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. DMD # 71092 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.) 1 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. DMD # 71092 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 2 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. DMD # 71092 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 3 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. DMD # 71092 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. 4 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. DMD # 71092 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 5 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. DMD # 71092 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- 6 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. DMD # 71092 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