RESUMO
PURPOSE: Current practices applied to mouse pharmacokinetic (PK) studies often use large numbers of animals with sporadic or composite sampling that inadequately describe PK profiles. The purpose of this work was to evaluate and optimize blood microsampling techniques coupled with dried blood spot (DBS) and LC-MS/MS analysis to generate reliable PK data in mice. In addition, the feasibility of cross-over designs was assessed and recommendations are presented. METHODS: The work describes a comprehensive evaluation of five blood microsampling techniques (tail clip, tail vein with needle hub, submandibular, retro-orbital, and saphenous bleeding) in CD-1 mice. The feasibility of blood sampling was evaluated based on animal observations, ease of bleeding, and ability to collect serial samples. Methotrexate, gemfibrozil and glipizide were used as test compounds and were dosed either orally or intravenously, followed by DBS collection and LC-MS/MS analysis to compare PK with various bleeding methods. RESULTS: Submandibular and retro-orbital methods that required non-serial blood collections did not allow for inter-animal variability assessments and resulted in poorly described absorption and distribution kinetics. The submandibular and tail vein with needle-hub methods were the least favorable from a technical feasibility perspective. Serial bleeding was possible with cannulated animals or saphenous bleeding in non-cannulated animals. CONCLUSIONS: Of the methods that allowed serial sampling, the saphenous method when executed as described in this report, was most practical, reproducible and provided for assessment of inter-animal variability. It enabled the collection of complete exposure profiles from a single mouse and the conduct of an intravenous/oral cross-over study design. This methodology can be used routinely, it promotes the 3Rs principles by achieving reductions in the number of animals used, decreased restraints and animal stress, and improved the quality of data obtained in mouse PK studies. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Assuntos
Coleta de Amostras Sanguíneas , Teste em Amostras de Sangue Seco , Genfibrozila/sangue , Glipizida/sangue , Metotrexato/sangue , Animais , Cromatografia Líquida , Estudos Cross-Over , Masculino , Camundongos , Espectrometria de Massas em TandemRESUMO
Breast cancer resistance protein (BCRP, ABCG2) is expressed in the hepatic canalicular membrane and mediates biliary excretion of xenobiotics including sulfate and glucuronide metabolites of some compounds. Hepatic Bcrp expression is sex-dependent, with higher expression in male mice. The hypothesis that sex-dependent Bcrp expression influences the hepatobiliary disposition of phase II metabolites was tested in the present study using acetaminophen (APAP) and the generated APAP glucuronide (AG) and sulfate (AS) metabolites in single-pass in situ perfused livers from male and female wild-type and Abcg(-/-) (Bcrp-deficient) mice. Pharmacokinetic modeling was used to estimate parameters governing the hepatobiliary disposition of APAP, AG, and AS. In wild-type mice, the biliary excretion rate constant was 2.5- and 7-fold higher in males than in females for AS and AG, respectively, reflecting male-predominant Bcrp expression. Sex-dependent differences in AG biliary excretion were not observed in Bcrp-deficient mice, and AS biliary excretion was negligible. Interestingly, sex-dependent basolateral excretion of AG (higher in males) and AS (higher in females) was noted in wild-type mice with a similar trend in Bcrp-deficient mouse livers, reflecting an increased rate constant for AG formation in male and AS formation in female mouse livers. In addition, the rate constant for AS basolateral excretion was increased significantly in female mouse livers compared with that in male mouse livers. It is interesting to note that multidrug resistance-associated protein 4 was higher in female than in male mouse livers. In conclusion, sex-dependent differences in conjugation and transporter expression result in profound differences in the hepatobiliary disposition of AG and AS in male and female mouse livers.
Assuntos
Acetaminofen/farmacocinética , Analgésicos não Narcóticos/farmacocinética , Fígado/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Algoritmos , Animais , Bile/metabolismo , Western Blotting , Peso Corporal , Feminino , Glucuronídeos/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Perfusão , Caracteres Sexuais , Sulfatos/metabolismoRESUMO
Previous studies implicated P-glycoprotein (P-gp) as the major transport protein responsible for the biliary excretion of fexofenadine (FEX). However, FEX biliary excretion was not impaired in P-gp- or breast cancer resistance protein (Bcrp)-knockout mice or multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The present study tested the hypothesis that species differences exist in the transport protein primarily responsible for FEX biliary excretion between mice and rats. Livers from Mrp2-knockout (Mrp2KO) mice and Mrp2-deficient (TR(-)) rats were perfused in a single-pass manner with 0.5 muM FEX. N-(4-[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (10 muM) was employed to inhibit P-gp and Bcrp. The biliary excretion rate of FEX was decreased 85% in Mrp2KO relative to wild-type mice (18.4 +/- 2.2 versus 122 +/- 34 pmol/min/g liver). In mice, more than 50% of FEX unbound intrinsic biliary clearance (CL(bile, int)(') = 3.0 ml/h/g liver) could be attributed to Mrp2 (Mrp2-dependent CL(bile, int)(') approximately 1.7 ml/h/g liver), with P-gp and Bcrp playing a minor role (P-gp- and Bcrp-dependent CL(bile, int)(') approximately 0.3 ml/h/g liver). Approximately one third of FEX CL(bile, int)(') was attributed to unidentified mechanisms in mice. In contrast to mice, FEX biliary excretion rate (245 +/- 38 and 250 +/- 25 pmol/min/g liver) and CL(bile, int)(') (9.72 +/- 2.47 and 6.49 +/- 0.68 ml/h/g liver) were comparable between TR(-) and control Wistar rats, respectively, suggesting that unidentified transport mechanism(s) can completely compensate for the loss of Mrp2 function in rats. Mrp2 clearly plays a major role in FEX biliary excretion in mice. In conclusion, remarkable species differences exist in FEX hepatobiliary transport mechanisms.
Assuntos
Bile/metabolismo , Antagonistas não Sedativos dos Receptores H1 da Histamina/farmacocinética , Fígado/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Terfenadina/análogos & derivados , Animais , Masculino , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Taxa de Depuração Metabólica , Camundongos , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ratos , Ratos Endogâmicos , Especificidade da Espécie , Terfenadina/farmacocinéticaRESUMO
A transdermal SARM has a potential to have therapeutic benefit through anabolic activity in muscle while sparing undesired effects of benign prostate hyperplasia (BPH) and liver-mediated decrease in HDL-C. 2-Chloro-4-[(2-hydroxy-2-methyl-cyclopentyl)amino]-3-methyl-benzonitrile 6 showed the desired muscle and prostate effects in a preclinical ORX rat model. Compound 6 had minimal effect on HDL-C levels in cynomolgus monkeys and showed human cadaver skin permeability, thus making it an effective tool for proof-of-concept studies in a clinical setting.
Assuntos
Anabolizantes/uso terapêutico , Antagonistas de Androgênios/uso terapêutico , Compostos de Anilina/uso terapêutico , Atrofia Muscular/tratamento farmacológico , Nitrilas/uso terapêutico , Administração Cutânea , Anabolizantes/administração & dosagem , Anabolizantes/síntese química , Antagonistas de Androgênios/administração & dosagem , Antagonistas de Androgênios/síntese química , Compostos de Anilina/administração & dosagem , Compostos de Anilina/síntese química , Animais , HDL-Colesterol/metabolismo , Humanos , Hipercolesterolemia/induzido quimicamente , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macaca fascicularis , Masculino , Modelos Moleculares , Nitrilas/administração & dosagem , Nitrilas/síntese química , Orquiectomia , Hiperplasia Prostática/induzido quimicamente , Ratos , Absorção Cutânea , Relação Estrutura-AtividadeRESUMO
The multidrug resistance proteins P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein 2 (Mrp2) are the three major canalicular transport proteins responsible for the biliary excretion of most drugs and metabolites. Previous in vitro studies demonstrated that P-gp transported macrolide antibiotics, including spiramycin, which is eliminated primarily by biliary excretion. Bcrp was proposed to be the primary pathway for spiramycin secretion into breast milk. In the present study, the contributions of P-gp, Bcrp, and Mrp2 to the biliary excretion of spiramycin were examined in single-pass perfused livers of male C57BL/6 wild-type, Bcrp-knockout, and Mrp2-knockout mice in the presence or absence of GF120918 (GW918), a P-gp and Bcrp inhibitor. Spiramycin was infused to achieve steady-state conditions, followed by a washout period, and parameters governing spiramycin hepatobiliary disposition were recovered by using pharmacokinetic modeling. In the absence of GW918, the rate constant governing spiramycin biliary excretion was decreased in Mrp2(-) knockout mice (0.0013 +/- 0.0009 min(-1)) relative to wild-type mice (0.0124 +/- 0.0096 min(-1)). These data are consistent with the approximately 8-fold decrease in the recovery of spiramycin in the bile of Mrp2-knockout mice and suggest that Mrp2 is the major canalicular transport protein responsible for spiramycin biliary excretion. Interestingly, biliary recovery of spiramycin in Bcrp-knockout mice was increased in both the absence and presence of GW918 compared to wild-type mice. GW918 significantly decreased the rate constant for spiramycin biliary excretion and the rate constant for basolateral efflux of spiramycin. In conclusion, the biliary excretion of spiramycin in mice is mediated primarily by Mrp2 with a modest P-gp component.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bile/metabolismo , Coccidiostáticos/farmacocinética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Espiramicina/farmacocinética , 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 , Animais , Canalículos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Interpretação Estatística de Dados , Técnicas In Vitro , Fígado/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Dinâmica não LinearRESUMO
This study characterized the hepatobiliary disposition of 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF), a model Abcc2/Mrp2 (canalicular) and Abcc3/Mrp3 (basolateral) substrate, in perfused livers from male C57BL/6 wild-type, Abcg2-/-, and Abcc2-/- mice. After single-pass liver perfusion with 1 muM CDF diacetate for 30 min and an additional 30-min perfusion with CDF-free buffer, cumulative biliary excretion of CDF in Abcg2-/- mice was significantly higher than in wild-type mice (65 +/- 6 and 47 +/- 15% of dose, respectively, p < 0.05), whereas CDF recovery in bile of Abcc2-/- mice was negligible. Cumulative recovery of CDF in perfusate was significantly higher in Abcc2-/- (90 +/- 8% of dose) relative to wild-type (35 +/- 11% of dose) mice. Compartmental pharmacokinetic analysis revealed that the rate constant for CDF biliary excretion was significantly increased in Abcg2-/- (0.061 +/- 0.005 min(-1)) compared with wild-type (0.039 +/- 0.011 min(-1)) mice. The rate constant governing the basolateral excretion of CDF was approximately 4-fold higher in Abcc2-/- (0.12 +/- 0.02 min(-1)) relative to wild-type (0.030 +/- 0.011 min(-1)) mice but was not altered in Abcg2-/- (0.031 +/- 0.004 min(-1)) mice. Hepatic Abcc3 protein levels, determined by immunoblot analysis, were approximately 60% higher in Abcc2-/- mice than in wild-type mice. In contrast, neither Abcc3 protein levels nor Abcc2 mRNA levels were altered in Abcg2-/- relative to wild-type mice. These data in knockout mouse models demonstrate that loss of expression and function of one canalicular transport protein may change the route and/or extent of excretion into bile or perfusate because of alterations in the function of other basolateral or canalicular transport proteins.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Bile/metabolismo , Fluoresceínas/farmacocinética , Fígado/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , RNA Mensageiro , Regulação para CimaRESUMO
The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(-/-), Abcg2(-/-), and Abcb1a(-/-)/Abcb1b(-/-) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also approximately 35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (> or =37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Acetaminofen/farmacocinética , Bile/metabolismo , Glucuronídeos/metabolismo , Harmina/análogos & derivados , Himecromona/análogos & derivados , Sulfatos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Harmina/farmacocinética , Himecromona/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Phenobarbital (PB) treatment impairs the biliary excretion of some organic anions. One mechanism may involve direct competition for biliary excretion by PB and/or a PB metabolite. Alternatively, PB may alter the expression and/or function of hepatic organic anion transport proteins. The role of multidrug resistance-associated protein 2 (Mrp2) in the biliary excretion of PB and metabolites was studied using isolated perfused livers (IPLs) from Wistar and Mrp2-deficient TR- rats. In normal livers, 4.19 +/- 0.53% of the PB dose was recovered in bile as PB metabolites [2.21 +/- 0.69% as 5-ethyl-5-(4-OH phenyl) barbituric acid (PBOH)-glucuronide; 1.98 +/- 0.09% as PBOH-sulfate]. In TR- livers, only PBOH-sulfate was recovered in bile (0.35 +/- 0.16% of dose) during the 2-h perfusion. Mrp2 message was increased (2.3-fold) by PB pretreatment (80 mg/kg i.p. x 4 days) but decreased to control values after a 48-h washout. Mrp2 protein was increased slightly in PB-treated livers and remained slightly elevated after a 24-h washout, but it was decreased significantly to 62 +/-7% of control values after a 48-h washout. The 120-min cumulative biliary excretion of the Mrp2 substrate 5-(and-6)-carboxy-2', 7'-dichlorofluorescein in IPLs from PB-treated rats after a 48-h washout was significantly lower than in vehicle-treated livers (66.3 +/- 9.2% versus 83.4 +/- 2.4% of the dose, respectively). These data support two mechanisms for impaired biliary excretion of some organic anions by PB treatment: 1) PBOH-glucuronide is a substrate for Mrp2 and may compete with other organic anions for biliary excretion and 2) Mrp2 protein expression and functional capacity is decreased 48 h after PB treatment.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Bile/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Fenobarbital/farmacologia , Animais , Bile/metabolismo , Proteínas de Transporte/genética , Glucuronídeos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Fenobarbital/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Hepatic disposition of 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) and its diacetate promoiety (CDFDA) was studied in isolated perfused rat livers. Livers from Wistar wild-type and multidrug resistance-associated protein (Mrp)2-deficient (TR(-)) rats were perfused with CDF in the presence or absence of probenecid. Probenecid decreased the recovery of CDF in bile approximately 4-fold in wild-type livers (65 +/- 8% versus 15 +/- 2% of dose over 2 h). In livers from TR(-) rats, CDF was not excreted into bile and probenecid decreased perfusate CDF concentrations in a concentration-dependent manner, in part due to inhibition of Mrp3. Plasma membrane vesicles from rat Mrp2- or Mrp3-transfected Sf9 cells were used to confirm that CDF is a substrate for Mrp2 and Mrp3; probenecid inhibited the transport of CDF by Mrp2 and Mrp3 in a concentration-dependent manner. CDF uptake in collagen sandwich-cultured rat hepatocytes was temperature-dependent and saturable (K(m) = 22 +/- 10 microM; V(max) = 97 +/- 9 pmol/min/mg protein). Uptake of CDF in sandwich-cultured rat hepatocytes was impaired significantly by bromosulfophthalein, a substrate for organic anion-transporting polypeptides (Oatps), but was not modulated by specific Oatp2 or organic anion transporter (Oat) substrates. CDFDA uptake was not saturable, temperature-dependent, or impaired by inhibitors. The hydrolysis of CDFDA to CDF is mediated by basic pH and esterases in biological media. CDFDA passively diffuses into hepatocytes where it is hydrolyzed to CDF. In contrast, CDF appears to be taken up by Oatp-mediated transport into rat hepatocytes and effluxed via Mrp2 into bile and via Mrp3 into sinusoidal blood.