Vinflunine, a new vinca alkaloid: cytotoxicity, cellular accumulation and action on the interphasic and mitotic microtubule cytoskeleton of PtK2 cells

Vinflunine, a newly synthesized derivative, possesses marked in vivo antitumor properties and, like other alkaloids, inhibits in vitro tubulin assembly at microM concentrations. However, in contrast to other vinca alkaloids, vinflunine exhibits relatively low in vitro cytotoxic potency. The aim of this report was to investigate whether the action(s) of vinflunine on the microtubule cytoskeleton could account for its cytotoxicity or if its cellular action requires another molecular target. Four vinca alkaloids used in cancer therapy and vinflunine were studied using PtK2 cells. Their activities on the most dynamic microtubules were investigated in mitosis and in interphase by evaluating the disturbance of the metaphase plate and the splitting of the diplosome, respectively. No correlation was observed between the cellular accumulation of these compounds and either their cytotoxicity or their action(s) on the microtubule cytoskeleton. In contrast, cytotoxicity, mitotic disturbance and diplosome splitting were observed in the nM range for vinblastine, vincristine, vindesine and vinorelbine, although these events occurred at 10 times higher concentrations in the case of vinflunine. Hence, dynamic modifications of both the mitotic and interphasic microtubule cytoskeleton are compatible with in vitro cytotoxicity of vinflunine, raising questions about the conventional biochemical screening of these vinca alkaloids.     

Efficacy and tolerability of vinorelbine in the cancer therapy

Vinorelbine (VRN) is one of the most representative compounds of its class: the vinca alkaloids. VRN interferes with microtubule assembly. VRN shows a better therapeutic index than the parent compound vincristine and vinblastine probably because of its higher affinity for mitotic microtubules. VNR high affinity for mitotic microtubules causes a high clinical efficacy for the treatment of non-small cell lung cancer (NSCLC) and for breast cancer (BC), together with a good tolerability at therapeutically effective doses. The vinca alkaloids are structurally similar compounds comprised of 2 multiringed units, vindoline and catharanthine. Unlike other vinca alkaloids, the catharanthine unit is the site of structural modification for VRN. The antitumor activity of VNR is thought to be due primarily to inhibition of mitosis at metaphase through its interaction with tubulin. Like other vinca alkaloids, VNR may also interfere with: 1) amino acid, cyclic AMP, and glutathione metabolism, 2) calmodulin-dependent Ca2?-transport ATPase activity, 3) cellular respiration, and 4) nucleic acid and lipid biosynthesis. The VNR is also characterized by improved hematologic tolerance and less neurotoxicity compared to parent compound. The aim of this review is 1) to explore the efficacy and tolerability of VNR in cancer therapy and 2) to examine the more recent approaches to improve the efficacy and tolerability of VNR in cancer therapy.     

Modifications in the "upper" velbenamine part of the Vinca alkaloids have major implications for tubulin interacting activities

Vinca alkaloids represent a chemical class of major interest in cancer chemotherapy. The lead compounds vinblastine and vincristine have been employed in clinical practice for more than thirty years and remain widely used to this day. Several hundred derivatives have been synthesised and evaluated for their pharmacological activities, the majority being modified in the vindoline moiety, bearing several reactive centers. These efforts led to the identification of the amido derivative vindesine, registered in Europe in 1980 and now available in several countries. Then novel chemistry permitted the semisynthesis of derivatives modified in the velbenamine "upper" part of the molecule, creating a new potential in the Vinca alkaloids medicinal chemistry: as a result, vinorelbine, obtained by C' ring contraction of anhydrovinblastine, and is now marketed worldwide. Several strategies aimed at the total synthesis of vinblastine derivatives have been investigated, giving the opportunity to design rationally certain compounds. Modifications in the D' ring appeared to induce dramatic changes in the tubulin interactions. These observations have been confirmed recently by the identification of unprecedented pharmacological properties exerted by the novel fluorinated Vinca alkaloid, vinflunine. This review will focus more specifically on derivatives which have been modified in the velbenamine part, with the aim of inducing different chemical and pharmacological properties.     

P-Glycoprotein mediated resistance to 5′-nor-anhydro-vinblastine (Navelbine®)

Summary Navelbine^? (NVB, vinorelbine tartrate) is a semisyntheticVinca alkaloid in which the catharanthine moiety contains an eight-membered ring in place of the nine-membered ring that is present in all naturally occurring members of the vinblastine group. This modification selectively reduces interaction with anoxalvs mititotic microtubules and may account for the lower neurotoxicity with improved antitumor activity that has been observed in clinical trials with breast, lung and ovarian cancer. We were interested in whether the structural modification in NVB would also alter the drug resistance profile. Specifically, our aim was to determine whether NVB, like vinblastine (VBL), participates in P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). NVB-resistant, murine P388 cells (P388/NVB), were derivedin vivo and used in conjunction with a battery of drug-resistant P388 cell linesin vivo and murine and human tumor cell linesin vitro to develop a resistance profile for NVB. P388/NVB bells were cross-resistant to drugs involved in MDR (doxorubicin, etoposide, amsacrine, vinblastine, vincristine and actinomycin D), but not to the alkylating agents, cyclophosphamide, carmustine, and cisplatin, or to the antimetabolites, 5-fluorouracil and methotrexate. P388/NVB cellular resistance to NVB was stable without drug pressure during continuous passagein vivo for more than ten weeks andin vitro for at least five weeks. These cells exhibited increased expression of P-gp, and a 30-fold level of resistance of NVBin vitro, which was completely reversable with verapamil. The MDR phenotype was confirmed in other tumor models. P388 tumors resistant to vinblastine, vincristine, doxorubicin, and etoposide were cross-resistant to NVBin vivo. Likewise, human KB carcinoma cells resistant to colchicine, MDA-A1^R breast carcinoma cells resistant to doxorubicin, and murine B16/F10 melanoma cells transfected with the humanmdr1 gene were cross-resistant to NVBin vitro. Finally, P388 cells resistantin vivo to melphalan, a drug that does not participate in MDR, were cross-resistant to NVB. Therefore, cellular resistance to NVB, like otherVinca alkaloids, may arise by a P-glycoprotein-mediated MDR mechanism, but may also involve non-MDR mechanisms in cells that do not overexpress P-gp.     

Efficacy of 5′-nor-anhydrovinblastine (vinorelbine), against freshly explanted clonogenic human tumor cells in vitro

Summary Vinorelbine (5-nor-anhydrovinblastine) is a semisynthetic vinca alkaloid currently undergoing extensive clinical evaluation. We have studied the antitumor effect of vinorelbine (final concentrations: 8.4–1000.0 ng/ml) against freshly explanted clonogenic cells from 102 human tumors using a capillary soft agar cloning system and have compared the compound's activity with vinblastine, vincristine, vindesine, paclitaxel, docetaxel, and other clinically used anticancer agents. Four specimens were excluded from further analyses (3 bacterial or fungal contamination, 1 benign histology). Fifty-one of the remaining 98 (52%) specimens had adequate colony formation in control capillaries. Vinorelbine showed concentration-dependent antitumor activity against a variety of solid rumors. At clinically relevant concentrations (0.1 × peak plasma concentrations in humans) vinorelbine inhibited 21 of 49 specimens (43%) and was as active as vinblastine, vincristine, vindesine, bleomycin, doxorubicin, 5-fluorouracil, mitomycin-C, cisplatin, methotrexate, and etoposide. However, paclitaxel (71% inhibition, p = 0.006) and docetaxel (78% inhibition, p = 0.002) were significantly more active than vinorelbine. Moreover, vinorelbine showed antitumor activity against several tumor types and in particular against breast cancer but also in non-small cell lung cancer. We conclude that vinorelbine has a wide spectrum of in vitro activity against freshly explanted human tumors and that the clinical activity of this compound against breast cancer and non-small cell lung cancer is reflected in vitro.     

Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells

Multidrug resistance (MDR), typified by resistance to Vinca alkaloids and anthracyclines, is a well characterized experimental phenomenon that may have some clinical correlates. Verapamil, chloroquine, and related drugs have been shown previously to be capable of enhancing anticancer drug cytotoxicity in multi-drug-resistant cells, but the mechanism(s) by which these agents do this is(are) unclear. Since these agents did not seem to have common features, we studied these and other compounds for their ability to "modulate" Vinca alkaloid resistance in order to determine whether they possessed any common chemical or physical features. In addition to verapamil, 24 compounds, consisting of indole alkaloids, lysosomotropic agents, and amines, were tested for their ability to enhance the cytotoxicity of vinblastine and/or vincristine in our human leukemic multidrug-resistant cell line, CEM/VLB100. Seventeen compounds that enhance the cytotoxicity of the Vinca alkaloids by more than 5-fold have been identified. These include quinolines (chloroquine, quinine, chinchonidine, and primaquine), acridines (acridine, acridine orange, and quinacrine), and indole alkaloids (yohimbine, corynanthine, reserpine, physostigmine, and the vindoline and catharanthine moieties of the Vinca alkaloids), as well as other alkaloids and amines (chlorpromazine, propranolol, atropine, and tryptamine). Vindoline, catharanthine, and quinacrine also enhanced the cytotoxicity of doxorubicin and teniposide in these cells, indicating that this "modulation" was not limited to Vinca alkaloids. We examined some well known lysosomotropic compounds (methylamine, epinephrine, suramin, and trypan blue) and found that they were not able to enhance the cytotoxicity of vincristine in the CEM/VLB100 cells, indicating that lysosomotropic activity per se is not required for modulator activity. Three-dimensional computer modeling permitted molecular comparisons of conformationally related congeners of vinblastine, vindoline, and verapamil and revealed three regions of structural homology. We measured the hydrophobicity (by oil/water partitioning) and calculated the molar refractivity (by the additive substituent constant method) of active and inactive compounds. We found that those cationic agents--verapamil, quinacrine, indole alkaloids, and quinolines--that were lipid soluble at physiologic pH and had similar molar refractivities were best able to enhance the cytotoxicity of the Vinca alkaloids in our multidrug-resistant cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of alpha-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of alpha-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. gamma-tubulin staining showed that cells treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.     

Cell fate after mitotic arrest in different tumor cells is determined by the balance between slippage and apoptotic threshold

Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x_L and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x_L gene was silenced in A549 cells, vincristine induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x_L or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x_L switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x_L/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types.     

Inhibition of tubulin polymerization by vitilevuamide, a bicyclic marine peptide, at a site distinct from colchicine, the vinca alkaloids, and dolastatin 10

Vitilevuamide, a bicyclic 13 amino acid peptide, was isolated from two marine ascidians, Didemnum cuculiferum and Polysyncranton lithostrotum. Vitilevuamide was cytotoxic in several human tumor cell lines, with LC(50) values ranging from 6 to 311nM, and analysis in a 25-cell line panel revealed a weak correlation with several taxol analogs. Vitilevuamide was strongly positive in a cell-based screen for inhibitors of tubulin polymerization. Vitilevuamide at 9 microg/mL (5.6 microM) had an effect equivalent to the maximal effect of colchicine at 25 microg/mL (62.5 microM). Vitilevuamide was active in vivo against P388 lymphocytic leukemia, increasing the lifespan of leukemic mice 70% at 30 microg/kg. We hypothesized that at least part of the cytotoxic mechanism of vitilevuamide was due to its inhibition of tubulin polymerization. Vitilevuamide was found to inhibit polymerization of purified tubulin in vitro, with an IC(50) value of approximately 2 microM. Cell cycle analysis showed that vitilevuamide arrested cells in the G(2)/M phase with 78% of treated cells tetraploid after 16hr. Therefore, vitilevuamide was tested for its ability to inhibit binding of known tubulin ligands. Vitilevuamide exhibited non-competitive inhibition of vinblastine binding to tubulin. Colchicine binding to tubulin was stabilized in the presence of vitilevuamide in a fashion similar to vinblastine. Dolastatin 10 binding was unaffected by vitilevuamide at low concentrations, but inhibited at higher ones. GTP binding was also found to be weakly affected by the presence of vitilevuamide. These results suggest the possibility that vitilevuamide inhibits tubulin polymerization via an interaction at a unique site.     

Antimitotic peptides and depsipeptides

Tubulin is the target for an ever increasing number of unusual peptides and depsipeptides that were originally isolated from a wide variety of organisms. Since tubulin is the major component of cellular microtubules, which maintain cell shape in interphase and form the mitotic spindle, most of these compounds are highly toxic to mammalian cells. These peptides and depsipeptides disrupt cellular microtubules and prevent formation of a functional spindle, resulting in the accumulation of cultured cells in the G2/M phase of the cell cycle through specific inhibition of mitosis. At the biochemical level, the compounds all inhibit the assembly of tubulin into polymer and, in the cases where it has been studied, strongly suppress microtubule dynamics at low concentrations. In most cases the peptides and depsipeptides inhibit the binding of vinblastine and vincristine to tubulin in a noncompetitive manner, inhibit tubulin-dependent GTP hydrolysis, and interfere with nucleotide turnover at the exchangeable GTP site on beta-tubulin. Most of the peptides and depsipeptides induce tubulin to form oligomers of aberrant morphology, including tubulin rings that vary in diameter depending on the (depsi) peptide under study. The purpose of this review is to give an overview of the cellular, biochemical, in vivo, and SAR aspects of this group of compounds. We also summarize initial efforts by computer modeling to decipher a pharmacophore among the diverse structures of these peptides and depsipeptides.     

10'-Fluorovinblastine and 10'-Fluorovincristine: Synthesis of a Key Series of Modified Vinca Alkaloids

A study on the impact of catharanthine C10 and C12 indole substituents on the biomimetic Fe(III)-mediated coupling with vindoline led to the discovery and characterization of two new and substantially more potent derivatives, 10'-fluorovinblastine and 10'-fluorovincristine. In addition to defining a pronounced and unanticipated substituent effect on the biomimetic coupling, fluorine substitution at C10', which minimally alters the natural products, was found to uniquely enhance the activity 8-fold against both sensitive (IC(50) = 800 pM, HCT116) and vinblastine-resistant tumor cell lines (IC(50) = 80 nM, HCT166/VM46). As depicted in the X-ray structure of vinblastine bound to tubulin, this site resides at one end of the upper portion of the T-shaped conformation of the tubulin-bound molecule, suggesting the 10'-fluorine substituent makes critical contacts with the protein at a hydrophobic site uniquely sensitive to steric interactions.     

Differential binding to the alpha/beta-tubulin dimer of vinorelbine and vinflunine revealed by nuclear magnetic resonance analyses

The binding of two antitumour alkaloids, vinorelbine and vinflunine, to the alpha/beta-tubulin dimer has been investigated at equilibrium by nuclear magnetic resonance (NMR) spectroscopy. Tubulin stability and assembly induced by these drugs has been checked under NMR experimental conditions, and tubulin spirals were found in majority. Then, using increasing ligand concentrations, the alkaloids were titrated against tubulin. A non-specific binding of both compounds to tubulin (K(d)>10(-5)M) was characterised by broad NMR ligand signal at 4 and 30 degrees. The tubulin dimer exhibited also 2.7 (sigma: 0.3) and 2.6 (sigma: 0.6) binding sites with a K(d)<10(-5)M for vinorelbine at 4 and 30 degrees, respectively. In contrast, if the tubulin dimer exhibited 2.7 (sigma: 0.2) binding sites for vinflunine at 4 degrees, these sites were not detected at 30 degrees. This NMR study revealed for the first time the presence of specific binding sites and a clear differential affinity of vinorelbine and vinflunine to the tubulin dimer at physiological temperatures which could possibly account for their differential cytotoxicity.     

Toxicology of vindesine (desacetyl vinblastine amide) in mice, rats, and dogs.

Comparative acute intravenous toxicity studies of vinblastine sulfate (VLB), vincristine sulfate (VCR), and vindesine in mice and rats indicated that vindesine was more toxic than VLB and less toxic than VCR. Rats were able to tolerate larger repeated doses of vindesine than dogs. Rats given intravenous doses totaling 0.15 mg/kg-wk vindesine for 3 months developed no remarkable signs of toxicity. Doses of 0.3 mg/kg-wk or greater produced anorexia, depressed blood cell counts, atrophic intestinal mucosa, inhibition of spermatogenesis, extramedullary hematopoiesis, and infections. Dogs were given total weekly intravenous doses of 0.04, 0.08, 0.1, or 0.16 mg/kg vindesine for 3 months. The only observed effect in the two lower dose groups was inhibition of spermatogenesis. Groups receiving 0.1 or 0.16 mg/kg developed leukopenia, slight erythropenia, inhibition of spermatogenesis, focal skeletal muscle degeneration, elevated lactic dehydrogenase, and an increase in bone marrow myeloid: erythroid ratio. No evidence of functional or structural changes in neural tissues was found. The above effects are common to animals given VCR at lower doses and for a shorter test period. It is therefore concluded that vindesine is less toxic in animals than VCR.     

Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine resistance by the flavonoid myricetin

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5+/-1.7 microM for MRP1 and 24.6+/-1.3 microM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1+/-0.1 microM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1+/-1.9 and 22.2+/-1.4 microM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 microM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6+/-0.5 and 5.8+/-0.5 microM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance.     

Cytotoxicity,Cell Cycle Kinetics and Morphonuclear-induced Effects of Vinca Alkaloid Anticancer Agents

The effects of four Vinca alkaloids (vinblastine, vincristine, vindesine and vinorelbine) on three neoplastic cell lines (the MXT mouse mammary cell line and the T24 and J82 bladder cell lines) were studied at three biological levels, i.e. cell proliferation, cell cycle kinetics and morphonuclear characteristics. These effects were studied by means of digital cell image analysis on Feulgen-stained nuclei. The aim of the present work was to characterize the effects specifically induced by Vinca alkaloids as compared with those obtained previously with other pharmacological classes of anticancer drugs. The results show that Vinca alkaloids inhibit the cell proliferation of neoplastic cell lines at a concentration of 10^***-8 M except in the case of the J82 cell line, for which only a slowing down of cell proliferation was observed. Concerning the cell cycle kinetics, the results show that the Vinca alkaloids induce an accumulation of cells in the mitosis phase. This accumulation of mitotic cells was maximal after 15 h incubation in the presence of the drugs. A study of the morphonuclear-induced effects of Vinca alkaloids showed that the variance of the optical density (VOD) is strongly influenced by these Vinca alkaloids. The development of the VOD was parallel with the development of the percentage of mitosis; thus, the VOD enabled the Vinca alkaloid-induced effects to be specifically characterized from a morphonuclear point of view. On the other hand, the results show that the mean value of the variance of the optical density was very highly correlated (P < 0***.001) with the efficiency of the Vinca alkaloids in terms of cytotoxicity. In clinical studies, the analysis of the development of this parameter would make it possible to assess the response to chemotherapy in the case of patients treated with Vinca alkaloids.     

Vinblastin-23-oyl amino acid derivatives: chemistry, physicochemical data, toxicity, and antitumor activities against P388 and L1210 leukemias

The dimeric alkaloids vinblastine (VLB) and vincristine (VCR) differ structurally only in the functional group on the dihydroindole nitrogen. The semisynthetic derivative vindesine (VDS) differs slightly from VLB by having an amide group instead of an ester group. However, these minor distinctions are responsible for profound differences in the oncolytic spectrum, potency, and toxicity of these compounds. Vinblastin-23-oyl amino acid derivatives were synthesized by linking amino acid carboxylic esters to the vinblastin-23-oyl moiety through an amide linkage. Studies were extended to explore the influence of the nature of the amino acid, the ester alkyl chain lengths, the stereoisomerism of the amino acid, or the reacetylation of the hydroxyl group (position O-4) of the vindoline moiety. The present study deals with the synthesis of 21 vinblastin-23-oyl amino acid derivatives, some of their physicochemical data, the acute toxicity in mice, and therapeutic activities of these derivatives against the P388 and L1210 leukemias in comparison with VDS, VBL, and VCR.     

A study of toxicity and comparative therapeutic efficacy of vindesine-prednisone vs. vincristine-prednisone in children with acute lymphoblastic leukemia in relapse

Summary Vindesine (des-acetyl Vinblastine) is a synthetic derivative of vinblastine, and was produced with the hope that it would have less neurotoxicity and hematopoietic toxicity than other vinca alkaloids.Phase I and II studies also demonstrated significant activity in lymphoid malignancies, especially Acute Lymphoblastic Leukemia (ALL). The present study was designed to compare therapeutic effectiveness of twice weekly vindesine (2 mg/M²/dose) plus Prednisone (60 mg/M²/dose) (Treatment 1) to weekly Vincristine (2 mg/M²/dose) plus Prednisone (60 mg/M²/day) (Treatment 2). All patients were less than 21 years of age, and had documented bone marrow relapse (blast count>25%). In 39 patients presumed sensitive to vincristine, there were 11 complete responses out of 20 patients (55%) randomized to receive vindesine/ prednisone and 7 complete responses out of 19 patients (37%) treated with Vincristine/Prednisone. In 37 patients resistant to vincristine, there were 7 complete responses (19%). Vindesine was more toxic than Vincristine. Major toxicities of vindesine included paraesthesias, peripheral neuropathy and ileus. Vindesine hematological toxicity appeared greater, but such toxicity is hard to assess in patients with bone marrow disease. In this study, vindesine and vincristine had similar efficacy, but vindesine use was associated with more toxicity.     

