Metabolic Characterization of Antifolate Responsiveness and Non-responsiveness in Malignant Pleural Mesothelioma Cells

Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM

Effect of Inhibition of the Lysophosphatidic Acid Receptor 1 on Metastasis and Metastatic Dormancy in Breast Cancer

Background Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated. Methods The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups. Results In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy. Conclusion The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor site

Japanese subpopulation analysis of MONARCH 2: phase 3 study of abemaciclib plus fulvestrant for treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer that progressed on endocrine therapy

BACKGROUND: This was a Japanese subpopulation analysis of MONARCH 2, a double-blind, randomized, placebo-controlled, phase 3 study of abemaciclib plus fulvestrant in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer (ABC). METHODS: Eligible women had progressed on (neo)adjuvant endocrine therapy (ET),  ≤ 12 months from end of adjuvant ET, or on first-line ET for ABC, and had not received chemotherapy for ABC. Patients were randomized 2:1 to receive abemaciclib or placebo plus fulvestrant. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), pharmacokinetics (PK), health-related quality of life (HRQoL), and safety. RESULTS: In Japan, 95 patients were randomized (abemaciclib, n = 64; placebo, n = 31). At final PFS analysis (February 14, 2017), median PFS was 21.2 and 14.3 months, respectively, in the abemaciclib and placebo groups (hazard ratio: 0.672; 95% confidence interval: 0.380-1.189). Abemaciclib had a higher objective response rate (37.5%) than placebo (12.9%). PK and safety profiles for Japanese patients were consistent with those of the overall population, without clinically meaningful differences across most HRQoL dimensions evaluated. The most frequent adverse events in the abemaciclib versus placebo groups were diarrhea (95.2 versus 25.8%), neutropenia (79.4 versus 0%), and leukopenia (66.7 versus 0%). At a second data cutoff (June 20, 2019), median OS was not reached with abemaciclib and 47.3 months with placebo (hazard ratio: 0.755; 95% confidence interval: 0.390-1.463). CONCLUSIONS: Results of the Japanese subpopulation were consistent with the improved clinical outcomes and manageable safety profile observed in the overall population. CLINICAL TRIAL REGISTRATION: NCT02107703; U.S. National Library of Medicine: https://clinicaltrials.gov/ct2/show/NCT02107703

Bioinformatics for Functional Analyses of Odorant Receptors

In order to delineate the functional aspects of odorant receptors as protein molecules, we here employed computational and biological approaches, each of which could eventually highlight different aspects of odorant receptors. We first characterized the non-redundant protein database to depict the general nature of proteins with a comprehensive database search strategy. We also employed a computational method called self-organizing map to understand possible relations of odorant receptors to other G-protein-coupled receptors. Furthermore, we have studied how to deliver foreign genes or proteins to olfactory sensory neurons in mice in vivo. Systematic synthesis of these computational and biological results could pave the way to our understanding of the whole nature of odorant receptors as protein molecules

Phase 3 trials of ixekizumab in moderate-to-severe plaque psoriasis

BACKGROUND Two phase 3 trials (UNCOVER-2 and UNCOVER-3) showed that at 12 weeks of treatment, ixekizumab, a monoclonal antibody against interleukin-17A, was superior to placebo and etanercept in the treatment of moderate-to-severe psoriasis. We report the 60-week data from the UNCOVER-2 and UNCOVER-3 trials, as well as 12-week and 60-week data from a third phase 3 trial, UNCOVER-1. METHODS We randomly assigned 1296 patients in the UNCOVER-1 trial, 1224 patients in the UNCOVER-2 trial, and 1346 patients in the UNCOVER-3 trial to receive subcutaneous injections of placebo (placebo group), 80 mg of ixekizumab every 2 weeks after a starting dose of 160 mg (2-wk dosing group), or 80 mg of ixekizumab every 4 weeks after a starting dose of 160 mg (4-wk dosing group). Additional cohorts in the UNCOVER-2 and UNCOVER-3 trials were randomly assigned to receive 50 mg of etanercept twice weekly. At week 12 in the UNCOVER-3 trial, the patients entered a long-term extension period during which they received 80 mg of ixekizumab every 4 weeks through week 60; at week 12 in the UNCOVER-1 and UNCOVER-2 trials, the patients who had a response to ixekizumab (defined as a static Physicians Global Assessment [sPGA] score of 0 [clear] or 1 [minimal psoriasis]) were randomly reassigned to receive placebo, 80 mg of ixekizumab every 4 weeks, or 80 mg of ixekizumab every 12 weeks through week 60. Coprimary end points were the percentage of patients who had a score on the sPGA of 0 or 1 and a 75% or greater reduction from baseline in Psoriasis Area and Severity Index (PASI 75) at week 12. RESULTS In the UNCOVER-1 trial, at week 12, the patients had better responses to ixekizumab than to placebo; in the 2-wk dosing group, 81.8% had an sPGA score of 0 or 1 and 89.1% had a PASI 75 response; in the 4-wk dosing group, the respective rates were 76.4% and 82.6%; and in the placebo group, the rates were 3.2% and 3.9% (P<0.001 for all comparisons of ixekizumab with placebo). In the UNCOVER-1 and UNCOVER-2 trials, among the patients who were randomly reassigned at week 12 to receive 80 mg of ixekizumab every 4 weeks, 80 mg of ixekizumab every 12 weeks, or placebo, an sPGA score of 0 or 1 was maintained by 73.8%, 39.0%, and 7.0% of the patients, respectively. Patients in the UNCOVER-3 trial received continuous treatment of ixekizumab from weeks 0 through 60, and at week 60, at least 73% had an sPGA score of 0 or 1 and at least 80% had a PASI 75 response. Adverse events reported during ixekizumab use included neutropenia, candidal infections, and inflammatory bowel disease. CONCLUSIONS In three phase 3 trials involving patients with psoriasis, ixekizumab was effective through 60 weeks of treatment. As with any treatment, the benefits need to be weighed against the risks of adverse events. The efficacy and safety of ixekizumab beyond 60 weeks of treatment are not yet known

Zebrafish-Based Screening Models for the Identification of Anti-Metastatic Drugs

Metastasis, a leading contributor to the morbidity of cancer patients, occurs through a multi-step process: invasion, intravasation, extravasation, colonization, and metastatic tumor formation. Each process is not only promoted by cancer cells themselves but is also affected by their microenvironment. Given this complexity, drug discovery for anti-metastatic drugs must consider the interaction between cancer cells and their microenvironments. The zebrafish is a suitable vertebrate animal model for in vivo high-throughput screening studies with physiological relevance to humans. This review covers the zebrafish model used to identify anti-metastatic drugs

Development of an in vivo tissue-engineered, autologous heart valve (the biovalve) : preparation of a prototype model

Objective: This study aimed to develop an autologous heart valve without using traditional in vitro tissue-engineering methods, which necessitate complicated cell management protocols under exceptionally clean laboratory facilities. Methods: An autologous heart valve construct composed of trileaflets was prepared using a specially designed mold. The mold was prepared by covering a silicone rod with a crown-shaped tubular polyurethane scaffold containing 3 horns. The mold was implanted in the dorsal subcutaneous space in Japan White rabbits for 4 weeks. After harvesting, the implanted trileaflet valve-shaped structure with an internal diameter of either 5 or 20 mm was obtained by trimming the membranous tissue formed between the horns located around the silicone rod. The valve substitute was examined both macroscopically and histologically. The tensile strength of the leaflets was measured to rupture. The degree of regurgitation in valve function was evaluated using a flow circuit by calculating the ratio of the regurgitation volume to the forward flow volume. Results: After implantation, the mold was completely covered with connective tissue consisting mostly of collagen and fibroblasts. Harvesting of the mold was straightforward, because there was little adhesion between the formed tissue and the native skin tissue. The trileaflet heart valve construct was obtained after withdrawing the inserted rods and trimming the membranous tissues formed between the horns of the scaffold. It was firmly attached to the scaffold, the interstices and surface of which revealed connective tissues composed of components similar to those of the leaflet tissue. Although the mechanical properties of the leaflet tissue were less efficient than those of the native porcine aortic valve leaflets, satisfactory valvular functions were demonstrated under pulsatile conditions using a flow circuit. No regurgitation was observed under retrograde hydrostatic pressures of up to 60 mm Hg, the physiologic pressure acting on the aortic valves during retrograde aortic flow. Conclusions: The biovalve, an autologous, in vivo tissue-engineered, trileaflet, valve-shaped construct, was developed using our novel in-body tissue architecture technology. The biovalve has the potential to be an ideal prosthetic heart valve, with excellent biocompatibility to the growth of the recipient’s heart

Plant Nitrogen Levels and Photosynthesis in the Supernodulating (Glycine max L. Merr.) Cultivar ‘Sakukei 4’

Supernodulating soybeans, having several times more nodules than normal genotypes, are expected to produce higher dry matter and grain yields through a higher nitrogen fixation potential. However, their growth and yield have been inferior to those of normal genotypes. We have recently developed the supernodulating genotype‘Sakukei 4’(formerly‘En-b0-1-2’, presently‘Kanto 100’), with improved growth and yield and a high nitrogen fixation potential. The objective of the present study was to examine the time course of changes in plant nitrogen content, leaf chlorophyll content (SPAD value), and photosynthetic rate of Sakukei 4 to reveal the source of its advantages in growth. The leaf nitrogen content after the flowering stage and the stem nitrogen content after the seed-filling stage were higher in Sakukei 4 than in the normal cultivar‘Enrei’. The SPAD values in Enrei and another normal cultivar,‘Tamahomare’, decreased rapidly after the seed-filling stage, whereas that in Sakukei 4 stayed high until the late seed-filling stage. Differences in photosynthetic rate and leaf SPAD value between Sakukei 4 and Enrei were negligible at the beginning of podding but became very clear at the seed-filling stage because of the drop in the values for Enrei. In Sakukei 4, a large amount of nitrogen might not be translocated from leaf to seed during the seed-filling stage because of the higher capability of the nodules to send fixed nitrogen to the growing seeds. Sakukei 4 could thus maintain a high photosynthetic rate and grain growth during the seed-filling stage