RESUMO
Transmembrane drug transporters can be important determinants of the pharmacokinetics, efficacy, and safety profiles of drugs. To investigate the potential cooperative and/or counteracting interplay of OATP1A/1B/2B1 uptake transporters and ABCB1 and ABCG2 efflux transporters in physiology and pharmacology, we generated a new mouse model (Bab12), deficient for Slco1a/1b, Slco2b1, Abcb1a/1b and Abcg2. Bab12 mice were viable and fertile. We compared wild-type, Slco1a/1b/2b1-/-, Abcb1a/1b;Abcg2-/- and Bab12 strains. Endogenous plasma conjugated bilirubin levels ranked as follows: wild-type = Abcb1a/1b;Abcg2-/- << Slco1a/1b/2b1-/- < Bab12 mice. Plasma levels of rosuvastatin and fexofenadine were elevated in Slco1a/1b/2b1-/- and Abcb1a/1b;Abcg2-/- mice compared to wild-type, and dramatically increased in Bab12 mice. Although systemic exposure of larotrectinib and repotrectinib was substantially increased in the separate multidrug transporter knockout strains, no additive effects were observed in the combination Bab12 mice. Significantly higher plasma exposure of fluvastatin and pravastatin was only found in Slco1a/1b/2b1-deficient mice. However, noticeable transport by Slco1a/1b/2b1 and Abcb1a/1b and Abcg2 across the BBB was observed for fluvastatin and pravastatin, respectively, by comparing Bab12 mice with Abcb1a/1b;Abcg2-/- or Slco1a/1b/2b1-/- mice. Quite varying behavior in plasma exposure of erlotinib and its metabolites was observed among these strains. Bab12 mice revealed that Abcb1a/1b and/or Abcg2 can contribute to conjugated bilirubin elimination when Slco1a/1b/2b1 are absent. Our results suggest that the interplay of Slco1a/1b/2b1, Abcb1a/1b, and Abcg2 could markedly affect the pharmacokinetics of some, but not all drugs and metabolites. The Bab12 mouse model will represent a useful tool for optimizing drug development and clinical application, including efficacy and safety.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Bilirrubina , Camundongos Knockout , Transportadores de Ânions Orgânicos , Animais , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Bilirrubina/sangue , Bilirrubina/metabolismo , Camundongos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Terfenadina/farmacocinética , Terfenadina/análogos & derivados , Masculino , Transporte Biológico , Rosuvastatina Cálcica/farmacocinética , Rosuvastatina Cálcica/farmacologia , Camundongos Endogâmicos C57BLRESUMO
Mammalian carboxylesterase 1 enzymes can hydrolyze many xenobiotic chemicals and endogenous lipids. We here identified and characterized a mouse strain (FVB/NKI) in which three of the eight Ces1 genes were spontaneously deleted, removing Ces1c and Ces1e partly, and Ces1d entirely. We studied the impact of this Ces1c/d/e deficiency on drug and lipid metabolism and homeostasis. Ces1c/d/e-/- mice showed strongly impaired conversion of the anticancer prodrug irinotecan to its active metabolite SN-38 in plasma, spleen and lung. Plasma hydrolysis of the oral anticancer prodrug capecitabine to 5-DFCR was also profoundly reduced in Ces1c/d/e-/- mice. Our findings resolved previously unexplained FVB/NKI pharmacokinetic anomalies. On a medium-fat diet, Ces1c/d/e-/- female mice exhibited moderately higher body weight, mild inflammation in gonadal white adipose tissue (gWAT), and increased lipid load in brown adipose tissue (BAT). Ces1c/d/e-/- males showed more pronounced inflammation in gWAT and an increased lipid load in BAT. On a 5-week high-fat diet exposure, Ces1c/d/e deficiency predisposed to developing obesity, enlarged and fatty liver, glucose intolerance and insulin resistance, with severe inflammation in gWAT and increased lipid load in BAT. Hepatic proteomics analysis revealed that the acute phase response, involved in the dynamic cycle of immunometabolism, was activated in these Ces1c/d/e-/- mice. This may contribute to the obesity-related chronic inflammation and adverse metabolic disease in this strain. While Ces1c/d/e deficiency clearly exacerbated metabolic syndrome development, long-term (18-week) high-fat diet exposure overwhelmed many, albeit not all, observed phenotypic differences.
Assuntos
Hidrolases de Éster Carboxílico , Síndrome Metabólica , Pró-Fármacos , Animais , Feminino , Masculino , Camundongos , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Inflamação , Irinotecano , Lipídeos , Mamíferos , Obesidade/metabolismoRESUMO
The mammalian carboxylesterase 1 (Ces1/CES1) family comprises several enzymes that hydrolyze many xenobiotic chemicals and endogenous lipids. To investigate the pharmacological and physiological roles of Ces1/CES1, we generated Ces1 cluster knockout (Ces1 -/- ) mice, and a hepatic human CES1 transgenic model in the Ces1 -/- background (TgCES1). Ces1 -/- mice displayed profoundly decreased conversion of the anticancer prodrug irinotecan to SN-38 in plasma and tissues. TgCES1 mice exhibited enhanced metabolism of irinotecan to SN-38 in liver and kidney. Ces1 and hCES1 activity increased irinotecan toxicity, likely by enhancing the formation of pharmacodynamically active SN-38. Ces1 -/- mice also showed markedly increased capecitabine plasma exposure, which was moderately decreased in TgCES1 mice. Ces1 -/- mice were overweight with increased adipose tissue, white adipose tissue inflammation (in males), a higher lipid load in brown adipose tissue, and impaired blood glucose tolerance (in males). These phenotypes were mostly reversed in TgCES1 mice. TgCES1 mice displayed increased triglyceride secretion from liver to plasma, together with higher triglyceride levels in the male liver. These results indicate that the carboxylesterase 1 family plays essential roles in drug and lipid metabolism and detoxification. Ces1 -/- and TgCES1 mice will provide excellent tools for further study of the in vivo functions of Ces1/CES1 enzymes.
RESUMO
Organic anion transporting polypeptide 2B1 (OATP2B1/SLCO2B1) facilitates uptake transport of structurally diverse endogenous and exogenous compounds. To investigate the roles of OATP2B1 in physiology and pharmacology, we established and characterized Oatp2b1 knockout (single Slco2b1-/- and combination Slco1a/1b/2b1-/-) and humanized hepatic and intestinal OATP2B1 transgenic mouse models. While viable and fertile, these strains exhibited a modestly increased body weight. In males, unconjugated bilirubin levels were markedly reduced in Slco2b1-/- compared to wild-type mice, whereas bilirubin monoglucuronide levels were modestly increased in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice. Single Slco2b1-/- mice showed no significant changes in oral pharmacokinetics of several tested drugs. However, markedly higher or lower plasma exposure of pravastatin and the erlotinib metabolite OSI-420, respectively, were found in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice, while oral rosuvastatin and fluvastatin behaved similarly between the strains. In males, humanized OATP2B1 strains showed lower conjugated and unconjugated bilirubin levels than control Slco1a/1b/2b1-deficient mice. Moreover, hepatic expression of human OATP2B1 partially or completely rescued the impaired hepatic uptake of OSI-420, rosuvastatin, pravastatin, and fluvastatin in Slco1a/1b/2b1-/- mice, establishing an important role in hepatic uptake. Expression of human OATP2B1 in the intestine was basolateral and markedly reduced the oral availability of rosuvastatin and pravastatin, but not of OSI-420 and fluvastatin. Neither lack of Oatp2b1, nor overexpression of human OATP2B1 had any effect on fexofenadine oral pharmacokinetics. While these mouse models still have limitations for human translation, with additional work we expect they will provide powerful tools to further understand the physiological and pharmacological roles of OATP2B1.
Assuntos
Bilirrubina , Transportadores de Ânions Orgânicos , Masculino , Camundongos , Humanos , Animais , Rosuvastatina Cálcica , Fluvastatina , Pravastatina , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Camundongos Transgênicos , Peptídeos/metabolismo , Ânions/metabolismo , Camundongos KnockoutRESUMO
Osimertinib is an irreversible EGFR inhibitor registered for advanced NSCLC patients whose tumors harbor recurrent somatic activating mutations in EGFR (EGFRm+) or the frequently occurring EGFR-T790M resistance mutation. Using in vitro transport assays and appropriate knockout and transgenic mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport osimertinib and whether they influence the oral availability and brain accumulation of osimertinib and its most active metabolite, AZ5104. In vitro, human ABCB1 and mouse Abcg2 modestly transported osimertinib. In mice, Abcb1a/1b, with a minor contribution of Abcg2, markedly limited the brain accumulation of osimertinib and AZ5104. However, no effect of the ABC transporters was seen on osimertinib oral availability. In spite of up to 6-fold higher brain accumulation, we observed no acute toxicity signs of oral osimertinib in Abcb1a/1b;Abcg2 knockout mice. Interestingly, even in wild-type mice the intrinsic brain penetration of osimertinib was already relatively high, which may help to explain the documented partial efficacy of this drug against brain metastases. No substantial effects of mouse Cyp3a knockout or transgenic human CYP3A4 overexpression on oral osimertinib pharmacokinetics were observed, presumably due to a dominant role of mouse Cyp2d enzymes in osimertinib metabolism. Our results suggest that pharmacological inhibition of ABCB1 and ABCG2 during osimertinib therapy might potentially be considered to further benefit patients with brain (micro-)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the tumor cells, without invoking a high toxicity risk.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Acrilamidas/metabolismo , Compostos de Anilina/metabolismo , Encéfalo/metabolismo , Animais , Disponibilidade Biológica , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Citocromo P-450 CYP3A/metabolismo , Cães , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Distribuição Tecidual/fisiologiaRESUMO
Ibrutinib (Imbruvica), an oral tyrosine kinase inhibitor (TKI) approved for treatment of B-cell malignancies, irreversibly inhibits the Bruton's tyrosine kinase (BTK). Its abundant metabolite, dihydrodiol-ibrutinib (ibrutinib-DiOH), which is primarily formed by CYP3A, has a 10-fold reduced BTK inhibitory activity. Using in vitro transport assays and genetically modified mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 and the multidrug-metabolizing CYP3A enzyme family can affect the oral bioavailability and tissue disposition of ibrutinib and ibrutinib-DiOH. In vitro, ibrutinib was transported moderately by human ABCB1 and mouse Abcg2 but not detectably by human ABCG2. In mice, Abcb1 markedly restricted the brain penetration of ibrutinib and ibrutinib-DiOH, either alone or in combination with Abcg2, resulting in 4.5- and 5.9-fold increases in ibrutinib brain-to-plasma ratios in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice relative to wild-type mice. Abcb1 and/or Abcg2 did not obviously restrict ibrutinib oral bioavailability, but Cyp3a deficiency increased the ibrutinib plasma AUC by 9.7-fold compared to wild-type mice. This increase was mostly reversed (5.1-fold reduction) by transgenic human CYP3A4 overexpression, with roughly equal contributions of intestinal and hepatic CYP3A4 metabolism. Our results suggest that pharmacological inhibition of ABCB1 during ibrutinib therapy might benefit patients with malignancies or (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of this transporter in the malignant cells. Moreover, given the strong in vivo impact of CYP3A, inhibitors or inducers of this enzyme family will likely strongly affect ibrutinib oral bioavailability and, thus, its therapeutic efficacy, as well as its toxicity risks.
Assuntos
Antineoplásicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Citocromo P-450 CYP3A/metabolismo , Pirazóis/farmacocinética , Pirimidinas/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenina/análogos & derivados , Administração Oral , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Citocromo P-450 CYP3A/genética , Cães , Feminino , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Piperidinas , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Distribuição TecidualRESUMO
Lorlatinib (PF-06463922) is a promising oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor currently in Phase III clinical trials for treatment of non-small-cell lung cancer (NSCLC) containing an ALK rearrangement. With therapy-resistant brain metastases a major concern in NSCLC, lorlatinib was designed to have high membrane and blood-brain barrier permeability. We investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the multispecific drug-metabolizing enzyme CYP3A in plasma pharmacokinetics and tissue distribution of lorlatinib using genetically modified mouse strains. In vitro, human ABCB1 and mouse Abcg2 modestly transported lorlatinib. Following oral lorlatinib administration (at 10 mg/kg), brain accumulation of lorlatinib, while relatively high in wild-type mice, was still fourfold increased in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice, but not in single Abcg2-/- mice. Lorlatinib plasma levels were not altered. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar increased the brain accumulation of lorlatinib in wild-type mice fourfold, that is, to the same level as in Abcb1a/1b;Abcg2-/- mice, without altering plasma exposure. Similar results were obtained for lorlatinib testis accumulation. In Cyp3a-/- mice, the plasma exposure of lorlatinib was increased 1.3-fold, but was then twofold reduced upon transgenic overexpression of human CYP3A4 in liver and intestine, whereas relative tissue distribution of lorlatinib remained unaltered. Our data indicate that lorlatinib brain accumulation is substantially limited by P-glycoprotein/ABCB1 in the blood-brain barrier, but this can be effectively reversed by elacridar coadministration. Moreover, oral availability of lorlatinib is markedly restricted by CYP3A4 activity. These insights may be used in optimizing the therapeutic application of lorlatinib.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Encéfalo/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Lactamas Macrocíclicas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Administração Oral , Aminopiridinas , Animais , Disponibilidade Biológica , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular , Cães , Feminino , Humanos , Lactamas , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Knockout , Projetos Piloto , PirazóisRESUMO
Encorafenib (LGX818) is a promising BRAFV600E inhibitor that has efficacy against metastatic melanoma. To better understand its pharmacokinetics, we studied its interactions with the multidrug efflux transporters ABCB1 and ABCG2 and the multidrug metabolizing enzyme CYP3A. In polarized MDCK-II cells, encorafenib was efficiently transported by canine and human ABCB1 and ABCG2 and by mouse Abcg2. Upon oral administration to wild-type, Abcb1a/1b-/-, Abcg2-/-, and Abcb1a/1b;Abcg2-/- mice, encorafenib was absorbed very quickly and to very high plasma levels, but without clear changes in oral availability between the strains. Upon oral or intravenous administration, encorafenib brain accumulation was markedly increased in Abcb1a/1b;Abcg2-/- mice and to a lesser extent in Abcb1a/1b-/- mice. However, absolute brain concentrations and brain-to-plasma ratios remained very low in all strains, indicating intrinsically poor brain penetration of encorafenib. Upon intravenous administration, Abcb1a/1b;Abcg2-/- mice showed somewhat reduced plasma elimination of encorafenib compared to wild-type mice, and lower accumulation of the drug in the intestinal tract, suggesting a limited role for these transporters in intestinal elimination of the drug. In Cyp3a-/- mice plasma levels of encorafenib were not markedly increased, suggesting a limited impact of Cyp3a on encorafenib oral availability. The low brain penetration of encorafenib might limit its efficacy against malignancies positioned behind a functional blood-brain barrier, but its oral bioavailability and distribution to other tested organs (liver, kidney, spleen, testis) was high.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacocinética , Encéfalo/metabolismo , Carbamatos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Sulfonamidas/farmacocinética , Transportadores de Cassetes de Ligação de ATP/genética , Administração Oral , Animais , Disponibilidade Biológica , Cães , Intestino Delgado/metabolismo , Células Madin Darby de Rim Canino , Camundongos Knockout , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Distribuição TecidualRESUMO
Ponatinib is an oral BCR-ABL1 inhibitor for treatment of advanced leukemic diseases that carry the Philadelphia chromosome, specifically containing the T315I mutation yielding resistance to previously approved BCR-ABL1 inhibitors. Using in vitro transport assays and knockout mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport ponatinib and whether they, or the drug-metabolizing enzyme CYP3A, affect the oral availability and brain accumulation of ponatinib and its active N-desmethyl metabolite (DMP). In vitro, mouse Abcg2 and human ABCB1 modestly transported ponatinib. In mice, both Abcb1 and Abcg2 markedly restricted brain accumulation of ponatinib and DMP, but not ponatinib oral availability. Abcg2 deficiency increased DMP plasma levels â¼3-fold. Cyp3a deficiency increased the ponatinib plasma AUC 1.4-fold. Our results suggest that pharmacological inhibition of ABCG2 and ABCB1 during ponatinib therapy might benefit patients with brain (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the malignant cells. CYP3A inhibitors might increase ponatinib oral availability, enhancing efficacy but possibly also toxicity of this drug.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/farmacologia , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Disponibilidade Biológica , Transporte Biológico/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/antagonistas & inibidores , Distribuição TecidualRESUMO
Ochratoxin A (OTA) is a fungal secondary metabolite that can contaminate various foods. OTA has several toxic effects like nephrotoxicity, hepatotoxicity, and neurotoxicity in different animal species, but its mechanisms of toxicity are still unclear. How OTA accumulates in kidney, liver, and brain is as yet unknown, but transmembrane transport proteins are likely involved. We studied transport of OTA in vitro, using polarized MDCKII cells transduced with cDNAs of the efflux transporters mouse (m)Bcrp, human (h)BCRP, mMrp2, or hMRP2, and HEK293 cells overexpressing cDNAs of the human uptake transporters OATP1A2, OATP1B1, OATP1B3, or OATP2B1 at pH7.4 and 6.4. MDCKII-mBcrp cells were more resistant to OTA toxicity than MDCKII parental and hBCRP-transduced cells. Transepithelial transport experiments showed some apically directed transport by MDCKII-mBcrp cells at pH7.4, whereas both mBcrp and hBCRP clearly transported OTA at pH6.4. There was modest transport of OTA by mMrp2 and hMRP2 only at pH6.4. OATP1A2 and OATP2B1 mediated uptake of OTA both at pH7.4 and 6.4, but OATP1B1 only at pH7.4. There was no detectable transport of OTA by OATP1B3. Our data indicate that human BCRP and MRP2 can mediate elimination of OTA from cells, thus reducing OTA toxicity. On the other hand, human OATP1A2, OATP1B1, and OATP2B1 can mediate cellular uptake of OTA, which could aggravate OTA toxicity.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Ocratoxinas/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Transporte Biológico , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Microbiologia de Alimentos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Cinética , Células Madin Darby de Rim Canino , Camundongos , Proteína 2 Associada à Farmacorresistência Múltipla , Ocratoxinas/toxicidade , Transdução Genética , TransfecçãoRESUMO
Afatinib is a highly selective, irreversible inhibitor of EGFR and HER-2. It is orally administered for the treatment of patients with EGFR mutation-positive types of metastatic NSCLC. We investigated whether afatinib is a substrate for the multidrug efflux transporters ABCB1 and ABCG2 and whether these transporters influence oral availability and brain and other tissue accumulation of afatinib. We used in vitro transport assays to assess human (h)ABCB1-, hABCG2- or murine (m)Abcg2-mediated transport of afatinib. To study the single and combined roles of Abcg2 and Abcb1a/1b in oral afatinib disposition, we used appropriate knockout mouse strains. Afatinib was transported well by hABCB1, hABCG2 and mAbcg2 in vitro. Upon oral administration of afatinib, Abcg2-/-, Abcb1a/1b-/- and Abcb1a/1b-/-;Abcg2-/- mice displayed a 4.2-, 2.4- and 7-fold increased afatinib plasma AUC0-24 compared with wild-type mice. Abcg2-deficient strains also displayed decreased afatinib plasma clearance. At 2h, relative brain accumulation of afatinib was not significantly altered in the single knockout strains, but 23.8-fold increased in Abcb1a/1b-/-;Abcg2-/- mice compared to wild-type mice. Abcg2 and Abcb1a/1b restrict oral availability and brain accumulation of afatinib. Inhibition of these transporters may therefore be of clinical importance for patients with brain (micro)metastases positioned behind an intact blood-brain barrier.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/farmacocinética , Radiossensibilizantes/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Afatinib , Animais , Transporte Biológico , Cães , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/metabolismo , Quinazolinas/administração & dosagem , Quinazolinas/metabolismo , Radiossensibilizantes/administração & dosagem , Radiossensibilizantes/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Distribuição TecidualRESUMO
The impact of OATP drug uptake transporters in drug-drug interactions (DDIs) is increasingly recognized. OATP1B1 and OATP1B3 are human hepatic uptake transporters that can mediate liver uptake of a wide variety of drugs. Recently, we generated transgenic mice with liver-specific expression of human OATP1B1 or OATP1B3 in a mouse Oatp1a/1b knockout background. Here, we investigated the applicability of these mice in OATP-mediated drug-drug interaction studies using the prototypic OATP inhibitor rifampicin and a good OATP substrate, the anticancer drug methotrexate (MTX). We next assessed the possibility of OATP-mediated interactions between telmisartan and MTX, a clinically relevant drug combination. Using HEK293 cells overexpressing OATP1B1 or OATP1B3, we estimated IC50 values for both rifampicin (0.9 or 0.3 µM) and telmisartan (6.7 or 7.9 µM) in inhibiting OATP-mediated MTX uptake in vitro. Using wild-type, Oatp1a/1b-/-, and OATP1B1- or OATP1B3-humanized transgenic mice, we found that rifampicin inhibits hepatic uptake of MTX mediated by the mouse Oatp1a/1b and human OATP1B1 and OATP1B3 transporters at clinically relevant concentrations. This highlights the applicability of these mouse models for DDI studies and may be exploited in the clinic to reduce the dose and thus methotrexate-mediated toxicity. On the other hand, telmisartan inhibited only human OATP1B1-mediated hepatic uptake of MTX at concentrations higher than those used in the clinic; therefore risks for OATP-mediated clinical DDIs for this drug combination are likely to be low. Overall, we show here that OATP1B1- and OATP1B3-humanized mice can be used as in vivo tools to assess and possibly predict clinically relevant DDIs.
Assuntos
Interações Medicamentosas/fisiologia , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Antineoplásicos/metabolismo , Benzimidazóis/metabolismo , Benzoatos/metabolismo , Transporte Biológico/fisiologia , Células HEK293 , Humanos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , TelmisartanRESUMO
We aimed to clarify the roles of the multidrug transporters ABCB1 and ABCG2 in oral availability and brain accumulation of ceritinib, an oral anaplastic lymphoma kinase (ALK) inhibitor used to treat metastatic non-small cell lung cancer (NSCLC) after progression on crizotinib. Importantly, NSCLC is prone to form brain metastases. Transport of ceritinib by human (h) ABCB1 or hABCG2 or mouse (m) Abcg2 was assessed in vitro. To study the single and combined roles of Abcb1a/1b and Abcg2 in ceritinib disposition in vivo, we used appropriate knockout mouse strains. Ceritinib was very efficiently transported by hABCB1, and efficiently by hABCG2 and mAbcg2 in vitro, and transport was specifically inhibited by the ABCB1 inhibitor zosuquidar and ABCG2 inhibitor Ko143, respectively. Absorption and 24-h oral availability were not significantly affected by the absence of Abcb1 and/or Abcg2, but the brain concentrations were greatly increased (>38-fold) in Abcb1a/1b(-/-) mice at 3 and 24h after oral administration of 20mg/kg ceritinib. The brain concentrations increased another â¼ 3-fold (to >90-fold) in Abcb1a/1b;Abcg2(-/-) mice, indicating that there was a significant additional effect of Abcg2-mediated transport of ceritinib as well in vivo. Overall, brain accumulation, but not the 24-h oral availability of ceritinib were profoundly restricted by Abcb1a/1b and Abcg2, with Abcb1a/1b being the dominant efflux protein. Our data suggest that coadministration of ceritinib with a dual ABCB1 and ABCG2 inhibitor may improve treatment of brain (micro) metastases positioned behind a functionally intact blood-brain barrier, and possibly also of tumors resistant to ceritinib due to ABCB1 or ABCG2 overexpression.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Pirimidinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sulfonas/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Quinase do Linfoma Anaplásico , Animais , Disponibilidade Biológica , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Crizotinibe , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Sulfonas/farmacologia , Distribuição Tecidual/fisiologiaRESUMO
We aimed to clarify the roles of the multidrug-detoxifying proteins ABCB1, ABCG2, ABCC2, and CYP3A in oral availability and brain accumulation of cabazitaxel, a taxane developed for improved therapy of docetaxel-resistant prostate cancer. Cabazitaxel pharmacokinetics were studied in Abcb1a/1b, Abcg2, Abcc2, Cyp3a, and combination knockout mice. We found that human ABCB1, but not ABCG2, transported cabazitaxel in vitro. Upon oral cabazitaxel administration, total plasma levels were greatly increased due to binding to plasma carboxylesterase Ces1c, which is highly upregulated in several knockout strains. Ces1c inhibition and in vivo hepatic Ces1c knockdown reversed these effects. Correcting for Ces1c effects, Abcb1a/1b, Abcg2, and Abcc2 did not restrict cabazitaxel oral availability, whereas Abcb1a/1b, but not Abcg2, dramatically reduced cabazitaxel brain accumulation (>10-fold). Coadministration of the ABCB1 inhibitor elacridar completely reversed this brain accumulation effect. After correction for Ces1c effects, Cyp3a knockout mice demonstrated a strong (six-fold) increase in cabazitaxel oral availability, which was completely reversed by transgenic human CYP3A4 in intestine and liver. Cabazitaxel markedly inhibited mouse Ces1c, but human CES1 and CES2 only weakly. Ces1c upregulation can thus complicate preclinical cabazitaxel studies. In summary, ABCB1 limits cabazitaxel brain accumulation and therefore potentially therapeutic efficacy against (micro)metastases or primary tumors positioned wholly or partly behind a functional blood-brain barrier. This can be reversed with elacridar coadministration, and similar effects may apply to ABCB1-expressing tumors. CYP3A4 profoundly reduces the oral availability of cabazitaxel. This may potentially be greatly improved by coadministering ritonavir or other CYP3A inhibitors, suggesting the option of patient-friendly oral cabazitaxel therapy.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacocinética , Química Encefálica , Carboxilesterase/sangue , Citocromo P-450 CYP3A/metabolismo , Taxoides/farmacocinética , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/análise , Hidrolases de Éster Carboxílico/metabolismo , Cães , Células Madin Darby de Rim Canino/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , Taxoides/administração & dosagem , Taxoides/análiseRESUMO
Recently, an efficient liver detoxification process dubbed "hepatocyte hopping" was proposed on the basis of findings with the endogenous compound, bilirubin glucuronide. According to this model, hepatocytic bilirubin glucuronide can follow a liver-to-blood shuttling loop via Abcc3 transporter-mediated efflux and subsequent Oatp1a/1b-mediated liver uptake. We hypothesized that glucuronide conjugates of xenobiotics, such as the anticancer drug sorafenib, can also undergo hepatocyte hopping. Using transporter-deficient mouse models, we show here that sorafenib-glucuronide can be extruded from hepatocytes into the bile by Abcc2 or back into the systemic circulation by Abcc3, and that it can be taken up efficiently again into neighboring hepatocytes by Oatp1a/1b. We further demonstrate that sorafenib-glucuronide excreted into the gut lumen can be cleaved by microbial enzymes to sorafenib, which is then reabsorbed, supporting its persistence in the systemic circulation. Our results suggest broad relevance of a hepatocyte shuttling process known as "hepatocyte hopping"-a novel concept in clinical pharmacology-for detoxification of targeted cancer drugs that undergo hepatic glucuronidation, such as sorafenib.
Assuntos
Glucuronídeos/metabolismo , Hepatócitos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Niacinamida/análogos & derivados , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Compostos de Fenilureia/farmacocinética , Animais , Feminino , Glucuronidase/metabolismo , Glucuronídeos/sangue , Humanos , Intestinos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , Niacinamida/sangue , Niacinamida/farmacocinética , Compostos de Fenilureia/sangue , Ratos , Células Sf9 , SorafenibeRESUMO
PURPOSE: Regorafenib is a novel multikinase inhibitor, currently approved for the treatment of metastasized colorectal cancer and advanced gastrointestinal stromal tumors. We investigated whether regorafenib is a substrate for the multidrug efflux transporters ABCG2 and ABCB1 and whether oral availability, brain and testis accumulation of regorafenib and its active metabolites are influenced by these transporters. METHODS: We used in vitro transport assays to assess human (h)ABCB1- or hABCG2- or murine (m)Abcg2-mediated active transport at high and low concentrations of regorafenib. To study the single and combined roles of Abcg2 and Abcb1a/1b in oral regorafenib disposition and the impact of Cyp3a-mediated metabolism, we used appropriate knockout mouse strains. RESULTS: Regorafenib was transported well by mAbcg2 and hABCG2 and modestly by hABCB1 in vitro. Abcg2 and to a lesser extent Abcb1a/1b limited brain and testis accumulation of regorafenib and metabolite M2 (brain only) in mice. Regorafenib oral availability was not increased in Abcg2(-/-);Abcb1a/1b(-/-) mice. Up till 2 h, metabolite M5 was undetectable in plasma and organs. CONCLUSIONS: Brain and testis accumulation of regorafenib and brain accumulation of metabolite M2 are restricted by Abcg2 and Abcb1a/1b. Inhibition of these transporters may be of clinical relevance for patients with brain (micro)metastases positioned behind an intact blood-brain barrier.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacocinética , Encéfalo/metabolismo , Proteínas de Neoplasias/metabolismo , Compostos de Fenilureia/farmacocinética , Piridinas/farmacocinética , Testículo/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Administração Oral , Animais , Antineoplásicos/sangue , Antineoplásicos/metabolismo , Transporte Biológico , Cães , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Compostos de Fenilureia/sangue , Compostos de Fenilureia/metabolismo , Piridinas/sangue , Piridinas/metabolismo , Distribuição Tecidual , TransfecçãoRESUMO
BACKGROUND: Rucaparib is a potent, orally available, small-molecule inhibitor of poly ADP-ribose polymerase (PARP) 1 and 2. Ongoing clinical trials are assessing the efficacy of rucaparib alone or in combination with other cytotoxic drugs, mainly in breast and ovarian cancer patients with mutations in the breast cancer associated (BRCA) genes. PURPOSE: We aimed to establish whether the multidrug efflux transporters ABCG2 (BCRP) and ABCB1 (P-gp, MDR1) affect the oral availability and brain penetration of rucaparib in mice. RESULTS: In vitro, rucaparib was efficiently transported by both human ABCB1 and ABCG2, and very efficiently by mouse Abcg2. Transport could be inhibited by the small-molecule ABCB1 and ABCG2 inhibitors zosuquidar and Ko143, respectively. In vivo, oral availability (plasma AUC0-1 and AUC0-24) and brain levels of rucaparib at 1 and 24 h were increased by the absence of both Abcg2 and Abcb1a/1b after oral administration of rucaparib at 10 mg/kg. CONCLUSIONS: Our data show to our knowledge for the first time that oral availability and brain accumulation of a PARP inhibitor are markedly and additively restricted by Abcg2 and Abcb1a/1b. This may have clinical relevance for improvement of rucaparib therapy in PARP inhibitor-resistant tumors with ABCB1 and/or ABCG2 expression and in patients with brain (micro)metastases positioned behind a functional blood-brain barrier.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Indóis/farmacocinética , Proteínas de Neoplasias/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Administração Oral , Animais , Disponibilidade Biológica , Transporte Biológico , Técnicas de Cultura de Células , Cães , Feminino , Humanos , Indóis/administração & dosagem , Indóis/sangue , Células Madin Darby de Rim Canino , Camundongos Knockout , Proteínas de Neoplasias/genética , Especificidade por Substrato , Distribuição TecidualRESUMO
Organic anion transporting polypeptides (human: OATPs and mouse: Oatps) are uptake transporters with important roles in drug pharmacokinetics and toxicity. We aimed to study the in vivo impact of mouse and human OATP1A/1B transporters on docetaxel plasma clearance and liver and intestinal uptake. Docetaxel was administered to Oatp1a/1b knockout and liver-specific humanized OATP1B1, OATP1B3 and OATP1A2 transgenic mice. Experiments were conducted with a low polysorbate 80 (2.8%) formulation, as 8% polysorbate somewhat inhibited docetaxel plasma clearance after intravenous administration. After intravenous administration (10 mg/kg), Oatp1a/1b knockout mice had an approximately threefold higher plasma area under the curve (AUC). Impaired liver uptake was evident from the significantly reduced (approximately threefold) liver-to-plasma AUC ratios. Absence of mouse Oatp1a/1b transporters did not affect the intestinal absorption of orally administered docetaxel (10 mg/kg), while the systemic exposure of docetaxel was again substantially increased owing to impaired liver uptake. Most importantly, liver-specific expression of each of the human OATP1B1, OATP1B3 and OATP1A2 transporters provided a nearly complete rescue of the increased plasma levels of docetaxel in Oatp1a/1b-null mice after intravenous administration. Our data show that one or more of the mouse Oatp1a/1b transporters and each of the human OATP1A/1B transporters can mediate docetaxel uptake in vivo. This might be clinically relevant for OATP1A/1B-mediated tumor uptake of docetaxel and for docetaxel clearance in patients in whom the transport activity of OATP1A/1B transporters is reduced owing to genetic variation or pharmacological inhibition, leading to potentially altered toxicity and therapeutic efficacy of this drug.
Assuntos
Antineoplásicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/fisiologia , Transportadores de Ânions Orgânicos/fisiologia , Taxoides/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Transporte Biológico , Química Farmacêutica , Docetaxel , Teste de Complementação Genética , Humanos , Absorção Intestinal , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Camundongos Knockout , Polissorbatos/administração & dosagem , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Taxoides/administração & dosagem , Taxoides/farmacocinéticaRESUMO
PURPOSE: To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. EXPERIMENTAL DESIGN: We used wild-type, Abcb1a/1b(-/-), Abcg2(-/-), Abcb1a/1b;Abcg2(-/-), and Cyp3a(-/-) mice to study everolimus oral bioavailability and brain accumulation. RESULTS: Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b(-/-)and Abcb1a/1b;Abcg2(-/-), but not Abcg2(-/-)mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (â¼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a(-/-), but not Abcb1a/1b(-/-), Abcg2(-/-), or Abcb1a/1b;Abcg2(-/-)mice, displayed highly (>5-fold) increased oral availability of everolimus. CONCLUSIONS: Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug-drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Neoplasias Encefálicas/tratamento farmacológico , Hidrolases de Éster Carboxílico/sangue , Sistema Enzimático do Citocromo P-450/fisiologia , Imunossupressores/farmacologia , Sirolimo/análogos & derivados , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Disponibilidade Biológica , Transporte Biológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Citocromo P-450 CYP3A , Everolimo , Humanos , Imunossupressores/farmacocinética , Camundongos , Camundongos Knockout , Sirolimo/farmacocinética , Sirolimo/farmacologia , Distribuição TecidualRESUMO
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 â¼ 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.