taxane and cephalomannine

The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.

Topics: Alkaloids; Amino Acids; Bridged-Ring Compounds; Cell Culture Techniques; Cyclodextrins; Indenes; Paclitaxel; Taxoids; Taxus; Transcription, Genetic

Three fluorescent probes 3a,3b, and 4 have been synthesized through conjugation of fluorescein and difluorescein groups to the 7-OH of C-2 modified paclitaxel and cephalomannine derivatives with very high affinity to microtubules. All these probes exhibited potent tubulin assembly promotion and tumor cell killing activities, thus may be useful as tools for the determination of thermodynamic parameters and exploration of ligand-microtubule interactions.

Topics: Anisotropy; Bridged-Ring Compounds; Chemistry, Pharmaceutical; Dose-Response Relationship, Drug; Drug Design; Fluorescein; Fluorescent Dyes; Ligands; Microtubules; Models, Chemical; Plant Extracts; Taxoids; Temperature; Thermodynamics; Tubulin

Taxanes exhibit a high tendency to epimerize at C-7 under physiological conditions. This study aimed to investigate the composite effect of C-7 configuration and other substructural elements on the metabolic properties of taxanes. Cephalomannine, 7-epi-cephalomannine, 10-deacetyl-paclitaxel, and 7-epi-10-deacetyl-paclitaxel were chosen as model compounds. In human liver microsomes, 7-epi-cephalomannine was subject to C-13 lateral chain (M-1) and diterpenoid core monohydroxylation (M-2), mediated by cytochrome P450 (CYP) 3A4 and CYP2C8, respectively. However, only one 7-epi-10-deacetyl-paclitaxel metabolite (M), monohydroxylated at taxane ring by CYP2C8, was detected. In comparison with cephalomannine, the catalytic efficiency of CYP2C8 for 7-epi-cephalomannine was about five-fold higher due to the decreased K(m). Although CYP2C8 showed a high capacity for metabolizing 7-epi-10-deacetyl-paclitaxel, 10-deacetyl-paclitaxel was hardly metabolized under the identical incubation conditions. In conclusion, C-7 configuration represents one of the most important structural determinants in taxanes metabolism.

Topics: Bridged-Ring Compounds; Chromatography, Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Docetaxel; Enzyme Inhibitors; Female; Humans; Hydroxylation; Isoenzymes; Kinetics; Male; Microsomes, Liver; Molecular Conformation; Paclitaxel; Recombinant Proteins; Taxoids

To investigate how taxane's substituents at C3' affect its metabolism, we compared the metabolism of cephalomannine and paclitaxel, a pair of analogs that differ slightly at the C3' position. After cephalomannine was incubated with human liver microsomes in an NADPH-generating system, two monohydroxylated metabolites (M1 and M2) were detected by liquid chromatography/tandem mass spectrometry. C4'' (M1) and C6alpha (M2) were proposed as the possible hydroxylation sites, and the structure of M1 was confirmed by (1)H NMR. Chemical inhibition studies and assays with recombinant human cytochromes P450 (P450s) indicated that 4''-hydroxycephalomannine was generated predominantly by CYP3A4 and 6alpha-hydroxycephalomannine by CYP2C8. The overall biotransformation rate between paclitaxel and cephalomannine differed slightly (184 vs. 145 pmol/min/mg), but the average ratio of metabolites hydroxylated at the C13 side chain to C6alpha for paclitaxel and cephalomannine varied significantly (15:85 vs. 64:36) in five human liver samples. Compared with paclitaxel, the major hydroxylation site transferred from C6alpha to C4'', and the main metabolizing P450 changed from CYP2C8 to CYP3A4 for cephalomannine. In the incubation system with rat or minipig liver microsomes, only 4''-hydroxycephalomannine was detected, and its formation was inhibited by CYP3A inhibitors. Molecular docking by AutoDock suggested that cephalomannine adopted an orientation in favor of 4''-hydroxylation, whereas paclitaxel adopted an orientation favoring 3'-p-hydroxylation. Kinetic studies showed that CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel due to an increased V(m). Our results demonstrate that relatively minor modification of taxane at C3' has major consequence on the metabolism.

Topics: Adult; Animals; Antineoplastic Agents; Biotransformation; Bridged-Ring Compounds; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Hydroxylation; Male; Microsomes, Liver; Middle Aged; Paclitaxel; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Swine; Swine, Miniature; Taxoids

Lx2-32c, a novel taxane derivative, is a semisynthetic analogue from cephalomannine. Its antitumor activity in vivo and in vitro was investigated in this study. Lx2-32c was cytotoxic (IC50=1.7+/-1.6nM) to various human tumor cell lines after 72h incubation. In vitro it enhanced the rate of tubulin polymerization in a dose-dependent manner and induced the bundling of microtubule in BGC-823 cells with the mode similar to that of paclitaxel. As determined by flow cytometry, after either 12 or 24h exposure, Lx2-32c caused BGC-823 cells G2/M phase arrest in a time- and dose-dependent manner. Moreover, we demonstrated that Lx2-32c had significant antitumor activity on BGC-823 (human gastric carcinoma) and A549 (human non-small cell lung carcinoma) xenograft in nude mice. These data suggest that Lx2-32c is a microtubule-stabilizing agent, which has significant antitumor activity in vitro and in vivo.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bridged-Ring Compounds; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Mice; Mice, Nude; Microtubules; Paclitaxel; Stomach Neoplasms; Taxoids; Tubulin; Xenograft Model Antitumor Assays

Baseline separation of 15 taxanes including paclitaxel (Taxol) was achieved on pentafluorophenyl (PFP) HPLC columns. Methods using aqueous acetonitrile gradients on each of two commercial PFP columns were developed that are suitable for the determination of potency, content uniformity, and degradation profile of the paclitaxel bulk drug and injectable dosage form. The elution order is apparently related to molecular size, the number of acetylated hydroxyl groups, and the substitution of a xylosyl group at the 7-position. The resolution of several of the taxanes is a sensitive function of the starting eluent composition and the programming rate, which requires optimization of the methods on both columns. Retention of 10 of the taxanes was studied over the temperature range from 30 to 70 degrees C, and enthalpies of transfer, delta H degree, were determined. Conversion of a hydroxy group to an acetyl group, which can interact more strongly with the fluorines on the PFP, has a large effect on the delta H degree, as does the addition of a xylosyl derivative to the 7-hydroxy. The methods developed for the injectable drug form allow good resolution of the taxanes from the excipient Cremophor EL, a polyethoxylated castor oil used with ethanol to solubilize the paclitaxel. The methods for the bulk drug were fully validated in terms of accuracy, precision, specificity including forced degradation, limits of detection and quantitation, and linearity and range.

Topics: Antineoplastic Agents, Phytogenic; Bridged-Ring Compounds; Chromatography, High Pressure Liquid; Dosage Forms; Fluorobenzenes; Paclitaxel; Phenols; Reference Standards; Reproducibility of Results; Taxoids; Thermodynamics