Succinate prodrugs as treatment for acute metabolic crisis during fluoroacetate intoxication in the rat

Abstract

Sodium fluoroacetate (FA) is a metabolic poison that systemically inhibits the tricarboxylic acid (TCA) cycle, causing energy deficiency and ultimately multi-organ failure. It poses a significant threat to society because of its high toxicity, potential use as a chemical weapon and lack of effective antidotal therapy. In this study, we investigated cell-permeable succinate prodrugs as potential treatment for acute FA intoxication. We hypothesized that succinate prodrugs would bypass FA-induced mitochondrial dysfunction, provide metabolic support, and prevent metabolic crisis during acute FA intoxication. To test this hypothesis, rats were exposed to FA (0.75 mg/kg) and treated with the succinate prodrug candidate NV354. Treatment efficacy was evaluated based on cardiac and cerebral mitochondrial respiration, mitochondrial content, metabolic profiles and tissue pathology. In the heart, FA increased concentrations of the TCA metabolite citrate (+ 4.2-fold, p < 0.01) and lowered ATP levels (- 1.9-fold, p < 0.001), confirming the inhibition of the TCA cycle by FA. High-resolution respirometry of cardiac mitochondria further revealed an impairment of mitochondrial complex V (CV)-linked metabolism, as evident by a reduced phosphorylation system control ratio (- 41%, p < 0.05). The inhibition of CV-linked metabolism is a novel mechanism of FA cardiac toxicity, which has implications for drug development and which NV354 was unable to counteract at the given dose. In the brain, FA induced the accumulation of β-hydroxybutyrate (+ 1.4-fold, p < 0.05) and the reduction of mitochondrial complex I (CI)-linked oxidative phosphorylation (OXPHOS_CI) (- 20%, p < 0.01), the latter of which was successfully alleviated by NV354. This promising effect of NV354 warrants further investigations to determine its potential neuroprotective effects.

Keywords: Cell-permeable succinate; Emergency medicine; Fluoroacetate; Mitochondria; Toxicity.

Fig. 1

Study design. This study was performed in three consecutive parts. a We first performed a dose–response titration with sodium fluoroacetate (FA) to determine the dose for intervention with the cell-permeable succinate prodrug NV354. The aim was to identify the lowest dose of FA which would induce significant hypotension and thus, mimic the cardiovascular dysfunction seen in patients exposed to FA. To this end, animals were divided into the following groups: a) Sham, b) FA 0.5 mg/kg, c) FA 0.75 mg/kg, and d) FA 1.25 mg/kg. FA was administered as a single, intravenous (IV) bolus dose to mimic an acute, one-time exposure. b As next step we performed control experiments with NV354 alone to confirm that NV354 did not induce any negative effects on hemodynamic parameters or mitochondrial respiration itself. NV354 treatment was initiated by a single IV injection (17 mg/kg), followed by continuous venous infusion (25 mg/kg/h) until the end of exposure (180 min). c At last, we evaluated the treatment efficacy of NV354 as treatment for acute FA intoxication in the established rat model of acute FA poisoning. For all three parts, rats were anesthetized, mechanically ventilated and temperature controlled to resemble intensive care unit settings. Mean arterial pressure and venous blood gases were monitored throughout the experiment. After exposure, animals were euthanized and heart and brain tissue were collected to evaluate mitochondrial function and -content, metabolomic profile, and tissue pathology

Fig. 2

Effect of sodium fluoroacetate (FA) and the cell-permeable succinate prodrug NV354 on mean arterial pressure (MAP). MAP change from baseline (%) following a exposure to increasing doses of FA to identify a relevant dose for intervention and b treatment of the selected dose of 0.75 mg/kg FA with the cell-permeable succinate prodrug NV354. Data are presented as median plus interquartile range with whiskers indicating minimal and maximal values. Kruskal–Wallis test or One-Way ANOVA was applied for analysis of differences of non-normally distributed and normally distributed data, respectively. To evaluate the effects of FA alone each FA dose was compared to sham whereas the treatment effect of the cell-permeable succinate prodrug NV354 was evaluated by comparison of every group with every other group. *p < 0.05 and **p < 0.01. n = 5–9

Fig. 3

Effect of the cell-permeable succinate prodrug NV354 on mean arterial pressure and cerebral and cardiac mitochondrial respiration of healthy rats. Animals received either no treatment (Sham, n = 6) or a single, intravenous bolus dose of NV354 at 17 mg/kg, followed by continuous venous infusion at 25 mg/kg/h (n = 3) for 180 min. a Mean arterial pressure was measured continuously over 180 min. Respiratory control ratios (RCR) reflective of (b) (g) mitochondrial complex I-linked metabolism, c, d, h, i convergent complex I + II-linked metabolism and (e) (j) complex II-linked metabolism were evaluated. (f) (k) Additionally, the phosphorylation system control ratio (OXPHOS/ETS) was calculated to inform about any limitation of oxidative phosphorylation by CV-linked metabolism. Data are expressed as median plus interquartile range with whiskers indicating minimal and maximal values. Time course analysis was performed using two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparison test and comparison to baseline (t₀min). Respiratory rates were statistically compared using a t test. p < 0.05 was considered to indicate significant differences. Definition of abbreviations: OXPHOS_CI = oxidative phosphorylation capacity linked to complex I metabolism; OXPHOS_CI+II = oxidative phosphorylation capacity linked to complex I and II metabolism; LEAK_CI+II = mitochondrial respiration dependent on the leakiness of the mitochondrial membrane in the presence of complex I and II-linked substrates; ETS_CI+II = electron transport system capacity linked to complex I + II metabolism; ETS_CII = electron transport system capacity linked to complex II metabolism; OXPHOS_CI+II/ETS_CI+II = phosphorylation system control ratio linked to complex I and II metabolism

Fig. 4

Treatment effect of the cell-permeable succinate prodrug NV354 on cardiac mitochondrial metabolism following acute exposure to sodium fluoroacetate (FA). Changes in a organic acids and the nucleotides, b ATP and c GTP were measured in the heart. To further characterize cardiac mitochondrial function, we used high-resolution respirometry of isolated heart mitochondrial to evaluate d oxidative phosphorylation capacity dependent on the TCA cycle metabolism of the complex I-linked substrates pyruvate and malate (OXPHOS_CI (PM)) and the e phosphorylation system control ratio (OXPHOS_CI+II/ETS_CI+II), which informs about CV-linked metabolism. Data are presented as median plus interquartile range with whiskers indicating minimal and maximal values. Kruskal–Wallis test or One-Way ANOVA was applied for analysis of differences of non-normally distributed and normally distributed data, respectively. The treatment effect of NV354 was evaluated by comparison of every group with every other group. *p < 0.05, **p < 0.01 and ***p < 0.001. n = 5–6

Fig. 5

Treatment effect of the cell-permeable succinate prodrug NV354 on cerebral mitochondrial metabolism following acute exposure to sodium fluoroacetate (FA). a Drug delivery of NV354 to the brain was confirmed by assessment of N-acetyl cysteamine (SNAC) levels in the tissue. NV354 is a prodrug of succinate which, upon metabolism, releases the thiol SNAC alongside succinate. SNAC therefore serves as a surrogate marker of drug deposition. Changes in the metabolites b β-hydroxybutyrate, c citrate, d ATP, and e GTP were measured in the brain. f We characterized cerebral mitochondrial function further by means of high-resolution respirometry of tissue homogenates and assessed oxidative phosphorylation capacity dependent on the metabolism of the complex I-linked substrates pyruvate, malate, and glutamate (OXPHOS_CI (PMG)). Data are presented as median plus interquartile range with whiskers indicating minimal and maximal values. Kruskal–Wallis test or One-Way ANOVA was applied for analysis of differences of non-normally distributed and normally distributed data, respectively. The treatment effect of NV354 was evaluated by comparison of every group with every other group. *p < 0.05 and **p < 0.01. n = 5–6