Conformation and energy investigation of microtubule longitudinal dynamic instability induced by natural products.

The natural products plinabulin, docetaxel, and vinblastine are microtubule targeting agents (MTAs). They have been used alone or in combination in cancer treatment. However, the exact nature of their effects on microtubule (MT) polymerization dynamics is poorly understood. To elucidate the longitudinal conformational and energetic changes during MT dynamics, a total of 140 ns molecular dynamic simulations combined with binding free energy calculations were performed on seven tubulin models. The results indicated that the drugs disrupted MT polymerization by altering both MT conformation and binding free energy of the neighboring tubulin subunits. The combination of plinabulin and docetaxel destabilized MT polymerization due to bending MT and weakening the polarity of tubulin polymerization. The new combination of docetaxel and vinblastine synergistically enhanced MT depolymerization and bending, while plinabulin and vinblastine had no synergistic inhibitory effects. The results were verified by the tubulin assembly assay. Our study obtained a comprehensive understanding of the action mechanisms of three natural drugs and their combinations on MT dynamic, provided theoretical guidance for new MTA combinations, and would promote the optimal use of MTA and contribute to developing new MTAs as anticancer agents.

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.

Fluorescent vinblastine probes for live cell imaging.

Herein we describe the synthesis of several fluorescent analogues of the clinically approved microtubule destabilizing agent vinblastine. The evaluated probes are the most potent described and provides the first example of uptake, distribution and live cell imaging using this well known antimitotic agent.

Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus.

We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon.

Reactivity of vinca alkaloids during water chlorination processes: Identification of their disinfection by-products by high-resolution quadrupole-Orbitrap mass spectrometry.

Concerns about the presence of anticancer drugs in the environment are rapidly increasing mainly due to their growing use in the developed countries and their known cytotoxic effects. Vinca alkaloids are widely used in cancer therapy; however, very scarce information is available on their occurrence, environmental fate and toxicological effects on aquatic organisms. Even less attention has been paid to their potential transformation products, which can exert higher toxicity than the parent compounds. Thus, in the present work, the reactivity of vincristine, vinblastine, vinorelbine and its metabolite 4-O-deacetyl vinorelbine during water chlorination processes has been investigated for the first time. Under the studied chlorination conditions, vincristine was fairly stable whereas vinblastine, vinorelbine and 4-O-deacetyl vinorelbine were quickly degraded. A total of sixty-five disinfection by-products were tentatively identified by ultra-high performance liquid chromatography coupled to high-resolution hybrid quadrupole-Orbitrap tandem mass spectrometry. Among them, twenty by-products corresponded to mono-chlorinated compounds, eight to di-chlorinated compounds and two to tri-chlorinated compounds, which may be of major environmental concern. Other disinfection by-products involved hydroxylation and oxidation reactions. Although the structures of these by-products could not be positively confirmed due to lack of commercial standards, their chemical formulas and product ions can be added to databases, which will allow their screening in future monitoring studies.

Simultaneous determination of vinblastine and its monomeric precursors vindoline and catharanthine in Catharanthus roseus by capillary electrophoresis-mass spectrometry.

Catharanthus roseus is an important dicotyledonous medicinal plant that contains various anticancer components, such as vinblastine (VLB) and its monomeric precursors (vindoline and catharanthine). A capillary electrophoresis-mass spectrometry (CE-MS) approach for the simultaneous determination of three components was developed in this work. Baseline separation for three components was achieved by using a running buffer consisting of 20 mM ammonium acetate and 1.5% acetic acid in <20 min. Quantification of three components was assigned in positive-ion mode at a protonated molecular ion [M+H](+). The CE-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The detection limits of VLB, catharanthine and vindoline are 0.8, 0.1 and 0.1 µg/mL, respectively. The precision was not more than 4.54% and the mean recovery of the analytes was 95.04-97.04%. The CE-MS method was successfully applied to determine VLB and its monomeric precursors in real sample C. roseus.

