The effectiveness of quality control samples in pharmaceutical bioanalysis.

The use of quality control (QC) samples in bioanalysis is well established and consistent with regulatory guidance. However, a systematic evaluation of whether QC samples serve the intended purpose of improving data quality has not been undertaken. The Translational and ADME Sciences Leadership Group (TALG) of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) conducted an evaluation to assess whether closer agreement is observed when comparing pharmacokinetic data from two passed runs, than when comparing data from failed and passed (retest) runs. Analysis of data collected across organizations, molecular types and analytical platforms, revealed that bioanalytical methods are very reproducible; and that QC samples improve the overall quality of pharmacokinetic concentration data and justifies their continued use.

Improved Pharmacokinetics with BAY 81-8973 Versus Antihemophilic Factor (Recombinant) Plasma/Albumin-Free Method: A Randomized Pharmacokinetic Study in Patients with Severe Hemophilia A.

BACKGROUND: BAY 81-8973 is a full-length, unmodified, recombinant human factor VIII (FVIII) for the treatment of hemophilia A. OBJECTIVE: The aim of this study was to compare the pharmacokinetic (PK) profile of BAY 81-8973 with antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM) PATIENTS/METHODS: In this phase I, open-label, crossover study, men aged 18-65 years with severe hemophilia A and ≥150 exposure days to FVIII were randomized to receive a single intravenous infusion of 50 IU/kg BAY 81-8973 or rAHF-PFM, followed by crossover to a single infusion of the other treatment. FVIII levels were measured in plasma over 48 h using one-stage and chromogenic assays. PK parameters, including area under the curve from time zero to the last data point (AUClast; primary outcome) and half-life (t ½) were calculated. A population PK model was developed to simulate various treatment scenarios. RESULTS: Eighteen patients were randomized and analyzed. Using both assays, geometric mean (coefficient of variation [%CV]) AUClast was significantly higher, and t ½ was significantly longer, for BAY 81-8973 versus rAHF-PFM (one-stage, AUClast: 1660 IU·h/dL [29.4] vs. 1310 IU·h/dL [29.0], p < 0.0001; one-stage, t ½: 14.5 [25.7] vs. 11.7 h [27.3], p < 0.0001). Simulations showed that median time to 1 IU/dL was approximately 27% longer for BAY 81-8973 versus rAHF-PFM over doses of 25-50 IU/kg; plasma levels >1 IU/dL could be maintained with 14.4 IU/kg BAY 81-8973 or 39.1 IU/kg rAHF-PFM 3×/week. CONCLUSIONS: BAY 81-8973 showed a superior PK profile versus rAHF-PFM. The same FVIII trough threshold level could be achieved with lower doses of BAY 81-8973 versus rAHF-PFM. ClinicalTrials.gov: NCT02483208.

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A.

Skeletal muscle mitochondrial uncoupling prevents diabetes but not obesity in NZO mice, a model for polygenic diabesity.

Induction of skeletal muscle (SM) mitochondrial stress by expression of uncoupling protein 1 (UCP1) in mice results in a healthy metabolic phenotype associated with increased secretion of FGF21 from SM. Here, we investigated whether SM mitochondrial uncoupling can compensate obesity and insulin resistance in the NZO mouse, a polygenic diabesity model. Male NZO mice were crossed with heterozygous UCP1 transgenic (tg) mice (mixed C57BL/6/CBA background) and further backcrossed to obtain F1 and N2 offspring with 50 and 75 % NZO background, respectively. Male F1 and N2 progeny were fed a high-fat diet ad libitum for 20 weeks from weaning. Blood glucose was reduced, and diabetes (severe hyperglycemia >300 mg/dl) was fully prevented in both F1- and N2-tg progeny compared to a diabetes prevalence of 15 % in F1 and 42 % in N2 wild type. In contrast, relative body fat content and plasma insulin were decreased, and glucose tolerance was improved, in F1-tg only. Both F1 and N2-tg showed decreased lean body mass. Accordingly, induction of SM stress response including FGF21 expression and secretion was similar in both F1 and N2-tg mice. In white adipose tissue, expression of FGF21 target genes was enhanced in F1 and N2-tg mice, whereas lipid metabolism genes were induced in F1-tg only. There was no evidence for induction of browning in either UCP1 backcross. We conclude that SM mitochondrial uncoupling induces FGF21 expression and prevents diabetes in mice with a 50-75 % NZO background independent of its effects on adipose tissue.

Uncoupling protein 1 expression in murine skeletal muscle increases AMPK activation, glucose turnover, and insulin sensitivity in vivo.

Uncoupling of oxidative phosphorylation represents a potential target for the treatment of hyperglycemia and insulin resistance in obesity and type 2 diabetes. The present study investigated whether the expression of uncoupling protein 1 in skeletal muscles of transgenic (mUCP1 TG) mice modulates insulin action in major insulin target tissues in vivo. Euglycemic-hyperinsulinemic clamps (17 pM x kg lean body mass(-1) x min(-1)) were performed in 9-mo-old hemizygous male mUCP1 TG mice and wild-type (WT) littermates matched for body composition. mUCP1 TG mice exhibited fasting hypoglycemia and hypoinsulinemia compared with WT mice, whereas fasting hepatic glucose production rates were comparable in both genotypes. mUCP1 TG mice were markedly more sensitive to insulin action compared with WT mice and displayed threefold higher glucose infusion rates, enhanced skeletal muscle and white adipose tissue glucose uptake, and whole body glycolysis rates. In the absence of alterations in plasma adiponectin concentrations, acceleration of insulin-stimulated glucose turnover in skeletal muscle of mUCP1 TG mice was accompanied by increased phosphorylated Akt-to-Akt and phosphorylated AMP-activated protein kinase (AMPK)-to-AMPK ratios compared with WT mice. UCP1-mediated uncoupling of oxidative phosphorylation in skeletal muscle was paralleled by AMPK activation and thereby stimulated insulin-mediated glucose uptake in skeletal muscle.

Dissociation of obesity and insulin resistance in transgenic mice with skeletal muscle expression of uncoupling protein 1.

We evaluated the effect of skeletal muscle mitochondrial uncoupling on energy and glucose metabolism under different diets. For 3 mo, transgenic HSA-mUCP1 mice with ectopic expression of uncoupling protein 1 in skeletal muscle and wild-type littermates were fed semisynthetic diets with varying macronutrient ratios (energy % carbohydrate-protein-fat): HCLF (41:42:17), HCHF (41:16:43); LCHF (11:45:44). Body composition, energy metabolism, and insulin resistance were assessed by NMR, indirect calorimetry, and insulin tolerance test, respectively. Gene expression in different organs was determined by real-time PCR. In wild type, both high-fat diets led to an increase in body weight and fat. HSA-mUCP1 mice considerably increased body fat on HCHF but stayed lean on the other diets. Irrespective of differences in body fat content, HSA-mUCP1 mice showed higher insulin sensitivity and decreased plasma insulin and liver triglycerides. Respiratory quotient and gene expression indicated overall increased carbohydrate oxidation of HSA-mUCP1 but a preferential channeling of fatty acids into muscle rather than liver with high-fat diets. Evidence for increased lipogenesis in white fat of HSA-mUCP1 mice suggests increased energy dissipating substrate cycling. Retinol binding protein 4 expression in white fat was increased in HSA-mUCP1 mice despite increased insulin sensitivity, excluding a causal role in the development of insulin resistance. We conclude that skeletal muscle mitochondrial uncoupling does not protect from the development of obesity in all circumstances. Rather it can lead to a "healthy" obese phenotype by preserving insulin sensitivity and a high metabolic flexibility, thus protecting from the development of obesity associated disturbances of glucose homeostasis.