Development of a plug and play ImmunoPCR technique for the analysis of biomolecules.

AIM: ImmunoPCR technology combines the advantages of specificity and robustness of a ligand binding assay with the amplification potential of PCR. We describe through three case studies a plug-and-play immuno polymerase chain reaction (iPCR) technique to measure biomolecules. RESULTS: Case Study 1 demonstrated feasibility of measurement of IgG1 in cerebrospinal fluid at the desired level of sensitivity with minimal cost and timelines of clinical assay implementation. Case Study 2 translated the iPCR protocol to measure multiple IgG1 analytes in cerebrospinal fluid. Case Study 3 demonstrated broad applicability of the technique to yet another analyte IL-6. CONCLUSION: The advantages of our iPCR approach were: lack of reliance on a single vendor for technology platform/software, minimal reliance on proprietary reagents and reduced method development times and cost.

Population Pharmacokinetics and Target Engagement of Natalizumab in Patients With Multiple Sclerosis.

Natalizumab (humanized immunoglobulin G4 antibody targeting alpha-4 integrins) is a highly efficacious treatment for relapsing-remitting multiple sclerosis (RRMS) that has been in clinical use since 2006. However, natalizumab pharmacokinetic (PK) characteristics and concentration alpha-4 integrin saturation relationships have not been well described in the scientific literature. Therefore, clinical data from 11 studies were pooled and analyzed to characterize the PK and pharmacodynamic (PD) properties of natalizumab in RRMS subjects. Natalizumab PK was best described using a 2-compartment model with linear first-order and Michaelis-Menten elimination. Subcutaneous absorption of natalizumab was characterized using first-order absorption with lag time. The relationship between natalizumab concentration and alpha-4 integrin saturation (PD) was best described by a direct response model with a sigmoidal effect on alpha-4 integrin saturation mediated by a maximum effect relationship with natalizumab concentrations. Covariate analysis showed that body weight, product formulations, and the presence of antinatalizumab antibodies were the main covariates affecting natalizumab PK, whereas age and formulations affected PD. The use of simulations based on a pharmacokinetic-pharmacodynamic model showed that covariates, although statistically significant, are not expected to have any clinical impact at the approved clinical dosing regimen of natalizumab (300 mg once every 4 weeks).

Evidence of activation of the Nrf2 pathway in multiple sclerosis patients treated with delayed-release dimethyl fumarate in the Phase 3 DEFINE and CONFIRM studies.

BACKGROUND: Delayed-release dimethyl fumarate (DMF) is an approved oral treatment for relapsing forms of multiple sclerosis (MS). Preclinical studies demonstrated that DMF activated the nuclear factor E2-related factor 2 (Nrf2) pathway. DMF and its primary metabolite monomethyl fumarate (MMF) were also shown to promote cytoprotection of cultured central nervous system (CNS) cells via the Nrf2 pathway. OBJECTIVE: To investigate the activation of Nrf2 pathway following ex vivo stimulation of human peripheral blood mononuclear cells (PBMCs) with DMF or MMF, and in DMF-treated patients from two Phase 3 relapsing MS studies DEFINE and CONFIRM. METHODS: Transcription of Nrf2 target genes NADPH:quinone oxidoreductase-1 (NQO1) and heme-oxygenase-1 (HO1) was measured using Taqman® assays. RNA samples were isolated from ex vivo-stimulated PBMCs and from whole blood samples of 200 patients each from placebo, twice daily (BID) and three times daily (TID) treatments. RESULTS: DMF and MMF induced NQO1 and HO1 gene expression in ex vivo-stimulated PBMCs, DMF being the more potent inducer. Induction of NQO1 occurred at lower DMF concentrations compared to that of HO1. In DMF-treated patients, a statistically significant induction of NQO1 was observed relative to baseline and compared to placebo. No statistical significance was reached for HO1 induction. CONCLUSION: These data provide the first evidence of Nrf2 pathway activation from two large pivotal Phase 3 studies of DMF-treated MS patients.