Better characterization of vinflunine pharmacokinetics variability and exposure/toxicity relationship to improve its use: Analyses from 18 trials.

AIMS: Vinflunine is a novel tubulin-targeted inhibitor indicated as a single agent for the treatment of bladder cancers after failure of prior platinum-based therapy. Its pharmacokinetics (PK) and pharmacodynamics (PD) have been independently characterized through several phase I and phase II studies. However, no global pharmacometric analysis had been conducted as yet. METHODS: Vinflunine concentrations and safety data from 18 phase I and phase II studies were used to conduct population PK and PK/PD analysis, using Nonmem. A four-compartment model was used to describe vinflunine PK and several covariates were tested to explain interindividual variability. In terms of PK/PD relationship, a semiphysiological population PK/PD model was applied to describe time course of absolute neutrophil counts (ANC) after vinflunine administration and logistic regression models were used to test the relationship between vinflunine exposure and toxicities. RESULTS: Vinflunine clearance is explained by creatinine clearance, body surface area and combination with PEGylated doxorubicin, leading to a decrease from 28.2 to 25.3% of the interindividual variability. When vinflunine dose is decreased, simulations of ANC time course (via a semiphysiological model) after vinflunine administration show a risk of neutropenia grade 3-4 at cycle 2 always lower than when dose is delayed. As an example, for moderate renal impaired patients, the risk is 42.1% when vinflunine is dosed at 320 mg m-2 once every 4 weeks vs. 23.3% for 280 mg m-2 once every 3 weeks. CONCLUSIONS: We propose for the first time a global comprehensive clinical pharmacological analysis for intravenous vinflunine that may help drive dose adjustment.

A label-free electrochemical biosensor based on tubulin immobilized on gold nanoparticle/glassy carbon electrode for the determination of vinblastine.

Vinblastine (VLB) is prescribed for a wide variety of cancers. Therefore, development of sensitive methods for early diagnosis is urgently required. In this work, a highly sensitive and label-free impedimetric biosensor was fabricated for the electrochemical detection of VLB. First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of a glassy carbon electrode (GCE). 3-Mercaptopropionic acid (MPA) was self-assembled over the AuNPs. Then, tubulin (TUB), as a receptor, was covalently immobilized at the AuNPs/GCE surface via carbodiimide coupling reaction using N-(3 dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) and N-hydroxy succinimide (NHS). The step-by-step modification process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of a redox probe [Fe(CN)6]3-/4-. The VLB concentration was measured through the increase of impedance values in the corresponding specific binding of VLB and TUB. The increased electron-transfer resistance (R et) values were proportional to the value of VLB concentrations in the range of 0.4 to 65.0 nmol L-1 with a detection limit of 8.4 × 10-2 nmol L-1 (SN-1 = 3). The practical analytical performance of the proposed method was demonstrated by determination of VLB in plant extracts and human serum samples with satisfactory recoveries.

Validated UHPLC-MS/MS assay for quantitative determination of etoposide, gemcitabine, vinorelbine and their metabolites in patients with lung cancer.

A fully valid UHPLC-MS/MS method was developed for the determination of etoposide, gemcitabine, vinorelbine and their metabolites (etoposide catechol, 2',2'-difluorodeoxyuridine and 4-O-deacetylvinorelbine) in human plasma. The multiple reaction monitoring mode was performed with an electrospray ionization interface operating in both the positive and negative ion modes per compound. The method required only 100 µL plasma with a one-step simple de-proteinization procedure, and a short run time of 7.5 min per sample. A Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) provided chromatographic separation of analytes using a binary mobile phase gradient (A, 0.1% formic acid in acetonitrile, v/v; B, 0.1% formic acid in water, v/v). Linear coefficients of correlation were >0.995 for all analytes. The relative deviation of this method was <10% for intra- and inter-day assays and the accuracy ranged between 86.35% and 113.44%. The mean extraction recovery and matrix effect of all the analytes were 62.07-105.46% and 93.67-105.87%, respectively. This method was successfully applied to clinical samples from patients with lung cancer.

Liquid chromatography mass spectrometry simultaneous determination of vindoline and catharanthine in rat plasma and its application to a pharmacokinetic study.

Vinblastine and vincristine, both of which are bisindole alkaloids derived from vindoline and catharanthine, have been used for cancer chemotherapy; their monomeric precursor molecules are vindoline and catharanthine. A simple and selective liquid chromatography mass spectrometry method for simultaneous determination of vindoline and catharanthine in rat plasma was developed. Chromatographic separation was achieved on a C18 (2.1 × 50 mm, 3.5 µm) column with acetonitrile-0.1% formic acid in water as mobile phase with gradient elution. The flow rate was set at 0.4 mL/min. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification. Mean recoveries were in the range of 87.3-92.6% for vindoline in rat plasma and 88.5-96.5% for catharanthine. Matrix effects for vindoline and catharanthine were measured to be between 95.3 and 104.7%. Coefficients of variation of intra-day and inter-day precision were both <15%. The accuracy of the method ranged from 93.8 to 108.1%. The method was successfully applied in a pharmacokinetic study of vindoline and catharanthine in rats. The bioavailability of vindoline and catharanthine were 5.4 and 4.7%, respectively.

Rapid and sensitive liquid chromatography coupled with electrospray ionization tandem mass spectrometry method for the analysis of paclitaxel, docetaxel, vinblastine, and vinorelbine in human plasma.

BACKGROUND: A rapid and sensitive analytical method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed for the determination of paclitaxel, docetaxel, vinblastine, and vinorelbine in human plasma. METHODS: A simple liquid-liquid extraction procedure was applied using only 100-µL plasma. Chromatographic separation of these anticancer drugs was achieved with an isocratic mobile phase consisting of acetonitrile/aqueous buffer (10 mmol/L ammonium acetate and 0.1% formic acid in 70:30, vol/vol) at a flow rate of 0.25 mL/min in a short time (4.5 minutes). RESULTS: The calibration curves for paclitaxel, docetaxel, vinblastine, and vinorelbine in spiked human plasma ranged from 25 to 2500, 10 to 1000, 10 to 1000, and 10 to 1000 ng/mL, respectively. The squares of the linear correlation coefficients were all more than 0.99. The intraday and interday relative standard deviations across 3 validation runs over the entire concentration range were less than 9.2%. CONCLUSIONS: The established method should be helpful for the pharmacokinetic monitoring of paclitaxel, docetaxel, vinblastine, and vinorelbine in the human plasma of non-small cell lung cancer patients.

A phase I and pharmacokinetic study of liposomal vinorelbine in patients with advanced solid tumor.

PURPOSE: This phase I study was performed to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of an untargeted liposomal formulation of vinorelbine (NanoVNB®) and to characterize its plasma pharmacokinetics in patients with advanced solid tumors which were refractory to conventional treatment or without an effective treatment. PATIENTS & METHODS: The study incorporated an accelerated titration design. Twenty-two patients with various solid tumors were enrolled. NanoVNB(®) was administered intravenously at doses of 2.2-23 mg/m(2) once every 14 days. Pharmacokinetic endpoints were evaluated in the first cycle. The safety profiles and anti-tumor effects of NanoVNB® were also determined. RESULTS: Skin rash was the DLT and the most common non-hematological toxicity. The MTD was 18.5 mg/m(2). Drug-related grade 3-4 hematological toxicities were infrequent. Compared with intravenous free vinorelbine, NanoVNB® showed a high C(max) and low plasma clearance. Of the 11 patients completing at least 1 post-treatment tumor assessment, 5 had stable disease. No responders were noted. CONCLUSION: NanoVNB® was well tolerated and exhibited more favorable pharmacokinetic profiles than free vinorelbine. Based on dose-limiting skin toxicity, further evaluation of NanoVNB® starting from 18.5 mg/m(2) as a single agent or in combination with other chemotherapeutic agents for vinorelbine-active malignancies is warranted.

A validated LC-MS/MS method for the determination of vinflunine in plasma and its application to pharmacokinetic studies.

A rapid, simple and sensitive LC-MS/MS method for the quantification of vinflunine in plasma was developed and validated. The analysis involved a simple liquid-liquid extraction. After making alkaline with NaOH, plasma was extracted with methyl tert-butyl ether and the organic extract was then evaporated and the residue was reconstituted in mobile phase. The reconstituted solution was injected into an HPLC system and was subjected to reverse-phase HPLC on a 5 µm ODS-3 column at a flow-rate of 0.2 mL/min. The mobile phase consisted of ammonium acetate (0.02 mol/L, pH = 3.0) and acetonitrile (20:80). Vinflunine was detected in the single ion monitoring mode using target ions at m/z 817.4/160.1/142.3 for vinflunine and m/z 447.2/128.3/112.1 for gefitinib (internal standard). Standard curves were linear over the concentration range of 5-1000 ng/mL. The mean predicted concentrations of the quality control samples deviated by less than 2% from the corresponding nominal values; the intra-assay and inter-assay precisions of the assay were within 7% relative standard deviation. The extraction recovery of vinflunine was more than 80%. The validated assay was applied to a pharmacokinetic study of vinflunine in plasma following the administration of a single vinflunine injection (2 mg/kg).

Sulfosalicylate mediates improved vinorelbine loading into LUVs and antineoplastic effects.

Liposomal vinorelbine formulation is desirable, as it might improve the therapeutic activity of vinorelbine. However, because of its lipophilic and membrane-permeable properties, vinorelbine is hard to be formulated into liposomes using conventional drug-loading technologies. To improve vinorelbine retention, ammonium salts of several anionic agents were employed to prepare liposomal vinorelbine formulations. It was found that 5-sulfosalicylate (5ssa) could form stable complexes with vinorelbine and stabilize entrapped vinorelbine. The resultant vesicles had an in vitro release t(1/2) of ~12.49 hours in NH(3)-containing media, which is longer than those of sulfate and phytate vesicles (~0.57 hours). The circulation half-life of vinorelbine after the injection of 5ssa vesicles into normal mice was ~13.01 hours, accounting for ~2-fold increase relative to that of sulfate vesicles. Improved drug retention correlated with enhanced antitumor efficacy. In the RM-1/c57 model, 5ssa vesicles were more efficacious than sulfate vesicles (P < 0.05). In RM-1/BDF1 and Lewis lung cancer/c57 models, antitumor efficacy was also considerably improved after vinorelbine encapsulation into 5ssa vesicles. For instance, in the RM/BDF1 model, liposomal vinorelbine was at least 4-fold more therapeutically active than free vinorelbine. Our results demonstrated that 5ssa could stabilize vinorelbine relative to other anions, resulting in the formulation with improved drug retention and efficacy. Improved vinorelbine retention might be associated with the formation of insoluble precipitate, which could be proved by precipitation study and decreased drug-release rate at a high D/L ratio.

Sensitive measurement of vinorelbine in dog plasma by liquid chromatography-electrospray ionization tandem mass spectrometry utilizing transitions from double-charged precursor ions.

A sensitive high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for measuring vinorelbine was developed. A 100 µL aliquot of plasma was spiked with deuterium-labeled internal standard and subjected to solid-phase extraction using an Oasis HLB µ-elution plate. Two microliters of the extracted samples was directly injected into LC/MS/MS. Chromatographic separation was achieved on a Capcell Pak C18 UG column (2 × 75 mm) with a gradient elution of methanol (mobile phase B) against 0.05% formic acid in aqueous 10 mm ammonium formate (mobile phase A). The LC flow rate was set to 0.28 mL/min and the gradient (solvent B concentration) was processed from 40 to 90%. In mass spectrometric detection, observation of the reaction from a double-charged precursor ion [M + 2H](2+) (m/z 390) to product ion m/z 122 provided very high sensitivity. The method was validated with a lower limit of detection of 0.2 ng/mL with 0.1 mL of plasma, and the method was used to determine the plasma pharmacokinetics of vinorelbine in dogs.

Transplacental transfer of anthracyclines, vinblastine, and 4-hydroxy-cyclophosphamide in a baboon model.

OBJECTIVE: The paucity of data on the fetal effects of prenatal exposure to chemotherapy prompted us to study transplacental transport of chemotherapeutic agents. METHODS: Fluorouracil-epirubicin-cyclophosphamide (FEC) and doxorubicin-bleomycin-vinblastine-dacarbazine (ABVD) were administered to pregnant baboons. At predefined time points over the first 25 h after drug administration, fetal and maternal blood samples, amniotic fluid (AF), urine, fetal and maternal tissues, and cerebrospinal fluid (CSF) were collected. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) were used for bioanalysis of doxorubicin, epirubicin, vinblastine, and cyclophosphamide. RESULTS: In nine baboons, at a median gestational age of 139 days (range, 93-169), FEC 100% (n = 2), FEC 200% (n=1), ABVD 100% (n = 5), and ABVD 200% (n = 1) were administered. The obtained ratios of fetal/maternal drug concentration in the different simultaneously collected samples were used as a measure for transplacental transfer. Fetal plasma concentrations of doxorubicin and epirubicin averaged 7.5 ± 3.2% (n = 6) and 4.0 ± 1.6% (n = 8) of maternal concentrations, respectively. Fetal tissues contained 6.3 ± 7.9% and 8.7 ± 8.1% of maternal tissue concentrations for doxorubicin and epirubicin, respectively. Vinblastine concentrations in fetal plasma averaged 18.5 ± 15.5% (n=9) of maternal concentrations. Anthracyclines and vinblastine were neither detectable in maternal nor in fetal brain/CSF. 4-Hydroxy-cyclophosphamide concentrations in fetal plasma and CSF averaged 25.1 ± 6.3% (n = 3) and 63.0% (n = 1) of the maternal concentrations, respectively. CONCLUSION: This study shows limited fetal exposure after maternal administration of doxorubicin, epirubicin, vinblastine, and 4-hydroxy-cyclophosphamide.

A simple and sensitive high-performance liquid chromatographic method for the determination of vinflunine and 4-O-deacetylvinflunine from human blood.

Vinflunine (VFL) is the first bifluorinated tubulin-targeted agent obtained through a semi synthetic process using superacidic chemistry. Pharmacologic models evidenced a high degree of activity from several cancer lines. The intravenous formulation of VFL (Javlor, Pierre Fabre Medicament, Boulogne, France) is registered for bladder cancer and is undergoing Phase III trials for nonsmall cell lung and breast cancer. To support most of the pharmacokinetic studies in humans, a sensitive high-performance liquid chromatography bioanalytical method coupled with ultraviolet detection was developed and validated for the simultaneous quantification of VFL and its active metabolite 4-O-deacetylvinflunine. The two compounds, together with 17-bromovinorelbine, used as an internal standard, were extracted from blood (1 mL) by a liquid-liquid process under basic conditions using diethyl ether. The organic phase was then back-extracted with HCl 0.1 mol/L. Analysis was performed through a cyano column and detection was set at 268 nm. Total analysis run time was less than 15 minutes. The assay was sensitive for the two compounds to at least 2 ng/mL and calibration curves were linear up to 200 ng/mL. The between-run imprecision and the mean inaccuracy were lower than 7% and 8.3%, respectively. Blood samples were stable when stored at -70°C over 24 months. The long-term reproducibility and the suitability of this analytical method were demonstrated through the analysis of about 6000 biologic samples during the clinical development of intravenous VFL. This method is adequately sensitive to monitor the blood concentrations observed at the recommended dose defined in a clinical setting.

Combination chemotherapy of vinorelbine and cisplatin: a phase I pharmacokinetic study in patients with metastatic solid tumors.

BACKGROUND: Vinorelbine (VRL)-cisplatin (CDDP) is an active doublet for advanced non-small cell lung cancer. CDDP has a narrow therapeutic index and may produce a cumulative nephrotoxicity over the treatment period. This study was to assess the risks of drug-drug interaction (DDI) over 3 consecutive cycles of VRL-CDDP combined treatments. PATIENTS AND METHODS: An open-label, nonrandomised, phase I study was carried out. Patients with normal hepatic/renal functions. D1: CDDP 100 mg/m2--D1, D8: oral VRL 60 mg/m2 q3w. Pharmacokinetics (PK) over the first 3 cycles. PK comparison between cycles and between study vs. literature. RESULTS: Thirteen patients were evaluable for safety and PK. Adverse events were those frequently observed with CDDP or VRL, and consisted of hematological toxicities, nausea, vomiting and constipation. Concerning VRL and CDDP PK, no difference was detected between the 3 administrations nor between the study and reference values. CONCLUSION: The absence of DDI between CDDP and oral VRL was demonstrated over 3 consecutive cycles of therapy.

Rapid and sensitive LC-MS method for pharmacokinetic study of vinorelbine in rats.

A rapid and sensitive LC-electrospray ionization-MS method was developed for determining vinorelbine in rat plasma. A 100 microL plasma sample was treated using a protein precipitation procedure and was chromatographed within 4 min using an Inertsil ODS-3 C(18 )(2.1 x 50 mm, 5 microm) column. The selected ion monitoring ions [M + H](+) were m/z 779 and m/z 811 for vinorelbine and vinblastine (internal standard), respectively. The method validation showed that the calibration curve for vinorelbine was linear over a concentration range of 1-1000 ng/mL with lower limit of quantification at 1 ng/mL. The method has been successfully applied to pharmacokinetics in rat plasma.

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.

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry.

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.

Evaluation of oral versus intravenous dose of vinorelbine to achieve equivalent blood exposures in patients with solid tumours.

Patient's preference is for oral chemotherapy when both oral and i.v. are available, provided that efficacy is equivalent. Reliable switch from oral to i.v. is possible if correspondence between respective doses has been established. Vinorelbine oral was developed as a line extension of VRL i.v. on the basis that similar AUCs result in similar activities. From a first crossover study on 24 patients receiving VRL 25 mg/m2 i.v. and 80 mg/m2 oral data extrapolation concluded on AUCs bioequivalence between Vinorelbine 30 mg/m2 i.v. and 80 mg/m2 oral. A new trial was performed to support this calculation. In a crossover design study on patients (PS 0-1) with advanced solid tumours (44% breast carcinoma), VRL was administered (30 mg/m2 i.v., 80 mg/m2 oral) with a standard meal and 5-HT3 antagonists, at 2 weeks interval. Pharmacokinetics was performed over 168 h and VRL was measured by LC-MS/MS. Statistics included bioequivalence tests. Forty-eight patients were evaluable for PK: median age 58 years (25-71), PS0/PS1: 20/28, M/F: 11/37. Mean AUCs were 1,230 +/- 290 and 1,216 +/- 521 ng/ml for i.v. and oral, respectively. The confidence interval of the AUC ratio (0.83-1.03) was within the required regulatory range (0.8-1.25) and proved the bioequivalence between the two doses. The absolute bioavailability was 37.8 +/- 16.0%, and close to the value from the first study (40%). Patient tolerability was globally comparable between both forms with no significant difference on either haematological or non-haematological toxicities (grade 3-4). This new study, conducted on a larger population, confirmed the reliable dose correspondence previously established between vinorelbine 80 mg/m2 oral and 30 mg/m2 i.v.

Development of a sensitive liquid chromatography method coupled with a tandem mass spectrometric detection for the clinical analysis of vinflunine and 4-O-deacetyl vinflunine in blood, urine and faeces.

A sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and fully validated for the simultaneous quantification of vinflunine (VFL) and its pharmacologically active metabolite, 4-O-deacetyl vinflunine (DVFL). The two compounds, as well as vinblastine (used as internal standard), were deproteinised from blood and faeces, analysed on a cyano type column and detected on a Micromass Quattro II system in the positive ion mode after ionisation using an electrospray ion source. In blood, linearity was assessed up to 200 ng/ml for vinflunine and 100 ng/ml for 4-O-deacetyl vinflunine. The lower limit of quantification was validated at 250 pg/ml for both compounds. In other biological media, the linearity was assessed within the same range; the limit of quantification was adjusted according to the expected concentration levels of each compound. This method was first developed in order to identify the structures and to elucidate the metabolic pathway of vinflunine. Thanks to its high sensitivity and specificity, the method has enabled the quantification of vinflunine and 4-O-deacetyl vinflunine in blood at trace levels, and has contributed to the knowledge of vinflunine metabolism by monitoring up to 10 metabolites.

A high performance liquid chromatography method for vinorelbine and 4-O-deacetyl vinorelbine: a decade of routine analysis in human blood.

A sensitive high performance liquid chromatographic method was developed and validated for the simultaneous quantification of vinorelbine and its active metabolite, 4-O-deacetyl vinorelbine, in human biological fluids. These two compounds together with vinblastine, used as internal standard, were extracted from blood and urine by a liquid-liquid process using diethyl ether, and followed by a back-extraction in acidic conditions. Then, they were analysed through a cyano column and detected in ultraviolet at 268 nm. The assay linearity was validated up to 2000 ng/ml. The lower limit of quantification was set at 2.5 ng/ml. The between-run precision and accuracy were always higher than 94%. Biological samples were stable when stored at -80 degrees C over 2 years. The long-term reproducibility and the suitability of this analytical method were demonstrated within the last decade through the analysis of about 7000 samples during the clinical development of i.v. and oral formulations of vinorelbine. Because vinorelbine binds mainly to platelets and blood cells and because this binding is rapidly reversible and highly influenced by environmental conditions, drug concentration in plasma may be highly influenced by the sampling conditions and the centrifugation process used to separate blood cells from plasma. Therefore, this method was developed in blood and then used for sample analyses in routine. The major benefit was that it was easy for nurses to directly collect blood instead of plasma and that reduced volume of sampling could be withdrawn from frail patients. Furthermore, the analysis in blood enabled to quantify vinorelbine and 4-O-deacetyl vinorelbine concentrations for a longer period of time, which resulted in a more accurate evaluation of pharmacokinetic parameters.

Monitoring Tumor Response after Liposomal Doxorubicin in Combination with Liposomal Vinorelbine Treatment Using 3'-Deoxy-3'-[18F]Fluorothymidine PET.

PURPOSE: Surgical resection is the standard treatment for localized colorectal cancer, which is the most common type of gastrointestinal cancer. However, over 40 % cases are diagnosed metastasized and apparently inoperable. Systemic chemotherapy provides an alternative to these patients. This study aims to evaluate the therapeutic potential of liposomal doxorubicin (lipoDox) in combination with liposomal vinorelbine (lipoVNB) in a CT-26 colon carcinoma-bearing mouse model. PROCEDURES: The in vitro cytotoxicity of Dox and VNB on CT-26 cancer cells was determined by MTT and colony formation assays. Mice were subcutaneously inoculated with 2 × 105 of CT-26 cells in the right hind flank. When tumor size reached 200 ± 50 mm3, mice were assigned to receive different treatment protocols. The pharmacokinetics, micro single-photon emission computed tomography/x-ray computed tomography imaging, biodistribution, and immunohistochemical staining studies were performed to survey the therapeutic efficacy of each regimen. RESULTS: Based on the results of pharmacokinetic study, co-administration of lipoDox and lipoVNB did not affect their individual systemic distribution, while lipoDox retained longer in blood than lipoVNB did. Superior tumor growth retardation was observed in the group received lipoDox plus lipoVNB administration (1 mg/kg each, namely D1V1) than those injected with lipoDox plus VNB (1 mg/kg each, namely D1fV1). No severe side effects were detected in each group. The tumor-to-muscle ratio (T/M) derived from 3'-dexoy-3'-[18F]fluorothymidine ([18F]FLT) micro positron emission tomography (PET) images of D1V1- and D1fV1-treated mice and the controls on day 7 was 6.88 ± 0.54, 7.50 ± 0.84, and 9.87 ± 0.73, respectively, suggesting that D1V1 is a more efficacious regimen against CT-26 xenografts. The results of proliferating cell nuclear antigen (PCNA) immunohistochemical staining were consistent with those findings obtained from [18F]FLT microPET imaging. CONCLUSION: This study demonstrated that lipoDox in combination with lipoVNB was more efficacious than clinically used regimen, lipoDox plus VNB, in the treatment of colon carcinoma and [18F]FLT-PET is a promising approach in monitoring the treatment outcome at early stage.

Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat.

We have evaluated a flow cytometric method that allows assessment of micronucleated reticulocytes (MN-RETs) in microliter quantities of peripheral blood and compared results using this assay with those of established microscopic methods of scoring bone marrow and peripheral blood from rats treated with well-characterized genotoxic agents. Young reticulocytes (RETs) are labeled with FITC-anti-CD71 (transferrin receptor) and micronuclei with propidium iodide (with RNase treatment). Red blood cells parasitized with Plasmodia serve as a calibration standard for DNA content. Microscopic scoring used acridine orange (AO) staining of methanol-fixed slides or supravital AO staining. The effect of the rat spleen on the parameters evaluated was determined by comparing age- and sex-matched normal and splenectomized rats treated with cyclophosphamide, cis-platin, or vinblastine under treatment conditions that established a steady-state frequency of MN-RETs in the bone marrow and peripheral blood compartments. The data demonstrate the sensitivity and reproducibility of the flow cytometric assay in the Sprague-Dawley rat, and comparative studies using identical blinded samples at multiple laboratories show that inter- and intra-laboratory reproducibility is much higher with the flow method than with the microscopic methods currently employed for regulatory studies. A significant effect of splenic selection against genotoxicant-induced MN-RETs was observed with each of the three scoring methodologies, despite the fact that the flow and supravital AO techniques restrict analysis to the youngest fraction of RETs. The high precision of flow-based measurements also demonstrated a slight but statistically significant level of selection against spontaneously arising MN-RET. Despite these spleen effects, assay sensitivity for blood-based analyses was maintained by the flow method as it was shown to have superior counting statistics, lower variability, and higher sensitivity than manual scoring. The data suggest that flow cytometric assessment of micronucleus induction can be integrated into routine toxicity testing, eliminating the need for a separate bioassay.