European Journal of Pharmacology 670 (2011) 534–540

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmacology

j ourna l homepage: www.e lsev ie r .com/ locate /e jphar

Cardiovascular Pharmacology

Direct formation of thienopyridine-derived nitrosothiols — Just add nitrite!

Shantu S. Bundhoo a, Richard A. Anderson a, Ewelina Sagan a, Nurudeen Hassan b, Andrew G. Pinder a,Stephen C. Rogers c, Keith Morris b, Philip E. James a,⁎a Section of Cardiology, Wales Heart Research Institute, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdomb The School of Applied Sciences, University of Wales Institute Cardiff, Llandaff Campus, Western Avenue, Cardiff CF 5 2YB, United Kingdomc The Department of Pediatrics, Washington University at St Louis, Missouri, MO 63110, United States

⁎ Corresponding author at: Section of Cardiology, WCardiff University School of Medicine, Heath Park, CardTel.: +44 29 2074 3512; fax: +44 29 2074 3500.

E-mail address: Jamespp@Cardiff.ac.uk (P.E. James).

0014-2999/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.ejphar.2011.09.022

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 April 2011Received in revised form 5 September 2011Accepted 11 September 2011Available online 19 September 2011

Keywords:NitrosothiolThienopyridineAnti-plateletNitric oxide

Thienopyridines (ticlopidine, clopidogrel and prasugrel) are pro-drugs that require metabolism to exhibit acritical thiol group in the active form that binds to the P2Y12 receptor to inhibit platelet activation and pre-vent thrombus formation in vivo. We investigated whether these thienopyridines participate in S-nitrosation(SNO) reactions that might exhibit direct anti-platelet behaviour. Optimum conditions for in vitro formationof thienopyridine–SNO formation were studied by crushing ticlopidine, clopidogrel or prasugrel into aqueoussolution and adding sodium nitrite, or albumin–SNO. Ozone-based chemiluminescence techniques were uti-lised to specifically detect NO release from the SNO produced. Effect on agonist-induced platelet aggregationwas monitored using light transmittance in a 96 well microplate assay. Pharmaceutical grade preparations ofticlopidine, clopidogrel and prasugrel were found to exhibit significant free thiol and formed SNO derivativesdirectly from anionic nitrite in water under laboratory conditions without the need for prior metabolism.Thienopyridine–SNO formation was dependent on pH, duration of mixing and nitrite concentration, withprasugrel–SNO being more favourably formed. The SNOmoiety readily participated in trans-nitrosation reac-tions with albumin and plasma. Prasugrel–SNO showed significantly better inhibition of platelet aggregationcompared with clopidogrel–SNO, however when compared on the basis of SNO concentration these wereequally effective (IC50=7.91±1.03 v/s 10.56±1.43 μM, ns). Thienopyridine-derived SNO is formed directlyfrom the respective base drug without the need for prior in vivometabolism and therefore may be an importantadditional contributor to the pharmacological effectiveness of thienopyridines not previously considered.

ales Heart Research Institute,iff CF14 4XN, United Kingdom.

rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Thienopyridine antiplatelet agents arewidely used for the secondaryprevention of cardiovascular disease, with clopidogrel being one ofthe most prescribed drugs worldwide (Top Ten Global Products, 2007)The use of these agents is a well established treatment for the preven-tion of ischaemic complications in patients undergoing percutaneouscoronary intervention and in the treatment of acute coronary syn-dromes (Hall et al. 1996; Mehta et al. 2001; Wiviott et al. 2007). Cur-rently there are three licenced oral preparations of thienopyridineantiplatelet agents; ticlopidine, clopidogrel and prasugrel.

These thienopyridines are all pro-drugs which require biotransfor-mation to their active metabolite via the family of CYP3 cytochromeP450-dependent isoenzymes (Kazui et al. 2010; Rehmel et al. 2006;Yoneda et al. 2004). These agents however differ significantly intheir metabolic pathways to their respective “active” metabolites.Ticlopidine and clopidogrel are metabolised in a two step process by

esterases and liver CYP3 enzymes and are subjected to competingmetabolic pathways to yield mainly inactive metabolites. Prasugrelactive metabolite is formed by a hydrolysis step and partly by theintestinal CYP3A enzymes (Farid et al. 2010). These different meta-bolic pathways have long been used to explain the observed varia-tions in potency of inhibition of platelet aggregation between theactive metabolites of the three drugs (Mega et al. 2009). Critically,the biotransformation leads to increased availability of a thiol groupwhich irreversibly binds to a cysteine residue of the P2Y12 receptorson platelets, inhibiting ADP-mediated platelet activation to result intheir pharmacological mode of action (Borja Ibanez et al., 2006).

In recent years attention has been given to properties of thieno-pyridines beyond their inhibition of platelet P2Y12. Clopidogrel andticlopidine exhibit vasomodulatory activity inmurine, rabbit and caninemodels (Yang and Fareed, 1997; Yang et al. 1998) and Jakubowski et al.(2005) have shown that clopidogrel (and its inactive metabolites) canstimulate guinea pig coronary artery to exhibit nitric oxide (NO)-dependent vasodilation. Oral administration of a single loading doseof clopidogrel to patients with stable coronary artery disease leads toa dose-dependent improvement of endothelial dysfunction which waspartly independent of platelet P2Y12 receptor inactivation (Warnholtzet al. 2008). Heitzer et al. (2006) have also reported the improvement

535S.S. Bundhoo et al. / European Journal of Pharmacology 670 (2011) 534–540

of NO bioavailability with clopidogrel in a similar patient group. Takentogether, these findings could point towards mechanisms of actionbeyond the inhibition of platelet receptor function and may also pointto direct effects of the parent drugs.

We hypothesised thienopyridines could formnitrosothiol derivatives(Thienopyridine–SNO) via the following mechanism:

Thienopyridine–SH þ Hþ þ NO−2 ↔Thienopyridine–SNO þ H2O

According to the established SNO biochemistry this would dependon the availability of a free thiol group, an acidic environment, and asource of NO+ (derived from either NO or nitrite (NO2

−))— conditionsthat would not be expected from the clinical grade drug preparationThienopyridine–SNO would have the potential to participate in all thereactions expected of native NO that could include direct inhibitionof platelet aggregation, with the added benefit that the NO “moiety” isprotected and is largely preserved from further reactive metabolism(Al-Sa'doni et al. 2000; Hogg, 2000).

These studies indeed show that thienopyridines exhibit free thioland form acidic aqueous solutions that can directly participate inthe formation of bioactive thienopyridine–SNO even without priormetabolism.

2. Materials and methods

2.1. Materials

Ticlopidine (Ticlopidin-ratiopharmTM ratiopharm GmbH, Ulm,Germany) tablets containing 250 mg ticlopidine hydrochloride,clopidogrel (PlavixTM Bristol-Myers Squibb, UK) tablets containing75 mg clopidogrel hydrogen sulphate and prasugrel (EfientTM DaiichiSankyo UK) tablets containing 10 mg prasugrel hydrochloride wereused in these studies. Purified clopidogrel sulphate (LKT Laboratories,Inc) was supplied as 98.6% pure. Ticlopidine hydrochloride (Sigma)was supplied as N99% pure. All other chemicals were purchased fromSigma other than HPLC grade water (Fisher).

2.2. Detection of reduced thiols (S–H)

Tablets of the commercially available clopidogrel, prasugrel orticlopidine were crushed and dissolved in HPLC grade water. After a10 min incubation at 37 °C, samples were neutralised to pH 7–7.5and loaded onto a 96-well plate in triplicate and 2.5 μg/ml ThioStarFluorescent Thiol Detection Reagent (Bioquote) added. In order tospecifically detect reduced thiol content, fluorescence was recorded5, 15 and 45 min after addition of the probe (excitation 380 nm andemission 510 nm). The corresponding concentration of free thiolwas calculated from a standard curve of reduced glutathione (GSH).All reagents were kept in the dark and on ice.

2.3. Thienopyridine–SNO production

Crushed thienopyridine tablets were dissolved in HPLC grade waterto yield a 10 mM basal suspension. To this NaNO2

− was added (10 to10,000 μM) and incubated for 10, 30, 60, 90 and 120 min at 37 °Cprior to neutralisation with NaOH (0.1 M) and immediate analysis by2C's ozone based chemiluminescence (2.5 - below). In experiments totest the effect of pH on thienopyridine–SNO formation, the pH of theaqueous solution was first adjusted to the test pH and only then nitritesolution added.

In experiments to test the effect of increasing incubation time andpH on thienopyridine–SNO yield, the final concentration of the thieno-pyridine solution was kept fixed at 10 mM and nitrite concentrationalso kept in slight excess (10.1 mM). This ensured a signal-noise ratioof N10:1 for all SNO analyses.

For platelet aggregation studies thienopyridine–SNO was preparedbymixing thienopyridine and nitrite for 10 min at 37 °C prior to neutra-lisation with NaOH (typically 10 μl of 0.1 M yielded a pH stable at7–7.5). This was passed through a filter paper (#42) in order to removeresidual debris and the filtrate passed through a 0.22 μm microporefilter in order to ensure minimal absorbance that would otherwiseinterfere with the transmittance measurement. The double filtratewas immediately analysed by 2C's for SNO content and the solution cor-rected on this basis for aggregation studies. A solution of thienopyridineonly (without addition of nitrite) was treated in exactly the same wayas a control. Nitrite controlswere also prepared inwhich thienopyridinewas omitted from the preparation.

2.4. Ozone based chemiluminescence

Assay reagents (5 ml) were placed in a glass purge vessel with arubber septum covered injection inlet. Oxygen-free nitrogen gaswas bubbled through the reagent mix, heated via a water bath on athermostatically controlled hotplate. The reaction vessel, linked to atrap containing 40 ml of 1 M NaOH, was connected to the NO analyser(Sievers NOA 280i, Analytix, UK) as we have described previously(Pinder et al. 2008). In our hands these assays are sensitive to ~1 nMNO generated from nitrite with precision better than ±10% betweenrepeat plasma samples.

2.5. Nitrosothiol (SNO) detection — “2C's”

Specific SNO detection (thienopyridine–SNO) was achieved usinga cleavage reagent of cuprous (I) chloride (0.1 mM) and cysteine(0.3 mM) at pH 7.2 refluxing at 50 °C (referred to as “2C's”) as wehave described in detail previously (Pinder et al. 2008). To ensure asteady pH of 7.2, the reagents were dissolved in phosphate bufferedsaline (PBS). This was also critical because thienopyridine solutiondespite being neutralised prior to injection was efficient at acidifyingthe reagent with time when 2Cs was made up in water. Standards ofacetyl-cysteine-SNO were prepared immediately prior to analysis byadding N-acetyl cysteine (1 mM) to acidified nitrite to achieve a slightexcess of the latter. A 1 in 200 dilution was read at 334 nm (usingε=727) to calibrate acetyl-cysteine-SNO production (NAC-SNO).Typically 200 μl injections were made to produce a standard curve,and in our hands we achieve a sensitivity of ~50 nM with a precisionof ±15%. Thienopyridine–SNO formation was also confirmed by U.V.spectrophotometry and the existence of a peak at ~334 nm.

Although 2Cs is essentially specific for SNO cleavage (Fang et al.1998), in some of these studies we utilised high nitrite concentrationsin order to improve thienopyridine–SNO yield. In control studies, wefound that 2Cs is capable of detecting NO from nitrite under theseconditions (refer to detailed supplemental information) and there-fore we conducted stringent control experiments to confirm SNOdetection. Throughout the Results section we show raw traces ofappropriate nitrite controls and where SNO concentrations arequoted, the nitrite AUC is already taken into account so that thefigure represents true SNO levels. Important to state also that thekinetic of SNO detection by 2Cs in the 50–1000 nM range (peakwithinmin) is very different to the low, drawn out peak observed from nitriteat 10 mM (10,000 excess), and importantly 2C's reagent was found tobe 100-fold more sensitive to SNO over nitrite.

2.6. Preparation of BSA-SNO

Equal volumes of bovine serum albumin (30 μM) and NAC-SNO(15 mM) were incubated for 30 min at 37 °C prior to separation on aSephadex G50 column. Fractions were analysed by spectrophotometry(60–400 nm; Jenway 6705 UV/vis spectrophotometer) to detect theBSA peak maxima at 280–300 nm and simultaneously measured on

Fig. 1. Typical NO detection by ozone based chemiluminescence of thienopyridine–SNOby “2C's” reagent. Both Prasugrel SNO and Clopidogrel SNO show immediate NOrelease from SNO reflected by a rapid rise and descent in the signal current(mV) detected. Ticlopidine does not generate a typical SNO signal, and when comparedwith the nitrite control (shown in black) confirms that ticlopidine when in solutiondoes not typically form SNO. The thienopyridine solution was kept fixed at 10 mMand nitrite added in slight excess (10.1 mM). Incubation was for 10 min at 37 °C priorto neutralisation and analysis.

536 S.S. Bundhoo et al. / European Journal of Pharmacology 670 (2011) 534–540

the Nitric Oxide Analyser using the 2C's cleavage reagent to detect the –SNO moiety of the BSA-SNO product.

Fig. 3. A Thienopyridine–SNO synthesis with varying nitrite concentration (1–100 mM)illustrating different SNO yield. Ticlopidine does not form SNO even at higher nitriteconcentrations. Prasugrel forms more SNO when compared to clopidogrel but thisreaches statistical significance at nitrite concentrationsN10 mM (*** Pb0.001) (n=4 foreach drug at each time point). The thienopyridine solution was kept fixed at 10mMand incubation was for 10 min at 37 °C. B: Thienopyridine–SNO signals generated whenthienopyridine (10 mM) is mixed with low nitrite concentrations (10, 100 and 1000 μM)for 10min at 37 °C. The raw signal (black) is smoothed (shown in light grey) to showSNO release and detection from clopidogrel–SNO. Note: The NOA signal values are ona logarithmic scale to illustrate the relative magnitude of the clopidogrel–SNO signals.

2.7. Transnitrosation reactions

BSA-SNO and clopidogrel, or clopidogrel–SNO and BSA, were mixed(in concentrations with a molar excess of the recipient) for 30 min at37 °C prior to separation on a Sephadex G50 column. Fractions wereanalysed by spectrophotometry and 2Cs NOA as above. Solvent extrac-tion was also performed (chloroform:methanol:water) and the non-aqueous fraction taken and diluted for Mass Spectrometry (MS) analysison a Agilent Ion Trap 6300 mass spectrometer (Agilent Technologies)operating in positive electrospray ionisation (ESI+) mode with selectedreaction monitoring (SRM). SRM transitions used for reference were:m/z 322→212 for unchanged clopidogrel, m/z 308→198 for the car-boxyl metabolite, and m/z 356→212 for the active metabolite.

Parallel studies were also conducted adding clopidogrel–SNO toundiluted human plasma (obtained fresh from a healthy volunteer)in approximately the same molar concentrations (based on albumincontent).

Fig. 2. Typical pH dependence of thienopyridine–SNO formation. All thienopyridinesgenerated SNO in an acidic medium provided that the pH was ≤4.0. SNO synthesisby prasugrel and ticlopidine is maximal at pH 3.0 whereas clopidogrel SNO synthesisis maximal at a pH of 2.0. Data shown obtained from one parent thienopyridine aqueoussolution in each case. The thienopyridine solution was kept fixed at 10 mM and nitriteconcentration was in slight excess (10.1 mM). Incubation was for 10 min at 37 °C.

2.8. Platelet aggregation

Platelet aggregation was monitored using light transmittance in a96 well microplate assay, essentially as described in detail by Bednar

Fig. 4. The proportion of formed thienopyridine–SNO remaining over time followingincubation of thienopyridine solution (10 mM) with nitrite in slight excess(10.1 mM) at 37 °C. Ticlopidine did not generate any SNO under these conditions.Both clopidogrel and prasugrel readily formed SNO. Clopidogrel–SNO synthesis wasmaximal when mixed with nitrite for 10 min but declined thereafter (t½~66 min).Prasugrel–SNO formation was sustained over a period of 2 h (n=3 experiments foreach drug).

Fig. 5. Typical experiment showing transfer of SNO from Clopidogrel SNO (~fraction 14)to (A) purified bovine albumin or (B) albumin within human plasma (~fraction 7). Eachfraction (0.5 ml) eluted from the G50 column was assessed by spectrophotometry(280 nm) reflecting protein content (dashed line) and by 2Cs ozone basedchemiluminescence for SNO content (solid grey or solid black). Confirmation of –SNO(either albumin or clopidogrel) was also obtained by UV spectrophotometry identifyinga peak at ~334 nm, and clopidogrel±SNO was confirmed at fraction 14 by massspectrometric identification of peaks at m/z 192 and 370 (versus 356 and 341, whichwould reflect clopidogrel active metabolite and carboxyl metabolite, respectively).

Fig. 6. Light transmission aggregometry of platelet rich plasma (PRP) in response to ADP(20 μM) and different concentrations of antagonists. Nitrite is a poor platelet inhibitor(IC50=66.11±7.3 mM) whereas clopidogrel–SNO, prasugrel–SNO and S-nitroso glutathi-one (GSNO;) are potent platelet inhibitors (IC50=10.56±1.43, 7.91±1.03 and 9.80±2.28 μM respectively, P=ns) (n=4 experiments×8 concentrations per drug).

Table 1Comparisons of the chemical structures, pH of thienopyridine solutions (dissolved inwater) and presence of free thiol in ticlopidine, clopidogrel and prasugrel. n=9 reflectsdata from 3 separate preparations for each drug, and 3 replicates per experiment.

Chemical structure Thienopyridinedrug

pH ofsolution

Free thiol(nM/mM of drug)(n=9)

Ticlopidinehydrochloride

5 66.7±1.8

Clopidogrelhydrogensulphate

2 131.0±9.1

Prasugrelhydrochloride

4 873.2±50.2

537S.S. Bundhoo et al. / European Journal of Pharmacology 670 (2011) 534–540

et al. (1995). Briefly, platelet rich plasma (PRP) was isolated by cen-trifugation (180×g; 10 min) of whole blood obtained from healthysubjects collected in citrate vacutainers (Fig. 6). 3 x 108 platelets/mlwere plated in 100 μl aliquots to wells (Greiner) that had previouslybeen loaded with agonist (ADP 5 or 20 μM) and phosphate bufferedsaline/antagonist (0.3–30 μM) and immediately placed within thespectrophotometer chamber (Fluostar Optima; BMGLabtech) incubatedat 37 °C, 5 cycles each of 30 s of linear shaking (5 mm at 115 per min)and 90 s reading, and the increase in transmittance (620 nm) overtimewas expressed relative to the PRP+nitrite control. S-nitroso gluta-thione (GSNO; 10 μM) was used as a standard antagonist and reflected100% SNO yield. Thienopyridine–SNO was administered on the basis ofSNO content measured for each drug by 2C's as described above. Totalaggregation was taken at the point of maximum transmittance. Inorder to investigate differences in the rate of aggregation and inhibitionby thienopyridine–SNO/nitrite/GSNO, log transmittance datawas plottedagainst time and a linear line of best fit expressed for each experiment.Rates were calculated between the starting point and the maximumtransmittance point within each experiment.

3. Results

3.1. Conditions for optimal thienopyridine–SNO production

All thienopyridine drugs showed the presence of a free thiol whendissolved in water as measured by the ThioStar assay. Prasugrelshowed the highest amount of free thiol followed by clopidogreland ticlopidine (Table 1). Addition of water to all the thienopyridinesresulted in an acidic solution; clopidogrel solution was most acidic(pH=2), followed by prasugrel (pH=4) and ticlopidine (pH=5).

Prasugrel and clopidogrel readily formed SNO when the drug andnitrite were mixed in the acidic solution formed. Fig. 1 shows typicalNOA signals obtained from clopidogrel–SNO and prasugrel–SNO com-pared to that observed from a 1 mM nitrite control (shown in black).Under similar conditions ticlopidine produced a signal indistinguishablefrom the nitrite control, confirming ticlopidine did not form significantamounts of SNO under these conditions. Purified clopidogrel sulphateexhibited similar properties to clopidogrel within PlavixTM, readily

538 S.S. Bundhoo et al. / European Journal of Pharmacology 670 (2011) 534–540

forming clopidogrel–SNO in similar proportions. Similarly, purifiedticlopidine hydrochloride was largely ineffective at producing SNO(unless conditions of pH and substrate concentration were forced —

see also below).Thienopyridine–SNO formation was dependent on the pH of the

solution formed. An acid medium promoted SNO synthesis in all threethienopyridines provided that the pH did not exceed 4.0. Althoughticlopidine did not generate SNO when in solution (pH of 5.0),ticlopidine–SNO synthesis was observed when the pH was forcedlower. Both ticlopidine and prasugrel SNO syntheses were maximalat pH 3.0 whereas clopidogrel SNO synthesis was maximal at pH 2.0(Fig. 2).

Thienopyridine–SNO formation increased with increasing nitrite:thienopyridine molar ratio (based on mixing for 10 min with thieno-pyridine solutions at the respective pH). Both clopidogrel and prasu-grel readily formed SNO when mixed with nitrite across a broadconcentration range, although prasugrel–SNO formation was greaterthan clopidogrel–SNO reaching statistical significance at nitriteconcentrationsN10 mM (Fig. 3A). Based on these data, Prasugrel–SNO formation was up to 1% of the parent drug. In order to mimicexpected in vivo concentrations of both reactants, we conductedexperiments down to 10 μM nitrite and 10 mM thienopyridine.Raw traces showing SNO detection under these conditions arealso shown (Fig. 3B). Thienopyridine–SNO formation reached aplateau at nitrite concentrationsN10 mM. For the majority of stabilityand pH studies illustrated herein, we utilised conditions which yieldedthienopyridine–SNO with a signal:noiseN10:1 to ensure the accuracyof the area-under-curve-analysis (nitrite=10.1 mM, thienopyridine=10mM).

3.2. Stability of thienopyridine–SNO

Fig. 4 shows the relative proportions of thienopyridine–SNOremaining at times post nitrite addition to the thienopyridine solution.This reflected the balance between thienopyridine–SNO formationand decay. Clopidogrel–SNO was maximal at 10 min however therelative proportion of SNO remaining after 2 h was significantlylower when compared to prasugrel–SNO (14% v/s 117% (Pb0.001)).Prasugrel–SNO was maximal at 30 min and was sustained over aperiod of 2 h.

Ticlopidine (native solution) did not form any SNO when mixedwith nitrite over a period of 2 h under these conditions.

The pH of the incubation mixture was not adjusted throughoutthese studies and therefore reflected the pH of the initial thienopyri-dine solution. The incubation pH was attained immediately and didnot change up to 240 min. Neutralisation was only carried out imme-diately prior to 2C's analysis.

The effect of pH on thienopyridine–SNO stability was assessed incontrol experiments in which thienopyridine–SNO was synthesised,residual nitrite removed from the sample (using acidified sulphanila-mide) and the neutralised sample injected into 2Cs reagent to detectan SNO signal. Injection of the same sample into acetic acid or NaOHas a reagent did not generate NO confirming thienopyridine–SNO isrelatively stable to pH change alone.

3.3. Interaction of thienopyridine–SNO with human plasma

We demonstrate transfer of SNO moiety from clopidogrel–SNO toalbumin and also to plasma protein (primarily albumin) (Fig. 5A andFig. 5B). This is clearly indicated by the transfer of SNO from laterfractions of the G50 column (Clopidogrel–SNO) to early fractions(BSA/plasma) as confirmed by spectrophotometry and 2C's analysis.Transfer of SNO moiety from albumin–SNO to clopidogrel was alsodemonstrable.

3.4. Thienopyridine–SNO inhibits platelet aggregation

When compared to the native drug alone, Prasugrel–SNO inhibitedADP-mediated platelet aggregation in PRP more effectively thanclopidogrel–SNO. In order to account for differences in SNO yield, datawas expressed in terms of SNO dose (rather than drug concentration).Concentration response curves showed prasugrel–SNO and clopidogrel–SNO to inhibited platelet aggregation equally (IC50 7.91±1.03 μM v/s10.56±1.43 μM, mean±S.E.M., n=4) (P=0.18) and was similar tothat found for GSNO (9.79±2.28 μM). Nitrite alone exhibited relativelypoor inhibition of aggregation (66.11±7.3 mM) that is consistentwith previous studies (Loscalzo, 1985). Thienopyridine controls wereineffective at inhibiting ADP-induced platelet aggregation. The rate ofeffectiveness of thienopyridine–SNO and time taken to reach maximuminhibition of ADP-induced aggregation were also similar (7.454+/−0.109 min vs. 7.409+/−0.135 min, respectively; P=0.8) when cor-rected for SNO yield.

4. Discussion

We report the novel nitrosation properties of the antiplateletagents ticlopidine, clopidogrel and prasugrel. We have shown thatthese thienopyridine drugs form a highly acidic aqueous solutionand exhibit sufficient free thiol in solution to provide ideal conditionsfor SNO formation in the presence of NO+ (such as from nitrite)under physiological conditions. We show thienopyridine–SNOparticipates in chemical reactions in human plasma expected ofnitrosothiol-containing compounds and exhibits direct and effectiveinhibition of platelet aggregation without the need for prior in vivometabolism.

We found significant variation in the nitrosation properties of theindividual thienopyridine drugs which were dictated largely by theextent of free thiol, pH dependence and the resulting stability of theSNO produced. Prasugrel exhibited the greatest SNO production,based largely on possessingmore free thiol and stability of the resultingSNO over 2 h. Clopidogrel also readily formed SNO; despite having lessfree thiol in aqueous solution (as measured by the Thiostar reaction),the low pH of the clopidogrel solution may have favoured increasedSNO formation. However, the resulting clopidogrel–SNO showedreduced stability (t½~66 min) when compared to prasugrel–SNO(stable for 240 min). Ticlopidine did not readily form SNO when inthe solution unless the pH was artificially reduced further to inducemore favourable conditions for SNO synthesis.

