Kohei Notoya

An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis.

Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-β–mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-β activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-β. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-β activity and disease, suggesting new therapeutic strategies for osteoarthritis.

αvβ3 Integrin ligands enhance volume-sensitive calcium influx in mechanically stretched osteocytes

We propose that specific osteocyte–matrix interactions regulate the volume-sensitive calcium influx pathway, which we have shown is mediated by stretch-activated cation channels (SA-Cat) and is essential for the stretch-activated anabolic response in bone. The current study measured the hypotonic swelling-induced increase in cytosolic calcium concentration, [Ca2+]i, in rat osteocytes, and found that cells adherent to different matrices behave differently. Osteopontin and vitronectin, matrix molecules that bind the αVβ3 integrin, induced larger responses to the hypotonic swelling than other matrix molecules that bind other integrins. Addition of echistatin, which is a soluble αVβ3 ligand, significantly enhanced the hypotonic [Ca2+]i increase in addition to inducing an immediate increase in [Ca2+]i by itself. These results strongly support the contention that αVβ3 integrin signaling in osteocytes interacts with that in mechanotransduction, which is downstream of SA-Cat.

A functional SNP in EDG2 increases susceptibility to knee osteoarthritis in Japanese

Osteoarthritis (OA) is the most common form of arthritis and is characterized by the gradual loss of articular cartilage. Several OA-susceptibility genes have been identified; however, there are few pharmaceutical targets that can be targeted with small-molecule compounds. To investigate whether a susceptibility gene for OA exists among G-protein-coupled receptors (GPCRs), we performed a stepwise association study for 167 single nucleotide polymorphisms (SNPs) in 44 GPCR genes that were present in cartilage. Through the stepwise association study, an SNP located in the promoter region of EDG2 [endothelial differentiation, lysophosphatidic acid (LPA) GPCR, 2] (-2,820G/A; rs10980705) showed significant association with knee OA in two independent populations (pooled P = 2.6 x 10(-5)). Luciferase and electrophoretic mobility shift assays indicate that this SNP exerts an allelic difference on transcriptional activity and DNA binding in synovial cells, with the susceptibility allele showing increased activity and binding. EDG2 encodes an LPA receptor dominantly expressed in the synovium. The LPA receptor increased the expression of inflammatory cytokines and matrix metalloproteases in synovial cells. Our findings suggest that the LPA-EDG2 signal is involved in the pathogenesis of OA via catabolic process.

Enhancement of fracture repair in rats with streptozotocin-induced diabetes by a single injection of biodegradable microcapsules containing a bone formation stimulant, TAK-778

The feasibility of using microcapsules containing a bone formation stimulant, (2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide (TAK-778) to enhance fracture repair was assessed in rats with streptozotocin-induced diabetes. The release profile of the microcapsules was designed to mimic a dosing regimen of multiple injections of TAK-778 solution. The solution was injected locally every third day from day 0 (the day of operation) to day 27 according to several dosing regimens, and fracture repair was assessed at day 28. The production of callus was most prominent when TAK-778 solution was injected so that 50-75% of the total dose (5 mg TAK-778/site) was administered during the first half of the treatment period. Thus, injectable microcapsules of 30 micrometer in mean diameter were prepared in order to release TAK-778 over 4 weeks using a biodegradable polymer, poly(d,l-lactic/glycolic) acid, with a copolymer ratio of 85:15 (mol/mol) and an average molecular weight of 14,000. A single local injection of the microcapsules markedly enhanced fracture repair, which resulted in recovery of destructive bending strength of the bone at day 28. Histologically, the injection of TAK-778 microcapsules stimulated both fibrous and cartilaginous proliferation and periosteal ossification in the callus at day 7; bony bridge formation was observed at day 28. At day 56, the callus was remodeled and cortical bony union was evidenced in the microcapsule-treated fractures compared with the controls, which showed only fibrous union.

Inhibitory effect of ipriflavone on pit formation in mouse unfractionated bone cells

SummaryEffects of ipriflavone (7-isopropoxyisoflavone) on osteoclast-induced bone resorption were evaluated using an unfractionated bone cell culture system containing mature osteoclasts from the femur and tibia of newborn mice. When cells were cultured for 4 days on dentin slices in the presence of 5% fetal bovine serum and 10−8 M 1α,25(OH)2D3, ipriflavone (3 x 10−7-3 x 10−5 M) inhibited pit formation and caused a decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). The lowest significant effect was observed at a concentration of 10−6 M. Unlike ipriflavone, calcitonin inhibited pit formation 4 days after the culture was started without affecting the number of TRAP-positive MNCs. Ipriflavone still inhibited pit formation when the culture period was 13 days, when new osteoclasts were expected to be formed. These findings suggest that ipriflavone inhibits new osteoclast formation and bone resorption at the cellular level.

Stimulatory effect of ipriflavone on formation of bone-like tissue in rat bone marrow stromal cell culture

SummaryThe effects of ipriflavone (IP) (10−5 M) on bone formation were studied in stromal cells from the femoral bone marrow of young adult rats cultured for 21 days in the presence of β-glycerophosphate and dexamethasone. Stereoscopic microscopy showed nodule formation after 14 days of culturing, and both the number and the size of the nodules increased with time. The alizarin-red-stained calcified area in the nodules in the IP group was nearly 4 times as large as that in the control after 21 days. Light and electron microscopy revealed the presence of many osteoblast-like cells with developed rough endoplasmic reticulum and Golgi apparatus in the nodules in the control group after 14 days, and a collagenous fibril network was seen among the cells. After 21 days, calcification of the dense collagenous fibril network and bone matrix-like tissue were observed in many nodules, resulting in the formation of bone-like tissue containing osteocyte-like cells. In the IP group, the collagenous fibril network area in the nodules was greater than that in the control after 14 days, and a further increase in both the dense collagenous fibril network area and calcified bone-like tissue area was observed after 21 days. These findings indicate that IP stimulates bone-like tissue formation in the rat bone marrow stromal cell culture, suggesting that the promotion of collagen production by osteoblasts is involved in the stimulation of bone-like tissue formation by IP.

