Lack of genetic association between OCT1, ABCB1, and UGT2B7 variants and morphine pharmacokinetics

Accepted Manuscript Lack of genetic association between OCT1, ABCB1, and UGT2B7 variants and morphine pharmacokinetics

LM Nielsen, E Sverrisdóttir, TB Stage, S Feddersen, K Brøsen, LL Christrup, AM Drewes, AE Olesen PII: DOI: Reference:

S0928-0987(16)30580-2 doi: 10.1016/j.ejps.2016.12.039 PHASCI 3856

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

21 August 2016 20 November 2016 31 December 2016

Please cite this article as: LM Nielsen, E Sverrisdóttir, TB Stage, S Feddersen, K Brøsen, LL Christrup, AM Drewes, AE Olesen , Lack of genetic association between OCT1, ABCB1, and UGT2B7 variants and morphine pharmacokinetics. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi: 10.1016/j.ejps.2016.12.039

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Lack of genetic association between OCT1, ABCB1, and UGT2B7 variants and morphine pharmacokinetics Nielsen LM*1,2, Sverrisdóttir E*2, Stage TB3, Feddersen S4, Brøsen K3, Christrup LL2, Drewes

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AM1,5 & Olesen AE1,2,5

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*These authors contributed equally to this work

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1. Mech-Sense, Department of Gastroenterology and Hepatology, Aalborg University Hospital, Aalborg, Denmark

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2. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,

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University of Copenhagen, Copenhagen, Denmark

3. Clinical Pharmacology, Department of Public Health, University of Southern Denmark,

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Odense, Denmark

Odense, Denmark

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4. Department of Clinical Biochemistry and Pharmacology, Odense University Hospital,

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5. Department of Clinical Medicine, Aalborg University, Aalborg, Denmark

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Running head: Lack of association between gene polymorphisms and morphine PK

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Corresponding author:

Anne Estrup Olesen, MSc (pharm), PhD Mech-Sense, Department of Gastroenterology & Hepatology Medicinerhuset, Mølleparkvej 4, 3rd floor

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Aalborg University Hospital

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DK-9000 Aalborg, Denmark

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Telephone: +45 9766 0535

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E-mail: [email protected]

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ABSTRACT Aim A high inter-individual variation in the pharmacokinetics and pharmacodynamics of morphine has been observed. Genetic polymorphisms in genes encoding the organic cation transporter isoform 1

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(OCT1), the efflux transporter p-glycoprotein (ABCB1), and the UDP-glucuronosyltransferase-2B7 (UGT2B7) may influence morphine pharmacokinetics and thus, also pharmacodynamics. The aim

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of this study was to evaluate the association between OCT1, ABCB1, and UGT2B7 variants, and

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morphine pharmacokinetics and -dynamics in healthy volunteers. Methods

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Pharmacokinetic and pharmacodynamic data were collected from a double-blinded, randomized,

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crossover trial in 37 healthy subjects. Pharmacokinetic data were analyzed in NONMEM®, and the time-concentration relationship of morphine, morphine-3-glucuronide, and morphine-6-glucuronide

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was parameterized as the transit compartment rate constant (ktr), clearance (CL), and volume of

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distribution (VD). The area under the plasma concentration-time curve (AUC0-150min) and the maximum plasma concentration (Cmax) were also calculated. Pharmacodynamic data were measured as pain tolerance thresholds to mechanical stimulation of the rectum and muscle, as well as tonic

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cold pain stimulation (“the cold pressor test” where hand was immersed in cold water). Six different

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single nucleotide polymorphisms in three different genes (OCT1 (n=22), ABCB1 (n=37), and UGT2B (n=22)) were examined. Results

Neither AUC0-150min, ktr, CL, nor VD were associated with genetic variants in OCT1, ABCB1, and UGT2B7 (all P>0.05). Similarly, the antinociceptive effects of morphine on rectal, muscle, and cold pressor tests were not associated with these genetic variants (all P>0.05).

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Conclusions In this experimental study in healthy volunteers, we found no association between different genotypes of OCT1, ABCB1, and UGT2B7, and morphine pharmacokinetics and pharmacodynamics. Nonetheless, due to methodological limitations we cannot exclude that

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associations exist.

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Keywords: Morphine, Interindividual variability, Pharmacokinetics, Pharmacogenetics, Healthy

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volunteers

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1. INTRODUCTION Morphine is a widely prescribed opioid for the treatment of severe acute and chronic pain. However, patients respond differently to morphine and this inter-individual variability is still a major challenge in the clinical use of morphine [1]. About 10-30 % of patients treated with

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morphine do not have adequate treatment outcome either because of inadequate analgesia or intolerable side effects [2]. Some of the inter-individual variability in the pharmacokinetic fate of

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morphine is caused by age, organ function, gender and co-morbidity [3]. However, gene variants

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relevant to opioid absorption, distribution, metabolism and excretion may also influence the

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pharmacokinetics and thus, the pharmacodynamics of morphine [3].

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Organic cation transporter 1 (OCT1) is expressed in the human hepatocytes and is involved in the distribution of organic cations, such as morphine [4]. OCT1 is highly genetically variable and five

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common reduced-function OCT1 single nucleotide polymorphisms (SNPs) are known in

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Caucasians [5, 6]. Around 10 % of Caucasians have substantially reduced or absent OCT1 activity and the OCT1 reduced-function allele limits the uptake and activity of drugs metabolized in the

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human liver [7–9]. Morphine has previously been shown to be an OCT1 substrate [10], and thus, it

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could be speculated that OCT1 reduced-function allele may affect morphine pharmacokinetics [11].

The efflux transporter P-glycoprotein, encoded by the ATP-binding cassette B1 (ABCB1) gene, have a major impact on the brain distribution of opioids such as morphine and its metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) [12, 13]. More than 50 SNPs and 3 insertion/deletion polymorphisms in ABCB1 have been suggested to affect the efflux activity of the transporter and thus the distribution of many drugs [14]. One polymorphism of interest is the SNP C3435T (rs1045642), that is associated with altered expression and function of P-glycoprotein,

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and consequently, this has led to altered pharmacokinetics and pharmacodynamics of different analgesics including morphine although the mechanisms are still unclear [15, 16]. The variant Tallele is found to be linked with lower P-glycoprotein expression and/or activity in the duodenum [17], natural killer cells [18], and renal parenchyma [19]. In animals, the lack of functional P-

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glycoprotein has been shown to have an important impact on analgesia after administration of opioids including morphine [20, 21]. A downregulation of the P-glycoprotein expression reduced

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the brain-to-blood transport of morphine and therefore, lowered P-glycoprotein expression

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significantly enhanced systemic morphine analgesia [21]. These findings indicate that genetic variation in P-glycoprotein may influence morphine absorption, distribution metabolism, and

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elimination due to it is located in the intestine, blood-brain-barrier, liver, and kidneys [22], and

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thereby, alter morphine pharmacokinetics and -dynamics.

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Morphine is primarily metabolized by UGT2B7 to 90 % M3G and 10 % M6G [1, 23]. In one study

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UGT2B7 variants have been shown to result in differences in glucuronidation activity [24]. However, the reported effect of UGT2B7 variants on glucuronidation activity is still inconsistent across studies. The UGT2B7 372 A>G (rs28365063) polymorphism has been reported to increase

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glucuronidation activity for morphine [25]. Additionally, the UGT2B7 -900 G>A (rs7438135)

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polymorphism has been associated with altered morphine pharmacokinetic in preterm infants and in young adults with sickle cell disease [26, 27].

Based on results from studies conducted so far, the influence of polymorphisms in OCT1, ABCB1, and UGT2B7 on clinical response seen after morphine administration is unclear. Investigation on the association between polymorphisms in these genes and morphine response in human

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experimental pain models might contribute with important knowledge, since confounders are limited compared to studies in patients [28, 29].

We hypothesized that genetic variation in OCT1, ABCB1, and UGT2B7 might influence the

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morphine pharmacokinetics and thereby, also influence morphine pharmacodynamics. This was investigated in a multi-modal, multi-tissue experimental pain setup in 40 healthy volunteers. The

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overall purpose of the study was 1) to evaluate associations between polymorphisms in OCT1,

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ABCB1 and UGT2B7, and the pharmacokinetics, measured as area under the plasma concentrationtime curve (AUC0-150min), the transit compartment rate constant (ktr), the clearance (CL), the volume

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of distribution (VD), and the maximum plasma concentration (Cmax) of morphine, M3G and M6G.

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And 2) to investigate if these polymorphisms were associated with morphine pharmacodynamics, measured as pain tolerance thresholds to rectal pressure stimulation, muscle pressure stimulation

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and the cold pressor test (immersion of the hand into ice water).

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2. MATERIAL AND METHODS 2.1 Study procedure The original study was conducted in the period November 2010 to April 2012. Pharmacokinetic and pharmacodynamics data were collected from this double-blinded, randomized, crossover trial with a

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washout period of at least 1 week between visits. Forty healthy Caucasians were randomized into

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four blocks (10 participants in each block receiving the same order of the study medication) to

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receive either oral morphine or placebo on day 1 or day 2. The participants received 30 mg (2 mg/mL) of oral morphine or 15 mL placebo. The North Denmark Region Committee on Health

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Research Ethics (reference no. N-20100046) and Danish Medicines Agency (reference no. 26124319) approved the study. The trial was registered at ClinicalTrials.gov with identifier number

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NCT01245244. Pharmacodynamic results of placebo versus morphine and population pharmacokinetic-pharmacodynamic models for morphine are presented in separate published papers

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[30, 31]. Furthermore, the genetic influence of mu opioid receptor (OPRM1), kappa opioid receptor

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(OPRK1), delta opioid receptor (OPRD1), and catechol-O-methyltransferase (COMT) on the pain sensitivity has been published (submitted) prior to the current publication. Genotyping of the

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ABCB1 SNP rs1045642 was carried out after the study was completed.

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An add-on study was performed to investigate the association between polymorphisms in genes encoding the transporter (OCT1) and the drug-metabolizing enzyme (UGT2B7), and morphine pharmacokinetics. This study was approved by The North Denmark Region Committee on Health Research Ethics (reference no N-20140018), and was carried out in the Research Laboratory at Mech-Sense, Department of Gastroenterology and Hepatology, Aalborg University Hospital, Aalborg, Denmark. The study was conducted in the period September 2014 to April 2015 and consisted of one study day, where two blood samples were collected with the purpose of genotyping

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for genetic variants in above mentioned genes. A research biobank of the genetic blood samples was established. 2.2 Study participants All 40 participants from the original study were contacted again if possible. Inclusion criteria were:

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1) age between 20 and 65 years; 2) opioid naïve; 3) no known allergy to morphine; 4) no ongoing

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participation in other drug studies; 5) not pregnant (all included woman were on safe contraceptive medication during the study); 6) no previous addictive behavior; 7) no previous pain causing

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diseases or psychiatric disorders. Exclusion criteria were not relevant for this study. Participants

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gave their informed consent before included in the study.

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2.3 Blood sampling and quantitative analysis of morphine and its metabolites Blood samples of 6 mL were collected in K2EDTA tubes at fixed times before and after drug

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administration (t = 0 (drug administration), 5, 10, 15, 30, 45, 60, 90, 120, and 150 min). Plasma was

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separated and kept at -80oC until analysis. The concentrations of morphine, M3G and M6G were determined using ultra-high-performance liquid chromatography/mass spectrometry as previously

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described [31].

2.4 Pharmacokinetic variables and data analysis

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The population pharmacokinetic model is presented in our previously publication [31]. Briefly, individual morphine concentration-time data were fitted to pharmacokinetic models using nonlinear mixed effects modelling (NONMEM version 7.2; ICON Plc). Morphine pharmacokinetics was best described with a one-compartment model, which was parameterized in terms of clearance (CL, L/min) and volume of distribution (VD, L). The absorption of morphine after administration in oral solution was described with a two compartment transit absorption model with a transit compartment rate constant (ktr, min-1). Individual predicted parameter estimates of ktr, CL, and VD for morphine,

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M3G, and M6G were used in the data analysis in the current publication. The AUC0-150 min of morphine was calculated in R [32], based on individual predicted concentrations. Cmax values were the maximum individual predicted concentrations. Parameter estimates are listed as medians with 25th to 75th percentile in Table 2.

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2.5 Pharmacodynamic variables and data analysis

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Data were collected from the original study and details of the experimental pain tests have been described previously [30]. In this study experimental pain testing in the healthy volunteers included

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rectal pressure stimulation with a bag, muscle pressure stimulation at the left forearm and the cold

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pressor test to left hand. These three tests were selected in order to limit multiple testing and they showed the smallest variation between subjects. The experimental tests were used as a “control”

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measurement to see if an altered morphine pharmacokinetics affect morphine pharmacodynamics. All stimulations were interrupted when the participants reported pain tolerance threshold (PTT) on a

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validated modified numerical rating scale from 0 to 10, where 5 = the pain detection threshold and 7

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= PTT [33].

Percentage change from baseline ((T60 min-baseline)/baseline*100%) for morphine (Δmor) and

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placebo (Δpla) effects in: 1) tolerated volume (mL) at PTT level for rectal stimulation; 2) tolerated

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pressure (kPa) at PTT level for muscle stimulation; and 3) area under the time-pain level curve (ice AUC) for the cold pressor test. Morphine response was calculated as difference between morphine and placebo effects (Δmor – Δpla) and presented as percentage points (PP). 2.6 DNA extraction and genotyping DNA was extracted from an aliquot of venous blood with the QIAsymphony DSP DNA Midi kit (Qiagen, Copenhagen, Denmark) using the QIAsymphonyTM SP instrument (Qiagen, Copenhagen, Denmark). Selected SNPs in OCT1 (rs12208357) and UGT2B7 (rs7438135 and rs28365063) were

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genotyped using predesigned TaqMan SNP genotyping assays on a StepOne Plus real-time instrument (Applied Biosystems, Foster City, California, USA) in accordance with the manufacturer’s protocol. The OCT1 SNPs rs72552763 and rs34130495 were genotyped by Sanger sequencing as previously described [34]. Genotyping for the ABCB1 SNP rs1045642 was carried

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out using sequence specific primers and polymerase chain reaction as described in [35]. Assay numbers and sequences of the primers used for genotyping are summarized in Supplementary Table

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S1. The three OCT1 SNPs are all located in the coding regions of the gene and lead to amino acid

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changes (rs12208357 Arg-Cys, rs34130495 Gly-Ser, rs72552763 Met-Del). Furthermore, the UGT2B7 rs28365063 (Arg-Arg) and ABCB1 rs1045642 (Ala-Ser) SNPs are placed in the coding

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regions of the gene, whereas the UGT2B7 rs7438135 SNP are not.

2.7 Statistical analysis and considerations

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All genotype frequencies were tested for Hardy-Weinberg equilibrium using Chi-square goodness-

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of-fit test (Haploview version 4.2, Broad Institute, Cambridge, USA). The demographic data are presented as medians with 25th to 75th percentile. A multivariate

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regression analysis was used to determine the effect of the genotypes on the pharmacokinetic parameters AUC0-150 min, ktr, CL, VD and Cmax of morphine. Additionally, the genetic associations

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with OCT1, UGT2B7 and ABCB1 genotypes and morphine response (Δmor – Δpla) for the pharmacodynamic variables (volume (mL), pressure (kPa) and ice AUC) were explored using multivariate regression analysis. All statistical analyses were accomplished in STATA 11.0 (StataCorp, Texas, US) and a P-value smaller than 0.05 was considered significant. P-values for the dominant (wt/wt vs. wt/v & v/v), recessive (v/v vs. wt/wt & wt/v), and additive (wt/wt vs. wt/v vs. v/v) genotype were calculated.

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A sample size was calculated for the pharmacodynamic outcomes of morphine for the primary endpoints [30]. However, a specific sample size calculation for the present study (secondary endpoint) was not accomplished since we aimed to include all 40 participants enrolled in the

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original study.

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3. RESULTS In the original study 39 completed the study (one male dropped out due to side effects). Two more participants were excluded due to methodical problems (no DNA from one male participant and no pharmacokinetic data from one female participant). Out of the 37 participants, 17 were women and

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20 men with a median age of 25 years old and these were genotyped for the ABCB1 SNP rs1045642. In the add-on study, we were in contact with 27 participants out of the 39 from the

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original study, but 5 rejected to participate. Hence, 22 participants from the original study gave

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written informed consent to provide an extra blood sample (13 women and 9 men) for the genetic association study of OCT1 and UGT2B7. Demographic characterization is shown in Table 1, and no

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significant differences were observed between the two groups.

