Use of [¹³C₁₈] Oleic Acid and Mass Isotopomer Distribution Analysis to Study Synthesis of Plasma Triglycerides In Vivo: Analytical and Experimental Considerations

We have previously reported on a liquid chromatography–mass spectrometry method to determine the disposition of [¹³C₁₈]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision, and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted, and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail, and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue; however, the present report provides investigators with the tools necessary to conduct such studies

Development of Anti-CD74 Antibody–Drug Conjugates to Target Glucocorticoids to Immune Cells

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody–drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology