The main objective of this study was the development of QSAR models for drug metabolism-related properties. Novel GALAS (Global, Adjusted Locally According to Similarity) modeling method was used, which is a combination of baseline global QSAR model and local similarity based corrections. GALAS modeling method allows forecasting the reliability of prediction thus defining the model applicability domain. Models predicting CYP3A4 inhibition and regioselectivity of metabolism in human liver microsomes were developed and validated using external test sets. In all cases the baseline models already showed acceptable results, and the overall accuracy of predictions increased after the similarity based corrections. Moreover, the numbers of mispredictions reduced significantly when only results of higher reliability were taken into account. However, the original models are applicable only for less than a half of external datasets. Since the similarity correction procedure of GALAS modeling method allows simple model training, the possibility to expand the applicability domain has been tested. The CYP3A4 inhibition model was successfully adapted to PubChem data and compounds with a novel chemical scaffold. After training the regioselectivity model new metabolism sites could be identified in compounds of new chemical class. Moreover, this model was adapted for human cytochrome P450 isoform profiling. / Pagrindinis šio darbo tikslas buvo kiekybinio struktūros ir aktyvumo ryšio modelių, prognozuojančių su vaistų metabolizmu susijusias savybes, kūrimas. Modeliai, prognozuojantys CYP3A4 slopinimą ir žmogaus kepenų mikrosomų katalizuojamo metabolizmo regioselektyvumą, buvo sukurti naudojant GALAS (angl. Global, Adjusted Locally According to Similarity; Globalus, lokaliai pakoreguotas pagal panašumą) modeliavimo metodą, kuris geba įvertinti prognozės patikimumą, taip apibrėždamas modelio pritaikymo sritį. Sukurtų modelių prognozės buvo tikrinamos naudojant eksperimentinius naujų cheminių junginių duomenis. Visų globalių modelių prognozės gerėjo po korekcijų pagal panašumą, o neteisingų spėjimų skaičius buvo ženkliai mažesnis tarp aukšto patikimumo prognozių. Visgi daugiau nei pusė išorinių duomenų nepatenka į šių modelių pritaikymo sritį. GALAS modeliai gali būti gana paprastai apmokomi, pridedant naujus duomenis į lokalią modelio dalį ir apskaičiuojant reikiamą korekciją. Po tokios apmokymo procedūros CYP3A4 slopinimo modelis prisitaikė prie PubChem duomenų bazės cheminių junginių ir taip pat prie vaistų, turinčių naują cheminį karkasą. Pridėjus naujų junginių ir apmokius regioselektyvumo modelį, jis pradėjo prognozuoti naujas metabolizmo vietas. Pastarasis modelis taip pat buvo pritaikytas atskirų fermentų katalizuojamo metabolizmo prognozavimui.

CYP2D6 metabolizes a range of centrally acting drugs, neurotoxins, and endogenous neurochemicals. Higher levels of brain, but not liver, CYP2D6 have been identified in alcoholics and smokers, suggesting exposure to ethanol and/or nicotine may induce brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression. METHODS: Monkeys were randomized into 4 groups of 10/group consisting of a control group, ethanol-only group, nicotine-only group, and a combined ethanol and nicotine group; treatments occurred for 64 days. RESULTS: Exposure to chronic ethanol and nicotine induced CYP2D across various brain regions and cell types, particularly when both drugs were given in combination. No changes in protein levels were observed in liver or in CYP2D mRNA levels in liver and brain. CONCLUSIONS: Ethanol and nicotine increase brain CYP2D levels, which may affect CNS drug response, neurodegeneration and personality among those exposed to alcohol and/or nicotine.

CYP2D6 metabolizes a range of centrally acting drugs, neurotoxins, and endogenous neurochemicals. Higher levels of brain, but not liver, CYP2D6 have been identified in alcoholics and smokers, suggesting exposure to ethanol and/or nicotine may induce brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression. METHODS: Monkeys were randomized into 4 groups of 10/group consisting of a control group, ethanol-only group, nicotine-only group, and a combined ethanol and nicotine group; treatments occurred for 64 days. RESULTS: Exposure to chronic ethanol and nicotine induced CYP2D across various brain regions and cell types, particularly when both drugs were given in combination. No changes in protein levels were observed in liver or in CYP2D mRNA levels in liver and brain. CONCLUSIONS: Ethanol and nicotine increase brain CYP2D levels, which may affect CNS drug response, neurodegeneration and personality among those exposed to alcohol and/or nicotine.

The kidneys have a significant role in drug elimination through both metabolic and excretory routes. Despite a recent paradigm shift towards systems pharmacology approaches, prediction of renal drug disposition using 'bottom-up' and mechanistic modelling approaches remains underdeveloped. Lack of 'gold-standard' in vitro assays and corresponding in vitro-in vivo extrapolation (IVIVE) approaches for prediction of renal metabolic (CLR,met) and excretory (CLR) clearances contribute to this. A comprehensive literature analysis of quantitative physiological data to inform renal IVIVE scaling factors and systems parameters relevant for physiologically based pharmacokinetic (PBPK) kidney models was initially performed to identify existing knowledge gaps. Following this, microsomal protein content in dog kidney cortex (MPPGK) and liver (MPPGL) were measured in 17 samples from the same animal. Mean dog MPPGK (44.0 mg/ g kidney) and MPPGL (63.6 mg/ g liver) obtained using glucose-6-phosphatase activity as the microsomal protein marker where systematically higher than when CYP content was used as the marker (33.9 mg/ g kidney and 41.1 mg/ g liver respectively). Dog MPPGK was lower than MPPGL, with no direct correlation between the organs. In addition to dog, MPPGK and cytosolic protein per gram kidney (CPPGK) were obtained from 31 human samples, which represent the largest dataset currently available. Mean human MPPGK (25.7 mg/ g kidney) and CPPGK (52.7 mg/ g kidney), were measured using glucose-6-phosphatase and glutathione-S-transferase activities as recovery markers, respectively. Activity of prepared kidney microsomes was assessed using mycophenolic acid glucuronidation as a marker. Novel scaling factor of 25.7 mg/ g kidney was applied for IVIVE of mycophenolic acid microsomal glucuronidation data, resulting in a 2-fold increase in scaled intrinsic clearance compared with data scaled by the commonly used literature MPPGK value (12.8 mg/ g kidney). In addition to the microsomal scaling factor, several elements of a modified stereology method were developed for quantifying human proximal tubule cellularity. The methods included implementation of a systematic uniform random sampling protocol and investigation of tinctorial and immunohistochemistry based staining approaches that could be used identify and count proximal tubule cells in histology sections. A range of mechanistic models for prediction of CLR via either tubular reabsorption or active secretion were developed. A novel 5-compartment model for prediction of tubular reabsorption and CLR from Caco-2 apparent permeability data was developed. This model accounted for relevant physiological complexities of the kidney, such as regional differences in tubular filtrate flow rates and tubular surface area, including consideration of the impact of microvilli. The model predicted the CLR of 45 drugs with overall good accuracy (geometric mean fold error of 1.96), although a systematic under-prediction was noted for basic drugs. The novel 5-compartment model represents an important addition to the IVIVE toolbox for physiologically-based prediction of renal tubular reabsorption and CLR and can be implemented in the more complex mechanistic kidney models, as shown in the case of prediction of urine flow dependent CLR of theophylline and caffeine. Final part of the Thesis focused on the refinement of digoxin PBPK kidney model and its ability to predict effect of aging and renal impairment on digoxin CLR. The analysis has identified that reducing either the proximal tubule cellularity or OATP4C1 abundance parameters in the mechanistic model recovers well observed reduced tubular secretion and CLR of digoxin in renal impairment populations whereas no effect of modification of P-gp abundance was observed. Conversely, reducing the proximal tubule cellularity, OATP4C1 abundance or P-gp abundance parameters in the model resulted in negligible change, decreased or increased accumulation of digoxin in proximal tubule cells, respectively. In conclusion, the current study provides to date the most comprehensive kidney microsomal and cytosolic metabolic scaling factors, together with revised database on renal physiological data necessary for quantitative prediction of renal drug disposition. Mechanistic modelling work shown here has highlighted a need for physiological data from different population groups to inform kidney model parameters, in order to improve the scope and utility of such models within the systems pharmacology paradigm.

Em face das recentes exigências das agências regulatórias quanto à aprovação de novos fármacos, os estudos de biotransformação têm-se tornado uma etapa indispensável para a identificação e otimização de compostos bioativos. O objetivo desses estudos é identificar, já nas fases iniciais da descoberta de fármacos, candidatos que apresentam propriedades indesejáveis como a (i) presença de metabólitos ativos ou tóxicos; (ii) inibição de enzimas metabolizadoras; (iii) depuração metabólica inadequada, entre outras. Neste estudo, foi realizada a caracterização metabólica e a identificação de possíveis inibidores das enzimas do citocromo P450 de oito promissores candidatos a fármacos, identificados através de ensaios virtuais como inibidores da TcGAPDH, Cruzaina e TcDHODH, todas do Trypanosoma cruzi, agente causador da doença de Chagas. Esses compostos foram testados contra as três principais isoformas do citrocromo P450: CYP 3A4, CYP 2D6 e CYP2C9. Os valores de IC50 de 1,4 µM e 1,3 µM contra a CYP2C9 foram encontrados para os compostos Nequimed53 e Nequimed125, enquanto o Nequimed42 inibiu a CYP 3A4 com um valor de IC50 de 7,12 µM. Posteriormente foi conduzida a caracterização metabólica dos compostos Nequimed53 e 125 com foco na identificação dos principais metabólitos, sítios de metabolismo e vias de biotransformação através da técnica de LC-ESI-QqTOF-MS. Para ambos os compostos, a biotransformação por microssomas extraídos de fígado de ratos deu-se preferencialmente por uma única via dependente de NADPH. No caso do Nequimed54, o metabólito formado apresentou uma variação da razão m/z de +16, indicando a ocorrência da hidroxilação do composto parental, enquanto que para o composto Nequimed125, o metabólito formado apresentou uma variação da razão m/z de -28, condizente com a perda de um fragmento etila do composto parental. / In the light of recent demands from regulatory agencies for the acceptance of new drugs, the biotransformation studies have become an essential step for the identification and optimization of bioactive compounds. The objective of these studies is to identify compounds that have undesirable properties such as (i) the presence of toxic or active metabolites, (ii) inhibition of metabolizing enzymes, (iii) excessive metabolic clearance, inter alia. In this study we characterized the metabolism and cytochrome P450 inhibition of eight compounds identified by virtual screening as inhibitors of TcGAPDH, Cruzain and TcDHODH which are of interest as targets for intervention in treatment of Chagas Disease. These compounds were tested against cytochrome P450 isoforms 3A4, 2D6 and 2C9. IC50 values of 1.4 µM and 1.3 µM against CYP 2C9 were observed for Nequimed53 and Nequimed125.while Nequimed42 inhibited CYP 3A4 with an IC50 of 7.1 µM. Subsequently, we characterized the in vitro metabolism of Nequimed53 and 125 with a focus on metabolite identification and biotransformation pathways using the LC-ESI-MS-QqTOF technique. For each, the biotransformation by rat liver microsomes occurred by a single NADPH-dependent pathway. For Nequimed54, the observed metabolite [M+16]+ indicated hydroxylation of parent compound. The metabolite [M-28]+ observed for Nequimed125 indicated desethylation of the parent compound.

Abstract Drug metabolite profiling and identification studies are nowadays regularly conducted with liquid chromatography (LC) coupled with mass spectrometry (MS) as an analytical tool. The speed, selectivity and sensitivity of modern LC–MS instruments have been significantly increased in recent years. Especially the use of ultra-high-performance LC (UHPLC) in combination with a modern high-resolution MS instrument offers high full scan detection sensitivity, mass accuracy and the detection of both expected and unexpected metabolites in a single LC–MS run. The present study showed that no single LC–MS conditions were suitable for the analysis of a large group of structurally diverse compounds. The testing of optimum conditions for each individual compound led to more high-quality data when chromatographic retention behavior and mass spectrometric ionization efficiency for in vitro metabolite profiling were considered. The developed LC–MS methods were applicable for measuring both the disappearance of the parent compound and the formation of metabolites. Tentative metabolite identification was based on the measured accurate mass time-of-flight (TOF) MS data. In the second part, a rapid and sensitive assay was designed and built for the trapping, screening and characterization of reactive metabolites in vitro. In total, 78 trapped reactive metabolite conjugates were detected and identified based on accurate mass data using 12 structurally different test compounds. The majority of the detected conjugates were reported for the first time. Amine-containing compounds, that formed methylated and cyanide-trapped products after CYP-mediated reaction steps in human liver microsomal (HLM) incubations, were studied further. The observed methylated cyano conjugates were shown to be experimental artifacts, i.e., metabonates. The study also describes the use of traditional high-performance LC (HPLC) and the more modern UHPLC coupled to time-of-flight, triple quadrupole and hybrid linear ion trap mass spectrometers in drug metabolism studies, and reviews on how to choose the most suitable LC–MS system for metabolite profiling purposes in drug discovery and early drug development. / Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on nykyaikana yleisesti käytetty analyysimenetelmä lääkeaineiden aineenvaihduntatuotteiden (metaboliittien) havaitsemisessa ja tunnistamisessa. Modernien LC–MS -laitteiden nopeus, selektiivisyys ja herkkyys ovat merkittävästi parantuneet viime vuosina. Käytettäessä ultrakorkean suorituskyvyn nestekromatografia (UHPLC) yhdessä nykyaikaisen korkean massaresoluution MS-laitteen kanssa on mahdollista havaita kaikki sekä odotetut että odottamattomat metaboliitit yhdellä kertaa. Tutkimalla suurta joukkoa rakenteellisesti erilaisia yhdisteitä voitiin todeta, että yksittäiselle yhdisteelle optimoidut mittausolosuhteet johtivat korkealaatuisempaan dataan kuin yleiset ei-optimoidut olosuhteet, kun arvioitiin sekä kromatografista piikin profiilia ja pidättymistä että ionisaatiotehokkuutta. Yksikään yksittäinen analyysiolosuhde ei myöskään soveltunut kaikille yhdisteille. Tutkimuksessa kehitetyillä LC–MS -analyysimenetelmillä tutkittiin sekä kanta-aineen häviämistä että metaboliatuotteiden muodostumista in vitro -menetelmillä. Alustava metaboliatuotteiden tunnistus perustui tarkan massan mittaukseen lentoaikamassaspektrometrillä (TOFMS). Tutkimustyön seuraavassa vaiheessa kehitettiin nopea ja herkkä analyysimenetelmä reaktiivisten metaboliittien pyydystämiseen, havaitsemiseen ja tunnistamiseen ihmisen maksamikrosomivalmisteista in vitro -menetelmin. 12 testiyhdisteelle havaittiin kaikkiaan 78 erilaista reaktiivisen metaboliitin konjugaatiotuotetta, jotka tunnistettiin tarkan massan perusteella. Suurin osa tunnistetuista konjugaatiotuotteista raportoitiin ensimmäistä kertaa. Amiineja sisältäville testiyhdisteille havaittiin muodostuvan sytokromi P450 (CYP) entsyymien katalysoimien reaktioiden välityksellä metyloituneita ja syanidianionilla konjugoituneita metaboliatuotteita. Tarkempien tutkimusten jälkeen näiden todettiin olevan koejärjestelyistä johtuvia artefaktoja, toisin sanoen metabonaatteja, eivätkä todellisia reaktiivisten metaboliittien konjugaatiotuotteita. Tässä tutkimuksessa arvioitiin myös perinteiseen korkean suorituskyvyn nestekromatografiin (HPLC) sekä uudempaan UHPLC-laitteistoon kytkettyjen lentoaika-, kolmoiskvadrupoli- ja hybridimallisten ioniloukkumassaspektrometrien soveltuvuutta aikaisen lääkekehitysvaiheen metaboliatutkimuksiin.

Abstract Liquid chromatography (LC) combined with mass spectrometry (MS) is a powerful tool for qualitative and quantitative analytics of organic molecules from various matrices, and the use of this hyphenated technique is very common in bioanalytical laboratories. In this study, LC/MS methods and the required sample preparation applications were developed for plant flavonoid and drug metabolism studies. The main focus was in developing methods to be used during cytochrome P450 (CYP) -specific drug interaction studies. Traditional high performance liquid chromatography (HPLC) and new, more efficient and faster ultra-performance liquid chromatography (UPLC) were utilized together with time-of-flight (TOF) and triple quadrupole (QqQ) mass spectrometry. In the flavonoid study, collision-induced radical cleavage of flavonoid glycosides was tested and observed to be a suitable tool for the structure elucidation of the 15 flavonol glycosides extracted from the medicinal plant Rhodiola rosea. Ten of these glycosides were previously unreported in the plant. Several unreported in vivo bupropion metabolites were identified from human urine when developing the method for the new and more extensive in vitro and in vivo N-in-one interaction cocktail assays. The qualified analysis methods developed here enable faster analysis for the N-in-one cocktail assays, in turn enabling a more efficient screening of drugs that affect CYP-enzyme activities. In the case of the human in vitro cocktail assay, fourteen compounds were analyzed using a single LC/MS/MS run. The method has proven to be very reliable and has been used in several interaction studies utilizing different sample matrices. The in vivo cocktail assay that was developed enables totally non-invasive sample collection from the patients, the urine sample being sufficient for the UPLC/MS/MS analysis of all target compounds. The last part of the study consisted of developing a specific and very sensitive UPLC/MS/MS method for the analysis of one of the in vivo cocktail analytes, the antidiabetic drug repaglinide, from human placenta perfusates. / Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on tehokas työväline kvalitatiivisessa ja kvantitatiivisessa analytiikassa, ja tätä tekniikkaa käytetään erityisesti bioalan laboratorioissa. Tässä väitöskirjatyössä kehitettiin ja sovellettiin LC/MS- ja näytteenkäsittelymenetelmiä kasvien flavonoidimetabolian ja lääkeaineiden metaboliatuotteiden tutkimukseen keskittyen erityisesti sytokromi P450 (CYP) -entsyymispesifisten lääkeaineiden interaktiotutkimuksiin tarvittaviin menetelmiin. Työssä hyödynnettiin perinteistä korkean erotuskyvyn nestekromatografiaa (HPLC) ja uutta, suorituskyvyltään vielä tehokkaampaa ja nopeampaa nestekromatografiaa (UPLC) yhdessä lentoaika- (TOF) ja kolmoiskvadrupolimassaspektrometrian (QqQ) kanssa. Tutkimustyön flavonoidimetaboliaan keskittyneessä osuudessa havaittiin törmäyksen aiheuttaman (CID) radikaalipilkkoutumisen soveltuvan lääkinnällisenä kasvina käytetystä ruusujuuresta (Rhodiola rosea) uutettujen viidentoista flavonoliglykosidin rakennemääritykseen. Kymmentä näistä löydetyistä glykosideista ei oltu aiemmin raportoitu ruusujuuresta. Tutkimustyön keskeisimpänä tavoitteena kehitettiin kvalifioidut LC/MS -analyysimenetelmät käytettäväksi aikaisempaa kattavampien in vitro ja in vivo -olosuhteiden N-in-one -tyyppisten CYP-entsyymi-interaktiotutkimusten analyyttisenä työkaluna. Näitä analyysimenetelmiä kehitettäessä löydettiin ja tunnistettiin ihmisen virtsasta aiemmin raportoimattomia metaboliitteja CYP2B6 -entsyymin malliaineena käytetyn bupropionin annostelun jälkeen. Kyseisten kehitettyjen analyysimenetelmien avulla CYP-entsyymien toimintaan vaikuttavien lääkeaineiden tutkiminen on aiempaa nopeampaa ja antaa yhdellä kertaa samasta tutkimuksesta entistä laaja-alaisempaa tietoa. In vitro -tutkimusta varten kehitetty LC/MS/MS -analyysimenetelmä on osoittautunut erittäin käyttökelpoiseksi lukuisissa interaktiotutkimuksissa, ja in vivo -tutkimusta varten kehitetty UPLC/MS/MS -analyysimenetelmä mahdollistaa täysin ei-invasiivisen näytteenoton potilaista. Tutkimustyön viimeisessä vaiheessa kehitettiin erittäin herkkä ja spesifinen UPLC/MS/MS -analyysimenetelmä CYP2C8-entsyymin toiminnan malliaineena käytetyn repaglinidin analysoimiseksi koejärjestelystä, jossa tutkitaan yhdisteiden kulkeutumista raskausaikana äidin ja sikiön verenkierron välillä istukan kautta.

No description available.

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards ‘normal’ liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.

Genetische Polymorphismen in den Enzymen CYP2D6, CYP2C19 und CYP2C9 beeinflussen die Pharmakokinetik medizinisch bedeutsamer Arzneimittel wie Antidepressiva, oraler Antidiabetika und nichtsteroidaler Antiphlogistika in erheblichem Ausmaß. In der Zukunft kann die Bestimmung genetischer Varianten bei Patienten zur Verbesserung der Arzneitherapie genutzt werden, jedoch nur dann, wenn klinische Konsequenzen wie konkrete Therapieempfehlungen aus den genetischen Daten abgeleitet werden können. An gesunden Probanden wurde die die Bedeutung der beiden Aminosäurevarianten des Enzyms CYP2C9, Arg144Cys (CYP2C9*2) und Leu359Ile (CYP2C9*3) für die Pharmakokinetik von Tolbutamid, Glibenclamid, Nateglinid, Diclofenac, Ibuprofen, Celecoxib und Fluvastatin untersucht. Die Analyse der pharmakokinetischen Parameter ergab eine erheblich erniedrigte Clearance für diese Substrate bei homozygoten Trägern der Allelvariante CYP2C9*3, wie sie etwa 1% in der Bevölkerung tragen. Um bioäquivalente Konzentrationsverläufe zu erreichen, müssten diese Patienten deutlich niedrigere Dosierungen (unter 50%) der meisten der untersuchten CYP2C9-Substrate erhalten. Hingegen zeigte die CYP2C9*2-Variante nur einen geringen Einfluss auf die Pharmakokinetik der untersuchten Medikamente. Für den Bereich der Therapie mit Antidepressiva und Antipsychotika sollte untersucht werden, inwieweit umfassende pharmakogenetisch begründete Therapieempfehlungen gegeben werden können. Eine systematische Analyse aller bisher publizierten Daten zum Einfluss von Polymorphismen von CYP2D6, CYP2C19 und CYP2C9 ergab, dass für die meisten gängigen Antidepressiva bereits Studien zur Bedeutung von Cytochrom-P450-Polymorphismen durchgeführt wurden. Für die beiden in Deutschland sehr häufig verwendeten Trizyklika Trimipramin und Doxepin dagegen lagen keine ausreichenden Daten vor. Beide Medikamente wurden deshalb bei Probanden getestet, die jeweils Träger eines oder zweier Allele mit defizienter oder herabgesetzter Enzymaktivität von CYP2D6, CYP2C19 oder CYP2C9 waren. Es ergab sich ein deutlicher Einfluss des CYP2D6-Genotyps, ein schwächerer von CYP2C19 und des Genotyps CYP2C9*3/*3. Eine Dosisreduktion für Langsam-Metabolisierer von CYP2D6 und etwas moderater für Langsam-Metabolisierer von CYP2C19, sowie für Träger des Genotyps CYP2C9*3/* erscheint für diese beiden Antidepressiva sinnvoll. Die eigenen Daten und die Daten für andere Antidepressiva aus der Literatur wurden dazu verwendet, eine Methode zur Ableitung von pharmakogenetisch basierten Dosierungsempfehlungen zu entwickeln. Auf dem Prinzip der Bioäquivalenz basierend wurden Dosierungsanpassungen für unterschiedliche Genotypen je nach Unterschieden in der Clearance von Substanzen errechnet. Durch diese Dosierungsanpassungen können zumindest theoretisch die durch herabgesetzte Enzymaktivität verursachten Unterschiede in den Plasmakonzentrationsverläufen von Medikamenten ausgeglichen werden. Dabei wurden aktive an der Arzneimittelwirkung teilhabende Metaboliten mit berücksichtigt. Auf Seiten der Pharmakodynamik wurden die vielen Studien zu genetischen Polymorphismen in Serotonin-, Dopaminrezeptoren und Transportern und auch zu anderen Kandidatengenen für die Antidepressiva-, und Antipsychotikawirkung analysiert. Jedoch lassen sich aus den teilweise geringen Einflüssen einzelner Genotypen auf die Arzneimittelwirkung derzeit noch keine pharmakodynamisch begründeten Therapieempfehlungen ableiten. Zusammenfassend lassen sich also bereits heute pharmakogenetisch basierte Dosierungsempfehlungen für viele Medikamente berechnen. Derartige Empfehlungen müssen prospektiv überprüft, validiert und angepasst werden. Auf Seiten der Zielmoleküle der Arzneimittelwirkung ist eine Ableitung genetisch basierter Therapieempfehlungen schwieriger. Das Ziel, konkrete Therapieempfehlungen aus genetischen Daten abzuleiten, ist eine notwendige Bedingung, um Pharmakogenetik in die klinische Praxis der Arzneitherapie einzuführen. / Genetic polymorphisms of the cytochrome P450 enzymes CYP2D6, CYP2C19 and CYP2C9 largely influence pharmacokinetics of clinically important drugs such as antidepressants, oral antidiabetics and nonsteroidal antiphlogistic drugs. Pharmacogenetic diagnostics is ready to be used for optimization of drug treatment in the future if concise recommendations for clinical decisions can be derived from the genetic data. Panel studies in healthy volunteers served to characterize the impact of the amino acid variants Arg144Cys (CYP2C9*2) and Leu359Ile (CYP2C9*3) in cytochrome P450 2C9 on pharmacokinetics of the oral antidiabetics tolbutamide, glyburide and nateglinide, of the nonsteroidal analgetic drugs diclofenac, ibuprofen, celecoxib, and of fluvastatin. Analysis of pharmacokinetic parameters revealed largely reduced oral clearances in homozygous carriers of the CYP2C9*3 allele for most of the substrates studied. About 1% in the general population are carriers of this genotype and patients should get about 50% lower doses of most of the CYP2C9 substrates in order to achieve similar plasma concentration versus time courses. In contrast, the CYP2C9*2 variant had little influence on pharmacokinetics of the CYP2C9 substrates. For antidepressant and antipsychotic drug therapy, the aim was to derive detailed dose recommendations from the large amount of data existing. Thus, a systematic analysis of all published data on the clinical influence of genetic polymorphisms in CYP2D6, CYP2C19 and CYP2C9 was performed and dose recommendations were given for many antidepressants. For two substances, the tricyclics doxepin and trimipramine, no pharmacogenetic data have been found, and therefore own clinical studies were performed. Healthy volunteers who were heterozygous and homozygous carriers of alleles with deficient activity of CYP2D6, CYP2C19 or CYP2C9 were tested for differences in metabolism and elimination of doxepin and trimipramine and compared to carriers of the wildtype. A significant influence of the CYP2D6 genotype was detected whereas CYP2C19 and the genotype CYP2C9*3/*3 had smaller influences. A significant dose reduction for CYP2D6 poor metabolizers and a smaller reduction for CYP2C19 poor metabolizers would be predicted from these data. According the principles of bioequivalence, calculation methods for pharmacogenetic based dose recommendations from clearance or drug concentration data were developed. Using all published data and the own study results, we developed dose adjustments for each genotype. These dose adjustments would allow to compensate for lower drug clearance caused by genetic variants. Active metabolites which contribute to overall drug effects were considered as well. With regard to target molecules of antidepressant and antipsychotic drug action, many studies have been performed on genetic polymorphisms in serotonin-, and dopamine receptors and in transporter molecules but as well in other candidate genes. However, it is not yet possible to derive therapeutic consequences based on these data. In conclusion, on the pharmacokinetic side of drug action, we are already able to give pharmacogenetic based therapeutic recommendations by adjusting the doses according to genotype. However, the benefit of pharmacogenetic dose adjustments has to be studied prospectively. With regard to the pharmacodynamic side of drug action, the situation is much more complex and we are not yet ready to give pharmacogenetics based therapeutic guidelines. However, this has to be a major goal in order to introduce pharmacogenetic diagnostic into clinical practice.