Development of Poly Unsaturated Fatty Acid Derivatives of Aspirin for Inhibition of Platelet Function S

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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/08/03/jpet.116.234781.DC1

1521-0103/359/1/134–141$25.00 http://dx.doi.org/10.1124/jpet.116.234781 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 359:134–141, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Development of Poly Unsaturated Fatty Acid Derivatives of Aspirin for Inhibition of Platelet Function s

Jahnabi Roy, Reheman Adili, Richard Kulmacz, Michael Holinstat, and Aditi Das Department of Chemistry (J.R.), Division of Nutritional Sciences, Departments of Comparative Biosciences, Biochemistry, and Bioengineering, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science (A.D.), University of Illinois at Urbana-Champaign, Urbana, Illinois; Division of Cardiovascular Medicine (M.H.), Department of Pharmacology (R.A., M.H.), University of Michigan Medical School, Ann Arbor, Michigan; and Department of Internal Medicine, Texas Health Science Center, McGovern Medical School, Houston, Texas (R.K.) Received April 27, 2016; accepted August 1, 2016 Downloaded from

ABSTRACT The inhibition of platelet aggregation is key to preventing particular, the aspirin-DHA anhydride displayed similar effective- conditions such as myocardial infarction and ischemic stroke. ness to aspirin. Platelet aggregation studies conducted in the Aspirin is the most widely used drug to inhibit platelet aggre- presence of various platelet agonists indicated that the aspirin-lipid gation. Aspirin absorption can be improved further to increase conjugates act through inhibition of the cyclooxygenase (COX)– jpet.aspetjournals.org its permeability across biologic membranes via esterification thromboxane synthase (TXAS) pathway. Hence, we performed or converting the carboxylic acid to an anhydride. There are detailed biochemical studies using purified COX-1 as well as TXAS several reports indicating that v-3 and v-6 fatty acids such as stabilized in nanoscale lipid bilayers of nanodiscs to confirm results linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic from the platelet aggregation studies. We show that although all of acid (DHA) separately inhibit platelet aggregation. Herein, we the aspirin conjugates act through the COX-TXAS pathway by synthesize anhydride conjugates of aspirin with linoleic acid, EPA, inhibiting COX-1, the parent fatty acids do not act via this pathway. and DHA to form aspirin anhydrides that are expected to have Finally, we studied the hydrolysis of these compounds in buffer and higher permeability across cellular membranes. These aspirin–fatty human plasma, and we demonstrate that all of the aspirin–fatty acid at ASPET Journals on September 28, 2021 acid anhydrides inhibited platelet aggregation in washed human conjugates hydrolyze to the parent molecules aspirin and fatty acid platelets and platelet-rich plasma in a dose-dependent manner. In in a controlled manner.

Introduction at this pH. This results in poor absorption of drugs with carboxylic acid moieties through lipid membrane barriers. Platelet adhesion and aggregation is important for main- Typically, nonsteroidal anti-inflammatory drugs such as taining normal hemostasis and is an essential component in aspirin also cause gastric toxicity because they inhibit COX-1, the morbidity and mortality associated with cardiovascular which is involved in maintaining the integrity of the gastroin- disease, including myocardial infarction and ischemic stroke. testinal epithelium. The general approach to resolve these side Therefore, the ability to modulate platelet function is of sig- effects is to esterify the carboxylic acid to produce lipophilic nificant clinical importance. Aspirin is currently the most prodrug forms. However, several aliphatic or aromatic esters of widely used therapeutic for inhibition of platelet activation. carboxylic acid drugs are not sufficiently labile in vivo to ensure Aspirin functions via irreversible acetylation of platelet cyclo- a suitably high rate and extent of conversion from the esterified oxygenase (COX)-1, resulting in inhibition of platelet-derived form. In addition, esters are highly susceptible to enzymatic thromboxane (TX) A2 formation. Aspirin has poor permeabil- hydrolysis in plasma and thus have differential rates of aspirin ity across biologic membranes at physiologic pH because of the liberation in different individuals. Moreover, aspirin has two presence of free carboxylic acid, which is significantly ionized major functional groups: the carboxylate end and the O-acetyl end. Because the O-acetyl end is the pharmacophore for the activity of aspirin, an aspirin ester derivative prodrug must This research was supported by the American Heart Association [Scientist hydrolyze faster at the carboxylate end than the O-acetyl end in Development Grant 15SDG25760064 (to A.D.) and Grants GM105671 and HL114405 (to M.H.)] and the University of Illinois at Urbana-Champaign order to be effective (Gilmer et al., 2002). Therefore, in this [Graduate College Travel Award and Department of Chemistry Graduate work, we synthesized aspirin anhydride derivatives with Fellowship (to J.R.)]. dx.doi.org/10.1124/jpet.116.234781. naturally occurring lipids and evaluated their platelet aggre- s This article has supplemental material available at jpet.aspetjournals.org. gation function.

ABBREVIATIONS: AA, arachidonic acid; AP, activating peptide; COX, cyclooxygenase; DCM, dichloromethane; DHA, docosahexaenoic acid; DMSO, dimethylsulfoxide; EPA, eicosapentaenoic acid; LA, linoleic acid; MSP, membrane scaffold protein; NTA, nitrilotriacetic acid; PAR, protease-activated receptor; PG, prostaglandin; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PRP, platelet-rich plasma; PUFA, polyunsaturated fatty acid; TX, thromboxane; TXAS, thromboxane synthase; U46619, (Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct- 1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid.

134 Aspirin Anhydrides to Reduce Platelet Aggregation 135

Scheme 1. Synthesis of aspirin-anhydride conjugates. Aspirin (acetylsalicylic acid) is first converted to the activated acyl chloride by refluxing with thionyl chloride (SOCl2) in benzene. This further reacts with the carboxylate on dietary fatty acids in the presence of pyridine in DCM to give LA anhydride (C1), EPA anhydride (C2), and DHA anhydride (C3).

Recent work has focused on the modification of drug molecules groups (Phang et al., 2013). Finally, we perform hydrolysis studies Downloaded from with lipids or lipid derivatives as a means to overcome their side to demonstrate that the aspirin-anhydride conjugates release effects and to facilitate their delivery. Specifically, the addition of aspirin and free fatty acids at a controlled rate in both buffer a hydrophobic lipid tail allows the drug molecule to cross the and plasma. hydrophobic plasma membrane and enter the cell (Bradley et al., 2001). Herein, we combine the two approaches of derivatizing Materials and Methods aspirin with polyunsaturated fatty acids (PUFAs) to keep aspi- Materials. Ampicillin, arabinose, chloramphenicol, isopropyl b-D-1- jpet.aspetjournals.org rin in a unionized form to facilitate facile transport across the thiogalactopyranoside, and Ni- nitrilotriacetic acid (NTA) resin were plasma membrane. The “hybrid-drug” approach of combining purchasedfromGoldBiotechnology (Olivette, MO). d-Aminolevulinic two drugs with similar therapeutic properties but different acid and hematin were obtained from Frontier Scientific (Logan, mechanisms has been gaining interest (Sparatore et al., 2011). UT). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was pur- For instance, there are several reports in the literature indicating chased from Avanti Polar Lipids (Alabaster, AL). Aspirin was purchased the inhibition of platelet aggregation by dietary fatty acids, from Sigma-Aldrich (St. Louis, MO). AA, EPA, DHA, and LA were v obtained from Cayman Chemical (Ann Arbor, MI). Protease-activated chiefly -3 fatty acids eicosapentaenoic acid (EPA) and docosa- at ASPET Journals on September 28, 2021 v receptors PAR4-activating peptide (AP) (AYPGKF) and PAR1-AP hexaenoic acid (DHA) and -6 fatty acid linoleic acid (LA) (Phang (SFLLRN) were purchased from GL Biochem (Shanghai, China). et al., 2013). The action of these lipids is thought to be through Thrombin was purchased from Enzyme Research Laboratories (South competitive inhibition of COX-1–mediated arachidonic acid (AA) Bend, IN). Collagen and ristocetin were purchased from Chrono-log metabolism. The metabolism of EPA and DHA by COX-1 leads to Corporation (Havertown, PA). U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3- the formation of alternative TX-like molecules that are less hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid] and ADP were also purchased from Sigma-Aldrich. The University of potent than TXA2 molecules with regard to platelet aggregation. Herein, we synthesized anhydride conjugates of dietary fatty Michigan Review Board approved this study and written informed acids LA, EPA, and DHA with aspirin for the inhibition of platelet consent was obtained from all participants prior to blood collection. Synthesis of Aspirin Chloride. Aspirin chloride (Scheme 1) was aggregation. We hypothesized that if the v-3 fatty acids and synthesized as previously mentioned (Abdellatif et al., 2009). Briefly, a aspirin reduce platelet aggregation separately, then the conju- 250-ml round-bottom flask was charged with aspirin (500 mg, gated moiety itself will have antiplatelet aggregatory properties 2.77 mmol) dissolved in benzene (69.5 ml, 0.04 M in aspirin) and through a synergistic or codrug effect. In addition, these aspirin- thionyl chloride (805 ml, 11.1 mmol). The mixture was refluxed for lipid conjugates will prevent the deleterious effects of the free 8 hours and the resulting solution was cooled. The solution was aspirin carboxylate group. The aspirin-lipid anhydride bond will concentrated in vacuo to yield a white solid that was taken into future be more susceptible to hydrolysis compared with the O-acetyl end. reactions without further purification (yield of 435 mg, 79%). Therefore, the compound will decompose to its carboxylic acid Synthesis of Anhydrides. A 7-ml vial containing aspirin chloride counterpart at a predictable rate and pattern depending on the (10 mg, 0.05 mmol) and fatty acid (16 mg, 0.05 mmol for DHA; 15 mg, 0.05 mmol and 14 mg, 0.05 mmol for EPA) was dissolved in dichloro- conjugated molecule, as shown previously with related compounds methane (DCM; 1 ml, 0.04 M). Pyridine (4 ml, 0.075 mmol) was added (Mizrahi and Domb, 2009). Furthermore, anhydrides are typically to the mixture, and the solution was stirred at room temperature less sensitive to enzymatic cleavage than esters or amides (Kumar for 4 hours. The reaction was then quenched with 1 ml 1 N HCl solution et al., 2002). Thus, the application of lipid anhydride conjugate is and vortexed. The organic layer was removed and the aqueous layer likely to increase bioavailability, be released in a controlled man- was re-extracted with 1 ml DCM. The combined organic layers were ner, and reduce gastric and mucosal toxicity. dried over sodium sulfate and concentrated in vacuo. The residual To our knowledge, this work is the first to report on the solid was recrystallized in diethyl ether to yield a white solid (yields of application of aspirin anhydride using PUFAs for reduction in 3.5 mg, 20% for DHA anhydride; 6 mg, 26% for EPA anhydride; and platelet aggregation. Here, we demonstrate that anhydride con- 17.5 mg, 79% for LA anhydride). Platelet Purification. Human whole blood was drawn from the jugates of aspirin with dietary v-3 fatty acids are potent inhibitors antecubital vein of healthy donors. Platelet-rich plasma (PRP) was of platelet aggregation. Furthermore, we biochemically examine obtained from whole blood by centrifugation at 200g for 10 minutes. – the effect of parent fatty acid on the COX-1 mediated TXA2 Leftover blood samples were centrifuged at 200g for 10 minutes to formation study because the previous literature focused mostly on obtain platelet-poor plasma. Washed human platelets were isolated the dietary supplementation of these fatty acids in controlled trial from whole blood and platelet aggregation studies were performed as 136 Roy et al. previously described (Yeung and Holinstat, 2012). Whole blood was times for 1 minute each using a sonicator. The solution was then drawn from the antecubital vein of untreated healthy donors or centrifuged at 35000 rpm in Ti 45 Beckman L8 70 MR Ultracentrifuge healthy donors who were given 81 mg/d aspirin orally for 7 days. for 1 hour and the pellet was resuspended in buffer A containing 2% Platelets were pelleted from PRP in the presence of anticoagulant Lubrol (MP Biomedicals, Santa Ana, CA) to solubilize TXAS. The citrate dextrose (2.5%) and apyrase (0.02 U/ml) by centrifugation at resuspended sample was centrifuged at 35,000 rpm for 1 hour and 2000g for 10 minutes and then resuspended in Tyrode’s buffer (12 mM the supernatant was loaded onto a Ni-NTA column. The column was

NaHCO3, 127 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 1 mM MgCl2, incubated for 2 hours with 5Â column volume of buffer A containing 8 5 mM glucose, and 10 mM HEPES) to a final concentration of 3.0 Â 10 10 mM histidine, 5 mM ATP, 10 mM MgCl2, and 150 mM KCl to separate platelets/ml. Washed platelets (250 ml) were separately incubated any copurifying GroEL (Joseph and Andreotti, 2008). TXAS was eluted with 2.5, 5, or 10 mM of compound C1, C2 or C3; or with the same using buffer A containing 0.2% Lubrol and 100 mM histidine. volume of dimethylsulfoxide (DMSO) for 5 minutes. Assembly of TXAS Nanodiscs. TXAS nanodiscs were assembled Washed Human Platelet Aggregation. Platelet aggregation from a mixture of TXAS, membrane scaffold protein MSP1D1, cholate, was induced by AA (5 mM), thrombin (1 nM), PAR4-AP (50 mM), and POPC lipids by removing the detergents using Amberlite (Sigma PAR1- AP (SFLLRN) (1 mM), ADP (1 mM), or U46619 (1 mM) and Aldrich, St. Louis, MO) (Bayburt et al., 2002). MSP1D1 was added the change in light transmission was recorded by an eight-channel to POPC (solubilized using sodium cholate) in a 65:1 ratio (lipids/MSP1D1) Chrono-log platelet aggregometer (Chrono-log Corporation) under and the solution was incubated at 4°C for 1 hour on a shaker. The stirring at 1200 rpm at 37°C. MSP1D1 gene was provided by Prof. Stephen G. Sligar, University of Platelet Aggregation in PRP. The platelet concentration in PRP Illinois, Urbana-Champaign. TXAS was then added in a 1:15 ratio Â 8

was adjusted to 3.0 10 platelets/ml using platelet-poor plasma from (TXAS/MSP1D1) and incubated for another 1 hour. Amberlite was Downloaded from the same donor. Then, 10 mM of C1, C2, and C3 compounds or DMSO added to remove detergents and initiate the formation of nanodiscs. in the same volume was separately incubated with 250 ml PRP for The TXAS nanodiscs were then purified using size exclusion chroma- 5 minutes. Platelet aggregation was induced by adding AA (5 mM), tography as previously mentioned (Das et al., 2014). ADP (1 mM), collagen (2 mg/ml), ristocetin (1 mM), or U46619 (1 mM) Coupled Assay with Secondary Lipids. TXAS converts pros- and the change in light transmission was recorded by a platelet taglandin (PG) H2 into TXA2 and 12-L-hydroxy-5,8,10-heptadecatrie- aggregometer as described above. noic acid and malondialdehyde as a side reaction (Diczfalusy et al.,

Expression and Purification of COX-1. An Sf9 cell pellet 1977). A coupled enzymatic assay was performed to measure the jpet.aspetjournals.org (8–10 g) from a 2-liter cell culture expression of COX-1 was suspended overall effect of a secondary lipid on the metabolization of AA. Briefly, in 40–45 ml buffer A (25 mM NaPO4, 20 mM imidazole, pH 7.4, and TXAS (50 nM) was added to COX-1 (10 nM) incubated with hematin 1 mM phenol) and 5 ml 10Â Sigma P2714 protease inhibitor was (0.5 mM) in Tris buffer (0.1 M, pH 7.4). AA (20 mM) and a secondary added. Cell clumps were resuspended by magnetic stirring. This substrate (20 mM) were then added, and the rates of malondialdehyde resuspension was homogenized by sonication and centrifuged at and 12-L-hydroxy-5,8,10-heptadecatrienoic acid formation were mon- 100,000g for 1 hour and the supernatant was removed. The pellet itored over 3 minutes at 268 nm and 234 nm, respectively. Absorbance was resuspended in approximately 45 ml buffer B (25 mM NaPO4, was then measured and the reaction mixture was quenched in 300 ml

100 mM NaCl, 20 mM imidazole, and 0.1 mM phenol, pH 7.4) using ethyl acetate. The amount of TXB2 formed was determined using at ASPET Journals on September 28, 2021 with a Dounce homogenizer. Then 10% Tween 20 solution was added a TXB2 EIA enzyme-linked immunosorbent assay kit (Cayman to a 1.5% final concentration and stirred for 1 to 2 hours followed by Chemical). centrifugation at 100,000g for 1 hour. The supernatant (S1) was removed and the pellet was resuspended in approximately 20 ml buffer B. S1 was mixed with 2.5–3 ml Ni-NTA agarose (Qiagen, Results Valencia, CA) prewashed with buffer C (25 mM NaPO4, 100 mM NaCl, 20 mM imidazole, 0.1 mM phenol, pH 7.4, and 0.1% Tween 20) and Synthesis of Aspirin Anhydride Derivatives. Synthe- shaken in a cold room for 2 hours. The mixture of S1 and Ni-NTA was sis of the fatty acid–aspirin derivatives was performed as poured into a column (approximately 1.5 Â 10 cm) and the flow though shown in Scheme 1. The free carboxylic acid in aspirin was was allowed to drain out. The column was washed with 10–15 ml first converted to its corresponding acid chloride by reacting buffer C and then with 10–15 ml buffer D (25 mM NaPO4, 300 mM with thionyl chloride in the presence of an amine base by NaCl, 20 mM imidazole, 0.1 mM phenol, pH 7.4, and 0.1% Tween 20). refluxing in benzene for 8 hours. The activated acid chloride The protein was eluted with 10 Â 0.75-ml aliquots of buffer E (25 mM was taken without further purification to react with the NaPO4, 100 mM NaCl, 200 mM imidazole, 0.1 mM phenol, pH 7.4, and carboxylic acid in EPA, DHA, and LA to form a mixed 0.1% Tween 20). The active fractions were pooled and concentrated. anhydride. The mixed anhydride was purified by recrystalli- The buffer was exchanged on a 10DG column eluted with 50 mM KPi, zation in diethylether to give .99% purity by 1H-nuclear pH 7.2, 50 mM NaCl, 0.01% NaN3, and 0.1% Tween 20 and 0.25-ml fractions were collected. The COX-1 activity of each fraction was magnetic resonance and high-resolution electrospray ioniza- assayed and active fractions were pooled. Protein was stored at 280°C tion mass spectrometry (Supplemental Figs. 10–12; Supple- with 25% glycerol. mental Table 1). Expression and Purification of Thromboxane Synthase. Lipid-Aspirin Conjugates Inhibit AA-Induced Plate- Thromboxane synthase (TXAS) was expressed and purified as pre- let Aggregation. The effect of fatty acid–aspirin derivative viously mentioned (Das et al., 2014). The gene for TXAS was obtained compounds C1, C2, and C3 on platelet function was studied from OriGene (Rockville, MD) and modified at the N terminus for in vitro using platelet aggregation at differing concentrations expression in Escherichia coli as described. Briefly, the cells were from 2.5 mMto10mM. As shown in Fig. 1, AA-induced (5 mM) grown in Terrific Broth (made in-house using peptone, tryptone and platelet aggregation was inhibited by all three compounds in a b yeast extract) and induced with 1 mM isopropyl -D-1-thiogalactopyr- dose-dependent manner. However, these compounds varied in anoside, 0.5 mM d-aminolevulinic acid, and 4 mg/l arabinose at an potency of inhibition, with C3 being the most potent followed optical density of 1.2. They were grown for 44 hours at 26°C and 160 rpm incubator speed. The cells were pelleted at 2800 g for 15 min. by C2 and C1. As seen from the aggregation curves, compound The harvested cells were resuspended in buffer A (0.1 M potassium C1 (LA anhydride) showed a slight decrease in platelet phosphate, pH 7.4, 10% glycerol, and 0.1 M sodium chloride) contain- aggregation, with a significant decrease at the 10-mM concen- ing 2 mM magnesium chloride, 1 mM phenylmethylsulfonyl fluoride, tration compared with the DMSO control (P 5 0.0158). In and 1 mg each of DNase and RNase for 1 hour and were lysed five contrast, C2 (the EPA anhydride) showed greater inhibition at Aspirin Anhydrides to Reduce Platelet Aggregation 137

Fig. 1. Effect of anhydrides on platelet aggregation in washed platelets by AA. The plots show the transmission of light- versus time for various concentrations (2.5 mM, 5 mM, and 10 mM) of LA anhydride (C1; A), EPA anhydride (C2; B), and DHA anhydride (C3; C) and the DMSO control. The reduction of light transmission indi- cates the inhibition of aggregation. All compounds show a concentration-dependent Downloaded from decrease in aggregation in the order of effectivity LA anhydride , EPA anhydride , DHA anhydride. Results show data from five separate trials. *P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001. jpet.aspetjournals.org at ASPET Journals on September 28, 2021 lower concentrations, with a significant decrease at 5 mM EPA, and DHA on AA-induced platelet aggregation is shown (P 5 0.0176) and a further decrease at 10 mM(P # 0.0001). in Supplemental Fig. 4. Although LA and EPA did not inhibit Compound C3 (DHA anhydride) showed the most significant platelet aggregation at concentrations of 2.5 mM, 5 mM, and inhibition of platelet aggregation, with inhibition at 2.5 mM 10 mM, DHA inhibited platelet aggregation at 2.5 mM. (P 5 0.0049) and 5 mM(P 5 0.0011) and almost complete In addition to AA, compounds C1, C2, and C3 did not inhibition of aggregation at 10 mM(P 5 0.0002). Similar attenuate platelet aggregation induced by various platelet results were observed for PRP (Fig. 2). At the 10-mM concen- agonists, including thrombin, PAR4-AP, PAR1-AP, ADP, or tration, compounds C2 and C3 showed a significant decrease U46619. As shown in Supplemental Figs. 1 and 2, a slight in platelet aggregation (P 5 0.1006 and P 5 0.0003 for C2 and albeit insignificant decrease was observed in platelet aggre- C3, respectively) (Fig. 2, A and B). However, C1 did not show a gation induced by thrombin, PAR4-AP, and PAR1-AP for all significant decrease in aggregation in PRP. The effect of LA, three compounds at the 10-mM concentration. No notable

Fig. 2. (A) Effect of 10-mM anhydrides on platelet aggregation as indicated by light transmission in PRP by 5 mM AA. (B) All compounds show a concentration-dependent decrease in aggregation in the following order of effectivity: LA anhydride (C1) , EPA anhydride (C2) , DHA anhydride (C3). Results show data from five separate trials. (C) Comparison of the inhibitory effect of C3 compound with aspirin. At 20 mM, the C3 compound completely inhibited washed human platelet aggregation similar to aspirin treatment (81 mg/d Â 7 days) tested by 5 mMAA. ****P , 0.0001. 138 Roy et al. inhibition in platelet aggregation was observed in PRP stimulated with ADP, collagen, ristocetin, or U46619 (Sup- plemental Figs. 1, D and E, and 2, B–D). Collectively, these observations indicate that the aspirin-lipid conjugates are acting through the inhibition of the COX-TXAS pathway. To understand the potency of inhibitory effects of fatty acid–aspirin derivative compounds on human platelet function, we compared the effectiveness of compound C3 against aspirin-treated controls. Figure 2C presents a comparison of aspirin-treated controls with compound C3 and DMSO controls in washed platelets, in which 20 mM compound C3 shows a similar level of inhibition of platelet function in response to AA (P , 0.0001) as aspirin-treated controls. The inhibition values are similar to those for aspirin-treated controls in which a treatment of 81 mg/d aspirin was administered for 7 days. Thus, compound C3 at the 20-mM concentration dis- plays similar effectiveness to aspirin. Inhibition of COX-1–TXAS Activity by Lipid-Aspirin Downloaded from Conjugates. Platelet aggregometry data show that inhibition by these compounds is effective only when it is induced by AA. Thus, the mechanism of action of the derivatives is likely through the COX-1–TXAS pathway. A coupled activity assay was performed to confirm the effects of the derivatives and their parent compounds on the COX-1–TXAS enzyme system. jpet.aspetjournals.org In this assay, COX-1 converts AA to PGH2, which is further converted by TXAS to TXA2.TXA2 is a proaggregatory molecule that initiates platelet aggregation. However, TXA2 is quickly converted to TXB by hydrolysis of the endoperoxide 2 Fig. 3. (A) TX formed in the presence of LA, EPA, and DHA anhydrides in bond to the diol form. TXB2 can be subsequently measured. a coupled assay and AA control with no inhibitor. Results show that We used nanodiscs to stabilize TXAS, which is a membrane although no significant reduction in TX formation is observed in the protein and tends to lose functionality in aqueous buffers presence of compound C1 (LA anhydride), a significant TX reduction is at ASPET Journals on September 28, 2021 seen in the presence of compounds C2 (EPA anhydride) and C3 (DHA typically used for protein assays. Therefore, it was incorpo- anhydride). (B) Effect of C1 (solid square, solid line), C2 (solid circle, rated into nanodiscs, which are lipid bilayers surrounded dashed line), and C3 (open square, dashed and dotted line) on COX activity by MSP (Supplemental Fig. 5A). We previously showed from 0 to 50 mM. All anhydrides show a concentration-dependent , that TXAS facilitates robust biophysical studies in nanodiscs inhibition of COX-1. *P 0.05. compared with naked protein in detergent solution (Das et al., 2014). The anhydrides and their parent molecules were subjected Since both aspirin and PUFAs are thought to affect COX-1 to the coupled activity assay in a 1:1 ratio with the substrate directly, we wanted to test the effects of these anhydrides on molecule AA. As observed in Fig. 3A, the EPA and DHA COX-1 activity. COX-1 converts AA into PGH2, which as anhydrides were potent in inhibiting the TX formed in a previously stated is highly unstable and decomposes to coupled assay system to a similar extent. The perceived PGF2a. Thus, a second reaction converting PGH2 into PGF2a reduction in TXA2 formation in both cases was approximately via action of tin (II) chloride was performed to completely 30%–35% of the control. However, the LA anhydride did not convert it to PGF2a, a more stable species. This was sub- show any significant decrease in the formation of TX. These sequently measured by an enzyme-linked immunosorbent observations are in agreement with the results obtained in the assay to determine the inhibition of COX activity by reduction aggregometry studies, which demonstrated that LA anhydride in product formation. The inhibition of product formation by had the least effect on reduction of platelet aggregation in COX was measured at concentrations from 0 to 50 mM and the washed platelets (Fig. 1). product formation relative to a control with no inhibitor was Furthermore, the parent molecules LA, EPA, and DHA by fitted using nonlinear regression analysis. Figure 3B shows themselves did not show any decrease in TXA2 formation in that all three anhydrides had a dose-dependent decrease in coupled assays (Supplemental Fig. 5B). EPA is known to be PGF2a formation, demonstrating less than 10% product metabolized to PGH3 and subsequently TXA3 (Fischer and formation, compared with the absence of inhibitor at 50 mM. Weber, 1983). Our results indicate that the rate of competitive However, the trend in the decline of product formation varied metabolism of these fatty acids is not sufficient to result in for the three derivatives. Whereas the decrease in product inhibition of AA metabolism by COX-1–TXAS. Therefore, it is formation was gradual for the LA anhydride, it was more evident that the effect of dietary unsaturated fatty acids on the dramatic initially for the EPA and DHA anhydrides. The EPA COX pathway of platelet aggregation is not significant. anhydride showed an initial decline to 50% activity at 2.5 mM Direct Inhibition of COX-1–TXAS Activity by Lipid- and then gradually declined to .10% at the 25-mM concen- Aspirin Conjugates. Observations in the coupled assay tration. The DHA anhydride showed an initial decrease to less indicated that the EPA and DHA anhydride conjugates reduce than 40% of total activity at 2.5 mM and then slowly declined to platelet aggregation by inhibiting the COX-1–TXAS pathway. approximately 10% at 25 mM. Interestingly, both EPA and Aspirin Anhydrides to Reduce Platelet Aggregation 139

DHA anhydrides showed similar effects at concentrations COX-1 compared with the EPA anhydride, which releases the above 10 mM. The IC50 values for the compounds were 7.5 mM, active species much faster. The DHA anhydride shows 5.8 mM, and 2.1 mM for the LA, EPA, and DHA anhydrides, hydrolysis trends similar to LA anhydride, whereby it is more respectively. gradual over the initial 5 minutes and plateaus to about 55% Direct Buffer Hydrolysis of the Lipid-Aspirin Conju- at 60 minutes. gates. To test the lability of the anhydrides in buffer, the rate Plasma Hydrolysis Studies. To investigate the release of of hydrolysis was measured by analyzing the remaining aspirin from the aspirin–fatty acid mixed anhydride in anhydride in phosphate-buffered saline at 37°C at different plasma, plasma buffered with phosphate buffer (pH 7.4) was time intervals. In all three cases, high-performance liquid equilibrated at 37°C and the anhydride was added. At various chromatography analysis of the aliquot demonstrated the time points, aliquots were withdrawn and the extracted presence of the original parent compound, aspirin, and the products were analyzed by high-performance liquid chroma- corresponding fatty acid, indicating the release of the aspirin tography. Figure 4B provides the plasma hydrolysis patterns active moiety and not of salicylic acid (Supplemental Figs. of all three anhydrides. Similar to the buffer hydrolysis, 7–9). As observed in Fig. 4A, all three compounds show pseudo–first-order kinetics can be observed in all three cases. pseudo–first-order kinetics of decomposition. The LA-aspirin All three compounds show a gradual decomposition over anhydride shows a gradual decomposition over 30 minutes, 2 hours, plateauing to approximately 35%–40% decomposi- leading to decomposition of approximately 60% of the drug tion. Note that the plasma hydrolysis of DHA is at a range species into its individual components. In contrast, the EPA- comparable to EPA. However, as seen in Supplemental Fig. 4, Downloaded from aspirin anhydride shows a sharp decomposition in the first DHA is a much better inhibitor of platelet aggregation in 5 minutes and plateaus around 60% decomposition after the human blood samples. It can be deduced that the effect of DHA initial hydrolysis. This trend provides an explanation for the and aspirin together on inhibiting aggregation is higher than significant differences in the activities of EPA and LA EPA and aspirin in combination. anhydrides in the COX activity assay as well as the jpet.aspetjournals.org COX-TXAS coupled activity assays, which were both carried Discussion out over shorter incubation times. The slower liberation of the LA anhydride results in a lesser effect on the inhibition of Platelet aggregation is triggered by a number of primary activators leading to positive feedback and potentiation of the activation signal. The COX-1–TXAS pathway is a well estab- lished positive feedback signal in the platelet. In this pathway, AA is metabolized by COX-1 to PGH2 followed by conversion of PGH2 to TXA2, which is a proaggregatory molecule (Paul et al., at ASPET Journals on September 28, 2021 1999). Activation of the platelet either through primary activation or feedback activation via COX-1 leads to activation of the integrin GPIIb/IIIa, resulting in platelet clot formation. The inhibition of platelet aggregation both ex vivo and in vivo can be aided by these molecules either directly inhibiting the COX-1–dependent feedback pathway or by directly blocking GPIIb/III integrins (Yeung and Holinstat, 2012). Aspirin is the most popular nonsteroidal anti-inflammatory drug for reducing pain, fever, and inhibition of platelet function. The free carboxylate group in aspirin remains an ionized species at physiologic pH and is poorly absorbed across cellular membranes. Hence, the derivatization of the free carboxylic acid to form more lipophilic species can facilitate transport across biomembranes. Herein, we have derivatized the carboxylate end of aspirin through anhydride formation with dietary fatty acids including EPA and DHA (v-3 fatty acids) as well as LA (v-6 fatty acid), which have been shown to reduce platelet aggregation through dietary supplementation in platelet aggregometry studies. Compared with esters, anhydrides show more controlled patterns of hydrolysis and decomposition that are enzyme independent. Therefore, we report the design of aspirin–PUFA mixed anhydrides that would exist in a unionized, lipophilic state as well as allow for a facile, more controlled release of the active species in plasma. In addition, recent studies on polyanhydrides as drug carriers Fig. 4. (A) Buffer hydrolysis of aspirin–fatty acid anhydride conjugates. show that anhydrides degrade in a controlled fashion and are (B) Plasma hydrolysis of aspirin–fatty acid anhydrides C3 (DHA biocompatible with human body tissues, including the brain anhydride) (dashed line, solid squares), C2 (EPA anhydride) (dotted line, (Domb et al., 1999). One rationale for the design of the hybrid solid circles), and C1 (LA anhydride) (solid line, open squares). The extent codrug is that the aspirin–fatty acid–anhydride prodrug of hydrolysis is in the order of C2 (EPA anhydride) . C3 (DHA anhydride) . C1 (LA anhydride). All compounds achieve a plateau after would hydrolyze to release not one but two active species, an hour of hydrolysis. which could both inhibit the COX enzyme. 140 Roy et al.

