Donald B. Kimmel

The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K.

Odanacatib is a potent, selective, and neutral cathepsin K inhibitor which was developed to address the metabolic liabilities of the Cat K inhibitor L-873724. Substituting P1 and modifying the P2 side chain led to a metabolically robust inhibitor with a long half-life in preclinical species. Odanacatib was more selective in whole cell assays than the published Cat K inhibitors balicatib and relacatib. Evaluation in dermal fibroblast culture showed minimal intracellular collagen accumulation relative to less selective Cat K inhibitors.

A Noninvasive, In Vivo Model for Studying Strain Adaptive Bone Modeling

We present a noninvasive, in vivo model for strain application in the tibiae of rats. The hind limb of each animal was placed into a device that applied four point bending to the tibia. Bending was applied in the medial-lateral direction causing compression on the lateral surface of the tibia and tension on the anteromedial surface. The peak strain magnitudes were estimated to be between 1600 and 3500 mu strain. In this pilot work, data were collected from 12 rats. The rats received either one cycle per day, four cycles per day, 12 cycles per day, 36 cycles per day, or 108 cycles per day of bending. The experimental (right) tibiae from all of the rats showed new bone formation after 12 days. The control (left) tibiae showed no new bone formation over this period. A better organized, dense bony reaction occurred in regions of lesser strains than in regions of higher strains, where there was a large accumulation of bone easily identified as woven. The organization and density of the newly formed bone appeared to be inversely related to the peak strains in the region. After 40 days of daily loading, the new bone area appeared to be more compact and better mineralized. However, bone formation was still occurring after 40 days. The results of this study suggest that woven bone formation occurred due to the bending stimulus and not due to pathology.

Mechanical loading stimulates rapid changes in periosteal gene expression

Although mechanical forces regulate bone mass and morphology, little is known about the signals involved in that regulation. External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein β-actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor-β (TGF-β). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc, which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF-β and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation. The altered pattern of gene expression observed in the present study supports previous evidence of increased periosteal cell proliferation seen both in vivo and in vitro following mechanical loading.

A comparison of iliac bone histomorphometric data in post-menopausal osteoporotic and normal subjects

Transilial bone biopsies following in vivo fluorochrome labeling were obtained from 90 women with postmenopausal osteoporosis and 34 healthy post-menopausal women. Standard histomorphometric data were collected from undecalcified sections. The distribution of values for both structural and remodelling indices was the same for each group. Bone volume was 35% lower (P less than 0.001), wall thickness was 12% lower (P less than 0.001), and trabecular thickness was 11% lower (P less than 0.02) in osteoporotics. Trabecular separation was 34% greater (P less than 0.001) and trabecular number was 36% lower (P less than 0.001) in osteoporotics. Biopsy core width was 11% less (P less than 0.02) and cortical width was 35-50% less (P less than 0.001) in osteoporotics. Static indices of remodelling, mineralizing surfaces, and mineral apposition rate were similar in the two groups. The absolute values for bone histomorphometric variables for both groups are similar to most published data. Osteoporotics had poorer bone structure, marked by decreased trabecular connectivity and thin cortices. There were no major differences in dynamic indices of remodelling. Since the histomorphometric data were distributed the same in both groups, special subsets of osteoporotic subjects not in the normal population did not exist.

Bone response to in vivo mechanical loading in two breeds of mice.

Abstract. We investigated the bone response to external loading in C57BL/6J and C3H/HeJ mice, both breeds with low and high bone density, respectively. An in vivo tibial four-point bending device previously used for application of measured external loads in rats was adapted for mice. It delivered a uniform medio-lateral bending moment to the region of the tibia located 1–5.5 mm proximal to the tibio-fibula junction. The right legs of six C57BL/6J [low bone density (LBD)] and C3H/HeJ [high bone density (HBD)] mice were externally loaded in the device for 36 cycles/day at 2 Hz, 6 days/week for 2 weeks at 9.3 ± 0.9 N force, inducing estimated lateral periosteal surface compressive strains of 5121 ± 1128 με in C3H/HeJ (HBD) mice (n = 6), significantly higher than the estimated 3988 ± 820 με in C57BL/6J mice (n = 6) (mean ± SD). In addition, C3H/HeJ HBD mice (n = 11) were externally sham (pad pressure or no bending) loaded in the device for 36 cycles/day at 2 Hz, 3 days/week for 3 weeks at 9.3 ± 0.9 N force. Calcein injections for bone labeling were given at the 10th and 3rd days before sacrifice. At the end of the experiment, all mice were killed and both tibiae were removed, fixed, embedded, and cross-sectioned through the loaded region. Both tibiae were measured for marrow area (Ma.Ar), cortical area (Ct.Ar), total area (Tt.Ar), cross-sectional moment of inertia (CSMI), and periosteal and endocortical woven bone surface (Wo.B/BS), single-labeled surface (sLS), double-labeled surface (dLS), and total formation surface (FS/BS). Differences in all variables due to breed and loading (both bending and sham-bending) were tested by two-way analysis of variance (ANOVA) (P < 0.05). Ma.Ar, Tt.Ar, and CSMI were greater in C57BL/6J (LBD) than in C3H/HeJ (HBD) mice. Periosteal and endocortical woven bone and formation surface were increased significantly more by loading (bending) in C57BL/6J than in C3H/HeJ mice. Periosteal woven bone response due to sham-bending or sham-loading was significantly lower than due to bending loads in the C3H/HeJ mice. We conclude that the bone response to external loading is greater in LBD mice than in HBD mice. The high bone density of C3H/HeJ (HBD) mice is related to breed-specific factors other than the response to loading.

Effects of prostaglandin E2 on production of new cancellous bone in the axial skeleton of ovariectomized rats

The effects of prostaglandin E2 (PGE2) were histomorphometrically evaluated in cancellous bone of the axial skeleton of ovariectomized, osteopenic rats. Four months following bilateral ovariectomy (OVX) and sham-ovariectomy (SHAM) at 3 months of age, rats received daily subcutaneous injections of PGE2 at 0, 0.3, 1.0, 3.0 and 6.0 mg/kg/day for 30 days. The undecalcified fourth lumbar vertebral bodies (LVB) were processed for static and dynamic bone histomorphometry. The OVX rats possessed a slightly osteopenic LVB (17% vs. 24% cancellous bone mass). In rats given PGE2 at 3 and 6 mg/kg/day for 30 days, bone turnover, lamellar bone mass, and formation of new woven bone trabeculae were increased. Observations supported the conclusion that PGE2 activates bone modeling and remodeling, and shifts bone balance in favor of formation. In OVX rats given 6 mg PGE2/kg/day, cancellous bone mass and trabecular numbers were restored to levels found in untreated SHAM rats. Cancellous bone mass in the LVB of SHAM rats given 3 and 6 mg PGE2/kg/day increased by 16% and 30% over that of control rats. In addition, PGE2 stimulated longitudinal bone growth in both OVX and SHAM rats, a response that differed from male rats.

Bone density, strength, and formation in adult cathepsin K (-/-) mice

Cathepsin K (CatK) is a cysteine protease expressed predominantly in osteoclasts, that plays a prominent role in degrading Type I collagen. Growing CatK null mice have osteopetrosis associated with a reduced ability to degrade bone matrix. Bone strength and histomorphometric endpoints in young adult CatK null mice aged more than 10 weeks have not been studied. The purpose of this paper is to describe bone mass, strength, resorption, and formation in young adult CatK null mice. In male and female wild-type (WT), heterozygous, and homozygous CatK null mice (total N=50) aged 19 weeks, in-life double fluorochrome labeling was performed. Right femurs and lumbar vertebral bodies 1-3 (LV) were evaluated by dual-energy X-ray absorptiometry (DXA) for bone mineral content (BMC) and bone mineral density (BMD). The trabecular region of the femur and the cortical region of the tibia were evaluated by histomorphometry. The left femur and sixth lumbar vertebral body were tested biomechanically. CatK (-/-) mice show higher BMD at the central and distal femur. Central femur ultimate load was positively influenced by genotype, and was positively correlated with both cortical area and BMC. Lumbar vertebral body ultimate load was also positively correlated to BMC. Genotype did not influence the relationship of ultimate load to BMC in either the central femur or vertebral body. CatK (-/-) mice had less lamellar cortical bone than WT mice. Higher bone volume, trabecular thickness, and trabecular number were observed at the distal femur in CatK (-/-) mice. Smaller marrow cavities were also present at the central femur of CatK (-/-) mice. CatK (-/-) mice exhibited greater trabecular mineralizing surface, associated with normal volume-based formation of trabecular bone. Adult CatK (-/-) mice have higher bone mass in both cortical and cancellous regions than WT mice. Though no direct measures of bone resorption rate were made, the higher cortical bone quantity is associated with a smaller marrow cavity and increased retention of non-lamellar bone, signs of decreased endocortical resorption. The relationship of bone strength to BMC does not differ with genotype, indicating the presence of bone tissue of normal quality in the absence of CatK.

Genetic Variations in Bone Density, Histomorphometry, and Strength in Mice

Abstract. The purpose of this study was to assess breed-related differences in bone histomorphometry, bone biomechanics, and serum biochemistry in three mouse breeds shown to differ in bone mineral density (BMD) (as measured by DXA) and bone mineral content (BMC). Femurs, tibiae, and sera were collected from 16-week-old C3H/HeJ {C3H}, C57BL/6J {BL6}, and DBA/2J {DBA}mice (n = 12/breed). Data collected included BMC and BMD (femora), histomorphometry of cancellous (distal femur) and cortical bone (diaphyseal tibiae and femora), bone strength (femora), and serum alkaline phosphatase (ALP). Consistent with previous reports, BMC and BMD were higher in C3H than in BL6 or DBA mice. The higher BMD in the C3H breed was associated with greater cancellous bone volume, cortical bone area, periosteal bone formation rate, biomechanical strength, and serum ALP. However, mid-diaphyseal total femoral and tibial cross-sectional area and moment of inertia were greatest in BL6, intermediate in C3H, and lowest in DBA mice. The specific distribution of cortical bone in C3H, BL6, DBA mice represents a difference in adaptive response to similar mechanical loads in these breeds. This difference in adaptive response may be intrinsic to the adaptive mechanism, or may be intrinsic to the bone tissue material properties. In either case, the bone-adaptive response to ordinary mechanical loads in the BL6 mice yields bones of lower mechanical efficiency (less stiffness per unit mass of bone tissue) and does not adapt as well as that of the C3H mice where the final product is a bone with greater resistance to bending under load. We suggest that the size, shape, and BMD of the bone are a result of breed-specific genetically regulated cellular mechanisms. Compared with the C3H mice, the lower BMD in BL6 mice is associated with long bones that are weaker because the larger cross-sectional area fails to compensate completely for their lower BMD and BMC.

Early Estrogen Replacement Therapy Reverses the Rapid Loss of Trabecular Bone Volume and Prevents Further Deterioration of Connectivity in the Rat

To evaluate the ability of estrogen replacement therapy (ERT) to prevent changes in trabecular bone volume (BV/TV) and connectivity beginning either at ovariectomy (OVX) or 5–13 days after OVX in adult female rats, the right proximal tibial was examined by three‐dimensional X‐ray tomographic microscopy (XTM) in vivo. Animals had XTM scans of the right tibia and then were randomized into six groups (n = 9). Groups 2–6 had bilateral (OVX), while group 1 was sham‐ovariectomized (OVXd) on day 0. Animals were treated with vehicle (groups 1 and 2) or 17β‐estradiol therapy (ERT) at 10 μ g/kg three times per week starting at days 0, 5, 8, and 13 post‐OVX (groups 3, 4, 5, and 6), until day 50 when they were rescanned by XTM and sacrificed. Trabecular bone structural variables were calculated from XTM data (BV/TVx and β1/BV/TVx) and standard histomorphometry. Trabecular bone volume (BV/TVx) and the trabecular connections per cubic millimeter of trabecular bone (β1/BV/TVx) were maintained in both sham‐OVXd animals and OVX animals given ERT from the time of OVX. However, OVX + vehicle–treated animals lost 54% BV/TVx and 46% β1/BV/TVx (p < 0.01 from day 0). BV/TVx and β1/BV/TVx decreased rapidly post‐OVX to −22% and −25% at day 13 (p < 0.01 from day 0). ERT initiated at day 5, 8, and 13 post‐OVX restored BV/TVx to baseline values at day 50 by modestly increasing trabecular plate thickness; however, β1/BV/TVx was reduced in all OVX groups when compared with their baseline values. ERT also caused a significant reduction in bone turnover compared with OVX + vehicle; however, resorption was suppressed more than formation. These results demonstrate that ERT can restore the lost trabecular bone, but not trabecular connectivity, that occurs soon after OVX by allowing bone formation to continue in previously activated bone remodeling units while suppressing the production of new remodeling units. This may be the mechanism by which prompt intervention with estrogen and other antiresorptive agents can restore bone mass that has been lost from the increase in remodeling space, and thereby reduce the risk of osteoporotic fractures in postmenopausal women.

Zoledronate Prevents the Development of Absolute Osteopenia Following Ovariectomy in Adult Rhesus Monkeys

This study assessed effects of the bisphosphonate zoledronate (ZLN) on bone density and biochemical markers of bone turnover in ovariectomized (OVX) adult female rhesus monkeys. Forty monkeys were randomly assigned to one control or four OVX groups. The control and one OVX group received saline, and the other three OVX groups received ZLN (0.5, 2.5, or 12.5 μg/kg) by a single weekly subcutaneous injection for 69 weeks. Bone mass of the total body (TB), lumbar spine (LS), distal and central radius (dual‐energy X‐ray absorptiometry), and skeletal turnover markers were measured at baseline and at 13, 26, 39, 52, and 69 weeks of treatment. Increased skeletal turnover and decreased bone mass (LS and TB) were demonstrable by 13 weeks post‐OVX. Maximal bone loss (7–8%) at these sites occurred by 39 weeks after OVX and persisted for the study duration. Long‐term ZLN treatment was well tolerated and prevented increased skeletal turnover and bone loss in a dose‐dependent fashion. Progressive turnover suppression was not observed with any ZLN dose. In conclusion, after OVX, adult rhesus monkeys develop persistent increased bone turnover and absolute osteopenia of the LS and TB, making them an outstanding model of skeletal behavior in perimenopausal women. These OVX‐related skeletal changes are dose‐dependently blocked by ZLN.