New variants in the Enpp1 and Ptpn6 genes cause low BMD, crystal-related arthropathy, and vascular calcification.

A large genome-wide, recessive, N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen was performed on a mixed C57BL/6J and C3H.SW-H2/SnJ mouse background to identify genes regulating bone mass. Approximately 6500 male and female G(3) hybrid mice were phenotyped at 8 and 10 wk of age by DXA analysis for evidence of changes in unadjusted or body weight-adjusted BMD or BMC. Phenodeviant lines were identified based on statistical criteria that included a false discovery rate (FDR) <20% and Z-score >2.8. Genome-wide mapping scans were initiated on 22 lines, with evidence of high or low BMD or BMC that deviated by approximately -30% to +50% from the means. Several lines were discontinued as showing lack of heritability, but two heritable lines were identified with narrow chromosomal regions that allowed sequencing of potential mutant candidate genes. Novel mutations were identified in the Enpp1 (C397S) gene on chromosome 10 (line 4482) and the Ptpn6 (I482F) gene on chromosome 6 (line 4489) that were both associated with low bone mass. In addition, the phenotype of the Enpp1 mice showed a striking joint disease and calcification of blood vessels including the aorta, myocardium, and renal arteries and capillaries. These results support a role for the Enpp1 gene in the pathogenesis associated with mineralization of articular cartilage and vascular calcification. This work confirms the utility of the chemical mutagenesis approach for identification of potential disease genes and confirms the role of Enpp1 and Ptpn6 in regulating mineralization and skeletal bone mass.

Experimental and statistical approaches in method cross-validation to support pharmacokinetic decisions.

A case study of experimental and statistical approaches for cross-validating and examining the equivalence of two ligand binding assay (LBA) methods that were employed in pharmacokinetic (PK) studies is presented. The impact of changes in methodology based on the intended use of the methods was assessed. The cross-validation processes included an experimental plan, sample size selection, and statistical analysis with a predefined criterion of method equivalence. The two methods were deemed equivalent if the ratio of mean concentration fell within the 90% confidence interval (0.80-1.25). Statistical consideration of method imprecision was used to choose the number of incurred samples (collected from study animals) and conformance samples (spiked controls) for equivalence tests. The difference of log-transformed mean concentration and the 90% confidence interval for two methods were computed using analysis of variance. The mean concentration ratios of the two methods for the incurred and spiked conformance samples were 1.63 and 1.57, respectively. The 90% confidence limit was 1.55-1.72 for the incurred samples and 1.54-1.60 for the spiked conformance samples; therefore, the 90% confidence interval was not contained within the (0.80-1.25) equivalence interval. When the PK parameters of two studies using each of these two methods were compared, we determined that the therapeutic exposure, AUC((0-168)) and C(max), from Study A/Method 1 was approximately twice that of Study B/Method 2. We concluded that the two methods were not statistically equivalent and that the magnitude of the difference was reflected in the PK parameters in the studies using each method. This paper demonstrates the need for method cross-validation whenever there is a switch in bioanalytical methods, statistical approaches in designing the cross-validation experiments and assessing results, or interpretation of the impact of PK data.

Kinetics of haematopoietic recovery after dose-intensive chemo/radiotherapy in mice: optimized erythroid support with darbepoetin alpha.

Despite its frequency and impact on clinical outcomes, anaemia in cancer patients remains poorly understood and suboptimally treated. The definition of optimum treatment schedules with erythropoietic agents requires a suitable model of chemotherapy-induced progressive anaemia. This study investigated novel strategies such as once-per-chemotherapy-cycle dosing, synchronization between erythroid supportive care and chemotherapy, and definition of the optimum timing of erythroid support. A murine model of carboplatin chemotherapy/radiotherapy (CRT)-induced anaemia was used, which caused progressive anaemia across multiple cycles. Weekly administration of recombinant human erythropoietin (rHuEPO) was effective, but the longer-acting darbepoetin alpha resulted in superior responses. In all animals, anaemia became progressive and more refractory across cycles because of accumulated bone marrow damage. Exploiting a specific enzyme-linked immunosorbent assay, which could distinguish between darbepoetin alpha and endogenous erythropoietin, the effect of CRT upon the pharmacokinetics of darbepoetin alpha showed that clearance of darbepoetin alpha, and presumably erythropoietin, was at least partially dependent on a chemotherapy-sensitive pathway. Scheduling data suggested that administration of erythropoietic agents prior to chemotherapy was more effective than administration after chemotherapy. There was no evidence that erythropoietic agents exacerbated anaemia, even when administered immediately prior to CRT in an attempt to "prime" erythroid cells for the effects of CRT.