The anabolic effect of human PTH (1–34) on bone formation is blunted when bone resorption is inhibited by the bisphosphonate tiludronate—is activated resorption a prerequisite for the in vivo effect of PTH on formation in a remodeling system?

Bone Vol. 16, No. 6 June 1995:603-610 ELSEVIER

The Anabolic Effect of Human PTH (1-34) on Bone Formation Is Blunted when Bone Resorption Is Inhibited by the Bisphosphonate Tiludronate Is Activated Resorption a Prerequisite for the In Vivo Effect of PTH on Formation in a Remodeling System? P. D. D E L M A S , l P. V E R G N A U D , 1 M. E. A R L O T , 1 P. P A S T O U R E A U , 1 P. J. M E U N I E R , 1 and M. H. L. N I L S S E N 2 I INSERM Unit 403, HOpital Ed. Herriot and Facult~ A. Carrel, Lyon, France 2 Karo Bio, Huddinge, Sweden

antiresorptive agents may not be effective and should be tested in an appropriate animal model before clinical trials in osteoporotic patients are undertaken. (Bone 16:603-610;

Parathyroid hormone (PTH) and its (1-34) fragment are stimulators of bone turnover that have an anabolic effect increasing trabecular bone mass when administered intermittently by daily subcutaneous injections. Its clinical use in osteoporosis, however, has been limited by the concomitant increased bone resorption and deleterious effect on cortical bone. To evaluate if a treatment combining PTH and a potent inhibitor of bone resorption would retain the anabolic effect of PTH without increasing bone resorption, we analyzed the effects of PTH (1-34) (500 IU/d) with or without the bisphosphonate tiludronate (1 mg/kg per day) for 3 months on biochemical and histological indices of bone turnover in old female sheep, an animal model which has a slow bone remodeling activity that resembles the one of elderly women. As expected, PTH (1-34) induced a significant increase of urinary pyridinoline and hydroxyproline (reflecting bone resorption), and of serum osteocalcin and alkaline phosphatase (reflecting bone formation), that were consistent with an increase of resorption and tetracycline-based formation of bone measured on iliac crest biopsy. In contrast, all biochemical and histological indices of bone turnover were decreased in sheep receiving tiludronate, a potent inhibitor of bone resorption. Surprisingly, in the combined therapy group, biochemical and histological indices of both resorption and formation did not differ from the control groups. Thus, the model of old sheep, which closely resembles the situation in old human, shows that the anabolic effect of PTH on bone is not maintained when PTH is coadministered with a bisphosphonate, in marked contrast to results noted in the growing rat. Because bisphosphonates are selective inhibitors of osteoclastic bone resorption that do not directly affect osteoblastic bone formation in vivo, these data suggest that the activation of bone resorption may be a prerequisite for the anabolic effect of PTH. Although tiludronate was the only bisphosphonate tested, our data also suggest that a combined PTlt-bisphosphonate therapy is not a valid strategy for osteoporotic patients. Combination regimens of anabolic and

1995)

Key Words: PTH; Bone resorption; Bone formation; Bisphosphonate; Osteoporosis.

Introduction Parathyroid hormone (PTH) is a major regulator of the rate of bone turnover. Patients with hypoparathyroidism have decreased bone turnover, whereas patients with primary hyperparathyroidism have an increase of bone turnover, as documented by biochemical markers of bone turnover and histomorphometric analysis of the iliac crest trabecular bone. 9'12'31 The chronic hypersecretion of PTH is responsible for an increased bone resorption, that is, of the osteoclast number and of eroded surfaces, and for an increased bone formation documented by extended tetracycline-labeled surfaces and by an increase of the wall width, reflecting an enhanced osteoblastic activity at the cell and at the tissue level. 12'31 In the rat, it has been shown that human PTH, given intermittently, stimulates bone growth, osteoprogenitor cells, and bone formation. 28 The increase of bone cell recruitment (osteoblasts and osteoclasts) and activity has also been demonstrated in vitro in organ culture. 22 The mechanism of action of PTH on bone, however, is not yet completely understood. Like for most hormones active on bone, specific receptors have been found on osteoblasts rather than osteoclasts. 4° Exposure of bone organ cultures to PTH causes the release of growth factor(s) into the medium. PTH augments the IGF-1 mRNA and protein production by fetal rat osteoblasts and anti-IGF-1 antibodies block the PTH-induced stimulation of collagen I synthesis by these cells. 3 In addition, PTH increases transforming growth factor-beta (TGF-[3) mRNA level by osteoblastlike cells. 4 Thus, the anabolic effect of PTH on bone is likely to be mediated by autocrine/paracrine growth factors such as insulinlike growth factor-I (IGF-1) and perhaps TGF-13, but the precise in vivo sequence of events is unknown. Because treatment of rats and greyhound dogs with either PTH or its 1-34 fragment, injected at low dose intermittently but

Address for correspondence and reprints: Dr. Pierre D. Delmas, H6pital

Ed. Herriot, Pavillon F, 69437 Lyon Cedex 03, France. © 1995 by ElsevierScienceInc.

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8756-3282/95/$9.50 8756-3282(95)00113-R

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P.D. Delmas et al. The anabolic effect of PTH is dependent on activation of resorption

not continuously, induced a marked increase of bone formation with neither hypercalcemia nor marked increased resorption, 14'16'20'26'33'38'42'43 PTH (1-34) has been suggested as a potential treatment of osteoporosis. Shen et al. 38 demonstrated in ovariectomized rats that a daily treatment with rat PTH (1-34) improves cancellous bone volume and bone mineral density when compared with the ovariectomized + vehicle group. This increase was due to an increased formation at the tissue level without further increase in osteoclast surfaces. Clinical trials with 400 to 500 IU of human FFH (1-34) given by daily subcutaneous (SC) injection over 6 to 12 months induces a marked increase of trabecular bone mass, documented by histomorphometry and quantitative computed tomography, of bone formation documented by bone histology, and by the accretion of 47Ca, but no change in total body retention of calcium, suggesting that the increase in trabecular bone mass occurs at the expense of a decrease of cortical bone. 2'15"18"19"3a'35"41 Because the absence of increase in the net calcium balance is likely to be due to increased bone resorption, the coadministration of an inhibitor of resorption might be beneficial, provided that the anabolic effect of PTH can be still obtained. Hock et al. 16 have shown that, in the rat, pretreatment with clodronate, a bisphosphonate that inhibits bone resorption, does not abolish the anabolic effect of PTH on bone formation, suggesting that stimulation of resorption is not a prerequisite for PTH-induced increased formation. In the present study, we have analyzed the biochemical and histological effect of PTH (1-34) with or without tiludronate, another bisphosphonate that blocks resorption 37 administered in the aged sheep, an animal that has a slow bone remodeling activity,5 with two specific aims: (1) to determine if activation of resorption is a prerequisite for the PTH-induced stimulation of formation, thus clarifying the in vivo mechanism of action of PTH in humans; and (2) to determine if the simultaneous administration of bisphosphonates and PTH (1-34) could be promising for the treatment of osteoporosis. Materials and Methods Animals and Treatment Twenty-eight crossbred Romanov-Limousin elderly female ewes (7.7 +- 0.9 years old) were studied at the National Institute of Agronomical Research (INRA, Theix, France). All were born and bred at INRA. They were divided into four groups of 7 animals that were matched by their weight (71.8 -+ 6.0 kg). One month before the beginning of the treatment, the animals were kept at a constant photoperiod to reduce the effect of the variations of the photoperiod on bone metabolism. Each group was randomly allocated to one of the four treatment groups: a control group (CT) was injected daily with a saline solution (0.9% NaC1 solution); one group (PTH group) received a daily injection of 500 IU of hPTH (1-34) (Karo Bio, Sweden); one group was treated with 1 mg/kg per day of the bisphosphonate tiludronate; and the last group (FFH + tiludronate group) was treated with a combination of PTH and tiludronate. All the compounds were injected SC during 3 months. Animals were fed ad libitum and the normal calcium intake was of 10 g per day. Blood Biochemical Measurements Blood samples were collected at days 0, 30, 60, and 90. Serum aliquots were stored at - 20°C until assayed. Serum osteocalcin, calcium, creatinine, phosphate, and alkaline phosphatase were

Bone Vol. 16, No. 6 June 1995:603-610 measured before and after l, 2, and 3 months of treatment. Serum calcium phosphate, and creatinine were measured by the standard colorimetric method. Serum osteocalcin was measured using a specific radioimmunoassay system for ovine osteocalcin that has been previously published. 32 This liquid phase radioimmunoassay uses a rabbit polyclonal antiserum at a final dilution of 1:40,000 against ovine osteocalcin purified from cortical sheep bone as a standard and as a 125I-labeled tracer. The sensitivity of the assay is 0.15 ng of osteocalcin and the intra- and interassay CV are 6% and 7%, respectively. The normal range for elderly sheep is 5.9 -+ 0.7 ng/mL. 32 Urine Biochemical Measurements Spot urinary samples were taken every month for calcium, hydroxyproline, creatinine, and pyridinoline assays. Urinary calcium and creatinine were measured by standard colorimetric techniques. Hydroxyproline was determined using a specific colorimetric assay on hydrolyzed urine samples 23 and corrected for creatinine excretion. Urinary excretion of pyridinium derivatives, hydroxylysylpyridinoline (Pyr) and lysylpyridinoline (D-Pyr), were measured according to a previously published method. 44 Briefly, the crosslinks are extracted from the hydrolyzed urine sample by cellulose chromatography, separated by reversed-phase HPLC, and identified by spectrofluorimetry. The area of the fluorescent peak was quantified by comparison with calibrated Pyr and D-Pyr external standards purified from human cortical bone. The intra- and interassay variations were less than 10% for Pyr and less than 15% for D-Pyr. 44 All measurements were performed in a blind fashion and the code was broken after completion of the study. Bone Histomorphometry The histomorphometric study was performed on transiliac bone biopsies taken prior to initiating the treatment (day 0) on the right side and at slaughter (day 90) on the left side. Before the first biopsy and before killing all animals received a double labeling with oxychlortetracycline (20 mg/kg body weight per day; 2 days on-12 days off-14 days on). The biopsies were processed undecalcified according to previously published m e t h o d s Y Five pairs of sections, 7 p,m thick, were prepared, the first one stained with Solochrome Cyanin R and the second unstained, both being consecutive. Two paired sections were separated by at least 40 ~xm. Five additional nonconsecutive sections were prepared for staining with Goldner's process. The measurements were performed in cortical and in cancellous bone according to previously published methods. 13"24"27 The nomenclature used was the one recommended by the ASBMR Histomorphometry Nomenclature Committee 3° with the results expressed in three dimensions. Values were not corrected for obliquity. The following parameters were measured or calculated. Structural parameters. Bone volume~tissue volume (BV/TV; %): percentage of a given bone area occupied by trabeculae, excluding the medullary space. It included both calcified and noncalcified osteoid tissue. Wall thickness (W.Th; Ixm): thickness of a complete cancellous bone packet. Cortical porosity (Ct.Po; %): mean of the porosity of the two cortices. Formation parameters. Osteoid volume~bone volume (OV/ BV; %): percentage of cancellous bone which is not calcified. Osteoid surface~bone surface (OS/B S; %): percentage of the total cancellous surface covered with osteoid. Osteoid thickness

Bone Vol. 16, No. 6 June 1995:603-610 (O.Th; ixm): average thickness of osteoid seams. Mineral apposition rate (MAR; i~rn/day): rate of progression of the mineralization front labeled twice by tetracycline. Double-labeled surface~bone surface (dLS/BS; %): extent of double-tetracyclinelabeled surfaces. Bone formation rate~bone surface (BFR/BS; txm3/Ixm2/day): rate of formation of mineralized bone per day [ d L S + (single LS/2) x MAR]. Adjusted apposition rate (Aj.AR; Ixm/day): average amount of mineralized bone made per day per unit of osteoid-covered surface BFR/OS. Mineralization lag time (Mlt; days): mean time between deposition and mineralization of any volume of osteoid averaged over the lifespan of osteoid seam (O.TtVAj.AR). Osteoid maturation time (days): mean time between the onset of deposition and the onset of mineralization at each forming site. Formation period (FP; days): time required to rebuild a new structural unit (W.Th/ Aj.AR). The active formation period [FP(a + ); days] represents the osteoblast lifespan (W.Wi/MAR). Activation frequency (Ac.f/year): probability that a new cycle of remodeling will be initiated at any point on the surface per year (BFR/BS)/W.Th).

Resorption parameters. Eroded surface~bone surface (ES/ BS; %): percentage of cancellous surface crenated by osteoclastic resorption whether active or inactive but without osteoblastic activity. Osteoclast surface~bone surface (Oc.S/BS; %): the percentage of bone surface covered with osteoclasts. Osteoclast number~bone surface (Oc.N/mm) is based on morphological identification. The histomorphometric parameters were measured with an automatic analyzer (Quantimet 520, Leica R) for bone volume and cortical porosity and with a semiautomatic image analyzer (IBAS 1, Kontron R) for the other parameters on three to five slides according to the interslide variance. 8 The measurements were performed on Goldner-stained sections for resorption parameters, on unstained sections for tetracycline-derived parameters, and on Solochrome Cyanin R-stained sections for the other parameters. The magnification used was x 80 except for osteoid volume and thickness ( x 125), for wall thickness and osteoclast number ( x 2 0 0 ) , and for mineral apposition rate (x320). The measurements were performed at randomly selected sites for the estimation of thickness, z4

P.D. Delmas et al. The anabolic effect of PTH is dependent on activation of resorption

605

4% change from controls 150 100 50 0 -50 30

60

90

DAYS

A% change from controls

!011 ~Hydr~ nel 30 PTH

60 I

90

DAYS

Tiludronate

Figure 1. Time-course change of urinary pyridinoline and hydroxyproline excretion in the treatment groups compared to controls. *p < 0.02 to 0.001 vs. controls. tion (Figure 1). The changes in urinary hydroxyproline, a less specific marker of bone resorption, showed a similar pattern, although the decrease in both tiludronate-treated groups was not significant at all time points (Figure 1). There was a two- to 4% change from controls 400.

I Osteocalcln

[

300.

100-

200.

Statistical Analysis The results are expressed as the mean --- one standard deviation (SD). According to the distribution of the variables and the homogeneity of variances, the following tests were used: either one-way analysis of variance or the Kruskal-Wallis analysis of variance for the comparison of the four groups; and either Student's t-test or the Mann-Whitney test for comparisons between two groups. 8 Because of the number of tests performed, the significance level was selected at p ~< 0.02.

Results Biochemical Parameters Age, weight, serum calcium, phosphate, and creatinine did not differ between groups at baseline and did not change significantly in either group during treatment at 30, 60, and 90 days, as assessed by two-factor analysis of variance, testing for both the effect of time and of treatment. PTH induced a significant twofold increase of urinary pyridinoline, a specific marker of bone resorption, while tiludronate, with or without PTH, significantly inhibited pyridinoline excre-

0 ~..L

-100

* 3O

60

90

DAYS

A% chan( e from controls

65-85- T

Phosphatase 1

45 25 -

s: -35 -"

T * 30 PTH

T

60 ~Tiludronate

90 /

DAYS

PTH + Tiludronate

Figure 2. Time-course change of serum osteocalcin and alkaline phosphatase in the treatment groups compared to controls. *p < 0.02 to 0.001 vs. controls.

606

P . D . Delmas et al. The anabolic effect of PTH is dependent on activation of resorption Osteoelast perimeter/BS

Osteoclast number/BS p < 0.02

/mm 0.2

% 0.50 -

0.1

0

0 -0.1

- 0.50

0.2

-1

m

p < 0.02 t p < 0.003 '

Controls

~

PTH

mTiludronate

[~]

PTH + Tiludronate

Figure 3. Changes with time of the osteoclast number (left panel) and osteoclast perimeter (right panel) in the four groups. Data are expressed as the mean of the absolute changes between the second (day 90) and the first (day 0) biopsies.

Bone Vol. 16, No. 6 June 1995:603~610 T h e static ( T a b l e 1) and d y n a m i c ( T a b l e 2) p a r a m e t e r s o f bone formation s h o w e d that P T H alone h a d a m a r k e d and significant anabolic effect: for e x a m p l e , a s e v e n f o l d e x t e n s i o n o f the tetracycline-labeled f o r m i n g surfaces in the cancellous bone o f the iliac crest. In contrast, b o n e f o r m a t i o n at the cellular level (adjusted apposition rate), and at the tissue level (bone f o r m a t i o n rate), was significantly inhibited by tiludronate. In the P T H + tiludronate group there w a s either no c h a n g e or a significant decrease o f bone formation (according to the histological p a r a m eter) w h e n c o m p a r e d to the controls ( F i g u r e 4). A s a conseq u e n c e o f these histological c h a n g e s , the activation f r e q u e n c y was m a r k e d l y increased (p < 0.005) in the P T H group and decreased (p < 0.02) in both the tiludronate a n d P T H + tiludronate groups ( F i g u r e 5).

Discussion fourfold increase o f s e r u m osteocalcin, a specific m a r k e r o f bone formation, in the P T H - t r e a t e d g r o u p t h r o u g h o u t the study, in contrast with no c h a n g e or e v e n a significant decrease in the tiludronate g r o u p s with or without P T H . S e r u m alkaline phosphatase, a less specific m a r k e r o f bone formation, s h o w e d similar c h a n g e s , a l t h o u g h o f lesser m a g n i t u d e ( F i g u r e 2).

Histological Parameters P T H i n d u c e d an increase in both parameters of bone resorption (osteoclast n u m b e r and perimeter), a l t h o u g h the increase did not r e a c h statistical s i g n i f i c a n c e w h e n c o m p a r e d to the control group. H o w e v e r , both p a r a m e t e r s were significantly decreased by tiludronate and by tiludronate + P T H treatment w h e n c o m pared to the P T H g r o u p ( F i g u r e 3).

W e f o u n d that P T H (1-34) i n d u c e d a m a r k e d increase in biochemical and histological indices o f bone turnover (formation and resorption) and tiludronate decreased bone turnover a s s e s s e d by the s a m e parameters. U n e x p e c t e d l y , the c o a d m i n i s t r a t i o n o f PTH (1-34) and tiludronate did not result in significant c h a n g e s o f bone turnover w h e n c o m p a r e d to the controls. T h u s , the anabolic effect of P T H (1-34) on bone f o r m a t i o n w a s c o m p l e t e l y abolished in the c o m b i n a t i o n therapy group. T h e s e findings have implications for the m e c h a n i s m o f P T H action and for possible c o m b i n a t i o n therapy in osteoporosis. T h e s e results in the s h e e p contrast with those obtained by H o c k et al. 16 in the rat. T h e dose o f h P T H (1-34) was equivalent in both studies, inducing a m a r k e d two- to threefold increase in bone formation. A l t h o u g h two b i s p h o s p h o n a t e s were u s e d , both have been s h o w n in vitro and in vivo to inhibit osteoclastic bone resorption. In p o s t m e n o p a u s a l w o m e n , tiludronate i n c r e a s e s

Table 1. Static histomorphometric parameters performed on iliac crest biopsies before (day 0) and after treatment (day 90) in the four treatment groups of ewes Parameter Bone volume/TV (%) Cortical porosity (%) Osteoid surface/BS (%) Osteoid volume/BV (%) Osteoid thickness (txm) Wall thickness (~m) Eroded surface/BS (%) Osteoclast surface/BS (%) Number of osteoclasts/BS (~/mm)

Day 0 90 0 90 0 90 0 90 0 90 0 90 0 90 0 90 0 90

Control 15.8 15.7 8.4 11.8 4.2 4.7 0.2 0.3 5.5 6.0 36.7 36.6 4.2 6.0 0.19 0.32 0.05 0.08

± 4.6 ± 1.5 ± 4.5 ± 5.6 ± 3.4 ÷ 3.5 -+ 0.2 ± 0.2 ± 0.6 ± 0.9 -+ 2.7 ± 2.7 ± 3.8 ± 2.6 ± 0.20 ± 0.29 ± 0.7 ± 0.7

PTH 15.4 16.6 12.5 15.6 6.4 30.7 0.3 3.0 4.9 8.3 34.8 39.1 2.2 10.0 0.30 0.82 0.05 0.17

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± -+ ±

2.2 5.8 6.6 6.6 6.9 20.3 c 0.4 2.6 a 0.8 2.8 2.6 3.4 1.9 5.9 0.37 0.93 0.08 0.18

Tiludronate 15.7 16.6 11.0 12.1 6.3 8.8 0.3 0.5 4.9 6.0 37.8 38.0 6.5 2.2 0.86 0.04 0.16 0.01

± ± -+ ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.1 3.6 9.1 7.0 4.1 6.2 d 0.2 0.4 a 0.8 0.9 2.9 2.9 4.4 l . l a'e 1.02 0.06 b'a 0.16 0.01 c'a

PTH + tiludronate 13.9 18.9 11.1 12.3 4.5 6.0 0.2 0.4 4.7 5.8 36.2 38.6 8.7 4.8 0.65 0.07 0.17 0.02

± 1.9 ± 5.3 ± 8.0 ± 7.5 ± 3.3 ± 3.8 ~ ± 0.2 ± 0.3 ~ ± 0.8 ± 0.9 ± 1.8 ± 5.3 -+ 5.9 ± 1.1 ± 0.53 ± 0.06 a ± 0.15 ± 0.01 b

Values are mean ± 1 SD; p values derived by nonparametric ANOVA using the Mann-Whitney test. TV: tissue Volume; BS: bone surface; BV: bone volume. ap < 0.02 difference vs. control. bp < 0.01 difference vs. control. Cp < 0.005 difference vs. control. dp < 0.02 difference vs. PTH. ep < 0.01 difference vs. PTH.

Bone Vol. 16, No. 6 June 1995:603~510

P.D. Delmas et al. The anabolic effect of PTH is dependent on activation of resorption

607

Table 2. Dynamic histomorphometric parameters performed on iliac crest biopsies before (day 0) and after treatment (day 90) in the four treatment groups

Parameter Mineral apposition rate (p-m/d) Double-labeled surfaee/BS (%) Bone formation rate/BS (~m3/ixm2/day) Adjusted apposition rate (p,m/day) Total formation period (days) Active formation period (days) Activation frequency (/year) Mineralization lag time (days) Osteoid maturation time (days)

0 90 0 90 0 90 0 90 0 90 0 90 0 90 0 90 0 90

Control

PTH

Tiludronate

PTH + tiludronate

0.78 ± 0.10 0.92 ± 0.10 1.8 ± 1.8 3.7 -+ 3.1 0.023 - 0.018 0.042 ± 0.034 0.588 ± 0.263 1.372 - 1.724 76 ± 38 75 ± 95 47 ± 6 40 -+ 5 0.23 -+ 0.18 0.41 ± 0.33 11 ± 5 12 ± 13 7 ± 2 7± 1

0.80 ± 0.23 1.13 --- 0.21a 3.4 ± 4.6 27.5 ± 16.7c 0.041 ± 0.038 0.379 ± 0.274 c 1.419 ± 1.675 1.648 ± 1.841 72 ± 61 40 ± 21 47 ± 15 36 ± 8 0.42 --- 0.38 3.70 ± 2.82 c 10--- 8 8 +-- 4 7±2 7±2

0.77 -+ 0.10 0.66 ± 0.15 a'r 5.5 --- 5.7 0.2 ± 0.2 b'f 0.058 -+ 0.049 0.006 ± 0.006 a'f 0.942 "4- 0.820 0.092 -+ 0.112 c'f 127 +- 167 993 ± 1007c'f 50 ± 8 60 - 15 a'cl 0.54 - 0.45 0.06 +- 0.06 a'a 17 ± 25 149 -+ 143 c'a 6-+2 9-+1 b

0.84 ± 0.20 0.84 ± 0.20 4.1 ± 3.3 0.3 ~ 0.2 a'f 0.064 ± 0.049 0.008 ± 0.006 f 1.496 ± 0.947 0.162 +- 0.125 b'e 38 ± 35 420 -+ 337 b'e 4 6 - 15 48 ± 11 0.64 ± 0.48 0.07 ± 0.04 a'e 5 --- 4 68 ± 60 b'e 6±1 7±2

Values are mean ± 1 SD; p values derived by nonparametric ANOVA using the Mann-Whitney test. BS = bone surface. ap < 0.02 difference vs. control. bp < 0.01 difference vs. control. ~p < 0.005 difference vs. control. ap < 0.01 difference of PTH vs. PTH + tiludronate. ep < 0.005 difference PTH vs. tiludronate. fp < 0.005 difference PTH vs. PTH + tiludronate.

bone mineral density of the spine by decreasing bone turnover in a way that is similar to other bisphosphonates. 36'37 The different sequences of treatment administration are also unlikely to explain the differing results. Hock et al. 16 pretreated the rats with large doses (4.7 mg/kg) of clodronate for 4 days prior to PTH administration for 12 days instead of giving both treatments simultaneously, but bisphosphonates have a long skeletal retention time and a marked persistent biological effect. In addition, the lack of increased bone turnover in the hPTH (1-34) + tiludronate group was documented in our study by biochemical markers at as soon as 2 weeks (not shown). Although the tiludronate dose was quite high, and did not " f r e e z e " the remodeling activity, as shown by the moderate decrease (30% to 50%) of the specific bone biomarkers, osteocalcin and pyridinoline, and by the persistence of active bone formation (assessed by tetracycline labeling) on the bone biopsies after 3 months of treatment, without mineralization defect. Thus, the differences in the two sets of results would appear to be related to differences in the animals used. The young rat is actively growing, with a predominant modeling activity and a remodeling activity restricted to some anatomical sites. In the young rat, PTH stimulates bone formation but has a controversial effect on trabecular bone resorption. With low doses of hPTH (3 I.tg per 100 g body weight) formation and resorption were both increased. 28 With higher doses (8 txg per 100 g body weight) the eroded surfaces were not different in controls and in PTH-treated animals. 16 Thus, activation of resorption does not appear to be necessary for the anabolic effect of PTH in the rat. This may explain that, in this model, blocking resorption with either clodronate or calcitonin does not influence PTH effect. In a study performed on 5-month-old rats, Shen et a l . 39 have shown that PTH given at various times after ovariectomy did not increase resorption activity in the trabecular envelope, despite a marked anabolic effect. When PTH was com-

bined with 17-13-estradiol in these ovariectomized rats, there was a decrease of bone resorption that was accompanied with a bone formation activity--assessed by histology and by serum osteocalcin--significantly lower than in the PTH-alone-treated group, suggesting some degree of coupling between resorption and formation changes induced by turnover activator (PTH) and inhibitors (estrogens) in the rat. These species differences in the PTH effects on bone turnover are of major interest for understanding the in vivo mechanism of action of this hormone. In contrast to the rat model, the aged ewe is characterized by a slow remodeling activity of the trabecular bone which is quite similar to that of elderly humans. In this model, fluoride salts stimulate6 and glucocorticosteroids inhibit the osteoblastic bone formation, such as in humans. 7 In this study, the treatment with hPTH (1-34) induced an increase of trabecular bone resorption and formation that resembled the histological effects observed in humans. Lower doses of tiludronate in the combination treatment might have resulted in an increase in bone formation, although it needs to be proven that bone formation can be uncoupled from bone resorption in humans treated with PTH. 19 It might be important to confirm our data in another large animal with low bone remodeling, such as the baboon. A bone remodeling cycle leads to a negative bone balance in adults. 21 •2 9 Indirect evidence suggests that PTH stimulates bone remodeling through a cycle of activation/resorption/formation lasting for 10 to 12 weeks in adults. In patients with primary hyperparathyroidism, bisphosphonate treatment will rapidly reduce hypercalcemia by decreasing resorption activity, followed after a few weeks by a reduction of the biochemical indices of bone formation, despite the persistence of elevated PTH secretion. In such patients, the suppression of PTH excess by removal of the parathyroid adenoma induces a rapid decrease of bone resorption, while the normalization of formation occurs several

608

P.D. Delmas et al. The anabolic effect of PTH is dependent on activation of resorption

Bone Vol. 16, No. 6 June 1995:603-610

Figure 4. Histological sections. (a) Animal treated with PTH (l-34) alone. Marked increased of osteoid seams (----,)covered with plump osteoblasts. (b) Animal treated with PTH + tiludronate. Reduced extension of osteoid surfaces. Goldner trichrome staining. weeks later, suggesting that the effects of PTH on formation are secondary to those on resorption, m,l~ More information on the mechanism of action of PTH on bone at the cell and molecular level is necessary to fully understand our observations. From the clinical perspective, our data do not support the hypothesis that the coadministration of PTH and bisphosphonates could be beneficial to patients with osteoporosis. The ad-

dition of bisphosphonate actually allowed blockage of PTHinduced bone resorption, but also blunted the anabolic effect of formation. All bisphosphonates seem to act on bone turnover in a similar way, and similar results would be expected with other bisphosphonates in our model, although results might have been different with a lower dose of bisphosphonate. Whether or not suppressing resorption with other agents such as estrogens or

B o n e Vol. 16, No. 6 June 1 9 9 5 : 6 0 3 - 6 1 0

P . D . Delmas et al. The anabolic effect o f P T H is dependent o n activation o f resorption

Bone formation rate/BS p < 0.003 p < 0.002 i if pm3/um2/d I

i

Activation frequency p < 0.002 p < 0.005 /year i ~1 i

11.

I

2 12. 13.

0 ANOVA p < 0.0001 m

Controls ~

PTH m

ANOVA p < 0.0001 Tiludronate ~

PTH +Tiludronate

F i g u r e 5. C h a n g e s with time o f the tetracycline-based b o n e formation rate (left panel) and o f the activation f r e q u e n c y (right panel) in the four groups. D a t a are expressed as the m e a n o f the absolute c h a n g e s between the second (day 90) a n d the first (day 0) biopsies. The A N O V A p value indicates significance of c h a n g e s between groups.

14. 15.

16.

17. c a l c i t o n i n w i l l p r e s e r v e t h e e f f e c t o f P T H o n f o r m a t i o n is n o t k n o w n . A r e c e n t s t u d y in p o s t m e n o p a u s a l o s t e o p o r o t i c w o m e n confirmed that daily subcutaneous injections of PTH increased both resorption and formation, and that the subsequent inhibition of bone resorption with calcitonin induced a decrease of bone f o r m a t i o n . 19 A d d i t i o n a l e x p e r i m e n t s c o m b i n i n g P T H a n d r e s o r p tion inhibitors are necessary. Finally, combination of bisphosp h o n a t e s w i t h o t h e r b o n e - f o r m i n g a g e n t s s u c h as I G F - 1 t a n d p r o s t a g l a n d i n s 2~ m i g h t b e e f f e c t i v e . In c o n c l u s i o n , c o m b i n a t i o n regimens of anabolic and antiresorptive agents should be first c a r e f u l l y a n a l y z e d in a d e q u a t e a n i m a l m o d e l s , s u c h as s h e e p a n d primates, before clinical trials in osteoporotic patients are undert a k e n . In a d d i t i o n t o t h e i r t h e r a p e u t i c c o n s e q u e n c e s , o u r o b s e r v a t i o n s h a v e i m p l i c a t i o n s f o r t h e u n d e r s t a n d i n g o f t h e in v i v o mechanisms of action of PTH on bone.

18.

19.

20.

21.

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Date Received: December 6, 1994 Date Revised: January 31, 1995 Date Accepted: January 31, 1995