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Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

There are virtually no analytical methods that describe determination of vinblastine and other vinca alkaloids in tumour tissue, albeit quantitative data on tumour drug amount is essential for maximal benefit of a particular anticancer treatment. The analytical method presented herein uses state-of-the-art sample preparation, separation and detection techniques to allow sensitive and selective determination of vinblastine in tumour tissue. After cryogenic grinding and sonication, tumour suspensions were extracted by Oasis MAX solid phase extraction and analysed for vinblastine with ultra-high performance liquid chromatography coupled to positive electrospray ionisation-high resolution mass spectrometric detection. The developed analytical method quantifies vinblastine down to 23 ng/g tumour tissue and shows satisfactory linearity (r(2)>0.99), precision (1.1-8.2%), accuracy (98%) and high selectivity with almost complete absence of matrix effects. The proposed method was found suitable to follow vinblastine levels in mice tumours and could be used to support preclinical pharmacologic studies., (© 2013 Elsevier B.V. All rights reserved.)

Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate.

Vinorelbine is a semi-synthetic vinca alkaloid with demonstrated high activities against various types of advanced cancer. To support a clinical pharmacokinetic study, a simple, rapid and sensitive method to determine vinorelbine in human plasma was developed using reversed phase liquid chromatography (LC) coupled with electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). Vinorelbine and vinblastine (the internal standard) were extracted from human plasma by one-step liquid-liquid extraction (LLE) with methyl-t-butyl ether. The chromatographic separation was achieved on a Spursil polar-modified C(18) column (50 mm×2.1 mm, 3 μm, Dikma Technologies) with an isocratic mobile phase of a 75:25 (v/v) acetonitrile-4 mmol/L ammonium formate (pH 3.0) mixture at a flow-rate of 0.4 mL/min. The MS/MS detection was performed in the positive ion multiple reaction monitoring (MRM) mode by monitoring the precursor→product ion transitions at m/z 779.4→122.0 and m/z 811.3→224.2 for vinorelbine and the internal standard, respectively. The assay was validated in the range 0.1-200 ng/mL (r>0.997), the lowest level of this range being the lower limit of quantification (LLOQ) based on 50 μL of plasma. The intra- and inter-day precisions were within 6.0%, while the accuracy was within ±4.7% of nominal values. Detection and quantification of both analytes within 2 min make this method suitable for high-throughput analyses. The method was successfully applied to evaluate the systemic pharmacokinetics of vinorelbine after a 20-min intravenous infusion of 25 mg/m(2) of vinorelbine to patients with metastatic breast cancer., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this work is to develop a highly sensitive assay system for P-gp-mediated transport by using two methods, induction of P-gp and short-term culture of Caco-2 cells. To induce P-gp in Caco-2 cells, cells were cultured in vinblastine-containing medium. The mRNA level of P-gp was approximately 7-fold higher in Caco-2 cells cultured with vinblastine (P-gp-induced Caco-2 cells) than in control cells. Western blot analysis showed a significant increase in P-gp expression. After cell differentiation, the mRNA level of P-gp was downregulated, however, P-gp-induced Caco-2 cells still possessed a 5.6-fold higher mRNA level of P-gp compared to control cells. Polarized transport of substrate drugs was greater in the monolayer of P-gp-induced cells than in that of control cells. Moreover, we found that P-gp expression in Caco-2 cells could be further enhanced by applying the higher concentration of vinblastine. Transport activity of P-gp in Caco-2 cells cultured with higher concentration of vinblastine was markedly higher than that in P-gp-induced Caco-2 cells and was comparable with that in MDR1-MDCKII cells. In conclusion, this study provided a stable and highly sensitive in vitro assay system that can identify compounds that are subject to P-gp-mediated efflux.

This phase I study assessed the safety, pharmacokinetics, and efficacy of gefitinib (IRESSA) combined with vinorelbine or vinorelbine/cisplatin in chemotherapy-naive patients with advanced non-small cell lung cancer (NSCLC). Patients received gefitinib 250 mg/day and vinorelbine (group A; n = 6) or vinorelbine/cisplatin (group B; n = 8). An additional set of group B patients (n = 9) received gefitinib 500 mg/day with vinorelbine/cisplatin. Adverse events were consistent with individual treatments of gefitinib (mild reversible rash, diarrhea) and chemotherapy (asthenia, fever, nausea, vomiting, constipation), although there was a higher than expected incidence of Common Toxicity Criteria grade 3 or 4 hematologic adverse events, specifically febrile neutropenia and neutropenia. Pharmacokinetic data suggested that neither of the chemotherapy regimens affected steady-state exposure to gefitinib and also that steady-state gefitinib did not alter exposure to vinorelbine or cisplatin. Objective, durable antitumor activity was observed: five partial responses (one in group A; four in group B) and six patients with stable disease (all in group B). The safety data demonstrated that gefitinib with vinorelbine or vinorelbine/cisplatin resulted in severe myelosuppression leading to an unacceptable rate of febrile neutropenia. This study does not support the concurrent administration of gefitinib and vinorelbine, with or without cisplatin, as a valid treatment for advanced NSCLC.

The influence of P-glycoprotein (P-gp) on intestinal absorption of drugs was investigated by comparison of the uptakes of two P-gp substrates, verapamil and vinblastine, using intestinal segments of wild-type and mdr1a/1b gene-deficient (mdr1a/1b(-/-)) mice, and Caco-2 cells. When [(3)H]vinblastine was injected into intestinal segments of wild-type mice, vinblastine was absorbed from duodenum and ileum, but not from jejunum. This difference among intestinal regions could not be explained by segmental differences of mdr1a mRNA expression. In Caco-2 cells, it was found that vinblastine had a high value of efflux/influx ratio (an index of affinity for P-gp) of 12.1, and a low permeability of less than 1 x 10(-6) cm/sec. The corresponding values for verapamil were 4.9 and 10.6 x 10(-6) cm/sec, respectively. After oral administration of [(3)H]vinblastine to mice, the maximum concentration (C(max)) and the area under the plasma concentration time-curve from time 0 to 24 hr (AUC(0-24 hr)) for mdr1a/1b(-/-) mice were 1.5 times greater than those for wild-type mice, while these parameters were not significantly different between the two strains in the case of [(3)H]verapamil. Therefore, P-gp substrates may be classified into at least two types, i.e., verapamil-type, for which the intestinal absorption is unaffected by P-gp, and vinblastine-type, for which the intestinal absorption is influenced by P-gp. Vinblastine-type P-gp substrates, with low permeability and high affinity for P-gp, would be unfavorable candidates for oral drugs.

Vinorelbine (VRL) is a particularly lipophilic member of the vinca alkaloids which, as a class of drugs, exhibit improved cytotoxicity and therapeutic activity through increased duration of exposure. Here, we describe and optimize a sphingomyelin/cholesterol (SM/Chol) liposome formulation of VRL to maximize in vivo drug retention, plasma circulation time, and therapeutic activity. VRL was efficiently encapsulated (>90%) into 100 nm liposomes using an ionophore-mediated loading method. VRL retention in SM/Chol liposomes after intravenous injection in mice was dependent on drug-to-lipid ratio (D/L), with higher D/L ratios exhibiting increased drug retention (0.3 > 0.2 > 0.1, wt/wt) and improved pharmacokinetics. Cryo-electron microscopic examination of a high D/L ratio formulation indicated that the intravesicular regions of these liposomes were electron dense compared with empty liposomes. The optimized, high D/L ratio SM/Chol VRL formulation showed promising activity against subcutaneous B16 melanoma tumors compared with VRL or SM/Chol formulations of vincristine or vinblastine. Finally, the stability of the formulation was excellent (<5% drug leakage, >99% intact VRL, no changes in liposome size after 1 year at 2-8 degrees C). The optimized drug retention properties of the SM/Chol formulation of VRL, combined with its promising antitumor activity and pharmaceutical stability, make this formulation an excellent candidate for future clinical development., (Copyright 2005 Wiley-Liss, Inc)

A vinblastine resistant cell line, KCVB2, was established by co-selecting the parental erythroleukemic cell line K562 with step-wise increased concentrations of vinblastine (Velban) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KCVB2 cells are 8-fold resistant to the selecting agent, vinblastine, but also exhibit significant resistance to other vinca alkaloids, including 14-fold resistance to vinorelbine, as well as 6-fold cross-resistance to paclitaxel. Doubling time and morphology were similar to the parental K562 cells. Rt-PCR analysis revealed no alterations in the expression of the mdr1 and MRP genes. Intracellular vinblastine accumulation was unchanged. Disruption of the mitotic spindles and multiple mitotic asters occurred in both cell lines but required higher concentrations of vinblastine in KCVB2 cells than in K562 cells. Significant differences were observed in the tubulin content of KCVB2 cells: reduction of total tubulin content, increased polymerized fraction of total tubulin, and overexpression of class III beta-tubulin which is expressed at very low levels in the parental K562 cells. K562 cells transfected with murine class III beta-tubulin did not display the resistance pattern observed in KCVB2 cells. Revertants of KCVB2 manifested reversion to parental drug sensitivity, an increase in total tubulin level, and a decrease in polymerized tubulin. In conclusion, the KCVB2 cell line displays a novel mechanism of resistance to both depolymerizing and stabilizing microtubule-targeted cytotoxins which does not involve altered cellular drug accumulation, but corresponds to alterations in the total tubulin content and polymerization status, and may involve an effect on microtubule dynamics.

The majority of cancer-related deaths are attributed to lung carcinoma. Age increases this incidence, which is also likely associated with physiologic modifications that affect drug pharmacokinetics and metabolism. Therefore, knowledge of pharmacokinetics in elderly patients is one of the major factors in deciding whether or not to reduce the dose to prevent toxicity. This phase II study was aimed at evaluating the influence of age on oral vinorelbine pharmacokinetics in elderly patients with non-small-cell lung cancer (NSCLC). Inclusion criteria were > 70 years of age; histologically or cytologically proven NSCLC; inoperable stage IIIB, IV, or delayed relapse of any stage becoming unresectable; Karnofsky performance status > 80%; and normal hematology and biochemistry. Blood-limited sampling at intervals of 1.5, 3, and 24 hours after dosing was performed during the first administration of oral vinorelbine at 60 mg/m2. Bayesian pharmacokinetic parameters were calculated through previously published nonlinear mixed-effect modeling (NONMEM), and compared with a reference population of 52 patients (age, 56 years 12) selected from vinorelbine pharmacokinetic database. There were 48 patients evaluable for pharmacokinetics out of the 52 elderly patients enrolled (age, 74 years 3). There was no difference between pharmacokinetic parameters, including the bioavailability factor evaluated by NONMEM, even without intravenous administration, and a similar interindividual variability (32%-33%) was observed between the 2 groups. Furthermore, no correlation between age (range, 31-82 years) and oral vinorelbine total clearance was observed in 100 patients pooled together. Therefore, no requirement for oral vinorelbine dose reduction was suggested from a pharmacokinetic standpoint.

Background: Vinflunine is a novel vinca alkaloid obtained by semi-synthesis using super-acidic chemistry to selectively introduce two fluorine atoms at the 20' position of vinorelbine. In human tumour xenografts, vinflunine showed definite antitumour activity in seven out of 11 tumours tested compared with three out of 11 for vinorelbine., Patients and Methods: In this phase I study, vinflunine was administered to 31 patients with advanced malignancies as a 10-min i.v. infusion every 3 weeks according to an escalating schedule of doses between 30 and 400 mg/m(2)., Results: Pharmacokinetic parameters and toxicities were assessed and, at 400 mg/m(2), three out of five patients experienced dose-limiting toxicity. At the maximum tolerated dose (MTD), i.e. 400 mg/m(2), the toxicity profile of vinflunine consisted mainly of mucositis, constipation and neutropenia of short duration. Vinflunine area under the curve increased as a proportion of the administered dose whereas no saturation of elimination was observed., Conclusion: The MTD of vinflunine was achieved at 400 mg/m(2) every 3 weeks. According to protocol rules, the recommended dose was established at 350 mg/m(2). A preliminary assessment of first patients included in early phase II trials led to reduction of the recommended dose to 320 mg/m(2) every 3 weeks for further development of vinflunine. Three partial responses (two in breast carcinoma, one in renal cell carcinoma) suggest that activity is likely to be seen in less heavily pretreated patient populations.

Background: A phase I dose-escalation study of a new formulation of oral vinorelbine was conducted to determine the maximum tolerated dose (MTD) of a once weekly regimen and preliminary pharmacokinetic profile in patients with advanced breast cancer (ABC). Twenty-six patients were treated at dose levels ranging from 60 to 100 mg/m2/week. Pharmacokinetics was assessed during the first administration., Patients and Methods: All patients had histologically confirmed locally advanced or metastatic breast cancer and had received no more than two prior chemotherapy regimens for ABC., Results: The MTD was 100 mg/m2/week due to the occurrence of dose-limiting neutropenia, nausea/vomiting and constipation in five of six patients. Toxicities at 80 mg/m2/week were manageable, neutropenia being the main toxicity (grade 3-4 seen in 10 of 13 patients). Nausea, vomiting and diarrhoea were common but rarely severe. Vinorelbine was rapidly absorbed with maximum blood concentration (Cmax) of 103.8 +/- 41.6 ng x ml(-1) observed 1.2 +/- 0.8 hours (Tmax) after administration of 80 mg/m2. Pharmacokinetic exposure increased linearly with dose. Area under the concentration-time curve (AUC) and concentration measured 24 hours after drug intake (C24h) were significantly correlated with depletion of neutrophils. Objective tumour responses were reported in 6 of the 14 evaluable patients treated at doses > or = 80 mg/m2/week., Conclusion: The safety profile of oral vinorelbine appears comparable to that of intravenous dosing. The recommended phase II dose is 80 mg/m2/week and requires regular monitoring of neutrophil counts.

Background: Vinorelbine is a vinca alkaloid obtained by hemisynthesis, which makes the molecule more lipophilic than the other vincas. An injectable formulation is already marketed for the treatment of non small cell lung cancer (NSCLC) and advanced breast cancer (ABC). A new oral form has been developed and its file registration is being submitted. As part of its development, a clinical study was conducted to determine the absolute bioavailability and pharmacokinetics of oral vinorelbine administered as softgel capsules, and to evaluate its safety profile compared with intravenous administration., Patients and Methods: Thirty-two patients with solid tumours were included in the study. Patients fasted and were randomised to receive vinorelbine on day 1, either as a 20 minute intravenous (i.v.) infusion of 25 mg/m2 or as softgel capsules at a dose of 80 mg/m2. Patients were treated with the alternate route after a one week wash-out period. Blood and urine samples for pharmacokinetic analysis were collected during each vinorelbine administration. Safety was assessed after each administration using the CALGB/expanded CTC classification., Results: Twenty-four patients were eligible for pharmacokinetic evaluation. Oral vinorelbine was rapidly absorbed at 80 mg/m2 (Tmax 1.4 +/- 0.7 h) and showed a bioavailability of 43 +/- 14, and close to 40% based on AUC(last) and AUC(inf), respectively. A bioequivalence analysis was conducted on dosage-normalised blood exposures. Equivalence was demonstrated between 80 mg/m2 oral and 30 mg/m2 i.v., and between 60 mg/m2 oral and 25 mg/m2 i.v. The inter-individual variability was equivalent for both routes (CV: 38% and 39% for oral and i.v., respectively). A correlation was found in both methods between AUClast and % nadir variation in white blood cells (WBC) and polymorphonuclears (PMN). More cases of neutropenia (all grades pooled), leucopenia (grades 3-4 only) and nausea (grades 2-3) were induced by 80 mg/m2 oral vinorelbine than by 25 mg/m2 i.v. The greatest intensity of these effects, following oral administration, probably reflects the higher, observed drug exposure., Conclusion: At therapeutic dosage levels, pharmacokinetic behaviour and safety profiles were similar for both routes. The absolute bioavailability of the oral vinorelbine (new, soft gelatine capsule) was close to 40%. Inter-individual variability in drug exposure was equivalent in both routes. The pharmacokinetic/pharmacodynamic (PK/PD) relationship in haematological toxicity was independent of the routes of administration. Reliable, corresponding doses between oral and i.v. vinorelbine were established, which will result in bioequivalent AUC.

We studied possible pharmacokinetic interactions between docetaxel (DTX) and vinorelbine (VNR) in patients affected by different types of cancer. Patients with metastatic breast cancer or recurrent head and neck cancer received the following schedules: Protocol A: 11 patients were i.v. infused for 1 h with DTX (80 mg/m2) at once, followed by VNR (25 mg/m2) as slow i.v. bolus; Protocol B: VNR (25 mg/m2) as a slow 10 min i.v. bolus was administered to 12 patients, immediately followed by 1 h i.v. infusion of DTX (80 mg/m2). In both schedules, VNR and DTX plasma concentrations versus time were analysed by HPLC obtaining the corresponding non-compartmental pharmacokinetic parameters. VNR appeared pharmacokinetically affected by the sequential administration of DTX, since with protocol B, Cmax and AUC were significantly higher and clearance lower than in protocol A. Moreover, a significant increase in the VNR plasma level was observed in correspondence with the peak plasma level of DTX. By contrast, Cmax, AUC and clearance of DTX did not vary in the two protocols. Also the number of neutrophils at nadir on day 8 of treatment varied significantly in the two schedules. In conclusion we observed altered pharmacokinetic parameters between protocol A (DTX, VNR) and protocol B (VNR/DTX). In particular, patients following protocol B seemed to be exposed to higher VNR plasma concentration and to higher haematological toxicity.

Background and Purpose: Anthracycline-containing regimens are widely used in advanced breast cancer. However, there is a need for new, non-anthracycline regimens that are active in patients for whom anthracyclines are contraindicated. The aim of this study was to determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs) and recommended doses of docetaxel and vinorelbine as first-line chemotherapy in patients with metastatic breast cancer. The pharmacokinetics of both drugs was also evaluated., Patients and Methods: Thirty-four women with first-line metastatic breast cancer were treated with docetaxel, 60-100 mg/m2 (day 1), and vinorelbine, 20-22.5 mg/m2 (days 1 and 5), repeated every three weeks and administered on an outpatient basis., Results: Two MTDs were determined: MTD1 was defined at the dose level using docetaxel 75 mg/m2, and vinorelbine 22.5 mg/m2 DLT being a grade 3 stomatitis that was more related to the dose of vinorelbine than that of docetaxel. Therefore, the study continued with a fixed dose of vinorelbine, 20 mg/m2, and docetaxel 85-100 mg/m2. MTD2 was defined at the dose level combining docetaxel, 100 mg/m2, and vinorelbine, 20 mg/m2; DLTs were grade 3 stomatitis and severe asthenia. Fluid retention was observed in 41% of patients but was never severe or a reason for patient discontinuation. In comparison with historical experience, Daflon 500 did not seem to increase the efficacy of the three-day corticosteroid premedication by further reducing the incidence or severity of fluid retention. No significant neurotoxicity was observed and no patient discontinued the study due to this site effect. Activity was observed at all dose levels and at all metastatic sites, with an overall response rate of 71% (95% CI: 52.0%-85.8%). The median time to progression was 31.4 weeks (95% CI: 12-48 weeks) and median survival was 15.6 months (95% CI: 2.6-26.6 months). The pharmacokinetics of docetaxel and vinorelbine were not modified between day 1 and day 3 when the two drugs were combined with the day 1 administration schedule used in this study., Conclusion: The recommended doses for phase II studies are docetaxel, 75 mg/m2 (day 1), plus vinorelbine, 20 mg/m2 (days 1 and 5), repeated every three weeks. At these doses, the combination was found to be active and well tolerated.

Combinatorial synthesis and high-throughput pharmacology screening have greatly increased compound throughput in modern drug-discovery programs. For CNS drugs, it is also important to determine permeability to the blood--brain barrier. Yet, given the increased pace of discovery, it difficult to conduct this screen in a timely fashion. In this presentation, we describe several improvements to an existing CNS permeability screen, the bovine brain microvessel endothelial cell (BBMEC) model. By implementation of these incremental process improvements, we have achieved a robust, facile screen for determination of CNS permeability of multiple compounds.

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid-liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250x4.6 mm I.D.) packed with 5 microm diameter particles as the stationary phase and a mobile phase of acetonitrile-80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m2 of this drug in patients with metastatic cancer.

Background: Vinorelbine and Doxil (liposomal doxorubicin) are active chemotherapeutic agents in metastatic breast cancer. A phase I study was designed to evaluate combination therapy., Patients and Methods: Thirty women with metastatic breast cancer were enrolled. Dose-limiting toxicity was determined through a dose escalation scheme, and defined for the first treatment cycle, only. Pharmacokinetic studies were performed during the first cycle of treatment., Results: In the first cohort of Doxil 30 mg/m2 day 1 and vinorelbine 25 mg/m2 days 1 and 8, patients experienced severe neutropenia. Vinorelbine administration was changed thereafter to days 1 and 15 of each cycle. Dose limiting toxicity was observed at Doxil 50 mg/m2 and vinorelbine 25 mg/m2. Doxil 40 mg/m2 and vinorelbine 30 mg/m2 was defined as the maximally tolerated dose. Few toxicities (principally neutro penia) were seen at this dose level, with the notable absence of significant nausea, vomiting, or alopecia. Though 63% of patients had received prior anthracycline-based chemotherapy, only one patient developed grade 2 cardiac toxicity. Pharmacokinetic studies revealed prolonged exposure to high doxorubicin concentrations for several days following Doxil administration., Conclusions: Combination chemotherapy with Doxil and vinorelbine affords treatment with two active drugs in women with metastatic breast cancer, and appears to have a favorable toxicity profile. A schedule of Doxil 40 mg/m2 day 1 and vinorelbine 30 mg/m2 days 1 and 15 given every 28 days is recommended for phase II studies.

The intracellular concentration of many steroids and xenobiotics is influenced by the membrane protein P-glycoprotein (Pgp). It has been inferred that the intracellular retention of many drugs that upregulate Pgp or modulate Pgp function might also be affected by Pgp. However, the ability of Pgp to influence the translocation of these drugs needs to be established to understand Pgp's influence upon their pharmacological effect. We utilized two approaches to determine the interaction of several agents with Pgp: (a) an in vitro system, LLC-PK1 cell lines and derivative LLC cell lines stably expressing on the apical membrane either mouse mdr1a or human MDR1 Pgp grown as polarized epithelium in transwell culture to measure translocation of radiolabeled drugs; and (b) an in vivo system, mdr1a nullizygous and wild-type animals, to compare the contribution of Pgp to in vivo distribution of radiolabeled drugs. In combination these complementary approaches identified erythromycin as a drug whose intracellular retention is influenced by Pgp, while the intracellular accumulation and tissue distribution of retinoic acid and benzo(a)pyrene were unaffected by Pgp., (Copyright 1998 Academic Press.)

MRP is a member of the ABC trafficking proteins thought to mediate the transport of glutathione S-conjugates and amphiphilic natural products. However, unlike P-glycoprotein, the biochemical mechanism by which MRP mediates the resistance to cytotoxic drugs is not clear. In this report, we describe the interactions of a quinoline-based drug, N-{4-[1-hydroxy-2-(dibutylamino)ethyl] quinolin-8-yl}-4-azidosalicylamide (IAAQ), with MRP. Our results demonstrate the ability of IAAQ to photoaffinity label a 190 kDa protein in resistant Small Cell Lung Cancer cells (H69/AR) but not in the parental H69 cells. The photoaffinity labeling of the 190 kDa protein with IAAQ was both saturable and specific. The identity of the 190 kDa protein, as MRP, was confirmed by immunoprecipitation with the monoclonal antibody, QCRL-1. Furthermore, a molar excess of LTC4, MK 571 or vinblastine inhibited the photoaffinity labeling of MRP with IAAQ in intact cells and plasma membranes. Cell growth and drug transport studies showed H69/AR cells to be less sensitive to and to accumulate less IAAQ than the parental H69 cells. In addition, MK 571 and doxorubicin increased the sensitivity to and the accumulation of IAAQ in H69/AR cells. Together, the results of this study show for the first time the direct binding of unaltered cytotoxic drug to MRP. Moreover, given the structural similarities between IAAQ and MK 571, we suggest that MK 571 modulates MRP-mediated resistance by direct binding to MRP., (Copyright 1997 Academic Press.)

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of vinorelbine in plasma is described. The technique was derived from that published by Debal for an assay of vinorelbine in cell culture medium. The modifications concern the preparation procedure for plasma samples (a two-step liquid-liquid extraction from plasma is described), optimization of the mobile phase composition, and use of a single C18 column. These changes resulted in an improved sensitivity and reproducibility of the assay and led to its feasibility for clinical pharmacokinetic studies. The range of the assay is 2 to 1000 ng/ml.

