Inhibition of Notch4 using Novel Neutralizing Antibodies Reduces Tumor Growth in Murine Cancer Models by Targeting the Tumor Endothelium.

Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed on tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancer. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms.

Safety of sarilumab in a Japanese population with rheumatoid arthritis by age group: Data from an interim analysis of a post-marketing surveillance study.

OBJECTIVES: Using data from a post-marketing surveillance, this interim subgroup analysis investigated the safety of sarilumab in younger (<65 years) and older patients (≥65 and ≥75 years) with rheumatoid arthritis. METHODS: During this interim analysis, patients who were treated with sarilumab in Japan were enrolled between June 2018-2021. Data collected by 12 January 2022 were analysed, with adverse drug events monitored over 52 weeks. RESULTS: Of 972 patients with available data, proportion of patients aged <65 years, ≥65 years and ≥75 years were 40.8%, 59.2% and 27.8%, respectively. Most patients (95.5%) received the standard 200 mg dose of sarilumab as the initial dose. Adverse drug reactions were reported in 24.6% of patients, with serious events accounting for 6.4% of cases. No malignancy and low incidences of adverse drug reactions of special interest were reported across all age groups (<65 years, 7.8%; ≥65 years, 8.2%; ≥75 years, 8.5%). When stratified by absolute neutrophil count above and below the lower limit of normal, there were no numerical differences in incidences of serious and non-serious infections between age groups. CONCLUSIONS: Regardless of age, sarilumab therapy was well tolerated by patients with rheumatoid arthritis, with no new safety signals reported in this study.

Safety and effectiveness of sarilumab in Japanese patients with rheumatoid arthritis refractory to previous treatments: An interim analysis of a post-marketing surveillance.

OBJECTIVES: An interim analysis of post-marketing surveillance data to assess the safety and effectiveness of sarilumab in Japanese patients with rheumatoid arthritis refractory to previous treatment. METHODS: The interim analysis included patients who initiated sarilumab therapy between June 2018 and January 2021. The primary objective of this surveillance was safety. RESULTS: In total, 1036 patients were enrolled and registered by 12 January 2021 (interim cut-off date). Of these, 678 were included in the safety analysis [75.4% female; mean age (± standard deviation) 65.8 ± 13.0 years]. Adverse drug reactions, defined as adverse events classified as possibly or probably related to sarilumab, were reported in 170 patients (incidence: 25.1%), with white blood cell count decreased (4.4%) and neutrophil count decreased (1.6%) most frequently reported. Serious haematologic disorders (3.4%) and serious infections (including tuberculosis) (2.5%) were the most frequently reported priority surveillance items. No malignant tumour was reported. An absolute neutrophil count (ANC) below the minimum standard value did not increase the incidence of serious infections. CONCLUSIONS: Sarilumab was well tolerated, and no new safety signals were noted in this analysis. There was no difference in the frequency of serious infections between patients with an ANC below or above normal.

Immunogenicity and Safety of a Two-Dose Series of a Meningococcal (Groups A, C, W, and Y) Polysaccharide Diphtheria Toxoid Conjugate Vaccine (Men-ACWY-D) in Healthy Japanese Adults.

The quadrivalent meningococcal polysaccharide diphtheria toxoid conjugate vaccine (Men-ACWY-D) has been licensed for use in Japan since 2014. An earlier registration study demonstrated the immunogenicity of a single dose in Japanese adults, wherein the immunogenicity against serogroup C was the lowest. The determination of the potential to increase the serogroup C response with a second dose was, therefore, of interest. This study (NCT02591290) evaluated the safety and immunogenicity of two doses administered 8 weeks apart to 60 healthy Japanese adults aged 20-55 years. Blood samples were collected at 28-35 days after vaccination. Immunogenicity endpoints included seroprotection and seroconversion rates. Safety assessments included systemic adverse events (AEs), non-serious AEs, and serious AEs. Fifty-eight participants (96.7%) completed the study. The seroprotection rates for serogroups A, C, W, and Y before vaccination were 76.8%, 26.8%, 26.8%, and 50.0%, respectively, increasing to 100%, 83.9%, 91.1%, and 96.4% and 100%, 92.9%, 94.6%, and 94.6%, respectively, after two doses. The seroconversion rates for the four serogroups were 100%, 93.8%, 97.1%, and 94.1%, respectively, after the first dose, and 100%, 96.9%, 100%, and 100%, respectively, after the second. The increase between the doses was insignificant, and there were no safety concerns. The two-dose series was well tolerated; however, the clinical benefits of a second dose within 8 weeks seemed to be low.

Gene expression analysis using a modified HiCEP method applicable to prokaryotes: a study of the response of Rhodococcus to isoniazid and ethambutol.

We established a novel method to analyze the gene expression of prokaryotes by modifying and optimizing the HiCEP (high coverage gene expression analysis) method, which was originally developed for eukaryotic gene expression profiling. Following the removal of abundant rRNA, the mRNA of prokaryotic cells was enzymatically polyadenylated and subjected to HiCEP analysis. This method was highly reproducible due to selective PCR that was performed by using adaptor specific primers. We confirmed induction of tipA and induction or suppression of cspA, which are genes that are obtained from distinctive actinomycetes and responded to thiostrepton and temperature stress, respectively. Further, we applied this method to explore the gene expression profile of Rhodococcus erythropolis in response to drugs that inhibit cell wall synthetic pathways, and we were able to identify 35 upregulated genes. Among these genes, we confirmed the upregulation of 22 genes by using RT-PCR (reverse transcriptase-polymerase chain reaction). Some of these genes are involved in the synthesis of mycolic acid and arabinogalactan, suggesting a cell response to drug treatment by regulation of the genes involved in cell wall synthesis. This method could prove to be a useful technique for gene expression analysis of prokaryotes, particularly nonmodel strains with unknown genome sequences.

Effects of fexofenadine hydrochloride in a guinea pig model of antigen-induced rhinitis.

Allergic rhinitis is an inflammatory disease of the nasal mucosa, induced by histamine, leukotrienes, and other substances released from mast cells. Fexofenadine hydrochloride, the active metabolite of terfenadine, is a novel, nonsedating antiallergic drug having H1 receptor antagonistic activity. Fexofenadine is effective for the treatment of allergic rhinitis. However, its mechanism of action in attenuating nasal congestion has not yet been elucidated. Therefore, we first examined the effects of fexofenadine on a guinea pig model of antigen-induced rhinitis. We also evaluated the effects of mepyramine, zafirlukast and ramatroban in this model; these drugs are an H1 receptor antagonist, a selective leukotriene antagonist and a selective thromboxane antagonist, respectively. Rhinitis was induced by ovalbumin (OVA) instillation into the nasal cavity of animals that had been sensitized by two earlier OVA injections (s.c. and i.p.). The nasal airway resistance was measured for 45 min after the challenge. Fexofenadine hydrochloride (20 mg/kg) and terfenadine (20 mg/kg) administered orally 70 min prior to the challenge significantly inhibited (fexofenadine, p < 0.001, terfenadine, p < 0.05) the increase in nasal airway resistance. Ramatroban (30 mg/kg) administered orally 60 min prior to the challenge also significantly inhibited (p < 0.05) the increase in nasal airway resistance. In contrast, mepyramine (3 mg/kg i.v.) and zafirlukast (3 mg/kg p.o.) failed to reduce the increase in nasal airway resistance. These results suggest that thromboxane may be involved in the increase in the nasal airway resistance in this model. Accordingly, fexofenadine may reduce the increase in nasal airway resistance by inhibiting the release of chemical mediators, including thromboxane, that are involved in the increase in nasal airway resistance in this model.

Differential expression of two catechol 1,2-dioxygenases in Burkholderia sp. strain TH2.

Burkholderia sp. strain TH2, a 2-chlorobenzoate (2CB)-degrading bacterium, metabolizes benzoate (BA) and 2CB via catechol. Two different gene clusters for the catechol ortho-cleavage pathway (cat1 and cat2) were cloned from TH2 and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that while both catechol dioxygenases (CatA1 and CatA2) were produced in BA-grown cells, CatA1 was undetectable when strain TH2 was grown on 2CB or cis,cis-muconate (CCM), an intermediate of catechol degradation. However, production of CatA1 during growth on 2CB or CCM was observed when cat2 genes were disrupted. The difference in the production of CatA1 and CatA2 was apparently due to a difference in inducer recognition by the regulators of the gene clusters. The inducer of CatA1 was found to be BA, not 2CB, by using a 2-halobenzoate dioxygenase gene (cbd) disruptant, which is incapable of transforming (chloro)benzoate. It was also found that CCM or its metabolite acts as an inducer for CatA2. When cat2 genes were disrupted, the growth rate in 2CB culture was reduced while that in BA culture was not. These results suggest that although cat2 genes are not indispensable for growth of TH2 on 2CB, they are advantageous.

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene.

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.