Underlying Immune Disorder May Predispose Some Transthyretin Amyloidosis Subjects to Inotersen-Mediated Thrombocytopenia.

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 10/L in 50% and ≥100 × 10/L in 80% of the subjects.

Generation of Macrophages from Cynomolgus-Monkey Bone Marrow as a Model to Evaluate Effects of Drugs on Innate Immunity.

Macrophages are innate immune cells that play important roles in various physiological and pathological processes. Evaluation of pro-inflammatory effects of drugs on macrophages has become commonplace in preclinical drug development prior to human clinical trials. Despite their body-wide distribution, tissue macrophages are often difficult to collect from large animals and humans in a noninvasive manner.

In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia.

Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance in vivo and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an in vitro flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mononuclear cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug.

High Throughput-Based Mitochondrial Function Assays by Multi-Parametric Flow Cytometry.

Mitochondrial dysfunction has been increasingly implicated as an important mechanism for chemical-induced toxicity. In the present unit, we describe a multi-parametric flow cytometry assay to assess the effects of drug or chemical-induced mitochondrial dysfunction in cells. Cells are cultured in a glucose-supplemented medium and exposed to increasing concentrations of various chemicals.

A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells.

Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media.

Troglitazone-induced intracellular oxidative stress in rat hepatoma cells: a flow cytometric assessment.

Background: Troglitazone (TRO), a thiazolidinedione (TZD) peroxisome proliferator-activated receptor gamma agonist, was recently withdrawn from the market because of rare but serious hepatotoxicity. Previous studies investigating the cytotoxicity of TRO in cultured rat hepatocytes have conjectured about the role of oxidative stress in TRO-induced hepatotoxicity. Therefore, we investigated whether TRO induces oxidative stress and, if so, the portion of the TRO molecule responsible for the induction of oxidative stress.