Hua Zhu Ke

FDA Guidelines and animal models for osteoporosis.

The recent FDA Guidelines For Preclinical and Clinical Evaluation of Agents Used in the Treatment or Prevention of Postmenopausal Osteoporosis (1994) delineate specific preclinical animal models to demonstrate the efficacy and safety of new, potential agents for osteoporosis therapy. The Guidelines recommend that agents be evaluated in two animal species, including the ovariectomized (OVX) rat and in a second non-rodent model. We have performed a series of studies to determine whether the recommended OVX rat models, endpoints, and study design adequately address the efficacy and safety of therapeutic agents for the treatment or prevention of osteoporosis. Our study results indicate that the rat OVX model mimics postmenopausal cancellous bone loss when examined over relatively short periods of time. These data illustrate that cancellous bone turnover increases following OVX and this increased bone turnover produces bone loss. Estrogen completely blocks the activation of bone turnover and bone loss. Thus, our data suggest that the rat OVX model in the proximal tibia, distal femur, and lumbar vertebrae mimics conditions in the postmenopausal woman and is suitable for the evaluation of potential therapeutic agents for the prevention of osteoporosis. However, when the duration of the studies extends to 12 months as suggested by the Guidelines, the indices of cancellous bone turnover return to the value of sham controls, although the trabecular bone volume remains lower than that of sham controls in OVX rats. Therefore, it is difficult to determine the effects of potential therapeutic agents on the bone turnover in estrogen deficient conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass

We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation ofOF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined thatOF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse.

Effects of CP-336,156, a New, Nonsteroidal Estrogen Agonist/Antagonist, on Bone, Serum Cholesterol, Uterus, and Body Composition in Rat Models.

We have discovered a new, nonsteroidal, potent estrogen agonist/antagonist, CP-336,156. CP-336,156 binds selectively and with high affinity to the human estrogen receptor-α with a half-inhibition concentration of 1.5 nm, which is similar to that seen with estradiol (4.8 nm). When given orally to immature (3-week-old) female Sprague-Dawley rats for 3 days at doses of 0.1, 1.0, 10, or 100 μg/kg·day, unlike 17α-ethynyl estradiol, CP-336,156 had no effect on uterine wet or dry weight. Similarly, no uterine hypertrophy was observed in aged (17-month-old) female rats treated (po) with CP-336,156 at 10 or 100 μg/kg·day for 28 days. We also found that CP-336,156 decreased total serum cholesterol and fat body mass and had no effect on lean body mass in these aged female rats. In 5-month-old ovariectomized (OVX) Sprague-Dawley female rats, CP-336,156 completely prevented OVX-induced increases in body weight gain, total serum cholesterol, and serum osteocalcin at doses between 10 and 1000 μg/kg·day after 4 weeks. At...

Comparative effects of droloxifene, tamoxifen, and estrogen on bone, serum cholesterol, and uterine histology in the ovariectomized rat model

The purpose of this study was to compare the effects of droloxifene (DRO), tamoxifen (TAM), and 17 alpha-ethynyl estradiol (EE) on bone mineral density, bone histomorphometry, total serum cholesterol, and uterine histology in the ovariectomized (ovx) rat model. Sprague-Dawley female rats at five months of age were sham-operated and treated orally with vehicle (n = 8), or ovx (n = 56) and treated (p.o.) with either vehicle, DRO at 0.1 or 1.0 mg/kg daily, TAM at 0.1 or 1 mg/kg daily, or EE at 3 or 30 micrograms/kg daily for 4 weeks. The uterine wet weight and uterine histologic parameters (cross-sectional tissue area, stromal thickness, and luminal epithelial thickness) were determined. Femoral and lumbar vertebral bone mineral density was determined ex vivo using dual energy x-ray absorptiometry. Static and dynamic cancellous bone histomorphometry was performed on double-labeled, undecalcified longitudinal sections from proximal tibial metaphyses. Furthermore, the changes in total serum cholesterol and body weight gain were also determined. Compared to sham controls, ovx for four weeks significantly decreased uterine weight (-72%), uterine cross-sectional tissue area (-74%), stromal thickness (-52%), and luminal epithelial thickness (-53%). ovx rats treated with EE at 30 micrograms/kg/day maintained these parameters at the levels of sham controls. Uterine weight and uterine cross-sectional tissue area in 3 micrograms/kg/day of EE treated ovx rats were higher than that of vehicle-treated ovx rats. In ovx rats treated with TAM at both 0.1 and 1 mg/kg/day, these parameters were significantly less than sham controls but significantly higher than ovx controls. DRO at 0.1 mg/kg/day had no effects on all above parameters. Uterine weight and cross-sectional tissue area in 1 mg/kg/day of DRO treated ovx rats was slightly but significantly higher than that in ovx controls. However, DRO at 1 mg/kg/day had no effects on uterine stromal thickness and luminal epithelial thickness compared to ovx controls. The ovx-induced decrease in femoral and lumbar vertebral bone mineral density was prevented by treatment with EE at 30 micrograms/kg/day, TAM at both 0.1 and 1 mg/kg/day, or DRO at 1 mg/kg/day. Similarly, the decrease in bone mass and the increase in bone resorption and bone turnover in proximal tibial metaphyses were prevented by treatment with EE at 30 micrograms/kg/day or TAM at both 0.1 and 1 mg/kg/day, or DRO at 1 mg/kg/day. Total serum cholesterol decreased significantly in ovx rats treated with either EE, DRO, or TAM at all dose levels compared to vehicle treated ovx controls (-32% to -56%). The ovx-induced body weight gain was completely prevented by EE at 30 micrograms/kg/day, and partially prevented by DRO at 1 mg/kg/day. TAM at both 0.1 and 1 mg doses caused a significant decrease in body weight compared to both sham and ovx controls. Our results indicated that DRO prevented ovx-induced bone loss and lowered total serum cholesterol with an ED50 less than 1 mg/kg/day. The bone protective and cholesterol lowering effects of DRO were comparable to those observed with TAM and EE. However, DRO differed from TAM and EE in its lack of significant estrogenic effects on uterine tissue at doses which were bone protective. These data suggest that DRO may be a significant alternative to EE and TAM for prevention and treatment of postmenopausal osteoporosis.

A novel, non-prostanoid EP2 receptor-selective prostaglandin E2 agonist stimulates local bone formation and enhances fracture healing.

CP‐533,536, a newly discovered, non‐prostanoid EP2 receptor‐selective PGE2 agonist, stimulates local bone formation and enhances fracture healing in rat models.

Lasofoxifene (CP-336,156) Protects Against the Age-Related Changes in Bone Mass, Bone Strength, and Total Serum Cholesterol in Intact Aged Male Rats

The purpose of this study was to evaluate if long‐term (6 months) treatment with lasofoxifene (LAS), a new selective estrogen receptor modulator (SERM), can protect against age‐related changes in bone mass and bone strength in intact aged male rats. Sprague‐Dawley male rats at 15 months of age were treated (daily oral gavage) with either vehicle (n = 12) or LAS at 0.01 mg/kg per day (n = 12) or 0.1 mg/kg per day (n = 11) for 6 months. A group of 15 rats was necropsied at 15 months of age and served as basal controls. No significant change was found in body weight between basal and vehicle controls. However, an age‐related increase in fat body mass (+42%) and decrease in lean body mass (−8.5%) was observed in controls. Compared with vehicle controls, LAS at both doses significantly decreased body weight and fat body mass but did not affect lean body mass. No significant difference was found in prostate wet weight among all groups. Total serum cholesterol was significantly decreased in all LAS‐treated rats compared with both the basal and the vehicle controls. Both doses of LAS treatment completely prevented the age‐related increase in serum osteocalcin. Peripheral quantitative computerized tomography (pQCT) analysis at the distal femoral metaphysis indicated that the age‐related decrease in total density, trabecular density, and cortical thickness was completely prevented by treatment with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day. Histomorphometric analysis of proximal tibial cancellous bone showed an age‐related decrease in trabecular bone volume (TBV; −46%), trabecular number (Tb.N), wall thickness (W.Th), mineral apposition rate, and bone formation rate‐tissue area referent. Moreover, an age‐related increase in trabecular separation (Tb.Sp) and eroded surface was observed. LAS at 0.01 mg/kg per day or 0.1 mg/kg per day completely prevented these age‐related changes in bone mass, bone structure, and bone turnover. Similarly, the age‐related decrease in TBV and trabecular thickness (Tb.Th) and the age‐related increase in osteoclast number (Oc.N) and osteoclast surface (Oc.S) in the third lumbar vertebral cancellous bone were completely prevented by treatment with LAS at both doses. Further, LAS at both doses completely prevented the age‐related decrease in ultimate strength (−47%) and stiffness (−37%) of the fifth lumbar vertebral body. These results show that treatment with LAS for 6 months in male rats completely prevents the age‐related decreases in bone mass and bone strength by inhibiting the increased bone resorption and bone turnover associated with aging. Further, LAS reduced total serum cholesterol and did not affect the prostate weight in these rats. Our data support the potential use of a SERM for protecting against the age‐related changes in bone and serum cholesterol in elderly men.

IL-1 receptor-associated kinase M is a central regulator of osteoclast differentiation and activation

Osteoporosis is a serious problem worldwide; it is characterized by bone fractures in response to relatively mild trauma. Osteoclasts originate from the fusion of macrophages and they play a central role in bone development and remodeling via the resorption of bone. Therefore, osteoclasts are important mediators of bone loss that leads, for example, to osteoporosis. Interleukin (IL)-1 receptor (IL-1R)–associated kinase M (IRAK-M) is only expressed in cells of the myeloid lineage and it inhibits signaling downstream of IL-1R and Toll-like receptors (TLRs). However, it lacks a functional catalytic site and, thus, cannot function as a kinase. IRAK-M associates with, and prevents the dissociation of, IRAK–IRAK-4–TNF receptor–associated factor 6 from the TLR signaling complex, with resultant disruption of downstream signaling. Thus, IRAK-M acts as a dominant negative IRAK. We show here that mice that lack IRAK-M develop severe osteoporosis, which is associated with the accelerated differentiation of osteoclasts, an increase in the half-life of osteoclasts, and their activation. Ligation of IL-1R or TLRs results in hyperactivation of NF-κB and mitogen-activated protein kinase signaling pathways, which are essential for osteoclast differentiation. Thus, IRAK-M is a key regulator of the bone loss that is due to osteoclastic resorption of bone.

Regulation of BMP-7 expression by retinoic acid and prostaglandin E2

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor‐beta (TGF‐β) gene superfamily of growth and differentiation factors. Members of the BMP family were originally cloned and characterized by their ability to induce ectopic bone formation. Of the various BMPs cloned, the bone inductive ability of BMP‐7 (OP‐1) and BMP‐2 has been well characterized. Both BMP‐7 and ‐2 have been shown to have clinical utility in the healing of non‐union fractures. However, in spite of the various advances in BMP research, the physiological regulation of BMPs is not well understood. Here we studied the expression of BMP‐7 by cloning a 4.6‐kB fragment of the human BMP‐7 promoter (hBMP‐7p) and placing it upstream of a luciferase reporter. The promoter reporter construct was stably transfected into different cell backgrounds and its regulation by various factors was investigated. We show that retinoic acid (RA) treatment results in an upregulation of the hBMP‐7p reporter activity. This regulation of the hBMP‐7p was further confirmed by Northern blot, PCR, and Western blot analyses, which showed an increase in both BMP‐7 mRNA and protein expression upon treatment with RA. We further show that RA specifically upregulates expression of osteocalcin via activation of BMP‐7 mRNA and protein in vitro. Similarly, prostaglandin E2 (PGE2) treatment increases BMP‐7 mRNA and protein levels, but does not transcriptionally activate the hBMP‐7p. Additionally, in vivo expression of BMP‐7 in bone was increased upon PGE2 treatment. In conclusion, RA and PGE2 upregulate BMP‐7 protein expression both in vitro and in vivo. J. Cell. Physiol. 190: 207–217, 2002.

Combination therapy for osteoporosis

Pharmaceutical combination compositions including certain estrogen agonists/antagonists and prostaglandins or prostaglandin agonists/antagonists. The compositions are useful for the treatment of bone disorders including osteoporosis.

A nonprostanoid EP4 receptor selective prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats.

CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia.