Analogue Of Hemiasterlin Research Articles

Abstract 1368: Preclinical evaluation of NC-6201, an antibody/drug-conjugated micelle incorporating novel hemiasterlin analogue E7974

Abstract Background: Antibody-drug conjugates (ADCs) have been recognized as a promising anticancer agent. There have been a lot of registered clinical trials for ADCs. However, not all ADC compounds were successful. To overcome the difficulties and drive the next generation of ADCs, we have recently developed Antibody/Drug-Conjugated Micelle (ADCM) system. ADCM is composed of polyethylene glycol-poly (amino acid derivative) polymers, which can form a micellar nanoparticle spontaneously in aqueous media with a diameter of 20-100 nm. Antibodies are attached to the surface of the nanoparticle, while payloads are incorporated in the inner core at a payload-to-antibody molecular ratio of 100-200. In this study, anti-EGFR monoclonal antibody NCAB001 and novel hemiasterlin analogue E7974 were used as a targeting sensor and a payload of the ADCM (NC-6201), respectively. Here we report the results of in vitro evaluation and in vivo efficacy and toxicity studies. Methods: NC-6201 was prepared as described previously (Japan Patent No.4538666) with slight modification. The antigen affinity and the cytotoxicity of NC-6201 were evaluated using a Biacore system and Cell Counting Kit-8, respectively. NC-6201 was administered intravenously to BALB/c-nu/nu mice xenografted with various human tumor cell lines. Tumor volumes and animal body weights were monitored 2 or 3 times a week. Also, the dose- and schedule-dependency of the antitumor effect were evaluated. Single-dosed toxicological studies of NC-6201 in SD rats and cynomolgus monkeys were performed. Results: NC-6201 showed similar antigen affinity to the unconjugated NCAB001 and had a broad range of in vitro cytotoxicity against a panel of tumor cells. NC-6201 potently suppressed tumor growth in nude mice bearing subcutaneous human tumor xenografts expressing EGFR, such as BxPC-3 (pancreas) and MDA-MB-231 (triple-negative breast) tumor models. The efficacious NC-6201 dose schedules were achieved at one tenth or two fifth of its MTD. In an EGFR-null tumor model, NC-6201 and untargeted micelle incorporating E7974 showed comparable tumor growth inhibition. Overall, NC-6201 at efficacious doses was well tolerated without significant body weight loss, indicating that NC-6201 has an excellent therapeutic window. Relative to E7974, NC-6201 showed an unaltered toxicity profile in rats and monkeys, and the potential for reduced toxicity and an improved therapeutic window. Conclusion: Based on these promising results, NC-6201 is advanced in our project pipeline as a clinical candidate for cancer therapy. Citation Format: Mitsunori Harada, Masami Tsuchiya, Ryusuke Miyazaki, Tadashi Inoue, Ryosuke Tanaka, Yuuki Yanagisawa, Masayoshi Ito, Yu Ito, Kenichiro Naito. Preclinical evaluation of NC-6201, an antibody/drug-conjugated micelle incorporating novel hemiasterlin analogue E7974. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1368.

Abstract 5525: Antibody/drug-conjugated micelle as a versatile platform technology for targeted tumor delivery

Abstract Background: Antibody-drug conjugates (ADCs) have been recognized as a promising anticancer agent, despite their stringent requirements of antibody affinity, internalization, payload potency, and linker stability. To drive the next generation of ADCs and improve versatility of the component combination, we have designed, prepared and recently developed Antibody/Drug-Conjugated Micelle (ADCM) system. ADCM is composed of polyethylene glycol-poly (amino acid derivative) polymers, which can form a micellar nanoparticle spontaneously in aqueous media with a diameter of 20-100 nm. Antibodies are attached to the surface of the nanoparticle, while payloads are incorporated in the inner core at a payload-to-antibody molecular ratio of 100-200. In this study, monoclonal antibody, trastuzumab or cetuximab, and anticancer drug, anthracycline antibiotic epirubicin or novel hemiasterlin analogue E7974, were used as a targeting sensor and a payload of the ADCM, respectively. Here we report the results of in vitro and in vivo studies. Methods: The ADCMs were prepared as described previously (Japan Patent No.4538666) with slight modification. To examine the cell binding behavior, flow cytometry analysis was performed using an Accuri C6 system. The antigen affinity was evaluated using a Biacore system. The cellular uptake was examined fluorescently using ADCMs employing Alexa 488-labeled antibodies. To compare the in vivo antitumor effects, ADCM, untargeted micelle and the payload were administered intravenously 3 times weekly at the same payload-equivalent dose to nude mice bearing human tumor xenografts with HER2 (NCI-N87 gastric or trastuzumab-resistant JIMT-1 breast tumor) or EGFR (BxPC-3 pancreatic or LS174T colon tumor) expression. Tumor volumes and animal body weights were monitored 2 or 3 times a week. Results: The ADCMs showed similar cell-binding behavior and antigen affinity to the original antibodies. They were found to be internalized more efficiently than the original antibodies. In vivo efficacy studies demonstrated that the ADCMs inhibited tumor growth most effectively compared to control formulations. The therapeutic indexes were significantly improved in terms of the maximum tolerated dose/ED50 (50% effective dose) compared to untargeted micelles and native payloads. These results suggest the potential clinical application of the ADCMs. Conclusion: These data showed improved versatility of the component combination and increased therapeutic indexes of ADCM system, indicating a promising next generation of ADCs. Citation Format: Mitsunori Harada. Antibody/drug-conjugated micelle as a versatile platform technology for targeted tumor delivery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2015-5525

Intravesical Chemotherapy of High-Grade Bladder Cancer with HTI-286, A Synthetic Analogue of the Marine Sponge Product Hemiasterlin

HTI-286 is a fully synthetic analogue of the natural tripeptide hemiasterlin that inhibits tubulin polymerization and has strong cytotoxic potential. In this study, we evaluate the inhibitory effects of HTI-286 on human bladder cancer growth, both in vitro and as an intravesical agent in an orthotopic murine model. Various bladder cancer cell lines were treated with HTI-286 and mitomycin C (MMC) in vitro. Human KU-7 bladder tumor cells that stably express firefly luciferase were inoculated in female nude mice by intravesical instillation and quantified using bioluminescence imaging. Mice with established KU-7-luc tumors were given HTI-286 or MMC intravesically twice a week for 2 h. Pharmacokinetic data was obtained using high-performance liquid chromatography-mass spectrometry analyses. In vitro, HTI-286 was a potent inhibitor of proliferation in all tested cell lines and induced marked increases in apoptosis of KU-7-luc cells even after brief exposure. In vivo, HTI-286 significantly delayed cancer growth of bladder tumors in a dose-dependent fashion. HTI-286, at a concentration of 0.2 mg/mL, had comparable strong cytotoxicity as 2.0 mg/mL of MMC. The estimated systemic bioavailability of intravesically given HTI-286 was 1.5% to 2.1% of the initial dose. Intravesical HTI-286 instillation therapy showed promising antitumor activity and minimal toxicity in an orthotopic mouse model of high-grade bladder cancer. These findings provide preclinical proof-of-principle for HTI-286 as an intravesical therapy for nonmuscle-invasive bladder cancer and warrant further evaluation of efficacy and safety in early-phase clinical trials.

A phase I trial of E7974 administered on days 1 and 15 of a 28-day cycle in patients with solid malignancies

2550 Background: E7974 is a synthetic hemiasterlin analogue exhibiting binding to a and β tubulin. It induces disruption of spindle formation and mitotic arrest characteristic of anti-tubulin cancer drugs. Unlike taxanes and vincas, E7974 is a poor substrate for the PgP drug efflux pump, potentially overcoming this common mechanism of drug resistance. E7974 demonstrates broad anti-tumor activity against a variety of human tumor xenografts. Methods: An accelerated phase I dose escalation design was used to determine the maximum tolerated dose (MTD) and pharmacokinetic (PK) profile of E7974 administered IV over 2–5 minutes on Days 1 and 15 of a 28-day cycle. Results: Seventeen patients (14 male, 3 female) with a median age of 59 yrs. (range: 42–77 yrs.) were treated at doses of 0.18 mg/m2, 0.25 mg/m2, 0.31 mg/m2, and 0.39 mg/m2. Patients with colorectal (6), esophageal (3), prostate (2), urothelial (2), NSCLC (2), and other (2) cancers were enrolled. At the 0.39 mg/m2 dose level, one patient experienced grade 3 neutropenia and 2 patients experienced grade 4 neutropenia. The 0.31 mg/m2 dose was declared the MTD. Additional drug related toxicities (grade 1–2) included fatigue (8 pts.), constipation (4 pts), neuropathy (3 pts.), diarrhea (2 pts.), flushing (2 pts.) and myalgia (1 pt.). One patient with esophageal cancer had a partial response at the 0.39 mg/m2 dose level and one patient with prostate cancer had >50% decline in PSA and significant clinical benefit for >4 months at the 0.31 mg/m2 dose level. Stable disease was seen in two patients, one with esophageal cancer (8 cycles) and one with colorectal cancer (4 cycles). The PK profile of E7974 was characterized by moderate to large distribution (Vss = 55 - 187 L), slow clearance (CL = 1.99 - 8.77 L/hr) and moderate to slow elimination (t1/2 = 11.9 - 31.6 hr). Approximately 26 - 85% of the administered dose of E7974 was recovered unchanged in the urine 48 hours post-dose. Conclusions: The recommended phase 2 dose of E7974 on days 1 and 15 of a 28-day schedule is 0.31mg/m2. Observed toxicities were manageable and reversible. No significant financial relationships to disclose.

A phase I trial of E7974 administered on days 1, 8, and 15 of a 28-day cycle in patients with solid malignancies

2543 Background: E7974 is a synthetic hemiasterlin analogue exhibiting binding to a and β tubulin. It induces disruption of spindle formation and mitotic arrest characteristic of anti-tubulin cancer drugs. Unlike taxanes and vincas, E7974 is a poor substrate for the PgP drug efflux pump, potentially overcoming this common mechanism of drug resistance. E7974 demonstrates broad anti-tumor activity against a number of human tumor xenografts. Methods: We conducted the initial Phase I trial using an accelerated dose escalation design to determine the maximum tolerated dose (MTD) and pharmacokinetic (PK) profile of E7974 administered IV over 2–5 minutes on Days 1, 8, and 15 of a 28-day cycle. Results: Sixteen patients (7 male, 9 female) with a variety of tumor types and a median age of 54 yrs.(range: 33–78 yrs) were treated at doses of 0.15 mg/m2, 0.21 mg/m2, and 0.29 mg/m2. In the accelerated phase, 0.15 mg/m2 and 0.21 mg/m2 were tolerated. At the 0.29 mg/m2 dose level, one patient experienced grade 4 neutropenia after Day 8. Expansion of the 0.21 mg/m2 dose level resulted in one patient experiencing grade 4 neutropenic fever. Two other patients experienced grades 2 and 3 neutropenia, resulting in delayed dosing beyond Cycle 1. Five additional patients were treated at 0.15 mg/m2; none experienced a DLT or dose delay, thus confirming 0.15 mg/m2 as the MTD. Other observed drug- related toxicities included mild to moderate fatigue, nausea, vomiting, constipation, neuropathy, and myalgias. The PK profile of E7974 can be characterized by moderate to large distribution (Vss = 50 - 133 L), slow clearance (CL = 1.4 - 9 L/hr) and moderate to slow elimination (t1/2 = 6.3 - 31 hr). Approximately 34 - 84% of the administered dose was recovered unchanged in the urine 48 hours post-dose. Stable disease was seen in one patient with colorectal cancer (6 cycles). The MTD cohort has been expanded to a total of 16 evaluable patients for confirmation of tolerability and PK. Conclusions: The recommended Phase 2 dose on Days 1, 8, and 15 of a 28-day schedule is 0.15 mg/m2. Observed toxicities were manageable and reversible. Enrollment is ongoing. [Table: see text]

151: Targeting Chemoresistant Prostate Cancer with HTI-286, a Synthetic Analogue of the Marine Sponge Product Hemiasterlin

151: Targeting Chemoresistant Prostate Cancer with HTI-286, a Synthetic Analogue of the Marine Sponge Product Hemiasterlin

Inhibition of hepatic tumor cell proliferationin vitroand tumor growthin vivoby taltobulin, a synthetic analogue of the tripeptide hemiasterlin

To investigate the inhibitory effects of taltobulin (HTI-286), a synthetic analogue of natural hemiasterlin derived from marine sponges, on hepatic tumor growth in vitro and in vivo. The potential anti-proliferative effects of HTI-286 on different hepatic tumor cell lines in vitro and in vivo were examined. HTI-286 significantly inhibited proliferation of all three hepatic tumor cell lines (mean IC50 = 2 nmol/L +/- 1 nmol/L) in vitro. Interestingly, no decrease in viable primary human hepatocytes (PHH) was detected under HTI-286 exposure. Moreover, intravenous administration of HTI-286 significantly inhibited tumor growth in vivo (rat allograft model). HTI-286 might be considered a potent promising drug in treatment of liver malignancies. HTI-286 is currently undergoing clinical evaluation in cancer patients.

Structure-Based Identification of the Binding Site for the Hemiasterlin Analogue HTI-286 on Tubulin

A binding mode of HTI-286, a synthetic analogue of the peptidic antimitotic agent hemiasterlin, to tubulin is proposed. The binding mode was derived from iterative docking experiments directed at regions of the tubulin interdimer interface that are believed to be consistent with all current experimental data regarding the HTI-286/tubulin interaction. These data include (1) competitive inhibition of the tubulin binding of the Vinca alkaloids and other antimitotic agents, (2) proximity to stretches of amino acid residues identified in two separate photoaffinity-labeling experiments, (3) structure-activity relationships for HTI-286 and its analogues, (4) saturation transfer difference nuclear magnetic resonance (NMR) experiments, and (5) NMR transfer nuclear Overhauser effect spectroscopy (NOESY) experiments that potentially identify the bioactive conformation. The predicted binding mode thus affords a means to understand the mode of action of hemiasterlin, HTI-286, and other closely related molecules.

Synthetic tubulin inhibitor HTI-286 and hepatocellular carcinoma: an in-vitro and and in-vivo study

4201 Background: HTI-286 (HTI) is a synthetic tubulin inhibitor and a hemiasterlin analogue. It is a potent inhibitor of cell growth with an additional advantage of circumventing the P-glycoprotein-mediated resistance, which hampers efficacy of several antimicrotubule agents. Aim of our study was to investigate in-vitro antiproliferative effect of HTI in several hepatic cancer cell lines (Morris hepatoma (MH), Hep G2 and Hep 3B) and on primary human hepatocytes. Furthermore tumor growth inhibition was analyzed in a rat in-vivo study. Methods: MTT assay was done to determine the doubling time of each cell line. Exponentially growing cells were seeded with different concentrations of HTI for several doubling times. We determined the cytotoxicity and IC(50) by the MTT method. Microtubule structure was visualized by alpha-tubulin-immunofluoresence (aTIF). All animal experiments were approved by the local ethic commission. MH were implanted into American cancer institute rats (ACI) and tumor growth was pursued with MR-imaging (MR). HTI was administered via tail injection at several time points. The tumor growth was followed for over a period of one month. Results: In MH, HepG2 and Hep3B cells HTI has shown an antiproliferative effect already after one doubling time (22, 48 and 55 hours). IC(50) in MH cells was around 1 nM. Cytotoxic effect of HTI rose with increasing concentration. Proliferation of primary human hepatocytes was not affected by the HTI-treatment. aTIF showed a significant difference in HTI-treated compared to non-treated cells. The in-vivo study revealed significant growth inhibition in HTI treated group compared to a saline solution treated group. Conclusions: Our in-vitro data show that HTI inhibits proliferation of hepatic tumor cells in-vitro and tumor growth in-vivo. aTIF confirmed disrupture of microtubule organization. Furthermore primary human hepatocytes do not seem to be affected by the HTI treatment. The data suggest HTI to be considered as a potent promising cytotoxic and antiproliferative drug in liver oncology especially as it circumvents the P-glycoprotein-mediated drug resistance, being responsible for resistance to taxanes and vinca alkaloids. No significant financial relationships to disclose.

Tumor Cells Resistant to a Microtubule-Depolymerizing Hemiasterlin Analogue, HTI-286, Have Mutations in α- or β-Tubulin and Increased Microtubule Stability

Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for P-glycoprotein, they are attractive candidates for cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of hemiasterlin, HTI-286 (HTI), was examined in cell populations derived from ovarian carcinoma (A2780/1A9) cells selected in HTI-286. 1A9-HTI-resistant cells (1A9-HTI(R) series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble tubulin in 1A9 parental and 1A9-HTI(R) cells corroborated the HTI cytotoxicity data. At 22 degrees C or 37 degrees C, in the absence of any drug, the percentage of polymerized microtubules for each of the 1A9-HTI(R) populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTI(R) populations were also more resistant to depolymerization at 4 degrees C and had more acetylated and detyrosinated (Glu-tubulin) alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTI(R) cell populations exhibited either a single nucleotide change in the M40 beta-tubulin isotype, S172A, or in two cell populations where no beta-tubulin mutation was detected, mutations in the Kalpha-1 alpha-tubulin isotype, S165P and R221H in one resistant cell population and I384V in another. Unlike reports of mutations resulting in reduced drug affinity, the experimental data and location of mutations are consistent with resistance to HTI-286 mediated by microtubule-stabilizing mutations in beta- or alpha-tubulin.

Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in α-tubulin

Abstract HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [3H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [14C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major β-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in α-tubulin that substitutes Ser for Ala12 near the nonexchangeable GTP-binding site of α-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala12 mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of α-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3.