Preclinical Development and Validation of ASP5354: A Near-Infrared Fluorescent Agent for Intraoperative Ureter Visualization.

PURPOSE: Iatrogenic ureteral injury (IUI) can complicate minimally invasive and open abdominopelvic surgery. The incidence of IUI is low and dependent on the type of surgery (< 10 %), but it is associated with high morbidity. Therefore, intraoperative visualization of the ureter is critical to reduce the incidence of IUI, and some methodologies for ureter visualization have been developed. Amongst these, near-infrared fluorescence (NIRF) visualization is thought to bring an advantage with real-time retroperitoneal visualization through the retroperitoneum. We investigated an indocyanine green (ICG) derivative, ASP5354, which emits NIRF at 820 nm when exposed to near-infrared light at a wavelength of 780 nm, in a rodent and porcine model. PROCEDURES: Wistar rats and Göttingen minipigs under anesthesia were laparotomized and then administered ASP5354 chloride intravenously at dose of 0.03 and 0.3 mg/kg for rats and 0.001 and 0.01 mg/kg for minipigs, respectively. Videos of the abdominal cavity in minipigs were taken using a near-infrared fluorescent camera (pde-neo) and assessed visually by three independent clinicians. Toxicological evaluation was demonstrated with cynomolgus monkeys. RESULTS: The proportion of animals whose ureters were visible up to 3 h after administration of ASP5354 chloride were 33 % at 0.001 mg/kg and 100 % at 0.01 mg/kg, respectively. In a toxicological study in cynomolgus monkeys, ASP5354 chloride demonstrated no significant toxicity, suggesting that 0.01 mg/kg provides an optimal dose when used clinically and could allow for ureter visualization during routine surgical procedures. CONCLUSIONS: The dose of 0.01 mg/kg provided an optimal dose for ureter visualization up to 3 h after administration. ASP5354 shows promise for ureter visualization during abdominopelvic surgery, which may potentially lower the risk of IUI.

Potential benefit of the cathepsin S inhibitor, ASP1617, as a treatment for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the dysregulation of various cell types and immunological pathways. Autoantibodies play an important role in its pathogenesis. The presence of autoantibodies suggests that self-antigen presentation through major histocompatibility complex (MHC) class II on antigen presenting cells is involved in the pathogenesis of autoimmune diseases, including SLE. Cathepsin S (CatS) is a key protease for antigen peptide loading onto lysosomal/endosomal MHC class II molecules through invariant chain degradation to promote antigen presentation. Inhibition of CatS is therefore expected to suppress antigen presentation via MHC class II, T and B cell activation, and antibody production from B cells. Here, we report the pharmacological profile of ASP1617, a novel CatS inhibitor. ASP1617 induced invariant chain accumulation and decreased the expression level of MHC class ΙΙ on the cell surface in both mouse and human B cells. Further, ASP1617 prevented DO11.10 mice T cell proliferation to ovalbumin antigen. We investigated the effects of ASP1617 and mycophenolate mofetil (MMF) on the development of lupus-like nephritis in NZB/W F1 mice, a widely used SLE mouse model. Oral administration of ASP1617 suppressed anti-dsDNA IgG, prevented progression of lupus-like glomerulonephritis, and significantly prevented proteinuria excretion. In contrast, MMF did not suppress anti-dsDNA IgG. Further, we found that plasma and/or urine CatS levels were increased in specimens from NZB/W F1 mice and several SLE patients. These results indicate that CatS may be an attractive therapeutic target for the treatment of SLE.

Full-length in meso structure and mechanism of rat kynurenine 3-monooxygenase inhibition.

The structural mechanisms of single-pass transmembrane enzymes remain elusive. Kynurenine 3-monooxygenase (KMO) is a mitochondrial protein involved in the eukaryotic tryptophan catabolic pathway and is linked to various diseases. Here, we report the mammalian full-length structure of KMO in its membrane-embedded form, complexed with compound 3 (identified internally) and compound 4 (identified via DNA-encoded chemical library screening) at 3.0 Å resolution. Despite predictions suggesting that KMO has two transmembrane domains, we show that KMO is actually a single-pass transmembrane protein, with the other transmembrane domain lying laterally along the membrane, where it forms part of the ligand-binding pocket. Further exploration of compound 3 led to identification of the brain-penetrant compound, 5. We show that KMO is dimeric, and that mutations at the dimeric interface abolish its activity. These results will provide insight for the drug discovery of additional blood-brain-barrier molecules, and help illuminate the complex biology behind single-pass transmembrane enzymes.

Tumor necrosis factor-alpha-nuclear factor-kappa B-signaling enhances St2b2 expression during 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia.

The mouse cholesterol sulfotransferase St2b2 contributes to epidermal differentiation by biosynthesizing cholesterol sulfate (CS) from cholesterol in the epidermis. 12-O-Tetradecanoylphorbol-13-acetate (TPA) causes epidermal hyperplasia, an abnormal increase in epidermal cell numbers resulting from aberrant cell differentiation and an increase in St2b2 protein levels. The mechanisms underlying enhanced St2b2 expression and the pathophysiologic significance of the increased expression are unclear, however. To verify whether increased St2b2 levels are necessary for TPA-induced epidermal hyperplasia, the effects of St2b2-specific small hairpin RNA (St2b2-shRNA) on hyperplasia were examined in mice. St2b2-shRNA clearly suppressed TPA-induced epidermal hyperplasia and the expression of a marker of epidermal differentiation, involucrin (INV). Interestingly, treating mouse epidermal cells with tumor necrosis factor-alpha (TNFα) increased St2b2 expression. Furthermore, treatment with TNFα-siRNA or anti-TNF receptor antibodies reduced the TPA-induced enhancement of St2b2 expression. Treatment with BAY 11-7082, a specific inhibitor of nuclear factor-kappa B (NF-κB), diminished TPA-induced St2b2 expression. These results suggested that enhancement of St2b2 expression by TPA treatment occurs mainly through the TNFα-NF-κB inflammatory signaling pathway, which in turn leads to increased CS concentrations in epidermal cells and hyperplasia.

[Comparative evaluation of 3D images reconstructed of MDCT or cone-beam CT: with special attention to depiction with abdominal IVR].

Diagnosis by CTHA (CT during hepatic arteriography) and CTAP (CT during arterio-portography) is indispensable in the treatment of hepatocellular carcinoma (HCC). An IVR-CT system makes it possible to perform accurate diagnosis and treatment of HCC in a short period of time. In recent years, attention has been drawn to cone-beam CT (CBCT) using a flat panel detector (FPD) angio-system, and the application of CBCT for CTAP and CTHA has been reported. However, it is well known that CBCT easily generates artifacts on the images, so it is necessary to use CBCT according to the intended purpose. The purpose of this study was to evaluate 3-D images reconstructed by MDCT or CBCT, image noise, and low-contrast resolution in a phantom with a tumor mimic model. From our results, CBCT images showed distortion and blurring, and it was difficult to visualize a tumor model of 7 mm or less. In addition CBCT can create only a small area of 3-D vascular mapping. In conclusion, it is considered that CBCT cannot be used in place of conventional CTHA or CTA for the treatment of HCC.

Association between renal dysfunction and mortality among inhabitants in the region around the Jinzu River basin polluted by cadmium.

A follow-up study was conducted on 5725 inhabitants (men 2858, women 2867) in and around the Jinzu River basin to determine the influence of environmental Cd exposure on mortality. In the Jinzu River basin, standardized mortality ratios (SMRs) investigated according to urinary findings (protein, glucose, and protein+glucose) were significantly low in the urinary protein-, glucose-, and protein+glucose-negative groups. SMRs calculated after dividing urinary protein- and glucose-positive status into two levels were lowest in the proteinuria- and glycosuria-negative groups and tended to be high in the higher positive groups. In the Jinzu River basin, Cox's hazard ratios were significantly higher for men and women in the urinary protein, glucose and protein+glucose-positive-groups. In the same analysis where the urinary protein- and glucose-positive subjects were divided into two levels, mortality was demonstrated to be higher in the groups with the greater degrees of proteinuria and glycosuria. In the Jinzu River water system almost all SMRs and Cox's hazard ratios showed statistical significance. This was not the case in the other water systems. Mortality of inhabitants with Cd-induced renal injury is increased in the Jinzu River basin.