Method for evaluating biological activity of pulmonary surfactant
By evaluating the improvement of respiratory compliance value of lung surfactants in the young rabbit model, the problems of complexity of extracting lung surfactants in animal tissues and the evaluation of the effectiveness of synthetic drugs in the prior art are solved, and the accurate assessment of the biological activity and efficacy of lung surfactants is achieved.
Patent Information
- Application Number
- CN202411988937.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-05-02
AI Technical Summary
Existing lung surfactants extracted from animal tissues have complexities in the production and sterilization methods and the possible immunological response problems, and the efficacy and activity of artificially synthesized lung surfactants need to be evaluated to ensure their effectiveness and safety.
Young rabbits with pulmonary surfactant deletion were used as experimental subjects to evaluate the efficacy and biological activity of the drug by administering the test lung surfactant and detecting the improvement of respiratory compliance under specific ventilation conditions.
This method has high accuracy, repeatability and sensitivity, and can effectively evaluate the biological activity and efficacy of lung surfactants, providing guarantees for drug production qualification and release.
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Figure CN119908660A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the technical field of drug efficacy and activity testing, and specifically relates to a method for evaluating the biological activity of pulmonary surfactant. Background Art
[0002] Neonatal respiratory distress syndrome (NRDS) is a common neonatal disease in clinical practice. The occurrence of this disease is closely related to the lack of pulmonary surfactant (PS). NRDS causes progressive atrophy of alveoli, so the children will have clinical manifestations such as dyspnea and cyanosis. NRDS progresses rapidly, and in severe cases it can cause respiratory failure, which in turn endangers the life safety of the child.
[0003] The human lungs are composed of numerous small air sacs, called alveoli, in which gases are exchanged between the blood and the air spaces of the lungs. In healthy individuals, this exchange is mediated by protein-containing surfactant complexes that prevent the lungs from collapsing at the end of expiration.
[0004] Modified natural surfactant drugs extracted from animal lungs can effectively treat NRDS. Currently, a variety of modified natural surfactant drugs have been approved for marketing. For example, poractanta (Curosurf TM ), calfactant extracted from calf lung lavage (Infasurf TM ), and chemically modified natural bovine lung extract beractant (Survanta TM , that is, Borattan).
[0005] However, lung surfactant drugs derived from animal tissues have many defects, such as the complexity of production and sterilization methods and the possibility of inducing immune responses in the body. Therefore, a variety of artificially synthesized lung surfactants have been successfully developed, such as sinapeptide phospholipid surfactant. However, for artificially synthesized lung surfactants, their own efficacy and activity and the difference in efficacy from lung surfactants extracted from animals need to be evaluated, and their effectiveness needs to be verified as a test standard for qualified drug production and release, to provide a guarantee for safe and effective clinical drug use. Summary of the invention
[0006] The purpose of the present application is to provide a method for evaluating the biological activity of pulmonary surfactant. This method uses young rabbits lacking pulmonary surfactant as test subjects, detects the improvement effect of the test pulmonary surfactant on the respiratory compliance value, and then evaluates the efficacy and biological activity of the test pulmonary surfactant.
[0007] In order to achieve the above invention purpose, the technical solution adopted in this application is:
[0008] In a first aspect, the present application provides a method for evaluating the biological activity of pulmonary surfactant, comprising the following steps:
[0009] S1. administering a lung surfactant to be tested to a lung surfactant-deficient young rabbit according to a preset dose to obtain a lung surfactant-deficient young rabbit;
[0010] S2, placing the test young rabbit after administration under test ventilation conditions, detecting and calculating the ratio of its respiratory compliance value to its body weight, and obtaining the test value of the test drug;
[0011] The test ventilation conditions include: maximum inspiratory pressure of 27±2 cmH2O, positive end-expiratory pressure of 0 cmH2O, ventilation flow of 8±2 L / min, respiratory rate of 30±2 times / min, exhalation time of 0.45±0.00s, and oxygen concentration of 100%.
[0012] In an optional implementation, the evaluation method further includes:
[0013] The operations of steps S1 and S2 are repeated using the positive control substance and the negative control substance respectively to obtain the positive control test value and the negative control test value.
