Hicks, L. | Search bioRxiv

bioRxiv2023

In-vitro Modeling of Intravenous Drug Precipitation by the Optical Spatial Precipitation Analyzer (OSPREY)

Intravenous (IV) administration of poorly water-soluble small molecule therapeutics can lead to precipitation during mixing with blood. This can limit characterization of pharmacological and safety endpoints in preclinical models. Most often, tests of kinetic and thermodynamic solubility are used to optimize the formulation for solubility prior to infusion in animals, but these do not capture the dynamic precipitation processes that take place during in-vivo administration. To better capture the fluid dynamic processes that occur during IV administration, we developed the Optical Spatial Precipitation AnalYzer (OSPREY) as a method to quantify the amount and size of compound precipitates in whole blood using a flow-through system that mimics IV administration. Here, we describe the OSPREY device and its underlying imaging processing methods. We then validate the ability to accurately segment particles according to their size using monodisperse suspensions of microspheres (diameter 50 to 425 microns). Next, we use a tool compound, ABT-737, to study the effects of compound concentration, vessel flow rate, compound infusion rate and vessel diameter on precipitation. Finally, we use the physiological diameter and flow rate of rat femoral vein and dog saphenous vein to demonstrate the potential of OSPREY to model in-vivo precipitation in a controlled, dynamic in-vitro assay. HighlightsO_LIProspective small molecule therapeutics are often solubility challenged when injected into whole blood at elevated concentrations for toxicology studies. C_LIO_LIImproved in-vitro solubility measurements in a flowing system are needed to better understand in-vivo intravenous precipitation C_LIO_LIOSPREY is a novel in-vitro flow-through system that quantifies solubility in whole blood C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/530827v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@15b28b4org.highwire.dtl.DTLVardef@1cd4845org.highwire.dtl.DTLVardef@43d919org.highwire.dtl.DTLVardef@61f978_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

De novo identification of CD4+ T cell epitopes

CD4+ T cells recognize peptide antigens presented on class II Major Histocompatibility Complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor (TCR) sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of Signaling and Antigen-presenting Bifunctional Receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Here, we demonstrate that SABR-IIs libraries presenting endogenous and post-translationally modified epitopes can be used for antigen discovery. Using SABR-II libraries in conjunction with single cell RNA sequencing, we de-convoluted multiple highly expanded TCRs from pancreatic islets of Non-Obese Diabetic (NOD) mice. We compounded antigen discovery by incorporating computational TCR similarity prediction metrics followed by experimental validation. Finally, we showed SABR-IIs presenting epitopes in class II HLA alleles can be used for antigen discovery for human CD4+ T cells. Taken together, we have developed a rapid, flexible, scalable, and versatile approach for the de novo identification of CD4+ T cell ligands from single cell RNA sequencing data using experimental and computational approaches.

immunology↗