Differential temporal effects of sclerostin antibody and parathyroid hormone on cancellous and cortical bone and quantitative differences in effects on the osteoblast lineage in young intact rats.

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague-Dawley rats were administered either vehicle, Scl-Ab (3 or 50mg/kg/week subcutaneously), or human PTH (1-34) (75 µg/kg/day subcutaneously) for 4 or 26 weeks. The 50mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume. Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.

Common handling procedures conducted in preclinical safety studies result in minimal hepatic gene expression changes in Sprague-Dawley rats.

Gene expression profiling is a tool to gain mechanistic understanding of adverse effects in response to compound exposure. However, little is known about how the common handling procedures of experimental animals during a preclinical study alter baseline gene expression. We report gene expression changes in the livers of female Sprague-Dawley rats following common handling procedures. Baseline gene expression changes identified in this study provide insight on how these changes may affect interpretation of gene expression profiles following compound exposure. Rats were divided into three groups. One group was not subjected to handling procedures and served as controls for both handled groups. Animals in the other two groups were weighed, subjected to restraint in Broome restrainers, and administered water via oral gavage daily for 1 or 4 days with tail vein blood collections at 1, 2, 4, and 8 hours postdose on days 1 and 4. Significantly altered genes were identified in livers of animals following 1 or 4 days of handling when compared to the unhandled animals. Gene changes in animals handled for 4 days were similar to those handled for 1 day, suggesting a lack of habituation. The altered genes were primarily immune function related genes. These findings, along with a correlating increase in corticosterone levels suggest that common handling procedures may cause a minor immune system perturbance.

A novel statistical algorithm for gene expression analysis helps differentiate pregnane X receptor-dependent and independent mechanisms of toxicity.

Genome-wide gene expression profiling has become standard for assessing potential liabilities as well as for elucidating mechanisms of toxicity of drug candidates under development. Analysis of microarray data is often challenging due to the lack of a statistical model that is amenable to biological variation in a small number of samples. Here we present a novel non-parametric algorithm that requires minimal assumptions about the data distribution. Our method for determining differential expression consists of two steps: 1) We apply a nominal threshold on fold change and platform p-value to designate whether a gene is differentially expressed in each treated and control sample relative to the averaged control pool, and 2) We compared the number of samples satisfying criteria in step 1 between the treated and control groups to estimate the statistical significance based on a null distribution established by sample permutations. The method captures group effect without being too sensitive to anomalies as it allows tolerance for potential non-responders in the treatment group and outliers in the control group. Performance and results of this method were compared with the Significant Analysis of Microarrays (SAM) method. These two methods were applied to investigate hepatic transcriptional responses of wild-type (PXR(+/+)) and pregnane X receptor-knockout (PXR(-/-)) mice after 96 h exposure to CMP013, an inhibitor of ß-secretase (ß-site of amyloid precursor protein cleaving enzyme 1 or BACE1). Our results showed that CMP013 led to transcriptional changes in hallmark PXR-regulated genes and induced a cascade of gene expression changes that explained the hepatomegaly observed only in PXR(+/+) animals. Comparison of concordant expression changes between PXR(+/+) and PXR(-/-) mice also suggested a PXR-independent association between CMP013 and perturbations to cellular stress, lipid metabolism, and biliary transport.