Cocktail-menetelmän kehittäminen ja optimointi keskeisimpien lääkeaineita metaboloivien sytokromi P450-entsyymien inhibition arviointiin in vitro

Sytokromi P450 (CYP) -entsyymien inhibitio on yksi yleisimmistä syistä haitallisille lääke-lääkeyhteisvaikutuksille. Erityisen haitallinen inhibitiomuoto on ajasta riippuva inhibitio (engl. time-dependent inhibition, TDI), jonka vaikutus voimistuu ajan kuluessa ja säilyy vielä lääkkeen lopettamisen jälkeenkin. Sekä suoraa että ajasta riippuvaa inhibitiota voidaan tutkia tehokkaasti ns. cocktail-menetelmän avulla, joka sisältää useita CYP-selektiivisiä koetinsubstraatteja samassa reaktioliuoksessa. Tämä menetelmä on hyödyllinen erityisesti lääkekehityksen ADME-tutkimuksissa, sillä se mahdollistaa perinteisiin menetelmiin verrattuna pienemmät kustannukset, vähemmän tarvittavia reagensseja sekä nopeamman toteutuksen. Lisäksi cocktail-menetelmää hyödyntämällä voidaan kuvata uusia diagnostisia CYP inhibiittoreita in vitro. Tämän erikoistyön tarkoituksena oli osallistua uuden cocktail-menetelmän kehittämiseen ja optimointiin. Menetelmä kehitettiin keskeisimpien lääkeaineita metaboloivien CYP-entsyymien tutkimiseksi suoran ja ajasta riippuvan inhibition suhteen in vitro hyödyntäen poolattuja ihmisen maksan mikrosomeja. Alustavien kokeiden perusteella koetinsubstraatit oli jaettu kahteen erilliseen cocktailiin merkittävien substraattien välisten interaktioiden välttämiseksi: cocktail I sisältää: takriini/CYP1A2, bupropioni/CYP2B6, amodiakiini/CYP2C8, tolbutamidi/CYP2C9 ja midatsolaami/CYP3A4 ja cocktail II sisältää: kumariini/CYP2A6, (S)-mefenytoiini/CYP2C19, dekstrometorfaani/CYP2D6 ja astemitsoli/CYP2J2. Ensimmäiseksi cocktailin inkubaatio-olosuhteet optimoitiin, minkä jälkeen määritettiin koetinreaktioiden kinetiikka, kineettiset parametrit (Km, Vmax) sekä koetinsubstraattien yhteensopivuus yhden tai kahden substraatin inkubaatioissa. Lopuksi sopivia substraattikonsentraatioita sekä cocktailien koostumuksia arviointiin saatujen tulosten avulla. Menetelmän optimoinnin tuloksena saavutettiin optimaaliset inkubaatio-olosuhteet toistaiseksi optimoimattomalle cocktailille II. Optimoiduissa inkubaatio-olosuhteissa kaikki koetinreaktiot noudattivat saturoituvaa, Michaelis-Menten-kinetiikkaa lukuun ottamatta takriinin 1-hydroksylaatiota (CYP1A2), jolla oli sen sijaan kaksivaiheinen (engl. biphasic) kinetiikka (Km1: 7.36, Km2: 517). Valitut koetinsubstraattien konsentraatiot olivat matalampia kuin reaktioita vastaavat Km-arvot tai niiden läheisyydessä lukuun ottamatta (S)-mefenytoiinin 4'-hydroksylaatiota (40 µM vs. Km 12,5 µM). (S)-mefenytoiinipitoisuutta ei kuitenkaan voitu pienentää riittävän metaboliittipitoisuuden muodostumisen takaamiseksi UHPLC-MS/MS-analyysiä varten. Koetinsubstraattien pari-inkubaatiot osoittivat tarpeen pienentää bupropionipitoisuutta alle 100 µM johtuen bupropionin CYP2C8:aa ja CYP3A4:tä estävistä vaikutuksista. Lisäksi cocktaileja täydentävänä testattiin klooratsoksatsoni/CYP2E1 ja testosteroni/CYP3A4, jotka osoittautuivat sopimattomiksi kummankin cocktailin osalta merkittävien yhteisvaikutusten vuoksi (>40% inhibitio). Ennen menetelmän käyttöönottoa joitakin muutoksia tarvitaan substraattikonsentraatioihin, minkä lisäksi tulisi harkita harvemmin käytettyjen CYP3A4- ja CYP2E1-koetinreaktioiden sopivuutta cocktailin kattavuuden parantamiseksi. Lopuksi menetelmä on myös validoitava hyödyntämällä tunnettuja ajasta riippuvia malli-inhibiittoreita. Tässä työssä tuotettiin menetelmän kehityksen lisäksi uutta tietoa cocktailien sisältämien koetinsubstraattien välisistä yhteisvaikutuksista sekä entsyymikinetiikasta, erityisesti vähän käytettyjen astemitsolin O-demetylaation (CYP2J2) ja takriinin 1-hydroksylaation (CYP1A2) osalta. Näitä tietoja voidaan hyödyntää esimerkiksi uusien cocktailien kehityksessä.Cytochrome P450 (CYP) enzyme inhibition is one of the most common reasons for adverse drug-drug interactions. An especially harmful form of inhibition is time-dependent inhibition (TDI) in which the inhibition potency increases over time and persists even after discontinuation of the drug. Both direct and time-dependent inhibition can be efficiently screened with the so-called cocktail method containing several CYP-selective probe substrates in a single reaction mixture. This method is practical especially in ADME studies of drug development, as it offers lower costs, consumption of fewer reagents and faster implementation in comparison to conventional methods. In addition, the cocktail method can be used to establish new diagnostic CYP inhibitors in vitro. The aim of this Master’s thesis was to participate in the development and optimization of a new cocktail assay method. The method was developed for screening of major drug-metabolizing CYP enzymes in vitro both in a direct and time-dependent manner using pooled human liver microsomes. Based on preliminary testing, included probe substrates were divided into two cocktails to avoid significant inter-substrate interactions: cocktail I containing tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4, and cocktail II containing coumarin/CYP2A6, (S)-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2. First, cocktail incubation conditions were optimized, followed by the determination of probe reaction kinetics, kinetic parameters (Km, Vmax) and inter-substrate interactions with single- or dual-substrate incubations. Finally, suitable probe substrate concentrations and the composition of cocktails was evaluated based on the obtained results. As a result of assay optimization, optimal incubation conditions for yet unoptimized cocktail II were established. In optimized incubation conditions, all probe reactions exhibited saturable Michaelis-Menten kinetics except for tacrine 1-hydroxylation (CYP1A2), which exhibited biphasic kinetics instead (Km1: 7.36, Km2: 517). The selected probe substrate concentrations were all below or near their respective Km values except for (S)-mephenytoin 4’-hydroxylation (40 µM vs. Km of 12.5 µM); however, its concentration could not be reduced in order to maintain sufficient metabolite formation for UHPLC-MS/MS-analysis. Dual-substrate incubation assays demonstrated a need for the reduction of bupropion concentration below 100 µM due to its inhibitory effects on CYP2C8 and CYP3A4. In addition, chlorzoxazone/CYP2E1 and testosterone/CYP3A4 were tested as complementary probe substrates for the cocktails; however, they proved to be unsuitable for both cocktails due to significant interactions (>40% inhibition). Prior to the deployment of the method, some adjustments of probe substrate concentrations are still required in addition to consideration of the suitability of less commonly used CYP3A4 and CYP2E1 probe reactions to improve cocktail coverage. Lastly, validation of the method with known time-dependent model inhibitors should also be conducted. Besides to improvement of the cocktails, new information was generated on inter-cocktail probe-probe interactions and enzyme kinetics of probe reactions, especially for the less-studied astemizole O-demethylation (CYP2J2) and tacrine 1-hydroxylation (CYP1A2). Generated information can be used, for example, in the development of new cocktails

An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes-application to establishing CYP2C8 inhibitor selectivity

We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-beta-glucuronide and clopidogrel acyl-beta-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/ CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-beta-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/ CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.Peer reviewe

An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes-application to establishing CYP2C8 inhibitor selectivity

We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-beta-glucuronide and clopidogrel acyl-beta-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/ CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-beta-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/ CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies