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Oral morphine pharmacokinetic in obesity: the role of P-glycoprotein, MRP2, MRP3, UGT2B7 and CYP3A4 jejunal contents and obesity-associated biomarkers Celia Lloret-Linares, Eisuke Miyauchi, Huilong Luo, Laurence Labat, Jean-Luc Bouillot, Christine Poitou, Jean-Michel Oppert, Jean-Louis Laplanche, Stephane Mouly, Jean-Michel Scherrmann, Yasuo Uchida, Masanori Tachikawa, Tetsuya Terasaki, Jean-François Bergmann, and Xavier Decleves Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00656 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 17, 2016

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Molecular Pharmaceutics

Oral morphine pharmacokinetic in obesity: the role of P-glycoprotein, MRP2, MRP3, UGT2B7 and CYP3A4 jejunal contents and obesity-associated biomarkers Running title: Morphine, intestine and obesity

Célia Lloret-Linares1,2 *, Eisuke Miyauchi3, Huilong Luo1,4, Laurence Labat1,4, Jean-Luc Bouillot5, Christine Poitou6, Jean-Michel Oppert6, Jean-Louis Laplanche1, Stéphane Mouly1,2, Jean-Michel Scherrmann1, Yasuo Uchida3, Masanori Tachikawa3, Tetsuya Terasaki3, Jean-François Bergmann1,2, Xavier Declèves1,4

1.

Inserm, UMR-S 1144 Université Paris Descartes-Paris Diderot, Variabilité de réponse aux psychotropes, France

2.

Assistance Publique-Hôpitaux de Paris, Hôpital Lariboisière, Therapeutic Research Unit, Department of Internal Medicine, Paris F-75010, France

3.

Membrane Transport and Drug Targeting Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan

4.

Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Pharmacokinetics and Pharmacochemistry Unit, Paris F-75014, France

5.

Assistance Publique-Hôpitaux de Paris, Hôpital Ambroise Paré, Université Versailles Saint Quentin, Department of Surgery, Boulogne, France

6.

Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière, Service de Nutrition, Université Pierre et Marie Curie, Institut cardiométabolisme et nutrition (ICAN), Paris F-75013, France

*Address all correspondence to: Dr Celia Lloret-Linares, MD, PhD Hôpital Lariboisière– Service de Médecine Interne A 2 rue Ambroise Paré-75010 Paris – France Tel: 33-1-49 95 81 27 ; Fax: 33-1-49 95 84 46 Email: [email protected] Word Count:3575 Tables: 3+supplemental table : 1; Figures: 1

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ABSTRACT

Objective: To study the association between the jejunal expression levels of P-gp, MRP2, MRP3, UGT2B7, CYP3A4, the ABCB1 c.3435C>T polymorphism and several obesity-associated biomarkers, and oral morphine and glucuronides pharmacokinetics in a population of morbid obese subjects. Methods: The pharmacokinetics of oral morphine (30mg) and its glucuronides was performed in obese patients candidate to bariatric surgery. A fragment of jejunal mucosa was preserved during surgery. Subjects were genotyped for the ABCB1 single nucleotide polymorphism (SNP) c.3435C>T. Results: The subjects were 6 males and 23 females, with a mean body mass index of 44.8 (35.4-61.9) kg/m2. The metabolic ratios AUC0-inf M3G/Morphine and AUC0-inf M6G/Morphine were highly correlated (rs=0.8, pT single nucleotide polymorphism and several obesity-associated biomarkers and oral morphine PK.


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MATERIAL AND METHODS Study Population

Morbidly obese volunteers involved in a medico-surgical obesity management program (Department of Nutrition, Pitié-Salpêtrière hospital, Paris, and Department of Digestive and metabolic surgery, Ambroise Paré hospital, Boulogne-Billancourt, France) and candidates for a Roux-en-Y gastric bypass (RYGB) were included in the Obesity and morphine (OBEMO) clinical study 14. Subjects with diabetes, renal or hepatic dysfunction, untreated obstructive sleep apnea syndrome, those usually treated with sedative or analgesic drug or with a history of allergy to morphine or opioids were not included. Their preoperative tests included a clinical chemistry check-up focusing on liver and renal function assessment (aspartate aminotransferase, AST; alanine aminotransferase, ALT; gamma-glutamyl transferase, GGT), determination of serum creatinine, insulin, inflammatory markers (interleukine-6 (IL6), ultrasensitive C-Reactive Protein (CRPus), orosomucoid), insulin and adipocytokines (adiponectin and leptin)

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. All subjects gave their written informed consent. The protocol

“OBEMO study” was approved by the Ethics Committee of Paris, France (CPP Ile de France I) and registered at the ClinicalTrials.gov website (EudraCT number 2009-010670-38, NCT00943969).

Oral morphine PK study

Study design A few days prior to RYGB surgery, each patient fasted overnight before being given a single oral dose of morphine sulphate solution (30 mg; Oramorph 5mL, Roxane Laboratories, Inc, Columbus, Ohio). The morphine plasma levels were measured in each of nine blood samples taken over the 12 hours post dose (0h, 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 12h). Additional samples were taken for some subjects during the first two hours. Plasma samples were separated and frozen at -20°C. Morphine, M3G and M6G quantification in plasma Morphine and its major metabolites (M3G and M6G) were analysed in plasma by a validated method using liquid chromatography mass spectrometry in tandem (LC-MS/MS) following solid phase extraction (SPE). Chromatographic separations were performed on a LC system (Accela system, Thermo Fisher) with Hypersil Gold® (Thermo Fisher) C18 column (2.1 × 100 mm, 3 µm) at a flow rate of 0.2 mL/min. The mobile phase consisted of 0.1% (v/v) formic acid water (A) and 0.1% (v/v) formic acid acetonitrile (B) with a gradient elution. Mass spectrometry was carried out in electrospray positive ionization (ESI) mode on a TSQ Quantum Ultra® system (Thermo Fisher). Selective Reaction Monitoring (SRM) transitions were 286.1→201.0 for morphine


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(m/z), 462.1→285.9 for M3G and M6G, 289.1→201.0 for morphine-d3 (internal standard (IS)), 465.1→288.9 for M3G-d3 (IS) and M6G-d3 (IS). Quantification methods were validated over the tested concentration ranges (0.15-200 ng/mL for morphine, 0.75-1000 ng/mL for M3G, 0.15-200 ng/mL for M6G). Intra- and inter-day precisions were less than 10%.

PK analysis The plasma morphine, M3G and M6G concentrations versus time data obtained from each individual subject were submitted to a non-compartmental pharmacokinetic analysis using WinNonlin® (version 4.1, Pharsight Corporation, Cary, NC, USA). The area under the plasma concentration–time curve (AUC0-Clast) from time zero to the last time point (Clast) where plasma levels for the three compounds of interest were adequately determined by the linear up and log down trapezoidal rule. Extrapolation of AUC (AUCClast-infinity) from the Clast to infinity was calculated from the Clast divided by the slope of the terminal elimination phase (ke) and was lower than 20% in all subjects and for all three compounds. AUC0-inf was calculated as the sum of AUC0-Clast and AUCClast-infinity. The residual AUC extrapolated from the Clast to infinity was calculated from Clast (predicted)/slope of the terminal phase determined using the 3 to 5 last sampling times. We also calculated the AUC from time zero to 4 hours after morphine dose to analyse the absorption phase. The terminal half-life (t1/2) of all compounds was computed from ln2/ke. The mass and molar ratios AUC0-inf M3G/Morphine and M6G/Morphine were calculated with AUC0-inf in ng.h/mL and in nmol.h/mL, respectively. Values for the maximum plasma concentration (Cmax) and time to Cmax (Tmax) were obtained directly from the observed individual plasma concentration–time curves.

Protein jejunal contents of transporters and enzymes

All subjects underwent RYGB performed by the same surgical team using the same laparoscopic technique, as previously described [14]. For 28 subjects, a fragment of jejunal mucosa located about 40-50 centimeters after the usual gastroduodenal junction (or ligament of Treitz) and considered as a surgical waste was preserved during surgery. Immediately after resection, the mucosal samples of the intestinal segments were snap frozen using liquid nitrogen and stored at -80°C. Protein levels of P-gp, MRP2, MRP3, CYP3A4 and UGT2B7 were chosen for close examination as they are located in the human small intestine and may be involved in the oral PK of morphine, M6G and M3G. The frozen jejunal tissues were homogenized using a Potter-Elvehjem homogenizer in hypotonic buffer [10mM Tris–HCl (pH 7.4), 10mM NaCl, 1.5mM MgCl2, 1mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (1% v/v, Sigma Chemical Co., St. Louis, MO)].


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The homogenates were subjected to nitrogen cavitation (450 psi for 15 min at 4°C) and centrifuged at 10,000 g for 10 min at 4°C twice. The supernatants were centrifuged at 100,000g for 40 min at 4°C. The resulting pellet was suspended in suspension buffer (10 mM Tris-HCl buffer (pH 7.4) containing 0.25M sucrose), and the part of the solution was obtained as the microsomal fraction for the protein quantification of CYP3A4 and UGT2B7. The remaining portion was layered on top of a 38% w/v sucrose solution and centrifuged at 100,000g for 40 min at 4°C. The turbid layer at the interface was collected and centrifuged at 100,000g for 40 min at 4°C. The resulting pellet was suspended in the suspension buffer and obtained as the plasma membrane fraction for the protein quantification of P-gp, MRP2, and MRP3. Protein concentrations were measured by the Lowry method using the DC protein assay reagent (Bio-Rad Laboratories, Hercules, CA). The jejunal protein amounts of the target molecules were determined by multiplexed selective/multiple reactions monitoring (SRM/MRM) in the LC-MS/MS system according to the previous reports [16, 17, 18, 19] with some modifications

16-18

. In brief, the LC-MS/MS system consisted of a NanoLC-Ultra 1Dplus system (Eksigent

Technologies, Dublin, CA, USA) coupled with a cHiPLC-nanoflex system (Eksigent Technologies) and a TripleTOF5600 (AB SCIEX, Framingham, MA, USA) equipped with a NanoSpray III ion source (AB SCIEX). The peptide samples prepared from the jejunal mucosa were separated and eluted from the column at a flow rate of 300 nL/min with a linear gradient as follows; 0.1% formic acid in water: 0.1% formic acid in acetonitrile: 100:0 for 40 min after injection for sample loading, 60:40 at 40 min, 0:100 at 41 min and up to 50 min, 100:0 at 50.1 min and up to 80 min. The SRM/MRM transitions for the quantification of each peptide were set as shown in supplemental Table 1.

Genotyping Analysis

Automated DNA purification was obtained from peripheral blood cells of the subjects using the Maxwell® 16 LEV Blood DNA Kit on a Maxwell 16 instrument (Promega, Fitchburg, Wisconsin, USA). Subjects were genotyped for the ABCB1 single nucleotide polymorphism (SNP) c.3435C>T (rs1045642) using allelicdiscrimination TaqMan® SNP Genotyping Assay (Assay ID C_7586657_20, Life Technologies, Carlsbad, CA, USA). Positive heterozygous and homozygous controls (defined by direct sequencing) and negative control (water) were systematically included in the experiments.

Statistics


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Statistical analysis was performed using JMP software from SAS institute, version 11. Quantitative data were presented as mean, standard deviation (SD) and minimum-maximum values. Quantitative data were compared using the Mann–Whitney test or the Wilcoxon test. The different correlations were estimated using the Spearman rank order correlation test (rs=correlation coefficient), and only the correlations with a p value of 0.05 or lower (p

Oral Morphine Pharmacokinetic in Obesity: The ... - ACS Publications

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