Mariët Ouwehand

Efficacy of novel P-glycoprotein inhibitors to increase the oral uptake of paclitaxel in mice

P-glycoprotein inhibitors can increase the oral bioavailability of paclitaxel. We have now explored the mechanisms that determine the efficacy of several novel P-glycoprotein inhibitors to increase the absorption of paclitaxel from the gut lumen of mice in both in vivo and in vitro experiments. The inhibitors studied were cyclosporin A, PSC 833, GF120918, LY335979 and R101933. Mass balance studies showed that GF120918 was the most effective inhibitor, resulting in almost complete uptake of paclitaxel. PSC 833 was slightly less effective, whereas cyclosporin A and LY335979 were moderately effective. R101933 had only marginal effects. These findings were in line with in vitro transport experiments using LLC-mdr1a cells. By studying the intra-intestinal kinetics of the agents we found that cyclosporin A, PSC 833 and GF120918 rapidly passed the stomach and traveled concurrently with paclitaxel through the intestines, whereas LY335979 and R101933 delayed stomach emptying. Moreover, these latter compounds appear to be more readily absorbed when released into the intestines thus reducing local intestinal concentrations. Due to their combined effects on absorption and metabolic elimination of paclitaxel, cyclosporin A and PSC 833 resulted in the highest paclitaxel levels in plasma. In conclusion, our models provide insight into the factors that determine the suitability of P-glycoprotein inhibitors to enable oral paclitaxel therapy and will be useful in selecting candidate inhibitors for clinical testing.

The co-solvent Cremophor EL limits absorption of orally administered paclitaxel in cancer patients

The purpose of this study was to investigate the effect of the co-solvents Cremophor EL and polysorbate 80 on the absorption of orally administered paclitaxel. 6 patients received in a randomized setting, one week apart oral paclitaxel 60 mg m–2 dissolved in polysorbate 80 or Cremophor EL. For 3 patients the amount of Cremophor EL was 5 ml m–2, for the other three 15 ml m–2. Prior to paclitaxel administration patients received 15 mg kg–1 oral cyclosporin A to enhance the oral absorption of the drug. Paclitaxel formulated in polysorbate 80 resulted in a significant increase in the maximal concentration (Cmax) and area under the concentration–time curve (AUC) of paclitaxel in comparison with the Cremophor EL formulations (P = 0.046 for both parameters). When formulated in Cremophor EL 15 ml m–2, paclitaxel Cmax and AUC values were 0.10 ± 0.06 μM and 1.29 ± 0.99 μM h–1, respectively, whereas these values were 0.31 ± 0.06 μM and 2.61 ± 1.54 μM h–1, respectively, when formulated in polysorbate 80. Faecal data revealed a decrease in excretion of unchanged paclitaxel for the polysorbate 80 formulation compared to the Cremophor EL formulations. The amount of paclitaxel excreted in faeces was significantly correlated with the amount of Cremophor EL excreted in faeces (P = 0.019). When formulated in Cremophor EL 15 ml m–2, paclitaxel excretion in faeces was 38.8 ± 13.0% of the administered dose, whereas this value was 18.3 ±15.5% for the polysorbate 80 formulation. The results show that the co-solvent Cremophor EL is an important factor limiting the absorption of orally administered paclitaxel from the intestinal lumen. They highlight the need for designing a better drug formulation in order to increase the usefulness of the oral route of paclitaxel

Cannulation of the jugular vein in mice: a method for serial withdrawal of blood samples

We have developed and validated a method that allows serial drawing of blood samples in freely moving mice using a cannula that is inserted via the jugular vein into the right atrium of the heart. The cannula was tunnelled subcutaneously to the head of the animal and attached to the skin by sutures, together with a metal spring, which was covered with PVC tubing for protection of the outer part of the cannula. Samples of blood up to 250 µl could be taken at serial time points. The blood volume in the circulation was maintained by replacement with an equal volume of blood obtained from donor animals. The applicability of this method of blood sampling for pharmacokinetic purposes was validated by comparing plasma concentrations–time curves in six cannulated animals after receiving an intravenous bolus dose of 10 mg/kg of the anti-cancer agent docetaxel versus the results in plasma samples obtained by cardiac puncture of non-cannulated mice. The presented method may lead to improved pharmacokinetic data produced from a reduced number of mice.

Trabectedin (ET-743, Yondelis™) is a substrate for P-glycoprotein, but only high expression of P-glycoprotein confers the multidrug resistance phenotype

SummaryTrabectedin (ET-743, Yondelis™) is a novel anticancer drug currently undergoing phase II and III investigations. There are various and conflicting reports whether trabectedin is a substrate for P-glycoprotein (P-gp), an important factor in drug disposition and multi-drug resistance (MDR). We have now unambiguously shown that trabectedin is a P-gp substrate by investigating vectorial transport over monolayers of LLC-PK1 pig kidney and Madine-Darby Canine kidney (MDCK) cells and the mdr1a and/or MDR1 transfected subclones. We further characterized the cytotoxic effects and cellular accumulation of trabectedin in these cell lines as well as in a panel of other cell lines with high or moderate expression levels of P-gp. Trabectedin displayed the typical MDR phenotype only in highly P-gp expressing cell lines, but not in cell lines with expression levels more closely conforming to clinical samples, suggesting that P-gp will not confer resistance to trabectedin in cancer patients.

Determination of cyclosporin A in human and mouse plasma by reversed-phase high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 227 nm has been validated for the determination of cyclosporin A in human and mouse plasma. The cyclosporin D analog PSC 833 was used as internal standard. Plasma samples were pretreated by liquid-liquid extraction with diethyl ether. A good chromatographic separation between cyclosporin A, the internal standard and two potentially interfering endogenous peaks was achieved using a stainless steel column packed with 5 microm Nova-Pak phenyl material operated at 72 degrees C, and a mobile phase consisting of acetonitrile-methanol-water (20:52:28, v/v/v). The calibration curve for cyclosporin A in human plasma was linear over the tested concentration range of 0.11 to 5.34 microM. Murine plasma samples (200 microl) were diluted up to a total volume of 500 microl with blank human plasma and the concentrations were read from the calibration curve prepared in human plasma. The lower limit of quantitation was 0.11 microM using 500 microl of human plasma and 0.28 microM using 200 microl of mouse plasma. The validation data showed that the assay is sensitive, selective and reproducible for determination of cyclosporin A. The applicability was demonstrated in a pharmacokinetic experiment where mice received oral cyclosporin A.