Eiji Sasaki

Neutralization of DHG, a New Depolymerized Holothurian Glycosaminoglycan, by Protamine Sulfate and Platelet Factor 4

The neutralization of depolymerized holothurian glycosaminoglycan (DHG), unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH) by protamine sulfate (PS) or platelet factor 4 (PF4) was studied. In in vitro studies, the prolongation of thrombin clotting time (TCT) by these glycosaminoglycans was completely neutralized by PS, whereas activated partial thromboplastin time (APTT) was relatively resistant to neutralization. In rats, prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT ex vivo. Heparin-cofactor-II-dependent antithrombin activity of DHG or UFH was neutralized by PF4 at a high concentration to the same extent; however, prolongation of the APTT by DHG was more resistant to neutralization by PF4 at a physiological plasma level than that by UFH. In conclusion, since the prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT, and since PF4 neutralized the antithrombin activity of DHG, these proteins may be useful as antidotes for DHG to prevent bleeding in case of an overdose.

TAS-303, a novel selective norepinephrine reuptake inhibitor that increases urethral pressure in rats, indicating its potential as a therapeutic agent for stress urinary incontinence

Stress urinary incontinence (SUI) is characterized by involuntary leakage associated with exertion, effort, sneezing, coughing, or lifting. Duloxetine, a serotonin norepinephrine reuptake inhibitor, is approved for the treatment of patients with SUI in some European countries, but not in the United States. There is currently no globally approved pharmacological drug for the treatment of patients with SUI. Therefore, a new pharmacological treatment option is required. TAS-303 [4-piperidinyl 2,2-diphenyl-2-(propoxy-1,1,2,2,3,3,3-day7)acetate hydrochloride] is a novel small-molecule selective norepinephrine reuptake inhibitor that displays significant norepinephrine transporter (NET) inhibitory activity toward the serotonin or dopamine transporters. In this report, we describe the pharmacological properties of TAS-303 and its effects on urethral function, using preclinical in vitro and in vivo studies. Radioligand-binding studies showed that TAS-303 selectively and potently inhibited [3H]norepinephrine binding to the human NET. Oral administration of TAS-303 (3 mg/kg) significantly increased norepinephrine levels in the plasma, whereas it did not significantly affect epinephrine, dopamine, and serotonin levels. TAS-303 (0.3, 1, and 3 mg/kg) dose-dependently increased basal urethral pressure in normal rats and leak point pressure in vaginal distention rats, exhibiting a maximal effect comparable to duloxetine. In the forced swimming test, TAS-303 (100 mg/kg) showed no significant effects on immobility time in rats, raising the possibility that this agent would have minimal central nervous system side effects at an effective dose for urethral function. These results demonstrate that TAS-303 has therapeutic potential for the treatment of patients with SUI.

Therapeutic effect of TAC-302, a cyclohexenoic fatty alcohol derivative, on bladder denervation-related storage and voiding dysfunctions in rats

To evaluate the therapeutic effect of TAC‐302, a cyclohexenoic fatty alcohol derivative, on bladder denervation‐related storage and voiding dysfunctions in rats with bladder outlet obstruction (BOO).

TAC-302 promotes neurite outgrowth of isolated peripheral neurons and prevents bladder denervation related bladder dysfunctions following bladder outlet obstruction in rats

To evaluate the ability of TAC‐302, a cyclohexenoic fatty alcohol derivative, to enhance neurite outgrowth in cultured rat dorsal root ganglion (DRG) neurons, and the preventive effects of TAC‐302 on bladder denervation‐related storage and voiding dysfunctions in rats with bladder outlet obstruction (BOO).

TAS05567, a Novel Potent and Selective Spleen Tyrosine Kinase Inhibitor, Abrogates Immunoglobulin-Mediated Autoimmune and Allergic Reactions in Rodent Models

Spleen tyrosine kinase (Syk) is involved in regulation of B-cell receptor (BCR) and Fc receptor downstream signal pathways. Syk plays an essential role in production of inflammatory mediators and differentiation in various immune cells and is therefore an attractive target for treating inflammatory conditions, such as autoimmune and allergic diseases. We identified TAS05567 as a highly selective Syk inhibitor and evaluated its therapeutic potential in animal models. In vitro biochemical assays were performed with available kinase assay panels. Inhibitory effects of TAS05567 on immune cells were analyzed by assessing the Syk downstream signaling pathway and production of inflammatory factors. In vivo effects of TAS05567 were evaluated in animal models of autoimmune diseases and antigen-specific IgE transgenic mice. TAS05567 inhibited only 4 of 191 kinases tested but inhibited Syk enzymatic activity with high potency. TAS05567 inhibited BCR-dependent signal transduction in Ramos cells, FcγR-mediated tumor necrosis factor-α production in THP-1 cells, and FcεR-mediated histamine release from RBL-2H3 cells. In rheumatoid arthritis models, TAS05567 suppressed hind-paw swelling in a dose-dependent manner compared with vehicle. Moreover, TAS05667 markedly reduced histopathologic scores in an established rat arthritis model. In a mouse immune thrombocytopenic purpura model, platelet counts were reduced with injection of anti-platelet antibody. TAS05567 prevented the platelet count decrease in a dose-dependent manner. Finally, TAS05567 treatment suppressed IgE-mediated ear swelling in vivo. Collectively, our data indicate TAS05567 is a selective Syk inhibitor and potential therapeutic candidate for treating humoral immune-mediated inflammatory conditions such as autoimmune and allergic diseases.

