Pierrette De Ron

Paving the Route to Plasma miR-208a-3p as an Acute Cardiac Injury Biomarker: Preclinical Rat Data Supports Its Use in Drug Safety Assessment

Drug-induced cardiac injury (DICI) detection remains a major safety issue in drug development. While circulating microRNAs (miRs) have emerged as promising translational biomarkers, novel early detection biomarkers of cardiotoxicity are needed. This work aims at evaluating whether a panel of putative cardiac injury plasma miRs could serve as early DICI biomarkers in a 4-day rat preclinical model. Out of a panel of 68 selected targets, we identified plasma miR-208a-3p as being significantly upregulated after single administration with either isoproterenol (ISO) or allylamine (AAM). This provides the first evidence of miR-208a-3p detection after AAM administration. Moreover, similarly to cardiac troponins (cTn), plasma miR-208a-3p expression profile appears to be compound-specific with most significant early changes occurring in ISO-treated rats. Overall, miR-208a-3p performance in detecting the severity of myocardial injury, as well as the magnitude of miR-208a-3p increase after ISO or AAM administration, were comparable to that of cTn. Our results highlight the importance of assessing the whole time-dependent profiles of miR expression. Hence, time course evaluation revealed plasma miR candidates whose expression was not stable across the duration of the study in the vehicle group, restricting their utility as cardiac injury-specific biomarkers. In light of these findings, miR-208a-3p has a potential to complement the existing biomarkers of cardiac injury specifically in the context of evaluating toxicity in a time-dependant manner. Assessment of miR-208a-3p in other DICI settings would strengthen its robustness as an early detection biomarker leading to a warranted extensive and rigorous validation.

Advantageous safety profile of a dual selective alpha2C agonist/alpha2A antagonist antinociceptive agent

A selective α2C‐adrenoceptor (AR) agonist was developed for the treatment of neuropathic pain. The objective was to dissociate analgesic activity from cardiovascular and sedative side effects commonly observed with nonselective agents. A 2‐amino‐oxazoline derivative (compound A), identified as a dual α2C‐AR agonist/α2A‐AR antagonist in in vitro‐binding assays, exhibited in vivo efficacy in rodent pain models. Its safety profile was compared with that of clonidine in six different in vivo models. Contrary to clonidine, compound A did not induce hypotension in pentobarbital‐anesthetized rats, in conscious spontaneous hypertensive rats, or in telemetered dogs. Both agents induced similar dose‐dependent decreases in heart rate in dogs and rats. In anesthetized pithed rats, clonidine showed dose‐dependent hypertension and inhibited electrical nerve stimulation‐induced tachycardia at doses close to its efficacious doses in the mouse formalin test, while compound A had much weaker vasoconstrictive and antichronotropic effects. Finally, in a mouse Irwin test, no sedation was observed with compound A at 30‐fold its ED50 in the mouse formalin test, while sedative effects of clonidine started from three‐fold its ED50. These data confirm the advantageous safety profile of the new dual α2C‐AR agonist/α2A‐AR antagonist agent vs. the nonselective agonist clonidine.

Inhibitory effects of sigma-1 ligands on handling-induced tachycardia in conscious tethered rats

We used conscious tethered Sprague‐Dawley rats to evaluate the cardiovascular effects of four sigma‐1 (σ1) agonists and five antagonists, given alone or in combination. All drugs were administered as a single intraperitoneal dose. The agonists were given at doses reported as efficacious in rodent cognition models, while the antagonists were administered at doses neutralizing agonist effects in vivo. Systolic blood pressure (SBP) and heart rate (HR) were continuously recorded for 20 min before and 60 min postadministration. Immediately after injection, a sudden, transitory increase in HR and SBP was noted in all animals, because of the stress induced by handling. For both parameters, a peak value (ΔHRmax and ΔSBPmax) and an area under the curve of changes from baseline over the period 5–20 min postinjection (ΔHR_AUC5–20 min and ΔSBP_AUC5–20 min) were calculated. Three of the four σ1 agonists (SKF‐10,047, dehydroepiandrosterone (DHEAS), Compound 14) significantly reduced ΔHR_AUC5–20 min value without changing ΔHRmax, while the fourth one, SA‐4503, had no significant effect. None of the antagonists (haloperidol, rimcazole, NE‐100, and BD1047) reduced, and even one (progesterone) enhanced the stress‐induced effects on HR. No changes in SBP were noted with any compound. When the antagonist NE‐100 was administered just before SKF‐10,047, it completely reversed the inhibitory effects of the σ1 agonist on HR increase. In conclusion, we demonstrated for the first time the involvement of σ1 receptors in the regulation of handling‐induced tachycardia in the conscious rat. Although additional investigations are needed to fully understand this role, it might offer new therapeutic perspectives to σ1 ligands in the cardiovascular sphere.

miR-21-5p as a potential biomarker of inflammatory infiltration in the heart upon acute drug-induced cardiac injury in rats

Investigation of genomic changes in cardiotoxicity can provide novel biomarkers and insights into molecular mechanisms of drug-induced cardiac injury (DICI). The main objective of this study was to identify and characterize dysregulated microRNAs (miRNAs) in the heart associated with cardiotoxicity. Wistar rats were dosed once with either isoproterenol (1.5 mg/kg, i.p), allylamine (100 mg/kg, p.o.) or the respective vehicle controls. Heart tissue was collected at 24 h, 48 h and 72 h post-drug administration and used for histopathological assessment, miRNA profiling, immunohistochemical analysis and in situ hybridization. Multiplex analysis of 68 miRNAs in the heart revealed a significant upregulation of several miRNAs (miR-19a-3p, miR-142-3p, miR-155-5p, miR-208b-3p, miR-21-5p) after isoproterenol and one miRNA (miR-21-5p) after allylamine administration. Localization of miR-21-5p was specific to inflammatory cell infiltrates in the heart after both treatments. Immunohistochemical analysis of Stat3, a known miR-21-5p regulator, also confirmed its upregulation in cardiomyocytes and inflammatory cell infiltrates. The toxicity signatures based on miRNA networks, identified in vivo, can potentially be used as mechanistic biomarkers as well as to study cardiotoxicity in vitro in order to develop sensitive tools for early hazard identification and risk assessment.

Fatty-Acid Binding Protein 4 (FABP4) as a Potential Preclinical Biomarker of Drug-Induced Kidney Injury

Identification of improved translatable biomarkers of nephrotoxicity is an unmet safety biomarker need. Fatty-acid-binding protein 4 (FABP4) was previously found to be associated with clinical renal dysfunction and was proposed as a biomarker of glomerular damage. The aim of this study was to evaluate FABP4 as a potential preclinical biomarker of drug-induced kidney injury (DIKI). Han-Wistar rats were dosed with cisplatin [2.5 mg/kg, single, intraperitoneally (i.p.)], puromycin (10 mg/kg, daily, i.p.) or N-phenylanthranylic acid [NPAA, 500 mg/kg, daily, per os (p.o.)] over a 28-day period to induce proximal tubule, glomerular or collecting duct injury, respectively. An increase in urinary FABP4 levels was observed on days 1 and 3 after NPAA treatment and on days 14, 21, and 28 after puromycin treatment, whereas cisplatin treatment had no effect. No significant changes were reported for plasma levels of FABP4 after any treatment. Interestingly, immunohistochemical analysis showed a marked decrease in FABP4 expression in the loop of Henle on day 7 after NPAA treatment and a complete loss of FABP4 expression on day 14 after puromycin treatment. The magnitude of increase in FABP4 urinary levels in response to NPAA and puromycin was higher than for established preclinical biomarkers serum creatinine, clusterin, or cystatin C. Our results suggest that FABP4 has the potential for preclinical application as a biomarker of DIKI.

Assessment of a Urinary Kidney MicroRNA Panel as Potential Nephron Segment-Specific Biomarkers of Subacute Renal Toxicity in Preclinical Rat Models

Drug-induced kidney injury (DIKI) remains a significant concern during drug development. Whereas FDA-endorsed urinary protein biomarkers encounter limitations including the lack of translatability, there is a considerable interest surrounding the application of microRNAs (miRNAs) in the renal biomarker space. Current knowledge about the value of these novel biomarkers for subacute preclinical rodent studies is still sparse. In this work, Wistar rats were treated with three nephrotoxic compounds-cisplatin (CIS, proximal tubule, 2.5 mg/kg, intraperitoneal [i.p.]), puromycin (PUR, glomerulus, 20/10 mg/kg, i.p.) and N-phenylanthranylic acid (NPAA, collecting ducts, 500 mg/kg, per os)-for up to 28 days to evaluate the performance of a panel of 68 urinary miRNAs as potential nephron segment-specific biomarkers. Out of these 68 kidney injury associated-miRNAs, our selection strategy ultimately revealed rno-miR-34c-5p significantly dysregulated after CIS single administration, and rno-miR-335 and rno-miR-155-5p significantly dysregulated after PUR treatment. In contrast, NPAA daily administration strongly altered the expression profile of 28 miRNAs, with rno-miR-210-3p displaying the most robust changes. A thorough evaluation showed that these miRNA candidates could complement urinary protein biomarkers to detect CIS- or PUR-induced kidney injury in a subacute setting, with a mechanistic (based on rno-miR-34c-5p) and/or a kidney injury detection potential. Our results also provide the first evidence that urinary miRNAs could enhance the detection of collecting duct damage. Overall, these data improve our understanding of the utility of urinary miRNAs as DIKI biomarkers in a subacute DIKI preclinical setting and support the value of using urinary biomarker panels comprising proteins and miRNAs.