Tubulin-binding Agents Research Articles

A novel palladium-mediated coupling approach to 2,3-disubstituted benzo(b)thiophenes and its application to the synthesis of tubulin binding agents.

[structure: see text]. Flexible, convergent access to 2,3-disubstituted benzo[b]thiophenes has been developed. The most concise approach involves sequential coupling of o-bromoiodobenzenes with benzylmercaptan and zinc acetylides to give benzyl o-ethynylphenyl sulfides which react with iodine to give 3-iodobenzo[b]thiophenes in a 5-endo-dig iodocyclization. These iodides can be further elaborated using palladium-mediated coupling and/or metalation techniques. This method has been applied to the synthesis of some novel tubulin binding agents.

Determinants of anti-vascular action by combretastatin A-4 phosphate: role of nitric oxide.

The anti-vascular action of the tubulin binding agent combretastatin A-4 phosphate (CA-4-P) has been quantified in two types of murine tumour, the breast adenocarcinoma CaNT and the round cell sarcoma SaS. The functional vascular volume, assessed using a fluorescent carbocyanine dye, was significantly reduced at 18 h after CA-4-P treatment in both tumour types, although the degree of reduction was very different in the two tumours. The SaS tumour, which has a higher nitric oxide synthase (NOS) activity than the CaNT tumour, showed ~10-fold greater resistance to vascular damage by CA-4-P. This is consistent with our previous findings, which showed that NO exerts a protective action against this drug. Simultaneous administration of CA-4-P with a NOS inhibitor, Nω-nitro-L-arginine (L-NNA), resulted in enhanced vascular damage and cytotoxicity in both tumour types. Administration of diethylamine NO, an NO donor, conferred protection against the vascular damaging effects. Following treatment with CA-4-P, neutrophil infiltration into the tumours, measured by myeloperoxidase (MPO) activity, was significantly increased. Levels of MPO activity also correlated with the levels of vascular injury and cytotoxicity measured in both tumour types. Neutrophilic MPO generates free radicals and may therefore contribute to the vascular damage associated with CA-4-P treatment. MPO activity was significantly increased in the presence of L-NNA, suggesting that the protective effect of NO against CA-4-P-induced vascular injury may be, at least partially, mediated by limiting neutrophil infiltration. The data are consistent with the hypothesis that neutrophil action contributes to vascular injury by CA-4-P and that NO generation acts to protect the tumour vasculature against CA4-P-induced injury. The protective effect of NO is probably associated with an anti-neutrophil action. © 2000 Cancer Research Campaign

Evaluation of antivascular and antimitotic effects of tubulin binding agents in solid tumor therapy.

Tubulin binding agents (TBAs) reduce tumor perfusion and inhibit mitosis of tumor cells in solid tumors, but it is not clear which effects contribute to the suppression of solid tumor growth. We evaluated the antivascular and antimitotic effects of several TBAs, combretastatin A‐4 (CS A‐4) phosphate, AC‐7700, a novel CS A‐4 derivative, colchicine, E7010, and vinblastine, on subcutaneous (s.c.) murine colon26 adenocarcinoma (c26). Tolerable doses of vinblastine and E7010 strongly inhibited tumor growth and induced mitotic arrest of tumor cells without affecting tumor perfusion. Colchicine had no effect on tumor growth and perfusion. When the injected dose was increased to the lethal range, however, these drugs markedly reduced tumor perfusion and caused necrosis of tumor tissue. Within the tolerable dose range, AC‐7700 both strongly suppressed tumor growth and reduced tumor perfusion, and CS A‐4 phosphate also exhibited a moderate antivascular effect. To evaluate the contribution of antivascular activity of TBAs to tumor growth suppression, excluding their direct cytotoxic effect on tumor cells, we established c26/acr, which is resistant to TBAs in vitro. Although E7010 showed a reduced suppressive effect on s.c. c26/acr tumor growth as compared with its effect on wild‐type c26, AC‐7700 remained potent against both cell lines. These results indicate that TBAs exert antivascular and antimitotic effects on solid tumors with marked differently effective dose ranges from agent to agent, and that the antivascular effect of TBAs inhibits solid tumor growth independently of the direct cytotoxic effect on tumor cells.

Synthesis, X-Ray Crystal Structure and Tubulin- Binding Properties of a Benzofuran Analogue of the Potent Cytotoxic Agent Combretastatin A4

The benzofuran (4), a ring-fused analogue of the potent antimitotic agent combretastatin A4 (1), has been prepared by a convergent route involving 5-endo-dig iodocyclization of o-hydroxytolan (5) as the key step. Compound (4), which has been characterized crystallographically as well as spectroscopically, is inactive as a tubulin-binding agent.

Synthesis and structure-activity profiles of A-homoestranes, the estratropones.

2-Methoxyestradiol, a mammalian metabolite of estradiol, has reported antiangiogenic activity which has been proposed to be mediated through interaction at the colchicine binding site on the tubulin monomer. Subsequent structure-activity studies of 2-methoxyestradiol have yielded highly potent steroidal inhibitors of tubulin polymerization. In an effort to probe the scope of binding at the colchicine binding site and the nature of the relationship between 2-methoxyestradiol and colchicine, a series of colchicine/2-methoxyestradiol hybrids was synthesized. These A-homoestrane hybrid systems, collectively termed estratropones, possessed an A-ring tropone system with the keto functionality at either the C-2, C-3, or C-4 position of the steroid nucleus. The estratropones were evaluated for their ability to inhibit the polymerization of tubulin using an in vitro purified bovine brain assay. Most of these hybrids inhibit polymerization with greater potency than either of the natural products. The most potent of these congeners possessed an approximate 5-fold enhancement of the activity of colchicine for the inhibition of tubulin polymerization. alpha-Substituents on the tropone ring showed varied effects on the activities for the two classes of estratropones studied in this regard, the C-3 oxo and the C-4 oxo species. The 3-substituted 4-oxoestratropones exhibited antitubulin activity according to Cl approximately Br > OCH3, whereas the 4-substituted 3-oxoestratropones exhibited activity according to OCH3 > Br approximately Cl. It is unclear if these substituent factors are purely electronic or steric effects or if the substituent operates indirectly by altering the conformation of the nonplanar troponoid ring. The estratropones represent a new class of tubulin binding agents with potential antiangiogenic utility.

Electrophysiological effects of CI-980, a tubulin binding agent, on guinea-pig papillary muscles.

1. The electrophysiological effects of CI-980, a new tubulin-binding agent that inhibits assembly of cytoplasmic microtubules, on transmembrane action potential characteristics were studied in right ventricular papillary muscles from guinea-pig hearts. 2. In papillary muscles driven at 1 Hz, CI-980 at concentrations > or = 10(-5) M produced a concentration-dependent increase in the maximum upstroke velocity (Vmax) and a lengthening of the action potential duration at 50% (APD50) and 90% (APD90) of repolarization without affecting the resting membrane potential. Prolongation of the APD90 was accompanied by a parallel lengthening of the effective refractory period (ERP) so that the ERP/APD90 ratio remained unaltered at all drug concentrations tested. 3. CI-980 exhibits a reverse use-dependent effect on APD90 values, that is, drug-induced APD90 prolongation become exagerated at slow rates and attenuated at fast rates. 4. CI-980 at concentrations > or = 10(-6) M lengthened the APD of the slow action potentials elicited by isoprenaline in papillary muscles depolarized by high K+ (27 mM) solution. 5. At 10(-5) M, CI-980 produced a small tonic Vmax block. However, in muscles driven at rates between 0.5 and 3 Hz it produced an exponential decline in Vmax (use-dependent Vmax block) which was augmented at higher rates of stimulation. At 3 Hz the onset kinetics of the use-dependent Vmax block was fitted by a monoexponential function with a K value 0.07 +/- 0.01 per AP. The recovery time constant (tau re) from the use-dependent Vmax block was prolonged from 21.6 +/- 2.6 ms to 3.5 +/- 0.2 s. 6. The curve relating membrane potential and Vmax was shifted by CI-980 (10(-5) M) in the hyperpolarizing direction by 2.3 +/- 1.1 mV. 7. It is concluded that in guinea-pig papillary muscles, CI-980 produces a use-dependent inhibition of Vmax and a reverse use-dependent prolongation of the ventricular action potential, thus exhibiting class I and class III antiarrhythmic actions, respectively. From the onset and offset kinetics of use-dependent Vmax block, CI-980 can be considered to be an intermediate kinetics (IA) Na+ channel blocker.

Single agent activity of rhizoxin in non-small-cell lung cancer: a phase II trial of the EORTC Early Clinical Trials Group

In a multicentre trial of the EORTC-Early Clinical Trials Group (ECTG) we treated 31 chemotherapy-naive patients with advanced non-small-cell lung cancer (NSCLC) with rhizoxin, a novel tubulin-binding agent. The drug was given as an i.v. bolus injection at 2 mg m-2 once every 3 weeks in an outpatient setting. Prophylactic antiemetics were not routinely given. Of the 29 eligible patients, nine had been treated surgically and three had received radiotherapy. The main toxic effects observed were stomatitis (34% of cycles) and neutropenia (41% of cycles). Neutropenic fever was rare (3% of cycles). Twenty-seven patients were evaluable for response. There were four partial responses (15%), while 13 patients (48%) showed stabilisation of their disease. The median duration of response was 7 months (range 6.0-10.7 months) and median survival from the start of rhizoxin treatment was 6 months (range 2-14.7 months). Rhizoxin as single agent shows activity in patients with advanced NSCLC.

Effects of chemotherapeutic and immunosuppressive drugs on the production of erythropoietin in human hepatoma cultures.

The effects of agents used in combination chemotherapy were studied on erythropoietin synthesis in cultures of the human hepatoma cell line, HepG2. Erythropoietin was measured by radioimmunoassay in the culture media after 24-h treatment periods. The RNA synthesis-inhibiting drugs daunorubicin, cyclophosphamide, ifosfamide, and cis-diamine-dichloroplatinum produced a dose-dependent decrease of the production of erythropoietin. Inhibition was also induced by the tubulin-binding agents vincristine and colchicine and the immunosuppressive agents azathioprine and cyclosporin A. The DNA synthesis-inhibiting drugs methotrexate and cytosine arabinoside, the antimycotics 5-fluorocytosine and amphotericin B, and glucocorticoids did not inhibit. Viability studies showed that the inhibition of erythropoietin production was partly correlated with cytotoxicity. These data could be relevant with respect to recombinant erythropoietin replacement therapy in tumor-associated anemia.

Cardiostimulatory and antiarrhythmic activity of tubulin-binding agents.

Rhythmic, spontaneously pulsating cardiac cells cultured from newborn rats are immediately stimulated to beat faster by addition of a number of tubulin-binding agents but not by their non-tubulin-binding analogues. The tubulin-binding agents tested include vinblastine, vincristine, navelbine, two analogs of vinblastine (S12362 and S12363), nocodazole, colchicine, and podophylotoxin. In addition to binding tubulin, all of the above agents also depolymerize microtubules. In contrast, taxol, a tubulin-binding agent that stabilizes microtubules, does not stimulate cardiac cells. Moreover, the immediate and ensuing cardiac stimulation by vinblastine at 0.05 microgram/ml is completely blocked by pre- and cotreatment with taxol at 1.0 microgram/ml. The time necessary to reverse the cardiostimulatory effect of vinblastine is significantly longer than that required for nocodazole, further implicating depolymerization of microtubules in the cardiac activity of these agents. All of the tubulin-binding agents tested (including taxol) also immediately reverse adriamycin-induced arrhythmias. By using a monoclonal antibody to alpha-tubulin, typical filamentous microtubules are visualized in cardiac muscle and cocultured non-muscle cells by immunofluorescence. When cells are treated for 2 hr with vinblastine at 0.05 microgram/ml, fluorescence is detected in cross-striated patterns in cardiac muscle cells. Overall, these data open the possibility of uncovering an additional relationship between cytoskeletal elements (other than actin and myosin) and the contractility of cardiac muscle. They also suggest an alternative mechanism for affecting cardiac cell function in vitro (namely, by tubulin-binding agents). If these agents are shown to be cardioactive in vivo, they may provide another approach to the treatment and management of cardiac arrhythmias.

Detection of Pleiotropic Drug Resistance by the Rapid Immunofluorescence Assay of Drug Effects on the Cell Skeleton

We describe the development of an immunofluorescent method for the detection of resistance to agents which affect the integrity of the cellular microtubular network. Three pleiotropic resistant MCF-7 human breast carcinoma cell lines mixed with vaginal adenocarcinoma cells were selected in serially increasing drug concentrations, and demonstrated a 30-fold increase in resistance to colchicine. Transport studies indicated that there was no difference in drug accumulation between the sensitive and resistant lines. The colchicine-binding capacity of cell extracts from sensitive and resistant cells was similar (Kd for sensitive cells was 1.9 x 10(-6) M and for resistant cells 1.58 x 10(-6) M). There were, however, significant differences in cytoskeletal morphology between sensitive and resistant cells. Drug-sensitive cells were mostly large (about 70 microns 2) and flattened. Their cytoplasm was filled with a microtubular network in which, in most of the cases, single fibers could be differentiated. Cells usually had a microtubule-organizing center and paracortical bundles of microtubules. In contrast, drug-resistant cells were mostly rounded and grew in clumps. In only 40% of these cells could single microtubular fibers be differentiated. Resistant cells lacked a microtubule-organizing center and had no clear paracortical bundles of microtubules. The tubulin-binding agents tested caused a sequence of morphological changes in sensitive cells. These changes included precipitation of tubulin and disappearance of cytoskeletal structure. Changes occurred initially within 3 h of incubation, but were expressed in all cells after 6 h. If, after 3 h of drug exposure, cells were subcultured in drug-free media, the cytoskeletal structure reformed within 10 h. Maximal recovery of cytoskeletal structure occurred 22 h after drug removal and was sustained up to 36 h. In contrast to changes observed in sensitive cells, drug exposure did not induce changes in the morphology of cytoskeleton in resistant cells. Cells from all three resistant lines reverted to sensitivity after 7 months of culture in drug-free media. This was first detected by immunofluorescence and then confirmed by cloning assay. Since the cytoskeletal disintegration of sensitive cells is readily detectable within a few hours of in vitro drug treatment, immunofluorescent imaging may have its clinical application in predicting the sensitivity/resistance to microtubule-binding agents.

ViVACCy—A Drug Schedule Based on G2 Blockade and Prolonged Infusion of Multiple Tubulin-Binding Agents A Pilot Study

A drug schedule has been devised based on a strategy of G2 blockade followed by prolonged infusion of tubulin-binding agents. The regimen consists of doxorubicin 32 mg/m2 i.v. and cyclophosphamide 320 mg/m2 i.v. on day 1 followed by vinblastine (0.3 to 1.2 mg/m2/day), cisplatin (3 to 12 mg/m2/day), and vincristine (0.04 to 0.16 mg/m2/day) by continuous intravenous infusion on days 5 to 12. Courses are repeated every 28 days. Eighteen patients with advanced solid tumors received 37 courses of chemotherapy in a pilot study to determine safe drug concentrations for the three-drug infusion for 7 days. Dose limiting toxicity was myelosuppression. Patients who received prior mitomycin-C experienced more profound thrombocytopenia than those who did not. Nonhematologic toxicities included mild nausea, vomiting, and transient elevations of serum alkaline phosphatase and serum creatinine. One patient with squamous cell carcinoma of the esophagus who erroneously received vincristine 0.8 mg/m2 instead of 0.08 mg/m2 for 4 1/2 days developed transient myalgia, ileus, and a transient peripheral neuropathy; the patient achieved a sustained complete remission for 15 months and died of unrelated causes. Minor responses and stable disease were seen in two patients with renal cell carcinoma (1 and 2.5 months), three patients with colorectal carcinoma (1.5, 2, and 4 months), and one patient with squamous cell carcinoma of the tongue (2 months). The ViVACCy drug regimen can be given without undue toxicity and may be active in solid tumors.

Functional expression of the Candida albicans β-tubulin gene in Saccharomyces cerevisiae

Expression of the β-tubulin-encoding gene ( TUB2) of Candida albicans has been examined in Saccharomyces cerevisiae. Overexpression of the TUB2 gene of C. albicans, as well as that of S. cerevisiae, was found to be lethal. Chromosomal integration of the C. albicans TUB2 gene into a strain in which the native TUB2 gene had been deleted led to functional complementation. The results demonstrate that correct splicing of the two introns present in the C. albicans TUB2 gene occurs in the heterologous host strain containing this gene. Such strains are supersensitive to the tubulin-binding agent benomyl, indicating that the natural resistance of C. albicans to benomyl is not related to the structure of its β-tubulin.

IV. Neurotoxicity of colchicine and other tubulin-binding agents: A selective vulnerability of certain neurons to the disruption of microtubules

Colchicine and certain other agents which disrupt microtubules and interfere with axonal and dendritic transport are highly toxic to certain CNS neurons. The present chapter summarizes our knowledge about this selective neurotoxicity. Injections of colchicine into several brain regions lead to the death of selected populations of neurons within those regions. Intra-hippocampal injections selectively destroy granule cells of the dentate gyrus; hippocampal pyramidal cells are essentially unaffected. Injections into the cerebellum, olfactory bulb, and caudate nucleus also destroy resident neurons. In these areas several cell types are vulnerable. Neurons of the cerebral cortex appear to be much less affected by colchicine, although some neurons of paleocortical regions are vulnerable. Colchicine does not appear to be an excitotoxin like kainic acid. The neurotoxicity of colchicine appears to be related to the destruction of microtubules, since other agents which disrupt microtubules have similar toxic effects, and since analogs of colchicine which do not disrupt microtubules are non-toxic. Colchicine may induce an autotoxic response which leads to neuronal death in certain populations due to the accumulation of some toxic cellular product which is normally transported by a microtubule-dependent process. The selective vulnerability of neurons to the neurotoxic effects of colchicine may be a model for system degenerations of the central nervous system in which certain subpopulations of neurons are selectively vulnerable to abnormal accumulations of metabolic products.

Effect of tubulin-binding agents on the infiltration of tumour cells into primary hepatocyte cultures.

The influence of tubulin-binding agents on the infiltration of murine MB6A lymphosarcoma and TA3 mammary carcinoma cells into primary rat hepatocyte cultures was studied. Colchicine, nocodazole and vinblastine reduced the number of infiltrating lymphosarcoma cells, probably by interfering with the adhesion of these cells to the exposed hepatocyte surface. However, the subsequent infiltration of cells that did adhere was not affected or even slightly stimulated. The reduced adhesion appears to be due to an effect on both the MB6A cells and the hepatocytes. In contrasts, adhesion of TA3 cells was not reduced and infiltration was markedly enhanced by these agents, due to an effect on the TA3 cells but probably not on the hepatocytes. These observations support previously described morphological evidence for differences between the infiltration mechanisms of the two tumour cell types. It is concluded that the system within the hepatocytes involved in adhesiveness of the exposed surface to MB6A cells is distinct from that mediating other types of adhesion. The tendency of TA3 cells to invaginate hepatocytes may be due to disturbances in tubulin-dependent processes.

Colchicine permeation is required for inhibition of concanavalin A capping in Chinese hamster ovary cells.

Several antimitotic, tubulin-binding agents, such as colchicine, colcemid, and podophyllotoxin, inhibit the capping of fluorescent-labeled concanavalin A in Chinese hamster ovary cells. By comparing the effects of these agents on parental cell lines on several independently selected colchicine-resistant mutants with decreased drug permeability, we have demonstrated that permeation of these drugs in required for inhibition of capping. These data support the hypothesis that these antimitotic agents interact with an intracellular component, probably microtubules, to prevent the directional movement of concanavalin A receptors on the surface membranes of Chinese hamster ovary cells.