Stimulation and inhibition of bone formation: use of peripheral quantitative computed tomography in the mouse in vivo.

The purpose of this study was to assess peripheral quantitative computed tomography (pQCT) imaging for measurement of volumetric bone mineral density (BMD) in vivo in mouse tibia following ovariectomy, and following treatment with 17 beta-oestradiol (E2). Two studies were undertaken. In study 1, three groups (n = 10) of mature mice were ovariectomized (OVX) or sham operated (SHAM); one of the OVX groups was dosed weekly with E2 (OVX.E2). Images of the proximal tibia were acquired on the day of surgery and at intervals following surgery until week 6. In study 2, four groups (OVX, SHAM, OVX.E2 and a SHAM group dosed with E2, SHAM.E2) of immature mice (n = 10) were imaged weekly up to 10 weeks post-surgery. Precision of pQCT for measurement of total (trabecular plus cortical) BMD was 2.4%, trabecular 5.2% and cortical 2.6%. In mature animals, significantly slower net bone formation was seen in OVX compared with SHAM animals using paired analysis with each animal as its own control. Group analysis detected no significant difference in BMD between SHAM and OVX at any time point. In immature animals, using paired analysis, with each animal as its own control, a significant difference between SHAM and OVX animals was detectable 3 weeks post-surgery (P < 0.05). As in study 1, group analysis of total BMD failed to detect any significant difference between SHAM and OVX at any time point. Treatment with E2 caused an easily-detected increase in BMD and led to osteopetrosis in both groups. The statistical power of this technique is adequate for testing antiresorptive or bone-forming therapies in the mouse.

Effects of an inhibitor of cathepsin L on bone resorption in thyroparathyroidectomized and ovariectomized rats.

The process of bone resorption by osteoclasts involves the dissolution of mineral salts and enzymatic degradation of the mainly collagenous extracellular matrix. Cysteine proteinases, which can efficiently degrade collagen at acidic pH, have been suggested to play an important role in the bone resorptive process. The cysteine proteinase cathepsin L is secreted by osteoclasts, and inhibitors of this enzyme can prevent bone resorption in vitro. The activity of acetyl-leu-leu-norleucinol (ALLN), a selective inhibitor of cathepsin L, was investigated in two models of bone resorption in vivo. In the first study, the ability of ALLN to inhibit bone resorption was investigated in Ro-13-6298 (arotinoid)-treated thyroparathyroidectomized (TPTX) rats. ALLN [100 mg/kg, intraperitoneally (i.p.)] inhibited hypercalcemia by 62.8% acutely (p < 0.001), compared to 94.9% (p < 0.001) inhibition by salmon calcitonin (sCT) (10 IU/kg, subcutaneously). In rats treated for 3 days with ALLN, arotinoid-induced reduction in cortical bone mineral density measured by peripheral quantitative computed tomography (pQCT) was inhibited by 86.4% (p < 0.05) in rats treated with ALLN 100 mg/kg, i.p., and by 82% in rats treated with 50 mg/kg, i.p. (p < 0.05). In a second study, the efficacy of ALLN was tested in a longitudinal study in ovariectomized (ovx) rats. Bone loss, measured by pQCT, was unaffected by treatment with ALLN. The bisphosphonate alendronate, however, inhibited bone loss in this model. These data demonstrate the ability of a cathepsin L inhibitor to inhibit bone resorption in arotinoid-treated TPTX rats, a process which may be dependent on the activity of cathepsin L-like cysteine proteinases. In contrast to its effects in TPTX rats, ALLN had no inhibitory activity on bone resorption in ovx rats. It is possible that in chronic bone resorption in ovx rats, the activity of other enzymes such as cathepsins OC-2 or K allows the process of resorption to continue even when cathepsin L is inhibited by ALLN. Further studies are required to determine why the activity of ALLN varies between different animal models. These data indicate that there may be variations in the effects of drugs in different animal models of bone resorption which should be considered when investigating novel antiresorptive therapies.

Effect of ovariectomy on magnetic resonance T2* in rat femur.

Measurements have been made in the rat femur in vivo and ex vivo by using an asymmetric spin echo technique of T2(1), the susceptibility contribution to T2*. The trabecular spacing in this study in rat bone is considerably less than in previous studies in the human. A significant increase in T2(1) was seen in vivo 3 mm proximal to the growth plate with ovariectomy (a model of osteopenia), from 8.1 +/- 0.7 to 10.0 +/- 0.6 ms. Parallel changes in trabecular bone mineral density measured by quantitative computed tomography were found. T2(1) was higher in living bones than in the same bones measured post mortem.

Regional analysis of bone mineral density in the distal femur and proximal tibia using peripheral quantitative computed tomography in the rat In vivo.

The use of peripheral quantitative computed tomography (pQCT) was investigated for the measurement of volumetric bone mineral density (BMD) in mg x cm-3. Two studies were undertaken. In the first study, the precision of pQCT in vivo and ex vivo was tested at 14 weeks postovariectomy (OVX). In the second study, the efficacy of a standard antiresorptive treatment, 17beta-estradiol (E2), was tested 6 weeks post-OVX. The precision for total (compact plus trabecular) BMD was 1.3-1.9%, and that for trabecular BMD was 2.4-2. 7%. There was excellent agreement between trabecular BMD measurements in vivo and ex vivo (r = 0.91). Significant reductions in trabecular BMD were observed in vivo at 14 and 6 weeks following ovariectomy in the femur, in each study. The loss of trabecular BMD depended on slice location, and varied from 0 to 22% at 6 weeks, and from 0 to 26% at 14 weeks (P < 0.001, at the affected locations). The antiresorptive effect of treatment was demonstrated in the 6-week study: there was no significant difference in BMD between sham-operated and E2-treated OVX rats.

Sustained release of salmon calcitonin in vivo from lactide: glycolide copolymer depots.

Studies were carried out to determine whether monolithic depot formulations, prepared using lactide:glycolide copolymers, could be used to administer salmon calcitonin (sCT) to rats in vivo. Formulations containing 2, 5, or 10% (w/w) sCT were administered subcutaneously to female Wistar strain rats. Release of sCT was determined by measurement of peptide in plasma using a specific radioimmunoassay and by measurement of residual sCT in the depots after recovery at postmortem. Plasma calcium concentrations and cumulative weight gain of the animals were used to measure pharmacological effects of the released sCT. Release of sCT from the depots was controlled by the copolymer and was sustained for periods up to 10 days. However, the release of sCT from the depots did not significantly alter plasma calcium concentrations, and effects on cumulative weight gain were small and transient. Peptide loading of the formulations was shown to modify sCT release. Maximal release of sCT from depots containing 10% peptide occurred over a 7 to 14-day period postadministration, with 5% sCT release occurred between days 11 and 14, and with 2% sCT, the period of maximal release was between days 11 and 18. Release of peptide from the depots was essentially complete by 21 days postadministration irrespective of the peptide loading. These data suggest that lactide:glycolide copolymer depots may have application for the convenient clinical administration of sCT in metabolic bone diseases.

Immunization with calcitonin gene-related peptide reduces the inflammatory response to adjuvant arthritis in the rat.

The influence of circulating antibodies to calcitonin gene-related peptide on the inflammatory response was examined in rats with adjuvant-induced arthritis. Rats were immunized with alpha calcitonin gene-related peptide conjugated to thyroglobulin, and circulating antibodies were identified by their capacity to bind radiolabelled rat alpha or human calcitonin gene-related peptide. In unimmunized rats and rats immunized with thyroglobulin alone, the secondary lesions (characterized as paw swelling, nodules on ears and tail, and inflamed nose) produced after adjuvant-induced arthritis were similar. However, at 21 days, when these lesions were maximal, the animals immunized with calcitonin gene-related peptide showed decreased numbers of lesions. An additional marker of disease activity, namely alpha 1 glycoprotein levels in plasma, was also measured. Again, plasma alpha 1 glycoprotein levels were similar in rats that were unimmunized or received thyroglobulin alone, but at 21 days were significantly reduced in animals immunized with calcitonin gene-related peptide. In contrast, the initial foot swelling seen in the first few days after injection of adjuvant was not significantly different in the various groups. The results suggest that antibodies to calcitonin gene-related peptide are able to reduce the severity of the adjuvant arthritis syndrome, and that this peptide contributes to the inflammatory response seen in the later stages of the disease model.