Synthesis of the mixed anhydrides was tested in platelet hydrophobicity of the species attached to the aspirin moiety. It aggregometry studies to analyze their antiaggregatory effects. is known that LA . DHA . EPA in terms of hydrophobicity. In All three compounds displayed a concentration-dependent general, the more hydrophobic a species, the slower the rate of decrease in platelet aggregation. However, the DHA anhy- hydrolysis. This observation can be further extended to control dride was the most potent (almost completely inhibiting ag- the rate of hydrolysis of these species by carefully tuning the gregation at the 10 mM concentration) followed by the EPA anhydride partner. In the future, the effect of C1–C3 com- and LA anhydrides, which showed a marginal decrease in the pounds on platelet adhesion and aggregation will be evaluated inhibition of aggregation. As expected from the mechanism of under arterial and venous shear conditions using an ex vivo inhibition of platelet aggregation studies, the decrease was flow chamber. The effect of C1–C3 compounds on thrombosis only observed in AA-induced platelet aggregation and was not and hemostasis will be evaluated and compared with similar observed for ADP, collagen, U46619, or thrombin, indicating existing reagents in vivo using real-time intravital microscopy that these molecules did not affect those pathways for platelet murine models of thrombosis and hemostasis (Reheman et al., aggregation. Furthermore, the inhibition of the DHA anhy- 2009; Wang et al., 2014). dride and aspirin-treated species showed comparable inhibition potentials. To understand the biochemical mechanism of action of these Conclusion molecules, an activity assay was performed on the COX-

We synthesized anhydrides of aspirin and dietary fatty Downloaded from – 1 TXAS system. It was observed that although the reduction acids to explore a hybrid-drug approach. Both aspirin and the in TXA2 was significant for the EPA and DHA anhydrides, the fatty acids in this study are inhibitors of platelet aggregation LA anhydride did not show a significant reduction, thereby and this conjugation addresses the side effects of aspirin. We following the similar trend of the platelet aggregometry stud- observed that all three compounds individually inhibit plate- ies. More interestingly, however, the parent fatty acids let aggregation in a dose-dependent manner in the effective- showed no inhibition in TX formation (Supplemental Fig. ness order of C3, C2, and C1. In addition, we showed that C3 jpet.aspetjournals.org 5B). Thus, it appears that although LA, EPA, and DHA may be is comparable to aspirin-treated samples. Furthermore, we able to compete for metabolism against AA, this is likely not showed that although all three molecules act through the the chief mechanism of action in preventing platelet aggrega- COX-TXAS pathway by inhibiting COX-1, the parent fatty v tion. Literature reports have suggested that -3 fatty acids acids do not act via this pathway. Finally, hydrolysis studies of reduce platelet aggregation by signaling through other path- the drugs in buffer as well as plasma conditions confirm the ways (Abeywardena and Head, 2001). Furthermore, a meta- release of aspirin and fatty acid separately, instead of the v analysis of the effect of -3 fatty acids on the inhibition of salicylate moiety, as is the case with several aspirin prodrugs. platelet aggregation suggests that the effect of their metabo- at ASPET Journals on September 28, 2021 lism is short term. This is not sufficient to result in significant Acknowledgments changes in plasma lipid composition (Driss et al., 1984; Gao The authors thank Susan Zelasko for performing the initial litera- et al., 2013). In addition, it is possible that this inhibition is ture review and Daniel McDougle and Navroop Gill for providing mediated through collagen-induced pathways (Tremoli et al., helpful comments on the manuscript. 1995). Next, we determined the effect of the compounds on COX-1 Authorship Contributions activity and we derived IC50 values for the anhydrides. IC50 Participated in research design: Roy, Adili, Holinstat, Das. values for the LA, EPA, and DHA anhydrides were 7.1, 5.8, Conducted experiments: Roy, Adili. and 2.1 mM, respectively. Literature reports suggest that the Contributed new reagents or analytic tools: Roy, Adili, Kulmacz, IC50 values of COX-1 inhibition are 93 mM, 13 mM, and 15 mM Holinstat, Das. for LA, EPA, and DHA, respectively. Furthermore, the IC50 Performed data analysis: Roy, Adili, Holinstat, Das. value of aspirin on COX-1 in the purified enzyme system was Wrote or contributed to the writing of the manuscript: Roy, Adili, 8 mM (Mitchell et al., 1993). These results suggest a greater Holinstat, Das. effect of the mixed anhydrides compared with their parent References molecules. Abdellatif KR, Chowdhury MA, Dong Y, Das D, Yu G, Velázquez CA, Suresh MR, Finally, the molecules were characterized by their hydro- and Knaus EE (2009) Dinitroglyceryl and diazen-1-ium-1,2-diolated nitric oxide lytic decomposition patterns, because hydrolysis is the key donor ester prodrugs of aspirin, indomethacin and ibuprofen: synthesis, biological evaluation and nitric oxide release studies. Bioorg Med Chem Lett 19:3014–3018. step in releasing both species. It was observed that aspirin and Abeywardena MY and Head RJ (2001) Longchain n-3 polyunsaturated fatty acids the corresponding fatty acid were the only species resulting and blood vessel function. Cardiovasc Res 52:361–371. Bayburt TH, Grinkova YV, and Sligar SG (2002) Self-assembly of discoidal phos- from the hydrolysis of the fatty acid–aspirin anhydride. This is pholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett 2: important, since it has been observed in several aspirin 853–856. Bradley MO, Swindell CS, Anthony FH, Witman PA, Devanesan P, Webb NL, Baker formulations that the O-acetyl group of aspirin hydrolyzes SD, Wolff AC, and Donehower RC (2001) Tumor targeting by conjugation of DHA to before the aspirin moiety is released from the prodrug, thereby paclitaxel. J Control Release 74:233–236. Das A, Varma SS, Mularczyk C, and Meling DD (2014) Functional investigations of only being as effective as salicylic acid and not aspirin. thromboxane synthase (CYP5A1) in lipid bilayers of nanodiscs. ChemBioChem 15: Furthermore, we observed that all three molecules show 892–899. – Diczfalusy U, Falardeau P, and Hammarström S (1977) Conversion of prostaglandin pseudo first-order decomposition kinetics in both buffer and endoperoxides to C17-hydroxy acids catalyzed by human platelet thromboxane plasma. In addition, the extent of hydrolysis in buffer and synthase. FEBS Lett 84:271–274. Domb AJ, Israel ZH, Elmalak O, Teomim D, and Bentolila A (1999) Preparation and plasma was comparable, indicating that enzyme hydrolysis is characterization of carmustine loaded polyanhydride wafers for treating brain not a significant pathway for the release of aspirin. It appears tumors. Pharm Res 16:762–765. Driss F, Vericel E, Lagarde M, Dechavanne M, and Darcet P (1984) Inhibition of that EPA hydrolyzes faster than DHA, which hydrolyzes platelet aggregation and thromboxane synthesis after intake of small amount of more rapidly than LA. This can be partly explained by the icosapentaenoic acid. Thromb Res 36:389–396. Aspirin Anhydrides to Reduce Platelet Aggregation 141

