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Endocrinology Vol. 139, No. 11 4642-4651
Copyright © 1998 by The Endocrine Society

ARTICLES

LY353381·HCl: An Improved Benzothiophene Analog with Bone Efficacy Complementary to Parathyroid Hormone-(1–34)

Masahiko Sato, George Q. Zeng, Ellen Rowley and Charles H. Turner

Lilly Research Laboratories, Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana 46285; and the Biomechanics and Biomaterials Research Center and Department of Orthopedic Surgery, Indiana University Medical Center (C.H.T.), Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Dr. Masahiko Sato, MC 797, Department of Endocrine Research, Lilly Corporate Center, Indianapolis, Indiana 46285. E-mail: sato_masahiko{at}lilly.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LY353381·HCl is a benzothiophene analog that is structurally related to raloxifene with potent selective estrogen receptor modulator activity in the ovariectomized rat model of postmenopausal osteoporosis. The effects of LY353381·HCl on bones, body weight, and uterine weight were evaluated in 7-month-old rats with osteopenia that was induced by ovariectomizing animals for 1 month before initiation of treatment with several agents individually, in combination, or in sequence. LY353381·HCl was administered daily by itself for 90 days, in combination with the amino-terminal fragment of PTH-(1–34) (PTH) for 90 days, or sequentially after PTH when PTH was discontinued after 45 days of treatment. Additionally, comparisons were made of animals treated with PTH alone, 17{alpha}-ethynyl estradiol alone, equine estrogens (Premarin) alone, raloxifene alone, or combinations of PTH and equine estrogens or raloxifene. Ovariectomy induced increases in the rate of bone turnover and body weight while decreasing bone mineral density, bone mineral content, bone strength, trabecular bone volume, trabecular thickness, trabecular number, and uterine weight. LY353381·HCl at 0.01–1 mg/kg had marginal effects on body weight and no effect on uterine weight compared with those in ovariectomized controls, in contrast to 17{alpha}-ethynyl estradiol or equine estrogens. LY353381·HCl prevented further bone loss due to ovariectomy in tibia, femora, and lumbar vertebra, like 17{alpha}-ethynyl estradiol but unlike equine estrogens. LY353381·HCl prevented the resorption of trabecular bone spicules, like 17{alpha}-ethynyl estradiol, but inhibited bone formation activity to a lesser extent than 17{alpha}-ethynyl estradiol. In this model, 17{alpha}-ethynyl estradiol appeared to be more efficacious after 3 months of treatment than equine estrogens in the proximal tibia metaphysis, suggesting efficacy differences between metabolites of 17ß-estradiol in bone. PTH at 10 µg/kg had no effect on body weight or uterine weight, but significantly increased bone mass to beyond those in sham-operated controls, baseline controls, and groups receiving other individual treatments at both axial and appendicular sites. The combination of LY353381·HCl and PTH increased bone mass at a faster rate and to a greater extent than PTH alone or the combinations of equine estrogens/PTH and raloxifene/PTH at trabecular bone sites. The LY353381·HCl/PTH combination improved bone mass and quality beyond any agent alone in regions enriched for cancellous bone, but was not significantly better than PTH alone on cortical bone. Additionally, when PTH was discontinued at 45 days, LY353381·HCl prevented the rapid loss of bone observed in controls. Therefore, LY353381·HCl appears to be useful by itself, in combination, or in sequence with PTH to replace lost bone in postmenopausal women.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS are currently the most prescribed therapy used to minimize bone loss and reduce fracture incidence in postmenopausal women (1, 2, 3, 4, 5). Although clearly efficacious, estrogens also substantially increase the incidence, but not the mortality, of endometrial cancer (6, 7, 8) and may increase the incidence of breast cancer (9, 10). Although epidemiological studies clearly show that the benefits of estrogen replacement therapy outweigh the risks of treatment for several years (11, 12), the real or perceived risks of ERT have motivated researchers to search for compounds with estrogen agonist activity in bone and serum cholesterol, but antagonist activity or no activity in reproductive tissues. One compound recently approved as prevention therapy for osteoporosis (13) and shown to have this selective pharmacology in estrogen target tissues is raloxifene (RA) (14, 15, 16). LY353381·HCl is a new benzothiophene analog with selective estrogen receptor modulator (SERM) activity similar to but not identical with that of RA (17, 18, 19).

A mechanistically different agent with superior efficacy in cancellous and cortical bone compartments is PTH (20). Specifically, intermittent sc treatment of aged, osteopenic, ovariectomized rats with human PTH-(1–34) has been shown to anabolically stimulate bone formation in cancellous and cortical bone compartments of multiple skeletal sites (21, 22, 23, 24, 25, 26, 27, 28). Significant clinical efficacy have been observed at multiple sites, including vertebra and proximal femur (29, 30, 31, 32, 33, 34). Recently, the combination of estrogen and PTH-(1–34) was shown to induce impressive gains in vertebra and hip, with significant reduction in fracture incidence in postmenopausal women (34).

The ovariectomized rat model has proven to be extremely useful in the mechanistic analysis of a variety of pharmacological agents with clinical potential to treat postmenopausal osteoporosis (35, 36, 37). Specifically, ovariectomized rats have been used for bone efficacy studies of estrogens (38, 39, 40) and SERMs, including tamoxifen (41, 42, 43), nafoxidine (18), RA (17, 18, 44, 45), droloxifene (46), and GW5638 (47). This model has permitted higher resolution analyses of dynamic and cellular processes for estrogen and SERMs with greater precision and accuracy than presently possible in clinical studies. These data have accurately predicted clinical efficacy in women for tamoxifen (48) and more recently RA (13). In addition, this model has been used to explore the benefits of the estrogen and PTH-(1–34) combination (49, 50).

In this study, LY353381·HCl efficacy in vivo was evaluated in mature ovariectomized rats with significant bone loss. That is, osteopenic rats were administered previously determined efficacious doses of LY353381·HCl, 17{alpha}-ethynyl estradiol (EE2), equine estrogens (Premarin, Wyeth-Ayerst, Radnor, PA), or RA and then compared with PTH-(1–34) (18, 19, 27). Additionally, LY353381·HCl was administered in combination with PTH-(1–34) or in sequence, after discontinuation of PTH-(1–34). The in vivo data suggest possible advantages for LY353381·HCl over EE2, equine estrogens, RA, or PTH-(1–34), alone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rat groups and dosing regimens
Six-month-old, virgin Sprague-Dawley female rats (Harlan, IN), weighing about 270 g, were maintained on a 12-h light, 12-h dark cycle at 22 C with ad libitum access to food (TD 89222 with 0.5% calcium and 0.4% phosphorus; Teklad, Madison, WI) and water. In the first experiment, a set of baseline intact controls (n = 7) was killed, and tissues were processed as described below. Bilateral ovariectomies were performed on the remaining rats, except on sham-operated controls, and then ovariectomized rats were randomized and permitted to lose bone mass for 1 month. At 7 months of age (1 month postsurgery), rats were grouped into treatment units of seven or eight to include 1) sham-operated controls (Sham), 2) ovariectomized controls (OVX), 3) ovariectomized and treated with LY353381·HCl (0.01, 0.3, and 1 mg/kg·day, orally; Eli Lilly & Co.) for 3 months, 4) ovariectomized and treated with PTH-(1–34) (PTH; 10 µg/kg·day, sc; Eli Lilly & Co.) for 3 months, 5) ovariectomized and treated with PTH (10 µg/kg·day, sc) plus LY353381·HCl (0.3 mg/kg·day, orally) for 3 months, 6) ovariectomized and treated with PTH (10 µg/kg·day, sc) for 45 days followed by vehicle for the remaining 45 days, 7) ovariectomized and treated with PTH (10 µg/kg·day, sc) for 45 days followed by LY353381·HCl (0.3 mg/kg·day, orally) for the remaining 45 days, and 8) ovariectomized treated with EE2 (Sigma Chemical Co.; 0.1 mg/kg·day, orally) for 3 months. The vehicle for oral administration by gavage was 1 µl/g BW 20% hydroxypropyl-ß-cyclodextrin (Aldrich Chemical Co., Milwaukee, WI). LY353381·HCl-treated rats were administered 0.01–1 mg/kg·day LY353381·HCl by gavage in 1 µl/g BW 20% cyclodextrin. Estrogen control rats were administered 0.1 mg/kg·day 17{alpha}-ethynyl estradiol by gavage. PTH (10 µg/kg·day) was administered by sc injection in an acidified saline vehicle (0.001 N HCl and 2% heat-inactivated rat serum in physiological saline; Butler Co., Columbus, OH). For dynamic histomorphometry, rats were injected with calcein (10 mg/kg; Sigma Chemical Co., St. Louis, MO) on days -15, -14, -3, and -2 before death.

In the second experiment, a set of baseline intact controls (n = 8) was killed as described below. At 1 month postsurgery, other treatment groups (n = 8) included 1) sham-operated controls (Sham), 2) ovariectomized controls (OVX), 3) ovariectomized and treated with equine estrogens (0.01–1 mg/kg·day, orally) for 3 months, 4) ovariectomized and treated with PTH (10 µg/kg·day, sc) for 3 months, 5) ovariectomized and treated with PTH (10 µg/kg·day, sc) plus equine estrogens (Premarin; 1 mg/kg·day, orally) for 3 months, 6) ovariectomized and treated with PTH (10 µg/kg·day, sc) for 45 days followed by vehicle for the remaining 45 days, and 7) ovariectomized and treated with PTH (10 µg/kg·day, sc) for 45 days followed by equine estrogens (1 mg/kg·day, orally) for the remaining 45 days.

In the third experiment, a set of baseline intact controls (n = 8) was killed, and treatment groups (n = 8) included 1) sham-operated controls (Sham), 2) ovariectomized controls (OVX), 3) ovariectomized and treated with Premarin; 1 mg/kg·day, orally, for 3 months, 4) ovariectomized and treated with RA (3 mg/kg·day, orally) for 3 months, 5) ovariectomized and treated with LY353381·HCl (0.3 mg/kg·day, orally) for 3 months, 6) ovariectomized and treated with PTH (10 µg/kg·day, sc) for 3 months, 7) ovariectomized and treated with PTH (10 µg/kg·day, sc) plus equine estrogens (1 mg/kg·day, orally) for 3 months, 8) ovariectomized and treated with PTH (10 µg/kg·day, sc) plus RA (3 mg/kg·day, orally) for 3 months, and 9) ovariectomized and treated with PTH (10 µg/kg·day, sc) plus LY353381·HCl (0.3 mg/kg·day, orally) for 3 months.

In the fourth experiment, treatment groups (n = 8) included 1) sham-operated controls (Sham), 2) ovariectomized controls (OVX), 3) ovariectomized and treated with LY353381·HCl (0.003, 0.03, 0.3, and 3 mg/kg·day, orally) for 3 months, and 4) ovariectomized and treated with EE2 (Sigma Chemical Co.; 0.1 mg/kg·day, orally) for 3 months. All animal procedures were reviewed before implementation by an internal animal welfare committee to ensure compliance with NIH guidelines.

Tissue collection
After treatment, anesthetized rats were subjected to cardiac puncture and killed by CO2 inhalation. Uteri were removed, and wet weights were determined on a Mettler balance to evaluate ovariectomy and efficacy of treatment with estrogen. Uteri were then fixed in 10% formalin, embedded in paraffin, and processed for histology. Tibia and femora were removed, cleaned of soft tissue, fixed in 50% ethanol-saline, and stored at 4 C. Vertebra L1–L6 were removed and analyzed by pQCT (L-4, microXCT, Stratec), histomorphometry (L-1), and biomechanics (L-5 and L-6).

X-Ray bone densitometry of excised rat bones
The metaphysis of proximal tibiae were scanned longitudinally from baseline at 0, 1, 2–2.5, and 4 months postsurgery, using a 960A pQCT loaded with Dichte software version 5.2 (Norland/Stratec, Fort Atkinson, WI), as described previously (44). Volumetric bone mineral density (BMD; milligrams per cm3), cross-sectional area (X-Area), voxel number, and mineral content (BMC; milligrams) were quantitated for the whole cross-section of the metaphysis. Sites of excised bones were analyzed at higher resolution, using a microXCT (Stratec). Specifically, the diaphysis of femora and L-4 vertebra were analyzed using voxel dimensions of 50 x 50 x 1000 and 70 x 70 x 1000 µm, respectively.

Histomorphometry
For histomorphometry, L-1 vertebra were trimmed using a low speed diamond saw (Buehler Ltd., Lake Bluff, IL) and fixed in 70% ethanol. Specimens were stained for 4 days in Villanueva osteochrome bone stain (Polysciences, Inc., Warrington, PA), destained, dehydrated in a graded series of alcohols, and defatted in acetone. L-1 vertebra were then infiltrated with methyl methacrylate, embedded in a 75 ml-19 ml-2.5 g mixture of methyl methacrylate:-dibutyl phthalate-benzoyl peroxide (Eastman Kodak Co., Rochester, NY), and polymerized at room temperature. Longitudinal sections (4 and 8 µm) were cut on a Reichert-Jung 2065 microtome (Magee Scientific, Inc., Dexter, MI). The 4-µm sections were stained with 6% silver nitrate (von Kossa stain) before coverslipping; the 8-µm thick sections were mounted unstained for dynamic measurements. Sections were glued onto slides dipped in 0.5% gelatin, dried overnight, and coverslipped with Eukitt.

Histomorphometric measurements were made using an Optiphot-2 fluorescence microscope (Nikon, Melville, NY) and a semiautomatic digitizing system (SummaSketch III, Summagraphics Co., Seymour, CT; KSS Image Analysis, KSS Scientific Consultants, Magna, UT) coupled to a PowerPC 7100/66 (Apple Computer, Cupertino, CA), using the image capture functions of NIH Image 1.59 (NIH, Bethesda, MD). For L-1, the entire marrow region within the cortical shell was measured to derive trabecular bone parameters. Specifically, measurements were made of cancellous bone volume (BV/TV; percentage), trabecular thickness (microns), number (per mm), separation (microns), mineralizing surface, mineral apposition rate, and bone formation rate (BFR), as previously described (51).

Biomechanical analyses
Femora were thawed before testing, and bone strength was measured on intact femora using a three-point bending test, as described by Turner and Burr (52). Load was applied midway between two supports that were 15 mm apart. The femora were positioned so the loading point was 7.5 mm proximal from the distal popliteal space, and bending occurred about the medial-lateral axis. Specimens were tested in a saline bath at 37 C. Each specimen was submerged in the saline bath for 3 min before testing to allow equilibration of temperature. Load displacement curves were recorded at a cross-head speed of 1 mm/sec using a servo-hydraulic materials testing machine (MTS Corp., Minneapolis, MN) and an x-y recorder (7090A, Hewlett-Packard Co., Palo Alto, CA). The measures of bone strength, ultimate load (Fu), stiffness, and work to failure (U) were calculated as described previously (27, 52).

Femoral neck strength was measured by mounting the proximal half of the femur vertically in a chuck and applying downward force at a rate of 1 mm/sec on the femoral head until the neck failed (52). The ultimate load was calculated as the maximum force sustained by the femoral neck. All tests were performed at room temperature using the MTS system.

The bone strength of the L6 vertebrae was measured after the posterior processes were removed and the ends of the centrum made parallel using a diamond wafering saw (Buehler Isomet, Evanston, IL). Ultimate stress ({varsigma}u), Young’s modulus (E), and toughness (u) for each vertebra were measured in compression at a load rate of 50 N/sec using the MTS machine. The compressive load was applied through a pivoting platen to correct for nonparallel alignment of the faces of the vertebral body (52). Specimens were tested in saline solution at 37 C. Ultimate stress was estimated as the maximum load divided by the gross cross-sectional area, {pi}AB/4, where A and B are the vertebral widths in the anterior-posterior and medial lateral directions. Stiffness was calculated as the maximum slope of the load displacement curve. Young’s modulus was calculated by multiplying stiffness times 4T/{pi}AB, where T is the specimen thickness. Toughness was calculated as the area under the load displacement curve divided by {pi}ABT/4.

Statistics
Data are presented as the mean ± SEM. Precision was calculated by averaging the coefficient of variation (variability), as defined by SD/mean for the specified rats. Group differences were assessed by ANOVA with pairwise contrasts examined using primarily Fisher’s protected least significant difference test (PLSD), where the significance level for the overall ANOVA was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Longitudinal analysis of LY353381·HCl effects in aged ovariectomized rats
The proximal tibial metaphysis was scanned longitudinally by pQCT for ovariectomized rats from baseline (Fig. 1Go). Ovariectomy significantly reduced volumetric BMD by 20% and 25% compared with Sham and Baseline values (P < 0.0001, by PLSD), respectively, by 1 month postsurgery (Fig. 1Go, A and B). Treatment was initiated at 1 month postovariectomy and continued for the following 3 months. In the first experiment (Fig. 1AGo), LY353381·HCl alone, PTH alone, LY353381·HCl plus PTH in combination, or PTH followed by LY353381·HCl in sequence were compared with OVX, Sham, and EE2 (0.1 mg/kg) controls (Fig. 1AGo). In Fig. 1BGo (Exp 3), a follow-up study was conducted with LY353381·HCl alone, equine estrogens alone, RA alone, PTH alone, and PTH in combination with LY353381·HCl, equine estrogens, or RA and compared with OVX and Sham. EE2 (0.1 mg/kg) and RA (3 mg/kg) prevented further bone loss due to ovariectomy and had BMD significantly greater than the OVX group at termination. All three doses of LY353381·HCl (0.01, 0.3, and 1.0 mg/kg) had BMD significantly greater than the OVX group and not different from the EE2 group, indicating that all three were efficacious doses. In Exp 2 and 3, equine estrogens at 0.1–1 mg/kg were shown to prevent bone loss at 30–45 days of treatment (75 days postovariectomy), but BMD was not significantly different from OVX at termination, unlike EE2. These data show that LY353381·HCl is able to prevent further reduction of bone induced by ovariectomy, like EE2 or RA but unlike equine estrogens.



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  		Figure 1. Longitudinal analysis of LY353381·HCl effects in a delayed dosing intervention model. Rats were ovariectomized (except for Sham) and scanned longitudinally in the proximal tibia metaphysis by pQCT. As indicated in A and B, treatment was initiated at 1 month postovariectomy and continued for the following 3 months. Specifically in A (Exp 1), treatments included LY353381·HCl (353381) alone, PTH (10 µg/kg, sc) alone, LY353381·HCl and PTH in combination (PTH+353381), PTH followed by vehicle (PTH/vehicle), or PTH followed by 353381 (PTH/353381) in sequence. These groups were compared with OVX controls, Sham controls, or EE2 (0.1 mg/kg) controls. Only the 0.3 mg/kg dose of 353381 is shown, but similar efficacy was observed for 0.01 and 1 mg/kg groups. Another study is shown in B (Exp 3); treatments included LY353381·HCl (353381; 0.3 mg/kg) alone, equine estrogens (Prem; 1 mg/kg) alone, RA (3 mg/kg) alone, PTH (10 µg/kg, sc) alone, PTH+353381, PTH+RA, and PTH plus equine estrogens (PTH+Prem). Plotted are the mean ± SE, with a group size of seven or eight. Significant differences from Sham and OVX are designated s and o, respectively (P < 0.05, by Fisher’s PLSD). Ovariectomy decreased BMD by 20–25% compared with Sham and Baseline values. LY353381·HCl and EE2 (0.1 mg/kg), but not equine estrogens, prevented further loss of BMD by termination compared with OVX. PTH increased BMD to significantly beyond OVX, Sham, and Baseline levels at termination. The combination of LY353381·HCl and PTH increased BMD significantly faster and to higher levels than either agent alone, whereas the equine estrogen and PTH combination was not different from PTH alone. Both LY353381·HCl and equine estrogens prevented loss of BMD after discontinuation of PTH at 45 days postovariectomy.

 
Rats administered PTH for 30–45 days regained BMD to Sham levels or slightly above (Fig. 1Go, A and B). However, when PTH was discontinued (PTH/vehicle), BMD (and BMC, data not shown) decreased to significantly below the values attained at 45 days. Sequential studies in which rats were treated with PTH for 45 days before switching to LY353381·HCl or equine estrogens for the remainder of the study showed that LY353381·HCl or equine estrogens were effective in preventing loss of BMD after discontinuation of PTH. That is, the BMD at termination of the study was not different from the PTH BMD attained after 45 days of treatment (Fig. 1AGo). Data from Exp 1 and 2 showed that LY353381·HCl, EE2 (data not shown) and equine estrogens (data not shown) can prevent the loss of bone after discontinuation of PTH treatment.

Animals treated continuously for 90 days with PTH (10 µg/kg) regained BMD to significantly beyond OVX, Sham, and Baseline group values or levels achieved by other individual treatments (Fig. 1Go, A and B). The combination of PTH and equine estrogens (1 mg/kg) mirrored the effects of PTH alone, with minor effects on kinetics during treatment. The combination of PTH and RA increased BMD above PTH in this study (Fig. 1BGo), but this was not reproduced in other studies. By contrast, the combination of LY353381·HCl (0.3 mg/kg) and PTH consistently increased BMD to levels higher than PTH alone or PTH plus equine estrogens at 30–45 and 90 days of treatment (P < 0.0001, by PLSD). This combination increased BMD significantly faster and to greater levels than any agent alone or any PTH combination in the proximal tibia, with the possible exception of PTH and RA, which was not always significantly different.

LY353381·HCl effects on body weight and uterine weight
Ovariectomy was confirmed to increase body weight above Sham values by 1 month postsurgery, as shown previously (18). Body weights of pretreatment ovariectomized controls (Pre-OVX; 1 month postsurgery) indicate weights at the initiation of treatment. Rats were weighed after treatment to ascertain dosing effects on the body weight gain due to ovariectomy. Body weights for Pre-OVX and OVX (4 months postsurgery) groups were not different (Fig. 2AGo). Rats treated with LY353381·HCl at 0.03–3 mg/kg were not reproducibly different from Pre-OVX or OVX controls, but were significantly heavier than Sham animals (Fig. 2Go, A and B). LY353381·HCl at 0.003 mg/kg was intermediate and not different from the effect of Sham or OVX. By contrast, EE2 and equine estrogens lowered body weight to below Pre-OVX and OVX levels to Sham levels. RA did not reduce body weight, and its effect was significantly greater than Sham but not different from that of OVX. Treatment with 10 µg/kg PTH alone had little effect compared with OVX and produced a body weight greater than that in the Sham group. Equine estrogens and to a lesser extent RA and LY353381·HCl reduced body weight in combination with PTH to lower than that in the OVX group. These data show that EE2 and equine estrogens are more efficacious than LY353381·HCl or RA in reducing the body weight gained by animals due to ovariectomy.



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  		Figure 2. LY353381·HCl effects on body weight. Body weights at termination (mean ± SE) are plotted in A (Exp 4) and B (Exp 3). Significant differences from Sham and OVX are designated s and o, respectively (P < 0.05, by Fisher’s PLSD). Ovariectomy increased body weight to above Sham levels. Treatment with LY353381·HCl (0.003, 0.03, 0.3, and 3 mg/kg) had little effect on body weight compared with pretreatment OVX (Pre-OVX; 1 month postsurgery) or OVX (4 months postsurgery). By contrast, EE2 lowered body weight from the pretreatment OVX levels to Sham levels. In B, equine estrogens (1 mg/kg; Prem) lowered body weight from pretreatment OVX levels to Sham levels. RA (1 mg/kg) and PTH (10 µg/kg) had no effect on body weight compared with OVX. PTH plus equine estrogens (PTH+Prem) also lowered body weight to Sham levels, whereas PTH plus RA and PTH plus 353381 had smaller effects.

 
Ovariectomy was confirmed to decrease the uterine wet weight of aged rats compared with that in the Sham group (Fig. 3Go). Uteri of animals treated with LY353381·HCl at 0.003–3 mg/kg from Exp 4 were not significantly different from but tended to be up to 26% heavier than OVX animals. LY353381·HCl uteri were significantly different from Sham and animals treated with 0.1 mg/kg EE2 or 0.01–1 mg/kg equine estrogens (data not shown), both of which increased uterine weight above OVX to Sham levels.



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  		Figure 3. LY353381·HCl effects on uterine weight. The wet weight of uteri were measured at termination for the groups indicated from Exp 4. The plotted data are the mean ± SEs with a group size of seven or eight. Ovariectomy was confirmed to decrease uterine wet weight compared with Sham values. Treatment with LY353381·HCl at 0.003–3 mg/kg had no significant effect on uterine weight compared with OVX values, whereas 0.1 mg/kg EE2 increased uterine weight above OVX toward Sham levels. Uterine effects similar to those of EE2 were observed for equine estrogens at 0.01–1 mg/kg (data not shown).

 
MicroCT analyzes of vertebrae and femora
Vertebra from Exp 3 were analyzed at higher resolution by microCT to clarify the effects of PTH alone and in combination with equine estrogens, RA, or LY353381·HCl in bone sites enriched with trabecular bone (Fig. 4Go). L-4 vertebra were analyzed cross-sectionally, using 70 x 70 x 1000-µm voxels, at termination. PTH was confirmed to restore BMD and BMC from OVX to Sham and Baseline levels. The PTH plus equine estrogen combination had efficacy similar to that of PTH alone. The PTH plus RA combination increased BMD but not BMC to significantly above OVX, Baseline, and Sham levels. LY353381·HCl plus PTH appeared to be more efficacious in vertebra than in other groups and increased BMD to above those in OVX, Baseline, Sham, PTH alone, and PTH plus equine estrogen combination groups. Cross-sectional areas for Sham, OVX and the treatment groups were not different, although the PTH groups were larger than Baseline, except for PTH plus RA (Fig. 4CGo). Similar efficacy data were obtained for the proximal tibia metaphysis (data not shown, but similar to Fig. 1Go, A and B). Therefore, the vertebra data taken together with the tibia data show that the LY353381·HCl plus PTH combination increased bone beyond these treatments alone or PTH combinations with equine estrogens or RA in regions enriched with cancellous bone.



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  		Figure 4. QCT analysis of PTH combination effects on vertebra. Excised L-4 vertebra were analyzed by microCT, using voxel dimensions of 70 x 70 x 1000 µm. Treatment groups included PTH (10 µg/kg) alone, PTH with equine estrogens (1 mg/kg; PTH+Prem), PTH with RA (1 mg/kg; PTH+RA), or PTH with LY353381·HCl (0.3 mg/kg; PTH+353381) from Exp 3. Plotted data are the mean ± SD. Significant differences from Sham and OVX are designated s and o, respectively (P < 0.05, by Fisher’s PLSD). L-4 from rats treated continuously with PTH regained BMD (A) and BMC (B) from OVX to Baseline and Sham levels. The effect of the combination of PTH+Prem was not different from that of PTH alone, whereas PTH+RA increased BMD but not BMC to above OVX, Baseline, and Sham levels. The combination of PTH+353381 increased BMD and BMC to levels above OVX, Baseline, and Sham levels. Cross-sectional areas (X-Area; C) for Sham, OVX, and the treatment groups were not different, although values in the PTH groups were larger than Baseline, except for PTH+RA.

 
MicroCT was also used to clarify the effects of PTH alone and PTH combinations with equine estrogens, RA, or LY353381·HCl in the cortical bone (Fig. 5Go). The midshaft of femora from the same animals was analyzed in cross-section at 50 x 50 x 1000 µm resolution at termination of the study. Sham BMC were 13% greater than Baseline, indicating that these animals were continuing to grow, although slowly (53). Ovariectomy over 4 months resulted in BMD and BMC 6% and 10%, respectively, lower than Sham values. PTH restored BMD and BMC to Sham levels, whereas the PTH combinations restored BMD, but not BMC, to Sham levels in this experiment. Reproducible differences between Sham or PTH at 10 µg/kg alone and these PTH combinations were not consistently observed for the femora midshaft. The cross-sectional area (X-Area) of the diaphysis for Sham, OVX, and the treatment groups were not significantly different (Fig. 5C).



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  		Figure 5. QCT analysis of PTH combination effects on cortical bone. The midshaft of excised femora were analyzed by microCT, using voxel dimensions of 50 x 50 x 1000 µm. Treatment groups included PTH (10 µg/kg) alone or combinations of PTH with equine estrogens (1 mg/kg; PTH+Prem), RA (1 mg/kg; PTH+RA), or LY353381·HCl (0.3 mg/kg; PTH+353381) from Exp 3. Plotted data are the mean ± SD. Significant differences from Sham and OVX are designated s and o, respectively (P < 0.05, by Fisher’s PLSD). BMD and BMC for Sham increased above Baseline levels, indicating growth with time. BMD and BMC for OVX were lower than Sham values, but not different from Baseline. BMD for PTH, PTH+Prem, PTH+RA, and PTH+353381 were greater than OVX values. However, the BMC for PTH alone was greater than the OVX value. Cross-sectional areas (X-Area) for Sham, OVX, and the treatment groups were not significantly different.

 
Static and dynamic histomorphometry of LY353381·HCl effects
Additional analyses of the effects of LY353381·HCl on L-1 vertebra from the first experiment were conducted by histomorphometry (Table 1Go). Ovariectomy decreased BV/TV, trabecular thickness, and trabecular number compared with Sham. As applied to osteopenic animals, LY353381·HCl and EE2 had little effect on BV/TV, trabecular thickness, or trabecular number, which were not different from OVX values at termination. However, PTH improved BV/TV and trabecular thickness above OVX values, whereas the combination of LY353381·HCl and PTH increased BV/TV significantly beyond Sham and PTH alone. Discontinuation of PTH decreased BV/TV and trabecular thickness to below Sham values, but switching to LY353381·HCl at 45 days prevented this loss.


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  		Table 1. Static and dynamic histomorphometry of L-1

 
Examination of dynamic parameters confirmed the ovariectomy stimulation of mineralized surface (MS/BS) and BFR (BFR/BS; Table 1Go). LY353381·HCl dose dependently decreased MS/BS and BFR to Sham and EE2 levels. However, the MAR for LY353381·HCl (0.01–1 mg/kg) was not different from that for OVX, whereas EE2 lowered MAR to below OVX, Sham, and LY353381·HCl (0.01–1 mg/kg) values. Limited dose-dependent effects were observed, as LY353381·HCl at 0.01 mg appeared to be as efficacious as 1 mg/kg for both static and dynamic parameters. PTH increased MS/BS, MAR, BFR/BS, and BFR/TV to significantly above Sham and OVX values. PTH in combination with LY353381·HCl increased MS/BS, BFR/BS, and BFR/TV to values above those with PTH alone. Discontinuation of PTH at 45 days lowered MS/BS, BFR/BS, and BFR/TV to OVX levels. Switching to LY353381·HCl at 45 days lowered MAR, BFR/BS, and BFR/TV to below those in the PTH/vehicle group. These data show that LY353381·HCl decreases bone turnover like EE2, but suppresses bone formation (MAR) to a lesser extent than EE2. Additionally, the PTH plus LY353381·HCl group had higher bone formation and mineralization activity than LY353381·HCl or PTH alone.

In an effort to further elucidate the mechanism, histomorphometry was conducted for the proximal tibia metaphysis from the third experiment with results largely similar to the L-1 vertebra data in Table 1Go and Fig. 6Go (data largely not shown). However, examination of the eroded perimeter (%Er.Pm) confirmed the dramatic increase in bone resorption activity induced by ovariectomy (Fig. 6Go). Equine estrogens and RA were confirmed to reduce %Er.Pm to Sham levels, as did LY353381·HCl. At 10 µg/kg PTH, %Er.Pm values were less than OVX values. Interestingly, LY353381·HCl reduced %Er.Pm in combination with PTH to levels significantly below those caused by PTH plus equine estrogens. Therefore, LY353381·HCl appears to be a potent inhibitor of resorption activity in vivo, by itself and in combination with PTH.



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  		Figure 6. Eroded perimeter in the proximal tibia metaphysis. The %Er.Pm was analyzed for the proximal tibia metaphysis from the third experiment. Plotted are the mean ± SE, with a group size of seven to nine. Significant differences from Sham and OVX values are designated s and o, respectively (P < 0.05, by Fisher’s PLSD). Ovariectomy was confirmed to increase eroded perimeter. Equine estrogens (Prem), RA, and LY353381·HCl reduced %Er to Sham levels. %Er values for PTH and PTH combinations were lower than OVX values, but not different from Sham values.

 
Biomechanical analysis of bone quality
The femora diaphyses from the first and second experiments were evaluated by three-point bending analysis of the midshaft (Tables 2Go and 3Go). Measurement of the cortical thickness for LY353381·HCl and EE2 showed that thickness for both were intermediate between Sham and OVX values. LY353381·HCl improved load to failure (Fu) and work to failure (U) to above OVX values and not different from Sham values at 1 mg/kg, as did EE2 at 0.1 mg/kg. Limited dose-dependent effects were observed, as LY353381·HCl at 0.01 mg appeared to be as efficacious as at 1 mg/kg. Interestingly, equine estrogens at 0.01–1 mg/kg did not have similar effects on Fu or U, suggesting subtle differences in efficacy between equine estrogens and EE2. PTH treatment for 90 days improved cortical thickness (t), Fu, stiffness (S), and U to significantly above OVX values. The LY353381·HCl plus PTH combination increased t, Fu, S, and U to significantly above OVX and Sham values, but were not different from PTH alone values (Table 2Go). The equine estrogens plus PTH combination increased t and S above OVX values. No difference in Young’s modulus were observed between groups (data not shown). Discontinuation of PTH after 45 days lowered cortical thickness to below the Sham value, but switching treatment to LY353381·HCl or equine estrogens prevented this decrease.


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  		Table 2. Effects of LY353381-HCl and PTH on the biomechanical parameters of the femora

 

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  		Table 3. Effects of Premarin and PTH on the biomechanical parameters of the femora

 
The ultimate load for the femora neck showed no differences between groups, except for PTH alone, PTH in sequence with equine estrogens, and PTH in combination with LY353381·HCl, which were all higher than OVX and Sham values (Tables 2Go and 3Go).

The mechanical properties of lumbar vertebra L-6 from Exp 1 and 3 were evaluated by compression testing. Ovariectomy reduced the strength ({varsigma}u) of vertebra by 33–37% compared with Sham values (Table 4Go). LY353381·HCl improved vertebral strength ({varsigma}u) and toughness to above OVX values, and they were not different from EE2 or Sham values. PTH treatment for 90 days increased {varsigma}u and toughness to above OVX and Sham levels. Discontinuation of PTH after 45 days lowered {varsigma}u to below Sham values, but switching treatment to LY353381·HCl prevented this decrease. Interestingly, PTH plus equine estrogens improved {varsigma}u above OVX and Sham values, but toughness was not different from that in the OVX group (Table 4Go). PTH plus LY353381·HCl improved {varsigma}u and toughness significantly above PTH alone or PTH plus equine estrogens. PTH plus RA had effects on {varsigma}u and toughness in between those of PTH alone and PTH plus LY353381·HCl. These collective data show advantages to LY353381·HCl, alone, in combination, or in sequence with PTH, especially in trabecular bone regions.


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  		Table 4. Effects of LY353381-HCl on the biomechanical parameters of the lumbar vertebra

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SERMs are nonsteroidal, synthetic compounds shown previously to bind with high affinity to the conventional estrogen receptor (14, 15, 16). These compounds belong to three major structural classes, consisting of triphenylethylenes, of which tamoxifen is the best studied example; dihydronaphthalenes, of which nafoxidine and CP336,156 are examples; and benzothiophenes, of which RA is the best studied example. SERMs are of considerable interest among investigators for their structure-based ability to function as estrogen agonists or estrogen antagonists depending on the tissue (18, 54, 55). Recently, different metabolites (analogs) of 17ß-estradiol were shown to regulate gene transcription in a manner similar to RA (56). These data suggest that SERMs may actually be mimicking the different spectra of estrogen agonist/antagonist properties, characteristic of different estrogen metabolites.

LY353381·HCl is the newest member of the benzothiophene family of SERMs, with potency advantages over RA in a prevention model of osteoporosis in ovariectomized rats (19). LY353381·HCl was reexamined in a delayed dosing intervention model of osteoporosis to determine whether LY353381·HCl has advantages over RA or estrogens in this model (57). Two estrogens used clinically, EE2 and equine estrogens (Premarin), were also evaluated for possible efficacy differences between estrogens in vivo in this model. The former was chosen over 17ß-estradiol, because in our hands 17ß-estradiol does not lower serum cholesterol levels in ovariectomized rats. Finally, human PTH-(1–34) was included as a positive control, as PTH was shown previously to rebuild bone in aged, ovariectomized rats (21, 22, 23, 24, 25, 26, 27, 28).

In this delayed dosing intervention model, LY353381·HCl at 0.003–3 mg/kg and PTH at 10 µg/kg had little effect on the increased body weight of ovariectomized rats. Previous data showed that LY353381·HCl and estrogen prevent the ovariectomy-induced gain in body weight (19) with administration immediately after ovariectomy. However, LY353381·HCl up to 3 mg/kg does not appear to reduce the body weight gained during the first month postovariectomy, even after 3 months of subsequent treatment. This is in contrast to subsequent treatment with 0.1 mg/kg EE2 or 0.01–1 mg/kg equine estrogens, which lowered the body weight of OVX rats to Sham levels. Additional studies are in progress to ascertain whether the body weight changes can be explained in terms of the agents’ effects on bone, lean tissue, or fat.

LY353381·HCl had marginal effects on the uterine weight of ovariectomized rats. PTH (10 µg/kg) alone, in combination, or in sequence with LY353381·HCl also showed no effect on uterine weight. These data confirm the absence of uterine stimulation for LY353381·HCl, similar to RA, but in contrast to EE2 or equine estrogens (17, 18, 19).

In the metaphysis of proximal tibiae, LY353381·HCl prevented the further loss of bone induced by ovariectomy. After 3 months of treatment (4 months postovariectomy), LY353381·HCl-treated rats had significantly more bone than ovariectomized controls, like the effects of RA and EE2 (57), but not equine estrogens. BMD for equine estrogens at 0.1–1 mg/kg were significantly greater than OVX values at 30–45 days, but not at termination, suggesting a loss of bone efficacy over the long term and subtle efficacy differences for equine estrogens compared with EE2 in the proximal tibia.

LY353381·HCl, EE2, and equine estrogen effects on the proximal tibia and vertebra were in marked contrast to the anabolic effects of PTH, which replaced lost trabecular bone to beyond Sham and Baseline levels (20, 21, 22, 23, 24, 25, 26, 27). Intermittent sc administration of PTH was confirmed to stimulate bone formation and thickening of existing trabecular bone spicules. The combination of LY353381·HCl and PTH was observed to significantly increase bone at a faster rate than PTH alone or the combination of PTH plus equine estrogens. This efficacy advantage was confirmed in strength and toughness analyses of vertebra. Therefore, proximal tibia and vertebra data taken together showed that LY353381·HCl appears to have an advantage over equine estrogens or RA in complementing the anabolic bone effects of PTH.

When PTH was discontinued at 45 days, ovariectomized rats lost bone rapidly. LY353381·HCl prevented this loss of bone in a manner similar to EE2 in both the proximal tibia and lumbar vertebra. Interestingly, equine estrogens appeared to be more efficacious in sequence with PTH in the femoral neck than LY353381·HCl in this model.

QCT and histomorphometric analyses showed that LY353381·HCl by itself prevents the ovariectomy-stimulated loss of trabecular bone in the appendicular and axial skeleton. That is, LY353381·HCl prevented the resorption of trabecular bone spicules induced by ovariectomy and prevented the resorption of bone accumulated during PTH administration that become susceptible to resorption with discontinuation of PTH. Therefore, LY353381·HCl appears to be a resorption inhibitor with marginal advantages over estrogens and RA (45, 58).

Dynamic histomorphometry showed that LY353381·HCl decreased bone turnover in a dose-dependent manner similar to RA and estrogens (45, 58). However, like RA, LY353381·HCl appears to suppress bone formation to a lesser degree than EE2 or equine estrogens. Therefore, part of the explanation for the enhanced bone accumulation observed for PTH plus LY353381·HCl may be that LY353381·HCl inhibits osteoclastic resorption activity with little effect on bone formation activity.

Biomechanical analyses confirmed the beneficial effects of PTH to strengthen the vertebra, femoral neck, and also cortical bone of osteopenic, ovariectomized rats (21, 22, 23, 24, 25, 26, 27, 28). LY353381·HCl plus PTH effects on the femora midshaft were not significantly different from those of PTH alone, possibly reflecting the inadequate power (seven or eight animals per group) of this study to discriminate between these two treatment regimens (27). Similar reasons are probably responsible for the inability to cleanly discriminate between LY353381·HCl plus PTH and RA plus PTH effects. Nevertheless, the cumulative bone data plus the lack of uterine stimulation observed for LY353381·HCl suggest a therapeutic advantage to LY353381·HCl over estrogens and other SERMs in the treatment of bone and possibly other tissues in postmenopausal women.


   Acknowledgments
 
The authors gratefully acknowledge the excellent technical assistance of Tongyu Wang, Tina Fuson, and Shawn Smith.

Received February 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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