Application of CYP3A4*1G gene in the preparation of products for evaluating the postoperative analgesic effect and dosage prediction of sufentanil combined with butorphanol
By detecting the polymorphism of the CYP3A4*1G gene and using primer sets for PCR amplification and pyrophosphate sequencing, the problem of individual differences in the use of sufentanil combined with butorphanol in postoperative analgesia was solved, personalized medication was achieved, and the analgesic effect and safety were improved.
Patent Information
- Application Number
- CN202411960185.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2044-12-30
AI Technical Summary
In the existing technology, there are individual differences in the use of sufentanil combined with butorphanol in postoperative analgesia treatment, which makes it impossible to accurately adjust the dosage, which may lead to unrelieved pain or serious adverse reactions, and lacks effective efficacy predictive evaluation indicators.
By detecting the polymorphism of the CYP3A4*1G gene and using primer sets for PCR amplification and pyrosequencing, the analgesic effect of sufentanil combined with butorphanol was evaluated and the dosage was predicted, providing personalized medication guidance.
The dosage of sufentanil combined with butorphanol was adjusted according to genotype differences, which improved the postoperative analgesic effect, reduced the occurrence of adverse reactions, and improved patient satisfaction and safety.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of the CYP3A4*1G gene in the preparation of a product for evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and for dosage prediction. Background Art
[0002] Pain is an unpleasant sensory and emotional experience caused by tissue damage or potential tissue damage, or a distressing experience with sensory, emotional, cognitive, and social dimensions. Pain can be categorized as acute or chronic based on the healing time of tissue damage and the duration of pain. Postoperative pain, which occurs immediately after surgery and includes both somatic and visceral pain, typically lasts no more than three to seven days. It is common in major thoracic and abdominal surgeries and procedures such as joint replacements that require prolonged functional training. Inadequately controlled postoperative pain can lead to a range of complications across various body systems, such as increased oxygen consumption affecting ischemic organs; triggering harmful spinal reflexes that reduce lung function; and reducing gastrointestinal motility and delaying gastrointestinal function recovery. Acute pain not only increases patient suffering and complications but, in severe cases, can also impair postoperative recovery and disease outcomes. Therefore, acute pain in the preoperative period remains a challenge that both physicians and patients must address. Proactive and effective analgesia is crucial for reducing complications, accelerating recovery, improving patient quality of life, and ultimately, lowering postoperative mortality. Currently, sufentanil combined with butorphanol is the most commonly used postoperative analgesia in clinical practice.
[0003] Sufentanil is a potent and highly selective μ opioid receptor agonist with analgesic potency 7-10 times that of fentanyl and less histamine release. However, high doses can cause adverse reactions such as respiratory depression, nausea, vomiting, dizziness, and itching, making its use as a single agent less effective. Butorphanol is a mixed opioid receptor agonist-antagonist that produces analgesia by stimulating central κ opioid receptors. Its analgesic effect is weaker than sufentanil, being 3-7 times that of morphine. However, it has a longer-lasting analgesic effect, weaker smooth muscle stimulation, and fewer gastrointestinal adverse reactions. It also antagonizes the μ2 receptor in sufentanil, suppressing sufentanil's addictive properties and adverse reactions such as respiratory depression. Furthermore, while μ receptor agonism provides better control of somatic pain, it is less effective for visceral pain, whereas κ receptor agonists are more effective for visceral pain. Therefore, the combination of sufentanil and butorphanol can act synergistically in clinical practice, enhancing analgesic efficacy while also allowing for a reduced dose of analgesic medication, thereby reducing the incidence of adverse reactions. Currently, sufentanil combined with butorphanol is widely used in clinical postoperative analgesia treatment.
[0004] Currently, clinical dosages of sufentanil and butorphanol are strictly determined based on weight, body surface area, or age. However, extensive clinical practice has revealed significant individual variability in the analgesic efficacy and adverse reactions of sufentanil combined with butorphanol. At the same dose concentration, some patients experience poor or even no analgesic response. A large clinical trial found that as many as 11.5% of patients are insensitive to opioid analgesia, requiring increased doses to achieve analgesic effects. This often leads to exacerbated pain, a more gradual progression of the disease, and even to chronic pain and drug addiction, impairing quality of life and, in severe cases, postoperative recovery and disease outcomes. Some patients experience severe adverse reactions such as respiratory depression, convulsions, nausea and vomiting at the same dose concentration, severely impacting prognosis and even life-threatening outcomes. Due to the lack of sensitive predictive indicators for analgesic efficacy, clinical dosing cannot be accurately determined, resulting in either insufficient dosage (failure to relieve pain symptoms) or excessive dosage (concomitant with numerous complications and adverse reactions). Finding out the reasons for this difference and searching for sensitive predictive evaluation indicators of efficacy are the key to clinical guidance of postoperative analgesia. Summary of the Invention
[0005] The present invention aims to address at least one of the technical problems existing in the aforementioned prior art. To this end, the present invention proposes 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.
[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 applications of the primer set and kit.
[0009] The present 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, genotyping of the CYP3A4*1G gene is performed to evaluate the postoperative analgesic effect of sufentanil combined with butorphanol and / or predict the postoperative dosage of sufentanil combined with butorphanol. When the patient's CYP3A4*1G genotype is detected to be AA, compared with patients with CYP3A4*1G genotypes of GA or 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 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, comprising the above 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, a use of the above primer set or kit is provided, wherein the use is 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 includes 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 method further comprises the step of evaluating the postoperative analgesic effect of sufentanil combined with butorphanol and / or predicting the postoperative dosage of sufentanil combined with butorphanol based on the genotyping results.
[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 includes:
[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 includes:
[0029]
[0030] Add water to make up to 50 μL.
[0031] In some embodiments of the present invention, the method for genotyping the amplified product is sequencing.
[0032] In some embodiments of the invention, the sequencing method comprises pyrosequencing.
[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 includes at least one of a detection plate, a test kit, and a detection chip.
[0038] According to some embodiments of the present invention, there are at least the following beneficial effects: the present invention has discovered for the first time that the CYP3A4*1G gene polymorphism 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 CYP3A4*1G gene typing related to the postoperative analgesic medication of the above-mentioned patients, guidance can be provided for the postoperative analgesic dosage of patients. DETAILED DESCRIPTION
[0039] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described 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 those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.
[0041] Example 1 Application of the CYP3A4*1G gene in evaluating the analgesic effect and dosage prediction of sufentanil combined with butorphanol after surgery
[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. 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 at Xiangya Second Hospital were enrolled. Inclusion criteria: 1) Patient age 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) Patients were conscious 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 or pulmonary insufficiency; 4) Adverse events during surgery that affect follow-up and assessment results.
[0047] 2. Methods
[0048] 1. Anesthesia and postoperative analgesia methods
[0049] All patients received a standardized postoperative analgesia regimen: Patient-controlled intravenous analgesia (PCIA) combined with sufentanil and butorphanol. Successful analgesia was defined as a NRS score of less than 3. Sufentanil and butorphanol consumption was recorded at 6, 24, and 48 hours after postoperative analgesia.
[0050] Postoperative analgesia was assessed using the VAS pain scale immediately after awakening, and at 2, 6, 8, 12, and 24 hours after surgery. A score of 0 indicated no pain, 1-3 indicated mild pain, 4-6 indicated moderate pain, and 7-10 indicated severe pain. A VAS score of ≤3 was considered effective analgesia. If the maximum hourly PCIA dose was reached but the VAS score remained >3, additional analgesics were administered 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 to assess adverse reactions such as nausea and vomiting, itching level, and respiratory depression.
[0052] 2. Genetic testing
[0053] Peripheral blood DNA from enrolled patients was genotyped for the CYP3A4*1G allele using pyrosequencing. (The CYP3A4 gene is located on human chromosome 7, 7q21.1-q22.1, and is approximately 27 kbp long. Its genetic structure comprises 13 exons and 12 introns. CYP3A4*1G (20230G>A) is a single nucleotide polymorphism (SNP) in intron 10 of CYP3A4, with a frequency of 22.1% to 37% in the Han Chinese population.) Preoperatively, 1 mL of peripheral venous blood was drawn from the patient and anticoagulated with ethylenediaminetetraacetic acid (EDTA). Genomic DNA from whole blood was extracted using the HiPure Blood DNA Mini Kit. DNA concentration and purity were determined using a nucleic acid analyzer. All DNA samples were maintained at an A260 / A280 ratio ≥ 1.8, indicating good purity. DNA was stored at -20°C.
[0054] The DNA fragment containing the polymorphic site was amplified by polymerase chain reaction (PCR) using primers. The primer sequences used in PCR were 5'-GTAATAGAAAGCAGATGAACC-3' (SEQ ID NO: 1) and 5'-TCACCCTGATGTCCAGCAGAA-3' (SEQ ID NO: 2).
[0055] The total PCR reaction volume was 50 μL, including 50 ng of genomic DNA, 10 μL of 5× PCR buffer, upstream and downstream primers at a final concentration of 0.2 μmol / L, 2 μL of 5 mmol / L dNTPs, and 1 unit of 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, for a total of 30 cycles; 72°C for 10 min.
[0057] Pyrosequencing was performed using a commercially available pyrosequencing kit. The method is briefly described as follows:
[0058] The CYP3A4*1G gene PCR product obtained by PCR amplification was added to the binding buffer and magnetic beads and incubated on a multi-functional oscillator at 1400 rpm / s for 10 minutes at room temperature. 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. The reaction plate was incubated at 80°C for 2 minutes and then removed from the plate and allowed to cool to room temperature.
[0059] The assay protocol for each reaction well was set according to the Pyro Mark ID instrument's operating instructions. The enzyme mix (E), fluorescent substrate (S), and four dNTPs contained in the Pyrophosphate Sequencing Kit were added to the reagent compartment. The reaction plate and reagent compartment were placed in the assay compartment for testing. Data analysis was performed using the analysis software provided with the Pyro Mark Q24 Pyrophosphate Sequencer to determine polymorphisms in the CYP3A4 gene.
[0060] Genotyping for CYP3A4*1G can be divided into wild-type homozygotes (CYP3A4*1 / *1, AA), mutant heterozygotes (CYP3A4*1 / *1G, GA), and mutant homozygotes (CYP3A4*1G / *1G, GG). CYP3A4*1G genotyping was performed blinded to the investigators throughout the study, and the results of the CYP3A4*1G genotyping test were publicly available upon completion of the study.
[0061] 3. Discovery and Verification
[0062] 1. Discovery Phase
[0063] After obtaining informed consent from 100 patients, 1 mL of peripheral venous blood was drawn, and whole-genomic DNA was extracted. The test was performed according to the above-mentioned method. The results showed that the mutation frequency of the CYP3A4*1G allele was 20%, which is consistent with the overall mutation rate of the Han Chinese. Clinical observations also found that to achieve the same analgesic effect, patients with CYP3A4*1G gene mutants (CYP3A4*1 / *1G, CYP3A4*1G / *1G) consumed less sufentanil and butorphanol than patients with the wild type (CYP3A4*1 / *1), which was a significant difference.
[0064] 2. Verification phase
[0065] 1 mL of peripheral venous blood was collected from 50 patients enrolled in the project experiment (completely different from the 100 patients mentioned above), 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 (x±s, μg)
[0071]
[0072] Table 4. Postoperative butorphanol consumption in the two groups (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 CYP3A4*1G allele mutation frequency was 20%.
[0075] The results of clinical observations on analgesic effects are shown in Tables 2-4. As can be seen from the table, to achieve the same analgesic effect (as shown in Table 2), the consumption of sufentanil and butorphanol in patients with CYP3A4*1G gene mutations (CYP3A4*1 / *1G, CYP3A4*1G / *1G) was lower than that in patients with wild type (CYP3A4*1 / *1) (as shown in Tables 3-4).
[0076] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.
Claims
1. Use of a primer set for amplifying the CYP3A4*1G gene in the preparation of a product for evaluating the analgesic effect of sufentanil combined with butorphanol after surgery and / or predicting the postoperative dosage of sufentanil combined with butorphanol, characterized in that: The CYP3A4*1G gene is CYP3A4(20230G>A); to achieve the same analgesic effect, patients with CYP3A4*1G gene mutants CYP3A4*1 / *1G and CYP3A4* 1G / *1G consume less sufentanil and butorphanol than patients with wild-type CYP3A4*1 / *1; the forward primer sequence of the primer set for amplifying the CYP3A4*1G gene is shown in SEQ ID NO:1, and the reverse primer sequence is shown in SEQ ID NO:
2.
2. The use according to claim 1, 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 as claimed in claim 1 to obtain an amplified product; and performing genotyping on the amplified product.
3. The use according to claim 2, characterized in that The reaction system of the PCR amplification comprises: 5× PCR buffer 1×; Forward primer 0.1-0.3 μM; Reverse primer 0.1-0.3 μM; Taq polymerase 0.5-2U; dNTP 0.1-0.3 mM; DNA 40-60ng; Add water to make up to 50 μL.
4. The use according to claim 2, characterized in that The PCR amplification reaction program 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, for a total of 28-32 cycles; 72°C for 8-12 min.