Osteogenic Effects of KY-054, a Novel Coumarin Derivative on Femur Cortical Bone in Ovariectomized Female Rats

Abstract

Osteoporosis is treated with oral and parenteral bone resorption inhibitors such as bisphosphonates, and parenteral osteogenic drugs including parathyroid hormone (PTH) analogues and anti-sclerostin antibodies. In the present study, we synthesized KY-054, a 4,6-substituted coumarin derivative, and found that it potently promoted osteoblast differentiation with an increase in alkaline phosphatase (ALP) activity at 0.01–1 µM in mouse-derived mesenchymal stem cells (ST2 cells) and rat bone marrow-derived mesenchymal stem cells (BMSCs). In the ovariectomized (OVX) rats, KY-054 (10 mg/kg/d, 8 weeks) increased plasma bone-type ALP activity, suggesting in vivo promoting effects on osteoblast differentiation and/or activation. In dual-energy X-ray absorption (DEXA) scanning, KY-054 significantly increased the distal and diaphyseal femurs areal bone mineral density (aBMD) that was decreased by ovariectomy, indicating its beneficial effects on bone mineral contents (BMC) and/or bone volume (BV). In micro-computed tomography (micro-CT) scanning, KY-054 had no effect on metaphysis trabecular bone loss and microarchitecture parameters weakened by ovariectomy, but instead increased metaphysis and diaphysis cortical bone volume (Ct.BV) and cortical BMC (Ct.BMC) without reducing medullary volume (Med.V), resulting in increased bone strength parameters. It is concluded that KY-054 preferentially promotes metaphysis and diaphysis cortical bone osteogenesis with little effect on metaphysis trabecular bone resorption, and is a potential orally active osteogenic anti-osteoporosis drug candidate.

INTRODUCTION

Osteoporosis is caused by an imbalance between bone formation via osteoblasts and resorption via osteoclasts. Thus, bone resorption inhibitors, such as oral and parenteral bisphosphonates, and osteogenesis activators, such as parenteral parathyroid hormone (PTH) and anti-sclerostin antibodies, are used for therapy of osteoporosis. Orally active osteogenic drugs are desirable, with some small synthetic molecules reported to exert osteogenic effects in vitro and in vivo. Benzothiopyran, benzofuran, and thienopyridine derivatives enhance osteoblast differentiation and increase alkaline phosphatase (ALP) activities, and also show osteogenic effects in rodent bone defect models and postmenopausal osteoporosis models.^1–3) However, none of these are successfully developed therapies; potentially due to problems regarding efficacy, pharmacokinetics and/or toxicity. On the other hand, various natural substances including coumarin, flavone, isoflavone, and phenol derivatives are reported to promote osteoblast differentiation.⁴⁾ Among them, coumarins attracted medicinal chemist’s attention as coumarin and its natural analogues have various physiological activities, thus coumarin is a useful therapeutic drug scaffold for various diseases including infection, cancer, diabetes, and inflammation.^5–7)

Simple coumarin derivatives, aesculetin, osthole, and scopolin (Fig. 1), which have 6-, 7- and/or 8-substituted structures, are reported to promote osteoblast differentiation, attenuate osteoclast differentiation,^8–12) and prevent trabecular bone loss in ovariectomized (OVX) rodents.^10,13–15) However, their osteogenic effects on cortical bone are undemonstrated; osteogenic drugs such as PTH and anti-sclerostin antibody increased cortical bone volume.^16,17) Furthermore, the effects of above-mentioned coumarins on osteoblast differentiation were observed at micromolar concentrations, comparable to or weaker than their inhibitory effects on osteoclast differentiation. Their preventive effects on trabecular bone loss may be due to inhibition of bone resorption, rather than enhancement of bone formation.^10,13,14)

Fig. 1. Chemical Structures of KY-054 and Natural Coumarins

In the present study, the novel 4, 6-substituted coumarin derivative KY-054 (Fig. 1) was synthesized, and found to show promoting effects on osteoblast differentiation at nanomolar concentrations, which were more potent than the above-mentioned coumarins. Thus, we investigated the effects of KY-054 on OVX rats, a postmenopausal osteoporosis model, at 3 and 10 mg/kg/d (per os (p.o.)) for 8 weeks. Micro-computed tomography (micro-CT) scanning showed that, unlike above coumarins, KY-054 increased femur cortical bone volume and bone strength parameters without affecting trabecular bone.

MATERIALS AND METHODS

Materials and Animals

KY-054 (Fig. 1), synthesized in our laboratories, was dissolved in dimethyl sulfoxide (DMSO) and diluted in alpha Modified Eagle Minimum Essential Medium (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 Center (Tsukuba, 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.

In Vitro Osteoblast Differentiation

Bone marrow mesenchymal stem cells (BMSCs) were isolated using a previously reported method.¹⁸⁾ BMSCs and ST2 cells were seeded on 96-well plates at a density of 4 × 10³ cells/well. Test compounds were added 24 h later and cultured for 4 d in α-MEM. Cells were then washed with phosphate buffered saline (PBS) (pH 7.4) and lysed in 50 µL saline containing 1% NP-40 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 1M NaOH after a 30-min incubation at 37 °C. ALP activities were assessed by measuring absorbance at 405 nm. Cell viability was monitored morphologically using a phase contrast microscope. Separately, cell viability analysis was performed using ATPlite™ (PerkinElmer, Inc., Waltham, MA, U.S.A.), according to the manufacturer’s instructions.

Plasma Concentrations of KY-054

Eight-week-old female F344/NSlc rats were orally administered KY-054 (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. Plasma concentrations of KY-054 were determined using a liquid chromatograph mass spectrometry (LC/MS/MS, API2000 QTRAP, AB SCIEX, MA, U.S.A) with a pump (G1312A; Agilent, CA, U.S.A) and autoinjector (G1329, Agilent).

OVX Rats

Eight-week-old female F344/NSlc rats were randomly allocated to 4 groups (n = 10); sham-operated group (Sham), OVX control group and KY-054 (3 and 10 mg/5 mL/kg/d)-treated group. Then, ovariectomy was performed as previously reported¹⁸⁾: rats were anesthetized using ketamine (37.5 mg/kg, intraperitoneally (i.p.)) and xylazine (7.5 mg/kg, i.p.), and underwent sham operation in the Sham group, and bilaterally ovariectomized in the OVX control group and KY-054-treated groups. Rats were orally administered the vehicle (0.5% MC) in Sham and OVX control groups, and KY-054 suspended in 0.5% MC in KY-054-treated groups for 8 weeks. Body weight and food consumption were monitored during the experiment period. The right femur was scanned using micro-computed tomographic equipment (micro-CT, R_μCT; Rigaku, Tokyo, Japan) under anesthesia with isoflurane 1 d before beginning administration and 1 d after final administration. After repeated administration, rats were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Fasting blood was collected from abdominal aorta. Then, rats were euthanized. Femurs were fixed with 70% ethanol and stored at 4 °C, then distal and diaphyseal femurs were scanned using dual-energy X-ray absorption (DEXA) (LaTheta; Aloka Co., Ltd., Tokyo, Japan) to determine areal bone mineral density (aBMD).

In Vivo Micro-CT Analyses

Micro-CT scanning data were analyzed using TRI/3D-BON software (RATOC, Tokyo, Japan).¹⁹⁾ In metaphysis trabecular bone, trabecular bone mineral content (Tb.BMC), trabecular bone tissue 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 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), cortical thickness (Ct.Th), cortical bone area (Ct.Ar), periosteal perimeter (Ps.Pm), and endocortical perimeter (Ec.Pm) were determined. For metaphysis and diaphysis cortical bone, as bone strength parameters, the major axis and minor axis section modules (IaSSI and IbSSI) and bone strength index (aBSI and bBSI, i.e., cross-sectional moment of inertia multiply Ct.TMD), reported to correlate to actual bone breaking strength,²⁰⁾ were also determined. Separately, the changes in principal parameters (ΔT.BV, ΔCt.BV, ΔMed.V, ΔCt.BMC, ΔCt.TMD, ΔPs.Pm, ΔEc.Pm, ΔΙaSSI, and ΔaBSI) during administration period were determined.

Bone-Specific ALP

Total plasma ALP activity was determined with colorimetric assay using p-nitrophenyl phosphate (pNNP) as substrate, which is catalyzed into p-nitrophenol (p-NP). Isozyme fractions in plasma ALP were determined by agarose gel electrophoresis, then bone-type ALP activity was calculated.

Statistical Analyses

All data are expressed as mean ± standard error of the mean (S.E.M.). Significance of difference was assessed using Dunnett’s multiple comparison tests. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, U.S.A.).

RESULTS

Osteoblast Differentiation

KY-054 (Fig. 1) increased concentration-dependently ALP activity over 0.01–1 µM in ST2 cells and rat BMSCs, with a maximal activity of 4.2-fold and 2.1-fold, respectively (Fig. 2). KY-054 had no cytotoxic effects over 0.01–1 µM in ST2 cells and BMSC in microscopic observations using a phase contrast microscope. In addition, cell viability analysis using ATPlite showed that KY-054 had no cytotoxicity over 0.01–1 µM in ST2 cells.

Fig. 2. Effects of KY-054 on Alkaline Phosphatase (ALP) Activities on ST2 Cells and Rat Bone Marrow-Derived Stem Cells (BMSC) Cultured for 4 d

BMSCs and ST2 cells were cultured for 4 d with and without KY-054, and then cellular ALP activity was determined. The % increase in ALP activity values compared to activities in DMSO-treated cells is shown. The values are the mean ± S.E.M. n = 3.

Plasma Concentration

The plasma concentration of KY-054 (10 mg/kg) reached a maximal level (3.63 ± 2.63 µM, n = 3) at 0.5 h after oral administration in healthy female F344/NSlc rats.

OVX Rats

Ovariectomy markedly reduced uterine weight and increased body weight, but had no effect on plasma bone-type ALP activities (Fig. 3). KY-054 did not affect uterine weight or body weight, but significantly increased plasma bone-type ALP activity at 10 mg/kg/d (Fig. 3).

Fig. 3. Effects of KY-054 on Final Body Weight, Uterus Weight and Plasma Bone-Type ALP Activity in Ovariectomized Rats

Rats were ovariectomized and orally administered KY-054 (3 mg or 10 mg/kg body weight) for 8 weeks. Values are the mean ± S.E.M. n = 8. ** p < 0.01 vs. Control, Dunnett’s multiple comparison test. Sham: Sham control group, Cont: OVX control group.

DEXA Analyses

Distal femur aBMD values were significantly smaller in the OVX control group than in the Sham group, indicating bone resorption by ovariectomy (Fig. 4). Distal femur aBMD values in KY-054 (3 and 10 mg/kg/d)-treated groups were significantly greater than those in OVX control group, but were still lower than those in the Sham group, indicating the attenuating effects of KY-054 on bone loss, or the increasing effects on BMC and/or BV. Diaphysis aBMD values in the OVX control group were not different from the Sham group. Diaphysis femur aBMD values in the KY-054 (10 mg/kg/d)-treated group were significantly greater than in the OVX control group, indicating the increasing effects of KY-054 on BMC and/or BV.

Fig. 4. Effects of KY-054 on Arial Bone Mineral Density (aBMD) Determined by DEXA Scanning in Ovariectomized Rats

Distal and diaphyseal femurs were scanned using dual-energy X-ray absorption (DEXA). Values are the mean ± S.E.M. n = 8. * p < 0.05, ** p < 0.01 vs. OVX control, Dunnett’s multiple comparison test. Sham: Sham control group, Cont: OVX control group.

In Vivo Micro-CT Analyses

Bone parameters before ovariectomy were not significantly different between OVX control group and other groups (data not shown).

After repeated administration, the metaphysis trabecular bone parameters Tb.BMC, Tb.TMD, BV/TV, Tb.BV, Tb.Th, and Tb.N were significantly smaller, while BS/BV, Tb.Sp, Tb.Pf, SMI, and Star Volume were greater in the OVX control group than the Sham group, indicating a reduction in trabecular bone volume, mineral contents, mineral density, number, thickness, tightness, and plate-likeness by ovariectomy-induced bone resorption (Table 1). These parameters were not significantly different between the OVX control group and KY-054-treated groups.

Table 1. Effects of KY-054 (3 and 10 mg/kg, 8-Weeks) on Trabecular Bone Parameters of Femur Metaphysis Determined by in Vivo Micro-CT Scanning in Ovariectomized Rats

	Sham	OVX control	OVX + KY-054 (mg/kg/d)
			3	10
Tb.BMC (mg)	1.20 ± 0.09**	0.41 ± 0.04	0.47 ± 0.04	0.47 ± 0.03
Tb.TMD (mg/mm3)	427.7 ± 7.6**	350.7 ± 5.6	365.1 ± 6.0	360.1 ± 7.9
BV/TV (%)	26.7 ± 1.5**	10.6 ± 0.9	11.1 ± 0.8	11.2 ± 0.5
Tb.BV (mm3)	2.79 ± 0.17**	1.18 ± 0.11	1.28 ± 0.09	1.31 ± 0.07
BS/BV (mm2/mm3)	18.49 ± 0.22**	22.01 ± 0.46	21.8 6 ± 0.38	21.74 ± 0.30
Tb.Th (µm)	108.3 ± 1.2**	91.2 ± 2.0	91.7 ± 1.6	92.1 ± 1.3
Tb.N (1/mm)	2.96 ± 0.14**	1.33 ± 0.09	1.39 ± 0.08	1.40 ± 0.06
Tb.Sp (µm)	235.6 ± 19.4**	687.9 ± 58.4	645.4 ± 49.5	630.3 ± 33.1
TBPf (1/mm)	3.53 ± 0.11**	6.02 ± 0.45	6.05 ± 0.37	5.69 ± 0.26
SMI	2.03 ± 0.05**	2.42 ± 0.06	2.42 ± 0.06	2.38 ± 0.04
Star Volume (mm3)	0.56 ± 0.08**	1.60 ± 0.05	1.60 ± 0.07	1.65 ± 0.05

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

In metaphysis cortical bone, Ct.BMC and Ct.Th values were significantly smaller with both Ct.TMD and Ec.Pm slightly smaller in the OVX control group than the Sham group, indicating slight cortical bone thinning by ovariectomy-induced bone resorption at the endocortical surface (Table 2). T.BV, Ct.BV, Ct.BMC, Ct.Ar, Ps.Pm, IaSSI, IbSSI, aBSI, and bBSI values were significantly greater, with Med.V, Ct.Th, and Ec.Pm slightly greater in KY-054 (10 mg/kg)-treated groups than the OVX control group, indicating that KY-054 enlarged bone, with increased cortical bone at the periosteal side with slightly increased Med.V, resulting in increased bone strength.

Table 2. Effects of KY-054 (3 and 10 mg/kg, 8-Weeks) on Cortical Bone Parameters of Femur Metaphysis Determined by in Vivo Micro-CT Scanning in Ovariectomized Rats

	Sham	OVX control	OVX + KY-054 (mg/kg/d)
			3	10
T.BV (mm3)	21.3 ± 0.22	21.6 ± 0.30	22.2 ± 0.30	22.7 ± 0.33*
Ct.BV (mm3)	8.8 ± 0.1	8.6 ± 0.1	8.8 ± 0.1	9.0 ± 0.1**
Med.V (mm3)	12.5 ± 0.15†	13.0 ± 0.24	13.4 ± 0.26	13.7 ± 0.22†
Ct.BMC (mg)	10.69 ± 0.14*	10.16 ± 0.13	10.54 ± 0.11†	10.77 ± 0.14**
Ct.TMD (mg/mm3)	1210.6 ± 8.5†	1186.1 ± 10.8	1191.7 ± 7.5	1192.1 ± 9.3
Ct.Th (µm)	372.5 ± 4.2*	354.7 ± 3.7	361.9 ± 3.9	365.4 ± 4.2†
Ct.Ar (mm2)	3.9 ± 0.1	3.8 ± 0.0	3.9 ± 0.0	4.0 ± 0.1*
Ps.Pm (µm)	11729 ± 66	11857 ± 83	12021 ± 80	12153 ± 86*
Ec.Pm (µm)	9415 ± 60†	9677 ± 91	9835 ± 93	9928 ± 75†
IaSSI (mm3)	2.71 ± 0.06	2.59 ± 0.06	2.72 ± 0.04	2.84 ± 0.08*
IbSSI (mm3)	3.16 ± 0.05	3.14 ± 0.06	3.31 ± 0.05†	3.43 ± 0.07**
aBSI (mm4 × mg/mm3)	6532.2 ± 205.6	6180.0 ± 134.3	6508.3 ± 125.4	6889.3 ± 202.9*
bBSI (mm4 × mg/mm3)	8060.7 ± 153.0	8019.9 ± 167.3	8642.2 ± 181.1*	8923.5 ± 176.7**

Mean ± S.E.M. n = 8. ^† p < 0.1, * p < 0.05, ** p < 0.01 vs. OVX control, Dunnett’s multiple comparison test.

In diaphysis, only Ct.TMD was significantly and slightly smaller in the OVX control group than Sham group. Ct.BMC and Ct.Th values were significantly greater, while Ct.BV, Ct.Ar, IbSSI, and bBSI slightly greater in the KY-054 (10 mg/kg)-treated group than OVX control group, indicating that KY-054 enlarged bone, with increased cortical bone at the periosteal side as in metaphysis. However, the effects in diaphysis were smaller than for metaphysis (Table 3).

Table 3. Effects of KY-054 (3 and 10 mg/kg, 8-Weeks) on Bone Parameters of Femur Diaphysis Determined by in Vivo Micro-CT Scanning in Ovariectomized Rats

	Sham	OVX control	OVX +  KY-054 (mg/kg/d)
			3	10
T.BV (mm3)	32.7 ± 0.4	33.1 ± 0.6	33.4 ± 0.5	34.2 ± 0.6
Ct.BV (mm3)	20.4 ± 0.2	20.3 ± 0.3	20.8 ± 0.2	21.2 ± 0.3†
Med.V (mm3)	12.3 ± 0.3	12.8 ± 0.3	12.6 ± 0.4	13.1 ± 0.29
Ct.BMC (mg)	28.14 ± 0.29	27.38 ± 0.28	28.11 ± 0.30	28.51 ± 0.34*
Ct.TMD (mg/mm3)	1382.1 ± 11.0*	1346.4 ± 11.1	1350.4 ± 10.8	1348.3 ± 6.0
Ct.Th (µm)	505.9 ± 4.0	500.5 ± 3.0	513.7 ± 4.7†	514.7 ± 3.9*
Ct.Ar (mm2)	3.74 ± 0.03	3.73 ± 0.05	3.84 ± 0.04	3.89 ± 0.06†
Ps.Pm (µm)	9103 ± 57	9149 ± 80	9201 ± 73	9300 ± 81
Ec.Pm (µm)	5780 ± 70	5863 ± 78	5841 ± 83	5940 ± 64
IaSSI (mm3)	1.86 ± 0.03	1.86 ± 0.04	1.88 ± 0.04	1.95 ± 0.05
IbSSI (mm3)	1.98 ± 0.04	2.02 ± 0 .06	2.07 ± 0.04	2.15 ± 0.05†
aBSI (mm4 × mg/mm3)	3688.1 ± 112.1	3609.9 ± 90.9	3633.8 ± 78.6	3818.1 ± 129.2
bBSI (mm4 × mg/mm3)	4526.3 ± 98.1	4542.0 ± 135.2	4781.1 ± 119.2	4936.7 ± 128.1†

Mean ± S.E.M. n = 8. ^† p < 0.1, * p < 0.05, ** p < 0.01 vs. OVX control, Dunnett’s multiple comparison test.

Changes in Cortical Bone Parameters during Administration Period

The above parameters in diaphysis were greater than for metaphysis, with very small differences between groups: individual deviations in rats may mask differences among groups in diaphysis. Therefore, changes in 9 selected parameters (ΔT.BV, ΔCt.BV, ΔMed.V, ΔCt.BMC, ΔCt.TMD, ΔPs.Pm, ΔEC.Pm, ΔIaSSI and ΔaBSI) between before ovariectomy and after repeated administration were analyzed.

In metaphysis cortical bone, the 9 selected parameters in the Sham group increased during 8-week administration period (Fig. 5). The ΔCt.BMC values were significantly smaller, while ΔMed.V values were significantly, and ΔEC.Pm values were slightly greater in the OVX group than Sham group. The ΔCt.BMC, ΔIaSSI, and ΔaBSI were significantly greater, with ΔT.BV, ΔCt.BV, and ΔPs.Pm slightly greater in the KY-054 (10 mg/kg)-treated group than OVX control group.

Fig. 5. Effects of KY-054 on Changes in Cortical Bone Parameters during Administration Period in Femur Metaphysis Determined by in Vivo Micro-CT Scanning in Ovariectomized Rats

Micro-CT scanning data were analyzed using TRI/3D-BON software. Values are the mean ± S.E.M. n = 8. ^† p < 0.1, * p < 0.05 vs. OVX control, Dunnett’s multiple comparison test. S: Sham control group, C: OVX control group.

For diaphysis cortical bone, the selected parameters, except Med.V and Ec.Pm in the Sham group, all increased during the 8-week administration period (Fig. 6). ΔCt.TMD was significantly smaller, and ΔCt.BMC and ΔaBSI slightly smaller in the OVX group than Sham group. The ΔT.BV, ΔCt.BV, ΔCt.BMC, ΔPs.Pm, ΔIaSSI, and ΔaBSI were significantly greater, while ΔMed.V and ΔEc.Pm slightly greater in the KY-054 (10 mg/kg)-treated group than OVX group.

Fig. 6. Effects of KY-054 on Changes in Cortical Bone Parameters during Administration Period in Femur Diaphysis Determined by in Vivo Micro-CT Scanning in Ovariectomized Rats

Micro-CT scanning data were analyzed using TRI/3D-BON software. Values are the mean ± S.E.M. n = 8. ^† p < 0.1, * p < 0.05, ** p < 0.01 vs. OVX control, Dunnett’s multiple comparison test. S: Sham control group, C: OVX control group.

DISCUSSION

For osteoporosis therapy, small molecule bone-resorption inhibitors such as bisphosphonates are used clinically, with small molecule osteogenic drugs not yet successfully developed. In the present study, KY-054, a novel synthetic coumarin derivative, was found to exert enhancing effects on osteoblast differentiation in mesenchymal stem cells, and yielded osteogenic effects in femur cortical bone in OVX rats.

KY-054 concentration-dependently increased ALP activity between 0.01–0.1 µM in mouse-derived mesenchymal stem cells (ST2 cells) and primary cultured rat BMSCs, indicating potent enhancing effects on osteoblast differentiation and/or activity. Aesculetin, osthole, and scopolin, natural coumarin derivatives, are also reported to enhance osteoblast differentiation at 5–10 µM, and prevent trabecular bone loss at 9–20 mg/kg/d in ovariectomized mice. This appears due to inhibition of bone resorption, rather than bone formation, as their inhibitory effect on osteoclast differentiation is more potent than influence on osteoblast differentiation.^8–15) The effect of KY-054 on osteoblast differentiation is more potent than these coumarins, and its plasma concentrations after oral administration at 10 mg/kg are much higher than the effective concentrations for influencing osteoblast differentiation. Thus, KY-054 was expected to have more potent osteogenic effects than the above-mentioned coumarins in OVX rats. In the present experiments, ovariectomy markedly decreased uterine weight and increased body weight, with no effect by KY-054 (3 and 10 mg/kg/d), indicating an absence of estrogenic effects, unlike coumestrol, an estrogenic coumarin derivative.²¹⁾ KY-054 significantly increased plasma bone-type ALP activity at 10 mg/kg/d, suggesting in vivo increases in osteoblast number and/or activity. Ovariectomy decreased aBMD at the distal but not diaphyseal femurs in DEXA scanning. KY-054 partly reversed aBMD at the distal decreased by ovariectomy, and increased aBMD at the diaphysis unchanged by ovariectomy, suggesting a role in bone formation rather than bone resorption inhibition.

In vivo micro-CT scanning data demonstrated that ovariectomy caused bone loss and micro-architectural weakness in trabecular bone as previously reported.^22,23) However, KY-054 had no significant effects on these OVX-induced changes in parameters. Bisphosphonates and many other compounds, including coumarins, prevent trabecular bone loss by bone resorption inhibition, and improve the weakened micro-architecture.^{10,13–15,24)} Osteogenic substances such as PTH and anti-sclerostin antibodies also improve trabecular bone loss and micro-architectural parameters, suggesting that their osteogenic effects surpass bone resorption and/or indirectly inhibit resorption.^25–27) KY-054 is suggested to have no inhibitory effects on bone resorption at the trabecular bone site, and its osteogenic effects are considered too weak to surpass the ovariectomy-induced trabecular bone resorption. However, it cannot be excluded that higher doses and/or longer administration of KY-054 may prevent trabecular bone loss and improve micro-architectural changes.

Analyses of in vivo micro-CT scanning data of the metaphysis and diaphysis showed that ovariectomy had little effect on cortical bone: slightly reducing Ct.BMC, Ct.Th, and Ec.Pm in the metaphysis, and Ct.TMD in the diaphysis. Estrogen-deficiency was reported to have little effect on cortical bone during short period after ovariectomy.²⁸⁾ In the metaphysis, KY-054 not only reversed Ct.BMC and Ct.Th, which were reduced by ovariectomy, but also increased T.BV, Ct.BV, Med.V, Ct.Ar, Ps.Pm, Ec.Pm, IaSSI, IbSSI, aBSI and bBSI, which were unaffected by ovariectomy. In diaphysis, KY-054 increased Ct.BV, Ct.BMC, Ct.Th, Ct.Ar, IbSSI and bBSI, which were unaffected by ovariectomy. These results suggest that KY-054 promoted osteogenesis at the peritoneal side and increased bone strength due to increased Ct.BV rather than Ct.TMD. This is supported by analyses of the changes in parameters between before ovariectomy and after administration.

As described in Results, for the Sham group, the metaphysis and diaphysis cortical bone parameters, except for the diaphysis Ec.Pm and Med.V, all increased during the administration period, probably by normal growth. Diaphysis Ec.Pm, and Med.V were unchanged over the administration period. At the age of 8-weeks, remodeling of endocortical surface may reach an equilibrium steady state, thereafter, bone may grow in a periosteal direction, resulting in unchanged bone marrow cavity. Ovariectomy decreased ΔCt.BMC and ΔaBSI, while enhanced ΔMed.V and ΔEc.Pm in the metaphysis, and slightly decreased ΔCt.BMC, ΔCt.TMD, and ΔaBSI in the diaphysis. KY-054 promoted bone growth with increased ΔT.BV, ΔCt.V, ΔCt.BMC, ΔPs.Pm, ΔIaSSI, and ΔaBSI in the metaphysis and ΔT.BV, ΔCt.V, ΔMed.V, ΔCt.BMC, ΔPs.Pm, ΔEc.Pm, ΔIaSSI, and ΔaBSI in the diaphysis. These results support that ovariectomy activated bone resorption at endocortical surfaces, and KY-054 accelerated bone growth, probably by osteogenesis at periosteal surfaces but not endocortical surfaces, resulting in enhanced bone strength. The osteogenic effects of KY-054 may be limited to the endocortical surface due to highly activated bone resorption by ovariectomy.

Clinically used osteogenic drugs, such as PTH and anti-sclerostin anti-body, increases bone formation in both side with a reduction of medullary volume.^16,17,27) EP4 selective prostaglandin agonist also enhances cortical bone formation at both the periosteal and endocortical sides with a reduction in medullary volume.²⁹⁾ Bone resorption inhibitor bisphosphonate effectively reverses the trabecular bone loss with slight cortical bone formation.²³⁾ Natural coumarins such as aesculetin, osthole, and scopolin enhance osteoblast differentiation, inhibit osteoclast differentiation, and prevented trabecular bone loss, probably by inhibition of bone resorption in OVX mice.^10,13,14) To our knowledge, there are no reports showing osteogenic effects of natural coumarins on cortical bone. These facts demonstrated that KY-054 is a unique synthetic coumarin derivative, which exerts osteogenic effects preferentially on cortical bone. Its osteogenic mode appears different to natural coumarin derivatives and clinically used drugs.

Combined and sequential treatment with resorption inhibitors and osteogenic drugs were experimentally and clinically demonstrated to have additive anti-osteoporosis effects.^29–35) KY-054 has osteogenetic effects preferentially on cortical bone at periosteal side, which is acceleration of normal bone growth, and had no serious toxicity at 10 mg/kg for 8-weeks in OVX rats (data not shown). Therefore, KY-054 is expected as an efficacious and safe oral anti-osteoporosis drug alone or in combination with resorption inhibitors. The present study showed the effects of KY-054 on cortical bone using in vivo micro-CT scanning in OVX rats, however the mechanism was not elucidated. Further studies are required to fully explore the osteogenic mechanism of KY-054.

In conclusion, KY-054, a novel synthetic coumarin derivative, increased serum bone type-ALP activity, and accelerated cortical bone formation at the periosteal side without a reduction of medullary volume, and increased the bone strength parameter. KY-054 is a potential candidate of an orally effective osteogenic drug for therapy of osteoporosis and bone fracture, and may have additive or synergistic therapeutic effects in combination with a bone resorption inhibitor.

Acknowledgments

The authors are grateful to Dr. Nobuhito Nango (Ratoc System Engineering Co., Ltd.), and Dr. Azusa Seki (Hamri Co., Ltd.) for useful discussions and carefully proofreading the manuscript.

Conflict of Interest

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

REFERENCES

• 1) Oda T, Notoya K, Gotoh M, Taketomi S, Fujisawa Y, Makino H, Sohda T. Synthesis of novel 2-benzothiopyran and 3-benzothiepin derivatives and their stimulatory effect on bone formation. J. Med. Chem., 42, 751–760 (1999).
• 2) Modukuri RK, Choudhary D, Gupta S, Rao KB, Adhikary S, Sharma T, Siddiqi MI, Trivedi R, Sashidhara KV. Benzofuran-dihydropyridine hybrids: a new class of potential bone anabolic agents. Bioorg. Med. Chem., 25, 6450–6466 (2017).
• 3) Saito K, Shinozuka T, Nakao A, Kunikata T, Nakai D, Nagai Y, Naito S. Discovery of 3-amino-4-{(3S)-3-[(2-ethoxyethoxy)methyl]piperidin-1-yl}thieno[2,3-b] pyridine-2-carboxamide (DS96432529): a potent and orally active bone anabolic agent. Bioorg. Med. Chem. Lett., 54, 128440 (2021).
• 4) An J, Yang H, Zhang Q, Liu C, Zhao J, Zhang L, Chen B. Natural products for treatment of osteoporosis: the effects and mechanisms on promoting osteoblast-mediated bone formation. Life Sci., 147, 46–58 (2016).
• 5) Pereira TM, Franco DP, Vitorio F, Kümmerle AE. Coumarin compounds in medicinal chemistry: some important exam-ples from the last years. Curr. Top. Med. Chem., 18, 124–148 (2018).
• 6) Stefanachi A, Leonetti F, Pisani L, Catto M, Carotti A. A natural, privileged and versatile scaffold for bioactive compounds. Molecules, 23, 250 (2018).
• 7) Annunziata F, Pinna C, Dallavalle S, Tamborini L, Pinto A. An overview of coumarin as a versatile and readilyaccessible scaffold with broad-ranging biological activities. Int. J. Mol. Sci., 21, 4618 (2020).
• 8) Na W, Kang MK, Park SH, Kim DY, Oh SY, Oh MS, Park S, Kang IJ, Kang YH. Aesculetin accelerates osteoblast differentiation and matrix-vesicle-mediated mineralization. Int. J. Mol. Sci., 22, 12391 (2021).
• 9) Tang DZ, Hou W, Zhou Q, Zhang M, Holz J, Sheu TJ, Li TF, Cheng SD, Shi Q, Harris SE, Chen D, Wang YJ. Osthole stimulates osteoblast differentiation and bone formation by activation of β-catenin-BMP signaling. J. Bone Miner. Res., 25, 1234–1245 (2010).
• 10) Park E, Kim J, Jin HS, Choi CW, Choi TH, Choi S, Huh D, Jeong SY. Scopolin attenuates osteoporotic bone loss in ovariectomized mice. Nutrients, 12, 3565 (2020).
• 11) Ma Y, Wang L, Zheng S, Xu J, Pan Y, Tu P, Sun J, Guo Y. Osthole inhibits osteoclasts formation and bone resorption by regulating NF-κB signaling and NFATc1 activations stimulated by RANKL. J. Cell. Biochem., 120, 16052–16061 (2019).
• 12) Lee SH, Ding Y, Yan XT, Kim YH, Jang HD. Scopoletin and scopolin isolated from artemisia iwayomogi suppress differentiation of osteoclastic macrophage RAW 264.7 cells by scavenging reactive oxygen species. J. Nat. Prod., 76, 615–620 (2013).
• 13) Liu M, Wang R, Li Y, Bai D, Pan J, Liu H, Wang S, Wu J, Sun G, Miao Q, Ju D, Liu L. Effect of esculetin on bone metabolism in ovariectomized rats. J. Tradit. Chin. Med., 38, 896–903 (2018).
• 14) Zhao D, Wang Q, Zhao Y, Zhang H, Sha N, Tang D, Liu S, Lu S, Shi Q, Zhang Y, Dong Y, Wang Y, Shu B. The naturally derived small compound Osthole inhibits osteoclastogenesis to prevent ovariectomy-induced bone loss in mice. Menopause, 25, 1459–1469 (2018).
• 15) Li XX, Hara I, Matsumiya T. Effects of osthole on postmenopausal osteoporosis using ovariectomized rats; comparison to the effects of estradiol. Biol. Pharm. Bull., 25, 738–742 (2002).
• 16) Brouwers JE, van Rietbergen B, Huiskes R, Ito K. Effects of PTH treatment on tibial bone of ovariectomized rats assessed by in vivo micro-CT. Osteoporos. Int., 20, 1823–1835 (2009).
• 17) Li X, Ominsky MS, Warmington KS, Morony S, Gong J, Cao J, Gao Y, Shalhoub V, Tipton B, Haldankar R, Chen Q, Winters A, Boone T, Geng Z, Niu QT, Ke HZ, Kostenuik PJ, Simonet WS, Lacey DL, Paszty C. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J. Bone Miner. Res., 24, 578–588 (2009).
• 18) Kubo M, Fukui M, Ito Y, Kitao T, Shirahase H, Hinoi E, Yoneda Y. Insulin sensitization by a novel partial peroxisome proliferator-activated receptor γ agonist with protein tyrosine phosphatase 1B inhibitory activity in experimental osteoporotic rats. J. Pharmacol. Sci., 124, 276–285 (2014).
• 19) Nango N, Kubota S, Horiguchi Y. Three-dimensional microstructural measurement for predicting the risk of osteoporotic fracture. Osteoporotic Fracture and Systemic Skeletal Disorders, 15, 129–160 (2022).
• 20) Ferretti JL, Capozza RF, Zanchetta JR. Mechanical validation of a tomographic (pQCT) index for noninvasive estimation of rat femur bending strength. Bone, 18, 97–102 (1996).
• 21) Tinwell H, Soames AR, Foster JR, Ashby J. Estradiol-type activity of coumestrol in mature and immature ovariectomized rat uterotrophic assays. Environ. Health Perspect., 108, 631–634 (2000).
• 22) Hsu PY, Tsai MT, Wang SP, Chen YJ, Wu J, Hsu JT. Cortical bone morphological and trabecular bone microarchitectural changes in the mandible and femoral neck of ovariectomized rats. PLOS ONE, 11, e0154367 (2016).
• 23) Brouwers JE, Lambers FM, Gasser JA, van Rietbergen B, Huiskes R. Bone degeneration and recovery after early and late bisphosphonate treatment of ovariectomized wistar rats assessed by in vivo micro-computed tomography. Calcif. Tissue Int., 82, 202–211 (2008).
• 24) Shuai B, Shen L, Yang Y, Ma C, Zhu R, Xu X. Assessment of the impact of zoledronic acid on ovariectomized osteoporosis model using micro-CT scanning. PLOS ONE, 10, e0132104 (2015).
• 25) Wang SP, Chen YJ, Hsu CE, Chiu YC, Tsai MT, Hsu JT. Intermittent parathyroid hormone treatment affects the bone structural parameters and mechanical strength of the femoral neck after ovariectomy-induced osteoporosis in rats. Biomed. Eng. Online, 21, 6 (2022).
• 26) Gittens SA, Wohl GR, Zernicke RF, Matyas JR, Morley P, Uludag H. Systemic bone formation with weekly PTH administration in ovariectomized rats. J. Pharm. Pharm. Sci., 7, 27–37 (2004).
• 27) Li X, Warmington KS, Niu QT, Asuncion FJ, Barrero M, Grisanti M, Dwyer D, Stouch B, Thway TM, Stolina M, Ominsky MS, Kostenuik PJ, Simonet WS, Paszty C, Ke HZ. Inhibition of sclerostin by monoclonal antibody increases bone formation, bone mass, and bone strength in aged male rats. J. Bone Miner. Res., 25, 2647–2656 (2010).
• 28) Zhang Y, Lai WP, Leung PC, Wu CF, Wong MS. Short- to mid-term effects of ovariectomy on bone turnover, bone mass and bone strength in rats. Biol. Pharm. Bull., 30, 898–903 (2007).
• 29) Ke HZ, Crawford DT, Qi H, Simmons HA, Owen TA, Paralkar VM, Li M, Lu B, Grasser WA, Cameron KO, Lefker BA, DaSilva-Jardine P, Scott DO, Zhang Q, Tian XY, Jee WS, Brown TA, Thompson DD. A nonprostanoid EP4 receptor selective prostaglandin E₂ agonist restores bone mass and strength in aged, ovariectomized rats. J. Bone Miner. Res., 21, 565–575 (2006).
• 30) Amugongo SK, Yao W, Jia J, Dai W, Lay YA, Jiang L, Harvey D, Zimmermann EA, Schaible E, Dave N, Ritchie RO, Kimmel DB, Lane NE. Effect of sequential treatments with alendronate, parathyroid hormone (1-34) and raloxifene on cortical bone mass and strength in ovariectomized rats. Bone, 67, 257–268 (2014).
• 31) Zhang C, Zhu J, Jia J, Guan Z, Sun T, Zhang W, Yuan W, Wang H, Leng H, Song C. Once-weekly parathyroid hormone combined with ongoing long-term alendronate treatment promotes osteoporotic fracture healing in ovariectomized rats. J. Orthop. Res., 39, 2103–2115 (2021).
• 32) Zhang L, Endo N, Yamamoto N, Tanizawa T, Takahashi HE. Effects of single and concurrent intermittent administration of human PTH (1-34) and incadronate on cancellous and cortical bone of femoral neck in ovariectomized rats. Tohoku J. Exp. Med., 186, 131–141 (1998).
• 33) Cosman F. Combination therapy for osteoporosis: a reappraisal. Bonekey Rep., 3, 518 (2014).
• 34) Lecart MP, Bruyere O, Reginster JY. Combination/ sequential therapy in osteoporosis. Curr. Osteoporos. Rep., 2, 123–130 (2004).
• 35) Zhang C, Song C. Combination therapy of PTH and antiresorptive drugs on osteoporosis: a review of treatment alternatives. Front. Pharmacol., 11, 607017 (2021).