J Lab Med 2014; aop

Review

Katharina M. Rentsch* An update on therapeutic monitoring and pharmacogenetic testing for the optimization of therapy with psychiatric

DOI 10.1515/labmed-2014-0032 Keywords: ; ; cyto- Received September 28, 2013; accepted January 28, 2014 chrome; pharmacogenetics; psychiatric medication.

Abstract: Therapeutic drug monitoring of psychiatric medication as well as pharmacogenetic testing is per- formed more and more frequently in numerous labora- Introduction tories. In this review, a summary of the literature in the years 2011 and 2012 has been completed. The guidelines The Therapeutic Drug Monitoring (TDM) of psychophar- of the German AGNP (Association for Neuropsychop- maceutical drugs is carried out in numerous laboratories harmacology and Pharmacopsychiatry) contain all the to an ever more frequent extent, as are pharmacogenetic information needed for the interpretation of drug concen- studies. Based on numerous studies that have shown that trations. The determination of in urine could pharmacogenetic polymorphisms, among other factors, be a marker for the assessment of the response of antide- cause large inter-individual differences in the pharma- pressants, and correlations between the occupancy of the cokinetics of psychopharmaceutical drugs, doctors who target receptors in the brain and drug concentration have prescribe such drugs increasingly order TDM tests. As part been established using positron emission tomography. of the diagnostics updates 2013 [1] a compilation of the The influence of age on drug concentrations has been literature on the above topics published in 2011 and 2012 controversially described, and additionally females have was prepared, and the individual papers and studies were always showed a slower and higher serum evaluated. This review article is based on this literature concentrations. Several liquid chromatography-mass compilation. spectrometry (LC-MS)/MS multi-analyte procedures for the quantification of psychiatric medication have been described. All methods showed good validation data, but there have always been some compounds with less good Basis for the therapeutic drug moni- validation results due to the fact that not all compounds toring of psychopharmaceutical of a multi-analyte procedure can be analyzed optimally. Pharmacogenetic testing is not routinely performed drugs prior to the prescription of psychiatric medication. This The Association for Neuropsychopharmacology and Phar- relies, among other things, on missing large randomized macopsychiatry (AGNP) published an update of its con- trials and the absence of standardized analytical meth- sensus guidelines on TDM in psychiatry at the end of 2011 ods, which allow the identification of the whole genetic [2]. These guidelines contained recommendations for all variability. steps of TDM with respect to psychoactive substances. The guidelines defined the term “therapeutic ref- erence range”. When falling below the lower limit, a response to treatment is improbable, and when the upper *Corresponding author: Katharina M. Rentsch, Clinical Chemistry, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland, limit is exceeded, any further therapeutic improvement Phone: +0041-61-265-42-36, Fax: +0041-61-265-53-33, E-Mail: becomes impossible. In addition, warning limits were [email protected] specified for laboratories, above which the doctor should 2 Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs be informed immediately that an overdose has occurred. before the next dose). Any order should always contain It was further specified for all drugs described whether information on the patient, the diagnosis, medication therapeutic drug monitoring was highly recommended, and therapeutic outcome. Laboratories should use a recommended, useful or maybe useful. The “highly rec- validated method for analysis, which is used to conduct ommended” category was set aside for all drugs for which both internal and external quality checks. The findings controlled clinical studies had demonstrated beneficial report should list the concentration of the drug (and any effects of TDM. In case of a sub- or supertherapeutic level, active metabolites), the unit, a reference range, as well as the dose should be adjusted until it is within the therapeu- an interpretation of the results. The ordering physician tic reference range again. The concentrations for drugs in should then incorporate the results into the further treat- the “recommended” category were arrived at by way of ment of the patient. If necessary, the dose may be adjusted plasma concentration measurements in connection with or the medication may be changed. Finally, recommenda- therapeutic doses of the drugs that were assigned to clini- tions on and help with interpretation were provided, as cal effects. The “useful” category contained therapeutic well as recommendations regarding the combination of reference ranges where drug concentrations were derived TDM with pharmacokinetic tests [2]. from pharmacokinetic studies. The “maybe useful” cat- These guidelines represent an extensive and widely- egory, finally, listed the drugs for which plasma concen- covered compilation of the literature published on this trations did not correlate with the clinical effects due to broad topic. They should be read by every laboratory the unique pharmacological effect of the drug or for which that analyzes psychopharmaceutical drugs and by every the dose could be clearly derived from clinical symptoms doctor ordering such analyses, and become part of clinical (e.g., sedatives inducing sleep) [2]. daily routine. In the case of psychopharmaceutical drugs, the drug concentration should be determined at the start of therapy or at the time of dose modifications if the therapeutic ref- erence range is well defined or if the therapeutic index Pharmacokinetic parameters of psy- is narrow. In the event of suspected non-compliance, a chopharmaceutical drugs determination of all psychopharmaceutical drugs may be useful, regardless of the recommendations for TDM. This The article by Patteet et al. [3] contains a compilation is also true if the therapeutic effect is absent. If the drug of the most important pharmacokinetic parameters of concentration is within the therapeutic reference range in common , which are necessary for the such a situation, a different treatment should be consid- determination of drug concentrations and also the inter- ered. In the presence of adverse drug reactions, a deter- pretation. The effect of the first-generation antipsychot- mination of the drug concentration can show whether ics was traced back mainly to the strong antagonism of the dose is too high and should be reduced. If a patient the D2 receptor. An antipsychotic effect was requires various drugs that are metabolized by the same achieved with an occupancy of the D2 receptor in the stri- cytochrome enzymes, interactions in terms of induction atum of 65%–70%; with an occupancy of > 80%, the risk or inhibition of the metabolism can occur relative to the of extrapyramidal side effects (EPS) increased. Thus, the individual drugs prescribed. If one of the drugs involved therapeutic window with a minimal risk of EPS was at a D2 affects the metabolism of another drug, the concentra- occupancy of between 65% and 80%. The second-genera- tion of the affected drug should be analyzed in order to tion antipsychotics were different mainly due to the block- detect levels of the active ingredient that are either too ing of various dopamine or serotonergic receptors (with high or too low. The working group of AGNP recommends the exception of and ). Here, too, an that the drug concentration should be determined every occupancy of the dopamine receptors of between 65% and 3–6 months in connection with long-term psychophar- 80% resulted in therapeutic success without adverse side maceutical treatment to prevent a recurrence. Apart from effects of the drug. was given as an exception, that, TDM should be performed for special patient groups, where a receptor occupancy of as little as 30%–40% was such as pregnant women, children and adolescents or associated with a therapeutic effect (Table 1). older patients [2]. According to the authors [3], the psychotic symptoms AGNP recommended TDM as routine monitoring change after the initiation of treatment with antipsychot- according to the above indications and also when specific ics only slowly. If there has been no improvement after 1–4 problems occur. Blood samples should always be taken weeks from the start of therapy, an increased dose or a as steady-state trough concentrations (i.e., immediately change of drug will have to be considered. Antipsychotics Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs 3

Table 1 Main pharmacokinetic parameters of antipsychotics (adapted from [3]).

Antipsychotic Therapeutic range, ng/mL Protein binding, % Half-life, h Bioavailability, %

Amisulpride 100–320 17 12–20 48 150–500 > 99 60–80 87 2–5 – 24 35 12–15 90 20–36 30 Clozapine 350–600 95 12–16 24–50 1–10 99 20–40 40 1–10 90 12–36 60–70 5–10 93 18–33 96 – > 99 12–37 9–19 20–80 93 30–60 60 20–60 74 23 28 0.6–2.4 – 8–12 60–80 15–20 – 23–43 50 100–400 – 17–22 – 100–500 83 7 70 20–60 89 3–24 70 50–100 50–100 55–90 75 Sulpiride 200–1000 40 8–14 25–35 4–50 4–50 15–24 44

lead to a number of adverse drug reactions, the most 140 important being EPS. These include Parkinson-like symp- 120 toms, dyskinesia, dystonia and akathisia. Sometimes it is 100 very difficult to make the differential diagnosis between 80 EPS, negative symptoms and depression. Tardive dyski- 60 nesia is an adverse drug reaction that is very severe and usually irreversible. It can result from long-term treatment 40 Plasma concentration with antipsychotics. A common endocrine effect of the 20 antagonism is an increase in prolactin 0 levels, associated with amenorrhea, erectile dysfunction, infertility and reduced libido [3]. With inducer With inhibitor Monotherapay Without inducerWithout inhibitor After dose increase After dose decreaseCombination therapy

Interactions and monitoring of Figure 1 Effect of inhibitors and inducers on the drug concentra- tion when administered either in monotherapy or when omitted in antipsychotic drugs combination therapy (adapted from [4]).

In the context of an individualized antipsychotic phar- macotherapy, many patients require more than one drug, because of their inadequate response to monotherapy. be analyzed with respect to their status as a substrate, Other patients may require, as a result of comorbidity, inhibitor, or inducer of (CYP) isoenzymes drugs from other pharmacological classes. But combina- (Table 2). Not only drugs affect the activity of CYP isoen- tions of drugs can lead to pharmacokinetic or pharmaco- zymes; for example grapefruit juice, too, is a potent inhibi- dynamic drug interactions. Pharmacokinetic interactions tor of CYP3A4. Psychopharmaceutical drugs themselves lead to an increase or a decrease in the drug concentration. have not been described as inducers of CYP isoenzymes. A The article by Hiemke et al. [4] summarized these issues very potent inducer of CYP1A2 and CYP3A4 is rifampicin, very nicely. The influence of inhibitors and inducers on the and St. John’s wort is a potent inducer of CYP3A4. drug concentration is illustrated in Figure 1. When looking For pharmacodynamic interactions, it is particularly at pharmacokinetic interactions, antipsychotics need to important to look at the effects on the dopamine D2, 4 Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs

Table 2 Antipsychotics with the enzymes involved in the degradation and/or whose activity is inhibited by the drug (adapted from [4]).

Drug Metabolizing enzymes Inhibiting enzymes

Amisulpride Not metabolized Aripiprazole CYP2D6, CYP3A4 Asenapine CYP1A2, NGT Unclear Bromperidol CYP3A4 CYP1A2, CYP2D6 CYP2D6 Clozapine CYP1A2, CYP2C19, CYP3A4 Flupentixol CYP2D6 CYP2D6 CYP2D6 CYP1A2, CYP2C19 Fluspirilene Unclear Haloperidol CYP2D6, CYP3A4 Iloperidone CYP2D6, CYP3A4 CYP1A2, CYP2D6 CYP2D6 Unclear CYP2D6 Olanzapine CYP1A2, CYP2D6, NGT Paliperidone Not metabolized CYP1A2, CYP2C19, CYP3A4 CYP1A2, CYP2C19 Perphenazine CYP1A2, CYP2C19, CYP2D6, CYP3A4 CYP2D6 Pimozide CYP1A2, CYP3A4 Pipamperone Unclear Unclear Quetiapine CYP3A4 Risperidone CYP2D6, CYP3A4 Sertindole CYP3A4, CYP2D6 Sulpiride Not metabolized CYP1A2, CYP2C19, CYP2D6, CYP3A4 CYP2D6 CYP3A4, aldehyde oxidase CYP1A2, CYP2D6, CYP3A4 Zuclopenthixol CYP2D6

