Roppei Yamada

Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer treatment

Protein disulfide isomerase (PDI), an endoplasmic reticulum chaperone protein, catalyzes disulfide bond breakage, formation, and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective, and safe small-molecule inhibitors of PDI. Here, we report a class of propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and MS, we established that PACMA 31, one of the most active analogs, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with the active site cysteines of PDI. We also showed that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent an important approach for the development of targeted anticancer agents for ovarian cancer therapy, and they can also serve as useful probes for investigating the biology of PDI-implicated pathways.

Biological evaluation of paclitaxel-peptide conjugates as a model for MMP2-targeted drug delivery

Paclitaxel (PTX) is a highly effective and cytotoxic agent widely used for the treatment of several solid tumors. However, PTX shows dose-limiting cytotoxicity and in most of the cases induces drug resistance followed by failure in treatments. To enhance the therapeutic index of a given drug, various drug delivery methods are being explored to systemically deliver sufficient amount of the drug to the desired site. In the present study, we designed and synthesized two PTX prodrugs by conjugating PTX at different sites with an octapeptide (AcGPLGIAGQ) that can be cleaved by MMP-2 at tumor sites. As a result, PTX is expected to be released in the tumor sites, absorbed by the tumor cells, and thereby inhibit the tumor growth. We evaluated the in vitro activities of the two drugs in a panel of drug-sensitive and -resistant cancer cell lines and their in vivo efficacy in a HT1080 fibrosarcoma mouse xenograft model that highly overexpress MMP2. Our in vitro results showed that the PTX-AcGPLGIAGQ conjugates inhibited cancer cell proliferation with higher activity compared with that observed for free PTX, both of which were mediated by an arrest of G2/M-phase of the cell cycle. Consistent with the in vitro results, treatment with PTX-octapeptide conjugate resulted in extensive areas of necrosis and a lower percentage of proliferating cells in xenograft tumor sections. Together, our results indicate the potential of the tumor-targeted delivery of PTX exploiting the specific recognition of MMP2 to reduce toxicity and selective killing of tumor cells.

Discovery and Preclinical Evaluation of a Novel Class of Cytotoxic Propynoic Acid Carbamoyl Methyl Amides (PACMAs)

Herein, we discovered a series of propynoic acid carbamoyl methyl-amides (PACMAs) with potent cytotoxicity against a panel of cancer cell lines. These compounds interrupted cell cycle progression at low micromolar concentrations and induced early and late stage apoptosis. A representative compound suppressed tumor growth without apparent toxicity in an MDA-MB-435 mouse xenograft model. We used a Kinexus 628-antibody microarray and the Ingenuity Pathway Analysis (IPA) bioinformatics tools to better understand their mechanisms. The IPA analysis revealed the initiation of Nrf2-mediated oxidative stress through modulating the expression of SOD1 and STIP1 by compound 1. The involvement of the oxidative stress pathway was further validated by measuring the levels of the PACMA-induced mitochondrial superoxide species. To our knowledge, this is the first report on the discovery and biological evaluations of PACMAs as anticancer agents. Their broad-spectrum in vitro cytotoxicity, possibly through an oxidative stress-mediated pathway, and in vivo efficacy warrant further preclinical investigations.

Synthesis and biological evaluation of novel hydrazide based cytotoxic agents

Recently, we designed and synthesized a series of pyrroloquinoxaline compounds with hydrazine moiety linking a nitrogen-containing polycyclic group to a heteroaroyl system. Several derivatives, with attractive drug-like properties, were identified as promising cytotoxic agents, showing excellent potency in a panel of cancer cell lines. In the current study, we synthesized a further 19 new analogues to optimize their physicochemical properties and assess a coherent mechanism of action. Several chemical modifications were made to the reference compounds by varying the fused-ring system and/or the heteroacyl moiety. To evaluate their in vitro activity, we tested these compounds in six human cancer cell lines derived from different origins. Among them, two compounds (Figure 1 and Figure 1) showed similar potency as the reference compounds with IC50 values in the sub-micromolar range in all cell lines tested. Furthermore, compound Figure 1 showed excellent in vivo efficacy in our preliminary human ovarian cancer mouse xenograft studies. Flow cytometric studies indicated that both derivatives interrupted cell cycle progression in colorectal cancer HCT116 cell lines and ovarian cancer SKOV-3 cells. Further mechanistic studies revealed that Figure 1 and Figure 1 were able to induce reactive oxygen species in SKOV-3 cells with apparently different kinetic patterns. Considering their cytotoxicity profiles in a variety of in vitro and in vivo cancer models, these hydrazide based compounds seem to have considerable potentials as novel chemotherapeutics.

Reduced expression of the AdipoR1 gene is correlated with venous invasion in colorectal cancer

Serum adiponectin concentrations are negatively correlated with body fat percentage and with the risk of colorectal cancer. However, few studies have examined the relationship between adiponectin receptor expression and colorectal cancer. We measured the expression levels of the AdipoR1 and AdipoR2 genes by quantitative real-time reverse-transcription polymerase chain reaction in 202 paired specimens of cancer tissue and adjacent normal mucosa obtained from patients with colorectal cancer. To evaluate the clinical significance of AdipoR1 and AdipoR2, correlations between the expression of these genes and clinicopathological features were examined. Both genes were expressed in colorectal cancer and in adjacent normal mucosa. The expression levels of the genes were significantly higher in cancer tissue than in normal mucosa (P<0.0001). Reduced expression of the AdipoR1 gene was correlated with venous invasion, but not with any other clinicopathological feature examined. Our findings suggest that reduced expression of the AdipoR1 gene may be a useful predictor of venous invasion.

Discovery of a novel quinoxalinhydrazide with a broad-spectrum anticancer activity

Previously, we discovered a novel class of salicylhydrazide compounds with remarkable activity in hormone-dependent and -independent human cancer cells. We then designed and synthesized numerous analogues. Among these analogues, a quinoxalinhydrazide compound, SC144, exhibited desirable physicochemical and drug-like properties and therefore, was selected for further preclinical investigation. In the present study, we evaluated the in vitro activity of SC144 in a range of drug-sensitive and -resistant cancer cell lines as well as its in vivo efficacy in MDA-MB-435 and HT29 mice xenograft models. The broad-spectrum cytotoxicity of SC144 is especially highlighted by its potency in ovarian cancer cells resistant to cisplatin, breast-cancer cells resistant to doxorubicin, and colon cancer cells resistant to oxaliplatin. Furthermore, its activity was independent of p53, HER-2, estrogen and androgen receptor expressions. We also examined the effect of SC144 on cell cycle progression and apoptosis in select cell lines. Considering its cytotoxicity profile in a variety of in vitro and in vivo cancer models as well as its effects on cell cycle regulation and apoptosis, SC144 appears to represent a promising agent for further clinical development.

A Phase II Study of S-1 Monotherapy as a First-line Combination Therapy of S-1 Plus Cisplatin as a Second-line Therapy, and Weekly Paclitaxel Monotherapy as a Third-line Therapy in Patients with Advanced Gastric Carcinoma: A Second Report

Background We have previousy reported on a Phase II study of S-1 monotherapy as a first line, combination therapy of S-1 plus cisplatin as a second line, and weekly paclitaxel monotherapy as a third line therapy in patients with advanced gastric carcinomas. The median survival time (MST) of patients over the whole course of treatment was not previously calculated because 12 out of 19 patients had not yet succumbed. Since then, we have calculated the MST for this study and herein report our findings. Patients and Methods Between 2002 and 2005, 19 patients were enrolled in this study. Chemotherapy consisted of either 60 mg/m2 of S-1 for 4 weeks at 6-week intervals, a combination of 60 mg/m2 S-1 for 3 weeks and 60 mg/m2 cisplatin on day 8 at 5-week intervals, or 60 mg/m2 paclitaxel at days 1, 8, and 15, at 4-week intervals. The regimens were repeated until the occurrence of unacceptable toxicities, disease progression, or patient noncompliance. The primary end point was the overall survival. Results The median survival time was 774 days. The response rates were 33.3% (3/9), 12.5% (1/8), and 0% (0/4) after the first, second, and third line chemotherapies, respectively. The major adverse hematological toxicity was leukopenia, which reached grades 3–4 in all lines of chemotherapy investigated. In addition, the major adverse non-hematological toxicity was anorexia, which reached grade 3–4 in second line chemotherapy, and no deaths were attributable to the adverse effects of the drugs. Conclusion This sequential therapy was an effective treatment for advanced gastric cancer with acceptable toxic side-effects. We considered this therapy to be effective because of the smooth transition to the next regimen.

Combination effects of SC144 and cytotoxic anticancer agents.

Previously, we synthesized a series of hydrazide class of compounds and examined their cytotoxicity in a number of cancer cell lines. Among these analogues, SC144 exhibited potent cytotoxicity against a panel of drug-sensitive and drug-resistant cancer cell lines. To further explore its therapeutic potentials in the combination settings, we evaluated the synergy between SC144 and selected conventional chemotherapeutic agents in in-vitro cancer cell models. SC144 showed synergism with both 5-fluorouracil and oxaliplatin when cotreated in colorectal cancer HT29 cells. Pretreatment with SC144 in oxaliplatin-resistant HTOXAR3 cells was more effective than oxaliplatin pretreatment. In addition, the combination of SC144 and paclitaxel exhibited synergism in MDA-MB-435 cells with a schedule-dependent block in cell cycle. In an MDA-MB-435 mouse xenograft model, coadministration of SC144 and paclitaxel delayed tumor growth in an SC144 dose-dependent manner. Evaluation of the pharmacokinetics of SC144 revealed that intraperitoneal administration of SC144 showed a two-compartmental pharmacokinetics elimination profile that was not observed in the oral dosing. In summary, these studies further validate SC144 as a novel anticancer agent and provide insights for developing combination therapies for both drug-sensitive and drug-resistant cancers.

Phase II Study of S-1 Monotherapy as a First-line, Combination Therapy of S-1 plus Cisplatin as a Second-line, and Weekly Paclitaxel Monotherapy as a Third-line Therapy in Patients with Advanced Gastric Carcinoma Phase II Study of S-1, S-1 plus Cisplatin, and Weekly Paclitaxel in Patients with Advanced Gastric Carcinoma

Background We conducted a pilot phase II study to evaluate the efficacy and safety of S-1 as a first-line, S-1 plus cisplatin as a second-line, and weekly paclitaxel as a third-line therapy for advanced gastric cancer. Patients and Methods Between 2002 and 2005, 19 patients were enrolled in this study. Chemotherapy consisted of either 60 mg/m2 of S-1 for 4 weeks at 6 weeks interval, a combination of 60 mg/m2 S-1 for 3 weeks and 60 mg/m2 cisplatin on day 8 at 5 weeks interval, or 60 mg/m2 paclitaxel at day 1, 8, 15, at 4 weeks interval. The regimen was repeated until the occurrence of unacceptable toxicities, disease progression, or patient refusal. The primary end point was the overall survival. Results The response rates were 33.3%, 12.5%, and 0% after the first, second, and third line chemotherapy, respectively. The mean overall survival time was 994 days. The median survival time could not be calculated because 12 out of 19 patients were still alive when the study was concluded. Regarding hematological toxicity, the major adverse effect was leukopenia, which reached grades 3–4 in all lines of chemotherapy investigated. In addition, regarding non-hematological toxicities, the major adverse effect was anorexia, which reached grade 3–4 in the second line chemotherapy, and no deaths were attributable to the adverse effects of the drugs. Conclusion This sequential therapy was an effective treatment for advanced gastric cancer with acceptable toxic side-effects. We considered this sequential therapy to be effective because of the smooth switch to the next regimen.

Abstract B38: Clinical applications of PDX/NOG models for personalized chemotherapy – possible use in chemo-sensitivity testing and clinical sequencing

Personalized medicine represents an ideal medical approach for cancer therapy. In the field of clinical oncology, personalized medicine or therapy involves the evolutionary expansion of conventional clinical approaches that progress from patient evaluation, differential diagnosis, to the treatment of diseases. A number of the complex techniques employed in personalized medicine, such as clinical genome sequencing, biochemical marker analyses, chemo-sensitivity testing, and cancer immunotherapy, require fresh, viable, and sufficient amounts of specimens for reliable estimations. Mice bearing patient-derived xenografts (PDXs) with clinical information (so-called “Cancer Xenopatients”) are remarkable systems in personalized medicine for cancer. We previously reported the rapid and efficient establishment of PDXs using NOG mice (PDX/NOG models, AACR2015 #1940, IJO 47 61-70 2015). NOG mice, NOD/Shi-scid/IL2Rγ null (NOG) mice derived from NOD/SCID mice with a common gamma chain, have multifunctional defects in natural killer cell activity, macrophage function, complement activity, and dendritic cell function in addition to the absence of functional T and B lymphocytes. NOG mice have been identified as the most appropriate immunodeficient host animal for the direct xenografting of fresh tumor tissue due to the preservation of cancer stem cells (CSCs). Fresh and valuable xenograft samples, similar to surgical samples with the preservation of CSC, are stably provided using PDX/NOG models. Moreover, human tissue (tumor) and mouse tissue (stroma) are clearly distinguished by immunohistochemical analysis or gene arrangement sequencing. In the present study, we discussed the possibility of using PDX/NOG model simulations for personalized cancer chemotherapy. We previously established 47 lines of gastrointestinal cancer xenografts. In these cases, clinical information regarding chemotherapy for donor patients was retrieved where possible. Collagen gel droplet-embedded culture-drug sensitivity tests (CD-DST) were performed on 16 lines of PDX/NOG. In 4 of these lines, CD-DST were successfully conducted on original surgical specimens. The results of CD-DST between original and PDX/NOG specimens generally correlated (R 2 =0.01-0.89). The results of CD-DST using PDX/NOG specimens were compatible with the clinical effects of anti-cancer drugs. Genome sequencing and interactome analyses, a comprehensive analysis of tumor-stroma interactions innovated by Professor Ishikawa S. at Tokyo Medical and Dental University, were also performed on mRNA from 17 lines of PDX/NOG. Our interactome analyses showed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene-expression level by distinguishing gene arrangements in human tissue (tumor) from mouse tissue (stroma). The EGF-EGFR or VEGFA-KDR interactions observed closely reflected the clinical effectiveness of an EGFR inhibitor (Cetuximab) or VEGF-A inhibitor (Bevacizumab) as well as the results of in vivo chemo-sensitivity tests using PDX/NOG. The results of CD-DST and sequencing in PDX/NOG appear to be reliable for clinical simulations of chemotherapy and will definitely assist in the selection of the most sensitive anti-cancer drug for each patient. The fast and efficient establishment of individual PDXs will contribute to personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Takayuki Isagawa, Akira Noguchi, Hidemitsu Sato, Akimune Hayashi, Haruhiko Cho, Manabu Shiozawa, Takeshi Kishida, Soichiro Morinaga, Tomoyuki Yokose, Makoto Katayama, Nobuo Takenaka, Mizuha Haraguchi, Naoki Miyao, Yuichi Tateishi, Kenji Kawai, Hiroshi Suemizu, Roppei Yamada, Yoshiyasu Nakamura, Kohzoh Imai, Daisuke Komura, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. Clinical applications of PDX/NOG models for personalized chemotherapy – possible use in chemo-sensitivity testing and clinical sequencing. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B38.