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. 2022 Apr;96(4):1055-1063.
doi: 10.1007/s00204-022-03235-z. Epub 2022 Feb 14.

An ex vivo perfused ventilated murine lung model suggests lack of acute pulmonary toxicity of the potential novel anticancer agent (-)-englerin A

Affiliations

An ex vivo perfused ventilated murine lung model suggests lack of acute pulmonary toxicity of the potential novel anticancer agent (-)-englerin A

Christian Schremmer et al. Arch Toxicol. 2022 Apr.

Abstract

(-)-Englerin A (EA), a potential novel anti-cancer drug, is a potent selective activator of classical transient receptor potential 4 and 5 (TRPC4, TRPC5) channels. As TRPC4 channels are expressed and functional in the lung endothelium, possible side effects such as lung edema formation may arise during its administration. Well-established in vivo rodent models for toxicological testing, however, rapidly degrade this compound to its inactive derivative, englerin B. Therefore, we chose an ex vivo isolated perfused and ventilated murine lung (IPVML) model to detect edema formation due to toxicants, which also reduces the number of incriminating animal experiments required. To evaluate the sensitivity of the IPVML model, short-time (10 min) drops of the pH from 7.4 down to 4.0 were applied, which resulted in linear changes of tidal volumes, wet-to-dry weight ratios and incorporation of FITC-coupled dextran particles from the perfusate. As expected, biological activity of EA was preserved after perfusion in the IPVML model. Concentrations of 50-100 nM EA continuously perfused through the IPVML model did not change tidal volumes and lung weights significantly. Wet-to-dry weight ratios were increased after perfusion of 100 nM EA but permeation of FITC-coupled dextran particles from the perfusate to the lung tissues was not significantly different. Therefore, EA shows little or no significant acute pulmonary toxicity after application of doses expected to activate target ion channels and the IPVML is a sensitive powerful ex vivo model for evaluating acute lung toxicity in accordance with the 3R rules for animal experimentation.

Keywords: Anti-cancer drug; Classical transient receptor potential 4 and 5 (TRPC4, TRPC5); FITC–dextran permeation assay; Lung edema; Tidal volume; Wet-to-dry weight ratio.

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Conflict of interest statement

David Beech is an inventor on a patent to develop EA analogues as anti-cancer therapeutics: U.S. Patent No. 11,098,054 issued August 24, 2021. This patent can remain in force until July 5, 2038. Compounds for Treating Cancer. Inventors: John Beutler, Antonio Echavarren, William Chain, David Beech, Zhenhua Wu, Jean-Simon Suppo, Fernando Bravo, Hussein Rubaiy.The other authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Experimental setup of the isolated perfused ventilated murine lung (IPVML) model (A). A freshly isolated lung heart preparation was perfused through the pulmonary artery and ventilated through the trachea. Twenty ml of solutions with Englerin A (EA, 50, 100 nM) or equilibrated to pH 6.0, pH 5.0 and pH 4.0 were relased from a reservoir in the perfusion solution. Lung weight and tidal volumes were continously quantified by in built weighing scales or the software, respectively. Time line of the experiment (B). After 15 min perfusion EA in solution or solutions of decreased pH were released from the reservoir. The pH was continously measured in the reservoir and adjusted for 10 min, if necessary. The outlet was closed after 4 min and the perfusion solutions circulated for another 50 min in a closed circuit
Fig. 2
Fig. 2
Lung weight gain after a short (10 min) drop of the pH to 6.0, 5.0 and 4.0 (A). Changes in tidal volumes after a short (10 min) drop of the pH to 6.0, 5.0 and 4.0 (B). Increases in wet-to-dry weight ratios after a short (10 min) drop of the pH to 6.0, 5.0 and 4.0 were quantified after the experiment (C). Data are means ± SEM. p values were calculated by two way ANOVA and are indicated by asterisks (*, p < 0.05; 2*, p < 0.01; 3*, p < 0.001, 4*, p < 0.0001)
Fig. 3
Fig. 3
Structural formula of (−)-englerin A (EA, provided by the manufacturer, see www.carlroth.com, A) and Ca2+ imaging experiments of HEK 293T cells heterologously expressing TRPC4 channels (TRPC4 transfected) or mock-transfected controls after application of EA (50 nM, 100 nM) before and after 1 h perfusion in the IPVLM model. One representative experiment (n > 3 cells) out of three is shown (B–E)
Fig. 4
Fig. 4
Lung weight gain after application of (−)-englerin A (EA, 50 and 100 nM) (A). Changes in tidal volumes after application of EA (50 and 100 nM) (B). Increases in wet-to-dry weight ratios after after application of EA (50 and 100 nM) (C). Data are means ± SEM. p values were calculated by two way ANOVA and are indicated by asterisks (*, p < 0.05; 2*, p < 0.01)
Fig. 5
Fig. 5
Tissue permeation of fluorescein isothiocyanate (FITC) dextrane particle after perfusion of lungs at pH 7.4 and after a short (10 min) drop of the pH to 4.0. Two representative overlays of FITC fluorescence and coresponding differential interference contrast (DIC) images showing lung tissues after perfusion of electrolyte solution (pH 7.4) and after a short (10 min) drop in pH (pH 4.0) are depicted (A). Two representative scans from total lungs at pH 7.4 and after a short (10 min) drop of the pH to 4.0 (B). Normalized integrated densities of lungs after a short (10 min) drop of the pH to 6.0, 5.0 and 4.0 or perfused with EA (50 and 100 nM) were normalized to lungs perfused with electrolyte solution at pH 7.4 and plotted as columns. Data are means ± SEM. p values were calculated by two way ANOVA and are indicated by asterisks (*, p < 0.05)

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