Main degrading enzymes are italicized. CYP, cytochrome P450; NGT, n-glucuronosyl transferase. Not metabolized = major portion of the drug is excreted unmetabolized. H2 and M1 receptors. If additive part of a successful therapy are generally better markers pharmacological effects on these target structures are for therapy optimization, since they reflect the pharma- generated, adverse drug reactions, such as EPS, dizziness, cokinetics and , that is, the effect of metabolic disorders with weight gain and heart problems, the drug. cognitive impairment, delirium or ventricular arrhyth- Nichkova et al. [5] have developed a test for determin- mias, may occur [4]. ing the concentration of serotonin in urine as a poten- The determination of drug concentrations in all cases tial biomarker for depression. Most antidepressants, of drug interactions is a valuable tool for dose adjustment. monoamine oxidase inhibitors and 5-hydroxytryptophan (5-HTP) have one or more biogenic as a biologi- cal target, such as serotonin. These are critical molecules Monitoring of in metabolic pathways that affect mood and depressive expression. Normally, serotonin secretion in urine reflects therapy with biomarkers or imaging the release from the enterochromaffin cells in the intes- techniques tine. But also the synthesis in the kidney and the release from platelets may also include a release from the brain. The determination of drug concentrations represents only In the case of neurological diseases, the concentration a surrogate marker for the assessment of an adequate drug of serotonin is partially increased in urine, plasma and/ dosage. Biomarkers that change their concentration as or platelets, which happens, for instance, with autism Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs 5 and when taking selective serotonin inhibitors effect occurred. For selective serotonin/ (SSRI). In depressed patients, the serotonin concentration reuptake inhibitors, only the binding of the drugs to the is reduced in the body fluids mentioned. SERT was examined in the PET studies. Typically, at least The authors of this study developed an ELISA test, 80% of the SERT were occupied at the therapeutically which enabled them to determine the concentration of effective plasma concentrations. Of the antide- serotonin in urine samples. The validation data showed pressants, only was tested for binding to good levels of precision and accuracy and a very good cor- the SERT. In this context, the PET studies found a binding relation with the LC-MS/MS method. For a small cohort of of 80% of the SERT at much lower doses and plasma con- 13 patients, they showed that depressed patients’ seroto- centrations than are normally used today. However, the nin levels in urine increased significantly following the therapeutic active principle of tricyclic antidepressants is start of antidepressant treatment with SSRIs from 39.2 ± 2.4 not an inhibition of the serotonin reuptake. Therefore, the μg/g creatinine to 183.4 ± 53.2 μg/g creatinine. A similar binding of clomipramine to SERT might represent only an picture emerged after treatment with 5-HTP [5]. epiphenomenon [6]. Since there is as yet no easy way in the laboratory to The selective norepinephrine reuptake inhibitors and monitor the success of an anti-depressive therapy, deter- dopamine reuptake inhibitors bind only to the norepi- mining serotonin in urine using an immunoassay would nephrine transporter (NET), and only in a small propor- be a technically simple way to obtain at least an indication tion to the dopamine transporter (DAT) [6]. A summary of of the pharmacodynamic effects. The results of the small the PET studies is shown in Table 3. patient study are promising, to the effect that serotonin may be a good biomarker for the diagnosis and/or therapy monitoring with SSRI and 5-HTP. Positron emission tomography (PET) technology is an Studies on factors influencing interesting way to represent the composition of the target the concentration of psychotropic receptors for psychoactive drugs in the brain. Grunder et al. [6] have summarized numerous PET studies in a drugs review article conclusively and clearly. The following two studies retrospectively examined the PET technology is already being used in drug develop- influence of age and gender on the serum concentrations ment to determine the doses for new drugs. In addition, of antidepressants. The patient groups varied in size, and the of psychotropic drugs, in combina- the results matched only partially. Both studies showed tion with PET, can represent important information about the relationship between blood concentrations of the drug and the occupied target molecules (e.g., receptors, trans- Table 3 Summary of the concentrations of psychotropic drugs from porters) over the course of time. This allowed for the pos- the PET studies (adapted from [6]). sibility of calculating the pharmacodynamic drug action in the brain, which constitutes the basis for developing Drug Therapeutic Risk of EPS, dosing strategies [6]. effect, ng/mL ng/mL Using aripiprazole, it has been shown that the Aripiprazole > 100a > 300 a dopamine D2 receptors have to be almost fully occu- Clozapine 350–600 a pied for an antipsychotic response. This is achieved Haloperidol 1–10 Olanzapine 15–20 > 50 > through plasma concentrations 100 ng/mL. There was Quetiapine 100–500a no upper limit at which the risk of EPS increased; the Risperidone 10–15 > 40–50 probability of side effects was based on individual sensi- Ziprasidone > 50a > 200–250 tivities. But it was unclear whether concentrations > 300 / 50–110a a ng/mL could yield any additional benefit at all. In the 120–500 Fluvoxamine Unclear case of clozapine, D2 receptor occupancy in the striatum 30–120a was relatively low at the recommended plasma concen- 10–150a trations between 350 and 600 ng/mL. The blockage of 30–120a D2 receptors in other brain areas, however, seemed to be 100–400a significantly higher [6]. Clomipramine Unclear For SSRIs, the serotonin transporter (SERT) had to be 225–1500 about 80% occupied by the drug, so that an anti-depressive aRecommended by AGNP [2]. 6 Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs that higher dose-corrected serum concentrations were or more drugs. In this study, 32,126 serum concentrations measured for some in elderly patients and of antidepressants in 17,930 patients were evaluated ret- that women had higher dose-corrected serum concentra- rospectively at the Center for Psychopharmacology in tions for a number of drugs. However, the same drugs in Oslo [8]. the two studies were not always assigned to the same cat- Only small differences in the ratio of concentration/ egories. These contradictory results may surely be attrib- dose were found in patients aged < 40 (controls) and uted to the collectives of various sizes and mainly to the 40–50. But in the oldest age group, the differences were retrospective approach of the two studies, because con- significantly higher when compared to the controls. The founding factors can never be ruled out in retrospective difference for citalopram, escitalopram, fluvoxamine, par- studies. oxetine and notriptyline was about two times greater than in the controls; approximately 1.5 times for , a) Effect of age and gender on serum concentrations clomipramine, duloxetine, , , ser- of antidepressants: The individuals examined as part of traline, and venlafaxine. Only in the case of fluoxetine clinical studies are usually selected carefully, and women and was no difference found relative to the and polymorbid patients are excluded, for example. But controls. In addition, for all medications except amitrip- in clinical practice, following admission, patients of either tyline, and fluvoxamine, significantly higher sex, of different ages and with various comorbidities are concentration/dose ratios were encountered among treated with multiple drugs (polypharmacy). In this study, women than in men [8]. 2891 serum concentrations of various antidepressants were The authors postulated a reduced hepatic blood flow studied retrospectively at the Department of Psychiatry, due to the reduced cardiac output, a reduced blood flow Psychosomatics and Psychotherapy of the University Hos- and reduced liver mass as well as decreasing kidney func- pital Wuerzburg [7]. tion as possible mechanisms behind their findings in the A high level of inter-individual variability in the oldest group of patients. Using the same assumptions, serum concentration was found for every drug at the same the increased bioavailability, too, could be explained by dose. Women had a significantly higher dose-corrected reduced pre-systemic elimination [8]. serum concentration of amitriptyline+nortriptyline, cit- As concerns the different concentration/dose ratios alopram, +nordoxepin and mirtazapine. No dif- between women and men, earlier studies already pos- ference was found for clomipramine+, tulated the lower body weight of women, the lower escitalopram, fluoxetine+norfluoxetine, and glomerular filtration rate and possibility of a changed sertraline [7]. expression of the cytochrome enzymes to explain the dif- Patients > 60 years exhibited significantly higher ferences [8]. dose-corrected serum concentrations than younger patients with respect to amitriptyline+nortriptyline, cit- c) Case report on a female patient with a resection of alopram, doxepin+nordoxepin, maprotiline, mirtazapine, the upper digestive system: This case described the and sertraline. This difference was not found in connec- psychopharmacotherapy of a very specific patient [9]. But tion with clomipramine+norclomipramine, escitalopram, similar changes are to be expected also in the much more fluoxetine+norfluoxetine [7]. frequently performed bariatric surgeries. So far, though, As a limitation of their study, the authors reported there have not been any studies on the changes in the that the concomitant medication could not be identi- pharmacokinetics of drugs in this patient group. fied and was therefore not considered in the analysis, An esophagogastrectomy and pancreaticoduodenec- although numerous studies have shown that the concomi- tomy had to be performed on a long-term depressed patient tant medication has a great impact on the pharmacokinet- following an attempted suicide with sodium hydroxide. ics of the drugs being studied. A second limitation was Following this procedure, the metabolic problems were dose-corrected data evaluation, the result of which was treated with insulin, pancreatic enzymes and nutritional that the dose-dependent changes in the metabolism were supplements, but treatment with antidepressants was not recorded [7]. unsuccessful, so that an electroconvulsive therapy had to be initiated [9]. b) Antidepressant serum concentrations in elderly During a depressive episode, contraindications for patients: Elderly patients take an average of 2–5 regu- electroconvulsive therapy led to another attempt, in an larly prescribed medications; 20%–50% of patients are inpatient setting, to administer oral antidepressants to the subject to polypharmacy, which is defined as taking five female patient; intravenous administration was refused by Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs 7 the patient. First, a daily dose of 175 mg of clomipramine drugs are selected deliberately because of their adverse drug was administered for a period of 4 weeks. The patient’s reactions (e.g., administering a sedative antidepressant to a serum concentration showed a concentration below the depressed patient suffering from insomnia). Although the quantification limit of the analytical method. Since mal- serum concentrations often vary greatly, TDM has shown absorption had to be considered in the extensive resec- to be rather useless in connection with most of the new tion of the upper digestive system, treatment was changed psychotropic drugs. Various studies have shown that > 75% to an oral solution of amitriptyline. Three days after this of patients with discontinue drug therapy modification, the plasma concentrations of amitriptyline within 18 months. For depressed patients it has been shown reached a trough level of 65 ng/mL, and a peak level of that > 50% of patients did not achieve remission, although 145 ng/ml 2 h after administering the drug. After 2 weeks, they were treated with two or more antidepressants. the mood of the patient improved significantly, and she The authors defined as the goal of pharmacogenetics was discharged with therapeutic serum concentrations of the ability to predict the benefit of a drug for a particular amitriptyline and nortriptyline [9]. patient in order to design an individually tailored therapy The authors pointed out that surgeries in the gastro- with improved efficiency and minimal adverse effects. The intestinal tract and on the pancreas might alter the phar- pharmacogenetic biomarkers can be used both for the pre- macokinetics of drugs. The efficacy of treatment may be diction of adverse drug reactions and for the selection of varied also depending on the pharmaceutical formula- the drug [14]. tion, according to the authors [9]. In psychopharmacogenetics, it is especially the cyto- chrome P450 enzymes CYP1A, CYP2C19, CYP2D6 and CYP3A that play a role. Polymorphisms in these genes result in an altered metabolism and changes in the drug LC-MS/MS analytical methods for concentrations of many psychotropic drugs. Depending the determination of several psy- on the constellation of the aforementioned genes, indi- viduals can be classed into “poor metabolizers” (PMs), chotropic drugs “extensive metabolizers” (EMs) and “ultrarapid metabo- lizers“ (UMs). The PM phenotype leads, for drugs metabo- Many laboratories today analyze psychopharmaceuticals lized by this enzyme, to a reduced degradation, while the by means of LC-MS/MS multi-analyte methods. In general, UM phenotype results in low serum concentrations due to these methods allow for a quick, highly sensitive and spe- accelerated elimination. cific determination of several psychopharmaceuticals. One study showed that CYP2D6 PMs had a risk of sig- Table 4 provides a summary of four of these analytical nificant adverse drug reactions about three times greater methods published in 2011 and 2012 [10–13]. The use of after taking risperidone, and about six times the risk of non-deuterated standards in LC-MS/MS methods is no discontinuing treatment due to side effects [14]. longer state-of-the-art and should be avoided. The valida- The identification of genes is another important area tion results were very good for all methods with the excep- of daily psychiatric routine for pharmacodynamics. Thus, tion of a few substances per method that were accepted genetic differences in the serotonin transporter (5-HTT) just barely in terms of accuracy and precision under the affected both the serotonin concentration and the avail- applicable guidelines. Unfortunately, when it comes to ability of the transporter as a target of antidepressant multi-analyte methods, this is something that must be therapy. In addition, the efficiency of the therapy and accepted, because no allowance can be made for specific the likelihood of the occurrence of adverse drug reactions requirements of an analyte when using these methods. were affected [14]. The use of pharmacogenetic tests in psychiatry is limited by different factors, according to the authors, such as the lack of knowledge about this topic and the lack of The role of pharmacogenetics in evidence-based data from randomized studies to demon- clinical psychiatry strate an improved outcome. The results of several studies and meta-analyses were often inconclusive, and many Lombard and Doraiswamy [14] have prepared a very nice analytical tests failed to detect all the genetic variability. review to illustrate the key points of pharmacogenetics in In addition, there is uncertainty regarding the account- psychiatry. Today medication for patients in psychiatry is ability of these tests, because the costs are generally not primarily selected on the basis of a process of trial and error: covered by health insurance [14]. 8 Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs , a , clozapine, , clozapine, a Hasselstrom [13] Hasselstrom Not analyzed Not 87.6%–120.9% < 17.9 Agilent Triple Quadrupole Quadrupole Triple Agilent 6410 SRM ESI, positive ESI, Formic acid, amm.acetate, amm.acetate, acid, Formic methanol Zorbax SB-C8 Zorbax Deuterated standard for each each for standard Deuterated analyte Protein precipitation Protein , , imipramine, desipramine, nortriptyline quetiapine, ziprasidone, ziprasidone, quetiapine, citalopram, amitriptyline, climipramine 13 mirtazapine, venlafaxine 13 mirtazapine,

Vecchione et al. [12] et al. Vecchione none 86.2%–114.5% < 15.6 API 3000 tandem mass spectrometer mass API 3000 tandem SRM Turbo ion spray, positive/negative ion spray, Turbo Acetic acid, , acetonitrile water, acid, Acetic Chromolith Speed ROD C18 Speed Chromolith Docosahexaenoic acid, acid, Docosahexaenoic Protein precipitation Protein 18 haloperidol, risperidone, lorazepam, lorazepam, risperidone, 18 haloperidol, escitalopram, aripiprazole, duloxetine, olanzapine, imipramine, fluvoxamine, sertraline, quetiapine, paroxetine, venlafaxine, clozapine, ziprasidone, acid valproic ,

, a , fluoxetine a Ansermot et al. [11] et al. Ansermot 7 citalopram < 13% 84.2%–109.6% < 18% Agilent Series 100 MSD 100 MSD Series Agilent quadrupole single SIM ESI, positive ESI, Amm. acetate, amm. Amm. acetate, acetonitrile hydroxide, XBridge C18 XBridge Deuterated standard for for standard Deuterated analyte each Solid phase extraction extraction phase Solid 10 mg support MCX Oasis fluvoxamine, paroxetine, paroxetine, fluvoxamine, sertraline

, a , b , clomipramine a , duloxetine, doxepin , duloxetine, a a , fluvoxamine, imipramine, maprotiline, maprotiline, imipramine, , fluvoxamine, a desipramine, desipramine, 27 amitriptyline, citalopram 27 amitriptyline, del Mar Ramirez Fernandez et al. [10] et al. Fernandez Ramirez Mar del mianserin, mirtazapine, moclobemide, moclobemide, mirtazapine, mianserin, sertraline, , nortriptyline, paroxetine, venlafaxine , Partially examined Partially 83.0%–120.4% < 18.1% Quattro Premier tandem mass spectrometer mass tandem Premier Quattro SRM ESI, positive ESI, Formic acid, amm.acetate, acetonitrile amm.acetate, acid, Formic Acquity UPLC BEH C18 UPLC Acquity 13 deuterated compounds 13 deuterated Liquid extraction in 1-chlorobutane extraction Liquid fluoxetine including 2 metabolites. Amm, ammonium; ESI, electrospray ionization; MS, mass spectrometry; SRM, selected reaction monitoring; SIM, single ion monitoring. single SIM, monitoring; reaction selected SRM, spectrometry; mass MS, ionization; electrospray ESI, Amm, ammonium; 2 metabolites. including b

