Distribution analysis of epertinib in brain metastasis of HER2-positive breast cancer by imaging mass spectrometry and prospect for antitumor activity.

Epertinib (S-222611) is a potent, reversible, and selective tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), human EGFR2 (HER2), and human EGFR4. We developed experimental brain metastasis models by intraventricular injection (intraventricular injection mouse model; IVM) of HER2-positive breast cancer (MDA-MB-361-luc-BR2/BR3) or T790M-EGFR-positive lung cancer (NCI-H1975-luc) cells. After a single oral administration, epertinib and lapatinib concentrations in brain metastatic regions were analyzed by quantitative imaging mass spectrometry. In the NCI-H1975 lung cancer IVM, the concentration of epertinib in brain metastasis was comparable to that of lapatinib. However, in the MDA-MB-361 breast cancer IVM, the concentration of epertinib in brain metastasis was >10 times higher than that of lapatinib. Furthermore, the epertinib tumor-to-normal brain ratio was ~4 times higher than that of lapatinib. Blood-tumor barrier (BTB) permeability was assessed in each brain metastatic region. In the lung cancer model, fluorescently labeled dextran was more highly detected in brain metastatic regions than in brain parenchyma. However, in breast cancer models, dextran fluorescence intensity in brain metastatic regions and brain parenchyma were comparable, suggesting that the BTB remained largely intact. Epertinib would be promised as a therapeutic agent for HER2-positive breast cancer with brain metastasis.

Improved Human Pharmacokinetic Prediction of Hepatically Metabolized Drugs With Species-Specific Systemic Clearance.

Accurate prediction of human pharmacokinetics (PK) is important for the choice of promising compounds in humans. As the predictability of human PK by an empirical approach is low for drugs with species-specific PK, the utility of a physiologically based pharmacokinetic (PBPK) model was verified using 16 hepatically metabolized reference drugs. After the prediction method for total clearance (CLtot) and distribution volume at steady state (Vdss) in the conventional PBPK model had been optimized, plasma concentrations following a single oral administration of each reference drug to healthy volunteers were simulated, and the prediction accuracy for human PK was compared between empirical approaches and the optimized PBPK model. In the drugs with low species-specific CLtot, there was little difference in predictability for maximum concentration (Cmax), time to maximum plasma concentration (Tmax), and area under the curve (AUC) (absolute average fold error: 1.3-2.4). In contrast, the optimized PBPK model predicted Cmax and AUC of the drugs with high species-specific CLtot with lower absolute average fold error (Cmax and AUC: 2.8 and 3.2, respectively) than those of the empirical approach (Cmax and AUC: 2.6-4.9 and 3.9-10.7, respectively). Therefore, the optimized PBPK model is useful for human PK prediction of drugs with species-specific CLtot.

Using Improved Serial Blood Sampling Method of Mice to Study Pharmacokinetics and Drug-Drug Interaction.

In pharmacokinetic evaluation of mice, using serial sampling methods rather than a terminal blood sampling method could reduce the number of animals needed and lead to more reliable data by excluding individual differences. In addition, using serial sampling methods can be valuable for evaluation of the drug-drug interaction (DDI) potential of drug candidates. In this study, we established an improved method for serially sampling the blood from one mouse by only one incision of the lateral tail vein, and investigated whether our method could be adapted to pharmacokinetic and DDI studies. After intravenous and oral administration of ibuprofen and fexofenadine (BCS class II and III), the plasma concentration and pharmacokinetic parameters were evaluated by our method and a terminal blood sampling method, with the result that both methods gave comparable results (ibuprofen: 63.8±4.0% and 64.4%, fexofenadine: 6.5±0.7% and 7.9%, respectively, in bioavailability). In addition, our method could be adapted to DDI study for cytochrome P450 and organic anion transporting polypeptide inhibition. These results demonstrate that our method can be useful for pharmacokinetic evaluation from the perspective of reliable data acquisition as well as easy handling and low stress to mice and improve the quality of pharmacokinetic and DDI studies.

Using improved serial blood sampling method of mice to study pharmacokinetics and drug-drug interaction.

In pharmacokinetic evaluation of mice, using serial sampling methods rather than a terminal blood sampling method could reduce the number of animals needed and lead to more reliable data by excluding individual differences. In addition, using serial sampling methods can be valuable for evaluation of the drug-drug interaction (DDI) potential of drug candidates. In this study, we established an improved method for serially sampling the blood from one mouse by only one incision of the lateral tail vein, and investigated whether our method could be adapted to pharmacokinetic and DDI studies. After intravenous and oral administration of ibuprofen and fexofenadine (BCS class II and III), the plasma concentration and pharmacokinetic parameters were evaluated by our method and a terminal blood sampling method, with the result that both methods gave comparable results (ibuprofen: 63.8 ± 4.0% and 64.4%, fexofenadine: 6.5 ± 0.7% and 7.9%, respectively, in bioavailability). In addition, our method could be adapted to DDI study for cytochrome P450 and organic anion transporting polypeptide inhibition. These results demonstrate that our method can be useful for pharmacokinetic evaluation from the perspective of reliable data acquisition as well as easy handling and low stress to mice and improve the quality of pharmacokinetic and DDI studies.

