Investigation into the Mechanism(s) That Leads to Platelet Decreases in Cynomolgus Monkeys During Administration of ISIS 104838, a 2'-MOE-Modified Antisense Oligonucleotide.

ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration. In response to continued dosing, PLT counts were decreased by 50%-90% by day 30 in all monkeys. PLT decreases were accompanied by 2- to 4.5-fold increases in immunoglobulin M(IgM), which were typified by a 2- to 5-fold increase in antiplatelet factor 4 (antiPF4) IgM and antiPLT IgM, respectively. Monocyte chemotactic protein 1 increased upon dosing of ISIS 104838, concomitant with a 2- to 6-fold increase in monocyte-derived extracellular vesicles (EVs), indicating monocyte activation but not PLT activation. Despite a 2- to 3-fold increase in von Willebrand factor antigen in all monkeys following ASO administration, only 2 monkeys showed a 2- to 4-fold increase in endothelial EVs. Additionally, a ∼60 - 80%% increase in PLT sequestration in liver and spleen was also observed. Collectively, these results suggest the overall increase in total IgM, antiPLT IgM and/or antiPF4 IgM, in concert with monocyte activation contributed to increased PLT sequestration in spleen and liver, leading to decreased PLTs in peripheral blood.

Melanoma Cell Invasive and Metastatic Potential Correlates with Endothelial Cell Reorganization and Tenascin Expression.

A direct correlation was observed between the invasive and metastatic potential of A375 melanoma cells and their expression of tenascin and ability to support endothelial cell adhesion and reorganization. Since the ability to metastasize and establish a neovasculature requires interaction of tumor cells with extracellular matrix and endothelial cells, we examined the potential of matrix proteins synthesized by three melanoma cell lines with low-A375P, medium-A375M and high-A375SM invasive and metastatic properties to induce adhesion and rearrangement of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs adhered to and reorganized into a network of connecting and aligned cells on wells conditioned by A375SM and A375M but not A375P cells. These changes in morphology suggested differences in matrix composition among the three melanoma cell lines. In comparison to low levels of substrate-bound fibronectin and laminin, increasingly higher levels of substrate-bound tenascin were synthesized by the A375P, A375M, A375SM cell lines. HUVEC adhesion and reorganization on A375-conditioned matrix was tenascin-dependent and could be inhibited with antibodies against human tenascin. HUVEC adhesion to A375SM-conditioned matrix and tenascin require α(v)ß(3) while reorganization may require α2ß1 as well. Our results suggest that tenascin plays a role in integrin-dependent adhesion and reorganization of HUVECs in response to the extracellular matrix of metastatic melanoma cells.