Fischer S and Weber PC (1983) Thromboxane A3 (TXA3) is formed in human Reheman A, Yang H, Zhu G, Jin W, He F, Spring CM, Bai X, Gross PL, Freedman J, platelets after dietary eicosapentaenoic acid (C20:5 omega 3). Biochem Biophys Res and Ni H (2009) Plasma fibronectin depletion enhances platelet aggregation and Commun 116:1091–1099. thrombus formation in mice lacking fibrinogen and von Willebrand factor. Blood Gao LG, Cao J, Mao QX, Lu XC, Zhou XL, and Fan L (2013) Influence of omega-3 113:1809–1817. polyunsaturated fatty acid-supplementation on platelet aggregation in humans: a Sparatore A, Santus G, Giustarini D, Rossi R, and Del Soldato P (2011) Therapeutic meta-analysis of randomized controlled trials. Atherosclerosis 226:328–334. potential of new hydrogen sulfide-releasing hybrids. Expert Rev Clin Pharmacol 4: Gilmer JF, Moriarty LM, Lally MN, and Clancy JM (2002) Isosorbide-based aspirin 109–121. prodrugs. II. Hydrolysis kinetics of isosorbide diaspirinate. Eur J Pharm Sci 16: Tremoli E, Maderna P, Marangoni F, Colli S, Eligini S, Catalano I, Angeli MT, 297–304. Pazzucconi F, Gianfranceschi G, Davi G, et al. (1995) Prolonged inhibition of Joseph RE and Andreotti AH (2008) Bacterial expression and purification of platelet aggregation after n-3 fatty acid ethyl ester ingestion by healthy volunteers. interleukin-2 tyrosine kinase: single step separation of the chaperonin impurity. Am J Clin Nutr 61:607–613. Protein Expr Purif 60:194–197. Wang Y, Reheman A, Spring CM, Kalantari J, Marshall AH, Wolberg AS, Gross PL, Kumar N, Langer RS, and Domb AJ (2002) Polyanhydrides: an overview. Adv Drug Weitz JI, Rand ML, Mosher DF, et al. (2014) Plasma fibronectin supports hemo- Deliv Rev 54:889–910. stasis and regulates thrombosis. J Clin Invest 124:4281–4293. Mitchell JA, Akarasereenont P, Thiemermann C, Flower RJ, and Vane JR (1993) Yeung J and Holinstat M (2012) Newer agents in antiplatelet therapy: a review. J Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and Blood Med 3:33–42. inducible cyclooxygenase. Proc Natl Acad Sci USA 90:11693–11697. Mizrahi B and Domb AJ (2009) Anhydride prodrug of ibuprofen and acrylic polymers. AAPS PharmSciTech 10:453–458. Address correspondence to: Dr. Aditi Das, Division of Nutritional Sciences, Paul BZ, Jin J, and Kunapuli SP (1999) Molecular mechanism of thromboxane A(2)- Departments of Comparative Biosciences, Biochemistry, and Bioengineering, induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors. J Center for Biophysics and Quantitative Biology, Beckman Institute for Biol Chem 274:29108–29114. Advanced Science, University of Illinois, Urbana-Champaign, 2001 South Phang M, Lincz LF, and Garg ML (2013) Eicosapentaenoic and docosahexaenoic acid Lincoln Avenue, 3836 VMBSB, Urbana, IL 61802. E-mail: aditidas@illinois. supplementations reduce platelet aggregation and hemostatic markers differen- edu tially in men and women. J Nutr 143:457–463. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 JPET#234781

Supplementary Information

Development of fatty acid derivatives of aspirin for inhibition of platelet aggregation

Jahnabi Roy c, Reheman Adili e, Richard Kulmacz f, Michael Holinstat e,g, and Aditi Das* a, b, d a Department of Comparative Biosciences, b Department of Biochemistry, Center for Biophysics and Quantitative Biology, Division of Nutritional Sciences, Department of Bioengineering c Department of Chemistry d Beckman Institute for Advanced Science, f The Department of Internal Medicine, Texas Health Science Center at Houston, McGovern Medical School, Houston, TX 77225-0708, e Department of Pharmacology, g Division of Cardiovascular Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, c, a University of Illinois Urbana-Champaign, Urbana IL 61801.

Journal: THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS (JPET) CONTENTS Fig S1: Inhibition of platelet aggregation by various inducers S2 Fig S2: Inhibition of platelet aggregation by various inducers -2 S3 Fig S3: TXB2 formed in platelet aggregometry samples S4 Fig S4: Inhibition of platelet aggregation by LA, EPA and DHA S5 Fig S5: Coupled assay schematic and results for parent fatty acids S6 Fig S6: Coupled assay for LA, EPA, DHA and aspirin S7 Fig S7: Representative HPLC trace of DHA anhydride hydrolysis S8 Fig S8: Representative HPLC trace of EPA anhydride hydrolysis S9 Fig S9: Representative HPLC trace of LA anhydride hydrolysis S10 Table 1: NMR and MS characterization of compounds S11 Fig S10: NMR of DHA anhydride S12 Fig S11: NMR of EPA anhydride S13 Fig S12: NMR of LA anhydride S14

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Supplemental figure 1: Inhibition of platelet aggregation in washed platelets by anhydrides (10 μM) in the presence of (A) 1nM Thrombin (B) 50 μM PAR4-AP (C) 1 μM PAR1-AP (D) 1 μM

ADP (E) 1 μM U46619 (thromboxane A2 analog). Results show no significant decrease in platelet aggregation using these agonists. The results show data from n=3.

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Supplemental Figure 2: Inhibition of platelet aggregation in platelet rich plasma by anhydrides (10 μM) in the presence of (A) 1 μM ADP (B) 2 μg/mL Collagen (C) 1 μM ristocetin (D) 1 μM

U46619 (thromboxane A2 analog). Results show no significant decrease in platelet aggregation using alternate agonists. Results show no significant decrease in platelet aggregation. The results show data from n=3 for ADP, collagen and ristocetin and n=2 for U46619.

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Supplemental figure 3: Figure shows amount of thromboxane B2 (TXB2) measured in platelet aggregometry samples using AA (5 μM) agonist and 10 μM of C1, C2 or C3, normalized to DMSO control. Compound C1, C2 and C3 all show reduction in TXB2 formation. ** p <0.01. Samples were tried for n=4.

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Supplemental figure 4: The effect of LA, EPA and DHA on platelet aggregation. Platelets were incubated with LA, EPA and DHA at concentrations of (A) 2.5 μM, (B) 5 μM and (C) 10 μM and aggregation was induced by 5μM AA. (D) The effect of DHA on AA (5μM) induced platelet aggregation at 0.25 μM, 0.5 μM and 1 μM concentration.

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Supplemental figure 5: (A) Schematic of coupled assay. Arachidonic acid (AA) converts to

Prostaglandin H2 (PGH2) in the presence of enzyme COX-1 (cyclooxygenase-1). PGH2 converts to Thromboxane A2 (TXA2) in the presence of TXAS (thromboxane synthase) that is stabilized in nanodiscs. (B) Thromboxane formed in the presence of LA, EPA and DHA in a coupled assay with TXAS in nanodisc and AA control with no inhibitor. Results show that while no significant reduction in thromboxane formation is observed in the presence of any of the dietary unsaturated fatty acids LA, EPA or DHA. (C) Thromboxane formed in the presence of LA, EPA and DHA in a coupled assay with TXAS solubilized in 0.53% β-octyl glucoside and AA control with no inhibitor. Results show that while no significant reduction in thromboxane formation is observed in the presence of any of the dietary unsaturated fatty acids LA, EPA or DHA.

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Supplemental figure 6: Thromboxane formed in the presence of LA, EPA, DHA and aspirin in a coupled assay with TXAS (thromboxane synthase) in nanodisc and AA control with no inhibitor. Results show that while no significant reduction in thromboxane formation is observed in the presence of any of the dietary unsaturated fatty acids LA, EPA or DHA. However a significant reduction (*p<0.05) is seen for aspirin.

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Supplemental figure 7: Representative HPLC trace of DHA anhydride hydrolysis. Peak at 2.57 mins is DHA anhydride (red line) and peak at 32 mins is DHA (blue line). The green line representing hydrolysis shows emergence of DHA as product

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Supplemental figure 8: Representative HPLC trace of EPA anhydride hydrolysis. Peak at 2.5 mins is EPA anhydride (red line) and peak at 28 mins is EPA (blue line). The green line representing hydrolysis shows emergence of EPA as product.

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Supplemental figure 9: Representative HPLC trace of LA anhydride hydrolysis. Peak at 2.7 mins is LA anhydride (red line) and peak at 34 mins is LA (blue line). The green line representing hydrolysis shows emergence of LA as product.

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Supplemental table 1: Characterization of compounds:

DHA- aspirin anhydride:

HR ESI-MS- 512.03 C31H38O5Na 1H NMR: 0.9 (t, 6H), 1.7 (t, 2H), 2.1 (m, 3H), 2.30 (s, 3H), 2.35 (t, 1H), 2.8 (m, 7H), 5.4 (m, 12 H), 7.05 (d, 1H), 7.3 (t, 1H), 7.55(t, 1H), 8.05(d, 1H)

EPA- aspirin anhydride

HR ESI MS- 487.24 C29 H36O5Na 1H NMR: 0.9 (t, 3H), 1.7 (t, 1H), 2.1 (m, 3H), 2.30 (s, 3H), 2.35 (t, 1H), 2.8 (m, 4H), 5.4 (m, 10 H), 7.05 (d, 1H), 7.3 (t, 1H), 7.55(t, 1H), 8.05(d, 1H)

LA aspirin anhydride:

HR ESI MS- 465.26 C27H38O5Na 1H NMR: 0.9 (t, 3H), 1.3 (m, 14 H), 1.6 (t, 2H), 2.0 (s, 3H), 2.01(m, 2H), 2.3 (m, 4H), 2.75(t, 2H), 5.3 (m, 4H), 7.05 (d, 1H), 7.3 (t, 1H), 7.55(t, 1H), 8.05(d , 1H)

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1 Supplemental figure 10: H NMR of DHA- aspirin anhydride in CDCl3

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1 Supplemental figure 11: H NMR of EPA- aspirin anhydride in CDCl3

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1 Supplemental figure 12: H NMR of LA- aspirin anhydride in CDCl3

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