Establishing a physiologically based pharmacokinetic framework for aldehyde oxidase and dual aldehyde oxidase-CYP substrates.

Aldehyde oxidase (AO) contributes to the clearance of many approved and investigational small molecule drugs, which are often dual substrates of AO and drug-metabolizing enzymes such as cytochrome P450s (CYPs). As such, the lack of established framework for quantitative translation of the clinical pharmacologic correlates of AO-mediated clearance represents an unmet need. This study aimed to evaluate the utility of physiologically based pharmacokinetic (PBPK) modeling in the development of AO and dual AO-CYP substrates.

Complementarity of two proteomic data analysis tools in the identification of drug-metabolising enzymes and transporters in human liver.

Several software packages are available for the analysis of proteomic LC-MS/MS data, including commercial ( Mascot/Progenesis LC-MS) and open access software ( MaxQuant). In this study, Progenesis and MaxQuant were used to analyse the same data set from human liver microsomes ( = 23). Comparison focussed on the total number of peptides and proteins identified by the two packages.

Challenges and Opportunities for In Vitro-In Vivo Extrapolation of Aldehyde Oxidase-Mediated Clearance: Toward a Roadmap for Quantitative Translation.

Underestimation of aldehyde oxidase (AO)-mediated clearance by current in vitro assays leads to uncertainty in human dose projections, thereby reducing the likelihood of success in drug development. In the present study we first evaluated the current drug development practices for AO substrates. Next, the overall predictive performance of in vitro-in vivo extrapolation of unbound hepatic intrinsic clearance (CL) and unbound hepatic intrinsic clearance by AO (CL) was assessed using a comprehensive literature database of in vitro (human cytosol/S9/hepatocytes) and in vivo (intravenous/oral) data collated for 22 AO substrates (total of 100 datapoints from multiple studies).

Proteomic quantification of receptor tyrosine kinases involved in the development and progression of colorectal cancer liver metastasis.

Introduction: Alterations in expression and activity of human receptor tyrosine kinases (RTKs) are associated with cancer progression and in response to therapeutic intervention.

Methods: Thus, protein abundance of 21 RTKs was assessed in 15 healthy and 18 cancerous liver samples [2 primary and 16 colorectal cancer liver metastasis (CRLM)] matched with non-tumorous (histologically normal) tissue, by a validated QconCAT-based targeted proteomic approach.

Results: It was demonstrated, for the first time, that the abundance of EGFR, INSR, VGFR3 and AXL, is lower in tumours relative to livers from healthy individuals whilst the opposite is true for IGF1R.

Toward systems-informed models for biologics disposition: covariates of the abundance of the neonatal Fc Receptor (FcRn) in human tissues and implications for pharmacokinetic modelling.

Biologics are a fast-growing therapeutic class, with intertwined pharmacokinetics and pharmacodynamics, affected by the abundance and function of the FcRn receptor. While many investigators assume adequacy of classical models, such as allometry, for pharmacokinetic characterization of biologics, advocates of physiologically-based pharmacokinetics (PBPK) propose consideration of known systems parameters that affect the fate of biologics to enable a priori predictions, which go beyond allometry. The aim of this study was to deploy a systems-informed modelling approach to predict the disposition of Fc-containing biologics.

Computational Studies on Selected Macrolides Active against Combined with the NMR Study of Tylosin A in Deuterated Chloroform.

Although many antibiotics are active against Gram-positive bacteria, fewer also show activity against Gram-negative bacteria. Here, we present a combination of in silico (electron ion-interaction potential, molecular docking, ADMET), NMR, and microbiological investigations of selected macrolides (14-membered, 15-membered, and 16-membered), aiming to discover the pattern of design for macrolides active against Gram-negative bacteria. Although the conformational studies of 14-membered and 15-membered macrolides are abundant in the literature, 16-membered macrolides, and their most prominent representative tylosin A, have received relatively little research attention.

Altered Bioavailability and Pharmacokinetics in Crohn's Disease: Capturing Systems Parameters for PBPK to Assist with Predicting the Fate of Orally Administered Drugs.

Backgrond And Objective: Crohn's disease (CD) is a chronic inflammatory bowel disease that affects a wide age range. Hence, CD patients receive a variety of drugs over their life beyond those used for CD itself. The changes to the integrity of the intestine and its drug metabolising enzymes and transporters (DMETs) can alter the oral bioavailability of drugs.

Quantitative Assessment of the Impact of Crohn's Disease on Protein Abundance of Human Intestinal Drug-Metabolising Enzymes and Transporters.

Crohn's disease affects the mucosal layer of the intestine, predominantly ileum and colon segments, with the potential to affect the expression of intestinal enzymes and transporters, and consequently, oral drug bioavailability. We carried out a quantitative proteomic analysis of inflamed and non-inflamed ileum and colon tissues from Crohn's disease patients and healthy donors. Homogenates from samples in each group were pooled and protein abundance determined by liquid chromatography-mass spectrometry (LC-MS).

Examining Physiologically Based Pharmacokinetic Model Assumptions for Cross-Tissue Similarity of Activity per Unit of Enzyme: The Case Example of Uridine 5'-Diphosphate Glucuronosyltransferase.

The default assumption during in vitro in vivo extrapolation (IVIVE) to predict metabolic clearance in physiologically based pharmacokinetics (PBPK) is that protein expression and activity have the same relationship in various tissues. This assumption is examined for uridine 5'-diphosphate glucuronosyltransferases (UGTs), a case example where expression and hence metabolic activity are distributed across various tissues. Our literature analysis presents overwhelming evidence of a greater UGT activity per unit of enzyme (higher k) in kidney and intestinal tissues relative to liver (greater than 200-fold for UGT2B7).

Proteomic quantification of perturbation to pharmacokinetic target proteins in liver disease.

Model-based assessment of drug pharmacokinetics in liver disease requires quantification of abundance and disease-related changes in hepatic enzymes and transporters. This study aimed to assess performance of three label-free methods [high N (HiN), intensity-based absolute quantification (iBAQ) and total protein approach (TPA)] against QconCAT-based targeted data in healthy and diseased (cancer and cirrhosis) liver tissue. Measurements were compared across methods and disease-to-control ratios provided a 'disease perturbation factor' (DPF) for each protein.

Quantitative Proteomics of Hepatic Drug-Metabolizing Enzymes and Transporters in Patients With Colorectal Cancer Metastasis.

The impact of liver cancer metastasis on protein abundance of 22 drug-metabolizing enzymes (DMEs) and 25 transporters was investigated using liquid chromatography-tandem accurate mass spectrometry targeted proteomics. Microsomes were prepared from liver tissue taken from 15 healthy individuals and 18 patients with cancer (2 primary and 16 metastatic). Patient samples included tumors and matching histologically normal tissue.

A family of QconCATs (Quantification conCATemers) for the quantification of human pharmacological target proteins.

We have developed a family of QconCAT standards for the absolute quantification of pharmacological target proteins in a variety of human tissues. The QconCATs consist of concatenated proteotypic peptides, are designed in silico, and expressed in E. coli in media enriched with [C] arginine and [C] lysine to generate stable isotope-labeled multiplexed absolute quantification standards.

Label-Free but Still Constrained: Assessment of Global Proteomic Strategies for the Quantification of Hepatic Enzymes and Transporters.

Building and refining pharmacology models require "system" data derived from tissues and in vitro systems analyzed by quantitative proteomics. Label-free global proteomics offers a wide scope of analysis, allowing simultaneous quantification of thousands of proteins per sample. The data generated from such analysis offer comprehensive protein expression profiles that can address existing gaps in models.

Selected Derivatives of Erythromycin B- and Anti-Malarial Studies.

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their molecular docking into segments of the exit tunnel of the apicoplast ribosome from .

Proteomics of colorectal cancer liver metastasis: A quantitative focus on drug elimination and pharmacodynamics effects.

Aims: This study aims to quantify drug-metabolising enzymes, transporters, receptor tyrosine kinases (RTKs) and protein markers (involved in pathways affected in cancer) in pooled healthy, histologically normal and matched cancerous liver microsomes from colorectal cancer liver metastasis (CRLM) patients.

Methods: Microsomal fractionation was performed and pooled microsomes were prepared. Global and accurate mass and retention time liquid chromatography-mass spectrometry proteomics were used to quantify proteins.

Non-uniformity of Changes in Drug-Metabolizing Enzymes and Transporters in Liver Cirrhosis: Implications for Drug Dosage Adjustment.

Liver cirrhosis is a chronic disease that affects the liver structure, protein expression, and overall metabolic function. Abundance data for drug-metabolizing enzymes and transporters (DMET) across all stages of disease severity are scarce. Levels of these proteins are crucial for the accurate prediction of drug clearance in hepatically impaired patients using physiologically based pharmacokinetic (PBPK) models, which can be used to guide the selection of more precise dosing.

Quantitative Proteomic Map of Enzymes and Transporters in the Human Kidney: Stepping Closer to Mechanistic Kidney Models to Define Local Kinetics.

The applications of translational modeling of local drug concentrations in various organs had a sharp increase over the last decade. These are part of the model-informed drug development initiative, adopted by the pharmaceutical industry and promoted by drug regulatory agencies. With respect to the kidney, the models serve as a bridge for understanding animal vs.

Proteomic Quantification of Changes in Abundance of Drug-Metabolizing Enzymes and Drug Transporters in Human Liver Cirrhosis: Different Methods, Similar Outcomes.

Model-based assessment of the effects of liver disease on drug pharmacokinetics requires quantification of changes in enzymes and transporters responsible for drug metabolism and disposition. Different proteomic methods are currently used for protein quantification in tissues and in vitro systems, each with specific procedures and requirements. The outcome of quantitative proteomic assays using four different methods (one targeted and three label-free) applied to the same sample set was compared in this study.

Hepatic Scaling Factors for In Vitro-In Vivo Extrapolation of Metabolic Drug Clearance in Patients with Colorectal Cancer with Liver Metastasis.

In vitro-in vivo extrapolation (IVIVE) linked with physiologically based pharmacokinetics (PBPK) modeling is used to predict the fates of drugs in patients. Ideally, the IVIVE-PBPK models should incorporate systems information accounting for characteristics of the specific target population. There is a paucity of such scaling factors in cancer, particularly microsomal protein per gram of liver (MPPGL) and cytosolic protein per gram of liver (CPPGL).

Inhibition of aggregation of amyloid-β through covalent modification with benzylpenicillin; potential relevance to Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is correlated with the misfolding and aggregation of amyloid- protein (Aβ). Here we report that the antibiotic benzylpenicillin (BP) can specifically bind to Aβ, modulate the process of aggregation and supress its cytotoxic effect, initially a reversible binding interaction, followed by covalent bonding between specific functional groups (nucleophiles) within the Aβ peptide and the -lactam ring. Mass spectrometry and computational docking supported covalent modification of Aβ by BP.

Liquid Biopsy Enables Quantification of the Abundance and Interindividual Variability of Hepatic Enzymes and Transporters.

Variability in individual capacity for hepatic elimination of therapeutic drugs is well recognized and is associated with variable expression and activity of liver enzymes and transporters. Although genotyping offers some degree of stratification, there is often large variability within the same genotype. Direct measurement of protein expression is impractical due to limited access to tissue biopsies.

Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein.

Mass spectrometry-based abundance atlas of ABC transporters in human liver, gut, kidney, brain and skin.

ABC transporters (ATP-binding cassette transporter) traffic drugs and their metabolites across membranes, making ABC transporter expression levels a key factor regulating local drug concentrations in different tissues and individuals. Yet, quantification of ABC transporters remains challenging because they are large and low-abundance transmembrane proteins. Here, we analysed 200 samples of crude and membrane-enriched fractions from human liver, kidney, intestine, brain microvessels and skin, by label-free quantitative mass spectrometry.

Quantification of Proteins Involved in Intestinal Epithelial Handling of Xenobiotics.

The intestinal epithelium represents a natural barrier against harmful xenobiotics, while facilitating the uptake of nutrients and other substances. Understanding the interaction of chemicals with constituents of the intestinal epithelium and their fate in the body requires quantitative measurement of relevant proteins in in vitro systems and intestinal epithelium. Recent studies have highlighted the mismatch between messenger RNA (mRNA) and protein abundance for several drug-metabolizing enzymes and transporters in the highly dynamic environment of the intestinal epithelium; mRNA abundances cannot therefore be used as a proxy for protein abundances in the gut, necessitating direct measurements.