Differential Morphological and Biochemical Recovery from Chemotherapy-Induced Peripheral Neuropathy Following Paclitaxel, Ixabepilone, or Eribulin Treatment in Mouse Sciatic Nerves.

The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.

Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs.

Peripheral neuropathy is a serious, dose-limiting side effect of cancer treatment with microtubule-targeting drugs. Symptoms present in a "stocking-glove" distribution, with longest nerves affected most acutely, suggesting a length-dependent component to the toxicity. Axonal transport of ATP-producing mitochondria along neuronal microtubules from cell body to synapse is crucial to neuronal function. We compared the effects of the drugs paclitaxel and ixabepilone that bind along the lengths of microtubules and the drugs eribulin and vincristine that bind at microtubule ends, on mitochondrial trafficking in cultured human neuronal SK-N-SH cells and on axonal transport in mouse sciatic nerves. Antiproliferative concentrations of paclitaxel and ixabepilone significantly inhibited the anterograde transport velocity of mitochondria in neuronal cells, whereas eribulin and vincristine inhibited transport only at significantly higher concentrations. Confirming these observations, anterogradely transported amyloid precursor protein accumulated in ligated sciatic nerves of control and eribulin-treated mice, but not in paclitaxel-treated mice, indicating that paclitaxel inhibited anterograde axonal transport, whereas eribulin did not. Electron microscopy of sciatic nerves of paclitaxel-treated mice showed reduced organelle accumulation proximal to the ligation consistent with inhibition of anterograde (kinesin based) transport by paclitaxel. In contrast, none of the drugs significantly affected retrograde (dynein based) transport in neuronal cells or mouse nerves. Collectively, these results suggest that paclitaxel and ixabepilone, which bind along the lengths and stabilize microtubules, inhibit kinesin-based axonal transport, but not dynein-based transport, whereas the microtubule-destabilizing drugs, eribulin and vincristine, which bind preferentially to microtubule ends, have significantly less effect on all microtubule-based axonal transport. Cancer Res; 76(17); 5115-23. ©2016 AACR.

Phase II trial of utidelone as monotherapy or in combination with capecitabine in heavily pretreated metastatic breast cancer patients.

BACKGROUND: The treatment of metastatic breast cancer (MBC) remains a great clinical challenge as drug resistance frequently develops. Alternative agents that can overcome drug resistance would offer new therapeutic options. The primary aim of this phase II study was to evaluate the efficacy and safety of utidelone as a monotherapy or in combination with capecitabine in metastatic breast cancer patients previously treated with and resistant to anthracyclines and taxanes. METHODS: In two open-label, noncomparative clinical studies, patients with metastatic breast cancer who previously received anthracycline- and/or taxane-containing regimens were given (1) 25 to 35 mg/m(2)/day intravenously infused utidelone, once daily for 5 days, in combination with 14 days of 2000 mg/m(2) capecitabine, divided in two equal daily oral doses or (2) 40 mg/m(2)/day intravenously infused utidelone, once daily for 5 days. These regimens were administered per each 21-day treatment cycle, and the maximum of treatment cycles allowed per protocol is 6. Objective response rate (ORR), progression-free survival (PFS), and tolerability were evaluated. RESULTS: In the combination study, 33 patients completed a median of 6 cycles of therapy, which was the highest cycles a trial patient could receive under the criteria of the study protocol. Efficacy was evaluated (n = 32) with an ORR of 42.4 % (FAS, 95 % CI, 26.6, 60.9) and a median PFS of 7.9 (FAS, 95 % CI, 6.1, 9.8) months. The monotherapy study (n = 63) resulted in an ORR of 28.57 % (FAS, 95 % CI, 18.4, 40.6) and a median PFS of 5.4 (FAS, 95 % CI, 2.9, 9.8) months. In both studies, common toxicities associated with utidelone administration included peripheral neuropathy, fatigue, myalgia, and arthralgia, but the toxicities were limited and manageable. Notably, very mild myelosuppression, low liver and renal toxicities, and very limited gastrointestinal toxic effect were observed, in contrast to other agents in the same class. CONCLUSIONS: Utidelone showed promising efficacy, tolerability, and advantageous safety profiles in the treatment of patients with advanced anthracycline/taxane-refractory metastatic breast cancer and may offer new treatment options to overcome drug resistance. TRIAL REGISTRATION: CHiCTR-TRC-13004205 , registered on August 15, 2013.

Nerve growth factor alters microtubule targeting agent-induced neurotransmitter release but not MTA-induced neurite retraction in sensory neurons.

Peripheral neuropathy is a dose-limiting side effect of anticancer treatment with the microtubule-targeted agents (MTAs), paclitaxel and epothilone B (EpoB); however, the mechanisms by which the MTAs alter neuronal function and morphology are unknown. We previously demonstrated that paclitaxel alters neuronal sensitivity, in vitro, in the presence of nerve growth factor (NGF). Evidence in the literature suggests that NGF may modulate the neurotoxic effects of paclitaxel. Here, we examine whether NGF modulates changes in neuronal sensitivity and morphology induced by paclitaxel and EpoB. Neuronal sensitivity was assessed using the stimulated release of calcitonin gene-related peptide (CGRP), whereas morphology of established neurites was evaluated using a high content screening system. Dorsal root ganglion cultures, maintained in the absence or presence of NGF, were treated from day 7 to day 12 in culture with paclitaxel (300nM) or EpoB (30nM). Following treatment, the release of CGRP was stimulated using capsaicin or high extracellular potassium. In the presence of NGF, EpoB mimicked the effects of paclitaxel: capsaicin-stimulated release was attenuated, potassium-stimulated release was slightly enhanced and the total peptide content was unchanged. In the absence of NGF, both paclitaxel and EpoB decreased capsaicin- and potassium-stimulated release and the total peptide content, suggesting that NGF may reverse MTA-induced hyposensitivity. Paclitaxel and EpoB both decreased neurite length and branching, and this attenuation was unaffected by NGF in the growth media. These differential effects of NGF on neuronal sensitivity and morphology suggest that neurite retraction is not a causative factor to alter neuronal sensitivity.

Identification of Synergistic, Clinically Achievable, Combination Therapies for Osteosarcoma.

Systemic therapy has improved osteosarcoma event-free and overall survival, but 30-50% of patients originally diagnosed will have progressive or recurrent disease, which is difficult to cure. Osteosarcoma has a complex karyotype, with loss of p53 in the vast majority of cases and an absence of recurrent, targetable pathways. In this study, we explored 54 agents that are clinically approved for other oncologic indications, agents in active clinical development, and others with promising preclinical data in osteosarcoma at clinically achievable concentrations in 5 osteosarcoma cell lines. We found significant single-agent activity of multiple agents and tested 10 drugs in all permutations of two-drug combinations to define synergistic combinations by Chou and Talalay analysis. We then evaluated order of addition to choose the combinations that may be best to translate to the clinic. We conclude that the repurposing of chemotherapeutics in osteosarcoma by using an in vitro system may define novel drug combinations with significant in vivo activity. In particular, combinations of proteasome inhibitors with histone deacetylase inhibitors and ixabepilone and MK1775 demonstrated excellent activity in our assays.

Characterization of and protection from neurotoxicity induced by oxaliplatin, bortezomib and epothilone-B.

