evaluation of clopidogrel conjugation metabolism: pk...

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1521-009X/44/9/14901497$25.00 http://dx.doi.org/10.1124/dmd.116.071092 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:14901497, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Evaluation of Clopidogrel Conjugation Metabolism: PK Studies in Man and Mice of Clopidogrel Acyl Glucuronide Simona Nicoleta Savu, Luigi Silvestro, Mariana Surmeian, Lina Remis, Yuksel Rasit, Simona Rizea Savu, and Constantin Mircioiu Department of Biopharmacy, Faculty of Pharmacy, University of Medicine and Pharmacy Carol Davila,Bucharest, Romania (S.N.S., C.M.); 3S-Pharmacological Consultation and Research GmbH, Harpstedt, Germany (S.N.S, L.S., S.R.S.); Pharma Serv International SRL, Bucharest, Romania (M.S.); Clinical Hospital of the Ministry of Health of the Moldavian Republic, Chisinau, The Moldavian Republic (L.R.); Pharmacology Department, National Institute for Chemical Pharmaceutical Research and Development (ICCF), Bucharest, Romania (Y.R.) Received April 28, 2016; accepted July 8, 2016 ABSTRACT The existence of a glucuronide conjugate of the major circulating clopidogrel metabolites, called clopidogrel acyl glucuronide (CAG), is already known. However, information regarding its pharmacokinetics (PK), metabolism, and clearance are modest. We investigated in vivo the potential CAG trans-esterification to clopidogrel (reaction occur- ring in vitro in particular conditions) by administering the metabolite to mice. Experiments were then carried out on men, clopidogrel administered alone or followed by activated charcoal intake (intestinal reabsorption blockade). Study objectives included: PK comparison of CAG, clopidogrel carboxylic acid (CCA), and clopidogrel in plasma, determination of their elimination patterns in urine and feces, and tracking of charcoal-induced changes in PK and/or urinary excretion that would indicate relevant enterohepatic recycling of CAG. In mice, CAG was rapidly hydrolyzed to CCA after oral administration, whereas by intravenous route metabolic conversion to CCA was delayed. No levels of clopidogrel were detected in mice plasma, excluding any potential trans-esterification or other form of back-conversion in vivo. PK experiments in man showed that CAG is hydrolyzed in the gastrointestinal tract (very low concentrations in feces), but there is no evidence of enterohepatic recirculation. Quantitation of the three moieties in stool samples accounted for only 1.2% of an administered dose, suggesting that other yet unknown metabolites/degradation products formed through metabolic processes and/or the activity of local microflora are mainly excreted by this route. In man CAG was confirmed as one of the major terminal metabolites of clopidogrel, with a PK behavior similar to CCA. Introduction Glucuronide conjugates represent one of the major types of phase II metabolites of xenobiotics. Since generally the biologic function of the aglycone is abolished by glucuronidation, conjugates are often consid- ered as metabolites of modest interest; however, a few compelling cases in which glucuronides maintain/increase the biologic function of their parent compound (Barua and Sidell, 2004; Ohno et al., 2008) suggest that further inquiry into their metabolic fate is warranted. In the particular case of clopidogrel, although the oxidative metab- olism is quite well known, the conjugative metabolism has not been studied in detail. In terms of phase I metabolism, it is known that two oxidative steps, mediated by multiple P450 cytochromes, are required for the conversion of clopidogrel to its active metabolite (Savi et al., 2000; Kazui et al., 2010). Interestingly, activation by the P450 system is rate-limited and ultimately a quantitatively minor metabolic pathway. In parallel, about 85% of the drug released from dosage form is converted to clopidogrel carboxylic acid (CCA) (von Beckerath et al., 2005; Ksycinska et al., 2006), which is subsequently conjugated to clopidogrel acyl glucuronide (CAG) (Silvestro et al., 2010)a quantitatively important metabolite that has not been studied in detail until now (see Fig. 1, schematic representation of clopidogrel metabolism). Although in vivo reactivity of CAG in particular remains to be clarified, it should be noted that acyl glucuronides of carboxylic acids are a class of conjugates generally prone to hydrolysis, molecular rearrangements, and interactions with cellular target molecules by covalent bindings (Ritter, 2000). So far, only binding to CYP2C8 has been demonstrated for CAG (Tornio et al., 2014), and it is unknown if the metabolite undergoes any type of metabolic conversion before being excreted from the human body. In vitro, reactivity of CAG has already been demonstrated. It was shown that in specific conditions it converts to parent clopidogrel by trans-esterification (Silvestro et al., 2011), a reaction sometimes occur- ring also during metabolic processes (Boyer and Petersen, 1992; Knights et al., 2000; Celli et al., 2007; Fujino et al., 2014). Should CAG contribute in vivo to any process resulting in back- conversion to clopidogrel, the amount reconstituted could be consider- able, because the exposure to CAG in man [on the basis of area under the curve from time zero to infinity (AUC 0inf )] is 500 times higher than that of clopidogrel (Silvestro et al., 2013)]; furthermore, the newly formed clopidogrel would be again available for metabolism by P450s and thus partly converted to the active metabolite. Although it is clear that the This work received financial support through the project entitled CEROCareer profile: Romanian Researcher, cofinanced by the European Social Fund for Sectoral Operational Programme Human Resources Development 20072013 [POSDRU/159/1.5/S/135760]. dx.doi.org/10.1124/dmd.116.071092. ABBREVIATIONS: AUC, area under the curve; CAG, clopidogrel acyl glucuronide; P450, cytochrome P450; HPLCMS/MS, high-performance liquid chromatographytandem mass spectrometry; K 2 EDTA, di-potassium ethylenediaminetetraacetic acid; PK, pharmacokinetics. 1490 at ASPET Journals on March 6, 2020 dmd.aspetjournals.org Downloaded from

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Page 1: Evaluation of Clopidogrel Conjugation Metabolism: PK ...dmd.aspetjournals.org/content/dmd/44/9/1490.full.pdf · Evaluation of Clopidogrel Conjugation Metabolism: PK Studies in Man

1521-009X/44/9/1490–1497$25.00 http://dx.doi.org/10.1124/dmd.116.071092DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1490–1497, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Evaluation of Clopidogrel Conjugation Metabolism: PK Studies inMan and Mice of Clopidogrel Acyl Glucuronide

Simona Nicoleta Savu, Luigi Silvestro, Mariana Surmeian, Lina Remis, Yuksel Rasit,Simona Rizea Savu, and Constantin Mircioiu

Department of Biopharmacy, Faculty of Pharmacy, University of Medicine and Pharmacy “Carol Davila,” Bucharest, Romania (S.N.S.,C.M.); 3S-Pharmacological Consultation and Research GmbH, Harpstedt, Germany (S.N.S, L.S., S.R.S.); Pharma Serv InternationalSRL, Bucharest, Romania (M.S.); Clinical Hospital of the Ministry of Health of the Moldavian Republic, Chisinau, The MoldavianRepublic (L.R.); Pharmacology Department, National Institute for Chemical Pharmaceutical Research and Development (ICCF),

Bucharest, Romania (Y.R.)

Received April 28, 2016; accepted July 8, 2016

ABSTRACT

The existence of a glucuronide conjugate of the major circulatingclopidogrel metabolites, called clopidogrel acyl glucuronide (CAG), isalready known. However, information regarding its pharmacokinetics(PK), metabolism, and clearance are modest. We investigated in vivothe potential CAG trans-esterification to clopidogrel (reaction occur-ring in vitro in particular conditions) by administering the metaboliteto mice. Experiments were then carried out on men, clopidogreladministeredalone or followed byactivated charcoal intake (intestinalreabsorption blockade). Study objectives included: PK comparison ofCAG, clopidogrel carboxylic acid (CCA), and clopidogrel in plasma,determination of their elimination patterns in urine and feces, andtracking of charcoal-induced changes in PK and/or urinary excretionthat would indicate relevant enterohepatic recycling of CAG. In mice,

CAGwas rapidly hydrolyzed toCCAafter oral administration,whereasby intravenous route metabolic conversion to CCA was delayed. Nolevels of clopidogrel were detected in mice plasma, excluding anypotential trans-esterification or other form of back-conversion in vivo.PK experiments in man showed that CAG is hydrolyzed in thegastrointestinal tract (very low concentrations in feces), but there isno evidence of enterohepatic recirculation. Quantitation of the threemoieties in stool samples accounted for only 1.2%of an administereddose, suggesting that other yet unknown metabolites/degradationproducts formed through metabolic processes and/or the activity oflocal microflora are mainly excreted by this route. In man CAG wasconfirmed as one of the major terminal metabolites of clopidogrel,with a PK behavior similar to CCA.

Introduction

Glucuronide conjugates represent one of the major types of phase IImetabolites of xenobiotics. Since generally the biologic function of theaglycone is abolished by glucuronidation, conjugates are often consid-ered as metabolites of modest interest; however, a few compelling casesin which glucuronides maintain/increase the biologic function of theirparent compound (Barua and Sidell, 2004; Ohno et al., 2008) suggestthat further inquiry into their metabolic fate is warranted.In the particular case of clopidogrel, although the oxidative metab-

olism is quite well known, the conjugative metabolism has not beenstudied in detail. In terms of phase I metabolism, it is known that twooxidative steps, mediated by multiple P450 cytochromes, are requiredfor the conversion of clopidogrel to its active metabolite (Savi et al.,2000; Kazui et al., 2010). Interestingly, activation by the P450 system israte-limited and ultimately a quantitatively minor metabolic pathway. Inparallel, about 85% of the drug released from dosage form is convertedto clopidogrel carboxylic acid (CCA) (von Beckerath et al., 2005;

Ksycinska et al., 2006), which is subsequently conjugated to clopidogrelacyl glucuronide (CAG) (Silvestro et al., 2010)—a quantitativelyimportant metabolite that has not been studied in detail until now (seeFig. 1, schematic representation of clopidogrel metabolism).Although in vivo reactivity of CAG in particular remains to be

clarified, it should be noted that acyl glucuronides of carboxylic acidsare a class of conjugates generally prone to hydrolysis, molecularrearrangements, and interactions with cellular target molecules bycovalent bindings (Ritter, 2000). So far, only binding to CYP2C8 hasbeen demonstrated for CAG (Tornio et al., 2014), and it is unknownif the metabolite undergoes any type of metabolic conversion beforebeing excreted from the human body.In vitro, reactivity of CAG has already been demonstrated. It was

shown that in specific conditions it converts to parent clopidogrel bytrans-esterification (Silvestro et al., 2011), a reaction sometimes occur-ring also during metabolic processes (Boyer and Petersen, 1992; Knightset al., 2000; Celli et al., 2007; Fujino et al., 2014).Should CAG contribute in vivo to any process resulting in back-

conversion to clopidogrel, the amount reconstituted could be consider-able, because the exposure to CAG inman [on the basis of area under thecurve from time zero to infinity (AUC0–inf)] is 500 times higher than thatof clopidogrel (Silvestro et al., 2013)]; furthermore, the newly formedclopidogrel would be again available for metabolism by P450s and thuspartly converted to the active metabolite. Although it is clear that the

This work received financial support through the project entitled “CERO—Careerprofile: Romanian Researcher”, cofinanced by the European Social Fund forSectoral Operational Programme Human Resources Development 2007–2013[POSDRU/159/1.5/S/135760].

dx.doi.org/10.1124/dmd.116.071092.

ABBREVIATIONS: AUC, area under the curve; CAG, clopidogrel acyl glucuronide; P450, cytochrome P450; HPLC–MS/MS, high-performanceliquid chromatography–tandem mass spectrometry; K2EDTA, di-potassium ethylenediaminetetraacetic acid; PK, pharmacokinetics.

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confirmation of such a pathway could only provide mechanistic insight(quantitative data on clopidogrel and its active metabolite is alreadyavailable in literature), the disposition of CAGwas considered importantknowledge to be gained, as any yet unknown intermediate reaction couldprove useful in understanding the large pharmacokinetic (PK) variabilityof clopidogrel and its active moiety.Rationale and Study Objectives. The present studies represent a

follow-up to previous work from which we reported the existence ofCAG and described its in vitro back-conversion to clopidogrel by trans-esterification (Silvestro et al., 2011). The main questions to clarify noware, “Can this happen by any means in vivo also?” and “What is themetabolic fate of this conjugate?”.First, in the absence of a CAG standard suitable for administration to

humans, we conducted a study in mice to determine if this metabolitemay back-convert to clopidogrel parent by trans-esterification or anotherreaction of the conjugated metabolite; the study was conducted on mice(C57BL) that have a similar glucuronidase tissue distribution to that ofman (Gad et al., 2007).Another important aspect to clarify was whether CAG undergoes

enterohepatic recycling, since mass balance studies conducted withradiolabeled clopidogrel in man (Lins et al., 1999) showed that recyclingoccurs, although themoieties involvedwere not identified. Plasma levelsof clopidogrel and its two main metabolites were compared in healthyvolunteers treated with clopidogrel alone or in combination withactivated charcoal; this bile-binding agent was administered accordingto a regimen designed to disrupt enterohepatic recycling, as alreadydescribed in literature (Elomaa et al., 2001; Wang et al., 2014) and haveminimal impact on clopidogrel absorption.In view of a more comprehensive understanding of their metabolism,

the determination of the main excretion route (urine and/or feces) forclopidogrel, CCA (as precursors) and CAG was also a set objective ofthe single -dose charcoal-interaction study in man.It is noteworthy that a human study was preferred owing to the

complex nature of the physiologic processes studied through PKdeterminations and the consideration that data gathered in any othermodel would be extremely difficult to extrapolate, raising concerns ofrelevance to a real clinical setting.

Materials and Methods

Standards, Reagents, and Medication

For the preparation of solutions for oral and intravenous administration inmice,clopidogrel acyl-b-D-glucuronide standards of adequate purity were purchasedfrom Toronto Research Chemicals (Toronto, Canada).

The internal standards used for high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) analytical determinations were:d3-clopidogrel hydrogen sulfate (SynFine Research, RichmondHill, ON, Canada),clopidogrel acyl-b- D-glucuronide (Toronto Research Chemicals Inc.), and13C6-clopidogrel carboxylic acid (Alsachim, Illkirch-Graffenstaden, France).Commercially available reagents of analytical grade purity were used forsample processing.

Plavix 75 mg tablets (Sanofi, Paris, France) from a commercial batch (AY171)were used. Medical-grade activated charcoal was also procured from the market(from Silcarbon Aktivkohle GmbH, Kirchhundem, Germany).

Intravenous and Oral Pharmacokinetics Study in Mice

Study Design and Sample Collection. All procedures used were inaccordance with the standards set forth in the eighth edition of Guide for theCare and Use of Laboratory Animals (National Academy of Sciences, TheNational Academies Press, Washington D.C.). Laboratory animals (C57BL/6male mice, weighing 206 4g, 256 1 day of age) were bred, raised, and cared forat the Cantacuzino National Institute of Research-Development for Microbiologyand Immunology (NIRDMIC) located in Bucharest, Romania. The experimentalpart was carried out in the Pharmacology Department of the National Institutefor Chemical Pharmaceutical Research and Development (ICCF) located inBucharest, Romania. The study was conducted according to a parallel design onan overall sample size of 71 laboratory animals (five per sampling point after eachmode of administration plus six animals treated with normal saline only in viewof obtaining blank plasma for preparation of analytical quality control samples).Animals randomized to the treatment arms, received in sterile conditions a dose of200 ml freshly prepared solution of 1.25 mg/ml clopidogrel acyl glucuronide innormal saline, either per os (through gavage) or intravenously, via tail-veininjection. Blood samples (150 ml) were collected in prechilled tubes containingdi-potassium ethylenediaminetetraacetic acid (K2EDTA) at 0.5, 1, 2, 4, 6, and8 hours after oral dosing or at 0.25, 0.5, 1, 2, 4, 6, and 8 hours after intravenousadministration. The samples were immediately immersed in water and icebath until centrifugation (performed at a nominal temperature of 4�C, 1500g fora duration of 10 minutes). The separated plasma was frozen at 270�C and

Fig. 1. Representation of clopidogrel metabolism.

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maintained at this temperature until analyzed. For sample processing and analysiswe used a slight modification of a method already published (Silvestro et al.,2011), as described below.

Extraction of Clopidogrel, Clopidogrel Carboxylic Acid, and ClopidogrelAcyl Glucuronide from Mice Plasma Samples. Plasma thawing was done onwet ice. Aliquots of 100 ml from postdose mice plasma samples were diluted with200 ml of ice-cold acetonitrile, spiked with 20 ml of internal standard mix inacetonitrile (d3-clopidogrel hydrogen sulfate, clopidogrel acyl-b-D-glucuronide,and 13C6-clopidogrel carboxylic acid, 200 ng/ml), vortexed for 3 minutes, andthen centrifuged for 5 minutes at 4000 rpm and 8�C. Supernatants (100 ml) werediluted with 100 ml ice-cold water containing 2% acetonitrile and 0.1% formicacid. The extracts were analyzed as described in the next paragraph.

Clopidogrel, Clopidogrel Carboxylic Acid, and Clopidogrel AcylGlucuronide Quantification. Six-point calibration curves were prepared in blankmice plasma (K2EDTA as anticoagulant) with concentrations ranging from 0.01 to100.00 ng/ml for clopidogrel and from 1.00 to 10,000.00 ng/ml for clopidogrel acylglucuronide and clopidogrel carboxylic acid. The quality control and calibrationcurve samples were also spiked with internal standard mix in acetonitrile(d3-clopidogrel hydrogen sulfate, clopidogrel acyl-b-D-glucuronide, and13C6-clopidogrel carboxylic acid, 200 ng/ml) and subsequently extracted inthe same manner described previously for study samples. Calibration curvesand quality control samples (three concentration levels and in triplicate)were analyzed during each analytical sequence. Decisions regarding theacceptance of sequences were taken according to well-established bio-analytical rules (FDA, 2013; EMA, 2011). No sequences had to be rejectedowing to quality control or calibration failure.

Human Oral Pharmacokinetics and Elimination Study

Study Design and Sample Collection. Six subjects were enrolled andcompleted the human PK and elimination study. Study population comprisedthree male and three nonpregnant, nonlactating female volunteers, 18 to 51 yearsold (mean age 32.17 6 14.48). The study was conducted at the Clinical Hospitalof the Ministry of Health of the Moldavian Republic located in Chisinau. Thestudy protocol was reviewed and approved by an Institutional Ethics Committeeand all 6 subjects enrolled were informed about the study medication andprocedures and gave consent for the participation in the study. Clinicalinvestigations were conducted according to the Declaration of Helsinkiprinciples and the medication administered consisted of a single oral dose ofreference-listed drug (Plavix 75 mg, procured from the market) per studyperiod. The design was two-way crossover: in one study period the subjectsreceived just clopidogrel and in the other they received clopidogrel plus aregimen consisting of 20 g activated charcoal suspended in 240 ml of water,given at 6.0, 12.0, 24.0, 36.0, 48.0 and 60.0 hours after dosing. Blood samples(4 ml) for the quantification of parent clopidogrel, clopidogrel acyl glucuro-nide, and clopidogrel carboxylic acid in plasma were collected in prechilledtubes containing K2EDTA as anticoagulant, at 1.0, 2.0, 6.0, 9.0, 24.0, 36.0,48.0, and 72.0 hours after dosing.

In the same study, urine was collected in both study periods up to 72 hourspostdose, and fecal matter was collected over the same interval but only whenclopidogrel was given without activated charcoal (as previous experience suggestedthat the presence of charcoal in stool samples would lead to ambiguous results).

Extraction of Metabolites from Biologic Samples. Before analysis, plasmasamples were thawed on wet ice, and 100-ml aliquots were spiked with 20 ml ofsolution of internal standard, which contained 200 ng/ml d3-clopidogrel hydrogensulfate, 200 ng/ml clopidogrel acyl-b-D-glucuronide, and 200 ng/ml 13C6-clopidogrel carboxylic acid in acetonitrile, and then diluted with 200 ml ice-cold acetonitrile. Afterward they were vortex for 3 minutes and centrifuged at4000 rpm and 8�C for 5 minutes. Supernatants (100 ml) were diluted with 100 mlice-cold water containing 2% acetonitrile and 0.1% formic acid.

Urine samples were collected during the time intervals 0–12 , 12–24 , 24–36,36–48, 48–60, and 60–72 hours postdose. The volume of each fresh urine samplewas measured and 50-ml aliquots were mixed with 100 ml of acetic acid 99.8%,vortexed for 2 minutes, and frozen at –20�C. To obtain a single representativeurinary excretion sample for each time interval, aliquots from individual sampleswere mixed in appropriate proportions according to initial sample volume. Beforeanalysis, samples (100 ml) were thawed on wet ice, spiked with 20 ml of internalstandard mix in acetonitrile (d3-clopidogrel hydrogen sulfate, clopidogrel acyl-

b-D-glucuronide, and 13C6-clopidogrel carboxylic acid 200 ng/ml), and then dilutedwith 200 ml ice-cold acetonitrile. Afterward they were vortexed for 3 minutes andthen centrifuged for 5 minutes at a nominal temperature of 8�C, with a speed of4000 rpm. A volume of 100 ml supernatant was separated and diluted with 100 mlice-cold water containing 2% acetonitrile and 0.1% formic acid.

Fresh fecal matter samples were frozen for storage at –20�C. Before analysis,samples were thawed on wet ice, weighed, and then diluted 1:10 (w/v) with anice-cold solution containing 50% acetonitrile and 1% formic acid, as follows:Samples were first vortexed for 2 minutes with one-fifth of the calculated volumeof the above solution for dilution and 250 mg of glass beads per gram of sample.The remaining volume of the solution was then added and the samples werevortexed again for 3 minutes and centrifuged at 4000 rpm and 8�C for 10 minutes.A volume of 100 ml of supernatant was recovered and processed in the samemanner as previously described for thawed urine samples.

Clopidogrel, Clopidogrel Carboxylic Acid, and Clopidogrel AcylGlucuronide Quantification. Six-point calibration curves were prepared inappropriate matrix (in blank plasma, blank urine, or blank fecal matter samples thatwere spiked with internal standard, processed, and diluted according to the sameprotocol previously described for study samples). The concentration ranges of thecalibration curveswere 0.01–100.00 ng/ml for clopidogrel and 1.00–10,000.00 ng/mlfor clopidogrel acyl glucuronide and clopidogrel carboxylic acid. Calibration curvesand quality control samples (three concentration levels in triplicate) were analyzedduring each analytical sequence. Decisions regarding the acceptance of sequenceswere made according to well-established bioanalytical rules (EMA, 2011; FDA,2013). No sequences had to be rejected owing to quality control or calibration failure.

HPLC–MS/MS Analysis. For the analytical determinations we used an HPLCbinary gradient (LC-20 AD chromatographic pumps) by Shimadzu-Japan with aCTC-PAL autosampler (model HTS) manufactured by CTC Analytics (Zwingen,Switzerland). The HPLC system was coupled with a triple quadrupole mass-spectrometer model API 5000 (mice PK samples) or API 6500 (human PK andelimination samples) with an atmospheric pressure electrospray ionization source(model TurboIonSpray), all manufactured by Applied Biosystems/MDS SCIEX(Concord, ON, Canada). Separations were performed on Ascentis ExpressRP-Amide columns (100 � 2.1 mm, 2.7 mm) produced by Supelco (Bellefonte,PA). The mobile phase used was a gradient of 0.1% formic acid and acetonitrileat a flow rate of 0.2 ml/min. The injection volume was 10 ml, the temperature ofthe autosampler 3�C, and the temperature of the chromatographic column 55�C.Quantitative data were acquired in multiple reaction monitoring (MRM)–positiveelectrospray ionization mode. The MRM transitions considered were 322.2/184.0for clopidogrel, 327.2/189.2 for clopidogrel-d3, 484.3/198.1 for clopidogrelacyl glucuronide, 308.2/95.0 for clopidogrel carboxylic acid, and 314.1/158.1for 13C6-clopidogrel carboxylic acid.

Software for Pharmacokinetic Evaluations and Statistics. Pharmacokineticparameters pertaining to the human PK study were determined and statisticallyanalyzed using SAS software (version 9.4; SAS Institute Inc., Cary, NC). For thedetermination of pharmacokinetic parameters from mean plasma concentration-versus-time curves constructed on mice data and for designing charts and graphs,Excel software was used (Microsoft Corporation, Redmond, WA).

Results

Mice PK and Metabolism Study. No concentration of clopidogrelparent above the lower limit of quantification (LLOQ) of the bio-analytical method was identified in any of the mice plasma samples,permitting the conclusion that either the concentrations were below0.01 ng/ml or, most probably, clopidogrel was not formed at all.As the only detected analytes (out of the three moieties screened), the

mean plasma concentration-versus-time profiles obtained for clopidog-rel acyl glucuronide and clopidogrel carboxylic acid after intravenousand oral administration of clopidogrel acyl glucuronide in mice arepresented in Fig. 2, A and B.In Fig. 2C we present in overlay mode and on ln-linear scale the

plasma concentration-versus-time curves of both metabolites after in-travenous and oral dosing.Pharmacokinetic parameters estimated for the two quantifiable

metabolites are presented in Table 1.

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The percentage ratio of oral versus intravenous AUCs within thesampling interval (0–8 hours) was estimated at 29.73%, suggesting thatclopidogrel acyl glucuronide undergoes extensive presystemic hydroly-sis resulting in the formation of the carboxylic acid derivative, notclopidogrel parent.Pharmacokinetic Data Gathered in the PK and Elimination

Study in Man. The concentration-versus-time profiles for parentclopidogrel, clopidogrel acyl glucuronide, and clopidogrel carboxylicacid obtained in human subjects following administration of clopidogrelwith and without charcoal are presented in Fig. 3.For the two metabolites the profiles are practically superimposable,

irrespective of charcoal intake, whereas for clopidogrel the circulatinglevels registered during the elimination phase were slightly increasedwhen charcoal was coadministered. Analysis of AUC data revealed that

the increase was not statistically significant [P value returned by theanalysis-of-variance (ANOVA) test for treatment effect was 0.055,above the 0.05 significance level].The main pharmacokinetic parameters estimated for clopidogrel and

its two metabolites are presented in Table 2.For clopidogrel parent the elimination half-life (t1/2) was 8.1 hours in

standard dosing conditions and 10.6 hours when charcoal was coadminis-tered; this differencewas found to be not statistically significant (paired t testreturned a value of 0.082, above the 0.05 significance level). For clopidogrelcarboxylic acid t1/2 was 7.8 hours for clopidogrel alone and 6.8 hours whencharcoal was coadministered, whereas for clopidogrel acyl glucuronide thesame t1/2 of 5.6 hours was estimated for both administration regimens.Elimination Data Gathered in the PK and Elimination Study in

Man. We found that about 15% of an administered clopidogrel dose

Fig. 2. Metabolites determined in plasma after administration of clopidogrel acyl glucuronide by intravenous (N = 35, parallel, 5 animals per sampling point) and oral route(N = 30 parallel, 5 animals per sampling point).

TABLE 1

PK parameters estimated for clopidogrel acyl glucuronide and clopidogrel carboxylic acid after intravenous and oraladministration of 200 ml of solution 1.25 mg/ml clopidogrel acyl glucuronide in mice

N = 35 parallel, 5 animals per sampling point.

Intravenous Administration Oral Administration

Cmax (6S.D.) AUC0–t (6S.D.) Tmax Cmax (6S.D.) AUC0–t (6S.D.) Tmax

ng/ml ng*h/ml h ng/ml ng*h/ml h

Clopidogrel acyl glucuronide 23454 (61755) 15425 (68645) 0.3 2280 (6331) 4586 (6807) 1.0Clopidogrel carboxylic acid 18395 (61382) 99265 (64980) 6.0 45000 (65207) 93660 (613806) 1.0

Cmax, peak analyte concentration.

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(calculated as micromolar ratios) is recovered in urine in the form of thequantified analytes (see Fig. 4). The longest recovery times were foundfor clopidogrel carboxylic acid (urinary excretion still ongoing in the 60–

72 hours postdose collection interval) and for clopidogrel acylglucuronide (recovered in urine up to 60 hours postdose). Forclopidogrel, only trace amounts were identified in urine (total recovery

Fig. 3. Plasma concentration-versus-time curves for the three analytes after administration of clopidogrel in human subjects (N = 6) with and without charcoal (linear-lineardisplay on A, C, E and ln-linear display on B, D, F).

TABLE 2

Main PK parameters determined in human volunteers (N = 6) for clopidogrel, clopidogrel carboxylic acid, and clopidogrel acyl glucuronide after oral dosing with Plavix75 mg with and without subsequent administration of activated charcoal (in a randomized, two-way crossover design study)

ParameterNo Charcoal(GeoMean)

With Charcoal (GeoMean)Charcoal/No Charcoal

Ratio (%)

Result of ANOVAfor Treatment as Fixed Effect

(P Value, Interpretation)

Clopidogrel Cmax (6S.D.) (ng/ml) 0.700 (60.402) 0.741 (60.343) 105.939% 7.51009E-01, N.S.AUC0–t (6S.D.) (ng*h/ml) 1.778 (61.559) 2.396 (60.982) 134.796% 5.53473E-02, N.S.

Clopidogrel carboxylic acid Cmax (6S.D.) (ng/ml) 2735.808 (6587) 2589.044 (6729) 94.635% 6.34597E-01, N.S.AUC0–t (6S.D.) (ng*h/ml) 9599.435 (64468) 10039.278 (61460) 104.582% 8.04498E-01, N.S.

Clopidogrel acyl glucuronide Cmax (6S.D.) (ng/ml) 428.937 (683) 419.236 (674) 97.738% 6.53555E-01, N.S.AUC0–t (6S.D.) (ng*h/ml) 1372.074 (6673) 1513.754 (6591) 110.326% 3.44311E-01, N.S.

ANOVA, analysis of variance; AUC0–t, area under the curve from time 0 until the last quantifiable point; Cmax, peak analyte concentration; N.S., not significant.

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well below 0.001 mM) up to 36 hours postdose, whereas, as expected,unchanged clopidogrel not absorbed from the intestine was mainlyrecovered in feces. Quantitation of the analytes in stool samplesaccounted for only 1.2% of an administered dose.The one-tailed paired t test was used to compare urinary excretion data

over the time intervals 0–12, 12–24, 24–36, 36–48, 48–60, and 60–72hours for the three analytes, after dosingwith clopidogrel with or withoutsubsequent administration of activated charcoal (see Fig. 5). It was foundthat the difference in amount recovered over the array of specified

intervals was not statistically significant (P values were 0.231 forclopidogrel, 0.488 for clopidogrel carboxylic acid, and 0.181 forclopidogrel acyl glucuronide).Urinary recovery by collection intervals for clopidogrel acyl glucu-

ronide is presented in Fig. 6A, and the amount of urine excreted withinthe intervals is depicted in Fig. 6B.No statistically significant difference in urinary recovery of clopidogrel

acyl glucuronide was identified in any of the collection intervals.

Discussion

The purpose of the present studies was to evaluate the pharmacoki-netics, metabolic fate, and elimination pattern of clopidogrel acylglucuronide, the main conjugated metabolite of clopidogrel. Sinceprevious in vitro data have demonstrated that CAG can undergo trans-esterification resulting in the formation of parent clopidogrel, emphasiswas placed on ascertaining if such a reaction could occur also in thein vivo setting. For each type of potential mechanistic conversion stud-ied (trans-esterification/hydrolysis, deconjugation during enterohepaticrecycling), a relevant physiologic model was chosen. For gaining insightinto the biodisposition of the metabolite (as such) and for identifyingthe reaction products derived from the activity of b-glucuronidaseand other hydrolases, a study was conducted in C57BL/6 mice ofproper age to ensure peak enzymatic activity (Peng et al., 2013). Foracquisition of quantitative data regarding the systemic availability andbalance between urinary and fecal recovery of CAG after oral dosingwith clopidogrel and for determining the likelihood of its involvementin enterohepatic recycling, the only clinically relevant option, giventhe complex metabolic processes involved, was to perform a study inman (Sörgel et al., 1989).Mice PK and Metabolism Study. After direct administration of

clopidogrel acyl glucuronide to mice by oral (gavage) and intravenous(tail vein) routes, HPLC–MS/MS analysis of postdose PK samples hasshown no generation of parent clopidogrel. While trans-esterification toclopidogrel did not take place in vivo, hydrolysis leading to theformation of the acidic derivative was the most important metabolicprocess observed for clopidogrel acyl glucuronide.Oral data have revealed a very fast metabolism of clopidogrel acyl

glucuronide within the first 2 hours from administration, probablyoccurring in the gastrointestinal tract by chemical degradation and/orenzymatic hydrolysis. The percentage ratio of oral versus intravenousAUCs estimated for the administered conjugated metabolite within thesampling interval (0–8 hours) was 29.73%.By intravenous route, as metabolism was restricted only to sys-

temic degradation of CAG, the rate of conversion to the carboxylic acidform was lower; specifically, while oral data showed that both the

Fig. 4. Total recovery of clopidogrel, clopidogrel acyl-glucuronide, and clopidogrelcarboxylic acid in urine and stool samples over 72 hours postdose after administrationof clopidogrel in human subjects (N = 6).

Fig. 5. Total recovery of clopidogrel, clopidogrel acyl-glucuronide, and clopidogrelcarboxylic acid in urine samples after administration of clopidogrel in humansubjects (N = 6) with or without activated charcoal.

Fig. 6. Recovery of clopidogrel acyl-glucuronide in urine (N = 6) displayed by collection intervals (A) and amount of urine excreted by collection intervals (B).

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administered clopidogrel acyl glucuronide and the formed clopidogrelcarboxylic acid reached peak levels simultaneously at 1 hour, followingtail injection the time lag until maximal plasma levels of clopidogrelcarboxylic acid was 6 hours and the peak concentrations reached were2.5 times lower than after oral dosing. Nevertheless, total exposure toclopidogrel carboxylic acid was almost identical irrespective of theadministration route of CAG (mean AUC ratio intravenous/per os was1.05), thus showing that systemic conversion is also very extensive. Thiswas to be expected considering that lysosomal and microsomal fractionsexpressing b-glucuronidase and esterases are widely expressed also inserum and organs other than the liver in the organism of C57BL/6 mice(Peng et al., 2013; Tegelström and Ryttman, 1981; Lusis and Paigen, 1977).Human PK Data. The use of activated charcoal as bile-binding agent

for the purpose of impeding enterohepatic recycling of xenobiotics isalready well established (Chyka et al., 2005; Stass et al., 2005; Taft,2009). Also, PK studies of interaction between drugs and activatedcharcoal have been used previously for determining if the activeingredient or related molecules undergo extensive recycling; reducedexposure coupled with accelerated elimination of the investigatedmolecule in the charcoal study arm are classic indicators of disconti-nuing/minimizing the recycling process (Sörgel et al., 1989; Elomaaet al., 2001; Wang et al., 2014). For unbiased results, the administrationschedule for activated charcoal must be individualized according to thebiopharmaceutical properties of the studied drug to ensure that admin-istration of the bile-binding agent does not also alter drug absorption. Forclopidogrel in particular, whereas the early time of the peak analyteconcentration (Tmax) can be misleading, it is important to note thatabsorption is slow and mainly occurs in the lower compartments of thegastrointestinal tract. With slow absorption and fast subsequent elimina-tion of the absorbed fraction (mainly through extensive metabolism andto a lesser extent the result of actual excretion), the equilibrium betweenthe two constants occurs much earlier than complete absorption of theprodrug. In fact, an in silico gastrointestinal simulation of regionalabsorption distribution of clopidogrel, recently published by our group,has shown that absorption only starts in the duodenum (33% of doseabsorbed) and is completed through significant contribution (30%) fromcecum and ascending colon (Savu et al., 2016). This behavior is quitetypical, since clopidogrel is a weak base characterized by a dissociationconstant (pKa) of 5.3 (NLM, 2012); therefore, it freely crosses cellmembranes in gastrointestinal compartments where the pH is greater than5.3. Considering these properties, administration of activated charcoalwas started at 6.0 hours after clopidogrel dosing so that any decreasedexposure possibly noted for the parent drug or the studied metabolites inthe charcoal arm could only be attributed to recycling impairment and notdecreased drug absorption.The fact that clopidogrel concentrations remained practically un-

changed irrespective of charcoal intake indicated that the administrationschedule for the bile-binding agent was correctly designed for theintended purpose and that clopidogrel (as such) is not involved in anyenterohepatic cycle.Considering the pharmacokinetic data obtained for clopidogrel acyl

glucuronide, with particular emphasis on elimination half-life (de-termined to be 5.6 hours irrespective of charcoal administration) andresults of the comparison carried out between plasma profiles of themetabolite generated in the presence and absence of activated charcoal(charcoal/no charcoal ratios of 0.98 for Cmax and 1.10 for AUC), it canbe concluded that any enterohepatic recycling of CAG that may occur isnot significant. The conclusion is supported also by the statistic testsapplied for comparison of the primary PK parameters of CAG in the twoadministration conditions (the ANOVA test checking for treatment asfixed-effect returned P values above the 0.05 significance level for bothCmax and AUC0–t data).

Human Elimination Data. On the basis of the knowledge acquired itcan be said that clopidogrel acyl glucuronide may be regarded as aquantitatively important yet terminal metabolite of the parent drug, notbeing capable of contributing to the regeneration of known moietieslinked to active metabolite formation. However, the potential of acylglucuronide to play other roles of significant importance in terms ofclopidogrel activity cannot be yet excluded.Quantitation of the analytes in stool samples accounted for only 1.2%

of an administered dose, quite far from the mass balance study resultspreviously reported in literature (Lins et al., 1999) that showed acumulative fecal recovery of radioactivity ranging from 35 to 57% aftersingle dosing with 75 mg of 14C-labeled clopidogrel. This fact stronglysuggests that other metabolites and/or degradation products not yetcharacterized are involved in this elimination process. The finding isconsistent with the report that twenty distinct metabolites of clopidogrelcan be identified in biological matrices (EMA, 2004).Urinary data confirm what we hypothesized on the basis of the

previously presented plasma PK results of same subjects, namely that theacyl glucuronide derivative does not undergo significant enterohepaticrecycling, if any. Should that have been the case, administration ofcharcoal would have accelerated elimination of the metabolite and notthe opposite. There is also no evidence that any of the three quantifiedmoieties is involved in enterohepatic recycling.To conclude, despite the high tendency observed for it in vitro, no

evidence was found to suggest that clopidogrel acyl glucuronide couldreconvert to parent clopidogrel in vivo by trans-esterification. Bycomparing of PK profiles for clopidogrel and the conjugated metabolitealone and in the presence of activated charcoal, it can also be stated that itis unlikely that clopidogrel acyl glucuronide would be capable ofreforming clopidogrel (as such) through participation in an enterohepaticcycle. So far it seems that the amount of clopidogrel converted bycarboxylesterase 1 to the inactive carboxylic acid (about 85% of anadministered dose) is not made again available for metabolization byP450s so that it might be oxidized and form the active thiol metabolite.

Acknowledgments

The authors would like to extend their gratitude to Angela Casarica,Department of Pharmaceutical Biotechnologies of ICCF, for her help in miceplasma processing and to Constanta Dulea and Adrian Ghita from Pharma ServInternational for the help granted concerning the HPLC–MS/MS analysis ofpharmacokinetic samples and respectively for aiding in the statistical analysis ofPK data.

Authorship ContributionsParticipated in research design: Savu, Silvestro, Rizea Savu, Mircioiu.Conducted experiments: Savu, Silvestro, Remis, Yuksel.Performed data analysis: Savu, Silvestro, Mircioiu.Wrote or contributed to the writing of the manuscript: Savu, Silvestro,

Surmeian, Mircioiu.

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Address correspondence to: Dr. Simona Nicoleta Savu, 52 Sabinelor Street, 5thDistrict, 050853 Bucharest, Romania. E-mail: [email protected]

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