Identification and quantification of aminophospholipid molecular species on the surface of apoptotic and activated cells

This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a cell-impermeable reagent (sulfo-NHS-biotin), and then they are isolated via a liquid:liquid extraction and quantified by reverse-phase liquid chromatography tandem MS (LC-MS/MS) against in-house-generated standards. This protocol describes a complementary method to existing assays that are not quantitative (e.g., annexin V flow cytometry), and it is applicable to the study of membrane reorganization in all cell types during apoptosis (e.g., during development, cancer, psychiatric disorders and other conditions, aging, vesiculation and cell division). The protocol takes ∼2–4 d, including the generation of standards

Dopamine D2 Occupancy as a Biomarker for Antipsychotics: Quantifying the Relationship with Efficacy and Extrapyramidal Symptoms

For currently available antipsychotic drugs, blockade of dopamine D2 receptors is a critical component for achieving antipsychotic efficacy, but it is also a driving factor in the development of extrapyramidal symptoms (EPS). To inform the clinical development of asenapine, generic mathematical models have been developed for predicting antipsychotic efficacy and EPS tolerability based on D2 receptor occupancy. Clinical data on pharmacokinetics, D2 receptor occupancy, efficacy, and EPS for several antipsychotics were collected from the public domain. Asenapine data were obtained from in-house trials. D2 receptor occupancy data were restricted to published positron emission tomography studies that included blood sampling for pharmacokinetics. Clinical efficacy data were restricted to group mean endpoint data from short-term placebo-controlled trials, whereas EPS evaluation also included some non-placebo-controlled trials. A generally applicable model connecting antipsychotic dose, pharmacokinetics, D2 receptor occupancy, Positive and Negative Syndrome Scale (PANSS) response, and effect on Simpson–Angus Scale (SAS) was then developed. The empirical models describing the D2–PANSS and D2–SAS relationships were used successfully to aid dose selection for asenapine phase II and III trials. A broader use can be envisaged as a dose selection tool for new antipsychotics with D2 antagonist properties in the treatment of schizophrenia