Serum benzylpenicillin levels during treatment for amatoxin poisoning: pharmacokinetic support for OATP1B3 inhibition

Introduction: Benzylpenicillin has long been included in treatment protocols for amatoxin poisoning based on experimental evidence suggesting inhibition of hepatocellular amatoxin uptake via OATP1B3. However, no human pharmacokinetic data have confirmed whether therapeutic regimens achieve serum concentrations sufficient to inhibit this transporter in vivo. Methods: We retrospectively analyzed serum benzylpenicillin concentrations in 16 patients with confirmed amatoxin poisoning treated at the Florence Poison Control Center between 2020 and 2024. All patients received continuous intravenous benzylpenicillin infusion (500,000 IU/kg/day for 48 h, followed by 250,000 IU/kg/day for 48 h) as part of a standardized multimodal protocol. Serum samples (n = 44) were collected during routine care and analyzed using LC-MS/MS. Concentrations were expressed in micromolar (µM) and compared with the reported OATP1B3 inhibitory concentration (IC50 = 25 µM). Results: During the first 48h, all serum samples exceeded the IC50 threshold, with mean concentrations of 265 ± 87 µM at 24 h and 241 ± 74 µM at 48 h. Across all samples, the median concentration was 176 µM (range 45-430 µM), and 93% exceeded 25 µM. Following dose reduction, mean concentrations declined but remained above the IC50 in 66% of samples. No adverse events attributable to benzylpenicillin were observed, and all patients survived to discharge without liver transplantation. Discussion: Although benzylpenicillin's clinical efficacy has yielded conflicting results across retrospective series, variability in dosing regimens and timing likely accounts for much of this inconsistency. The present findings demonstrate that continuous infusion maintains pharmacologically relevant exposure throughout the critical window of amatoxin hepatocellular uptake, supporting target engagement in vivo. The reduced-dose regimen sustained inhibitory concentrations in most samples, suggesting dose optimization is feasible without compromising pharmacological activity. Conclusion: Continuous intravenous infusion of benzylpenicillin achieves serum concentrations exceeding the in vitro OATP1B3 inhibitory threshold in patients with amatoxin poisoning, providing human pharmacokinetic support for its proposed antidotal mechanism and its continued inclusion in multimodal treatment protocols.