Significant differences were observed between the extent of freethiol detected in the thienopyridine aqueous solutions (as measuredby Thiostar reagent) and the extent of thienopyridine–SNO formed(measured by specific cleavage of SNO). For example, the data forprasugrel shows only ~0.1% free thiol in the aqueous solution, whereason addition of 10 mMnitrite up to ~1% prasugrel–SNOwas synthesised.We concluded that this difference may be explained in terms of themethodology; the Thiostar is stable at neutral pH only and assuch can detect the level of free thiol at neutral pH. Importantly,this result confirms the existence of free thiol even in the thienopyridinesolutions — a fact not previously considered in the literature.However, this may not specifically relate to the extent or capacity forS-nitrosylation since the reaction pH could govern the availability ofsubstrate and reaction rates. Higher doses (N10 mM) of nitrite did notyield proportionate increases in thienopyridine–SNO. This could betaken to reflect saturation of available thiol since nitrite in acid shouldyield proportionate and excess NO availability under these conditions.

The biochemistry and properties of S-nitrosothiols are welldocumented (Hogg et al. 1996; Padgett and Whorton, 1995). Thepotential for donating NO+ groups (that essentially possesses allthe properties of native NO) results in potent anti-aggregatory andvasodilatory properties under appropriate conditions (Loscalzo,1985, Megson et al. 2000). Here, we show that thienopyridine–SNO

539S.S. Bundhoo et al. / European Journal of Pharmacology 670 (2011) 534–540

exhibit potent anti-aggregatory capacity. Prasugrel–SNO yield wasgreater than clopidogrel–SNO under similar substrate conditions.When assessed on the basis of SNO content however, the capacityto inhibit platelet aggregation was similar and not significantlydifferent from a standard such as GSNO. Prasugrel or clopidogrel(without SNO) were both ineffective inhibitors of aggregation,whereas we found nitrite to be a relatively poor inhibitor of plateletaggregation in agreement with previous studies (Megson et al. 2000).

In addition to direct effects on platelets, nitrosothiol activationand deactivation of signal proteins are emerging as an importantmechanism of redox control of cell function (Lander, 1996). Transni-trosation between nitrosothiols and reduced thiol groups is also likelyimportant to the way thiol redox regulates signal transduction.Protein nitrosothiols and the complexes they form with haem/Fe2+,termed dinitrosothiol iron complexes (Keese et al. 1997) prolongthe biological half life of NO by providing a reservoir or a pool ofNO. The discovery of albumin–SNO and other protein–SNO inhuman blood and tissues (Stamler et al., 1988, 1992) ignited a fieldof research spanningmore than15 years demonstrating the physiologicalsignificance of this SNO reserve in human physiology and patho-physiology (Stamler et al. 1993). Based on our laboratory findings,thienopyridine–SNOmay participate in similar reactions and contributeto this store.

The relevance of thienopyridine–SNO formation in humans is asyet unknown. Certainly physiological conditions ideally match thelaboratory studieswe showhere. Oral loading of clopidogrel (typically600 mg) prior to coronary intervention is bound for reaction withnitrite in the saliva and stomach where concentrations are typically20 μM and 0.6 μM, respectively (McKnight et al. 1997). Furthermore,the acidic environment of the stomach (~30 ml typicallybpH 4) inaddition to the acidic thienopyridine solution (~50–60 mM) couldfavour nitrosothiol formation. It is noteworthy that we show invitro thienopyridine–SNO formation with nitrite and thienopyridineconcentrations lower than this (10 μM and 10 mM respectively).Direct measurement of thienopyridine–SNO in patient plasmawould be ideal but is likely to be fraught with the practical difficultyassociatedwithmeaningful detection of themultitude of thienopyridinemetabolites, complicated by SNO chemistry. In addition, transnitrosationreactions (as shown here) may hinder or enhance absorption fromthe stomach and it may be necessary to measure the total RSNO poolin tissue and plasma in order to gain accurate insight.

The importance of thienopyridine–SNO in terms of biologicaleffectiveness of the drug in vivo is also speculative, yet it is noteworthythat these reactions occur with the parent drug without need for priorin vivo metabolism. The effectiveness of thienopyridines in patientshas largely been discussed in terms of variation in patient metabolismand activation of the drug to its active form, yet the potential for directthienopyridine–SNO formation could be important. In terms of the“ideal” conditions for SNO production, it is also interesting that theco-administration of some proton pump inhibitors to patients (suchas omeprazole) has also been shown to reduce thienopyridine efficacyon platelet function (Gilard et al., 2008; Gurbel et al. 2008), a findingthat to date has been attributed to competition for p450 cytochromemetabolism (Fock et al. 2008; Li et al. 2004) but could clearly influenceSNO formation by neutralising stomach pH. Remarkably, the extent ofactive thienopyridine found at peak in human plasma following oralloading is ~0.01% of the inactive drug present at the same time, yetthis is sufficient to produce irreversible clinically relevant inhibition ofplatelet activation. Based on the data we have shown, ~1% of the parentdrug can potentially be nitrosated, which could amount to significantlevels systemically.

5. Conclusion

Clinical grade thienopyridine drugs exhibit free thiol and form anacidic solution, ideal conditions for thienopyridine nitrosothiol

production. Prior metabolism of the drug is not necessary and thenitrosothiol formed exhibits potent and direct anti-platelet activity.Furthermore, the formation of thienopyridine–SNO may not affectthe ability of thienopyridine to irreversibly bind P2Y12 receptors.Indeed the targeted delivery of thienopyridine and simultaneousdelivery of SNO to platelets may point to a dual antiplatelet mechanismand may explain some of the vasomodulatory effects previouslyobserved (Heitzer et al. 2006; Jakubowski et al. 2005; Warnholtzet al. 2008).

Conflict of interest

None.

Acknowledgements

S.B. was funded by a Clinical Research Fellowship, Cardiff and ValeUniversity Health Board. A.G.P. was supported by a British HeartFoundation Studentship.

Appendix A. Supplementary data

Supplementary data to this article can be found online at doi:10.1016/j.ejphar.2011.09.022.

References

Al-Sa'doni, H.H., Khan, I.Y., Poston, L., Fisher, I., Ferro, A., 2000. A novel family of S-nitro-sothiols: chemical synthesis and biological actions. Nitric Oxide 4, 550–560.

Bednar, B., Condra, C., Gould, R.J., Connolly, T.M., 1995. Platelet aggregation monitoredin a 96 well microplate reader is useful for evaluation of platelet agonists and an-tagonists. Thromb. Res. 77, 453–463.

Borja Ibanez, G.V., Juan, J., Badimon, J.J., 2006. Pharmacology of thienopyridines: ratio-nale for dual pathway inhibition. Eur. Heart J. 8 (Suppl.), G3–G9.

Fang, K., Ragsdale, N.V., Carey, R.M., MacDonald, T., Gaston, B., 1998. Reductive assaysfor S-nitrosothiols: implications for measurements in biological systems. Biochem.Biophys. Res. Commun. 252, 535–540.

Farid, N.A., Kurihara, A., Wrighton, S.A., 2010. Metabolism and disposition of the thie-nopyridine antiplatelet drugs ticlopidine, clopidogrel, and prasugrel in humans.J. Clin. Pharmacol. 50, 126–142.

Fock, K.M., Ang, T.L., Bee, L.C., Lee, E.J., 2008. Proton pump inhibitors: do differences inpharmacokinetics translate into differences in clinical outcomes? Clin. Pharmaco-kinet. 47, 1–6.

Gilard, M., Arnaud, B., Cornily, J.C., Le Gal, G., Lacut, K., Le Calvez, G., Mansourati, J., Mot-tier, D., Abgrall, J.F., Boschat, J., 2008. Influence of omeprazole on the antiplateletaction of clopidogrel associated with aspirin: the randomized, double-blind OCLA(Omeprazole CLopidogrel Aspirin) study. J. Am. Coll. Cardiol. 51, 256–260.

Gurbel, P.A., Lau, W.C., Tantry, U.S., 2008. Omeprazole: a possible new candidateinfluencing the antiplatelet effect of clopidogrel. J. Am. Coll. Cardiol. 51, 261–263.

Hall, P., Nakamura, S., Maiello, L., Itoh, A., Blengino, S., Martini, G., Ferraro, M., Colombo,A., 1996. A randomized comparison of combined ticlopidine and aspirin therapyversus aspirin therapy alone after successful intravascular ultrasound-guidedstent implantation. Circulation 93, 215–222.

Heitzer, T., Rudolph, V., Schwedhelm, E., Karstens, M., Sydow, K., Ortak, M.,Tschentscher, P., Meinertz, T., Böger, R., Baldus, S., 2006. Clopidogrel improves sys-temic endothelial nitric oxide bioavailability in patients with coronary artery dis-ease: evidence for antioxidant and antiinflammatory effects. Arterioscler.Thromb. Vasc. Biol. 26, 1648–1652.

Hogg, N., 2000. Biological chemistry and clinical potential of S-nitrosothiols. Free Radic.Biol. Med. 28, 1478–1486.

Hogg, N., Singh, R.J., Kalyanaraman, B., 1996. The role of glutathione in the transportand catabolism of nitric oxide. FEBS Lett. 382, 223–228.

Jakubowski, A., Chlopicki, S., Olszanecki, R., Jawien, J., Lomnicka, M., Dupin, J.P., Grygle-wski, R.J., 2005. Endothelial action of thienopyridines and thienopyrimidinones inthe isolated guinea pig heart. Prostaglandins Leukot. Essent. Fatty Acids 72,139–145.

Kazui, M., Nishiya, Y., Ishizuka, T., Hagihara, K., Farid, N.A., Okazaki, O., Ikeda, T., Kuri-hara, A., 2010. Identification of the human cytochrome P450 enzymes involved inthe two oxidative steps in the bioactivation of clopidogrel to its pharmacologicallyactive metabolite. Drug Metab. Dispos. 38, 92–99.

Keese, M.A., Bose, M., Mulsch, A., Schirmer, R.H., Becker, K., 1997. Dinitrosyl–dithiol–iron complexes, nitric oxide (NO) carriers in vivo, as potent inhibitors of humanglutathione reductase and glutathione-S-transferase. Biochem. Pharmacol. 54,1307–1313.

Lander, H.M., 1996. Cellular activation mediated by nitric oxide. Methods 10, 15–20.Li, X.Q., Andersson, T.B., Ahlstrom, M., Weidolf, L., 2004. Comparison of inhibitory ef-

fects of the proton pump-inhibiting drugs omeprazole, esomeprazole,

540 S.S. Bundhoo et al. / European Journal of Pharmacology 670 (2011) 534–540

lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activi-ties. Drug Metab. Dispos. 32, 821–827.

Loscalzo, J., 1985. N-Acetylcysteine potentiates inhibition of platelet aggregation by ni-troglycerin. J. Clin. Investig. 76, 703–708.

McKnight, G.M., Smith, L.M., Drummond, R.S., Duncan, C.W., Golden, M., Benjamin, N.,1997. Chemical synthesis of nitric oxide in the stomach from dietary nitrate inhumans. Gut 40, 211–214.

Mega, J.L., Close, S.L., Wiviott, S.D., Shen, L., Hockett, R.D., Brandt, J.T., Walker, J.R., Antman,E.M.,Macias,W.L., Braunwald, E., Sabatine,M.S., 2009. Cytochrome P450 genetic poly-morphisms and the response to prasugrel: relationship to pharmacokinetic, pharma-codynamic, and clinical outcomes. Circulation 119, 2553–2560.

Megson, I.L., Sogo, N., Mazzei, F.A., Butler, A.R., Walton, J.C., Webb, D.J., 2000. Inhibitionof human platelet aggregation by a novel S-nitrosothiol is abolished by haemoglo-bin and red blood cells in vitro: implications for anti-thrombotic therapy. Br. J.Pharmacol. 131, 1391–1398.

Mehta, S.R., Yusuf, S., Peters, R.J., Bertrand, M.E., Lewis, B.S., Natarajan, M.K., Malmberg,K., Rupprecht, H., Zhao, F., Chrolavicius, S., Copland, I., Fox, K.A., 2001. Effects ofpretreatment with clopidogrel and aspirin followed by long-term therapy in pa-tients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet358, 527–533.

Padgett, C.M., Whorton, A.R., 1995. Regulation of cellular thiol redox status by nitricoxide. Cell Biochem. Biophys. 27, 157–177.

Pinder, A.G., Rogers, S.C., Khalatbari, A., Ingram, T.E., James, P.E., 2008. The measure-ment of nitric oxide and its metabolites in biological samples by ozone-basedchemiluminescence. Meth. Mol. Biol. 476, 11–28.

Rehmel, J.L., Eckstein, J.A., Farid, N.A., Heim, J.B., Kasper, S.C., Kurihara, A., Wrighton,S.A., Ring, B.J., 2006. Interactions of two major metabolites of prasugrel, a thie-nopyridine antiplatelet agent, with the cytochromes P450. Drug Metab. Dispos.34, 600–607.

Stamler, J.S., Cunningham, M., Loscalzo, J., 1988. Reduced thiols and the effect of intra-venous nitroglycerin on platelet aggregation. Am. J. Cardiol. 62, 377–380.

Stamler, J.S., Simon, D.I., Osborne, J.A., Mullins, M.E., Jaraki, O., Michel, T., Singel, D.J.,Loscalzo, J., 1992. S-nitrosylation of proteins with nitric oxide: synthesis and char-acterization of biologically active compounds. Proc. Natl Acad. Sci. USA 89,444–448.

Stamler, J.S., Osborne, J.A., Jaraki, O., Rabbani, L.E., Mullins, M., Singel, D., Loscalzo, J.,1993. Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J. Clin. Investig. 91, 308–318.

Top Ten Global Products, 2007. IMS Midas Monthly. IMS Health Inc., London (UK).December 2007. Available online: www.imshealth.com/deployedfiles/imshealth/Global/Content/StaticFile/Top_Line_Data/Top10GlobalProducts.pdf.

Warnholtz, A., Ostad, M.A., Velich, N., Trautmann, C., Schinzel, R., Walter, U., Munzel, T.,2008. A single loading dose of clopidogrel causes dose-dependent improvement ofendothelial dysfunction in patients with stable coronary artery disease: results of adouble-blind, randomized study. Atherosclerosis 196, 689–695.

Wiviott, S.D., Braunwald, E., McCabe, C.H., Montalescot, G., Ruzyllo, W., Gottlieb, S.,Neumann, F.J., Ardissino, D., De Servi, S., Murphy, S.A., Riesmeyer, J., Weerakkody,G., Gibson, C.M., Antman, E.M., TRITON-TIMI 38 Investigators, 2007. Prasugrel ver-sus clopidogrel in patients with acute coronary syndromes. N Engl J. Med. 357,2001–2015.

Yang, L.H., Fareed, J., 1997. Vasomodulatory action of clopidogrel and ticlopidine.Thromb. Res. 86, 479–491.

Yang, L.H., Hoppensteadt, D., Fareed, J., 1998. Modulation of vasoconstriction by clopi-dogrel and ticlopidine. Thromb. Res. 92, 83–89.

Yoneda, K., Iwamura, R., Kishi, H., Mizukami, Y., Mogami, K., Kobayashi, S., 2004. Iden-tification of the active metabolite of ticlopidine from rat in vitro metabolites. Br. J.Pharmacol. 142, 551–557.