Charge-tagging liquid chromatography–mass spectrometry methodology targeting oxysterol diastereoisomers

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods. Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography – mass spectrometry with multistage fragmentation

Characterizing the plasma protein binding profiles of chemistry diversified antisense oligonucleotides in human and mouse plasma using an ultrafiltration method

IntroductionPlasma protein binding plays a significant role in influencing the pharmacokinetic and pharmacodynamic properties of drugs. This study focuses on examining two pairs of sequence-matched ASOs: phosphorodiamidate morpholino oligomers (PMOs) and 2’-O-methoxyethyl/phosphorothioate (MOE/PS)-modified ASOs, to assess their plasma protein binding profiles.MethodsThe binding of both PMO and MOE/PS-modified ASOs was investigated using an ultrafiltration method combined with hybridization electrochemiluminescence, allowing for the measurement of the unbound fraction (fu) in both mouse and human plasma. To further characterize the interaction between ASOs and plasma proteins, individual binding measurements were taken for five major proteins in human plasma: human serum albumin, α1-acid glycoprotein, human γ-globulin, low-density lipoprotein, and high-density lipoprotein.ResultsThe results showed a notable difference in plasma protein binding between the two types of ASOs, with MOE/PS-modified ASOs exhibiting significantly higher binding compared to PMOs. The fu, plasma values revealed no significant species difference between mouse and human plasma. Additionally, a saturation point for fu, plasma was observed in MOE/PS-modified ASOs at concentrations above 1 μM, whereas PMOs did not show saturation even at concentrations up to 10 μM. Notably, human γ-globulins were found to have a predominant binding affinity for both MOE/PS and PMO ASOs at physiological concentrations, surpassing human serum albumin, the most abundant plasma protein.DiscussionThe results suggest that the chemistries of the ASOs, particularly their modifications, are key determinants of their binding profiles. The study also highlights the important, though previously overlooked, role of human γ-globulins in the plasma protein binding of ASOs. This could have implications for understanding ASO distribution and tissue disposition, which may inform the development and optimization of ASO-based therapies

Cholesterolomics: An update

Cholesterolomics can be regarded as the identification and quantification of cholesterol, its precursors post squalene, and metabolites of cholesterol and of its precursors, in a biological sample. These molecules include 1,25-dihydroxyvitamin D3, steroid hormones and bile acids and intermediates in their respective biosynthetic pathways. In this short article we will concentrate our attention on intermediates in bile acid biosynthesis pathways, in particular oxysterols and cholestenoic acids. These molecular classes are implicated in the aetiology of a diverse array of diseases including autoimmune disease, Parkinson's disease, motor neuron disease, breast cancer, the lysosomal storage disease Niemann-Pick type C and the autosomal recessive disorder Smith-Lemli-Opitz syndrome. Mass spectrometry (MS) is the dominant technology for sterol analysis including both gas-chromatography (GC)-MS and liquid chromatography (LC)-MS and more recently matrix-assisted laser desorption/ionisation (MALDI)-MS for tissue imaging studies. Here we will discuss exciting biological findings and recent analytical improvements

A Phase 1, Randomized, Double‐Blind, Placebo‐Controlled, Single Ascending Dose Trial of AWZ1066S, an Anti‐ Wolbachia Candidate Macrofilaricide

AWZ1066S has been developed as a potential treatment for the neglected tropical diseases lymphatic filariasis and onchocerciasis. AWZ1066S targets the Wolbachia bacterial endosymbiont present in the causative nematode parasites. This phase 1, first‐in‐human study aimed to assess the safety and pharmacokinetics of AWZ1066S in healthy human participants. In a randomized double‐blind, placebo‐controlled, single ascending dose study, healthy adults received a single oral dose of AWZ1066S (or placebo) and were followed up for 10 days. The planned single doses of AWZ1066S ranged from 100 to 1600 mg, and each dose was administered to a cohort of 8 participants (6 AWZ1066S and 2 placebo). In total 30 people participated, 18 (60%) female, median age 30.0 years (minimum 20, maximum 61). The cohorts administered 100, 200, 300, and 400 mg of AWZ1066S progressed unremarkably. After single 700‐mg doses all 4 participants developed symptoms of acute gastritis and transient increases in liver enzymes. The severity of these adverse events ranged from mild to severe, with 1 participant needing hospital admission. Pharmacokinetic analysis indicated that AWZ1066S is rapidly absorbed with predictable pharmacokinetics. In conclusion, safety concerns prevented this study from reaching the human exposures needed for AWZ1066S to be clinically effective against lymphatic filariasis and onchocerciasis

Effect of cholesterol on the dipole potential of lipid membranes

The membrane dipole potential, ψd, is an electrical potential difference with a value typically in the range 150 – 350 mV (positive in the membrane interior) which is located in the lipid headgroup region of the membrane, between the linkage of the hydrocarbon chains to the phospholipid glycerol backbone and the adjacent aqueous solution. At its physiological level in animal plasma membranes (up to 50 mol%), cholesterol makes a significant contribution to ψd of approximately 65 mV; the rest arising from other lipid components of the membrane, in particular phospholipids. Via its effect on ψd, cholesterol may modulate the activity of membrane proteins. This could occur through preferential stabilization of protein conformational states. Based on its effect on ψd, cholesterol would be expected to favour protein conformations associated with a small local hydrophobic membrane thickness. Via its membrane condensing effect, which also produces an increase in ψd, cholesterol could further modulate interactions of polybasic cytoplasmic extensions of membrane proteins, in particular P-type ATPases, with anionic lipid headgroups on the membrane surface, thus leading to enhanced conformational stabilization effects and changes to ion pumping activity.Australian Research Counci

ダイオキシン ノ タイシャ ニ カカワル ホニュウ ドウブツ シトクロム P450 ノ コウゾウ ト キノウ ノ カイセキ

京都大学0048新制・課程博士博士(農学)甲第12357号農博第1538号新制||農||923(附属図書館)学位論文||H18||N4115(農学部図書室)24193UT51-2006-J349京都大学大学院農学研究科食品生物科学専攻(主査)教授 井上 國世, 教授 吉川 正明, 教授 村田 幸作学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA