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 con- jugates. Aspirin (acetylsalicylic acid) is first converted to the activated acyl chloride by refluxing with thionyl chloride (SOCl2) in ben- zene. 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