JOURNAL OF CHILD AND ADOLESCENT PSYCHOPHARMACOLOGYVolume 6, Number 2, 1996Mary Ann Liebert, Inc.Pp. 119-131

Correlated Changes in Symptoms and NeurotransmitterIndices during Maintenance Treatment with Clozapine or

Conventional Neuroleptics in Adolescents and YoungAdults with Schizophrenia

EBERHARD SCHULZ, M.D., CHRISTIAN FLEISCHHAKER, andHELMUT E. REMSCHMIDT, M.D., Ph.D.

ABSTRACT

A study of 40 young patients (age 14-22 years) with DSM-III-R schizophrenia (without sub-stance abuse) was conducted following a mean of 3.4 years of neuroleptic treatment. Afterfailing on conventional agents in clinical trials lasting a mean of 2 years, 20 patients were

prospectively maintained on open-label clozapine (mean 324 mg daily), and another 20 pa-tients continued on typical neuroleptics (mean 465 mg chlorpromazine-equivalents daily).Patients were then sampled for biochemical measures and assessed for psychopathology(Brief Psychiatric Rating Scale, Scales for the Assessment of Positive/Negative Symptoms)on six occasions at consecutive 6-week intervals during maintenance treatment on clozapineor conventional neuroleptics. There were 22-fold interindividual differences in clozapine lev-els and also high intraindividual differences over time. Maintenance dosage was linearly re-

lated to plasma levels of clozapine and its metabolites. Prolactin levels were elevated withtypical neuroleptics but not clozapine. Blood levels of serotonin, methoxyhydroxyphenyl-glycol (MHPG), norepinephrine, and epinephrine (but not dopamine) were significantlyhigher in clozapine-treated patients than in conventionally treated patients. Higher serotoninlevels were associated with significantly fewer negative symptoms, whereas higher MHPGlevels were correlated with less depression. These findings suggest involvement of norepi-nephrine and serotonin in the pathophysiology of schizophrenia (with depression associatedwith lower MHPG levels and negative symptoms associated with lower serotonin levels) andin the therapeutic actions of clozapine. Speculatively, a treatment strategy of targeting spe-cific neurotransmitter systems might be based on the presence of specific symptoms in ado-lescents and young adults with schizophrenia.

INTRODUCTION

Clozapine is a neuroleptic medication with high affinity for dopamine Di, D2, and D4, serotonin 5-HTi and 5-HT2, a-i-adrenergic, muscarinic, and histamine Hi receptors (Coward 1992, Farde and

Nordstrom 1992, Kuoppamäki et al. 1993, Meltzer 1992a). Clozapine is considered to be an atypical neu-

Department of Child and Adolescent Psychiatry, Philipps University, Marburg, Germany.

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roleptic because of its clinical characteristics, such as relatively low risk of acute extrapyramidal side ef-fects, tardive dyskinesia, and hyperprolactinemia (Casey 1989, Gudelsky et al. 1989, Kane 1992, Liebermanet al. 1986). Based on its clinical and endocrine effects, clozapine is currently an important option in themanagement of patients with treatment-resistant schizophrenia.

Despite the well-established efficacy of atypical neuroleptics in short-term and maintenance treatment ofschizophrenia and treatment-refractory schizophrenia (Kane et al. 1988) in adults, adolescents have not beenexamined as rigorously. Several uncontrolled studies have provided evidence that clozapine might be aneffective therapy for children and adolescents with schizophrenia (Birmaher et al. 1992, Frazier et al. 1994,Jacobsen et al. 1994, Levkovitch et al. 1994, Mozes et al. 1994, Remschmidt et al. 1992, 1994a, Schulz etal. 1994, Siefen and Remschmidt 1986). Nonetheless, about 30% of adolescents with schizophrenia do notrespond to clozapine in open-label treatments (Remschmidt 1992).

Measurement of biogenic amines and their metabolites in cerebrospinal fluid (CSF) and plasma is con-

sidered a promising tool to help elucidate the mechanisms of action of neuroleptics (Kahn and Davidson1993). Only a few studies have been conducted, all in adults, that investigate differences in the neuro-chemical effects of clozapine and conventional neuroleptic drugs (Ackenheil 1989, Banki 1978, Breier etal. 1994, Green et al. 1993, Pickar et al. 1992).

There are no previous reports on the short- or long-term effects of clozapine on catecholamine or in-doleamine measures in children or adolescents with schizophrenia. The aim of this study is to investigate(1) the relationship of clozapine dose and serum levels in adolescents, (2) the relationships of plasma cloza-pine levels with circulating concentrations of monoamine neurotransmitters, and (3) the relationships ofplasma monoamine levels with changes in psychopathology in a sample of adolescents with chronic schiz-ophrenia during maintenance treatment with clozapine.

METHODS

SubjectsThe study included all 40 patients with DSM-III-R criteria of schizophrenic or schizoaffective psychosis

in a rehabilitation center ("Leppermühle") for adolescents with schizophrenia. The data collection was per-formed at 6-week intervals during the time period between May 1991 and October 1992. During the study,20 patients with schizophrenia received clozapine, and the other 20 patients were treated with standard neu-

roleptic medications.According to DSM-III-R criteria, the following subtypes of schizophrenic psychoses were found in the

total sample of 40 subjects: paranoid type (17 males, 77%; 10 females, 55%), disorganized type (5 males,22%; 4 females, 22%), residual type (1 female, 5%), and schizoaffective disorder (3 females, 16%). Thediagnoses and gender distribution (22 males, 18 females) revealed no significant differences, as determinedby chi-square or Fisher's test.At the beginning of the prospective investigation, the age range of the total sample was 14-22 years.

There were no statistically significant differences (Chi-square or Fisher's test) between the two treatment

groups with respect to gender or age distribution (total sample 19.1 ± 2.2 years, clozapine group 19.5 ±2.1 years, conventionally treated group 18.8 ± 2.3 years).At the start of the investigation, the course of the illness showed the following distributions, using DSM-

III-R criteria: subchronic 12.5% (n = 5), cnronic 72.5% (n = 29), chronic with acute exacerbation 7.5%(n = 3), and remitted 7.5% (n = 3). Of the sample of 40 young patients, 32 patients (80%) were already ina chronic state (with or without acute exacerbation).Intellectual functioning by standard intelligence tests (Wechsler Intelligence Scale or Culture Fair Test)

showed a high IQ level (IQ 115-129) in 5% of patients (n = 2), normal range (IQ 85-114) in 58% (n =

23), low normal (IQ 70-84) in 35% (n = 14), and mild mental retardation (IQ 50-69) in 2% (n = 1).InstrumentsTo assess premorbid adaptation and precursor symptoms of schizophrenia, we administered the Instrument

for Retrospective Assessment of the Onset of Schizophrenia (IRAOS, Hafner et al. 1990), a semistructuredinterview that retrospectively examines schizophrenic and schizotypic precursors and other symptoms be-

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fore the first manifestation of schizophrenia. The interrater reliability of the IRAOS was found to be satis-factory, with kappa values varying between 0.62 and 1.00 (Hafner 1992). This instrument was modified byour group for investigating children and adolescents and their parents (Remschmidt et al. 1994b). For thisstudy, the IRAOS was administered by experienced child psychiatrists, who directly interviewed the par-ents. In addition, information was obtained from the past medical records.To identify symptoms that may be present during the premorbid stage of schizophrenia, a checklist was

developed out of a factor analysis involving more than 2000 patients (Remschmidt et al. 1994b). The symp-toms could be classified as "internalizing" and "externalizing," similar to the categories derived byAchenbach's analysis of normative data for youths on the Child Behavior Checklist (Achenbach andEdelbrock 1983). Examples of "internalizing" symptoms are mutism, mental slowness, social isolation, gen-eralized anxiety, specific phobias, and obsessive-compulsive symptoms. The "externalizing" dimension con-

sisted of items such as hypermotoric behavior, antisocial behavior, aggression, and school refusal.Positive and negative symptoms of schizophrenia were evaluated using the Scale for the Assessment of

Negative Symptoms (SANS) and the Scale for the Assessment of Positive Symptoms (SAPS) developedby Andreasen (1982, 1984a,b). The interrater reliability of both the SANS and the SAPS was found to begood, with kappa values of 0.8 (Andreasen 1982, Andreasen et al. 1991, Moscarelli et al. 1987), with theexception of the SANS item "attention" (kappa value 0.67). As described elsewhere (Remschmidt et al.1991), the attribution of attentional impairment by the SANS to negative symptoms (rather than also to pos-itive symptoms) seems to be problematic, and so we excluded this item from the rating scale. The sum-

mary scores for the negative and positive symptoms ratings were calculated according to Andreasen (1982).In addition to the evaluation of positive and negative symptoms, the BriefPsychiatric Rating Scale (BPRS,

Overall and Gorham 1962) was employed to measure symptomatology and outcome during the prospectiveinvestigation. The BPRS has been shown to be effective for the evaluation of outcome in treatment stud-ies of schizophrenia (Kane et al. 1988, Beckmann et al. 1992, Meltzer 1991), and its use in follow-up stud-ies of schizophrenic patients is well established (Thiemann et al. 1987, Bell et al. 1992, Deister and Marneros1993). In our study, the BPRS Total Score and the BPRS Depressive Score (including items 1, 2, 5, and9) were employed for statistical analysis.

The clinical ratings were performed by an experienced child and adolescent psychiatrist at the same timesas the pharmacological investigations, i.e., at 6-week intervals.

Medication treatment

Indications for clozapine treatment were nonresponse, aggravation of target symptoms, or severe side ef-fects during treatment with conventional neuroleptic medication.

These adolescents were first treated for schizophrenia with conventional neuroleptic medications at age15.7 ± 1.5 years, and clozapine (Leponex®) was initially administered as neuroleptic monotherapy at age17.5 ± 2.0 years.At the beginning of the prospective investigation, the patients had already been receiving clozapine treat-

ment for 24 ± 15 months. The adolescents receiving conventional neuroleptic therapy were treated withhaloperidol (n = 9,22.5%), levomepromazine (n = 5,12.5%), fluphenazine (n = 4,10%), flupentixole (n =

3, 7.5%), chlorprothixene (n = 2, 5%), promethazine (n = 1, 2.5%), perazine (n = 1, 2.5%), and thiorid-azine (n = 1, 2.5%). Of the 20 patients receiving typical neuroleptic agents, 7 received monotherapy and13 were treated with more than one drug.During the investigation, 65% of the clozapine-treated group (13/20) received monotherapy, but only 4

patients (20%) treated with typical neuroleptic medication received monotherapy. In the clozapine-treatedgroup, 4 patients (20%) required comedication with sympathomimetics or /3-blockers to manage side ef-fects (hypotonia, tachycardia). In the conventionally treated group, 15 patients (75%) experienced side ef-fects that led to comedication with j3-blockers, sympathomimetics, biperiden, or laxatives.

The calculation of neuroleptic dose equivalents was performed according to Rey et al. (1989) and Schulzet al. (1989), who related clozapine to chlorpromazine at a ratio of 1:1. Using this ratio, the clozapine-treated patients received a daily oral dose of 324 ±178 mg equivalents of chlorpromazine, and the con-

ventionally treated patients received daily oral doses of 465 ± 317 mg chlorpromazine-equivalents.121

SCHULZ ET AL.

Clinical symptomsAll of the 40 youths in the sample exhibited evidence of psychopathology in their premorbid function-

ing, according to the findings of the modified IRAOS. Except for 4 patients who exhibited exclusively ex-

ternalizing symptomatology, all of the other adolescents were characterized by predominantly internalizingsymptoms. A first peak for the diagnosis of a mental disorder was observed at the age of 6 years. A sec-ond peak occurred at a mean age of 11.5 years, when signs and symptoms were already appearing relatedto the onset of schizophrenic psychosis (attentional impairment, decreased functioning at school, social with-drawal, emotional disturbances).The mean age at diagnosis of DSM-III-R schizophrenia for this sample was 14.8 years. At that age, the

patients experienced characteristic psychotic symptoms. Interestingly, they were first hospitalized with thediagnosis of schizophrenia at a mean age of 15.6 years. Thus, at the beginning of our investigation, the pa-tients showed a mean duration of schizophrenia of 4.3 years. Table 1 summarizes some data about pre-morbid functioning and the course of the disorder, as evaluated at the beginning of the investigation.Assessment of clozapine and its major metabolites

Measurements of serum levels of clozapine andmajormetabolites were performed at consecutive 6-weekintervals on a sample of 20 of the adolescent schizophrenics (13 male, 7 female; age 19.5 ±2.1 years).On the day of blood sampling, after 15 min at rest, a butterfly needle was inserted into a forearm vein

and a 10 mL blood sample was collected into serum monovettes (Sarstedt, Germany). Blood samples were

generally taken prior to the morning dose, about 12 h after the prior administration (Riederer and Laux1992). Blood sampling was performed under strict steady-state conditions, i.e., when 4-5 half-life intervalshad elapsed since the last dosage change.After coagulation at ambient temperature for 15 min, the blood samples were centrifuged for 10 min at

2500g and 4°C, and serum was stored in micro test tubes (Eppendorf, Hamburg, Germany) at -80°C un-til analyzed.

