The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

Abstract

<p>Antisense oligonucleotides (AON) delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS) AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a <em>foamy</em> appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized <em>in vitro</em> using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1<em>α </em>and TNF<em>α</em>, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON<em> in vitro</em>, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the <em>in vivo </em>phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.</p

Clinical Expert Panel on Monitoring Potential Lung Toxicity of Inhaled Oligonucleotides: Consensus Points and Recommendations

“…This is consistent with the fact that oligonucleotides stain blue with hematoxylin and eosin (Levin et al, 1998). This macrophage staining, with no or little evidence of activation, nor other inflammatory cell accumulation, would typically be considered a non-adverse adaptive mechanism in an otherwise healthy animal lung (Lewis et al, 2002;Mosser and Edwards, 2008;Brasey et al, 2011). Whether this finding might be considered adverse-particularly in patients with established lung disease-is not known, because this response also shows reversibility upon termination lends credence to the response being non adverse, whereas fibrosis and metaplasia, described at high inhaled ON doses in animals, showed only partial reversibility in the recovery time allotted (Guimond et al, 2008; FDA personal communication, 2011).…”

“…1). Structure-function relationships of lung disease have been amply demonstrated, in particular using various rodent models (Costa et al, 1986). Similarly, the use of human lung biopsy tissue to help validate some of the more promising, less invasive clinical techniques, such as sputum induction, exhaled gases or breath condensates collection, and measurement of pulmonary function, was also encouraged.…”

Toxicity of orally inhaled drug formulations at the alveolar barrier: parameters for initial biological screening

“…In addition to inducing morphological changes, amiodarone impaired phagocytosis of J774A.1 cells, leaving the reaction to endotoxin challenge unchanged (Hoffman et al, 2015). Brasey et al, by contrast, reported only induction of morphological changes in RAW264.7 cells by amiodarone without impairment of phagocytosis (Brasey et al, 2011). It cannot be excluded that cell lines differ in their sensitivity to drug-induced PLD.…”

In Vitro Multiparameter Assay Development Strategy toward Differentiating Macrophage Responses to Inhaled Medicines

“…The strategy combines a variety of techniques, which have been used previously, often separately, to characterize FMΦ. These include cellular imaging to quantify morphometry and phospholipid accumulation, , toxicogenomic approaches, − and functional assays. − …”