Application of CYP3A4*1G gene in preparation of product for evaluating postoperation analgesic effect and dosage prediction of sufentanil compounded butorenol

Abstract

The invention discloses an application of a CYP3A4 * 1G gene in preparation of a product for evaluating the postoperative analgesic effect and dosage prediction of sufentanil compounded with butorenol. According to the invention, the gene polymorphism of CYP3A4 * 1G is found to be one of genetic factors causing sufentanil compounded butornori postoperative analgesic effect and dosage use difference individualized difference for the first time, and anesthetists can determine the dosage of analgesic drugs for postoperative patients according to genetic characteristic difference, so that the satisfaction degree of anesthesia and postoperative analgesia of the patients is improved; therefore, the CYP3A4 * 1G gene can be used as a marker for evaluating the postoperative analgesic effect and dosage use of sufentanil compounded with butoenorphan, and detection is carried out through related genes of postoperative analgesic medication of a patient and CYP3A4 * 1G genotyping, so that guidance can be provided for the postoperative analgesic medication dosage of the patient.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of CYP3A4*1G gene in preparing a product for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and dosage prediction. Background Art

[0002] Pain is an unpleasant feeling and emotional experience caused by tissue damage or potential tissue damage, or a painful experience with sensory, emotional, cognitive and social levels. According to the healing time of tissue damage and the duration of pain, pain can be divided into acute pain and chronic pain. Postoperative pain is acute pain that occurs immediately after surgery, including somatic pain and visceral pain, which usually lasts no more than 3 to 7 days. It is common in thoracic and abdominal surgeries with large trauma and joint replacement surgeries that require a long period of functional training. If postoperative pain is not adequately controlled, it will bring a series of complications to various systems of the body, such as: increasing oxygen consumption and affecting ischemic organs; triggering harmful spinal reflex arcs and reducing lung function; reducing gastrointestinal motility and delaying gastrointestinal function recovery. Acute pain will not only increase the patient's pain and complications, but also affect postoperative recovery and disease outcome in severe cases. Therefore, acute pain in the preoperative period is still a problem and challenge that both doctors and patients must face. Actively taking effective analgesia is a key link in reducing complications, accelerating recovery, improving the quality of life of patients, and thus reducing postoperative mortality. At present, sufentanil combined with butorphanol is the most commonly used postoperative analgesia method in clinical practice.

[0003] Sufentanil is a potent and highly selective μ opioid receptor agonist with an analgesic effect 7 to 10 times that of fentanyl and less histamine release. However, excessive doses can cause adverse reactions such as respiratory depression, nausea, vomiting, dizziness and itching, making its use alone unsatisfactory. Butorphanol is a mixed opioid receptor agonist-antagonist that produces analgesia by stimulating central κ opioid receptors. Its analgesic effect is weaker than that of sufentanil, 3 to 7 times that of morphine; however, its analgesic effect lasts for a long time, its excitatory effect on smooth muscle is weak, and its gastrointestinal adverse reactions are few. It can also antagonize μ2 receptors in sufentanil, inhibiting adverse reactions such as addiction and respiratory depression caused by sufentanil. In addition, when μ receptors are excited, somatic pain is better controlled, but visceral pain is poorly treated, while κ receptor agonists are better for visceral pain. Therefore, sufentanil combined with butorphanol can play a synergistic role in clinical application, improve the analgesic effect, and reduce the dosage of analgesics, thereby reducing the incidence of adverse reactions. Currently, sufentanil combined with butorphanol is widely used in clinical postoperative analgesia.

[0004] At present, the dosage of sufentanil and butorphanol is strictly determined by weight, body surface area or age in clinical practice. However, a large number of clinical practices have found that different patients have obvious individual differences in the analgesic effect and adverse reactions of sufentanil combined with butorphanol. At the same dosing concentration, some patients have poor or even no analgesic effect. A large-sample clinical trial found that the proportion of patients who are insensitive to opioid analgesia is as high as 11.5%. To achieve analgesic effect, the dose needs to be increased, which often leads to increased pain, disease progression, and even chronic pain and drug addiction, reducing the quality of life of patients. In severe cases, it affects postoperative recovery and disease outcome. Some patients experience severe adverse reactions such as respiratory depression, convulsions, nausea and vomiting at the same dosing concentration, which seriously affects the prognosis of patients and even endangers their lives. However, due to the lack of sensitive predictive evaluation indicators for the efficacy of analgesia, clinical applications cannot accurately grasp the drug dosage, which can easily lead to insufficient dosage, inability to relieve the patient's pain symptoms, or excessive dosage, resulting in many complications and adverse reactions. Finding out the reasons for this difference and searching for sensitive predictive evaluation indicators for efficacy are the key to current clinical guidance of postoperative analgesia. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of CYP3A4*1G gene in the preparation of a product for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol.

[0006] The present invention also provides a primer set for amplifying the CYP3A4*1G gene.

[0007] The present invention also provides a kit.

[0008] The present invention also provides the application of the primer set and the kit.

[0009] The invention also provides a product.

[0010] According to one aspect of the present invention, the use of the CYP3A4*1G gene in the preparation of a product for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol is proposed.

[0011] In some embodiments of the present invention, the postoperative analgesic effect of sufentanil combined with butorphanol and / or the postoperative dosage of sufentanil combined with butorphanol are evaluated by genotyping the CYP3A4*1G gene. When the patient's CYP3A4*1G genotype is detected to be AA, compared with patients with CYP3A4*1G genotypes of GA and GG, more sufentanil and butorphanol are consumed to achieve the same analgesic effect.

[0012] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned CYP3A4*1G gene is provided.

[0013] In some embodiments of the present invention, the forward primer sequence of the primer set is shown as SEQ ID NO:1, and the reverse primer sequence is shown as SEQ ID NO:2.

[0014] According to a third aspect of the present invention, a kit is provided, the kit comprising the above-mentioned primer set.

[0015] In some embodiments of the present invention, the kit further comprises PCR buffer, dNTPs, and PCR enzyme.

[0016] In some embodiments of the invention, the PCR enzyme comprises a thermostable DNA polymerase.

[0017] In some embodiments of the present invention, the kit further comprises supporting reagents for pyrophosphate sequencing.

[0018] According to a fourth aspect of the present invention, an application of the above primer set or kit is provided, wherein the application is application in CYP3A4*1G genotyping.

[0019] In some embodiments of the present invention, the use is for preparing a product for CYP3A4*1G genotyping.

[0020] In some embodiments of the present invention, the application is an application for preparing a product for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol.

[0021] In some embodiments of the present invention, the method for using the product comprises the following steps: extracting DNA from the patient to be tested, performing PCR amplification using the above primer set to obtain an amplified product; and performing genotyping on the amplified product.

[0022] In some embodiments of the present invention, the step of evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol according to the genotyping results is also included.

[0023] In some embodiments of the present invention, the DNA is peripheral blood DNA.

[0024] In some embodiments of the present invention, the reaction system of PCR amplification comprises:

[0025]

[0026]

[0027] Add water to make up to 50 μL.

[0028] In some embodiments of the present invention, the reaction system of PCR amplification comprises:

[0029]

[0030] Add water to make up to 50 μL.

[0031] In some embodiments of the present invention, the method for genotyping the amplification product is sequencing.

[0032] In some embodiments of the invention, the sequencing method comprises pyrophosphate sequencing.

[0033] In some embodiments of the present invention, the reaction procedure of the PCR amplification includes: 92-97°C, 8-12 min; entering the cycling stage: 92-97°C 8-12 min, 58-62°C 12-17 s, 70-75°C 25-35 s, a total of 28-32 cycles; 72°C for 8-12 min.

[0034] In some embodiments of the present invention, the reaction procedure of the PCR amplification includes: 95°C, 10 min; entering the cycling stage: 95°C for 10 min, 60°C for 15 s, 72°C for 30 s, for a total of 30 cycles; 72°C for 10 min.

[0035] In some embodiments of the present invention, when the patient's CYP3A4*1G genotype is detected to be AA, compared with patients whose CYP3A4*1G genotype is GA or GG, more sufentanil and butorphanol are consumed to achieve the same analgesic effect.

[0036] According to a fifth aspect of the present invention, a product is provided, which contains the above-mentioned primer set or kit for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol.

[0037] In some embodiments of the present invention, the product comprises at least one of a test plate, a test kit, and a test chip.

[0038] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention discovers for the first time that the genetic polymorphism of CYP3A4*1G is one of the genetic factors that cause individual differences in the postoperative analgesic effect and dosage of sufentanil combined with butorphanol. Anesthesiologists can determine the amount of analgesics used by postoperative patients based on differences in genetic characteristics, thereby improving patients' satisfaction with anesthesia and postoperative analgesia. Therefore, the CYP3A4*1G gene can be used as a marker for evaluating the postoperative analgesic effect and dosage of sufentanil combined with butorphanol. By detecting the genetic typing of the CYP3A4*1G gene related to postoperative analgesia in the above-mentioned patients, guidance can be provided for the dosage of postoperative analgesics in patients. DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0040] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0041] Example 1 Application of CYP3A4*1G gene in evaluating the postoperative analgesic effect and dosage prediction of sufentanil combined with butorphanol

[0042] This example provides the application of the CYP3A4*1G gene in evaluating the postoperative analgesic effect and dosage prediction of sufentanil combined with butorphanol, and the specific verification steps are as follows:

[0043] 1. Sample

[0044] This study was approved by the hospital ethics committee, and the patients signed informed consent.

[0045] Inclusion criteria: 150 patients undergoing thoracic and abdominal surgery under general anesthesia in Xiangya Second Hospital were enrolled, with the following inclusion criteria: 1) patients aged between 18 and 65 years; 2) body mass index: 18-23 kg / m2; 3) ASA surgical classification: I-II; 4) preoperative laboratory test results showed that cardiac function, liver function, and renal function were within the normal range; 5) no liver enzyme inducers or inhibitors were taken within 30 days before surgery; 7) autonomous consciousness and voluntarily signed the informed consent before the start of the study; 8) no history of opioid abuse.

[0046] Exclusion criteria: 1) Patients with a history of peripheral neuropathy and mental illness, chronic pain, and long-term opioid use; 2) Patients with uncontrolled diabetes, hypertension, heart disease, chronic kidney disease, chronic active viral hepatitis, or related diseases; 3) Major organ failure such as decompensated heart failure and pulmonary insufficiency; 4) Adverse events that affect follow-up and assessment results during surgery.

[0047] 2. Methods

[0048] 1. Anesthesia and postoperative analgesia methods

[0049] All patients were assigned to a unified postoperative analgesia method: All enrolled patients received patient-controlled intravenous analgesia (PCIA) combined with sufentanil and butorphanol for postoperative analgesia. Successful analgesia was defined as an NRS score of less than 3 points. The consumption of sufentanil and butorphanol at three time points after postoperative analgesia was recorded: 6h, 24h, and 48h.

[0050] Postoperative analgesia assessment: VAS pain score was used for assessment immediately after awakening, 2h, 6h, 8h, 12h, and 24h after surgery. 0 was painless, 1-3 was mild pain, 4-6 was moderate pain, and 7-10 was severe pain. A VAS score of ≤3 was defined as effective analgesia. If the maximum PCIA hourly dose was reached but the VAS score was still >3, other analgesics were added and the patient was excluded from the trial.

[0051] Adverse reaction assessment: All enrolled patients were followed up throughout the analgesia process and within 48 hours after analgesia, and adverse reactions such as nausea and vomiting, itching level, and respiratory depression were evaluated.

[0052] 2. Genetic testing

[0053] Pyrosequencing was used to perform genotyping of the CYP3A4*1G allele (CYP3A4 gene is located on human chromosome 7, 7q21.1-q22.1, about 27 kbp long, and its gene structure includes 13 exons and 12 introns. CYP3A4*1G (20230G>A) is a single nucleotide polymorphism (SNP) in intron 10 of CYP3A4, and its frequency in the Han Chinese population is 22.1% to 37%). 1 mL of peripheral venous blood was drawn from the patient before surgery and anticoagulated with ethylenediaminetetraacetic acid (EDTA). The HiPure Blood DNAMini Kit was used to extract whole blood genomic DNA, and the DNA concentration and purity were determined by a nucleic acid analyzer to ensure that all DNA samples had an A260 / A280 of ≥1.8 and good DNA purity, and were stored at -20°C for later use.

[0054] The polymerase chain reaction (PCR) was used to amplify the DNA fragment containing the polymorphic site using primers. The primer sequences used in the PCR were 5'-GTAATAGAAAGCAGATGAACC-3' (SEQ ID NO: 1) and 5'-TCACCCTGATGTCCAGCAGAA-3' (SEQ ID NO: 2).

[0055] The total volume of the PCR reaction was 50 μL, including 50 ng genomic DNA, 10 μL 5× PCR buffer, upstream and downstream primers with a final concentration of 0.2 μmol / L, 2 μL 5 mmol / L dNTPs and 1 unit TaKaRa HotStart Taq polymerase. Nuclease-free ultrapure water was used to adjust the reaction volume to 50 μL.

[0056] PCR reaction time: 95°C, 10 min; entering the cycling stage: 95°C 10 min, 60°C 15 s, 72°C 30 s, a total of 30 cycles; 72°C for 10 min.

[0057] Pyrosequencing was performed using a commercially available pyrosequencing kit, and the method is briefly described as follows:

[0058] After adding the binding buffer and magnetic beads to the CYP3A4*1G gene PCR product obtained by the above PCR amplification, the mixture was incubated at 1400 rpm / s at room temperature for 10 minutes on a multifunctional oscillating mixer. After denaturation and washing, the beads bound to the single-stranded purified sequencing template were released into a mixture of sequencing primers and annealing buffer, and the reaction plate was placed at 80°C, taken out after 2 minutes, and naturally cooled to room temperature.

[0059] According to the Pyro Mark ID instrument operating instructions, set the detection program for each reaction well, add the enzyme mixture (E), fluorescent substrate (S) and four bases (dNTP) contained in the pyrophosphate sequencing kit to the reagent compartment, put the reaction plate and reagent compartment into the detection compartment, and perform the test. Data analysis The analysis software supporting the Pyro Mark Q24 pyrophosphate sequencer was used for SNP analysis to determine the polymorphism of the CYP3A4 gene.

[0060] According to genotyping, CYP3A4*1G can be divided into wild homozygous type (CYP3A4*1 / *1, AA), mutant heterozygous type (CYP3A4*1 / *1G, GA), and mutant homozygous type (CYP3A4*1G / *1G, GG). The CYP3A4*1G genotyping was kept blind to the researchers during the study, and the CYP3A4*1G genotyping test results were announced after the study was completed.

[0061] 3. Discovery and Verification

[0062] 1. Discovery

[0063] After obtaining informed consent from 100 patients, 1 mL of peripheral venous blood was drawn and whole genome DNA was extracted for testing according to the above-mentioned detection method. The results showed that the mutation frequency of the CYP3A4*1G allele was 20%, which was consistent with the overall mutation rate of the Han Chinese. Clinical observations also found that to achieve the same analgesic effect, the consumption of sufentanil and butorphanol in patients with CYP3A4*1G gene mutants (CYP3A4*1 / *1G, CYP3A4*1G / *1G) was less than that in patients with wild type (CYP3A4*1 / *1), which was significantly different.

[0064] 2. Verification phase

[0065] 1 mL of peripheral venous blood was collected from 50 patients (completely different from the 100 patients mentioned above) enrolled in the project experiment, and whole genome DNA was extracted. The above-mentioned method was used for CYP3A4 genotyping and analgesic effect detection.

[0066] Table 1. CYP3A4 gene test results and allele frequencies

[0067]

[0068] Table 2. Comparison of VAS scores of postoperative pain between the two groups

[0069]

[0070] Table 3. Postoperative sufentanil consumption in the two groups of patients (x±s, μg)

[0071]

[0072] Table 4. Postoperative butorphanol consumption in the two groups of patients (x±s, μg)

[0073]

[0074] The results of CYP3A4*1G gene polymorphism detection are shown in Table 1. As can be seen from the table, there were 32 cases of wild homozygous type (CYP3A4*1 / *1, AA), 13 cases of mutant heterozygous type (CYP3A4*1 / *1G, GA), and 6 cases of mutant homozygous type (CYP3A4*1G / *1G, GG). The mutation frequency of CYP3A4*1G allele was 20%.

[0075] The results of clinical observations on analgesic effects are shown in Tables 2-4. It can be seen from the table that, to achieve the same analgesic effect (as shown in Table 2), the consumption of sufentanil and butorphanol in patients with CYP3A4*1G gene mutants (CYP3A4*1 / *1G, CYP3A4*1G / *1G) is less than that in patients with wild type (CYP3A4*1 / *1) (as shown in Tables 3-4).

[0076] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of CYP3A4*1G gene in the preparation of products for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol.

2. Primer set for amplifying CYP3A4*1G gene.

3. The primer set according to claim 2, characterized in that The forward primer sequence of the primer set is shown as SEQ ID NO: 1, and the reverse primer sequence is shown as SEQ ID NO:

2.

4. A kit, characterized in that: The kit comprises the primer set according to any one of claims 2 to 3.

5. The kit according to claim 4, characterized in that The kit also includes PCR buffer, dNTPs, and PCR enzyme.

6. Use of the primer set according to any one of claims 2 to 3 or the kit according to any one of claims 4 to 5 in any of the following: (1) CYP3A4*1G genotyping; (2) Preparation of products for CYP3A4*1G genotyping; (3) Application in the preparation of products for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol.

7. The use according to claim 6, characterized in that: The method for using the product comprises the following steps: extracting DNA from a patient to be tested, performing PCR amplification using the primer set described in any one of claims 2 to 3 to obtain an amplified product; and performing genotyping on the amplified product.

8. The use according to claim 7, characterized in that: The reaction system of the PCR amplification comprises: Add water to make up to 50 μL.

9. The use according to claim 7, characterized in that: The reaction procedure of the PCR amplification includes: 92-97°C, 8-12min; entering the cycle stage: 92-97°C 8-12min, 58-62°C 12-17s, 70-75°C 25-35s, a total of 28-32 cycles; 72°C for 8-12min.

10. A product, characterized in that The product contains the primer set according to any one of claims 2-3 or the kit according to any one of claims 4-5.

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