Discovery of RXFP2 genetic association in resistant hypertensive men and RXFP2 antagonists for the treatment of resistant hypertension.

Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women.

In vivo biodistribution and pharmacokinetics of sotrovimab, a SARS-CoV-2 monoclonal antibody, in healthy cynomolgus monkeys.

Purpose: Sotrovimab (VIR-7831), a human IgG1κ monoclonal antibody (mAb), binds to a conserved epitope on the SARS-CoV-2 spike protein receptor binding domain (RBD). The Fc region of VIR-7831 contains an LS modification to promote neonatal Fc receptor (FcRn)-mediated recycling and extend its serum half-life. Here, we aimed to evaluate the impact of the LS modification on tissue biodistribution, by comparing VIR-7831 to its non-LS-modified equivalent, VIR-7831-WT, in cynomolgus monkeys.

Overcoming challenges associated with the bioanalysis of cysteine-conjugated metabolites in the presence of antibody-drug conjugates.

Investigations have shown that for the antibody-drug conjugate (ADC) belantamab mafodotin, concentrations of the cysteine-conjugated metabolite, Cys-mcMMAF, were overestimated in the presence of the ADC during sample processing when utilizing a historical SPE method. A new assay was developed utilizing an acidic protein precipitation to remove the ADC early in the extraction process, thus eliminating the risk of overestimating Cys-mcMMAF in the presence of belantamab mafodotin. experiments demonstrated a linear relationship between the concentration of belantamab mafodotin and the release of Cys-mcMMAF.

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.

Toward best practices in data processing and analysis for intact biotherapeutics by MS in quantitative bioanalysis.

Aim: Typically, quantitation of biotherapeutics from biological matrices by LC-MS is based on a surrogate peptide approach to determine molecule concentration. Recent efforts have focused on quantitation of the intact protein molecules or larger mass subunits of monoclonal antibodies. To date, there has been limited guidance for large or intact protein mass quantitation for quantitative bioanalysis.

A whole-molecule immunocapture LC-MS approach for the in vivo quantitation of biotherapeutics.

Aim: Large-molecule biotherapeutic quantitation in vivo by LC-MS has traditionally relied on enzymatic digestion followed by quantitation of a 'surrogate peptide' to infer whole-molecule concentration. MS methods presented here measure the whole molecule and provide a platform to better understand the various circulating drug forms by allowing for variant quantitation.

Results: An immunocapture LC-MS method for quantitation of a biotherapeutic monoclonal antibody from human plasma is presented.

Modify on the fly: triple quad to high resolution in support of a dermal clinical study requiring an ultra low LLOQ.

Background: FTIH studies can be challenging due to the varying dosing regimens and rapid data delivery. Chemists are asked to provide ultra-low limits of quantitation to provide an understanding of patient efficacy and safety in order to progress drug development. In a recent dermal study it became necessary to reduce the LLOQ of a small molecule drug from 50 to 1 pg/ml due to reductions in the dose and surface area of drug application.

Application of high-resolution MS for development of peptide and large-molecule drug candidates.

Background: For quantitative bioanalysis utilizing MS, the instrument of choice is typically a triple quadruple mass spectrometer. However, advances in high-resolution MS have allowed sensitivity and dynamic ranges to approach that of triple quadrupole instruments.

Results: A matrix-free protein digest, a digested therapeutic protein and the intact peptide therapeutic liraglutide were each analyzed on high-resolution and triple quadrupole mass spectrometers with data compared.

Evaluation of organic anion transporting polypeptide 1B1 and 1B3 humanized mice as a translational model to study the pharmacokinetics of statins.

Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography-tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins.

Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS.

Background: The quantification of proteins and peptides in in vivo samples is a critical part of supporting the drug development process for biotherapeutics. LC-MS/MS using tandem quadrupole mass spectrometers is well established as the technology of choice for the quantification of small-molecule drugs and their metabolites in biological fluid. The application of accurate mass MS for quantification in a DMPK environment has attracted considerable interest in recent years.

Application of DBS for the quantitative assessment of a protein biologic using on-card digestion LC-MS/MS or immunoassay.

Background: Dried blood spot (DBS) sampling has recently gained popularity in the bioanalysis community for quantitation of small molecules. Since the pharmaceutical industry continues to increase investment in biopharmaceuticals, DBS technologies were investigated to determine if immunoassay and/or LC-MS/MS techniques would be amenable for quantitation of a large protein therapeutic (>70 kDa).

Results: Methods were successfully qualified for the protein therapeutic utilizing DBS technology.

Utility of dried blood spot sampling and storage for increased stability of photosensitive compounds.

Background: Compound stability remains a major point of concern within pharmaceutical development. In attempts to minimize degradation, scientists may utilize acidification of samples prior to storage, dark chambers, decreased freezer temperatures and a variety of other stabilization techniques. All of these steps require additional procedures, increased costs and increased validation steps.

Application of DBS for quantitative assessment of the peptide Exendin-4; comparison of plasma and DBS method by UHPLC-MS/MS.

Background: An investigation was performed in order to establish if dried blood spots (DBS) could be applied to the quantitation of biopharmaceuticals in biological matrices and perform equivalently in terms of accuracy, precision and stability to traditional plasma methods.

Results: A method was successfully validated for the peptide Exendin-4 (39 amino acids in length) utilizing DBS technology. The validated DBS method resulted in a more sensitive and simplistic method than an existing monkey plasma method and required tenfold less sample volume.

Absolute quantification of a therapeutic domain antibody using ultra-performance liquid chromatography-mass spectrometry and immunoassay.

Background: Domain antibodies (dAbs; ∼10-15 kDa) are made up of the variable heavy chain or the variable light chain of the antibody structure, and retain binding capability. dAbs have proved difficult to detect in plasma using immunoassay without specific antibodies raised against the dAb.

Results: A sensitive and selective UPLC-MS/MS method for the absolute quantification of a dAb in monkey plasma was developed (range: 1 to 500 ng/ml) without the need for a specific capture antibody.

Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma.

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane.

Development of an in vivo preclinical screen model to estimate absorption and first-pass hepatic extraction of xenobiotics. II. Use of ketoconazole to identify P-glycoprotein/CYP3A-limited bioavailability in the monkey.

The effect of P-glycoprotein (Pgp) and/or CYP3A on the disposition of xenobiotics has been extensively investigated and is often of interest during drug discovery lead optimization. We have previously described a monkey pharmacokinetic screen to rapidly estimate absorption and first-pass extraction. In the present work, this monkey screen has been expanded to include an assessment of Pgp/CYP3A effects on absorption and first-pass extraction, using ketoconazole as a prototypic dual Pgp/CYP3A inhibitor.