CN115006398A - Application of bosentan in preparation of medicine for treating tourniquet pain of clinical patient - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of bosentan in preparation of a medicine for treating tourniquet pain of a clinical patient. The invention also discloses a verification method, which comprises the following processes: animal grouping: 10 adult male wild type C57BL/6J mice were selected, housed under standard conditions and provided free access to water and food; preparing a peripheral vascular pain model: preparing peripheral vascular pain models for the control group and the administration group, and returning the mice which are successfully prepared into cages for normal feeding; randomly selecting 5 mice to be injected with a control solvent in the abdominal cavity as a control group when the peripheral vascular pain is 4 weeks, and taking the other 5 mice to be injected with bosentan in the abdominal cavity as an administration group; the Von Frey test was performed on the mice of the control group and the administration group of the aforementioned peripheral vascular pain model. The verification result shows that the treatment of tourniquet pain (bandage pain) is facilitated by blocking the ET-1 pathway with bosentan.

Description

Application of bosentan in preparation of medicine for treating tourniquet pain of clinical patient

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of bosentan in preparation of a medicine for treating tourniquet pain of a clinical patient.

Background

Tourniquets are often used to provide a virtually bloodless environment to minimize surgical bleeding, thereby facilitating orthopedic surgery under general, local, or regional anesthesia. Tourniquet inflation is often associated with intra-and post-operative pain. Patients who have received sufficient anesthesia at the surgical site still experience post-operative tourniquet pain. Tourniquet pain, generally refers to a painful reaction of the limb that occurs as a result of excessive pressure and time on the tourniquet. After prolonged use of the tourniquet, the patient experiences a sensation of soreness, distension and pain, either locally or remotely, which are described by different patients as burning, cramping, heaviness and numbness, causing discomfort to the patient. Meanwhile, most patients also have systemic emotional reactions, such as cold sweat, dysphoria, restlessness, anxiety and other mental changes and cardiovascular system reactions, and even by means of drugs such as anesthesia, the adverse reactions of tachycardia, hypertension and the like of the patients cannot be effectively relieved, so that huge resistance is caused to subsequent treatment and operation. At present, few research reports on the pain of the tourniquet, the research of the pain mechanism and the research and development of pain drugs are available.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems or the defects in the prior art, the invention provides the application of bosentan in preparing the medicine for treating clinical patient tourniquet pain (bandage pain), so as to solve the problems in the background technology.

To achieve the above object, the present invention provides the use of bosentan in the preparation of a medicament for treating tourniquet pain (bandage pain) in a clinical patient.

Further, bosentan is an ET-1 type A receptor/ET-1 type B receptor mixed antagonist.

Further, other medically acceptable agents are added into the medicine.

Further, bosentan is an intervention drug.

The embodiment of the present invention additionally proposes a method for verifying bosentan for non-therapeutic purposes in the treatment of clinical patient tourniquet pain (bandage pain), characterized in that it comprises the following steps:

(1) animal grouping: 10 adult male wild type C57BL/6J mice were selected, housed under standard conditions and provided free access to water and food; randomly selecting 5 mice to be injected with a control solvent in the abdominal cavity as a control group, and taking the other 5 mice to be injected with bosentan in the abdominal cavity as an administration group;

(2) preparing a peripheral vascular pain model: preparing a peripheral vascular pain model for the control group and the administration group in the step (1), and putting the mice which are successfully prepared back into a cage for feeding as usual;

(3) systemic administration (intraperitoneal injection): randomly selecting 5 mice to be injected with a control solvent in the abdominal cavity as a control group when the peripheral vascular pain of the mice is 4 weeks, and taking the other 5 mice to be injected with bosentan in the abdominal cavity as an administration group;

(4) the Von Frey test was performed on the peripheral vascular pain mice of the solvent control group and the administration group in step (3).

Further, in the step (4), the following process is included: measuring the basic value of mechanical pain before molding and the mechanical pain 4 weeks after molding, then administering the drug to two groups of mice, and measuring the mechanical pain 1, 3 and 24 hours after administration; measurement of mechanical pain was determined by the paw withdrawal threshold of mice on Von Frey cilia stimulation; i.e. the mice were placed in boxes on an overhead wire mesh and allowed to acclimate inside for 30 minutes; their hind feet were then stimulated with a series of Von Frey cilia (0.16, 0.40, 0.60, 1.00 and 2.00 grams) with logarithmic growth in stiffness; cilia are perpendicular to the plantar surface of the mouse hind paw, and a 50% withdrawal threshold is determined using the Dixon's up-down method.

Further, in step (3), the administration group: the mice in the drug intervention group were injected with 10 mg/kg bosentan in the abdominal cavity, and the control group was given the same dose of physiological saline.

The technical scheme of the invention has the following beneficial effects:

(1) the ET-1 plays a certain role in inflammatory pain and neuropathic pain, and through research of a team of inventors, the ET-1, namely, bosentan which is a non-selective endothelin receptor antagonist, is found to have the effect of relieving tourniquet pain (bandage pain) of a clinical patient for the first time. Tourniquet pain (bandage pain) is a manifestation of vascular pain, and the invention also proves that bosentan has a certain effect of treating vascular pain in a peripheral vascular pain animal model.

(2) The invention adopts an antagonist to block an endothelin (ET-1) signal channel, in particular relates to a non-selective endothelin receptor antagonist, namely bosentan, 89 patients receiving unilateral total knee joint replacement under general anesthesia at auxiliary hospital of Xuzhou medical university are selected as research objects, and bosentan and placebo treatment are given in two groups. Tourniquet pain (bandage pain) is a vascular pain that is modeled by ligating the superficial blood vessels of the hind legs of mice to create a peripheral vascular pain model, and then using pharmacological methods to verify the effect of bosentan in peripheral vascular pain. The results show that blocking the ET-1 pathway with bosentan is beneficial for the treatment of blood-borne pain (bandage pain).

Drawings

FIG. 1 is a graph of mechanical pain sensitivity in mice injected intraperitoneally with bosentan to relieve peripheral vascular pain in accordance with an embodiment of the present invention.

FIG. 2 is a graph of the improvement in tourniquet pain in clinical patients by blocking ET-1 signaling in an embodiment of the invention.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

The invention adopts the following technical scheme:

example 1 animal experiments

(1) Animal grouping: 10 adult male Wild Type (WT) C57BL/6J mice were selected, and the mice (2-5/cage) were housed under standard conditions (21-24 ℃ C., 60% humidity, 12:12-h light/dark cycle) and provided free access to water and food. Randomly selecting 5 mice to be injected with control solvent in the abdominal cavity as a control group, and the other 5 mice to be injected with bosentan in the abdominal cavity as an administration group. In this step, the control solvent injection and the intraperitoneal bosentan injection were not performed, and only grouped.

(2) Preparing a peripheral vascular pain model: preparing peripheral vascular pain models for the administration group and the control group in the step (1), wherein the preparation process comprises the following steps: after anesthetizing the mice with 1% sodium pentobarbital (100 mg/kg, i.p.), the medial thigh of the left hind limb was shaved and the mice were placed in a supine position, where the great saphenous vein and saphenous artery were clearly visible through the skin; after conventional sterilization, a longitudinal incision of about 0.5 cm was made with ophthalmic scissors along the medial side of the blood vessel about 0.5 cm and half of the tibia; the great saphenous vein (inside) and the saphenous artery and saphenous nerve (outside) accompanying the great saphenous vein (inside) can be seen under an operation microscope, mucous membranes between the arteriovenous and the nerve are carefully separated by using the tip of an ophthalmic scissors, and then the artery and the vein are completely ligated by using a 6-0 line to serve as a peripheral vascular pain model group; suturing the skin; after surgery, the animals were recovered by placing them on an electric blanket and then returned to their cages as usual.

(3) Systemic administration (intraperitoneal injection): and (3) performing systemic administration on 10 peripheral vascular pain model mice prepared in the step (2), and performing intraperitoneal administration of bosentan and solvent control on an administration group and a control group respectively at 4 weeks of the peripheral vascular pain model mice. The left little finger and the third finger of the left hand grasp the tail of the mouse, the other three fingers grasp the neck and back skin of the mouse, and the right hand holds a 1 ml syringe and pierces the needle into the skin slightly to the left or slightly to the right of the leucorrhea. After the needle head reaches the subcutaneous part, the needle head is pushed forward by about 3-5 mm, and then the needle head of the injector is enabled to be 45 degrees with the skin to penetrate into the abdominal cavity, and the falling feeling is generated. Then fixing the needle, keeping the needle tip still, withdrawing the pintle, and slowly injecting the liquid medicine if no backflow matter exists. Animal model bosentan administration method: the main intervention drug "bosentan" in the present invention is a purification chemical reagent, provided by Sigma company. The research result of the invention shows that bosentan is an ET-1A type receptor/ET-1B type receptor mixed antagonist and is a medicament for treating pulmonary hypertension. In this section, "bosentan" was applied to prophylactic intervention in peripheral vascular pain animal models, and the improved effect of "bosentan" on peripheral blood vessels was observed. The administration method comprises the following steps: the mice in the drug intervention group were injected with 10 mg/kg bosentan in the abdominal cavity, and the control group was given the same dose of physiological saline.

(4) Von Frey experiments were performed to test mechanical pain: the Von Frey test was performed on peripheral angiodynia mice in the solvent control group and the administration group. The basal value of mechanical pain before molding and mechanical pain 4 weeks after molding were measured, and then two groups of mice were administered with the mechanical pain measured 1, 3, and 24 hours after administration. Mechanical pain measurements were determined by the paw withdrawal threshold of mice on Von Frey cilia stimulation. I.e. the mice were placed in a box on an overhead wire mesh and allowed to acclimate inside for 30 minutes. Their hind feet were then stimulated with a series of Von Frey cilia (0.16 g, 0.40 g, 0.60 g, 1.00 g, and 2.00 g) with logarithmic growth in stiffness. Cilia are perpendicular to the plantar surface of the mouse hind paw, and a 50% withdrawal threshold is determined using the Dixon's up-down method.

Example 2 clinical study

Clinical study subjects: 89 patients (age: 59 ± 11 years) who received unilateral total knee arthroplasty under general anesthesia at the subsidiary hospital of xu zhou medical university were selected between 2017 and 2019, month 3. The inclusion criteria are as follows: a. american Society of Anesthesiologists (ASA) grades II-III; b. requiring the lower limb tourniquet to be inflated during the operation; c. the age is 18-75 years old; d. agreeing to voluntarily provide signed informed content. The following exclusion criteria were used: a. no tourniquet was used during surgery, b. emergency surgery, c. tourniquet was used during the last three months, d. bilateral total knee replacement, e. tourniquet was inflated for less than 60 minutes or more than 90 minutes, f. there was a history of daily analgesic administration, g. liver or kidney dysfunction, h. severe myocardial disease (coronary heart disease, heart failure or severe arrhythmia), i. coagulation dysfunction, j. diabetes, k. history of drug or alcohol abuse, l. pneumonia, asthma or chronic obstructive pulmonary disease, j. pre-operative hypotension (systolic blood pressure < 90) and k. mmHg pregnancy or recent childbirth. In calculating the sample size, the present invention used a 36% ratio of rescue analgesia, as determined in the preliminary experiments of the present invention. Thus, using 42 subjects per group, a 25% reduction between groups can be detected at a significance level of 5% and 80% power. Considering the 10% drop-out rate, the present invention estimates that 45 patients are required per group.

The clinical administration method comprises the following steps: random sequences were generated by Stata 12.0. Each subject was randomly assigned to bosentan or placebo control groups at a ratio of 1: 1. The assignment of groups was double-blind by researchers, patients and statistical analysts. The concealment of the assignment is achieved by placing the random sequence used to determine the assignment of groups in an opaque sealed envelope, which is opened 3 hours prior to surgery. All patients fasted for 8 hours prior to surgery. Patients in the bosentan group took bosentan (125 mg, tablets) orally 2 hours before surgery and 6 hours after surgery. Patients in the placebo control group received placebo at the same time point.

The clinical curative effect is as follows: the primary observation is the change in VAS scores at different time points after surgery. Secondary observational indices included the dose of propofol and remifentanil used in surgery, the number of PCIA compressions, the incidence of remedial analgesia, the concentration of ET-1 in plasma, and the incidence of side effects.

The effect of a non-selective endothelin receptor antagonist, bosentan, on the treatment of tourniquet pain (bandage pain) was analyzed by the above steps with the following results:

i, 44 patients in the bosentan group (fig. 2A) who used bosentan prophylactically two hours prior to surgery and acted 6 hours post-surgery, and as a result, were found to have a significant improvement in post-operative tourniquet-associated pain in bosentan-treated patients compared to placebo-controlled patients, as assessed by evaluation of Visual Analog Scale (VAS) scores (fig. 2B and 2C).

Ii, the number of patient-controlled intravenous analgesia Pumps (PCIA) used by patients treated with bosentan of the invention and the incidence of remedial analgesia were significantly reduced (fig. 2D and 2E).

The invention adopts a pharmacological method to verify the effect of bosentan in peripheral vascular pain. Bosentan is a non-specific ET-1 blocking agent, and when bosentan is administered systemically (in an abdominal cavity), mechanical pain sensitivity is improved 3 hours after administration, as shown in figure 1, the mechanical pain base value of two groups of mice, the mechanical pain threshold (pre) before 4 weeks of peripheral vascular pain administration and the mechanical pain 1, 3 and 24 hours after administration are respectively measured, and as can be seen from the figure, the mechanical pain sensitivity is obviously relieved in the bosentan administration group compared with a solvent control group.

The invention uses an antagonist to block an endothelin (ET-1) signal channel, in particular to a non-selective endothelin receptor antagonist, namely bosentan, and 89 patients who receive unilateral total knee joint replacement under general anesthesia at auxiliary hospital of Xuzhou medical university are selected as research objects and treated by bosentan and placebo in two groups. Tourniquet pain (bandage pain) is a vascular pain that is modeled by ligating the superficial blood vessels of the hind legs of mice to establish a peripheral vascular pain model, and then the pharmacological approach was used to verify the effect of bosentan in peripheral vascular pain. Blocking the ET-1 pathway with bosentan is indicated to be beneficial for the treatment of blood-borne pain (bandage pain).

While the foregoing is directed to the preferred embodiment of the present invention, it will be appreciated by those skilled in the art that various changes and modifications may be made therein without departing from the principles of the invention as set forth in the appended claims.

Claims (7)

1. Application of bosentan in preparation of medicine for treating tourniquet pain of clinical patients.

2. Use of bosentan according to claim 1, wherein bosentan is an ET-1A receptor/ET-1B receptor mixed antagonist, for the preparation of a medicament for the treatment of tourniquet pain in a clinical patient.

3. Use of bosentan according to claim 1 for the preparation of a medicament for the treatment of clinical patient tourniquet pain, wherein a pharmaceutically acceptable additional agent is added to said medicament.

4. Use of bosentan in the preparation of a medicament for treating tourniquet pain in a clinical patient according to claim 1, wherein bosentan is an intervention drug and wherein bosentan treats tourniquet pain by blocking the ET-1 pathway.

5. A method for verifying, for non-therapeutic purposes, bosentan in the treatment of clinical patient tourniquet pain, comprising the steps of:

(1) animal grouping: selecting 10 adult male wild type C57BL/6J mice, feeding the mice under standard conditions and providing free drinking and eating, randomly selecting 5 mice as a control group, and selecting the other 5 mice as administration groups;

(2) preparing a peripheral vascular pain model: preparing a peripheral vascular pain model for the control group and the administration group in the step (1), and putting the mice which are successfully prepared back into a cage for feeding as usual;

(3) systemic administration (intraperitoneal injection): when the peripheral vascular pain of the mice is 4 weeks, injecting a control solvent into the abdominal cavity of the selected mice of 5 control groups, and injecting bosentan into the abdominal cavity of the other 5 administration groups;

(4) the Von Frey test was performed on peripheral angiodynia mice in the solvent control group and the administration group in step (3).

6. The method for verifying bosentan for non-therapeutic purposes in treating tourniquet pain in a clinical patient according to claim 5, wherein step (4) comprises the following steps: measuring the basic value of mechanical pain before molding and the mechanical pain 4 weeks after molding, then administering the drug to two groups of mice, and measuring the mechanical pain 1, 3 and 24 hours after administration; measurement of mechanical pain was determined by the paw withdrawal threshold of mice on Von Frey cilia stimulation; i.e. the mice were placed in boxes on an overhead wire mesh and allowed to acclimate inside for 30 minutes; their hind paw was then stimulated with a series of Von Frey cilia of logarithmic growth in stiffness, 0.16 g, 0.40 g, 0.60 g, 1.00 g and 2.00 g in order; cilia are perpendicular to the plantar surface of the mouse hind paw, and a 50% withdrawal threshold is determined using the Dixon's up-down method.

7. The method of claim 5 for verification of bosentan in the treatment of tourniquet pain in a clinical patient for a non-therapeutic purpose, wherein: in step (1), the administration group: the mice in the drug intervention group were injected with 10 mg/kg bosentan in the abdominal cavity, and the control group was given the same dose of physiological saline.

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