Matrix-assisted laser desorption/ionization-ion mobility separation-mass spectrometry imaging of vinblastine in whole body tissue sections.

During early-stage drug development, drug and metabolite distribution studies are carried out in animal tissues using a range of techniques, particularly whole body autoradiography (WBA). While widely employed, WBA has a number of limitations, including the following: expensive synthesis of radiolabeled drugs and analyte specificity and identification. WBA only images the radiolabel. MALDI MSI has been shown previously to be advantageous for imaging the distribution of a range of drugs and metabolites in whole body sections. Ion mobility separation (IMS) adds a further separation step to imaging experiments; demonstrated here is MALDI-IMS-MS whole body imaging of rats dosed at 6 mg/kg i.v. with an anticancer drug, vinblastine and shown is the distribution of the precursor ion m/z 811.4 and several product ions including m/z 793, 751, 733, 719, 691, 649, 524, and 355. The distribution of vinblastine within the ventricles of the brain is also depicted. Clearly demonstrated in these data are the removal of interfering isobaric ions within the images of m/z 811.4 and also of the transition m/z 811-751, resulting in a higher confidence in the imaging data. Within this work, IMS has shown to be advantageous in both MS and MS/MS imaging experiments by separating vinblastine from an endogenous isobaric lipid.

Attempt to detect natural anticancer compounds--protein binding through precursor ion scan and MS/MS measurements.

A 2'-succinyltaxol-bovine serum albumin (BSA) conjugate was prepared as an antigen to produce an anti-taxol monoclonal antibody by immunizing mice. Formation of a linkage between hapten and protein is usually confirmed by the UV or fluorescamine method. However, it was difficult to confirm the binding of 2'-succinyltaxol to BSA by these methods owing to the similar UV absorption maxima of 2'-succinyltaxol (273 nm) and BSA (280 nm). In the present study, we therefore conducted a mass spectrometric analysis using the precursor ion scan and MS/MS techniques to confirm the formulation of the 2'-succinyltaxol-BSA conjugate in the following way: The conjugate was subjected to thermal denaturalization, dithiothreitol (DTT)-reduction, iodoacetamide-alkylation and trypsin-digestion, affording a peptide fragment mixture. This was then analyzed by electrospray ionization (ESI)-MS in the positive mode by scanning the peaks containing a mass of 854 corresponding to taxol. The detected peaks were in turn subjected to MS/MS measurements. Among them, a peak at m/z 1247.4 was found to be a peptide fragment containing Lys (epsilon-2'-succinyltaxol), demonstrating the formulation of the 2'-succinyltaxol-BSA conjugate. In order to confirm the feasibility of this analytical method, the deacetylvinblastine (deacetylVLB)-BSA antigen which produced the anti-VLB monoclonal antibody (MAb-10-A9), was subjected to the same analytical treatment as above, giving a peak at m/z 851.3 originating from a Lys (epsilon-deacetylVLB). Thus, this new method could serve as an additional tool for confirmation of the formation of hapten-protein conjugates which are difficult to detect by the above spectrophotometric methods.

[A study on the hairy root culture and antitumor alkaloids production of Catharanthus roseus].

OBJECTIVE: To establish transformation system and obtain alkaloids from the hairy root of Catharanthus roseus. METHOD: Hairy roots were obtained by infecting the different explants of C. roseus. Culture conditions of hairy root were optimized. RESULT: The best transformation condition was leaf infected by two-day's pre-culture and two-day's co-culture and additional A(S) (hydroxyacetosyringone) 100 mg x L(-1). The inducing rate of hairy root was up to 86.25%. The best condition of hairy root culture was MS medium with sucrose as carbon material and lactalbumin as nitron material. The analysis result showed that the contents of total alkaloids in hairy roots were higher than explants and calli. CONCLUSION: Hairy root of C. roseus will be useful for the production of active components in C. roseus.

Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines.

Cell viability assays are important tools in oncological research and clinical practice to assess the tumor cell sensitivity of individual patients. The purpose of this study was to demonstrate the comparability of 3 widely used assays (MTT, ATP, calcein assays) by principal component analysis. The study included 4 different cytostatics (cisplatin, docetaxel, doxorubicin, vinblastine) and 3 different human cancer cell lines (MCF-7, A2780, doxorubicin resistant A2780adr). Ninety-three percent of the total variance of all variables included in the principal component analysis (resulting from 3 cell lines and 3 assays) could be explained by 1 principal component. Factor loadings were > 0.937 except for the variable MTT-A2780adr, which was 0.872. These results indicate the similarity of the 3 assays. A 2nd principal component analysis included literature data and showed accordance of data from this study and the literature. The MTT assay was further improved as a high-throughput screening-capable assay. The ATP assay is able to detect effects of cytostatics already after 1 h incubation. The determination of resistance factors allowed to differentiate cytostatics into P-gp or non-P-gp substrates. In conclusion, this study provides improved microplate reader-based cell viability assays and sets a statistically solid basis for a future comparison of data obtained in different laboratories by any of the 3 assays.

Sensitive and rapid spectrophotometric methods for determination of anticancer drugs.

Sensitive, rapid, and simple spectrophotometric methods were developed for determination of the anticancer drugs vinblastine sulfate (VBS) and vincristine sulfate (VCS), which belong to the class of vinca alkaloids. The first method is based on the reaction of VBS and VCS with diazotized dapsone, forming yellow azo products with absorption maxima at 430 nm. The colored species obey Beer's law in the concentration range of 0.5-24 microg/mL for VBS and 0.5-12 microg/mL for VCS. The second method describes the reaction of VBS and VCS with iron(III) and subsequent reaction with ferricyanide in hydrochloric acid medium to yield blue products with absorption maxima at 750 nm. The Beer's law range for this method is 0.1-4 microg/mL for VBS and 0.5-10 microg/mL for VCS. With both methods, colored species were stable for 1 h. The methods are simple and reproducible and are applied for determination of VBS and VCS in pharmaceutical formulations. Commonly encountered pharmaceuticals added as excipients do not interfere in the analysis and the results obtained in the analysis of dosage forms agree well with the labeled contents.

Monitoring the production yields of vincristine and vinblastine in Catharanthus roseus from somatic embryogenesis. Semiquantitative determination by flow-injection electrospray ionization mass spectrometry.

A semiquantitative determination of two bis-indole antitumor alkaloids, vincristine and vinblastine, has been performed by flow-injection electrospray ionization mass spectrometric analysis of the extracts of Catharanthus roseus. Leaves and flowers of two different phenotypes (pink flower and white flower) obtained from somatic embryogenesis were thus examined and compared with the field-grown mother plant. Different amounts of vincristine and vinblastine were detected depending on the examined samples.

Yeast cells expressing differential levels of human or yeast DNA topoisomerase II: a potent tool for identification and characterization of topoisomerase II-targeting antitumour agents.

PURPOSE: To identify and characterize the specificity and potency of topoisomerase II-interacting antitumour drugs in an in vivo model utilizing the yeast Saccharomyces cerevisiae. METHODS: Four yeast transformants were selected for the expression of either human or yeast DNA topoisomerase II at different, biologically relevant, levels under the tight control of promoters of various strengths. RESULTS: Analyses of 24 drugs permitted their classification into three distinct groups, depending on whether they induced topoisomerase II-related cytotoxicity (etoposide), showed nonspecific cytotoxicity (camptothecin), or exerted no cytotoxicity at all (vinorelbine). Within the first group different patterns of action were distinguishable: (1) classical topoisomerase II expression-dependent cytotoxicity for both species of enzyme (e.g. etoposide, amsacrine, doxorubicin, actinomycin D), although amsacrine and TOP 53 were more active, respectively, on human and yeast topoisomerase II; and (2) compounds that appeared to poison only one species of topoisomerase II with, for example, genistein and the bisdioxopiperazine ICRF-193 lethally targeting only the human type, and mitoxantrone only the yeast isozyme. Three of the 16 known topoisomerase II inhibitors tested were not correctly identified with this assay, possibly owing to restricted cell wall permeability or to the absence of correct processing pathways such as, for example, in the case of the prodrug etopophos. CONCLUSION: This methodology, in vivo in yeast, selected for a large range of potent topoisomerase II-targeting anticancer agents. Of particular interest in this yeast model, and in contrast to yeast topoisomerase II, human topoisomerase II was shown to confer dominant sensitivity in the presence of the series of bisdioxopiperazine derivatives tested. This assay therefore has the potential easily to identify and characterize the potency and specificity of synthesized anticancer drugs with modified original chemical structures or those present, for example, in natural plant extracts or marine organisms.

Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line.

P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood-brain barrier (BBB). Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine. When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased. When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels. Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level. RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.

Clinical pharmacokinetics of vinorelbine.

Vinorelbine (5'-noranhydrovinblastine) is a recently developed semisynthetic anticancer drug which belongs to the Catharanthus alkaloid family. Its mechanism of action is only partially known but it is assumed that it acts, like vinblastine and vincristine, as an antimicrotubule agent arresting cell division in mitosis. Clinically, vinorelbine has mainly shown activity in the treatment of advanced non-small-cell lung cancer and the treatment of metastatic breast cancer. Early pharmacokinetic data were obtained with radioactive assays (radio-immunoassay or 3H-labelled vinorelbine), then with more selective high performance liquid chromatographic techniques. Vinorelbine is usually administered intravenously but there has also been some experimentation with an oral formulation. The bioavailability of a liquid filled gelatin capsule ranges between 12 and 59% with a mean value of 27% [standard deviation (SD) 12%]. Vinorelbine is rapidly absorbed with peak serum concentration reached within 2 hours. In vitro, vinorelbine is mainly distributed into the blood cells, especially platelets (78%) and lymphocytes (4.8%) The unbound blood fraction is around 2%. In lung tissue vinorelbine concentrations are much higher than in serum, by up to 300-fold 3 hours after administration. Little is known about the biotransformation of vinorelbine. Desacetylvinorelbine is considered to be a minor metabolite and is only found in urine fractions, representing 0.25% of the injected dose. Urinary excretion of vinorelbine is low, accounting for less than 20% of the dose. Faecal elimination has been demonstrated in 2 patients who were administered 3H-labelled vinorelbine; the amount of radioactivity recovered in the faeces was 33.9 and 58.4% for the 2 patients, respectively. The pharmacokinetic profile of vinorelbine is often described as a 3-compartment model characterised by a long terminal half-life (t1/2) that varies between 20 and 40 hours and a large apparent volume of distribution (Vd) of around 70 L/kg. Systemic clearance ranges between 72.54 and 89.46 L/h (1209 and 1491 ml/min) when determined by high performance liquid chromatography and is higher than that reported by radioimmunoassay [46.2 L/h (770 ml/min)]. This could be due to the greater specificity of the chromatographic method. Vinorelbine has been administered by continuous intravenous infusion over 4 days. Steady-state was reached and the concentrations obtained were above the in vitro IC50 (concentration of drug causing 50% inhibition). The effect of liver disease on vinorelbine pharmacokinetics has been studied in patients with breast cancer. Patients with massive secondary liver disease had a lower systemic clearance than those who have no liver disease or a lesser invasion. In children, vinorelbine seems to display a shorter t1/2 (14.7 hours) than that found in adults. In addition, the systemic clearance is highly variable [from 12 to 93.96 L/h/m2 (200 to 1566 ml/min/m2)]. Vinorelbine is often co-administered with cisplatin in the treatment of advanced non-small-cell lung cancer. The disposition of the alkaloid is not altered by concurrent administration of cisplatin.

Stability of vinblastine sulphate in 0.9% sodium chloride in polypropylene syringes.

The stability of vinblastine sulphate diluted in 0.9% sodium chloride solution for injection was studied. Vinblastine sulphate was reconstituted with 0.9% sodium chloride solution for injection to concentration of 1.0 mg/mL and stored in polypropylene syringes at 25 degrees C +/- 1 degree C protected from light. On different days the solutions were analysed and the vinblastine concentration was determined by high-performance liquid chromatography. An high-pressure liquid chromatographic method is described for the quantitative determination of vinblastine in the presence of its degradation products. The degradation of vinblastine was studied by examining the percentage changes from the theoretical concentrations for each solution. The results of these studies indicate that vinblastine solutions in 0.9% sodium chloride solution for injection (1 mg/mL) in polypropylene syringes at 25 degrees C +/- 1 degree C protected from light are stable for up to one month.

Interaction between vinblastine and ionizing radiation in the mouse MO4 fibrosarcoma in vivo.

The effect of combining vinblastine (VLB) and ionizing radiation (IR) on tumor response was investigated in CDF1 mice bearing the MO4 mouse fibrosarcoma. Favorable interactions were assumed to occur if VLB treatment caused accumulation of cells in metaphase (M-)phase at the time of IR. High pressure liquid chromatography (HPLC) measurements showed that VLB administered at 2.0 mg/kg (0.4 maximal tolerated dose, MTD) was taken up rapidly by the tumor in vivo, and that sufficient concentrations of VLB were achieved to cause blocking of tumor cells in M-phase. However, different treatment schedules of the combination of 2.5 mg/kg VLB (0.5 MTD) and 10 Gy IR resulted in additive tumor responses. The best therapeutic response was observed when IR was given 6 hours after intravenous injection of VLB. In order to increase the therapeutic response, we attempted to enhance the accumulation of cells in M-phase by treating mice with VLB at the MTD. The maximum percentage of tumor cells that could be accumulated in mitosis by a single intravenous bolus of VLB at the MTD was around 13%. The results show that this will probably be insufficient for significant IR enhancement.

Plasma pharmacokinetics, tissue disposition, excretion and metabolism of vinleucinol in mice as determined by high-performance liquid chromatography.

We investigated the pharmacokinetics of the experimental semisynthetic vinca alkaloid vinleucinol (VileE; O4-deacetyl-3-de(methoxycarbonyl)-3-[[[1-ethoxycarbonyl-2- methylbutyl]amino]carbonyl]-vincaleukoblastine). The study was performed in male FVB mice receiving 10.5 mg/kg VileE i.v. or p.o. Plasma, urine, faeces and tissue samples were analysed by a selective method based on ion-exchange normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection and liquid-liquid extraction for sample clean-up. Apart from the parent drug, two other metabolic compounds were detected. One of these metabolites is vinleucinol acid (VileA; O4-deacetyl-3-de(methoxycarbonyl)-3-[[[1-carboxyl-2- methylbutyl]amino]carbonyl]-vincaleukoblastine), which possesses no cytotoxic activity. The structure proposed for the second metabolite (VileX) was based on tandem mass spectrometry (MS-MS) and infrared (IR) spectroscopy data. Metabolization of VileE to VileX must occur in the amino acid moiety of the molecule, with a (beta- or gamma-) lactone ring being formed after oxidation of the (beta- or gamma) carbon of the amino acid. VileX is a major metabolite, which is excreted in faeces and urine after i.v. administration and accounting for up to 23% of the administered dose. The activity of VileX against cultured L1210 cells is four times that of the parent drug VileE and comparable with that of vinblastine (VBL). At 48 h after administration of VileE, the concentration of VileX exceeds that of the parent drug in many tissues. These findings indicate that the metabolite VileX may be at least largely responsible for the activity observed against xenografts in mice after administration of the parent drug, VileE.