Interaction of fused-pyrimidine nucleoside analogs with human concentrative nucleoside transporters: High-affinity inhibitors of human concentrative nucleoside transporter 1

Human concentrative nucleoside transporters (hCNTs) mediate electrogenic secondary active transport of physiological nucleosides and nucleoside drugs into cells. Six fused-pyrimidine ribonucleosides and one 2′-deoxynucleoside were assessed for their abilities to inhibit [³H]uridine transport in the yeast Saccharomyces cerevisiae producing recombinant hCNT1, hCNT2 or hCNT3. Six of the analogs inhibited hCNT1 with K_i values < 1 μM whereas only two analogs inhibited hCNT3 with K_i values < 1 μM and none inhibited hCNT2. To assess if the inhibitory analogs were also permeants, currents evoked were measured in oocytes of Xenopus laevis producing recombinant hCNT1, hCNT2 or hCNT3. Significant inward currents, indicating permeant activity, were generated with (i) three of the analogs in hCNT1-producing oocytes, (ii) none of the analogs in hCNT2-producing oocytes and (iii) all of the analogs in hCNT3-producing oocytes. Four were not, or were only very weakly, transported by hCNT1. The thienopyrimidine 2′-deoxynucleoside (dMeThPmR, 3) and ribonucleoside (MeThPmR, 4) were the most active inhibitors of uridine transport in hCNT1-producing oocytes and were an order of magnitude more effective than adenosine, a known low-capacity transport inhibitor of hCNT1. Neither was toxic to cultured human leukemic CEM cells, and both protected CEM cell lines with hCNT1 but not with hENT1 against gemcitabine cytotoxicity. In summary, dMeThPmR (3) and MeThPmR (4) were potent inhibitors of hCNT1 with negligible transportability and little apparent cytotoxicity, suggesting that pending further evaluation for toxicity against normal cells, they may have utility in protecting normal hCNT1-producing tissues from toxicities resulting from anti-cancer nucleoside drugs that enter via hCNT1.     

Microtubulin binding sites as target for developing anticancer agents

Microtubules (MTs) play important and diverse roles in eukaryotic cells. Their function and biophysical properties have made alpha-and beta-tubulin, the main components of MTs, the subject of intense study. Interfering with normal MT dynamics, for example, by the addition of tubulin ligands, can cause the cell great distress and affect MT stability and functions, including mitosis, cell motion and intracellular organelle transport. It has been shown in the literature that tubulin is an important target molecule for developing anticancer drugs. Tubulin binding molecules have generated considerable interest after the successful introduction of the taxanes into clinical oncology and the widespread use of the vinca alkaloids vincristine and vinblastine. These compounds inhibit cell mitosis by binding to the protein tubulin in the mitotic spindle and preventing polymerization into the MTs. This mode of action is also shared with other natural agents eg colchicine and podophyllotoxin. However various tubulin isotypes have shown resistance to taxanes and other MT agents. Therefore, there is a strong need to design and develop new natural analogs as antimitotic agents to interact with tubulin at sites different from those of vinca alkaloids and taxanes. This minireview provides SAR on several classes of antimitotic agents reported in the literature. The structures and data given are essential to the scientists who are involved in drug design and development in the field of anticancer drugs.     

Discovery of allosteric inhibitors of kinesin spindle protein (KSP) for the treatment of taxane-refractory cancer: MK-0731 and analogs

Current cancer chemotherapy relies heavily on cytotoxic agents, such as the taxanes and Vinca alkaloids, that interfere with the cellular machinery required for cell division and divert the cell down a pathway of programmed cell death. These antimitotic agents, or spindle poisons, target the mitotic spindle by binding to tubulin, a protein required not only for mitosis but also for structural integrity and proper function of healthy, terminally differentiated cells. To avoid side effects attributed to this nonselective mechanism of action, new targets in the mitotic pathway that act only in dividing cells were sought and a leading candidate to emerge from these efforts was kinesin spindle protein (KSP or HsEg5). KSP is a molecular motor protein that is expressed only during mitosis and controls the formation of a functional mitotic spindle. Inhibition of KSP causes mitotic arrest followed by cell death in malignant cells and thus has the potential to become a novel chemotherapeutic strategy with the potential for reduced toxicity. This article summarizes efforts carried out at Merck to discover potent, selective and water soluble KSP inhibitors that culminated in the discovery of MK-0731, the second KSP inhibitor to enter clinical trials. Of special focus in this article is how an HTS lead was optimized in apparently divergent directions, but these disparate leads converged in the design of compounds that overcame P-glycoprotein efflux and hERG channel activity, two issues that required considerable optimization within our program.