Introduction

Drug Induced Liver Injury (DILI) accounted for 11% of acute liver failures in the USA in 2013¹ and causes withdrawal of otherwise successful drugs when it appears in clinical studies. The few specific biomarkers available suffer from poor sensitivity to the degree of liver damage, or manifest after irreversible damage. Drug-Drug Interactions (DDIs) occur when one drug alters the pharmacokinetics of another drug and is a common problem for those taking multiple medications. New methods to assess transport function in vivo are needed.

Gadoxetate is a clinically used contrast agent transported into the hepatocyte by Organic Anion Transporter Protein (OATP1/Oatp1) and excreted by Multidrug Resistance Associated protein (MRP2/Mrp2). Gadoxetate DCE-MRI consequently provides MR biomarkers with unique molecular specificity, exquisite sensitivity to transporter inhibition, and immediate availability in man. It was recently shown to detect acute effects of a clinical dose of rifampicin (RIF) on Oatp1 function in rats².

The aim of the current study was to examine whether gadoxetate DCE-MRI can measure the effects of clinical and high dose RIF after acute and chronic treatment.

Methods

This study was approved by the Ethical Committee on Animal Experiments in Gothenburg, Sweden.

Rifampicin (Orifarm, 300 mg capsules) was suspended in an 85% sugar solution (vehicle) for oral administration. Gadoxetate (Primovist, 0.25 mmol/ml, Bayer) was diluted 1:5 in saline, and dosed at 0.5 ml/kg i.v.

Male rats (Wistar Han, ~300g) were separated in three groups (n=4/group): vehicle (sugar solution), clinical RIF dose (10 mg/kg) and high RIF dose (67 mg/kg). The dose of 67 mg/kg was predicted to be the highest dose for which plasma AUC and Cmax were expected to be within the observed clinical range³. All animals received oral vehicle on day -3 (baseline) followed by once daily group specific oral dosing on days 1-4. Dosing was preceded by 2h fasting.

Rats were imaged at 4 timepoints. Baseline and acute imaging were performed 3h after oral dosing. The 3h interval was chosen for maximum liver RIF concentration (pilot study, data not shown). Day 2 and Day 5 follow-up imaging were performed 24h after the previous dose, i.e. immediately before the 2^nd dose on day 2, and 24h after the last dose on day 5 (Figure 1).

Isoflurane anesthesia (~1.8-2.2% to maintain ~60 breaths/min) was used and breathing and temperature monitored (SA Instruments). The tail vein was cannulated to draw blood and administer contrast agent.
Animals were imaged at 7T (Bruker Biospec, PV 5.1) using a 72-mm transmit/receive volume coil. Two 2D IntraGateFLASH images covered spleen and liver, with navigator positioned over the heart (TR/TE 36/1.5 ms, FA 30°, matrix 192×192, FOV 60×60 mm², slice thickness/separation 1.5/~5 mm).
Retrospective triggering was used to create frames of 1min resolution from the quiescent 70% of the respiratory cycle. Total acquisition time was 30 min. Contrast was injected during 13 sec, starting 4:49 min into the acquisition. Rats were sacrificed by cardiac puncture under anesthesia immediately after the experiment.

The mean signal from manually drawn regions in liver and spleen were used to calculate the change in relaxation rates induced by gadoxetate using the inverted signal equation for a spoiled gradient-echo in steady state. ROIs were drawn to exclude visible vasculature and collecting ducts. Hepatocellular uptake rate (k_he) was then determined as described by Ziemian et al.⁴, with literature values for precontrast relaxation rates and volume fractions⁵.

The repeated measures ANOVA analysis with Bonferroni post hoc testing was used to evaluate acute and chronic effects of RIF on uptake rates (IBM SPSS Statistics, Armonk, USA).

Results & Discussion

No adverse effects of rifampicin treatment or imaging were detected by clinical observation.

Figure 2 shows an example of gadoxetate uptake in liver and spleen, and the group mean deltaR₁ over the DCE-MRI experiments. The effects of rifampicin on uptake rate are shown in Figure 3.

The acute high dose had a statistically significant inhibitory effect on the mean k_he at day 1 (reduced by 85%, p=0.025). k_he was not significantly different from baseline in the chronic dose setting (reduced by 61% and 42% on day 2 and 5, respectively, p>0.05). The clinical dose effects on k_he were not statistically significant (p>0.05), although there was a tendency towards inhibition at day 1. No statistically significant effects on k_he were seen in the vehicle treated group .

More than n=4/group may be required to further understand chronic dosing effects and dose dependency, and 24h may be too short to allow complete rifampicin clearance 24h post dosing. Studies to evaluate hepatic rifampicin clearance and/or increased post dosing DCE-MRI interval are currently planned.

Conclusion

This is the first study to examine the effects of both clinical and high dose rifampicin after chronic dosing.

Our results demonstrate the potential of gadoxetate DCE-MRI to non-invasively assess drug-induced inhibition of hepatocellular transport and Drug-Drug interactions. The method also shows sensitivity to dose-dependent effects.

The uptake rate tended to return to baseline values in the chronic dosing regime.

Acknowledgements

The research leading to these results received funding from the Innovative Medicines Initiatives 2 Joint Undertaking under grant agreement No 116106. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA