EVALUATION OF CLOPIDOGREL CONJUGATION METABOLISM: …dmd.aspetjournals.org/content/dmd/early/2016/07/11/dmd.116.071092.full.pdf · DMD # 71092 1 TITLE PAGE EVALUATION OF CLOPIDOGREL

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TITLE PAGE

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, Constantin Mircioiu

University of Medicine and Pharmacy "Carol Davila", Faculty of Pharmacy, Department of

Biopharmacy, Bucharest, Romania (S.N.S., M.C.);

3S-Pharmacological Consultation & Research GmbH, Koenigsbergerstrasse 1 – 27243 Harpstedt,

Germany (S.N.S, L.S., S.R.S.);

Pharma Serv International SRL., 52 Sabinelor Street, 5th District, 050853 Bucharest, Romania (M.S.);

Clinical Hospital of the Ministry of Health of the Moldavian Republic, 51 Puskin Street, MD-2005

Chisinau, The Moldavian Republic (L.R.)

National Institute for Chemical Pharmaceutical Research and Development (ICCF), Pharmacology

Department, 112 Vitan Avenue, 3rd District, 031299 Bucharest, Romania (Y. R.)

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092

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RUNNING TITLE PAGE

Running title: PK STUDIES IN MAN AND MICE OF CLOPIDOGREL ACYL

GLUCURONIDE

Corresponding author:

Simona Nicoleta Savu

Address: 52 Sabinelor Street, 5th District, 050853 Bucharest, Romania

Mobile phone: +40 758 109 202

E-mail: [email protected]

Document statistics:

Abstract - 242

Introduction - 748

Discussion - 1297

Tables - 2

Figures - 6

References - 34

Nonstandard abbreviations:

AUC0-t - area under the curve from time 0 until the last quantifiable point

AUC0-inf - area under the curve from time 0 to infinite

CAG - clopidogrel acyl glucuronide

CCA – clopidogrel carboxylic acid


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Cmax - peak analyte concentration

CYP 450 - Cytochromes P450

HPLC-MS/MS - High-performance liquid chromatography - Tandem Mass

Spectrometry

ICCF - National Institute for Chemical Pharmaceutical Research and

Development

i.v. – intravenous

K2EDTA - di-potassium ethylenediaminetetraacetic acid

LLOQ – lower limit of quantification

MRM - multiple reactions monitoring

N. A. - not applicable

N. S. - not significant

PK - pharmacokinetics

QC - quality control

SD - standard deviation

t1/2 - plasma half life

Tmax - time of the peak analyte concentration

UGTs - UDP-glucuronosyltransferases


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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 PK, metabolism and clearance are modest. We investigated

the potential in vivo CAG trans-esterification to clopidogrel (reaction occurring in

vitro in particular conditions) by administering the metabolite to mice. Experiments

were then carried-out on men, administering clopidogrel alone or followed by

activated charcoal intake (intestinal reabsorption blockade). Here, 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 entero-hepatic recycling of CAG. In mice, CAG was rapidly hydrolyzed to

CCA after oral administration while by i.v. 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 gastro intestinal tract (very

low concentrations in feces) but there is no evidence of entero-hepatic 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.


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INTRODUCTION

Glucuronide conjugates represent one of the major types of phase II

metabolites of xenobiotics. Since generally the biological function of the aglycone is

abolished by glucuronidation, conjugates are often considered as metabolites of

modest interest; however, few compelling cases in which glucuronides

maintain/increase the biological function of their parent compound, [Baruna et. al.,

2004; Ohno et. al., 2008] suggest that further inquiry into their metabolic fate is

warranted.

In the particular case of clopidogrel, while the oxidative metabolism 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 CYP450

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 CAG [Silvestro et. al., 2010] - a quantitatively important

metabolite that has not been studied in detail until now [Figure 1, schematic

representation of clopidogrel metabolism].

Though 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

was demonstrated for CAG [Tornio et. al., 2014] and it is unknown if the metabolite


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undergoes any type of metabolic conversion before being excreted from the human

body.

