Charlotte E. Hotchkiss

Levormeloxifene prevents increased bone turnover and vertebral bone loss following ovariectomy in cynomolgus monkeys.

Levormeloxifene, a nonsteroidal selective estrogen receptor modulator (SERM), has been evaluated for its effects on bone in cynomolgus monkeys (Macaca fascicularis). Adult female monkeys were imported from Indonesia and randomized into six groups of 25-28 animals each (n = 158). Animals in one group were sham ovariectomized (sham) and received vehicle. Animals in the remaining five groups were ovariectomized and received either vehicle (ovx); 17beta-estradiol at 0.016 mg/kg (est); or levormeloxifene at 0.5 (L1), 1 (L2), or 5 (L3) mg/kg. Lumbar spine and whole body bone mass were measured by dual-energy X-ray absorptiometry (DXA) pretreatment and at 6 and 12 months following the initiation of treatment. Bone mass at the femoral neck was measured by peripheral quantitative computed tomography (pQCT) at 0 and 12 months. Serum markers of bone turnover, including bone-specific alkaline phosphatase (BSAP), osteocalcin (BGP), tartrate-resistant acid phosphatase (TRAP), and urinary collagen C-terminal extension peptides (CrossLaps), were measured at 0, 6, and 12 months. Ovariectomy resulted in an increase in these markers; the increase was prevented by estradiol or levormeloxifene. Estradiol or levormeloxifene inhibited loss of lumbar spine bone mineral density (BMD) following ovariectomy compared with untreated monkeys (ovx -5.0%; sham -0.4%; est +0.2%; L1 -3.6%, L2 -2.0%, L3 -2.5%). Estradiol, but not levormeloxifene, prevented loss of BMD at the femoral neck (ovx -7.4%; sham -3.1%; est -3.6%; L1 -8.0%, L2 -6.5%, L3 -7.8%), and whole body bone mineral content (BMC) (ovx -7.6%; sham -1.9%, est -2.9%; L1 -6.2%, L2 -6.1%, L3 -6.7%). Bone loss at each site was correlated with bone turnover as measured by serum and urine biomarkers. There was no dose effect of levormeloxifene.

Use of peripheral quantitative computed tomography for densitometry of the femoral neck and spine in cynomolgus monkeys (Macaca fascicularis)

Quantitative computed tomography (QCT) allows for the separate densitometric examination of cortical and cancellous bone in vivo. With the new peripheral QCT (pQCT) instrument (the Norland/Stratec XCT-3000A), we evaluated the clinically relevant axial sites of spine and femoral neck in nonhuman primates in vivo. The reproducibility was good (coefficient of variation [CV] <3% at both sites for cortical, trabecular, and total bone mineral density [BMD]; CV 3%-7% for bone mineral content [BMC] and cross-sectional bone area). One hundred sixty intact female cynomolgus monkeys (M. fascicularis) were scanned at the femoral neck. There was less variability among monkeys in cortical BMD (mean 802 mg/mL, CV 6%) as opposed to trabecular BMD (mean 334 mg/mL, CV 28%) or transition zone BMD (mean 457 mg/mL, CV 12%). Scans were performed on lumbar vertebrae (L-4, L-5, and L-6) from five monkeys in vivo and ex vivo. Removal of soft tissue increased measured BMD. Decreasing voxel size from 0.4 mm to 0.2 mm increased measured BMD by diminishing the partial volume effect. Factor analysis demonstrated the expected relationships between pQCT parameters and physical measurement of bone mass and volume ex vivo. Preliminary results in eight ovariectomized and eight reproductively intact monkeys revealed a lower transition zone BMD at the femoral neck, and lower total BMD of the vertebral body in estrogen-deficient animals.

The anesthetic isoflurane decreases ionized calcium and increases parathyroid hormone and osteocalcin in cynomolgus monkeys

The effects of anesthetics on calcium metabolism in cynomolgus monkeys were studied. Eight adult female cynomolgus monkeys were used in a crossover design. Blood was collected from each of the monkeys at four timepoints: (1) while conscious; (2) following induction of anesthesia with ketamine, ketamine and atropine, isoflurane, or no anesthetic; (3) at 30 min; and (4) 120 min thereafter. Four experiments were performed with a 1 week washout period between sessions, such that each monkey received each treatment. Potassium was lower in anesthetized monkeys than in those that remained conscious. Cortisol, although high, did not differ among anesthetic treatments. Ketamine and ketamine/atropine did not consistently affect ionized calcium or parathyroid hormone (PTH) concentrations. Isoflurane decreased ionized calcium (0.05 mmol/L), and increased PTH and osteocalcin twofold. The serum inorganic fluoride concentration was higher in monkeys anesthetized with isoflurane than with ketamine/atropine, which may partially account for the decrease in ionized calcium with isoflurane. The increases in PTH and osteocalcin are presumably secondary to the decrease in ionized calcium.

Evaluation of a nonhuman primate model to study circadian rhythms of calcium metabolism

We evaluated primate models for the study of circadian rhythms in calcium and bone metabolism. Blood and urine were collected from two cynomolgus macaques every 4 h for 24 h. Studies were initiated at three different clock times to separate the effects of repeated experimental sampling from circadian effects. Also, samples were collected from seven monkeys at times of expected maxima and minima. Some parameters exhibited the expected circadian rhythm with increases at night (serum total calcium) or in the early morning (urinary collagen cross-links). Others displayed the effects of the experimental procedure, either increasing (urinary creatinine and phosphorus) or decreasing (osteocalcin, urinary calcium) with repeated sampling. Serum phosphorus, cortisol, and type I procollagen were influenced by both clock time and experimental procedures. Alkaline phosphatase and parathyroid hormone did not show any differences with time or sampling. This data is consistent with findings in humans that bone resorption increases at night and that endogenous corticosteroids decrease bone formation. The usefulness of the monkey model is limited by the physiological stress of sample collection in these subjects.We evaluated primate models for the study of circadian rhythms in calcium and bone metabolism. Blood and urine were collected from two cynomolgus macaques every 4 h for 24 h. Studies were initiated at three different clock times to separate the effects of repeated experimental sampling from circadian effects. Also, samples were collected from seven monkeys at times of expected maxima and minima. Some parameters exhibited the expected circadian rhythm with increases at night (serum total calcium) or in the early morning (urinary collagen cross-links). Others displayed the effects of the experimental procedure, either increasing (urinary creatinine and phosphorus) or decreasing (osteocalcin, urinary calcium) with repeated sampling. Serum phosphorus, cortisol, and type I procollagen were influenced by both clock time and experimental procedures. Alkaline phosphatase and parathyroid hormone did not show any differences with time or sampling. This data is consistent with findings in humans that bone resorption increases at night and that endogenous corticosteroids decrease bone formation. The usefulness of the monkey model is limited by the physiological stress of sample collection in these subjects.