Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective assay qualifications. Conclusion The one-stage clotting assay with the STA-Cephascreen APTT reagent, the ROX Factor IX chromogenic assay and the BIOPHEN Factor IX chromogenic assay are considered to be qualified for the measurement of N9-GP in 3.2% (0.109 m) citrated human plasma.

The improvement of long-term transplant organ and patient survival remains a critical challenge following kidney transplantation. Proteomics and biochemical profiling (metabolomics) may allow for the detection of early changes in cell signal transduction regulation and biochemistry with high sensitivity and specificity. Hence, these analytical strategies hold the promise to detect and monitor disease processes and drug effects before histopathological and pathophysiological changes occur. In addition they will identify enriched populations and enable individualize drug therapy. However, proteomics and metabolomics have not yet lived up to such high expectations. Renal transplant patients are highly complex, making it difficult to establish cause-effect relationships between surrogate markers and disease processes. Appropriate study design, adequate sample handling, storage and processing, quality and reproducibility of bioanalytical multi-analyte assays, data analysis and interpretation, mechanistic verification and clinical qualification (=establishment of sensitivity and specificity in adequately powered prospective clinical trials) are important factors for the success of molecular marker discovery and development in renal transplantation. However, a newly developed and appropriately qualified molecular marker can only be successful if it is realistic that it can be implemented in a clinical setting. The development of combinatorial markers with supporting software tools is an attractive goal.

Enhancement of calcineurin inhibitor nephrotoxicity by sirolimus (SRL) is limiting the clinical use of this drug combination. We compared the dose-dependent effects of the structurally related everolimus (EVL) and sirolimus (SRL) alone, and in combination with cyclosporine (CsA), on the rat kidney. Lewis rats were treated by oral gavage for 28 days using a checkerboard dosing format (0, 3.0, 6.0 and 10.0 CsA and 0, 0.5, 1.5 and 3.0 mg/kg/day SRL or EVL, n = 4/dose combination). After 28 days, oxidative stress, energy charge, kidney histologies, glomerular filtration rates, and concentrations of the immunosuppressants were measured along with 1H-magnetic resonance spectroscopy (MRS) and gas chromatography- mass spectrometry profiles of cellular metabolites in urine. The combination of CsA with SRL led to higher urinary glucose concentrations and decreased levels of urinary Krebs cycle metabolites when compared to controls, suggesting that CsA+SRL negatively impacted proximal tubule metabolism. Unsupervised principal component analysis of MRS spectra distinguished unique urine metabolite patterns of rats treated with CsA+SRL from those treated with CsA+EVL and the controls. SRL, but not EVL blood concentrations were inversely correlated with urine Krebs cycle metabolite concentrations. Interestingly, the higher the EVL concentration, the closer urine metabolite patterns resembled those of controls, while in contrast, the combination of the highest doses of CsA+SRL showed the most significant differences in metabolite patterns. Surprisingly in this rat model, EVL and SRL in combination with CsA had different effects on kidney biochemistry, suggesting that further exploration of EVL in combination with low dose calcineurin inhibitors may be of potential benefit.

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