Preclinical antitumor activity of S-222611, an oral reversible tyrosine kinase inhibitor of epidermal growth factor receptor and human epidermal growth factor receptor 2.

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are validated molecular targets in cancer therapy. Dual blockade has been explored and one such agent, lapatinib, is in clinical practice but with modest activity. Through chemical screening, we discovered a novel EGFR and HER2 inhibitor, S-222611, that selectively inhibited both kinases with IC50 s below 10 nmol/L. S-222611 also inhibited intracellular kinase activity and the growth of EGFR-expressing and HER2-expressing cancer cells. In addition, S-222611 showed potent antitumor activity over lapatinib in a variety of xenograft models. In evaluations with two patient-oriented models, the intrafemoral implantation model and the intracranial implantation model, S-222611 exhibited excellent activity and could be effective against bone and brain metastasis. Compared to neratinib and afatinib, irreversible EGFR/HER2 inhibitors, S-222611 showed equivalent or slightly weaker antitumor activity but a safer profile. These results indicated that S-222611 is a potent EGFR and HER2 inhibitor with substantially better antitumor activity than lapatinib at clinically relevant doses. Considering the safer profile than for irreversible inhibitors, S-222611 could be an important option in future cancer therapy.

Apparent differences in mechanisms of harmol sulfate biliary excretion in mice and rats.

Previous experiments demonstrated that the biliary excretion of harmol sulfate (HS) was mediated by breast cancer resistance protein (Bcrp) and not by multidrug resistance-associated protein (Mrp)2 or P-glycoprotein in mice. However, recent reports suggested that species differences in hepatic canalicular transport mechanisms for a given substrate exist between mice and rats. In the present study, biliary excretion of HS was examined in perfused livers from mice and rats in the absence or presence of the P-glycoprotein and Bcrp inhibitor 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). As expected, in mouse liver perfusions, the biliary excretion of HS was decreased approximately 3.5-fold by GF120918, consistent with previous reports of Bcrp-mediated HS biliary excretion. However, despite sufficient hepatic unbound concentrations of GF120918 to achieve extensive inhibition of Bcrp, the biliary excretion of HS was not decreased significantly in wild-type (50 +/- 12 versus 41 +/- 6%) or TR(-) (18 +/- 2 versus 16 +/- 3%) Wistar rats. In summary, biliary excretion of HS was mediated by a GF120918-sensitive mechanism in mice, previously elucidated as Bcrp. In contrast, the pathway responsible for HS biliary excretion in rats was not impaired by GF120918. Thus, transport mechanism(s) responsible for harmol sulfate biliary excretion appear to differ between mice and rats.

Multidrug resistance-associated protein 2 is primarily responsible for the biliary excretion of fexofenadine in mice.

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.

Biotransformation and transport of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in bile duct-cannulated wild-type and Mrp2/Abcc2-deficient (TR ) Wistar rats.

The role of uptake and efflux transport proteins in the tissue distribution of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites is largely unknown. Carbonyl reduction of NNK results in formation of the carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which in rats is glucuronidated to the non-toxic NNAL-O-glucuronide. Previous in vitro studies showed that NNAL-O-glucuronide is a substrate for the human ATP-binding cassette transport proteins multidrug resistance protein (MRP)1 (ABCC1) and MRP2 (ABCC2). To investigate the influence of Mrp2 deficiency on NNK biotransformation and biliary excretion, [(3)H]NNK was administered intravenously to bile duct-cannulated wild-type (WT) and Mrp2-deficient (TR(-)) Wistar rats; plasma, bile and urine samples were collected for 5 h and analyzed by high-pressure liquid chromatography with radiochemical detection. The total radioactivity recovered in WT and TR(-) bile was 12 and 7% of the dose, respectively. NNAL-O-glucuronide accounted for 87% of the radioactivity in WT bile but was not detected in TR(-) bile. Urinary recovery of 1-(3-pyridyl)-1-butanol-4-carboxylic acid (hydroxy acid), NNAL-O-glucuronide and NNAL-N-oxide from 2-5 h was greater in TR(-) compared with WT rats. NNK plasma clearance was significantly higher in TR(-) (115 +/- 23 ml/min/kg) compared with WT (48 +/- 13 ml/min/kg) rats. A higher concentration and/or earlier appearance of hydroxy and 1-(3-pyridyl)-1-butanone-4-carboxylic acids, NNAL-N-oxide and NNK-N-oxide, and decreased NNK and NNAL concentrations in TR(-) plasma suggested increased cytochrome P450 biotransformation in TR(-) rats. The total recovery of hydroxy acid in bile and urine was significantly higher in TR(-) compared with WT rats. Thus, Mrp2 is responsible for the biliary excretion of NNAL-O-glucuronide and Mrp2 deficiency results in increased formation of carcinogenic NNK metabolites.

The important role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in mice.

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.

Evaluation of the role of multidrug resistance-associated protein (Mrp) 3 and Mrp4 in hepatic basolateral excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3-/- and Abcc4-/- mice.

Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3(-/-) and Abcc4(-/-) mice using a cassette dosing approach. In the livers of Abcc3(-/-) and Abcc4(-/-) mice, the basolateral excretory clearance of acetaminophen sulfate was reduced approximately 20 and approximately 20%, 4-methylumbelliferyl sulfate was reduced approximately 50 and approximately 65%, and harmol sulfate was decreased approximately 30 and approximately 45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by approximately 96, approximately 85, and approximately 40%, respectively, in the livers of Abcc3(-/-) mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.

Differential involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in biliary excretion of 4-methylumbelliferyl glucuronide and sulfate in the rat.

The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR(-)) Wistar rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-micromol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in wild-type rat IPLs (25 +/- 3 micromol) but was negligible in TR(-) livers (0.4 +/- 0.1 micromol); coadministration of the Bcrp and P-glycoprotein inhibitor GF120918 [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] had no effect on 4MUG biliary excretion in wild-type rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient rat IPLs. Recovery of 4MUS in bile was approximately 50 to 60% lower in both control TR(-) (149 +/- 8 nmol) and wild-type IPLs with GF120918 coadministration (176 +/- 30 nmol) relative to wild-type control livers (378 +/- 37 nmol) and was nearly abolished in TR(-) IPLs in the presence of GF120918 (13 +/- 8 nmol). These changes were the result of decreased rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.

Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites.

The liver is the primary site of drug metabolism in the body. Typically, metabolic conversion of a drug results in inactivation, detoxification, and enhanced likelihood for excretion in urine or feces. Sulfation, glucuronidation, and glutathione conjugation represent the three most prevalent classes of phase II metabolism, which may occur directly on the parent compounds that contain appropriate structural motifs, or, as is usually the case, on functional groups added or exposed by phase I oxidation. These three conjugation reactions increase the molecular weight and water solubility of the compound, in addition to adding a negative charge to the molecule. As a result of these changes in the physicochemical properties, phase II conjugates tend to have very poor membrane permeability, and necessitate carrier-mediated transport for biliary or hepatic basolateral excretion into sinusoidal blood for eventual excretion into urine. This review summarizes sulfation, glucuronidation, and glutathione conjugation reactions, as well as recent progress in elucidating the hepatic transport mechanisms responsible for the excretion of these conjugates from the liver. The discussion focuses on alterations of metabolism and transport by chemical modulators, and disease states, as well as pharmacodynamic and toxicological implications of hepatic metabolism and/or transport modulation for certain active phase II conjugates. A brief discussion of issues that must be considered in the design and interpretation of phase II metabolite transport studies follows.

Altered hepatobiliary disposition of 5 (and 6)-carboxy-2',7'-dichlorofluorescein in Abcg2 (Bcrp1) and Abcc2 (Mrp2) knockout mice.

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.

Liver-specific distribution of rosuvastatin in rats: comparison with pravastatin and simvastatin.

Rosuvastatin is a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. The liver is the target organ for the lipid-regulating effect of rosuvastatin; therefore liver-selective uptake of this drug is a desirable property. The aim of this study was to investigate, and compare with pravastatin and simvastatin, the tissue-specific distribution of rosuvastatin. Bolus intravenous doses (5 mg/kg) of radiolabeled rosuvastatin, pravastatin, and simvastatin were administered to rats, and initial uptake clearance (CL(uptake)) in various tissues was calculated. Hepatic CL(uptake) of rosuvastatin (0.885 ml/min/g tissue) was significantly (p < 0.001) larger than that of pravastatin (0.703 ml/min/g tissue), and rosuvastatin was taken up by the hepatic cells more selectively and efficiently than pravastatin. Hepatic CL(uptake) of simvastatin (1.24 ml/min/g tissue) was significantly larger than that of rosuvastatin (p < 0.01) and pravastatin (p < 0.001). However, adrenal CL(uptake) of simvastatin (1.55 ml/min/g tissue) was larger than hepatic CL(uptake), and simvastatin was distributed to other tissues more easily than rosuvastatin. Microautoradiography of the liver, spleen, and adrenal was undertaken 5 min after administration of the study drugs; distribution was quantified by counting the number of silver grains. After administration of rosuvastatin and pravastatin, silver grains were distributed selectively in the intracellular space of the liver, but more rosuvastatin (3.3 +/- 1.0 x 10(5) particles/mm(2)) than pravastatin (2.0 +/- 0.3 x 10(5) particles/mm(2)) tended to distribute to the liver. Simvastatin was less liver-specific (it also distributed to the spleen and adrenal). The results of this study indicated that rosuvastatin was taken up by hepatic cells more selectively and more efficiently than pravastatin and simvastatin.