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--- Table 1 near here --The pharmacokinetic parameters according to genotype are provided in Table 2.No significant

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differences in the parameters were seen between the different OCT1, ABCB1, and UGT2B7

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genotypes. Neither the pharmacokintics parameters obtain for M3G and M6G could not be

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demonstrated to vary according to genotype (all P-values > 0.05, data not presented here). --- Table 2 near here ---

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Morphine pharmacodynamics in the healthy volunteers according to genotype are provided in Table 3. No impact of OCT1, ABCB1, and UGT2B7 genotypes on the pharmacodynamic response to morphine could be demonstrated. --- Table 3 near here ---

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4. DISCUSSION Our results did not support the hypothesis that genetic variation in OCT1, ABCB1, and UGT2B7 influence morphine pharmacokinetics or -dynamics. 4.1 Methodological considerations

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The main weakness of this study is the explorative nature of the analysis, meaning that no sample

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size calculation was performed. However, previous experimental studies with a relative low number

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of participants have been sufficiently powered to explore the variation in pharmacokinetics between groups in comparable settings [11, 36]. Evaluating morphine plasma concentrations for only 150

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minutes after administration may exclude some important information about morphine metabolism and elimination. In addition, only a limited number of polymorphisms in three genes were

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genotyped. Thus, it cannot be excluded that other genes may affect morphine pharmacokinetics. Lastly, in genetic association studies the interaction between genes (epistasis) may complicate the

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4.2 OCT1

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be considered for future studies.

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analysis and interpretation. Interaction between genes were not included in this analysis but should

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In humans, the drug transporter OCT1 is primary localized at the basolateral membrane of epithelial cells in the liver [37] and therefore, less morphine is expected to be transported into the hepatocytes in participants with OCT1 reduced-function allele. Supporting the theory, a study has shown that in healthy Caucasian volunteers carrying one or two OCT1 reduced-function alleles a significant higher morphine AUC0-24h and a insignificant higher Cmax [11]. However, our results could neither confirm nor exclude that OCT1 reduced-function alleles have an influence on any morphine pharmacokinetic parameters.

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Both AUC and Cmax are highly dependent on the timing of bloodsampling, especially in studies with sparse sampling. In the study by Tzvetkov et al. [11], where higher values for AUC and Cmax were seen in volunteers with one or two OCT1 reduced-function alleles, blood samples were only collected every 30 min for the first 2 hours. The recorded Cmax was therefore the morphine

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concentration after 30 min for all individuals, while the actual Cmax most likely occurred before or after 30 min for most subjects. In our study, six blood samples were collected during the first 60

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min after administration and the absorption was described with a transit compartment constant (ktr),

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which was 0.226 min-1 (mean transit time = 22 min). The inter-individual variability related to the absorption parameter (ktr) was 52% CV, and thus the absorption duration varied greatly between

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individuals. We were not able to identify a difference in absorption rate related to OTC1

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polymorphism, probably due to small sample size. However, the absorption phase was much better characterised in our study than in the study by Tzvetkov et al. [11], where Cmax due to sparse

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sampling was estimated to be the same for all subjects and thus not reflecting the true inter-

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individual variability. The difference in Cmax values might therefore be related to inter-individual variability not explained by OTC1 polymorphism. The same applies for the AUC values, where the number and timing of samples is also important for an accurate estimation of the AUC. The total

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drug exposure might be the same for two individuals with very different absorption profiles, but if

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that difference in absorption is not captured due to sparse sampling, the AUC’s might be significantly different. When investigating covariate effects (including genetics) on absorption, it is crucial to have as many bloodsamples during the absorption phase as possible. Furthermore, nonlinear mixed effect modelling is superior to non-compartmental analysis as all data are analysed simultaneously to determine the typical population parameters as well as quantifying interindividual, inter-occasion, and random variability. Moreover, non-linear mixed effects modelling does not require a rich data set, which is crucial for non-compartmental analysis.