Including metabolite; metabolite; Including Specific drugs: drugs: Specific names and Numbers Overview of four different LC-MS/MS multi-variate methods for psychotropic drugs. psychotropic for methods multi-variate LC-MS/MS different four of 4 Overview Table a Matrix effects Matrix Accuracy Imprecision Mass spectrometer Mass MS detection MS Ionization Mobile phase HPLC column HPLC Internal standards Internal Sample extraction Sample Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs 9

Pharmacogenetics in psychiatry – individually dosed, but would also have to be identi- fied as such because of the genetic basis for effects and the path from research to clinical side effects. Numerous studies have examined possible practice genetic markers for weight gain induced by antipsychot- ics. This represents a significant public health problem The publication of Malhotra et al. [15] shows very clearly and can be easily determined as a phenotype. In the why pharmacogenetic tests have not played a major role second-generation antipsychotics (especially clozapine, in the clinical practice of psychiatry so far. Due to the poor olanzapine, quetiapine), weight gain is the most common data from clinical studies, there is now too little solid data adverse drug reaction, but is also observed in many first- to propagate the routine use of tests in psychiatry as well generation antipsychotics. In a recent study, weight gain as to achieve a settlement with health insurers. was examined in 139 pediatric patients receiving for the The DRD2 gene encodes the dopamine D2 receptor, first time risperidone, aripiprazole or quetiapine. A muta- which is the common target structure of all approved tion in the MC4R (melanocortin 4 receptor) gene was antipsychotics. A meta-analysis has demonstrated that identified, which occurred more frequently in patients functional polymorphisms in the DRD2 promoter region, with weight gain. The results were confirmed in two inde- which modulate gene expression, significantly affect the pendent cohorts [15]. efficiency of antipsychotic treatment. The same has also The study of genetic influences on psychiatric drugs been found for the serotonin transporter, which repre- therapy is often performed retrospectively and on patients sents the common target structure of serotonin reuptake already having undergone intensive treatment. Thus, inhibitors. Carriers of certain genetic constellations with various studies looked at the HTR2C C-759T polymorphism reduced protein expression accounted only for about half (serotonin receptor ) and the role it plays in the weight to two-thirds of the therapeutic success in comparison to gain caused by antipsychotic drugs. A meta-analysis non-carriers of this mutation. Even though these effects showed that the odds ratio was much higher for the few were proved in a statistically significant manner, they had studies with patients undergoing their initial treatment insufficient sensitivity and specificity, thus preventing than in studies with patients who had been treated with them from entering clinical practice [15]. antipsychotics for longer periods of time [15]. The absence of convincing data from pharmacogenetic Clozapine-induced agranulocytosis is a dreaded studies hampered the development of clinical pharmaco- adverse effect of this drug, which prevents its wide genetic tests. In order for such tests to be approved by the application. In the human leukocyte antigen, a mutation FDA, they must have a higher sensitivity and specificity was detected (HLA-DQB1, 667G > C) that was encoun- than is the case today. Thus, only the Roche AmpliChip R tered with high specificity in patients who developed CYP450 test is approved by the FDA at the moment, which agranulocytosis as a result of clozapine, but only 21.5% examines 27 alleles in CYP2D6 and 3 alleles in CYP2C19. of the affected patients carried this mutation, result- The approval was granted due to the fact that the CYP450 ing in a small sensitivity for the biomarker. Given the genotype can affect drug action and safety [15]. various studies on this subject, one must assume that a A problem of pharmacogenetic studies in psychia- risk profile should be created, instead of determining a try has to do with their clinical endpoints, which were single polymorphism [15]. defined by assessing clinical symptoms. The detection of clinical symptoms depends on the high level of subjectiv- ity of patients. Another problem is the non-compliance of patients. Especially the detection of adverse drug reac- Examples of current tions is negatively affected by patients not taking their pharmaco­genetic studies drugs, so that the statistical power is greatly reduced, making it necessary to study a much larger collective. a) CYP2D6: The work of Muller et al. [17] is a study on the So far, there have been no prospective pharmacogenetic use of AmpliChip as part of a pilot study, and represents studies in psychiatry [15]. a very typical pharmacogenetic study with a relatively In a second review article by the same authors, small collective of previously treated patients. Up to 20% numerous studies were summarized that examined the of schizophrenic patients do not respond to the initially genetic influences on life-threatening adverse drug reac- administered antipsychotic medication, and 20%–30% tions or those crucial to health policy [16]. In an indi- of patients who responded to treatment suffer a relapse vidualized therapy, a drug would have to be not only during maintenance therapy. As for obsessive-compulsive 10 Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs disorders, only 40%–60% of patients exhibit an adequate The study was able to highlight some trends regarding response to treatment with antidepressants. the link between the CYP2D6 genotype and response to The pharmacogenetic studies have been performed therapy, even though they were not conclusive. Based on with the AmpliChip CYP450 from Roche, which was the results, the authors recommended the determination able to detect 33 alleles (including 7 duplications) in the of the CYP genotype in specific patients and pointed out CYP2D6 gene, and three alleles in the CYP2C19 gene. The that further prospective and randomized studies should CYP2C19*17 gene, which leads to the ultrarapid metabo- be performed. lizer phenotype could not be determined. The CYP2D6 gene is highly polymorphic with at least 80 known single b) CYP1A2: Dobrinas et al. [18] investigated the influence nucleotide polymorphisms (SNPs) and several variations of polymorphisms of cytochrome P450 oxidoreductase on in the number of gene copies present. The frequency of CYP1A2 activity and inducibility resulting from smoking. single polymorphisms varies greatly across different CYP1A2 is one of the major cytochrome enzymes in the ethnic groups (see Table 5). The CYP2C19 gene has eight liver, and is involved in the metabolism of endogenous known mutations, with the two known inactive forms compounds and commonly used xenobiotics. It has been CYP2C19*2 and *3 [17]. shown in several studies that the activity of CYP1A2 is In the first collective, 35 schizophrenic patients were highly variable. Smoking induces CYP1A2 activity, espe- studied, 23 of whom were resistant to treatment and were cially through the polycyclic hydrocarbon compounds treated with risperidone or haloperidol for an average of contained in smoke. Therefore, many of the 11.4 weeks, and 12 had tardive dyskinesia. In the second drugs metabolized by CYP1A2 showed decreased plasma collective, 39 patients were recruited with obsessive-com- concentrations in smokers. In return, the drug concen- pulsive disorder: they had undergone at least one thera- trations increased during smoking cessation to partially peutic trial with selective serotonin reuptake inhibitors or toxic concentrations. The authors showed that smokers clomipramine in sufficient doses during > 10 weeks [17]. exhibit on average a 1.55-fold increase in CYP1A2 activ- In the group of schizophrenic patients, three CYP2D6 ity compared to nonsmokers, but with very high inter- PMs, two CYP2D6 UMs, 22 CYP2D6 EMs and eight patients individual variability. This meant that the individual with an intermediate phenotype (IMs) were identified. All variation of CYP1A2 activity covered the range from 1.0 patients were CYP2C19 EMs. The CYP2D6 genotype had (no change) to 7.3. no significant effect on the response to treatment in ther- In the present study, the authors investigated the apy-resistant patients and no significant influence on the influence of a polymorphism in cytochrome P450 oxi- development of tardive dyskinesia [17]. doreductase (POR) on CYP1A2 activity while smoking In the group of patients with obsessive-compulsive and after a smoke cessation of 4 weeks. During the disorders, there were 32/39 patients who were CYP2D6 smoking phase, none of the detected POR polymorphism EMs, two patients were UMs, and one patient was PM. All had any significant effect on CYP1A2 activity. During the patients had an EM phenotype with respect to CYP2C19. smoke cessation, a multi-variate analysis failed to show The statistical analysis between the CYP2D6 metabolizer any effect on CYP1A2 activity, but did find a haplotype to status and the response to treatment did not yield any have a tendency toward lower CYP1A2 activity. Concomi- statistically significant results. In the exploratory analy- tant medications that induce or inhibit CYP1A2 and that sis of all drug trials in these patients, there was a signifi- the patients took had no effect on CYP1A2 activity and cant effect of the CYP2D6 phenotype on the response to inducibility [18]. therapy; the two UMs did not respond to 9/11 trials with selective serotonin reuptake inhibitors [17]. Conflict of interest statement Table 5 Frequencies of the individual CYP2D6 polymorphisms in different ethnic groups (adapted from [17]). Author contributions: All the authors have accepted responsibility for the entire content of this submitted Ethnic Group Poor Extensive Ultra rapid manuscript and approved submission. metabolizers metabolizers metabolizers Research funding: None declared. (PM) (EM) (UM) Employment or leadership: None declared. Caucasians (Europeans) 5%–10% 90% 1%–2% Honorarium: None declared. Africans –40% Competing interests: The funding organization(s) played Asians 1%–2% 1%–2% no role in the study design; in the collection, analysis, and Rentsch: TDM and pharmacogenetic testing of psychopharmaceutical drugs 11 interpretation of data; in the writing of the report; or in the metabolites in plasma with ultraperformance liquid chromato­ decision to submit the report for publication. graphy-tandem mass spectrometry. Ther Drug Monit 2012;34: 11–24. 11. Ansermot N, Brawand-Amey M, Eap CB. 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