Purpose: Highly water soluble and highly metabolized statins, cerivastatin (CEST) and fluvastatin (FLST), are categorized as class 1 drugs in the Biopharmaceutical Drug Distribution Classification System (BDDCS) and according to BDDCS are considered to not exhibit in vivo significant transporter based drug-drug interactions (DDIs). In contrast, the Extended Clearance Classification System suggests that both CEST and FLST will have exhibit transporter DDIs mediated organic anion transporting polypeptides (OATPs), as these drugs are acids with a molecular weight greater than 400 Da. We note that no significant changes in pharmacokinetics were observed for clinically relevant doses of FLST in subjects with OATP genetic variants or following co-administration of OATP inhibitors. No such human studies are available for CEST due to its removal from the market. Here we report the results for CEST and FLST of in vitro rat and human hepatocyte studies with the OATP inhibitor rifampin (RIF) as well as in vivo rat studies of the same potential interaction.
Methods: Drug uptake studies: Uptake properties of CEST and FLST were evaluated in rodent Oatp (Oatp1a1, 1a4 and 1b2) or human OATP (OATP1B1, 1B3 and 2B1) overexpressing HEK293 cells, in freshly isolated Sprague Dawley (SD) rat hepatocytes and cryopreserved human hepatocytes (purchased from BD). Drug uptake studies were performed for 1 min at 37˚C with CEST (100 nM) or FLST (600 nM) in 5x10E+06 plated HEK293 or suspended hepatocytes. To inhibit the uptake transport, the OATP inhibitor rifampicin (RIF, 50 µM) was co-administered. Drug uptake kinetics were evaluated in suspended hepatocytes over a wide concentration range (1-400 µM) for both CEST and FLST, calculating Michaelis-Menten (Km and Vmax) and passive diffusion (PS) parameters.
Drug metabolism studies: CEST (100 nM) or FLST (600 nM) was incubated in pooled SD rats and pooled human microsomes (both from BD) at 37˚C for 60 min after a 15 min preincubation. Diclofenac and ketoconazole were used to inhibit metabolism; RIF was also evaluated for effects on CEST and FLST metabolism.
In vivo pharmacokinetic studies: CEST (2 nmole/kg) or FLST (100 nmole/kg) was administered intravenously to 220-240 g male Sprague Dawley rats. The Oatp inhibitor RIF (250 µmole/kg) or control saline was also administered intravenously 5 min before CEST or FLST. Blood (150 µL) was withdrawn at appropriate time points up to 24 hours. Plasma was separated from blood and saved for drug analysis.
Drug analyses: All the samples were treated with acetonitrile and CEST and FLST concentrations were quantified by Shimadzu HPLC-Sciex API5000 tandem mass spectrometry via multiple ion monitoring mode.
Data analyses: The in vitro data were plotted by GraphPad Prism and the pharmacokinetic analyses were performed using Certara Phoenix WinNonlin.
Results: Drug uptake studies: Evaluating the effect of inhibiting human OATPs and rat Oatps in HEK293 transfected cells for CEST and FLST, RIF significantly inhibited the uptake of CEST 31.8-40.8% for rat and 29.8-49.8% for human transporters; inhibition of FLST was 33.1-48.5% for rat and 30.3-48.2% for human transporters. In the CEST hepatocyte study, Km (µM), intrinsic clearance (Vmax/Km, mL/min/10E+06cells) and PS (mL/min/10E+06cells) were 6.88, 4.51x10E-02 and 3.86x10E-02, respectively, for rat and 23.3, 4.27x10E-02 and 3.21x10E-02, respectively, for human. In the FLST hepatocyte study, Km, Vmax/Km and PS were 14.7, 4.40x10E-02 and 4.68x10E-02, respectively, for rat and 52.4, 3.28x10E-02 and 4.10 x10E-02, respectively, for human.
Drug metabolism studies: Intrinsic clearance (µL/min/mg protein) of CEST in rat and human microsomes were 15.1 and 32.0, respectively, and these were not changed by RIF (13.7 and 31.1). Intrinsic clearance of FLST in rat and human microsomes were 23.1 and 199, respectively, and these were decreased by RIF (17.8 and 159).
In vivo pharmacokinetic studies: AUC of intravenous administered CEST alone was 9.06±5.03 nM•hr, while CEST with pre-administration of RIF was 7.03±0.93 nM•hr (n=3). These values were not significantly different (p=0.718). There was no significant difference in other pharmacokinetic parameters such as total clearance, t1/2, MRT, and Vss. AUC of intravenous administered FLST alone was 197±102 nM•hr and FLST with RIF was 310±86 nM•hr (n=5). These values were also not significantly different (p=0.096). Other pharmacokinetic parameters such as t1/2, MRT, and Vss were not altered by RIF, however the total clearance tended to decrease.
Conclusion: The BDDCS class 1 statins, CEST and FLST, were substrates of rodent and human OATPs in the cell systems, and uptake was significantly inhibited by the OATP inhibitor RIF. Similar Vmax/Km and PS parameters in the hepatocyte uptake studies suggested that the contribution of hepatic transporters and passive diffusion for CEST and FLST distribution were similar. In vivo intravenous study showed that OATP inhibitor RIF did not significantly affect either CEST or FLST pharmacokinetics. However, since there are large species differences between rat and human in terms of metabolism and uptake of CEST and FLST, we are unable to conclude whether the BDDCS Class 1 drugs CEST and FLST are affected by drug transporters in humans.