Introduction

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Platelet aggregation is an important component of the hemostatic mechanism that prevents undesired bleeding, in which a platelet plug forms at the site of injury to the blood vessel, leading to cessation of bleeding. Several aggregation pathways have been described (Packham, 1993), one of which is the thromboxane pathway (Hamberg et al., 1974, 1975; Diczfalusy and Hammarstrom, 1979; Hammarstrom and Diczfalusy, 1980). The active mediator in this pathway is thromboxane A₂, a powerful unstable pro-aggregating substance and a vasoconstrictor of blood vessels. It is formed in platelets from arachidonic acid, a fatty acid present in membranes, by the enzyme, cyclooxygenase, through an intermediate, prostaglandin endoperoxide, common to all the prostaglandins and thromboxane. On the other hand extreme cases of aggregation can lead to serious outcome as in septic shock and thrombosis (Parellada and Planas, 1977; Randall and Wilding, 1982; Fiedler et al., 1989; Silver et al., 1995; Wolkow et al., 1997; Zaitsu et al., 1999). By comparison, NSAIDs such as aspirin reduce platelet aggregation through inhibition at the early stage of cyclooxygenase within the platelet but, also, in other cells/tissues, such as the blood vessel wall, prevent the formation of all prostaglandins, some of which are anti-aggregatory and therefore beneficial, e.g., prostacyclin and prostaglandin E₂ (Vane, 1978; Willis, 1978; Bunting et al., 1983; Harada et al., 1998). Therefore, proper management of thromboxane-mediated disease is desirable at the level of thromboxane (synthesis and/or receptor action), as this leaves the beneficial prostaglandins in place (Bunting et al., 1983). Recently, this approach has identified a new class of compounds, oxazolecarboxamide-substituted alkenoic acids with dual TSI/TRA activities (Takeuchi et al., 1998).

During the course of our studies on the metabolic conversion of arachidonic acid, we discovered a novel pathway with products, hepoxilins, that showed biological actions on a variety of systems (Pace-Asciak et al., 1983; Pace-Asciak and Martin, 1984; Pace-Asciak, 1994). Since these compounds were unstable biologically, we prepared by total chemical synthesis a family of analogs, PBTs, that were both chemically and biologically suitable for in vivo studies (Demin and Pace-Asciak, 1993). Indeed, we found that different compounds within this family acted on different in vivo systems, e.g., insulin secretion (Pace-Asciak et al., 1999), decrease in plasma glucose (unpublished observations), or inhibition of lung fibrosis (Pace-Asciak et al., 2000). During more comprehensive screening for in vitro biological actions, we discovered that one of these analogs, PBT-3, potently inhibited collagen-evoked aggregation of human platelets through selective blockade of the thromboxane synthetic pathway (Reynaud et al., 2001). The present results demonstrate a far more important and powerful action of this compound as an antagonist of the TP receptor revealing a more likely mechanism for its inhibitory actions on platelet aggregation.

	Experimental Procedures

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Materials. The hepoxilin analogs, PBT-1, -2, -3, and -4, were prepared as previously described (Demin and Pace-Asciak, 1993). Collagen (Chrono-Par) was purchased from Chronolog Corp. (Havertown, PA). ADAM reagent (9-anthryldiazomethane) was from Research Organics Inc. (Cleveland, OH). U46619, I-BOP, and ¹²⁵I-BOP were from Cayman Chemical (Ann Arbor, MI).

Isolation of Human Platelets. Healthy human subjects who had not taken NSAIDs for at least 2 weeks were used. Blood was drawn into plastic syringes containing citric acid-sodium citrate-dextrose (9:1, v/v). It was immediately centrifuged at 23°C at 200g for 15 min. The platelet-rich plasma was transferred into fresh plastic tubes and centrifuged at 400g for 5 min. The supernatant was discarded and the platelet pellet was resuspended in fresh medium containing 137 mM NaCl, 1 mM KCl, 0.4 mM NaH₂PO₄, 5.5 mM glucose, 20 mM HEPES, and 1 mM CaCl₂, pH 7.4, and allowed to stand at room temperature for 30 min. The platelet count was adjusted to 350 × 10⁶ cells with medium to make 0.5 ml/assay/cuvette for each measurement.

Measurement of Platelet Aggregation. Appropriate calibration of the platelet aggregation profiler (model PAP-4C, Bio/Data Corp., Horsham, PA) for 0% and 100% transmission was carried out with a sample of platelet suspension and cell-free medium, respectively. A total of 0.5 ml of platelet suspension was added to siliconized glass tubes (four samples at a time) and heated with magnetic stirring (900 rpm) to 37°C for 1 min in the aggregometer. Either vehicle alone (1 µl ethanol) or PBT analog at various concentrations in ethanol (1 µl) was added, followed by agonist 2 min later (either collagen at 2 µg/0.5 ml or the thromboxane receptor agonist, U46619, at 10 ng/0.5 ml, or I-BOP at 2 ng/0.5 ml). The response was recorded for the next 5 min. In experiments addressing whether levels of endogenously produced thromboxane A₂ play a role in the inhibition of aggregation by PBT-3, platelets were treated with aspirin (20 µg/0.5 ml), followed either by collagen (in which aggregation was inhibited) or the thromboxane agonists, I-BOP or U46619 (which resulted in aggregation); PBT-3 was added after aspirin but before I-BOP or U46619 at the above-mentioned doses, resulting in inhibition of aggregation.

Binding of ¹²⁵I-BOP to Platelets. Washed platelets were prepared as described above, except that the platelet suspension was made up at a concentration of 10 × 10⁶ cells/0.5 ml (Dorn, 1991) in a clear medium (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM sodium-free HEPES, and 10 mM glucose, pH 7.3). The binding assay involved the addition of radioligand (30,000 cpm ¹²⁵I-BOP) to all tubes in triplicates, containing either various concentrations of unlabeled I-BOP (10⁹-10⁷ M) or PBT-3 (10⁹-10⁷ M) or U46619 (10⁹-10⁷ M) in 1 µl ethanol. Additional tubes containing excess unlabeled I-BOP were included to assess the extent of nonspecific binding. Binding was allowed to take place during 30 min at 37°C; free radioligand was removed by rapid vacuum filtration through Whatman (Maidstone, UK) GF/B glass fiber filters prewashed with clear medium. The tubes and the filters were rapidly washed with ice-cold clear medium (three times with 3 ml). The radioactivity on the filters containing the ligand-receptor complexes was counted in an LKB (Uppsala, Sweden) Compugamma CS counter.

Measurement of Adenyl Cyclase Activity. Human washed platelets (350 × 10⁶ cells) in 1 ml of assay buffer were preincubated during 10 min at 37°C. Dimethyl sulfoxide (1 µl) alone (control) or containing various concentrations of PBT-1, -2, -3, and -4 (2.8 × 10⁷-10⁵ M final concentration) was added, and the cells were incubated for a further 5 min. The reaction was stopped by the addition of 12% trichloroacetic acid, and the samples were sonicated (four times for 3 sec). The samples were left on ice for 60 min to extract cAMP. After centrifugation at 2500g for 15 min, the supernatants were transferred and extracted five times with 5 ml of water-saturated diethyl ether to remove the trichloroacetic acid. The aqueous phase was transferred and lyophilized. cAMP was measured using specific double antibody radioimmunoassay kits with ¹²⁵I-labeled cAMP according to the instructions by the manufacturer (Amersham Biosciences, Piscataway, NJ). Results are expressed in picomoles/350 million cells. Experiments were performed in triplicate for each point and repeated twice.

Measurement of COX-1 and COX-2 Activity. COX-1 and COX-2 enzyme preparations were purchased from Cayman Chemical (Ann Arbor, MI). Preliminary studies established that 40 U of COX-1 or 20 U of COX-2 could convert about 70% of ¹⁴C-AA [specific activity 55 mCi/mmol; Ontario Isotopes, Flamborough, ON, Canada; 100,000 cpm were diluted with 0.5 µg unlabeled arachidonic acid (Cayman Chemicals)/1 ml assay] into products in vitro. Different amounts of PBT-3 (10 µg and 20 µg) were added and the conversion of AA into products was assessed during a 10-min reaction in 1 ml phosphate buffer at 37°C. After extraction, products were assessed by thin layer chromatography (Silica Gel G, ethyl acetate/acetic acid, 99:1, v/v). After development, the plates were scanned for radioactive products with a Berthold thin layer chromatography radiochromatogram scanner (PerkinElmer Instruments, Norwalk, CT), and the radioactivity was quantified by scraping zones of silica gel, placing in scintillation vials, elution with 1 ml of methanol/water (1:1, v/v), and addition of scintillation medium. Radioactivity was determined with conventional counting in a beta scintillation counter (Beckman LS 3800; Beckman Coulter, Inc., Fullerton, CA).

Measurements of Platelet-Derived Eicosanoids. Measurement of eicosanoids formed by platelets during treatment with collagen or the TP receptor agonists in the presence or absence of PBT-3 was carried out by HPLC after appropriate derivatization with a fluorescent tag (ADAM), which forms a fluorescent ester (Demin et al., 1995). The method was adapted to measure the following compounds: TxB₂, HHT, 12-HETE, and AA. The platelet suspension at the end of the experiment was mixed with ethyl acetate, 100 ng of prostaglandin B₁ was added as internal standard, and the mixture was acidified to pH 3 with 0.1 N HCl. After centrifugation, the organic layer was separated, washed twice with water to neutrality, and evaporated to dryness. The residue was resuspended in ethyl acetate and half of the sample was taken for derivatization. It was diluted to 0.2 ml with ethyl acetate containing 20 µg ADAM reagent and was left in the dark for 2 h. The solvent was then evaporated and the residue was acetylated with a solution of pyridine/acetic anhydride (3:1, v/v) for 16 h at 23°C. The reagents were evaporated to dryness and the residue was resuspended in acetonitrile. One-tenth of the sample was used for HPLC analysis. Dose-response curves for varying amounts of test compounds were generated and the data were expressed as percentage of inhibition of control representing agonist-induced platelet aggregation. Each point was investigated three times and statistical analysis of the data was carried out (see below).

Chromatography. Analysis of the anthryl (ADAM)-acetate derivatives of TxB₂, HHT, and 12-HETE (AA only forms an ADAM derivative) in the extracted platelet samples was carried out on a Hewlett Packard (Palo Alto, CA) (1100 series) HPLC to which was attached a Shimadzu (Kyoto, Japan) fluorescent detector (RF-10AXL). The detector was operated with excitation at 364 nm, emission at 411 nm. Chromatographic separation of the compound was carried out on a Waters (Milford, MA) C18 Novapak column (3.9 × 300 mm) using acetonitrile/water (80:20) at injection and after 10 min programmed with a linear gradient to 100% acetonitrile during 20 min.

Statistical Analysis. Values stated are the mean ± S.D. of the number of observations (n) indicated. Analysis of statistical significance was performed using Student's t test involving the Macintosh StatView software program. Inhibition data (Figs. 3 and 4) were fitted to a line of best fit through a Michaelis-Menten-like hyperbolic treatment with a Kaleidagraph statistical software package.

	Results

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PBT Analogs Inhibit Aggregation Evoked by TP Receptor Activation in Washed Human Platelets in Vitro. Dose-related aggregation curves for I-BOP, a potent thromboxane receptor agonist, indicated that at a concentration of 2 ng/0.5 ml, it caused approximately 70% aggregation (data not shown). This dose was therefore chosen because it represented a point at which inhibition curves for the test compounds could be most sensitive. We tested four related PBT analogs (PBT-1 to -4) on the I-BOP-evoked aggregation of human platelets. Figure 1A shows aggregometer curves for all four compounds at 50 ng each. These data show that PBT-3 was clearly more active than the other three analogs in inhibiting aggregation. Figure 1B shows a comparison of two concentrations (20 and 50 ng/0.5 ml) of the four compounds, clearly resolving PBT-3 as the more active of the compounds, although all four compounds appear to inhibit aggregation evoked by I-BOP. PBT-3 is about 5-fold more active than the other analogs and is about 500 times more active than the native hepoxilins (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   A, aggregometer curves showing the effects of four PBT analogs (50 ng each/0.5 ml of platelet suspension containing 350 × 106 cells) on the aggregation of human washed platelet suspensions evoked by I-BOP (2 ng). PBTs were added 2 min before I-BOP. B, inhibition by PBTs at two concentrations (20 and 50 ng/0.5 ml) of the aggregation of human washed platelets evoked by I-BOP (2 ng).

Dose-Related Inhibition by PBT-3 of I-BOP- and U-46619-Evoked Aggregation in Washed Human Platelets in Vitro. Fig. 2A shows aggregation responses in human washed platelet suspensions evoked by I-BOP and the inhibition of this response by different doses of PBT-3 added 2 min before the addition of I-BOP. Figure 2B shows aggregation responses of human washed platelets challenged with U46619 and their inhibition by different amounts of PBT-3. Quantitative data for these experiments are shown in Fig. 3, demonstrating an IC₅₀ for inhibition of aggregation evoked by I-BOP of 0.6 × 10⁷ M PBT-3. PBT-3 also inhibited the aggregation evoked by the agonist, U46619, with an IC₅₀ of 7 × 10⁷ M (Fig. 3). Collagen evokes the aggregation of human platelets through the formation of thromboxane A₂. PBT-3 dose dependently prevented collagen-evoked aggregation of platelets with an IC₅₀ of 8 × 10⁷ M (Reynaud et al., 2001). Analysis of the thromboxane formed in these experiments indicated that collagen-evoked formation of thromboxane was blocked by PBT-3 with an IC₅₀ of 4 × 10⁷ M (Reynaud et al., 2001). In separate studies we showed that I-BOP-evoked or U46619-evoked aggregation was not accompanied by thromboxane formation (data not shown); hence PBT-3 inhibition of the action of these two thromboxane mimetics is due to direct inhibition at the TP receptor level and is not dependent on endogenous formation (or blockade) of thromboxane A₂. These experiments clearly show that the primary action of PBT-3 is at the level of the TP receptor, although at higher concentrations thromboxane formation is also inhibited (Reynaud et al., 2001).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Aggregation curves showing the pro-aggregatory actions of the thromboxane receptor agonists, I-BOP (A) and U46619 (B) in washed human platelet suspensions and the inhibition of aggregation by various amounts of PBT-3 added 2 min before the addition of the agonist. Values shown are typical of three separate experiments; quantitative data are shown in Fig. 3.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Dose-response curves showing the inhibition by PBT-3 of the action of the thromboxane receptor agonists, I-BOP and U46619, and of collagen on the in vitro aggregation of washed human platelets. Values represent data from three separate experiments ± S.D.

Competition of I-BOP Binding to Platelets by PBT-3. Additional confirmation that inhibition of aggregation by PBT-3 occurs at the level of the TP receptor was obtained through competition binding studies with ¹²⁵I-BOP as ligand. This reagent has been used as a specific agonist for the TP receptor. If PBT-3 antagonizes the aggregation of platelets caused by I-BOP (as shown in Figs. 1-3), we thought that it must compete for the TP receptor. Figure 4 shows that PBT-3 competes for the binding of ¹²⁵I-BOP in a dose-dependent way. Competition curves are shown for I-BOP itself (IC₅₀ = 0.5 × 10⁹ M), PBT-3 (IC₅₀ = 8.1 × 10⁹ M), and U46619 (IC₅₀ = 4.1 × 10⁹ M), another known TP receptor agonist. PBT-3 is about 16-fold less active in competing with I-BOP for ¹²⁵I-BOP binding to the platelet TP receptor, but about equal to U46619. This study demonstrates that PBT-3 antagonizes I-BOP binding to the TP receptor, and together with its inhibition of aggregation evoked by the two TP receptor agonists, I-BOP and U46619, provides evidence that PBT-3 acts as a TP receptor antagonist.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Antagonism of 125I-BOP binding to washed human platelets by PBT-3, I-BOP, and U46619 (n = 3 separate experiments; values represent the mean ± S.D.). Note the similarity of potency of PBT-3 and U46619, a known TP receptor agonist.

Inhibition of Platelet Aggregation by PBT-3 Is Independent of Endogenous Thromboxane Synthesis. To establish whether endogenous formation of thromboxane A₂ plays a role in the action of PBT-3 in inhibiting the aggregation process rather than the action of thromboxane, i.e., at the TP receptor, we carried out experiments in which platelets were treated with aspirin (to block endogenous thromboxane formation) followed by the agonist, U46619 (which still causes aggregation). Figure 5 shows an aggregation profile of such an experiment. Both collagen and U46619 cause platelets to aggregate (see Fig. 5, lines 1 and 2, respectively). Aspirin greatly reduced collagen-evoked aggregation (Fig. 5, early part of line 3). We chose a dose of 20 µg aspirin for this study from earlier dose-response studies (not shown), because this dose blocked collagen effects almost completely, demonstrating that collagen-induced aggregation is mediated through the formation of endogenous thromboxane. Conversely, aspirin at this dose did not block U46619-evoked aggregation (Fig. 5, later part of line 3), demonstrating that aspirin did not interfere with the TP receptor or the cascade of events initiated by U46619. Addition of PBT-3 to aspirin-treated platelets before the addition of U46619 resulted in a blockade of the aggregation induced by U46619 (Fig. 5, line 4; compare with line 3). This finding, together with the binding data of Fig. 4, confirms that PBT-3 caused inhibition of the action of U46619 at the TP receptor level and is independent of the formation of endogenous thromboxane.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Platelet aggregation curves (typical of three separate experiments) demonstrating actions of PBT-3 at the TP receptor, independent of endogenous thromboxane formation. The study was carried out with normal platelets to which acetylsalicylic acid (ASA) was added (see above) to block endogenous formation of thromboxane and challenging with collagen (much reduced aggregation showing inhibitory effects of acetylsalicylic acid) or the TP receptor agonist, U46619 (positive aggregation because this agonist bypasses the requirement for thromboxane formation). PBT-3 inhibited the U46619-evoked aggregation in the presence of acetylsalicylic acid, indicating an action on the TP receptor. Arrows indicate the time of addition of the various substances as reflected in the legend. Note curve 1, which shows the integrity of platelets to respond to collagen in the absence of acetylsalicylic acid.

Effects of PBT Analogs on Platelet Cyclic AMP Levels. Figure 6 shows the effects of the four PBT analogs on platelet cyclic AMP levels. All four compounds caused a stimulation of adenyl cyclase activity, but this occurred at concentrations much larger than those required for antagonism of I-BOP binding to the TP receptor; in addition, there was little discrimination between the four PBT analogs in stimulating cyclic AMP formation. This suggests that the mode of action of the PBT analogs, but especially of PBT-3, in inhibiting platelet aggregation could only partly be ascribed to stimulation of cyclic AMP formation. Antagonism of the TP receptor is a more likely and effective mechanism of action of PBT-3 in preventing aggregation of platelets evoked by TP receptor activation.

View larger version (52K): [in this window] [in a new window]   Fig. 6.   Dose-related effects of four PBT compounds on platelet cyclic AMP levels. Washed human platelets were incubated with the compounds shown for 5 min, and the cells were extracted and measured by radioimmunoassay for cyclic AMP as described under Experimental Procedures.

Lack of Inhibition by PBT-3 of COX-1, COX-2, 12-LOX, and Plase A₂. Several experiments were carried out to investigate whether PBT-3 affected several enzymes involved in the generation of various eicosanoids or whether it acted to block selectively thromboxane formation subsequent to the actions of PBT-3 at the TP receptor. The data are summarized in Table 1. Neither COX-1, COX-2, 12-LOX, nor Plase A₂ is inhibited by relatively large amounts of PBT-3, i.e., about 3 to 4 log doses greater than that required to antagonize the TP receptor. In contrast, PBT-3 inhibited significantly TxB₂ formation in platelets (Table 1, last column).

	Discussion

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Because thromboxane A₂ is a powerful aggregating substance and a powerful constrictor of blood vessels, the control of its formation in the body is an important requirement when its formation/action goes astray as in episodes of thrombosis or in states of septic shock in which thromboxane formation is believed to be exacerbated. Normal hemostasis requires a balance between thromboxane, formed by platelets, and prostacyclin, a powerful anti-aggregating substance formed by endothelial cells in the wall of the blood vessel. Indeed, in disease states in which prostacyclin levels are abrogated, the tendency to thrombosis is a serious matter to be reckoned with. The most common way to control these events is with high doses of NSAIDs, which inhibit the formation of thromboxane and other prostaglandins by blocking the precursor, PGH₂, from being formed by the enzyme cyclooxygenase. Aspirin has been a drug of choice because of its popular use, and because it is cheap to produce and is generally well tolerated by most people. However, aspirin sensitivity has been noted, leading to asthma (Szczeklik et al., 1977, 2001), Reye's syndrome (Baldwin 2000), and Down's syndrome (Ebadi and Kugel, 1970), as well as gastric ulcers (Konturek et al., 1981; Brzozowski et al., 2001), a common side effect. For the latter reason "super aspirins" have been designed which reduce the incidence of gastric lesions. Two such drugs are Celebrex (celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide) and VIOXX (rofecoxib, 4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone) (Everts et al., 2000; Urban, 2000). These two drugs appear to selectively inhibit COX-2 (COX-1 is present in platelets), an enzyme that produces the thromboxane precursor, PGH₂; however, these drugs do not affect formation of thromboxane (formed via COX-1) in platelets. Hence, the super aspirins do not inhibit platelet-derived thromboxane, the compound involved in the aggregation process.

The results reported herein show novel anti-aggregating effects of a new family of eicosanoids which selectively inhibit the action of thromboxane at the TP receptor level, with secondary actions on thromboxane synthase at higher concentrations (Scheme 1). Human platelets appear to predominantly express the TP form of the two TP isoforms, suggesting that PBT-3 may be binding to this isoform, resulting in the blockade of both I-BOP- and U46619-evoked aggregation (Walsh et al., 2000). Interestingly, whereas 8-epi-prostaglandin F₂ competes for I-BOP binding in human platelets with 1000-fold less effectiveness than I-BOP itself (Kinsella et al., 1997), PBT-3 is only 16-fold less active than I-BOP (see Fig. 4), making it far more effective than 8-epi-prostaglandin F₂ as an antagonist to the TP receptor in platelets. The PBT compounds do not block thromboxane formation at the level of COX-1 or COX-2 (Table 1), hence allowing the formation of PGH₂ and its redirection into the beneficial prostaglandins, PGE₂ and PGI₂. Additional studies have shown that PBT-3 increased cAMP formation by washed human platelets but required a concentration greater than 0.5 × 10⁶ M (Fig. 6), several log concentrations greater than the concentration required to inhibit I-BOP binding or action in platelets, suggesting that activation of adenyl cyclase represents a minor pathway of PBT-3 action. In vivo studies have shown that the PBT analogs reported herein are active, safe, and well tolerated in rodent models of diabetes and in inflammatory models of lung fibrosis (Pace-Asciak et al., 2000).

View larger version (14K): [in this window] [in a new window]   Scheme 1.   Scheme describing TSI/TRA actions of PBT-3 reported in this study. Note that the main effect of PBT-3 is at the TP receptor as shown in this study with minor actions on TSI and cAMP formation, resulting in an overall inhibition of platelet aggregation. Arrow line width indicates level of activity. (+), activation; (), inhibition; X, no effect (see Table 1).

Although interest in the development of combined TSI/TRA drugs is not new, the many drugs that have been developed, ranging in effectiveness within the concentrations of 10⁴ to 10⁸ M, have not proven clinically successful (Boehm et al., 1996; Moncada et al., 1977; Needleman et al., 1977). A recent addition to this group of drugs has been made with the development of substituted -phenyl--(3-pyridyl) alkenoic acids (Takeuchi et al., 1998). These drugs were shown to be as effective as TRAs with an IC₅₀ of 55 × 10⁹ M. The compounds were found active in washed platelet suspensions and in ex vivo models, but were less active on platelet-rich plasma, possibly due to plasma binding or metabolism. Our compounds display similar potency (IC₅₀ 8 × 10⁹ M), yet we have found them to be active in vivo in models of inflammation and diabetes, suggesting that although the PBTs may bind to albumin, they are still effective in those models. We believe that the mechanism of action of the PBT compounds described in our studies may be through their ability to potently antagonize the TP receptor.