Varespladib (LY315920) Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation

Abstract

Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2) activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib showed high-level secretory PLA2 (sPLA2) inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases) could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite.

Keywords: LY315920; LY333013; antidote; envenomation; field treatment; inhibitor; methyl-varespladib; pre-referral; snakebite; varespladib.

Figure 1

In vitro dose-response curves for varespladib and its orally bioavailable prodrug methyl-varespladib tested against 20 medically important snake venoms. While demonstrating high degrees of potency against snake venoms, neither varespladib nor methyl-varespladib showed high degrees of potency against bee venom sPLA2 (positive control). N = 1 run unless otherwise specified number of replicates. Error bars signify s.d. a. Agkistrodon brevicaudus blomhoffi, b. Bungarus caeruleus, c. B. fasciatus, d. Crotalus adamanteus, e. Crotalus atrox, f. Crotalus scutulatus, g. Dendroaspis polylepis, h. Echis carinatus, i. Laticauda semifasciata, j. Micrurus fulvius, k. Naja naja atra, l. Naja naja kaouthia, m. Naja naja naja, n. Notechis scutatus scutatus, o. Ophiophagus hannah, p. Oxyuranus scutellatus, q. Pseudechis australis, r. Vipera berus, s. Daboia russelli, t. Crotalus durissus terrificus, u. Bee venom (Apis mellifera) purified sPLA2 positive (+) control.

Figure 2

Pretreatment with varespladib protects against M. fulvius envenomation. (a) Five of 5 (100%) of mice given 4 mg/kg SC injections of M. fulvius venom died quickly with previously described paralytic and hemorrhagic complications. Zero of 5 (0%) of mice pre-treated with varespladib (4 mg/kg) several minutes before venom injection died within 8 h; (b) from a different experiment with methyl-varespladib, but exemplary of coral snake bite syndrome and effect of the study treatments: Left, untreated mouse 2 h after venom administration showing effects of venom including (i) postural weakness; (ii) vasodilation (ears) and (iii) ptosis; Right, methyl-varespladib treated mouse. Both mice have piloerection.

Figure 2

Pretreatment with varespladib protects against M. fulvius envenomation. (a) Five of 5 (100%) of mice given 4 mg/kg SC injections of M. fulvius venom died quickly with previously described paralytic and hemorrhagic complications. Zero of 5 (0%) of mice pre-treated with varespladib (4 mg/kg) several minutes before venom injection died within 8 h; (b) from a different experiment with methyl-varespladib, but exemplary of coral snake bite syndrome and effect of the study treatments: Left, untreated mouse 2 h after venom administration showing effects of venom including (i) postural weakness; (ii) vasodilation (ears) and (iii) ptosis; Right, methyl-varespladib treated mouse. Both mice have piloerection.

Figure 3

In vivo protection and rescue of V. berus envenomed mice by varespladib. (a) Venom and varespladib injected simultaneously into the subcutaneous space outlived controls (venom + excipient) N = 7 each group; (b) mice injected with lethal doses of venom just prior to SC administration of varespladib outlived controls (N = 7 each). Those injected with varespladib alone showed no signs of toxicity (N = 2); (c) varespladib administered SC or IV at a contract laboratory using the same lots of venom (8 mg/kg SC) and drug (8 mg/kg IV in lateral tail vein) following venom administration resulted in significant survival benefit with 5 of 5 IV treated animals surviving 24 h. 5 of 5 mice treated with SC varespladib outlived excipient only treated controls (N = 5 each group Survival: Controls 237 ± 92 min; Treated 1440 min. p < 0.001).

Figure 4

Rats given lethal doses of M. fulvius venom subcutaneously (SC) were treated with a single dose of Varespladib 8 mg/kg intravenously (IV) or excipient (control). (a) Plot showing survival benefit (Excipient n = 6, Varespladib n = 12). Dose of venom indicated inside the red dots indicate 4 mg/kg and 8 mg/kg venom doses for which there was no apparent difference in time to death (287 ± 110 min vs. 240 ± 68, respectively; p = 0.32); (b) snake venom-induced rise in sPLA2 activity is suppressed by varespladib (N = 3 each group; * p < 0.001); time of varespladib addition post venom-addition is noted by “T =” for each group; (c) treatment with varespladib prevented intravascular hemolysis in the same 12 animals. Tubes shown are blood drawn at the 4 h time point.