CN109298102B - In-vitro evaluation method for drug pulmonary metabolism characteristics - Google Patents
In-vitro evaluation method for drug pulmonary metabolism characteristics Download PDFInfo
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Abstract
The invention provides an in-vitro evaluation method for pulmonary metabolism characteristics of a medicament, which comprises the following steps: (1) preparing a lung subcellular tissue; (2) respectively preparing a positive quality control system, a negative quality control system, a control group reaction system and a test group reaction system by adopting the lung subcellular tissue obtained in the step (1), incubating, culturing, and centrifuging to obtain a supernatant; (3) respectively detecting the parent content of the positive substrate in the supernatant of the positive quality control system and the parent content of the parent of the negative quality control system in the step (2) and the parent content of the substance to be detected in the supernatants of the control group reaction system and the test group reaction system; the positive substrate of the positive quality control system comprises any one or the combination of at least two of 2-aminofluorene, 4-methoxy-1,8-naphthalimide, phenacetin or 4-sweet potato scab diol. The evaluation method provided by the invention has stable effect, is a perfect evaluation method aiming at pulmonary drug delivery, and provides methodological support for research and development of new drugs and drug interaction.
Description
Technical Field
The invention belongs to the field of biological medicine, relates to a method for evaluating medicine safety, particularly relates to a method for evaluating the pulmonary metabolic characteristics of a medicine in vitro, and particularly relates to a method for evaluating the pulmonary metabolic characteristics of an inhalant medicine in vitro.
Background
The guidelines for drug interaction (CFDA) state food and drug administration, national institute of health and drug administration (2012) in the people's republic of china states: the metabolism of a new drug should be determined during drug development and the interaction between the drug and other drugs should be studied as part of the evaluation of safety and efficacy. Since the elimination of oral and intravenous drugs or their metabolites is usually carried out in the liver, the tissues selected by the current in vitro study methods of drug metabolism are mostly the liver and its subcellular tissues.
With the continuous development of medicine, inhalation therapy is recommended by the World Health Organization (WHO) and Europe and America as the first-choice therapy for respiratory diseases such as asthma, COPD and the like, the lung has large absorption surface area and rich capillary vessel network, so that the pulmonary administration has quick response, the inhalation administration has small irritation, the use is convenient, the patient compliance is good, and the method is suitable for patients needing long-term treatment. Therefore, research and development of novel inhalation drugs are important approaches to the treatment of respiratory diseases and partial systemic action drugs. However, the inhalation preparation drug is administrated through the lung, the liver first-pass effect is avoided, and the inhalation preparation drug directly acts on lung tissues, so that the research on the drug metabolism characteristics of the drug by selecting liver cells and subcellular tissues thereof is not completely applicable, the current research on the metabolism characteristics and drug interaction of the drug is limited to the molecular mechanism research of specific compound metabolism paths, the personalized research scheme has no universality, the method has poor stability and high probability of false positive and false negative, and the in-vitro research on the drug lung metabolism characteristics is lack of a perfect technical scheme.
(Aune T, et al. deacylation to 2-aminofluorene as a major metabolic reaction in the micro metabolic of 2-acetylaminofluorene to multigenic products in preliminary from the branched and lever [ J ]. Cancer Research,1985,45(11Pt2):5859.) it was disclosed that rabbit lung microsomes have different metabolic activities for 2-acetamidofluorene and 2-aminofluorene, and that the metabolic activity of the lung is higher than that of the liver, suggesting that some drugs may be more metabolically active in the lung, whereas for such drugs the use of liver metabolic criteria is more inaccurate and fails to assess the metabolism of the drug in vivo.
Therefore, it is of great significance to provide an in vitro research method for evaluating pulmonary metabolism characteristics of drugs, particularly inhalation drugs.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides an in-vitro evaluation method for the pulmonary metabolic characteristics of the medicine, specifically selects a positive substrate and the use concentration thereof, establishes the in-vitro evaluation method for the pulmonary metabolic characteristics, improves the sensitivity and stability of the evaluation method, reduces the probability of false positive and false negative in the evaluation process, and provides a methodological support for the evaluation and research of the medicine safety.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for in vitro assessment of pulmonary metabolic profile of a drug, said method comprising the steps of:
(1) preparing a lung subcellular tissue;
(2) respectively preparing a positive quality control system, a negative quality control system, a control group reaction system and a test group reaction system by adopting the lung subcellular tissue obtained in the step (1), incubating, culturing, and centrifuging to obtain a supernatant;
(3) respectively detecting the parent content of the positive substrate in the supernatant of the positive quality control system and the parent content of the parent of the negative quality control system in the step (2) and the parent content of the substance to be detected in the supernatants of the control group reaction system and the test group reaction system;
the positive substrate of the positive quality control system comprises any one or the combination of at least two of 2-aminofluorene, 4-methoxy-1,8-naphthalimide, phenacetin or 4-sweet potato scab diol.
According to the invention, the lung subcellular tissue is matched with a positive quality control system and a negative quality control system, the positive quality control system verifies that an enzyme reaction system in the lung subcellular tissue can work normally, and the negative quality control system is used for indicating that non-enzymatic metabolism of a positive substrate does not exist in the system, so that the reaction system can be used for evaluating the drug metabolic stability and simultaneously reducing the probability of false positive and false negative; the control group reaction system and the test group reaction system can identify the metabolic characteristics of the drug or the compound to be detected in the lung and are used for researching drug interaction, the control group reaction system verifies the stability of the substance to be detected, and the judgment of the self-degradation of the substance to be detected on the metabolic result is excluded. According to the research of the inventor, 2-aminofluorene (2-aminofluorene), 4-methoxy-1,8-naphthalimide (4-methoxy-1,8-naphthalimide), phenacetin (phenacetin) or 4-sweet potato scar mycetogluca diol (4-ipomoenol) are selected to be more suitable as positive substrates for evaluating the pulmonary metabolism characteristics of the medicine.
Preferably, the positive substrate is 2-aminofluorene.
In the invention, the positive quality control system is used for detecting whether the enzyme reaction system in the lung subcellular tissue works normally, and is of great importance for the subsequent evaluation process. The research of enzyme systems in lung tissues is not complete, the types and contents of enzymes contain various uncertainties, and different species have large differences. Therefore, the selection of a positive substrate with universality and sensitivity is critical. The inventor shows through a large number of researches that CYP450 enzyme systems with higher expression level in lung are CYP1A2 and CYP4B1, and the expression activity in different species (human, monkey, rat, dog) is similar. Thus, substrates for CYP1A2 and CYP4B1 are preferably considered as positive substrates for the evaluation. As an evaluation system, in determining a positive probe substrate, various factors are considered, including (1) the specificity of the substrate: whether the substrate is specifically metabolized by a metabolic enzyme; (2) sensitivity: the degree of sensitivity of the enzyme to metabolism of the substrate; (3) stability of the compound: stability of the compound in a biological matrix; (4) detectability: the detection limit of the substrate in LC-MS-MS detection. Although the substrates of CYP1A2 and CYP4B1 are currently studied more, most positive substrates do not meet the above requirements.
For example, some positive substrates suitable for animals are not suitable for evaluation in human lung s9 because of species differences between the enzyme systems in human lung and animals (e.g., rabbits), and some positive substrates, although acting on both human and animal lung, are not enzymatically degraded and are not stable themselves and can not be used to evaluate the metabolism of drugs or compounds in the lung. Through a large amount of screening, the invention discovers that the 2-aminofluorene has strong specificity to lung tissues, is chemically stable, has small species difference and good verification effect, can be used for a positive quality control system, and ensures that an enzyme system in a lung subcellular tissue can be used for evaluating the metabolism of a drug or a compound in the lung.
Preferably, the final concentration of the 2-aminofluorene is 0.05-0.2. mu.M, and may be, for example, 0.05. mu.M, 0.1. mu.M, 0.15. mu.M, or 0.2. mu.M.
In the invention, the concentration is the final concentration of the 2-aminofluorene in the reaction system, and the inventor finds that the 2-aminofluorene has the best effect in the concentration range by adjusting the mass concentration ratio of the method system to the positive substrate. The metabolic capacity of lung tissue is lower because the expression level of metabolic enzymes of the lung tissue is far lower than that of liver tissue; therefore, as an evaluation system, if the concentration of the positive substrate is too high and exceeds the metabolic capability of the enzyme, the working state of the enzyme cannot be judged; if the substrate concentration is too low, the detection error is large, and the result is unstable.
Preferably, step (1) is followed by a dilution step, specifically comprising: the protein mass concentration of the dilution to lung subcellular tissue is 0.5-4mg/mL, and may be, for example, 0.5mg/mL, 0.8mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL or 4 mg/mL.
Preferably, the lung subcellular tissue comprises lung S9 or lung microsomes.
Preferably, the protein concentration of the lung S9 is 2-4mg/mL, and may be, for example, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, or 4 mg/mL.
Preferably, the protein concentration of the lung microsomes is 0.5-2mg/mL, and may be, for example, 0.5mg/mL, 1mg/mL, 1.5mg/mL, or 2mg/mL, preferably 0.5-1 mg/mL.
In the invention, the effect is best in the mass concentration range of the lung subcellular tissue protein, the reaction system is more sensitive when the lung S9 is diluted to the protein concentration of 2-4mg/mL aiming at different optimum concentrations of different lung subcellular tissues, the reaction system is high in sensitivity when the lung microsome is 0.5-2mg/mL, the enzyme activity is improved even if the protein concentration is too high, but the impurity content is increased, non-enzyme system degradation is caused, the detection difficulty is increased, and the like.
Preferably, the positive quality control system in the step (2) comprises a positive substrate, lung subcellular tissues and NADPH coenzyme.
In the invention, the positive quality control system is used for detecting whether the enzyme reaction system in the lung subcellular tissue works normally or not, and the enzyme reaction system is proved to be normal through the verification of a positive substrate.
Preferably, the final concentration of the NADPH coenzyme is between 1 and 2mM, preferably 1 mM.
In the invention, the final concentration of the NADPH coenzyme in the reaction system is in the range, so that the reaction of the enzyme in the lung subcellular tissue and a positive substrate can be effectively promoted, and whether the enzyme reaction system works normally or not is verified.
Preferably, the negative quality control in the step (2) comprises a positive substrate, lung subcellular tissues and a Tris buffer.
According to the in-vitro evaluation method, the accuracy of the in-vitro evaluation method is improved by optimizing and adjusting the composition and the proportion of the negative quality control system, the influence of non-enzymatic degradation on a reaction result is eliminated, and the false positive probability is reduced.
Preferably, the mass concentration of the Tris buffer is 0.1 mM.
In the invention, the quality concentration of the Tris buffer solution is adapted to the whole in-vitro evaluation method, and the method can be used for preparing the NADPH coenzyme and adjusting the volume of a negative quality control reaction system and a control reaction system.
Preferably, the incubation time in step (2) is 60-240min, for example 60min, 90min, 120min, 150min180min, 210min or 240min, preferably 120 min.
In the invention, after the positive quality control system, the negative quality control system, the control group reaction system and the test group reaction system are respectively incubated for 120min, the content of the parent body of the positive substrate in the positive quality control system and the content of the parent body of the negative substrate in the negative quality control system before and after incubation are compared, the metabolism condition of the lung subcellular tissue to the positive substrate is judged, and then whether the enzyme reaction system in the lung subcellular tissue works normally is judged; and (3) observing whether the lung subcellular tissue has a significant metabolic effect on the substance to be detected by comparing the parent content of the substance to be detected in the reaction system of the control group and the reaction system of the test group before and after incubation, and further judging the metabolic characteristics of the lung on the substance to be detected.
Preferably, the control reaction system in step (2) comprises the analyte, the lung subcellular tissue and a Tris buffer.
Preferably, the test panel reaction system includes an analyte, lung subcellular tissue, and an NADPH coenzyme.
Preferably, the final concentration of the analyte in the system is 0.1-10. mu.M, and may be, for example, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 1.5. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, or 10. mu.M.
In the invention, the reaction system of the test group is used for detecting the metabolism condition of the lung subcellular tissue to the substance to be detected. If the substance is a substrate of metabolic enzyme in lung, the enzyme system in lung subcellular tissue acts on the substance in the presence of NADPH coenzyme, and the chemical structure of the substance is changed, so as to judge the metabolic characteristics of the substance in lung.
Preferably, the addition of the NADPH coenzyme or Tris buffer solution in the positive quality control system, the negative quality control system, the control group reaction system and the experimental group reaction system comprises a pre-incubation step.
In the invention, after a positive substrate or an object to be detected is added into the lung subcellular tissue, the system needs to be pre-incubated for 3-8min, and then corresponding NADPH coenzyme or Tris buffer solution is added for mixing. The pre-incubation process can simulate the in vivo situation, activate the metabolic enzyme system in the lung subtype cell tissue, obtain energy by reducing NADPH into NADP, thereby carrying out metabolic activity, and if the pre-incubation is not carried out, the energy supply of the reaction system can be reduced, thereby influencing the evaluation result.
Preferably, the pre-incubation time is 3-8min, for example, 3min, 4min, 5min, 6min, 7min or 8min, preferably 5 min.
Preferably, the method specifically comprises the following steps:
(1) preparing lung subcellular tissue, measuring the protein content, and diluting the lung subcellular tissue with 0.1mM Tris buffer solution until the concentration of the protein is 0.5-4 mg/mL;
(2) respectively preparing a positive quality control system, a negative quality control system, a control group reaction system and a test group reaction system by using the lung subcellular tissue obtained in the step (1), wherein the positive quality control system comprises a positive substrate and the lung subcellular tissue which are uniformly mixed and pre-incubated for 3-8min, then adding NADPH coenzyme and uniformly mixing, the final concentration of the positive substrate is 0.05-0.2 mu M, and the final concentration of the NADPH coenzyme is 1-2 mM; the negative quality control system comprises uniformly mixing a positive substrate and the lung subcellular tissue, pre-incubating for 3-8min, adding a Tris buffer solution, and uniformly mixing, wherein the final concentration of the positive substrate is 0.05-0.2 mu M; the control group reaction system comprises uniformly mixing the substance to be detected and the lung subcellular tissue, pre-incubating for 3-8min, adding Tris buffer solution, and uniformly mixing, wherein the final concentration of the substance to be detected is 0.1-10 mu M; the reaction system of the test group comprises uniformly mixing the substance to be tested and the lung subcellular tissue, pre-incubating for 3-8min, adding NADPH coenzyme, and uniformly mixing, wherein the final concentration of the NADPH coenzyme is 1-2mM, and the final concentration of the substance to be tested is 0.1-10 μ M; respectively incubating the positive quality control system, the negative quality control system, the control group reaction system and the test group reaction system for 60-240 min; adding precooled methanol after the incubation is finished, stopping metabolic reaction of each system, precipitating cell protein, mixing the sample evenly by vortex for 15-25s, centrifuging at 12000rpm for 3-8min, and taking supernatant;
(3) detecting the supernatant in the step (2) by using a liquid chromatography-mass spectrometer LC-MS/MS (liquid chromatography-mass spectrometer) relatively and quantitatively, and respectively detecting the parent content of the positive substrate in the supernatant of the positive quality control system and the parent content of the parent of the control group of the parent of the;
the positive substrate of the positive control comprises any one of or a combination of at least two of 2-aminofluorene, 4-methoxy-1,8-naphthalimide, phenacetin or 4-sweet potato scab diol.
Preferably, the evaluation criteria of the method are: (1) the maternal content of the test substance has a significant difference after incubation compared with 0min, which indicates that the test substance can be metabolized in the lung; (2) the content of the parent body of the substance to be detected has no significant difference when being compared with 0min after incubation, which indicates that the substance to be detected is stable in the lung and is not metabolized; the significant difference was p < 0.05.
According to the invention, whether an enzyme system in the lung subcellular tissue normally works is verified through a positive quality control system and a negative quality control system, so that whether the lung subcellular tissue can be used for evaluating the metabolic characteristics of a medicament or an object to be tested in the lung is further determined, the residual rate of a positive substrate after incubation in the positive quality control system is obviously different when being compared with 0min, and the residual rate of the positive substrate after incubation in the negative quality control system is not obviously different when being compared with 0min, so that the normal work of the enzyme reaction system in the lung subcellular tissue is indicated; the metabolism condition of the substance to be detected in lung tissue is judged by comparing the residual amount of the parent of the substance to be detected before and after incubation, if the content of the parent of the substance to be detected after incubation is obviously reduced, the substance to be detected can be metabolized by the lung, and if the content of the parent of the substance to be detected after incubation is not obviously different from that before incubation, the substance to be detected is stable in the lung.
In a second aspect, the present invention provides a use of a lung subcellular tissue positive substrate for assessing a metabolic characteristic of a drug in the lung, said use employing a method as described in the first aspect.
Preferably, the positive substrate comprises 2-aminofluorene.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a perfect in vitro method for evaluating the metabolic characteristics of a drug or a substance to be tested in the lung, and the evaluation method has stable effect and is a preferable evaluation method aiming at pulmonary drug delivery; provides an important research method for the interaction research of the direct lung administration or the lung-targeted drug.
(2) The in-vitro evaluation method reduces the probability of false positive or false negative through a perfect quality control system, and improves the accuracy of the method;
(3) the method specifically selects the positive substrate and the use concentration thereof, and is matched with the whole evaluation method, so that the in vitro evaluation method has high universality, is suitable for detecting various objects to be detected and judging the metabolic characteristics of the objects in the lung;
(4) the in vitro evaluation method provided by the invention is simple, saves manpower and material resources and is convenient to popularize.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following embodiments further illustrate the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments.
Experimental Material
(1) Preparing a 0.15M KCl solution: dissolving 11.18g KCl in 1L water to obtain 0.15M KCl solution;
(2) preparing 1M Tris-Acetate: 121.14g Tris is dissolved in 1L water to obtain 1M Tris solution;
(3) preparing a 1M KCl solution: dissolving 74.56g of KCl in 1L of water to obtain a 1M KCl solution;
(4) preparation of 100mM EDTA solution: dissolving 37.2g of EDTA in 1L of water to obtain 100mM EDTA solution;
(5) preparing 20mM BHT Stock, and dissolving 44.1mg of butyl Sodium hydroxyurea in 10mL of water to obtain 20mM BHT Stock-20 degrees for storage;
(6) preparing a buffer solution A: 100Ml of 1M Tris-Acetate solution was added to 100Ml of 1M KCl solution and 10Ml of 100mM EDTA solution was added to make 1L volume. Obtaining the product; note that: buffer solution A1 mL of the previous buffer solution A was added with 1uL of 20mM BHT solution;
(7) preparing a buffer solution C: 10ml of 1M Tris-Acetate solution was added to 10ml of 100mM EDTA solution and 200ml of glycerol to make a volume of 1L. Adjusting the pH value to 7.4, and storing at 4 ℃;
the materials used in the following examples are not limited to those listed above, and other similar materials may be substituted, and the apparatus may be operated under the conventional conditions or the instructions without specifying the specific conditions, and those skilled in the art should understand the knowledge about the use of conventional materials and apparatuses.
EXAMPLE 1 preparation of animal Lung subcellular tissue
The animal is fasted overnight, the animal is anesthetized by 30mg/kg of isoflurane or 3% pentobarbital sodium aqueous solution through intraperitoneal injection, the abdominal cavity and the thoracic cavity are opened by surgical scissors, the abdominal aorta is clamped by hemostatic forceps, the left ventricle of the heart is cut, a 50mL syringe (with physiological saline or 0.15M potassium chloride solution for precooling) is inserted, the solution is quickly injected for washing until the lung is milk white, the lung tissue is taken out and placed in a cold 0.15M potassium chloride (or 0.9% sodium chloride) solution beaker, the beaker is soaked and washed for 2 to 3 times according to the solid-liquid volume ratio of 2:3, after the lung tissue is washed for 2 times again by using a buffer solution A, the cleaned lung tissue and the buffer solution A are taken and placed in a centrifuge tube according to the solid-liquid volume ratio of 2:3, the centrifuge tube is sheared, a high-speed homogenizer is used for homogenizing, and the homogenate is collected and placed in the centrifuge tube. Precooling to 4 ℃ by using a centrifuge, placing the collected lung homogenate in the centrifuge, centrifuging for 30min at 9000G, and filtering by using double-layer gauze to obtain filtrate, namely the lung S9. The obtained S9 was transferred to an ultra-high speed centrifuge tube, equilibrated with buffer solution A, and centrifuged at 100000G for 1 hour, with the temperature of the centrifuge controlled to 4 ℃. After centrifugation is finished, removing supernatant to obtain lung microsomes; adding the buffer solution C in a ratio of 1:1, uniformly transferring the mixture into a new centrifugal tube, and freezing and storing the obtained liquid for later use after determining the protein content.
EXAMPLE 2 in vitro evaluation of the Metabolic characteristics of Testosterone in the Lung
(1) After thawing the beagle lung S9 in a 37 ℃ water bath, diluting the lung S9 to 3mg/mL by using Tris buffer;
(2) respectively preparing a positive quality control system, a negative quality control system, a control group reaction system and a test group reaction system, wherein the positive quality control system comprises uniformly mixing 2-aminofluorene and lung S9 for pre-incubation for 5min, adding NADPH coenzyme, and uniformly mixing according to the volume that 2-aminofluorene, lung S9 and NADPH coenzyme are respectively 25 muL, 25 muL and 50 muL, the final concentration of 2-aminofluorene is 0.05 muM, the final concentration of NADPH coenzyme is 1mM, and the protein content of lung S9 is 3 mg/mL; the negative quality control system comprises the steps of uniformly mixing 2-aminofluorene and lung S9, pre-incubating for 5min, adding 0.1mM Tris buffer solution, and uniformly mixing according to the volume ratio of 25 muL, 25 muL and 50 muL of 2-aminofluorene, lung S9 and Tris buffer solution respectively, wherein the final concentration of the 2-aminofluorene is 0.05 muM, and the protein content of lung S9 is 3 mg/mL; the control group reaction system comprises the steps of uniformly mixing testosterone and lung S9, pre-incubating for 5min, adding 0.1mM Tris buffer solution, and uniformly mixing according to the volume ratio of 25 muL, 25 muL and 50 muL of the testosterone, lung S9 and Tris buffer solution respectively, wherein the final concentration of the testosterone is 0.1 muM, and the protein content of lung S9 is 3 mg/mL; the reaction system of the test group comprises the steps of uniformly mixing testosterone and lung S9, pre-incubating for 5min, adding NADPH coenzyme, and uniformly mixing according to the volume ratio that the testosterone, the lung S9 and the NADPH coenzyme are respectively 25 muL, 25 muL and 50 muL, wherein the final concentration of the NADPH coenzyme is 1mM, the final concentration of the testosterone is 0.1 muM, and the protein content of the lung S9 is 3 mg/mL; adding the positive quality control system, the negative quality control system, the control group reaction system and the test group reaction system into 48-pore plates, and respectively placing the plates in an incubator at 37 ℃ and 5% CO2Incubating for 120min under the condition that 3 systems are parallel, and recording the parent content of 2-aminofluorene or testosterone in each system at 0 min;
(3) adding precooled methanol after the incubation is finished, stopping metabolic reaction of each system, precipitating cell proteins, mixing the samples evenly for 20s in a vortex manner, centrifuging at 12000rpm for 5min, and taking supernatant;
(4) and (3) relatively and quantitatively detecting the parent content of the 2-aminofluorene or testosterone in the supernatant in the step (3) by using a liquid chromatography-mass spectrometer (LC-MS/MS), and calculating the corresponding parent residual rate, wherein the LC-MS/MS detection results of the four systems are shown in tables 1 and 2.
TABLE 1 detection results of positive and negative quality control systems
TABLE 2 test results of control group reaction system and test group reaction system
As shown in Table 1, 2-aminofluorene showed no decrease in metabolism at 120 minutes without addition of coenzyme NADPH; however, 2-aminofluorene showed a 16% reduction upon addition of NAPDH coenzyme, and the data reached statistically significant differences (p ═ 0.0263), suggesting that the present system can be used to assess drug metabolic stability.
As can be seen from table 2, no significant reduction in the parent was found for compound testosterone at 120 minutes without the addition of a coenzyme; however, after the addition of NADPH coenzyme, the residual amount of 0.1 μ M testosterone was 69.6%, and the metabolism was 30.4%, and the data reached a statistically significant difference (p ═ 0.0045), and it could be judged that the compound could be metabolized in the lung of this species.
In conclusion, the in vitro method for evaluating the metabolic characteristics of the drug or the substance to be tested in the lung has stable effect, is an evaluation method aiming at pulmonary administration, can effectively evaluate the metabolic characteristics of the compound or the drug to be tested in the lung, and provides method support for drug interaction research or new drug research and development.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (23)
1. An in vitro evaluation method for pulmonary metabolism characteristics of a drug, comprising the steps of:
(1) preparing a lung subcellular tissue;
(2) respectively preparing a positive quality control system, a negative quality control system, a control group reaction system and a test group reaction system by adopting the lung subcellular tissue obtained in the step (1), incubating, culturing, and centrifuging to obtain a supernatant;
(3) respectively detecting the parent content of the positive substrate in the supernatant of the positive quality control system and the parent content of the parent of the negative quality control system in the step (2) and the parent content of the substance to be detected in the supernatants of the control group reaction system and the test group reaction system;
the positive substrate of the positive quality control system comprises any one or the combination of at least two of 2-aminofluorene, 4-methoxy-1,8-naphthalimide, phenacetin or 4-sweet potato scab diol.
2. The method of claim 1, wherein the positive substrate is 2-aminofluorene.
3. The method according to claim 2, wherein the final concentration of the 2-aminofluorene is 0.05-0.2 μ M.
4. The method according to claim 1, characterized in that step (1) is followed by a dilution step, in particular comprising: the dilution is that the protein concentration of the lung subcellular tissue is 0.5-4 mg/mL.
5. The method of claim 1, wherein the lung subcellular tissue comprises lung S9 or lung microsomes.
6. The method of claim 5, wherein the lung S9 protein mass concentration is 2-4 mg/mL.
7. The method according to claim 5, wherein the protein mass concentration of the lung microsomes is 0.5-2 mg/mL.
8. The method of claim 7, wherein the lung microsome has a protein mass concentration of 0.5-1 mg/mL.
9. The method of claim 1, wherein the positive control system of step (2) comprises a positive substrate, lung subcellular tissue, and an NADPH coenzyme.
10. The method according to claim 9, characterized in that the final concentration of NADPH coenzyme is 1-2 mM.
11. The method according to claim 10, characterized in that the final concentration of NADPH coenzyme is 1 mM.
12. The method of claim 1, wherein the negative quality control of step (2) comprises a positive substrate, lung subcellular tissue and Tris buffer.
13. The method of claim 1, wherein the control reaction system of step (2) comprises the analyte, the lung subcellular tissue and the Tris buffer.
14. The method of claim 1, wherein the test panel reaction system comprises the test agent, lung subcellular tissue, and an NADPH coenzyme.
15. The method of claim 14, wherein the final concentration of the test agent is 0.1-10 μ Μ.
16. The method of claim 1, wherein the incubation of step (2) is for a period of 60-240 min.
17. The method of claim 16, wherein the incubation of step (2) is performed for 120 min.
18. The method of any one of claims 9, 12, 13 or 14, wherein the NADPH-coenzyme or Tris buffer is added to the positive control system, the negative control system, the control reaction system and the experimental reaction system prior to the addition of the NADPH-coenzyme or Tris buffer.
19. The method of claim 18, wherein the pre-incubation time is 3-8 min.
20. The method of claim 19, wherein the pre-incubation time is 5 min.
21. The method according to claim 1, characterized in that it comprises in particular the steps of:
(1) preparing lung subcellular tissue, measuring the protein content, and diluting the lung subcellular tissue with 0.1mM Tris buffer solution until the concentration of the protein is 0.5-4 mg/mL;
(2) respectively preparing a positive quality control system, a negative quality control system, a control group reaction system and a test group reaction system by using the lung subcellular tissue obtained in the step (1), wherein the positive quality control system comprises a positive substrate and the lung subcellular tissue which are uniformly mixed and pre-incubated for 3-8min, then adding NADPH coenzyme and uniformly mixing, the final concentration of the positive substrate is 0.05-0.2 mu M, and the final concentration of the NADPH coenzyme is 1-2 mM; the negative quality control system comprises uniformly mixing a positive substrate and the lung subcellular tissue, pre-incubating for 3-8min, adding a Tris buffer solution, and uniformly mixing, wherein the final concentration of the positive substrate is 0.05-0.2 mu M; the control group reaction system comprises uniformly mixing the substance to be detected and the lung subcellular tissue, pre-incubating for 3-8min, adding Tris buffer solution, and uniformly mixing, wherein the final concentration of the substance to be detected is 0.1-10 mu M; the reaction system of the test group comprises uniformly mixing the substance to be tested and the lung subcellular tissue, pre-incubating for 3-8min, adding NADPH coenzyme, and uniformly mixing, wherein the final concentration of the NADPH coenzyme is 1-2mM, and the final concentration of the substance to be tested is 0.1-10 μ M; respectively incubating the positive quality control system, the negative quality control system, the control group reaction system and the test group reaction system for 60-240 min; adding precooled methanol after the incubation is finished, stopping metabolic reaction of each system, precipitating cell protein, mixing the sample evenly by vortex for 15-25s, centrifuging at 12000rpm for 3-8min, and taking supernatant;
(3) detecting the supernatant in the step (2) by using a liquid chromatography-mass spectrometer LC-MS/MS (liquid chromatography-mass spectrometer) relatively and quantitatively, and respectively detecting the parent content of the positive substrate in the supernatant of the positive quality control system and the parent content of the parent of the control group of the parent of the;
the positive substrate of the positive control comprises any one of or a combination of at least two of 2-aminofluorene, 4-methoxy-1,8-naphthalimide, phenacetin or 4-sweet potato scab diol.
22. The method according to claim 1, wherein the evaluation criteria of the method are: (1) the maternal content of the test substance has a significant difference after incubation compared with 0min, which indicates that the test substance can be metabolized in the lung; (2) the content of the parent body of the substance to be detected has no significant difference when being compared with 0min after incubation, which indicates that the substance to be detected is stable in the lung and is not metabolized; the significant difference was p < 0.05.
23. Use of a lung subcellular tissue positive substrate for assessing a metabolic characteristic of a drug in the lung, using the method of any of claims 1-22.
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