0013-7227/91/1291-0092$03.00 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 1 Printed in U.S.A.
Salmon Calcitonin Prevents Cyclosporin-A-Induced High Turnover Bone Loss* B. STEINf, M. TAKIZAWA, I. KATZ, I. JOFFE, J. BERLIN, M. FALLON$, AND S. EPSTEIN Albert Einstein Medical Center, University of Pennsylvania (J.B.), and Jefferson Medical College (M.F.), Philadelphia, Pennsylvania 19141
ABSTRACT. Cyclosporin-A (CsA) has greatly influenced the outcome of organ transplantation and has also been effective in the treatment of many autoimmune diseases. Unfortunately, it has deleterious effects on bone remodelling, causing a high turnover bone loss, with bone resorption exceeding bone formation. Salmon calcitonin (SCtn) has been shown to inhibit bone resorption in high turnover states such as Paget's disease and postmenopausal osteoporosis. In an attempt to attenuate the high turnover bone remodelling caused by CsA alone, we studied the bone mineral effects of CsA in combination with SCtn in male Sprague-Dawley rats. Group A (n = 20) received vehicle as control, group B (n = 20) received CsA (15 mg/kg BW) by daily gavage and SCtn vehicle sc, group C (n = 20) received SCtn (1.3 IU/kg BW) daily sc and CsA vehicle, and
group D (n = 20) received a combination of CsA and Ctn daily, as described above. Rats were bled weekly for determination of circulating biochemical bone parameters. Eight rats from each group were killed on day 14 (short term), and the remaining rats were killed on day 28 (long term). Tibiae were removed for bone histomorphometry after death, which revealed a reduction of trabecular bone volume and an increase in osteoclast number induced by CsA alone. These changes were significantly attenuated by the combination of CsA and SCtn to resemble the histomorphometry of the control group. The inhibition of osteoclast number by SCtn is the most plausible mechanism by which the combination therapy attenuates the high turnover bone loss induced by CsA alone. (Endocrinology 129: 92-98,1991)
C
YCLOSPORIN-A (CsA) is a novel cyclic endecapeptide which selectively inhibits T-helper lymphocyte proliferation in response to alloantigen stimulation. This contributes to the success of this immune suppression in transplantation therapy and autoimmune diseases (1). In spite of its immunological specificity, it also has a variety of nonimmunological toxic side-effects, such as nephrotoxicity, hypertension, and hepatotoxicity (1). We have previously reported that the administration of CsA to male Sprague-Dawley rats in vivo affects bone mineral metabolism, causing severe osteopenia associated with a high bone-remodelling state (2). This deleterious and serious side-effect has already been reported in renal and cardiac transplant patients treated with CsA as the principal immunosuppressant agent (3-5). Calcitonin (Ctn) has been used for the treatment of
high bone-remodelling states such as Paget's disease and postmenopausal osteoporosis. Both synthetic animal and human Ctn are extensively administered to patients with Paget's disease of bone, where it improves the clinical manifestations as well as the biochemical and histological abnormalities (6-9). More recently, synthetic salmon Ctn (SCtn) has been reported to be effective in the treatment of postmenopausal osteoporosis in both short and long term studies (10-14). Recently, Mazzuoli et al. (15) reported that administration of SCtn to ovariectomized women prevents estrogen deficiency-induced bone loss, inhibiting skeletal resorption and allowing osteogenesis to occur normally. This has been confirmed in vitro, where Ctn has been shown to inhibit osteoclast function and bone resorption (16, 17). We, therefore, hypothesized that SCtn (in doses equivalent to those given in clinical situations) could favorably modify the effect of CsA-induced high turnover bone loss and, thus, have important clinical benefit for the prevention of posttransplantation bone disease.
Received December 7, 1990. Address requests for reprints to: Sol Epstein, M.D., Albert Einstein Medical Center, York And Tabor Roads, Philadelphia, Pennsylvania 19141. * This work was supported by Sandoz, Inc. (East Hanover, NJ), and Rhone-Polenc-Rorer Pharmaceutical Corp. (Fort Washington, PA). t Presented with the Mercke Sharpe and Dohme Young Investigator Award for the abstract of this work at the American Society of Bone and Mineral Research, Atlanta, GA, August 1990. $ Deceased.
Materials and Methods Animals Eighty 10-week-old male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 320-350 g, 92
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SCtn AND CsA
93 BONE FORMATION
BONE MASS
20
dM
"D Q)
Q>
—
C
15
5
CO*! O 0)
v. 2 10
is •
»J» 10 — o
t^
0 (d)
(o)
0)
I
1.0
"o 0.8
DCM
8>
Z E E O "5 CO
O (b)
i
BONE FORMATION
0)
1 < a, So CD Q :
0.8 0.4 0.2
c =
0.0
(e)
Control
E
CsA
5K
SCtn
CO
O (c) TABLE 1. Bone histomorphometry on day 14
BV/TV OV OC TLC MAR
II:
BONE FORMATION
BONE RESORPTION
FIG. 1. Bone histomorphometry in the control, CsA, SCtn, and combination CsA and SCtn groups in male SpragueDawley rats, a, Percent trabecular bone volume; b, osteoclast number; c, osteoid volume; d, percent tetracycline labeled surface; e, mineral appositional rate after the tibiae were removed after double tetracycline labeling. *, P < 0.05. All values are the mean ± SEM.
h
Group A
Group B
Group C
Group D
18.6 ± 0.4 2.5 ± 0.2 1.2 ± 0.1 9.0 ± 0.05 0.65 ± 0.03
10.2 ± 1.2° 2.9 ± 0.2° 2.8 ± 0.3° 15.1 ± 0.5° 0.83 ± 0.02"
18.4 ± 2.4 1.3 ±0.1 1.7 ± 0.2 8.9 ± 0.6 0.59 ± 0.2
16.9 ± 0.6 1.9 ± 0.1 1.5 ± 0.2 9.2 ± 0.37 0.57 ± 0.4
Group A, Control; group B, CsA; group C, SCtn; group D, CsA plus SCtn. All values are the mean ± SEM. BV/TV, Percent bone volume; OV, percent osteoid volume; OC, osteoclast count (number per mm2); TLS, percent tetracycline-labeled surface; MAR, mineral appositional rate (microns per day). " P < 0.05 vs. other groups.
i
CsA + SCtn
were used. All rats were housed under similar conditions and maintained on a diet of rat chow (calcium, 2.09%; phosphorous, 0.89%; vitamin D, 5000 IU/kg diet) obtained from Teklad (Madison, WI) and provided water ad libitum. CsA CsA, in a solution containing 100 mg CsA/ml and 10% alcohol by volume, was kindly provided by Sandoz, Inc. (East Hanover, NJ). The CsA solution was appropriately diluted in an alcohol-olive oil vehicle to obtain a concentration of 1.5 mg/ 100 pi SCtn SCtn (Calcimar) was kindly provided by Rhone-PolencRorer Pharmaceutical Corp. (Fort Washington, PA). The SCtn
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94
SCtn AND CsA
Endo-iogi Vol 129 • No 1
FIG. 2. Photomicrographs of proximal rat tibia-metaphyseal bone; trichromestained undecalcified 7-/im thick histological sections (original magnification, X31). a, Control; b, CsA; c, SCtn; d, CsA and SCtn. In the control rats, the proximal tibial medullary space (M) contains long ribbons of mineralized trabecular bone (arrowheads). In the CsA-treated rats, the bone volume is reduced, resulting in small trabecular bone islands (arrowheads). In the SCtn rats, the mineralized trabecular bone is similar to that in controls (arrowheads), and in the rats treated with the combination of CsA and SCtn, the mineralized trabecular bone (arrowheads) is preserved and similar to that in controls.
was appropriately diluted in normal saline to obtain a concentration of 0.13 IU/100 /d. Experimental protocol The rats were randomly divided into four equal groups and administered different agents daily as follows: group A (n = 20) received olive oil-based vehicle of CsA by gavage and the SCtn
saline diluent sc in equivalent volume per kg BW, group B (n = 2 °) received CsA (15 mg/kg BW) by gavage and SCtn vehicle sc, group C (n = 20) received SCtn (1.3 IU/kg BW) sc and CsA vehicle by gavage, and group D (n = 20) received a combination of CsA and SCtn daily, administered as described above. Eight r a ts from each group were killed on day 14 (short term), and the remaining rats were killed on day 28 (long term), All rats received tetracycline hydrochloride (Achromycin,
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SCtn AND CsA 210 180
• • — A O
• • A O
Control CsA SCtn CsA + SCtn
150
O -
120
Vol. 129, No. 1 Printed in U.S.A.
Salmon Calcitonin Prevents Cyclosporin-A-Induced High Turnover Bone Loss* B. STEINf, M. TAKIZAWA, I. KATZ, I. JOFFE, J. BERLIN, M. FALLON$, AND S. EPSTEIN Albert Einstein Medical Center, University of Pennsylvania (J.B.), and Jefferson Medical College (M.F.), Philadelphia, Pennsylvania 19141
ABSTRACT. Cyclosporin-A (CsA) has greatly influenced the outcome of organ transplantation and has also been effective in the treatment of many autoimmune diseases. Unfortunately, it has deleterious effects on bone remodelling, causing a high turnover bone loss, with bone resorption exceeding bone formation. Salmon calcitonin (SCtn) has been shown to inhibit bone resorption in high turnover states such as Paget's disease and postmenopausal osteoporosis. In an attempt to attenuate the high turnover bone remodelling caused by CsA alone, we studied the bone mineral effects of CsA in combination with SCtn in male Sprague-Dawley rats. Group A (n = 20) received vehicle as control, group B (n = 20) received CsA (15 mg/kg BW) by daily gavage and SCtn vehicle sc, group C (n = 20) received SCtn (1.3 IU/kg BW) daily sc and CsA vehicle, and
group D (n = 20) received a combination of CsA and Ctn daily, as described above. Rats were bled weekly for determination of circulating biochemical bone parameters. Eight rats from each group were killed on day 14 (short term), and the remaining rats were killed on day 28 (long term). Tibiae were removed for bone histomorphometry after death, which revealed a reduction of trabecular bone volume and an increase in osteoclast number induced by CsA alone. These changes were significantly attenuated by the combination of CsA and SCtn to resemble the histomorphometry of the control group. The inhibition of osteoclast number by SCtn is the most plausible mechanism by which the combination therapy attenuates the high turnover bone loss induced by CsA alone. (Endocrinology 129: 92-98,1991)
C
YCLOSPORIN-A (CsA) is a novel cyclic endecapeptide which selectively inhibits T-helper lymphocyte proliferation in response to alloantigen stimulation. This contributes to the success of this immune suppression in transplantation therapy and autoimmune diseases (1). In spite of its immunological specificity, it also has a variety of nonimmunological toxic side-effects, such as nephrotoxicity, hypertension, and hepatotoxicity (1). We have previously reported that the administration of CsA to male Sprague-Dawley rats in vivo affects bone mineral metabolism, causing severe osteopenia associated with a high bone-remodelling state (2). This deleterious and serious side-effect has already been reported in renal and cardiac transplant patients treated with CsA as the principal immunosuppressant agent (3-5). Calcitonin (Ctn) has been used for the treatment of
high bone-remodelling states such as Paget's disease and postmenopausal osteoporosis. Both synthetic animal and human Ctn are extensively administered to patients with Paget's disease of bone, where it improves the clinical manifestations as well as the biochemical and histological abnormalities (6-9). More recently, synthetic salmon Ctn (SCtn) has been reported to be effective in the treatment of postmenopausal osteoporosis in both short and long term studies (10-14). Recently, Mazzuoli et al. (15) reported that administration of SCtn to ovariectomized women prevents estrogen deficiency-induced bone loss, inhibiting skeletal resorption and allowing osteogenesis to occur normally. This has been confirmed in vitro, where Ctn has been shown to inhibit osteoclast function and bone resorption (16, 17). We, therefore, hypothesized that SCtn (in doses equivalent to those given in clinical situations) could favorably modify the effect of CsA-induced high turnover bone loss and, thus, have important clinical benefit for the prevention of posttransplantation bone disease.
Received December 7, 1990. Address requests for reprints to: Sol Epstein, M.D., Albert Einstein Medical Center, York And Tabor Roads, Philadelphia, Pennsylvania 19141. * This work was supported by Sandoz, Inc. (East Hanover, NJ), and Rhone-Polenc-Rorer Pharmaceutical Corp. (Fort Washington, PA). t Presented with the Mercke Sharpe and Dohme Young Investigator Award for the abstract of this work at the American Society of Bone and Mineral Research, Atlanta, GA, August 1990. $ Deceased.
Materials and Methods Animals Eighty 10-week-old male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 320-350 g, 92
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 June 2015. at 13:53 For personal use only. No other uses without permission. . All rights reserved.