AIM: To characterize neurotoxicity induced by oxaliplatin, bortezomib, and epothilone-B as well as protection against their neurotoxicity using an in vitro model. MATERIALS AND METHODS: Neurotoxicity was evaluated using the neurite outgrowth method in PC12 rat pheochromo-cytoma cells differentiated towards a mature neuronal phenotype, while neuroprotection was explored by simultaneous exposure to 0.5 mM amifostine. The potential markers of neuronal differentiation, cyclin-B2 (Ccnb2) and baculoviral inhibitor of apoptosis repeat-containing 5 (Birc5), were evaluated by quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Bortezomib, epothilone-B, and oxaliplatin reduced neurite length to 68%, 78% and 66%, respectively (p<0.05). The percentage of neurite-forming-cells (discriminating neurotoxicity from general cytotoxicity) decreased from 70% (control) to 55% (bortezomib), 46% (epothilone-B), and 51% (oxaliplatin). Amifostine was neuroprotective against oxaliplatin-induced neurotoxicity, increasing both neurite length and neurite-forming-cells. Quantitative-RT-PCR showed a 2.7-fold decrease in Ccnb2 expression in differentiated PC12 vs. undifferentiated cells. CONCLUSION: Oxaliplatin, bortezomib, and epothilone-B are neurotoxic in the PC12 model. Amifostine has a neuroprotective effect only against oxaliplatin-induced neurotoxicity, suggesting that these compounds have different mechanisms of neurotoxicity.

Activation of apoptotic pathway in normal, cancer ovarian cells by epothilone B.

The epothilones, a new class of microtubule-targeting agents, seem to be a very promising alternative to the current strategy of cancer treatment. We have analyzed the aspects of epothilone B (Epo B) on cellular metabolism of tumor (OV-90) and normal (MM 14) ovarian cells. The observed effects were compared with those of paclitaxel (PTX), which is now a standard for the treatment of ovarian cancer. The results provide direct evidence that Epo B is considerably more cytotoxic to human OV-90 ovarian cancer cells than PTX. We have found, that antitumor efficacy of this new drug is related to its apoptosis-inducing ability, which was confirmed during measurements typical markers of the process. Epo B induced changes in morphology of cells, mitochondrial membrane potential and cytochrome c release. Also a slight increase of the intracellular calcium level was observed. Moreover, we have found that ROS production, stimulated by Epo B, is directly involved in the induction of apoptosis via mitochondrial pathway.

Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious, painful and dose-limiting side effect of cancer drugs that target microtubules. The mechanisms underlying the neuronal damage are unknown, but may include disruption of fast axonal transport, an essential microtubule-based process that moves cellular components over long distances between neuronal cell bodies and nerve terminals. This idea is supported by the "dying back" pattern of degeneration observed in CIPN, and by the selective vulnerability of sensory neurons bearing the longest axonal projections. In this study, we test the hypothesis that microtubule-targeting drugs disrupt fast axonal transport using vesicle motility assays in isolated squid axoplasm and a cell-free microtubule gliding assay with defined components. We compare four clinically-used drugs, eribulin, vincristine, paclitaxel and ixabepilone. Of these, eribulin is associated with a relatively low incidence of severe neuropathy, while vincristine has a relatively high incidence. In vesicle motility assays, we found that all four drugs inhibited anterograde (conventional kinesin-dependent) fast axonal transport, with the potency being vincristine=ixabepilone>paclitaxel=eribulin. Interestingly, eribulin and paclitaxel did not inhibit retrograde (cytoplasmic dynein-dependent) fast axonal transport, in contrast to vincristine and ixabepilone. Similarly, vincristine and ixabepilone both exerted significant inhibitory effects in an in vitro microtubule gliding assay consisting of recombinant kinesin (kinesin-1) and microtubules composed of purified bovine brain tubulin, whereas paclitaxel and eribulin had negligible effects. Our results suggest that (i) inhibition of microtubule-based fast axonal transport may be a significant contributor to neurotoxicity induced by microtubule-targeting drugs, and (ii) that individual microtubule-targeting drugs affect fast axonal transport through different mechanisms.

Exposure-response relationship of the synthetic epothilone sagopilone in a peripheral neurotoxicity rat model.

Since peripheral sensory neuropathy is the major, clinically relevant side effect of sagopilone we investigated the general and peripheral neurotoxicity of sagopilone administered intravenously with different doses (1.2 and 2.4 mg/kg) and schedules in 48 Wistar rats and we performed in parallel a pharmacokinetic/pharmacodynamic (PK/PD) study. A trend toward a different peripheral neurotoxicity could be assessed after 2 weeks of treatment (bolus > 30-min infusion > 3-h infusion) with both doses of sagopilone. Although sagopilone concentrations in peripheral nerve tissue above 100 ng/g were associated with a reduction in nerve conduction velocity (NCV), a clear dose-dependence of this reduction on the level of systemic exposure to sagopilone was not observed. The PK/PD evaluation revealed no consistent effect of the infusion duration on serum PK parameters or the PD read-out NCV. Sagopilone concentrations in brain, sciatic nerve, liver, and kidney were higher after bolus compared to infusion, but there were no influence of infusion duration on these concentrations. No correlation between sagopilone concentrations in any organ/tissue with NCV changes was detected. This study evidences that the PD of sagopilone is not the main determinant of the onset and severity of sagopilone-induced peripheral neurotoxicity in the investigated clinically-relevant dose range, thus indicating that further investigation might identify neuronal-specific mechanisms of action able to drive a focused strategy to prevent peripheral neurotoxicity without reducing the anticancer effectiveness of the epothilones.

Comparison of neuropathy-inducing effects of eribulin mesylate, paclitaxel, and ixabepilone in mice.

Chemotherapy-induced neurotoxicity is a significant problem associated with successful treatment of many cancers. Tubulin is a well-established target of antineoplastic therapy; however, tubulin-targeting agents, such as paclitaxel and the newer epothilones, induce significant neurotoxicity. Eribulin mesylate, a novel microtubule-targeting analogue of the marine natural product halichondrin B, has recently shown antineoplastic activity, with relatively low incidence and severity of neuropathy, in metastatic breast cancer patients. The mechanism of chemotherapy-induced neuropathy is not well understood. One of the main underlying reasons is incomplete characterization of pathology of peripheral nerves from treated subjects, either from patients or preclinically from animals. The current study was conducted to directly compare, in mice, the neuropathy-inducing propensity of three drugs: paclitaxel, ixabepilone, and eribulin mesylate. Because these drugs have different potencies and pharmacokinetics, we compared them on the basis of a maximum tolerated dose (MTD). Effects of each drug on caudal and digital nerve conduction velocity, nerve amplitude, and sciatic nerve and dorsal root ganglion morphology at 0.25 × MTD, 0.5 × MTD, 0.75 × MTD, and MTD were compared. Paclitaxel and ixabepilone, at their respective MTDs, produced significant deficits in caudal nerve conduction velocity, caudal amplitude and digital nerve amplitudes, as well as moderate to severe degenerative pathologic changes in dorsal root ganglia and sciatic nerve. In contrast, eribulin mesylate produced no significant deleterious effects on any nerve conduction parameter measured and caused milder, less frequent effects on morphology. Overall, our findings indicate that eribulin mesylate induces less neuropathy in mice than paclitaxel or ixabepilone at equivalent MTD-based doses.

Phase II trial of sagopilone, a novel epothilone analog in metastatic melanoma.

BACKGROUND: Sagopilone is a novel fully synthetic epothilone with promising preclinical activity and a favourable toxicity profile in phase I testing. METHODS: A phase II pharmacokinetic and efficacy trial was conducted in patients with metastatic melanoma. Patients had measurable disease, Eastern Cooperative Oncology Group performance status 0-2, adequate haematological, and organ function, with up to 2 previous chemotherapy and any previous immunotherapy regimens. Sagopilone, 16 mg m⁻², was administered intravenously over 3 h every 21 days until progression or unacceptable toxicity. RESULTS: Thirty-five patients were treated. Sagopilone showed multi-exponential kinetics with a mean terminal half-life of 64 h and a volume of distribution of 4361 l m⁻² indicating extensive tissue/tubulin binding. Only grade 2 or lower toxicity was observed: these included sensory neuropathy (66%), leukopenia (46%), fatigue (34%), and neutropenia (31%). The objective response rate was 11.4% (one confirmed complete response, two confirmed partial responses, and one unconfirmed partial response). Stable disease for at least 12 weeks was seen in an additional eight patients (clinical benefit rate 36.4%). CONCLUSION: Sagopilone was well tolerated with mild haematological toxicity and sensory neuropathy. Unlike other epothilones, it shows activity against melanoma even in pretreated patients. Further clinical testing is warranted.

Polymeric micelles for parenteral delivery of sagopilone: physicochemical characterization, novel formulation approaches and their toxicity assessment in vitro as well as in vivo.

PURPOSE: The block copolymers PEG(2000)-b-PLA(2200), PEG(2000)-b-PCL(2600) and PEG(5000)-b-PCL(5000) have been currently identified as optimal solubilizing agents for Sagopilone, a poorly water-soluble anticancer drug. In the present study, the stability, formulation feasibility and in vitro as well as in vivo toxicity were evaluated. METHODS: Dispersion media, storage conditions, and dilutions were varied for stability assessment. The critical micelle concentration (CMC) was determined using a fluorescent probe technique. Lyophilizates and polymeric films were investigated as formulation options. Furthermore, the toxicity was studied in vitro and in vivo using HeLa/MaTu cells and a nude mouse model, respectively. RESULTS: A drug-polymer ratio as low as 1:20 (w/w) was sufficient to solubilize Sagopilone effectively and to obtain stable dispersions (24h: drug content >or= 95%). Although the micelles exhibited a similar thermodynamic stability (CMC: 10(-7)-10(-6)M), PEG-b-PCL micelles were kinetically more stable than PEG(2000)-b-PLA(2200) (24h at 37 degrees C: drug content >or= 90% compared to 30%, respectively). Lyophilization of PEG-b-PCL micelles and storage stability of solid drug-loaded PEG(2000)-b-PLA(2200) films (3m, 6 degrees C: drug content of (95.6+/-1.4)%) were demonstrated for the first time. The high antiproliferative activity has been maintained in vitro (IC(50)<1 nM). Carrier-associated side effects have not been observed in vivo and the maximum tolerated dose of micellar Sagopilone was determined to be 6 mg/kg. CONCLUSION: The results of this study indicate that polymeric micelles, especially PEG-b-PCL micelles, offer excellent potential for further preclinical and clinical cancer studies using Sagopilone.

Synthesis and biological evaluation of epothilone A dimeric compounds.

The preparation and biological evaluation of a novel series of dimeric epothilone A derivatives (1-6) are described. Two types of diacyl spacers were introduced to establish the various dimeric epothilone A constructs. The effect of these compounds on tubulin polymerization and their cytotoxicity against four different cancer cell lines are reported. Several of the newly synthesized compounds inhibit endothelial cell differentiation and endothelial cell migration that are key steps of the angiogenic process.

Experimental epothilone B neurotoxicity: results of in vitro and in vivo studies.

Epothilones are a novel class of microtubule-targeting anticancer agents that are neurotoxic. In this study, we investigated the epothilone B toxic effect in vitro and we characterized in vivo the general and neurological side effects of epothilone B administration in Wistar and Fischer rats. The in vitro experiments made it possible to explore a wide concentration range (0.1 nM-1 muM) and evidenced a dose-dependent effect of epothilone B exposure on neuron neurite elongation. This dose-dependent neurotoxic effect was confirmed in both in vivo studies performed on two different rat strains at the neurophysiological, behavioral and pathological levels in the dose range 0.25-1.5 mg/kg iv weekly x 4 weeks and tubulin hyper-polymerization was demonstrated in sciatic nerve specimens. These are the first studies of the neurological effects of epothilone B and they can provide a basis for extending pre-clinical investigation to other members of the epothilone family.

Tubulin: a target for antineoplastic drugs into the cancer cells but also in the peripheral nervous system.

Since the introduction into clinical practice of vinca alkaloids, tubulin has become a key and well-established target of modern antineoplastic chemotherapy. When taxanes became available their broad spectrum of activity was striking and opened up new horizons for cancer patients' treatment. However, taxanes' susceptibility to drug resistance caused by the drug efflux pump protein, P-glycoprotein, is not infrequent and their use may be limited by poor solubility, synthetic problems and toxicity. The epothilones are a new class of chemotherapeutic agents with a mechanism of action similar to that of taxanes, but different enough to escape, for example, the multidrug resistance caused by P-glycoprotein. Moreover, the epothilones (that are strong promoters of tubulin polymerization) have significant antitumor activity against human cancer cells that are taxane-resistant, express the multidrug resistance gene MDR-1, and have acquired tubulin mutations. Finally, starting from the natural molecules, several synthetic analogues have been developed. Besides their antineoplastic efficacy, all the antitubulin drugs share a common toxicity on the peripheral nervous system and peripheral neurotoxicity is a major, potentially dose-limiting side effect also of the epothilones. The current knowledge regarding the features of epothilones' peripheral neurotoxicity and their comparison with taxanes will be reviewed.

Phase I trial and pharmacokinetic study of ixabepilone administered daily for 5 days in children and adolescents with refractory solid tumors.

PURPOSE: The objectives of this phase I trial were to determine the maximum-tolerated dose (MTD), toxicity profile, dose-limiting toxicities (DLTs), pharmacokinetics, and preliminary response rate for ixabepilone, a microtubule-stabilizing agent, administered intravenously daily for 5 days in children and adolescents. PATIENTS AND METHODS: Patients >or= 2 and 5 days and grade 3 fatigue) were observed in two of three patients receiving 10 mg/m(2)/d. The MTD of ixabepilone administered daily for 5 days every 21 days was 8 mg/m(2)/d. Myelosuppression, GI, and hepatic toxicities were common non-DLTs. Peripheral neuropathy was uncommon. Ixabepilone clearance was 475 +/- 247 mL/min/m(2), volume of distribution at steady-state was 12.2 +/- 5.4 L/kg, and half-life was 14 hours. CONCLUSION: The recommended dose of ixabepilone for phase II trials in solid tumors is 8 mg/m(2)/d daily for 5 days every 21 days. This dose is 33% higher than the MTD in adults receiving the same dosing schedule. Pharmacokinetic parameters in children and adolescents were highly variable but similar to adults.

Total synthesis and evaluation of C25-benzyloxyepothilone C for tubulin assembly and cytotoxicity against MCF-7 breast cancer cells.

The total synthesis of C25-benzyloxy epothilone C is described. A sequential Suzuki-Aldol-Yamaguchi macrolactonization strategy was utilized employing a novel derivatized C8-C12 fragment. The C25-benzyloxy analog exhibited significantly reduced biological activity in microtubule assembly and cytotoxicity assays. Molecular modeling simulations indicated that excessive steric bulk in the C25 position may reduce activity by disrupting key hydrogen bonds that are crucial for epothilone binding to beta-tubulin.

Cell-based and biochemical structure-activity analyses of analogs of the microtubule stabilizer dictyostatin.

Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. The naturally occurring compound (-)-dictyostatin binds to MTs, causes cell cycle arrest in G(2)/M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. In this study, we examined a series of dictyostatin analogs to probe biological and biochemical structure-activity relationships. We used a high-content multiparameter fluorescence-based cellular assay for MT morphology, chromatin condensation, mitotic arrest, and cellular toxicity to identify regions of dictyostatin that were essential for biological activity. Four analogs (6-epi-dictyostatin, 7-epi-dictyostatin, 16-normethyldictyostatin, and 15Z,16-normethyldictyostatin) retained low nanomolar activity in the cell-based assay and were chosen for analyses with isolated tubulin. All four compounds were potent inducers of MT assembly. Equilibrium binding constant (K(i)) determinations using [(14)C]epothilone B, which has a 3-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epi-dictyostatin and 7-epi-dictyostatin displaced [(14)C]epothilone B with K(i) values of 480 and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (K(i) values of 4.55 and 4.47 muM, respectively), consistent with previous reports showing that C16-normethyldictyostatin loses potency in paclitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of beta-tubulin. Finally, we developed a set of quantitative structure-activity relationship equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogs.

A phase Ib and pharmacokinetic trial of patupilone combined with carboplatin in patients with advanced cancer.

PURPOSE: Patupilone is a microtubule-targeting chemotherapeutic agent with clinical activity in a broad range of taxane-sensitive/resistant tumor types. The present phase Ib study examined the safety/tolerability and pharmacokinetics of patupilone in combination with carboplatin in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Patients with advanced cancer received patupilone via a 5- to 10-min i.v. infusion at doses of 3.6 to 6.0 mg/m(2) q3w, immediately followed by carboplatin area under the curve (AUC) 5 or 6 mg/mL/min. RESULTS: Of the 37 patients enrolled, the majority previously received taxanes (81%) and/or platinum-containing drugs (97.3%). The maximum tolerated dose (MTD) of patupilone with carboplatin AUC 6 was 4.8 mg/m(2); additional patients were enrolled to consolidate experience at this dose. Of the 22 patients who received the MTD, the most common nonhematologic adverse events were fatigue in six (27.3%) and diarrhea, nausea, vomiting, abdominal pain, and neuropathy in one each (4.5%; all grade 3); hematologic toxicities included two patients (9.1%) with grade 3 neutropenia. The pharmacokinetics of patupilone were similar to those in a previous study of patupilone monotherapy. Of the 26 patients with recurrent platinum-sensitive ovarian cancer, tumor response was assessable by response evaluation criteria in solid tumors in 17; 1 patient (6%) achieved a complete response, and 10 (59%) achieved a partial response. CONCLUSIONS: The combination of patupilone 4.8 mg/m(2)/carboplatin AUC 6 was well tolerated and showed antitumor activity similar to established regimens in patients with recurrent platinum-sensitive ovarian cancer. The optimal dose for this regimen is currently being further refined in phase II trials.

Epothilone induced cytotoxicity is dependent on p53 status in prostate cells.

BACKGROUND: Epothilones are a promising class of drugs in clinical trials of prostate cancer that target the microtubules, similar to taxanes, and induce apoptosis in taxane resistant tumors. The tumor suppressor p53 is one important molecular mechanism of chemotherapy resistance that in some studies predicted tumor sensitivity to paclitaxel. We hypothesized that epothilone induced cytotoxicity would be influenced by the status of p53 in prostate cells. METHODS: LNCaP, DU145, and a transformed rat prostate (RP) epithelial cell line with a temperature sensitive mutant p53 (val 135) were studied for the effect of epothilone on cell viability, cell cycle, and cell cycle checkpoint proteins. RESULTS: Epothilone had greater cytotoxicity in p53 mutant cancer cells compared to wild type cells. We confirmed our findings by creating a transformed RP epithelial cell line with a temperature sensitive mutant p53 (val 135). Using a tetrazolium (MTT) assay we found that epothilone (100 nM) decreased cell viability in RP cells by 90% with mutant p53 compared to 45% with wild type p53 (P < 0.01). Epothilone induced G2/M arrest in 50% of cells with mutant p53 compared to 25% with wild type p53 (P < 0.01). To begin to understand mechanism of epothilone induced G2/M arrest, we assessed cell cycle checkpoint proteins. We found that the effect to enhance G2/M cell cycle arrest was associated with dephosphorylation of cdc2 in both p53 wild type and p53 mutant RP cells. CONCLUSIONS: These results demonstrate that epothilone is more active against transformed prostate epithelial cells with mutant compared to wild type p53. Epothilone is capable of dephosphorylation of cdc2 in both p53 wild type and mutant cells, which is associated with G2/M cell cycle arrest. These data provide a basis for further study of p53, and the phosphorylation status of cdc-2, as markers for epothilone sensitivity in clinical studies.