Analyzing the Effects of Psychotropic Drugs on Metabolite Profiles in

Analyzing the Effects of Psychotropic Drugs on Metabolite Profiles in Rat Brain Using 1H NMR Spectroscopy Gerard A. McLoughlin,†,# Dan Ma,‡,# Tsz M. Tsang,† Declan N. C. Jones,§ Jackie Cilia,§ Mark D. Hill,§ Melanie J. Robbins,§ Isabel M. Benzel,§ Peter R. Maycox,§ Elaine Holmes,† and Sabine Bahn*,‡ Department of Biomolecular Medicine, Division of SORA, Faculty of Medicine, Imperial College, London, SW7 2AZ, U.K., Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, U.K., and Psychiatry CEDD, New Frontiers Science Park, GlaxoSmithKline, Third Avenue, Harlow, CM19 5AW, U.K. Received October 21, 2008

The mechanism of action of standard drug treatments for psychiatric disorders remains fundamentally unknown, despite intensive investigation in academia and the pharmaceutical industry. So far, little is known about the effects of psychotropic medications on brain metabolism in either humans or animals. In this study, we investigated the effects of a range of psychotropic drugs on rat brain metabolites. The drugs investigated were haloperidol, clozapine, olanzapine, risperidone, aripiprazole (antipsychotics); valproate, carbamazapine (mood stabilizers) and phenytoin (antiepileptic drug). The relative concentrations of endogenous metabolites were determined using high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy. The results revealed that different classes of psychotropic drugs modulated a range of metabolites, where each drug induced a distinct neurometabolic profile. Some common responses across several drugs or within a class of drug were also observed. Antipsychotic drugs and mood stabilizers, with the exception of olanzapine, consistently increased N-acetylaspartate (NAA) levels in at least one brain area, suggesting a common therapeutic response on increased neuronal viability. Most drugs also altered the levels of several metabolites associated with glucose metabolism, neurotransmission (including glutamate and aspartate) and inositols. The heterogenic pharmacological response reflects the functional and physiological diversity of the therapeutic interventions, including side effects. Further study of these metabolites in preclinical models should facilitate the development of novel drug treatments for psychiatric disorders with improved efficacy and side effect profiles. Keywords: NMR • antipsychotic drug • mood stabilizer • antiepilepsy • metabolites • rat

Introduction Both schizophrenia and bipolar disorder are highly prevalent, debilitating and poorly understood psychiatric disorders. A number of medications are currently used in the clinical management of these two mental disorders,1,2 including typical and atypical antipsychotic drugs (such as haloperidol and clozapine, respectively),3,4 used predominantly in the management of schizophrenia, and mood stabilizers (such as valproic acid5 and carbamazapine6,7), used in the maintenance treatment of bipolar disorder. Valproic acid and carbamazapine are also anticonvulsants, as is phenytoin, and are licensed for the treatment of epilepsy,8,9 and several atypical antipsychotics * To whom correspondence should be addressed. Dr, Sabine Bahn M.D., Ph.D. MRCPsych, Cambridge Centre for Neuropsychiatric Research, Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT. Tel: +44 1223 334151. Fax: +44 1223 334162. E-mail: [email protected]. † Imperial College. ‡ University of Cambridge. # These authors contributed equally to this work. § GlaxoSmithKline. 10.1021/pr800892u CCC: $40.75

 2009 American Chemical Society

have been found to have mood stabilizing properties.10 Unfortunately, the pharmacological management of schizophrenia and bipolar disorder represents a “hit and miss” approach. This is most likely due to a combination of both a lack of knowledge regarding the pathophysiology of schizophrenia and bipolar disorder, as well as a lack of full understanding of the mechanism of action of current drug regimes. The development of new antipsychotic drugs for treatment of these psychotic disorders is an important ongoing area of research. This is critical since the response of patients to existing medications can be variable and often includes severe side effects such as weight gain, type II diabetes and tardive dyskinesia.11 Therefore, the most appropriate drugs for individual patients requires careful consideration. Numerous genetic and some functional genomic studies have been undertaken aiming to elucidate the etiology of psychiatric disorders and psychotropic drug action.12-15 However, so far, hardly any studies have explored neuroactive drugassociated metabolic changes. Metabolites are the final products of metabolic pathways directly reflecting biological activities Journal of Proteome Research 2009, 8, 1943–1952 1943 Published on Web 02/02/2009

research articles of a particular tissue. In the brain, their relative concentrations provide insights into neural function and structural integrity. Measuring metabolic alterations in brain tissue following psychotropic drug treatment could, therefore, provide useful information relating to the mechanism of action of drugs and the biochemical pathology of psychiatric disorders. Studies using magnetic resonance spectroscopy (MRS) investigating schizophrenia in patients have identified changes in various brain metabolites, such as a reduction in NAA,16-19 a marker for neuronal integrity, and increased lactate levels,20 an important metabolite reflecting the metabolic status of neurons and astrocytes. MRS studies on bipolar disorder patients have also found regional abnormalities of NAA, choline and glutamate/glutamine in the brain.21 NMR spectroscopybased metabonomics studies in postmortem human brain and rat have also identified metabolic perturbations associated with psychiatric disorders and mood stabilizers including changes in creatine, myo-inositol, and neurotransmitters GABA and glutamate.22 NMR spectroscopy-based metabonomics can be used to monitor a wide range of metabolites in biological samples.23-25 Similar to MRS, this technique can be applied to a large variety of tissues and biofluids, but possesses far greater sensitivity than MRS, thereby enabling improved spectral resolution. NMR spectroscopy has been used extensively in studies on schizophrenia,14,25-27 bipolar disorder22 and other diseases.28 In this study, we used high-resolution 1H NMR spectroscopy to explore and compare, for the first time, metabolite profiles in different brain regions of the rat after treatment with a wide range of antipsychotics (typical, haloperidol; atypical, clozapine, olanzapine,29 risperidone,30 aripiprazole31), mood stabilizers (valproate, carbamazapine) and an antiepileptic drug (phenytoin32). Many of the metabolic alterations that were identified relate to neuronal viability, energy metabolism, neurotransmission and myelin function. Interestingly, both antipsychotics and mood stabilizing agents consistently increased markers associated with neuronal viability/activity (especially NAA). Furthermore, the different drugs and drug classes also showed differential effects on different brain regions. The observed neurochemical alterations and region-specific metabolite patterns are described here and discussed in relation to therapeutic drug effects, as well as side effects.

Material and Methods Animal Drug Treatment and Sample Collection. Adult male Wistar rats weighing 280-300 g (Charles River, Margate, U.K.) were housed 4/cage with access to food (Harlan, Bicester, U.K.) and water ad libitum and were maintained under a 12-h light/ dark cycle (lights on at 0600 h). All experiments complied with the United Kingdom Animals (Scientific Procedures) Act, 1986 and the ethical policies of GlaxoSmithKline (GSK). One of seven drugs or vehicle only (control) was orally administered by gavages in dose volumes of 2 mL/kg once a day for 21 days, n ) 8 rats per treatment group, as follows: vehicle (0.5 mg/mL of tartaric acid in 1% methylcellulose water at 2 mL/kg), haloperidol (1 mg/kg [Sigma, Dorset, U.K.]), clozapine (20 mg/kg), olanzapine (2 mg/kg), risperidone (1 mg/ kg) aripiprazole (40 mg/kg) (Medicinal Chemistry, GSK, Harlow, U.K.), sodium valproate (150 mg/kg), or carbamazapine (20 mg/ kg) (Sigma, U.K.). An eighth drug, phenytoin (Sigma, U.K.), was orally administered (n ) 8) at 40 mg/kg. These doses were chosen based on previous studies conducted at GSK which suggested biological activity.33 1944

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McLoughlin et al. Two hours following dosing on day 21, rats were decapitated. Brains were immediately dissected on ice to obtain the prefrontal cortex, defined as the anterior portion of the frontal cortex (up to 2.15 mm rostral from Bregma),34 the dorsal striatum (dissected from both hemispheres using punches from a 2 mm slice, approximately 2.15-0.10 mm rostral from Bregma)34 and the hippocampus. Tissues were snap frozen in liquid nitrogen and stored frozen at -80 °C until use. Preparation of Tissue Extracts from Rat Brain. Aqueous components from frozen tissue samples were extracted using the same methods as described previously.22 For lipid extraction, a 0.5 mL chloroform/methanol (3:1) solution was added to the pellets. Samples were vortex mixed and left for 10 min to allow extraction of the lipophilic metabolites. Samples were then centrifuged at 4800g for 10 min, after which the supernatants were collected and left overnight to allow evaporation of the chloroform and methanol. For 1H NMR analysis, samples were reconstituted in a 0.5 mL solution of 3:1 deuteratedchloroform/deuterated-methanol. All extractions were performed under randomized conditions. 1 H NMR Spectroscopic Analysis of Brain Tissue Extracts. Tissue extracts were transferred into 5 mm diameter NMR tubes and loaded onto a Bruker AV600 spectrometer (Bruker Avance, Bruker GmBH, Rheinestennen, Germany) and spectrally acquired using the first increment of the NOESY pulse sequence (RD, π/2-t1-π/2-tm-π/2-Acq; TR ) 3 s) using the same parameters as described previously.22 All experiments were performed under blind and randomized conditions. Multivariate Data Analysis. Spectral data were reduced into 0.04 ppm spectral buckets using previously described methods22,24 and exported into SIMCA P (version 11.0, Umetrics AB, Umeå, Sweden). Principal components analysis (PCA) was applied to the data to discern the presence of inherent similarities of spectral profiles. To identify metabolites differentiating treatment groups from control, projection to latent structure discriminant analysis (PLS-DA) was employed.35 Spectra were further analyzed using an integrated orthogonally filtered PLS-DA algorithm (O-PLS-DA)36 on full-resolution spectral data, in order to remove confounding variation and focus solely on the effect of drug treatment. The advantages of analysis on full-resolution data is that it facilitates the diagrammatic representation and identification of the subtlest metabolic changes that attribute to class separation detected by O-PLS-DA, which maximizes biomarker differentiation between two or more classes. However, where chemical shifts are apparent, as a consequence of pH differences, bucketed data may be more appropriate as this approach accommodates small shifts in peak registration. Furthermore, an added confidence was brought by using two different statistical approaches.

Results Multivariate Analysis of 1H NMR Spectroscopic Data from Brain Tissue Extracts. Two representative 1H NMR spectra from the frontal cortex, one from a drug-treated group (aripiprazole) the other from a control group, are shown in Figure 1a labeled with the metabolites differentiating each treatment group. Metabolites found to be altered following a given drug intervention are highlighted in blue (decreased) and red (increased). The PLS-DA scores plot in Figure 1b shows the separation between the drug-treated group and controls, while the O-PLS-DA loadings plot in Figure 1c shows which metabolites are most influential to

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Figure 1. Metabolite changes observed in the frontal cortex of rats following treatment with aripiprazole as compared with control rats. (a) Two representative 1H NMR spectra from frontal cortex from an aripiprazole-treated and control rat. Key discriminatory brain metabolites are indicated. Metabolites found to be increased by aripiprazole treatment are labeled in red, while those found to be decreased are marked in blue. (b) A PLS-DA scores plot (R2X ) 0.47; R2Y ) 0.72; Q2 ) 0.36) shows clear separation between NMR spectra from aripiprazole-treated and control samples. (c) An O-PLS-DA loadings coefficient plot generated from the analysis of the NMR spectra. The plot shows spectral descriptors presenting differences between treatment groups whereby the direction of change is indicated by signal orientation (positive coefficient values correspond to spectral regions positively covarying with drug treatment, while negative coefficient values correspond to spectral regions positively covarying with controls). The color of the signal is proportional to the difference between the drug treated and control animals (based on r2 values). In this plot, the most significant difference between the two treatments is in the concentration of NAA, which is increased in brain extracts of aripiprazole treated animals.

the separation. The goodness of fit (R2X, R2Y) and the goodness of prediction Q2Y values for each PLS-DA and O-PLS-DA model comparing the effect of each of the eight drugs with the control profile for the three neuroanatomical regions are provided in Table 1. Where models showing a clear difference between drug-treated and control rats could be constructed and cross-validated (with Q 2 > 0.3 in either analysis), the metabolites responsible for the difference were identified in the corresponding loadings plot. The Q2Y value is a statistically validated version of the R2Y value and indicates the fraction of the class-related data set that can be successfully predicted by a model. While any Q2Y value > 0 is of predictive relevance, our analysis of these data showed that, in general, the degree of separation between control and drug treated groups after 7-fold crossvalidation using either PLS-DA or O-PLS-DA declined when the Q2Y value of the model fell below 0.3. To maintain comparability between models of different drugs and brain regions, a Q2Y value > 0.3 was used as a cutoff for all models. Region-Specific Effects of the Drugs. The overall analysis revealed that each of the drugs tested affected at least one brain region, while clozapine (Figure 2) and haloperidol induced changes in all three brain regions (Table 1). The frontal cortex was the region most prominently affected, with metabolite changes being observed following treatment with all antipsychotic and mood stabilizing drugs, but not with the anticonvulsant phenytoin (Table 1). Notably, the striatum was the only brain area where phenytoin induced metabolite changes. The mood stabilizer carbamazapine, which is effective as an anti-

convulsant, also induced changes in the striatum. The fewest drug-induced metabolic effects were observed in the hippocampus where altered metabolites were only identified following treatment with haloperidol, clozapine and valproate. In all, the various psychotropic drugs altered the levels of 15 different metabolites, to varying degrees, in a drug-dependent manner. There were similarities in the perturbations caused by each drug or class, particularly within each brain region; however, each treatment ultimately exhibited a specific metabolic profile (Table 2). Metabolite Changes in the Frontal Cortex. As shown in Table 2, levels of NAA were increased by treatment with the antipsychotics haloperidol, risperidone and aripiprazole, and the two mood stabilizers valproate and carbamazapine. A reduction in the levels of its precursor metabolites acetate and aspartate was also consistently observed. The two remaining antipsychotics, clozapine and olanzapine, which did not induce alterations in NAA, acetate or aspartate, were the only drugs to cause a reduction in the levels of glutamate in the cortex. However, the antipsychotics haloperidol, clozapine, and olanzapine, along with valproate and carbamazapine, all led to a reduction in glutamine levels. Haloperidol, clozapine and olanzapine induced an increase in lactate levels, while risperidone caused a reduction. The list of metabolite changes is provided in Table 2. Metabolite Changes in the Hippocampus. NAA levels in the hippocampus were increased by haloperidol, clozapine and valproate, while haloperidol alone also caused decreased levels of N-acetylaspartylglutamate (NAAG). Similarly to the frontal cortex, aspartate levels were reduced by all three of the Journal of Proteome Research • Vol. 8, No. 4, 2009 1945

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Table 1. Rat Brain Regions Affected by Psychotropic Medications O-PLS-DA on full resolution data 2

PLS-DA on bucketed data

drug

2

2

RX

RY

Q

Haloperidol* Clozapine Olanzapine Risperidone* Aripiprazole Valproate Carbamazapine* Phenytoin Haloperidol* Clozapine Olanzapine Risperidone Aripiprazole Valproate* Carbamazapine Phenytoin Haloperidol* Clozapine* Olanzapine Risperidone Aripiprazole* Valproate Carbamazapine* Phenytoin*

0.48 0.79 0.95 0.48 0.66 0.20 0.48 0.76 0.71 0.57 0.30 0.58 0.66 0.23 0.20

0.35 0.81 0.95 0.48 0.54 0.71 0.28 0.86 0.63 0.68 0.90 0.86 0.63 0.96 0.71

0.18 0.51 0.69 0.24 0.39 0.43 0.03 0.71 0.45 0.44 0.35 0.11 0.36 0.58 0.38

2

RX

R 2Y

Q2

brain region

0.47 0.70 0.68 0.39 0.47 0.50 0.55 0.76 0.82 0.35 0.18 0.79 0.24 0.37 0.56

0.70 0.85 0.78 0.82 0.72 0.78 0.74 0.78 0.92 0.36 0.59 0.98 0.36 0.40 0.76

0.34 0.53 0.36 0.35 0.36 0.48 0.34 0.27 0.76 0.06 0.10 0.74 -0.10 0.15 0.27

Frontal Cortex

Hippocampus

Striatum

a Statistical values are given for drug treated vs control spectral data sets analyzed using the multivariate data analysis techniques PLS-DA and O-PLS-DA on bucketed and full resolution data respectively. (*) Denotes those data sets for which predictive models (with Q2 > 0.3) showing separation could be built using only one of either PLS-DA or O-PLS-DA. (-) Denotes no significant model showing separation observed. R2X refers to the fraction of the data set explained by the model; R2Y refers to the fraction of the class-correlated (i.e., drug-treated or control) data explained by the model; Q2 refers to the predictive ability of the model.

aforementioned drugs, but acetate levels remained unchanged. All three drugs also reduced glutamate levels, while only haloperidol and valproate reduced glutamine levels. Lactate levels were changed by haloperidol, clozapine and carbamazapine (see Table 2). Metabolite Changes in the Striatum. As in the frontal cortex and hippocampus, haloperidol and clozapine also showed consistent effects on the metabolite profile of the striatum (Table 1). However, striatal NAA levels were only increased by aripiprazole. Acetate levels were reduced by haloperidol and aripiprazole, while lactate levels were increased by haloperidol and clozapine. The striatum was the only region in which GABA was altered, where haloperidol and carbamazapine increased its levels. The striatum was also the only region in which phenytoin caused an effect (Table 1). Phenytoin was the only drug that decreased NAA levels among all the drug treatments in any brain region. Phenytoin and carbamazapine both increased striatal levels of NAAG and reduced concentrations of glutamate and glutamine (see Table 2). Lipid Extracts. Analysis of lipid extract spectral profiles did not reveal any significant differences in any brain region between control and drug-treated animals (data not shown).

Discussion In this study, we employed high-resolution 1H NMR spectroscopy to compare the metabolic effects of a wide range of antipsychotic drugs, mood stabilizers as well as an antiepileptic drug. We investigated different regions of the rat brain with regard to drug-induced metabolic changes and identified both distinct and shared neurometabolic profiles in association with different drug treatments. These drug-related metabolic profiles 1946


provide novel insights into the mechanism of action and associated therapeutic effects of diverse classes of various psychoactive drugs. 1. Drug Pharmacology and Region-Specific Effects of the Drugs. The pharmacological basis of antipsychotic drug action is thought to be exerted through antagonistic action at various neurotransmitter receptors.2,37,38 Typical antipsychotic drugs are thought to predominantly block dopamine D2 receptors in subcortical brain regions, while atypical antipsychotic drugs exhibit a preferentially affinity to both D2 and 5-HT 2A receptors in the cortex.2,38 Aripiprazole is a D2 partial agonist in contrast to the other antipsychotics which are D2 antagonists.39 The exploration of region-specific metabolite patterns in rat brain following subchronic antipsychotic drug treatment may reveal further detailed responses in neural metabolism that are linked to therapeutic drug action. In this study, we found that each drug caused region-specific metabolic effects and both haloperidol and clozapine induced significant changes in all three brain regions investigated. Clozapine is arguably the most effective antipsychotic drug showing effectiveness in otherwise treatment-resistant schizophrenia patients and ameliorating positive, negative and cognitive symptoms. Its effect on metabolites in multiple brain regions might be associated with this superior effectiveness. The remaining atypical antipsychotic drugs predominantly induced changes in the frontal cortex. This is consistent with the observation that atypical antipsychotics preferentially increase cortical dopamine efflux40 and may explain their effect on negative and cognitive symptoms.11 The lower efficiency of haloperidol on initial treatment and its lack of effect on negative and cognitive symptoms, despite


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Figure 2. Multivariate statistical analysis of NMR spectra generated from extracts of the frontal cortex, hippocampus, and striatum of rats treated with clozapine as compared to control rats. (a) A PLS-DA scores plot (R2X ) 0.76; R2Y ) 0.53; Q2 ) 0.40) showing movement in metabolic space caused by tissue region and clozapine treatment. The second principal component t[2] shows separation between tissue regions, while the third t[3] and fourth t[4] principal components combined show separation between clozapine-treated and control rats. (b) O-PLS-DA loadings coefficient plots generated from analysis of spectra from different regions for clozapine-treated rats as compared with control rats. The plots show spectral descriptors presenting differences between treatment groups, whereby the direction of change is indicated by signal orientation (see Figure 1). The color of the signal is proportional to the difference between the drug treated and control animals (based on r 2 values). In the frontal cortex plot, the most significant difference between the two treatments is in the clozapine-induced decrease in the relative concentration of glutamate.

altering metabolism in all three brain regions, might reflect its primary focus on D2 antagonism. These drug-specific metabolic changes could reflect the fact that each compound reacts to varying degrees with different receptor sites localized in different cells and brain regions.2 Although valproate and carbamazapine possess anticonvulsant properties, they are predominantly used for the treatment and relapse prevention of bipolar affective disorder. The pharmacological basis of their mechanistic action has been investigated; some studies have found that mood stabilizers alter GABAergic neurotransmission, modify the monoamine system9,41,42 and induce neuroprotective effects.43,44 In the present study, valproate and carbamazapine both induced metabolic changes in the frontal cortex, while the latter also affected striatal metabolism. Both brain regions are known to be involved in mood regulation.45 Phenytoin, which is also an antiepileptic drug, but is not licensed for the treatment of bipolar disorder, only induced metabolic changes in the striatum. We have also used phenytoin as a negative control in cellular assays where it exerts a distinct profile in comparison with drugs that are mood stabilizers and anticonvulsants (data not shown). The overlap of the metabolic effects of atypical

antipsychotics and mood stabilizing drugs is of particular interest as several atypical antipsychotics have been licensed for the treatment of bipolar disorder, although not always for the same symptoms.46 2. Drug Effects on Neuronal Function. 2.1. Neuronal Viability and NAA. NAA is a marker of neuronal viability and activity, although its functional role in the brain is not fully understood.47,48 NAA has been suggested to act as an organic osmolyte in the nervous system, a source of acetate for myelin lipid synthesis in oligodendrocytes, and a precursor of NAAG. It is also thought to be involved in energy metabolism in neuronal mitochondria.48 NAA and NAAG are primarily synthesized in neurons. Upon release, NAA is taken up by oligodendrocytes and NAAG by astrocytes where both molecules are hydrolyzed.47,48 NAA is a prominent metabolite that has consistently been found to be reduced in the brains of schizophrenia and bipolar disorder patients.16-18,21,49 This change is thought to be more profound in first-episode patients compared to chronically medicated patients.50,51 A negative correlation between the severity of symptoms and NAA levels in the frontal cortex of schizophrenics may suggest that NAA could be an indicator of Journal of Proteome Research • Vol. 8, No. 4, 2009 1947

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Table 2. Summary of the Metabolite Changes Caused by Oral Treatment with Different Psychotropic Drugs in Three Different Rat Brain Regionsa antipsychotics haloperidol

mood stabilizers clozapine

olanzapine

risperidone

anticonvulsant

aripiprazole

valproate

carbamazapine

phenytoin

v V V V v v V

v V V V V V V v V

v V V V v -

-

NAA NAAG Acetate Aspartate Glutamate Glutamine GABA Taurine Alanine Lactate Creatine Myo-inos Scyllo-inos Choline Phosphocholine

v V V V V v V v V

V V V v v V

V V v V v v V

(a) Frontal Cortex v V V V V V V v v V


v V V V V V V V V V -

v V V v v V -

-

(b) Hippocampus -

-

v V V V V V V -

-

-


V v v v V V v -

v v v v v -

-

(c) Striatum -

v V V V v v V

-

v V V v v V v -

V v V V V v v -

a Key: (v) increases, (V) decreases, (-) no change. NAA (N-acetyl-aspartate), NAAG (N-acetyl-aspartyl-glutamate), GABA (γ-amino butyric acid), Myo-inos (Myo-inositol), scyllo-inos (scyllo-inositol). (a) Frontal cortex; (b) hippocampus; (c) striatum.

disease severity.52 Szule et al.53 reported that positive symptoms before drug treatment correlated positively with NAA levels in the frontal cortex and negatively with NAA levels in the temporal lobe of schizophrenia patients. Other studies have observed no change in NAA levels in chronically medicated schizophrenia and bipolar patients which may be attributed to the normalizing effect of drug treatment, although this remains a controversial assumption.51,54 The effects of antipsychotic exposure on NAA and other metabolites have been examined in both human and animal studies by MRS. Manic adolescents treated with olanzapine showed an increase in NAA levels in the prefrontal cortex.55 Risperidone-induced increases in NAA levels have been reported in the thalamus of schizophrenia patients.53,56 In rats treated for 6 months with haloperidol, no changes were observed in multiple brain regions,57 and short-term (1 week) treatments of haloperidol, clozapine, or olanzapine did not 1948


change NAA levels in rat brains.58 However, a study using HPLC found NAA levels to be increased in rat striatum after exposure to haloperidol for 6 months.59 With regard to mood stabilizers, chronic treatment with lithium, but not sodium valproate, has been shown to increase cortical NAA levels in euthymic bipolar patients.44 Valproate has, however, been observed to be able to increase the NAA/ choline ratio in the hippocampus of bipolar patients.50 By contrast, we reported that neither lithium nor valproate altered NAA levels in the frontal cortex of chronically treated rats.22 These conflicting results might reflect differences in dosage, duration of treatment, and the treatment method. In the present study, we observed a consistent up-regulation in NAA levels by all drugs investigated, with the exception of olanzapine and phenytoin, in at least one brain region. This effect was most noticeable in the prefrontal cortex (5 out of 7 antipsychotics/mood stabilizers). This consistent increase in

Metabonomic Study of Psychotropic Drugs in Rat Brain NAA levels suggests that various classes of psychoactive drugs may improve neuronal viability and activity which is likely to be impaired in the schizophrenia and bipolar disorder brain. Interestingly, the anticonvulsant phenytoin was the only drug in our study associated with a decrease in NAA levels. NAA has been shown to be reduced in epilepsy patients in different brain regions.60,61 The striatum is not likely to be a prominent target for antiepileptic drugs; therefore, this decrease may not be very informative with regard to its mechanism of action and may even be related to the drugs side effects. This finding may also provide some insight into why, unlike the other anticonvulsants, phenytoin is not effective in the treatment of bipolar disorder. 2.2. Energy Metabolism. An increasing number of studies suggest that abnormalities in brain energy metabolism and mitochondrial function are associated with the pathogenesis of schizophrenia and bipolar disorder.14,54,62,63 Novel insights into the biochemistry and physiology of cerebral energy metabolism indicate that the regulation of brain energy metabolism is under the control of an intimate dialogue between astrocytes and neurons. Lactate is no longer viewed as a mere end product of anaerobic glycolysis, but as an oxidative substrate for brain energy metabolism and a possible alternative to glucose under certain conditions. It is the major product of cerebral glycolysis and a substrate for the mitochondrial tricarboxylic acid cycle.64 Recent studies have revealed increased lactate levels in the brain of schizophrenia and bipolar disorder patients suggesting a disease-related glycolytic shift.27,65 This may indicate a disturbed neuronal glucose metabolism associated with the psychiatric disease pathophysiology. There may be several possible reasons for this observation including an abnormal surge in the rate of glycolysis, an increase in anaerobic metabolism (due to impaired blood supply or impaired mitochondrial function), a decrease in the usage of lactate by neurons, or perhaps some other abnormality in energy metabolism. In the present study, we found that three of the antipsychotic drugs (haloperidol, clozapine and olanzapine) increased lactate levels in most of the brain regions investigated, while risperidone decreased levels in two brain regions. Of the mood stabilizers, only valproate altered lactate concentrations and only in the hippocampus. This suggests that antipsychotics may specifically target pathways related to glucose metabolism. Some studies have shown that energy consumption is mainly devoted to glutamatergic neurotransmission and that glutamateglutamine cycling is coupled to glucose oxidation in the cerebral cortex.66-68 As its synthesis is closely linked to glucose metabolism, glutamate may be indirectly involved in the regulation of brain energy metabolism. Indeed, glutamate/Na+ uptake into astrocytes is a key trigger signal for glucose utilization.66,68 In the present study, we found that nearly all drugs investigated, with the exception of risperidone and aripiprazole, decreased glutamate levels in at least one of the three brain regions analyzed, which is opposite to the changes that have been observed in schizophrenia and bipolar disorder patients.66 The creatine/phosphocreatine cycle is closely linked to ATP production and utilization, functioning as an alternative energy source. The reported increase in creatine levels in the brain of schizophrenia and bipolar disorder patients is consistent with impairment in brain energy production/consumption.69,70 We found that haloperidol, olanzapine, risperidone, aripiprazole and valproate reduced creatine levels in rat brain, again

research articles suggesting that their respective drug actions may compensate for underlying alterations in energy metabolism. However, clozapine showed an increase in creatine levels in both the frontal cortex and the striatum, indicating that it has a possible alternate effect on energy metabolism. 2.3. Multiple Effects on Neurotransmitter Metabolism. Glutamate is the main excitatory neurotransmitter in the brain; the glutamate hypothesis is one of the most prominent explanations on molecular mechanisms of schizophrenia.13 The glutamate-glutamine cycle between neuron and astrocyte is central to normal brain function and the rate of this cycle is indicative of glutamatergic activity.71 Increased levels of glutamine have previously been found using 1H MRS in the left anterior cingulate and thalamic regions of first-episode schizophrenia patients,72 while decreased levels of glutamate and/or glutamine have been found in chronic or medicated patients.16,72 In addition, a previous metabonomic study in our laboratory showed increased levels of glutamate in postmortem bipolar brain.22 In this study, we found that glutamate and glutamine levels were reduced in at least one brain region following treatment with the antipsychotics haloperidol, clozapine, olanzapine, both mood stabilizers and the anticonvulsant phenytoin. This is consistent with in vivo observations in chronically medicated schizophrenia patients,73 and opposite to the observation in our bipolar disorder study.22 Aspartate is another important excitatory neurotransmitter that is associated with glutamate neurotransmission. We found aspartate levels to be consistently reduced in at least in one brain region following treatment with all drugs with the exception of olanzapine. GABA is a major inhibitory neurotransmitter which has been linked with the mechanism of action of both mood stabilizers and antiepileptics. In this study, GABA concentrations were increased in the striatum by the mood stabilizer/antiepileptic carbamazapine. Atypical antipsychotics did not affect GABA levels, but an increase was induced by the typical antipsychotic haloperidol in the striatum. This might reflect the pharmacological and therapeutic differences between typical and atypical antipsychotics. Acetylcholine is synthesized in neurons from acetyl-Co A and choline and has been reported to be involved in the mechanism of action of atypical antipsychotic drugs74,75 and anticonvulsants.76 We found choline levels were consistently up-regulated by all drugs with the exception of valproate, indicating a possible preferential effect on acetylcholine metabolism by antipsychotic drugs. 2.4. Oligodendrocyte Function. NAA and the NAA/NAAG pathway are involved in myelin function and synthesis, where NAA provides acetate for oligodendrocyte membrane formation.48 Impairment in myelin-associated functions has previously been linked to the neuropathologies of schizophrenia and bipolar disorder.19,49,77 We found consistent increases in NAA levels in at least one brain area caused by antipsychotic drugs and mood stabilizers, with the exception of olanzapine, while a similarly consistent decrease in acetate levels was observed with the exception of olanzapine and clozapine. Levels of aspartate, a precursor to NAA, were also consistently reduced where NAA levels were increased. These changes reflect the involvement of psychotropic drugs in multiple steps of the NAA/NAAG pathway. Myo-inositol has several naturally occurring isomers including scyllo-inositol, and is known to be a marker of membrane turnover. It is more abundant in glia than in neurons and so Journal of Proteome Research • Vol. 8, No. 4, 2009 1949

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can be considered as a marker for glial integrity. Some studies have shown altered myo-inositol levels in the frontal cortex of bipolar and schizophrenic patients.22,79 Chronic sodium valproate treatment has also been found to reduce myo-inositol levels while increasing the concentration of inositol monophosphates in rat brain.22,80 In this study, we found a consistent reduction of myo-inostitol in different brain regions following treatment with haloperidol, clozapine, risperidone and valproate, and consistently elevated levels of scyllo-inositol in all but the haloperidol- and phenytoin-treated rat groups. These findings suggest that antipsychotics and mood stabilizers may be involved in the modulation of glial structure through inositol metabolism. 3. Drug Effects on Brain Lipids. Abnormalities in the brain lipid metabolism of schizophrenic81 and bipolar disorder82 patients could potentially bring about changes in membrane structure and function leading to altered cell signaling molecules and the perturbed behavior of neurotransmitter systems.83 Antipsychotic drugs have been reported to be able to normalize membrane lipids observed in drug-treated schizophrenic patients.84,85 By contrast, no significant effects on membrane lipid spectral profiles were observed for all 8 drugs examined in this study. This is consistent with a study by Levant et al.86 which reported that similar haloperidol and clozapine treatment in rats did not show disturbances in neuronal membrane phospholipids or fatty acid composition. Thus, the present analyses indicate that antipsychotic drugs might not directly alter brain lipid metabolism in normal adult rats following subchronic treatment with these particular eight drugs at the treatment dosages and durations used in this study. Emerging technologies may allow for improved sensitivity providing a more conclusive determination of brain lipid changes in the future; however, it may simply be the case that antipsychotic drugs only ameliorate defective lipid metabolism in the brains of a subset of psychiatric patients.

Conclusion In this study, we explored and compared the effects of a wide range of antipsychotic drugs, mood stabilizers and an antiepileptic drug on their respective metabolite profiles in several regions of the rat brain. We found that antipsychotics and mood stabilizers affect multiple metabolites showing both common and distinct profiles. All drugs, except olanzapine, consistently altered levels of the neuronal marker NAA, suggesting that they modulate NAA associated pathways, which in turn may improve disease-associated deficits in neuronal viability and myelin dysfunction. Furthermore, most of the investigated drugs modulate glucose/energy metabolism, glutamate neurotransmission and glial cell functioning as illustrated by alterations in the levels of lactate, creatine, glutamate, glutamine, myo- and scyllo-inositols. Since the response of patients to existing medications can be variable and often includes severe side effects, increased insight into the mechanism of current antipsychotic drugs and the development of new therapeutic approaches for the treatment of schizophrenia and bipolar disorder is of great importance. Further investigations surrounding the metabolic markers identified in this study should help to elucidate the mechanism of action of these drugs and help to explain the molecular basis of their therapeutic effects and associated side effects.

Acknowledgment. This research was supported by the Stanley Medical Research Institute (SMRI). G. A. McLoughlin 1950


was supported by METAGRAD (Astra Zeneca, Unilever, and Servier). Dr. D. Ma is supported by SMRI. We thank Dr. J. Huang for his scientific advice.

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