The role of OATP transporters in the active uptake of drugs into hepatocytes

Active transport processes in the basolateral (sinusoidal) membrane of hepatocytes have an important role in the hepatic clearance and overall disposition for several types of drugs. Organic anion transporting polypeptides (OATPs) expressed in the sinusoidal membrane have been shown to mediate the sodium-independent hepatic uptake of broad range of drugs and they have been associated with clinically relevant drug-drug interactions (DDIs) and genetic polymorphisms. The literature review focuses on sinusoidal OATP transporters and on the pharmacokinetic effects of OATP-mediated hepatic uptake. In addition, current methods to investigate the interactions between drugs and transporters are discussed, with the emphasis on methods applicable to study uptake transporters. The aim of the experimental part of the master's thesis was to determine if two clinically used drugs, entacapone and fluvastatin, are actively transported from blood into rat and human hepatocytes, and to assess the role of OATP transporters in the hepatic uptake of the drugs in comparison with known OATP substrates, estrone 3-sulfate (E3S) and taurocholic acid and broad OATP inhibitor rifamycin SV. The uptake kinetics of compounds of interest were determined in freshly isolated and cryopreserved rat hepatocytes and in cryopreserved human hepatocytes using the oil-spin method. Uptake clearances (CLuptake) via active uptake (CLactive) and passive diffusion (Pdiff) were calculated from the initial uptake data over a 1 - 200 µM and 1 - 50 µM concentration range for entacapone and fluvastatin, respectively. The half-maximal inhibitor concentration (IC50) of E3S uptake transport was determined for entacapone in a competitive uptake experiment over a 10 - 400 µM concentration range. Fluvastatin uptake showed active saturable transport kinetics in rat hepatocytes with a Km value of 6 µM, whereas entacapone uptake in rat hepatocytes was somewhat linear and did not inhibit E3S uptake at clinically significant concentrations, with an IC50 value of 240 µM. Significantly lower hepatic uptake of taurocholate and entacapone was observed between rat and human hepatocytes, indicating species differences in hepatic uptake processes, although cryopreservation may have had an effect on the noticed difference. The results suggest that murine Oatp transporters do not have a significant contribution to hepatic uptake of entacapone. However, this should be confirmed with future studies with more repetitions and a reliable quantification method.Maksasolujen basolateraalisen (sinusoidaalisen) solukalvon aktiivisilla kuljetusmekanismeilla on suuri vaikutus monien lääkeaineiden maksapuhdistumaan sekä laajemmin farmakokineettisiin jakautumis- ja eliminaatiovaiheisiin. Sinusoidaalikalvolla ilmentyvien orgaanisia anioneita kuljettavien polypeptidien (engl. organic anion transporting polypeptides, OATP) on huomattu kuljettavan maksasoluihin laajasti erityyppisiä lääkeaineita. Monien kliinisesti merkittävien lääke-lääkeyhteisvaikutusten on havaittu liittyvän OATP-kuljetinproteiinien estyneeseen tai rajoittuneeseen toimintaan. Samoin useat geneettiset polymorfismit vaikuttavat OATP-substraattien farmakokinetiikkaan, tehoon ja toksisuuteen. Kirjallisuuskatsauksessa tarkastellaan sinusoidaalisten OATP-kuljetinproteiinien vaikutusta lääkkeiden farmakokinetiikkaan sekä tarjolla olevia menetelmiä kuljetinproteiinien tutkimiseen lääkekehityksessä. Erikoistyön tavoitteena oli selvittää, onko fluvastatiinin ja entakaponin soluunotto aktiivista rotan ja ihmisen maksasoluissa, sekä arvioida OATP-kuljetinproteiinien merkitystä näiden lääkkeiden maksasoluunotossa. Lääkeaineiden soluunottokinetiikkaa tutkittiin tuoreissa eristetyissä rotan maksasoluissa sekä pakastetuissa rotan ja ihmisen maksasoluissa käyttäen öljyn läpi -sentrifugointimenetelmää. Soluunoton alkunopeus määritettiin entakaponi-pitoisuuksilla 1 -200 µM ja fluvastatiinipitoisuuksilla 1 - 50 µM. Määritetyistä arvoista arvioitiin aktiivisen (CLactive) ja passiivisen (Pdiff) mekanismin osuudet lääkeaineiden soluunotossa. Lisäksi rotan Oatp-kuljetinproteiinien merkitystä entakaponin soluunottoon tutkittiin pitoisuusalueella 10- 400 µM kilpailevassa soluunottokokeessa Oatp-substraatti estroni-3-sulfaatin kanssa. Fluvastatiinin soluunotto rotan maksasoluihin oli saturoituvaa, ja laskennallinen Km-arvo oli 6 µM. Sitä vastoin entakaponin soluunotto oli lineaarista tutkitulla pitoisuusalueella, eikä estroni-3-sulfaatin soluunotto estynyt kliinisesti merkittävillä entakaponipitoisuuksilla IC50-arvon (puolet maksimaalisesta inhibitiopitoisuudesta) ollessa 240 µM. Entakaponin soluunotto oli huomattavasti alhaisempaa ja hitaampaa ihmisen maksasoluissa verrattuna rotan maksasoluihin, mikä viittaa lajien välisiin eroihin soluuottomekanismeissa. Havainto voi kuitenkin selittyä tuoreiden ja pakastettujen solujen välisillä eroilla. Kokonaisuudessaan tulokset viittaavat siihen, että entakaponi ei ole fluvastatiinin tavoin rotan Oatp-kuljetinproteiinien substraatti. Tulosten luotettavuus tulisi kuitenkin vahvistaa uusissa kokeissa käyttäen rinnakkaisia määrityksiä ja luotettavaa menetelmää näytteiden lääkeainepitoisuuksien määrittämiseksi

Hydroxychloroquine is Metabolized by Cytochrome P450 2D6, 3A4 and 2C8, and Inhibits Cytochrome P450 2D6, while its Metabolites also Inhibit Cytochrome P450 3A in vitro

This study aimed to explore the cytochrome P450 (CYP) metabolic and inhibitory profile of hydroxychloroquine (HCQ). Hydroxychloro-quine metabolism was studied using human liver microsomes (HLMs) and recombinant CYP enzymes. The inhibitory effects of HCQ and its metabolites on nine CYPs were also determined in HLMs, us-ing an automated substrate cocktail method. Our metabolism data in-dicated that CYP3A4, CYP2D6, and CYP2C8 are the key enzymes involved in HCQ metabolism. All three CYPs formed the primary me-tabolites desethylchloroquine (DCQ) and desethylhydroxychloro-quine (DHCQ) to various degrees. Although the intrinsic clearance (CLint) value of HCQ depletion by recombinant CYP2D6 was > 10-fold higher than that by CYP3A4 (0.87 versus 0.075 mu l/min/pmol), scaling of recombinant CYP CLint to HLM level resulted in almost equal HLM CLint values for CYP2D6 and CYP3A4 (11 and 14 mu l/min/mg, respec-tively). The scaled HLM CLint of CYP2C8 was 5.7 mu l/min/mg. Data from HLM experiments with CYP-selective inhibitors also suggested rela-tively equal roles for CYP2D6 and CYP3A4 in HCQ metabolism, with a smaller contribution by CYP2C8. In CYP inhibition experiments, HCQ, DCQ, DHCQ, and the secondary metabolite didesethylchloroquine were direct CYP2D6 inhibitors, with 50% inhibitory concentration (IC50) val- ues between 18 and 135 mu M. HCQ did not inhibit other CYPs. Further- more, all metabolites were time-dependent CYP3A inhibitors (IC50 shift 2.2-3.4). To conclude, HCQ is metabolized by CYP3A4, CYP2D6, and CYP2C8 in vitro. HCQ and its metabolites are reversible CYP2D6 inhibi- tors, and HCQ metabolites are time-dependent CYP3A inhibitors. These data can be used to improve physiologically-based pharmacokinetic models and update drug-drug interaction risk estimations for HCQ.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.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