Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2)

Abstract

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR⁻ rats were perfused with FLAP (30 μM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR⁻ rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR⁻ rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 μM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.

Introduction

Flavopiridol (FLAP; NSC 649890, L86-8275, HMR 1275), is a selective inhibitor of cyclin dependent kinases (cdk1, cdk2, cdk4, cdk7) presently undergoing clinical phase II trials Carlson et al., 1996, De-Azevedo et al., 1996, Sedlacek, 2001, Shapiro et al., 2001. FLAP exerts pronounced antitumor activity in a variety of cell types including human breast Carlson et al., 1996, Worland et al., 1993, prostate (Drees et al., 1997), hematopoietic (Parker et al., 1998), lung (Bible and Kaufman, 1996), and head and neck cancer cells (Patel et al., 1998), and also in human tumor xenografts models including colon and prostate carcinomas Czech et al., 1995, Sedlacek et al., 1996, Arguello et al., 1998. Clinical trials with FLAP as a single agent or in combination with anticancer drugs also showed tumor responses in most phase I and phase II studies on different types of progressive tumors refractory to conventional treatment Thomas et al., 1997, Senderowicz et al., 1998, Innocenti et al., 2000, Shapiro et al., 2001. FLAP undergoes extensive metabolism in the rat liver to two monoglucuronides M1 and M2 mainly excreted into bile (Jäger et al., 1998). Pronounced glucuronidation followed by biliary elimination could be also observed in cancer patients as indicated by high glucuronide levels in plasma and enterohepatic circulation (Lush et al., 1997). Moreover, the main side effect, diarrhea, is also linked to biliary excretion of FLAP glucuronides (Innocenti et al., 2000) (Fig. 1).

Another toxicity of note during FLAP treatment is the induction of reversible conjugated hyperbilirubinemia (grade 2 or greater). It was observed in up to 22% of patients at doses of 78 mg/m²/day × 3 and lasted less than 48 hours postinfusion (Senderowicz et al., 1998). So far the molecular mechanism of FLAP-induced conjugated hyperbilirubinemia has not been elucidated. Conjugated bilirubin excretion into bile, however, is mediated with high affinity by the ATP-dependent transporter Mrp2. Hence, our hypothesis was that FLAP glucuronides may also be actively transported across the canalicular membrane into bile via Mrp2. This hypothesis is supported by previous studies from our lab showing a clear preference of Mrp2 for the biliary excretion of glucuronides from a structurally similar flavonoid, genistein. Addition of genistein to an unconjugated bilirubin containing rat liver perfusion medium also gradually decreased biliary excretion of bilirubin-conjugates by 76% based on a competition for this canalicular anion carrier (Jäger et al., 1997). FLAP-conjugates may therefore also act as competitive inhibitors of this transporter by modifying the hepatic disposition of bilirubin glucuronides. A possible interaction on the level of the enzymatic pathway between FLAP and bilirubin, causing hyperbilirubinemia can be excluded as bilirubin is glucuronidated selectively by the UDP-glucuronosyltransferase UGT1A1, whereas UGT1A9 is the major UGT involved in hepatic FLAP conjugation Hagenauer et al., 2001, Ramirez et al., 2002.

Because of the clinical importance of biliary FLAP elimination, we studied whether the excretion of FLAP and its glucuronides in the isolated perfused liver is dependent on Mrp2. For this approach, the release of FLAP and FLAP-glucuronides into bile and the perfusion medium were monitored in mutant TR⁻ rats lacking a functional Mrp2 at the canalicular membrane Jansen et al., 1987, Kitamura et al., 1992, Elferink and Groen, 2002. Mrp2-competent Wistar rats acted as controls. Additionally, we investigated whether FLAP can influence the hepatic excretion of the Mrp2 substrate bilirubin in control rats.

Although FLAP induced hyperbilirubinemia was described in humans, we chose the rat model for our investigations as the human and the rat orthologs of MRP2 exhibit very similar transport characteristics for conjugated bilirubin. Furthermore, contrary to in vivo studies, the isolated perfused rat liver is a good model to study the metabolism and biliary elimination of drugs since pharmacokinetic variables, such as renal clearance and drug distribution, are eliminated.