Roger Phipps

Risedronate Preserves Trabecular Architecture and Increases Bone Strength in Vertebra of Ovariectomized Minipigs as Measured by Three‐Dimensional Microcomputed Tomography

Risedronate reduces the risk of new vertebral fractures up to 70% within 1 year of treatment in patients with osteoporosis. Both increases in bone mass and preservation of bone architecture are thought to contribute to antifracture effects. Our objectives were to determine the effects of risedronate on trabecular bone mass and architecture and to determine the relative contributions of mass and architecture to strength in the vertebra of ovariectomized (OVX) minipigs. The minipigs were OVX at 18 months of age and were treated daily for 18 months with either vehicle or risedronate at doses of 0.5 mg/kg per day or 2.5 mg/kg per day. The three‐dimensional (3D) bone architecture of the L4 vertebral cores of Sinclair S1 minipigs was evaluated by 3D microcomputed tomography (μCT). Compared with the OVX control, the vertebral bone volume (bone volume/tissue volume [BV/TV]) was higher in both treated groups (p < 0.05). The architectural changes were more significant at the 2.5‐mg/kg dose and were more prevalent at the cranial‐caudal ends compared with the midsection. At the higher dose, the trabecular thickness (Tb.Th), trabecular number (Tb.N), and connectivity were higher, and marrow star volume (Ma.St.V) and trabecular separation (Tb.Sp) were lower (p < 0.05). The trabecular separation variation index(TSVI), a new measure to approximate structural variations, was smaller in the 2.5‐mg/kg‐treated group (p < 0.05). In this group, a significant preservation of trabeculae orthogonal to the cranial‐caudal axis was confirmed by a decrease in the degree of anisotropy (DA) and an increase in the percent Cross‐strut (%Cross‐strut; p < 0.05). Both normalized maximum load (strength) and normalized stiffness of the same vertebral cores were higher in the 2.5‐mg/kg risedronate group compared with the OVX group (p < 0.05). BV/TV alone could explain 76% of the variability of the bone strength. The combination of bone volume and architectural variables explained >90% of the strength. The study showed that risedronate preserved trabecular architecture in the vertebra of OVX minipigs, and that bone strength is tightly coupled to bone mass and architecture.

Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42, and Rho GTPases

N‐BPs, which inhibit bone resorption by preventing prenylation of small GTPases, unexpectedly cause the accumulation of GTP‐bound, unprenylated Rho family GTPases in macrophages and osteoclasts. In macrophages, this also leads to sustained, Rac‐mediated activation of p38. The antiresorptive activity of N‐BPs may therefore be caused at least in part, by the accumulation of unprenylated small GTPases, causing inappropriate activation of downstream signaling pathways.

Exosome Delivered Anticancer Drugs Across the Blood-Brain Barrier for Brain Cancer Therapy in Danio Rerio

PurposeThe blood–brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model.Materials and MethodsFour types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model.ResultsFour exosomes in 30–100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker.ConclusionsBrain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

Effects of 3- and 5-Year Treatment With Risedronate on Bone Mineralization Density Distribution in Triple Biopsies of the Iliac Crest in Postmenopausal Women

Long‐term effects of risedronate on bone mineralization density distribution in triple transiliac crest biopsies of osteoporotic women were evaluated. In this double‐blinded study, 3‐ and 5‐year treatment with risedronate increased the degree and homogeneity of mineralization without producing hypermineralization. These changes at the material level of bone could contribute to risedronates antifracture efficacy.

Bone material properties in trabecular bone from human iliac crest biopsies after 3- and 5-year treatment with risedronate.

Long‐term effects of risedronate on bone mineral maturity/crystallinity and collagen cross‐link ratio in triple iliac crest biopsies of osteoporotic women were evaluated. In this double‐blinded study, 3‐ and 5‐year treatment with risedronate arrested the tissue aging encountered in untreated osteoporosis and in osteoporosis treated with other antiresorptives. This effect may be contributing to risedronates antifracture efficacy.

Nitrogen-containing bisphosphonates induce apoptosis of Caco-2 cells in vitro by inhibiting the mevalonate pathway: a model of bisphosphonate-induced gastrointestinal toxicity.

Bisphosphonates have become an important addition to the pharmacological armamentarium against postmenopausal osteoporosis. One of the major side effects of oral therapy with some nitrogen-containing bisphosphonates appears to be gastrointestinal (GI) intolerability, particularly esophageal irritation and ulceration. Because nitrogen-containing bisphosphonates can cause apoptosis in a variety of cell types in vitro, by inhibiting the mevalonate pathway, we hypothesized that the effect of these agents on the GI tract may be due to apoptosis or inhibition of growth of gut epithelial cells. A comparison between clodronate, etidronate, pamidronate, alendronate, and risedronate demonstrated that only the nitrogen-containing bisphosphonates were effective at inducing apoptosis or inhibiting proliferation of Caco-2 human epithelial cells in vitro, at concentrations of between 10 and 1000 micromol/L. The ability of nitrogen-containing bisphosphonates to cause apoptosis and inhibit Caco-2 cell proliferation was due to inhibition of the mevalonate pathway, because the addition of farnesol, oxidized low-density lipoprotein (LDL) cholesterol, or especially geranylgeraniol suppressed the effects. Furthermore, pamidronate, alendronate, and risedronate inhibited protein prenylation in Caco-2 cells, as determined by analysis of the processing of Rap1A, a prenylated small GTPase. These studies suggest that the effects of nitrogen-containing bisphosphonates observed in the GI tract may be due to inhibition of proliferation or apoptosis of gut epithelial cells, following loss of prenylated proteins and sterols.

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral.

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and β-APN treated animals were fed additionally with β-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2–L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that β-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p < 0.05). Further, compression tests revealed a significant negative impact of β-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, β-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of β-APN, despite a similar mineral content. In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.

Treatment with risedronate or alendronate prevents hind-limb immobilization-induced loss of bone density and strength in adult female rats.

Immobilization leads to rapid loss of bone mass and mechanical competence, and long-term immobilization or repeated periods of short-term immobilization can have serious skeletal consequences and may lead to increased fracture liability. The aim of the present preclinical study was, therefore, to assess whether two antiresorptive agents, risedronate (Ris) or alendronate (Aln), would be capable of preventing immobilization-induced loss of bone mass and strength in rats. The study was designed as a dose-response study, and the site-specific effects of immobilization and of treatment are described. Four-month-old virgin female Sprague-Dawley rats were divided into eight groups with 12 animals in each group: (1) immobilized (Imm) control; (2) normal control; (3) Imm + Ris 0.1 mg/kg body weight/day (b.w./day); (4) Imm + Ris 0.2 mg/kg b.w./day; (5) Imm + Ris 1.0 mg/kg b.w./day; (6) Imm + Aln 0.2 mg/kg b.w./day; (7) Imm + Aln 1.0 mg/kg b.w./day; and (8) Imm + Aln 2.0 mg/kg b.w. /day. In groups 1 and 3-8, the right hind leg was immobilized with an elastic bandage. The study period was 28 days. The effects of unilateral hind-limb immobilization and of treatment were determined by dual-energy X-ray absorptiometry (DEXA) measurements on tibiae and by biomechanical testing of femora at three different sites: diaphysis; femoral neck; and distal metaphysis. Bilateral measurements were performed (on the immobilized and nonimmobilized legs). Immobilization induced a significant loss of bone mineral density (BMD) at the proximal tibial metaphysis, but no change at the mid-diaphysis. Furthermore, immobilization induced a loss of bone strength at the two femoral metaphyses, but no change was seen in three-point bending of the diaphysis. Both risedronate and alendronate treatment showed a dose-dependent protection against the immobilization-induced loss of bone density and strength at the metaphyses. We conclude that, in rats, short-term hind-limb immobilization affects only the metaphyses and that no changes are seen in the diaphysis. Both risedronate and alendronate can prevent immobilization-induced bone loss at the metaphyses. The present study confirms the importance of examining several skeletal sites when testing the efficacy of therapeutic agents.

Effect of temporal changes in bone turnover on the bone mineralization density distribution: a computer simulation study.

The heterogeneous distribution of mineral content in trabecular bone reflects the continuous renewal of bone material in bone remodeling and the subsequent increase in mineral content in the newly formed bone packets. The bone mineralization density distribution (BMDD) is typically used to describe this nonuniform mineral content of the bone matrix. Our mathematical model describes changes of the BMDD of trabecular bone as a function of bone resorption and deposition rates and the mineralization kinetics in a newly formed bone packet. Input parameters used in the simulations were taken from experimental studies. The simulations of the time evolution of the BMDD after increase in bone turnover (perimenopausal period) resulted in a shift of the BMDD toward lower values of the mineral content. Transiently, there was a broadening of the BMDD configuration partly showing two peaks, which points to a strongly heterogeneous distribution of the mineral. Conversely, when the remodeling rate was reduced (antiresorptive therapy), the BMDD shifted toward higher values of the mineral content. There was a transient narrowing of the distribution before broadening again to reach the new steady state. Results from this latter simulation are in good agreement with measurements of the BMDD of patients after 3 and 5 yr of treatment with risedronate. Based on available experimental data on bone remodeling, this model gives reliable predictions of changes in BMDD, an important factor of bone material quality. With the availability of medications with a known effect on bone turnover, this knowledge opens the possibility for therapeutic manipulation of the BMDD.

Prevention of Bone Loss in Ovariectomized Rats by Combined Treatment With Risedronate and 1α,25‐Dihydroxyvitamin D3

Bisphosphonates inhibit bone loss through inhibition of osteoclast‐mediated bone resorption. At low doses, vitamin D metabolites can prevent bone loss in models of osteopenia in rats by an antiresorptive effect, while at high doses they also stimulate osteoblast activity and show an anabolic effect. Therefore, combined therapy with bisphosphonates and vitamin D analogs might be expected to be more effective than either treatment alone. It was the aim of this study to compare the efficacy of risedronate and of the naturally occurring vitamin D hormone 1α,25‐dihydroxyvitamin D3 (calcitriol), alone and in combination, for the prevention of ovariectomy‐induced bone loss in rats. One hundred ten female 4‐month‐old Sprague‐Dawley rats were used for this experiment. Ninety rats were bilaterally ovariectomized (OVX), 10 rats were sham‐operated (SHAM), and 10 rats were killed at the time of surgery as a baseline control. Groups of rats (10 rats/group) received vehicle or daily doses of 0.1 mg or 0.5 mg of risedronate or 0.05 μg or 0.1 μg of calcitriol/kg body weight, alone and in combination. Both compounds were administered orally via gavage, commencing on the day after surgery. Although estrogen deficiency‐induced bone loss was prevented by individual prophylactic administration of risedronate or calcitriol, OVX rats treated with a combination of risedronate and calcitriol had higher bone mineral density (BMD), cancellous bone area (B.Ar), and bone strength in long bones and vertebrae compared with rats receiving risedronate alone. Furthermore, calcitriol enhanced the suppressive effects of risedronate on osteoclast number and partially counteracted the suppressive effects of risedronate on bone formation and histomorphometric indices of osteoblast team performance. Risedronate did not reduce the anabolic effect of calcitriol, and at the high dose it normalized hypercalcemia in calcitriol‐treated OVX rats. Therefore, this study in OVX rats suggests that combined therapy with bisphosphonates and vitamin D analogs may offer advantages over the treatment with bisphosphonates or vitamin D analogs alone.