PK studies in man and mice of clopidogrel acyl glucuronide

Transcription

DMD Fast Forward. Published on July 11, 2016 as DOI: 10.1124/dmd.116.071092
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TITLE PAGE
EVALUATION OF CLOPIDOGREL CONJUGATION
METABOLISM: PK STUDIES IN MAN AND MICE OF
CLOPIDOGREL ACYL GLUCURONIDE
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.)
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Downloaded from dmd.aspetjournals.org at ASPET Journals on October 13, 2016
Simona Nicoleta Savu, Luigi Silvestro, Mariana Surmeian, Lina Remis,
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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: simona.duna@diss.ro
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
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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i.v. – intravenous
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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
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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activated charcoal intake (intestinal reabsorption blockade). Here, study objectives
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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
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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warranted.
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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].
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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Should CAG participate in vivo to any process resulting in back-conversion to
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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
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 charcoalinteraction 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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volunteers treated with clopidogrel alone or in combination with activated charcoal;
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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: d3clopidogrel hydrogensulfate (SynFine Research, Canada), clopidogrel Acyl-β-D-
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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glucuronide (TRC, Canada) and 13C6-clopidogrel carboxylic acid (Alsachim, France).
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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 prechilled 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
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 (d3clopidogrel hydrogensulfate, clopidogrel Acyl-β-D-glucuronide and
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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 icecold 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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immediately immersed in water and ice bath until centrifugation (performed at a
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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
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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
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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[FDA, 2013; EMA, 2011]. No sequences had to be rejected due to quality control or
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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).
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 d3clopidogrel 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, 3648h; 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-Dglucuronide and
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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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Extraction of metabolites from biological samples.
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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
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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for dilution and 250 mg glass beads per gram of sample. The remaining volume of the
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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
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
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C6-
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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rate of 0.2 mL/min. The injection volume was 10 μL, the temperature of the
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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
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 (08 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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concentration versus time profiles obtained for clopidogrel acyl glucuronide and
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during the elimination phase were slightly increased when charcoal was coadministered. 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
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,
15
conditions and 10.6 hours when charcoal was co-administered; this difference was
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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
No statistically significant difference in urinary recovery of clopidogrel acyl
glucuronide was identified in any of the collection intervals.
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depicted in Figure 6-B.
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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-
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
17
esterification/hydrolysis, deconjugation during entero-hepatic recycling), a relevant
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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,
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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following tail injection the time lag till maximal plasma levels of clopidogrel
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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
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 bilebinding 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
19
occurs much earlier than complete absorption of the prodrug. In fact, an in silico
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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
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,
20
above the 0.05 significance level for both Cmax and AUC0-t data).
DMD # 71092
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
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.
21
charcoal, it can also be stated that it is unlikely that clopidogrel acyl glucuronide
DMD # 71092
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.
22
DMD # 71092
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.
23
DMD # 71092
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7430, revised 2012/04/2. Available online at:
DMD # 71092
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].
29
DMD # 71092
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
(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)
30
administration of clopidogrel in human subjects (N=6) with and without charcoal
DMD # 71092
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
Oral administration
(N = 35, parallel, 5 animals per
(N= 30, parallel, 5 animals per
sampling point)
sampling point)
Cmax
AUC0-t
Cmax
AUC0-t
[±SD]
[±SD]
(ng/mL)
(ng*h/mL)
2280
4586
[±331]
[±807]
45000
93660
[± 5207]
[±13806]
Tmax
Clopidogrel acyl
[±SD]
[±SD]
(ng/mL)
(ng*h/mL)
23454
15425
(h)
Tmax
0.3
glucuronide
[±1755]
[±8645]
Clopidogrel
18395
99265
1.0
6.0
carboxylic acid
[±1382]
[±4980]
31
(h)
1.0
Intravenous administration
DMD # 71092
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
Parameter
Charcoal
With
Charcoal Charcoal ratio
(GeoMean) (GeoMean)
0.700
0.741
(ng/mL)
[±0.402]
[±0.343]
AUC0-t [±SD]
1.778
2.396
(ng*h/mL)
[±1.559]
[±0.982]
Clopidogrel
Cmax [±SD]
2735.808
2589.044
carboxylic acid
(ng/mL)
[±587]
[±729]
AUC0-t [±SD] 9599.435
Clopidogrel acyl
glucuronide
[±4468]
[±1460]
Cmax [±SD]
428.937
419.236
(ng/mL)
[±83]
[±74]
(ng*h/mL)
[±673]
effect
(p-Value, interpretation)
105.939%
7.51009E-01, N. S.
134.796%
5.53473E-02, N. S.
94.635%
6.34597E-01, N. S.
104.582%
8.04498E-01, N. S.
97.738%
6.53555E-01, N.S.
110.326%
3.44311E-01, N. S.
10039.278
(ng*h/mL)
AUC0-t [±SD] 1372.074
(%)
Treatment as fixed
1513.754
[±591]
N.S., not significant
32
Cmax [±SD]
Clopidogrel
Charcoal/No
DMD Fast Forward. Published on July
as DOI:
COOCH
3 10.1124/dmd.116.071092
H 11, 2016
N
S
Cl
Clopidogrel
CYP450
H
esterase
O
COOCH3
N
N
O
OH
S
Cl
Clopidogrel carboxylic metabolite
S
Cl
2-oxo-Clopidogrel
CYP450
UDP-glucuronosyltransferase
PON 1
H
HOOC
COOCH3
H
N
HS
HOOC
HS
COOCH3
H
N
O
O
Cl
Thiol active
metabolite
Cl
Clopidogrel "endo"
thiol metabolite
O
CO2H
OH
OH
OH
N
S
Cl
Clopidogrel acyl glucuronide
Figure 1

PK studies in man and mice of clopidogrel acyl glucuronide

Transcription

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