Male mice treated with combined anti-fibrotic therapeutics, IPW5371 and tadalafil, are predisposed to adverse cardiovascular events

Abstract

Fibrosis is a pathological process with few therapeutic options. Experimental molecules are being developed to counteract the fibrotic effects through TGFβ receptor inhibition. Additionally, phosphodiesterase 5 (PDE5) inhibitors also have anti-fibrotic effects; however, the mechanism of action remains unresolved. IPW5371 is an example of an experimental TGFβ-mediated anti-fibrotic compound, and tadalafil is an example of a PDE5 inhibitor. Irradiation increases the frequency of fibrotic lesions, driven by the activation of the TGFβ pathway. We hypothesized that the TGFβ receptor and PDE5 inhibitor agents would be additive in their ability to prevent fibrosis development in tissues in a sub-lethal whole-body irradiation mouse model. However, the combined use of anti-fibrotic agents, tadalafil and IPW5371, caused increased male mouse mortality associated with ascending and thoracic aortic rupture compared to mice that only received one of the drugs. Following histopathological analysis of the mouse hearts, we also observed that irradiation protected against lesions caused by the combination therapy as non-irradiated male mice had significantly worse outcomes as compared to irradiated male mice, substantiating the drug-drug interaction independent of the radiation effects. This important drug interaction needs further investigation as these agents are developed for anti-fibrosis therapy, and PDE5 inhibitors are commonly prescribed to male patients.

Keywords: anti-fibrosis agents; aortopathy; drug interaction; high-fat and high-fructose diet; irradiation; sex differences; vasculopathy.

FIGURE 1

Overview of study designs. Timing (T) of the study is indicated by the numerical marking of weeks (0–40). (A) Study 1: confirmation of irradiation-induced pro-fibrotic signaling. Mice were exposed to Western diet and sham or sub-lethal whole-body irradiation (7Gy) to induce persistent inflammation and fibrosis. (B) Anti-fibrotic therapeutic intervention study. Mice were randomized into treatment groups to evaluate anti-fibrotic agents (tadalafil and/or IPW5371 or control) to inhibit diet- and irradiation-induced effects. (C) Anti-fibrotic intervention evaluation in non-irradiated male mice. Follow-up study to assess the effects of the drug interaction in non-irradiated male mice.

FIGURE 2

(A) Body weight in grams and (B) food intake as average grams/mouse/day was comparable across all groups of mice in studies 1 and 3, which is indicative that drug exposure did not change caloric consumption and weight gain. There were no significant sex by group interactions (NS = non-significant at p > 0.05), and so male and female mice are shown as combined data in these graphs. Means with standard error bars are shown. GP, Group.

FIGURE 3

Sub-lethal whole-body irradiation (7Gy) leads to persistently increased transforming growth factor β (TGF-β) in the liver 6 months after exposure. Means with standard error bars are shown. N = 7/group; **p < 0.01.

FIGURE 4

Survival curves for irradiated mice kept on Western diet and treated with experimental anti-fibrotic therapies. Sub-lethal whole-body irradiation was performed on day 30 of diet consumption, and the study end was 365 days later. Pathological outcomes were measured in a subset evaluated at 180 days. (A) Male and female mice together showed that the combination of IPW5371 and tadalafil caused a significantly higher mortality (p < 0.001). (B) The excess risk of death was sex-specific, with only male mice exposed to this drug combination having a significantly higher probability of death (p < 0.001). Female mice had no group differences in survival (data not shown).

FIGURE 5

Representative images from observed cardiovascular pathology in male C57BL/6N mice. The combined administration of anti-fibrotic therapeutics IPW5371 (TGFβ-mediated anti-fibrotic drug) and tadalafil (PDE5 inhibitor) led to increased mortality associated with ascending and thoracic aortic inflammation, fibrosis, and rupture compared to mice that only received one of the drug treatments. Abbreviations: HE = hematoxylin and eosin. MTC = Masson’s trichrome (A). Animal #105, tadalafil + IPW5371 treatment. Heart base, ascending aorta, and inflammation. The focally thickened and hypercellular wall (arrow) has transmural fibroplasia with a mixed inflammatory cellular infiltrate. HE stain. Scale bar = 20 μm. (B) Animal #105, tadalafil + IPW5371 treatment. Heart base, ascending aorta, inflammation. Higher magnification of (A). widely spaced, mixed inflammatory cellular infiltrate within a loosely arranged fibroblastic tissue stroma shows obscured thickened vessel wall (arrow). HE stain. Scale bar = 8 μm. (C) Animal #168, tadalafil + IPW5371 treatment. Heart base, aorta, inflammation with necrosis. The arrow shows a focus of necrosis and thinning of the vessel wall. HE stain. Scale bar = 20 μm. (D) Animal #168, tadalafil + IPW5371 treatment. Heart base, aorta, inflammation. Higher magnification of (C) a layer of hypereosinophilic material (fibrin; arrow) mixed with necrotic cellular debris and mixed inflammatory cellular infiltrate shows smudging of the thin vessel wall. HE stain. Scale bar = 8 μm. (E) Animal #167, tadalafil + IPW5371 treatment. Heart base, aorta, and inflammation. Transmural mixed inflammatory cellular infiltrates obscure the walls of the vessel (arrow). A thin layer of fibrin covers the thin-walled vessel (arrowheads). HE stain. Scale bar = 20 μm. (F) Animal #167, tadalafil + IPW5371 treatment. Heart base, aorta, and inflammation. Higher magnification of the wall of the aorta in (E) loosely arranged, mixed inflammatory cells and spaced fibroblasts shows obscured vessel wall. A thin layer of fibrin (arrows) mixed with necrotic cellular debris (arrowheads) covers the denuded endothelial surfaces (arrow). The asterisk (*) is in the lumen of the vessel. HE stain. Scale bar = 8 μm. (G) Animal #110, tadalafil + IPW5371 treatment. Thoracic aorta, rupture. There is focal rupture (arrow) of the aorta. From the borders of the ruptured vessel and along the adjacent, irregularly thickened walls of the aorta, there is widespread fibroplasia (arrowheads, blue color). The asterisk (*) is on the wall of the unaffected part of the vessel. MTC stain. Scale bar = 7 mm. (H) Animal #110, tadalafil + IPW5371 treatment. Thoracic aorta, rupture. Higher magnification of the wall of the aorta in (G) widely spaced, mixed inflammatory cellular infiltrate within a loosely arranged fibroblastic tissue stroma (blue color) shows obscured disrupted vessel walls. The arrows point to a focus of fibrinous necrosis. MTC stain. Scale bar = 8 μm.

FIGURE 6

No pharmacokinetic (PK) or pharmacodynamics interaction was observed between anti-fibrotic compounds. (A) TGF-β pathway inhibition, measured by activation of SMAD2/3, was selectively reduced with IPW5371 (30 mg/kg; male and female mice n = 6–8/group). (B) Tissue concentrations of IPS5371 were dose-proportional at 10 and 30 mg/kg doses with no effect of tadalafil co-administration (male mice, n = 4–6/group). (C) Plasma concentrations of IPW5371 confirm no PK interaction of agents (male mice, n = 4–6/group). Means with standard error bars are shown.

FIGURE 7

The combination of anti-fibrotic agents was more lethal in control Western diet-fed male mice (p < 0.001) as compared to similarly treated mice that had sub-lethal whole-body irradiation.