Mild but significant elevation of creatine kinase (CK) was detected in DOX pigs after the 2nd treatment (P<0.05 in comparison with MYO pigs), which was absent after the 3rd treatment, showing the insensitiveness of CK as a cardiotoxicity biomarker (Supplementary material online, Figure S1). A.J. The most pronounced difference in the RNA-sequencing analysis is the induction and repression of ISGs by MYO, and DOX, respectively (Figure5). Moreover, the animals were healthy at the time of the study start, and lacking conventional risk factors. Myocardial samples were stained for fibrosis, ki67, and caspase activity (Supplementary material online). snoRNAs are non-coding transcripts that guide nucleotide modifications of other RNAs. We performed cMRI on a 1.5T Siemens Avanto Syngo B17 scanner (Erlangen, Germany) with a phased-array coil and a vector ECG system. In AngII induced fibrosis, syndecan-1 mediates profibrotic signalling through TGF-/SMAD signalling.25. Collagen 1 and a number of collagen-regulating genes (SerpinE1 and H1, P4HA1, and PLOD3) were more strongly induced after DOX treatment. Liposomal DOX was prepared directly before the injection according to the manufacturers instructions. Oxford University Press is a department of the University of Oxford. Weichselbaum RR, Ishwaran H, Yoon T, Nuyten DSA, Baker SW, Khodarev N, Su AW, Shaikh AY, Roach P, Kreike B, Roizman B, Bergh J, Pawitan Y, van de Vijver MJ, Minn AJ. Two weeks after the third and final treatment cycle, the drug concentration in myocardial samples was still lower after application of MYO compared to DOX. These results confirm the comprehensive pharmacokinetic data collected during pre-clinical and clinical development of Myocet,29,30 and the translational value of the pig study. Corresponding author. differences between regions of interest: LV and RV in DOX vs. CO and MYO vs. CO) a linear model for each gene was fitted and the estimated coefficients and standard errors for these contrasts were computed. Department of Cardiology, Medical University of Vienna. For quantitative evaluation of myocardial fibrosis, LE diastolic phase images were obtained after injection of 0.05mmol/kg contrast medium using an inversion recovery prepared, gradient-echo MRI sequence. Data analyses and interpretations were performed by an experienced observer who was blinded to the randomization and to results. We aimed to evaluate transcriptomic responses to anthracyclines with different cardiotoxicity profiles in a translational large animal model for identifying potential alleviation strategies. A direct comparison of gene clusters (Figure5 and Supplementary material online, Table S3) shows consistently stronger expression of interferon-responsive genes after MYO treatment. (A and B) Cardiac magnetic resonance imaging (cMRI) with late enhancement, short-axis view. These include procollagen peptidase enhancer PCOLCE, the plasminogen converting enzymes tPA (PLAT) and uPA (PLAU) and its inhibitor PAI-1 (SerpinE1), matrix metalloproteinases (MMPs)-2, 11, 14, and 15, and tissue inhibitors of metalloproteinases (TIMPs)-1 and 3 (Figure4). Bottom row: Representative image of western blot of cleaved caspase-3 and tubulin as loading control, with significantly higher caspase-3 activity in DOX as compared to MYO samples. Copyright 2022 European Society of Cardiology. The downstream signal transducers SMAD was changed non-significantly, except for a downregulation of SMAD5. Proteinprotein interactions of differentially expressed genes between MYO and DOX groups in the left (LV) and right ventricle (RV). Although the extent of upregulation of fibrosis-associated genes was lower after Myocet, similar caution and in particular close observation of cardiac function is advisable. Twenty-three domestic pigs (Sus scrofa, female large whites 302kg, 3 months old) were randomized into four groups receiving either DOX (group DOX, n=6), EPI (group EPI, n=6), Myocet (group MYO, n=6), or physiologic saline (group CO, n=5) in doses equivalent to human treatment regimens (60mg/m2 body surface area DOX and MYO, 100mg/m2 EPI) as a single 1-h intravenous infusion every 21days (at Days 1, 22, and 43). Interferon-stimulated genes (ISGs) are induced upon certain degrees of DNA damage and can mediate pro-survival signals.18 Among those genes, we found altered expression between DOX and MYO of IFIT1 and 2, ISG15, OAS2, and Poly(ADP-ribose)-polymerases (PARP) 1, 9, and 14. Using 3D volumetry, end-diastolic (EDV), end-systolic volumes (ESV), global LV EF were automatically calculated on short-axis cine MRI images. This work was supported by TEVA ratiopharm, which provided NPL-doxorubicin and an unrestricted grant, but was not involved in the study protocol, data acquisition, data analysis, or the writing of the manuscript. This is reflected by lower expression of the apoptosis marker activated caspase-3 in MYO animals (Figure7). Several of those are incompletely characterized in Sus scrofa databases and some might be of interest for further investigations. We found slight upregulation of the -3 and -5 subunits in the MYO and DOX group, respectively, without changes of -1, -11, and -1; -3 was not detected in the data set. In pigs, transcriptional activation of several matrix metalloproteinases was found after DOX administration.8 It has been shown that DOX triggers several signalling pathways, such as the MAPK, p53, Jak-STAT, Wnt, MAPK/p53, or PPAR pathways, which might all be involved in DOX-associated cardiomyopathy.9,10 Proposed strategies for mitigation of cardiotoxicity include iron chelation,11 VEGF-B gene therapy,12 stimulation of oxidative phosphorylation,9 modulation of DNA damage and oxidative stress,10 or targeting an RNA-binding protein.13 However, a comprehensive high throughput transcriptomic screening of genes or proteins in a translational large animal model of cardiotoxicity had not yet been performed. Functional clusters of dysregulated genes include apoptosis regulation, proto-oncogenes and oncogenes, cellular homeostasis and DNA repair, collagen synthesis, metabolism, and cytoskeleton (Figure4 and Supplementary material online, Table S1). The weight of the MYO and control pigs was significantly higher after the first treatment until the end of the experiments compared to the pigs in the DOX group, indicating better general health of these animals (Figure 1B). doxorubicin anticancer randomized carcinoma multicenter encapsulated metastatic liposome Activation of this subset of ISGs, as seen after liposomal DOX treatment, induces a pro-survival cell response. Their downstream target, the central guardian against genomic mutations, TP53, was slightly upregulated only in MYO. *P<0.05. We also focused on the expression of genes previously associated with anthracycline toxicity (Supplementary material online, Figure S8). COL1A1 and COL1A2 were activated more strongly in the DOX than in the MYO group, while COL3A1 showed significant induction only in the RV of MYO. Differences between treatment and control groups were tested for normality with ShapiroWilk, and parametric data were evaluated for statistical significance using one-way ANOVA tests with Bonferroni post hoc corrections. Levels of TnI (C) and NT-proBNP (D) increased during the experiment and were in the pathologic range in all DOX and MYO animals. The KaplanMeier survival analysis was calculated for all groups. Intracellular localization of TSPO is similar in both groups, with a distinct punctuate pattern indicative for mitochondrial localization, with higher signal intensity in MYO group (green). Supplementary material online, Figure S2 shows representative histological images of the LV and RV in DOX, MYO, and EPI groups with myocardial tissue fibrosis. Current means to mitigate anthracycline-induced cardiotoxicity are limited.5 On the molecular level, the cytostatic effect of anthracyclines is attributed to DNA intercalation, DNA binding and cross-linking, inhibition of topoisomerase II, and induction of apoptosis. The weights of the MYO pigs were significantly higher than those of DOX pigs after the first treatment until the end of the experiments indicating better general health (B). Representative pictures show stronger interstitial collagen deposition after DOX treatment. Pulsed wave Dopplers of diastolic function were recorded by measurements of mitral E and A waves and the E/e ratio by using 4-chamber view. This is mainly due to refined therapy by chemotherapeutics, such as anthracyclines, immunotherapies, other specific treatments, and/or targeted tumour excision or irradiation.1 Unfortunately, 1075% of cancer survivors suffer from chronic health issues in later life, including heart failure, vascular or valve diseases, and other cardiac complications, caused by toxicity of many chemotherapeutics.2 Anthracyclines are one of the most frequently used anticancer drugs. Cardinale D, Colombo A, Lamantia G, Colombo N, Civelli M, De Giacomi G, Rubino M, Veglia F, Fiorentini C, Cipolla CM. Strong increase of expression was observed for the selected interferon-responsive genes. Liposomal encapsulation of DOX (Myocet) provides a certain protective effect against cardiotoxicity by reducing myocardial drug accumulation. A difference was considered statistically significant at P<0.05. This confirms a concentration-dependent effect on interferon-responsive genes. Plasma concentrations upon application of liposomal DOX were 6- and 14-fold higher than after infusion of the free drug directly after completion of the first infusion, and 10 min later, respectively (Figure 7A), indicating faster clearance of the free drug compound. as Myocet (TLC D-99; liposome-encapsulated doxorubicin citrate) compared with conventional doxorubicin when given in combination with cyclophosphamide in patients with metastatic breast cancer, Distinct poly(I-C) and virus-activated signaling pathways leading to interferon- production in hepatocytes, Efficacy and cardiotoxicity of liposomal doxorubicin-based chemotherapy in advanced breast cancer: a meta-analysis of ten randomized controlled trials, Pharmacokinetics of liposomal doxorubicin (TLC-D99; Myocet) in patients with solid tumors: an open-label, single-dose study, Preclinical toxicology study of liposome encapsulated doxorubicin (TLC D-99): comparison with doxorubicin and empty liposomes in mice and dogs, Doxorubicin induced heart failure: phenotype and molecular mechanisms, Comparison of safety and toxicity of liposomal doxorubicin vs. conventional anthracyclines: a meta-analysis, The DNA damage response arouses the immune system, Topoisomerase II inhibitors induce DNA damage-dependent interferon responses circumventing Ebola virus immune evasion, An interferon-related gene signature for DNA damage resistance is a predictive marker for chemotherapy and radiation for breast cancer, Mitochondrial translocator protein (TSPO) ligands prevent doxorubicin-induced mechanical dysfunction and cell death in isolated cardiomyocytes, Update on cardiotoxicity of anti-cancer treatments, The Author(s) 2019.