CN109298102A - A kind of in-vitro evaluation method of drug pulmonary metabolism feature - Google Patents
A kind of in-vitro evaluation method of drug pulmonary metabolism feature Download PDFInfo
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- CN109298102A CN109298102A CN201811466514.0A CN201811466514A CN109298102A CN 109298102 A CN109298102 A CN 109298102A CN 201811466514 A CN201811466514 A CN 201811466514A CN 109298102 A CN109298102 A CN 109298102A
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Abstract
The present invention provides a kind of in-vitro evaluation methods of drug pulmonary metabolism feature, and described method includes following steps: (1) preparing lung subcellular organization；(2) positive quality control system, negative quality control system, control group reaction system and test group reaction system are prepared using lung subcellular organization obtained by step (1) respectively, are incubated for culture, centrifuging and taking supernatant；(3) the parent content of the parent content and control group reaction system and the determinand in test group reaction system supernatant of difference detecting step (2) described positive quality control system and the positive substrate in negative quality control system supernatant；The positive substrate of the positive quality control system includes 2- aminofluorene, 4- methoxyl group -1,8-naphthalimide, any one of phenacetin or 4- isopropanol or at least two combination.Evaluation method effect stability of the invention is the perfect evaluation method for pulmonary administration, is that new drug development and drug interaction provider's science of law are supported.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of evaluation method of drug safety, and in particular to a kind of drug lung
The in-vitro evaluation method of the pulmonary metabolism feature of the in-vitro evaluation method more particularly to imbedibility drug of portion's metabolic characteristics.
Background technique
According to state food pharmaceuticals administration general bureau of the People's Republic of China (PRC) (CFDA), " guidance of drug interaction is former
Then " (2012) are pointed out: the metabolism of new drug should be determined in drug development process, mutual between the medicine and other drugs
Effect should be used as safety and a part of efficiency evaluation is studied.Since oral and intravenous (IV) drug or its metabolism produce
The elimination of object is usually to be metabolized in liver, therefore tissue selected by the study in vitro of drug metabolism at present is most
For liver and its subcellular organization.
With the continuous development of medicine, Inhalation in Treating is recommended as asthma, COPD etc. by the World Health Organization (WHO) and America and Europe
The preferred therapy of respiratory disease, pulmonary absorption surface area is big, capillary network is abundant so that pulmonary administration work compared with
Fastly, smaller by inhalation irritation, easy to use, good patient compliance is applicable to that the patient of long-term treatment need to be carried out.
Therefore, the novel imbedibility drug of research and development is the important channel for treating respiratory disease and part general action drug.
However, imbedibility preparation medicine avoids liver first-pass effect, directly acts on lung tissue by pulmonary administration, it is therefore, right
It carries out drug metabolism characteristics research and liver cell and its subcellular organization is selected not fully to be applicable in, at present to its metabolic characteristics and
The study limitation of drug interaction does not have in the Study on Molecular Mechanism of particular compound metabolic pathway, personalized research approach
There is universality, and the stability of method is poor, false positive and probability of false negative are high, the in vitro study for drug pulmonary metabolism feature
Still lack perfect technical solution.
(Aune T,et al.Deacetylation to 2-aminofluorene as a major initial
reaction in the microsomal metabolism of 2-acetylaminofluorene to mutagenic
products in preparations from rabbit lung and liver[J].Cancer Research,1985,
45 (11Pt2): it is different for the metabolic activity of 2-acetamidofluorene with 2- aminofluorene 5859.) to disclose rabbit lung microsome, and lung
Metabolic activity be higher than the metabolic activity of liver, indication some drugs may be metabolized more vigorous in lung, and such drug be adopted
It is then mostly inaccurate with hepatic metabolism evaluation criterion, it can not correctly assess the metabolic condition of drug in vivo.
Therefore it provides a kind of evaluation drug, the study in vitro of especially imbedibility drug pulmonary metabolism feature has
Significance.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provides a kind of the external of drug pulmonary metabolism feature
Evaluation method, specific selection positive substrate and its use concentration, establish the in-vitro evaluation method of pulmonary metabolism feature, raising is commented
The sensitivity and stability of valence method reduce false positive and probability of false negative in evaluation procedure, grind for Drug safety assessment
Study carefully the support of provider's science of law.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of in-vitro evaluation method of drug pulmonary metabolism feature, the method includes such as
Lower step:
(1) lung subcellular organization is prepared；
(2) positive quality control system, negative quality control system, control are prepared using lung subcellular organization obtained by step (1) respectively
Group reaction system and test group reaction system are incubated for culture, centrifuging and taking supernatant；
(3) difference detecting step (2) described positive quality control system and the positive substrate in negative quality control system supernatant
The parent content of parent content and control group reaction system and the determinand in test group reaction system supernatant；
The positive substrate of the positive quality control system includes 2- aminofluorene, 4- methoxyl group -1,8-naphthalimide, it is non-that
Any one of western fourth or 4- isopropanol or at least two combination.
The present invention is tested by lung subcellular organization, cooperation positive quality control system and negative quality control system, positive quality control system
Enzyme reaction system in card lung subcellular organization can work normally, and negative quality control system is for showing positive substrate in system not
There are non-enzymatic metabolism, illustrate that reaction system of the invention can be used for evaluating drug metabolism stability, while reducing false positive
With the probability of false negative；Control group reaction system and test group reaction system can identify drug or compound to be measured in the generation of lung
Thank to feature, for studying drug interaction, control group reaction system verifies the stability of determinand itself, excludes its self drop
Judgement of the solution to Metabolic products.It is wherein studied through inventor, selects 2- aminofluorene (2-aminofluorene), methoxyl group -1 4-,
8- naphthalimide (4-methoxy-1,8-naphthalimide), phenacetin (phenacetin) or 4- isopropanol (4-
Ipomeanol positive substrate) is more suitable for for evaluating drug pulmonary metabolism feature.
Preferably, the positive substrate is 2- aminofluorene.
In the present invention, positive quality control system is for detecting the whether normal work of enzyme reaction system in the lung subcellular organization
Make, it is most important to subsequent evaluation process.Enzyme system research in lung tissue is not perfect, and the type and content of enzyme include a variety of
Uncertainty, different genera differ greatly.Therefore selection has generality and the positive substrate of sensitivity is crucial.Inventor is logical
Cross a large number of studies show that, in lung the higher CYP450 enzyme system of expression quantity be CYP1A2 and CYP4B1, and different genera (people,
Monkey, mouse, dog) in expression activity it is more similar.Therefore positive bottom of the substrate of CYP1A2 and CYP4B1 as evaluation is paid the utmost attention to
Object.As a kind of appraisement system, when determining positive control probe substrate, many factors of worrying too much are integrated, the spy including (1) substrate
Anisotropic: whether the substrate is special to be metabolized by a certain metabolic enzyme；(2) sensibility: sensitivity of the enzyme to the substrate utilization；
(3) stability of compound: stability of the compound in bio-matrix；(4) detectability: substrate is when LC-MS-MS is detected
Detection limit.Although the substrate research of current CYP1A2 and CYP4B1 is more, most of positive substrate does not meet above want
It asks.
Such as since there are species variations for the enzyme system in people's lung and animal (such as rabbit), so some are suitable for the sun of animal
Property substrate be just not suitable for evaluating in people's lung s9, although also some positive substrates can act on people's lung and animal lung simultaneously,
But the positive substrate, there are Non-enzymatic Degradation, itself is with regard to unstable, it is impossible to be used in the metabolism of evaluation drug or compound in lung.
The present invention has found 2- aminofluorene to the high specificity of lung tissue, chemical stabilization, species difference are small, verifying by largely screening
Effect is good, can be used for positive quality control system, it is ensured that enzyme system can be used for evaluating drug or compound in lung in lung subcellular organization
In metabolism.
Preferably, final concentration of 0.05-0.2 μM of the 2- aminofluorene, such as can be 0.05 μM, 0.1 μM, 0.15 μM
Or 0.2 μM.
In the present invention, the concentration is the final concentration of 2- aminofluorene in the reaction system, and inventor is by adjusting method body
System and the mass concentration of positive substrate match, and discovery 2- aminofluorene effect in the concentration range is best.Due to lung tissue generation
Thank to expression of enzymes amount far below hepatic tissue, therefore its metabolic capability is relatively low；Therefore, as appraisement system, if positive substrate
Excessive concentration has exceeded the metabolic capability of enzyme, and will lead to can not judge enzyme working condition；If concentration of substrate is too low, detection
Error it is big, unstable result.
Preferably, dilution is further included the steps that after step (1), is specifically included: described to be diluted to be diluted to lung subcellular
The albumen quality concentration of tissue be 0.5-4mg/mL, such as can be 0.5mg/mL, 0.8mg/mL, 1mg/mL, 1.5mg/mL,
2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL or 4mg/mL.
Preferably, the lung subcellular organization includes lung S9 or lung microsome.
Preferably, the protein concentration of the lung S9 be 2-4mg/mL, such as can be 2mg/mL, 2.5mg/mL, 3mg/mL,
3.5mg/mL or 4mg/mL.
Preferably, the protein concentration of the lung microsome be 0.5-2mg/mL, such as can be 0.5mg/mL, 1mg/mL,
1.5mg/mL or 2mg/mL, preferably 0.5-1mg/mL.
In the present invention, effect is best in the lung subcellular organization albumen quality concentration range, sub- thin for different lungs
The optimum concentration of born of the same parents' tissue is different, and reaction system is sensitiveer when lung S9 is diluted to a protein concentration of 2-4mg/mL, and lung microsome
The reaction system high sensitivity in 0.5-2mg/mL, although protein concentration is excessively high to improve enzymatic activity, impurity content is also therewith
Increase, will cause non-enzyme system degradation, increase detection difficulty etc..
Preferably, step (2) the positive quality control system includes positive substrate, lung subcellular organization and NADPH coenzyme.
In the present invention, positive quality control system is led to for detecting whether enzyme reaction system in lung subcellular organization works normally
Cross the verifying of positive substrate, it was demonstrated that enzyme reaction system is normal.
Preferably, the final concentration of 1-2mM of the NADPH coenzyme, preferably 1mM.
In the present invention, the final concentration of NADPH coenzyme in the reaction system can effectively facilitate lung subcellular in the range
Whether enzyme and positive substrate reactions in tissue, verifying enzyme reaction system work normally.
Preferably, step (2) the negative Quality Control includes positive substrate, lung subcellular organization and Tris buffer.
In the present invention, by optimizing and revising the composition and proportion of negative quality control system, in-vitro evaluation side of the invention is improved
The accuracy of method excludes influence of the Non-enzymatic Degradation to reaction result, reduces false positive probability.
Preferably, the mass concentration of the Tris buffer is 0.1mM.
In the present invention, the mass concentration of Tris buffer is adapted to entire in-vitro evaluation method, can be used for matching for NADPH coenzyme
System adjusts the volume of negative Quality Control and control group reaction system.
Preferably, the time of step (2) described incubation be 60-240min, such as can be 60min, 90min, 120min,
150min180min, 210min or 240min, preferably 120min.
In the present invention, by positive quality control system, negative quality control system, control group reaction system and test group reaction system point
Fu Yu not be after 120min, comparison is incubated for front and back, and the parent content of positive substrate, sentences in positive quality control system, negative quality control system
Disconnected lung subcellular organization judges the whether normal work of enzyme reaction system in lung subcellular organization to the metabolic condition of positive substrate
Make；By comparing the parent content for being incubated for determinand in cross-reference group reaction system and test group reaction system, observation lung is sub-
Whether cell tissue has significant metabolism to determinand, and then judges lung to the metabolic characteristics of determinand.
Preferably, step (2) the control group reaction system includes determinand, lung subcellular organization and Tris buffer.
Preferably, the test group reaction system includes determinand, lung subcellular organization and NADPH coenzyme.
Preferably, final concentration of 0.1-10 μM in system of the determinand, such as can be 0.1 μM, 0.5 μM, 1 μ
M, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or 10 μM.
In the present invention, test group reaction system is for detecting lung subcellular organization to the metabolic condition of determinand.If should
Determinand is the substrate of metabolic enzyme in lung, then the enzyme system in lung subcellular organization acted in the presence of NADPH coenzyme to
Object is surveyed, the chemical structure of determinand is changed, to judge determinand in the metabolic characteristics of lung.
Preferably, described in positive quality control system, negative quality control system, control group reaction system and experimental group reaction system
NADPH coenzyme or Tris buffer include the steps that preincubate before being added.
It, need to be by system preincubate 3-8min, later after positive substrate or determinand is added in lung subcellular organization in the present invention
It adds corresponding NADPH coenzyme or Tris buffer is mixed.Situation in preincubate process analog body activates lung hypotype
Metabolism enzyme system in cell tissue obtains energy by the way that NADPH is reduced to NADP, to carry out metabolic activity, if not into
Row preincubate may be decreased the energy supply of reaction system, influence evaluation result.
Preferably, the time of the preincubate be 3-8min, such as can be 3min, 4min, 5min, 6min, 7min or
8min, preferably 5min.
Preferably, the method specifically comprises the following steps:
(1) lung subcellular organization is prepared, protein content is measured, is diluted lung subcellular organization with 0.1mM Tris buffer
Concentration to albumen is 0.5-4mg/mL；
(2) positive quality control system, negative quality control system, control are prepared using lung subcellular organization obtained by step (1) respectively
Group reaction system and test group reaction system, wherein positive quality control system includes mixing positive substrate and lung subcellular organization in advance
It is incubated for 3-8min, NADPH coenzyme is added and mixes, final concentration of 0.05-0.2 μM of the positive substrate, the end of NADPH coenzyme
Concentration is 1-2mM；Negative quality control system includes that positive substrate and lung subcellular organization are mixed preincubate 3-8min, is added
Tris buffer mixes, and final concentration of 0.05-0.2 μM of the positive substrate；Control group reaction system include by determinand and
Lung subcellular organization mixes preincubate 3-8min, adds Tris buffer and mixes, and final concentration of 0.1-10 μM of determinand；Examination
Testing a group reaction system includes that determinand and lung subcellular organization are mixed preincubate 3-8min, adds the mixing of NADPH coenzyme,
The final concentration of 1-2mM of NADPH coenzyme, final concentration of 0.1-10 μM of determinand；By positive quality control system, negative Quality Control body
System, control group reaction system and test group reaction system are incubated for 60-240min respectively；The methanol of pre-cooling is added after incubation,
The metabolic response of each system, and sedimentation cell albumen are terminated, after sample vortex is mixed 15-25s, 12000rpm is centrifuged 3-
8min takes supernatant；
(3) LC-MS instrument LC-MS/MS relative quantification detecting step (2) described supernatant is used, respectively detecting step (2)
The parent content and control group reaction system of the positive quality control system and the positive substrate in negative quality control system supernatant
With the parent content of the determinand in test group reaction system supernatant；
The positive substrate of the positive control includes 2- aminofluorene, 4- methoxyl group -1,8-naphthalimide, phenacetin
Any one of 4- isopropanol or at least two combination.
Preferably, the evaluation criterion of the method are as follows: (1) the parent content of determinand when 0min after incubation than having
Significant difference indicates that the determinand can be metabolized in lung；(2) the parent content of determinand after incubation than 0min when
There was no significant difference, indicates that the determinand is stablized in lung, is not metabolized；The significant difference is p < 0.05.
In the present invention, by positive quality control system and negative quality control system, enzyme system in the lung subcellular organization is verified
Whether work normally, and then determines whether the lung subcellular organization can be used for evaluating the metabolism of drug or determinand in lung
Feature, there were significant differences when positive substrate surplus ratio ratio 0min after being incubated in positive quality control system, and in feminine gender quality control system
Without significant difference when positive substrate surplus ratio ratio 0min after being incubated for, indicate that the work of enzyme reaction system is just in lung subcellular organization
Often；Metabolic condition of the determinand in lung tissue is judged by comparing the parent surplus for being incubated for front and back determinand, if after being incubated for
The parent content of determinand significantly reduces, and shows that the determinand can be metabolized by lung, if after being incubated for the parent content of determinand with
No significant difference before being incubated for, indicates that the determinand is more stable in lung.
Second aspect, the present invention provide it is a kind of using lung subcellular organization positive substrate for evaluating drug in pulmonary metabolism
The application of feature, the application is using method as described in relation to the first aspect.
Preferably, the positive substrate includes 2- aminofluorene.
Compared with prior art, the invention has the following beneficial effects:
(1) present invention provides the in-vitro method of the metabolic characteristics of one kind perfect evaluation drug or determinand in lung, this
The evaluation method effect stability of invention is the preferred evaluation method for pulmonary administration；It is directly administered for lung, or with lung
Important research method is provided for the repercussion study of the drug of target spot.
(2) in-vitro evaluation method of the invention reduces false positive or probability of false negative by perfect quality control system, improves
The accuracy of method；
(3) method specific selection positive substrate of the invention and its use concentration, cooperate entire evaluation method so that this
The in-vitro evaluation method universality of invention is high, is suitable for detecting various determinands, judges its metabolic characteristics in lung；
(4) in-vitro evaluation method provided by the invention is simple, uses manpower and material resources sparingly, convenient for promoting.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below by way of specific embodiment come into
One step illustrates technical solution of the present invention, but the present invention is not limited in scope of embodiments.
Experimental material
(1) prepare 0.15M KCl solution: 11.18g KCl is dissolved in 1L water up to 0.15M KCl solution；
(2) 1M Tris-Acetate:121.14g Tris is prepared to be dissolved in 1L water up to 1M Tris solution；
(3) prepare 1M KCl solution: 74.56g KCl is dissolved in 1L water up to 1M KCl solution；
(4) prepare 100mM EDTA solution: 37.2g EDTA is dissolved in 1L water up to 100mM EDTA solution；
(5) 20mM BHT Stock, 44.1mg Butylated Sodium Hydroxytoluene are prepared and is dissolved in 10mL
Water is up to the preservation of 20mM BHT Stock-20 degree；
(6) it prepares buffer solution A liquid: taking 100Ml 1M Tris-Acetate liquid that the KCl solution of 100ml 1M is added to add 10mL
100mM EDTA solution is settled to 1L.To obtain the final product；Note: buffer solution A liquid adds 1uL 20mM BHT solution using preceding 1mL buffer solution A；
(7) it prepares buffer C liquid: taking 10ml 1M Tris-Acetate liquid that 10ml 100mM EDTA solution is added to add 200ml
Glycerol is settled to 1L.PH is adjusted to 7.4,4 degree of preservations；
The material used in following embodiment be not limited to it is above-mentioned enumerate, can be substituted with other same type of material, instrument is not specified
The routinely condition or operation instructions of actual conditions operate, and those skilled in the art should grasp using conventional material and instrument
Relevant knowledge.
The preparation of 1 animal lung subcellular organization of embodiment
Animal overnight fasting, with isoflurane or 3% amobarbital sodium water solution 30mg/kg intraperitoneal injection of anesthesia, operating scissors
Knife opens abdominal cavity and thoracic cavity, haemostatic clamp clamp abdominal aorta, cuts off heart left ventricle, is inserted into the chlorine with physiological saline or 0.15M
The 50mL syringe (solution pre-cooling) for changing potassium solution is rapidly injected solution flushing, until lung is creamy white, takes lung tissue in cold
In potassium chloride (or 0.9% sodium chloride) solution beaker of 0.15M, in solid-liquid volume ratio 2:3 ratio soaking and washing 2-3 times, use
After buffer solution A cleans 2 times again, takes clean lung tissue and buffer solution A to go in centrifuge tube with solid-liquid volume ratio 2:3, cut
It is broken, it is homogenized with high-speed homogenization machine, collects homogenate in centrifuge tube.Centrifuge is cooled to 4 degree in advance, and the lung homogenate liquid of collection is placed in
In centrifuge, 30min is centrifuged with 9000G, is filtered with double gauze, obtained filtrate is lung S9.Obtained S9 is gone to super
In high speed centrifugation pipe, after buffer solution A liquid balance, with 100000G centrifugation 1 hour, the control of centrifuge temperature was 4 degree.It has been centrifuged
Cheng Hou, removal supernatant obtain lung microsome；Buffer C liquid is added with 1:1, is transferred in new centrifuge tube, obtains after uniform
Liquid assay protein content after, can freeze spare.
