Mari Nakata

Effects of serotonin 5-HT3 receptor antagonists on stress-induced colonic hyperalgesia and diarrhoea in rats: a comparative study with opioid receptor agonists, a muscarinic receptor antagonist and a synthetic polymer.

Abstract  In this study, we examined the effects of serotonin (5‐HT)3 receptor antagonists (5‐HT3RAs) including ramosetron, alosetron, and cilansetron on colonic nociceptive threshold in rats. Furthermore, we established a restraint stress‐induced colonic hyperalgesia model in rats, and compared the inhibitory effects of 5‐HT3RAs on restraint stress‐induced colonic hyperalgesia and diarrhoea with those of loperamide, trimebutine, tiquizium and polycarbophil. The colonic nociceptive threshold was measured as the balloon pressure at the time the rat showed a nociceptive response during colonic distension by an intrarectally inserted balloon. Oral administration of ramosetron (3–30 μg kg−1), alosetron (30–300 μg kg−1), or cilansetron (30–300 μg kg−1) increased the colonic nociceptive threshold in a dose‐dependent manner in non‐stressed rats. Restraint stress for 1 h significantly decreased the colonic nociceptive threshold, but ramosetron (0.3–3 μg kg−1), alosetron (3–30 μg kg−1), cilansetron (3–30 μg kg−1) and trimebutine (100–1000 mg kg−1) significantly inhibited the decrease in the threshold. Loperamide (3–30 mg kg−1), tiquizium (100–1000 mg kg−1) and polycarbophil (1000 mg kg−1) did not affect the restraint stress‐induced decrease in the colonic nociceptive threshold. All drugs tested in this study showed dose‐dependent inhibition of restraint stress‐induced diarrhoea in rats. These results indicate that, unlike existing antidiarrhoeal and spasmolytic agents, 5‐HT3RAs have inhibitory effects on colonic nociception, and prevented restraint stress‐induced both diarrhoea and hyperalgesia at almost the same doses in rats. This suggests that the 5‐HT3RAs may be useful in ameliorating both colonic hyperalgesia and diarrhoea in patients with irritable bowel syndrome.

YM155, a selective survivin suppressant, inhibits tumor spread and prolongs survival in a spontaneous metastatic model of human triple negative breast cancer.

Metastatic triple negative breast cancer [TNBC, with negative expression of estrogen and progesterone receptors and no overexpression of HER2/neu (ErbB-2)] remains a major therapeutic challenge because of its poor overall prognosis and lack of optimal targeted therapies. Survivin has been implicated as an important mediator of breast cancer cell growth and dysfunctions in apoptosis, and its expression correlates with a higher incidence of metastases and patient mortality; thus, survivin is an attractive target for novel anti-cancer agents. In previous studies, we identified YM155 as a small molecule that selectively suppresses survivin expression. YM155 inhibits the growth of a wide range of human cancer cell lines. Tumor regression induced by YM155 is associated with decreased intratumoral survivin expression, increased apoptosis and a decreased mitotic index. In the present study, we evaluated the antitumor efficacy of YM155 both in vitro and in vivo using preclinical TNBC models. We found that YM155 suppressed survivin expression, including that of its splice variants (survivin 2B, δEx3 and 3B), resulting in decreased cellular proliferation and spontaneous apoptosis of human TNBC cells. In a mouse xenograft model, continuous infusion of YM155 led to the complete regression of subcutaneously established tumors. Furthermore, YM155 reduced spontaneous metastases and significantly prolonged the survival of animals bearing established metastatic tumors in the MDA-MB-231-Luc-D3H2-LN orthotopic model. These results suggest that the survivin-suppressing activity of YM155 may offer a novel therapeutic option for patients with metastatic TNBC.

Antitumor effects of YM155, a novel survivin suppressant, against human aggressive non-Hodgkin lymphoma.

