In Vivo Pharmacokinetic and Pharmacodynamic Properties of the Antiarrhythmic Moleculeent-Verticilide

Abstract

The unnatural verticilide enantiomer (ent-verticilide) is a selective and potent inhibitor of cardiac ryanodine receptor (RyR2) calcium release channels and exhibits antiarrhythmic activity in a murine model of catecholaminergic polymorphic ventricular tachycardia (CPVT). To determine verticilide's pharmacokinetic and pharmacodynamic properties in vivo, we developed a bioassay to measure nat-and ent-verticilide in murine plasma and correlated plasma concentrations with antiarrhythmic efficacy in a mouse model of CPVT. nat-Verticilide rapidly degraded in plasma in vitro, showing >95% degradation within five minutes, whereas ent-verticilide showed <1% degradation over six hours. Plasma was collected from mice following intraperitoneal administration of ent-verticilide at two doses (3 mg/kg, 30 mg/kg). Peak plasma concentration (C max ) and area under the plasma-concentration time curve (AUC) scaled proportionally to dose and the half-life was 6.9 hr for the 3 mg/kg dose and 6.4 hr for the 30 mg/kg dose.Antiarrhythmic efficacy was examined using a catecholamine challenge protocol at time points ranging from 5 to 1440 min after intraperitoneal dosing. ent-Verticilide inhibited ventricular arrhythmias as early as 7 min after administration in a concentration-dependent manner, with an estimated potency (IC 50 ) of 266 ng/mL (312 nM) and an estimated maximum inhibitory effect of 93.5%. Unlike the FDA-approved pan-RyR blocker dantrolene, the RyR2-selective blocker entverticilide (30 mg/kg) did not reduce skeletal muscle strength in vivo. We conclude that entverticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development.

ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic inCasq2−/−Mice

“…5B and (Batiste et al, 2019)). As reported for ent-1 (Blackwell et al, 2023), we also observe a clear dose-dependent effect of ent-B1 in vivo (Fig. 5).…”

“…ent-B1's combination of sub-micromolar potency and partial channel inhibition is ideal for our target, as a stronger inhibition of RyR2 could be fatal given its central role in physiologic excitation-contraction coupling. Although peak plasma concentration was comparable to ent-1, our pharmacokinetic study revealed a more rapid systemic clearance of ent-B1 (t 1/2 = 45 min, table 1) relative to ent-1 (415 min) in mice (Blackwell et al, 2023). Given the short half-life, ent-B1 would only be useful in acute clinical scenarios unless the drug formulation is modified to produce a sustainedrelease profile.…”

“…All protocols and procedures were compliant with Animal care and Use Application approved by the Institution Animal Care and Use Committee of Pharmaron, Inc., (protocol # PH-DMPK-VUMC-22-003) following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Based on our previous study with ent-1 (Blackwell et al, 2023), an ent-B1 dose of 3 mg/kg (drug/body weight) was used. Three male CD1 mice, age 6-8 weeks, were selected for the study.…”

“…Our hit compound ent-1 had antiarrhythmic efficacy in vivo in a single dose study at 3 and 30 mg/kg i.p. in Casq2 -/mice (Blackwell et al, 2023). We tested the same doses of ent-B1 in a triple-crossover design with each mouse receiving vehicle (DMSO), 3 mg/kg, and 30 mg/kg with a oneweek washout period between experiments.…”

ent-Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice

“…All protocols and procedures were compliant with Animal care and Use Application approved by the Institution Animal Care and Use Committee of Pharmaron, Inc., following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Based on our previous study with ent-1 (Blackwell et al, 2023), an ent-B1 dose of 3 mg/kg (drug/body weight) was used. Three CD1 mice (all males) 6-8 weeks old were selected for the study.…”