sPLA2/secretory phospholipase A2 (IC50 = 9 nM)

Varespladibmethyl, also known as LY333013, is a prodrug of methanol [1]. When used against 28 medically poisonous snake venom species from six continents, varespladib and its sidewall bioavailable prodrug methylvarespladib (methyl-Varespladib) exhibit high levels of fatal PLA2 (sPLA2) at nanomolar and picomolar concentrations. inhibiting impact [2].

---

Inhibition of sPLA2 Activities in Vitro [2]
In examining drugs that could be repurposed for snakebite, we found that varespladib (LY315920) and methyl-varespladib (LY333013) inhibited the sPLA2 activity of large arrays of snake venoms in vitro using chromogenic assays, but did not show great activity against bee venom used as a standard, positive control (Table 1 and Figure 1). Additionally surprising was the observation that the IC50 of varespladib and methyl-varespladib for essentially all snake venoms tested was significantly lower than values ever reported for inhibition of mammalian, including human, sPLA2 [2].

Efficacy. At week 1 of treatment methyl-varespladib/LY333013, 1000 mg/day, was superior to placebo in the proportion of patients achieving an ACR20 response (p = 0.064) and a dose-response relationship was observed (p = 0.058) (Figure 1). A statistically significant dose-response relationship was observed for CRP at week 1 (p = 0.046). However, ACR20 response rates increased in all treatment groups at study weeks 4 and 8, with loss of difference from placebo. Exploratory analyses showed a trend towards a dose-dependent relationship for ACR20 response (p = 0.077) among patients older than 65 years. Analyses of secondary endpoints, including time to achieve an ACR20 response, time to treatment failure, and time to achieve a 20% improvement in patient-reported pain or global arthritis status did not differ among treatment groups. An ACR50 response was achieved by 9/56 (16.1%) patients treated with placebo, and by 10/60 (16.7%), 5/59 (8.5%), and 5/61 (8.2%) patients treated with methyl-varespladib/LY333013 50, 250, and 1000 mg/day, respectively. Changes among the treatment groups in ACR response components were generally small or inconsistent and not statistically significant. TJC increased at weeks 8 and 12 in the LY333013, 250 and 1000 mg/day groups, and SJC did so at week 12. The investigators’ global assessment reflected these findings, but the patients’ global and pain assessments did not. Exploratory analyses did not suggest a treatment response in terms of presence of RF, number of DMARD used, or specific DMARD used at study entry. Notably, only 18% of patients were taking NSAID at study entry, in contrast to 48% using low-dose corticosteroids. Corticosteroid use appeared to reduce achievement of ACR20 in the LY333013 1000 mg/day group [1].

---

The demographic characteristics of the treatment groups were similar. Dose-response relationships were found for ACR20 responses (p = 0.058) and reductions in C-reactive protein (p = 0.058) at week 1. The proportions of patients with an ACR20 response subsequently increased in all study groups including the placebo group at weeks 4 and 8, and the initial treatment benefit was lost. Adverse events were generally mild in severity and not associated with treatment. Conclusion: Treatment with methyl-varespladib/LY333013 for 12 weeks was well tolerated but ineffective as an adjunct to DMARD treatment of active RA [1].

---

ApoE−/− Standard Model [3]
The body weights of animals in control or A-002 (varespladib methyl/LY333013/S-3013-treated groups were similar at time 0 (vehicle: 27.2 ± 2.6, 30 mg/kg A-002: 26.7 ± 2.3, and 90 mg/kg A-002: 27.3 ± 2.5). Body weight increased over the 16 weeks on a Western diet by approximately 155%, 150%, and 141%, for the vehicle, 30 mg/kg A-002 (varespladib methyl/LY333013/S-3013, and 90 mg/kg A-002 groups, respectively (Fig. 2). There was no statistically significant difference in body weights between the groups using a repeated-measures 2-way ANOVA where time and treatment were the variables.

Plasma cholesterol levels at the beginning of the study were not significantly different between groups. However, after 1 month of twice a day treatment with A-002 (varespladib methyl/LY333013/S-3013, either 30 or 90 mg/kg doses, total cholesterol was significantly decreased (Fig. 3) compared with the control group. This effect remained consistent throughout the 4 months of treatment. Plasma cholesterol changed after 4 months of diet per treatment by +15%, −10%, and −12% for vehicle, 30 mg/kg A-002, and 90 mg/kg A-002 groups, respectively (Fig. 3). There was no apparent dose-response effect on plasma cholesterol concentrations.

Treatment with A-002 (varespladib methyl/LY333013/S-3013 had a significant effect on plaque content expressed as percent occupancy of the aortic luminal surface by atherosclerotic plaques. Vehicle-treated mice had approximately 12.6% ± 0.7% plaque coverage, whereas mice treated with 30 mg/kg A-002 twice a day had 6.3% ± 0.6% plaque coverage and mice treated with 90 mg/kg A-002 twice a day had 6.7% ± 0.8% plaque coverage. These represent significant decreases in plaque content in each of the A-002 treatment groups compared with the treatment group receiving only the formulation vehicle (P < 0.05) (Fig. 4).

---

Angiotensin II ApoE−/− Model [3]
Angiotensin II formulated in water twice a day or in 5% acacia twice a day resulted in similar aortic plaque coverage (18% ± 3.3% and 14.4% ± 4.8%, respectively, Fig. 5). A-002 (varespladib methyl/LY333013/S-3013 (30 mg/kg) significantly reduced the plaque coverage of the aorta (8% ± 3% vs. 18% ± 3.3% observed with angiotensin II infusion without drug treatment, P < 0.025). The background amount of atherosclerosis in the absence of angiotensin II (3.8% ± 0.6%, subcutaneous saline pump and water twice a day) was significantly lower than that with angiotensin II infusion (18% ± 3.3%, P < 0.025). Representative in face images are shown in figure 6.

Aortic aneurysm rate was assessed in each group of mice. In the absence of angiotensin II infusion, no aneurysms were observed. Infusion of angiotensin II formulated in water resulted in a 25% incidence of aneurysm and infusion of angiotensin II formulated in the acacia vehicle caused a 22.2% incidence of aneurysm. A-002 (varespladib methyl/LY333013/S-3013 treatment (30 mg/kg twice a day) in the mice infused with angiotensin II formulated in acacia prevented aneurysm formation completely (Prob>ChiSq = 0.0096) (Table 1).

In Vitro Experiments [2]
Experiments were performed to assess sPLA2 activity using the 1,2-dithio analog of diheptanoyl phosphatidylcholine. sPLA2 catalyzes the hydrolysis of phospholipids at the sn-2 position yielding a free fatty acid and a lysophospholipid. The release of arachidonic acid from membrane phospholipids by PLA is believed to be a key step in the control of eicosanoid production within the cell. The Bee Venom PLA2 Control was a 100 μg/mL solution of bee venom PLA2 was supplied as a positive control from kits. Assay optimization, screening and dose response measurements were performed at the Yale Center for Molecular Discovery. Experiments were performed in an assay buffer containing 25 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 100 mM KCl, 0.3% Triton X-100 (Fluka) and 454 µM DTNB and plated into clear, Non-Treated 384-well plates. Venoms were reconstituted in 1× phosphate-buffered saline to a concentration of 10,000 µg/mL. Crude, unfractionated lyophilized venom purchased from Sigma (E. carinatus and D. russelli) or the Miami Serpentarium (all others) was used in all cases. Varespladib and methyl-varespladib/LY333013 were purchased from Chemietek and dissolved in DMSO for in vitro experiments and bicarbonate/dextrose for in vivo experiments. The activity of venoms with 0.375 mM 1,2-bis(heptanoyl) Glycerophosphocholine, the sPLA2 substrate, was selected based on kinetic enzymatic assays conducted at room temperature. Concentrations of venom was selected for screening and potency studies in which high sPLA2 activity was observed relative to any background activity of no venom control wells, and for which there was negligible substrate depletion at 60 min. The range in final concentrations for venoms used in assays was 0.0037–5 µg/mL, demonstrating large differences among venoms in the proportion or relative sPLA2 activity for this substrate. For 13 elapid venoms final concentrations ranged from 0.0037 µg/mL for M. fulvius to 100 µg/mL for Dendroaspis polylepis (mean concentration 7.8 ± 28 µg/mL; median concentration 0.1 µg/mL) and for 15 viper venoms a range of 0.033 µg/mL, e.g., V. berus and 25 µg/mL Calloselasma rhodostoma (mean concentration 2.47 ± 6.35 µg/mL; median concentration 1 µg/mL). Piloting collections used in the first phases of screening were selected by the inventor or from libraries of known compounds and natural products available at the time of experiments on selected venoms including: NIH Clinical Collection, GenPlus, Pharmakon, Bioactive lipids, Protease Inhibitors, Procured Drugs and FDA Approved Drugs libraries. The GenPlus (the NINDS Custom Collection) from MicroSource Discovery Systems contains 960 compounds. Varespladib outperformed all compounds in the library confirming (within the limits of the screening libraries) its superior potency as an sPLA2 inhibitor. No molecule in these collections totaling more than 4000 distinct chemical entities was found to be within two orders of magnitude potency of varespladib and results are not within the scope of this manuscript. For inhibitor and dose-response testing, 10 µL of snake venom or bee venom (+control) was added to assay plates using a multichannel pipetman or a multidrop dispenser. Compounds from the chemical libraries or prepared serial dilution master plates dissolved in DMSO were added to assay plates using a pin tool to transfer 20 nL of compounds. Final DMSO concentrations in the assay are 0.1%. Substrate was then added in 10 µL for a final assay volume of 20 µL. Controls populations were included on each plate in replicate wells. The negative control wells were vehicle (DMSO-only) with no small molecule compound. The positive control to simulate full venom activity inhibition were wells in which no venom was added, and assay buffer added in its place. Assay signals were measured at initiation and after 60 min of reaction time at room temperature. Signals were quantified on the Tecan infiniTe M1000 plate reader measuring absorbance at 405 nm. Signals at initiation were subtracted from the signals at 60 min. These background-corrected values were normalized to the mean of replicate negative and positive control wells within the plate. To define the normalization scale, the mean of the negative control well signals, representing full venom activity, was normalized to 100% effect and the mean of positive control well signals, representing complete inhibition of venom activity, was normalized to 0% effect. And wells within the plate were scaled accordingly. These calculations were performed in MicroSoft Excel. Data were transferred to GraphPad Prism (6th edition, 2014, La Jolla, CA, USA) plotted and fit to models, such that IC50 or EC50 values could be determined. Tests of significance were calculated by Student’s t and all others were descriptive.

---

sPLA2 Inhibition [3]
The intrinsic activity of A-001 on the inhibition of sPLA2 group V and X enzymes was measured according to a chromogenic method described elsewhere.

Two hundred and fifty-one patients with active RA despite treatment with one or more disease modifying antirheumatic drugs (DMARD) received oral doses of LY333013 (50, 250, and 1000 mg) or placebo once daily for 12 weeks. Concomitant low-dose glucocorticoids (< or = 10 mg/day prednisone equivalent) were allowed. Clinical improvement was assessed using the response criteria of the American College of Rheumatology (ACR20), and safety was evaluated with respect to adverse events and laboratory test abnormalities. [1]

---

\n\n Animal in Vivo Studies [2]
\nM. fulvius and V. berus venoms had the highest sPLA2 activity in vivo and and after successful pilot survival studies was chosen for the Non-GLP study in rats and V. berus for mouse studies. CD-1 mice and Sprague-Dawley Rats with implanted jugular venous catheters were used. 18 Sprague-Dawley rats weighing between 183 and 214 g at the time of the study had jugular vein cannulas surgically implanted by the supplier. Rats were randomly assigned to six treatment groups (n = 3 each) and received snake venom with and without varespladib. Animals were monitored for signs of toxicity for approximately 24 h. Blood samples (without anticoagulant) were collected from each rat prior to dose administration and post dose administration at approximately 30 min, 1 h and 4 h. Per protocol, nominal blood collection times were pre-dose administration and post-varespladib administration at 30 min ± 1 min., 1 h ± 1 min., and 4 h ± 5 min. There were no deviations from these specifications except for animals that died prior to the last scheduled blood collect ion at 4 h. Blood was processed to serum and analyzed by the AILAC certified contract research organization to determine sPLA2 activity validated beforehand with rat serum for quality control. Surviving animals were euthanized following the 24-h observation. Tissues were grossly examined but not collected for further processing. Justification for the use of the mouse in this study is based on the premise that animal testing is an appropriate and ethical prerequisite to testing new drugs in humans, and that data obtained from nonclinical animal models will have relevance to the behavior of the test material in humans. Because of the complex interactions that occur in vivo, an in vitro system does not provide sufficient information for evaluation of a compound’s in vivo activities. It was expected that the number of animals used in this study would provide a large enough sample for scientifically meaningful results while using the fewest possible animals to achieve that result. The intravenous route was chosen to maximize the bioavailability of varespladib. All experiments were designed to insure 100% mortality in control animals within the expected ½ life of the test drug in order to produce clear results using the lowest number of animal.

---

\n

---

\n\nsPLA2 Group IIA Transgenic Model [3]
\nA transgenic mouse model with human sPLA2 group IIA under inducible mouse metallothionein promoter control in a C57BL/6J ApoE+/+ background was used to evaluate the inhibition of phospholipase A2 activity in serum from animals dosed with methyl-varespladib/LY333013/A-002 as described by Fox et al.21 Animals were administered a single oral dose of methyl-varespladib/LY333013/A-002 (1 or 3 mg/kg) or vehicle (5% acacia), and enzymatic activity22 was measured in samples at baseline before treatment and 0.5, 2, and 4 hours after dosing.\n

---

\n\nApoE−/− Standard Model [3]
\nThe ApoE−/− model was developed as described elsewhere.23 Male ApoE−/− mice (129/Ola × C57BL/6J, multiple generations) were used at an age of 6-8 weeks. Mice were fed ad libitum a high-fat diet (21% fat: 0.15% cholesterol and 19.5% casein) for 2 weeks to allow time to adjust to the diet. Mice were randomized into groups based on plasma triglyceride and total cholesterol levels using the GroupOptimizer V211.xls program. Animals were treated for 16 weeks with 30 mg/kg twice a day or 90 mg/kg twice a day methyl-varespladib/LY333013/A-002. Compounds were formulated in 0.2 mL of 5% acacia (10 mL/kg body weight) and administered by oral gavage twice a day at 7 am and 3 pm.\n

---

\n\nApoE−/− Angiotensin II Accelerated Model [3]
\nMale ApoE−/− mice (129/Ola × C57BL/6J, multiple generations) were used at an age of 6-8 weeks. In this model, angiotensin II was administered by 0.7 mg kg−1 d−1 subcutaneous infusion to induce faster atherosclerosis development associated with aneurysm formation.24 Mice were fed ad libitum a high-fat diet (21% fat: 0.15% cholesterol and 19.5% casein) for 2 weeks to allow time to adjust to the diet. The animals were then randomized into groups based on plasma triglyceride and total cholesterol levels using the GroupOptimizer V211.xls program and were treated for 4 weeks with 30 mg/kg twice a day of methyl-varespladib/LY333013/A-002 as described in the standard ApoE−/− model.\n\n

---

\nProcedures. Patients meeting the inclusion and exclusion criteria at an initial entry visit were reassessed at their baseline visit to assure that they continued to meet the entry criteria for active RA. The patients completed the Stanford Health Assessment Questionnaire (HAQ)20, including a 100 mm horizontal visual analog scale (VAS) for pain (0 = no pain; 100 = extreme pain), and assessed the global activity of their arthritis on a 100 mm VAS (0 = very well; 100 = very poorly) at each visit. The investigators were instructed in a standardized 28-joint technique21 for counting the number of swollen (SJC) and tender joints (TJC) at each visit, and they judged global arthritis activity on a 100 mm VAS (0 = very well; 100 = very poorly). Safety was assessed on the basis of adverse events reported at each visit (baseline and after 1, 4, 8, and 12 weeks of treatment), findings on physical examination, and laboratory evaluations. Samples for population pharmacokinetics and/or CRP were obtained at baseline and subsequent visits. Study medications. The study medications were prepared in identicalappearing white tablets containing 0, 50, 125, or 250 mg of methyl-varespladib/LY333013. Patients took 2 tablets twice daily from blister cards that corresponded to assigned dose levels of 0 (placebo), 50, 250, and 1000 mg/day of methyl-varespladib/LY333013. The 50 and 250 mg/day dose groups received active medication only in the morning dose. Randomization to treatment was blocked and stratified by site. Treatment assignments were managed by an interactive telephone voice response system. Treatment identities were provided to the investigators in a sealed envelope, but except for emergencies warranting it, unblinding did not occur until after the last study visit. Unblinded patients were discontinued from study participation unless there was a compelling ethical reason for their continuation in the study. Compliance to the treatment regimen was monitored by counts of medication in the returned blister cards. In the event that the blister cards were lost, medication use was assessed by patient report. Statistical analysis. The p values for the demographic, RA baseline disease features and medications, and adverse event data (Tables 1-4) were computed for continuous variables from the rank-transformed analysis of variance model with terms for dose and pooled investigative site; p values for categorical variables were computed from the Cochran-Mantel-Haenszel test stratified by pooled investigative site. The primary efficacy evaluation utilized the ACR Definition of Improvement22 at the 20% level (ACR20). Treatment was deemed a failure if, at the end of 4 weeks of treatment, the patient’s condition had worsened by > 20% relative to baseline or, after 8 weeks, there had been < 10% improvement or, after 12 weeks, there had been < 20% improvement. These patients were discontinued from study treatment and were classified as non-responders with respect to any definition of improvement. Efficacy analysis was performed on all randomized patients who received at least one dose of study medication and who had at least one efficacy assessment at the 4, 8, or 12 week visits [intention-totreat (ITT) group]. Missing data were handled using a last observation carried forward analysis. Adverse event analysis was performed on all patients who received at least one dose of study medication. Assuming that 30% of patients receiving placebo would achieve an ACR20 response compared to 50% of patients receiving LY333013, 1000 mg/day, 59 patients per treatment group would provide 80% probability of detecting a linear doseresponse relationship at a one-sided α = 0.1 using a logistic regression model with dose as the explanatory variable. Pharmacokinetic measurements. High performance liquid chromatography/mass spectroscopic methods were used to measure the active metabolite of LY333013. Results were evaluated according to the reported time of the last 4 doses of study medication and the assigned dose. A 2- compartment distribution model was used with assumption of first-order elimination. Based on previous studies, it was known that plasma clearance is affected by bioavailability, which in turn is reduced by increasing dose. These factors were included in the model evaluating individual apparent oral clearance and inter-individual variability in this parameter. Systemic exposure was estimated based on dose and apparent oral clearance for all patients with available pharmacokinetics data.[1]

Treatment exposure. Mean steady-state plasma concentrations of the active metabolite of methyl-varespladib/LY333013 (LY315920) were estimated to be 222 ng/ml for the 50 mg/day dose group, 627 ng/ml for the 250 mg/day dose group, and 1445 ng/ml for the 1000 mg/day dose group. No significant interaction was observed between LY315920 concentrations and combination therapy with methotrexate (MTX), sulfasalazine, and hydroxychloroquine. 91% of patients in the efficacy analysis received 80% to 119% of the prescribed dose of the study drug. [1] Methyl-varespladib/LY333013 is a rapidly absorbed, orally bioavailable prodrug of varespladib that can be administered orally (e.g., in the form of an oral solution), so that even people with limited or no skills can begin treatment outside of a hospital. Methyl-varespladib/LY333013 is metabolized to varespladib, so while the intravenous formulation is unlikely to be used in practice, our proof-of-concept study focused on the parent compound. Inhibition of snake venom sPLA2 using 3-substituted indole compounds may provide an opportunity to develop effective field treatments for snakebites; both approaches could be rapidly developed with project support and industry collaboration. Our results suggest that further investigation into the efficacy of verapalafil and methylverapalafil in a wider range of snake species is necessary to determine whether they can become an important component of the long-desired first-line field treatment for snakebites—venom antagonists. [2]

---

Plasma Concentrations and Dosage Selection[3]
Figure 1 shows the plasma concentrations of A-001 in serum after a single oral dose of methylverapalafil/LY333013/A-002. A-001 was detectable in samples from all treated mice at doses of 10, 30, and 90 mg/kg. Throughout the dosing period, the plasma concentrations of A-001 at the two highest doses (30 and 90 mg/kg A-002) were higher than the IC50 values in the sPLA2 V and X groups, respectively.

---

The secretory phospholipase A2 (sPLA2) enzyme family is associated with inflammatory diseases and tissue damage, including atherosclerosis. A-001 is a novel sPLA2 enzyme inhibitor discovered through structure-based drug design, while A-002 (varespladib methyl/LY333013/S-3013) is a highly bioavailable oral prodrug currently undergoing clinical development. A-001 inhibits sPLA2 groups IIA, V, and X in humans and mice with IC50 values in the low nanomolar range. In transgenic mice overexpressing human sPLA2 group IIA in a C57BL/6J background, A-002 (1 mg/kg) increased serum A-001 concentrations and inhibited PLA2 activity. In addition, surface analysis was used to evaluate the effect of A-002 on atherosclerosis in two ApoE−/− mouse models. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg, twice daily for 16 weeks) reduced the degree of aortic atherosclerosis. The degree of atherosclerosis was reduced by 50% (P < 0.05). Plasma total cholesterol was significantly reduced within 1 month (P < 0.05) and remained at a low level throughout the study period. (2) In an accelerated atherosclerosis model induced by angiotensin II aortic lesions and aneurysms, A-002 (30 mg/kg, twice daily) reduced the degree of aortic atherosclerosis by approximately 40% (P < 0.05) and aneurysm formation (P = 0.0096). Therefore, A-002 can effectively and significantly reduce total cholesterol, atherosclerosis and aneurysm formation in both ApoE−/− mouse models. [3]

Safety. Of the 251 patients who received at least one treatment with the study drug, 182 reported at least one adverse event (AE). The most common AEs included rhinitis (21.9%), headache (19.5%), cough (13.5%), diarrhea (9.2%), nausea (8.8%), fever (8.4%), dizziness (6.4%), abdominal pain (6.0%), sinusitis (6.0%), and pharyngitis (5.6%). The incidence of these AEs did not differ statistically between the treatment groups (Table 4). Overall, the type, severity, and distribution of AEs were similar across the treatment groups. The overall proportion of adverse events (AEs) in the placebo group (60.3%) was slightly lower than in the 50 mg/day methyl-varespladib/LY333013 group (69.4%), 250 mg/day group (79.0%), and 1000 mg/day group (81.3%, overall p = 0.043). However, when analyzing AEs by organ system, only the cardiovascular system showed a statistically significant difference among the treatment groups (p = 0.033). Cardiovascular AEs did not appear to be dose-independent; the incidence in the 50 mg/day and 1000 mg/day methyl-varespladib/LY333013 groups (16.1% and 15.6%, respectively) was higher than in the 250 mg/day methyl-varespladib/LY333013 group (3.2%) and the placebo group (4.3%). The incidence of upper respiratory tract infections was higher in the LY333013 treatment group and higher in the placebo group. These differences were considered not clinically significant. Six patients discontinued study treatment due to non-serious adverse events: all patients were receiving LY333013 (Table 5). These adverse events included: dizziness (50 mg group); dyspepsia, nausea, and rash (250 mg group); fatigue and somnolence (1000 mg group). Serious adverse events occurred in 3 patients in the placebo group and 4 patients in the LY333013 group; the placebo group included septic arthritis, pneumonia, and stroke, while the LY333013 group included chest pain (50 mg, 250 mg, and 1000 mg groups) and diverticulum perforation (50 mg group). The latter patient had a history of diverticulitis, and all 3 patients with chest pain had a history of cardiovascular disease. One patient (250 mg group) underwent coronary angioplasty. Patients who experienced serious adverse events (such as pneumonia and colonic perforation) discontinued study treatment. The investigators assessed that only the latter serious adverse event was likely related to the study treatment. No patients died during the study. No clinically significant changes were observed in laboratory parameters, vital signs, or electrocardiogram records. According to the National Cancer Institute Common Toxicity Criteria (CTC) classification, only serum AST and calcium levels differed between the placebo group and the LY333013 group (p = 0.049). No patients had a CTC grade 4 abnormality for any analyte; the highest CTC grade for serum calcium was grade 2. Elevated serum AST was more common in the LY333013 1000 mg/day group; the highest value in this group was 122 IU. [1]

[1]. A randomized, double-blinded, placebo-controlled clinical trial of LY333013, a selective inhibitor of group II secretory phospholipase A2, in the treatment of rheumatoid arthritis. J Rheumatol. 2005 Mar;32(3):417-23.

[2]. Varespladib (LY315920) Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation. Toxins (Basel). 2016 Aug 25;8(9). pii: E248.

[3]. Varespladib (A-002), a Secretory Phospholipase A2 Inhibitor, Reduces Atherosclerosis and Aneurysm Formation in ApoE−/− Mice J Cardiovasc Pharmacol. 2009 Jan;53(1):60-5.

Varespladib methyl ester (or methyl-Varespladib/LY333013) is a methyl ester of Varespladib formed by the condensation of its carboxyl group with methanol. It is a potential treatment for snakebite poisoning, particularly for snakebites where toxicity depends on phospholipase A2 (PLA2) activity. It has dual action as a prodrug, anti-inflammatory, antidote, and EC 3.1.1.4 (phospholipase A2) inhibitor. It is a methyl ester, aromatic ether, benzene compound, indole compound, and primary amide compound. Its function is related to that of Varespladib. Varespladib methyl ester has been studied for the treatment of acute coronary syndrome. Studies have shown that Varespladib methyl treatment significantly improves lipoprotein and inflammation levels. [Pharmaceutical Indications] It has been studied for the treatment of atherosclerosis and coronary artery disease.

---

Mechanism of Action
A-002 is an orally administered, potent secretory phospholipase SPL2 (SPL2) inhibitor, available in groups IIA, V, and X. Atherosclerosis is an arterial disease caused by inflammation and plaque buildup in the inner walls of blood vessels. This buildup can lead to swelling and eventual rupture of the vessels. Studies have shown elevated SPL2 levels in patients with both stable and unstable coronary artery disease. Elevated levels of this enzyme have been shown to predict an increased risk of future cardiovascular events such as heart attacks and strokes.

---

Varespladib methyl is a methyl ester formed by the condensation of the carboxyl group of varespladib with methanol. It is a potential treatment for snakebite poisoning, with toxicity depending on the action of PLA2. It can be used as a prodrug, anti-inflammatory agent, antidote, and EC 3.1.1.4 (phospholipase A2) inhibitor. It is a methyl ester, aromatic ether, benzene compound, indole compound, and primary amide. Its function is related to varespladib. Varespladib methyl has been investigated for the treatment of acute coronary syndrome. Studies have shown that Varespladib methyl treatment significantly improves lipoprotein and inflammation levels. VARESPLADIB METHYL is a small molecule drug that has completed Phase III clinical trials (covering all indications) and has three investigational indications. Despite calls for improved prevention and treatment of snakebites for over a century, snakebites remain a neglected medical problem in developing countries, causing up to 125,000 deaths annually. It is estimated that 75% of snakebite deaths occur outside of hospitals. Since phospholipase A2 (PLA2) activity is a crucial component of snake venom toxicity, we are searching for candidate PLA2 inhibitors through direct drug testing. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib/LY333013, exhibited high levels of secretory phospholipase A2 (sPLA2) inhibition against 28 medicinally significant snake venoms from six continents at nanomolar and picomolar concentrations. In vivo proof-of-concept studies of varespladib showed that it significantly improved the survival rate of mice against lethal doses of yellow-bellied viper (Micrurus fulvius) and European viper (Vipera berus) venom and inhibited venom-induced sPLA2 activity in rats attacked with 100% lethal doses of yellow-bellied viper venom. The rapid development and application of broad-spectrum PLA2 inhibitors, whether used alone or in combination with other small-molecule snake venom inhibitors (e.g., metalloproteinase inhibitors), holds promise for filling a critical gap in the treatment process from pre-diagnosis to hospitalization. Lowering the threshold for clinical trials of small molecule therapies that have been tested for safety and reused is a potentially cost-effective way to fill the gap in pre-referral treatment for snakebites. [2] The secretory phospholipase A2 (sPLA2) enzyme family is associated with inflammatory diseases and tissue damage, including atherosclerosis. A-001 is a novel sPLA2 enzyme inhibitor discovered through structure-based drug design, while methyl-varespladib/LY333013/A-002 is a highly bioavailable oral prodrug currently in clinical development. A-001 inhibits sPLA2 groups IIA, V and X in humans and mice with IC50 values in the low nanomolar range. In transgenic mice overexpressing human sPLA2 group IIA under the C57BL/6J background, A-002 (1 mg/kg) resulted in elevated serum A-001 levels and inhibition of PLA2 activity. In addition, surface analysis was used to evaluate the effect of A-002 on atherosclerosis in two ApoE−/− mouse models. (1) In a high-fat diet model, A-002/methyl-varespladib/LY333013 (30 and 90 mg/kg, twice daily for 16 weeks) reduced the degree of aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was significantly reduced at 1 month (P < 0.05) and remained at a low level throughout the study period. (2) In an accelerated atherosclerosis model induced by angiotensin II aortic lesions and aneurysms, A-002 (30 mg/kg, twice daily) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and aneurysm formation (P = 0.0096). Therefore, A-002 can effectively and significantly reduce total cholesterol, atherosclerosis and aneurysm formation in both ApoE−/− mouse models. [3]

C22H22N2O5

394.427

394.153

C, 66.99; H, 5.62; N, 7.10; O, 20.28

172733-08-3

Varespladib;172732-68-2; 172733-42-5 (Varespladib sodium)

9886917

White to off-white solid powder

3.172

1

5

9

29

604

0

CCC1=C(C2=C(N1CC3=CC=CC=C3)C=CC=C2OCC(=O)OC)C(=O)C(=O)N

VJYDOJXJUCJUHL-UHFFFAOYSA-N

InChI=1S/C22H22N2O5/c1-3-15-20(21(26)22(23)27)19-16(24(15)12-14-8-5-4-6-9-14)10-7-11-17(19)29-13-18(25)28-2/h4-11H,3,12-13H2,1-2H3,(H2,23,27)

methyl 2-(1-benzyl-2-ethyl-3-oxamoylindol-4-yl)oxyacetate

A002; LY-333013; S-3013; A002; A-002; LY333013; Varespladib methyl; 172733-08-3; LY-333,013; Varespladib methyl [USAN]; LY333,013; Varespladib methyl ester; S3013

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~253.54 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (12.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)