No relation between docetaxel administration route and high-grade diarrhea incidence.

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.

Ritonavir inhibits intratumoral docetaxel metabolism and enhances docetaxel antitumor activity in an immunocompetent mouse breast cancer model.

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.

Severe lactic acidosis in a diabetic patient after ethanol abuse and floor cleaner intake.

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.

Taxanes: old drugs, new oral formulations.

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.

P-glycoprotein and cytochrome P450 3A act together in restricting the oral bioavailability of paclitaxel.

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.

P-glycoprotein, multidrug-resistance associated protein 2, Cyp3a, and carboxylesterase affect the oral availability and metabolism of vinorelbine.

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.

Brain accumulation of sunitinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by oral elacridar and sunitinib coadministration.

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.

Toxicokinetics of a dipyridamole (Persantin) intoxication: case report.

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.

Lethal morphine intoxication in a patient with a sickle cell crisis and renal impairment: case report and a review of the literature.

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.

Hepatic clearance of reactive glucuronide metabolites of diclofenac in the mouse is dependent on multiple ATP-binding cassette efflux transporters.

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.

Breast cancer resistance protein and P-glycoprotein limit sorafenib brain accumulation.

Sorafenib is a second-generation, orally active multikinase inhibitor that is approved for the treatment of patients with advanced renal cell carcinoma and patients with unresectable hepatocellular carcinoma. We studied active transport of sorafenib in MDCK-II cells expressing human P-glycoprotein (P-gp/ABCB1) or ABCG2 (breast cancer resistance protein) or murine Abcg2. Sorafenib was moderately transported by P-gp and more efficiently by ABCG2 and Abcg2. Because sorafenib is taken orally, we orally administered sorafenib to wild-type, Abcb1a/1b(-/-), Abcg2(-/-), and Abcb1a/1b;Abcg2(-/-) mice, completely lacking functional Abcb1a/1b, Abcg2, or both, respectively, and we studied plasma pharmacokinetics and brain accumulation. The systemic exposure on oral administration was not different among all strains. However, brain accumulation was 4.3-fold increased in Abcg2(-/-) mice and 9.3-fold increased in Abcb1a/1b;Abcg2(-/-) mice. Moreover, when wild-type mice were treated with sorafenib in combination with the dual P-gp and ABCG2 inhibitor elacridar, brain accumulation was similar to that observed for Abcb1a/1b;Abcg2(-/-) mice. These results show that the brain accumulation of sorafenib is primarily restricted by ABCG2. This contrasts with previous studies using shared ABCG2 and P-gp substrates, which all suggested that P-gp dominates at the blood-brain barrier, and that an effect of ABCG2 is only evident when both transporters are absent. Interestingly, for sorafenib, it is the other way around, that is, ABCG2, and not P-gp, plays the dominant role in restricting its brain accumulation. Clinically, our findings may be relevant for the treatment of renal cell carcinoma patients with central nervous system relapses, as a dual ABCG2 and P-gp inhibitor might improve the central nervous system entry and thereby the therapeutic efficacy of sorafenib.

Individual and combined roles of CYP3A, P-glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) in the pharmacokinetics of docetaxel.

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.

P-glycoprotein (P-gp/Abcb1), Abcc2, and Abcc3 determine the pharmacokinetics of etoposide.

PURPOSE: Despite the extensive use of etoposide for the treatment of different malignant neoplasms, its main pharmacokinetic determinants are not completely defined. We aimed to study the impact of P-glycoprotein (P-gp/ABCB1) and the multidrug resistance proteins ABCC2 (MRP2) and ABCC3 (MRP3) on the pharmacokinetics of etoposide. EXPERIMENTAL DESIGN: Abcb1a/1b(-/-), Abcc2(-/-), Abcc3(-/-), Abcb1a/1b;Abcc2(-/-), and Abcc2;Abcc3(-/-) mice were used to investigate the separate and combined impact of P-gp, Abcc2, and Abcc3 on the in vivo behavior of etoposide. RESULTS: P-gp restricted the oral (re)uptake of unchanged etoposide, and mediated its excretion across the gut wall. In contrast, hepatobiliary excretion was almost entirely dependent on Abcc2. Yet, complete loss of Abcc2 did not result in elevated liver or plasma concentrations of etoposide. Instead, Abcc2(-/-) mice displayed an increased hepatic formation of etoposide glucuronide, which was secreted via Abcc3 from the liver to the blood circulation and eliminated with the urine. Combination Abcc2;Abcc3(-/-) mice had highly increased accumulation of etoposide glucuronide in their livers, whereas both single knockouts did not, indicating that Abcc2 and Abcc3 provide alternative pathways for the hepatic elimination of etoposide glucuronide. CONCLUSIONS: P-gp, ABCC2, and ABCC3 significantly affect the pharmacokinetics of etoposide and/or etoposide glucuronide. Variation in transporter expression or activity may explain the high variation in oral availability of etoposide (25-80%) among cancer patients. However, despite the fact that substantial variations in transporter activity can occur, we believe that cancer patients are often relatively protected from etoposide toxicity due to overlapping functions of these transporters in the elimination of etoposide.

Absence of both cytochrome P450 3A and P-glycoprotein dramatically increases docetaxel oral bioavailability and risk of intestinal toxicity.

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 i.v. The drug-metabolizing enzyme cytochrome P450 3A (CYP3A) and the drug transporter P-glycoprotein (P-gp; MDR1) are considered to be major determinants of docetaxel pharmacokinetics. It has been hypothesized that CYP3A and P-gp work synergistically in limiting the systemic exposure to many orally ingested drugs. However, it has been difficult to examine this interplay in vivo. We therefore generated mice lacking all CYP3A and P-gp genes. Although missing two primary detoxification systems, Cyp3a/Mdr1a/1b(-/-) mice are viable, fertile, and without spontaneous abnormalities. When orally challenged with docetaxel, a disproportionate (>70-fold) increase in systemic exposure was observed compared with the increases in single Cyp3a(-/-) (12-fold) or Mdr1a/1b(-/-) (3-fold) mice. Unexpectedly, although CYP3A and P-gp collaborated extremely efficiently in lowering docetaxel exposure, their individual efficacy was not dependent on activity of the other protein. On reflection, this absence of functional synergism makes biological sense, as synergism would conflict with a robust detoxification defense. Importantly, the disproportionate increase in docetaxel exposure in Cyp3a/Mdr1a/1b(-/-) mice resulted in dramatically altered and lethal toxicity, with severe intestinal lesions as a major cause of death. Simultaneous inhibition of CYP3A/P-gp might thus be a highly effective strategy to improve oral drug bioavailability but with serious risks when applied to drugs with narrow therapeutic windows.

Pharmacokinetic assessment of multiple ATP-binding cassette transporters: the power of combination knockout mice.

A TP-binding cassette (ABC) multidrug transporters are cellular efflux pumps with broad and often widely overlapping substrate specificities. They can have a major impact on the pharmacokinetics and hence overall pharmacological behavior of many drugs. To study their separate roles and functional overlap, or complementarity, a collection of mice deficient in two or more ABC transporters has been generated. This review discusses recent findings obtained with these models, focusing on pharmacokinetic studies with a number of clinically relevant drugs. In addition, the characterization of these mice and some physiological aspects of ABC multidrug transporters are addressed.

The bioanalysis of vinorelbine and 4-O-deacetylvinorelbine in human and mouse plasma using high-performance liquid chromatography coupled with heated electrospray ionization tandem mass-spectrometry.

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.

Brain accumulation of dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by elacridar treatment.

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.

Liquid chromatography-tandem mass spectrometric assay for sorafenib and sorafenib-glucuronide in mouse plasma and liver homogenate and identification of the glucuronide metabolite.

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.

Physiological and pharmacological roles of ABCG2 (BCRP): recent findings in Abcg2 knockout mice.

The multidrug transporter ABCG2 (BCRP/MXR/ABCP) can actively extrude a broad range of endogenous and exogenous substrates across biological membranes. ABCG2 limits oral availability and mediates hepatobiliary and renal excretion of its substrates, and thus influences the pharmacokinetics of many drugs. Recent work, relying mainly on the use of Abcg2(-/-) mice, has revealed important contributions of ABCG2 to the blood-brain, blood-testis and blood-fetal barriers. Together, these functions indicate a primary biological role of ABCG2 in protecting the organism from a range of xenobiotics. In addition, several other physiological functions of ABCG2 have been observed, including extrusion of porphyrins and/or porphyrin conjugates from hematopoietic cells, liver and harderian gland, as well as secretion of vitamin B(2) (riboflavin) and possibly other vitamins (biotin, vitamin K) into breast milk. However, the physiological significance of these processes has been difficult to establish, indicating that there is still a lot to learn about this intriguing protein.

Transport of diclofenac by breast cancer resistance protein (ABCG2) and stimulation of multidrug resistance protein 2 (ABCC2)-mediated drug transport by diclofenac and benzbromarone.

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.