A high-performance liquid chromatographic method was developed for the determination in plasma (400-microliters sample) of a vinca alkaloid, vinorelbine. The analysis was performed by using an octadecylsilane column and heptanesulfonic acid as ion-pairing agent. This method used a new internal standard, teniposide, that permitted a good compromise between sensitivity and retention times (10.6 and 15.5 min for teniposide and vinorelbine, respectively). After a liquid-liquid extraction with diethyl ether, the extracts were injected into a reversed-phase system. The extraction efficiency was approximately 80% for both vinorelbine and the internal standard. The mobile phase was phosphate buffer (pH 3)-acetonitrile-methanol (50:30:20, v/v/v). Using coulometric detection, the limit of detection in plasma (400 microliters) was 1 ng/ml. The intra-assay coefficients of variation were 10.95, 3.80 and 5.71% for 5, 500 and 1000 ng/ml, respectively, and the inter-assay coefficients of variation were 20.14, 14.27 and 10.67% for 5, 500 and 1000 ng/ml, respectively. A linear response was observed for the plasma calibration graph in the ranges 2.5-50 and 50-1000 ng/ml. This method was used to follow the time course of the concentration of vinorelbine in rabbit plasma after a single intravenous dose of vinorelbine (30 mg/m2) and seems to be suitable for studying the pharmacokinetics of vinorelbine in rabbit.

A human liver plasma membrane model for the evaluation of the specific binding and transport processes of drugs presenting high hepatic clearance such as vinca alkaloids was developed. Uptake of the two structural antitumor analogs, navelbine (NVB) and vincristine (VCR), which exhibit wide variabilities in their respective pharmacokinetic parameters and antitumor spectra, was investigated. The high yield, the enzymatic profile and the retention of physiologic transport capacities, as demonstrated by taurocholate uptake, revealed that this membrane preparation was well suited for studies of hepatic drug transport systems. For both drugs two distinct processes were observed: mainly membrane binding and transport. NVB was found to bind to the membrane vesicles more intensively than VCR, but the transport processes were almost identical. However only NVB uptake seems to involve Na(+)-dependent processes. These significant differences may be related to the respective lipophilicity of the drugs. The more lipophilic molecule (NVB) presents the highest uptake, which is presumably at the origin of its greatest distribution volume in vivo.

A high-performance liquid chromatographic method is described for the determination of vinblastine in various normal mouse tissues, such as lung, heart, liver, kidney and muscles, and in implanted MO4 tumours. Vincristine was used as the internal standard. Freshly obtained mouse tissue or tumour tissue was frozen at -20 degrees C and then lyophilized. After lyophilisation, the dry tissues were pulverized and homogeneously mixed, and an aliquot was suspended in 0.1 M hydrochloric acid. The drugs of interest were then isolated from this suspension using ion-pair extraction at pH 3 with octylsulphate as counter-ion. The obtained extracts were analysed on a reversed-phase system with a cyanopropyl stationary phase. The detection limit was 1 ng/l in plasma and 10 ng/g in tissue. The extraction recoveries of vincristine and vinblastine were between 45 and 67%, and there were no interferences from blank components. Preliminary pharmacokinetic data for different mouse tissues and tumour implanted in muscle tissue are presented.

A high-performance liquid chromatographic method is described for separating and determining navelbine and possible metabolites in plasma, cell culture medium and MO4 cells. Navelbine is extracted from these fluids by ion-pair extraction with sodium octylsulphate as the counter-ion at pH 3. The system uses a cyano column as the stationary phase and a mobile phase of acetonitrile-0.12 M phosphate buffer (pH 3) (60:40, v/v). Application of the method to a study of the pharmacokinetic behaviour of navelbine in MO4 mouse fibrosarcoma cells is reported.

A high-performance liquid chromatographic procedure, including sample pretreatment, is presented for the analysis of the experimental antitumour drug vintriptol in plasma. The sample pretreatment involved liquid-liquid extraction of the buffered (pH 3) sample with chloroform. Vinblastine was used as internal standard. Separation was achieved on a Hypersil ODS (5 microns) column with a mobile phase of acetonitrile-phosphate buffer. Electrochemical detection (at +0.70 V) was used, giving a detection limit of 2 micrograms/l. The applicability of the assay was demonstrated in a pharmacokinetic study with eight cancer patients who received 45 or 50 mg/m2 vintriptol in a phase I study. A three-compartment model was used to fit the plasma concentration-time curves. Pharmacokinetic parameters are presented.

