Use of adenylyl cyclase inhibitor in preparation of drug for treating perioperative pain

Abstract

The present invention relates to use of an adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating perioperative pain. The perioperative period comprises a preoperative period, an intraoperative period, and a postoperative period. The drug comprises an active ingredient for gastrointestinal or non-gastrointestinal administration, and a suitable pharmaceutical organic or inorganic inert carrier material. In the drug for treating perioperative pain, the adenylyl cyclase inhibitor represented by formula (1) or the pharmaceutically acceptable salt thereof is the only active ingredient. The drug of the present invention can relieve the stresses of undergoing surgery, has a therapeutic effect on pain in the entire perioperative period, and does not have side effects such as addiction.

Description

Use of an adenylyl cyclase inhibitor for the manufacture of a medicament for the treatment of perioperative pain

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 202410993544.6, filed on July 24, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of medicine, in particular to the use of an adenylyl cyclase inhibitor or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of perioperative pain. BACKGROUND

[0004] Perioperative period is a whole process around surgery, starting from the patient's decision to accept surgical treatment, to the surgical treatment until the basic recovery, including a period of time before, during and after surgery.

[0005] Postoperative pain is a kind of acute pain, and the pain will be more obvious after the decline of the anesthetic efficacy. At present, more attention is paid to the analgesic treatment during this period. After surgery, not only the pain of the wound, many patients will be nervous, and dare not to take a deep breath and cough, which will lead to pulmonary infection and cardiovascular complications and other conditions. Some patients will feel pain before surgery, especially for patients who have undergone non-first surgery (second or multiple surgeries).

[0006] The perioperative analgesia guidelines point out that the first is to emphasize more perioperative analgesia, rather than postoperative analgesia, because although the pain occurs after surgery, the postoperative pain is derived from two aspects, one is the damage to the tissue during the operation, such as the damage to the skin, muscle, bone and nerve caused by cutting and pulling, which is a major component, and after these stimuli cause tissue damage, a series of inflammatory reactions will occur, these inflammatory mediators will stimulate the peripheral nerve endings, and then produce pain, which involves secondary inflammatory pain, these two components constitute the main source of postoperative pain. These two factors will cause peripheral and central sensitization, after peripheral and central sensitization, a little stimulus that originally does not produce pain can now produce severe pain, and the intensity of the pain that was originally very light will be very high now, and the duration will be very long. At present, it is mainly advocated to give appropriate measures before surgery to reduce trauma stress, but generally auxiliary work such as preoperative psychological guidance is carried out.

[0007] The current analgesics mainly include: non-steroidal anti-inflammatory analgesics, central analgesics and narcotic analgesics, these drugs not only have great side effects, but also have a great possibility of addiction, cause damage to human health, and preoperative use even more aggravate the anxiety of patients, and are not suitable for preoperative use. Therefore, a drug which can be used throughout the perioperative period, especially for preoperative administration, is urgently needed. SUMMARY

[0008] In order to solve the above problems, the present application provides a drug which can be used preoperatively, can relieve surgical tension, has a therapeutic effect on pain throughout the perioperative period, and has no side effects such as addiction.

[0009] In order to achieve the above-mentioned object, the present application relates to the use of an adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating perioperative pain, said perioperative period including preoperative, intraoperative and postoperative, said drug comprising an active ingredient for gastrointestinal or non-gastrointestinal administration, and a suitable pharmaceutically organic or inorganic inert carrier material;

[0010] In the drug for treating perioperative pain, the adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof is the only active ingredient.

[0011] Optionally, the drug is administered preoperatively.

[0012] Optionally, the surgery is a non-first surgery.

[0013] Optionally, the suitable pharmaceutically organic or inorganic inert carrier material includes one or more of water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil and polyalkylene glycol.

[0014] Optionally, the dosage form of the drug is a tablet, suppository or capsule.

[0015] Optionally, the dosage form of the drug is a solution, suspension or emulsion.

[0016] Optionally, the drug contains a preservative, a stabilizer, a wetting agent or an emulsifying agent.

[0017] Optionally, the drug contains a salt for changing osmotic pressure, or a buffer auxiliary material.

[0018] Optionally, the drug for treating perioperative pain is a drug for treating perioperative pain without addiction.

[0019] The second aspect of the present application provides the use of an adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating perioperative pain and anxiety, said perioperative period including pre-operation, intra-operation and post-operation, said medicament comprising an active ingredient for gastrointestinal or non-gastrointestinal administration, and a suitable pharmaceutically organic or inorganic inert carrier material;

[0020] In the medicament for treating perioperative pain, the adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof is the only active ingredient.

[0021] Optionally, the medicament for treating perioperative pain and anxiety is a non-addictive medicament for treating perioperative pain and anxiety.

[0022] Through the above technical solution, the present application provides the use of an adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof as the only active ingredient in the manufacture of a medicament for treating perioperative pain, and the present application can reduce the heart rate during operation and achieve better analgesic effect in the management of perioperative pain; and the secretion of adrenal cortex is reduced after postoperative recovery, thereby reducing psychological burden. BRIEF DESCRIPTION OF DRAWINGS

[0023] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, and together with the specific embodiments described below, serve to explain the present application, but do not constitute a limitation on the present application. In the drawings:

[0024] FIG. 1 is a pain score from 0-4h after the first administration in Example 2.

[0025] FIG. 2 is a total pain score trend in Example 2.

[0026] FIG. 3 is a pain score comparison at the best time point of drug efficacy in Example 2.

[0027] FIG. 4 is a pain score reduction value after three administrations in four groups in Example 2.

[0028] FIG. 5 is the effect of NB001 on the percentage of the duration of entering the open arm of the SNL model rats in Example 4 (Mean ± SD, n = 10).

[0029] FIG. 6 is the effect of NB001 on the percentage of the number of entering the open arm of the SNL model rats in Example 4 (Mean ± SD, n = 10).

[0030] FIG. 7 is the effect of NB001 on the percentage of pain threshold of the SNL model rats in Example 5 (Mean ± SD, n = 10).

[0031] FIG. 8 is the residence time of animals in the A box at D9 in Example 6.

[0032] Figure 9 is the residence time of each group of animals in the B box at D9 in Example 6.

[0033] Figure 10 is the number of shuttles of each group of animals at D9 in Example 6.

[0034] Figure 11 is the conditioned place preference score of each group of animals at D9 in Example 6. DETAILED DESCRIPTION

[0035] The specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the present disclosure, and are not intended to limit the present disclosure.

[0036] In the present application, the term "perioperative pain" refers to a series of reactions caused by surgical stimulation (including surgical trauma, tissue damage or inflammatory response) during the entire process from preoperative preparation to postoperative rehabilitation (usually covering 24 hours before surgery to 7 days after surgery), which is manifested in physiology, psychology and behavior, and needs to be treated acute pain.

[0037] The term "perioperative pain and anxiety" is based on perioperative pain, and simultaneously intervenes in the pathological anxiety state (such as preoperative fear and postoperative delirium) induced by surgical stress, forming a "physiological- psychological" dual symptom set.

[0038] The term "non-addictive drug for treating perioperative pain and anxiety" refers to the complete avoidance of addiction risk on the basis of achieving pain-anxiety dual intervention, and meeting the long-term surgical rehabilitation needs.

[0039] The first aspect of the present application provides the use of an adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating perioperative pain, wherein the perioperative period includes preoperative, intraoperative and postoperative periods, and the medicament comprises an active ingredient for gastrointestinal or non-gastrointestinal administration, and a suitable pharmaceutically inert organic or inorganic carrier material;

[0040] In the medicament for treating perioperative pain, the adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof is the only active ingredient.

[0041] In an embodiment of the present application, the medicament is administered before surgery.

[0042] The principles of the currently clinically used analgesic drugs are as follows:

[0043] 1. Non-steroidal anti-inflammatory drugs, such as ibuprofen sustained-release capsules, indomethacin tablets, diclofenac sodium sustained-release tablets, which act by inhibiting the synthesis of prostaglandins in the periphery, reducing the excitability of pain receptors, and at the same time inhibiting the synthesis of bradykinin and other pain-causing substances. This class of drugs is only effective for moderate pain, such as headache, toothache, muscle pain, joint pain, neuralgia, moderate intensity postoperative pain, and early pain in cancer patients; 2. Opioid analgesics, such as morphine, pethidine hydrochloride tablets, fentanyl citrate injection, etc. The principle of this class of drugs is to act on the opioid receptors in the central nervous system to produce analgesia. This class of drugs is mainly used for the treatment of severe pain, such as trauma, and pain in patients with advanced cancer; 3. Racemic anisodine tablets or belladonna tablets, which act mainly by relieving gastrointestinal smooth muscle spasm to achieve analgesic effect, and are usually used to treat abdominal pain.

[0044] The inventors used the above currently used analgesic drugs for the perioperative period, i.e. the same as the present application, and found that there was no difference in analgesic effect between the administration before surgery and no administration, and in fact, the patients became more anxious. Moreover, the above currently used analgesic drugs not only have large side effects, but also have a high possibility of addiction, causing harm to human health.

[0045] The adenylyl cyclase inhibitor (AC1 inhibitor) represented by formula (1) of the present application specifically binds to AC1, not only inhibits the activity of AC1, regulates synaptic plasticity changes, but also inhibits the expression of AC1. That is, by preoperative administration, the expression of AC1 can be inhibited, memory can be reduced, trauma stress response can be reduced, and pain sensitivity can be reduced. Therefore, the AC1 inhibitor has important clinical significance for perioperative analgesia.

[0046] Studies have shown that in the brain area related to pain in patients with chronic pain, especially in the anterior cingulated cortex (ACC) brain area, adenylate cyclase (AC) and its downstream cyclic adenosine monophosphate / protein kinase A (cAMP / PKA) signaling pathway are highly activated, which triggers a series of synaptic plasticity changes. The AC1 inhibitor reduces the level of cyclic adenosine monophosphate (cAMP) in the anterior cingulated cortex (ACC) tissue by inhibiting the activity of AC1, and reduces the N-methyl-D-aspartic acid receptor (NMDA) NR2A and NR2B subunits and phosphorylation sites (NR2B serine 1303 and tyrosine 1472 sites) in the ACC, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor GluA1 subunit and the GluA1 phosphorylation level (serine 831 and 845 sites), which plays a role in reducing or eliminating neuropathic pain or inflammatory pain.

[0047] The present application carries out pain management during the perioperative period (starting to take the adenylate cyclase inhibitor of formula (1) described in the present application before surgery) than after surgery, which will reduce the heart rate during the operation, and the analgesic effect is better; after waking up after surgery, it will reduce the secretion of adrenal cortex and reduce the psychological burden. That is, taking it before surgery (especially for patients who are more nervous about surgery and have psychological pain), it can well reduce the pain and nervousness of patients during surgery, and has a therapeutic effect on the whole perioperative pain.

[0048] In an embodiment of the present application, the surgery is a non-first surgery.

[0049] In an embodiment of the present application, the suitable pharmaceutical organic or inorganic inert carrier material includes one or more of water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil and polyalkylene glycol.

[0050] In an embodiment of the present application, the dosage form of the drug is a tablet, a suppository or a capsule.

[0051] In an embodiment of the present application, the dosage form of the drug is a solution, a suspension or an emulsion.

[0052] In one embodiment of the present application, the medicament comprises a preservative, a stabilizer, a wetting agent, or an emulsifying agent.

[0053] In one embodiment of the present application, the medicament comprises a tonicity- modifying salt, or a buffer.

[0054] In one embodiment of the present application, the medicament for treating perioperative pain is a medicament for treating perioperative pain without addiction.

[0055] The second aspect of the present application provides the use of an adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating perioperative pain and anxiety, wherein the perioperative period comprises pre-operation, intra-operation, and post-operation, and the medicament comprises an active ingredient for gastrointestinal or non-gastrointestinal administration, and a suitable pharmaceutically inert organic or inorganic carrier material.

[0056] In the medicament for treating perioperative pain, the adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof is the only active ingredient.

[0057] In one embodiment of the present application, the medicament for treating perioperative pain and anxiety is a medicament for treating perioperative pain and anxiety without addiction.

[0058] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited in any way by the following examples.

[0059] The raw materials used in the following examples are commercially available unless otherwise specified. Experimental drug NB001 is an adenylyl cyclase inhibitor represented by formula (1) and is provided by Zhejiang Yongzhan Pharmaceutical Technology Co., Ltd. AOA089 is a chewable tablet prepared from an adenylyl cyclase inhibitor represented by formula (1) and is provided by Zhejiang Yongzhan Pharmaceutical Technology Co., Ltd.

[0060] Example 1 Evaluation of the preoperative and postoperative analgesic effect of oral NB001 in cats

[0061]

Test Process

[0062] This study is a randomized, parallel controlled clinical trial, and the test period lasts about 2 weeks. Only the dispensers and the drug administrators know the information about the grouping and administration of animals, and the clinical examination and efficacy evaluation personnel are unaware of the administration information and grouping of animals. After the end of the test, the analysis and statistics of the test data are carried out by the research director and the statistician, and the dispensers and the efficacy evaluation personnel do not participate.

[0063] Six healthy female cats were selected as test animals (Table 1). The test animals were adaptively fed for 3 days before surgery (D-3), and the cats meeting the inclusion criteria were randomly grouped (see Table 2). According to the order of selection, the cats were randomly divided into two groups: the recommended dose group (H) and the negative dose group (C), with 3 cats in each group. The cats in the recommended dose group (H) were orally administered with the test drug 3 days before surgery, once a day for 6 days before and after surgery. The cats in the negative dose group (C) were not treated before surgery, and were orally administered with the test drug 1 day after surgery, once a day for 3 days, with an interval of 24 hours between each administration. The same treatment was performed on the cats in both groups during the anesthesia process, and no pain management was performed during the surgery.

[0064] Table 1 Basic information and grouping of test cats

[0065] Table 2 Dose of test drug and test grouping of test cats

[0066] The test period was defined as D-2 to D3, and the surgery day was defined as D0. The test schedule is shown in Table 3.

[0067] Table 3 Test schedule Note: T0: 3 hours before surgery; T1: first incision during surgery; T2: uterine traction during surgery; T3: ligation during surgery; T4: 1 hour after surgery; T5: second administration 24 hours after T0; T6: score 3 hours after the second administration; T7: third administration 48 hours after T0; T8: last administration 72 hours after T0; T9: 3 hours after the last administration.

[0068] Clinical indicators were detected every day during the administration period. Blood was collected 3 hours before surgery (T0) to detect the serum cortisol content. Heart rate, respiratory rate, and blood pressure (systolic pressure) were monitored 6 hours before surgery (T0), at the first incision during surgery (T1), uterine traction during surgery (T2), and ligation during surgery (T3). Pain was assessed according to the pain score table at anesthesia awakening (1 hour after surgery, T4), T6, and T9, and heart rate, respiratory rate, and blood pressure (systolic pressure) were monitored. Blood was collected to detect the serum cortisol content. Adverse events were also recorded.

[0069] During the test period, no death occurred, and no test cat withdrew from the test.

[0070]

Effect evaluation

[0071] Primary indicator: Glasgow Feline Pain Scale (CMPS-Feline)

[0072] The Glasgow Composite Pain Scale (CMPS-Feline) can be applied quickly and reliably in a clinical setting and has been designed as a clinical decision tool for acute pain in cats. It consists of 28 descriptor options in 7 behavioural categories. In each category, the descriptors are numerically ordered according to their associated pain severity, and the person performing the assessment selects the descriptor that best fits the cat's behaviour / condition in each category. The pain score is the sum of the rank scores. The maximum score for the 7 categories is 20, and a pain score of 5 / 20 is recommended for intervention.

[0073] Other indicators: serum cortisol and heart rate

[0074] Serum cortisol detection and analysis: The detection animal option of the endocrine instrument is selected for the cat, and the serum is detected according to the instrument prompt steps, and the test cat number is sequentially performed.

[0075] Heart rate is measured by sphygmomanometer.

[0076] Data collection is carried out with each test cat as a test unit, and data analysis is carried out in the group.

[0077] The data included in the analysis of this test at least include:

[0078] (1) Heart rate, respiratory rate and systolic blood pressure of each test cat at each time point during D-2~D3 and adverse reactions;

[0079] (2) Serum cortisol content at T0, T4, T6;

[0080] (3) Pain score at T4, T6, T9;

[0081] The statistical analysis software used is IBM SPSS22.0. After summarizing and arranging the test data, the mean and standard deviation of the quantitative data are obtained, and the t test is used for analysis. The analgesic effect of the test drug before and after operation is comprehensively evaluated.

[0082]

Test Results

[0083] I. Preoperative analgesic results of test cats

[0084] Heart rate

[0085] The specific data of heart rate collected at each time point during the test are shown in Table 4. The results of statistical analysis of heart rate are shown in Table 5.

[0086] The results are as follows:

[0087] Table 4 Test cat heart rate detection results (times / min)

[0088] Table 5 Test cat heart rate results

[0089] The results of the inter-group comparison showed that there was no significant difference in the heart rate of each group at D-2, D-1 and T0. After the test animals were anesthetized (T1, T2, T3), the heart rate was lower than that at T0. After the operation, the results of the test animals in group H at 1 h after anesthesia recovery (T4) had no significant difference compared with T0, and the results of the test animals in group C at 1 h after anesthesia recovery (T4) were lower than T0, but had no significant difference. The heart rate was an observation of safety after administration, and there was no significant difference between groups and within groups before and after the operation, indicating that the drug was safe.

[0090] The results of the inter-group comparison showed that there was no significant difference in the heart rate of each group at D-2, D-1, T0 and T4. The heart rate of the test animals in group H after anesthesia (T1, T2, T3, T4) was lower than that in group C. It was indicated that taking the test drug before the operation could reduce the physiological impact of intraoperative pain on the test animals.

[0091] 2. Pain score results

[0092] In order to evaluate the analgesic effect of preoperative administration, the test cats were given a pain score at 1 h after the operation (T4). The specific results are shown in Table 6. The results after statistical analysis are shown in Table 7.

[0093] Table 6 Pain score results of test cats

[0094] Table 7 Pain score results of test cats

[0095] After inter-group comparison, the pain score of group H at T4 was lower than that of group C, and the pain score of group H at T4 was already lower than 5, and pain management was not needed. It was indicated that taking the drug before the perioperative operation had an analgesic effect on postoperative pain, and pain management was not needed.

[0096] 3. Cortisol results

[0097] Blood was collected from the test cats at T0, T4 and T6, serum was separated, and the serum cortisol content was measured. The specific results are shown in Table 8. The results after statistical analysis are shown in Table 9.

[0098] Table 8 Serum cortisol results of test cats (ng / mL)

[0099] Table 9 Serum cortisol content results of test cats

[0100] After inter-group comparison, the cortisol content of groups H and C at T4 was significantly higher than that at T0.

[0101] From the numerical values, it can be seen that the cortisol content of group H T4 is 30% lower than that of group C. The increase of cortisol is a group of clinical symptoms caused by the abnormal regulation of hypothalamic-pituitary-adrenal axis and the excessive secretion of glucocorticoids in adrenal cortex. Perioperative pain management (starting medication before surgery) can greatly reduce the secretion of adrenal cortex and reduce psychological burden after postoperative recovery compared with postoperative pain management.

[0102] II. Postoperative analgesia results of test cats

[0103] 1. Heart rate detection results of test cats

[0104] The specific data of heart rate collected at each time point during the period are shown in Table 4. The results of statistical analysis of heart rate at 3h after the first postoperative administration and 3h after the last administration (T6, T9) are shown in Table 10.

[0105] Table 10 Heart rate results of test cats

[0106] After intragroup comparison, the heart rates of groups H and C at T6 and T9 were lower than those at T0 and T4.

[0107] After intergroup comparison, there was no significant difference in heart rate between groups H and C at T6 and T9.

[0108] 2. Pain score results

[0109] To evaluate the analgesic effect of preoperative administration, the test cats were given pain scores at 1h after surgery (T4), 4h after administration on the second postoperative day (T6), and 4h after the last administration (T9). The specific results are shown in Table 6. The results after statistical analysis are shown in Table 11.

[0110] Table 11 Pain score results of test cats

[0111] After intragroup comparison, the pain scores of groups H and C at T6 and T9 were lower than those at T4, and there was no significant difference between T6 and T9.

[0112] After intergroup comparison, the pain score of group H at T4 was lower than that of group C, and there was no significant difference between T6 and T9, and both were lower than 5, so no pain management was needed. It is shown that starting medication before surgery during the perioperative period has better analgesic effect on postoperative pain than taking medication only after surgery.

[0113] 3. Cortisol results of test cats

[0114] Blood was collected from test cats at T0, T4 and T6, serum was separated, and serum cortisol content was measured. The specific results are shown in Table 8. The results after statistical analysis are shown in Table 12.

[0115] Table 12 Serum Cortisol content results of test cats

[0116] By intra-group comparison, the cortisol content of H group and C group at T4 was significantly higher than that at T0; the cortisol content at T6 was significantly lower than that at T4, but there was no significant difference between T0 and T6.

[0117] By inter-group comparison, from the numerical values, the cortisol content of H group at T4 was about 30% lower than that of C group, and the cortisol content of H group at T6 was also lower than that of C group.

[0118] In summary, the following conclusions are drawn from this test:

[0119] (1) After 3 days of continuous administration before surgery, the intraoperative pain can be effectively relieved, and the analgesic effect is better than that of postoperative administration, which can also reduce the secretion of adrenal cortex and psychological burden;

[0120] (2) The results of 3 days of continuous administration after surgery show that 3 days of continuous administration after surgery can effectively relieve postoperative pain.

[0121] The results of Example 1 prove that NB001 can effectively treat perioperative pain.

[0122] Example 2 Analgesic effect of AOA089 on pain caused by soft tissue injury in cats

[0123] 1. Experimental drugs

[0124] Experimental group: AOA089 chewable tablets, 50 mg / tablet;

[0125] Positive control: Meloxicam oral suspension (for cats) (Shanghai Hanwei, 0.2 mL for the first dose per 1 kg of body weight, and 0.1 mL for the maintenance dose, directly fed, once a day, for 3-4 days).

[0126] 2. Experimental animals

[0127] 25 healthy cats, all over 6 months old.

[0128] 3. Animal pain model

[0129] After excluding infectious diseases such as feline distemper, the cats were adaptively fed for 7 days before modeling. Blood was collected for preoperative examination (blood routine, preoperative biochemistry) one day before modeling.

[0130] Preoperative:

[0131] a. Fasting for 8 hours, water for 4 hours;

[0132] b. Preoperative examination, assessment of surgical anesthesia risk: preoperative biochemistry, blood routine.

[0133] c. Induction of anesthesia, a. Record body weight; b. Cat forelimb implantation of indwelling needle; c. Routine sedation and anesthesia; f. Preoperative and postoperative prohibition of analgesics such as analgin and the like (except for the test drug and meloxicam after operation).

[0134] Intraoperative:

[0135] a. Incision positioning: 1 cm backward from the navel hole, opening on the back;

[0136] b. Sterilization of the operation site: after hair removal and cleaning of the cat's abdomen, routine sterilization is performed;

[0137] c. Fixation of the wound dressing: fixation at the surgical incision site, only the operation site is exposed;

[0138] d. Sterilization of the wound;

[0139] e. Incision of the skin along the midline of the abdomen 1 cm backward from the navel hole, incision 8 cm. Bluntly separate the subcutaneous tissue to see the abdominal muscles, and cut the abdominal white line. After checking that the abdominal organs are normal, continuously suture to close the abdominal cavity, and nodularly suture the skin;

[0140] f. Anesthesia monitoring throughout the operation by a dedicated person, monitoring heart rate, respiration, blood oxygen and other indicators.

[0141] Postoperative:

[0142] Postoperative administration of long-acting antibiotic Kangweining (0.1 mL / kg), intravenous infusion of lactated Ringer's solution 50 mL.

[0143] The animals wear a neck ring to prevent the animals from licking the wound, and the modeling is completed after the animals wake up.

[0144] Note: Prevent falling before anesthesia wakes up after operation; food can be taken 6 hours after complete recovery, and the wound should be kept dry.

[0145] After the modeling is completed, random grouping is performed, and according to the tag number, EXCEL is used to generate a random number sequence to fill in the administration group, positive control group and negative control group. The grouping treatment is shown in Table 13.

[0146] Table 13 Grouping and treatment of experimental animals

[0147] 4. Observation index

[0148] (1) Pain baseline value:

[0149] When the cat wakes up 6 hours after operation, the visual analogue scale (VAS) and the Glasgow Cat Comprehensive Pain Scale (CMPS-FS) are used to evaluate the degree of cat pain, which is the baseline value.

[0150] (2) Administration time:

[0151] After the baseline evaluation, the drug was administered at the prescribed dose. Fifteen minutes after administration, the cat was observed for vomiting, and if vomiting occurred, the administration was repeated.

[0152] (3) Onset and offset of analgesia

[0153] Pain was evaluated at 1 h, 2 h, 3 h, 4 h, 5 h, 7 h, 9 h, 12 h, 15 h, 18 h, 21 h, and 24 h after the first administration, and if the pain score was the same at two consecutive time points, the analgesia was considered to have disappeared. The positive control and the test drug were administered again every 24 h after the first administration, and the scores were recorded 4 h after the second administration and 4 h after the third administration (the optimal effect time was found to be 4 h after administration according to the pre-experiment).

[0154] Note: If the pain score was close to the baseline value at two consecutive time points after the initial administration, rescue analgesia was immediately performed, and the drug was considered to be ineffective at that dose.

[0155] 5. Experimental results

[0156] (1) Onset and offset of analgesia

[0157] As shown in Figure 1, which represents the pain score from 0-4 h after the first administration, the pain score of the negative group changed little. The meloxicam group began to exert an analgesic effect around 2 h after administration, and the AOA089 high-dose and medium-dose groups began to exert an analgesic effect around 1 h after administration. The pain score of the AOA089 low-dose group changed little, and the pain score was the same at 1 h and 2 h after administration, so the dose was considered to be ineffective, and rescue analgesia was performed at 7 h after the first administration. The subsequent analysis will not involve the AOA089 low-dose group.

[0158] (2) Comparison of analgesic effects

[0159] As shown in Figure 2, which represents the total pain score trend, the second and third administrations were performed 24 h and 48 h after the first administration, respectively. As shown in Figure 2, the pain score of the negative group decreased due to its own healing ability, but the rate and amplitude of the decrease were less than those of the administration groups. This indicates that the AOA089 high-dose and medium-dose groups have analgesic effects, and the pain score continues to decrease with an increase in the number of administrations, with the largest decrease in the pain score occurring after the second administration. The analgesic ability of the AOA089 high-dose group is superior to that of the positive drug meloxicam.

[0160] As shown in Figure 3, which represents the pain score at the best time point of drug efficacy, every four columnar bars are a group, from left to right, in turn, the negative group, the meloxicam group, the AOA089 high-dose group, and the AOA089 medium-dose group. From the pain score, the baseline pain scores of the four groups of cats had no significant difference. At the best time of drug efficacy after the first administration (4h after administration), the pain scores of the three administration groups were significantly lower than that of the negative group, indicating that the three drugs had significant analgesic effects, but at this time, the pain scores of the three administration groups had no significant difference; at the best time of drug efficacy after the second administration and the third administration (4h after administration), the pain scores of the three administration groups had significant differences, which were that the AOA089 high-dose group was significantly lower than the meloxicam group, and the meloxicam group was significantly lower than the AOA089 medium-dose group (p<0.05). The results show that after one administration, the analgesic effects of the three drugs have no significant difference; after two administrations and three administrations, the analgesic effect of the AOA089 high-dose group is significantly better than that of meloxicam (p<0.05).

[0161] As shown in Figure 4, which represents the pain score reduction value of the four groups after three administrations, every four columnar bars are a group, from left to right, in turn, the negative group, the meloxicam group, the AOA089 high-dose group, and the AOA089 medium-dose group. At the best time of drug efficacy after the second and third administrations (4h after administration), the pain relief degree of the AOA089 high-dose group was significantly higher than that of the meloxicam group.

[0162] 6、Conclusion

[0163] (1) The optimal dose of AOA089 is 40mg / kg;

[0164] (2) At this dose, the onset time of AOA089 (about 1h) is faster than that of meloxicam (about 2h);

[0165] (3) Under the condition of only one administration, the analgesic effect of the AOA089 at this dose on cats has no significant difference with that of meloxicam;

[0166] (4) From the second administration, the analgesic effect of the AOA089 high-dose on cats is better than that of meloxicam.

[0167] In summary, the AOA089 high-dose group has a good analgesic effect on the pain caused by soft tissue injury in cats.

[0168] The results of Example 2 prove that AOA089 can effectively treat perioperative pain.

[0169] Example 3 Oral NB001 Preoperative and Postoperative Analgesic Effect Evaluation Test Report for Dogs

[0170] This study is a randomized, parallel controlled clinical trial, the administration and efficacy evaluation are separated, the test cycle lasts about 2 weeks. The information of animal grouping and administration is only known by the dispensing person and the administration personnel, the clinical examination and efficacy evaluation personnel are unaware of the animal administration information and grouping. After the end of the test, the analysis and statistics of the test data are carried out by the research person in charge and the statistician.

[0171] Six healthy female dogs were selected as test animals (Table 14), and the test animals were adaptively fed for 3 days before surgery (D-3), and the dogs meeting the inclusion criteria were randomly grouped (see Table 15), and randomly divided into 2 groups according to the order of selection, recommended dose group (H) and negative dose group (C), 3 in each group. The recommended dose group (H) orally administered the test drug 3 days before surgery, 1 time / day for 6 days before and after surgery, and the negative dose group (C) did not do anything before surgery, and orally administered the test drug 1 time / day for 3 days after surgery, with an interval of 24 hours between each administration, and the two groups were treated the same during the anesthesia process, and no pain management was performed during the surgery.

[0172] Table 14 Basic information and grouping of test dogs

[0173] Table 15 Drug dosage and test grouping of test dogs

[0174] Clinical indicators were detected every day during the administration period, and blood was collected 6 hours before surgery (T0) to detect serum cortisol content. Heart rate, respiratory rate, and blood pressure (systolic pressure) were monitored at 6 hours before surgery (T0), the first incision of surgery (T1), uterine traction during surgery (T2), and ligation during surgery (T3). Pain assessment was performed according to the pain score table at 1 hour after anesthesia (T4), T6, and T9, and heart rate, respiratory rate, and blood pressure (systolic pressure) were monitored, and blood was collected to detect serum cortisol content. Adverse events were also recorded.

[0175] During the test period, no death occurred, and no test dog withdrew from the test.

[0176] During the test period, the pain scores, serum cortisol content, heart rate, respiration, and systolic pressure of the test dogs were compared between and within groups, and the analgesic effect of the test drug on the dogs before and after surgery was comprehensively evaluated.

[0177] The test time was defined as D-2 to D3, and the surgery day was defined as DO. The test schedule is shown in Table 16.

[0178] Table 16 Test schedule Note: T0: 6 hours before surgery; T1: first incision; T2: uterine traction; T3: ligation; T4: 1 hour after surgery; T5: second administration after T0-24 hours; T6: score 3 hours after second administration; T7: third administration after T0-48 hours; T8: last administration after T0-72 hours; T9: 3 hours after last administration.

[0179] The recommended dose group (H) was administered continuously for 6 days from D-2 to D3. DO was administered 6-8 hours before surgery. The negative dose group (C) was not treated before surgery, and oral administration of the test drug was started 1 day after surgery, continuously for 3 days, with a 24-hour interval between each administration.

[0180]

Effect evaluation

[0181] Primary endpoint: Glasgow canine pain scale short form (CMPS-FS)

[0182] CMPS-SF is quick and reliable in clinical use, and has been developed as one of the clinical decision tools for intervention in acute pain in dogs. It contains 30 description options in 6 behavior categories, including mobility. The evaluator selects the description that best fits the dog's behavior / state in each category. It is very important to strictly follow the description on the questionnaire. The pain score is the sum of the scores in each category, and the higher the score, the more severe the pain. The total score is 24 points, and if mobility cannot be evaluated, it is 20 points. A pain score of 6 / 24 or 5 / 20 is recommended for intervention.

[0183] Other indicators: serum cortisol and heart rate

[0184] Serum cortisol detection and analysis: Select the dog as the detection animal option of the endocrine instrument, and detect the serum according to the instrument prompt steps, and perform them in order according to the test dog number.

[0185] Heart rate is measured by sphygmomanometer.

[0186] In order to avoid the influence of the research results, any other drugs that may affect or mask the postoperative pain symptoms (such as corticosteroids and analgesics other than pre-specified rescue analgesics) are not allowed during the study period. Record all co-administration of each dog unrelated to surgical procedures.

[0187] Data collection is performed for each test dog as a test unit, and data analysis is performed for the group to which it belongs.

[0188] The data included in the analysis of this test at least includes:

[0189] (1) Heart rate, respiratory rate, and systolic blood pressure of each test dog at each time point during D-2 to D3, and adverse reactions;

[0190] (2) Serum cortisol content at T0, T4, T6;

[0191] (3) Pain score of T4, T6, T9;

[0192] The statistical analysis software used was IBM SPSS 22.0. After summarizing and sorting the test data, the mean and standard deviation of the quantitative data were calculated, and the t-test was used for analysis. Three indicators were analyzed to comprehensively evaluate the analgesic effect of the tested veterinary drugs before and after surgery.

[0193]

Test Results

[0194] I. Preoperative analgesic results of test dogs

[0195] 1. Heart rate results of test dogs

[0196] The specific data of heart rate collected at each time point during the test are shown in Table 17. The results of statistical analysis of heart rate are shown in Table 18.

[0197] Table 17 Heart rate test results of test dogs (times / min)

[0198] Table 18 Heart rate results of test dogs

[0199] The intragroup comparison results showed that there was no significant difference in heart rate of D-2, D-1 and T0 of each group. After the test animals were anesthetized (T1, T2, T3), the heart rate of the C group was higher than that of T0; the heart rate of the H group was lower than that of T0 at T1, T2 and T3. After the operation was completed, the results of the C group test animals 1 hour after anesthesia recovery (T4) were higher than those of T0, but there was no significant difference, and the results of the H group test animals 1 hour after anesthesia recovery (T4) were lower than those of T0, with no significant difference.

[0200] The intergroup comparison results showed that there was no significant difference in heart rate of D-2, D-1 and T0 of the H group and the C group. The heart rate of the C group after anesthesia (T1, T2, T3, T4) was higher than that of the H group. It is shown that preoperative administration of the tested drug can reduce the physiological impact of intraoperative pain on experimental animals.

[0201] 2. Pain score results of test dogs

[0202] To evaluate the analgesic effect of preoperative administration, the test cats were given a pain score 1 hour after the operation (T4). The specific results are shown in Table 19. The results of statistical analysis are shown in Table 20.

[0203] Table 19 Pain score results of test dogs Note: The pain score criteria are detailed in Section 10.4.1.

[0204] Table 20 Pain score results of test dogs

[0205] The pain score of T4 of C group was higher than that of H group. It was indicated that the drug taken before operation had analgesic effect on postoperative pain, and pain management was not needed.

[0206] 3. Cortisol results of test dogs

[0207] Blood was collected from test dogs at T0, T4 and T6, and serum was separated to measure the content of serum cortisol. The results are shown in Table 21. The results after statistical analysis are shown in Table 22.

[0208] Table 21. Results of serum cortisol of test dogs (ng / mL)

[0209] Table 22. Results of serum cortisol content of test dogs

[0210] It can be seen from the intragroup comparison that the cortisol content of T4 of H group and C group was significantly higher than that of T0.

[0211] It can be seen from the intergroup comparison that the cortisol content of T4 of H group was about 10% lower than that of C group. Pain management during the perioperative period (starting with preoperative medication) can reduce the secretion of adrenal cortex to a certain extent after postoperative recovery, and reduce the psychological burden.

[0212] II. Postoperative analgesic results of test dogs

[0213] 1. Heart rate detection results of test dogs

[0214] The specific data of heart rate collected at each time point during the period are shown in Table 17. The results after statistical analysis of heart rate at 3h after the first postoperative day and 3h after the last administration (T6, T9) are shown in Table 23.

[0215] Table 23. Results of heart rate of test dogs

[0216] It can be seen from the intragroup comparison that the heart rate of T6 and T9 of H group and C group was lower than that of T0 and T4.

[0217] It can be seen from the intergroup comparison that there was no significant difference between the heart rate of T6 and T9 of H group and C group.

[0218] 2. Pain score results

[0219] In order to evaluate the analgesic effect of preoperative administration, the test dogs were given pain score at 1h after operation (T4), 4h after administration on the second postoperative day (T6), and 4h after the last administration (T9). The specific results are shown in Table 19. The results after statistical analysis are shown in Table 24.

[0220] Table 24 Results of pain score of test dogs

[0221] It can be seen from the comparison within the group that the pain scores of H group and C group at T6 and T9 are lower than that at T4, and there is no significant difference between T6 and T9.

[0222] Through comparison between groups, the pain scores of H group at T4, T6 and T9 are lower than that of C group. It is indicated that taking the drug before surgery in the perioperative period has better analgesic effect on postoperative pain than taking the drug only after surgery.

[0223] 3. Results of cortisol of test dogs

[0224] Blood was collected from test dogs at T0, T4 and T6, serum was separated, and the content of serum cortisol was measured. The specific results are shown in Table 21. The results after statistical analysis are shown in Table 25.

[0225] Table 25 Results of serum cortisol content of test dogs

[0226] Through comparison within the group, the cortisol content of H group and C group at T4 is significantly higher than that at T0; there is no significant difference between T6 and T0 of H group and C group.

[0227] Through comparison between groups, there is no significant difference in the cortisol content between H group and C group at T0. There is also no significant difference in the cortisol content between H group and C group at T4, and the cortisol content of H group at T6 is lower than that of C group.

[0228] Based on the above test results, the following conclusions can be drawn:

[0229] (1) According to the results of this test, the operation after continuous administration for 3 days before surgery can relieve intraoperative pain, and the analgesic effect is better than that of administration only after surgery;

[0230] (2) The results of continuous administration for 3 days after surgery show that continuous administration for 3 days after surgery can effectively relieve intraoperative pain.

[0231] The results of Example 3 prove that NB001 can effectively treat perioperative pain.

[0232] Example 4 Anti-anxiety effect of NB001 on L5 / L6 nerve ligated rats

[0233] (1) Method: 90 male SD rats, weighing 200 ± 20 g, randomly selected 10 as the sham operation group, the remaining 80 rats after L5 / L6 nerve ligation to screen the pain sensitivity, select 60 qualified rats into the experiment. The rats were randomly divided into model group, gabapentin 100 mg / kg and NB001 20, 10, 5, 2.5 mg / kg dose group, the specific grouping as shown in Table 26:

[0234] Table 26

[0235] The drug preparation and administration method is as follows:

[0236] 100 mg / kg gabapentin: 100 mg (1 grain) is ground and added to 10 mL of 1% CMC-Na suspension, and then orally gavaged, with a dose of 1 mL / 100 g body weight;

[0237] 20 mg / kg NB001: 100 mg is ground and added to 50 mL of 1% CMC-Na solution, and then orally gavaged, with a dose of 1 mL / 100 g body weight;

[0238] 10 mg / kg NB001: 25 mL of the above 20 mg / kg NB001 solution is added to 25 mL of 1% CMC-Na and mixed, then orally gavaged, with a dose of 1 mL / 100 g body weight;

[0239] 5 mg / kg NB001: 10 mL of the above 20 mg / kg NB001 solution is added to 30 mL of 1% CMC-Na and mixed, then orally gavaged, with a dose of 1 mL / 100 g body weight;

[0240] 2.5 mg / kg NB001: 5 mL of the above 20 mg / kg NB001 solution is added to 35 mL of 1% CMC-Na and mixed, then orally gavaged, with a dose of 1 mL / 100 g body weight;

[0241] After 8 days of administration once a day, two hours later, the elevated plus maze was used to test the anxiolytic effect of NB001.

[0242] (2) Model preparation:

[0243] Anesthetize the rats with ether, lie down on the operating table, fix the limbs, slightly pad the waist, make a longitudinal midline incision along the back skin at the level of L4-S2 spinous process, separate the skin and muscle layer by layer, fully expose to the L6 transverse process, use hemostatic forceps to bite off the L6 transverse process, separate the left L5 and L6 nerves, use 6-0 silk to ligate the nerve roots, stop bleeding, suture the muscle and skin layer by layer, avoid postoperative infection, and perform screening test 10 days after operation. Sham operation only exposes the left L5 and L6 nerves without ligation.

[0244] (3) Test method:

[0245] The rats were orally administered with drugs once a day for 8 days. Two hours after the last administration, the experimental animals were placed in the central area of the maze with their heads facing the open arms. The video monitor was turned on to record the number of times the experimental animals entered the open arms and closed arms and the duration of entering each arm within 5 minutes. After recording, the experimental animals were returned to the feeding cage. Meanwhile, the maze was cleaned and wiped with 75% alcohol to eliminate the influence of animal odor on subsequent experimental animals. The animal movement trajectory tracking system automatically calculated the time and number of times the animals entered each open arm.

[0246] (4) Statistical method:

[0247] Number of open arm entries % = number of open arm entries / (number of open arm entries + number of closed arm entries) x 100%;

[0248] Open arm residence time % = open arm residence time / (open arm residence time + closed arm residence time) x 100%.

[0249] The data are expressed as Mean ± SD, and the significance analysis was performed using T test, with p < 0.05 being statistically significant.

[0250] (5) Results:

[0251] As shown in Figure 5, after modeling, the percentage of the duration of entering the open arms of the rats was significantly reduced to 5.93%, compared with the blank control group P < 0.01. After treatment with drugs, the percentage of the duration of entering the open arms of the rats treated with 100 mg / kg gabapentin was 15.53%, compared with the model group P < 0.05. The percentage of the duration of entering the open arms of the rats treated with 2.5 mg / kg NB001 was 9.24%, compared with the model group P > 0.05. The percentage of the duration of entering the open arms of the rats treated with 5 mg / kg NB001 was 13.91%, compared with the model group P > 0.05. The percentage of the duration of entering the open arms of the rats treated with 10 mg / kg NB001 was 25.56%, compared with the model group P < 0.05. The percentage of the duration of entering the open arms of the rats treated with 20 mg / kg NB001 was 25.59%, compared with the model group P < 0.05, and the dose-effect relationship was obvious.

[0252] As shown in Figure 6, the percentage of the number of times that the modeling rats entered the open arm was significantly reduced to 19.42%, P<0.05 compared with the blank control group; after drug treatment, the percentage of the number of times that the rats entered the open arm was increased to 26.99% for 100 mg / kg gabapentin, P>0.05 compared with the model group; the percentage of the number of times that the rats entered the open arm was increased to 18.14% for 2.5 mg / kg NB001, P>0.05 compared with the model group; the percentage of the number of times that the rats entered the open arm was increased to 20.25% for 5 mg / kg NB001, P>0.05 compared with the model group; the percentage of the number of times that the rats entered the open arm was increased to 27.06% for 10 mg / kg NB001, P>0.05 compared with the model group; the percentage of the number of times that the rats entered the open arm was increased to 43.55% for 20 mg / kg NB001, P<0.05 compared with the model group, and the dose-effect relationship was obvious.

[0253] (6) Conclusion:

[0254] The elevated plus maze model is developed from the conflict state of the exploration characteristics of animals to new and strange environment and the fear of the high open arm. After the rats are put into the maze, they will actively explore the open arm, but they are afraid of the high open environment in the open arm. Antianxiety drugs can increase the percentage of the number of times that the rats enter the open arm and the percentage of the time that the rats stay in the open arm. NB001 has the effect of increasing the percentage of the duration that the SNL model rats enter the open arm and the percentage of the number of times that the rats enter the open arm, and the initial effective dose is 10 mg / kg.

[0255] Example 5 Analgesic effect of NB001 on L5 / L6 nerve ligation induced neuropathic pain in rats

[0256] (1) Methods:

[0257] Ten male SD rats weighing 170±10 g were randomly selected as the sham operation group, and the remaining 80 rats were subjected to L5 / L6 nerve ligation for pain sensitivity screening, and 60 qualified rats were selected for the experiment. The rats were randomly divided into a model group, a 100 mg / kg gabapentin group, and 20, 10, 5, and 2.5 mg / kg NB001 dose groups, as shown in Table 27:

[0258] Table 27

[0259] The drug preparation and administration method is as follows:

[0260] 100 mg / kg gabapentin: 100 mg (1 pill) was ground and suspended in 10 mL of 1% CMC-Na, and then orally gavaged, with a dosing volume of 1 mL / 100 g of body weight;

[0261] 20mg / kg NB001: 100mg was added to 50mL 1% CMC-Na and mixed, then orally gavaged, the administration volume was 1mL / 100g body weight;

[0262] 10mg / kg NB001: 25mL of the above 20mg / kg NB001 solution was added to 25mL 1% CMC-Na and mixed, then orally gavaged, the administration volume was 1mL / 100g body weight;

[0263] 5mg / kg NB001: 10mL of the above 20mg / kg NB001 solution was added to 30mL 1% CMC-Na and mixed, then orally gavaged, the administration volume was 1mL / 100g body weight;

[0264] 2.5mg / kg NB001: 5mL of the above 20mg / kg NB001 solution was added to 35mL 1% CMC-Na and mixed, then orally gavaged, the administration volume was 1mL / 100g body weight;

[0265] Once a day. The electronic Von Frey test was used to test the rats' foot pain threshold two hours after single administration, continuous 3-day administration, and continuous 7-day administration.

[0266] (2) Model preparation:

[0267] The rats were anesthetized with ether, placed prone on the operating table, the limbs were fixed, the waist was slightly elevated, a longitudinal midline incision was made along the back skin at the level of the L4-S2 spinous process, the skin and muscle were separated layer by layer, and the L6 transverse process was fully exposed. After the L6 transverse process was removed with hemostatic forceps, the left L5 and L6 nerves were separated, the nerve roots were ligated with 6-0 silk, hemostasis was performed, and the muscle and skin were sutured layer by layer to avoid postoperative infection. The screening test was performed 10 days after the operation. The sham operation only exposed the left L5 and L6 nerves without ligation.

[0268] (3) Test method:

[0269] Pain test: Electronic Von Frey was used in a quiet room with a temperature maintained at 20-25°C. The rats were placed in a glass box for 15 minutes, and the Electronic Von Frey probe was vertically stimulated on the middle of the rat's hind foot. When the rat showed a positive response such as foot withdrawal or foot lifting, the instrument value was read, and the test was repeated twice with a 30s interval between each stimulation. The average of the three test results was taken as the rat's pain threshold.

[0270] Note: After L5 / L6 nerve ligation, 80 rats were screened for pain sensitivity, and 60 qualified rats were selected for the experiment.

[0271] Screening criteria: After 10 days of rest, the rats were subjected to the pain test procedure described above, and the probe grams of 3 times of lifting foot were recorded and calculated to select the animals with a pain threshold percentage decrease of 40-80% as qualified animals. According to the percentage decrease, the animals were numbered and randomly divided into six groups, 10 animals in each group.

[0272] Experimental method: The rats in each group were orally administered the next day after grouping, and the administration volume was 1 mL / 100 g. The rats in each group were placed in the test box for 15 minutes after 105 minutes of administration, and the test procedure was the same as above. After the test, the rats were returned to the animal room, and the test was performed on the fourth and seventh days after continuous daily administration, respectively, and the test method was the same as above.

[0273] (4) Statistical method:

[0274] The left hind limb and right hind limb of each rat were divided to obtain a relative pain threshold. Pain threshold % = (left hind limb pain threshold / right hind limb pain threshold) x 100%;

[0275] The data are expressed as Mean ± SD, and the significance analysis is performed by one-way ANOVA using IBM SPSS Statistics Version 21, and then Dunnett is used to compare the differences between the model group and the experimental group, and p<0.05 is considered statistically significant.

[0276] (5) Experimental results:

[0277] As shown in Figure 7, after modeling, the pain threshold of the left hind limb of the rats was significantly reduced, and the pain threshold was reduced to 46.14% (P<0.01 compared with the blank control group, P<0.01 compared with 100 mg / kg gabapentin); after single administration, 2.5 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rats to 56.95% (P>0.05 compared with the model group, P<0.05 compared with 100 mg / kg gabapentin); 5 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rats to 66.54% (P<0.05 compared with the model group, P>0.05 compared with 100 mg / kg gabapentin); 10 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rats to 65.71% (P<0.05 compared with the model group, P>0.05 compared with 100 mg / kg gabapentin); 20 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rats to 70.6% (P<0.05 compared with the model group, P>0.05 compared with 100 mg / kg gabapentin).

[0278] After 4 days of administration, the pain threshold of the model increased slightly, and the pain threshold decreased to 61.70% (P < 0.01 compared with the blank control group, P < 0.05 compared with 100 mg / kg gabapentin); 2.5 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 68.46% (P > 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin); 5 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 72.40% (P > 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin); 10 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 75.74% (P < 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin); 20 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 77.46% (P < 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin).

[0279] After 7 days of administration, the pain threshold of the model increased slightly, and the pain threshold decreased to 61.84% (P < 0.05 compared with 100 mg / kg gabapentin); 2.5 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 69.02% (P > 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin); 5 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 63.13% (P > 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin); 10 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 76.3% (P < 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin); 20 mg / kg of NB001 increased the pain threshold percentage of the left hind limb of the rat to 76.79% (P < 0.05 compared with the model group, P > 0.05 compared with 100 mg / kg gabapentin).

[0280] (6) Conclusion:

[0281] NB001 has analgesic effect on SNL model rats, and the effective initial dose of single administration is 5 mg / kg of rats, and the effective initial dose of multiple administration is 10 mg / kg of rats. These results suggest that the repeated use of NB001 can produce significant analgesic effect.

[0282] The results of Examples 4 and 5 demonstrate that NB001 can effectively treat perioperative pain and anxiety.

[0283] Example 6 evaluates the psychotropic dependence of NB001 in a rat conditioned place preference experiment

[0284] This example is intended to observe whether the positive control morphine hydrochloride injection and the test product NB001 can induce conditioned place preference in SD rats, and to evaluate the psychotropic dependence of the test product and compare it with morphine (morphine hydrochloride injection, batch number: 130112-2).

[0285] Experimental animals: Animal species: SD rats. Animal level: SPF level. Gender and number: 120 were purchased, and 72 were screened for formal experiment, half male and half female. Animal age: 6-7 weeks old when received. Animal weight range: 180-220 g when received. Animal source: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0286] 1. Method:

[0287] (1) Screening and grouping: On D-3 (the third day before the experiment) and D-2 (the second day before the experiment), the quarantine qualified animals were placed in the C box of the conditioned place preference box, and the baffles of the A box and the B box were opened, allowing them to freely move for 30 minutes every day, so that the animals could adapt to the environment of the conditioned place preference box.

[0288] On D-1 (the day before the experiment), the animals were placed in the C box, and the baffles of the A box and the B box were opened, allowing them to freely move for 15 minutes. The time each animal spent in the A box and the B box was counted, and the animals were randomly divided into 5 groups according to the results, including: negative control group (group 1), positive control group (group 2, 10 mg / kg), low-dose test product group (group 3, 5 mg / kg), medium-dose test product group (group 4, 10 mg / kg), high-dose test product group (group 5, 20 mg / kg), and test product + morphine synergistic group (group 6, 10+10 mg / kg), with 12 in each group, half male and half female.

[0289] (2) Dosing method:

[0290] The animals in the negative control group (group 1) were given 0.9% sodium chloride injection throughout the experiment.

[0291] The animals in the positive control group (group 2) were given morphine injection in the morning and 0.9% sodium chloride injection in the afternoon on D1, D3, D5, and D7. On D2, D4, D6, and D8, they were given 0.9% sodium chloride injection in the morning and morphine hydrochloride injection in the afternoon.

[0292] The animals in the test product groups (groups 3-5) were given test product NB001 in the morning and 0.9% sodium chloride injection in the afternoon on D1, D3, D5, and D7. On D2, D4, D6, and D8, they were given 0.9% sodium chloride injection in the morning and test product NB001 in the afternoon.

[0293] Test article + morphine coordination group (group 6) animal D1, D3, D5, D7 daily morning to give test article NB001 and positive control hydrochloric acid morphine injection, afternoon to give 0.9% sodium chloride injection. D2, D4, D6, D8 daily morning to give 0.9% sodium chloride injection, afternoon to give test article NB001 and positive control hydrochloric acid morphine injection.

[0294] Group 1, 3, 4, 5 is oral gavage (including test article NB001 and 0.9% sodium chloride injection), group 2 is subcutaneous administration (including hydrochloric acid morphine injection and 0.9% sodium chloride injection). Group 6 test article is oral gavage, hydrochloric acid morphine injection is subcutaneous administration, non-companion medicine box training before gavage and subcutaneous injection to give equal volume of 0.9% sodium chloride injection.

[0295] Among them, D1 represents the first day of the experiment, D2 represents the second day of the experiment, D3 represents the third day of the experiment, D4 represents the fourth day of the experiment, D5 represents the fifth day of the experiment, D6 represents the sixth day of the experiment, D7 represents the seventh day of the experiment, D8 represents the eighth day of the experiment.

[0296] (3) Condition position preference training: D1-D8 for condition position preference training. Specifically as follows:

[0297] D1 morning group 2-group 5 animals are given test article or positive control at least 5 min after putting into A box (companion medicine box) and closing the baffle, so that the animal stays in A box for 30 min, afternoon to give 0.9% sodium chloride injection at least 5 min after putting into B box (non-companion medicine box) and closing the baffle, so that the animal stays in B box for 30 min. D2 morning to give 0.9% sodium chloride injection, afternoon to give test article or positive control. D3, D5, D7 training program is the same as D1. D4, D6, D8 training program is the same as D2.

[0298] Group 6 animal D1 morning oral administration of test article and subcutaneous administration of positive control at least 5 min after putting into A box (companion medicine box) and closing the baffle, so that the animal stays in A box for 30 min, afternoon respectively oral and subcutaneous administration of equal volume of 0.9% sodium chloride injection at least 5 min after putting into B box (non-companion medicine box) and closing the baffle, so that the animal stays in B box for 30 min. D2 morning to give 0.9% sodium chloride injection, afternoon to give test article and positive control. D3, D5, D7 training program is the same as D1. D4, D6, D8 training program is the same as D2.

[0299] Group 1 animals D1, D3, D5, D7 were placed into A box and the baffle was closed at least 5 min after administration of 0.9% sodium chloride injection in the morning, and the animals were allowed to stay in the A box for 30 min. The animals were placed into B box and the baffle was closed at least 5 min after administration of 0.9% sodium chloride injection in the afternoon, and the animals were allowed to stay in the B box for 30 min. D2, D4, D6, D8 were placed into B box and the baffle was closed at least 5 min after administration of 0.9% sodium chloride injection in the morning, and the animals were allowed to stay in the B box for 30 min. The animals were placed into A box and the baffle was closed at least 5 min after administration of 0.9% sodium chloride injection in the afternoon, and the animals were allowed to stay in the A box for 30 min.

[0300] (4) Condition position preference test: D9, the animals in groups 1-6 were placed into C box and the baffles of A and B boxes were opened, and the residence time of the animals in A and B boxes, the number of shuttling and the condition position preference score (A box residence time-B box residence time) were counted.

[0301] 2. Results:

[0302] (1) General clinical observation: No abnormalities were observed.

[0303] (2) Condition position preference test: On D-1, the A box residence time, B box residence time and the number of shuttling of the animals in each group were close, and no significant difference was observed.

[0304] As shown in Figure 8, from left to right in turn are the negative control group, the positive control group (10 mg / kg), NB001 (5 mg / kg), NB001 (10 mg / kg), NB001 (20 mg / kg), and the NB001 + morphine synergistic group. On D9 (the ninth day of the experiment), the A box residence time of the negative control group was 313.2 ± 83.1 s, and that of the positive control group was 447.1 ± 112.1 s, which was statistically different from that of the negative control group (P < 0.01). The A box residence times of the low, medium and high dose groups of the test product were 327.6 ± 66.6, 279.8 ± 76.4 and 323.0 ± 69.9 s, respectively, which were not statistically different from that of the negative control group. The A box residence time of the test product + morphine synergistic group was close to that of the positive control group (406.5 ± 62.4 s).

[0305] As shown in Figure 9, from left to right in turn are the negative control group, the positive control group (10 mg / kg), NB001 (5 mg / kg), NB001 (10 mg / kg), NB001 (20 mg / kg), and the NB001 + morphine synergistic group. On D9, compared with the negative control group, the B box residence time of the positive control group and the test product + morphine synergistic group decreased to a certain extent, and no significant change was observed in the other groups.

[0306] As shown in Figure 10, from left to right are negative control group, positive control group (10 mg / kg), NB001 (5 mg / kg), NB001 (10 mg / kg), NB001 (20 mg / kg), and NB001 + morphine synergistic group. At D9, the negative control group had 26.1 ± 6.3 times. Morphine had a certain inhibitory effect on the number of shuttle times (positive control group 16.6 ± 3.3 times, test product + morphine synergistic group 19.1 ± 5.7 times), while the test product tended to increase the number of shuttle times (30.9 ± 11.3-31.9 ± 8.3 times).

[0307] As shown in Figure 11, from left to right are negative control group, positive control group (10 mg / kg), NB001 (5 mg / kg), NB001 (10 mg / kg), NB001 (20 mg / kg), and NB001 + morphine synergistic group. At D9, the negative control group had a conditioned place preference score of 13.4 ± 159.0 s. The positive control group was 227.5 ± 158.9 s, greater than 100 s and statistically different from the negative control group (P < 0.01). The low, medium and high dose groups of the test product were 33.6 ± 112.3, -77.8 ± 137.1 and 32.3 ± 136.8 s, respectively, and were not statistically different from the negative control group and were all less than 100 s. The test product and morphine had a tendency to reduce the conditioned place score (176.6 ± 64.9 s vs 227.5 ± 158.9 s) when used synergistically, but there was no statistical difference between the two.

[0308] 3. Conclusion:

[0309] Under the conditions of this experiment, 10 mg / kg of morphine hydrochloride injection can induce conditioned place preference in SD rats (i.e., can cause psychological dependence). The test product NB001 at 5-20 mg / kg has no psychotropic effect on SD rats.

[0310] Example 7 Evaluation of the physical dependence of NB001 by natural withdrawal experiment in rats

[0311] Objective: This experiment aims to confirm whether the withdrawal of the test product NB001 after 4 weeks of continuous administration will cause withdrawal symptoms in SD rats, in order to evaluate its physical dependence.

[0312] Morphine hydrochloride injection, batch number: 130112-2.

[0313] Experimental animals: Species: SD rats. Animal level: SPF level. Gender and number: 86 were purchased, 72 were selected for formal experiment, all were male. Animal age: 6-7 weeks old when received. Animal weight range: 180-220 g when received. Animal source: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0314] 1. Method:

[0315] (1) Screening and grouping: 72 SD rats were randomly divided into 6 groups, namely, negative control group (group 1), positive control group (group 2, 5-40 mg / kg), low (group 3, 5 mg / kg), medium (group 4, 10 mg / kg), and high (group 5, 20 mg / kg) dose groups of the test product, and morphine + test product synergistic group (group 6, morphine 5-40 mg / kg, test product 10 mg / kg), with 12 rats in each group.

[0316] (2) Administration method:

[0317] The animals in the negative control group (group 1) were given 0.9% sodium chloride injection.

[0318] The animals in the positive control group (group 2) were given positive control product morphine hydrochloride injection.

[0319] The animals in the test product groups (groups 3-5) were given the test product NB001.

[0320] The animals in the test product + morphine synergistic group (group 6) were given the test product NB001 and positive control product morphine hydrochloride injection simultaneously every day.

[0321] Groups 1, 3, 4, and 5 were administered by gavage, group 2 was administered subcutaneously, and group 6 was administered by gavage for the test product and subcutaneously for morphine hydrochloride injection.

[0322] All animals were administered twice a day from D1 to D28. The morphine in groups 2 and 6 was increased in dosage every week, with W1 being 5 mg / kg, W2 being 10 mg / kg, W3 being 20 mg / kg, and W4 being 40 mg / kg. The test product was a fixed dose and was not increased. From D29 to D31, the administration was stopped.

[0323] (3) Index detection

[0324] General clinical observation: twice a day.

[0325] Body weight: all animals were weighed once before grouping, and once a day on D7, D14, D21, and D28. On D29, D30, and D31, the animals were weighed at around 9:00 and 17:00 each day.

[0326] Food intake: the food intake of animals in each cage was measured every day on D29, D30, and D31. The specific method was to weigh the feed of animals in each cage at a fixed time point every day on D29, D30, D31, and D32, and the difference between the previous day and the next day was taken as the food intake of the previous day.

[0327] Body temperature: the body temperature of all animals was measured once in the morning and once in the afternoon on D29, D30, and D31.

[0328] Behavior: D29, D30, D31, once a day, place the animals in the open field test device, free activity for 10 min. Video and software statistics total distance, average speed, central activity time, central activity distance.

[0329] 2、Results:

[0330] (1) General clinical observation: part of the animals in the positive control group (group 2) and the test product + morphine synergistic group (group 6) showed typical morphine withdrawal symptoms such as soft stool, wet dog shaking, etc. on D29-D31, and the rest showed no abnormalities.

[0331] (2) As shown in Table 28, body weight and food intake: during D1-D28, the body weight of the animals in the negative control group increased steadily, and the body weight of the animals in the positive control group was significantly inhibited, which was statistically different from that of the negative control group. No significant effect on body weight gain was observed in the low, medium and high dose groups of the test product. The low dose of the test product inhibited the increase of body weight, and there was a statistical difference compared with the negative control group on D28. The body weight increase of the morphine + test product synergistic group was similar to that of the positive control group.

[0332] As shown in Table 29, during D29-D31, the body weight of the animals in the negative control group and the test product group showed no significant change, and the body weight of the animals in the positive control group and the morphine + test product synergistic group decreased significantly.

[0333] As shown in Table 30, during D29-D32, the food intake of the animals in the negative control group was stable and showed no significant change. Compared with the negative control group, the food intake of the animals in the positive control group was significantly inhibited, but it increased on the third day after drug withdrawal (D31-D32). There was no difference in food intake between the animals in the medium and high dose groups of the test product and the negative control group, and no significant change was observed during the drug withdrawal period. The food intake of the animals in the low dose group of the test product was inhibited compared with the negative control group on the first day (D29-D30) and the second day (D30-D31) after drug withdrawal (P<0.05, P<0.01), and it increased on the third day. The food intake of the animals in the morphine + test product synergistic group changed similarly to the positive control group.

[0334] Table 28 Body weight changes of animals in each group during D1-D28 Note: **, ***: P<0.01, 0.001 vs negative control group.

[0335] Table 29 Body weight changes of animals in each group during the withdrawal period Note: ++, +++: P<0.01, 0.05 vs D28 body weight.

[0336] Table 30 Changes in average food intake of animals in each group during the withdrawal period Note: *, **, ***: P<0.05, 0.01, 0.001 vs. negative control group. +++: P<0.001 vs. D29-D30 food intake.

[0337] (3) Body temperature: As shown in Table 31, the body temperature of the rats changed periodically, and the general trend was that the body temperature was higher in the morning and lower in the afternoon, which was related to the biological rhythm of rodents.

[0338] The body temperature change trend of the positive control group animals on D29 was opposite to that of the negative control group (lower body temperature in the morning and higher body temperature in the afternoon), and was basically consistent with that of the negative control group on D30 and D31. The body temperature change trend of the low-dose test product group was basically consistent with that of the negative control group. The body temperature of the medium- and high-dose test product groups was slightly higher than that of the negative control group throughout the withdrawal period, and the body temperature change trend was consistent with that of the negative control group. The body temperature change trend of the morphine + test product synergistic group on D29 and D30 was consistent with that of the positive control group, and the body temperature was stable on D31, which was different from that of the other groups. The body temperature in the morning on D31 was lower than that of the positive control group, and there was a statistically significant difference.

[0339] Table 31 Body temperature change of animals in each group during the withdrawal period Note: *, **, ***: P<0.05, 0.01, 0.001 vs. negative control group. #: P<0.05 vs. positive control group.

[0340] (4) Behavior: As shown in Tables 32-34, the total activity distance of animals in the open field represented the increase or decrease of their exploration behavior. As shown by the total activity distance change of the negative control group animals from D29 to D31, with the increase of the test number, the animals tended to adapt and familiarize with the experimental field, and their exploration behavior gradually decreased.

[0341] The positive control group animals showed obvious spontaneous activity inhibition on D29 and D30 compared with the negative control group. On D31, the inhibition was weakened, and there was no statistically significant difference compared with the negative control group. Compared with the negative control group, the animals in the low- and medium-dose test product groups showed a slight activity decrease trend on D30, but there was no statistically significant difference. On D29 and D31, no similar phenomenon was observed. The animals in the high-dose test product group showed an activity decrease trend on D31 in addition to D30, but there was no statistically significant difference. The total activity distance of the morphine + test product synergistic group was close to that of the negative control group.

[0342] Since all animals were tested for the same time, the change of the average speed was the same as the total activity distance.

[0343] From the central zone activity time of D29-D31, it can be seen that the central zone activity time gradually decreased with the adaptation of the animals to the environment. The central zone activity time of the positive control group was slightly higher than that of the negative control group at D29, but no statistical difference was found. The central zone activity time gradually decreased at D30 and D31 and was close to that of the negative control group. The central zone activity time of each test product group was lower than that of the negative control group, but no statistical difference was found. The central zone activity time of the morphine + test product synergistic group was close to that of each test product group.

[0344] Except for the positive control group, the change trend of the central zone activity distance of each group of animals was basically consistent with that of the central zone activity time.

[0345] Table 32 Statistical data of D29 open field test Note: *, **, ***: P < 0.05, 0.01, 0.001 vs. negative control group.

[0346] Table 33 Statistical data of D30 open field test

[0347] Table 34 Statistical data of D31 open field test

[0348] 3. Conclusion:

[0349] Under the experimental conditions, the injection of morphine hydrochloride at a dose of 5-40 mg / kg per week with a gradual increase in dose and continuous administration for 4 weeks followed by drug withdrawal can cause typical opiate receptor withdrawal reactions, such as weight loss and decrease in spontaneous activity. The injection of the test product NB001 at a fixed dose of 5-20 mg / kg for 4 weeks followed by drug withdrawal did not show obvious withdrawal reaction symptoms.

[0350] The results of Examples 4-7 prove that NB001 can effectively treat perioperative pain and anxiety and has no addictive property.

Claims

1. Use of an adenylyl cyclase inhibitor represented by the formula (1) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of perioperative pain, characterized in that, The perioperative period includes preoperative, intraoperative and postoperative periods. The medicaments comprise active ingredients for gastrointestinal or non-gastrointestinal administration, and suitable pharmaceutically organic or inorganic inert carrier materials. wherein the drug for treating perioperative pain is a drug for treating perioperative pain in which the adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof is the only active ingredient.

2. Use according to claim 1, characterized in that, The drug is administered before surgery.

3. Use according to claim 1, characterized in that, The surgery is a non-first surgery.

4. Use according to claim 1, characterized in that, The suitable pharmaceutically organic or inorganic inert carrier materials include one or more of water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, and polyalkylene glycols.

5. The use according to claim 1, characterized in that, The drug is in the form of a tablet, suppository, or capsule.

6. Use according to claim 1, characterized in that, The drug is in the form of a solution, suspension, or emulsion.

7. Use according to claim 1, characterized in that, The drug contains a preservative, stabilizer, wetting agent, or emulsifying agent.

8. The use according to claim 1, characterized in that, The drug contains a bulking agent that changes the osmotic pressure or a buffer.

9. The use according to claim 1, characterized in that, The drug for treating perioperative pain is a drug for treating perioperative pain that does not have addictive properties.

10. Use of an adenylyl cyclase inhibitor represented by the formula (1) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of perioperative pain and anxiety, characterized in that, The perioperative period includes pre-, intra- and post-surgery, and the medicaments comprise active ingredients for gastrointestinal or non-gastrointestinal administration, and suitable pharmaceutically organic or inorganic inert carrier materials; wherein the drug for treating perioperative pain is a drug for treating perioperative pain in which the adenylyl cyclase inhibitor represented by formula (1) or a pharmaceutically acceptable salt thereof is the only active ingredient.

11. Use according to claim 10, characterized in that, The drug for treating perioperative pain and anxiety is a drug for treating perioperative pain and anxiety that does not have addictive properties.

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