Discovery of 4,6- and 5,7-Disubstituted Isoquinoline Derivatives as a Novel Class of Protein Kinase C ζ Inhibitors with Fragment-Merging Strategy.

Two chemical series of novel protein kinase C ζ (PKCζ) inhibitors, 4,6-disubstituted and 5,7-disubstituted isoquinolines, were rapidly identified using our fragment merging strategy. This methodology involves biochemical screening of a high concentration of a monosubstituted isoquinoline fragment library, then merging hit isoquinoline fragments into a single compound. Our strategy can be applied to the discovery of other challenging kinase inhibitors without protein-ligand structural information. Furthermore, our optimization effort identified the highly potent and orally available 5,7-isoquinoline 37 from the second chemical series. Compound 37 showed good efficacy in a mouse collagen-induced arthritis model. The in vivo studies suggest that PKCζ inhibition is a novel target for rheumatoid arthritis (RA) and that 5,7-disubstituted isoquinoline 37 has the potential to elucidate the biological consequences of PKCζ inhibition, specifically in terms of therapeutic intervention for RA.

Discovery of Novel Transient Receptor Potential Vanilloid 4 (TRPV4) Agonists as Regulators of Chondrogenic Differentiation: Identification of Quinazolin-4(3 H)-ones and in Vivo Studies on a Surgically Induced Rat Model of Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease characterized by joint destruction and loss of cartilage. There are many unmet needs in the treatment of OA and there are few promising candidates for disease-modifying OA drugs, particularly, anabolic agents. Here, we describe the identification of novel quinazolin-4(3 H)-one derivatives, which stimulate chondrocyte cartilage matrix production via TRPV4 and mitigate damaged articular cartilage. We successfully identified the water-soluble, highly potent quinazolin-4(3 H)-one derivative 36 and studied its intra-articular physicochemical profile to use in in vivo surgical OA model studies. Compound 36·HCl provided relief from OA damage in a rat medial meniscal tear (MT) model. Specifically, 36·HCl dose-dependently suppressed cartilage degradation and enhanced the messenger RNA expression of aggrecan and SOX9 in cartilage isolated from MT-operated rat knees compared with knees treated with vehicle. These results suggest that 36 induces anabolic changes in articular cartilage and consequently reduces OA progression.

Uptake and metabolism of mizoribine, an immunosuppressant, in L5178Y-R mouse lymphoma cells in vitro and peripheral blood mononuclear cells of rats and kidney transplant recipients in vivo.

The cellular uptake of mizoribine (MZR), an immunosuppressant, and metabolism of MZR to MZR-5'- monophosphate (MZRP), an active metabolite, were evaluated in L5178Y-R mouse lymphoma cells and peripheral blood mononuclear cells (PBMCs) of rats and kidney transplant recipients (KTRs, n = 22). Real-time PCR analysis revealed the expression of ENT1 and ENT2 mRNAs, but not of CNTs, in L5178Y-R cells and rat's PBMCs. In L5178Y-R cells, the uptake of MZR was suppressed by adenosine, a substrate for ENT1 and ENT2, but not by 5-(4-nitrobenzyl)-6-thioinosine (0.1 µM), an ENT1 inhibitor. Saturable metabolism of MZR to MZRP was observed. In rats, peak plasma concentrations of MZR and peak concentrations of MZR and MZRP in PBMCs were observed 3 h after oral administration. MZR disappeared from PBMCs in parallel with plasma MZR, but the disappearance of MZRP from PBMCs appeared to be slow. In KTRs, the mean plasma concentration of MZR 3-4 h after ingestion was 3.14 µg/ml and the mean MZRP concentration in PBMCs was 16.8% of MZR, reflecting the involvement of ENT in the uptake of MZR. A linear relationship was observed between plasma MZR concentrations ranging from 1 to 6 µg/ml and PBMC's MZRP concentrations ranging from 90 to 200 ng/ml.

Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study.

This study examined the potential for osteogenesis via regenerative medicine using autologous tissues (umbilical cord (UC) and umbilical cord blood (UCB)) in nude mice. The study was designed to provide the three elements required for regenerative medicine (cell, scaffold, and growth factor) and autoserum for culture by means of autologous tissues. Mesenchymal stromal cells were obtained from UC (UC-MSCs). Fibrin, platelet-rich-plasma, and autoserum were obtained from UCB as scaffold, growth factor and serum for culture respectively. UC-MSCs were obtained from Wharton jelly and cultured with UCB-derived fibrin (UCB-fibrin) for 3-4 weeks to induce their differentiation into osteoblasts. They were implanted subcutaneously into the dorsum of male nude mice for 6 weeks prior to undergoing assessment. The assessments performed were haematoxylin and eosin, and alizarin red staining, immunohistochemical staining of human mitochondria, scanning electron microscopy, scanning electron microscopy with energy dispersive X-ray spectrometry and real-time reverse transcriptase-polymerase chain reaction to assess the expressions of osteoblast markers. Consequently, the differentiation of UC-MSCs into osteoblasts and the production of hydroxyapatite were verified. This study suggested the possible formation of bone tissue using biomedical materials obtained from UC and UCB.

Osteogenic potential of human bone marrow-derived mesenchymal stromal cells cultured in autologous serum: a preliminary study.

PURPOSE: As part of the authors' research on potential osteogenesis by filling bone defects with human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) in patients with cleft lip and palate, they examined the cytoproliferative potential and cytobiological activity of hBM-MSCs in vitro and their osteogenic potential in vivo without performing osteoinduction. MATERIALS AND METHODS: The hBM-MSCs were collected from iliac cancellous bone and then used in primary culture, followed by 2 subcultures using an autologous serum (AS)-containing medium and a fetal bovine serum (FBS)-containing medium. Cytoproliferative potential and cytobiological activity as expressed by bone markers (alkaline phosphatase and osteocalcin) in hBM-MSCs cultured in the AS-containing medium (AS-cultured hBM-MSCs) and the FBS-containing medium (FBS-cultured hBM-MSCs) were examined in vitro, and the osteogenic potential of AS- and FBS-cultured hBM-MSCs was examined in mice. RESULTS: On day 6 of the second subculture, the number of hBM-MSCs per milliliter of specimen from 8 pediatric patients was significantly larger (P < .05) in FBS-cultured compared with AS-cultured hBM-MSCs. The alkaline phosphatase activity of hBM-MSCs was significantly greater (P < .05) when cultured in the AS-containing medium compared with the FBS-containing medium. The in vivo study showed the formation of an osteoid-like matrix rather than definite bone tissue. CONCLUSIONS: 1) FBS is appropriate for the cytoproliferation of hBM-MSCs; 2) the AS-containing medium is likely to have a good possibility of inducing the differentiation of hBM-MSCs; and 3) AS-cultured hBM-MSCs contain a group of cells that spontaneously differentiate into an osteoid-like matrix without performing osteoinduction.

Reconstruction of the umbilicus using a reverse fan-shaped flap.

The goal of the reconstruction for umbilical absence is to obtain a natural three-dimensional appearance of the umbilicus with minimal operative scarring. This paper presents two cases of umbilical reconstruction using a reverse fan-shaped flap. In both cases, the umbilicus was lost during surgical procedures on the abdominal wall when the patients were newborns. We performed this technique in both cases. This technique is simple and safe. With this technique, a permanent umbilical depth and ring can be obtained without any complications.