[0014] In an optional embodiment, the positive control is a lung surfactant extracted from animal tissue;
[0015] Optionally, the positive control is bovine lung surfactant beractant;
[0016] Optionally, the dosage range of the bovine lung surfactant beractant is 2-8 ml / kg, preferably 4 ml / kg. Before administration, beractant needs to be taken out from 4°C and restored to room temperature for use.
[0017] In an optional embodiment, the negative control is air;
[0018] Optionally, the dosage range of air is 2.9 to 11.6 ml / kg, preferably 5.8 ml / kg.
[0019] In an optional embodiment, when the increase value of the positive control test value relative to the negative control test value is not less than 600%, it is determined that the performance of the experimental system of the evaluation method meets the requirements.
[0020] In an optional embodiment, when the increase value of the test drug value relative to the negative control test value is not less than 320%, and the coefficient of variation between the positive control test value and the test drug value after three repetitions is not greater than 30%, and the coefficient of variation between the positive control test value and the test drug value after six repetitions is not greater than 35%, the drug activity of the test pulmonary surfactant is determined to be good.
[0021] In an optional embodiment, the baby rabbit is a baby New Zealand White rabbit with a gestation period of 26.5±0.5 days.
[0022] In an optional embodiment, the tested lung surfactant is a synthetic lung surfactant;
[0023] Optionally, the lung surfactant to be tested is a sinapeptide phospholipid surfactant, and the sinapeptide phospholipid surfactant comprises dipalmitoylphosphatidylcholine, palmitoleoylglycerophosphoglycerol sodium salt, palmitic acid and sinapeptide;
[0024] Optionally, the dosage range of the Sinaputide phospholipid surfactant is 2.9-11.6 ml / kg, preferably 5.8 ml / kg. Before administration, the Sinaputide phospholipid surfactant needs to be taken out from 4°C and rewarmed at 42-46°C for 15 minutes.
[0025] In a second aspect, the present application provides the application of the above-mentioned evaluation method in the quality control of lung surfactant products.
[0026] Based on the above technical solution, this application has at least the following beneficial effects:
[0027] The method for evaluating the biological activity of pulmonary surfactant provided in the present application selects young rabbits lacking pulmonary surfactant as experimental subjects, detects the improvement effect of the tested pulmonary surfactant on the respiratory compliance value of the young rabbits' lungs through pulmonary administration, and evaluates whether the drug efficacy and biological activity meet the requirements based on the experimental data results. The method has high accuracy, repeatability and sensitivity, simple operating steps and wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0029] Figure 1 Graph of linearity test results in Example 2 of the present application. DETAILED DESCRIPTION
[0030] In order to further explain the technical means and results taken by the present application to achieve the predetermined invention purpose, the specific implementation methods, technical solutions and features of the present application are described in detail below with preferred embodiments. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.
[0031] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.
[0032] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.
[0033] Example 1 Activity Evaluation Experiment System Performance Verification Experiment (Only Negative Group and Positive Group)
[0034] Experimental materials: New Zealand pregnant rabbits with a gestational age of 26.5 days (rabbits weighing less than 23 g cannot be used in the experiment), boratan inhalation suspension, and 1 mL syringe.
[0035] Experimental method: The young rabbits were placed on their backs, with their necks raised to an appropriate height. A tracheal tube (depth of 4-5 mm) was inserted through the mouth and fixed with a 4-0 line. The rewarmed Bolatan inhalation suspension was extracted with a 1 mL syringe and given to the young rabbits at a dose of 4 ml / kg through the tracheal tube as the positive group. A certain volume of air (5.8 ml / kg) was given to the young rabbits in the negative group with a syringe.
[0036] After administration, the young rabbits were quickly placed in the chamber of the detection system. Each chamber had a cannula, and each cannula was connected to a multi-hole ventilation pipe connected to a ventilator to ensure that the respiratory settings and pressure of each animal were consistent. The positive group required 6 young rabbits, and the negative group (only air) required at least 4 young rabbits. No more than three young rabbits from the same litter of the same batch of drugs could be administered consecutively, and the experimental sequence should be randomly sorted according to the litter of animals for testing. During the experiment, the ventilation conditions of the young rabbits were maintained as follows: maximum inspiratory pressure of 27±2cmH2O, positive end-expiratory pressure ventilation of 0cmH2O, ventilation flow of 8±2L / min, respiratory rate of 30±2 times / min, expiratory time of 0.45±0.00s, and oxygen concentration of 100%.
[0037] After the data collection of the young rabbit is completed, the young rabbit is taken out of the test chamber. The average positive growth value E% and CV% value of three runs are calculated. If the average positive growth value E% (the growth percentage of the respiratory compliance value to body weight ratio CRS / kg of the positive group relative to the CRS / kg of the negative group) is > 600%, and the three-run CV% is < 30%, the performance verification experiment is passed. The test is divided into three runs, and the final result is the average of the three runs, as shown in Table 1.
[0038] Table 1 Experimental results of three runs of Example 1
[0039]
[0040]
[0041] It can be seen from Table 1 that the activity evaluation experimental system of the present application has good performance.
[0042] Example 2 Linearity Experiment
[0043] Experimental materials: New Zealand pregnant rabbits at 26.5 days of gestation (rabbits weighing less than 23 g cannot be used for the experiment), bolatan inhalation suspension, sinapeptide phospholipid surfactant (concentrations are 30, 10, 5, 2.5, 1, and 0.5 mg / ml, respectively), and 1 ml syringe.
[0044] Experimental method: The rabbits were placed on their backs, with their necks raised to an appropriate height. A tracheal tube (depth of 4-5 mm) was inserted through the mouth and fixed with a 4-0 line. The rewarmed Bolatan inhalation suspension was extracted with a 1 ml syringe and given to the rabbits at a dose of 4 ml / kg through the tracheal intubation, as the positive group; the Sinaputide group was given to the rabbits at a dose of 5.8 ml / kg, and a certain volume of air (5.8 ml / kg) was given to the rabbits in the negative group with a syringe.
[0045] After administration, the young rabbits were quickly placed in the chamber of the detection system. Each chamber had a cannula, and each cannula was connected to a multi-hole ventilation tube connected to a ventilator to ensure that the breathing settings and pressure of each animal were consistent. The positive group and the test group (different concentration groups) required 6 young rabbits each, and the negative group (only air) required at least 4 young rabbits. The same batch of drugs should not be given to more than three animals in the same litter, and the experimental sequence composed of three groups should be randomly sorted according to the litter of animals for testing. During the test, the ventilation conditions of the young rabbits were maintained as follows: maximum inspiratory pressure of 27±2cmH2O, positive end-expiratory pressure ventilation of 0cmH2O, ventilation flow of 8±2L / min, respiratory rate of 30±2 times / min, exhalation time of 0.45±0.00s, and oxygen concentration of 100%.
[0046] After the data collection of the young rabbits is completed, the young rabbits are taken out of the chamber of the detection system. The average growth value E% of the positive group (the growth percentage of the respiratory compliance value to body weight ratio CRS / kg of the positive group relative to the CRS / kg of the negative group) must be >600%, and the CV% of the positive group must be <30% during the three runs. The growth results of the test groups (different concentration groups) relative to the negative group are summarized and calculated, and after square root conversion, a linear curve is drawn to evaluate the linear results (R is required to be 2 >0.95).
[0047] Experimental results: The growth value E% of the test group (different concentration groups) (the growth percentage of the respiratory compliance value to body weight ratio CRS / kg of the test group relative to the negative group CRS / kg) was taken as the average value of all runs, and finally a linear plot was made with the concentration (square root) and E% value to calculate R 2 Values, as shown in Table 2, Table 3 and Figure 1 shown.
[0048] Table 2 Sample concentration and growth value E% in the test group
[0049] Sample concentration (mg / ml) 0.5 1.0 2.5 5.0 10.0 30.0 Sample E% 12 79 146 146 349 499
[0050] Table 3 Sample concentration (square root) and growth value E% in the test group
[0051]
[0052]
[0053] From Table 2, Table 3 and Figure 1 It can be seen that the linearity of the activity evaluation experimental system of the present application is good.
[0054] Example 3 Activity Evaluation Test of Sinaputide Phospholipid Surfactant
[0055] Experimental materials: New Zealand pregnant rabbits at 26.5 days of gestation (rabbits weighing less than 23 g cannot be used in the experiment), bolata inhalation suspension, sinapeptide phospholipid surfactant, and 1 ml syringe.
[0056] Test method: The young rabbits were placed on their backs, with their necks raised to an appropriate height, and a tracheal tube (depth of 4-5 mm) was inserted through the mouth and fixed with a 4-0 line. The rewarmed Bolatan inhalation suspension was extracted with a 1ml syringe, and administered to the young rabbits at a dose of 4ml / kg through the tracheal intubation, as the positive group; the Sinaputide group was administered to the young rabbits at a dose of 5.8ml / kg, and a certain volume of air (5.8ml / kg) was administered to the young rabbits in the negative group with a syringe.
[0057] After administration, the young rabbits were quickly placed in the chamber of the detection system. Each chamber had a cannula, and each cannula was connected to a multi-hole ventilation tube connected to a ventilator to ensure that the breathing settings and pressure of each animal were consistent. The positive group and the test group each required 6 young rabbits, and the negative group (only air) required at least 4 young rabbits. The same batch of drugs should not be given to more than three animals in the same litter, and the experimental sequence of three groups should be randomly sorted according to the litter of animals for testing. During the test, the ventilation conditions of the young rabbits were maintained as follows: the maximum inspiratory pressure was 27±2cmH2O, the positive end-expiratory pressure ventilation was 0cmH2O, the ventilation flow was 8±2L / min, the respiratory rate was 30±2 times / min, the exhalation time was 0.45±0.00s, and the oxygen concentration was 100%.
[0058] After the data collection of the young rabbits is completed, the young rabbits are taken out of the test chamber. The average growth value E% of the positive group (the growth percentage of the respiratory compliance value and body weight ratio CRS / kg of the positive group relative to the negative group CRS / kg) must be >600%, and the average growth value E% of the test group (the growth percentage of the respiratory compliance value and body weight ratio CRS / kg of the test group relative to the negative group CRS / kg) must be >320%. During the three runs, the CV% of the positive group and the test group must be <30%, and the biological activity of the sample meets the requirements. The test was divided into three runs, and the final result was the average of the three runs, as shown in Table 4.
[0059] Table 4 Experimental results of three runs of Example 3
[0060]
[0061]
[0062] Example 4 Activity Evaluation Test of Sinaputide Phospholipid Surfactant Degradation Sample (25°C, 4 Weeks)
[0063] Experimental materials: New Zealand pregnant rabbits at 26.5 days of gestation (rabbits weighing less than 23 g cannot be used in the experiment), bolata inhalation suspension, sinapeptide phospholipid surfactant (degraded), and 1 ml syringe.
[0064] Experimental method: The rabbits were placed face up, with their necks raised to an appropriate height, and a tracheal tube was inserted through the mouth (depth 4-5 mm), and fixed with 4-0 thread. The rewarmed Bolatan inhalation suspension was extracted with a 1ml syringe, and administered to the rabbits at a dose of 4ml / kg through the tracheal tube, as the positive group; the Sinaputide group (degraded) was administered to the rabbits at a dose of 5.8ml / kg, and a certain volume of air (5.8ml / kg) was administered to the rabbits in the negative group with a syringe.
[0065] After administration, the young rabbits were quickly placed in the chamber of the detection system. Each chamber had a cannula, and each cannula was connected to a multi-hole ventilation tube connected to a ventilator to ensure that the breathing settings and pressure of each animal were consistent. The positive group and the test group (degradation) required 6 young rabbits each, and the negative group (only air) required at least 4 young rabbits. The same batch of drugs should not be given to more than three animals in the same litter, and the experimental sequence of three groups should be randomly sorted according to the litter of animals for testing. During the test, the ventilation conditions of the young rabbits were maintained as follows: maximum inspiratory pressure of 27±2cmH2O, positive end-expiratory pressure ventilation of 0cmH2O, ventilation flow of 8±2L / min, respiratory rate of 30±2 times / min, exhalation time of 0.45±0.00s, and oxygen concentration of 100%.
[0066] After the data collection of the young rabbits is completed, the young rabbits are taken out of the test chamber. The average growth value E% of the positive group (the growth percentage of the respiratory compliance value and body weight ratio CRS / kg of the positive group relative to the negative group CRS / kg) must be >600%, the average growth value E% of the test group (the growth percentage of the respiratory compliance value and body weight ratio CRS / kg of the test group relative to the negative group CRS / kg) <320%, the CV% of the positive group and the test group must be <30% in three runs, and the CV% of the positive group and the test group must be <35% in six runs (the test group results failed in three runs, and three more runs were performed), then the bioactivity of the degradation sample meets the requirements. The test was divided into six runs, and the final result was the average of the six runs, as shown in Table 5.
[0067] Table 5 Experimental results of six runs of Example 4
[0068]
[0069] It can be seen that the method of the present application can accurately evaluate the biological activity of fresh samples or degraded samples of sinapeptide phospholipid surfactant, and the method has good repeatability and sensitivity.
[0070] The above is only a preferred specific implementation of the present application; however, the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved ideas of the present application within the technical scope disclosed in the present application, which should be included in the protection scope of the present application.
Claims
1. A method for evaluating the biological activity of pulmonary surfactant, characterized in that: The steps include: S1. administering a lung surfactant to be tested to a lung surfactant-deficient young rabbit according to a preset dose to obtain a lung surfactant-deficient young rabbit; S2, placing the test young rabbit after administration under test ventilation conditions, detecting and calculating the ratio of its respiratory compliance value to its body weight, and obtaining the test value of the test drug; The test ventilation conditions include: maximum inspiratory pressure of 27±2 cmH2O, positive end-expiratory pressure of 0 cmH2O, ventilation flow of 8±2 L / min, respiratory rate of 30±2 times / min, exhalation time of 0.45±0.00s, and oxygen concentration of 100%.
2. The evaluation method according to claim 1, characterized in that: The evaluation method further comprises: The operations of steps S1 and S2 are repeated using the positive control substance and the negative control substance respectively to obtain the positive control test value and the negative control test value.
3. The evaluation method according to claim 2, characterized in that: The positive control substance is a lung surfactant extracted from animal tissue; Optionally, the positive control is bovine lung surfactant beractant; Optionally, the dosage range of the bovine lung surfactant beractant is 2 to 8 ml / kg.
4. The evaluation method according to claim 2, characterized in that: The negative control substance is air; Optionally, the dosage range of air is 2.9 to 11.6 ml / kg.
5. The evaluation method according to any one of claims 2 to 4, characterized in that: In the case where the increase value of the positive control test value relative to the negative control test value is not less than 600%, it is determined that the performance of the experimental system of the evaluation method meets the requirements.
6. The evaluation method according to claim 5, characterized in that: When the increase value of the test value of the test drug relative to the negative control test value is not less than 320%, and the coefficient of variation between the positive control test value and the test value of the test drug after three repetitions is not greater than 30%, and the coefficient of variation between the positive control test value and the test value of the test drug after six repetitions is not greater than 35%, it is determined that the drug activity of the test pulmonary surfactant is good.
7. The evaluation method according to claim 1, characterized in that: The young rabbits are New Zealand white rabbits whose gestation days are 26.5±0.5 days.
8. The evaluation method according to claim 1, characterized in that: The tested lung surfactant is an artificially synthesized lung surfactant; Optionally, the lung surfactant to be tested is a sinapeptide phospholipid surfactant, and the sinapeptide phospholipid surfactant comprises dipalmitoylphosphatidylcholine, palmitoleoylglycerophosphoglycerol sodium salt, palmitic acid and sinapeptide; Optionally, the dosage range of the sinapeptide phospholipid surfactant is 2.9 to 11.6 ml / kg.
9. Use of the evaluation method according to any one of claims 1 to 8 in the quality control of pulmonary surfactant products.