ABSTRACT
Docetaxel (Taxotere(®)) is currently used intravenously as an anticancer agent and is primarily metabolized by Cytochrome P450 3A (CYP3A). The HIV protease inhibitor ritonavir, a strong CYP3A4 inhibitor, decreased first-pass metabolism of orally administered docetaxel. Anticancer effects of ritonavir itself have also been described. We here aimed to test whether ritonavir co-administration could decrease intratumoral metabolism of intravenously administered docetaxel and thus increase the antitumor activity of docetaxel in an orthotopic, immunocompetent mouse model for breast cancer. Spontaneously arising K14cre;Brca1(F/F) ;p53(F/F) mouse mammary tumors were orthotopically implanted in syngeneic mice lacking Cyp3a (Cyp3a(-/-)) to limit ritonavir effects on systemic docetaxel clearance. Over 3 weeks, docetaxel (20 mg/kg) was administered intravenously once weekly, with or without ritonavir (12.5 mg/kg) administered orally for 5 days per week. Untreated mice were used as control for tumor growth. Ritonavir treatment alone did not significantly affect the median time of survival (14 vs. 10 days). Median time of survival in docetaxel-treated mice was 54 days. Ritonavir co-treatment significantly increased this to 66 days, and substantially reduced relative average tumor size, without altering tumor histology. Concentrations of the major docetaxel metabolite M2 in tumor tissue were reduced by ritonavir co-administration, whereas tumor RNA expression of Cyp3a was unaltered. In this breast cancer model, we observed no direct antitumor effect of ritonavir alone, but we found enhanced efficacy of docetaxel treatment when combined with ritonavir. Our data, therefore, suggest that decreased docetaxel metabolism inside the tumor as a result of Cyp3a inhibition contributes to increased antitumor activity.
Subject(s)
Antineoplastic Agents/pharmacology , Cytochrome P-450 CYP3A Inhibitors/pharmacology , HIV Protease Inhibitors/pharmacology , Mammary Neoplasms, Experimental/drug therapy , Ritonavir/pharmacology , Taxoids/pharmacology , Animals , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/physiology , Docetaxel , Drug Synergism , Female , Immunocompetence , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Taxoids/metabolismABSTRACT
Paclitaxel is avidly transported by P-glycoprotein (P-gp/MDR1/ABCB1). This results in low oral bioavailability, which can be boosted by coadministration of P-gp inhibitors. Unlike paclitaxel, docetaxel is extensively metabolized by CYP3A4 and its oral bioavailability can be enhanced in mice and humans by coadministration of the potent CYP3A inhibitor ritonavir. Unexpectedly, ritonavir also enhances the oral bioavailability of paclitaxel in humans. We aimed to resolve the mechanism underlying this enhancement. Using mice lacking Cyp3a and/or P-gp, we investigated the combined and separate restricting roles of Cyp3a and P-gp in the oral bioavailability of paclitaxel, and the boosting effect of ritonavir. CYP3A4-humanized mice were used for translation to the human situation. P-gp had a dominant effect (11.6-fold, p < 0.001) over Cyp3a (<1.5-fold, n.s.) in limiting plasma concentrations of oral paclitaxel. However, in the absence of P-gp, Cyp3a decreased paclitaxel plasma concentrations twofold (p < 0.001). Coadministered ritonavir inhibited Cyp3a-mediated metabolism, but not P-gp-mediated transport of paclitaxel. Owing to the dominant effect of P-gp, ritonavir enhanced only paclitaxel plasma concentrations in P-gp-deficient mice. Mouse liver microsomes metabolized paclitaxel far less efficiently than human or CYP3A4-transgenic liver microsomes, revealing much lower efficiency of paclitaxel metabolism by mouse than by human CYP3As. Accordingly, ritonavir could enhance the oral bioavailability of paclitaxel in CYP3A4-humanized mice, despite the fact that these mice are P-gp-proficient. Our results show that CYP3A4 inhibition most likely underlies the boosting effect of ritonavir on oral paclitaxel bioavailability in humans. Furthermore, CYP3A4-humanized mice allow improved understanding of CYP3A4-mediated paclitaxel metabolism in humans.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents, Phytogenic/pharmacokinetics , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 Enzyme System/metabolism , Enzyme Inhibitors/pharmacology , Paclitaxel/pharmacokinetics , Ritonavir/pharmacology , Taxoids/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Administration, Oral , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/blood , Area Under Curve , Biological Availability , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/deficiency , Cytochrome P-450 Enzyme System/genetics , Docetaxel , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/therapeutic use , Humans , Infusions, Intravenous , Intestinal Absorption , Male , Mice , Mice, Knockout , Paclitaxel/administration & dosage , Paclitaxel/blood , Ritonavir/administration & dosage , Ritonavir/therapeutic use , Taxoids/administration & dosage , Taxoids/bloodABSTRACT
We investigated the interactions of the anticancer drug vinorelbine with drug efflux transporters and cytochrome P450 3A drug-metabolizing enzymes. Vinorelbine was transported by human multidrug-resistance associated protein (MRP) 2, and Mrp2 knockout mice displayed increased vinorelbine plasma exposure after oral administration, suggesting that Mrp2 limits the intestinal uptake of vinorelbine. Using P-glycoprotein (P-gp), Cyp3a-, and P-gp/Cyp3a knockout mice, we found that the absence of P-gp or Cyp3a resulted in increased vinorelbine plasma exposure, both after oral and intravenous administration. Surprisingly, P-gp/Cyp3a knockout mice displayed markedly lower vinorelbine plasma concentrations than wild-type mice upon intravenous administration but higher concentrations upon oral administration. This could be explained by highly increased formation of 4'-O-deacetylvinorelbine, an active vinorelbine metabolite, especially in P-gp/Cyp3a knockout plasma. Using wild-type and Cyp3a knockout liver microsomes, we found that 4'-O-deacetylvinorelbine formation was 4-fold increased in Cyp3a knockout liver and was not mediated by Cyp3a or other cytochrome P450 enzymes. In vitro incubation of vinorelbine with plasma revealed that vinorelbine deacetylation in Cyp3a and especially in P-gp/Cyp3a knockout mice but not in P-gp-deficient mice was strongly up-regulated. Metabolite formation in microsomes and plasma could be completely inhibited with the nonspecific carboxylesterase (CES) inhibitor bis(4-nitrophenyl) phosphate and partly with the CES2-specific inhibitor loperamide, indicating that carboxylesterase Ces2a, which was appropriately up-regulated in Cyp3a and especially in P-gp/Cyp3a knockout liver was responsible for the 4'O-deacetylvinorelbine formation. Such compensatory up-regulation can complicate the interpretation of knockout mouse data. Nonetheless, P-gp, Mrp2, Cyp3a, and Ces2a clearly restricted vinorelbine availability in mice. Variation in activity of their human homologs may also affect vinorelbine pharmacokinetics in patients.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , Antineoplastic Agents/pharmacokinetics , Carboxylesterase/metabolism , Cytochrome P-450 CYP3A/metabolism , Vinblastine/analogs & derivatives , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Administration, Oral , Animals , Biological Availability , Biological Transport , Carboxylesterase/antagonists & inhibitors , Cytochrome P-450 CYP3A/genetics , Dogs , Female , Humans , In Vitro Techniques , Loperamide/pharmacology , Madin Darby Canine Kidney Cells , Mice , Mice, Knockout , Microsomes, Liver/metabolism , Multidrug Resistance-Associated Protein 2 , Nitrophenols/pharmacology , Up-Regulation , Vinblastine/metabolism , Vinblastine/pharmacokinetics , Vinorelbine , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
Sunitinib is an orally active, multitargeted tyrosine kinase inhibitor which has been used for the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. We aimed to investigate the in vivo roles of the ATP-binding cassette drug efflux transporters ABCB1 and ABCG2 in plasma pharmacokinetics and brain accumulation of oral sunitinib, and the feasibility of improving sunitinib kinetics using oral coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. We used in vitro transport assays and Abcb1a/1b(-/-) , Abcg2(-/-) and Abcb1a/1b/Abcg2(-/-) mice to study the roles of ABCB1 and ABCG2 in sunitinib disposition. In vitro, sunitinib was a good substrate of murine (mu)ABCG2 and a moderate substrate of human (hu)ABCB1 and huABCG2. In vivo, the systemic exposure of sunitinib after oral dosing (10 mg kg(-1) ) was unchanged when muABCB1 and/or muABCG2 were absent. Brain accumulation of sunitinib was markedly (23-fold) increased in Abcb1a/b/Abcg2(-/-) mice, but only slightly (2.3-fold) in Abcb1a/b(-/-) mice, and not in Abcg2(-/-) mice. Importantly, a clinically realistic coadministration of oral elacridar and oral sunitinib to wild-type mice resulted in markedly increased sunitinib brain accumulation, equaling levels in Abcb1a/1b/Abcg2(-/-) mice. This indicates complete inhibition of the blood-brain barrier (BBB) transporters. High-dose intravenous sunitinib could saturate BBB muABCG2, but not muABCB1A, illustrating a dose-dependent relative impact of the BBB transporters. Brain accumulation of sunitinib is effectively restricted by both muABCB1 and muABCG2 activity. Complete inhibition of both transporters, leading to markedly increased brain accumulation of sunitinib, is feasible and safe with a clinically realistic oral elacridar/sunitinib coadministration.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , ATP-Binding Cassette Transporters/physiology , Acridines/administration & dosage , Antineoplastic Agents/administration & dosage , Brain/metabolism , Indoles/administration & dosage , Pyrroles/administration & dosage , Tetrahydroisoquinolines/administration & dosage , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Biological Transport , Blood-Brain Barrier/drug effects , Brain/drug effects , Dogs , Drug Resistance, Multiple , Humans , Indoles/blood , Indoles/pharmacokinetics , Male , Mice , Mice, Knockout , Pyrroles/blood , Pyrroles/pharmacokinetics , Sunitinib , Tissue DistributionABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Diclofenac/metabolism , Glucuronides/metabolism , Liver/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Angiogenic Proteins/physiology , Animals , Bile/metabolism , Diclofenac/toxicity , Liver/drug effects , Mice , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/physiologyABSTRACT
Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can, therefore, only be administered intravenously. The drug-metabolizing enzyme CYP3A and the drug transporter MDR1 (P-glycoprotein) are major determinants of docetaxel pharmacokinetics. In vitro studies have indicated that docetaxel is also a substrate for the drug transporter MRP2, but the in vivo importance of MRP2 for docetaxel is currently unknown. We, therefore, investigated the role of MRP2 in the pharmacokinetics of docetaxel by utilizing Mrp2(-/-) mice. We also generated and characterized Cyp3a/Mdr1a/b/Mrp2(-/-) combination knockout mice to get more insight into how these drug-handling systems work together in determining docetaxel pharmacokinetics. The systemic exposure in Mrp2(-/-) mice was not significantly different from wild-type, after either oral or intravenous administration. Strikingly, however, in Cyp3a/Mdr1a/b/Mrp2(-/-) mice, systemic docetaxel exposure was increased 166-fold after oral administration when compared with wild-type mice, and 2.3-fold when compared with Cyp3a/Mdr1a/b(-/-) mice. Interestingly, this 166-fold increase was disproportionate compared with that for the separate Cyp3a (12-fold) or Mdr1a/b/Mrp2 (4-fold) knockouts. The oral bioavailability was increased to 73% in the Cyp3a/Mdr1a/b/Mrp2(-/-) strain, versus only 10% in wild-type mice. Our data thus indicate that in the absence of CYP3A and Mdr1a/b activity, Mrp2 has a marked impact on docetaxel pharmacokinetics. These findings could have important implications for improving the oral bioavailability and reducing the variability in docetaxel exposure.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , ATP-Binding Cassette Transporters/physiology , Cytochrome P-450 Enzyme System/physiology , Multidrug Resistance-Associated Proteins/physiology , Taxoids/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B , Administration, Oral , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Biological Availability , Cytochrome P-450 CYP3A , Docetaxel , Male , Mice , Mice, Knockout , Multidrug Resistance-Associated Protein 2 , Taxoids/pharmacology , Tissue DistributionABSTRACT
PURPOSE: Imatinib, a BCR-ABL tyrosine kinase inhibitor, is a substrate of the efflux transporters P-glycoprotein (P-gp; ABCB1) and ABCG2 (breast cancer resistance protein), and its brain accumulation is restricted by both transporters. For dasatinib, an inhibitor of SCR/BCR-ABL kinases, in vivo interactions with P-gp and ABCG2 are not fully established yet. EXPERIMENTAL DESIGN: We used Abcb1a/1b(-/-), Abcg2(-/-), and Abcb1a/1b;Abcg2(-/-) mice to establish the roles of P-gp and ABCG2 in the pharmacokinetics and brain accumulation of dasatinib. RESULTS: We found that oral uptake of dasatinib is limited by P-gp. Furthermore, relative brain accumulation, 6 hours after administration, was not affected by Abcg2 deficiency, but absence of P-gp resulted in a 3.6-fold increase after oral and 4.8-fold higher accumulation after i.p. administration. Abcb1a/1b;Abcg2(-/-) mice had the most pronounced increase in relative brain accumulation, which was 13.2-fold higher after oral and 22.7-fold increased after i.p. administration. Moreover, coadministration to wild-type mice of dasatinib with the dual P-gp and ABCG2 inhibitor elacridar resulted in a similar dasatinib brain accumulation as observed for Abcb1a/1b;Abcg2(-/-) mice. CONCLUSIONS: Brain accumulation of dasatinib is primarily restricted by P-gp, but Abcg2 can partly take over this protective function at the blood-brain barrier. Consequently, when both transporters are absent or inhibited, brain uptake of dasatinib is highly increased. These findings might be clinically relevant for patients with central nervous system Philadelphia chromosome-positive leukemia, as coadministration of an inhibitor of P-gp and ABCG2 with dasatinib might result in better therapeutic responses in these patients.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Acridines/pharmacology , Antineoplastic Agents/pharmacokinetics , Brain/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Tetrahydroisoquinolines/pharmacology , Thiazoles/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/metabolism , Biological Transport , Cell Line , Dasatinib , Dogs , Mice , Mice, Knockout , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/metabolism , Pyrimidines/blood , Pyrimidines/metabolism , Thiazoles/blood , Thiazoles/metabolismABSTRACT
Oral administration of docetaxel in combination with the CYP3A4 inhibitor ritonavir is used in clinical trials to improve oral bioavailability of docetaxel. Diarrhea was the most commonly observed and dose-limiting toxicity. This study combined preclinical and clinical data and investigated incidence, severity and cause of oral docetaxel-induced diarrhea. In this study, incidence and severity of diarrhea in patients were compared to exposure to orally administered docetaxel. Intestinal toxicity after oral or intraperitoneal administration of docetaxel was further explored in mice lacking Cyp3a and mice lacking both Cyp3a and P-glycoprotein. In patients, severity of diarrhea increased significantly with an increase in AUC and Cmax (P = .035 and P = .025, respectively), but not with an increase in the orally administered dose (P = .11). Furthermore, incidence of grade 3/4 diarrhea after oral docetaxel administration was similar as reported after intravenous docetaxel administration. Intestinal toxicity in mice was only observed at high systemic exposure to docetaxel and was similar after oral and intraperitoneal administration of docetaxel. In conclusion, our data show that the onset of severe diarrhea after oral administration of docetaxel in humans is similar after oral and intravenous administration of docetaxel and is caused by the concentration of docetaxel in the systemic blood circulation. Mouse experiments confirmed that intestinal toxicity is caused by a high systemic exposure and not by local intestinal exposure. Severe diarrhea in patients after oral docetaxel is reversible and is not related to the route of administration of docetaxel.
Subject(s)
Antineoplastic Agents/adverse effects , Diarrhea/chemically induced , Docetaxel/adverse effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Administration, Intravenous , Administration, Oral , Adult , Aged , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Area Under Curve , Cytochrome P-450 CYP3A/genetics , Diarrhea/physiopathology , Docetaxel/administration & dosage , Docetaxel/pharmacokinetics , Female , Humans , Incidence , Injections, Intraperitoneal , Male , Mice , Mice, Knockout , Middle Aged , Severity of Illness IndexABSTRACT
Diclofenac is an important analgesic and anti-inflammatory drug, widely used for treatment of postoperative pain, rheumatoid arthritis, and chronic pain associated with cancer. Consequently, diclofenac is often used in combination regimens and undesirable drug-drug interactions may occur. Because many drug-drug interactions may occur at the level of drug transporting proteins, we studied interactions of diclofenac with apical ATP-binding cassette (ABC) multidrug efflux transporters. Using Madin-Darby canine kidney (MDCK)-II cells transfected with human P-glycoprotein (P-gp; MDR1/ABCB1), multidrug resistance protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) and murine Bcrp1, we found that diclofenac was efficiently transported by murine Bcrp1 and moderately by human BCRP but not by P-gp or MRP2. Furthermore, in Sf9-BCRP membrane vesicles diclofenac inhibited transport of methotrexate in a concentration-dependent manner. We next used MDCK-II-MRP2 cells to study interactions of diclofenac with MRP2-mediated drug transport. Diclofenac stimulated paclitaxel, docetaxel, and saquinavir transport at only 50 microM. We further found that the uricosuric drug benzbromarone stimulated MRP2 at an even lower concentration, having maximal stimulatory activity at only 2 microM. Diclofenac and benzbromarone stimulated MRP2-mediated transport of amphipathic lipophilic drugs at 10- and 250-fold lower concentrations, respectively, than reported for other MRP2 stimulators. Because these concentrations are readily achieved in patients, adverse drug-drug interactions may occur, for example, during cancer therapy, in which drug concentrations are often critical and stimulation of elimination via MRP2 may result in suboptimal chemotherapeutic drug concentrations. Moreover, stimulation of MRP2 activity in tumors may lead to increased efflux of chemotherapeutic drugs and thereby drug resistance.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Benzbromarone/pharmacology , Diclofenac/pharmacokinetics , Multidrug Resistance-Associated Proteins/physiology , Neoplasm Proteins/physiology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Animals , Cell Line , Diclofenac/pharmacology , Dogs , Drug Interactions , Humans , Kidney/cytology , Kidney/metabolism , Multidrug Resistance-Associated Protein 2ABSTRACT
The first bioanalytical assay for the simultaneous determination of sorafenib and sorafenib-glucuronide in mouse plasma and liver homogenate was developed and validated. In addition, the structure of the glucuronide metabolite was elucidated. The quantitative assay started with addition of isotopically labeled internal standards to a 20 microl sample volume and protein precipitation with acetonitrile, the supernatant was diluted with water and injected into the chromatographic system. A polar embedded reversed-phase column with gradient elution using formic acid in water-acetonitrile was used. The eluate was transferred into an electrospray interface with positive ionization and the analytes were detected and quantified using triple quadrupole mass spectrometry. The assay was validated in the ranges 10-5000 ng/ml for sorafenib and 1-500 ng/ml for sorafenib-glucuronide, the lowest levels of these ranges (10 and 1 ng/ml) being the lower limits of quantification (LLQ). Within day precisions were 2-8%, between day precisions 2-10% (both excluded the LLQ level of the glucuronide) and accuracies were between 89% and 106%. Both analytes were chemically stable under all relevant conditions. The assay was successfully applied in pilot in vivo pharmacokinetic studies with sorafenib in mice.
Subject(s)
Antineoplastic Agents/analysis , Benzenesulfonates/analysis , Chromatography, Liquid , Glucuronides/analysis , Liver/chemistry , Pyridines/analysis , Tandem Mass Spectrometry , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Benzenesulfonates/blood , Benzenesulfonates/chemistry , Benzenesulfonates/metabolism , Female , Glucuronides/blood , Glucuronides/chemistry , Glucuronides/metabolism , Liver/metabolism , Male , Mice , Niacinamide/analogs & derivatives , Phenylurea Compounds , Pilot Projects , Pyridines/blood , Pyridines/chemistry , Pyridines/metabolism , Reproducibility of Results , Sensitivity and Specificity , SorafenibABSTRACT
A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4-O-deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 microL plasma aliquot was protein precipitated with acetonitrile-methanol (1:1, v/v) containing the internal standard vinorelbine-d3 and 20 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 x 2.1 mm i.d. Xbridge C(18) column using isocratic elution with 1 mm ammonium acetate-ammonia buffer pH 10.5-acetonitrile-methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4-O-deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 microL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4-O-deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre-)clinical pharmacologic studies with vinorelbine in humans and mice.
Subject(s)
Antineoplastic Agents, Phytogenic/blood , Chromatography, High Pressure Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Vinblastine/analogs & derivatives , Animals , Calibration , Hot Temperature , Humans , Mice , Reference Standards , Reproducibility of Results , Vinblastine/blood , VinorelbineABSTRACT
The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1'-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.
Subject(s)
Anti-Anxiety Agents/metabolism , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 Enzyme System/metabolism , Midazolam/metabolism , Animals , Cytochrome P-450 CYP3A/genetics , DNA, Complementary/biosynthesis , Data Interpretation, Statistical , Mice , Mice, Knockout , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Up-Regulation/drug effectsABSTRACT
The first liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of diclofenac, 4'-hydroxy-diclofenac, 5-hydroxy-diclofenac and diclofenac-acyl-glucuronide in mouse plasma, using a simple sample pre-treatment procedure, was developed and validated. Analytes in plasma were stabilized using acetic acid and ascorbic acid. After addition of the internal standard D(4)-diclofenac to a 10 microl sample volume and protein precipitation with acetonitrile, the supernatant was supplemented with an equal volume of water and injected into the chromatographic system. A polar embedded reversed-phase column with gradient elution using formic acid and ammonium acetate in water-methanol were used. The eluate was totally transferred into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the ranges 10-5000 ng/ml for 4'-hydroxy-diclofenac and 20-10,000 ng/ml for the other analytes, the lowest levels of these ranges (10 or 20 ng/ml) being the lower limits of quantification. Within day precisions were < or = 10%, between day precisions < or = 13% and accuracies were between 90 and 108%. The analytes were chemically stable under all relevant conditions. The assay was successfully applied in a pharmacokinetic study with diclofenac in mice.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/blood , Diclofenac/blood , Tandem Mass Spectrometry/methods , Animals , Calibration , Half-Life , Male , Mice , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.
Subject(s)
Antineoplastic Agents, Phytogenic/blood , Chromatography, High Pressure Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Vinblastine/analogs & derivatives , Animals , Edetic Acid/chemistry , Humans , Mice , Reference Standards , Sensitivity and Specificity , Vinblastine/blood , VinorelbineABSTRACT
Fast and sensitive liquid chromatography-tandem mass spectrometric assays for the determination of salinomycin in mouse plasma, liver, brain and small intestinal contents and in OptiMEM cell culture medium, were developed and validated using simple sample pre-treatment procedures. Tissue samples were homogenized with phosphate buffered saline or, for high levels in liver, with human plasma. After addition of monensin as the internal standard to plasma, homogenate or culture medium and acetonitrile extraction for tissue and plasma, the diluted medium or the supernatant was directly injected into the isocratic chromatographic system using a polar embedded reversed-phase column and formic acid in water-acetonitrile as the eluent. The eluate was completely led into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assays were successfully validated in the ranges 10-2000 ng/ml for OptiMEM cell culture medium, 1-2000 ng/ml for plasma and 3-2000 ng/g in liver brain and small intestinal contents. At the lowest levels, the intra-day precisions were < or =9%, inter-day precisions were < or =14% and accuracies were between 91 and 112%. The analytes were chemically stable under all relevant conditions and the assays were applied in different in vitro transport studies and in pharmacokinetic and tissue distribution studies with salinomycin in mice.
Subject(s)
Anti-Bacterial Agents/metabolism , Chromatography, Liquid/methods , Pyrans/metabolism , Tandem Mass Spectrometry/methods , Animals , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/isolation & purification , Brain/metabolism , Culture Media , Intestine, Small/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred Strains , Pyrans/blood , Pyrans/isolation & purification , Reference Standards , Sensitivity and SpecificityABSTRACT
PURPOSE: P-glycoprotein (P-gp; ABCB1) efficiently transports lipophilic amphipathic drugs, including the widely used anticancer drug paclitaxel (Taxol). We found previously that human multidrug resistance protein 2 (MRP2; ABCC2) also transports paclitaxel in vitro, and although we expected that paclitaxel pharmacokinetics would be dominated by P-gp, the effect of Mrp2 was tested in vivo. EXPERIMENTAL DESIGN: We generated and characterized Mdr1a/1b/Mrp2(-/-) mice, allowing assessment of the distinct roles of Mrp2 and Mdr1a/1b P-gp in paclitaxel pharmacokinetics. RESULTS: Surprisingly, the effect of Mrp2 on i.v. administration of paclitaxel was as great as that of P-gp. The area under plasma concentration-time curve (AUC)i.v. in both Mrp2(-/-) and Mdr1a/1b(-/-) mice was 1.3-fold higher than in wild-type mice, and in Mdr1a/1b/Mrp2(-/-) mice, a 1.7-fold increase was found. In spite of this similar effect, Mrp2 and P-gp had mostly complementary functions in paclitaxel elimination. Mrp2 dominated the hepatobiliary excretion, which was reduced by 80% in Mrp2(-/-) mice. In contrast, P-gp dominated the direct intestinal excretion, with a minor role for Mrp2. The AUCoral of paclitaxel was 8.5-fold increased by Mdr1a/1b deficiency but not affected by Mrp2 deficiency. However, in the absence of Mdr1a/1b P-gp, additional Mrp2 deficiency increased the AUCoral another 1.7-fold. CONCLUSIONS: Thus far, Mrp2 was thought to mainly affect organic anionic drugs in vivo. Our data show that Mrp2 can also be a major determinant of the pharmacokinetic behavior of highly lipophilic anticancer drugs, even in the presence of other efficient transporters. Variation in MRP2 activity might thus directly affect the effective exposure to paclitaxel, on i.v. administration, but also on oral administration, especially when P-gp activity is inhibited.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Paclitaxel/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacokinetics , Area Under Curve , Biological Transport , Mice , Mice, Knockout , Multidrug Resistance-Associated Protein 2 , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
An intoxication with drugs, ethanol or cleaning solvents may cause a complex clinical scenario if multiple agents have been ingested simultaneously. The situation can become even more complex in patients with (multiple) co-morbidities. A 59-year-old man with type 2 diabetes mellitus (without treatment two weeks before the intoxication) intentionally ingested a substantial amount of ethanol along with ~750 mL of laminate floor cleaner containing citric acid. The patient was admitted with severe metabolic acidosis (both ketoacidosis and lactic acidosis, with serum lactate levels of 22 mM). He was treated with sodium bicarbonate, insulin and thiamine after which he recovered within two days. Diabetic ketoacidosis and lactic acidosis aggravated due to ethanol intoxication, thiamine deficiency and citrate. The high lactate levels were explained by excessive lactate formation caused by the combination of untreated diabetes mellitus, thiamine deficiency and ethanol abuse. Metabolic acidosis in diabetes is multi-factorial, and the clinical situation may be further complicated, when ingestion of ethanol and toxic agents are involved. Here, we reported a patient in whom diabetic ketoacidosis was accompanied by severe lactic acidosis as a result of citric acid and mainly ethanol ingestion and a possible thiamine deficiency. In the presence of lactic acidosis in diabetic ketoacidosis, physicians need to consider thiamine deficiency and ingestion of ethanol or other toxins.
Subject(s)
Acidosis, Lactic/chemically induced , Acidosis/chemically induced , Alcoholic Intoxication/complications , Citric Acid/poisoning , Acidosis/drug therapy , Acidosis, Lactic/drug therapy , Diabetes Mellitus, Type 2/physiopathology , Humans , Insulin/therapeutic use , Male , Middle Aged , Severity of Illness Index , Sodium Bicarbonate/therapeutic use , Thiamine/therapeutic use , Thiamine Deficiency/complicationsABSTRACT
Oral administration of anticancer drugs is most often preferred over intravenous administration, as it is convenient for patients, prevents hospitalisation and reduces costs of the therapy. However, the oral route is often hampered by low oral bioavailability, for instance of the taxanes paclitaxel and docetaxel. Limited oral bioavailability can be due to pharmaceutical as well as pharmacological reasons. Taxanes are poorly water-soluble drugs and do not sufficiently dissolve when administered in their crystalline form. Furthermore, affinity for drug transporters highly expressed in the epithelial layer of the gastro-intestinal tract, such as the drug efflux pump P-glycoprotein (P-gp, ABCB1), and presystemic elimination by the cytochrome P450 (CYP) metabolic enzymes, especially CYP3A4, present in liver and gut wall, further hamper oral application of these important anticancer drugs. Preclinical studies with knockout mice lacking functional Pgp and CYP3A4 metabolic enzymes show a significant increase in the bioavailability of orally applied taxanes. Enhancement of oral bioavailability of both taxanes was shown also in wild-type mice using P-gp and CYP3A4 blockers such as cyclosporine A (CsA) and ritonavir (RTV). Subsequently, in clinical studies enhancement of the oral bioavailability of paclitaxel and docetaxel was established when administered orally in combination with CsA or ritonavir. Initially, in preclinical and clinical studies drinking solutions based on the intravenous formulations were applied for oral administration of taxanes. Because these solutions had several disadvantages, solid pharmaceutical formulations of paclitaxel and docetaxel were developed. Clinical studies with these novel formulations in combination with ritonavir are currently ongoing at our Institute.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Chemistry, Pharmaceutical/methods , Taxoids/administration & dosage , Taxoids/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/pharmacology , Cytochrome P-450 CYP3A Inhibitors , Humans , Taxoids/pharmacologyABSTRACT
We report a case of a 51-year-old woman who was admitted to the hospital after ingestion of large doses of dipyridamole (12 g), temazepam (1 g) and oxazepam (0.2 g) with suicidal intent. The highest dipyridamole concentration that was measured in serum was 9.2 mg/L, which was paralleled by impaired platelet activation. For temazepam and oxazepam, peak serum concentrations were 8.5 and 1.3 mg/L, respectively. The patient was treated with activated charcoal, magnesium sulfate and aminophylline and could be discharged in good physical condition within 17 hours. This is the first report that provides toxicokinetic data and a corresponding pharmacodynamic effect after an intoxication with dipyridamole.
Subject(s)
Dipyridamole/pharmacokinetics , Dipyridamole/poisoning , Platelet Aggregation Inhibitors/pharmacokinetics , Platelet Aggregation Inhibitors/poisoning , Suicide, Attempted , Anti-Anxiety Agents/poisoning , Dipyridamole/blood , Female , Humans , Middle Aged , Oxazepam/poisoning , Platelet Aggregation Inhibitors/blood , Temazepam/poisoningABSTRACT
Morphine-6-glucuronide, the active metabolite of morphine, and to a lesser extent morphine itself are known to accumulate in patients with renal failure. A number of cases on non-lethal morphine toxicity in patients with renal impairment report high plasma concentrations of morphine-6-glucuronide, suggesting that this metabolite achieves sufficiently high brain concentrations to cause long-lasting respiratory depression, despite its poor central nervous system penetration. We report a lethal morphine intoxication in a 61-year-old man with sickle cell disease and renal impairment, and we measured concentrations of morphine and morphine-6-glucuronide in blood, brain and cerebrospinal fluid. There were no measurable concentrations of morphine-6-glucuronide in cerebrospinal fluid or brain tissue, despite high blood concentrations. In contrast, the relatively high morphine concentration in the brain suggests that morphine itself was responsible for the cardiorespiratory arrest in this patient. Given the fatal outcome, we recommend to avoid repeated or continuous morphine administration in renal failure.