In this study, we attempted to determine the importance of hepatic uptake transporters for the disposition of the widely-used, narrow therapeutic index, anticoagulant warfarin. The results of our clinical study demonstrated that co-administration of the potent OATP inhibitor rifampin, given as a single intravenous dose, had no effect on the AUC0–12h and Cmax values of S- and R- warfarin or S-warfarin's hydroxyl metabolite. Accordingly, OATP uptake transport is likely not clinically significant for the disposition of warfarin and will provide no further insight into mechanisms of warfarin drug-drug interactions or genetic variation important in warfarin dose selection.
Rifampin was chosen as a model OATP inhibitor to block the in vivo
OATP-mediated hepatic uptake of warfarin(11
). Because of rifampin's shorter t1/2
(2.9 ±0.9 h(19
)) relative to S- and R- warfarin (39h and 49h respectively, as determined in our clinical study) and because we could not give rifampin as multiple doses dues to its metabolic enzyme induction effect, rifampin was only present in plasma for a portion of warfarin's concentration-time curve.OATP inhibition and any potential effect on warfarin pharmacokinetics occurred at most during the time of rifampin presence, and therefore, outcome measures for our study needed to be suitably selected to capture this period of OATP inhibition. Previous reports have shown that after a single 600 mg intravenous dose of rifampin to healthy volunteers, rifampin was present for up to 12–24 hours in plasma. (11
) Assuming a similar time of exposure for rifampin in our clinical study, the best parameters to capture the potential effects of OATP inhibition on warfarin pharmacokinetics were the AUC0–12h
. No effect on either of these pharmacokinetic parameters for S- or R-warfarin were seen in the presence of rifampin suggesting OATP transport of warfarin is not clinically important.
Two previous clinical studies by our laboratory using a similar study design as used here demonstrated significant changes in the pharmacokinetics of atorvastatin and glyburide with hepatic OATP inhibition by rifampin(11
). The AUC of atorvastatin was 680% higher and the AUC of glyburide 130% higher in rifampin vs. control phases. In those clinical studies rifampin also produced a large decrease in the Vss
/F for both drugs (atorvastatin, 93% decrease; glyburide, 60% decrease). This decrease in Vss
/F likely demonstrated inhibition of uptake transporter mechanisms important for drug distribution in tissues. In our current study we did not see a change in Vss
/F for S- or R-warfarin on rifampin days (), which we would have predicted if OATP transport processes were important for warfarin distribution.
In contrast to our clinical study, our in vitro
data showed that warfarin uptake into hepatocytes was inhibited by rifampin in both rats and humans () presumably via OATP uptake transporter inhibition. The magnitude of warfarin uptake inhibition (23% in rat hepatocytes and 34% in human hepatocytes) was modest at the concentration of rifampin (100 μM) used for the hepatocyte uptake studies. The 100 μM concentration of rifampin used in our hepatocyte uptake studies is significantly higher than peak plasma concentrations that were likely achieved in our healthy volunteers after a single IV dose of 600 mg rifampin (17–20μM (11
)) and could explain the disagreement between our in vitro
and in vivo
results, but the in vitro
effects were similar to what we observed in vitro
with glyburide. We cannot rule-out the possibility that higher plasma concentrations of rifampin would result in additional OATP inhibition and therefore alter warfarin pharmcokinetics, although the clinical relevance would be questionable. The discordance between our in vitro
and in vivo
data highlights the importance of translational research in drug transporter research and the need to verify the clinical relevance of all preclinical findings using appropriately designed clinical trials.
Our healthy volunteer clinical data are in line with the numerous genome wide association studies (GWAS) undertaken to identify polymorphisms in the human genome that may be important in warfarin dose selection. In these GWAS studies, no common variants in hepatic uptake transporters or any other drug transporters were found to be significant in determining the warfarin dose requirements of patients.(2
) Genetic variants of a transporter will not produce a significant signal in a GWAS if the drug is not a substrate for the transporter of interest since changes in transporter function will not alter the drug's pharmacokinetics and thus dose requirements. This negative predictive value has now been demonstrated nicely for warfarin and OATP uptake transporters through the results of our clinical study and the above mentioned GWASs. In contrast, for drugs that are known substrates of uptake or efflux transporters including OTAP1B1 and OATP1B3, established polymorphisms in the corresponding transporter have been shown to alter drug pharmacokinetics in human clinical studies.(13
Intravenous administration of rifampin conceivably minimized any potential confounding interactions in the intestine at either the enzyme or transporter level although the lack of an interaction cannot be fully confirmed. By giving rifampin as a single dose, we attempted to avoid the well-described and clinically relevant CYP induction effect seen with multiple dosing.(16
) Induction of both CYP2C9(24
) and CYP3A4 (25
), the major metabolizing enzymes for S- and R-warfarin respectively, has been described after multiple dose rifampin. The effect of multiple dose rifampin on warfarin pharmacokinetics was highlighted in a clinical trial of four healthy men.(24
) After 3 days of pretreatment with 300 mg oral rifampin twice per day, a three-fold increase in R-warfarin clearance and a two-fold increase in S-warfarin clearance were seen. Interestingly, our clinical study using only single dose rifampin immediately prior to warfarin likely demonstrates a mild enzyme inductive effect indicated by the small but significant decrease in AUC0–∞
for S- and R-warfarin (15% decrease and 25% decrease respectively) and terminal t1/2
for R-warfarin (25% decrease) with rifampin treatment. The increase in Cmax
and AUC for the R-warfarin metabolite 10-hydroxywarfarin and the increase in the AUC ratio of 10-hydroxywarfarin to R-warfarin further supports induction of R-warfarin metabolism via CYP3A4. The pharmacokinetics of the specific S-warfarin metabolite 7-hydroxywarfarin did not change with rifampin treatment and therefore is not congruent with induction of S-warfarin metabolism via CYP2C9.
The pharmacokinetic effect of enzyme induction appeared to be delayed since early in the time course S- and R-warfarin concentration-time curves, AUC0–12h
, and Cmax
were similar with and without rifampin. Such a delay is expected based on the mechanism of rifampin enzyme induction through activation of the nuclear transcription factor pregnane X receptor (PXR).(23
) A previous clinical study examining the time-course of CYP enzyme induction with multiple-dose rifampin using verapamil as a probe substrate reported a half-life of 1 day for the increase in CYP enzyme activity.(29
) CYP induction after single dose rifampin has previously been shown with a single dose of 1200 mg oral rifampin given 12 hours before oral nifedipine, resulting in a 64% decrease in the AUC0–∞
) Nonetheless, the potential induction of warfarin metabolism by single-dose rifampin in our study does not alter the interpretation of the primary objective of this study as any induction was small (especially for the more biologically active S-warfarin) and it occurred late in the time-course after the primary endpoints (0–12 hours).
We purposely selected a warfarin dose for this study (7.5mg) that would provide adequate drug levels for pharmacokinetic analysis, yet in favor of subject safety produce minimal anticoagulation. Our nominal increase in anticoagulation was similar to a previous healthy volunteer study that also used a single 7.5mg dose of oral warfarin.(31
) The addition of rifampin had no effect on the warfarin pharmacodynamic response as measured by INRmax
and AUC0–96h, INR
() and is in general agreement with our pharmacokinetic data. However, the study was neither designed nor powered to detect a difference in pharmacodynamics.
In conclusion, our single dose clinical study of the effect of rifampin on warfarin pharmacokinetics in healthy volunteers suggeststhat hepatic uptake via OATP may not be clinicallyimportant in the disposition for this highly metabolized, narrow therapeutic index, anticoagulant. Single-dose rifampin may induce metabolizing enzymes such as CYP2C9 and CYP3A4.