Osteoblastgenic and Osteogenic Effects of KY-273 with CDK8/19 Inhibitory Activity in Bone Marrow Mesenchymal Stem Cells and Female Rats

Abstract

Osteoporosis is caused by imbalance between osteogenesis and bone resorption, thus, osteogenic drugs and resorption inhibitors are used for treatment of osteoporosis. The present study examined the effects of (R)-4-(1-hydroxyethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (KY-273), a diphenyl ether derivative, on CDK8/19 activity, osteoblast differentiation and femoral bone using micro-computed tomography in female rats. KY-273 potently inhibited CDK8/19 activity, promoted osteoblast differentiation with an increase in alkaline phosphatase (ALP) activity, and gene expression of type I collagen, ALP and BMP-4 in mesenchymal stem cells (ST2 cells). In female rat femur, ovariectomy decreased metaphyseal trabecular bone volume (Tb.BV), mineral content (Tb.BMC), yet had no effect on metaphyseal and diaphyseal cortical bone volume (Ct.BV), mineral content (Ct.BMC) and strength parameters (BSPs). In ovaries-intact and ovariectomized rats, oral administration of KY-273 (10 mg/kg/d) for 6 weeks increased metaphyseal and diaphyseal Ct.BV, Ct.BMC, and BSPs without affecting medullary volume (Med.V), but did not affect Tb.BV and Tb.BMC. In ovariectomized rats, alendronate (3 mg/kg/d) caused marked restoration of Tb.BV, Tb.BMC and structural parameters after ovariectomy, and increased metaphyseal but not diaphyseal Ct.BV, Ct.BMC, and BSPs. In ovaries-intact and ovariectomized rats, by the last week, KY-273 increased bone formation rate/bone surface at the periosteal but not the endocortical side. These findings indicate that KY-273 causes osteogenesis in cortical bone at the periosteal side without reducing Med.V. In conclusion, KY-273 has cortical-bone-selective osteogenic effects by osteoblastogenesis via CDK8/19 inhibition in ovaries-intact and ovariectomized rats, and is an orally active drug candidate for bone diseases such as osteoporosis in monotherapy and combination therapy.

INTRODUCTION

Bones are continuously remodeled by bone formation via osteoblasts and bone resorption via osteoclasts. This process is governed by complicated crosstalk between osteoclasts and osteoblasts via various humoral factors. An imbalance between bone formation and resorption leads to osteoporosis in both the aged people and postmenopausal women. To mitigate these conditions, osteoporosis is treated with bone resorption inhibitors, such as oral and parenteral bisphosphonates, and parenteral osteogenic agents, such as parathyroid hormone (PTH) and anti-sclerostin antibodies. Orally active osteogenic drugs have been a desirable target, and a great number of small molecules have been reported to exert in vitro osteoblastogenic effects and in vivo osteogenic effects.^1–6) Unfortunately, these have not been yet developed as effective and safe anti-osteoporotic drugs. Among them, benzothiopyran, benzofuran, and coumarin derivatives are suggested to enhance osteoblast differentiation via BMP potentiation.^1–4) One lactone derivative reportedly promotes osteoblast differentiation via stimulating Wnt/LPR5/β-catenin signaling.⁵⁾ Thienopyridine derivatives are considered to exerts osteoblastogenic effects through CDK8 inhibition, since their CDK8 inhibitory activities are correlated with their osteoblast differentiation-promoting activities measured as alkaline phosphatase (ALP) activities in mesenchymal stem cells.⁶⁾ CDK8 is a positive transcription regulator, maintaining stemness and dedifferentiation in cancer and stem cells.^7–9) It may also function similarly with bone marrow mesenchymal stem cells. We endeavored to find a new chemotype of small molecules with CDK8 inhibitory and osteoblastogenic activity in bone marrow mesenchymal cells, and anti-osteoporotic effects in female ovariectomized (OVX) rats. We reported the preliminary results that (R)-4-(1-hydroxyethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (KY-273), a diphenyl ether derivative, inhibits CDK8, promotes osteoblast differentiation, and increases femur cortical bone in OVX rats at the symposium in the annual meeting of the Pharmaceutic Society of Japan (2018)¹⁰⁾; however, further study is needed to more clearly understand its effects. These effects, for the diphenyl ether derivatives, appear mediated by CDK8 inhibition due to the correlation between osteoblast marker ALP activity and CDK inhibition, reflecting behavior reported for the thienopyridine derivatives.^6,10)

The present study clarifies and characterizes the effects of KY-273 on CDK8/19 activity and osteoblast differentiation, and investigates its osteogenic effects on femur cortical bone and trabecular bone in ovaries-intact and OVX rats using micro-computed tomography (micro-CT) and histological analyses. KY-273 was shown to promote osteoblast differentiation with increased ALP activity and gene expression of type I collagen, ALP, and BMP-4 in mouse mesenchymal cells (ST2 cells) alongside increases in femur cortical bone volume and mineral contents via osteogenesis at the periosteal side, but not trabecular bone, in ovaries-intact and OVX rats.

MATERIALS AND METHODS

Materials and Animals

KY-273 was synthesized in our laboratories. It was dissolved in dimethyl sulfoxide (DMSO) and diluted in Minimum Essential Medium alpha Media (α-MEM) for in vitro study, and suspended in 0.5% methyl cellulose (MC) solution for in vivo study. ST2 cells were purchased from the Riken BioResource Research Center (Tsukuba, Japan) and subcultured in RPMI-1640 (Nissui, Tokyo, Japan). Female F344/NSlc rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Animals were housed under conditions with controlled temperature, humidity, and light exposure (12-h light–dark cycle) and provided ad libitum access to commercial standard rodent chow (CE2; CLEA Japan, Tokyo, Japan) and tap water. Animals were handled in accordance with the “Guidelines for Animal Experimentation” approved by The Japanese Pharmacological Society with all procedures approved by the Animal Ethical Committee of Kyoto Pharmaceutical Industries, Ltd.

CDK8 Kinase Assay

CDK8 activity was measured using the QSS Assist CDK8 enzyme-linked immunosorbent assay (ELISA) Kit (Carna Bioscience, Kobe, Japan) following the manufacturer’s protocol. The effects of KY-273 from 0.3 nM–1 µM on CDK8 activity were determined. To 96-well plate coated with streptavidin at room temperature was added 10 µL of KY-273 solution (1.2 nM–4 µM), 10 µL of ATP/substrate/MgCl₂ solution, and 20 µL of CDK8/cyclin C. After 30 min, the wells were washed four times each with 200 µL/well wash buffer then 200 µL/well blocking buffer added and incubated at room temperature for 30 min. The well solution was discarded and 100 µL/well primary antibody solution added, followed by incubation at room temperature for 30 min. The well solution was then discarded, and wells washed four times with 200 µL/well of wash buffer. Then, 100 µL/well of horseradish peroxidase (HRP)-labeled secondary antibody solution was added and incubated at room temperature for 30 min. The wells were washed four times with 200 µL/well wash buffer, then 100 µL/well of chromogenic reagent was added, incubated at room temperature for 5 min, and 100 µL/well of color reaction stopping reagent was added. The absorbance at 450 nm was measured using a microplate reader, and IC₅₀ values were calculated.

Separately, the effects of KY-273 (3–100 nM) on the CDK8 activity were examined in the presence of substrate (250 nM) and ATP (10–100 µM), and ATP (30 µM) and substrate (125–500 nM) were determined. Then, the data were analyzed by Lineweaver–Burk plot.

Other CDK Kinase Assay

CDK7, CDK8, CDK9 and CDK19 were classified into transcriptional CDK, and CDK2, CDK3, CDK4, CDK5 and CDK6 into cell-cycle related CDK.¹¹⁾ The effects of KY-273 on CDK7 and CDK9 activity were examined using QSS Assist STK ELISA Kit (Carna Bioscience). The effects of KY-273 (1 µM) on CDK2, CDK3, CDK4, CDK5, and CDK6 were determined with in vitro mobility shift assay (Carna Bioscience).

In Vitro Osteoblast Differentiation: ALP Activities

Osteoblast differentiation was investigated using ST2 cells derived from mouse bone marrow mesenchymal stem cells as previously reported.¹²⁾ ST2 cells were seeded on 96-well plates at a density of 4 × 10³ cells/100 µL/well and cultured for 24 h in α-MEM, osteoblast differentiation medium. Then, KY-273 was added and further cultured for 1, 2, 4, and 6 d. Cells were then washed with phosphate buffered saline (PBS) (pH 7.4) and lysed in 50 µL of 10 mM MgCl₂ solution containing 1% Triton X-100 for the measurement of ALP activity. The reaction was initiated by the addition of 50 µL of 10 mM p-nitrophenyl phosphate (pNPP) in 50 mM ethanolamine and quenched by the addition of 50 µL of 1 M NaOH after a 30-min incubation at 37 °C. ALP activities were assessed by measuring absorbance at 405 nm.

In Vitro Osteoblast Differentiation: mRNA Expression

ST2 cells were seeded on 96-well plates at a density of 4 × 10³ cells/100 µL/well and cultured for 24 h in α-MEM. To this was then added KY-273 and cultured for either 6 or 24 h. ST2 cells were lysed using Trizol (Life Technologies, Carlsbad, CA, U.S.A) with RNA extraction using a combination of Trizol and High Pure RNA Isolation Kit (Roche, Mannheim, Germany). The RNA absorbance at 260 nm was measured to calculate RNA concentration. RNA samples were converted to cDNA using PrimeScript RT reagent kit (TaKaRa-Bio, Kusatsu, Japan). Real-time quantitative PCR assays were performed with the Light Cycler 480 (Roche) using FastStart Essential DNA Probes Master and primer (Roche). Primer sequences were designed using the Universal ProbeLibrary (Roche): Col1a1, 5′-AGACATGTTCAGCTTTGTGGAC-3′ and 5′-GCAGCTGACTTCAGGGATG-3′; and ALP, 5′-AATGAGGTCACATCCATCCTG-3′ and 5′-CACCCGAGTGGTAGTCACAA-3′; and BMP4, 5′-GAGGAGTTTCCATCACGAAGA-3′ and 5′-GCTCTGCCGAGGAGATCA-3′; and 18s rRNA forward, 5′-GGTGCATG GCCGTTCTTA-3′ and reverse, 5′-TCGTTCGTTATCGGAATTAACC-3′. Gene expression levels for Type I collagen, ALP, and BMP-4 in ST2 cells were normalized to a geometric mean of 18s ribosomal RNA (rRNA). Data were analyzed using ΔΔCT methods.

Plasma Concentrations in Female Rats

Non-operated and ovariectomized 12-week-old female rats were orally administered KY-273 (3 and 10 mg/5 mL/kg) suspended in 0.5% MC solution. Blood was taken from the jugular vein at 0.25, 0.5, 1, 3, 5, 8, and 24 h after administration. KY-273 plasma concentrations were determined using LC/MS/MS (QTRAP5500, AB Sciex, Framingham, MA, U.S.A) with a pump (Nexera X2, LC-30AD, Shimadzu, Kyoto, Japan) and autoinjector (Nexera X2, SIL-30AC, Shimadzu).

Ovaries-Intact and OVX Rats: 6-Week Experiments

Twelve-week-old female F344/NSlc rats were randomly allocated to 5 groups (n = 10); ovaries-intact-control, ovaries-intact-KY-273-treated, OVX-control, OVX-KY-273-treated and OVX-alendronate-treated. Then, ovariectomy was performed as previously reported.¹²⁾ Rats were anesthetized using ketamine (37.5 mg/kg, intraperitoneally (i.p.)) and xylazine (7.5 mg/kg, i.p.), and underwent sham operation for the ovaries-intact-control and ovaries-intact-KY-273-treated groups. The OVX-control, OVX-KY-273-treated, and OVX-alendronate-treated groups were bilaterally ovariectomized. For 6 weeks, rats were orally administered vehicle (0.5% MC, 5 mL/kg/d), KY-273 (10 mg/5 mL/kg/d) suspended in 0.5% MC, and alendronate (3 mg/5 mL/kg/d) suspended in 0.5% MC. Body weight and food consumption were monitored during the experiment period. The right femur was scanned, under anesthesia with isoflurane, using micro-CT (R_μCT; Rigaku, Tokyo, Japan) 1 d before initiating administration and on the final day of administration. To assess new bone formation (bone formation rate/bone surface, BFR/BS) all rats received a subcutaneous injection of calcein (10 mg/kg), a fluorescent calcium chelator, at 7 and 2 d before the final day. After repeated administration, rats were deeply anaesthetized with pentobarbital sodium (50 mg/kg, i.p.), fasting blood collected from the abdominal aorta, and then euthanized. Femurs were then isolated and fixed with 70% ethanol and stored at 4 °C.

OVX Rats: 12-Week

Twelve-week-old female F344/NSlc rats were randomly allocated to 4 groups (n = 8); ovaries-intact-control, OVX-control, and OVX-KY-273 (1 and 10 mg/kg/d)-treated groups. Sham-operations and ovariectomies were performed, and either vehicle (0.5% MC) or KY-273 (1 or 10 mg/5 mL/kg/d) administered over 12 weeks. The right femur was scanned using micro-CT under anesthesia 1 d before beginning administration, then at the 4th, 8th, and 12th weeks (the final administration day).

In Vivo Micro-CT Analyses

Micro-CT data analyses used TRI/3D-BON software (RATOC, Tokyo, Japan).^13,14) In metaphysis trabecular bone, the trabecular bone mineral content (Tb.BMC), trabecular bone tissue volumetric mineral density (Tb.TMD), bone volume fraction (BV/TV), trabecular bone volume (Tb.BV), specific bone surface (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular bone pattern factor (TBPf), structure model index (SMI), and marrow space star volume (Star Volume) were determined. In both the metaphysis and diaphysis cortical bone, total bone volume (T.BV), cortical bone volume (Ct.BV), medullary volume (Med.V), cortical BMC (Ct.BMC), cortical tissue volumetric BMD (Ct.TMD), periosteal perimeter (Ps.Pm), and endocortical perimeter (Ec.Pm) were determined. As metaphysis and diaphysis cortical bone strength parameters, the major and minor axis section modules (IaSSI and IbSSI) and bone strength index (aBSI and bBSI, i.e., the cross-sectional moment of inertia multiplied by the Ct.TMD), reported to correlate to actual bone breaking strength, were also determined.¹⁴⁾ In the 6-week experiments, principal cortical bone parameter changes (ΔCt.BV, ΔCt.BMC, ΔIaSSI, and ΔIbSSI) were determined for the administration period. For the 12-week experiments, Ct.BV and Ct.BMC were calculated before treatment, and at the 4-, 8-, and 12-week points of the administration period.

Bone Histology

Isolated and fixed bones were embedded in methyl methacrylate with 10 µm sections prepared and stained using toluidine blue. BFR/BS in diaphysis cortical bone was then evaluated using fluorescence microscopy and OsteoMeasure software (OsteoMetrics, Decatur, GA, U.S.A.).

Statistical Analyses

All data are expressed as mean ± standard error of the mean (S.E.M.). Significance of difference was assessed by one-way or repeated measures one-way ANOVA followed by Bonferroni’s or Dunnett’s multiple comparison tests. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, U.S.A.).

RESULTS

CDK8 Inhibition

KY-273 inhibited CDK8 and CDK19 enzyme activity in a concentration-dependent manner over 1–1000 nM, but had no effect on CDK7 and CDK9 activities. IC₅₀ of CDK8 and CDK19 were 29.1 and 44.7 nM, respectively (Figs. 1A, B). Lineweaver-Burk plot showed that KY-273 is an ATP-competitive and substrate-noncompetitive inhibitor (Fig. 1C). KY-273 had little effect on CDK2, CDK3, CDK4, CDK5 and CDK6 even at 1000 nM (Fig. 1D).

Fig. 1. Effects of KY-273 on CDKs Enzyme Activities

Osteoblast Differentiation

KY-273 concentration- and time-dependently increased ALP activities for 0.1–10 µM at 2, 4, and 6 d, yielding 2.3-, 4.5-, and 5.5-fold increases over non-treated levels, respectively (Fig. 2). KY-273 concentration-dependently enhanced mRNA expression of Type I collagen for 24 h, ALP for 24 h, and BMP-4 for 6 h (Fig. 3).

Fig. 2. Effects of KY-273 on ALP Activities in ST2 Cells

ALP activity levels in DMSO-treated cells were taken as 100%. Values are the mean ± S.E.M. n = 4. ** p < 0.01 vs. Control, repeated measures one-way ANOVA followed by Dunnett’s multiple comparison test.

Fig. 3. Effects of KY-273 on mRNA Expression of Type I Collagen, ALP and BMP-4

The values were calculated relative to the mean of the control. KY-273 was treated for 24 h for Type I Collagen and ALP, and for 6 h for BMP-4. Values are the mean ± S.E.M. n = 4. * p < 0.05, ** p < 0.01 vs. Control, repeated measures one-way ANOVA followed by Dunnett’s multiple comparison test.

Plasma Concentrations in Female Rats

KY-273 plasma concentration after oral administration at 10 mg/5 mL/kg (p.o.) reached a maximum of 3.5 µM at 0.5 h in non-operated rats, and 8.8 µM at 0.5 h in OVX rats. In both, the plasma concentration of KY-273 exceeded effective concentrations for CDK8 inhibition and osteoblast differentiation promotion for at least over 8 h.

Ovaries-Intact and OVX Rats

In OVX rats, body weight was significantly increased, with uterine weight markedly and equally reduced, indicating successful ovariectomy. KY-273 at 10 mg/kg/d and alendronate at 3 mg/kg/d for 6 weeks had little effect on body weight and uterine weight (Fig. 4), indicating no estrogenic effects.

Fig. 4. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on Final Body Weight and Uterus Weight in Ovaries-Intact and Ovariectomized Female Rats

Values are the mean ± S.E.M. n = 10. ^††p < 0.01 vs. ovaries-intact-control, * p < 0.05 vs. OVX-control, one-way ANOVA followed by Bonferroni’s multiple comparison test. Intact: ovaries-intact, Cont: control, KY: KY-273, ALN: Alendronate.

In Vivo Micro-CT Analyses in 6-Week Experiments

Bone parameters before ovariectomy were not significantly different among ovaries-intact and OVX groups (data not shown).

After repeated administration, the femur metaphysis trabecular bone parameters were similar between the ovaries-intact-control and ovaries-intact-KY-273-treated groups. All measured parameters worsened in the OVX-control compared to the ovaries-intact-control group. In the OVX-control group, Tb.BV, Tb.BMC, Tb.TMD, BV/TV, Tb.Th, and Tb.N were significantly smaller, while BS/BV, Tb.Sp, TBRf, SMI, and star volume were greater, indicating reductions in trabecular bone volume, mineral density, number, thickness, tightness, and plate-likeness due to ovariectomy-induced bone resorption (Table 1). These parameters were not significantly different between the OVX-control and OVX-KY-273-treated groups. However, in the OVX-alendronate-treated group, these parameters were all significantly improved compared to the OVX-control group, indicating restoration from ovariectomy-induced trabecular bone destruction.

Table 1. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on Femur Metaphyseal Trabecular Bone Parameters Analyzed by in vivo Micro-CT Scanning in Ovaries-Intact and Ovariectomized Rats

	Ovaries-Intact		OVX
	Control	KY-273	Control	KY-273	Alendronate
Tb.BV (mm3)	3.4 ± 0.1	3.5 ± 0.2	1.5 ± 0.1††	1.8 ± 0.1	3.7 ± 0.3**
Tb.BMC (mg)	1.3 ± 0.1	1.3 ± 0.1	0.5 ± 0.1††	0.6 ± 0.0	1.4 ± 0.1**
Tb.TMD (mg/mm3)	398.9 ± 4.4	384.1 ± 3.8	337.6 ± 5.0††	326.9 ± 3.3	367.1 ± 6.5**
BV/TV (%)	32.4 ± 1.1	32.5 ± 1.1	13.3 ± 1.0††	13.9 ± 0.9	30.7 ± 1.7**
BS/BV (mm2/mm3)	17.2 ± 0.3	17.0 ± 0.2	20.6 ± 0.4††	20.5 ± 0.5	17.7 ± 0.3**
Tb.Th (µm)	116.4 ± 1.8	118.1 ± 1.8	97.3 ± 1.9††	97.8 ± 2.1	113.3 ± 2.0**
Tb.N (1/mm)	3.4 ± 0.1	3.3 ± 0.1	1.6 ± 0.1††	1.6 ± 0.1	3.2 ± 0.2**
Tb.Sp (µm)	183.5 ± 11.1	189.8 ± 8.0	551.5 ± 38.6††	536.1 ± 37.0	208.1 ± 17.3**
TBPf (1/mm)	2.0 ± 0.2	1.9 ± 0.2	4.6 ± 0.5††	4.7 ± 0.3	2.5 ± 0.3**
SMI	1.8 ± 0.0	1.7 ± 0.0	2.3 ± 0.1††	2.2 ± 0.1	1.8 ± 0.1**
Star Volume (mm3)	0.4 ± 0.0	0.3 ± 0.0	1.3 ± 0.1††	1.5 ± 0.1	0.5 ± 0.1**

Mean ± S.E.M. n = 10. ^††p < 0.01 vs. ovaries-intact-control, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Bonferroni’s multiple comparison test.

In femur metaphyseal cortical bone, measured parameters were not significantly different between the ovaries-intact and OVX-control groups. In the ovaries-intact-KY-273-treated group, Ct.BV, Ct.BMC, IbSSI, and bBSI were significantly greater than in the ovaries-intact-control group (Table 2). For the OVX-KY-273-treated group, T.BV, Ct.BV, Med.V, Ct.BMC, Ec.Pm, IaSSI, IbSSI, aBSI, and bBSI were significantly greater than the OVX-control group. These parameters, except for bBSI, were significantly greater in the OVX-alendronate-treated group than the OVX-control group.

Table 2. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on Femur Metaphyseal Cortical Bone Parameters Analyzed by in vivo Micro-CT Scanning in Ovaries-Intact and Ovariectomized Rats

	Ovaries-Intact		OVX
	Control	KY-273	Control	KY-273	Alendronate
T.BV (mm)	20.8 ± 0.2	21.8 ± 0.3	21.4 ± 0.4	23.8 ± 0.2**	23.3 ± 0.4**
Ct.BV (mm3)	8.6 ± 0.1	9.2 ± 0.1††	8.5 ± 0.1	9.1 ± 0.1**	9.2 ± 0.1**
Med.V (mm3)	12.3 ± 0.2	12.7 ± 0.2	12.9 ± 0.4	14.7 ± 0.2**	14.1 ± 0.3**
Ct.BMC (mg)	8.3 ± 0.1	9.0 ± 0.1††	8.2 ± 0.1	8.7 ± 0.1**	8.8 ± 0.1**
Ct.TMD (mg/mm3)	965.4 ± 4.6	982.1 ± 4.4	958.7 ± 6.4	958.0 ± 4.9	960.0 ± 4.6
Ps.Pm (µm)	12797 ± 307	12095 ± 154	13221 ± 406	12811 ± 173	12445 ± 103
Ec.Pm (µm)	9121 ± 152	9560 ± 91	9293 ± 283	10205 ± 117**	10050 ± 121*
IaSSI (mm3)	2.6 ± 0.0	2.7 ± 0.0	2.7 ± 0.0	2.9 ± 0.0**	3.0 ± 0.1**
IbSSI (mm3)	3.1 ± 0.0	3.3 ± 0.0†	3.1 ± 0.1	3.5 ± 0.0**	3.4 ± 0.1**
aBSI (mm4 × mg/mm3)	5297 ± 130	5518 ± 120	5429 ± 69	6000 ± 74**	6020 ± 124**
bBSI (mm4 × mg/mm3)	6486 ± 156	7239 ± 138††	6813 ± 139	7699 ± 104**	7390 ± 225

Mean ± S.E.M. n = 10. ^†p < 0.05, ^††p < 0.01 vs. ovaries-intact-control, * p < 0.05, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Bonferroni’s multiple comparison test.

In femur diaphysis, no parameters were significantly different between the ovaries-intact and OVX-control groups. For the ovaries-intact-KY-273-treated group, Ct.BV, Ct.BMC, IaSSI, IbSSI, and bBSI were significantly greater than for the ovaries-intact control group (Table 3). For the OVX-KY-273-treated group, T.BV, Ct.BV, Ct.BMC, IaSSI, IbSSI, aBSI, and bBSI were significantly greater than for the OVX-control group, indicating that KY-273 increased cortical bone without affecting Med.V. Between the OVX-control and OVX-alendronate-treated group, bone parameters were similar.

Table 3. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on Femur Diaphyseal Cortical Bone Parameters Analyzed by in vivo Micro-CT Scanning in Ovaries-Intact and Ovariectomized Rats

	Ovaries-Intact		OVX
	Control	KY-273	Control	KY-273	Alendronate
T.BV (mm)	31.0 ± 0.3	32.3 ± 0.4	32.2 ± 0.2	33.6 ± 0.3*	32.2 ± 0.5
Ct.BV (mm3)	19.1 ± 0.2	20.2 ± 0.2††	19.8 ± 0.2	21.0 ± 0.2**	20.1 ± 0.3
Med.V (mm3)	11.9 ± 0.1	12.1 ± 0.2	12.4 ± 0.1	12.6 ± 0.2	12.1 ± 0.3
Ct.BMC (mg)	20.6 ± 0.2	21.8 ± 0.2††	21.1 ± 0.3	22.6 ± 0.1**	21.7 ± 0.3
Ct.TMD (mg/mm3)	1077 ± 3	1083 ± 5	1065 ± 11	1073 ± 5	1079 ± 5
Ps.Pm (µm)	8821 ± 43	9002 ± 53	9010 ± 27	9186 ± 45	8998 ± 79
Ec.Pm (µm)	5674 ± 34	5733 ± 46	5794 ± 31	5832 ± 39	5718 ± 59
IaSSI (mm3)	1.7 ± 0.0	1.8 ± 0.0†	1.8 ± 0.0	1.9 ± 0.0**	1.8 ± 0.0
IbSSI (mm3)	1.9 ± 0.0	2.0 ± 0.0†	2.0 ± 0.0	2.1 ± 0.0**	2.0 ± 0.1
aBSI (mm4 × mg/mm3)	2496 ± 53	2704 ± 59	2628 ± 44	2913 ± 53**	2695 ± 72
bBSI (mm4 × mg/mm3)	3220 ± 59	3579 ± 77†	3487 ± 67	3819 ± 59*	3568 ± 132

Mean ± S.E.M. n = 10. ^†p < 0.05, ^††p < 0.01 vs. ovaries-intact-control, * p < 0.05, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Bonferroni’s multiple comparison test.

Changes in Cortical Bone Parameters for 6 Weeks

Changes in selected bone parameters (ΔTb.BV and ΔTb.BMC in trabecular, ΔCt.BV, ΔCt.BMC, ΔaBSI, and ΔbBSI in cortical bone) between before and after repeated administration were analyzed in femur metaphysis and diaphysis (Figs. 5–7).

Fig. 5. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on Changes in Trabecular Bone Parameters of Femur Metaphysis Analyzed by in vivo Micro-CT Scanning in Ovaries-Intact and Ovariectomized Female Rats

Values are the mean ± S.E.M. n = 10. ^††p < 0.01 vs. ovaries-intact-control, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Bonferroni’s multiple comparison test. Intact: ovaries-intact, Cont: Control, KY: KY-273, ALN: Alendronate.

Fig. 6. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on Changes in Cortical Bone Parameters of Femur Metaphysis Analyzed by in vivo Micro-CT Scanning in Ovaries-Intact and Ovariectomized Female Rats

Values are the mean ± S.E.M. n = 8. ^††p < 0.01 vs. ovaries-intact-control, * p < 0.05, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Bonferroni’s multiple comparison test. Intact: ovaries-intact, Cont: Control, KY: KY-273, ALN: Alendronate.

Fig. 7. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on Changes in Cortical Bone Parameters of Femur Diaphysis Analyzed by in vivo Micro-CT Scanning in Ovaries-Intact and Ovariectomized Female Rats

Values are the mean ± S.E.M. n = 8. ^†p < 0.05, ^††p < 0.01 vs. ovaries-intact-control, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Bonferroni’s multiple comparison test. Intact: ovaries-intact, Cont: Control, KY: KY-273, ALN: Alendronate.

In the metaphyseal trabecular bone, ΔTb.BV and ΔTb.BMC were comparable between the ovaries-intact and ovaries-intact-KY-273-treated groups. The increase in these parameters observed in the ovaries-intact groups shifted to a decrease in the OVX control group. The decrease in both parameters was similar between the OVX control and OVX-KY-273-treated groups. However, in the OVX-alendronate-treated group, the decrease was reversed, returning to levels observed in the ovaries-intact group (Fig. 5).

For the metaphyseal cortical bone, parameters between the ovaries-intact-control and OVX-control group were comparable. In the ovaries-intact-KY-273-treated group, ΔCt.BV, ΔCt.BMC, and ΔbBSI were significantly and ΔaBSI only slightly greater than in the ovaries-intact-control group. In the OVX-KY-273-treated group, ΔCt.BV, ΔaBSI, and ΔbBSI were significantly and ΔCt.BMC only slightly greater than in the OVX-control group, in the OVX-alendronate-treated group, these values were significantly greater than in the OVX-control group (Fig. 6).

For diaphyseal cortical bone, all parameters were similar between the ovaries-intact-control and OVX-control groups. In the ovaries-intact-KY-273-treated group, ΔCt.BV, ΔCt.BMC, ΔaBSI, and ΔbBSI were significantly greater than in the ovaries-intact-control group. In the OVX-KY-273-treated group, ΔCt.BV, ΔCt.BMC, ΔaBSI, and ΔbBSI were significantly greater than in the OVX-control group, but these were comparable between the OVX-control and OVX-alendronate-treated groups (Fig. 7).

BFR/BS in Diaphysis at the Last Week

In the ovaries-intact-KY-273-treated group, BFR/BS at the diaphysis periosteal side was significantly greater, while BFR/BS at the endocortical side was significantly smaller compared to the ovaries-intact control group (Fig. 8). Between the ovaries-intact and OVX-control groups, there was no difference in BFR/BS. However, in the OVX-KY-273-treated group, BFR/BS at the periosteal side was significantly greater than OVX-control group, a significant difference not observed at the endocortical side (Fig. 8).

Fig. 8. Effects of KY-273 (10 mg/kg/d, p.o. 6 Weeks) and Alendronate (3 mg/kg/d, p.o., 6 Weeks) on BFR/BS in Periosteal and Endocortical Side

Values are the mean ± S.E.M. n = 10. ^†p < 0.05, ^††p < 0.01 vs. Intact control, ** p < 0.01 vs. OVX control, one-way ANOVA followed by Bonferroni’s multiple comparison test. Intact: ovaries-intact, Cont: Control, KY: KY-273, ALN: Alendronate.

In Vivo Micro-CT Analyses in 12-Week Experiments

Bone parameters before ovariectomy were similar among ovaries-intact and OVX groups (data not shown).

After repeated administration, the femur metaphysis trabecular bone parameters all worsened in the OVX-control group compared to the ovaries-intact-control group. These parameters were not significantly different between the OVX-control and OVX-KY-273-treated groups (data not shown).

In the metaphyseal cortical bone, only IbSSI and bBSI were significantly greater in the OVX-control than ovaries-intact-control group. All parameters were similar between the OVX-control and OVX-KY-273 (1 mg/kg/d)-treated group (Table 4). In the OVX-KY-273 (10 mg/kg/d)-treated group, T.BV, Ct.BV, Ct.BMC, IaSSI, IbSSI, aBSI, and bBSI were significantly greater than in OVX-control group, indicating that KY-273 at 10 mg/kg/d enlarged bone by increasing cortical bone and bone strength, without affecting medullary volume.

Table 4. Effects of KY-273 (1 and 10 mg/kg/d, p.o. 12 Weeks) on Femur Metaphyseal Cortical Bone Parameters Analyzed by in vivo Micro-CT Scanning in Ovariectomized Rats

	Ovaries-Intact control	OVX control	OVX + KY-273 (mg/kg/d)
			1	10
T.BV (mm3)	20.2 ± 0.3	21.3 ± 0.4	21.7 ± 0.3	22.5 ± 0.4*
Ct.BV (mm3)	8.5 ± 0.1	8.8 ± 0.1	9.0 ± 0.1	9.6 ± 0.1**
Med.V (mm3)	11.6 ± 0.3	12.5 ± 0.3	12.7 ± 0.3	12.9 ± 0.3
Ct.BMC (mg)	8.7 ± 0.2	8.9 ± 0.1	9.3 ± 0.2	9.8 ± 0.1**
Ps.Pm (µm)	11408 ± 86	11757 ± 110	11867 ± 84	12075 ± 116
Ec.Pm (µm)	9053 ± 112	9449 ± 136	9503 ± 110	9571 ± 127
IaSSI (mm3)	2.5 ± 0.0	2.6 ± 0.0	2.7 ± 0.0	2.9 ± 0.1**
IbSSI (mm3)	3.0 ± 0.1*	3.2 ± 0.0	3.3 ± 0.1	3.6 ± 0.1**
aBSI (mm4 × mg/mm3)	4866 ± 126	5206 ± 140	5403 ± 138	5894 ± 220*
bBSI (mm4 × mg/mm3)	6434 ± 147*	7007 ± 123	7482 ± 170	8262 ± 202**

Mean ± S.E.M. n = 8. * p < 0.05, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Dunnett’s multiple comparison test.

In diaphyseal cortical bone, parameters were unchanged between the OVX-control and ovaries-intact-control groups, suggesting no influence of ovariectomy on cortical bone, even over a longer period. All parameters were significantly greater in OVX-KY-273 (10 mg/kg/d)-treated, and only bBSI in OVX-KY-273 (1 mg/kg/d)-treated group than OVX-control group, indicating that KY-273 at 10 mg/kg/d increased bone formation by osteogenesis at the periosteal side with increased cortical bone, medullary volume and bone strength (Table 5).

Table 5. Effects of KY-273 (1 and 10 mg/kg/d, p.o. 12 Weeks) on Femur Diaphyseal Cortical Bone Parameters Analyzed by in vivo Micro-CT Scanning in Ovariectomized Rats

	Intact	OVX control	OVX + KY-273 (mg/kg/d)
			1	10
T.BV (mm3)	31.1 ± 0.3	32.2 ± 0.4	33.1 ± 0.2	35.4 ± 0.3**
Ct.BV (mm3)	19.8 ± 0.2	20.3 ± 0.2	20.7 ± 0.2	21.9 ± 0.3**
Med.V (mm3)	11.3 ± 0.2	11.9 ± 0.2	12.4 ± 0.1	13.5 ± 0.2**
Ct.BMC (mg)	22.6 ± 0.3	22.5 ± 0.2	23.1 ± 0.2	24.3 ± 0.4**
Ps.Pm (µm)	8903 ± 41	9021 ± 47	9141 ± 34	9444 ± 48**
Ec.Pm (µm)	5558 ± 55	5673 ± 49	5790 ± 27	6030 ± 47**
IaSSI (mm3)	1.7 ± 0.0	1.8 ± 0.0	1.8 ± 0.0	2.0 ± 0.0**
IbSSI (mm3)	1.9 ± 0.0	2.0 ± 0.0	2.1 ± 0.0	2.3 ± 0.0**
aBSI (mm4 × mg/mm3)	2740 ± 49	2793 ± 69	2914 ± 56	3344 ± 75**
bBSI (mm4 × mg/mm3)	3493 ± 47	3665 ± 64	3923 ± 64*	4375 ± 85**

Mean ± S.E.M. n = 8. * p < 0.05, ** p < 0.01 vs. OVX-control, one-way ANOVA followed by Dunnett’s multiple comparison test.

Ct.BV and Ct.BMC, in the metaphysis and diaphysis, gradually increased over 12 weeks (Fig. 9). In the OVX-KY-273 (10 mg/kg/d)-treated group, Ct.V and Ct.BMC were significantly larger than in the OVX-control group in metaphysis at the 8- and 12-week points, and in diaphysis at the 4-, 8-, and 12-week points.

Fig. 9. Effects of KY-273 (10 mg/kg/d, p.o. 12 Weeks) on Cortical Bone Parameters of Femur Metaphysis and Diaphysis Analyzed by in vivo Micro-CT Scanning in Ovariectomized Rats

Values are the mean ± S.E.M. n = 8. * p < 0.05, ** p < 0.01 vs. OVX control, one-way ANOVA followed by Dunnett’s multiple comparison test.

DISCUSSION

In the present study, we examined the effects of KY-273 on CDK8/19 activity, osteoblast differentiation in ST2 cells, and femoral bone in female ovaries-intact and OVX rats.

KY-273 potently and concentration-dependently inhibited both CDK8 and CDK19, a paralogue of CDK8, yet did not affect other transcriptional CDKs, CDK7 and CDK9, and cell cycle related CDKs, CDK2, CDK3, CDK4, CDK5 and CDK6 activities. Furthermore, as KY-273 does not affect 30 other serine/threonine kinases, it seems a selective CDK8/19 inhibitor.¹⁰⁾

KY-273 concentration-dependently and potently increased ALP activity in 2-, 4-, and 6-d treatments in ST2 cells, maximally to 5.5-fold. KY-234, within 24 h in ST2 cells, also enhanced mRNA expression of type I collagen, ALP, and BMP-4, osteoblast markers. We briefly presented preliminary results showing that KY-273 increased ALP activity for 4 d in ST2 and rat bone marrow mesenchymal stem cells, and enhanced mineralization for 13 d in ST2 cells.¹⁰⁾ Furthermore, CDK8/19 knockdown enhanced osteoblast differentiation, and CDK8 inhibition corelated with increased ALP activity for thienopyridine derivatives and diphenyl ether derivatives, including KY-273.^6,10) Given these similarities, KY-273 is thought to promote osteoblast differentiation via CDK8 inhibition, followed by gene expression for osteoblastogenesis signaling molecules. KY-273 had little effect on adipocyte or osteoclast differentiation in preliminary experiments.¹⁰⁾ Benzothiopyran, benzofuran, coumarin derivatives, and estradiol are all reported to promote osteoblast differentiation via BMP-2 and BMP-4 upregulation.^1–4,15) Cellular BMP-4 is required for maintenance and stimulation of osteoblast differentiation.¹⁶⁾ Interestingly, KY-273 rapidly enhanced BMP-4 gene expression during the 6 h treatment, thus CDK8 inhibition may promote osteoblast differentiation by potentiation of BMP-4 signaling. However, how KY-273 promoted osteoblast differentiation via CDK8 inhibition remains unclear. Further study is necessary to fully elucidate the role of CDK8 in osteoblastogenesis. Also required for this is a comprehensive investigation into the impact of CDK8 inhibition by KY-273 on mesenchymal stem cells, and progenitor co-cultures of osteoblasts and osteoclasts.

The plasma concentration of KY-273 after oral administration at 10 mg/kg reached 3.5 µM, which exceeded its CDK8 inhibitory and osteoblast-differentiation promoting concentrations. After administration of KY-273, an oxidized metabolite 4-acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide, which also has osteoblastogenic activity, was detected, but its C_max was about 10-fold lower than that of KY-273 (0.40 µM).¹⁰⁾ Thus, orally administered KY-273 was expected to exert the osteogenic effects via osteoblastogenic activity in rats. The effects of KY-273 on femoral bone parameters were examined using micro-CT in female ovaries-intact and OVX rats. In the ovaries-intact group, KY-273 increased metaphyseal and diaphyseal Ct.BV, Ct.BMC, and bone strength parameters (BSPs), yet had no effect on metaphyseal Tb.BV, Tb.BMC, and other trabecular bone parameters. These indicate that, in the ovaries-intact group, KY-273 exerted selective osteogenic effects on diaphyseal and metaphyseal cortical bone, but not on metaphyseal trabecular bone. Bone parameter changes (ΔCt.BV, ΔCt.BMC, and ΔBSPs) between initial and final scans demonstrated that KY-273 accelerated metaphyseal and diaphyseal cortical bone growth. Furthermore, by the last week of experiment, KY-273 increased BFR/BS on the periosteal side, but decreased it on the endocortical side, suggesting cortical bone growth at periosteal side. These findings indicate that the selective cortical bone osteogenic effects of KY-273 are likely due to osteoblastogenesis activation rather than osteoclast inhibition, as bone resorption was not as strongly activated in the ovaries-intact group. However, possibility that KY-273 inhibits osteoclasts in cortical bone but not trabecular bone cannot be currently excluded.

In OVX rats, ovariectomy markedly decreased both Tb.BV and Tb.BMC, and worsened trabecular bone parameters. KY-273 had no effects on metaphyseal trabecular bone parameters, whereas alendronate significantly improved them. Also, ovariectomy had no effect on bone parameters in metaphyseal and diaphyseal cortical bone. Femur diaphysis is mostly unaffected by ovariectomy.¹⁷⁾ KY-273 increased metaphyseal and diaphyseal T.BV, Ct.BV, Ct.BMC, and BSPs, which were not affected by ovariectomy. Alendronate increased metaphyseal Ct.BV, Ct.BMC, and BSPs, but had no effect on diaphyseal cortical bone. Alendronate, a bisphosphonate, is a well-known inhibitor of bone resorption, inducing bone restoration in osteoporosis.^18,19) It increased metaphyseal cortical bone probably by inhibition of bone resorption. Indeed, osteoclastic bone resorption seems more active in the metaphysis than diaphysis. Taken together, KY-273 increased diaphyseal cortical bone, which was not affected by ovariectomy and not influenced by alendronate, and did not affect trabecular bone, which is destructed by ovariectomy and improved by alendronate, indicating that unlike alendronate, KY-273 showed an osteogenic effect rather than bone-resorption inhibition.

In the context of the 6-week bone growth study, ovariectomy had no effect on metaphyseal and diaphyseal cortical bone growth, but reversed trabecular bone growth. KY-273 accelerated metaphyseal and diaphyseal cortical bone growth, but did not affect the reduced trabecular bone growth. Alendronate accelerated the metaphyseal but not diaphyseal cortical bone growth, and completely reversed metaphyseal trabecular bone growth to levels observed for the ovaries-intact control group. Ovariectomy had no effect on BFR/BS on the periosteal side in diaphyseal cortical bone, but slightly reduced it at the endocortical side. KY-273 markedly increased BFR/BS at the periosteal side but not the endocortical side. These findings indicate that KY-273 preferentially increases cortical bone in the ovaries-intact and OVX groups without reducing medullary cavity and affecting trabecular bone. To our knowledge, there are few reports on cortical selective osteogenic compounds. Among these are KY-054, a synthetic coumarin derivative with similar cortical bone selective osteogenic effects as KY-273, and osaterone acetate, a new synthetic steroid that increases femoral diaphyseal bone volume, promotes periosteal bone formation, and enhances physical strength without influencing metaphysis bone.^12,20) The effect of KY-273 is remarkably different to those of clinically used osteogenic drugs, such as PTH and anti-sclerostin, which increase cortical and trabecular bone while reducing the medullary cavity.^21–23)

KY-273 may, for longer term treatments, increase both cortical bone and trabecular bone with reducing the medullary cavity. To explore this, KY-273 was administered for a 12-week period to OVX rats, investigating the effect on bone parameters. KY-273 increased metaphyseal Ct.BV, Ct.BMC, and BSPs without affecting Med.V, and increased all diaphyseal cortical bone parameters. However, little effect was observed on trabecular bone parameters. KY-273 was demonstrated to enlarge bone in the periosteal direction with increased Ct.BV, Ct.BMC, BSPs, Med.V, Ps.Pm, and Ec.Pm for the longer treatment period. During the 12-week treatment period, the metaphyseal and diaphyseal Ct.BV and Ct.BMC gradually and similarly increased in the ovaries-intact- and OVX-control groups, and both parameters were constantly enhanced in the OVX-KY-273-treated group. The growth in ovaries-intact and OVX rats with and without KY-273 almost plateaued at the 12-week mark (24 weeks-old), suggesting KY-273 may not induce excessive bone enlargement.

From these results, we postulate that KY-273 promotes osteoblast differentiation via CDK8 inhibition, which then enhances osteogenesis preferentially at the cortical bone periosteal side. In OVX mice, KY-065, also a CDK8 inhibitor, promoted osteoblast differentiation and mitigated trabecular bone loss by suppressing osteoclastogenesis and bone resorption via bone marrow mesenchymal stem cells; however, its effects on diaphyseal cortical bone were not examined.²⁴⁾ CDK8 inhibition by locally administered Senexin A and B suppress osteoclastogenesis, promoting osteoblast mineralization and cancellous bone healing in rat tibiae.²⁵⁾ The underlying reasons for the divergent effect of KY-273 compared to other CDK inhibitors, and why KY-273 has no effect on trabecular bone, remain to be clarified. This difference may be due to species, experiment model, dose, and administration route differences. As mentioned above, it cannot be excluded that KY-273 selectively inhibits osteoclastogenesis and bone resorption in cortical bone, resulting in osteogenesis. Further histological immunostaining and mRNA expression analyses are needed to better understand the mechanisms behind the osteogenic bone remodeling effects of KY-273, including osteoblast and osteoclast localization and activity.

In terms of bone structure, cortical bone volume significantly surpasses that of trabecular bone, with the former playing a substantial role in determining bone strength. Thus, KY-273 may be an orally effective drug candidate for osteoporosis, bone defects, fractures, and osteogenesis imperfecta. Combination therapies using osteogenic drugs such as PTH and a resorption inhibitor like bisphosphonate are widely recommended as safe and efficacious therapies.^26–32) Given the cortical bone selective osteogenic activity of KY-273, it appears an excellent candidate for combination therapies with a trabecular bone-oriented drug such as bisphosphonate.

In conclusion, KY-273 is a selective CDK8/19 inhibitor with osteoblastogenic activity in mesenchymal stem cells, and osteogenic effects in ovaries-intact and OVX rats on metaphysis and diaphysis cortical bone without reducing Med,V and affecting trabecular bone. These properties highlight it as an excellent orally active drug candidate for mono- and combination therapies for bone diseases.

Conflict of Interest

M.Y., Y.S., Y.I., M.F., H.K. Y.S., T.K. and H.S. are employees of Kyoto Pharmaceutical Industries, Ltd.; E.H. has no conflicts of interest.

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