Masanori Shindo

A polymorphism upstream from the human paraoxonase (PON1) gene and its association with PON1 expression

Human serum paraoxonase (PON1) is an esterase that has been shown to decrease the susceptibility of lipoproteins to lipid peroxidation. We found a polymorphism of cytosine/thymidine (-108C/T, the number is from the ATG codon) in the upstream region of the PON1 gene. The luciferase activity was lower in the -108T allele than in the -108C allele. The serum PON1 concentrations in 132 normal subjects were as follows: -108CC>-108CT and>-108TT genotypes. The polymorphism upstream from the PON1 gene is associated with transcription of the PON1 gene and the serum PON1 concentration.

Discovery of a potent and short-acting oral calcilytic with a pulsatile secretion of parathyroid hormone.

Short-acting oral calcilytics, calcium-sensing receptor (CaSR) antagonists, have been considered as alternatives for parathyroid hormone (PTH), an injectable bone anabolic drug used in the treatment of osteoporosis. Previously, we identified aminopropandiol 1, which transiently stimulated endogenous PTH secretion in rats. However, the inhibition of cytochrome P450 (CYP) 2D6 and the low bioavailability of 1 remain to be solved. Attempts to change the physicochemical properties of the highly lipophilic amine 1 by introduction of a carboxylic acid group as well as further structural modifications led to the discovery of the highly potent biphenylcarboxylic acid 15, with a markedly reduced CYP2D6 inhibition and a significantly improved bioavailability. Compound 15 evoked a rapid and transient elevation of endogenous PTH levels in rats after oral administration in a dose-dependent manner at a dose as low as 1 mg/kg. The PTH secretion pattern correlated with the pharmacokinetic profile and agreed well with that of the exogenous PTH injection which exerts a bone anabolic effect.

IL-4, but not vitamin D3, induces monoblastic cell line UG3 to differentiate into multinucleated giant cells on osteoclast lineage

The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)‐4 and 1α,25(OH)2 vitamin D3 (D3) are known to induce MGC formation from monocytes/macrophages. D3 is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL‐4 as an inhibitor. Previously, we showed that IL‐4‐induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite‐resorptive activity in the presence of M‐CSF without stroma cells. In this study, we examined the effects of D3 and/or IL‐4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D3‐induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal‐R) expression, hydroxyapatite‐resorptive activity, and bone‐resorptive activity. A low concentration of D3 synergistically stimulated IL‐4‐induced TRAP‐positive MGC formation, whereas a high concentration of D3 inhibited it. When IL‐4 was added on day 7 of the 2‐week culture with D3, TRAP positivity reached maximum. On the other hand, delayed addition of D3 on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2‐week culture in the presence of D3, it increased further in the second week following the addition of IL‐4 on day 7. Furthermore, IL‐4‐induced, or IL‐4‐ and D3‐induced MGCs differentiated into functional osteoclasts with bone‐resorptive activity following coculture with osteoblastic cells, whereas D3‐induced MGCs did not acquire bone‐resorptive activity even after coculture with osteoblastic cells in the presence of D3. These findings suggest that IL‐4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D3 had only a “supportive” effect on this differentiation. IL‐4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells. J. Cell. Physiol. 182:214–221, 2000.

New aminopropandiol derivatives as orally available and short-acting calcium-sensing receptor antagonists

Synthesis and structure-activity relationship studies on a new aminopropandiol class of derivatives as calcium-sensing receptor antagonists are described. Modification of the phenolic moiety of a calcilytic compound NPS 2143 led to the identification of an orally available compound (R,R)-31 which demonstrated a rapid and transient stimulation of PTH release in rats.

A Novel Synthetic Triazolotriazepine Derivative JTT-606 Inhibits Bone Resorption by Down-Regulation of Action and Production of Bone Resorptive Factors

In the search for a new class of bone‐sparing agents, we have conducted random screening of the domestic chemical library using 45Ca release assay from prelabeled cultured neonatal mouse calvariae and identified a novel synthetic triazolotriazepine JTT‐606 as a candidate for a potent inhibitor of bone resorption. JTT‐606 inhibited 45Ca release dose dependently not only in the control calvarial culture but also in the stimulated cultures by interleukin‐1α (IL‐1α), fibroblast growth factor 2 (FGF‐2), and parathyroid hormone (PTH). JTT‐606 also inhibited both basal and stimulated osteoclast‐like (OCL) cell formation in the coculture of mouse osteoblastic cells and bone marrow cells dose dependently, indicating its inhibitory effect on osteoclast differentiation. Ex vivo OCL cell formation by cultured bone marrow cells collected from ovariectomized (OVX) mice also was decreased dose dependently by in vivo application of JTT‐606 to a level similar to that from sham‐operated mice. Furthermore, JTT‐606 inhibited resorbed pit formation by isolated mature osteoclasts as well as by unfractionated bone cells derived from rabbit long bones in the control and FGF‐2–stimulated cultures dose dependently, indicating both the direct and the indirect actions of JTT‐606 on mature osteoclast function. In addition, JTT‐606 reduced production of IL‐1α, tumor necrosis factor α (TNF‐α), IL‐6, and granulocyte‐macrophage colony–stimulating factor (GM‐CSF) in the human peripheral blood mononuclear cell culture. In vivo analyses of mature OVX rats revealed that the application of JTT‐606 for 12 weeks increased the BMD of the lumbar spine and decreased the levels of serum osteocalcin and urine deoxypyridinoline to levels similar to those of 17β‐estradiol–treated OVX rats. We propose that JTT‐606 may inhibit both osteoclast differentiation and function by down‐regulating both the action and the production of bone resorptive factors. It is speculated that JTT‐606 could be a potent agent for the treatment of osteopenic disorders with elevated osteoclastic bone resorption.