SCtn AND CsA
93 BONE FORMATION
BONE MASS
20
dM
"D Q)
Q>
—
C
15
5
CO*! O 0)
v. 2 10
is •
»J» 10 — o
t^
0 (d)
(o)
0)
I
1.0
"o 0.8
DCM
8>
Z E E O "5 CO
O (b)
i
BONE FORMATION
0)
1 < a, So CD Q :
0.8 0.4 0.2
c =
0.0
(e)
Control
E
CsA
5K
SCtn
CO
O (c) TABLE 1. Bone histomorphometry on day 14
BV/TV OV OC TLC MAR
II:
BONE FORMATION
BONE RESORPTION
FIG. 1. Bone histomorphometry in the control, CsA, SCtn, and combination CsA and SCtn groups in male SpragueDawley rats, a, Percent trabecular bone volume; b, osteoclast number; c, osteoid volume; d, percent tetracycline labeled surface; e, mineral appositional rate after the tibiae were removed after double tetracycline labeling. *, P < 0.05. All values are the mean ± SEM.
h
Group A
Group B
Group C
Group D
18.6 ± 0.4 2.5 ± 0.2 1.2 ± 0.1 9.0 ± 0.05 0.65 ± 0.03
10.2 ± 1.2° 2.9 ± 0.2° 2.8 ± 0.3° 15.1 ± 0.5° 0.83 ± 0.02"
18.4 ± 2.4 1.3 ±0.1 1.7 ± 0.2 8.9 ± 0.6 0.59 ± 0.2
16.9 ± 0.6 1.9 ± 0.1 1.5 ± 0.2 9.2 ± 0.37 0.57 ± 0.4
Group A, Control; group B, CsA; group C, SCtn; group D, CsA plus SCtn. All values are the mean ± SEM. BV/TV, Percent bone volume; OV, percent osteoid volume; OC, osteoclast count (number per mm2); TLS, percent tetracycline-labeled surface; MAR, mineral appositional rate (microns per day). " P < 0.05 vs. other groups.
i
CsA + SCtn
were used. All rats were housed under similar conditions and maintained on a diet of rat chow (calcium, 2.09%; phosphorous, 0.89%; vitamin D, 5000 IU/kg diet) obtained from Teklad (Madison, WI) and provided water ad libitum. CsA CsA, in a solution containing 100 mg CsA/ml and 10% alcohol by volume, was kindly provided by Sandoz, Inc. (East Hanover, NJ). The CsA solution was appropriately diluted in an alcohol-olive oil vehicle to obtain a concentration of 1.5 mg/ 100 pi SCtn SCtn (Calcimar) was kindly provided by Rhone-PolencRorer Pharmaceutical Corp. (Fort Washington, PA). The SCtn
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 June 2015. at 13:53 For personal use only. No other uses without permission. . All rights reserved.
94
SCtn AND CsA
Endo-iogi Vol 129 • No 1
FIG. 2. Photomicrographs of proximal rat tibia-metaphyseal bone; trichromestained undecalcified 7-/im thick histological sections (original magnification, X31). a, Control; b, CsA; c, SCtn; d, CsA and SCtn. In the control rats, the proximal tibial medullary space (M) contains long ribbons of mineralized trabecular bone (arrowheads). In the CsA-treated rats, the bone volume is reduced, resulting in small trabecular bone islands (arrowheads). In the SCtn rats, the mineralized trabecular bone is similar to that in controls (arrowheads), and in the rats treated with the combination of CsA and SCtn, the mineralized trabecular bone (arrowheads) is preserved and similar to that in controls.
was appropriately diluted in normal saline to obtain a concentration of 0.13 IU/100 /d. Experimental protocol The rats were randomly divided into four equal groups and administered different agents daily as follows: group A (n = 20) received olive oil-based vehicle of CsA by gavage and the SCtn
saline diluent sc in equivalent volume per kg BW, group B (n = 2 °) received CsA (15 mg/kg BW) by gavage and SCtn vehicle sc, group C (n = 20) received SCtn (1.3 IU/kg BW) sc and CsA vehicle by gavage, and group D (n = 20) received a combination of CsA and SCtn daily, administered as described above. Eight r a ts from each group were killed on day 14 (short term), and the remaining rats were killed on day 28 (long term), All rats received tetracycline hydrochloride (Achromycin,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 June 2015. at 13:53 For personal use only. No other uses without permission. . All rights reserved.
SCtn AND CsA 210 180
• • — A O
• • A O
Control CsA SCtn CsA + SCtn
150
O -
120
