A biomarker assay validation approach tailored to the context of use and bioanalytical platform.

It is widely acknowledged by the bioanalytical and biomarker community that biomarker assay validations should be fit-for-purpose depending on the context of use. The challenge is how to consistently apply these principles in teams responsible for measuring a disparate array of biomarkers, often on multiple analytical platforms, at various stages of the drug discovery and development pipeline and across diverse biology focus areas. To drive consistency, while maintaining the necessary flexibility to allow validations to be driven by scientific rationale and taking into consideration the context of use and associated biological and (pre)analytical factors, a framework applicable across biomarker assays was developed.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs ( - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.

Pharmacokinetic interaction of brivaracetam on other antiepileptic drugs in adults with focal seizures: Pooled analysis of data from randomized clinical trials.

Objective: To assess the effect of brivaracetam (BRV) on steady-state plasma concentrations of commonly prescribed antiepileptic drugs (AEDs).

Methods: Data were pooled from five randomized, double-blind, placebo-controlled efficacy studies (NCT00175929, NCT00175825, NCT00490035, NCT00464269, and NCT01261325) in which adults with refractory epilepsy, and receiving stable doses of 1-2 AEDs, initiated adjunctive treatment with BRV (or placebo) for up to 12 weeks, following a 4-8 week baseline period. Concentrations of carbamazepine, carbamazepine epoxide, clobazam, clonazepam, lacosamide, lamotrigine, levetiracetam, oxcarbazepine (MHD), phenobarbital, phenytoin, pregabalin, topiramate, valproic acid and zonisamide, were measured during baseline and during BRV or placebo evaluation periods.

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches.

European Bioanalysis Forum recommendation on the best practices to demonstrate processed sample stability.

The European Bioanalysis Forum is providing a proposal on experiments to be conducted if processed samples are to be stored for a period of time, either after an initial injection or if processed samples are to be stored for a period of time prior to their first injection. Using survey information and extensive discussion an insight was gained as to whether the current practices employed during method validation were conducted to scientifically validate actual sample handling conditions or to simply meet a perceived regulatory expectation. The goal of this report is to recommend the adoption of scientifically justified validation experiments that exactly reproduce and processed test sample handling and storage procedures and to introduce some uniformity across the industry.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 2 - Hybrid LBA/LCMS and input from regulatory agencies).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10 WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity.

Cross-Species Differences in the Preclinical Pharmacokinetics of CT7758, an α4β1/α4β7 Integrin Antagonist.

CT7758, a carboxylate containing α4β1/α4/β7 integrin antagonist, was characterized for its pharmacokinetic profile in various in vitro and in vivo assays in support of clinical development. The oral bioavailability of CT7758 was 4% in mice, 2% in rats, 7-55% in dogs, and 0.2% in cynomolgus monkeys.

Anti-ictogenic and antiepileptogenic properties of brivaracetam in mature and immature rats.

Objective: Brivaracetam (BRV) is a new antiepileptic drug candidate rationally designed for high affinity and selectivity for the synaptic vesicle protein 2A. This study explored anti-ictogenic and antiepileptogenic effects of BRV in rats at different stages of development.

Methods: Using a rapid kindling model in P14, P21, P28, and P60 rats, we studied two doses of BRV: 10 and 100 mg/kg injected intraperitoneally 30 min before afterdischarge assessment.

N-alkylprotoporphyrin formation and hepatic porphyria in dogs after administration of a new antiepileptic drug candidate: mechanism and species specificity.

A new antiepileptic synaptic vesicle 2a (SV2a) ligand drug candidate was tested in 4-week oral toxicity studies in rat and dog. Brown pigment inclusions were found in the liver of high-dose dogs. The morphology of the deposits and the accompanying liver changes (increased plasma liver enzymes, increased total hepatic porphyrin level, decreased liver ferrochelatase activity, combined induction, and inactivation of cytochrome P-450 CYP2B11) suggested disruption of the heme biosynthetic cascade.

In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium.

Background: At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consortium did not form a dedicated team to investigate the spot homogeneity. However, two teams performed experiments that produced results relating to sample homogeneity.

Results: The data, which were produced via two different approaches (a radiolabeled and a nonradiolabeled approach), are highly complementary and demonstrate clear effects on sample inhomogeneity due to the substrate type, compound and hematocrit levels.

(123)I-Interleukin-2 uptake in squamous cell carcinoma of the head and neck carcinoma.

Introduction: Information obtained on the IL-2 receptor status of tumour infiltrating lymphocytes in patients suffering from squamous cell carcinoma of the head and neck (SSCHN) before and after IL-2 treatment may lead to a better understanding of the immunological changes and related kinetics induced at the tumour level and ultimately to a strategy that allows selection of those patients that will benefit from IL-2 therapy. This study set out to assess the relationship between (123)I-IL2 single-photon emission computed tomography (SPECT) findings and the presence of IL-2 receptors (CD25 staining) on tumour-infiltrating lymphocytes as well as on SCCHN tumour cells in patients suffering from SCCHN.

Materials And Methods: Seventeen consecutive patients (12 men; mean age, 57 years) highly suspected to suffer from SSCHN were prospectively included in the study.

Optimization by experimental design of precursor synthesis and radiolabeling of 2-iodo-L-phenylalanine, a novel amino acid for tumor imaging.

Various radiolabeled amino acids show promising results in tumor detection, as applied in the management of cancer patients. We synthesized the precursor 2-iodo-L-phenylalanine for easier kit labeling of [123/125I]- 2-iodo-L-phenylalanine, using the Cu1+ -assisted nucleophilic halogen exchange. Precursor synthesis was optimized by experimental design: Eight parameters were initially screened by a quarter fractional design.

Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis.

Objective: The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA.

Methods: Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production.

Valine-based biphenylsulphonamide matrix metalloproteinase inhibitors as tumor imaging agents.

Among matrix metalloproteinases (MMPs), the subfamily of gelatinases (MMP-2, MMP-9) is of particular interest due to their ability to degrade type IV collagen and other non-fibrillar collagen domains and proteins such as fibronectin and laminin. Whilst malignant cells often over-express various MMPs, the gelatinases have been most consistently detected in malignant tissues and associated with tumor growth, metastatic potential and angiogenesis. Radiosynthesis of carboxylic (1') and hydroxamic (2') MMPIs resulted in radiochemical yields of 70 +/- 5% (n = 6) and 60 +/- 5% (n = 4), respectively.

Tryptophane-based biphenylsulfonamide matrix metalloproteinase inhibitors as tumor imaging agents.

Aim: As a part of our efforts to use small organic matrix metalloproteinase (MMP) inhibitors with improved characteristics for the diagnosis and treatment of different kinds of tumor tissues, biphenylsulfonamide analogues were synthesized. This study reports on the in vivo biodistribution of iodine-123-labeled biphenylsulfonide and analogues in A549 lung carcinoma inoculated into athymic mice and the evaluation of their suitability as imaging agents using a single photon emission computed tomography (SPECT) camera.

Methods: The radioiodinated carboxylic and hydroxamic MMP inhibitors 2-(4'- [(123)I]iodobiphenyl-4-sulfonylamino)-3-(1H-indol-3-yl)-propionic acid (1') and 2-(4'-[(123)I]iodobiphenyl-4- sulfonylamino)-3-(1H-indol-3-yl)-propionamide (2') were synthesized by electrophilic aromatic substitution of the tributylstannyl derivatives.

In vitro and in vivo evaluation of [123I]-VEGF165 as a potential tumor marker.

One of the research challenges in oncology is to develop new biochemical methods for noninvasive tumor therapy evaluation to determine whether the chemotherapeutics is effective. Vascular endothelial growth factor (VEGF) was labeled with radioiodine and evaluated in vitro as well as in vivo, using A2058, a melanoma cell line overexpressing VEGFR-1 and -2. Saturation binding analysis with [(125)I]-VEGF resulted in a K(d) of 0.

In vivo evaluation of [(123)I]-3-(4-iodobenzyl)-1,2,3,4-tetrahydro-8-hydroxychromeno[3,4-c]pyridin-5-one: a presumed dopamine D4 receptor ligand for SPECT studies.

[(123)I]-3-(4-iodobenzyl)-1,2,3,4-tetrahydro-8-hydroxychromeno[3,4-c]pyridin-5-one ([(123)I]-ITCP), a presumed radioligand for visualization of the dopamine D4 receptor by single photon emission computed tomography, was evaluated in vivo in mice and rabbits. This new radioiodinated tracer exhibited high brain uptake (3.64% injected dose per gram of tissue at 10 min p.

Synthesis, radiosynthesis, in vitro and preliminary in vivo evaluation of biphenyl carboxylic and hydroxamic matrix metalloproteinase (MMP) inhibitors as potential tumor imaging agents.

Excess matrix degradation is one of the hallmarks of cancer and is an important factor in the process of tumor progression. It is implicated in invasion, metastasis, growth, angiogenesis and migration. Many characteristics of matrix metalloproteinases (MMPs) make them attractive therapeutic and diagnostic targets.

In vivo evaluation of [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benzamide: a potential sigma receptor ligand for SPECT studies.

In this study, in vivo evaluation in mice and rabbits of [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benzamide ([123I]-BPB), a potential radioligand for visualisation of the sigma receptor by single photon emission computed tomography (SPECT), is reported. The compound possesses appropriate lipophilicity (log P=2.2) and binds sigma-1 and sigma-2 receptors (pKi=6.

New radioiodinated carboxylic and hydroxamic matrix metalloproteinase inhibitor tracers as potential tumor imaging agents.

Several studies have demonstrated a positive correlation between tumor progression and expression of extracellular proteinases such as matrix metalloproteinases (MMPs). MMP-2 and MMP-9 have become attractive targets for cancer research because of their increased expression in human malignant tumor tissues of various organs, providing a target for medical imaging techniques. Radioiodinated carboxylic and hydroxamic MMP inhibitors 2-(4'-[(123)I]iodo-biphenyl-4-sulfonylamino)-3-(1H-indol-3-yl)-propionic acid (9) and 2-(4'-[(123)I]iodo-biphenyl-4-sulfonylamino)-3-(1H-indol-3-yl)-propionamide (11) were synthesized by electrophilic aromatic substitution of the tributylstannyl derivatives and resulted in radiochemical yields of 60% +/- 5% (n = 3) and 70% +/- 5% (n = 6), respectively.