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Effect of ipriflavone on glucocorticoid-induced osteoporosis in rats
Life Sciences, Vol. 38, pp. 951-958 Printed in the U.S.A.
Pergamon Press
EFFECT OF IPRIFLAVONE ON GLUCOCORTICOID-INDUCED OSTEOPOROSIS IN RATS
Iwao Yamazaki*, Akio Shino*, Yasuyoshi Shimizu**, Ryoichi Tsukuda*, Y o s h i h i r o Shirakawa* and Masako K i n o s h i t a * * Biology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., 17-85, Jusohonmachi 2-chome, Yodogawaku, Osaka 532, Japan ** NRI Life Science, 4-17, Kajiwara 4-chome, Kanagawa-ken 247, Japan (Received in final form December 16, 1985) Summary Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, was administered orally for 12 weeks to male rats with prednisoloneinduced osteoporosis. Microdensitometric analysis of a roentgenograph of the femurs revealed that ipriflavone increased the density of the distal metaphysis dose-dependently and tended to increase the density of the diaphysis. It also inhibited dose-dependently the decreases in the mechanical strength of the tibia, breaking strain and breaking energy, and the fractional content of ash in femurs. These results indicate that ipriflavone markedly suppresses bone resorption at the metaphysis where the content of trabecular bone with a rapid turnover rate is high, and possibly inhibits bone reduction at the diaphysis. Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, synthesized by Chinoin Pharmaceutical and Chemical Works Ltd., Hungary, is one of the isoflavone derivatives that occur widely in nature and are regarded as growth promotion factors in animals. This compound is devoid of estrogenic activity but increases the uterotropic and calcitonin secreting activities of estrogen (I). Calcitonin is known to suppress bone resorption directly (2) and is also presumed to stimulate bone formation (3). When ipriflavone was added to the medium of fetal rat bone culture carried out following the method of Raisz (4), it also acted directly on bone to suppress resorption by itself or in the presence of estrogen (M. Tsuda, personal communication). Therefore, this compound is expected to be effective in treating osteopenic disease including osteoporosis. It is well known that stimulated secretion of endogenous glucocorticoid (5) in Cushing disease or chronic administration of glucocorticoid in cases of chronic rheumatoid arthritis or asthma (6-9) leads to osteoporosis very frequently. The present studies were designed to assess the prophylactic effect of ipriflavone on glucocorticoid-induced osteoporosis in rats. Materials and Methods Animals : Male Sprague-Dawley rats, 7 weeks of age, were purchased from Japan CLEA Co. They were housed separately in plastic cages and fed a commercial diet 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
952
Effect of Ipriflavone on Osteoporosis
Vol. 38, No. i0, 1986
(CE-2, Japan CLEA) and drinking water ad libitum, in an air-conditioned environment (23 ± 2°C, 55 ± 10% humidity). The calcium, phosphorus, and vitamin D contents of the diet were 1.2%, 1.1%, and 2.0 IU/g, respectively. Compounds used : Ipriflavone was synthesized in our Chemical Development Laboratories and suspended in water containing 1% hydroxypropyl cellulose (HPC-L, Nippon Soda Co., Japan). Sodium prednisolone succinate (Soluble prednisolone) was purchased from Shionogi and Co., Ltd. and dissolved in water just before use. Procedures for administering the compounds and for collecting serum and bone specimens : Administrations of ipriflavone, 25, I00, and 400 mg/kg body weight/ day, orally, and prednisolone, 80 mg/kg body weight/time, twice a week into the rump muscle alternately, were started at 8 weeks of age and continued for 12 weeks. On the day after the last dosing of ipriflavone, the animals were anesthetized with ether and blood was withdrawn from the abdominal aorta. The separated serum was stored at -20°C until used for calcium, inorganic phosphorus, calcitonin, and parathyroid hormone (PTH) determinations. Both femurs were removed for x-ray analysis and mineral content determinations. The left tibia was also removed, immersed in liquid paraffin, and used to estimate breaking properties within 1 week. Methods for takin$ the bone roentgenograph and for microdensitometric analysis : The right femur of each rat was placed sagitally on a soft x-ray film (Fuji Softex film FG, Fuji Photofilm Co., Japan) and a roentgenograph was taken with 35 kVp, 3 mA, 165 sec, at 65 cm. Microdensitometric analysis was performed following the method of Inoue et al. (i0). Using an interactive image analyser system, IBAS (Carl Zeiss, West Germany), microdensitometry to detect the density of transverse sections of the femur was applied at two locations : 0.18/1 (distal metaphysis) and 0.43/1 (diaphysis) from the edge of the distal end of the femur on the roentgenograph. Seven kinds of volumetric variables, cortical diameter outside (D), cortical diameter inside (d), cortical thickness index (CTI = (D-d)/D), mean density integrated area/cortical diameter outside (~GS/D), mean density of both cortical regions (AGSmax), density of marrow region (AGSmin), and bone length were estimated. Bone length was measured on the line connecting the trochanter major and the edge of the condylus lateralis. Measurements of breaking properties of bone : The breaking properties of the left tibia were measured following the method of Ezawa et al. (ii). Using a universal test instrument of Instron type (Intesco model 205, Intesco Co., Japan), a 3point bending test was carried out; a tibia was carefully positioned at each site on the two support noses standing 17 mm apart and a load was applied at the branching point of the tibia and fibula with a cross head speed of 200 mm/min; the load-deflection curve was recorded on the X-Y recorder transduced by a displacement indicator. Breaking strain and breaking energy were calculated from the curve. Bone configuration was simulated as an elliptical column at the loading point. Measurement of bone mineral content : The measurement was performed at Sumika Chemical Analysis Service Ltd. (Japan). The left femur was defatted by immersing it twice in chloroform:ethanol (2:1) for 3 hr each. After it was placed in a oven and heated at 120°C for 8 hr under vacuum on magnesium perchlorate, its dry weight was determined. It was further heated in an electric hearth for 30 min at each of 250, 400, and 600°C, and finally at 800°C for 1 hr; and cooled in a desiccator for 30 min; the ashed weight was then determined. The calcium and phosphorus contents of the ashed bone were determined by titration (12) and colorimetric methods (13), respectively. Determinations
of serum calcium and inorganic phosphorus
: To determine
serum
Vol. 38, No. i0, 1986
Effect of Ipriflavone on Osteoporosis
953
calcium, a spectrophotometric method (14), using the metal complexing dye, orthocresolphthalein complexon, was applied; a commercial kit, Calcium C-test Wako (Wako Pure Chemical Industries, Ltd., Japan) was used. To determine serum inorganic phosphorus, a spectrophotometric method (iS), using molybdenum hydrochloride, was applied; a commercial kit, Phospha B-test Wako (Wako Pure Chemical Industries, Ltd.) was used. Determinations of serum calcitonin and PTH : Serum calcitonin was determined using a commercial radioimmunoassay kit for human calcitonin (Daiichi Radioisotope Labs., Japan)(16) by the double antibody technique. It was confirmed beforehand that the dilution curve of the rat thyroid extract paralleled the standard curve for human calcitonin of the radioimmunoassay kit. Serum PTH was determined using a commercial radioimmunoassay kit for human PTH (hPTH-~-kit, Immuno Nuclear, USA)(17), which is said to be highly immunocrossreactive to rat PTH, by the double antibody technique. Statistical analysis : The statistical significance of differences between means was determined by Dunnett's test (18). Results Body weight : Prednisolone suppressed body weight increase. Ipriflavone, combined with prednisolone, did not influence the effect of prednisolone on body weight. No abnormal behavior or change in appearance was observed in the rats. Microdensitometric analysis of the femur (TABLE I) : Bone length was not changed by prednisolone or ipriflavone combined with prednisolone. At the distal metaphysis, prednisolone reduced bone density (~GS/D) and ipriflavone increased this bone index dose-dependently when administered in combination with prednisolone. However, the cortical diameter outside (D) was not influenced by these compounds. At the diaphysis, prednisolone significantly reduced cortical thickness index (CTI) by slightly decreasing the cortical diameter outside (D) and a slightly increasing the cortical diameter inside (d); ipriflavone did not change these measurements. Prednisolone significantly decreased the density of the marrow region (&GSmin) and bone density (~GS/D); these changes recovered dosedependently after treatment with ipriflavone combined with prednisolone, although the increase in bone density was statistically non-significant. Breaking properties of tibia (TABLE II) : Prednisolone caused a slight, nonsignificant, decrease in both the breaking strain and breaking energy of tibia. Ipriflavone induced a slight but not significant increase in the breaking strain and a significant dose-dependent increase in breaking energy. Mineral content of femur (TABLE If) : Prednisolone produced a slight, non-significant, decrease in the fractional contents of ash, calcium, and phosphorus on the basis of dry weight of femur. Ipriflavone produced a slight increase in phosphorus content and a marked increase in ash content at higher doses. Serum calcium, inorganic phosphorus, calcitonin, and PTH (TABLE III) : The compounds did not influence serum calcium level and only prednisolone slightly increased inorganic phosphorus level. Ipriflavone increased the calcitonin level significantly and decreased PTH insignificantly; prednisolone did not influence these levels. Discussion Excess glucocorticoid appears to induce osteopenia to a greater extent in
80
80
80
80
0
25
i00
400
8
8
8
8
8
39.2±0.2
39.6±0.3
39.4±0.3
39.5±0.2
39.8±0.3
Femoral length (mm)
6.27±0.10
6.58±0.10
6.41±0.05
6.30±0.07
6.51±0.13
D (nun)
4.83±0.09
4.69±0.07
4.78±0.07
4.88±0.08
D (tam)
1.88±0.10"* 4.69±0.10
1.78±0.04"
1.53±0.05
1.47±0.08
1.72±0.05"
ZGS/D
Distal metaphysis
3.48±0.10
3.59±0.08
3.54±0.07
3.56±0.06
3.S0±0.06
d (mm)
0.26±0.01
0.26±0.00
0.25±0.01
0.26±0.00
0.28±0.01"*
CTI
1.86±0.07"
1.71±0.07
1.63±0.04
1.63±0.08
1.97±0.05"*
AGSmin
~GS/D
2.30±0.08
2.18±0.06
2.09±0.03
2.12±0.06
2.36±0.06*
Osteoporosis
Values are expressed as means ± SEM. 0.05, ** p < O.01 (Dunnett's test).
