Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

Varespladib (VPL) was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A2 (PLA2). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA2-dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA2, caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA2 in vitro, with VPL abolishing the PLA2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001–1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 ‘v/w’) in rats, with antivenom alone producing no protective action on this parameter. In conclusion, VPL does not inhibit other major enzymatic groups of L. m. rhombeata venom, with its high PLA2 antagonize activity efficaciously preventing the venom-induced coagulation disturbances.

was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A 2 (PLA 2 ). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA 2dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA 2 , caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA 2 in vitro, with VPL abolishing the PLA 2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001-1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 'v/w') in rats, with antivenom alone producing no protective action on this parameter. In

INTRODUCTION
Snakes of Lachesis genus are represented by three species found in Central America (L. stenophrys, L. melanocephala, and L. acrochorda) and one in South America (L. muta), with the latter being recognized as two subspecies distributed in the Amazon river basin ( L. muta muta) and Atlantic rainforest ( L. muta rhombeata) in Brazil (Costa and Bérnils, 2018;Nogueira et al., 2019;Diniz-Sousa et al., 2020). Together, these snakes are responsible by the second most frequent cases of snakebites in Americas, being exceeded only by Bothrops snakes (Magalhães et al., 2019;Diniz-Sousa et al., 2020).
Polyvalent antivenom ( anti-Bothrops/Lachesis serum) therapies comprise the main therapeutic options to treat the systemic envenomation by Lachesis snakes (Madrigal et al., 2017;Solano et al., 2018). Recently, several studies have demonstrated the value of varespladib, a PLA 2 inhibitor drug (Lewin et al., 2016;Salvador et al., 2019), concerning its suppressive action on the biological effects of Elapidae and Viperidae venoms (Bittenbinder et al., 2018;Lewin et al., 2018;Wang et al., 2018;Zinenko et al., 2020;Gutiérrez et al., 2020a), including their toxins Salvador et al., 2021). However, there are not reports about the action of varespladib, as a stand-alone therapy and/or combined with antivenoms, on the toxic effects caused by Lachesis venoms. In the present study, we have investigated the efficiency of this drug on some aspects of the envenomation by Lachesis muta rhombeata venom using in vitro and in vivo approaches for enzymatic, coagulant and haemorrhagic activities of this venom. We have also assessed an eventual synergic mechanism of action by varespladib when combined with a commercial antivenom used to treat envenomations by Lachesis in Brazil.

Reagents
Varespladib (LY-315920) was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States) and anti-Bothrops/Lachesis serum was from Butantan Institute (São Paulo, SP, Brazil); varespladib was dissolved in DMSO prior to use, whereas the antivenom was provided ready for injection and maintained under refrigeration. Azocasein (A2765), Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (B4875) and 4-nitro-3-octanoyloxy-benzoic acid (N1646) substrates were also from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States). Lachesis m. rhombeata venom was provided by Center for Biological Studies and Research of the Pontifical Catholic University of Goiás (PUC Goiás, Goiânia, GO, Brazil) through Dr Nelson J. Silva Jr. A lyophilized pool of venom obtained from one female adult snake was stored at −20°C and dissolved in ultrapure water prior to use.

Animals
Wistar rats (300-350 g; 2-3 months old) obtained from Central Bioterium of the University of Western São Paulo (UNOESTE, Presidente Prudente, SP, Brazil) were housed in plastic cages (3 animals/cage) with a wood-shaving substrate, at 23 ± 1°C on a 12-h light/dark cycle with lights on at 6 a.m. The animals had free access to food and water. The experimental procedures were approved by an institutional Committee for Ethics in Animal Use (CEUA/UNOESTE, Protocol No. 6713/2021) and were done according to the general ethical guidelines for animal use established by the Brazilian Society of Laboratory Animal Science (SBCAL) and Brazilian Federal Law No. 11.794 of October 8, 2008, in conjunction with the guidelines for animal experiments established by the Brazilian National Council for Animal Experimentation (CONCEA).

Caseinolytic Activity
Caseinolytic activity was determined through colorimetric assay in a SpectraMax 340 multiwell plate reader (Molecular Devices, San Jose, CA, United States) using Azocasein as substrate, essentially as described elsewhere (Torres-Bonilla et al., 2020). The standard assay mixture contained 90 μl of substrate (212 mM Azocasein), 10 μl of reaction buffer (0.05 M Tris-HCl, 1 mM CaCl 2 , pH 8.0) and 10 μl of sample [venom alone (1 mg/ml) or venom (1 mg/ml) pre-incubated (for 30 min at 37°C) with varespladib (0.001-1 mM)] in a final volume of 110 μl. The mixture was incubated for 90 min at 37°C and then the reaction was terminated adding 200 μl of TCA 5% for 5 min at room temperature; the mixture was centrifuged (5 min at 8.000 g) and 150 μl of supernatant was transferred to the multiwell plate containing the same volume of NaOH (0.5 M). Finally, the absorbance was read at 440 nm via endpoint mode, with one activity unit being defined as an increase of absorbance of 0.001/min.

Haemorrhagic Activity
Hemorrhagic activity was performed according to (Theakston and Reid, 1983). Male Wistar rats were anesthetized with sodium thiopental (1.8 mg/kg, i.p.) and their dorsal region was trichotomized in order to set the injection sites. Initially, some doses of L. m. rhombeata venom (0.01, 0.05, 0.1, 0.5 and 1 mg/kg) were tested in order to find the minimum haemorrhagic dose (0.5 mg/kg), which was used to investigate the neutralizing action of antivenom (antivenom:venom ratio of 1:3 'v/w') and varespladib (1 mM). Protocol: after injection of venom (i.d.), the animals were subsequently treated with antivenom and/or varespladib via an intraperitoneal injection, followed by monitoring period of 24 h. Control sites were injected with 0.9% NaCl, DMSO (varespladib solvent) or antivenom. After 24 h, the rats were euthanized in saturated atmosphere with CO 2 , the dorsal skin was removed and the subcutaneous hemorrhagic halos in the inner surface were measured through ImageJ software (National Institute of Health, Bethesda, Maryland, United States).

Statistical Analysis
All results (enzymatic, coagulant and haemorrhagic) were expressed as the mean ± SDM and statistical comparisons were done using Student's t test or ANOVA followed by the Tukey-Kramer test, with p < 0.05 indicating significance. Data were analyzed using SAS University Edition software (SAS Institute Inc., Cary, NC, United States).

Inhibitory Action of Varespladib on the Main Enzymatic Groups of L. m. rhombeata Venom
In colorimetric assays, L. m. rhombeata (1 mg/ml) exhibited moderate enzymatic activity for PLA 2 which was promptly abolished from 0.01 mM of varespladib; the lowest concentration of varespladib (0.001 mM) did not produce inhibitory effect on the PLA 2 activity of this venom ( Figure 1A). Caseinolytic ( Figure 1B) and esterasic ( Figure 1C) activities were not affected by any of these concentrations of varespladib tested on artificial substrates (0.001-1 mM).

Inhibitory Action of Varespladib on the Coagulant Effect of L. m. rhombeata Venom in Rat Citrated Plasma
In rat citrated plasma, L. m. rhombeata venom (1 mg/ml) exhibited procoagulant action on the aPPT (intrinsic pathway) and PT (extrinsic pathway), decreasing in approximately 56.5 and 55.7% these times, respectively (p < 0.05 compared to basal values for both, n 6) (Figures 2A,B). L. m. rhombeata venom (1 mg/ml)induced procoagulant action (aPTT and PT) was not prevented by pre-incubating venom with antivenom (antivenom:venom ratio of 1:3 'v/w') alone for 30 min at 37°C before clotting assay; however, varespladib (1 mM) alone significantly prevented the venom-induced procoagulant action for PT, with approximately 16.4% of decreasing being verified (p < 0.05 compared to venom alone, n 6), and it partially avoided the venom-induced procoagulant action for aPTT, being observed approximately 26.9% of decreasing; the combination of both agents successfully contributed to avoid both intrinsic and extrinsic disorders in rat citrated plasma, resulting in 8.4 and 18.6% of decreasing for PT and aPTT, respectively (p < 0.05 compared to venom alone, n 6) ( Figure 2A). In assays FIGURE 2 | Action of varespladib on the procoagulant activity of L. m. rhombeata venom in rat citrated plasma in vitro. (A) Antivenom did not prevent the venom-induced procoagulant action applying the pre-incubation protocol, whereas varespladib alone avoided the procoagulant action for PT and its association with antivenom successfully prevented both intrinsic and extrinsic disorders. (B) Antivenom did not prevent the venom-induced procoagulant action applying the direct exposure protocol, whereas varespladib alone or combined with antivenom produced minor protective action on both coagulant pathways even combined with antivenom. Bars represent the mean ± SDM (n 6). ◆ p < 0.05 compared to control, ◊ p < 0.05 compared to venom alone and *p < 0.05 compared to antivenom alone.
Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 812295 performed immediately after the exposure L. m. rhombeata venom (1 mg/ml) to antivenom (antivenom:venom ratio of 1: 3 'v/w') and/or varespladib (1 mM), both of agents slightly delayed the L. m. rhombeata venom (1 mg/ml)-induced procoagulant action for PT and aPTT; under this experimental condition, varespladib alone or combined with antivenom were more effective in avoiding only the venom-induced procoagulant action for PT, with approximately 23.3% of decreasing being verified using both agents ( Figure 2B).
Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 812295 2018; Oliveira et al., 2020;Gutiérrez et al., 2020a;Dashevsky et al., 2021;Kazandjian et al., 2021;Silva-Carvalho et al., 2021) and Viperidae (Lewin et al., 2016;Wang et al., 2018;Youngman et al., 2020;Zinenko et al., 2020;Gutiérrez et al., 2020a;Liu et al., 2021) snakes. However, there is no report associating the efficacy of varespladib with toxicological aspects of Lachesis venoms. Based on this premise, we have unprecedentedly investigated the action of varespladib on the enzymatic, coagulant and haemorrhagic activities of Lachesis muta rhombeata venom to determine its efficiency as a single pharmacological tool or combined with a commercial antivenom used in Brazil. In summary, we have demonstrated that varespladib used as a single pharmacological tool abolishes the PLA 2 activity of L. m. rhombeata venom at low concentrations, without affecting the catalytic activity for proteases (metalloprotease and serino protease) of the venom, indicating a very specific inhibitory activity; its high PLA 2 antagonistic activity was reflected on the venom-induced procoagulant action, mostly interfering on the extrinsic pathway disorders produced by venom in rat citrated serum (pre-incubation protocol), whereas the drug did not prevent the haemorrhagic activity induced by L. m. rhombeata venom in rats. The association of varespladib with a commercial antivenom used in Brazil to treat envenomations by Lachesis spp. did not produce important synergic actions on the procoagulant (direct incubation protocol) and haemorrhagic effects induced by L. m. rhombeata venom; such interaction resulted in a major prevention of the venom-induced intrinsic and extrinsic coagulant disorders seen under pre-incubation protocol.
Although the procoagulant action of Viperidae venoms has been mostly associated with the presence of serine proteases in these venoms (Gutiérrez et al., 2021), varespladib can partially prevent the procoagulant action of L. m. rhombeata venom, indicating an eventual role of PLA 2 in these processes. Accordingly, varespladib also contributes to prevent coagulating disorders induced by other groups of snake venoms, e.g., Bothrops (Viperidae-Crotalinae), Daboia, Echis, Oxyuranus, Naja, Pseudechis and Bitis spp., which exhibit high PLA 2 activity (Bittenbinder et al., 2018;Xie et al., 2020;Youngman et al., 2020;Zdenek et al., 2020). On the other hand, varespladib does not affect the L. m. rhombeata venom-induced subcutaneous haemorrhage in rats, reflecting its disability in antagonizing the metalloproteases of this venom, since local and systemic haemorrhagic actions produced by Viperidae venoms are mainly mediated by this family of toxins (Escalante et al., 2011;Seo et al., 2017). In addition, although an Asp49 PLA 2 (LmrTX) with anticoagulant activity has been already isolated from L. m. rhombeata venom (Damico et al., 2012), it does not appear to contribute expressively with the subcutaneous haemorrhage induced in rats, as reported in this study.
In Brazil, envenomations by L. m. muta and L. m. rhombeata are treated with anti-Bothrops/Lachesis serums, with their rescue action being dependent on early i.v. administration (Pla et al., 2013;Madrigal et al., 2017;Solano et al., 2018). There are some negative factors that resulting in deaths by accidents involving Lachesis snakes in Brazil: 1) limited availability of antivenoms, 2) difficulties in accessing health services in certain regions of the country, and 3) lack of a specific anti-Lachesis serum. Consequently, other types of antivenoms, e.g., anti-Bothrops serum and anti-Bothrops/Crotalus serum, have been inaccurately recommended to treat envenomations by L. muta in the absence of anti-Bothrops/Lachesis serum (Magalhães et al., 2019;Muniz et al., 2021). These challenges involving the treatment of envenomations by Lachesis snakes strengthen the search for therapeutically useful adjuncts, with varespladib rising as a plausible tool.
In conclusion, varespladib abolishes efficiently the PLA 2 activity of L. m. rhombeata venom at low concentrations and does not affect other majority enzymatic groups of this venom, e.g., metalloproteases and serine proteases. Varespladib alone might partially prevent the procoagulant effect of L. m. rhombeata venom, with its combination with antivenom avoiding alterations in both intrinsic and extrinsic pathways. Varespladib does not reduce the subcutaneous haemorrhage formation induced by L. m. rhombeata venom in rats due to the lack of inhibitory action on the proteases of the venom. The association of varespladib with a recommended polyvalent antivenom does not produce synergic action on the venom-induced haemorrhagic action but it helps to prevent the venom-induced coagulation disorders.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the Article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Committee for Ethics in Animal Use of University of Western São Paulo (CEUA/UNOESTE, Protocol No. 6713/2021).

AUTHOR CONTRIBUTIONS
RMBN and RSF contributed to conception and design of the study. PGG, DRP, and NLV organized the database. LFA performed the statistical analysis. PGG, DRP, and KAT-B wrote the first draft of the manuscript. NJSJ and SH wrote sections of the manuscript. KM-Z, EGR, and RSF contributed to manuscript revision, read, and approved the submitted version.