Delayed LY333013 (Oral) and LY315920 (Intravenous) Reverse Severe Neurotoxicity and Rescue Juvenile Pigs from Lethal Doses of Micrurus fulvius (Eastern Coral Snake) Venom

Abstract

Objective: There is a clear, unmet need for effective, lightweight, shelf-stable and economical snakebite envenoming therapies that can be given rapidly after the time of a snake's bite and as adjuncts to antivenom therapies in the hospital setting. The sPLA2 inhibitor, LY315920, and its orally bioavailable prodrug, LY333013, demonstrate surprising efficacy and have the characteristics of an antidote with potential for both field and hospital use.

Methods: The efficacy of the active pharmaceutical ingredient (LY315920) and its prodrug (LY333013) to treat experimental, lethal envenoming by Micrurus fulvius (Eastern coral snake) venom was tested using a porcine model. Inhibitors were administered by either intravenous or oral routes at different time intervals after venom injection. In some experiments, antivenom was also administered alone or in conjunction with LY333013.

Results: 14 of 14 animals (100%) receiving either LY315920 (intravenous) and/or LY333013 (oral) survived to the 120 h endpoint despite, in some protocols, the presence of severe neurotoxic signs. The study drugs demonstrated the ability to treat, rescue, and re-rescue animals with advanced manifestations of envenoming.

Conclusions: Low molecular mass sPLA2 inhibitors were highly effective in preventing lethality following experimental envenoming by M. fulvius. These findings suggest the plausibility of a new therapeutic approach to snakebite envenoming, in this example, for the treatment of a coral snake species for which there are limitations in the availability of effective antivenom.

Keywords: Micrurus fulvius; PLA2; antidote; antivenom; coral snake; envenoming; inhibitor; neurotoxicity; phospholipase A2; snakebite.

Figure 1

Kaplan Meier curves of lethality in pigs receiving various doses of M. fulvius venom.

Figure 2

LY315920 (Intravenous) and LY3333013 (Oral) alone or in combination completely abrogated lethality induced by M. fulvius venom in different protocols: IV Rescue (A), IV to Oral transition (B) and Oral only and antivenom rescue (C) (see description of the various treatment protocols in the Materials and Methods section).

Figure 3

Evidence of systemic envenoming prior to initiation of treatment is illustrated by the incoagulability of blood. In this example, coagulopathy was corrected within a short time after oral administration of LY333013. Sonoclot (A) and Thromboelastography (TEG) (B). (C): A typical presentation of venom-induced gross intravascular hemolysis was frequently observed during the experiments. Samples correspond to plasma from pigs injected with 0.5 mg/kg venom and collected at times 0 min (1), 30 min (2), 1 h (3), 4 h (4) and 8 h (5). Notice the evident hemolysis in the sample at 1 h.

Figure 4

LY315920-treated animals showed a general preservation of hemostasis compared to controls. (A) Platelet counts remained normal but platelet function (B) was inhibited by venom. Platelet function was restored and maintained in the presence of drug. (C) Activated clotting time, (D) Prothrombin Time and (E) Creatine kinase largely remained within normal levels when animals were treated with sPLA2 inhibitors. Creatinine concentrations were normal for all subjects (F). Reference ranges: Platelet count 200–400 10³/µL, Platelet Function 2–4.8 Units, ACT 69–221 s, PT 10.5–13.5 s, CK 100–400 IU/L, Creatinine 0.1-2 mg/dL.

Figure 5

Clinical score in envenomed animals. All animals that received the test drug avoided or recovered from neurotoxicity. When rescue antivenom was not efficacious, the drug resolved severe neurotoxicity (Protocol C.4).