Accepted Manuscript Title: Pharmacogenetic Aspects of Tramadol Pharmacokinetics and Pharmacodynamics After a Single Oral Dose Author: Salumeh Bastami Pernilla Haage Robert Kronstrand Fredrik C. Kugelberg Anna-Lena Zackrisson Srinivas Uppugunduri PII: DOI: Reference:
S0379-0738(14)00096-6 http://dx.doi.org/doi:10.1016/j.forsciint.2014.03.003 FSI 7534
To appear in:
FSI
Received date: Revised date: Accepted date:
31-10-2013 20-2-2014 2-3-2014
Please cite this article as: S. Bastami, P. Haage, R. Kronstrand, F.C. Kugelberg, A.-L. Zackrisson, S. Uppugunduri, Pharmacogenetic Aspects of Tramadol Pharmacokinetics and Pharmacodynamics After a Single Oral Dose, Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pharmacogenetic Aspects of Tramadol Pharmacokinetics and Pharmacodynamics After a Single Oral Dose
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Salumeh Bastami*1, Pernilla Haage*1,2, Robert Kronstrand1,2, Fredrik C. Kugelberg1,2, Anna-Lena Zackrisson2, Srinivas Uppugunduri3
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* Both authors contributed equally to this work
Affiliations
an
1 Department of Medical and Health Sciences, Division of Drug Research, Linköping University, Linköping, Sweden
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2 National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden
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3 Department of Clinical and Experimental Medicine, Linköping University, Department of Clinical Chemistry, County Council of Östergötland, Linköping, Sweden.
Anna-Lena Zackrisson
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Corresponding author
Ac ce p
National Board of Forensic Medicine Department of Forensic Genetics and Forensic Toxicology Artillerigatan 12 SE 587 58 Linköping, Sweden
E-mail: [email protected] Phone: +46 13 252153 Fax: +46 13 136005
Page 1 of 23
Pharmacogenetic Aspects of Tramadol Pharmacokinetics and Pharmacodynamics After a Single Oral Dose
ip t
Abstract
The major purpose of this study was to elucidate if genotyping can facilitate interpretations of tramadol (TRA) in
cr
forensic case work, with special regard to the estimation of the time of drug intake and drug related symptoms
(DRS). The association between genetic polymorphisms in CYP2D6, OPRM1 and ABCB1 and pharmacokinetic
us
and pharmacodynamic properties of TRA was studied. Nineteen healthy volunteers were randomized into two groups receiving a single dose of either 50 or 100 mg of orally administrated TRA. Blood samples were collected
an
prior to dosing and up to 72 h after drug intake. The subjects were asked to report DRS during the experimental day. We found a positive correlation between the metabolic ratio of O-desmethyltramadol (ODT) to TRA and the
M
time after drug intake for both CYP2D6 intermediate metabolizers and extensive metabolizers. For the only poor metabolizer with detectable ODT levels the metabolic ratio was almost constant. Significant associations were found between the area under the concentration-time curve (AUC) and three of the investigated ABCB1 single
d
nucleotide polymorphisms for TRA, but not for ODT and only in the 50 mg dosage group. There was great
te
interindividual variation in DRS, some subjects exhibited no symptoms at all whereas one subject both fainted and vomited after a single therapeutic dose. However, no associations could be found between DRS and investigated
Ac ce p
polymorphisms. We conclude that the metabolic ratio of ODT/TRA may be used for estimation of the time of drug intake, but only when the CYP2D6 genotype is known and taken into consideration. The influence of genetic polymorphisms in ABCB1 and OPRM1 requires further study.
Key Words Tramadol, Pharmacokinetics, Pharmacodynamics, CYP2D6, ABCB1, OPRM1.
Page 2 of 23
1. Introduction The use of tramadol (TRA) for treatment of moderate to severe pain has been increasing steadily during the last decades. Unfortunately, abuse of this drug is also becoming more common. Further, development of addiction to TRA in association with analgesic treatment within the recommended dose range is another alarming trend. A
ip t
history of abuse or use of a drug of abuse seems to be an important risk factor [1]. TRA has also become a more common cause of death in drug addicts with a similar trend for increase in overdose cases [2, 3].
cr
Analysis of drugs of abuse is a common feature of forensic investigations and correct interpretation of the measured concentrations is important in both post mortem and human performance toxicology. Accurate
us
estimation of the time of drug intake and expected drug effects from a certain dose or concentration are also
an
frequent issues in drug-facilitated crimes.
TRA has a dual mechanism of action, acting as a µ-opioid receptor agonist as well as a serotonin and
M
norepinephrine reuptake inhibitor. The cytochrome P450 (CYP) enzyme CYP2D6 is involved in the formation of the active metabolite O-desmethyltramadol (ODT). In comparison to TRA, ODT is a significantly more potent μopioid agonist [4]. The concentration of the parent compound alone is often not sufficient to make an accurate
d
estimation of the time of drug intake. The ratio between metabolite and parent compound is generally used to
te
indicate a recent acute intake, e.g. to diagnose suspected acute overdoses. It has earlier been shown, for some substances other than TRA, that the ratio between metabolite and parent compound also can be helpful in a more
Ac ce p
accurate estimation of the time of intake [5, 6]. The amount of ODT formed is largely dependent on the CYP2D6 genotype but also on the time of intake. It is however unclear if the metabolic ratio (MR) of ODT/TRA is a useful indicator of the time of TRA intake.
There is considerable variation in the interindividual response to the same dose of opioids, including TRA, with respect to both therapeutic and adverse effects. Development of tolerance due to prolonged use of opioids in addition to genetic factors could partly explain some of these differences. Several genes and polymorphisms have been studied in this respect. Reduced or absent metabolite formation with reduced analgesic effect have been observed after TRA administration in CYP2D6 poor metabolizers (PMs) [7], whereas ultrarapid metabolizers (UMs), are instead associated with quicker analgesic effects, but with higher risk for adverse effects [8].
Page 3 of 23
The ABCB1 gene encodes P-glycoprotein (P-gp), which is located in the blood-brain barrier and gut and is responsible for the cellular efflux of a variety of drugs [9]. The most common single nucleotide polymorphisms (SNPs) in the coding region are C1236T, G2677T and C3435T [10]. C3435T, the most studied SNP, has been associated with both increased and decreased expression of P-gp [9]. An altered expression in gut could potentially
ip t
change the pharmacokinetics of the drugs being substrates of this transporter. A decreased expression and functionality of P-gp has been suggested in homozygous for the 3435T allele [11], possibly leading to higher
cr
bioavailability and subsequently a higher concentration of TRA in blood. Although P-gp is of importance for the pharmacokinetic and pharmacodynamic properties of a number of substances, including opioids [9, 12], its
us
importance for TRA remains unknown. A clinical study suggested that TRA is a substrate of P-gp [13]. In contrast, an in vitro study and one in vivo study in rat have demonstrated that TRA and ODT are not P-gp
an
substrates [14, 15]. A recent study showed no association between the C3435T polymorphism and pain relief in patients receiving TRA [16]. Taken together, the importance of P-gp for the pharmacokinetic and
M
pharmacodynamics properties of TRA still remains to be corroborated.
Genetic polymorphisms in OPRM1 have been associated with an altered pain threshold and opioid requirements.
d
Results from clinical studies have shown that patients’ homozygous for the wild-type of the most common SNP
te
A118G require less opioids than those with the other allelic variants, AG and GG [17, 18]. It has also been shown that the 118G allele is associated with a more severe clinical outcome in emergency department patients with acute
Ac ce p
drug overdose [19]. There is only limited information regarding the influence of OPRM1 A118G polymorphism on TRA efficacy [20].
We undertook a human study to elucidate if genotyping can facilitate interpretations of TRA in forensic case work, with special regard to the estimation of the time of drug intake and drug related symptoms (DRS). Our study had the following specific aims:
1) Investigate if the metabolic ratio (MR) of ODT/TRA is a useful indicator of the time of TRA intake. 2) Determine the association between polymorphisms in the ABCB1 gene (SNPs G1199A, C1236T, G2677T, C3435T) and pharmacokinetic parameters of TRA. 3) Study the association between polymorphisms in the CYP2D6, OPRM1 (SNP A118G) or ABCB1 (SNPs G1199A, C1236T, G2677T, C3435T) gene and DRS.
Page 4 of 23
2. Methods 2.1 Study participants and study design Twenty healthy volunteers were recruited through advertisements. Nineteen subjects (nine males, ten females)
ip t
aged 18 years or older (mean 25.4±4.3) completed the study, whereas one subject did not show up on the experimental day due to unspecified illness. Participants were informed and examined by a physician before
cr
inclusion in the study. Questions of present or previous drug use were asked. Previous or concurrent use of opioids or drugs known of interacting with TRA were exclusion criteria. Demographic data such as age, gender, height
us
and weight were noted. Seven of the female participants were taking oral contraceptives. None of the participants stated upon question that they were pregnant or breast-feeding. Written informed consent was collected from each
an
participant. The study was approved by the Regional Ethical Review Board in Linköping (No: 2011/337-31). The subjects were randomized into two groups receiving a single dose of either 50 or 100 mg of orally administered TRA (Tramadol HEXAL, Sandoz). During the experimental day, blood samples were obtained from a peripheral
M
venous catheter, Insyte W in combination with a Mandrin (Becton Dicinson AB), which was inserted in the forearm. The blood was collected in labeled 7 ml heparinized collection tubes using a Vacutainer Luer-Lok Access
d
Device (Becton Dicinson AB). Blood samples were collected prior to dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5,
te
6, 7, 8, 10, 24, 48 and 72 h. Samples were stored at –80 ºC pending analysis. The participants were exhorted to eat breakfast according to their usual routines. Lunch was bought from a nearby restaurant and served around noon.
Ac ce p
Fruit, some biscuits, tea, coffee, juice and water were available during the whole day. The subjects were requested to fill in a form regarding their experience of DRS during the experimental day in conjunction to the last blood sampling on the first day (10 h). Seven questions about nausea, dizziness, headache, vomiting, dry mouth, sweating and fatigue were posed. A scale between zero to five were used, where zero was no symptoms at all and five was worst imaginable symptoms.
2.2 Quantitation of tramadol and O-desmethyltramadol in whole blood 2.2.1 Chemicals and reagents Acetonitrile (gradient grade), methanol (gradient grade) and formic acid (98 %) were purchased from Merck (Darmstadt, Germany). Ammonium formate (98 %) and ethanol (95 %) were purchased from Fluka (Basel, Switzerland) and Kemetyl (Haninge, Sweden) respectively. Water used was first purified with a MilliQ-water purifying system (Millipore Corporation, Bedford, MA, USA). Reference substances used for making calibrators and quality controls (QCs), i.e. cis-tramadol and O-desmethyl-cis-tramadol were purchased from Cerilliant
Page 5 of 23
(Austin, TX, USA). Tramadol-13c-d3 and O-desmethyl-cis-tramadol-d6, used for making the internal standard were also purchased from Cerilliant.
2.2.2 Instrumentation
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High performance liquid chromatography was performed on a 1290 Infinity LC instrument (Agilent Technologies), using a 2.1x100 mm Zorbax Eclipse Plus C18 RRHD column with 1.8 μm particles. A guard filter
cr
with 0.2 μm particles (Waters) was also used. Column temperature was set to 60 ˚C. Mass detection (MS/MS) was performed on an AT6460 instrument (Agilent Technologies) with an electrospray interface, using positive
us
ionization. Mobile phase A consisted of 0.05 % formic acid in 10 mM ammonium formate while mobile phase B consisted of 0.05 % formic acid in methanol. Gradient elution with a total run time of 8 min was used. The total
an
flow rate was 0.5 ml/min. The software used was Masshunter (Agilent Technologies). Identification criteria were based on transition ratios. Both TRA and ODT exhibit a very strong base peak of 58.1 and few other fragments, which have abundance less than 10 % of base peak. In accordance with European recommendations (EUD
M
2002/657/EC) an acceptance limit of 50 % was used. The following transitions were used for TRA, ODT, tramadol-13c-d3, and O-desmethyl-cis-tramadol-d6, respectively: 264.2 → 58.1; 246.1, 250.1 → 58.1; 232.1,
2.2.3 Sample preparation
te
d
268.2 → 58.1 and 256.2 → 64.2. The underlined fragments were used for quantification.
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25 μl of internal standard (tramadol-13c-d3 and O-desmethyl-cis-tramadol-d6, 4.0 μg/ml) and 1 ml of protein precipitation solvent (0.075 % formic acid in acetonitrile:ethanol; 90:10 v/v) were added to 0.5 g of whole blood. The samples were then mixed for 10 minutes and centrifuged for another 10 minutes (5000 rpm, 5 ˚C). 100 μl of the supernatant was finally transferred to a vial for LC-MS-MS analysis. The injection volume was 3 μl. A blank sample, a low (100 ng/g T, 40 ng/g ODT) and a high (2000 ng/g T and ODT) QC were run within each batch of samples.
2.2.4 Method characteristics Calibration models were evaluated by analysis of six replicates at nine levels from 10 to 3000 ng/g blood and were found to be best fitted by quadratic equations using a 1/X weighting. Calibrators and QCs were made by adding a volume of standard to 0.5 g of bovine whole blood. Absence of substances capable of interacting with the quantitative analysis was confirmed by using the described method prior to use. TRA and ODT were added in the
Page 6 of 23
same concentration for each calibrator. Lower limit of quantitation (LLOQ) was assessed by running five replicates at lower and lower concentrations and defined as the concentration where the imprecision was less than 25 % and the accuracy was 75-125 %. LLOQ was found to be 10 ng/g for ODT, and 20 ng/g for TRA. The method imprecision and accuracy was evaluated by analysis of triplicates of controls at four levels during eight days
ip t
(n=24). The total imprecision for TRA was 2.7 % (at 40 ng/g), 2.5 % (at 200 ng/g), 5.0 % (at 600 ng/g), and 3.5 % (at 2000 ng/g). The accuracy was between 90.4 and 94.7 % at all levels. The total imprecision for ODT was 2.8 %
cr
(at 40 ng/g), 2.2 % (at 200 ng/g), 4.4 % (at 600 ng/g), and 3.8 % (at 2000 ng/g). The accuracy was between 88.0 and 94.2 % at all levels. The method is currently enrolled in the Nordquant PT scheme and has presented with
us
good results.
2.3 Genotyping
an
Genotyping analyses were performed at the Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden and at the Department of Clinical Chemistry, County Council of Östergötland, Linköping, Sweden.
M
Genomic DNA was extracted using NorDiag Arrow (Autogen, Holliston, MA, USA). The extracted DNA was stored frozen at –20 ºC until analyzed.
d
Genotyping of CYP2D6 includes three SNPs; CYP2D6*3 (rs35742686), CYP2D6*4 (rs3892097) and CYP2D6*6 (rs5030655) as well as determination of copy number variation (CNV), which includes identification
te
of whole gene deletion (CYP2D6*5) and multiple gene copies (CYP2D6xN). These methods have earlier been
Ac ce p
described [21, 22] but some minor modifications were made. In brief, the PCR amplification was performed in a total volume of 10 µl using ~5 ng human genomic DNA, 0.2 µl of 20 µM forward and reverse primers (Invitrogen, Lidingö, Sweden), 5 µl 2x HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany) and for the CNV reactions 2 µl Q-solution was also needed (Qiagen, Hilden, Germany). Primer sequences are shown in Supplement 1. The PCR reactions were carried out on a Gene Amp PCR System 9700 (Applied Biosystems) with an initial denaturation step at 95 °C for 5 min, thereafter 40 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, followed by a final ex tension step at 72 °C for 10 min. Alleles not carrying any of the determined polymorphisms were classified as *1 (wild-type). The outcomes of the genotype analysis were categorized into four groups; individuals carrying no active gene (i.e. carrier of only the *3, *4, *5 or *6 alleles, also known as poor metabolizers, PMs), individuals carrying one active gene (i.e. carrier of *1 in combination with one of the alleles *3, *4, *5 or *6, also known as intermediate metabolizers, IMs), individuals with two active genes (i.e. carrier of two *1 alleles, also known as extensive metabolizers, EMs) and individuals carrying more than two active genes (i.e. carrier of multiple *1 alleles, also known as ultrarapid metabolizers, UMs).
Page 7 of 23
The PCR reactions for OPRM1 A118G, (rs1799971) and ABCB1 G1199A (rs2229109), ABCB1 C1236T (rs1128503), ABCB1 G2677T/A (rs2032582) and ABCB1 C3435T (rs1045642) were carried out on a Mastercycler ep (Eppendorf, Hamburg, Germany). For each PCR reaction, 2 µl 10× PCR buffer, 2 mM MgCl2, 0.5 U HotStar Taq polymerase (Qiagen, Hilden, Germany), 125 µM dNTPs (VWR International, Stockholm, Sweden), 5 pmol of
ip t
each forward and reverse primer (Biomers, Ulm, Germany), 20 – 50 ng DNA sample and nuclease free water were mixed to a final volume of 20 µl. The PCR reaction was initiated with 15 min of enzyme activation at 95 °C
cr
followed by 50 cycles at 95 °C for 30 s, 63 °C for 45 s, and 72 °C for 60 s. The reaction was finished with a 5 min final extension step at 72 °C followed by 4 °C incubation. Primer sequences are shown in Supplement 1.
us
Pyrosequencing was carried out on a Q96 MD according to the manufacturer’s recommendations (Qiagen, Hilden, Germany). Briefly, to 10 µl PCR products a mix containing 2 µl steptavidine-coated sepharose beads
an
(Amersham Biosciences, Piscataway, NJ, USA), 40 µl 1x binding buffer (10 mM Tris-HCl, 2M NaCl, 1mM EDTA, 0.1% Tween 20, pH 7.6) and 28 µl water was added. The samples were shaken on a thermomixer (Eppendorf, Hamburg, Germany) at 1400 rpm for 5 min. Biotinylated single-stranded DNA (ssDNA) was prepared
M
on a Vacuum Prep Workstation according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The ssDNA was hybridised to 15 pmol sequencing primer in 12 µl 1x annealing buffer (200 mM Tris-acetate and 50
d
mM MgAc2, pH 7.6) at 80 ºC for 2 min using a block thermostat (Grant Instrument, Cambridge, UK). A SNP
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Reagent containing substrate, enzyme and dNTP mixtures (Qiagen, Hilden, Germany) were added to a reagent cartridge, and the pyrosequencing reaction was carried out according to the dispensation orders shown in
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Supplement 1.
2.4 Data analysis
The area under the concentration-time curve (AUC0-10) was calculated using the linear trapezoidal method. For AUC0-∞ the area was extrapolated to infinity using the logarithmic trapezoidal method. Since the extrapolated area exceeded 20% of the total AUC0-∞ we opted to always use AUC0-10 for subsequent group comparisons. AUC therefore refers to the AUC0-10 values throughout this article. Peak blood concentrations (Cmax) and corresponding times (tmax) of TRA and ODT were read directly from the data. Metabolic ratios of ODT/TRA are in the present study abbreviated MR according to the following: MR = CODT/CTRA, AUC MR = AUCODT/AUCTRA, Cmax MR = Cmax ODT/Cmax TRA. Statistical analysis was performed by using the SPSS statistical program (Version 19.0 for Windows; IBM SPSS). Non-parametric analysis method was used to compare the pharmacokinetics parameters in subjects taking 50 and 100 mg TRA. Mann-Whitney test was also used to compare allelic variation in genes between groups. The
Page 8 of 23
hypothetical effect of allelic variation on AUC MR and Cmax MR was estimated by regression analysis. Significance level was set at P
S0379-0738(14)00096-6 http://dx.doi.org/doi:10.1016/j.forsciint.2014.03.003 FSI 7534
To appear in:
FSI
Received date: Revised date: Accepted date:
31-10-2013 20-2-2014 2-3-2014
Please cite this article as: S. Bastami, P. Haage, R. Kronstrand, F.C. Kugelberg, A.-L. Zackrisson, S. Uppugunduri, Pharmacogenetic Aspects of Tramadol Pharmacokinetics and Pharmacodynamics After a Single Oral Dose, Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pharmacogenetic Aspects of Tramadol Pharmacokinetics and Pharmacodynamics After a Single Oral Dose
cr
ip t
Salumeh Bastami*1, Pernilla Haage*1,2, Robert Kronstrand1,2, Fredrik C. Kugelberg1,2, Anna-Lena Zackrisson2, Srinivas Uppugunduri3
us
* Both authors contributed equally to this work
Affiliations
an
1 Department of Medical and Health Sciences, Division of Drug Research, Linköping University, Linköping, Sweden
M
2 National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden
d
3 Department of Clinical and Experimental Medicine, Linköping University, Department of Clinical Chemistry, County Council of Östergötland, Linköping, Sweden.
Anna-Lena Zackrisson
te
Corresponding author
Ac ce p
National Board of Forensic Medicine Department of Forensic Genetics and Forensic Toxicology Artillerigatan 12 SE 587 58 Linköping, Sweden
E-mail: [email protected] Phone: +46 13 252153 Fax: +46 13 136005
Page 1 of 23
Pharmacogenetic Aspects of Tramadol Pharmacokinetics and Pharmacodynamics After a Single Oral Dose
ip t
Abstract
The major purpose of this study was to elucidate if genotyping can facilitate interpretations of tramadol (TRA) in
cr
forensic case work, with special regard to the estimation of the time of drug intake and drug related symptoms
(DRS). The association between genetic polymorphisms in CYP2D6, OPRM1 and ABCB1 and pharmacokinetic
us
and pharmacodynamic properties of TRA was studied. Nineteen healthy volunteers were randomized into two groups receiving a single dose of either 50 or 100 mg of orally administrated TRA. Blood samples were collected
an
prior to dosing and up to 72 h after drug intake. The subjects were asked to report DRS during the experimental day. We found a positive correlation between the metabolic ratio of O-desmethyltramadol (ODT) to TRA and the
M
time after drug intake for both CYP2D6 intermediate metabolizers and extensive metabolizers. For the only poor metabolizer with detectable ODT levels the metabolic ratio was almost constant. Significant associations were found between the area under the concentration-time curve (AUC) and three of the investigated ABCB1 single
d
nucleotide polymorphisms for TRA, but not for ODT and only in the 50 mg dosage group. There was great
te
interindividual variation in DRS, some subjects exhibited no symptoms at all whereas one subject both fainted and vomited after a single therapeutic dose. However, no associations could be found between DRS and investigated
Ac ce p
polymorphisms. We conclude that the metabolic ratio of ODT/TRA may be used for estimation of the time of drug intake, but only when the CYP2D6 genotype is known and taken into consideration. The influence of genetic polymorphisms in ABCB1 and OPRM1 requires further study.
Key Words Tramadol, Pharmacokinetics, Pharmacodynamics, CYP2D6, ABCB1, OPRM1.
Page 2 of 23
1. Introduction The use of tramadol (TRA) for treatment of moderate to severe pain has been increasing steadily during the last decades. Unfortunately, abuse of this drug is also becoming more common. Further, development of addiction to TRA in association with analgesic treatment within the recommended dose range is another alarming trend. A
ip t
history of abuse or use of a drug of abuse seems to be an important risk factor [1]. TRA has also become a more common cause of death in drug addicts with a similar trend for increase in overdose cases [2, 3].
cr
Analysis of drugs of abuse is a common feature of forensic investigations and correct interpretation of the measured concentrations is important in both post mortem and human performance toxicology. Accurate
us
estimation of the time of drug intake and expected drug effects from a certain dose or concentration are also
an
frequent issues in drug-facilitated crimes.
TRA has a dual mechanism of action, acting as a µ-opioid receptor agonist as well as a serotonin and
M
norepinephrine reuptake inhibitor. The cytochrome P450 (CYP) enzyme CYP2D6 is involved in the formation of the active metabolite O-desmethyltramadol (ODT). In comparison to TRA, ODT is a significantly more potent μopioid agonist [4]. The concentration of the parent compound alone is often not sufficient to make an accurate
d
estimation of the time of drug intake. The ratio between metabolite and parent compound is generally used to
te
indicate a recent acute intake, e.g. to diagnose suspected acute overdoses. It has earlier been shown, for some substances other than TRA, that the ratio between metabolite and parent compound also can be helpful in a more
Ac ce p
accurate estimation of the time of intake [5, 6]. The amount of ODT formed is largely dependent on the CYP2D6 genotype but also on the time of intake. It is however unclear if the metabolic ratio (MR) of ODT/TRA is a useful indicator of the time of TRA intake.
There is considerable variation in the interindividual response to the same dose of opioids, including TRA, with respect to both therapeutic and adverse effects. Development of tolerance due to prolonged use of opioids in addition to genetic factors could partly explain some of these differences. Several genes and polymorphisms have been studied in this respect. Reduced or absent metabolite formation with reduced analgesic effect have been observed after TRA administration in CYP2D6 poor metabolizers (PMs) [7], whereas ultrarapid metabolizers (UMs), are instead associated with quicker analgesic effects, but with higher risk for adverse effects [8].
Page 3 of 23
The ABCB1 gene encodes P-glycoprotein (P-gp), which is located in the blood-brain barrier and gut and is responsible for the cellular efflux of a variety of drugs [9]. The most common single nucleotide polymorphisms (SNPs) in the coding region are C1236T, G2677T and C3435T [10]. C3435T, the most studied SNP, has been associated with both increased and decreased expression of P-gp [9]. An altered expression in gut could potentially
ip t
change the pharmacokinetics of the drugs being substrates of this transporter. A decreased expression and functionality of P-gp has been suggested in homozygous for the 3435T allele [11], possibly leading to higher
cr
bioavailability and subsequently a higher concentration of TRA in blood. Although P-gp is of importance for the pharmacokinetic and pharmacodynamic properties of a number of substances, including opioids [9, 12], its
us
importance for TRA remains unknown. A clinical study suggested that TRA is a substrate of P-gp [13]. In contrast, an in vitro study and one in vivo study in rat have demonstrated that TRA and ODT are not P-gp
an
substrates [14, 15]. A recent study showed no association between the C3435T polymorphism and pain relief in patients receiving TRA [16]. Taken together, the importance of P-gp for the pharmacokinetic and
M
pharmacodynamics properties of TRA still remains to be corroborated.
Genetic polymorphisms in OPRM1 have been associated with an altered pain threshold and opioid requirements.
d
Results from clinical studies have shown that patients’ homozygous for the wild-type of the most common SNP
te
A118G require less opioids than those with the other allelic variants, AG and GG [17, 18]. It has also been shown that the 118G allele is associated with a more severe clinical outcome in emergency department patients with acute
Ac ce p
drug overdose [19]. There is only limited information regarding the influence of OPRM1 A118G polymorphism on TRA efficacy [20].
We undertook a human study to elucidate if genotyping can facilitate interpretations of TRA in forensic case work, with special regard to the estimation of the time of drug intake and drug related symptoms (DRS). Our study had the following specific aims:
1) Investigate if the metabolic ratio (MR) of ODT/TRA is a useful indicator of the time of TRA intake. 2) Determine the association between polymorphisms in the ABCB1 gene (SNPs G1199A, C1236T, G2677T, C3435T) and pharmacokinetic parameters of TRA. 3) Study the association between polymorphisms in the CYP2D6, OPRM1 (SNP A118G) or ABCB1 (SNPs G1199A, C1236T, G2677T, C3435T) gene and DRS.
Page 4 of 23
2. Methods 2.1 Study participants and study design Twenty healthy volunteers were recruited through advertisements. Nineteen subjects (nine males, ten females)
ip t
aged 18 years or older (mean 25.4±4.3) completed the study, whereas one subject did not show up on the experimental day due to unspecified illness. Participants were informed and examined by a physician before
cr
inclusion in the study. Questions of present or previous drug use were asked. Previous or concurrent use of opioids or drugs known of interacting with TRA were exclusion criteria. Demographic data such as age, gender, height
us
and weight were noted. Seven of the female participants were taking oral contraceptives. None of the participants stated upon question that they were pregnant or breast-feeding. Written informed consent was collected from each
an
participant. The study was approved by the Regional Ethical Review Board in Linköping (No: 2011/337-31). The subjects were randomized into two groups receiving a single dose of either 50 or 100 mg of orally administered TRA (Tramadol HEXAL, Sandoz). During the experimental day, blood samples were obtained from a peripheral
M
venous catheter, Insyte W in combination with a Mandrin (Becton Dicinson AB), which was inserted in the forearm. The blood was collected in labeled 7 ml heparinized collection tubes using a Vacutainer Luer-Lok Access
d
Device (Becton Dicinson AB). Blood samples were collected prior to dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5,
te
6, 7, 8, 10, 24, 48 and 72 h. Samples were stored at –80 ºC pending analysis. The participants were exhorted to eat breakfast according to their usual routines. Lunch was bought from a nearby restaurant and served around noon.
Ac ce p
Fruit, some biscuits, tea, coffee, juice and water were available during the whole day. The subjects were requested to fill in a form regarding their experience of DRS during the experimental day in conjunction to the last blood sampling on the first day (10 h). Seven questions about nausea, dizziness, headache, vomiting, dry mouth, sweating and fatigue were posed. A scale between zero to five were used, where zero was no symptoms at all and five was worst imaginable symptoms.
2.2 Quantitation of tramadol and O-desmethyltramadol in whole blood 2.2.1 Chemicals and reagents Acetonitrile (gradient grade), methanol (gradient grade) and formic acid (98 %) were purchased from Merck (Darmstadt, Germany). Ammonium formate (98 %) and ethanol (95 %) were purchased from Fluka (Basel, Switzerland) and Kemetyl (Haninge, Sweden) respectively. Water used was first purified with a MilliQ-water purifying system (Millipore Corporation, Bedford, MA, USA). Reference substances used for making calibrators and quality controls (QCs), i.e. cis-tramadol and O-desmethyl-cis-tramadol were purchased from Cerilliant
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(Austin, TX, USA). Tramadol-13c-d3 and O-desmethyl-cis-tramadol-d6, used for making the internal standard were also purchased from Cerilliant.
2.2.2 Instrumentation
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High performance liquid chromatography was performed on a 1290 Infinity LC instrument (Agilent Technologies), using a 2.1x100 mm Zorbax Eclipse Plus C18 RRHD column with 1.8 μm particles. A guard filter
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with 0.2 μm particles (Waters) was also used. Column temperature was set to 60 ˚C. Mass detection (MS/MS) was performed on an AT6460 instrument (Agilent Technologies) with an electrospray interface, using positive
us
ionization. Mobile phase A consisted of 0.05 % formic acid in 10 mM ammonium formate while mobile phase B consisted of 0.05 % formic acid in methanol. Gradient elution with a total run time of 8 min was used. The total
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flow rate was 0.5 ml/min. The software used was Masshunter (Agilent Technologies). Identification criteria were based on transition ratios. Both TRA and ODT exhibit a very strong base peak of 58.1 and few other fragments, which have abundance less than 10 % of base peak. In accordance with European recommendations (EUD
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2002/657/EC) an acceptance limit of 50 % was used. The following transitions were used for TRA, ODT, tramadol-13c-d3, and O-desmethyl-cis-tramadol-d6, respectively: 264.2 → 58.1; 246.1, 250.1 → 58.1; 232.1,
2.2.3 Sample preparation
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268.2 → 58.1 and 256.2 → 64.2. The underlined fragments were used for quantification.
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25 μl of internal standard (tramadol-13c-d3 and O-desmethyl-cis-tramadol-d6, 4.0 μg/ml) and 1 ml of protein precipitation solvent (0.075 % formic acid in acetonitrile:ethanol; 90:10 v/v) were added to 0.5 g of whole blood. The samples were then mixed for 10 minutes and centrifuged for another 10 minutes (5000 rpm, 5 ˚C). 100 μl of the supernatant was finally transferred to a vial for LC-MS-MS analysis. The injection volume was 3 μl. A blank sample, a low (100 ng/g T, 40 ng/g ODT) and a high (2000 ng/g T and ODT) QC were run within each batch of samples.
2.2.4 Method characteristics Calibration models were evaluated by analysis of six replicates at nine levels from 10 to 3000 ng/g blood and were found to be best fitted by quadratic equations using a 1/X weighting. Calibrators and QCs were made by adding a volume of standard to 0.5 g of bovine whole blood. Absence of substances capable of interacting with the quantitative analysis was confirmed by using the described method prior to use. TRA and ODT were added in the
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same concentration for each calibrator. Lower limit of quantitation (LLOQ) was assessed by running five replicates at lower and lower concentrations and defined as the concentration where the imprecision was less than 25 % and the accuracy was 75-125 %. LLOQ was found to be 10 ng/g for ODT, and 20 ng/g for TRA. The method imprecision and accuracy was evaluated by analysis of triplicates of controls at four levels during eight days
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(n=24). The total imprecision for TRA was 2.7 % (at 40 ng/g), 2.5 % (at 200 ng/g), 5.0 % (at 600 ng/g), and 3.5 % (at 2000 ng/g). The accuracy was between 90.4 and 94.7 % at all levels. The total imprecision for ODT was 2.8 %
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(at 40 ng/g), 2.2 % (at 200 ng/g), 4.4 % (at 600 ng/g), and 3.8 % (at 2000 ng/g). The accuracy was between 88.0 and 94.2 % at all levels. The method is currently enrolled in the Nordquant PT scheme and has presented with
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good results.
2.3 Genotyping
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Genotyping analyses were performed at the Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden and at the Department of Clinical Chemistry, County Council of Östergötland, Linköping, Sweden.
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Genomic DNA was extracted using NorDiag Arrow (Autogen, Holliston, MA, USA). The extracted DNA was stored frozen at –20 ºC until analyzed.
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Genotyping of CYP2D6 includes three SNPs; CYP2D6*3 (rs35742686), CYP2D6*4 (rs3892097) and CYP2D6*6 (rs5030655) as well as determination of copy number variation (CNV), which includes identification
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of whole gene deletion (CYP2D6*5) and multiple gene copies (CYP2D6xN). These methods have earlier been
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described [21, 22] but some minor modifications were made. In brief, the PCR amplification was performed in a total volume of 10 µl using ~5 ng human genomic DNA, 0.2 µl of 20 µM forward and reverse primers (Invitrogen, Lidingö, Sweden), 5 µl 2x HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany) and for the CNV reactions 2 µl Q-solution was also needed (Qiagen, Hilden, Germany). Primer sequences are shown in Supplement 1. The PCR reactions were carried out on a Gene Amp PCR System 9700 (Applied Biosystems) with an initial denaturation step at 95 °C for 5 min, thereafter 40 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, followed by a final ex tension step at 72 °C for 10 min. Alleles not carrying any of the determined polymorphisms were classified as *1 (wild-type). The outcomes of the genotype analysis were categorized into four groups; individuals carrying no active gene (i.e. carrier of only the *3, *4, *5 or *6 alleles, also known as poor metabolizers, PMs), individuals carrying one active gene (i.e. carrier of *1 in combination with one of the alleles *3, *4, *5 or *6, also known as intermediate metabolizers, IMs), individuals with two active genes (i.e. carrier of two *1 alleles, also known as extensive metabolizers, EMs) and individuals carrying more than two active genes (i.e. carrier of multiple *1 alleles, also known as ultrarapid metabolizers, UMs).
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The PCR reactions for OPRM1 A118G, (rs1799971) and ABCB1 G1199A (rs2229109), ABCB1 C1236T (rs1128503), ABCB1 G2677T/A (rs2032582) and ABCB1 C3435T (rs1045642) were carried out on a Mastercycler ep (Eppendorf, Hamburg, Germany). For each PCR reaction, 2 µl 10× PCR buffer, 2 mM MgCl2, 0.5 U HotStar Taq polymerase (Qiagen, Hilden, Germany), 125 µM dNTPs (VWR International, Stockholm, Sweden), 5 pmol of
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each forward and reverse primer (Biomers, Ulm, Germany), 20 – 50 ng DNA sample and nuclease free water were mixed to a final volume of 20 µl. The PCR reaction was initiated with 15 min of enzyme activation at 95 °C
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followed by 50 cycles at 95 °C for 30 s, 63 °C for 45 s, and 72 °C for 60 s. The reaction was finished with a 5 min final extension step at 72 °C followed by 4 °C incubation. Primer sequences are shown in Supplement 1.
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Pyrosequencing was carried out on a Q96 MD according to the manufacturer’s recommendations (Qiagen, Hilden, Germany). Briefly, to 10 µl PCR products a mix containing 2 µl steptavidine-coated sepharose beads
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(Amersham Biosciences, Piscataway, NJ, USA), 40 µl 1x binding buffer (10 mM Tris-HCl, 2M NaCl, 1mM EDTA, 0.1% Tween 20, pH 7.6) and 28 µl water was added. The samples were shaken on a thermomixer (Eppendorf, Hamburg, Germany) at 1400 rpm for 5 min. Biotinylated single-stranded DNA (ssDNA) was prepared
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on a Vacuum Prep Workstation according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The ssDNA was hybridised to 15 pmol sequencing primer in 12 µl 1x annealing buffer (200 mM Tris-acetate and 50
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mM MgAc2, pH 7.6) at 80 ºC for 2 min using a block thermostat (Grant Instrument, Cambridge, UK). A SNP
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Reagent containing substrate, enzyme and dNTP mixtures (Qiagen, Hilden, Germany) were added to a reagent cartridge, and the pyrosequencing reaction was carried out according to the dispensation orders shown in
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Supplement 1.
2.4 Data analysis
The area under the concentration-time curve (AUC0-10) was calculated using the linear trapezoidal method. For AUC0-∞ the area was extrapolated to infinity using the logarithmic trapezoidal method. Since the extrapolated area exceeded 20% of the total AUC0-∞ we opted to always use AUC0-10 for subsequent group comparisons. AUC therefore refers to the AUC0-10 values throughout this article. Peak blood concentrations (Cmax) and corresponding times (tmax) of TRA and ODT were read directly from the data. Metabolic ratios of ODT/TRA are in the present study abbreviated MR according to the following: MR = CODT/CTRA, AUC MR = AUCODT/AUCTRA, Cmax MR = Cmax ODT/Cmax TRA. Statistical analysis was performed by using the SPSS statistical program (Version 19.0 for Windows; IBM SPSS). Non-parametric analysis method was used to compare the pharmacokinetics parameters in subjects taking 50 and 100 mg TRA. Mann-Whitney test was also used to compare allelic variation in genes between groups. The
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hypothetical effect of allelic variation on AUC MR and Cmax MR was estimated by regression analysis. Significance level was set at P