Significantly different from the prednisolone
treated control
: * 0.01 < p <
Bone density of the femurs in a roentgenograph was measured at points 0.18/1 (distal metaphysis) and 0.43/1 (diaphysis) from the edge of the distal end. D : cortical diameter outside, d : cortical diameter inside, ~GS/D : mean density integrated area/cortical diameter outside, CTI : cortical thickness index = (D-d)/D, AGSmax : mean density of both cortical regions, AGSmin : density of marrow region.
i.m., twice a week, for 12 solution daily for 12 weeks.
3.77±0.12
3.65±0.07
3.76±0.07
3.71±0.09
3.87±0.04
AGSmax
Diaphysis
Analysis of the Femurs of Male Rats with Prednisolone-induced Treated with Ipriflavone for 12 Weeks
I
Male Sprague-Dawley rats (8 weeks old) were treated with prednisolone (Pred), 80 mg/kg, weeks. Rats received orally ipriflavone (Ipri) suspended in 1% hydroxypropyl cellulose
0
Dose No. of of Pred rats
0
Dose of Ipri
Microdensitometric
TABLE
~D GO
O
Z O
LO OO
O
O
O
O
rt
O
O
O
H
O
~'h
of Pred
0
80
80
80
80
of Ipri
0
0
25
i00
400
8
8
8
8
8
rats
of
No.
1.44 + 0.07
1.68 -+ 0.23
1.37 + 0. I0
1.29 -+ 0.06
1.66 ± 0.13
Breaking strain (x 10 -2)
21.5 + 2.0*
20.4 + 2.3*
14.2 ± 1.4
14.0 + 1.4
17.6 + 3.9
Breaking energy (kg.mm/cm 3)
65.5 ± 0.4*
65.8 + 0.3*
64.4 ± 0.4
63.7 -+ 0.6
65.0 ± 0.6
(ash/dry wt)
Ash
25.8 + 0.i
25.9 ± 0. i
25.6 ± 0.2
25.3 -+ 0.3
25.6 ± 0.3
(Ca/dry wt)
(Ca/ash)
39.5 ± 0.i
39.4 + 0.i
39.8 + 0.i
39.7 + 0. i
39.4 ± 0.i
Calcium
Values are expressed as means ± SEM. 0.05, ** p < 0.01 (Dunnett's test).
Osteoporosis
17.8 ± 0.0
17.8 + 0.0
17.8 ± 0.0
17.7 + 0.i
17.8 ± 0.0
(P/ash)
Significantly different from the prednisolone treated control : * 0.01 < p <
11.7 ± 0.i*
11.7 ± 0.i**
11.5 + 0. I
11.3 + 0.i
11.6 ± 0.i
(P/dry wt)
Phosphorus
Fractional contents in femur (%)
and Mineral Contents of Femurs of Male Rats with Prednisolone-induced Treated with Ipriflavone for 12 Weeks
Rats and treatment with prednisolone and ipriflavone were the same as those in Table I.
Dose
Dose
of Tibiae
Breaking properties of tibia
Breaking Properties
TABLE II
ka %a
i~. g~
0
0
0
0
0
0 (D
m
H
O
rt
~h
r~
ko oo
O
O
Z
OO
<~ O
956
Effect of Ipriflavone on Osteoporosis
Vol. 38, No. i0, 1986
TABLE I I I Serum C o n c e n t r a t i o n o f C a l c i u m , I n o r g a n i c P h o s p h o r u s , C a l c i t o n i n , i n Male R a t s w i t h P r e d n i s o l o n e - i n d u c e d Osteoporosis Treated with Ipriflavone f o r 12 Weeks
Dose of Ipri
Dose of Pred
No. of rats
Calcium (mg/dl)
Inorganic phosphorus (mg/dl)
Calcitonin (pg/ml)
a n d PTH
PTH (p mol/l)
0
0
8
10.2 _+ 0.2
6.1 +_ 0.i*
124.8 _+ 43.5
110.9 +
7.2
0
80
8
10.2 _+ 0.I
6.8 ,+ 0.i
128.1 +
24.8
104.0 -+ 8.1
25
80
8
10.2 -+ 0.i
6.5 _+ 0.3
216.7 _+ 40.5
121.4 ,+ 12.7
i00
80
8
10.3 -+ 0.i
6.8 +_ 0.2
455.0 _+ 122.2"*
97.7 -+ 6.5
400
80
8
10.3 -+ 0.2
6.7 -+ 0.I
429.6 _+ 59.9*
81.2 +
5.9
Rats and treatment with prednisolone and ipriflavone were the same as those in TABLE I . V a l u e s a r e e x p r e s s e d a s m e a n s ,+ SEM. S i g n i f i c a n t l y different from the predniso l o n e t r e a t e d c o n t r o l : * 0 . 0 1 < p < 0 . 0 5 , ** p < 0 . 0 1 ( D u n n e t t ' s t e s t ) .
trabecular bones than in cortical bones (7). In the long bones, the action of the hormone is more prominent at the metaphyseal site containing a relatively large proportion of trabecular bone than at the diaphyseal site composed primarily of cortical bone (8). In the present studies, bone density was reduced by prednisolone at both the distal metaphysis and diaphysis, but the extent of reduction was somewhat larger at the former site. However, prednisolone appeared to reduce the cortical bone mass to some extent because it reduced the cortical thickness index and the density of marrow region at the diaphysis. In the prednisolone-induced osteopenia ipriflavone increased bone density at the distal metaphys~s dose-dependently. The highest dose of ipriflavone increased the density of the marrow region at the diaphysis. Furthermore, ipriflavone dose-dependently increased the mechanical strength, especially the breaking energy, of the tibia at the diaphysis. Ipriflavone also increased the fractional content of ash (ash weight/dry bone weight) in the femur. These results indicate that ipriflavone markedly suppresses bone resorption at the metaphysis where the content of rapid turnover trabecular bone is high, and slightly, but definitely, inhibits bone reduction at the diaphysis The mechanism whereby glucocorticoids increases bone resorption is somewhat complex. Secondary hyperparathyroidism results from negative calcium balance (19) due to the action of glucocorticoids in directly inhibiting enteral calcium absorption (20, 21), in inhibiting the production of active vitamin D (22), and in causing hypercalciuria (23, 24). Furthermore, glucocorticoids directly stimulate PTH secretion from the parathyroid tissue (25). These actions increase blood PTH and lead to increased bone resorption. Glucocorticoids have also direct actions on bones to suppress bone formation (26) and to accelerate bone
Vol. 38, No. i0, 1986
Effect of Ipriflavone on Osteoporosis
957
resorption (24). On the other hand, glucocorticoids suppress pituitary ACTH secretion, which in turn, lead, to reduced adrenal androstenedione production which may decrease testosterone and estrogen secretion from the adrenal gland (27). Ipriflavone accelerates calcitonin secretion in the presence of small amount of estrogen by increasing the sensitivity of the thyroid gland to blood calcium (i), although the possibility that the effect of ipriflavone on calcitonin level resulted from decreased serum clearance cannot be excluded. Also experimental procedures, such as ether anesthesia at blood collection, might influence serum calcitonin levels, but both control and each experimental groups were treated in an identical manner to eliminate such disturbances. Ipriflavone, by itself or in combination with estrogen, also suppresses bone resorption in vitro determined by Raisz's bone culture technique (M. Tsuda, personal communication). The testes of mammals have an aromatase activity (28) and small amount of estrogen is secreted from the testes by conversion from androgen (29). Thus, in the present studies, ipriflavone administered orally, in addition to directly suppressing
bone r e s o r p t i o n , a c c e l e r a t e d c a l c i t o n i n s e c r e t i o n i n the p r e s e n c e of small amount of e s t r o g e n i n male r a t s , which, i n t u r n , s u p r e s s e d bone r e s o r p t i o n induced by p r e d n i s o l o n e . I n c i d e n t a l l y , i p r i f l a v o n e has n e i t h e r an a n d r o g e n i c n o r an androgen p o t e n t i a t i n g a c t i v i t y ( u n p u b l i s h e d ) , a l t h o u g h androgen a l s o s u p r e s s e s bone r e s o r p t i o n (30). Acknowledgements We are grateful to Mrs. Masako Suzuki, Mrs. Hitomi Hamajo, Mr. Tadashi Miwa, and Mr. Haruhiko Yamaguchi for technical assistance; to Mr. Yuzo Hayakawa for advice on measuring the breaking properties of the bone; and to Drs. Shintaro Kikuchi, Hisashi Iwatsuka and J. R. Miller for comments on the manuscript. References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12.
13. 14. 15. 16. 17.
18.
I. YAMAZAKI, Program of the 7th International Congress of Endocrinology, Quebec City, Canada, p. 1628 (Abstract), Excerpta Medica, Amsterdam (1984). D. ATKINS and M. PEACOCK, J. Endocrinol. 64 573-583 (1975). M. EGUCHI, E. SHIOTA, T. YAMAGUCHI, S. HASHIGUCHI, H. KAWAMURA and K. SHIBATa, Bone Metab. 15 72-80 (1982). L. G. RAISZ, J. Clin. Invest. 44 i03-i16 (1965). M. L. SUSSMAN and B. COPELMAN, Radiology 39 288-292 (1942). F. DEMARTINI, A.W. GROKOEST and C. RAGEN, J. Am. Med. Assoc. 149 750-752 (1952). T. J. HAHN, Arch. Intern. Med. 138 882-885 (1978). T. J. HAHN, V. C BOISSEAU and L. V. ARIOLI, J. Clin. Endocrinol. Metab. 39 274-282 ( 1 9 7 4 ) . A. D. ADINOFF and J. R. HOLLISTER, New Engl. J. Med. 309 265-268 (1983). T. INOUE, K. KUSHIDA, S. MIYAMOTO, Y. SUMI, and H. ORIMO, J. Jpn. Orthop. Assoc. 57 1923-1936 (1983). I. EZAWA, R. OKADA, Y. NOZAKI, and E. OGATA, J. Jpn. Soc. Food Nutr. 32 329-535 ( 1 9 7 9 ) . D. H. COPP, B. A. CHENEY and N. M. STOKOE, J. Lab. C l i n . Med. 61 1029-1037 (1963). C. H. FISKE and Y. SABBAROW, J. Biol. Chem. 66 375-400 (1925). H. V. CONNERTY and A. R. BRIGGS, Am. J. Clin. Path. 45 290-296 (1966). H. GOLDENBERG and A. FERNANDEZ, Clin. Chem. 12 871-882 (1966). S. MORIMOTO, Y. OKADA, T. ONISHI, M. TSUJI, and Y. KUMAHARA, Clin. Endocrinol. (Japan) 28 313-319 (1980). B. A. ROOS, A. W. LINDELL, D. C. ARON, J. W. ORF, M. YOOM, M. B. HUBER, J. PENSKY, J. ELLIS, and P. W. LAMBERT, J. Clin. Endocrinol. Metab. 53 709-721 (1981). C . W . DUNN~ETT, Biometrics 20 482-491 (1964).
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19. 20. 21.
22. 23. 24. 25. 26. 27. 28. 29. 30.
Effect of Ipriflavone
on Osteoporosis
Vol. 38, No. i0, 1986
R. F. FUCIK, J. C. KUKREJA, G. K. HARGIS, E. N. BOWSER, W. J. HENDERSON, and G. A. WILLIAMS, J. Clin. Endocrinol. Metab. 40 152-155 C1975). J. J. FEHER and R. H. WASSERMAN, Endocrinology 104 547-551 (1979). M. J. FAVUS, M. W. WALLING, and D. V. KIMBERG, J. Clin. Invest. 52 16801685 (1973). E. SEEMAN, R. KUMAR, G. G. HUNDER, M. SCOTT, H. HEATH 111, and B. L. RIGGS, J. C l i n . I n v e s t . 66 664-669 (1980). H. CAAKE, Acta E n d o c r i n o l . 34 60-64 (1960). A. CANIGGIA, R. NUTI, F. LORE, and A. VATTIMO, J. S t e r o i d Biochem. 15 153-161 (1981). W. AU, Science 193 1015-1017 (1976). E. CANALIS, Endocrinology 112 931-939 (1983). R. CRILLY, D. H. MARSHALL, M. CAWOOD, and B. E. C. NORDIN, J. R. Soc. Med. 71 733-736 (1978). J. A. CANICK, A. MAKRIS, G.L. GUNSALUS, and K. J. RYAN, Endocrinology 104 28S-288 (1979). F. H. DE JONG, A. H. HEY and H. J. VAN DER MOLEN, J. Endocrinol. 57 277284 (1973). F. W. LAFFERTY, G. E. SPENCER, and O. H. PEARSON, Am. J. Med. 36 514-528 (1964).
Pergamon Press
EFFECT OF IPRIFLAVONE ON GLUCOCORTICOID-INDUCED OSTEOPOROSIS IN RATS
Iwao Yamazaki*, Akio Shino*, Yasuyoshi Shimizu**, Ryoichi Tsukuda*, Y o s h i h i r o Shirakawa* and Masako K i n o s h i t a * * Biology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., 17-85, Jusohonmachi 2-chome, Yodogawaku, Osaka 532, Japan ** NRI Life Science, 4-17, Kajiwara 4-chome, Kanagawa-ken 247, Japan (Received in final form December 16, 1985) Summary Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, was administered orally for 12 weeks to male rats with prednisoloneinduced osteoporosis. Microdensitometric analysis of a roentgenograph of the femurs revealed that ipriflavone increased the density of the distal metaphysis dose-dependently and tended to increase the density of the diaphysis. It also inhibited dose-dependently the decreases in the mechanical strength of the tibia, breaking strain and breaking energy, and the fractional content of ash in femurs. These results indicate that ipriflavone markedly suppresses bone resorption at the metaphysis where the content of trabecular bone with a rapid turnover rate is high, and possibly inhibits bone reduction at the diaphysis. Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, synthesized by Chinoin Pharmaceutical and Chemical Works Ltd., Hungary, is one of the isoflavone derivatives that occur widely in nature and are regarded as growth promotion factors in animals. This compound is devoid of estrogenic activity but increases the uterotropic and calcitonin secreting activities of estrogen (I). Calcitonin is known to suppress bone resorption directly (2) and is also presumed to stimulate bone formation (3). When ipriflavone was added to the medium of fetal rat bone culture carried out following the method of Raisz (4), it also acted directly on bone to suppress resorption by itself or in the presence of estrogen (M. Tsuda, personal communication). Therefore, this compound is expected to be effective in treating osteopenic disease including osteoporosis. It is well known that stimulated secretion of endogenous glucocorticoid (5) in Cushing disease or chronic administration of glucocorticoid in cases of chronic rheumatoid arthritis or asthma (6-9) leads to osteoporosis very frequently. The present studies were designed to assess the prophylactic effect of ipriflavone on glucocorticoid-induced osteoporosis in rats. Materials and Methods Animals : Male Sprague-Dawley rats, 7 weeks of age, were purchased from Japan CLEA Co. They were housed separately in plastic cages and fed a commercial diet 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
952
Effect of Ipriflavone on Osteoporosis
Vol. 38, No. i0, 1986
(CE-2, Japan CLEA) and drinking water ad libitum, in an air-conditioned environment (23 ± 2°C, 55 ± 10% humidity). The calcium, phosphorus, and vitamin D contents of the diet were 1.2%, 1.1%, and 2.0 IU/g, respectively. Compounds used : Ipriflavone was synthesized in our Chemical Development Laboratories and suspended in water containing 1% hydroxypropyl cellulose (HPC-L, Nippon Soda Co., Japan). Sodium prednisolone succinate (Soluble prednisolone) was purchased from Shionogi and Co., Ltd. and dissolved in water just before use. Procedures for administering the compounds and for collecting serum and bone specimens : Administrations of ipriflavone, 25, I00, and 400 mg/kg body weight/ day, orally, and prednisolone, 80 mg/kg body weight/time, twice a week into the rump muscle alternately, were started at 8 weeks of age and continued for 12 weeks. On the day after the last dosing of ipriflavone, the animals were anesthetized with ether and blood was withdrawn from the abdominal aorta. The separated serum was stored at -20°C until used for calcium, inorganic phosphorus, calcitonin, and parathyroid hormone (PTH) determinations. Both femurs were removed for x-ray analysis and mineral content determinations. The left tibia was also removed, immersed in liquid paraffin, and used to estimate breaking properties within 1 week. Methods for takin$ the bone roentgenograph and for microdensitometric analysis : The right femur of each rat was placed sagitally on a soft x-ray film (Fuji Softex film FG, Fuji Photofilm Co., Japan) and a roentgenograph was taken with 35 kVp, 3 mA, 165 sec, at 65 cm. Microdensitometric analysis was performed following the method of Inoue et al. (i0). Using an interactive image analyser system, IBAS (Carl Zeiss, West Germany), microdensitometry to detect the density of transverse sections of the femur was applied at two locations : 0.18/1 (distal metaphysis) and 0.43/1 (diaphysis) from the edge of the distal end of the femur on the roentgenograph. Seven kinds of volumetric variables, cortical diameter outside (D), cortical diameter inside (d), cortical thickness index (CTI = (D-d)/D), mean density integrated area/cortical diameter outside (~GS/D), mean density of both cortical regions (AGSmax), density of marrow region (AGSmin), and bone length were estimated. Bone length was measured on the line connecting the trochanter major and the edge of the condylus lateralis. Measurements of breaking properties of bone : The breaking properties of the left tibia were measured following the method of Ezawa et al. (ii). Using a universal test instrument of Instron type (Intesco model 205, Intesco Co., Japan), a 3point bending test was carried out; a tibia was carefully positioned at each site on the two support noses standing 17 mm apart and a load was applied at the branching point of the tibia and fibula with a cross head speed of 200 mm/min; the load-deflection curve was recorded on the X-Y recorder transduced by a displacement indicator. Breaking strain and breaking energy were calculated from the curve. Bone configuration was simulated as an elliptical column at the loading point. Measurement of bone mineral content : The measurement was performed at Sumika Chemical Analysis Service Ltd. (Japan). The left femur was defatted by immersing it twice in chloroform:ethanol (2:1) for 3 hr each. After it was placed in a oven and heated at 120°C for 8 hr under vacuum on magnesium perchlorate, its dry weight was determined. It was further heated in an electric hearth for 30 min at each of 250, 400, and 600°C, and finally at 800°C for 1 hr; and cooled in a desiccator for 30 min; the ashed weight was then determined. The calcium and phosphorus contents of the ashed bone were determined by titration (12) and colorimetric methods (13), respectively. Determinations
of serum calcium and inorganic phosphorus
: To determine
serum
Vol. 38, No. i0, 1986
Effect of Ipriflavone on Osteoporosis
953
calcium, a spectrophotometric method (14), using the metal complexing dye, orthocresolphthalein complexon, was applied; a commercial kit, Calcium C-test Wako (Wako Pure Chemical Industries, Ltd., Japan) was used. To determine serum inorganic phosphorus, a spectrophotometric method (iS), using molybdenum hydrochloride, was applied; a commercial kit, Phospha B-test Wako (Wako Pure Chemical Industries, Ltd.) was used. Determinations of serum calcitonin and PTH : Serum calcitonin was determined using a commercial radioimmunoassay kit for human calcitonin (Daiichi Radioisotope Labs., Japan)(16) by the double antibody technique. It was confirmed beforehand that the dilution curve of the rat thyroid extract paralleled the standard curve for human calcitonin of the radioimmunoassay kit. Serum PTH was determined using a commercial radioimmunoassay kit for human PTH (hPTH-~-kit, Immuno Nuclear, USA)(17), which is said to be highly immunocrossreactive to rat PTH, by the double antibody technique. Statistical analysis : The statistical significance of differences between means was determined by Dunnett's test (18). Results Body weight : Prednisolone suppressed body weight increase. Ipriflavone, combined with prednisolone, did not influence the effect of prednisolone on body weight. No abnormal behavior or change in appearance was observed in the rats. Microdensitometric analysis of the femur (TABLE I) : Bone length was not changed by prednisolone or ipriflavone combined with prednisolone. At the distal metaphysis, prednisolone reduced bone density (~GS/D) and ipriflavone increased this bone index dose-dependently when administered in combination with prednisolone. However, the cortical diameter outside (D) was not influenced by these compounds. At the diaphysis, prednisolone significantly reduced cortical thickness index (CTI) by slightly decreasing the cortical diameter outside (D) and a slightly increasing the cortical diameter inside (d); ipriflavone did not change these measurements. Prednisolone significantly decreased the density of the marrow region (&GSmin) and bone density (~GS/D); these changes recovered dosedependently after treatment with ipriflavone combined with prednisolone, although the increase in bone density was statistically non-significant. Breaking properties of tibia (TABLE II) : Prednisolone caused a slight, nonsignificant, decrease in both the breaking strain and breaking energy of tibia. Ipriflavone induced a slight but not significant increase in the breaking strain and a significant dose-dependent increase in breaking energy. Mineral content of femur (TABLE If) : Prednisolone produced a slight, non-significant, decrease in the fractional contents of ash, calcium, and phosphorus on the basis of dry weight of femur. Ipriflavone produced a slight increase in phosphorus content and a marked increase in ash content at higher doses. Serum calcium, inorganic phosphorus, calcitonin, and PTH (TABLE III) : The compounds did not influence serum calcium level and only prednisolone slightly increased inorganic phosphorus level. Ipriflavone increased the calcitonin level significantly and decreased PTH insignificantly; prednisolone did not influence these levels. Discussion Excess glucocorticoid appears to induce osteopenia to a greater extent in
80
80
80
80
0
25
i00
400
8
8
8
8
8
39.2±0.2
39.6±0.3
39.4±0.3
39.5±0.2
39.8±0.3
Femoral length (mm)
6.27±0.10
6.58±0.10
6.41±0.05
6.30±0.07
6.51±0.13
D (nun)
4.83±0.09
4.69±0.07
4.78±0.07
4.88±0.08
D (tam)
1.88±0.10"* 4.69±0.10
1.78±0.04"
1.53±0.05
1.47±0.08
1.72±0.05"
ZGS/D
Distal metaphysis
3.48±0.10
3.59±0.08
3.54±0.07
3.56±0.06
3.S0±0.06
d (mm)
0.26±0.01
0.26±0.00
0.25±0.01
0.26±0.00
0.28±0.01"*
CTI
1.86±0.07"
1.71±0.07
1.63±0.04
1.63±0.08
1.97±0.05"*
AGSmin
~GS/D
2.30±0.08
2.18±0.06
2.09±0.03
2.12±0.06
2.36±0.06*
Osteoporosis
Values are expressed as means ± SEM. 0.05, ** p < O.01 (Dunnett's test).
Significantly different from the prednisolone
treated control
: * 0.01 < p <
Bone density of the femurs in a roentgenograph was measured at points 0.18/1 (distal metaphysis) and 0.43/1 (diaphysis) from the edge of the distal end. D : cortical diameter outside, d : cortical diameter inside, ~GS/D : mean density integrated area/cortical diameter outside, CTI : cortical thickness index = (D-d)/D, AGSmax : mean density of both cortical regions, AGSmin : density of marrow region.
i.m., twice a week, for 12 solution daily for 12 weeks.
3.77±0.12
3.65±0.07
3.76±0.07
3.71±0.09
3.87±0.04
AGSmax
Diaphysis
Analysis of the Femurs of Male Rats with Prednisolone-induced Treated with Ipriflavone for 12 Weeks
I
Male Sprague-Dawley rats (8 weeks old) were treated with prednisolone (Pred), 80 mg/kg, weeks. Rats received orally ipriflavone (Ipri) suspended in 1% hydroxypropyl cellulose
0
Dose No. of of Pred rats
0
Dose of Ipri
Microdensitometric
TABLE
~D GO
O
Z O
LO OO
O
O
O
O
rt
O
O
O
H
O
~'h
of Pred
0
80
80
80
80
of Ipri
0
0
25
i00
400
8
8
8
8
8
rats
of
No.
1.44 + 0.07
1.68 -+ 0.23
1.37 + 0. I0
1.29 -+ 0.06
1.66 ± 0.13
Breaking strain (x 10 -2)
21.5 + 2.0*
20.4 + 2.3*
14.2 ± 1.4
14.0 + 1.4
17.6 + 3.9
Breaking energy (kg.mm/cm 3)
65.5 ± 0.4*
65.8 + 0.3*
64.4 ± 0.4
63.7 -+ 0.6
65.0 ± 0.6
(ash/dry wt)
Ash
25.8 + 0.i
25.9 ± 0. i
25.6 ± 0.2
25.3 -+ 0.3
25.6 ± 0.3
(Ca/dry wt)
(Ca/ash)
39.5 ± 0.i
39.4 + 0.i
39.8 + 0.i
39.7 + 0. i
39.4 ± 0.i
Calcium
Values are expressed as means ± SEM. 0.05, ** p < 0.01 (Dunnett's test).
Osteoporosis
17.8 ± 0.0
17.8 + 0.0
17.8 ± 0.0
17.7 + 0.i
17.8 ± 0.0
(P/ash)
Significantly different from the prednisolone treated control : * 0.01 < p <
11.7 ± 0.i*
11.7 ± 0.i**
11.5 + 0. I
11.3 + 0.i
11.6 ± 0.i
(P/dry wt)
Phosphorus
Fractional contents in femur (%)
and Mineral Contents of Femurs of Male Rats with Prednisolone-induced Treated with Ipriflavone for 12 Weeks
Rats and treatment with prednisolone and ipriflavone were the same as those in Table I.
Dose
Dose
of Tibiae
Breaking properties of tibia
Breaking Properties
TABLE II
ka %a
i~. g~
0
0
0
0
0
0 (D
m
H
O
rt
~h
r~
ko oo
O
O
Z
OO
<~ O
956
Effect of Ipriflavone on Osteoporosis
Vol. 38, No. i0, 1986
TABLE I I I Serum C o n c e n t r a t i o n o f C a l c i u m , I n o r g a n i c P h o s p h o r u s , C a l c i t o n i n , i n Male R a t s w i t h P r e d n i s o l o n e - i n d u c e d Osteoporosis Treated with Ipriflavone f o r 12 Weeks
Dose of Ipri
Dose of Pred
No. of rats
Calcium (mg/dl)
Inorganic phosphorus (mg/dl)
Calcitonin (pg/ml)
a n d PTH
PTH (p mol/l)
0
0
8
10.2 _+ 0.2
6.1 +_ 0.i*
124.8 _+ 43.5
110.9 +
7.2
0
80
8
10.2 _+ 0.I
6.8 ,+ 0.i
128.1 +
24.8
104.0 -+ 8.1
25
80
8
10.2 -+ 0.i
6.5 _+ 0.3
216.7 _+ 40.5
121.4 ,+ 12.7
i00
80
8
10.3 -+ 0.i
6.8 +_ 0.2
455.0 _+ 122.2"*
97.7 -+ 6.5
400
80
8
10.3 -+ 0.2
6.7 -+ 0.I
429.6 _+ 59.9*
81.2 +
5.9
Rats and treatment with prednisolone and ipriflavone were the same as those in TABLE I . V a l u e s a r e e x p r e s s e d a s m e a n s ,+ SEM. S i g n i f i c a n t l y different from the predniso l o n e t r e a t e d c o n t r o l : * 0 . 0 1 < p < 0 . 0 5 , ** p < 0 . 0 1 ( D u n n e t t ' s t e s t ) .
trabecular bones than in cortical bones (7). In the long bones, the action of the hormone is more prominent at the metaphyseal site containing a relatively large proportion of trabecular bone than at the diaphyseal site composed primarily of cortical bone (8). In the present studies, bone density was reduced by prednisolone at both the distal metaphysis and diaphysis, but the extent of reduction was somewhat larger at the former site. However, prednisolone appeared to reduce the cortical bone mass to some extent because it reduced the cortical thickness index and the density of marrow region at the diaphysis. In the prednisolone-induced osteopenia ipriflavone increased bone density at the distal metaphys~s dose-dependently. The highest dose of ipriflavone increased the density of the marrow region at the diaphysis. Furthermore, ipriflavone dose-dependently increased the mechanical strength, especially the breaking energy, of the tibia at the diaphysis. Ipriflavone also increased the fractional content of ash (ash weight/dry bone weight) in the femur. These results indicate that ipriflavone markedly suppresses bone resorption at the metaphysis where the content of rapid turnover trabecular bone is high, and slightly, but definitely, inhibits bone reduction at the diaphysis The mechanism whereby glucocorticoids increases bone resorption is somewhat complex. Secondary hyperparathyroidism results from negative calcium balance (19) due to the action of glucocorticoids in directly inhibiting enteral calcium absorption (20, 21), in inhibiting the production of active vitamin D (22), and in causing hypercalciuria (23, 24). Furthermore, glucocorticoids directly stimulate PTH secretion from the parathyroid tissue (25). These actions increase blood PTH and lead to increased bone resorption. Glucocorticoids have also direct actions on bones to suppress bone formation (26) and to accelerate bone
Vol. 38, No. i0, 1986
Effect of Ipriflavone on Osteoporosis
957
resorption (24). On the other hand, glucocorticoids suppress pituitary ACTH secretion, which in turn, lead, to reduced adrenal androstenedione production which may decrease testosterone and estrogen secretion from the adrenal gland (27). Ipriflavone accelerates calcitonin secretion in the presence of small amount of estrogen by increasing the sensitivity of the thyroid gland to blood calcium (i), although the possibility that the effect of ipriflavone on calcitonin level resulted from decreased serum clearance cannot be excluded. Also experimental procedures, such as ether anesthesia at blood collection, might influence serum calcitonin levels, but both control and each experimental groups were treated in an identical manner to eliminate such disturbances. Ipriflavone, by itself or in combination with estrogen, also suppresses bone resorption in vitro determined by Raisz's bone culture technique (M. Tsuda, personal communication). The testes of mammals have an aromatase activity (28) and small amount of estrogen is secreted from the testes by conversion from androgen (29). Thus, in the present studies, ipriflavone administered orally, in addition to directly suppressing
bone r e s o r p t i o n , a c c e l e r a t e d c a l c i t o n i n s e c r e t i o n i n the p r e s e n c e of small amount of e s t r o g e n i n male r a t s , which, i n t u r n , s u p r e s s e d bone r e s o r p t i o n induced by p r e d n i s o l o n e . I n c i d e n t a l l y , i p r i f l a v o n e has n e i t h e r an a n d r o g e n i c n o r an androgen p o t e n t i a t i n g a c t i v i t y ( u n p u b l i s h e d ) , a l t h o u g h androgen a l s o s u p r e s s e s bone r e s o r p t i o n (30). Acknowledgements We are grateful to Mrs. Masako Suzuki, Mrs. Hitomi Hamajo, Mr. Tadashi Miwa, and Mr. Haruhiko Yamaguchi for technical assistance; to Mr. Yuzo Hayakawa for advice on measuring the breaking properties of the bone; and to Drs. Shintaro Kikuchi, Hisashi Iwatsuka and J. R. Miller for comments on the manuscript. References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12.
13. 14. 15. 16. 17.
18.
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Effect of Ipriflavone
on Osteoporosis
Vol. 38, No. i0, 1986
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