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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, Downloaded from
Yuksel Rasit, Simona Rizea Savu, Constantin Mircioiu
University of Medicine and Pharmacy "Carol Davila", Faculty of Pharmacy, Department of dmd.aspetjournals.org
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.) at ASPET Journals on September 23, 2021
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.)
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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 Downloaded from
Mobile phone: +40 758 109 202
E-mail: [email protected] dmd.aspetjournals.org
Document statistics:
Abstract - 242
Introduction - 748 at ASPET Journals on September 23, 2021
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 Downloaded from i.v. – intravenous
K2EDTA - di-potassium ethylenediaminetetraacetic acid dmd.aspetjournals.org LLOQ – lower limit of quantification
MRM - multiple reactions monitoring
N. A. - not applicable at ASPET Journals on September 23, 2021
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 Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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 Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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]. Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021
(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 Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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- Downloaded from 13 glucuronide (TRC, Canada) and C6-clopidogrel carboxylic acid (Alsachim, France).
Commercially available reagents of analytical grade purity were used for sample
processing. dmd.aspetjournals.org
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). at ASPET Journals on September 23, 2021
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 Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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-
13 clopidogrel hydrogensulfate, clopidogrel Acyl-β-D-glucuronide and C6-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,
13 clopidogrel Acyl-β-D-glucuronide and C6-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 Downloaded from [FDA, 2013; EMA, 2011]. No sequences had to be rejected due to quality control or calibration failure.
Human Oral Pharmacokinetics and Elimination Study dmd.aspetjournals.org
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 at ASPET Journals on September 23, 2021 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). Downloaded from 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- dmd.aspetjournals.org clopidogrel hydrogensulfate, 200 ng/mL clopidogrel Acyl-β-D-glucuronide and 200
13 ng/mL C6-clopidogrel carboxylic acid in acetonitrile, and then diluted with 200 μL
ice-cold acetonitrile. Afterwards they were vortex for 3 minutes and centrifuged at at ASPET Journals on September 23, 2021
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-
13 glucuronide and C6-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 Downloaded from 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 dmd.aspetjournals.org was recovered and processed in the same manner as previously described for thawed urine samples.
Clopidogrel, clopidogrel carboxilic acid and clopidogrel acyl glucuronide at ASPET Journals on September 23, 2021 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 Downloaded from 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 dmd.aspetjournals.org 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
13
glucuronide; 308.2/95.0 for clopidogrel carboxylic acid and 314.1/158.1 for C6- at ASPET Journals on September 23, 2021 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 Downloaded from 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. dmd.aspetjournals.org
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. at ASPET Journals on September 23, 2021
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 Downloaded from 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 dmd.aspetjournals.org 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. at ASPET Journals on September 23, 2021
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 Downloaded from depicted in Figure 6-B.
No statistically significant difference in urinary recovery of clopidogrel acyl dmd.aspetjournals.org glucuronide was identified in any of the collection intervals.
at ASPET Journals on September 23, 2021
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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- Downloaded from esterification/hydrolysis, deconjugation during entero-hepatic recycling), a relevant physiological model was chosen. For gaining insight into the biodisposition of the dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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, Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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 Downloaded from 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% dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021
[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 Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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,
20 DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092 This article has not been copyedited and formatted. The final version may differ from this version.
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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 Downloaded from 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 dmd.aspetjournals.org 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. at ASPET Journals on September 23, 2021
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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. Downloaded from
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at ASPET Journals on September 23, 2021
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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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at ASPET Journals on September 23, 2021
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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 Downloaded from
(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 dmd.aspetjournals.org 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 at ASPET Journals on September 23, 2021 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 Oral administration
(N = 35, parallel, 5 animals per (N= 30, parallel, 5 animals per Downloaded from
sampling point) sampling point)
Cmax AUC0-t Cmax AUC0-t Tmax Tmax [±SD] [±SD] [±SD] [±SD] dmd.aspetjournals.org
(ng/mL) (ng*h/mL) (h) (ng/mL) (ng*h/mL) (h)
Clopidogrel acyl 23454 15425 2280 4586 0.3 1.0 glucuronide [±1755] [±8645] [±331] [±807] at ASPET Journals on September 23, 2021
Clopidogrel 18395 99265 45000 93660 6.0 1.0 carboxylic acid [±1382] [±4980] [± 5207] [±13806]
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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)
Result of ANOVA for No With Charcoal/No Treatment as fixed Parameter Charcoal Charcoal Charcoal ratio effect (GeoMean) (GeoMean) (%) (p-Value, interpretation) Downloaded from
Cmax [±SD] 0.700 0.741 Clopidogrel 105.939% 7.51009E-01, N. S. (ng/mL) [±0.402] [±0.343]
AUC0-t [±SD] 1.778 2.396 dmd.aspetjournals.org 134.796% 5.53473E-02, N. S. (ng*h/mL) [±1.559] [±0.982]
Clopidogrel Cmax [±SD] 2735.808 2589.044 94.635% 6.34597E-01, N. S. carboxylic acid (ng/mL) [±587] [±729] at ASPET Journals on September 23, 2021
AUC0-t [±SD] 9599.435 10039.278 104.582% 8.04498E-01, N. S. (ng*h/mL) [±4468] [±1460]
Clopidogrel acyl Cmax [±SD] 428.937 419.236 97.738% 6.53555E-01, N.S. glucuronide (ng/mL) [±83] [±74]
AUC0-t [±SD] 1372.074 1513.754 110.326% 3.44311E-01, N. S. (ng*h/mL) [±673] [±591]
N.S., not significant
32 DMD Fast Forward. Published on JulyH 11, 2016COOCH as DOI:3 10.1124/dmd.116.071092 This article has not been copyedited and formatted. The final version may differ from this version. N
S Cl Clopidogrel
CYP450 esterase
O OH H COOCH3 N N O S S Cl Cl 2-oxo-Clopidogrel Clopidogrel carboxylic metabolite CYP 450 UDP-glucuronosyltransferase
PON 1 CO2H COOCH H H 3 OH O OH N O O H COOCH3 HOOC OH HOOC HS
Cl Downloaded from N Thiol active N metabolite HS Cl S Cl Clopidogrel "endo" Clopidogrel acyl glucuronide thiol metabolite Figure 1 dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021