Measurements of clozapine, clozapine N-oxide, and /V-desmethylclozapine were conducted using theHPLC method developed by our group (Schulz et al. 1995). The method entails deproteinization and sub-sequent high-performance liquid chromatography with electrochemical detection.Clozapine, /V-desmefhylclozapine, and clozapine N-oxide were kindly donated by Sandoz Pharmaceutical

(Basel, Switzerland). Imipramine was obtained from Ciba-Geigy (Basel, Switzerland). HPLC-grade ace-tonitrile (Merck, Darmstadt, Germany) and methanol (Baker, Deventer, Netherlands) were used without fur-

Table 1. Characteristics of the Sample of 40 YoungPatients with Chronic Schizophrenia

Gender ratio (male : female) 22:18Age at first signs and symptoms

related to schizophrenia 11.5 ± 4.4 yearsAge at diagnosis of schizophrenia 14.8 ± 1.8 yearsFirst hospitalization becauseof schizophrenia 15.6 ± 1.8 years

Number of hospitalizationsClozapine-treated group (n = 20) 3.1 ± 2.4Conventionally treated group (n = 20) 2.2 ± 0.8

Age at first treatment withstandard neuroleptics 15.7 ± 1.5 years

Age at first treatment withclozapine 17.5 ± 2.0 years

Time period between startingfirst neuroleptic treatment andfirst starting clozapine 1.8 ± 1.7 years

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CLOZAPINE, NEUROTRANSMITTERS, AND SYMPTOMS IN SCHIZOPHRENIA

ther purification. Potassium dihydrogen phosphate was obtained from Merck, and the precipitation solutionfor HPLC analysis (Order No. 3005) was made by Chromsystems (Munich, Germany). Water was purifiedby a Milli-Q water processing system (Millipore, Eschborn, Germany).Clozapine, clozapine N-oxide, and A/-desmethylclozapine were dissolved at a concentration of 1 mg of

free base per mL methanol, and then diluted to final concentrations using HPLC eluent. Stock solutionswere stored in the dark at

—

80°C for several weeks, and no instabilities were observed in either serum sam-

ples of clozapine-treated patients or serum samples spiked with medication. The latter were prepared bymixing blank serum with standard solutions to obtain final concentrations of 1-1200 ng/mL clozapine, N-desmethylclozapine, and clozapine N-oxide, respectively.Prior to extraction, 200 pL of serum was supplemented with 100 pL of internal standard solution (100

ng/mL) and 50 pL of the precipitation solution. Protein precipitation was performed with the precipitationsolution for HPLC analysis by salting out. The samples were vortexed for 5 min, then centrifuged for 10min at 6000g, and 20 pL of the supernatant was injected for HPLC analysis.

Chromatographie separation was accomplished using a RP-select-B 5-pm column (125 X 4 mm i.d.) andprecolumn, including Manu CART "4" (Merck, Darmstadt, Germany), in conjunction with an electro-chemical detector (Waters 460) with a glassy-carbon working electrode. The system was connected on-lineto a Waters Expert-EASE laboratory data system (Waters, Eschborn, Germany). The glassy-carbon elec-trode was set at +0.9 V potential versus a Ag/AgCl reference electrode.The eluent consisted of 15% acetonitrile, 45% methanol, and 40% HPLC-grade water (v/v), adjusted to

pH 6.6 with 0.1 M potassium dihydrogen phosphate. The flow rate was 1 mL/min at 50°C. Prior to use,the HPLC eluent was filtered through a 0.45-/xm filter (Satorius, Göttingen, Germany) and degassed bysonication. Given a signal-to-noise ratio of 5:1, the method shows sufficient sensitivity to accurately quan-titate clozapine, clozapine N-oxide, and A/-desmethylclozapine at 0.5, 5.0, and 0.5 ng/mL, respectively. Theinterassay and intraassay coefficients of variation were lower than 10 and 6%, respectively, for clozapine,/V-desmethylclozapine, and clozapine N-oxide. None of the blood samples fell into the undetectable range.

HPLC analysis of biogenic amines and metabolitesThe biochemical probes were obtained using standard procedures for prestudy diet, blood collection, and

storage of biogenic amines (Lake and Ziegler 1985, Pluto and Bürger 1988, Weier et al. 1986, Ganhao etal. 1991, Boomsma et al. 1993).

Plasma catecholamines, plasma level ofmethoxyhydroxyphenylglycol (MHPG), and serum serotonin con-centrations were measured by HPLC kits (Catechol-Kit No. 5000, Serotonin-Kit No. 3000) obtained fromChromsystems Corporation (Munich, Germany). The kits consist of a mobile phase, calibration standard,internal standard [dihydroxybenzylamine (DHBA)], precipitation solution, extraction buffer, wash solvent,Cig RP column, and cartridges containing A1203. MHPG was obtained from Sigma (St. Louis, MO).Water was purified by a Milli-Q water processing system (Millipore, Eschborn, Germany).For chromatography of the biogenic amines, analytical pump (Waters 510), automatic sample injector

(Satellite WISP 700), electrochemical detector (Waters 460) with a glassy-carbon working electrode(Waters), and a column compartment (Waters) were used. The system was connected on-line to WatersExpert-EASE laboratory data system on a microvax with LAC/E and SIM (Waters).For the determination of the catecholamines, blood was drawn from an antecubital vein and immediately

injected into EGTA Kabevetten (KABE Labortechnik, Nümbrecht/Elsenroth, Germany). The samples were

centrifuged at 2500g at 4°C, and the supernatant was transferred into micro test tubes (Eppendorf, Hamburg,Germany) and stored at

—

80°C until analysis.For extraction, 0.5 mL extraction buffer was injected into cartridges containing A1203. The cartridges

were vortexed and subsequently adjusted with 1 mL of plasma and 50 mL of internal standard solution (600pg DHBA). The cartridges were shaken upside down for 10 min and then aspirated under low vacuum

through the cartridges. The residue was washed three times using 1 mL of wash solvent. After this proce-dure, 120 mL of extraction buffer was added, then the sample was vortexed for 30 sec and eluted into amicrovial by centrifugation for 1 min at 2000g. An aliquot of 20 mL was injected into the Chromatograph.HPLC separation was obtained by using a Ci8 RP column and a mobile phase with a flow rate of 0.9 mL/minat 25°C.

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For the determination of plasma catecholamines, electrochemical detection was amperometric at E = 0.60V. For pMHPG, E = 0.720 V. An internal standardization of DHBA was used with every sample.The retention times were 3.1 min for MHPG, 4.5 min for norepinephrine, 5.5 min for epinephrine, 8.0

min for DHBA, and 13.0 min for dopamine. For computer calculations, Expert Ease (Waters, Division ofMillipore, Eschborn, Germany) laboratory software was used.Within a 10-1000 pg/mL range, linear relationships were obtained for electrochemical detection. The re-

covery rates were 68% for MHPG, 72% for norepinephrine, 70% for epinephrine, 73% for DHBA, and68% for dopamine. Recovery rates related to the internal standard amount were found to be between 90and 98%. The method shows sufficient sensitivity to accurately quantitate norepinephrine, epinephrine, anddopamine at 5 pg/mL and MHPG at 50 pg/mL. The interassay coefficient of variation was below 10%.Blood samples for serotonin determination were collected in serum-monovettes (Sarstedt, Germany) by

venipuncture. After coagulation at ambient temperature, the samples were centrifuged at 2500g (4°C), trans-ferred to micro test tubes (Eppendorf, Hamburg, Germany), and stored at

—

80°C until analysis. Before de-termination, 200 pL of serum was supplemented with 100 pL of internal standard solution (100 ng/mL, N-methylserotonin) and 50 pL of precipitation solution. The micro test tubes were vortexed for 5 min andcentrifuged for 10 min at 6000g. The supernatant was directly transferred into microvials (Waters, Eschborn,Germany). An aliquot of 20 pL was injected into the Chromatograph.HPLC separation was accomplished using a Cig RP column and a mobile phase with a flow rate of 0.9

mL/min at 25°C. Electrochemical detection was amperometric at E = 0.60 V. The retention times were 4.0min for serotonin and 5.0 min for A/-methylserotonin. For computer calculations, Expert Ease (Waters,Division of Millipore, Eschborn, Germany) laboratory software was used. Within a 10-1000 ng/mL range,linear relationships were obtained for electrochemical detection. The recovery rates were 86% for serotoninand 88% for A/-methylserotonin. Recovery rates related to the internal standard amount were found to be98%. The method shows sufficient sensitivity to accurately quantitate serotonin at 1 ng/mL. The interassaycoefficient of variation was less than 10%.

Measurement ofplasma prolactinPlasma prolactin levels were determined at the Department of Endocrinology and Metabolism, Center of

Internal Medicine, Philipps University in Marburg, Germany (Prof. Dr. H. Kaffarnik). The measurementswere obtained by ELISA (IMX- Prolactin-Test, Abbott Corp., Wiesbaden, Germany).

Statistical analysisSeveral group comparisons were conducted using chi-square and Fisher's exact test. Pearson correlation,

linear regression analysis, and median test were also employed. Significance was set at p < 0.05.Intraindividual variations are presented as coefficients of variation and expressed in percentages:

(SD/mean) X 100.Normal range values of the biogenic amines derived from data obtained by Chromsystems Corp. (Munich,

Germany), which were obtained by the HPLC method used in our study.For group comparisons, the following ranges were defined as physiological: serum serotonin 128.4-173.7

ng/mL, plasma MHPG 4.4—6.0 ng/mL, plasma norepinephrine 198.6-268.7 pg/mL, plasma epinephrine76.0-102.9 pg/mL, and plasma dopamine 77-116.4 pg/mL. Because of normal distribution, t tests could beapplied to the statistical analysis of serum serotonin levels. In case of the plasma catecholamines, a non-

parametric statistic had to be used (median test).

RESULTS

No significant alterations were found in complete blood counts or other clinical laboratory data betweenthe clozapine-treated and the conventionally treated patients.In the 20 adolescents who were treated with oral doses of clozapine varying from 75 to 600 mg daily

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CLOZAPINE, NEUROTRANSMITTERS, AND SYMPTOMS IN SCHIZOPHRENIA

(mean ± SD = 307 ± 160 mg), serum concentrations of clozapine and its metabolites ranged widely: forclozapine, the range was 24-732 ng/mL, for A/-desmethylclozapine 10-795 ng/mL, and for clozapine N-oxide levels 5-237 ng/mL. Serum levels across six consecutive measurements, determined at 6-week in-tervals, were clozapine 251 ± 140 ng/mL, /V-desmethylclozapine 263 ±146 ng/mL, and clozapine N-ox-ide 46 ± 38 ng/mL.During the prospective trial, 16 patients received intraindividually fixed doses of clozapine. In this group,

the individual mean serum levels of clozapine and its metabolites were calculated from the six consecutivemeasurements and were found to be highly correlated with the administered fixed dosage of the neurolep-tic (Pearson correlation): clozapine r = 0.82, p = 0.0001; A/-desmethylclozapine r = 0.81, p = 0.0001;clozapine N-oxide r = 0.91, p = 0.0001.

The 16 patients receiving fixed doses of clozapine were selected for the calculation of intra- and in-terindividual variations in serum levels of clozapine and its metabolites. A strong linear relationshipbetween clozapine dose and the measured serum level was observed in data analysis, and up to 22-foldinterindividual variation in serum levels was revealed. The prospective investigation also demonstratedmarked intraindividual variability of the measured drug concentrations. The intraindividual variabilities of6 consecutive serum level measurements in the 16 patients receiving fixed clozapine doses were 36 ± 20%(range 10-76%) for clozapine, 31 ±21% (range 9-84%) for A/-desmethylclozapine, and 55 ± 16% (range34-85%) for clozapine N-oxide.

Serum levels of clozapine and its two major metabolites were correlated with the plasma levels of thebiogenic amines in these 16 patients. Of the biogenic amines examined, only plasma norepinephrine levelsshowed a significant correlation with the measured serum clozapine levels. Figure 1 shows a regressionanalysis plot for the relationship between the serum levels of clozapine and plasma norepinephrine levels(Pearson correlation coefficient r = 0.52, p = 0.04). The correlation between A/-desmethylclozapine andplasma norepinephrine levels was highly significant (r = 0.64, p = 0.008), whereas clozapine N-oxide failedto show a significant correlation (r = 0.47, p = 0.07).

1600

EQ.

"53>_i(0Ew

Q_LUZ

100 200 300 400 500

Clozapine Serum Level [ng/ml]600

FIG. 1. Relationship between serum clozapine levels and plasma norepinephrine levels. Individual mean values were

calculated from six measurements at consecutive 6-week intervals (n = 16). Regression analysis plot with confidencelimits (hatched lines indicate 95% confidence level). Pearson correlation coefficient r = 0.52, p = 0.04. NE, norepi-nephrine.

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Individual mean values for the biochemical measures (prolactin, serotonin, MHPG, norepinephrine, ep-inephrine), calculated from values at the 6 time points over 36 weeks, demonstrate a significant differencebetween the clozapine-treated patients and the group receiving typical neuroleptic drugs (see Table 2). Overthe course of treatment, the plasma levels of norepinephrine, MHPG, and epinephrine and the serum lev-els of 5-HT were significantly increased in the clozapine-treated group as compared with the convention-ally treated patients. The conventionally treated group exhibited elevated levels of prolactin, whereas theclozapine-treated group demonstrated prolactin levels in the normal range values (<20 ng/mL).Interestingly, patients whose serotonin levels were 50% higher than the upper limit of the normal range

had significantly (median test/? = 0.041) lower scores of negative symptoms on the SANS (Table 3). Also,a significant relationship was found between plasma MHPG and depressive symptomatology (as assessedby BPRS). Patients with plasma MHPG levels that were 50% above the upper normal range limit showedsignificantly (median test p = 0.029) lower scores of depression (see Table 4).

DISCUSSION

The aims of this study were to evaluate the relationships between (1) clozapine dosage and the plasmaclozapine levels, (2) plasma clozapine levels and circulating concentrations of biogenic amines, (3) cloza-pine treatment and conventional neuroleptic therapy in their effects on prolactin levels, and (4) biogenicamine levels and psychopathology measures during clozapine treatment.

Circulating clozapine concentrationsOur measurements of serum clozapine levels in adolescent patients with schizophrenia are in good agree-

ment with results from studies in adults (Haring et al. 1990, Lovdahl et al. 1991, Perry et al. 1991). For ex-ample, using liquid chromatography with photodiode array detection, Volpicelli et al. (1993) reported serum

clozapine levels of ranging from 33 to 528 ng/mL in 25 adult patients receiving fixed doses of clozapine(mean 269 mg daily). This result is similar to our findings in 20 adolescents, whose levels ranged from 24to 732 ng/mL.Also, as in most studies of clozapine in adult patients (Ackenheil 1989, Bondesson and Lindström 1988,

Chung et al. 1993, Gold et al. 1990, Haring et al. 1988, Lovdahl et al. 1991, Volpicelli et al. 1993), wefound a significant relationship of dose to serum level. In the dose range used, serum concentrations ofclozapine and its two metabolites exhibited a strong linear relationship to oral clozapine dose.With respect to interindividual variations in serum clozapine levels, Ackenheil (1989) reported up to a

Table 2. Biochemical Measures in the Two Treatment Groups: Clozapine vs. Typical Neuroleptics3

Typicalneuroleptics

n = 20Clozapinen = 20 Statistics

Prolactin (ng/mL)

Serotonin (ng/mL)

MHPG (ng/mL)

Norepinephrine (pg/mL)

Epinephrine (pg/mL)

Dopamine (pg/mL)

26.7 ± 16.0

146 ± 77.1

2.09 ± 1.28

445 ± 201

83.0 ± 58.2

122 ± 62.0

11.2 ± 6.12

237 ± 110

3.98 ± 0.94

754 ± 292

104 ± 51.4

153 ± 91.1

X2 = 5.02, df = 1,p = 0.025

t test with unequal variancesi2 = -3.0, df = 34,p = 0.005

X2 = 14.04, df = 1,p = 0.0002

X2 = 6.24, df = 1,p = 0.012

X2 = 6.24, df = 1,p = 0.012

n.s.

"Individual values (mean ± standard deviation) were calculated from six measurements at consecutive 6-week inter-vals.

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CLOZAPINE, NEUROTRANSMITTERS, AND SYMPTOMS IN SCHIZOPHRENIA

Table 3. Serum Levels of Serotonin and Negative Symptoms in 40 Young Patientswith Chronic Schizophrenia3

Serum serotoninlevel

Numberof

subjects

Negativesymptoms(SANS)

Mediantest

df= 1

Less than or equal to260 ng/mL

Greater than260 ng/mL

28

12

7.13 ± 2.77

5.14 ± 3.45 0.041

"Individual values (mean ± standard deviation) for negative symptoms were calculated from six measurements at con-secutive 6-week intervals.

20-fold interindividual variation in an adult sample. This finding is congruent with the up to 22-fold in-terindividual variability observed in our sample of adolescents.

Thus, in both adolescents and adults, it appears that blood clozapine concentrations show similar rangeand interindividual variability, and there is a significant relationship of dose and serum level.

Prolactin levels

Our findings confirm that in adolescents as in adults, typical neuroleptic agents differ markedly from theatypical neuroleptic clozapine by increasing plasma prolactin levels during maintenance treatment. Clozapinedid not increase plasma prolactin levels in our adolescent sample during maintenance treatment, consistentwith clinical data and animal studies (Ackenheil 1989, Albinsson et al. 1993, Gudelsky et al. 1987, 1989,Lieberman et al. 1986, Meltzer 1989, 1992b).

Basal prolactin secretion is under inhibitory dopaminergic control but is stimulated by serotonin agonists(Meltzer 1992a,b). Interactions between the dopaminergic and serotonergic neurotransmitter systems maybe relevant to understanding functional mechanisms involved in the etiology of schizophrenic psychosesand the mechanism of action of atypical antipsychotic drugs (Meltzer 1992a).

Neurotransmitter indicesResults in these adolescents demonstrate that during maintenance treatment, clozapine differs from typ-

ical neuroleptics by increasing serum serotonin levels as well as plasma norepinephrine, epinephrine, andMHPG levels. Banki (1978) first described elevated total blood serotonin concentrations due to clozapinetreatment in adults. To the best of our knowledge, this finding has not been previously replicated. In ouradolescent sample, we can confirm that serum serotonin levels were significantly increased in the cloza-pine-treated patients in comparison to adolescents receiving conventional neuroleptic drugs.

Table 4. Plasma MHPG Levels and BPRS-Derived Depression Scores in 40 Young Patients withChronic Schizophrenia3

PlasmaMHPG level

Number ofsubjects

Depressivesymptoms

Median testdf= 1

Less than2.93 ng/mL

Greater thanor equal to2.93 ng/mL

20

20

10.9 ± 3.97

8.28 ± 3.29 p = 0.029

"Individual values (mean ± standard deviation) for negative symptoms were calculated from measurements at consec-utive 6-week intervals.

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The finding of markedly increased plasma norepinephrine levels in adolescent patients during mainte-nance clozapine treatment concurs with the (mostly) short-term studies in adult patients, which report cloza-pine-induced increases in plasma and/or CSF levels of norepinephrine in adults with schizophrenia(Ackenheil 1989, Breier et al. 1994, Green et al. 1993, Lieberman et al. 1991, Pickar et al. 1992, Sarafoffet al. 1979).

Despite the ongoing uncertainty about the proportion of plasma MHPG that derives from the brain, it isclear that plasma measurements of MHPG reflect norepinephrine metabolism and are continuing to be pur-sued as clinical research tools (Green et al. 1993).

Symptoms in adolescent schizophreniaThe relationship between plasma MHPG levels and depressive symptomatology, and the relationship be-

tween serum serotonin level and negative symptoms, reemphasizes the potential role of noradrenergic me-

tabolism and of serotonergic status in the pathophysiology of schizophrenia. The data in this samplesuggest that in adolescents with schizophrenia, negative symptoms may be reflected by lower serotonin lev-els and depressive symptoms may be associated with lower MHPG levels. Although a role of the dopamin-ergic system is often highlighted in schizophrenic psychosis, it may be suggested that both the noradren-ergic and the serotonergic system are also involved as potential targets of atypical antipsychotic drug action.These findings need further elucidation, including investigation of possible relationships between plasmamonoamine levels with observed clinical responses and side effects.Our study was based on the open clinical trials of young patients with chronic schizophrenia who were re-

ceiving maintenance treatment and who had been "naturally selected" into two treatment groups. It is note-worthy that the clozapine-treated patients were initially nonresponders to typical neuroleptic drugs; i.e., dur-ing prior treatments with conventional neuroleptics, they had exhibited higher rating scores on both positiveand negative symptoms. In contrast, during the prospective study, no significant differences between the clin-ical ratings of the two treatment groups were found. Thus, clozapine appeared more effective in neuroleptic-resistant patients than conventional agents were in the general population of adolescents with schizophrenia.

Because plasma monoamine levels appeared possibly related to clinical symptoms, it would be valuableto know whether the difference in the plasma concentrations of biogenic amines observed between the twotreatment groups is partially due to clinical differences of the patients in each treatment group. Unfortunately,the open clinical study design did not allow us to investigate this issue, because some patients received con-

comitant medications to manage side effects of the neuroleptic drugs. However, we have preliminary datafrom an independent study (unpublished) that suggest that concurrent medications did not account for thedifferences in plasma monoamines observed between two similar treatment samples. Additional studies are

needed before firm conclusions can be inferred about the relationship of clinical symptoms and bioaminemeasurements, and further work would be needed to assess the speculation that drug treatment of schizo-phrenia might be enhanced by targeting medication choice to clinically observed symptoms or clinicallyassayed bioamine levels. At present, such a targeted clinical approach has not been empirically tested.

General monitoring of the blood levels of neuroleptic drugs is also not suitable for application in clinicaltreatment, though such measurements might become useful in selected aspects of drug management (e.g., com-pliance, genetic polymorphisms). However, drug level monitoring of clozapine in relation to biogenic aminesseems to be a promising research tool and calls for further prospective controlled clinical trials.

ACKNOWLEDGMENTS

The authors wish to thank Mrs Regina Stöhr, CTA, for her excellent and skillful technical assistance.

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Address reprint requests to:Eberhard Schulz, MD.

Department of Child and Adolescent PsychiatryPhilipps University

Hans Sachs Strasse 6D-35-033 Marburg, Germany

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