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Organic anion-transporting polypeptides (OATPs) are important drug uptake transporters, mediating distribution of substrates to several pharmacokinetically relevant organs. Doxorubicin is a widely used anti-cancer drug extensively studied for its interactions with various drug transporters, but not OATPs. Here, we investigated the role of OATP1A/1B proteins in the distribution of doxorubicin. In vitro, we observed ∼ 2-fold increased doxorubicin uptake in HEK293 cells overexpressing human OATP1A2, but not OATP1B1 or OATP1B3. In mice, absence of Oatp1a/1b transporters led to up to 2-fold higher doxorubicin plasma concentrations and 1.3-fold higher plasma AUC. Conversely, liver AUC and liver-to-plasma ratios of Oatp1a/1b(-/-) mice were 1.4-fold and up to 4.1-fold lower than in wild-type mice, respectively. Decreased doxorubicin levels in the small intestinal content reflected those in the liver, indicating a reduced biliary excretion of doxorubicin in Oatp1a/1b(-/-) mice. These results demonstrate important control of doxorubicin plasma clearance and hepatic uptake by mouse Oatp1a/1b transporters. This is unexpected, as the fairly hydrophobic weak base doxorubicin is an atypical OATP1A/1B substrate. Interestingly, transgenic liver-specific expression of human OATP1A2, OATP1B1 or OATP1B3 could partially rescue the increased doxorubicin plasma levels of Oatp1a/1b(-/-) mice. Hepatic uptake and bile-derived intestinal excretion of doxorubicin were completely reverted to wild-type levels by OATP1A2, and partially by OATP1B1 and OATP1B3. Thus, doxorubicin is transported by hepatocyte-expressed OATP1A2, -1B1 and -1B3 in vivo, illustrating an unexpectedly wide substrate specificity. These findings have possible implications for the uptake, disposition, therapy response and toxicity of doxorubicin, also in human tumors and tissues expressing these transporters., (© 2014 UICC.)

Organic anion-transporting polypeptides (OATPs) mediate the liver uptake and hence plasma clearance of a broad range of drugs. For rosuvastatin, a cholesterol-lowering drug and OATP1A/1B substrate, the liver represents both its main therapeutic target and its primary clearance organ. Here we studied the impact of Oatp1a/1b uptake transporters on the pharmacokinetics of rosuvastatin using wild-type and Oatp1a/1b-null mice. After oral administration (15 mg/kg), intestinal absorption of rosuvastatin was not impaired in Oatp1a/1b-null mice, but systemic exposure (area under the curve) was 8-fold higher in these mice compared with wild-type. Although liver exposure was comparable between the two mouse strains (despite the increased blood exposure), the liver-to-blood ratios were markedly decreased (>10-fold) in the absence of Oatp1a/1b transporters. After intravenous administration (5 mg/kg), systemic exposure was 3-fold higher in Oatp1a/1b-null mice than in the wild-type mice. Liver, small intestinal, and kidney exposure were slightly, but not significantly, increased in Oatp1a/1b-null mice. The biliary excretion of rosuvastatin was very fast, with 60% of the dose eliminated within 15 minutes after intravenous administration, and also not significantly altered in Oatp1a/1b-null mice. Rosuvastatin renal clearance, although still minor, was increased ∼15-fold in Oatp1a/1b-null males, suggesting a role of Oatp1a1 in the renal reabsorption of rosuvastatin. Absence of Oatp1a/1b uptake transporters increases the systemic exposure of rosuvastatin by reducing its hepatic extraction ratio. However, liver concentrations are not significantly affected, most likely due to the compensatory activity of high-capacity, low-affinity alternative uptake transporters at higher systemic rosuvastatin levels and the absence of efficient alternative rosuvastatin clearance mechanisms.

Organic anion-transporting polypeptides (OATPs) mediate the hepatic uptake of many drugs. Hepatic uptake is crucial for the therapeutic effect of pravastatin, a cholesterol-lowering drug and OATP1A/1B substrate. We aimed to gain empirical insight into the relationship between OATPs and pravastatin pharmacokinetics and toxicity. We therefore compared the distribution and toxicity of pravastatin in wild-type and Oatp1a/1b-null mice. Intestinal absorption of pravastatin was not affected by Oatp1a/1b absence, but systemic plasma exposure (AUC) increased up to 30-fold after oral bolus administration. This increased plasma exposure resulted from reduced hepatic uptake, as evident from 10 to 100-fold lower liver-to-plasma concentration ratios. However, the reductions in liver exposure were far smaller (<2-fold) than the increases in plasma exposure. Reduced pravastatin liver uptake in Oatp1a/1b-null mice was more obvious shortly after intravenous administration, with 8-fold lower biliary pravastatin excretion. Although mice chronically exposed to pravastatin for 60 days evinced little muscular toxicity, Oatp1a/1b-null mice displayed 10-fold higher plasma concentrations and 8-fold lower liver concentrations than wild-type mice. Thus, Oatp1a/1b transporters importantly control the hepatic uptake of pravastatin. Activity-reducing human OATP1B polymorphisms may therefore both reduce pravastatin therapeutic efficacy in the liver and increase systemic toxicity risks, thus compromising its therapeutic index in a two-edged way.

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.

Diclofenac is an important analgesic and anti-inflammatory drug that is widely used for the treatment of postoperative pain, rheumatoid arthritis, and chronic pain associated with cancer. Diclofenac is extensively metabolized in the liver, and the main metabolites are hydroxylated and/or glucuronidated conjugates. We show here that loss of multidrug resistance protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) in mice results in highly increased plasma levels of diclofenac acyl glucuronide, after both oral and intravenous administration. The absence of Mrp2 and Bcrp1, localized at the canalicular membrane of hepatocytes, leads to impaired biliary excretion of acyl glucuronides and consequently to elevated liver and plasma levels. Mrp2 also mediates the biliary excretion of two hydroxylated diclofenac metabolites, 4'-hydroxydiclofenac and 5-hydroxydiclofenac. We further show that the sinusoidal efflux of diclofenac acyl glucuronide, from liver to blood, is largely dependent on multidrug resistance protein 3 (MRP3/ABCC3). Diclofenac acyl glucuronides are chemically instable and reactive, and in patients, these metabolites are associated with rare but serious idiosyncratic liver toxicity. This might explain why Mrp2/Mrp3/Bcrp1(-/-) mice, which have markedly elevated levels of diclofenac acyl glucuronides in their liver, display acute, albeit very mild, hepatotoxicity. We believe that the handling of diclofenac acyl glucuronides by ATP binding cassette transporters may be representative for the handling of acyl glucuronide metabolites of many other clinically relevant drugs.

Purpose: ABCC2 (MRP2) and ABCG2 (BCRP) transport various endogenous and exogenous compounds, including many anticancer drugs, into bile, feces, and urine. We investigated the possibly overlapping roles of Abcg2 and Abcc2 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX)., Experimental Design: We generated and characterized Abcc2;Abcg2(-/-) mice, and used these to determine the overlapping roles of Abcc2 and Abcg2 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX., Results: Compared with wild-type, the plasma areas under the curve (AUC) for MTX were 1.6-fold and 2.0-fold higher in Abcg2(-/-) and Abcc2(-/-) mice, respectively, and 3.3-fold increased in Abcc2;Abcg2(-/-) mice. The biliary excretion of MTX was 23-fold reduced in Abcc2;Abcg2(-/-) mice, and the MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-MTX were not significantly altered in Abcg2(-/-) mice, but the areas under the curve were 6.2-fold and even 12.4-fold increased in Abcc2(-/-) and Abcc2;Abcg2(-/-) mice, respectively. This indicates that Abcc2 compensates for Abcg2 deficiency but that Abcg2 can only partly compensate for Abcc2 absence. Furthermore, 21-fold decreased biliary 7OH-MTX excretion in Abcc2;Abcg2(-/-) mice and substantial 7OH-MTX accumulation in the liver and kidney were seen. We additionally found that in the absence of Abcc2, Abcg2 mediated substantial urinary excretion of MTX and 7OH-MTX., Conclusions: Abcc2 and Abcg2 together are major determinants of MTX and 7OH-MTX pharmacokinetics. Variations in ABCC2 and/or ABCG2 activity due to polymorphisms or coadministered inhibitors may therefore substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.

Human organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter that can transport a wide variety of drugs. In the present study, we have generated and characterized a transgenic mouse model with specific and functional expression of human OATP1B1 (SLCO1B1) in the liver. Immunohistochemical staining revealed basolateral localization of transgenic OATP1B1 in the liver, whereas no expression of OATP1B1 was found in the kidney and small intestine. Using this transgenic model, the in vivo role of human OATP1B1 in the disposition of the anticancer drug methotrexate (MTX) was studied. In mice on a semisynthetic diet, the area under the plasma concentration-time curve for intravenous methotrexate in SLCO1B1 transgenic mice was 1.5-fold decreased compared with wild-type mice. Furthermore, the amount of MTX in the liver was markedly higher ( approximately 2-fold) in the SLCO1B1 transgenic mice compared with wild-type mice, resulting in 2- to 4-fold higher liver-plasma ratios of MTX. Some murine liver Slco genes were markedly down-regulated on the semisynthetic diet compared with a standard diet, which probably reduced murine Oatp-mediated MTX uptake in the liver and therefore facilitated detection of the function of the transgenic OATP1B1. Taken together, these data demonstrate a marked and possibly rate-limiting role for human OATP1B1 in MTX elimination in vivo. Variation in OATP1B1 activity due to genetic polymorphisms, drug-drug interactions, and possibly dietary conditions may therefore play a role in the severity of MTX-related toxicity. SLCO1B1 transgenic mice could be a useful tool in studying the in vivo role of human OATP1B1 in drug pharmacokinetics.

CYP3A4 is an important xenobiotic metabolizing enzyme. We previously found that CYP2C55 is highly up-regulated in Cyp3a(-/-) mice. Here, we have further investigated the mechanism of regulation of CYP2C55 and other detoxifying systems in Cyp3a(-/-) mice. Induction studies with prototypical inducers demonstrated an important role for the nuclear receptors PXR and CAR in the up-regulation of CYP2C55. Subsequent diet-switch experiments revealed that food-derived xenobiotics are primarily responsible for the increased induction of CYP2C55, as well as of several other primary detoxifying systems in Cyp3a(-/-) mice. Our data suggest that CYP3A normally metabolizes food-derived activators of PXR and/or CAR, explaining the increased levels of such activators in Cyp3a(-/-) mice and subsequent up-regulation of a range of detoxifying systems. Interestingly, our studies with tissue-specific CYP3A4 transgenic Cyp3a(-/-) mice revealed that not only hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression of detoxifying systems to near wild-type levels. Apparently, intestinal CYP3A4 can limit the hepatic exposure to food-derived activators of nuclear receptors, thereby regulating the expression of a range of detoxifying systems in the liver. This broad biological effect further emphasizes the importance of intestinal CYP3A activity and could have profound implications for the prediction of drug exposure.

Cytochrome P450 3A4 (CYP3A4) is a major determinant of the metabolism of many drugs, including important anticancer drugs, with sometimes profound impact on therapeutic efficacy and toxic side effects. To study in vivo CYP3A(4) functions, we have generated and characterized transgenic mice with functional expression of human CYP3A4 cDNA in the liver. Two transgenic lines displayed substantial, physiologically relevant and stable CYP3A4 levels in liver and moderate levels in kidney, but not in small intestine. The mice did not display obvious physiological abnormalities. The CYP3A4 substrate drugs midazolam and cyclosporin A were used to test functional activity of CYP3A4 in liver. The area under the plasma concentration versus time curve (AUC) of intravenously administered midazolam (30 mg/kg) was 2.2-fold decreased in the transgenic mice compared with wild-type (5.45 +/- 0.21 versus 11.7 +/- 0.46 microg . h ml(-1); P < 0.01), and early formation of the primary metabolite 1-hydroxymidazolam was about 2-fold increased, demonstrating the functionality of CYP3A4 in the liver. Similarly, following intravenous administration of cyclosporin A (20 mg/kg), CYP3A4 transgenic mice displayed a reduced plasma AUC compared with wild-type (24.3 +/- 0.66 versus 35.8 +/- 0.53 microg . h ml(-) (1); P < 0.01). Thus, midazolam and cyclosporin A, compounds with markedly different clearance rates and half-lives, both demonstrated clearly accelerated kinetics in the CYP3A4 transgenic mice. We expect that this CYP3A4 transgenic model will provide a useful tool to study the impact of CYP3A4 on drug levels, especially when combined with other transgenic and knockout strains.

Nitrofurantoin is a commonly used urinary tract antibiotic prescribed to lactating woman. It is actively transported into human and rat milk by an unknown mechanism. Our group has demonstrated an important role of the breast cancer resistance protein (BCRP/ABCG2) in the secretion of xenotoxins into the milk. This ATP-binding cassette drug efflux transporter extrudes xenotoxins from cells in intestine, liver, mammary gland, and other organs, affecting the pharmacological and toxicological behavior of many compounds. We investigated whether Bcrp1 is involved in the pharmacokinetic profile of nitrofurantoin and its active secretion into the milk. Using polarized cell lines, we found that nitrofurantoin is efficiently transported by murine Bcrp1 and human BCRP. After oral administration of 10 mg/kg nitrofurantoin, the area under the plasma concentrationtime curve in Bcrp1 knockout mice was almost 4-fold higher than in wild-type mice (148.8 +/- 30.4 versus 37.5 +/- 6.8 min x microg/ml); and after i.v. administration (5 mg/kg), 2-fold higher (139.2 +/- 22.0 versus 73.9 +/- 9.0 min x microg/ml). Hepatobiliary excretion of nitrofurantoin was almost abolished in Bcrp1 knockout mice (9.6 +/- 3.2 versus 0.2 +/- 0.1% in wild-type and Bcrp1 knockout mice, respectively). The milk-to-plasma ratio of nitrofurantoin was almost 80 times higher in wild-type compared with Bcrp1 knockout lactating female mice (45.7 +/- 16.2 versus 0.6 +/- 0.1). Nitrofurantoin elimination via milk was quantitatively even higher than hepatobiliary elimination. We conclude that Bcrp1 is an important determinant for the bioavailability of nitrofurantoin and the main mechanism involved in its hepatobiliary excretion and secretion into the milk.

The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter present in the liver and other tissues that affects the pharmacological behavior of many compounds. To assess the possible role of BCRP in sex-dependent pharmacokinetics, we studied the in vivo disposition of several murine Bcrp1 substrates in male and female wild-type and Bcrp1 knockout mice. After oral administration of the antibiotic nitrofurantoin, the area under the plasma concentration-time curve in wild-type female mice was approximately 2-fold higher than in wild-type male mice. Moreover, after i.v. administration of nitrofurantoin, the antiulcerative cimetidine, the anticancer drug topotecan, and the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the plasma levels in wild-type female mice were all significantly higher than those in wild-type male mice. Analysis of the expression of murine Bcrp1 in several pharmacokinetically important tissues showed that only the hepatic Bcrp1 expression was higher in male mice compared with female mice. In line with this difference, the hepatobiliary excretion for nitrofurantoin and PhIP was, respectively, 9-fold higher and approximately 2-fold higher in male compared with female wild-type mice. No significant sex differences were observed in plasma levels or hepatobiliary excretion for any of the tested compounds in Bcrp1-/- mice, indicating that Bcrp1 was the main cause of the sex difference in wild-type mice. Analysis of hepatic expression of human BCRP also indicated a higher expression in men compared with women. In conclusion, sex-dependent expression of BCRP/Bcrp1 in the liver may be a cause of sex-specific variability in the pharmacokinetics of BCRP substrates, with potential impact on the clinical-therapeutic applications and toxicity risks of drugs.