Materials and Methods

In Vivo Study.

Unlabeled and ¹⁴C-labeled BIBR 277 (181.65 MBq/mmol, radiochemical purity 98.5%) were synthesized by Boehringer Ingelheim Pharma KG (Biberach, Germany). Unlabeled and¹⁴C-labeled BIBR 277 glucuronide (177.93 MBq/mmol, radiochemical purity 99.2%) were isolated from rat bile by Nemoto & Co., Ltd. (Tokyo, Japan). BIBR 277 glucuronide was stable in plasma at 37°C for 30 min and at −20°C for 3 weeks. The in vivo study was performed according to the method previously reported (Sasabe et al., 1998). BIBR 277 and its glucuronide in plasma and bile were separated by TLC (1-butanol:acetic acid:distilled water = 4:1:1). For the analysis of differences in the plasma concentration profiles between the two strains, repeated measures analysis of variance was performed.

Microsomal Glucuronidation.

The incubation mixture (170 μl) contained microsomes (0.5 mg/ml), 0.1 M MES buffer (pH 6.0), 10 mM MgCl₂, 0.2 mg/ml Brij 58, 5 mM saccharic acid-1,4-lactone, and 0.5 mM AMP. The mixture was incubated for 10 min on ice. Then, 10 μl of BIBR 277 solution in DMSO was added and preincubated at 37°C for 2 min. After this, 10 μl of UDP glucuronic acid solution (final concentration, 3 mM) was added to initiate glucuronidation. The Michaelis-Menten equation was fitted to the initial reaction velocity to obtain the Michaelis constant (K_m) and maximum glucuronidation velocity (V_max).

Transport Study Using Bile Canalicular Membrane Vesicles.

Unlabeled and ³H-labeled DNP-SG (50 MBq/mmol, radiochemical purity 99.4%) were synthesized as described previously (Kobayashi et al.,1990). Transport study was performed by the method previously reported (Sasabe et al., 1998). The inhibition constant (K_i) was obtained by fitting the following equation to the data:V_(+I)/V_(−I) = 1/(1 + I/K_i); whereV_(+I) andV_(−I) represent the transport velocity in the presence and absence of inhibitor, respectively; I is the inhibitor concentration.

Results and Discussion

Due to a genetic defect in cMOAT/MRP2, the biliary excretion of several glucuronide conjugates of xenobiotics has been reported to be much lower in EHBR or TR⁻ (Chu et al., 1997,Sasabe et al., 1998, Loe et al., 1996, Jedlitschky et el., 1997). However, the biliary excretion of BIBR 277 glucuronide was similar in both SDRs and EHBR (Fig. 1B), suggesting that at least part of BIBR 277 glucuronide transport across the bile canalicular membrane is mediated by a transport system other than cMOAT/MRP2. Although some glucuronides, such as morphine 6-glucuronide (Huwyler et al., 1996) and estradiol 17β-d-glucuronide (Vore et al., 1996), have been suggested to be transported by P-glycoprotein, there have been no reports suggesting the major role of such a transporter in the biliary excretion of glucuronides. It should also be noted that the plasma concentration of BIBR 277 glucuronide in EHBRs was 2 to 7 times higher than in SDRs although this difference was not statistically significant (P > .05, Fig. 1A). There are at least two possible explanations of this: one is that BIBR 277 glucuronide is partially transported by cMOAT/MRP2, and, due to the reduction in its excretion into bile in EHBR, it is accumulated both in liver and plasma. This possibility is supported by the present finding that the disappearance of BIBR 277 glucuronide (1.3 mg/kg) from plasma was delayed in EHBR compared with SDRs: the plasma radioactivity was 5350 (n = 2), 2340 ± 365, 960 ± 153, 189 ± 37, 80.5 ± 21.5, and 53.8 ± 8.5 ng eq. of BIBR 277 glucuronide/ml at 1, 3, 5, 10, 15, and 20 min in SDRs (n = 3) whereas that in EHBRs (n = 4) was 5190 ± 1640, 3290 ± 673, 2050 ± 432, 733 ± 225, 200 ± 51.1, and 99.0 ± 17.4 ng/ml at 1, 3, 5, 10, 20, and 30 min. Cumulative radioactivity excreted into the bile in SDRs and EHBRs was 64.4 ± 15.6 and 45.6 ± 9.04, and 75.3 ± 19.5 and 66.7 ± 10.2% of the dose at 20 and 120 min, respectively. The biliary clearance based on these profiles was 12.8 ± 1.7 and 5.2 ± 1.9 ml/min/kg in SDRs and EHBR, respectively. In addition, the ATP-dependent uptake of DNP-SG, a typical substrate for cMOAT/MRP2, by canalicular membrane vesicles was inhibited by BIBR 277 and its glucuronide in a concentration-dependent manner with K_i values of 3.44 ± 0.37 and 1.81 ± 0.30 μM, respectively, suggesting that both BIBR 277 and BIBR 277 glucuronide have an affinity for cMOAT/MRP2.

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Figure 1

Plasma concentrations (A) and biliary excretion (B) of BIBR 277 (circles) and its glucuronide (triangles) after i.v. administration of [¹⁴C]BIBR 277 (1 mg/kg) to SDRs (closed symbols) and EHBR (open symbols).

Each point and vertical bar represent the mean ± S.D. (n = 3 and 4 for panels A and B, respectively).

Even if we assume that half the BIBR 277 glucuronide excretion was impaired in EHBR due to cMOAT/MRP2 deficiency, the increase in the plasma glucuronide concentraion in EHBR appears to be much more than double that in SDRs (Fig. 1A). Another possible explanation for such an increase in plasma glucuronide concentration might be enhancement of the glucuronidation of BIBR 277 in EHBR. TheK_m values for the glucuronidation of BIBR 277 in the liver microsomes were similar between SDRs and EHBR (9.66 and 10.6 μM, respectively), whereas theV_max value was about 2.5 times higher in EHBR than SDRs (279 and 709 pmol/min/mg in SDRs and EHBR, respectively). Considering that the glucuronidation in EHBR is 2 to 3 times higher than in SDRs, whereas the biliary clearance of the glucuronide in EHBR is half that in SDRs, it appears reasonable that the biliary excretion of BIBR 277 glucuronide is similar in both rat strains after i.v. administration of the parent compound. In conclusion, BIBR 277 glucuronide transport across the bile canalicular membrane is mediated by both cMOAT/MRP2 and another transporter that is present in EHBR.