Treatment of drug-resistant human KB carcinoma cells (KB-V1) with 0.2 microM phorbol 12-myristate 13-acetate (PMA) resulted in increases of 4-fold in both membrane-associated protein kinase C activity and phosphorylation of P-glycoprotein. The response was essentially complete after 30 min and was relatively stable, since both of these parameters remained elevated above basal levels in cells exposed to PMA for 24 hours. In contrast, long-term PMA treatment of drug-sensitive KB-3 cells caused complete depletion of protein kinase C. The rate of accumulation of [3H]vinblastine in KB-V1 cells was 0.8 +/- 0.1 pmol/mg/30 min in the absence, and 1.9 +/- 0.2 pmol/mg/30 min in the presence, of 20 microM verapamil. Preincubation of cells with PMA resulted in a time-dependent decrease, up to 60% after 24 hours, in both of these values. These results suggest that protein kinase C mediated phosphorylation stimulates the drug transport activity of P-glycoprotein.

One major feature of multidrug resistance is the reduced cellular level of drugs maintained by MDR cells. Although there is now strong evidence that drugs are actively pumped out of MDR cells, transport experiments have indicated decreased initial rates of influx at the earliest times at which measurements could be made. We have used a pulsed quench-flow apparatus to study transport characteristics of colchicine resistant MDR cells on a very fast time scale. A rapid association of daunomycin with drug sensitive cells occurred within 0.11 sec. This association is virtually absent in MDR cells. In efflux experiments performed on the same rapid time scale, greater than 50% of daunomycin efflux occurred within 0.1 sec. No substantial efflux from B1, drug sensitive cells was observed. On the other hand, vinblastine accumulation by both cell types was similar for approx. 10 seconds. Thus, kinetically, not all drugs are handled in a similar fashion by MDR cells. The pulsed quench-flow apparatus was useful in making fast time measurements of drug influx and efflux and in demonstrating the differences between drug recognition patterns by MDR cells.

Navelbine (NVB) (5'-noranhydrovinblastine) is a new semi-synthetic vincaalkaloid (VA) exhibiting a high affinity for tubulin and considerable anticancer activity in patients. A better hematologic tolerance and a weak neurotoxicity have been reported for this drug as compared to other VA. Moreover, NVB presents a relatively high bioavailability and a good digestive tolerance, thus offering original perspectives for the treatment of ambulatory cancer patients. A clinical pharmacokinetic study of NVB was carried out on 12 patients after oral administration of the drug. The pharmacokinetic parameters were similar to those of intravenous administration and also showed a high interindividual variability. Studies on the in vitro metabolism of NVB using hepatic microsomal fractions from 22 different donors demonstrated the formation of 3 metabolites. The biotransformation rate quantitatively varies from one human liver specimen to another, a fact which could be, in part, at the origin of the interindividual variability of the therapeutic response.

The reversed-phase high-performance liquid chromatography with electrochemical detection was used to quantify plasma and urine levels of vinblastine, vincristine, vindesine and a metabolite of vinblastine, desacetylvinblastine. Sample clean-up consisted of solid-phase extraction with a Bond Elut CN column. The extracts were separated on a Hypersil ODS column. The mobile phase consisted of a mixture of methanol and 10 mM phosphate buffer (pH 7.0). The limit of sensitivity using electrochemical detection was 100 pg on-column for all compounds with a signal-to-noise ratio of 3. Quantification of the compounds in human plasma and urine was possible down to 1 ng/ml (ca. 1 pmol). Pharmacokinetic results show that the sensitivity of the method is adequate for drug monitoring in clinical research.

Vinca alkaloids (VA) represent a family of closely related molecules, which includes vincristine (VCR), vinblastine (VLB), vindesine (VDS) and navelbine (NVB), a new synthetic VA presently in phase II clinical trial. Development of sensitive and specific analytical tools (polyclonal and monoclonal antibodies) enabled us to investigate the kinetic behavior of VDS and NVB after IV bolus or long term administration. Following IV bolus injection, the mean pharmacokinetic parameters are: total plasma clearance: 0.53 l/h-1kg-1, 0.72 l/h-1kg-1 and apparent elimination half-life: 23.2 h, 39.5 h for VDS and NVB, respectively. Chronic treatment reveals time- and dose-dependence relationships and detailed observations of individual kinetics demonstrate an important interindividual variability for both drugs. Renal excretion of VDS and NVB is low (from 5 to 12% of the total dose), suggesting the important role of the liver in their rapid elimination.