YM155, a novel small-molecule that down-regulates survivin, exhibits broad, potent antitumor activity against a range of human tumors. We evaluated the activity of YM155 in aggressive non-Hodgkin lymphoma. In a number of diffuse large B-cell lymphoma lines, YM155 exhibited 50% growth inhibition with values between 0.23 and 3.9 nM. Within in vivo xenograft models, continuous infusion of YM155 eradicated large, established subcutaneous WSU-DLCL-2 and Ramos tumors, with sustained efficacy observed through 4 cycles of YM155 therapy. YM155 increased survival significantly versus rituximab in disseminated Ramos models. This study suggests that YM155 may represent an effective treatment for aggressive lymphomas.

Pharmacological profile of ramosetron, a novel therapeutic agent for IBS.

Abstract.Ramosetron is a potent and selective serotonin (5-HT)3 receptor antagonist that has been shown to affect abnormal colonic function and abdominal pain in animals. Ramosetron (0.3 to 100 µg/kg, p.o.) has been found to significantly suppress abnormal defecation induced by conditioned-fear stress (CFS), restraint stress, corticotropin releasing factor (CRF) and 5-HT in rats and mice, and these effects were more potent than those of alosetron, cilansetron or loperamide. On the other hand, ramosetron (3,000 µg/kg, p. o., once daily for 7 days) did not inhibit normal defecation in dogs while tiquizium significantly inhibited it. Ramosetron (3 to 100 µg/kg, p. o.) also significantly prevented CFS-induced acceleration of colonic transit and CRF-induced abnormal water transport in rats, respectively. Moreover, ramosetron (0.3 to 3 µg/kg, p. o.) significantly suppressed restraint stress-induced decrease in colonic pain threshold, an effect not observed with loperamide. These results indicate that ramosetron produce beneficial clinical effects on IBS symptoms.

Sepantronium Bromide (YM155) Enhances Response of Human B-cell non-Hodgkin Lymphoma to Rituximab

In the treatment of B-cell non-Hodgkin lymphoma (B-NHL) rituximab improves long-term survival in combination with conventional chemotherapy. However, because the majority of patients with B-NHL eventually relapse, the development of more effective therapies is needed. Here, we evaluated the antitumor effects of a combination treatment involving sepantronium bromide (YM155), a first-in-class survivin suppressant, and rituximab in B-NHL xenograft mice models. To determine the efficacy of the combination treatment, YM155- and rituximab-treated B-NHL cell xenografted mice were monitored for tumor size and survival and subjected to 2′-deoxy-2′-18F-fluoro-d-glucose (18F-FDG) and 3′-18F-fluoro-3′-deoxythymidine (18F-FLT) positron emission tomography (PET) imaging. In addition, the cell proliferation status of excised tumors was examined by Ki-67 immunohistochemistry. In DB, WSU-DLCL-2, and Mino xenograft-bearing mice, the combination treatment of YM155 and rituximab induced significant tumor growth inhibition and tumor regression compared with either single agent. On day 3 after the initiation of treatment a significant decrease in both 18F-FDG and 18F-FLT tumor uptake from pretreatment levels was observed in combination treatment groups. The Ki-67 proliferation index was significantly decreased on day 3 in the xenograft models treated with combination treatment, suggesting that the combination of YM155 and rituximab reduced cell proliferation and glucose metabolism. Furthermore, compared with monotherapy, combination treatment prolonged survival times of severe combined immunodeficient mice with disseminated WSU-FSCCL and Jeko B-NHL tumors. Our findings demonstrate that YM155 and rituximab combination treatment enhances antitumor activity in B-NHL xenografts, and 18F-FLT and 18F-FDG PET imaging may allow the early functional evaluation of treatment responses in patients with B-NHL.

Effects of serotonin 5-HT3 receptor antagonists on CRF-induced abnormal colonic water transport and defecation in rats

The effects of corticotropin releasing factor (CRF) and serotonin (5-HT)(3) receptor antagonists on intestinal water transport are not well understood. Hence, we established a CRF-induced abnormal water transport model in rat colon, and evaluated the effects of 5-HT(3) receptor antagonists including ramosetron, alosetron, and cilansetron, and the antidiarrheal agent loperamide, in this model. In addition, the effects of 5-HT(3) receptor antagonists and loperamide on abnormal defecation induced by CRF in rats were examined. Colonic water transport was measured in colonic loops in conscious rats. Centrally administered CRF (3-30 microg/kg) markedly decreased colonic fluid loss, whereas oral administration of ramosetron (3, 30 microg/kg), alosetron (300 microg/kg), cilansetron (300 microg/kg), or loperamide (3 mg/kg) significantly inhibited it. Ramosetron (1-10 microg/kg), alosetron (10-100 microg/kg), cilansetron (10-100 microg/kg), or loperamide (0.3-3 mg/kg) also showed dose-dependent inhibition of CRF-induced defecation in rats. These results suggest that 5-HT(3) receptors are involved in both abnormal colonic water transport and defecation induced by CRF, and that the inhibitory effects of 5-HT(3) receptor antagonists on CRF-induced abnormal defecation partly result from their ameliorating action on colonic water transport.

Antitumor efficacy and biodistribution of liposomal sepantronium bromide (YM155), a novel small-molecule survivin suppressant

Sepantronium bromide (YM155) exhibits time-dependent antitumor activity, although the plasma half-life of YM155 after a bolus intravenous (i.v.) administration is very short. Therefore, greater antitumor efficacy is obtained by continuous infusion than by bolus i.v. administration. In the present study, we attempted to liposomalize YM155 to obtain a longer circulation time than that achieved by bolus i.v. administration and yet retain sufficient antitumor activity. Encapsulation of YM155 in polyethylene glycol-coated liposomes extended the half-life of the drug, and high tumor accumulation of the drug was observed. Bolus i.v. administration of liposomal YM155 by a weekly administration regimen showed antitumor activity comparable to that obtained by the continuous infusion without severe toxicity in a murine xenograft model. Therefore, this liposomal formulation can be a new dosage form of YM155 that achieves sufficient efficacy and safety and is a more convenient administration regimen for users. It should be noted that liposomal YM155 showed unexpectedly high accumulation in the kidneys. This is a specific finding for liposomal YM155, offering important information for the consideration of the potential toxicity of liposomal YM155.

Abstract A227: Antitumor activities of ASP3026 against EML4-ALK-dependent tumor models.

EML4-ALK is an oncogenic fusion kinase, which was first identified in non-small cell lung cancer (NSCLC), and is regarded as an attractive therapeutic target for treating a subpopulation of NSCLC patients. Crizotinib, which is inhibitor for MET and ALK, was recently approved by FDA (26 August 2011) for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK)-positive as detected by an FDA-approved test. We synthesized and screened chemical compounds utilizing an ALK kinase inhibition assay aimed at the EML4-ALK target for drug discovery, and found ASP3026, a novel and selective inhibitor for the ALK kinase. ASP3026 potently inhibited ALK kinase activity and was more selective than crizotinib in a Tyr-kinase panel. In an anchorage independent in vitro cell growth assay, ASP3026 inhibited the growth of NCI-H2228, a human NSCLC tumor cell line endogenously expressing EML4-ALK variant 3 and that of 3T3 cells expressing EML4-ALK variant 1, 2 and 3. The plasma and tumor concentrations of ASP3026 in mice xenografted with NCI-H2228 tumor were determined using high-performance liquid chromatography-tandem mass spectrometry. Significant tumor penetration was observed. The antitumor activities were evaluated using mice bearing subcutaneous NCI-H2228 tumor xenografts. ASP3026, (daily oral dosing for 14 days) induced dose dependent anti-tumor effects starting at 1 mg/kg with marked regression at 10, 30 and 100 mg/kg. Body weights were unaffected. Crizotinib, (twice daily oral dosing) was less potent, with growth inhibition at 10 mg/kg, and tumor regression at 30 mg/kg. A dose of 100 mg/kg of crizotinib was poorly tolerated. Resistance mutations in ALK kinase domain against crizotinib were reported following sequence analysis of tumor cells derived from crizotinib-relapsed patients. The position of the mutation is the so-called gatekeeper mutation and is thought to be one of the causes of crizotinib relapse. In an EML4-ALK driven tumor model with gatekeeper mutation, ASP3026 showed potent anti-tumor effects while crizotinib was ineffective even at 100 mg/gk qd. In summary, ASP3026 has a broad safety margin and inhibitory activity at the gatekeeper mutation. Therefore, ASP3026 may still effective in EML4-ALK fusion positive NSCLC patients, that have relapsed to crizotinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A227.

