Association Of UGT1A9Gene Polymorphisms With Propofol Pharmacodynamics Of Target-Controlled Infusion And Pharmacokinetics Of Single Injection

Background and ObjectivePropofol is a widely used clinical rapid-acting intravenous general anesthetics, it has many advantages:rapid onset, short-acting, rapid recovery, large range of safty, fewer adverse reactions, etc. It quickly become the first choice of clinical anesthesia sedative drugs, so it is used for anesthesia induction, maintenance, and also ICU ward for sedation commonly. But there is a big demand and sedative effects of differences in individual who used it for sedation. Study confirmed that genetic factors can alter metabolism enzyme activity, thus affect the drug pharmacokinetics and pharmacodynamics, resulting in sedative effects of individual differences. Therefore, to observe the association of UGT1A9gene polymorphisms with propofol pharmacodynamics of target-controlled infusion and pharmacokinetics of single injection in benign breast tumor resection patients under general anesthesia and to achieve reasonable safety for clinical anesthetics use and provide a theoretical basis are the purpose of this study. Materials and Methods1. SubjectsTotal150cases, American Society of Anesthesiologists (ASA) physical status: I or II,20～49years, and body mass index in the normal range (1±20%), undergoing selective benign breast tumor resection were included. Exclusion criteria: history of alcohol abuse, smoking, psychiatric disease, diabetes mellitus, significant cardiovascular diease, hepatic or renal dysfunction, chronic analgesia use, recently used liver drug metabolizing inducers or inhibitors, and patients with long-term use of sedative and hypnotic drugs. Subjects were divided into three groups according to the genotypes:wild-type homozygotes, mutant heterozygous, homozygous mutant. The protocol of this study was approved by the Ethical Committee of the First Affiliated Hospital of Zhengzhou University, and informed consent was obtained from all patients enrolled in this study.2. Anesthesia2.1Pharmacodynamic sectionAll patients using a uniform method of TIVA, administer propofol by target controlled infusion (TCI), induction of anesthesia:plasma concentration of propofol TCI is3μg/ml, intravenous remifentanil and cis-atracurium after the patients lost their consciousness, skilled anesthetists insert Laryngeal mask and mechanical ventilation. Anesthesia was maintained with propofol TCI3μg-ml-1and remifentanil and change the maintenance dose to ensure the patients maintain stable vital signs. We disable the cis-atracurium and remifentanil before the end of surgery30min and5min, stop the propofol TCI at the end of the surgery and intravenous atropine and neostigmine antagonistic muscle relaxants, assessment of the propofol sedation effects at the same time, removal of the laryngeal mask until the patients’conscious and breathing recovery good.Record time, effect-site concentration and BIS value after stop injecting of propofol to the patient’s OAA/S (the Observer’s Assessment of Alertness/Sedation Scale) was4, record the time and effect-site concentration when patients recover their consciousness (BIS value back to80). 2.2Pharmacokinetic sectionAll30subjects use a standardized general anesthesia technique. General anesthesia was induced with propofol2mg/kg, remifentanil1.5μg/kg and cis-atracurium0.15mg/kg within60s, insert Laryngeal mask and mechanical ventilation, main end-tidal partial pressure of carbon dioxide at35～45mmHg. Remifentanyl (0.1-0.2μg/kg/min) and sevoflurane (1%-2%) were used for anesthesia maintenance during the operation. We withdrawal drugs and antagonize muscle relaxants at the end of the surgery.3. Evaluation of propofol sedation effectsObserve and record the time, BIS value and effect-site concentration after stop propofol target controlled infusion to patients OAA/S was4points, time and effect-site concentration of patient recovery of consciousness, to regard awareness convalescent evaluation as propofol indicators of sedative effect after target-controlled infusion.4. Genotyping assaysCollect2ml venous blood samples from all patients in this study. DNA was extracted from leukocytes in peripheral blood using DNA extraction kit, genotyping of UGT1A91399C>T-1818T>C and-1887T>G alleles was conducted by polymerase chain reaction (PCR) and direct sequence anaylsis.5. Collect blood samplesIn the first part, blood samples were collected when patients BIS value was back to80after stop propofol target controlled infusion.In the second part,3ml blood samples were taken after propofol injection through ulnar vein at the time of1,3,5,10,15,30,45,60,90,120,240,360min. After3000r/min centrifugal plasma lOmin, shifted the upper to2ml EP tube and stored at-80℃refrigerator. We use high performance liquid chromatography to detect blood concentration of propofol. Propofol plasma concentration was processed using DAS pharmacokinetic software by the Chinese Pharmacological Society prepared by the commission in this study, selecting the best compartment model, drawing blood drug concentration-time curves and obtaining pharmacokinetic parameters. Statistical analysisSPSS17.0software was used for statistical analysis(SPSS Inc, Chicago, IL, USA). The Chi-square test was used to verify Hardy-Weinberg equilibrium. Data for numerical variables are expressed as mean±standard deviation (x±s). One-way analysis of variance (ANOVA) was used to assess whether significant differences exist among the three genotypes. Data for the time, effect-site concentration and BIS value were compared using one-way analysis of variance with post hoc Bonferroni correction, multiple comparisons was performed before and after adjusted for age, height, weight of the patients and duration of anesthesia. Propofol standard curve regression equation using linear regression and correlation processing, amount of data used by a single factor analysis of variance. A P<0.05(two-sided) was considered statistically significant.Results1. General informationEvery three groups according to UGT1A9SNP of patient’s age, weight, height, anesthesia time, medication, blood loss, fluid volume, liver’s and kidneys function and others have no significant difference in the two parts of this artical (P>0.05).2. Distribution of UGT1A9alleleThe frequency of UGT1A91399C>T,-1818T>C,-1887T>G allele in Han Chinese patients with breast surgery was52.3%,41%,9.3%. The allele frequency was in Hardy-Weinberg equilibrium (P>0.05), which indicates that the patient pool was likely representative of the population being studied. The allelic frequency of UGT1A9I399C>T in our study was similar to that reported in the literature of the Chinese Han (55.0%)(P>0.05), but lower than the Japanese population (64.4%) and higher than the Caucasus people(38%), and the difference have statistically significant(P<0.05).3. Sedation effects of propofol TCI in patients are quite differentThe results showed that there is a big individual different in propofol efficacy. Stopping propofol TCI injection to patients OAA/S was4, the time1-18mina difference of18times, the effect-site concentration1.2～2.9μg-ml-1(2.2±0.4μg-ml-1) a difference of2.5times, BIS values of51～80(69±7) a difference of1.9times; patients with BIS>80time2～29min a difference of15times, the effect-site concentration0.9～2.8μg·ml-1(1.9±0.4μg-ml-1) a difference of3.3times. Indicators of efficacy between the maximum and minimum values show large individual differences.4. Association of UGT1A9gene polymorphism with propofol TCI sedation effectAccording to the genotype of UGT1A9I399C>T,-1818T>C,-1887T>G, patients were divided into three genotypes of homozygous wild group, heterozygous and mutant homozygous group. No statistical differences in general information among every three genotype groups (P>0.05). No statistical differences in propofol sedative effects after stopping propofol TCI to the patients OAA/S was4among the every three genotype groups (P>0.05). After age, weight, height and duration of anesthesia as a covariate analysis of covariance, there were no significant difference in propofol sedative effects too (P>0.05). Propofol sedative effect indicators after stop TCI, compared with UGT1A9I399C/C-1818C/T group, I399C/T-1818C/T group and I399C/T-1818T/T group patients had a long time and low effect-site concentration when OAA/S was4, and had a long time to BIS value back to80, the difference between groups was statistically significant(P<0.05).5. High performance liquid chromatographic determination of propofol concentration in plasmaPropofol and internal standard retention times were11.1min and7.1min in standard chromatograms, which indicated that they are completely separated. Detection range of propofol concentration is0.04～25μg. ml-1, and0.01～g-ml-1is the minimum detection limits. Propofol three detection rate concentrations of0.1,1and25μg-ml-1were97.17%,103.79%, and98.18%, respectively, and RSD<5%. RSD of day precision was1.72%-2.34%and inter-day precision was2.18%-4.38%.6. Pharmacokinetic parameters of propofolBlood propofol concentration-time data curve were identified by DAS soft ware, and in line with the three compartment open model features. Primary ph armacokinetic parameters:UGT1A9I399C/C, C/T, T/T T1/2α(1.31±0.llvsl.28±0.11vs1.45±0.10)min,T1/2p(29.88±3.00vs19.68±1.28vs21.59±1.18)min,T1/2γ(209.65±31.4 5vs56.76±13.15vs215.85±20.36)min,AUC0â†'t(111.38±3.71vs110.89±2.64vs108.45±3.28)μg·min-1·ml-1,CL(0.0152±0.0004vs0.0151±0.0003vs0.015l±0.0005)ml·kg-1·min-1, Cmax (8.71±0.62vs8.88±0.64vs8.52±0.83) mg/l, there was no statistically signific ant differences among the three groups about pharmacokinetic parameters(P>0.05). UGT1A9-1818T/T, T/C, C/C T1/2α(1.35±0.12vsl.33±0.08vsl.43±0.16)min, T1/2β(26.71±2.22vs21.84±1.64vs20.00±1.25)min, T1/2γ(209.52±26.75vs184.98±16.93vsl94.58±28.64)min, AUC0â†'t(113.808±3.659vs107.342±2.641vs111.959±3.204)μ g·min-1·ml-1, CL(0.0146±030005vs0.l55±0.0003vs0.015±0.0003)ml·kg-1·min-1,Cmax (8.56±0.75v58.68±0.71vs0.1046±0.0058) mg/1, there was no statistically signific ant differences among the three groups about pharmacokinetic parameters(P>0.05). UGT1A9-1887(T/TvsTG/GG) T1/2α(1.16±0.22vsl.39±0.34)min, T1/2β(24.65±8.62vs22.23±5.83)min, T1/2γ(194.02±48.34vsl92.78±71.90)min, AUC0â†'t(0.131±0.022vs0.146±0.021)μg·min-1·ml-1, CL(0.0150±0.0012vs0.0151±0.0013)ml·kg-1·min-1,max为(8.87±0.63vs8.66±0.74) mg/1, there was no statistically significant difference s between the two groups (P>0.05).Our results show that UGT1A9polymorphism was not one of the genetic factors that lead to propofol pharmacodynamics and pharmacokinetics individual differences. Epigenetics, interaction of multiple single nucleotide polymorphisms (SNPs), and sample size are all factors that influence the results. To exclude confounding factors that impact our results, we will increase the sample size and study metabolic enzyme gene polymorphism on the metabolism of propofol in vitro single cell experiments in late study. Explore the reasons for individual differences of propofol, to achieve gene-oriented individualized anesthesia.Conclusion1. The frequencies of UGT1A91399OT,-1818T>C,-1887T>G allele in Chinese Han benign breast lumpectomy female patients are52.33%(157/300),41%(123/300),9.33%(28/300) respectively.2. There are large individual differences of propofol sedative effects in patients with consciousness recovering after stopping target-controlled infusion of propofol, UGT1A91399OT,-1818T>C,-1887T>G polymorphism have no significant effect on this and were not one of genetic markers that can affect propofol sedation effects.3. Propofol plasma concentration was determined by high performance liquid chromatography, which is a simple, accurate and reproducible method that can be used in clinical blood propofol concentration monitoring and pharmacokinetic studies.4. Three groups of propofol concentration-time curves meet the three compartment model, UGT1A9gene polymorphisms have no significant effects on the propofol pharmacokinetic characteristics after single intravenous injection.