Efflux transporters in drug disposition during pregnancy.

Evidence-based dose selection of drugs in pregnant women has been lacking due to challenges in studying maternal-fetal pharmacokinetics. Hence, many drugs are administered off-label during pregnancy based on data obtained from non-pregnant women. During pregnancy, drug transporters play an important role in drug disposition along with known gestational age-dependent changes in physiology and drug-metabolizing enzymes.

Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671.

Comparing Multiple Reaction Monitoring and Sequential Window Acquisition of All Theoretical Mass Spectra for the Relative Quantification of Barley Gluten in Selectively Bred Barley Lines.

Celiac disease (CD) is a disease of the small intestine that occurs in genetically susceptible subjects triggered by the ingestion of cereal gluten proteins for which the only treatment is strict adherence to a life-long gluten-free diet. Barley contains four gluten protein families, and the existence of barley genotypes that do not accumulate the B-, C-, and D-hordeins paved the way for the development of an ultralow gluten phenotype. Using conventional breeding strategies, three null mutations behaving as recessive alleles were combined to create a hordein triple-null barley variety.

O-Methyltransferase is shared between the pentose phosphate and shikimate pathways and is essential for mycosporine-like amino acid biosynthesis in Anabaena variabilis ATCC 29413.

The parent core structure of mycosporine-like amino acids (MAAs) is 4-deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7-phosphate by the enzymes 2-epi-5-epivaliolone synthase (EVS) and O-methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway-specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway.

Investigation of the protein alkylation sites of the STAT3:STAT3 inhibitor Stattic by mass spectrometry.

STAT3 (Signal Transducer and Activator of Transcription factor 3) is constitutively active in a wide range of human tumours. Stattic is one of the first non-peptidic small molecules reported to inhibit formation of the STAT3:STAT3 protein dimer complex. A mass spectrometry method has been developed to investigate the binding of Stattic to the un-phosphorylated STAT3βtc (U-STAT3) protein.

Site-specific PEGylation at histidine tags.

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines.

Structure-activity relationships of monomeric C2-aryl pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumor agents.

A comprehensive SAR investigation of the C2-position of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) monomer antitumor agents is reported, establishing the molecular requirements for optimal in vitro cytotoxicity and DNA-binding affinity. Both carbocyclic and heterocyclic C2-aryl substituents have been studied ranging from single aryl rings to fused ring systems, and also styryl substituents, establishing across a library of 80 analogues that C2-aryl and styryl substituents significantly enhance both DNA-binding affinity and in vitro cytotoxicity, with a correlation between the two. The optimal C2-grouping for both DNA-binding affinity and cytotoxicity was found to be the C2-quinolinyl moiety which, according to molecular modeling, is due to the overall fit of the molecule in the DNA minor groove, and potential specific contacts with functional groups in the floor and walls of the groove.

Purification, characterisation and identification of acidocin LCHV, an antimicrobial peptide produced by Lactobacillus acidophilus n.v. Er 317/402 strain Narine.

In the last two decades, antimicrobial peptides (AMPs) have been gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Lactobacillus acidophilus n.v.

Discovering oxysterols in plasma: a window on the metabolome.

While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging.

Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge.

The covalent conjugation of a functionalized poly(ethylene glycol) (PEG) to multiple nucleophilic amine residues results in a heterogeneous mixture of PEG positional isomers. Their physicochemical, biological, and pharmaceutical properties vary with the site of conjugation of PEG. Yields are low because of inefficient conjugation chemistry and production costs high because of complex purification procedures.

Site-specific PEGylation of native disulfide bonds in therapeutic proteins.

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.

Analysis of derivatised steroids by matrix-assisted laser desorption/ionisation and post-source decay mass spectrometry.

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude.