Method for evaluating biological activity of pulmonary surfactant
By detecting the respiratory compliance-improving effect of lung surfactants in a juvenile rabbit model, the problem of validating the effectiveness of synthetic lung surfactants was solved, achieving highly accurate and sensitive activity evaluation, which is suitable for the quality control of lung surfactant products.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- ZHAOKE PHARMA HEFEI
- Filing Date
- 2025-12-30
- Publication Date
- 2026-07-09
AI Technical Summary
The efficacy of existing synthetic lung surfactants is not clearly distinguished from that of animal-derived lung surfactants, and their effectiveness needs to be verified to ensure safe and effective clinical use.
Using young rabbits lacking lung surfactant as experimental subjects, the efficacy and bioactivity of the tested lung surfactant were evaluated by measuring its effect on improving respiratory compliance under test ventilation conditions.
A highly accurate, repeatable, and sensitive method for evaluating the bioactivity of lung surfactants is provided, which is suitable for the quality control of lung surfactant products.
Smart Images
Figure CN2025147494_09072026_PF_FP_ABST
Abstract
Description
A method for evaluating the bioactivity of pulmonary surfactants Technical Field
[0001] This application belongs to the field of drug efficacy and activity testing technology, specifically relating to a method for evaluating the bioactivity of pulmonary surfactant. Background Technology
[0002] Neonatal respiratory distress syndrome (NRDS) is a common neonatal disease that is closely related to a deficiency of pulmonary surfactant (PS). NRDS causes progressive alveolar collapse, leading to symptoms such as difficulty breathing and cyanosis. NRDS progresses rapidly, and in severe cases, it can cause respiratory failure, endangering the infant's life.
[0003] The human lungs consist of numerous small air sacs called alveoli, through which gases are exchanged between the blood and the air spaces in 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 surfactants extracted from animal lungs can effectively treat NRDS. Currently, several modified natural surfactants have been approved for marketing. For example, poractanta (Curosurf) derived from pig lungs... TM ), calfactant (Infasurf) extracted from calf lung lavage fluid TM ), and chemically modified natural bovine lung extract beractant (Survanta) TM (i.e., Boratan).
[0005] However, lung surfactant drugs derived from animal tissues have many drawbacks, such as the complexity of production and sterilization methods and the potential to induce immune responses in the body. Therefore, several synthetic lung surfactants have been successfully developed, such as cinalopeptide phospholipid surfactant. However, the efficacy and activity of synthetic lung surfactants, as well as their differences in efficacy compared to animal-derived lung surfactants, need to be evaluated. Their effectiveness needs to be verified as a testing standard for drug production qualification and release, ensuring safe and effective clinical use. Summary of the Invention
[0006] The purpose of this application is to provide a method for evaluating the bioactivity of pulmonary surfactants. This method uses young rabbits lacking pulmonary surfactants as test subjects to detect the effect of the tested pulmonary surfactants on improving respiratory compliance, thereby evaluating the efficacy and bioactivity of the tested pulmonary surfactants.
[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:
[0008] In a first aspect, this application provides a method for evaluating the bioactivity of pulmonary surfactants, comprising the following steps:
[0009] S1. The test lung surfactant was administered to young rabbits lacking lung surfactant at a preset dose to obtain the test young rabbits after administration.
[0010] S2. Place the test rabbits after drug administration under test ventilation conditions, detect and calculate the ratio of their respiratory compliance value to their body weight, and obtain 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 rate of 8±2 L / min, respiratory rate of 30±2 breaths / min, expiratory time of 0.45±0.00s, and oxygen concentration of 100%.
[0012] In an optional implementation, the evaluation method further includes:
[0013] Steps S1 and S2 were repeated using positive and negative controls respectively to obtain positive and negative control test values.
[0014] In one 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 removed from 4°C and brought to room temperature.
[0017] In one alternative implementation, the negative control is air;
[0018] Optionally, the dosage of air administration ranges from 2.9 to 11.6 ml / kg, preferably 5.8 ml / kg.
[0019] In one alternative implementation, the experimental system performance of the evaluation method is determined to meet the requirements if the increase in the positive control test value relative to the negative control test value is not less than 600%.
[0020] In an optional embodiment, the test lung surfactant is determined to have good drug activity if the increase in 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 drug test value is not greater than 30% after three repetitions, and the coefficient of variation between the positive control test value and the test drug test value is not greater than 35% after six repetitions.
[0021] In one alternative implementation, the baby rabbit is a New Zealand White rabbit with a gestation period of 26.5 ± 0.5 days.
[0022] In one optional embodiment, the tested lung surfactant is a synthetic lung surfactant;
[0023] Optionally, the tested lung surfactant is a cinnaptide phospholipid surfactant, which includes dipalmitoyl phosphatidylcholine, palmitoyl glycerol phosphate sodium salt, palmitic acid, and cinnaptide.
[0024] Optionally, the dosage range of the cinalopeptide phospholipid surfactant is 2.9~11.6 ml / kg, preferably 5.8 ml / kg. Before administration, the cinalopeptide phospholipid surfactant needs to be removed from 4°C and rewarmed at 42~46°C for 15 min.
[0025] Secondly, this application provides the application of the above-mentioned evaluation method in the quality control of pulmonary surfactant products.
[0026] Based on the above technical solution, this application has at least the following beneficial effects:
[0027] The method for evaluating the bioactivity of pulmonary surfactant provided in this application selects young rabbits lacking pulmonary surfactant as experimental subjects. The method detects the effect of the tested pulmonary surfactant on the improvement of the young rabbits' lung respiratory compliance by administering the drug to the lungs. The efficacy and bioactivity of the drug are evaluated based on the experimental data. This method has high accuracy, repeatability and sensitivity, simple operation steps and wide applicability. Attached Figure Description
[0028] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0029] Figure 1 shows the linear experimental results in Embodiment 2 of this application. Detailed Implementation
[0030] To further illustrate the technical means and results adopted by this application to achieve the intended inventive purpose, the following preferred embodiments are used to describe in detail the specific implementation methods, technical solutions, and features according to this application. Specific features, structures, or characteristics in the various embodiments described below can be combined in any suitable form.
[0031] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.
[0032] The present application will be further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.
[0033] Example 1: Performance verification experiment of the activity evaluation experimental system (negative and positive groups only)
[0034] Experimental materials: New Zealand pregnant rabbits with a gestation period of 26.5 days (young rabbits weighing less than 23g cannot be used for the experiment), boraxane inhalation suspension, and 1mL syringe.
[0035] Experimental Method: Young rabbits were placed face up with their necks elevated at an appropriate height. An endotracheal tube (4-5 mm deep) was inserted through their mouths and secured with 4-0 suture. A 1 mL syringe was used to draw up rewarmed boralotinib suspension, which was then administered to the young rabbits via the endotracheal tube at a dose of 4 ml / kg. This group served as the positive control group. The negative control group was administered a specific volume of air (5.8 ml / kg) via syringe.
[0036] After drug administration, the young rabbits were quickly placed in the testing system compartments. Each compartment contained a cannula, and each cannula was connected to a multi-port ventilation tube for the ventilator to ensure consistent breathing settings and pressure for each animal. The positive group required 6 young rabbits, and the negative group (air-only) required at least 4 young rabbits. No more than three rabbits from the same litter should be administered the same batch of drug consecutively, and the experimental sequence should be randomly ordered according to the litter number. During the experiment, the ventilation conditions for the young rabbits were maintained as follows: maximum inspiratory pressure 27±2 cmH2O, positive end-expiratory pressure 0 cmH2O, ventilation flow rate 8±2 L / min, respiratory rate 30±2 breaths / min, expiratory time 0.45±0.00s, and oxygen concentration 100%.
[0037] After data collection from the young rabbits was completed, the rabbits were removed from the testing chamber. The average positive growth rate (E%) and CV% (CV%) of the three runs were calculated. If the average positive growth rate (E%) (the percentage increase in respiratory compliance to body weight ratio (CRS / kg) of the positive group relative to the CRS / kg of the negative group) > 600% and the CV% of the three runs < 30%, the performance verification experiment was considered successful. The experiment consisted of three runs, and the final result was the average of the three runs, as shown in Table 1.
[0038] Table 1 Results of three runs of Example 1
[0039]
[0040] As can be seen from Table 1, the activity evaluation experimental system of this application has good performance.
[0041] Example 2: Linear Experiment
[0042] Experimental materials: New Zealand pregnant rabbits with a gestation period of 26.5 days (young rabbits weighing less than 23g could not be used for the experiment), boraxane inhalation suspension, cinapeptide phospholipid surfactant (concentrations of 30, 10, 5, 2.5, 1, and 0.5 mg / ml, respectively), and 1ml syringes.
[0043] Experimental Methods: Young rabbits were placed face up with their necks elevated at an appropriate height. An endotracheal tube (4-5 mm deep) was inserted through the mouth and secured with 4-0 suture. A 1 ml syringe was used to draw up rewarmed boralotinic suspension, which was then administered to the young rabbits via the endotracheal tube at a dose of 4 ml / kg (positive group). The cinalatropin group was administered to the young rabbits at a dose of 5.8 ml / kg. The negative group was administered a certain volume of air (5.8 ml / kg) via syringe.
[0044] After drug administration, the young rabbits were quickly placed in the testing system compartments. Each compartment contained a cannula, and each cannula was connected to a multi-port ventilation tube for the ventilator to ensure consistent breathing settings and pressure for each animal. Six young rabbits were required for each positive group and test group (different concentration groups), and at least four young rabbits were required for the negative group (air only). No more than three animals from the same litter should be given the same batch of drug, and the experimental sequence of three groups should be randomly ordered according to the litter size. During the experiment, the ventilation conditions for the young rabbits were maintained as follows: maximum inspiratory pressure 27±2 cmH2O, positive end-expiratory pressure 0 cmH2O, ventilation flow rate 8±2 L / min, respiratory rate 30±2 breaths / min, expiratory time 0.45±0.00s, and oxygen concentration 100%.
[0045] After data collection from the young rabbits is completed, the young rabbits are removed from the testing system chamber. The average growth rate (E%) of the positive group (the percentage increase in respiratory compliance value to body weight ratio CRS / kg relative to the negative group CRS / kg) must be >600%, and the CV% of the positive group must be <30% after 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 transformation, a linear curve is plotted, and the linearity results are evaluated (R² > 0.95 is required).
[0046] Experimental Results: The increase in E% (the percentage increase in respiratory compliance to body weight ratio (CRS / kg) of the test group relative to the negative group (CRS / kg)) for the test group (different concentration groups) was calculated as the average of all runs. A linear plot was then plotted using the concentration (square root) and E% values to calculate R. 2 The values are shown in Tables 2 and 3 and Figure 1.
[0047] Table 2. Sample concentration and growth rate (E%) of the test group
[0048]
[0049] Table 3. Sample concentration (square root) and growth rate (E%) of the test group
[0050]
[0051] As can be seen from Tables 2 and 3 and Figure 1, the activity evaluation experimental system of this application exhibits good linearity.
[0052] Example 3: Evaluation Test of the Activity of Cinnaptide Phospholipid Surfactant
[0053] Experimental materials: New Zealand pregnant rabbits with a gestation period of 26.5 days (young rabbits weighing less than 23g cannot be used for the experiment), boraxane inhalation suspension, cinapeptide phospholipid surfactant, and a 1ml syringe.
[0054] Experimental method: Young rabbits were placed face up with their necks elevated at an appropriate height. An endotracheal tube (4-5 mm deep) was inserted through the mouth and secured with 4-0 suture. A 1 ml syringe was used to draw up rewarmed boralotinic suspension, which was then administered to the young rabbits via the endotracheal tube at a dose of 4 ml / kg (positive group). The cinalatropin group was administered to the young rabbits at a dose of 5.8 ml / kg. The negative group was administered a certain volume of air (5.8 ml / kg) via syringe.
[0055] After drug administration, the young rabbits were quickly placed in the testing system compartments. Each compartment contained a cannula, and each cannula was connected to a multi-port ventilation tube for the ventilator to ensure consistent breathing settings and pressure for each animal. Six young rabbits were required for each of the positive and test groups, and at least four young rabbits were required for the negative group (air-only). No more than three animals from the same litter should be given the same batch of drug, and the experimental sequence of three groups should be randomly ordered according to the litter number of animals. During the experiment, the ventilation conditions for the young rabbits were maintained as follows: maximum inspiratory pressure 27±2 cmH2O, positive end-expiratory pressure 0 cmH2O, ventilation flow rate 8±2 L / min, respiratory rate 30±2 breaths / min, expiratory time 0.45±0.00s, and oxygen concentration 100%.
[0056] After data collection from the young rabbits, they were removed from the testing chamber. The average growth rate (E%) of the positive group (the percentage increase in respiratory compliance to body weight ratio (CRS / kg) relative to the negative group's CRS / kg) needed to be >600%, and the average growth rate (E%) of the test group (the percentage increase in respiratory compliance to body weight ratio (CRS / kg) relative to the negative group's CRS / kg) needed to be >320%. The CV% of both the positive and test groups needed to be <30% across the three runs for the sample to meet the bioactivity requirements. The experiment was conducted in three runs, and the final result was the average of the three runs, as shown in Table 4.
[0057] Table 4 Results of three runs of the experiment in Example 3
[0058]
[0059] Example 4: Activity Evaluation Test of Cinnaptide Phospholipid Surfactant Degradation Samples (25℃, 4 weeks)
[0060] Experimental materials: New Zealand pregnant rabbits with a gestation period of 26.5 days (young rabbits weighing less than 23g cannot be used for the experiment), boraxane inhalation suspension, cinapeptide phospholipid surfactant (degraded), and a 1ml syringe.
[0061] Experimental Methods: Young rabbits were placed face up with their necks elevated at an appropriate height. An endotracheal tube (4-5 mm deep) was inserted through the mouth and secured with 4-0 suture. A 1 ml syringe was used to draw up rewarmed boralotinic suspension, which was then administered to the young rabbits via the endotracheal tube at a dose of 4 ml / kg (positive group). The cinalatropin group (degraded) was administered to the young rabbits at a dose of 5.8 ml / kg. The negative group was administered a certain volume of air (5.8 ml / kg) via syringe.
[0062] After drug administration, the young rabbits were quickly placed in the testing system compartments. Each compartment contained a cannula, and each cannula was connected to a multi-port ventilation tube for the ventilator to ensure consistent breathing settings and pressure for each animal. Six young rabbits were required for each of the positive and test (degradation) groups, and at least four young rabbits were required for the negative group (air only). No more than three animals from the same litter should be given the same batch of drug. The experimental sequence of three groups should be randomly ordered according to the litter number of animals. During the experiment, the ventilation conditions for the young rabbits were maintained as follows: maximum inspiratory pressure 27±2 cmH2O, positive end-expiratory pressure 0 cmH2O, ventilation flow rate 8±2 L / min, respiratory rate 30±2 breaths / min, expiratory time 0.45±0.00s, and oxygen concentration 100%.
[0063] After data collection from the young rabbits, they were removed from the testing chamber. The average growth rate (E%) of the positive group (the percentage increase in respiratory compliance to body weight ratio (CRS / kg) relative to the negative group's CRS / kg) must be >600%, and the average growth rate (E%) of the test group (the percentage increase in respiratory compliance to body weight ratio (CRS / kg) relative to the negative group's CRS / kg) must be <320%. The CV% of both the positive and test groups must be <30% after three runs, and <35% after six runs (if the test group's results are unsatisfactory after three runs, three more runs must be performed). For these conditions to be met, the bioactivity of the degraded samples must be satisfactory. The experiment consisted of six runs, and the final result was the average of the six runs, as shown in Table 5.
[0064] Table 5 Results of six runs of Example 4
[0065]
[0066] It is evident that the method of this application can accurately evaluate the bioactivity of fresh or degraded samples of cinapeptide phospholipid surfactant, and the method has good repeatability and sensitivity.
[0067] The above description is merely a preferred embodiment of this application; however, the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and its improved concept, should be covered within the scope of protection of this application.
Claims
1. A method for evaluating the bioactivity of pulmonary surfactants, characterized in that, Includes the following steps: S1. The test lung surfactant was administered to young rabbits lacking lung surfactant at a preset dose to obtain the test young rabbits after administration. S2. Place the test rabbits after drug administration under test ventilation conditions, detect and calculate the ratio of their respiratory compliance value to their body weight, and obtain 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 rate of 8±2 L / min, respiratory rate of 30±2 breaths / min, expiratory 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 also includes: Steps S1 and S2 were repeated using positive and negative controls respectively to obtain positive and negative control test values.
3. The evaluation method according to claim 2, characterized in that, The positive control was 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 2-8 ml / kg.
4. The evaluation method according to claim 2, characterized in that, The negative control was air; Optionally, the dosage of air administration ranges from 2.9 to 11.6 ml / kg.
5. The evaluation method according to any one of claims 2 to 4, characterized in that, If the increase in the positive control test value relative to the negative control test value is not less than 600%, the experimental system performance of the evaluation method is deemed to meet the requirements.
6. The evaluation method according to claim 5, characterized in that, The test lung surfactant is deemed to have good drug activity if the increase in 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 drug test value is not greater than 30% after three repetitions, and the coefficient of variation between the positive control test value and the test drug test value is not greater than 35% after six repetitions.
7. The evaluation method according to claim 1, characterized in that, The baby rabbits were New Zealand White rabbits with a gestation period of 26.5 ± 0.5 days.
8. The evaluation method according to claim 1, characterized in that, The tested lung surfactant was a synthetic lung surfactant; Optionally, the tested lung surfactant is a cinnaptide phospholipid surfactant, which includes dipalmitoyl phosphatidylcholine, palmitoyl glycerol phosphate sodium salt, palmitic acid, and cinnaptide. Optionally, the dosage range of the cinapeptide phospholipid surfactant is 2.9~11.6 ml / kg.
9. The application of the evaluation method according to any one of claims 1 to 8 in the quality control of pulmonary surfactant products.