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

Authors

  • Daichi Chikazu,

    1. Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
    2. Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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  • Masanori Shindo,

    1. Central Pharmaceutical Research Institute, Japan Tobacco, Incorporated., Osaka, Japan
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  • Toshiki Iwasaka,

    1. Central Pharmaceutical Research Institute, Japan Tobacco, Incorporated., Osaka, Japan
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  • Mika Katagiri,

    1. Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
    2. Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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  • Noriyo Manabe,

    1. Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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  • Tsuyoshi Takato,

    1. Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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  • Kozo Nakamura,

    1. Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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  • Hiroshi Kawaguchi M.D., Ph.D.

    Corresponding author
    1. Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
    • Department of Orthopaedic Surgery Faculty of Medicine University of Tokyo Hongo 7–3-1, Bunkyo Tokyo 113-8655, Japan
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Abstract

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.

INTRODUCTION

Bone is a complex component of the body that provides mechanical support, a site for hematopoiesis, and a storehouse for calcium. In the normal state, bone mass is maintained by the closely coupled activity of osteoclasts and osteoblasts. Uncoupling of these activities and thus the balance between bone resorption and bone formation results in generalized and local bone loss such as osteoporosis, hyperparathyroidism, and rheumatoid arthritis (RA). These disorders are extremely diffused, affecting a large number of individuals worldwide, and are characterized by an up-regulation of bone resorption that exceeds bone formation. Cytokines and growth factors have been suggested as playing important roles in the pathogenesis of these disorders. Interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to be involved in the pathogenesis of osteoporosis.(1–8) Fibroblast growth factor 2 (FGF-2) also is known to be a potent stimulator of bone resorption and to contribute to the joint destruction in RA patients.(9–13)

Among the individuals that may develop osteoporosis, women are considered at high risk because of the estrogen deficiency that abruptly occurs at menopause. At present, postmenopausal osteoporosis is treated with a restricted number of drugs, including estrogens, calcitonin, sodium fluoride, calcium supplementation, and bisphosphonates. However, the use of these drugs is limited by specific problems concerning either their real efficacy or the incidence of unwanted side effects. In the search for a new class of bone-sparing agents, we conducted random screening of domestic chemical library using 45Ca release assay from prelabeled cultured neonatal mouse calvariae and identified a novel synthetic triazolotriazepine derivative, 6-(4-chlorophenyl)-1-methyl-4-(3-pyridylmethyl)-4H-2,3,4,5,10b-pentaazabenzo[e]azulene monohydrochloride (C22H17N6ClHCl; JTT-606) as a candidate for a potent inhibitor of bone resorption. In this study, the effects of JTT-606 on both the action and the production of bone resorptive factors were investigated using several culture systems. Because osteoclastic bone resorption is constituted of two steps, osteoclast differentiation and mature osteoclast function, the effects of JTT-606 were analyzed from these two aspects using different culture systems. Well-known 45Ca release assay from prelabeled cultured neonatal mouse calvariae reflects the effect on the overall bone resorption through both osteoclast differentiation and mature osteoclast function. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast-like (OCL) cell formation assay in the coculture of mouse osteoblastic cells and bone marrow cells reflects the effect on osteoclast differentiation. The recently established resorbed pit formation assay by mature osteoclasts purified from rabbit long bones (more than 99% purity) reflects the direct effect on mature osteoclast function.(14) The effect of JTT-606 on cytokine production was determined by measuring the cytokine concentrations in the culture medium of human peripheral blood mononuclear cells. Further, in vivo actions of JTT-606 on bone mineral density (BMD), biochemical markers of bone turnover, and mechanical properties were investigated and compared with those of 17β-estradiol (E2) in ovariectomized (OVX) rats.

MATERIALS AND METHODS

Experimental animals

All animal experiments were performed according to the guidelines of the International Association for the Study of Pain.(15) In addition, the experimental work was approved by the animal care and use committees of Tokyo University and Japan Tobacco. Neonatal, 5-week-old and 8-week-old ddY mice were obtained from Shizuoka Laboratories Animal Center (Shizuoka, Japan). Ten-day-old Japanese white rabbits were purchased from Saitama Experimental Animal (Saitama, Japan). Ten-month-old or 11-month-old female F334 rats and 6-week-old male SD rats were obtained from Charles River Japan Laboratories (Shiga, Japan), and all were acclimatized for 1 week before the experiment. During the acclimatization and experimentation, the animals were housed in a 12-h light/dark cycle (lights on from 7 a.m. to 7 p.m.) at 23 ± 3°C and 55 ± 15% relative humidity with free access to commercial laboratory food and water.

Materials

JTT-606 was synthesized at the Central Pharmaceutical Research Institute of Japan Tobacco, Inc. (Osaka, Japan). Human recombinant FGF-2 was obtained from Scios Nova (Mountain View, CA, U.S.A.). The 1-hydroxyethylidine-1,1-bisphosphonic acid (etidronate) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and lipo-polysaccharide (LPS) was purchased from DIFCO Laboratories (Detroit, MI, U.S.A.). Other chemicals were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.).

Ca release assay from cultured neonatal mouse calvariae

Bone resorption was measured as the release of previously incorporated 45Ca into neonatal mouse calvariae as previously described.(16) Briefly, timed pregnant mother ddY mice (6-7 weeks old) were injected with 0.05 mCi 45Ca on the 16th day of gestation. Seven-day-old neonatal mouse calvariae were dissected and precultured for 16-24 h in BGJb medium containing 100 μg/ml l-ascorbic acid phosphate and 1 mg/ml bovine serum albumin (BSA). They were further cultured in the medium in the presence and absence of IL-1α (10 ng/ml), FGF-2 (10−8 M), or parathyroid hormone (PTH; 10−8 M) with or without JTT-606 (10-1000 nM) in 24-multiwell dishes on a rocking platform for 96 h with a medium change after 48 h. 45Ca in medium and trichloroacetic acid extracts of bone were determined by liquid scintillation counting, and the percentage of 45Ca released into the medium during 96 h of culture was calculated.

OCL cell formation assay in the coculture of mouse osteoblastic cells and bone marrow cells

Mouse primary osteoblastic cells were harvested from calvariae of neonatal ddY mice. Calvariae were digested with 1 ml of trypsin/EDTA (Gibco BRL, Rockville, MD, U.S.A.) containing 10 mg collagenase (Sigma, St. Louis, MO, U.S.A.; type 7) for 10 minutes × 5 times, and cells from fractions 2-5 were pooled and grown to confluence in α modified Eagle medium (α-MEM) containing 10% fetal bovine serum (FBS). These osteoblastic cells (3 × 104 cells/well) were cocultured with bone marrow cells (1 × 106 cells/well) prepared from tibias and femurs of 8-week-old ddY mice in 24-multiwell dishes with α-MEM containing 10% FBS in the presence of IL-1α (10 ng/ml), FGF-2 (10−8 M), or PTH (10−8 M), with or without JTT-606 (10-1000 nM). After 7 days of culture, the cells were fixed with 3.7% (vol/vol) formaldehyde in phosphate-buffered saline (PBS) and ethanol-acetone (50:50 [vol/vol]), and stained at pH 5.0 in the presence of l(+)-tartaric acid using naphthol AS-MX phosphate (Sigma, St. Louis, MO, U.S.A.) in N,N-dimethyl formamide as the substrate. TRAP-positive multinucleated cells containing more than three nuclei were counted as OCL cells.

Cell count in bone marrow of OVX mouse femurs

Eight-week-old female ddY mice were subjected to either dorsal OVX or sham operation under general anesthesia with diethyl ether. JTT-606 (3, 10, and 30 mg/kg per day, per oral [po]) in 0.5% methylcellulose, vehicle (methylcellulose) alone (po), or E2 (50 μg/kg per week, 2 times/week, subcutaneously [sc]) in 5% benzyl alcohol/95% corn oil (vol/vol) was administered to OVX mice starting 1 day after the operation. Vehicle alone (po) also was administered to sham-operated mice. After 4 weeks of treatment, mice were killed by cervical dislocation. To count the number of bone marrow cells, bilateral femurs were excised and their epiphyses were cut off at the same place to minimize variations in bone size. Bone marrow was flushed from both ends of bones with 1 ml of PBS. The number of cells were counted after diluting the flushed PBS 1:100 (vol/vol) in 0.01 M acetic acid to lyse red blood cells. Smears were made of the residual leukocytes and stained by Wright's staining method using Hemo-Tek 1000 (Miles, Inc., Elkhart, IN, U.S.A.).

OCL cell formation in ex vivo bone marrow cell culture from femurs of OVX mice

The bone marrow cells taken from femurs as described above were harvested, and the same number of cells (1 × 106 cells/well) from every treatment group was cultured in 24-multiwell dishes in α-MEM containing 10% FBS and 1,25-hydroxyvitamin D3 [1,25(OH)2D3; 10−8 M]. After 7 days of culture, cells were stained with TRAP and the number of OCL cells was counted as described above.

Resorbed pit formation assay by purified mature osteoclasts and unfractionated bone cells from rabbit long bones

Long bones from 10-day-old rabbits were minced with scissors and agitated with a vortex mixer. An aliquot of unfractionated bone cells was seeded onto 0.24% collagen gel (Nitta Gelatin, Tokyo) coated on 100-mm tissue culture dishes and incubated. Four hours later, nonadherent cells were washed off and osteoclasts were then removed from the gels with 0.1% collagenase solution (Wako Pure Chemical Co., Osaka, Japan). By staining with TRAP, we ascertained that more than 99% of isolated cells were pure osteoclasts. Isolated osteoclasts (150 cells/well) or unfractionated bone cells (5 × 104 cells/well) were cultured on a dentine slice placed in each well of 96-multiwell dishes. JTT-606 (10-1000 nM) and/or FGF-2 (10−8 M) were added to the cultures at 1 h after the seeding. After 24 h of culture, cells on dentine slices were removed in 1 N NH4OH solution and stained with 0.5% toluidine blue for 1 minute. Total area of pits on the dentine slice was estimated under a light microscope with a micrometer using an image analyzer (System Supply Co., Nagano, Japan).

Cytokine concentrations in human peripheral blood mononuclear cell culture

Human peripheral blood mononuclear cells were isolated from blood obtained from four healthy volunteers (one female and three males, 25-37 years, average 30.5 years) and cultured with 5% FBS/RPMI in the presence of LPS (10 μg/ml) and JTT-606 (0.03-10 μM). After 24 h of culture, concentrations of IL-1α, TNF-α, IL-6, and GM-CSF in the culture media were measured by ELISA (Quantikine; R&D Systems, Minneapolis, MN, U.S.A.), and the data from each volunteer were pooled.

Analyses of BMD, biochemical markers, and mechanical properties in OVX rats

Ten to 11-month-old female F334 rats were subjected to either dorsal OVX or sham operation under anesthesia with pentobarbital sodium (40 mg/kg) injection. JTT-606 (0.3, 1.0, and 3.0 mg/kg per day, po), vehicle alone (po), or E2 (50 μg/kg per time or 100 μg/kg per time, 2 times/week, sc) was administered to OVX rats starting at 1 week after the operation. Vehicle alone also was administered to sham-operated rats. After 12 weeks of treatment, rats were killed by taking blood from abdominal aorta under anesthesia with diethyl ether, and the lumbar spine and bilateral femurs were harvested. BMD of the fourth and fifth lumbar spine was measured using a dual-energy X-ray absorptiometer (DCS-600R, Aloka, Tokyo), and the mechanical properties of the femoral shaft were determined by the three-point bending test (AGS-50A, Shimadzu, Kyoto, Japan). Serum osteocalcin level was measured by radioimmunoassay (RIA; Biochemical Technologies, Inc., CA, U.S.A.). Urine was collected for 8 h and deoxypyridinoline cross-links in urine were measured by high-performance liquid chromatography (HPLC) after HCL hydrolysis and normalized by creatinine concentration determined by the Jaffe method (MBC, Tokyo, Japan).

Histological analyses of proximal tibias of normal young rats

High doses of JTT-606 (30 mg/kg per day and 100 mg/kg per day, po), vehicle alone (po), or etidronate (40 mg/kg per day, sc) in normal saline buffer was administered to 6-week-old male SD rats for 7 days. For labeling, rats were injected sc with 20 mg/kg of calcein on the fifth day and seventh day. Animals were killed on the eighth day with diethyl ether. Excised tibias were fixed with 100% ethanol, embedded in methyl methacrylate, and sectioned in 6-μm slices using a microtome (ROTARY-ONE, Pharmacia LKB, Uppsala, Sweden). Histological features at the proximal tibias were shown by calcein fluorescence. The width of the growth plate was measured under a light microscope with a micrometer using an image analyzer (System Supply Co., Nagano, Japan).

Figure FIG. 1..

Effect of JTT-606 on 45Ca release from cultured neonatal mouse calvariae in the presence and absence of resorptive factors. Calvariae of 7-day-old mice born from timed pregnant mother mice injected with 0.05 mCi 45Ca were dissected and cultured in the presence and absence of JTT-606 (10, 100, and 1000 nM), IL-1α (10 ng/ml), FGF-2 (10−8 M), or PTH (10−8 M) for 4 days with a medium change at 2 days. 45Ca in medium and trichloroacetic acid extracts of bone were determined by liquid scintillation counting, and the percentage of 45Ca released into the medium during 96 h of culture was calculated. Data are expressed as means (symbols) ± SEMs (error bars) for 8-10 cultures/group. *p < 0.01, significant inhibition of JTT-606 as compared with the culture without JTT-606.

Statistical analysis

Results are expressed as mean ± SEM. Group differences were analyzed using one-way analysis of variance (ANOVA) and subsequent mean comparison by Dunnett's test.

RESULTS

Effect of JTT-606 on 45Ca release from cultured calvariae

Bone resorptive factors, IL-1α, FGF-2, and PTH stimulated 45Ca release from prelabeled cultured neonatal mouse calvariae 2.2-, 2.7-, and 2.9-fold over the control culture, respectively. Addition of JTT-606 (10-1000 nM) dose-dependently inhibited 45Ca release both in the control culture and in the cultures stimulated by these factors (Fig. 1). The highest concentration of JTT-606 (1000 nM) reduced stimulated 45Ca release to a level similar to that of the control culture. Because this assay reflects the overall bone resorption activity through both osteoclast differentiation and mature osteoclast function, we further investigated the effects of JTT-606 on these two activities separately.

Figure FIG. 2..

Effect of in vitro application of JTT-606 on OCL cell formation in the coculture in the presence and absence of resorptive factors. Primary osteoblastic cells (3 × 104 cells/well) from neonatal mouse calvariae and bone marrow cells (1 × 106 cells/well) from 8-week-old mouse long bones were cocultured in the presence and absence of JTT-606 (10, 100, and 1000 nM), IL-1α (10 ng/ml), FGF-2 (10−8 M), or PTH (10−8 M). After 7 days of culture, TRAP-positive multinucleated cells containing more than three nuclei were counted as OCL cells. Data are expressed as means (symbols) ± SEMs (error bars) for 7-10 cultures/group. *p < 0.01, significant inhibition of JTT-606 as compared with the culture without JTT-606.

Effects of in vitro and in vivo application of JTT-606 on osteoclast differentiation

In vitro effect of JTT-606 on osteoclast differentiation was determined by OCL cell formation in the co-culture of mouse osteoblastic cells and bone marrow cells (Fig. 2). JTT-606 (10-1000 nM) decreased OCL cell formation both in the control culture and in the cultures stimulated by IL-1α, FGF-2, and PTH. The highest concentration of JTT-606 (1000 nM) abrogated OCL cell formation in all cultures.

In vivo effects of JTT-606 (3, 10, and 30 mg/kg per day, po) on cell number and osteoclast differentiation in bone marrow of femurs of OVX mice were further investigated. OVX increased the bone marrow cell number 1.4-fold over that of sham-operated mice (Fig. 3, top). JTT-606 did not affect the bone marrow cell number of the OVX femur while E2 decreased it. When the same number of bone marrow cells from every group was seeded and cultured in the presence of 1,25(OH)2D3, OCL cell formation from marrow cells of OVX mice was increased 1.4-fold over that of sham-operated mice (Fig. 3, bottom). In vivo application of JTT-606 decreased OCL cell formation in the ex vivo marrow cell culture dose dependently with significant effects at 10 mg/kg per day and 30 mg/kg per day up to levels similar to or lower than those of sham-operated mice and E2 (100 μg/kg per week, sc)-applied OVX mice. These results indicate that both in vitro and in vivo applications of JTT-606 down-regulate osteoclast differentiation under bone resorptive conditions.

Figure FIG. 3..

Effects of in vivo application of JTT-606 on the total bone marrow cell number in the femur (top) and OCL cell formation in the ex vivo marrow cell culture (bottom) from OVX mice. JTT-606 (3, 10, and 30 mg/kg per day, po), vehicle (po), or E2 (50 μg/kg per week, 2 times/week, sc) was administered to 8-week-old OVX mice starting at 1 day after the operation. After 4 weeks of treatment, the number of bone marrow cells collected from bilateral femurs was counted. The same number of marrow cells (1 × 106 cells/well) from every treatment group was seeded and cultured in the presence of 1,25(OH)2D3 (10−8 M). After 7 days of culture, TRAP-positive multinucleated cells containing more than three nuclei were counted as OCL cells. Data are expressed as means (bars) ± SEMs (error bars) for 8 mice/group. #p < 0.05 and *p < 0.01 versus OVX vehicle.

Effect of JTT-606 on mature osteoclast function

The direct effect of JTT-606 (10-1000 nM) on mature osteoclast function was determined by measuring the resorbed pit area on a dentine slice made by isolated osteoclasts from rabbit long bones (Fig. 4, top) and was compared with the overall (direct and indirect) effect determined by the pit area made by unfractionated bone cells from the same origin (Fig. 4, bottom). Among the bone resorptive factors used in 45Ca release and OCL cell formation assays, only FGF-2 is known to act directly on mature osteoclasts and to have its receptor on them.(12,17) In fact, FGF-2 (10−8 M) increased the pit area resorbed by isolated osteoclasts up to 1.9-fold and that by unfractionated bone cells up to 7.5-fold, suggesting both direct and indirect actions of FGF-2 as previously reported.(12) JTT-606 dose-dependently inhibited the resorbed pit formation not only by unfractionated bone cells but also by isolated osteoclasts both in control and in FGF-2-stimulated cultures. These results indicate that JTT-606 inhibits bone resorption by reducing mature osteoclast function as well as osteoclast differentiation and that JTT-606 can target both osteoclasts and osteoblasts/stromal cells, which support osteoclastic bone resorption.

Figure FIG. 4..

Effect of JTT-606 on resorbed pit formation by isolated osteoclasts (top) and by unfractionated bone cells (bottom) from rabbit long bones in the presence and absence of FGF-2. Bone cells were extracted from long bones of 10-day-old rabbits and were seeded onto collagen gel. Nonadherent cells were washed off and osteoclasts were then removed from the gels with collagenase solution. Isolated osteoclasts (150 cells/well) or unfractionated bone cells (5 × 104 cells/well) were cultured on a dentine slice with or without JTT-606 (10-1000 nM) and/or FGF-2 (10−8 M). After 24 h of culture, total area of pits on the dentine slice was estimated. Data are expressed as means (symbols) ± SEMs (error bars) for 6 cultures/group. *p < 0.01, significant inhibition of JTT-606 as compared with the culture without JTT-606.

Effect of JTT-606 on cytokine production from cultured human peripheral blood mononuclear cells

In addition to inhibitory effects of JTT-606 on bone resorptive activities of cytokines, the effects on the production of the putative bone resorptive cytokines, IL-1α, TNF-α, IL-6 and GM-CSF, which have been reported to be involved in bone loss in osteoporosis with estrogen deficiency, were investigated using the culture of human peripheral blood mononuclear cells obtained from four healthy volunteers (Fig. 5).(1–8) JTT-606 (0.03-10 μM) dose-dependently inhibited the production of all of these cytokines in this culture stimulated by LPS. We assume that JTT-606 inhibits not only the action but also the production of bone resorptive factors.

Figure FIG. 5..

Effect of JTT-606 on cytokine production from cultured human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were isolated from blood obtained from four healthy volunteers (one female and three males, 25-37 years, average 30.5 years), and cultured in the presence of LPS (10 μg/ml) with or without JTT-606 (0.03-10 μM). After 24 h of culture, concentrations of IL-1α, TNF-α, IL-6, and GM-CSF in the culture media were measured by ELISA, and the data from each volunteer were pooled. Data are expressed as means (symbols) ± SEM (error bars) for 24 cultures/group (6 cultures/group per volunteer). *p < 0.01, significant inhibition of JTT-606 as compared with the culture without JTT-606.

Table Table 1.. In Vivo Effects of JTT-606 on Bone Density, Bone Turnover, and Mechanical Properties in Mature OVX Rats
  OVX
   JTT-606 (mg/kg per day)E2 (μg/kg per week)
 ShamVehicle0.313100200
  1. Values are the mean ± SEM for 8-15 rats/group.

  2. *P < 0.01 and P < 0.05 versus OVX vehicle.

BMD (mg/cm2) Biochemical markers67.7 ± 1.2*55.3 ± 1.164.0 ± 0.7*62.7 ± 1.2*63.0 ± 1.0*62.6 ± 1.1*62.5 ± 1.9*
Serum osteocalcin (ng/ml)28.9 ± 1.2*37.5 ± 1.034.5 ± 1.433.8 ± 1.029.0 ± 1.0*23.2 ± 0.6*24.6 ± 0.8*
Urine deoxypyridinoline (nmol/mmol creatinine)17.5 ± 2.0*26.2 ± 1.026.9 ± 1.527.4 ± 1.420.2 ± 0.8*13.0 ± 0.6*12.3 ± 0.1*
Mechanical properties Ultimate load (N)89.6 ± 2.482.7 ± 2.487.2 ± 2.786.7 ± 2.687.4 ± 0.986.3 ± 2.287.3 ± 2.4
Stiffness (N/mm)278.8 ± 8.1254.1 ± 7.5266.1 ± 4.4269.2 ± 5.9271.6 ± 4.2273.5 ± 6.7270.1 ± 6.2

In vivo effects of JTT-606 on bone density, bone turnover, and mechanical properties in mature OVX rats

We further examined in vivo effects of JTT-606 on bone of 10- to 11-month-old rats with skeletal maturity (Table 1). BMD of the fourth and fifth lumbar spine was decreased in OVX rats by 18.3% compared with that of sham-operated rats at 13 weeks after the operation. Subcutaneous injection of E2 (50 μg/kg per time and 100 μg/kg per time, 2 times/week) for the last 12 weeks increased the BMD of OVX rats to levels not significantly different from that of sham-operated rats. JTT-606 (0.3, 1.0, and 3.0 mg/kg per day, po) administered for the same period also increased the BMD of OVX rats to levels similar to those of E2. Significant effect was seen at 0.3 mg/kg per day but no further increase was seen at higher concentrations. The regulation of bone turnover was investigated by measuring the biochemical markers: serum osteocalcin for bone formation and urine deoxypyridinoline for bone resorption. The levels of both markers were higher in OVX rats by 29.8% and 49.7%, respectively, than in sham rats, indicating an elevated bone turnover by OVX. JTT-606 at 3.0 mg/kg per day significantly decreased the elevated levels of these markers (22.7% in osteocalcin and 22.9% in deoxypyridinoline) although the inhibitions were smaller than those by E2. We also examined the effect of JTT-606 on mechanical properties of the femur as determined by the three-point bending at its midshaft. The stiffness was decreased by OVX; however, both JTT-606 and E2 failed to increase the mechanical properties in OVX rats significantly.

Figure FIG. 6..

In vivo effect of high doses of JTT-606 on mineralization of the proximal tibias of young normal rats (original magnification, ×40). High doses of JTT-606 (30 mg/kg per day and 100 mg/kg per day, po), vehicle alone (po), or etidronate (40 mg/kg per day, sc) was administered to 6-week-old male rats for 7 days. Histological features are shown by calcein fluorescence, and the impairment of mineralization was determined by the growth plate width expressed as means (bars) ± SEMs (error bars) for 4-5 rats/group. *p < 0.01 versus OVX vehicle.

In vivo effect of high doses of JTT-606 on mineralization of the proximal tibias of young normal rats

We examined whether or not high doses of JTT-606 would impair the normal mineralization because JTT-606 possibly decreased bone turnover in OVX rats like bisphosphonates (Fig. 6). Histological analysis of the proximal tibias of 6-week-old normal rats revealed that high doses of JTT-606 (30 mg/kg per day and 100 mg/kg per day, po) did not impair the mineralization determined by the growth plate width. Etidronate (40 mg/kg per day, sc), in contrast, impaired mineralization causing the increase in the growth plate width to 3.9-fold over that of vehicle-treated rats.

DISCUSSION

In this study using several in vitro and ex vivo culture systems, we showed that an originally synthesized triazolo-triazepine derivative JTT-606 down-regulated the action and the production of bone resorptive factors and inhibited osteoclast differentiation and function. In vivo application of JTT-606 also increased the bone mass and decreased the bone turnover in OVX rats.

JTT-606 showed antibone resorptive actions in mouse, rabbit, and rat cell cultures, indicating no differences among these species. In addition, effects of JTT-606 were not cell specific because this agent appears to target marrow cells, mature osteoclasts, monocytes, and osteoblasts/stromal cells. Although the mechanism of actions of triazolotriazepine derivatives has not yet been well studied, a benzotriazepine derivative 5-methyl-10,11-dihydro-5H-pyrrolo [1,2b] [1,2,5] benzotriazepine-11-acetic acid is known to be a nonsteroidal anti-inflammatory drug (NSAID), which inhibits prostaglandin (PG) production.(18) PGs are well-known as mediators of bone resorption and have been suggested to contribute to bone loss in several osteopenic disorders.(19) In fact, bone resorptive factors used in this study, IL-1α, FGF-2, and PTH are known to stimulate PG production mainly through cyclooxygenase-2 (COX-2) induction in osteoblastic cells.(9,20) However, because JTT-606 did not alter COX-2 messenger RNA (mRNA) level as determined by reverse-transcription polymerase chain reaction (RT-PCR) or PG production as determined by ELISA induced by these resorptive factors in mouse primary cultured osteoblastic cells (data not shown), it probably inhibits bone resorption by mechanisms independent of PG production. A characteristic action of JTT-606 is the inhibition of mature osteoclast function that is not seen by NSAIDs. Mature osteoclast function is known to be dependent on src-kinase activity and FGF-2 stimulation of osteoclast function is regulated by mitogen-activated protein (MAP) kinase signalings (Chikazu D and Kawaguchi H, unpublished observation, February 2000).(21,22) Hence, JTT-606 might regulate the intracellular signalings through tyrosine kinase pathways in osteoclasts as well as in other cells.

Osteoporosis actually is caused not only by increased bone resorption, but also by a substantial decrease in bone formation. Given this recognition, a molecule that inhibits bone resorption while at the same time stimulating bone formation is an ideal candidate as an antiosteopenic drug. Although high doses of JTT-606 appear to increase the trabecular bone mass at the proximal tibias of young rats (Fig. 6), this effect probably is not caused by an anabolic action. Bone growth is accompanied by high bone turnover at this age of rats, and if resorption is inhibited, new bone spicules will accumulate on unresorbed old spicules, resulting in an increase in trabecular bone mass. Hence, this increase is likely to be caused by the inhibitory effect of JTT-606 on bone resorption, but not its anabolic effect. In fact, JTT-606 did not affect cell proliferation or alkaline phosphatase (ALP) activity of primary cultured mouse osteoblastic cells (data not shown).

To study in vivo actions of JTT-606, we used 10- to 11-month-old rats that are older than those used in previous studies on OVX rats.(23–27) Kalu et al. analyzed the rate of bone growth in rats and revealed that it is very high and rapid from 1 to 3 months of age, is more gradual from 3 to 6 months, and after that then becomes minimal.(28,29) By 12 months, all the bone parameters are reported to reach plateau levels with no further significant change up to 24 months of age. Considering this observation, we chose a skeletally mature rat model because human postmenopausal bone loss starts after the attainment of skeletal maturity. However, preventive effects of not only JTT-606 but also E2 on bone density of OVX rats did not achieve the levels of sham-operated rats, and their effects were not dose dependent (Table 1). It is true that previous reports using younger rats (3–6 months old) have shown that bone density is fully restored by E2 with amounts similar to or lower than those used in this study.(23–26) It is supposed that JTT-606 and E2 might have exhibited stronger effects if we used immature rat models as shown in Fig. 6, although its clinical relevance is rather difficult to refer to. Another issue of in vivo JTT-606 action is the lack of significant effects on mechanical properties of femurs. This may be caused by the insufficient decrease in mechanical properties in OVX rats as compared with sham-operated rats at 12 weeks after the surgery. A previous report has shown that neither OVX nor E2 replacement had a significant effect on tibial mechanical properties at 2 months after the surgery on 6-, 12-, and 30-month-old rats.(30) Another report also has shown that the decrease in femoral bone strength by OVX is not seen before 9 months after the surgery on 6-month-old rats.(27) Because mechanical properties of rat vertebrae are reported to be significantly reduced by OVX and restored by E2 replacement at 6 months after surgery, further studies not only on long bones but also on vertebrae that are rich in cancellous bones and for longer observation periods might possibly reveal the stronger action of JTT-606 on bone strength.(31)

Taken together, the results found here first show that JTT-606 is a promising new class of agent that can be tested for its bone mass protective role. Of course, this study, although allowing a rapid and reliable screening of this agent for its bone resorption inhibiting effect without disturbing the mineralization, is not sufficient to establish whether it can be proposed as an effective and safe pharmacologic agent in humans. Further in vivo studies using several animal models are needed to establish whether this agent may be appropriate for clinical studies.

Acknowledgements

We are grateful to Ms. Akiko Hasegawa at Central Pharmaceutical Research Institute, Japan Tobacco, Inc. for her expert technical assistance. Funded by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture (09307033 [H.K.] and 10470302 [K.N.]) and Bristol-Myers Squibb/Zimmer Unrestricted Research Grant (K.N.).

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