EFFECTS OF MIRABEGRON (YM178) ON NON-MICTURITION CONTRACTIONS IN THE BLADDER OF CONSCIOUS RATS WITH BLADDER OUTLET OBSTRUCTION (BOO)

patients within the development cohort was 26 vs. 71 months in the external validation cohort (P<0.001). In overall survival analyses, the median values were 21 vs. 32 months for respectively the development and the external validation cohort (P<0.001). Three variables (age, stage and surgical status) were included in the nomograms predicting cancer-specific and overall mortality. In the external validation cohort, the nomograms achieved between 72 and 80% accuracy for prediction of ACC-specific or overall mortality at 1 to 5 years after either surgery or diagnosis of ACC for non-surgical patients. CONCLUSIONS: Our models are the first standardized and individualized prognostic tools for patients with ACC. Their accuracy was confirmed within a large external population-based cohort of ACC patients.

Abstract 2821: Anti-tumor activity of ASP3026, – A novel and selective ALK inhibitor -

EML4-ALK is an oncogenic fusion kinase which was first identified in non-small cell lung cancer (NSCLC), and is regarded as an attractive therapeutic target for treating a subpopulation of NSCLC patients. We synthesized and screened chemical compounds utilizing an ALK kinase inhibition assay aimed at the EML4-ALK target for drug discovery, and found ASP3026, a novel and selective inhibitor for the ALK kinase. ASP3026 inhibited ALK kinase activity at an IC 50 value of 3.5 nmol/L, and showed more selective ALK inhibition in a Tyr-kinase panel than PF02341066. In an anchorage independent in vitro cell growth assay, ASP3026 inhibited the growth of NCI-H2228, a human NSCLC tumor cell line endogenously expressing EML4-ALK variant 3, with an IC 50 value of 64.8 nmol/L. This growth inhibition was accompanied with the decrease in phosphorylation of EML4-ALK protein, indicating that ASP3026 exerts its anti-proliferative activity through ALK kinase inhibition. The plasma and tumor concentrations of ASP3026 in mice xenografted with NCI-H2228 tumor after a 5-day repeated oral dosing of ASP3026 (10 mg/kg once daily) were determined using high-performance liquid chromatography-tandem mass spectrometry. Tmax values were 4 h in plasma and tumors. Cmax values at the corresponding doses were, respectively, 875 nmol/mL and 15500 nmol/g. A decrease of phophorylated EML4-ALK was confirmed 4 hours after a single administration of ASP3026 at 10 mg/kg by Western-blot analysis. The antitumor activities were evaluated using mice bearing subcutaneous NCI-H2228 tumor xenografts. ASP3026, administered as twice daily oral dosing for 14 days, induced dose dependent anti-tumor effects starting at 1 mg/kg with strong regression at 10, 30 and 100 mg/kg. No influence on body weights was observed in all dose range of ASP3026 treated-mice. In contrast, PF02341066 at twice daily oral dosing resulted in growth inhibition of NCI-H2228 xenografted tumors at 10 mg/kg, and tumor regression at 30 mg/kg. In addition, 100 mg/kg of PF02341066 was intolerable in this model. These results suggest that ASP3026 is a novel and selective ALK inhibitor, which is orally active, and will possibly target NSCLC patients possessing the EML4-ALK fusion. We are starting phase I clinical trials of ASP3026 in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2821. doi:10.1158/1538-7445.AM2011-2821