In vitro, reactivity of CAG has been already demonstrated. It was shown that

in specific conditions it converts to parent clopidogrel by trans-esterification

[Silvestro et. al., 2011], a reaction sometimes occurring also during metabolic

processes [Boyer et. al. 1992; Knights et. al., 2000; Celli et. al., 2007; Fujino et. al.,

2014].

Should CAG participate in vivo to any process resulting in back-conversion to

clopidogrel, the amount reconstituted could be considerable being the exposure to

CAG in man (based on AUC0-inf), 500 times higher than that of clopidogrel [Silvestro

et. al., 2013]); furthermore, the newly formed clopidogrel would be again available

for metabolism by CYPs and thus partly converted to the active metabolite. While it is

clear that the confirmation of such a pathway could only provide mechanistic insight

(quantitative data on clopidogrel and its active metabolite being already available in

literature), the disposition of CAG was considered important knowledge to be gained

as any yet unknown intermediate reaction could prove useful in understanding the

large PK variability of clopidogrel and its active moiety.

Rationale and study objectives

The present studies represent a follow-up to previous work in which we

reported the existence of CAG and described its in vitro back-conversion to

clopidogrel by trans-esterification [Silvestro et. al., 2011]. The main questions to

clarify now are “Can this by any means happen also in vivo?” and “Which is the

metabolic fate of this conjugate?”.

First, in the absence of a CAG standard suitable for administration to humans,

we conducted a study in mice in order to determine if this metabolite may back-


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convert to clopidogrel parent by trans-esterification or another reaction of the

conjugated metabolite; the study was conducted on mice (C57BL) having a similar

glucuronidase tissue distribution to that of man [Gad, 2007].

Another important aspect to clarify is if CAG undergoes enterohepatic

recycling since mass balance studies conducted with radiolabeled clopidogrel in man

[Lins, 1999] showed that recycling occurs without identifying the moieties involved.

Plasma levels of clopidogrel and its 2 main metabolites were compared in healthy

volunteers treated with clopidogrel alone or in combination with activated charcoal;

this bile-binding agent was administered according to a regimen designed to disrupt

enterohepatic recycling, as already described in literature [Elomaa et. al., 2001; Wang

et. al., 2014], and have minimal 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) for CAG, clopidogrel

and CCA (as precursors) was also a set objective of the single dose charcoal-

interaction study in man.

It is noteworthy that a human study was preferred due to the complex nature

of the physiological processes studied through PK determinations and the

consideration that data gathered in any other model would be extremely difficult to

extrapolate, raising concerns of relevance in a real clinical setting.


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MATERIALS AND METHODS

Standards, reagents and medication

For the preparation of solutions for oral and intravenous administration in mice,

clopidogrel Acyl-β-D-glucuronide standards of adequate purity were purchased from

Toronto Research Chemicals, Canada.

The internal standards used for HPLC-MS/MS analytical determinations were: d3-

clopidogrel hydrogensulfate (SynFine Research, Canada), clopidogrel Acyl-β-D-

glucuronide (TRC, Canada) and 13C6-clopidogrel carboxylic acid (Alsachim, France).

Commercially available reagents of analytical grade purity were used for sample

processing.

Plavix 75 mg tablets (Sanofi) from a commercial batch [AY171] were used. The

medical grade activated charcoal was also procured from the market (from Silcarbon

Aktivkohle GmbH, Germany).

Intravenous and Oral Pharmacokinetics Study in Mice

Study design and sample collection. All the procedures used were in

accordance with the standards set forth in the eighth edition of Guide for the Care and

Use of Laboratory Animals (National Academy of Sciences, The National Academies

Press, Washington D.C.). Laboratory animals (C57BL/6 male mice, weighting 20 ±

4g, 25 ± 1 days of age) were bred, raised and cared for at the Cantacuzino National

Institute of Research-Development for Microbiology and Immunology (NIRDMIC)

located in Bucharest, Romania. The experimental part was carried out in the

Pharmacology Department of the National Institute for Chemical Pharmaceutical

Research and Development (ICCF) located in Bucharest, Romania. The study was

conducted according to a parallel design on an overall sample size of 71 laboratory

animals (5 per sampling point after each mode of administration plus 6 animals


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treated with normal saline only in view of obtaining blank plasma for preparation of

analytical quality control samples). Animals randomized to the treatment arms,

received in sterile conditions a dose of 200 µL freshly prepared solution of 1.25mg/ml

clopidogrel acyl glucuronide in normal saline, either per os (through gavage) or

intravenously, via tail vein injection. Blood samples (150 μL) were collected in pre-

chilled tubes containing K2EDTA at 0.5, 1, 2, 4, 6 and 8 hours after oral dosing or at

0.25, 0.5, 1, 2, 4, 6 and 8 hours after intravenous administration. The samples were

immediately immersed in water and ice bath until centrifugation (performed at a

nominal temperature of 4 °C, 1500 G-force for a duration of 10 minutes). The

separated plasma was frozen at −70 °C and maintained at this temperature until

analyzed. For sample processing and analysis we used a slight modification of a

method already published [Silvestro et. al., 2011], as described below.

Extraction of clopidogrel, clopidogrel carboxilic acid and clopidogrel acyl

glucuronide from mice plasma samples. Plasma thawing was done on wet ice.

Aliquots of 100 μL from post-dose mice plasma samples were diluted with 200 μL of

ice-cold acetonitrile, spiked with 20 μL of internal standard mix in acetonitrile (d3-

clopidogrel hydrogensulfate, clopidogrel Acyl-β-D-glucuronide and 13C6-clopidogrel

carboxylic acid, 200 ng/mL), vortexed for 3 minutes and then centrifuged for 5

minutes with 4000 rpm at 8 °C. Supernatants (100 μL) were diluted with 100 μL ice-

cold water containing 2% acetonitrile and 0.1% formic acid. The extracts were

analyzed as described in the next paragraph.

Clopidogrel, clopidogrel carboxilic acid and clopidogrel acyl glucuronide

quantification. Six-point calibration curves were prepared in blank mice plasma

(K2EDTA as anticoagulant) with concentrations ranging from 0.01 to 100.00 ng/mL

for clopidogrel and from 1.00 to 10000.00 ng/ml for clopidogrel acyl glucuronide and


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clopidogrel carboxylic acid. The quality control and calibration curve samples were

also spiked with internal standard mix in acetonitrile (d3-clopidogrel hydrogensulfate,

clopidogrel Acyl-β-D-glucuronide and 13C6-clopidogrel carboxylic acid, 200 ng/mL)

and subsequently extracted in the same manner described previously for study

samples. Calibration curves and QC samples (three concentration levels and in

triplicate) were analyzed during each analytical sequence. Decisions regarding the

acceptance of sequences were taken according to well-established bioanalytical rules

[FDA, 2013; EMA, 2011]. No sequences had to be rejected due to quality control or

calibration failure.

Human Oral Pharmacokinetics and Elimination Study

Study design and sample collection.

Six subjects were enrolled and completed the human PK and elimination

study. Study population was comprised of 3 male and 3 non-pregnant, non-lactating

female volunteers, 18 to 51 years old (mean age 32.17 ± 14.48). The study was

conducted at the Clinical Hospital of the Ministry of Health of the Moldavian

Republic located in Chisinau. The Study Protocol was reviewed and approved by an

Institutional Ethics Committee and all 6 subjects enrolled were informed about the

study medication and procedures and gave consent for the participation in the study.

Clinical investigations were conducted according to the Declaration of Helsinki

principles and the medication administered consisted of a single oral dose of

reference-listed drug (Plavix 75 mg, procured from the market) per study period. The

design was two-way cross-over: in one study period the subjects received just

clopidogrel and in the other they received clopidogrel plus a regimen 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


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parent clopidogrel, clopidogrel acyl glucuronide and clopidogrel carboxylic acid in

plasma were collected in pre-chilled tubes 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 hours post-dose

while fecal matter was collected over the same interval but only when clopidogrel

was given without activated charcoal (as previous experience thought, presence of

charcoal in stool samples leads to ambiguous results).

Extraction of metabolites from biological samples.

Before analysis, plasma samples were thawed on wet ice, and 100 μL aliquots were

spiked with 20 μL solution of internal standard which contained 200 ng/mL d3-

clopidogrel hydrogensulfate, 200 ng/mL clopidogrel Acyl-β-D-glucuronide and 200

ng/mL 13C6-clopidogrel carboxylic acid in acetonitrile, and then diluted with 200 μL

ice-cold acetonitrile. Afterwards they were vortex for 3 minutes and centrifuged at

4000 rpm and 8 °C for 5 minutes. Supernatants (100 μL) were diluted with 100 μL

ice-cold water containing 2% acetonitrile and 0.1% formic acid.

Urine samples were collected during the time intervals 0-12h, 12-24h, 24-36h, 36-

48h; 48-60h and 60-72h post-dose. The volume of each fresh urine sample was

measured and 50 ml aliquots were mixed with 100 μL acetic acid 99.8%, vortexed for

2 minutes and frozen at -20°C. In order to obtain a single representative urinary

excretion sample for each time interval, aliquotes from individual samples were

mixed in approporiate proportions according to initial sample volume. Before

analysis, samples (100 μL) were thawed on wet ice, spiked with 20 μL of internal

standard mix in acetonitrile (d3-clopidogrel hydrogensulfate, clopidogrel Acyl-β-D-

glucuronide and 13C6-clopidogrel carboxylic acid 200 ng/mL), and then diluted with

200 μL ice-cold acetonitrile. Afterwards they were vortexed for 3 minutes and then


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centrifuged for 5 minutes at a nominal temperature of 8 °C, with a speed of 4000 rpm.

A volume of 100 μL supernatant was separated and diluted with 100 μL ice-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 an ice-cold

solution containing 50% acetonitrile and 1% formic acid, as follows: samples were

first vortexed for 2 minutes with 1/5 of the calculated volume of the above solution

for dilution and 250 mg glass beads per gram of sample. The remaining volume of the

solution was then added and the samples were vortexed again for 3 minutes and

centrifuged at 4000 rpm and 8 °C for 10 minutes. A volume of 100 μL supernatant

was recovered and processed in the same manner as previously described for thawed

urine samples.

Clopidogrel, clopidogrel carboxilic acid and clopidogrel acyl glucuronide

quantification. Six-point calibration curves were prepared in appropriate matrix (in

blank plasma, blank urine, or blank fecal matter samples which were spiked with

internal standard, processed and diluted according to the same protocol previously

described for study samples). The concentration ranges of the calibration curves were

0.01 to 100.00 ng/mL for clopidogrel and 1.00 to 10000.00 ng/ml for clopidogrel acyl

glucuronide and clopidogrel carboxylic acid. Calibration curves and QC samples

(three concentration levels in triplicate) were analyzed during each analytical

sequence. Decisions regarding the acceptance of sequences were taken according to

well-established bio-analytical rules [FDA, 2013; EMA, 2011]. No sequences had to

be rejected due to quality control or calibration failure.

HPLC/MS/MS Analysis. For the analytical determinations we used a HPLC

binary gradient (LC-20 AD chromatographic pumps) by Shimadzu - Japan with a


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CTC-PAL autosampler (model HTS) manufactured by CTC Analytics, Switzerland.

The HPLC system was coupled with a triple quadrupole mass-spectrometer model

API 5000 (mice PK samples) or API 6500 (human PK and elimination samples) with

an atmospheric pressure electrospray ionization source (model TurboIonSpray), all

manufactured by Applied Biosystems-Sciex - Canada. Separations were performed on

Ascentis Express RP-Amide columns (100×2.1 mm, 2.7 μm) produced by Supelco.

The mobile phase used was a gradient of 0.1% formic acid and acetonitrile at a flow

rate of 0.2 mL/min. The injection volume was 10 μL, the temperature of the

autosampler 3°C and the temperature of the chromatographic column 55°C.

Quantitative data were acquired in multiple reactions monitoring (MRM) positive

electrospray ionization mode. The MRM transitions considered were 322.2/184.0 for

clopidogrel; 327.2/189.2 for clopidogrel-d3; 484.3/198.1 for clopidogrel acyl

glucuronide; 308.2/95.0 for clopidogrel carboxylic acid and 314.1/158.1 for 13C6-

clopidogrel carboxylic acid.

Software for pharmacokinetic evaluations and statistic. Pharmacokinetic

parameters pertaining to the human PK study were determined and statistically

analyzed using SAS software (version 9.4; SAS Institute Inc., Cary, NC - USA). For

the determination 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 - USA).


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RESULTS

1. Mice PK and metabolism study

No concentration of clopidogrel parent above the LLOQ of the bioanalytical

method was identified in any of the mice plasma samples, permitting to conclude that

either the concentrations were below 0.01 ng/mL or, most likely, 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 clopidogrel acyl glucuronide and

clopidogrel carboxylic acid after intravenous and oral administration of clopidogrel

acyl glucuronide in mice, are presented in Figure 2, Charts A and B.

In Chart C of Figure 2 we present in overlay mode and on ln-linear scale the plasma

concentration versus time curves of both metabolites after intravenous and oral

dosing.

Pharmacokinetic parameters estimated for the two quantifiable metabolites are

presented in Table 1 below:

The percentage ratio of oral versus intravenous AUCs within the sampling interval (0-

8 hours) was estimated at 29.73%, suggesting that clopidogrel acyl glucuronide

undergoes extensive pre-systemic hydrolysis resulting in the formation of the

carboxylic acid derivative, not clopidogrel parent.

2. Pharmacokinetic data gathered in the PK and elimination study in man

The concentration versus time profiles for parent clopidogrel, clopidogrel acyl

glucuronide and clopidogrel carboxylic acid obtained in human subjects following

administration of clopidogrel with and without charcoal are presented in Figure 3.

For the two metabolites the profiles are practically superimposable,

irrespective of charcoal intake, while for clopidogrel the circulating levels registered


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during the elimination phase were slightly increased when charcoal was co-

administered. Analysis of AUC data revealed that the increase was not statistically

significant (p-value returned by the 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 below:

For clopidogrel parent the elimination half-life (t½) was 8.1 hours in standard dosing

conditions and 10.6 hours when charcoal was co-administered; this difference was

found to be not statistically significant (paired T-test applied returned a value of

0.082, above the 0.05 significance level). For clopidogrel carboxylic acid t½ was 7.8

hours for clopidogrel alone and 6.8 hours when charcoal was co-administered while

for clopidogrel acyl glucuronide the same t½ of 5.6 hours was estimated for both

administration regimens.

3. Elimination data gathered in the PK and elimination study in man

We found that about 15% of an administered clopidogrel dose (calculated as

µM ratios) is recovered in urine in the form of the quantified analytes (see Figure 4).

The longest recovery times were found for clopidogrel carboxylic acid (urinary

excretion still ongoing in the 60 to 72 hours post-dose collection interval) and for

clopidogrel acyl glucuronide (recovered in urine up to 60 hours post-dose). For

clopidogrel, only trace amounts were identified in urine (total recovery well below

0.001 microM) up to 36 hours post dose while, as expected, unchanged clopidogrel

not absorbed from the intestine was mainly recovered in feces. Quantitation of the

analytes in stool samples accounted 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 0-12h, 12-24h, 24-36h, 36-48h; 48-60h and 60-72h for the three analytes,


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after dosing with clopidogrel with or without subsequent administration of activated

charcoal (see Figure 5). It was found that the difference in amount recovered over the

array of specified intervals was not statistically significant (p values were 0.231 for

clopidogrel, 0.488 for clopidogrel carboxylic acid and 0.181 for clopidogrel acyl

glucuronide).

Urinary recovery by collection intervals for clopidogrel acyl glucuronide is

presented in Figure 6-A, while the amunt of urine excreted within the intervals is

depicted in Figure 6-B.

No statistically significant difference in urinary recovery of clopidogrel acyl

glucuronide was identified in any of the collection intervals.


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DISCUSSION

The purpose of the present studies was to evaluate the pharmacokinetics,

metabolic fate and elimination pattern of clopidogrel acyl glucuronide, the main

conjugated metabolite of clopidogrel. Since previous in vitro data have demonstrated

that CAG can undergo trans-esterification resulting in the formation of parent

clopidogrel, emphasis was put on ascertaining if such a reaction could occur also in

the in vivo setting. For each type of potential mechanistic conversion studied (trans-

esterification/hydrolysis, deconjugation during entero-hepatic recycling), a relevant

physiological model was chosen. For gaining insight into the biodisposition of the

metabolite (as such) and for identifying the reaction products derived from the

activity of beta-glucuronidase and other hydrolases, a study was conducted in

C57BL/6 mice of proper age to ensure peak enzymatic activity [Peng et. al., 2013].

For acquisition of quantitative data regarding the systemic availability and balance

between urinary and fecal recovery of CAG after oral dosing with clopidogrel and for

determining the likelihood of its involvement in enterohepatic recycling, the only

clinically relevant option, given the complex metabolic processes involved, was to

perform a study in man [Sörgel et. al., 1989].

Mice PK and metabolism study: After direct administration of clopidogrel

acyl glucuronide to mice by oral route (gavage) and intra-venous route (tail vein),

HPLC/MS-MS analysis of post-dose PK samples has shown no generation of parent

clopidogrel. While trans-esterification to clopidogrel did not take place in vivo,

hydrolysis leading to the formation of the acidic derivative was the most important

metabolic process observed for clopidogrel acyl glucuronide.

Oral data have revealed a very fast metabolism of clopidogrel acyl glucuronide within

the first 2 hours from administration, probably occurring in the GI tract by chemical


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degradation and/or enzymatic hydrolysis. The percentage ratio of oral versus i.v.

AUCs estimated for the administered conjugated metabolite within the sampling

interval (0-8 hours) was of 29.73%.

By intravenous route, as metabolism was restricted only to systemic degradation of

CAG, the rate of conversion to the carboxylic acid form was lower; specifically, while

oral data showed that both the administered clopidogrel acyl glucuronide and the

formed clopidogrel carboxylic acid reached peak levels simultaneously at one hour,

following tail injection the time lag till maximal plasma levels of clopidogrel

carboxylic acid was of 6 hours and the peak concentrations reached were 2.5 times

lower than after oral dosing. Nevertheless, total exposure to clopidogrel carboxylic

acid was almost identical irrespective of the administration route of CAG (mean AUC

ratio i.v./p.o. was 1.05), thus showing that systemic conversion is also very extensive

(as was to be expected considering that lysosomal and microsomal fractions

expressing beta-glucuronidase and esterases are widely expressed also in serum and

organs other than the liver in the organism of C57BL/6 mice [Peng et. al., 2013;

Tegelstrom et. al. 1981; Lusis et. al., 1977]).

Human PK data: The use of activated charcoal as bile-binding agent for the

purpose of impeding enterohepatic recycling of xenobiotics is already well

established [Stass et. al., 2005; Taft, 2009; AACT, 2005]. Also, PK-interaction

studies between drugs and activated charcoal have been used previously for

determining if the active itself or related molecules undergo extensive recycling;

reduced exposure coupled with accelerated elimination of the investigated molecule

in the charcoal study arm are classic indicators of discontinuing/minimizing the

recycling process [Sörgel et. al., 1989; Elomaa et. al., 2001; Wang et. al., 2014]. For

unbiased results, the administration schedule for activated charcoal must be


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individualized according to the biopharmaceutical properties of the studied drug to

ensure that administration of the bile-binding agent does not also alter drug

absorption. For clopidogrel in particular, while the early Tmax can be misleading, it is

important to note that absorption is slow and mainly occurs in the lower

compartments of the gastrointestinal tract. With slow absorption and fast subsequent

elimination of the absorbed fraction (mainly through extensive metabolism and to a

lesser extent due to actual excretion), the equilibrium between the two constants

occurs much earlier than complete absorption of the prodrug. In fact, an in silico

gastrointestinal simulation of regional absorption distribution of clopidogrel, recently

published by our group, has shown that absorption only starts in the duodenum (33%

of dose absorbed) and is completed through significant contribution (30%) from

caecum and ascending colon [Savu et. al., 2016]. This behaviour is quite typical,

since clopidogrel is a weak base characterized by a dissociation constant (pKa) of 5.3

[US National Library of Medicine, 2012], therefore freely crossing cell membranes in

gastro-intestinal compartments where the pH is greater than 5.3. Considering these

properties, administration of activated charcoal was started at 6.0 hours after

clopidogrel dosing so that any decreased exposure possibly noted for the parent drug

or the studied metabolites in the charcoal arm could only be attributed to recycling

impairment and not decreased drug absorption.

The fact that clopidogrel concentrations remained practically unchanged

irrespective of charcoal intake indicated that the administration schedule for the bile-

binding agent was correctly designed for the intended purpose and that clopidogrel (as

such) is not involved in any enterohepatic cycle.

Considering the pharmacokinetic data obtained for clopidogrel acyl

glucuronide, with particular emphasis on elimination half-life (determined to be 5.6


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hours irrespective of charcoal administration) and results of the comparison carried

out between plasma profiles of the metabolite generated in the presence and absence

of activated charcoal (charcoal/no charcoal ratios of 0.98 for Cmax and 1.10 for AUC),

it can be concluded that any entero-hepatic recycling of CAG possibly occurring is

not significant. The conclusion is supported also by the statistic tests applied for

comparison of the primary PK parameters of CAG in the two administration

conditions (the ANOVA test checking for treatment as fixed effect returned p-values

above the 0.05 significance level for both Cmax and AUC0-t data).

Human elimination data: Based on the knowledge acquired it can be said

that clopidogrel acyl glucuronide may be regarded as a quantitatively important yet

terminal metabolite of the parent drug, not being capable of contributing to the

regeneration of known moieties linked to active metabolite formation. However, the

potential of acyl glucuronide to play other roles of significant importance in terms of

clopidogrel 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 results previously reported

in literature [Lins et. al., 1999] that showed a cumulative fecal recovery of

radioactivity ranging from 35 to 57% after single dosing with 75 mg of 14C-labeled

clopidogrel. This fact strongly suggests that other metabolites and/or degradation

products not yet characterized are involved in this elimination process. The finding is

consistent with the SmPC report that twenty distinct metabolites of the clopidogrel

can be identified in biological matrices.

Urinary data confirm what we hypothesized based on the previously presented plasma

PK results of same subjects, namely that the acyl glucuronide derivative does not

undergo significant entero-hepatic recycling, if any. Should that have been the case,


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administration of charcoal would have accelerated elimination of the metabolite and

not the opposite. There is also no evidence that any of the three quantified moieties is

involved in entero-hepatic recycling.

To conclude, despite the high tendency observed for it in vitro, no evidence

was found to suggest that clopidogrel acyl glucuronide could reconvert to parent

clopidogrel in vivo by trans-esterification. Based on comparison of PK profiles for

clopidogrel and the conjugated metabolite alone and in the presence of activated

charcoal, it can also be stated that it is unlikely that clopidogrel acyl glucuronide

would be capable of reforming clopidogrel (as such) through participation in an

entero-hepatic cycle. So far it seems that the amount of clopidogrel converted by

carboxylesterase 1 to the inactive carboxylic acid (about 85% of an administered

dose) is not made again available for metabolisation by CYPs so that it might be

oxidized and form the active thiol metabolite.


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ACKNOWLEDGEMENTS

The authors would like to extend their gratitude to Angela Casarica, from the

Department of Pharmaceutical Biotechnologies of ICCF for her help in mice plasma

processing and to Constanta Dulea and Adrian Ghita from Pharma Serv International

for the help granted concerning the HPLC/MS-MS analysis of pharmacokinetic

samples and respectively for aiding in the statistical analysis of PK data.


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AUTHORSHIP CONTRIBUTIONS

Participated 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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FOOTNOTES

This work received financial support through the project entitled "CERO – Career

profile: Romanian Researcher", cofinanced by the European Social Fund for Sectoral

Operational Programme Human Resources Development 2007-2013

[POSDRU/159/1.5/S/135760].


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LEGENDS FOR FIGURES

Figure 1. Representation of clopidogrel metabolism

Figure 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)

Figure 3. Plasma concentration vs. time curves for the three analytes after

administration of clopidogrel in human subjects (N=6) with and without charcoal

(linear-linear display on charts 3-A, C, E and ln-linear display on charts 3-B, D, F)

Figure 4. Total recovery of clopidogrel, clopidogrel acyl-glucuronide and clopidogrel

carboxylic acid in urine and stool samples over 72h post dose after administration of

clopidogrel in human subjects (N=6)

Figure 5. Total recovery of clopidogrel, clopidogrel acyl-glucuronide and clopidogrel

carboxylic acid in urine samples after administration of clopidogrel in human subjects

(N=6) with or without activated charcoal

Figure 6. Recovery of clopidogrel acyl-glucuronide in urine (N=6) displayed by

collection intervals (6-A) and amount of urine excreted by collection intervals (6-B)


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TABLES

TABLE 1

PK parameters estimated for clopidogrel acyl glucuronide and clopidogrel carboxylic acid after

intravenous and oral administration of 200µL solution 1.25mg/ml clopidogrel acyl glucuronide in mice

Intravenous administration

(N = 35, parallel, 5 animals per

sampling point)

Oral administration

(N= 30, parallel, 5 animals per

sampling point)

Cmax

[±SD]

AUC0-t

[±SD] Tmax

Cmax

[±SD]

AUC0-t

[±SD] Tmax

(ng/mL) (ng*h/mL) (h) (ng/mL) (ng*h/mL) (h)

Clopidogrel acyl

glucuronide

23454

[±1755]

15425

[±8645] 0.3

2280

[±331]

4586

[±807] 1.0

Clopidogrel

carboxylic acid

18395

[±1382]

99265

[±4980] 6.0

45000

[± 5207]

93660

[±13806] 1.0


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TABLE 2

Main pharmacokinetic parameters determined in human volunteers (N=6) for clopidogrel, clopidogrel

carboxylic acid and clopidogrel acyl glucuronide after oral dosing with Plavix 75mg with and without

subsequent administration of activated charcoal (in a randomized, two-way cross-over design study)

Parameter

No

Charcoal

(GeoMean)

With

Charcoal

(GeoMean)

Charcoal/No

Charcoal ratio

(%)

Result of ANOVA for

Treatment as fixed

effect

(p-Value, interpretation)

Clopidogrel Cmax [±SD]

(ng/mL)

0.700

[±0.402]

0.741

[±0.343] 105.939% 7.51009E-01, N. S.

AUC0-t [±SD]

(ng*h/mL)

1.778

[±1.559]

2.396

[±0.982] 134.796% 5.53473E-02, N. S.

Clopidogrel

carboxylic acid

Cmax [±SD]

(ng/mL)

2735.808

[±587]

2589.044

[±729] 94.635% 6.34597E-01, N. S.

AUC0-t [±SD]

(ng*h/mL)

9599.435

[±4468]

10039.278

[±1460] 104.582% 8.04498E-01, N. S.

Clopidogrel acyl

glucuronide

Cmax [±SD]

(ng/mL)

428.937

[±83]

419.236

[±74] 97.738% 6.53555E-01, N.S.

AUC0-t [±SD]

(ng*h/mL)

1372.074

[±673]

1513.754

[±591] 110.326% 3.44311E-01, N. S.

N.S., not significant


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N

S

Clopidogrel

Cl

H

N

S Cl

H

O

2-oxo-Clopidogrel

Thiol active metabolite

N

S ClClopidogrel carboxylic metabolite

esteraseCYP450

UDP-glucuronosyltransferase

N

S ClClopidogrel acyl glucuronide

CYP450

N

ClHOOC

HS

COOCH3HH

Clopidogrel "endo" thiol metabolite

PON 1

COOCH3

COOCH3

N

ClHS

COOCH3HHOOCOO

OOH

OHOH

CO2H

OHO

Figure 1


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EVALUATION OF CLOPIDOGREL CONJUGATION METABOLISM: …dmd.aspetjournals.org/content/dmd/early/2016/07/11/dmd.116.071092.full.pdf · DMD # 71092 1 TITLE PAGE EVALUATION OF CLOPIDOGREL