Nonclinical pharmacology and toxicology of the first biosimilar insulin glargine drug product (BASAGLAR®/ABASAGLAR®) approved in the European Union.

Basaglar®/Abasaglar® (Lilly insulin glargine [LY IGlar]) is a long-acting human insulin analogue drug product granted marketing authorisation as a biosimilar to Lantus® (Sanofi insulin glargine [SA IGlar]) by the European Medicines Agency. We assessed the similarity of LY IGlar to the reference drug product, European Union-sourced SA IGlar (EU-SA IGlar), using nonclinical in vitro and in vivo studies. No biologically relevant differences were observed for receptor binding affinity at either the insulin or insulin-like growth factor-1 (IGF-1) receptors, or in assays of functional or de novo lipogenic activity. The mitogenic potential of LY IGlar and EU-SA IGlar was similar when tested in both insulin- and IGF-1 receptor dominant cell systems. Repeated subcutaneous daily dosing of rats for 4 weeks with 0, 0.3, 1.0, or 2.0 mg/kg LY IGlar and EU-SA IGlar produced mortalities and clinical signs consistent with severe hypoglycaemia. Glucodynamic profiles of LY IGlar and EU-SA IGlar in satellite animals showed comparable dose-related hypoglycaemia. Severe hypoglycaemia was associated with axonal degeneration of the sciatic nerve; the incidence and severity were low and did not differ between LY IGlar and EU-SA IGlar. These results demonstrated no biologically relevant differences in toxicity between LY IGlar and EU-SA IGlar.

In Vivo and In Vitro Characterization of Basal Insulin Peglispro: A Novel Insulin Analog.

The aim of this research was to characterize the in vivo and in vitro properties of basal insulin peglispro (BIL), a new basal insulin, wherein insulin lispro was derivatized through the covalent and site-specific attachment of a 20-kDa polyethylene-glycol (PEG; specifically, methoxy-terminated) moiety to lysine B28. Addition of the PEG moiety increased the hydrodynamic size of the insulin lispro molecule. Studies show there is a prolonged duration of action and a reduction in clearance. Given the different physical properties of BIL, it was also important to assess the metabolic and mitogenic activity of the molecule. Streptozotocin (STZ)-treated diabetic rats were used to study the pharmacokinetic and pharmacodynamic characteristics of BIL. Binding affinity and functional characterization of BIL were compared with those of several therapeutic insulins, insulin AspB10, and insulin-like growth factor 1 (IGF-1). BIL exhibited a markedly longer time to maximum concentration after subcutaneous injection, a greater area under the concentration-time curve, and a longer duration of action in the STZ-treated diabetic rat than insulin lispro. BIL exhibited reduced binding affinity and functional potency as compared with insulin lispro and demonstrated greater selectivity for the human insulin receptor (hIR) as compared with the human insulin-like growth factor 1 receptor. Furthermore, BIL showed a more rapid rate of dephosphorylation following maximal hIR stimulation, and reduced mitogenic potential in an IGF-1 receptor-dominant cellular model. PEGylation of insulin lispro with a 20-kDa PEG moiety at lysine B28 alters the absorption, clearance, distribution, and activity profile receptor, but does not alter its selectivity and full agonist receptor properties.

A phase I dose-escalation study to a predefined dose of a transforming growth factor-ß1 monoclonal antibody (TßM1) in patients with metastatic cancer.

Transforming growth factor ß (TGF-ß) plays an important role in cancer. Monoclonal antibodies (mAb) designed to specifically block the TGF-ß ligands, are expected to inhibit tumor progression in patients with metastatic cancer. TßM1 is a humanized mAb optimized for neutralizing activity against TGF-ß1. The objective of this clinical trial was to assess the safety and tolerability of TßM1 in patients with metastatic cancer. In this phase I, uncontrolled, non-randomized, dose-escalation study, 18 eligible adult patients who had measurable disease per RECIST and a performance status of ≤ 2 on the ECOG scale were administered TßM1 intravenously over 10 min at doses of 20, 60, 120 and 240 mg on day 1 of each 28-day cycle. Safety was assessed by adverse events (as defined by CTCAE version 3.0) and possible relationship to study drug, dose-limiting toxicities and laboratory changes. Systemic drug exposure and pharmacodynamic (PD) parameters were assessed. TßM1 was safe when administered once monthly. The pharmacokinetic (PK) profile was consistent with a mAb with a mean elimination half-life approximately 9 days. Although anticipated changes in PD markers such as serum VEGF, bFGF and mRNA expression of SMAD7 were observed in whole-blood, suggesting activity of TßM1 on the targeted pathway, these changes were not consistent to represent a PD effect. Additionally, despite the presence of an activated TGF-ß1 expression signature in patients' whole blood, the short dosing duration did not translate into significant antitumor effect in the small number of patients investigated in this study.

Deciphering ADME genetic data using an automated haplotype approach.

OBJECTIVE: To investigate the utility of statistical tools in translating Affymetrix Drug Metabolizing Enzyme and Transporter (DMET) Assay single-nucleotide polymorphisms (SNPs) into common consensus star alleles. METHODS: DMET SNP data from clinical trials in different ethnicities were pooled for analyses. Three different statistical methods, PHASE, Bayesian, and expectation-maximization (EM), were first assessed by comparing the consistency of calling CYP2D6 alleles among 1108 Asians and 55 Caucasians. Subsequently, the performance of EM in deriving haplotype calls was evaluated against the Affymetrix Translation Table for CYPs 2B6, 2C19, 2C9, and 3A4/5 in 582 Asians, 296 Caucasians, and 369 Africans. Selected DNA samples were sequenced to verify the EM-predicted haplotype calls. RESULTS: PHASE, Bayesian, and EM methods showed a similar CYP2D6 star allele call rate. The EM method, with a 0.99 posterior probability cutoff, was chosen for further evaluation because of its low false-positive call rate. Haplotype calls obtained with the EM method were consistent with the Affymetrix Translation Table more than 95% of the time for all five CYPs, except for the CYP2B6 calls in the African descents (83%). In addition, the EM method was superior to the Translation Table-only approach in resolving complex haplotype patterns, identifying novel haplotypes in CYP2B6 and CYP3A5, and determining genotype calls in the presence of missing SNP data. CONCLUSION: A statistical method such as EM could be used to augment the translation of DMET assay SNP data into star alleles, especially for complex genes, to facilitate full utilization and interpretation of clinical pharmacogenetics data.

Haplotype-based pharmacogenetic analysis for longitudinal quantitative traits in the presence of dropout.

We propose a variety of methods based on the generalized estimation equations to address the issues encountered in haplotype-based pharmacogenetic analysis, including analysis of longitudinal data with outcome-dependent dropouts, and evaluation of the high-dimensional haplotype and haplotype-drug interaction effects in an overall manner. We use the inverse probability weights to handle the outcome-dependent dropouts under the missing-at-random assumption, and incorporate the weighted L(1) penalty to select important main and interaction effects with high dimensionality. The proposed methods are easy to implement, computationally efficient, and provide an optimal balance between false positives and false negatives in detecting genetic effects.

Multiplex assay for comprehensive genotyping of genes involved in drug metabolism, excretion, and transport.

BACKGROUND: Drug metabolism is a multistep process by which the body disposes of xenobiotic agents such as therapeutic drugs. Genetic variation in the enzymes involved in this process can lead to variability in a patient's response to medication. METHODS: We used molecular-inversion probe technology to develop a multiplex genotyping assay that can simultaneously test for 1227 genetic variants in 169 genes involved in drug metabolism, excretion, and transport. Within this larger set of variants, we performed analytical validation of a clinically defined core set of 165 variants in 27 genes to assess accuracy, imprecision, and dynamic range. RESULTS: In a test set of 91 samples, genotyping accuracy for the core set probes was 99.8% for called genotypes, with a 1.2% no-call (NC) rate. The majority of the core set probes (133 of 165) had < or = 1 genotyping failure in the test set; a subset of 12 probes was responsible for the majority of failures (mainly NC). Genotyping results were reproducible upon repeat testing with overall within- and between-run variation of 1.1% and 1.4%, respectively-again, primarily NCs in a subset of probes. The assay showed stable genotyping results over a 6-fold range of input DNA. CONCLUSIONS: This assay generates a comprehensive assessment of a patient's metabolic genotype and is a tool that can provide a more thorough understanding of patient-to-patient variability in pharmacokinetic responses to drugs.

The minimum significant ratio: a statistical parameter to characterize the reproducibility of potency estimates from concentration-response assays and estimation by replicate-experiment studies.

The authors show by illustration that procedures used to validate the reliability of single-concentration high-throughput screens such as the signal window and Z' factor do not ensure sufficient reliability in potency estimates from concentration response assays. They develop the minimum significant ratio as a statistical parameter to characterize the fold change between 2 compounds run in the same experiment that can be considered a real difference and use this parameter to characterize the reliability of the assay. They adapt methods described by Bland and Altman to develop a simple set of 2 experiments to estimate the minimum significant ratio and show that this protocol can identify assays that lack reproducibility. The methods are then extended to validate the equivalency of the same assay run by multiple laboratories.

Precision profiling and components of variability analysis for Affymetrix microarray assays run in a clinical context.

Although gene expression profiling using microarray technology is widely used in research environments, adoption of microarray testing in clinical laboratories is currently limited. In an attempt to determine how such assays would perform in a clinical laboratory, we evaluated the analytical variability of Affymetrix microarray probesets using two generations of human Affymetrix chips (U95Av2 and U133A). The study was designed to mimic potential clinical applications by using multiple operators, machines, and reagent lots, and by performing analyses throughout a period of several months. A mixed model analysis was used to evaluate the relative contributions of multiple factors to overall variability, including operator, instrument, run, cRNA/cDNA synthesis, and changes in reagent lots. Under these conditions, the average probeset coefficient of variation (CV) was relatively low for present probesets on both generations of chips (mean coefficient of variation, 21.9% and 27.2% for U95Av2 and U133A chips, respectively). The largest contribution to overall variation was chip-to-chip (residual) variability, which was responsible for between 40 to 60% of the total variability observed. Changes in individual reagent lots and instrumentation contributed very little to the overall variability. We conclude that the approach demonstrated here could be applied to clinical validation of Affymetrix-based assays and that the analytical precision of this technique is sufficient to answer many biological questions.

Post-transcriptional regulation of macrophage ABCA1, an early response gene to IFN-gamma.

Interferon-gamma (IFN-gamma) down-regulates receptors associated with reverse cholesterol transport including ABCA1. In the present study, the kinetics and mechanism of ABCA1 down-regulation were determined in mouse peritoneal macrophages. IFN-gamma decreased ABCA1 mRNA 1h following IFN-gamma addition and was maximally reduced by 3h. Down-regulation was protein synthesis dependent and involved post-transcriptional processes. ABCA1 message had a T(1/2) of 115 min in actinomycin treated cells that was reduced to a T(1/2) of 37 min by IFN-gamma. The decrease in message stability was also associated with a rapid loss of ABCA1 protein, significant 3h following IFN-gamma addition. The kinetics of ABCA1 message and protein decrease was consistent with the early IFN-gamma-induced changes in Stat1 phosphorylation and nuclear translocation observed in these cells. Therefore, ABCA1 can be considered as an early response gene to macrophage activation by IFN-gamma with down-regulation occurring by message destabilization.

A four component coupling strategy for the synthesis of D-phenylglycinamide-derived non-covalent factor Xa inhibitors.

A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.