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Effect of DPC 333 [(2R)-2-{(3R)-3-Amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide], a Human Tumor Necrosis Factor -Converting Enzyme Inhibitor, on the Disposition of Methotrexate: A Transporter-Based Drug-Drug Interaction Case Study

Preclinical Candidate Optimization-Metabolism and Pharmacokinetics (G.L.) and Chemistry (J.D., C.P.D., T.M.), Bristol-Myers Squibb Company, Pennington, New Jersey; AstraZeneca Plc, Waltham, Massachusetts (C.E.G.); School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (H.X., K.L.R.B.); Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut (H.H., L.-S.G); GlaxoSmithKline, King of Prussia, Pennsylvania (L.E.R.); Quest Pharmaceutical Services, Inc., Newark, Delaware (H.S.); and Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts (F.W.L.).

(Received November 16, 2006; accepted February 26, 2007)

	Abstract

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DPC 333 [(2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide] is a potent human tumor necrosis factor -converting enzyme inhibitor with potential therapeutic implications for rheumatoid arthritis. Methotrexate (MTX), a drug for the treatment of rheumatoid arthritis, is eliminated primarily unchanged via renal and biliary excretion in humans as well as in rats and dogs. The objective of the present study was to investigate the potential effect of DPC 333 on the disposition of MTX. In dogs, DPC 333 administered orally at 1.7 mg/kg 15 min before the intravenous administration of [¹⁴C]MTX (0.5 mg/kg) did not alter the plasma concentration-time profile of MTX; however, the total amount of radioactivity excreted in urine increased from 58.7% to 92.2% of the dose, and the renal clearance increased from 1.8 ml/min/kg to 2.9 ml/min/kg, suggesting a decrease in MTX disposition via biliary excretion. The biliary excretion of MTX was investigated in isolated perfused livers prepared from wild-type and TR^- [multidrug resistance-associated protein 2 (Mrp2)-deficient] Wistar rats in the absence and presence of DPC 333. Mrp2-mediated biliary excretion of MTX was confirmed with 95.8% and 5.1% of MTX recovered in the bile of wild-type and TR^- Wistar rats, respectively. DPC 333 at an initial perfusate concentration of 50 µM completely blocked the biliary excretion of MTX, but not the clearance from perfusate, in both wild-type and TR^- rats. These results suggest that the enhanced renal elimination of MTX may be due to the potent inhibition of biliary excretion and active renal reabsorption by DPC 333 and/or its metabolites.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Chemical structures of DPC 333 and methotrexate.

Biliary excretion of MTX is mediated by MRP2 (Masuda et al., 1997). MRP2 (ABCC2), previously referred to as canalicular multispecific organic anion transporter (cMOAT), is an important ATP-binding cassette transporter (Ito et al., 1997; Xiong et al., 2000; Gerk and Vore, 2002). This apical membrane-bound transport protein is highly expressed in the canalicular membrane, and is also present in intestine, kidney, and the blood-brain barrier (Paulusma et al., 1996; Ito et al., 1997; Masuda et al., 1997; Xiong et al., 2000; Gerk and Vore, 2002). MRP2 plays a critical role in the biliary excretion of certain endogenous anionic compounds and many xenobiotics. Bilirubin glucuronide, for example, is excreted into bile via MRP2. Many drugs and their glucuronide-, glutathione-, and sulfate-conjugates are substrates for MRP2 (Paulusma et al., 1996; Ito et al., 1997; Masuda et al., 1997; Xiong et al., 2000; Gerk and Vore, 2002). MRP2 deficiency causes hyperbilirubinemia in humans (Dubin-Johnson syndrome), as well as in TR^- rats and Eisai hyperbilirubinemic rats (EHBR) (Jansen et al., 1985; Paulusma et al., 1996; Ito et al., 1997; Iyanagi et al., 1998). Biliary excretion of MTX is markedly inhibited by MRP2 inhibitors such as probenecid (Furst, 1995) and becomes insignificant in EHBR (Masuda et al., 1997).

Renal elimination of MTX is a combination of glomerular filtration, active renal proximal tubular secretion, and reabsorption (Liegler et al., 1969; Huang et al., 1979; Williams and Huang, 1982), with high capacity for elimination of excess MTX to compensate for impaired biliary excretion. Notably, active secretion of MTX is predominant in humans and monkeys, whereas active reabsorption is predominant in dogs (Liegler et al., 1969; Huang et al., 1979; Williams and Huang, 1982). Active reabsorption also occurs in rats and rabbits (Iven and Brasch, 1988, 1990; Statkevich et al., 1993). At high concentrations of MTX, renal elimination is greatly enhanced either through more active secretion in humans and monkeys or less active reabsorption in dogs, rats, and rabbits (Liegler et al., 1969; Huang et al., 1979; Williams and Huang, 1982; Iven and Brasch 1988, 1990; Statkevich et al., 1993). Folate binding protein 1 (FBP1), MRP2, and organic anion transporter (OAT) proteins, which are expressed in the proximal renal tubule, are involved in the active reabsorption (Birn et al., 2005) and secretion of MTX (Uwai et al., 1998, 2000; Van Aubel et al., 2000).

DPC 333 (Fig. 1), a tumor necrosis factor -converting enzyme inhibitor and a novel anti-inflammatory drug candidate for treatment of rheumatoid arthritis, is likely to be coadministered with MTX in patients. In the present study, the potential effect of DPC 333 on the disposition of MTX elimination was investigated using beagle dogs in vivo and isolated perfused rat livers in vitro. DPC 333 remarkably enhanced the renal elimination of MTX in dogs and potently inhibited the biliary excretion in isolated perfused rat livers. However, DPC 333 did not significantly alter the plasma concentration-time profile of MTX in dogs in vivo.

	Materials and Methods

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Chemicals. MTX [methotrexate; (+)-amethopterin] and aminopterin were purchased from Sigma-Aldrich (St. Louis, MO). [¹⁴C]MTX (55 mCi/mmol in ethanol/water 1:1, 0.1 mCi/ml, purity >98% by HPLC) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). DPC 333 [(2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide] (Fig. 1) was prepared by Bristol-Myers Squibb Company (Pennington, NJ).

Animals. For the in vivo pharmacokinetic and renal elimination of MTX studies, three male beagle dogs (6.3, 6.5, and 7.4 kg) were purchased from Marshall Farms (North Rose, NY). Dogs were allowed to acclimate for 2 weeks before experimentation. For isolated perfused rat liver studies, male wild-type (250 g; Charles River Laboratories, Raleigh, NC) and TR^- (250 g; in-house breeding colony) Wistar rats were used. Male retired breeder rats (Wistar; Charles River Laboratories) were used as blood donors. Rats were maintained on a 12-h light/dark cycle with access to chow and water ad libitum. Rats were allowed to acclimate for at least 5 days before experimentation.

Renal Elimination and Pharmacokinetics of MTX in Beagle Dogs. Three beagle dogs were dosed intravenously with 0.5 mg/kg [¹⁴C]MTX (2.5 mg/ml and 0.70 mCi/mmol). One milliliter of blood was taken before the dosing and at each of the following times: 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, and 36 h. Plasma (50 µl) in duplicate was used for measurement of [¹⁴C]MTX by liquid scintillation counting. Urine was collected before dosing and continuously up to 48 h. A 100-µl aliquot of urine samples in duplicate was used for measurement of [¹⁴C]MTX by liquid scintillation counting.

The same beagle dogs were reused to test the effect of DPC 333 on the pharmacokinetics and renal elimination of MTX after a 1-week washout. DPC 333 was dosed orally at 1.7 mg/kg 15 min before the intravenous administration of [¹⁴C]MTX. The blood and urine samples were collected and [¹⁴C]MTX was determined as described above. MTX is minimally metabolized to 7-hydroxy MTX in vivo by hepatic aldehyde oxidase (Lui et al., 1985; Nuernberg et al., 1990; Seideman et al., 1993; Chladek et al., 1997, 1998); the total radioactivity measured by liquid scintillation counting was mainly from [¹⁴C]MTX and was considered as the concentration of MTX.

Isolated Perfused Rat Livers. Rat livers were isolated from male wild-type and TR^- Wistar rats. After anesthesia (ketamine, 60 mg/kg, and xylazine, 12 mg/kg, i.p), the bile duct and portal vein were cannulated and the liver was perfused in situ with oxygenated Krebs-Ringer-bicarbonate buffer maintained at 37°C. After the liver was transferred to a 37°C perfusion chamber, perfusion was continued with 80 ml of recirculating oxygenated Krebs-Ringer-bicarbonate buffer containing 1% dextrose (w/v) and 20% heparinized (1000 U/ml) male rat blood (v/v) at a constant flow rate of 20 ml/min. Sodium taurocholate was constantly infused into the perfusate reservoir at a rate of 2 ml/h (30 µmol/h). The liver and perfusate were allowed to equilibrate for 15 min before addition of 1 ml containing 6.0 mM MTX with or without 4.0 mM DPC 333. The liver was perfused for 120 min. Bile was collected continuously and 0.5-ml perfusate samples were obtained at 15-min intervals. All samples were frozen at -20°C until assayed. Liver viability was determined by bile flow rate (0.8 and 0.13 µl/min/g liver for wild-type and TR^- Wistar rats, respectively), constant inflow perfusion pressure (<15 cm of H₂O), and the release of lactate dehydrogenase from the liver (<0.1 I.U./g liver/h; assayed with Sigma lactate dehydrogenase diagnostic kit, catalog number 500) (Brouwer and Thurman 1996; Xiong et al., 2000, 2002).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Effect of DPC 333 on the plasma concentration-time profile of MTX in beagle dogs. In the first week, three beagle dogs were dosed with [14C]MTX (0.5 mg/kg i.v.). In the second week, the dogs received an oral dose of DPC 333 (1.7 mg/kg) and 15 min later, [14C]MTX (0.5 mg/kg) was administered. Blood samples were collected and analyzed as described under Materials and Methods.

Determination of MTX in Bile. A 20-µl aliquot of bile sample was mixed with 40 µl of 15 µM aminopterin (internal standard) in 10 mM ammonium formate buffer (pH 7.4). Ten microliters of this mixture was injected onto an Agilent 1100 HPLC system equipped with a Metachem Polaris C18-A column (100 x 2.0 mm, 5 µ; Varian, Inc.). Mobile phase A was 10 mM ammonium formate buffer (pH 7.4) containing 5% acetonitrile and mobile phase B was 10 mM ammonium formate buffer (pH 7.4) containing 90% acetonitrile. The flow rate was 0.3 ml/min. The initial mobile phase composition was 0% B for 3 min, ramped to 40% B over 3 min and held for 7 min, then ramped to 70% B over 1 min and held for 1 min, returned to the initial condition over the next 0.01 min, and reequilibrated for 5 min. The retention time was 2.04 min and 4.20 min for aminopterin and MTX, respectively. The compounds were detected by MS using a Finnigan LCQ mass spectrometer (Thermo Electron) operated in the positive ion mode, and peak area was measured over 439 to 443 amu at 2.04 min for aminopterin, and 453 to 457 amu at 4.20 min for MTX. A standard curve was generated and used to quantify MTX in the study samples.

Data Analysis. MTX pharmacokinetic parameters were determined with noncompartmental analysis using the computer software, Kinetica 4.2 (Innaphase, Philadelphia, PA). The area under the MTX concentration versus time curve (AUC_0-) was determined using a combination of linear and log-linear trapezoidal summations. Additional pharmacokinetic parameters were also generated including clearance (CL), steady-state volume of distribution (V_ss), mean residence time, and elimination half-life (t). The paired data from n = 3 beagle dogs in the absence and presence of pretreatment with DPC 333 were compared using Student's t test at an = 0.05 level of significance.

Renal clearance (CL_renal) was calculated by the timed-interval method: CL_renal = U_t1-t2/AUC_t1-t2, where U_t1-t2 was the amount (in the unit of nmol/kg) of MTX excreted unchanged in the urine during the time interval t1 to t2 and AUC_t1-t2 was the area under the plasma concentration time curve between t1 and t2.

	Results

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Effect on Pharmacokinetics of MTX in Beagle Dogs. After intravenous administration, MTX was found to have a total body CL (CL_total) of 3.1 ± 0.1 ml/min/kg, a V_ss of 0.5 ± 0.1 l/kg, and a t of 2.5 ± 0.4 h (Table 1). DPC 333 (1.7 mg/kg), which was dosed orally 15 min before the intravenous administration of [¹⁴C]MTX (0.5 mg/kg), did not significantly affect the plasma concentration-time profile of MTX in beagle dogs (Fig. 2). No statistically significant differences were observed in the absence and presence of DPC 333 (Table 1).

Effect on Renal Elimination of MTX in Beagle Dogs. In the absence of DPC 333, the mean cumulative (0- to 48-h) recovery of [¹⁴C]MTX in urine was 58.7 ± 1.8% of the dose (Fig. 3; Table 2). Most of the renal elimination of MTX occurred in the first 12 h (55.2% of the dose). Because MTX is minimally metabolized to 7-hydroxy MTX in dogs in vivo (Henderson et al., 1965a; Huang et al., 1979), it was inferred that the biliary clearance of MTX in beagle dogs at this dose level contributed approximately 40% of the total clearance. When DPC 333 was dosed orally at 1.7 mg/kg 15 min before intravenous administration of [¹⁴C]MTX, the mean cumulative renal elimination of MTX in 48 h was significantly increased to 92.2 ± 14.0% of the dose (Table 2). The cumulative renal elimination of MTX was enhanced remarkably by DPC 333 in the first 2 h (48.7% versus 8.1% of the dose; Table 2); in contrast, between 2 and 48 h, DPC 333 did not increase further the renal elimination of MTX (43.5% versus 50.6% of the dose in the presence and absence of DPC 333, respectively). It is noteworthy that in the presence of DPC 333, the renal clearance (CL_renal) of MTX was increased significantly from 0.4 ± 0.1, 1.6 ± 0.4, and 1.8 ± 0.1 ml/min/kg to 2.9 ± 0.5, 3.0 ± 0.5, and 2.9 ± 0.4 ml/min/kg, in the time periods of 0 to 2 h, 0 to 8 h, and 0 to 48 h, respectively (Table 2).

View larger version (17K): [in this window] [in a new window]   FIG. 3. Effect of DPC 333 on the renal elimination of MTX in beagle dogs. Experiments were performed as described in the legend to Fig. 2. Urine was collected before the dose and continuously up to 48 h. Data represent the mean ± S.D. of n = 3.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Bile flow rate of isolated perfused rat livers. Livers isolated from male wild-type (WT) or TR- Wistar rats were perfused at 20 ml/min for 120 min with 80 ml of recirculating perfusate containing MTX at an initial concentration of 75 µM in the absence (n = 2) or presence (n = 2) of DPC 333 at an initial concentration of 50 µM. Bile was collected continuously at 5-, 10-, or 15-min intervals. Data represent the average of n = 2 livers in each group.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effect of DPC 333 on the biliary excretion of MTX in isolated perfused rat livers. Livers isolated from male wild-type (A) or TR- (B) Wistar rats were perfused at 20 ml/min for 120 min with 80 ml of recirculating perfusate containing MTX at an initial concentration of 75 µM in the absence (n = 2) or presence (n = 2) of DPC 333 at an initial concentration of 50 µM. Bile was collected continuously at 5-, 10-, or 15-min intervals. Concentrations of MTX in bile were determined based on quantitative liquid chromatography/MS analysis as described under Materials and Methods. Data represent the average of n = 2 livers in each group.

Effect on Biliary Excretion of MTX in Isolated Perfused Rat Livers. In the isolated perfused rat livers, the cumulative (0- to 120-min) biliary excretion of MTX (75 µM in 80 ml of recirculating perfusate) was 95.8% in wild-type rat livers (Fig. 5A), but only 5.1% in TR^- rat livers (Fig. 5B). This report demonstrates that TR^- rat livers dispose of MTX very differently from the wild-type livers. These results also confirmed that MTX is a Mrp2 substrate and that Mrp2 plays a critical role (Masuda et al., 1997; Borst et al., 2000), whereas other transporters only play a minor role in the biliary excretion of MTX.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Effect of DPC 333 on the disposition of MTX in isolated perfused rat livers. Livers isolated from male wild-type and TR- Wistar rats were perfused as described in the legend to Fig. 4. Data represent the average of n = 2 livers in each group.

Effect on MTX Disposition in Isolated Perfused Rat Livers. In the absence of DPC 333, the remaining concentrations of MTX in the perfusate of TR^- and wild-type rat livers after the 120-min perfusion were 25.8 and 3.1 µM, respectively (Fig. 6). The clearance of MTX from the recirculating perfusate was much less extensive in TR^- (0.7 ml/min) compared with wild-type rat livers (3.4 ml/min; Table 3). In the presence of DPC 333, the remaining concentrations of MTX in the perfusate of TR^- and wild-type rat livers were 23.8 and 2.0 µM, respectively (Fig. 6); the clearance of MTX from the recirculating perfusate was 0.8 and 4.0 ml/min, respectively. Clearly, DPC 333 did not affect the perfusate concentrations of MTX in TR^- (0.7 versus 0.8 ml/min) or in wild-type (3.4 versus 4.0 ml/min) rat livers (Table 3).

	Discussion

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MTX is an excellent substrate to use to demonstrate the role of transporters in drug disposition and excretion. This therapeutic agent is minimally metabolized to 7-hydroxy MTX in vivo by hepatic aldehyde oxidase (Lui et al., 1985; Nuernberg et al., 1990; Seideman et al., 1993; Chladek et al., 1997, 1998) but is a substrate for multiple transport proteins. Reduced folate carrier 1 (RFC1, solute carrier 19A1), cloned from humans, murine, and other species, facilitates the absorption from the intestine and distribution in other tissues (Rajgopal et al., 2001). In the liver, Mrp2 is predominantly responsible for the biliary excretion of MTX (Masuda et al., 1997), whereas Mrp3, which is expressed on the sinusoidal membrane of hepatocytes, effluxes MTX from hepatocytes into blood (Hirohashi et al., 1999; Kool et al., 1999; Borst et al., 2000). In the kidney, Mrp2 and Oats contribute to the active renal tubular secretion of MTX (Saito et al., 1996; Uwai et al., 1998, 2000; Van Aubel et al., 2000), whereas FBP1 likely handles the active reabsorption of MTX, just as that for folic acid (Birn et al., 2005). The functional activities of MRP2 and FBP1 have been demonstrated to affect the elimination of MTX. For instance, high plasma concentrations, a long half-life, and slow clearance of MTX were observed in MRP2-deficient patients (Hulot et al., 2005) and in EHBR (Masuda et al., 1997), and in the presence of the Mrp2 inhibitors probenecid (Iven and Brasch, 1988; Ueda et al., 2001) and penicillin G (Iven and Brasch 1990). Increased renal clearance of MTX was observed in the presence of cephalosporins, ceftriaxone, sulfamethoxazole, indomethacin, and flurbiprofen (Iven and Brasch, 1988, 1990; Statkevich et al., 1993).

In the absence of DPC 333, the cumulative renal excretion of MTX in beagle dogs was 58.7% of the total dose, consistent with a previous report from another laboratory (Henderson et al., 1965a). Since MTX undergoes minimal metabolism in vivo, this result suggests that biliary excretion contributes approximately 40% to its total elimination. In the presence of DPC 333, the cumulative renal excretion of MTX was increased to 92.2% of the total dose, consistent with impaired biliary excretion of MTX mediated by Mrp2. Therefore, it was hypothesized that DPC 333 or its metabolite(s) inhibited Mrp2.

The present study confirmed the critical role of Mrp2 in the biliary excretion of MTX in rat livers reported previously by another laboratory (Masuda et al., 1997). In isolated perfused rat livers, 95.8% of MTX administered to the recirculating perfusate was recovered in the bile of wild-type rats, but only 5.1% of the dose was recovered in the bile of TR^- rats. MTX perfusate concentrations declined more rapidly in wild-type rat livers. This outcome could be attributed to Mrp2 deficiency on the canalicular membrane and up-regulation of Mrp3 on the basolateral membrane of TR^- rat hepatocytes. MTX is also a substrate of Mrp3, which effluxes MTX back into the perfusate in the present study or into blood stream in vivo (Hirohashi et al., 1999; Kool et al., 1999; Borst et al., 2000). The up-regulation of Mrp3 in TR^- rats (Xiong et al., 2002) and EHBR (Akita et al., 2001) compensates for Mrp2 deficiency on the canalicular membrane.

In the present study, DPC 333 almost completely blocked the biliary excretion of MTX in the isolated perfused livers from wild-type rats. Even though only a small percentage of the MTX dose was excreted into bile of TR^- rat livers, DPC 333 almost completely abolished MTX biliary excretion. This latter result suggests that the residual biliary excretion of MTX in TR^- rat livers is due to one or more other canalicular transport proteins that are inhibited by DPC 333. Notably, DPC 333 did not significantly change the bile flow rates of isolated perfused rat (both wild-type and TR^-) livers. In beagle dogs, the cumulative renal excretion of MTX was 58.7% and 92.2%, respectively, in the absence and presence of DPC 333. This difference may be attributed to potent inhibition of MTX biliary excretion in the presence of DPC 333. The remarkable reduction in the biliary excretion of MTX in the presence of DPC 333 was likely due to inhibition of Mrp2 by DPC 333 or its metabolites, and not because of inhibition of MTX uptake into hepatocytes, as demonstrated by the lack of effect of DPC 333 on the initial MTX perfusate concentration-time profiles in the isolated perfused liver studies.

Glucuronide conjugates of DPC 333 most likely inhibited Mrp2 rather than direct inhibition by the parent compound itself. The cumulative biliary excretion of DPC 333 was less than 0.4% of the total dose administered to the recirculating perfusate, and no difference was observed between wild-type and TR^- Wistar rat livers (data not shown), consistent with the previous finding that DPC 333 was a P-glycoprotein rather than a Mrp2 substrate (data not shown). Glucuronidation represents the major metabolic pathway of DPC 333 elimination in rats and beagle dogs in vivo; more than 55% of absorbed DPC 333 was biotransformed into the glucuronide conjugate in a mass balance study in rats, and the glucuronide conjugate of DPC 333 was found at high concentrations in the rat bile (data not shown). The glucuronide conjugate of DPC 333 would likely be a substrate of Mrp2 and Mrp3.

Renal elimination of MTX is the sum of glomerular filtration, active tubular secretion, and reabsorption. Glomerular filtration is a passive process and is unlikely to be affected by DPC 333 or its metabolites. In the absence of DPC 333, the renal clearance of MTX in beagle dogs (from 0 to 48 h) was 1.8 ± 0.1 ml/min/kg. With correction for canine plasma protein binding (36.9%) (Lui et al., 1985), the renal clearance of MTX (2.9 ± 0.2 ml/min/kg) was lower than the glomerular filtration rate determined in beagle dogs (4.4 ± 1.2 ml/min/kg) using inulin (Lefebvre et al., 1997). Our observation indicates that active renal tubular reabsorption of MTX is significant in dogs, consistent with a previous report (Huang et al., 1979). In the presence of DPC 333, the renal clearance of MTX (throughout the whole experiment) was increased to 2.9 ± 0.4 ml/min/kg. With correction for protein binding, the renal clearance of MTX (4.6 ± 0.6 ml/min/kg) approximated the glomerular filtration rate. This increase was likely due to inhibition of active renal tubular reabsorption. The active reabsorption of MTX may be mediated by FBP1 (Birn et al., 2005), similar to folic acid (a MTX analog); high concentrations of folic acid block the renal tubular reabsorption of MTX (Huang et al., 1979). Furthermore, it is possible that high concentrations of MTX inhibit the active reabsorption of MTX, similar to folic acid, and consequently enhance MTX renal elimination. Further studies are required to determine whether the increased renal elimination of MTX observed in the present study in the presence of DPC 333 was due to inhibition of FBP1 by DPC 333 (and/or its metabolites) or by MTX itself.

In healthy humans, the impact of DPC 333 on the pharmacokinetics of MTX should be minimal because the biliary excretion of MTX in humans is insignificant; as previously reported, approximately 5% and 90% of a MTX dose was recovered in human feces and urine, respectively (Henderson et al., 1965b). However, caution should be exercised when DPC 333 and MTX are administered concomitantly to individuals with impaired renal function.

In summary, DPC 333 significantly enhanced the renal elimination, but did not markedly alter the plasma concentration-time profile, of MTX in beagle dogs. DPC 333 almost completely blocked the biliary excretion of MTX in isolated perfused rat livers, likely due to inhibition of Mrp2 by the glucuronide conjugates of DPC 333.

	Footnotes

 
doi:10.1124/dmd.106.013946.

H.X. and K.L.R.B. were funded in part by Grant R01 GM41935 from the National Institutes of Health.

ABBREVIATIONS: MTX, methotrexate; amu, atomic mass unit(s); AUC, area under the plasma concentration-time curve; CL, clearance; DPC 333, (2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide; EHBR, Eisai hyperbilirubinemic rats; FBP, folate binding protein; HPLC, high-performance liquid chromatography; MRP, multidrug resistance-associated protein; t, half-life; TR^-, Mrp2-deficient Wistar rats; V_ss, steady-state volume of distribution.

Address correspondence to: Dr. Gang Luo, Bristol-Myers Squibb Company, 311 Pennington Rockyhill Road, Pennington, NJ, 08534. E-mail: gang.luo{at}bms.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Akita H, Suzuki H, and Sugiyama Y (2001) Sinusoidal efflux of taurocholate is enhanced in Mrp2-deficient rat liver. Pharm Res (NY) 18: 1119-1125.

Birn H, Spiegelstein O, Christensen E, and Finnell R (2005) Renal tubular reabsorption of folate mediated by folate binding protein 1. J Am Soc Nephrol 16: 608-615.[Abstract/Free Full Text]

Borst P, Evers R, Kool M, and Wijnholds J (2000) A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst 92: 1295-1302.[Abstract/Free Full Text]

Bressolle F, Bologna C, Kinowski J, Sany J, and Combe B (1998) Effects of moderate renal insufficiency on pharmacokinetics of methotrexate in rheumatoid arthritis patients. Ann Rheum Dis 57: 110-113.[Abstract/Free Full Text]

Brouwer KLR and Thurman RG (1996) Isolated persfused liver, in Models for Assessing Drug Absorption and Metabolism (Borchardt RT, Smith PL, and Wilson G eds) pp 161-186. Plenum Press, New York.

Chladek J, Martinkova J, Simkova M, Vaneckova J, Koudelkova V, and Nozickova M (1998) Pharmacokinetics of low doses of methotrexate in patients with psoriasis over the early period of treatment. Eur J Clin Pharmacol 53: 437-444.[CrossRef][Medline]

Chladek J, Martinkova J, and Sispera L (1997) An in vitro study on methotrexate hydroxylation in rat and human liver. Physiol Res 46: 371-379.[Medline]

Furst D (1995) Practical clinical pharmacology and drug interactions of low-dose methotrexate therapy in rheumatoid arthritis. Br J Rheumatol 34 (Suppl 2): 20-25.[Medline]

Gerk P and Vore M (2002) Regulation of expression of the multidrug resistance-associated protein 2 (MRP2) and its role in drug disposition. J Pharmacol Exp Ther 302: 407-415.[Abstract/Free Full Text]

Henderson E, Adamson R, Denham C, and Oliverio V (1965a) The metabolic fate of tritiated methotrexate. I. Absorption, excretion, and distribution in mice, rats, dogs and monkeys. Cancer Res 25: 1008-1017.[Medline]

Henderson E, Adamson R, and Oliverio V (1965b) The metabolic fate of tritiated methotrexate. II. Absorption and excretion in man. Cancer Res 25: 1018-1024.[Medline]

Hirohashi T, Suzuki H, and Sugiyama Y (1999) Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3). J Biol Chem 274: 15181-15185.[Abstract/Free Full Text]

Huang K, Wenczak B, and Liu Y (1979) Renal tubular transport of methotrexate in the rhesus monkey and dog. Cancer Res 39: 4843-4848.[Abstract/Free Full Text]

Hulot J, Villard E, Maguy A, Morel V, Mir L, Tostivint I, William-Faltaos D, Fernandez C, Hatem S, Deray G, et al. (2005) A mutation in the drug transporter gene ABCC2 associated with impaired methotrexate elimination. Pharmacogenet Genomics 15: 277-285.[Medline]

Ito K, Suzuki H, Hirohashi T, Kume K, Shimizu T, and Sugiyama Y (1997) Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR. Am J Physiol 272: G16 -G22.[Medline]

Iven H and Brasch H (1988) The effects of antibiotics and uricosuric drugs on the renal elimination of methotrexate and 7-hydroxymethotrexate in rabbits. Cancer Chemother Pharmacol 21: 337-342.[Medline]

Iven H and Brasch H (1990) Cephalosporins increase the renal clearance of methotrexate and 7-hydroxymethotrexate in rabbits. Cancer Chemother Pharmacol 26: 139-143.[Medline]

Iyanagi T, Emi Y, and Ikushiro S (1998) Biochemical and molecular aspects of genetic disorders of bilirubin metabolism. Biochim Biophys Acta 1407: 173-184.[Medline]

Jansen PL, Peters WH, and Lamers WH (1985) Hereditary chronic conjugated hyperbilirubinemia in mutant rats caused by defective hepatic anion transport. Hepatology 5: 573-579.[Medline]

Kool M, van der Linden M, de Haas M, Scheffer G, de Vree J, Smith A, Jansen G, Peters G, Ponne N, Scheper R, et al. (1999) MRP3, an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci USA 96: 6914-6919.[Abstract/Free Full Text]

Lefebvre H, Laroute V, Alvinerie M, Schneider M, Vinclair P, Braun J, and Toutain P (1997) The effect of experimental renal failure on tolfenamic acid disposition in the dog. Biopharm Drug Dispos 18: 79-91.[CrossRef][Medline]

Liegler D, Henderson E, Hahn M, and Oliverio V (1969) The effect of organic acids on renal clearance of methotrexate in man. Clin Pharmacol Ther 10: 849-857.[Medline]

Lui C, Lee M, and Chiou W (1985) Urinary pH and urine flow independent renal clearance of methotrexate in dogs. J Pharmacokinet Biopharm 13: 159-171.[CrossRef][Medline]

Masuda M, I'izuka Y, Yamazaki M, Nishigaki R, Kato Y, Ni'inuma K, Suzuki H, and Sugiyama Y (1997) Methotrexate is excreted into the bile by canalicular multispecific organic anion transporter in rats. Cancer Res 57: 3506-3510.[Abstract/Free Full Text]

Nuernberg B, Koehnke R, Solsky M, Hoffman J, and Furst D (1990) Biliary elimination of low-dose methotrexate in humans. Arthritis Rheum 33: 898-902.[Medline]

Paulusma C, Bosma P, Zaman G, Bakker C, Otter M, Scheffer G, Scheper R, Borst P, and Oude Elferink R (1996) Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science (Wash DC) 271: 1126-1128.[Abstract]

Rajgopal A, Sierra E, Zhao R, and Goldman I (2001) Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities. Am J Physiol 281: C1579-C1586.

Saito H, Masuda S, and Inui K (1996) Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney. J Biol Chem 271: 20719-20725.[Abstract/Free Full Text]

Schnabel A and Gross WL (1994) Low-dose methotrexate in rheumatic diseases-efficacy, side effects, and risk factors for side effects. Semin Arthritis Rheum 23: 310-327.[CrossRef][Medline]

Seideman P, Beck O, Eksborg S, and Wennberg M (1993) The pharmacokinetics of methotrexate and its 7-hydroxy metabolite in patients with rheumatoid arthritis. Br J Clin Pharmacol 35: 409-412.[Medline]

Statkevich P, Fournier D, and Sweeney K (1993) Characterization of methotrexate elimination and interaction with indomethacin and flurbiprofen in the isolated perfused rat kidney. J Pharmacol Exp Ther 265: 1118-1124.[Abstract/Free Full Text]

Steinberg S, Campbell C, Bleyer W, and Hillman R (1982) Enterohepatic circulation of methotrexate in rats in vivo. Cancer Res 42: 1279-1282.[Abstract/Free Full Text]

Ueda K, Kato Y, Komatsu K, and Sugiyama Y (2001) Inhibition of biliary excretion of methotrexate by probenecid in rats: quantitative prediction of interaction from in vitro data. J Pharmacol Exp Ther 297: 1036-1043.[Abstract/Free Full Text]

Uwai Y, Okuda M, Takami K, Hashimoto Y, and Inui K (1998) Functional characterization of the rat multispecific organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney. FEBS Lett 438: 321-324.[CrossRef][Medline]

Uwai Y, Saito H, and Inui K (2000) Interaction between methotrexate and nonsteroidal anti-inflammatory drugs in organic anion transporter. Eur J Pharmacol 409: 31-36.[CrossRef][Medline]

Van Aubel R, Masereeuw R, and Russel F (2000) Molecular pharmacology of renal organic anion transporters. Am J Physiol 279: F216-F232.

Weinblatt M, Coblyn J, Fox D, Fraser P, Holdsworth D, Glass D, and Trentham D (1985) Efficacy of low-dose methotrexate in rheumatoid arthritis. N Engl J Med 312: 818-822.[Abstract]

Williams W and Huang K (1982) Renal tubular transport of folic acid and methotrexate in the monkey. Am J Physiol 242: F484-F490.[Medline]

Xiong H, Suzuki H, Sugiyama Y, Meier P, Pollack G, and Brouwer KLR (2002) Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment. Drug Metab Dispos 30: 962-969.[Abstract/Free Full Text]

Xiong H, Turner K, Ward E, Jansen P, and Brouwer KLR (2000) Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(-) rats. J Pharmacol Exp Ther 295: 512-518.[Abstract/Free Full Text]

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