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4.3 ABCB1 The efflux transporter P-glycoprotein exerts its function in various tissues. It has been suggested that homozygotes of the ABCB1 3435T allele are good morphine responders whereas hetero- and homozygotes of the C allele are moderate responders [38]. Hence, it has been speculated whether

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the absorption of morphine is reduced in homozygotes of the C allele due to increased efflux of morphine in the gut. Consequently, reducing the bioavailability of morphine. The efflux of

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morphine from the gut epithelial cells is mainly expected to affect the absorption of morphine, but

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the transit compartment rate constant showed no significant difference between genotypes. We showed that the AUC0-150min of morphine was found slightly increased in homozygotes and

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heterozygotes for the 3435C allele, but these results were not statistically significant. The same

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trend was observed in another study of postoperative patients, where morphine concentrations 24 hours after administration were compared [39]. Thus, ABCB1 3435C allele may have a weak impact

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on morphine pharmacokinetics, but further studies are needed to confirm this association.

4.4 UGT2B7

Glucuronidation activity may also influence the effect of morphine. The difference in AUC0-150 min

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was not significantly different among the UGT2B7 -900G>A (rs7438135) genotypes. Nonetheless,

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the trend was consistent with results from another study in patients with sickle cell disease [27], where homozygous and heterozygous carriers of the G allele had higher morphine AUC than homozygous carriers of the A allele. This indicates that the G allele may be associated with reduced glucuronidation of morphine. A rather limited number of studies have investigated morphine pharmacokinetics and the genetic influence of the UGT2B7 372A>G (rs28365063) polymorphism. Contradictory results have been published with regard to the effect of UGT2B7 polymorphism on metabolism of antiepileptic drugs [40, 41], but to our knowledge no studies exist for morphine

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population pharmacokinetics. The results from the present study did not provide any further evidence regarding UGT2B7 polymorphism and association to morphine pharmacokinetics.

4.5 Pain tolerance thresholds for experimental induced pain

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Genetic variation in genes encoding drug transporters, such as OCT1 and ABCB1, and the drug-

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metabolizing enzyme UGT2B7 may have an indirect influence on morphine pharmacodynamics by changing the pharmacokinetics of the drug. In the present study we were unable to demonstrate an

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association between these polymorphisms and the antinociceptive effect of morphine. Literature

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investigating this association is limited. Twin studies have shown that up to 60% of the variation in the response to experimental pain is genetically inherited [42–44]. It has been suggested that genetic

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variation influences different types of pain and therefore, a genetic association in one experimental

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pain modality may not be present in another [45].

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This study was explorative and we recommend for future larger scale studies to: 1) include several candidate genes e.g. OCT1, ABCB1 and UGT2B7; 2) investigate several SNPs in same candidate

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gene; 3) include other pain modalities (we only included pressure in multiple tissues) [46] ; 4) include test in different tissues; 5) more frequent sampling during the first hour and sampling for six

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hours or more to enable more precise estimation of morphine absorption and elimination; 6) analysis of the influence of genetic polymorphism using nonlinear mixed effect modelling approach on parameters such as ktr/ka, CL, and VD instead of AUC and Cmax, which allows for a more integrated analysis of the pharmacokinetic and/or pharmacodynamic data and the pharmacogenetic data than ANOVA analysis [47].

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4.6 Conclusion In conclusion, no statistically significant influence of OCT1, ABCB1, and UGT2B7 genotypes on morphine pharmacokinetics or pharmacodynamics could be demonstrated. Nonetheless, due to methodological limitations we cannot exclude that associations between genetic variations in OCT1,

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ABCB1, and UGT2B7, and morphine pharmacokinetics and -dynamics exist.

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5. ACKNOWLEDGEMENT

The authors wish to thank laboratory technician Susanne Hillbrandt for invaluable technical

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assistance.

6. FINANCIAL DISCLOSURE

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This work was supported by funding from the Innovation Fund Denmark - Individuals, Disease and

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Society. TBS has held unrelated paid lectures for Eisai, Orifarm, Novartis and Astellas-Pharma.

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TABLES Table 1 Demographic characterization of the original study (n=37) and the present study (n=22) Demographic characteritics

Number

Median

25-75 percentile

Age at inclusion (years)

25

24-26

Height (m)

1.8

1.74-1.85

Weight (kg)

78

72-87

BMI (kg/m2)

24.7

22.8-26.3

Age at inclusion (years)

25

25-26.8

Height (m)

1.8

1.73-1.85

78.5

72.3-87

Original study (n=37):

Present study (n=22):

Weight (kg) 2

BMI (kg/m )

25.1

23.7-26.1

2

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BMI: Body Mass Index=weight/height

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13:9

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Gender (women:men)

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Gender (women:men)

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ACCEPTED MANUSCRIPT Table 2 Median with 25th and 75th percentile trough pharmacokinetics of the healthy volunteers treated with morphine according to genotype Gene OCT1 (n=22)

ABCB1 (n=37)

dbSNP Diplotypes for RF

rs1045642 (wt=T)

n

wt/wt Median(25-75th percentile)

AUC0-150min (min* ng/L)

14

1395 (1177 to 1502)

5

1180 (915 to 1582)

3

ktr (min-1)

14

0.2 (0.1 to 0.3)

5

0.2 (0.1 to 0.3)

3

CL (L/min)

14

17 (14.5 to 20.9)

5

17.7 (17 to 27.5)

3

VD (L)

14

1421 (1000 to 1683)

5

1933 (918 to 2054)

Cmax (ng/ml)

14

15.7 (13.2 to 20.2)

5

AUC0-150min (min* ng/L)

12

1500 (1121 to 1702)

20

ktr (min-1)

12

0.2 (0.2 to 0.3)

M

CL (L/min)

12

VD (L)

12

Parameter

UGT2B7 (n=22)

rs7438135 (wt=A)

12

n

v/v Median(25-75th percentile)

Pvalues

1312 (1216 to 1597)

0.69

T P

I R

0.2 (0.1 to 0.4)

0.72

17 (11.2 to 18.1)

0.40

3

1650 (875 to 1659)

0.61

13.1 (11.3 to 22.4)

3

15 (13.7 to 17.5)

0.65

1398 (1200 to 1624)

5

1311 (1229 to 1582)

0.51

20

0.2 (0.1 to 0.3)

5

0.3 (0.2 to 0.3)

0.91

16 (11 to 20.8)

20

16.2 (13.1 to 19.3)

5

17 (17 to 17.7)

0.83

1223 (831 to 1835)

20

1406 (970 to 1706)

5

1398 (1265 to 1658)

0.95

17.7 (12.8 to 22)

20

15.7 (13.4 to 19.5)

5

15.2 (15.0 to 18.1)

0.43

D E

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E C

Cmax (ng/ml)

n

wt/v Median(25-75th percentile)

C S U

N A

AUC0-150min (min* ng/L)

2

1129 (1042 to 1216)

15

1448 (940 to 1597)

5

1341 (1275 to 1489)

0.32

ktr (min-1)

2

0.3 (0.2 to 0.3)

15

0.2 (0.1 to 0.3)

5

0.2 (0.1 to 0.3)

0.93

CL (L/min)

2

21.3 (18.1 to 24.5)

15

17.2 (15.2 to 23.6)

5

14.5 (12.8 to 15.5)

0.47

VD (L)

2

1633 (1616 to 1650)

15

1113 (918 to 1933)

5

1490 (1352 to 1683)

0.63

Cmax (ng/ml)

2

14 (13.7 to 14.3)

15

17.5 (12.3 to 22.4)

5

14.7 (13.2 to 16.7)

0.45

C A

27

ACCEPTED MANUSCRIPT rs28365063 (wt=A)

AUC0-150min (min* ng/L)

15

1448 (1184 to 1582)

7

1180 (940 to 1498)

0

-

0.15

ktr (min-1)

15

0.2 (0.1 to 0.4)

7

0.3 (0.2 to 0.3)

0

-

0.92

CL (L/min)

15

15.6 (13.6 to 18.8)

7

18.1 (17.2 to 27)

0

-

0.05

VD (L)

15

1352 (930 to 1683)

7

1650 (1095 to 1933)

0

Cmax (ng/ml)

15

16.7 (13.2 to 22.4)

7

13.7 (12.3 to 18.6)

0

I R

T P -

0.40

-

0.40

Diplotypes for RF: reduced-function of the SNPs rs12208357, rs34130495 and rs72552763, dbSNP: single nucleotide polymorphism database identification, wt = wildtype, v = variant, P-values are for the dominant genotype (wt/wt vs. wt/v & v/v) (P-values for the recessive and additive genotype are not shown – all P > 0.05),

C S U

AUC0-150min = area under the plasma concentration-time curve from morphine administration to 150 minutes after, ktr= transit compartment rate constant, CL = clearance, VD = volume of distribution, Cmax = the maximum plasma concentration

N A

D E

M

T P E

C C

A

28

ACCEPTED MANUSCRIPT Table 3 Median with 25th and 75th percentile trough pharmacodynamics of the healthy volunteers treated with morphine according to genotype wt/wt Gene

dbSNP

OCT1 (n=22)

Diplotypes for RF

ABCB1 (n=37)

rs1045642 (wt=T)

Parameter

rs7438135 (wt=A)

v/v Median(25-75th percentile)

P-values

2

8.7 (2.5 to 14.8)

0.75

3

1.3 (-2.9 to 6.7)

0.82

-8.9 (-17.3 to -3)

3

-4.5 (-11.6 to -0.5)

0.32

19

4.4 (-15.3 to 32.9)

4

38.5 (13.8 to 72.3)

0.85

13.2 (-3.8 to 33.2)

20

13.4 (22.0 to 28.1)

5

1.3 (-26.4 to 6.3)

0.94

12

-9.1 (-14.5 to -1.7)

17

-7.0 (-16.3 to -2.0)

3

-3.5 (-8.8 to 0.4)

0.81

2

2.4 (-10 to 14.8)

14

25.9 (2.5 to 47.3)

5

-8.5 (-18.5 to -4.4)

0.92

Muscle pressure stimulation (PP)

2

8.7 (6.7 to 10.7)

15

1.3 (-12.7 to 40.8)

5

34.9 (21.7 to 43.1)

0.71

ice AUC for cold pressor test (PP)

2

-5.3 (-17.6 to 6.9)

11

-3.6 (-7.1 to -0.5)

5

-8.0 (-11.6 to -0.9)

0.98

n Rectal pressure stimulation (PP)

14

3.4 (-10 to 44.5)

5

25.6 (3.5 to 51.7)

Muscle pressure stimulation (PP)

14

23.1 (-4.7 to 40.8)

5

-5.3 (-24.6 to 43.9)

ice AUC for cold pressor test (PP)

13

-5.3 (-8 to 1.3)

2

Rectal pressure stimulation (PP)

12

1.43 (-7.2 to 16.1)

Muscle pressure stimulation (PP)

12

C C

A

Median(25-75 percentile)

D E

M

n

T P

I R

C S U

N A

T P E

Rectal pressure stimulation (PP)

n

th

Median(25-75 percentile)

ice AUC for cold pressor test (PP) UGT2B7 (n=22)

wt/v th

29

ACCEPTED MANUSCRIPT rs28365063 (wt=A)

Rectal pressure stimulation (PP)

14

15.8 (-8.5 to 44.5)

7

3.5 (-10.0 to 66.3)

0

-

0.82

Muscle pressure stimulation (PP)

15

11.1 (-12.7 to 43.1)

7

10.7 (-24.6 to 29.4)

0

-

0.53

ice AUC for cold pressor test (PP)

14

-6.2 (-11.6 to -0.9)

4

-6.2 (-16.6 to 4.4)

-

0.99

I R

T P 0

All dynamic variables are presented as morphine response (Δmorphine – Δplacebo) presented as percentage points ( ) where Δmorphine and Δplacebo are calculated as percentage change from baseline ((T60 min-baseline)/baseline*100%), Diplotypes for RF: reduced-function of the SNPs rs12208357, rs34130495 and rs72552763, dbSNP: single nucleotide polymorphism database identification, wt = wildtype, v = variant, P-values are for the dominant genotype (wt/wt vs. wt/v & v/v) (P-values for the recessive and additive genotype are not shown – all P > 0.05), ice AUC for the cold pressor test: area under time-pain level curve for the cold pressor test.

C S U

N A

D E

M

T P E

C C

A

30

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Graphical abstract

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