Similarities and differences between the effects of ipriflavone and vitamin K on bone resorption and formation in vitro

The effects of ipriflavone and vitamin K on bone metabolism were examined using a culture system. Vitamin K1 and vitamin K2 (10(-7)M-10(-5)M) inhibited both the activation of mature osteoclasts and the formation of new osteoclasts without affecting the growth of progenitor cells in cultures of mouse unfractionated bone cells. The inhibitory effects of vitamin K on bone resorption were similar to those of ipriflavone and were not affected by the vitamin K antagonist warfarin. When ipriflavone was added to the culture medium in combination with vitamin K2, an additive inhibitory effect on bone resorption was observed. An additive effect was also observed in organ cultures of mouse calvaria. On the other hand, ipriflavone, but neither vitamin K1 nor vitamin K2, stimulated cellular alkaline phosphatase (ALP) activity on rat bone marrow stromal cells under culture conditions in which cells subsequently form mineralized bone-like tissue. Vitamin K1 and vitamin K2 also did not modulate the stimulatory effect of ipriflavone on the ALP activity of the cells. These results suggest that the inhibitory effects of vitamin K on bone resorption are similar to those of ipriflavone through mechanisms that may be independent of the gamma-carboxylation system, while the effects of vitamin K on osteoblast phenotype expression are different from those of ipriflavone.

Enhancement of osteogenesis in vitro by a novel osteoblast differentiation-promoting compound, TAK-778, partly through the expression of Msx2.

TAK-778 [(2R,4S)-(-)-N-(4-Diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide: mw 505.52], a novel compound promoting osteoblast differentiation, promotes osteogenesis in vitro and enhances bone formation during skeletal repair in vivo. In this study, we further evaluated the effects of TAK-778 on the differentiation of cultured bone marrow stromal cells into osteoblasts in the presence of dexamethasone, paying particular attention to the expression of transcription factors involved in regulating osteoblast differentiation. Treatment of TAK-778 (10(-7)-10(-5) M) for 4 h resulted in an increase in the mRNA expression of Msx2, but not Cbfa1 or Dlx5. This transcriptional alteration preceded the changes in other markers related to the osteoblast phenotype, such as alkaline phosphatase and osteocalcin mRNA. The transfection of Msx2-antisense in the cells caused a significant reduction in the levels of alkaline phosphatase mRNA expression induced by TAK-778. These results suggest that TAK-778 promotes osteoblast differentiation partly through the expression of Msx2, a homeobox-related gene.

Increase in femoral bone mass by ipriflavone alone and in combination with 1α-hydroxyvitamin D3 in growing rats with skeletal unloading

We assessed the possibility that ipriflavone treatment might result in bone restoration in immobilized rats. We also investigated the effect of combined treatment with ipriflavone and vitamin D3 on the bone. Male Sprague-Dawley rats, 6 weeks of age, were subjected to unilateral sciatic neurectomy. Three weeks after the operation, ipriflavone (100 mg/kg), 1α-hydroxyvitamin D3 [1α(OH)D3, 25 ng/kg], or both ipriflavone and 1α(OH)D3 were orally administered every day for 12 or 24 weeks. After 12 weeks of treatment, only the group receiving combined treatment with ipriflavone and 1α(OH)D3 showed increases in total femur calcium content (+16.4%, compared with the control). After 24 weeks, both animals treated with ipriflavone alone and those that had received the combination of ipriflavone and 1α(OH)D3 showed significant increases in femur calcium content (+18.0% and +23.8%, respectively). In these treatment groups, X-ray analysis revealed an increase in bone mineral density over the entire length of the femur, and an increase in cortical diameter at the midshaft without affecting medullary width. Administration of 1α(OH)D3 (25 ng/kg) alone had no effect. Body weight, femur length, and serum markers of calcium and bone metabolism were not affected in any group. We evaluated the relationship between ipriflavone and vitamin D3 in bone cells in a culture system using rat bone marrow stromal cells in which the cells subsequently form mineralized bone-like tissue. Continuous treatment with ipriflavone (10−5 M) for 21 days resulted in an increase in osteocalcin secretion, and enhanced its response to 1α,25-dihydroxyvitamin D3 (10−11 M-10−8 M). These findings indicate that ipriflavone treatment increases the femoral bone mass in immobilized rats. In addition, a low dose of 1α(OH)D3, which did not induce hypercalcemia, in combination with ipriflavone, augmented the stimulatory effect of ipriflavone alone on the bone mass, possibly due to a direct effect of each agent on osteoblastic cells.

Novel and potent calcium-sensing receptor antagonists: Discovery of (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate (TAK-075) as an orally active bone anabolic agent

The calcium-sensing receptor antagonist (CaSR) has been recognized as a promising target of anabolic agents for treating osteoporosis. In the course of developing a new drug candidate for osteoporosis, we found tetrahydropyrazolopyrimidine derivative 1 to be an orally active CaSR antagonist that stimulated transient PTH secretion in rats. However, compound 1 showed poor physical and chemical stability. In order to work out this compounds chemical stability and further understand its in vivo efficacy, we focused on modifying the 2-position of the tetrahydropyrazolopyrimidine. As a result of chemical modification, we discovered (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate 10m (TAK-075), which showed improved solubility, chemical stability, and in vivo efficacy. Furthermore, we describe that evaluating the active metabolite is important during repeated treatment with short-acting CaSR antagonists.