Identification of Cha o 3 homolog Cry j 4 from Cryptomeria japonica (Japanese cedar) pollen: Limitation of the present Japanese cedar–specific ASIT

BACKGROUNDnAbout one-third of the Japanese population suffers from Japanese cedar pollinosis, which is frequently accompanied by Japanese cypress pollinosis. Recently, a novel major Japanese cypress pollen allergen, Cha o 3, was discovered. However, whether a Cha o 3 homolog is present in Japanese cedar pollen remains to be determined.nnnMETHODSnWestern blot analysis was performed using Cha o 3-specific antiserum. In addition, cloning of the gene encoding Cry j 4 was conducted using total cDNA from the male flower of Japanese cedar trees. Allergen potency and cross-reactivity were investigated using a T-cell proliferation assay, basophil activation test, and ImmunoCAP inhibition assay.nnnRESULTSnA low amount of Cha o 3 homolog protein was detected in Japanese cedar pollen extract. The deduced amino acid sequence of Cry j 4 showed 84% identity to that of Cha o 3. Cross-reactivity between Cry j 4 and Cha o 3 was observed at the T cell and IgE levels.nnnCONCLUSIONSnCry j 4 was discovered as a counterpart allergen of Cha o 3 in Japanese cedar pollen, with a relationship similar to that between Cry j 1-Cha o 1 and Cry j 2-Cha o 2. Our findings also suggest that allergen-specific immunotherapy (ASIT) using Japanese cedar pollen extract does not induce adequate immune tolerance to Cha o 3 due to the low amount of Cry j 4 in Japanese cedar pollen. Therefore, ASIT using Cha o 3 or cypress pollen extract coupled with Japanese cedar pollen extract is required in order to optimally control allergy symptoms during Japanese cypress pollen season.

Abstract PR09: TAS3681, a new type androgen receptor antagonist, suppresses ligand-independent AR activation through its AR downregulation activity

Background: Inhibitors of androgen receptor (AR) signaling axis, such as enzalutamide and abiraterone, represent important advances in the management of castration-resistant prostate cancer (CRPC). However, resistance to these drugs is still occurring. Recent reports suggest that there are several mechanisms of resistance. Ligand independent AR activation such as induction of AR splice variants or c-Myc expression is a major emerging mechanism of CRPC progression. There is an urgent need for developing a novel therapy to address these unresolved issues. Methods: For assay of androgen-independent AR transactivation, prostate cancer cells were transiently transfected with androgen-responsive reporter gene construct. The transfected cells were treated with growth factor and cytokine in steroid-depleted media, and luciferase activity was measured. To evaluate the effect of TAS3681 on AR and c-Myc protein expression, prostate cancer cells were treated with TAS3681 in steroid-depleted media. AR and c-Myc protein levels were determined by Western blot. Real-time PCR was used to analyze the mRNA levels of c-Myc and c-Myc target gene. Chromatin immunoprecipitation was performed to determine the enrichment of AR at the element. Results: TAS3681 dose-dependently reduced AR protein levels in prostate cancer cells. In contrast to enzalutamide, TAS3681 suppressed androgen-independent AR transactivation by growth factor and cytokine. In prostate cancer cells which express full-length AR and splice variant AR-v7, TAS3681 suppressed AR-v7 target gene expression through downregulation of AR-v7 occupancy at the enhancer. Moreover, TAS3681 reduced expression of c-Myc, critical driver of androgen-independent mechanisms of prostate cancer progression, via AR downregulation activity. In addition, real-time PCR assay showed the suppression of c-Myc and c-Myc target gene mRNA levels by TAS3681 but not by enzalutamide. Conclusion: Our findings suggest that TAS3681, a new type AR antagonist with AR downregulation activity, has a potential to overcome ligand-independent AR activation and could be a candidate of breakthrough therapy for resistance to current AR pathway target drugs. Citation Format: Daisuke Kajiwara, Kazuhisa Minamiguchi, Masanao Seki, Hiroya Mizutani, Hiroki Aoyagi, Shigeo Okajima, Eiji Sasaki, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS3681, a new type androgen receptor antagonist, suppresses ligand-independent AR activation through its AR downregulation activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR09.