Impact Of CYP2D6～*10 Polymorphism On Tramadol Metabolism And Postoperative Analgesia

Tramadol is a centrally acting synthetic non - opioid analgesic. It produces analgesia effect by combining synergistically activating opioid receptor and inhibiting the neuronal reuptake of monoaminergic materials. Tramadol has a low incidence of adverse effects, particularly of respiratory depression, cardiovascular depression and abuse potential. Therefore it enjoys increasingly attention in therapy of acute pain resulting from operation and other reasons, and also for the management of chronic pain. Great interindividual differences were observed in the effects of tramadol, so it is very important to find the genetic reason about the differences. By illuminating the genetic factor of individual tramadol pharma-codynamics, we can predict individual patient reaction, therefore eventually implement individual rational administration.Tramadol is mainly metabolised in liver by O - demethylation to O - dem-ethyltramadol (M1 ) and N - demethylation to N - demethyltramadol ( M2). Ml is the main analgesic effective metabolite of tramadol and has been demonstrated to have an affinity to μ - opioid receptors that is approximately 300 times greater than that of the parent compound. Thus, it is largely responsible for opioid receptor mediated analgesia.The 0 - demethylation of tramadol to metabolite M1 is catalyzed by the liver enzyme cytochrome P450 2D6 (CYP2D6). CYP2D6 is a highly polymorphic isoenzyme of the cytochrome P450 - system and there were four different pheno-types in population;poor metabolism ( PM ) , intermediate metabolism (IM ) , extensive metabolism ( EM ) and ultraextensive metabolism ( UEM ). CYP2D6 enzyme activity mainly depends on genetic control, but not environmental fac-tors.CYP2D6 gene locates on chromosome 22ql3. 1, up to now CYP2D6 gene polymorphism is found to be more than 70 types, and its incidence varies significantly from race to race. CYP2D6 genetic polymorphism is responsible for the four phenotypes. EM is the phenotype of health adults;UEM has extra duplicated CYP2D6 * 2 gene, it has extra high CYP2D6 enzyme activity, and is very rare. PM dues to allelomorphic gene mutation of CYP2D6*3,*4,*5,*6,*15, and it occurs 5 - 10% in Caucasians, only 1% in Asian. IM results from allelomorphic gene mutation of CYP2D6 * 10 in typical Asian. Despite the low frequency of PMs of CYP2D6 substrates in the Asian populations, they have been found to carry a high frequency (50% ) of the C188 to T188 mutation in exon 1, which causes a Pro^—>Ser amino acid substitution leading to a form of an unstable enzyme with lower metabolic activity. This mutation is relatively rare in Caucasian populations.CYP2D6 * 10 polymorphism may affect the transformation of tramadol and its metabolite Ml, therefore affects tramadol clinical analgesic effect. CYP2D6* 10 polymorphism could be the genetic reason of tramadol metabolism and clinical analgesic effect difference in Chinese individual and the whole race. There is no report on this subject by now.This study focus on the effects of CYP2D6 * 10 genetic polymorphism on tramadol pharmacokinetics and postoperative analgesia, therefore unveils the genetic feature of tramadol clinical effect in Chinese, which eventually leads to individual rational administration .Materials1. Chemicals and reagentsTramadol hydrochloricde is a product of Grunenthal (Germany). Tramadol hydrochloricde and Ml standard were gifts from Grunenthal (Germany). The PCR primers, PCR reagents and Marker were purchased from Takara (Japan). Hph I restriction enzyme is the product of NEB ( U. S. A. ).2. InstrumentationThe HPLC system (Agilent, USA);The zorbax SB—C18 analytical column (Agilent, U. S. A. );UV 300 spectrophotometer ( Pye - unicam/spectronic, England);Power Pac 1000 electrophoresis apparatus (Bio - Rad,U. S. A. );Gene Amp PCR System 9700 PCR amplification device (Perkin - Elmer, U. S. A. );BIS 303PC gel image analyzing system (DNR,Israel);ABI 3700 DNA sequencing device (ABI, U. S. A. )Methods1. Impact of CYP2D6 * 10 polymorphism on tramadol pharmacokinet-icsNineteen healthy volunteers were chosen in this study and 2ml venous blood anti - coagulated by sodium citrate were taken for DNA extraction and genoty-ping. Tramadol (1.5mg/kg) was slowly intravenously injected. Then at Oh (premedication) , 0.25h, 0.5h, lh, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, 16h, 24h and 32h, 3ml venous blood were taken and anti - coagulated by heparin for the plasma concentration measurement of tramadol and Ml.High - performance liquid chromatography ( HPLC) was used to measure the plasma concentrations of tramadol and Ml. The fluorescence detection wavelength were as follows, Ex =2l6nm, Em =308nm.Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) was used for CYP2D6'10 genotyping: DNA was extracted by Phenol/Chloroform extraction and then PCR amplification was done. The PCR amplification products were added Hph I restriction enzyme and incubation at 37 °C for 16 h. The agarose gel electrophoresis for the observation and DNA sequencing were done for the confirmation.2. Impact of CYP2D6 * 10 polymorphism on the analgesia and blood concentration of tramadol after the postoperative continuous intravenous injectionOne hundred and thirty - nine cases, ASA I ~ II , were chosen, who were picked up for the general anesthesia in the Urinary Surgery Department of the First Affiliated Hospital of China Medical University from October, 2005 to De-cember, 2005. Seventeen cases were randomly picked up from 139 cases for the variation curve measurement of plasma drug concentration.A loading dose of 1. 5mg/kg tramadol was slowly intravenous injected 30min before termination of surgery. Then the continuous intravenous injection of tramadol at the rate of 18mg/ was given for 48h for analgesia.Two - milliliter venous blood was taken before the operation for DNA extraction and genotyping. Twenty hours after the operation 3ml venous blood was taken for the plasma concentration of tramadol and Ml. In 17 cases, who were picked up for the variation curve measurement of drug concentration, 3ml venous blood were respectively taken at Oh (premedication) , 0.25h, 0.5h, lh, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, 16h, 24h, 32h, 40h and 48h for the blood drug concentration measurement of tramadol and Ml. Then visual analogue scales (VAS) evaluation were respectively made lh, 6h, 24h and 48h after the operation.3. Impact of CYP2EK> * 10 polymorphism on the postoperative patient -controlled analgesia (PCA)Two hundred and twelve cases, ASA I ~ II , were chosen, who were picked up for the general amesthesia in the Thoracic Surgery Department of the First Affiliated Hospital of China Medical University from October, 2005 to March, 2006.With 100ml total volume (tramadol 15 mg/ml) , PCA devices were programmed to a 1. 5 ml bolus dose, and a lock - out time of 12 min. Furthermore, for the first 24 h, a basal infusion rate of 1.2 ml/h was administered, and then 0. 8ml/h for the next day.About 30 min before termination of surgery, each patient received a loading dose of tramadol 1. 5mg/kg. In the recovery room, patients with pain intensities of more than 4 on the VAS received a further loading dose of tramadol, up to a total maximum dose of 3 nig/kg body weight. If analgesia under rest remained insufficient ( VAS > 4) , rescue medication fentanyl i. v. was given until pain scores were reduced to less than 4. If analgesia was insufficient or the patient was not satisfied with pain management, rescue medication was used during PCA period.VAS evaluations were respectively made 6h, 24h and 48h after the operation. All adverse events and their possible relation to study medication were recorded throughout the study. After discontinuation of PC A treatment, patients were asked to fill in a final questionnaire focussing on patients'satisfaction with pain management. A patient was assessed as non - responder if he needed rescue medication after titration of the loading dose or during the 48 - h PCA period and/or gave a negative rating in the final questionnaire. 4. pharmacokinetic analysis and statistical treatment Data was expressed as mean standard deviation. The pharmacodynamic parameters were calculated using the noncompartmental method, with the aid of DAS1.0 software. Statistical analyses were performed with the SPSS11.5 software. ANOVA analysis and F test were used in the inter - group comparisons of the measurement data, and the chi - square test was used in the inter - group comparisons of the numeration data. The Kruskal - Wallis test was used for the comparisons of the pharmacokinetic parameters. P <0. 05 was considered as the significant difference.Results1. Analysis of Tradamol and Ml plasma concentrationsHPLC result showed that sample concentration had good linear relation with peak area. The limit of quantitation of Ml and tradamol was separately 5ng/ml and 12. 5ng/ml. In our study, Ml and tramadol were totally separated with internal standard bisoprolol and suffered no endogenous material intervention. Retention time of Ml, tramadol and internal standard bisoprolol was separately 2. 9min^6. 8minA15. 5min. Degree of accuracy, precision and extraction recovery rate fitted the demand of biological sample detection. Tramadal and Ml plasma samples were stable while stored at -20T! for 30 days, difference of results was within ±10%.2. CYP2D6*10 genotypingAll PCR products were specific 433bp fragments . PCR products of CYP2D6 wild type had one Hph I catalytic site, and was separated into 362bpand 71bp segments by enzymolysis;the C,88—>T mutation on CYP2D6 * 10 leads to a new catalytic site, and its PCR product was detached into there segments, 262bpN100bp and 71 bp. Therefore wild homozygote (w/w) had two segments 362bp and 71bp, while mutation homozygote ( m/m) had three segments 262bpN100bp and 71bp, heterozygote (w/m) had four segments 362bp^262bpN lOObp and 71bp. DNA sequence analysis showed that CYP2D6* 10 polymorphic site of type w/w, m/m, m/w by PCR - RFLP detection were C, T, C and T respectively.3. Impact of CYP2D6 * 10 polymorphism on tramadol pharmacokinet-icsNineteen volunteers were assigned into 3 groups according to their genotypes : 5 cases were wild homozygote w/w, 8 cases were heterozygote m/w, and 6 cases were mutation homozygote m/m.The half - life (t1/2) of tramadol was 7. 3 ± 2. lhN9. 3 ± 2. 5h and 12. 5 ± 3. 6h in w/w group^m/w group and m/m group respectively. The difference has the statistical meaning (P = 0.022). Compared with the w/w group, t1/2 and mean residence time (MRT) in m/m group respectively increased by 71.2% and 47. 1% ,and the difference has the statistical meaning (P values were 0.014 and 0.047 respectively).The area under the plasma concentration - time curve (AUC) of Ml, the main active metabolite of tramadol, significantly decreased( P = 0. 018). The metabolic ratios in plasma ( MRs) in w/w group N m/w group and m/m group were respective 0. 377 ±0. 044^0. 311 ±0. 048 and 0. 198 ±0. 036, which had the meaning of the transformation ratio of tramadol into Ml. Compared with the w/w group, the MRs in the m/w group and the m/m group all significantly decreased and there was significant difference (P<0.001).Among all groups, there were no significant differences as to the plasma clearance (CL) of tramadol and peak concentration (CnuU[)^peak time ( Tmax) ,, MRT of Ml.4. Impact of CYP2D6 * 10 polymorphism on the analgesia and blood concentration of tramadol after the postoperative continuous intravenous injectionOne hundred and thirty four patients completed the observation and were assigned into 3 groups according to their genotypes: 26 cases were wild homozy-gote w/w, 73 cases were heterozygote m/w, and 35 cases were mutation homo-zygote m/m. In 17 cases who were picked up for the variation curve measurement of drug concentration, 4 cases were w/w type, 8 cases were m/w type and 5 cases were m/m type.In 17 cases who were picked up for the variation curve measurement of drug concentration, the mean plasma concentration of tramadol and Ml in all groups reached the homeostatic I6h after operation. In w/w group, m/w group and w/ w group, the time of mean plasma concentration of Ml reaching the homeostatic were 8h, 12h and 16h respectively. Compared with w/w group, the homeostatic concentration of Ml significantly decreased at 6h, 8h, 12h, 16h, 24h and 32h after operation (P <0. 05). There was no statistical difference in m/w group.Compared with w/w group, Ml plasma concentration and Ml/tramadol ratio in m/m group were significantly lower than w/w group ( P was 0.022 and 0. 019 respectively), but no statistical difference was observed in m/w group. There was no statistical difference was observed in tramadol plasma concentrations among three groups. In m/m group, the VAS scores lh and 6h after operation were significantly higher than those of w/w group. In m/w group, the VAS scores 6h after operation were also significantly higher than those of w/w group, but the lh VAS scores no statistical difference. There was no statistical difference of postoperative 24h and 48hVAS score among the three groups.5. Impact of CYP2D6 * 10 polymorphism on tramadol postoperative PCAFive out of 212 patients failed in the study. Two hundred and seven patients were assigned into 3 groups according to their genotypes: 44 cases were wild homozygote w/w, 112 cases were heterozygote m/w, and 51 cases were mutation homozygote m/m.Compared with w/w group, the loading doses and 6,12, 24h postoperative doses of tradamol in m/m group were significantly higher , increase was 25. 4% N16.6% J5.4% and 11. 9% respectively (p value was 25. 4% J6. 6% ^ 15.4% and 11.9% respectively). There was no statistical difference in m/wgroup. There was no statistical difference was observed in 48h cumulated dose a-mong all three groups .In m/m group, the VAS scores right after general anesthesia and 6h after operation were significantly higher than those of w/w group,. but no statistical difference was observed in the m/w group. There was no statistical difference of postoperative 24h and 48h VAS score among the three groups.In w/w group ^ m/w group and m/m group, non - responders of tramadol postoperative PCA was 12 (27. 3% ) ^34 (30. 3% ) and 21 (41. 2% ) respectively. There was a increasing tendency according to the number of mutation al-leles, but no statistical difference among the three groups was observed.DiscussionIn this study the single intravenous injection of 1. 5mg/kg tramadol was given in the healthy people in different genotype groups of CYP2D6 * 10, then the observation of the pharmacokinetics of tramadol and the main active metabolite Ml was made. The results were as follows: The C188—*T mutation of CYP2D6* 10 increased the t1/2 and MRT of tramadol and decreased the AUC of Ml and MR, which had the meaning of the transformation ratio of tramadol into Ml. After the continuous intravenous infusion of 18mg/h tramadol in the postoperative patients for analgesia, the CYP2D6 * 10 gene mutation increased the time of Ml to reach the homeostatis and decreased its homeostatic concentration during the continuous infusion of tramadol. It is CYP2D6 that catalyzes the transformation of tramadol into Ml. The results above suggested that the C188—>-T mutation of the extron 1 of CYP2D6 gene, which commonly existed in Chinese, decreased the activity of CYP2D6 enzyme, so as to slowed down the internal metabolism of tramadol and the formation of its main active metabolite Ml. Therefore the polymorphism of CYP2D6 * 10 is the significant factor to effect the pharmacokinetics of tramadol and is one of the hereditary factors to produce the individual differences of pharmacokinetics of tramadol. As the occurrence frequency of CYP2D6 * 10 allele is up to 51% , the polymorphism of CYP2D6' 10 may produce the entirely decreasing metabolism of tramadol in Chinese. Therefore theresults in this study also confirmed that CYP2D6 * 10 polymorphism was one important factor to produce the Chinese population differences in tramadol metabolism.In the clinical pharmacodynamics study of the postoperative continuous intravenous analgesia and PC A analgesia of tramadol in different CYP2D6 * 10 genotype groups, it was found that during the postoperative continuous intravenous analgesia of tramadol the VAS scores in m/m group lh and 6h after the operation were significantly higher than in w/w group, and the VAS score in m/w group 6h after the operation was also significantly higher than in w/w group. During the PCA analgesia of tramadol, the dosage 24h after the operation and the VAS scores 6h after the operation in m/m group were significantly higher than in w/w group. It was confirmed by the results that the CYP2D6* 10 allele mutation slowed down the transformation of tramadol to its main active metabolite Ml, so as to decrease the analgesia of tramadol. It was illustrated that CYP2D6 * 10 polymorphism was the impact factor to produce the Chinese individual differences of tramadol analgesic pharmacodynamics.Through the optimization of the experiment condition, the established HPLC, which was used for the simultaneous determination of the plasma concentrations of tramadol and Ml ,had the advantages of high sensitivity and specificity, easy manipulation, high efficiency of sample extraction , perfect stability and less impurity interference, and has met the requirement of biological sample analysis in the internal drug concentration measurement and clinical pharmaco-dynamic study. The plasma samples of tramadol and Ml in this study were preserved under - 20X, and were kept stable characters, so that the data of the measured blood drug concentrations were reliable.The detected CYP2D6 * 10 polymorphism was a kind of single nucleotide polymorphism (SNP). The adopted PCR -RFLP was a commonly used method to detect SNP. The results in the study were clear to distinguish, and through DNA sequence analysis, the actual typing results were identical to the theoretical design. PCR - RFLP was a reliable method for CYP2D6 * 10 typing.At present, the studies of the impact of CYP2D6 * 10 polymorphism on tramadol pharmacokinetics were only reported in Malaysia population. As the genepolymorphism existed significant inter - ethnic differences, even in the same race, there might also be differences between different populations. The study results in Malaysia population cannot be used to illustrate Chinese individual differences of tramadol metabolism. Furthermore the simultaneous measurement of Ml plasma concentration wasnt done in the study, so the impact of CYP2D6 * 10 polymorphism on Ml wasn' t illustrated. There was no report about the impact of CYP2D6 * 10 polymorphism on tramadol pharmacodynamics by now. In this study, the impact of CYP2D6* 10 polymorphism on the pharmacokinetics and pharmacodynamics of tramadol and its metabolite Ml was firstly observed in Chinese population, which confirmed that CYP2D6 * 10 polymorphism was one important genetic factor to impact on the individual and inter - ethnic differences of tramadol metabolism and postoperative analgesia.Conclusion1. The C—>T mutation of CYP2D6* 10 allele slowed down the internal metabolism and transformation of tramadol to its main active metabolite Ml, and increased the plateau concentration time of Ml in the continuous intravenous analgesia of tramadol, and decreased the plateau concentration of Ml. CYP2D6 * 10 polymorphism was an important genetic factor to produce individual and inter -ethnic differences of tramadol pharmacokinetics in Chinese.2. The C—>T mutation of CYP2D6*10 allele decreased the early effects of postoperative continuous intravenous analgesia and PCA analgesia of tramadol, and increased the required amount of tramadol in the early postoperative stage. CYP2D6 * 10 polymorphism was one of the factors to produce individual difference of postoperative analgesia of tramadol in Chinese.