels, whereas %NDMC showed a weaker negative correlation with clozapine dose. The mean %NDMC value was 37.0% +/- 16.8%, with high variability between subjects. Repeated measurements in subjects treated with the same dose of clozapine showed a high within-subject variability of %NDMC. Our results suggest a high degree of between-subject and within-subject variability in the metabolism of clozapine in vivo. Direct administration of NDMC may be preferable to reliably achieve sufficient plasma levels of this compound.

Drs. Ghaznavi and Bhagwagar Reply

Back to table of contents Previous article Next article Letters to the EditorFull AccessDrs. Ghaznavi and Bhagwagar ReplySHARMIN GHAZNAVI M.D., Ph.D.ZUBIN BHAGWAGAR M.D., Ph.D., M.R.C.Psych.,SHARMIN GHAZNAVI M.D., Ph.D.Search for more papers by this authorZUBIN BHAGWAGAR M.D., Ph.D., M.R.C.Psych.Search for more papers by this author,Published Online:1 Dec 2008https://doi.org/10.1176/appi.ajp.2008.08071105rAboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: We thank Dr. Caetano for expressing interest in our article. To summarize, we reported on a patient who was treated with clozapine, 750 mg/day, and valproic acid, 1500 mg/day, for several years without incident. The first episode of clozapine-induced neutropenia occurred 1 month following the addition of donepezil to our patient’s treatment regimen. Dr. Caetano raises two related points regarding this initial episode of neutropenia. First, donepezil does not activate/inhibit enzymes that influence clozapine or valproic acid (1A2, 2D6, 3A4). Second, it is possible that valproic acid was responsible for increased clozapine concentration, leading to neutropenia. We agree that valproic acid can lead to increased concentrations of clozapine (1) , which we noted in our article. In fact, we raised the possibility that the combination of clozapine and valproic acid may confer a greater risk of later onset neutropenia in some patients. However, the fact that our patient was treated for years with the combination of clozapine and valproic acid without incident suggests that we cannot dismiss the setting in which our patient developed neutropenia, namely the addition of donepezil. To further address this point as well as the comment by Dr. Caetano pertaining to donepezil’s effect on the P450 isoenzymes 2D6 and 3A4, donepezil, to the best of our knowledge, is metabolized by the isoenzymes 2D6 and 3A4 (2) , which are also responsible for the metabolism of clozapine. Thus, it is possible that donepezil may have led to increased serum levels of clozapine and placed our patient at greater risk for developing neutropenia. At the time of the second clozapine rechallenge, our patient was being treated with risperidone, and clozapine was added to the regimen. Given that risperidone is known to increase serum levels of clozapine (3) , we concluded that the combination of clozapine and risperidone may have been responsible for neutropenia in our patient on that occasion. Thus, although we cite that risperidone alone carries a risk for neutropenia (4) , we do not feel that the drug was the primary offender and agree with Dr. Caetano that risperidone likely increased clozapine serum levels that led to neutropenia in our patient, and we appreciate the opportunity to clarify this point. Finally, Dr. Caetano suggests that lamotrigine should be avoided because it increases clozapine serum concentration. Indeed, there has been a case report of a threefold increase in serum concentration with the addition of lamotrigine (5) . However, Wong and Delva (6) , in their review of treatment for clozapine-induced seizures, recommended lamotrigine for its relative lack of effect on serum clozapine levels. We would therefore advise caution when prescribing lamotrigine as a secondary prophylaxis for clozapine-induced seizures rather than avoiding one of two (gabapentin) agents that are effective for clozapine-induced seizures and which demonstrate a relative lack of interactions with clozapine compared with valproic acid (6) . New Haven, Conn.The authors’ disclosures accompany the original article.This letter (doi: 10.1176/appi.ajp.2008.08071105r) was accepted for publication in August 2008.

DISRUPTED SMALL-WORLD FUNCTIONAL INTERCONNECTIVITY IN SCHIZOPHRENIA

Although clozapine is the only antipsychotic agent to have demonstrated superior efficacy in treatment-refractory schizophrenia, one- to two-thirds of patients do not respond adequately despite acceptable dosages and plasma levels. Moreover, a significant number of patients stop the therapy for various reasons, including its side effects, many of which are thought to be related to its active metabolite, norclozapine. However, combining clozapine with the SSRI antidepressant fluvoxamine decreases norclozapine formation by inhibiting the CYP450 1A2 isoenzyme. Lowering norclozapine levels in this way while maintaining therapeutic clozapine levels increases the clozapine: norclozapine ratio; the potential benefits include both a reduction of such side effects as sedation, weight gain, metabolic disturbances, and neutropenia, and an increase in efficacy. The optimal ratio of clozapine to norclozapine has not yet been defined, but a ratio of two or more implies that saturation of clozapine metabolism has been reached. We hypothesize that co-administration of clozapine and fluvoxamine at dosages that will produce therapeutic plasma levels of clozapine and a clozapine: norclozapine ratio of two or more will increase efficacy and tolerability of clozapine therapy in treatment-resistant schizophrenic patients.

Evaluation of HepaRG Cells as an in Vitro Model for Human Drug Metabolism Studies

HepaRG cells, a newly developed human hepatoma cell line, differentiate into hepatocyte-like morphology by treatment with dimethyl sulfoxide (DMSO). The expression of cytochrome P450 (P450) enzymes, transporter proteins, and transcription factors was stable in differentiated HepaRG cells over a period of 6 weeks when cultured with DMSO. Compared with human hepatocytes, expression of P450 in HepaRG cells was in general lower with the exception for a considerably higher expression of CYP3A4 and CYP7A1. The expression of P450s generally decreased when DMSO was removed from the medium, whereas transporters and liver-specific factors were unaffected. The relative mRNA content of drug-metabolizing P450s displayed the highest resemblance between human hepatocytes and differentiated HepaRG cells 1 day after removal of DMSO from the medium. The metabolism of midazolam, naloxone, and clozapine in HepaRG cells was similar to human hepatocytes, indicating the function of CYP3A4, CYP1A2, and UDP-glucuronosyltransferase enzymes. However, the metabolism of 7-ethoxycoumarin and dextromethorphan was low, confirming low levels of CYP2E1 and CYP2D6 in HepaRG cells. The P450 probe substrates indicate a decrease in CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activities in HepaRG cells 1 day after removal of DMSO from the medium. The activities were then relatively stable in DMSO-free medium for up to 14 days. Based on the stable expression of liver-specific functions over a long period in culture, the relative mRNA content of drug-metabolizing P450s, and metabolic properties, HepaRG cells provide a valuable in vitro model for human drug metabolism studies.

Activation of T-Cells from Allergic Patients and Volunteers by p-Phenylenediamine and Bandrowski's Base

Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction.

P.3.d.015 CYP1A2 polymorphisms and clozapine metabolism in Chinese psychotic patients

The expression and activity of CYP1A1 were examined in fresh, small-sized lung biopsy specimens from nine human subjects. CYP1A1 transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis of total lung RNA. CYP1A2 transcripts were detected in the RNA samples as well, and bioactivation of 2-aminofluorene (2-AF) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), a CYP1A2-preferential activity, was catalyzed by the lung S9 fractions also. Two major bands were detected in the whole homogenate by western blot analysis using CD3, a mouse anti rat CYP1A1 monoclonal that cross-reacts with rat CYP1A2 as well as with human CYP1A1 and CYP1A2. S9 fractions from the tissues catalyzed the bioactivation of benzo[a]pyrene (B[a]P), a CYP1A1-preferential activity, to mutagens in the Ames assay. Our findings are in agreement with the known presence of CYP1A1 in the human lung, and provide strong evidence for the expression of catalytically functional CYP1A2 in the tissue.