In-vitro evaluation method of 2 testosterone of embodiment in pulmonary metabolism feature
(1) lung S9 is diluted to 3mg/mL with Tris buffer after 37 DEG C of water-baths are thawed by beasle dog lung S9；
(2) positive quality control system, negative quality control system, control group reaction system and test group reaction system are prepared respectively,
Wherein positive quality control system includes that 2- aminofluorene and lung S9 are mixed preincubate 5min, NADPH coenzyme is added, according to 2- amino
Fluorenes, lung S9 and NADPH coenzyme are respectively that the volume mixture of 25 μ L, 25 μ L and 50 μ L are uniform, the 2- aminofluorene it is final concentration of
0.05 μM, the protein content of the final concentration of 1mM of NADPH coenzyme, lung S9 are 3mg/mL；Negative quality control system includes by 2- amino
Fluorenes and lung S9 mix preincubate 5min, the Tris buffer of 0.1mM are added, according to 2- aminofluorene, lung S9 and Tris buffer
The volume ratio of respectively 25 μ L, 25 μ L and 50 μ L are uniformly mixed, and final concentration of 0.05 μM of the 2- aminofluorene, the albumen of lung S9
Content is 3mg/mL；Control group reaction system includes that testosterone and lung S9 are mixed preincubate 5min, and the Tris for adding 0.1mM is slow
Fliud flushing is uniformly mixed, the end of testosterone according to the volume ratio that testosterone, lung S9 and Tris buffer are respectively 25 μ L, 25 μ L and 50 μ L
Concentration is 0.1 μM, and the protein content of lung S9 is 3mg/mL；Test group reaction system includes that testosterone and lung S9 are mixed preincubate
5min adds NADPH coenzyme, and the volume ratio for being respectively 25 μ L, 25 μ L and 50 μ L according to testosterone, lung S9 and NADPH coenzyme is mixed
It closes uniformly, the final concentration of 1mM of NADPH coenzyme, final concentration of 0.1 μM of testosterone, the protein content of lung S9 is 3mg/mL；It will be positive
Property quality control system, negative quality control system, control group reaction system and test group reaction system 48 orifice plates are added, respectively in incubator
37 DEG C, 5%CO2Under the conditions of be incubated for 120min, each system 3 are parallel, record when 0min 2- aminofluorene or testosterone in each system
Parent content；
(3) methanol of pre-cooling is added after being incubated for, the metabolic response of each system, and sedimentation cell albumen are terminated, by sample
After this vortex mixes 20s, 12000rpm is centrifuged 5min, takes supernatant；
(4) with 2- aminofluorene or testosterone in LC-MS instrument LC-MS/MS relative quantification detecting step (3) described supernatant
Parent content and calculate corresponding parent surplus ratio, the LC-MS/MS testing result of four systems is shown in Tables 1 and 2.
1 positive quality control system of table and negative quality control system testing result
2 control group reaction system of table and test group reaction system testing result
As shown in table 1, when not adding coenzyme NADP 11,2- aminofluorene did not show that metabolism is reduced at 120 minutes；
However, 2- aminofluorene, which is shown, reduces 16%, and data have reached the difference (p=of statistical significance after adding NAPDH coenzyme
0.0263), illustrate that this system can be used for evaluating drug metabolism stability.
As shown in Table 2, for compound testosterone, when not adding coenzyme, which did not sent out in 120 minutes parents
Now significantly reduce；But after adding NADPH coenzyme, 0.1 μM of testosterone surplus is 69.6%, and metabolism 30.4%, data reach
The significant difference (p=0.0045) of statistical significance, it can be determined that the compound can be metabolized in the kind lung.
To sum up, the in-vitro method effect stability of metabolic characteristics of the evaluation drug or determinand that the present invention mentions in lung is
For the evaluation method of pulmonary administration, energy effective evaluation untested compound or drug are drug phase interaction in pulmonary metabolism feature
It is supported with research or new drug development providing method.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of in-vitro evaluation method of drug pulmonary metabolism feature, which is characterized in that described method includes following steps:
(1) lung subcellular organization is prepared；
(2) lung subcellular organization obtained by step (1) is used to prepare positive quality control system, negative quality control system, control group respectively anti-
System and test group reaction system are answered, culture, centrifuging and taking supernatant are incubated for；
(3) parent of difference detecting step (2) described positive quality control system and the positive substrate in negative quality control system supernatant
The parent content of content and control group reaction system and the determinand in test group reaction system supernatant；
The positive substrate of the positive quality control system includes 2- aminofluorene, 4- methoxyl group -1,8-naphthalimide, phenacetin
Any one of 4- isopropanol or at least two combination.
2. the method according to claim 1, wherein the positive substrate is 2- aminofluorene.
3. method according to claim 1 or 2, which is characterized in that final concentration of 0.05-0.2 μM of the 2- aminofluorene.
4. method according to any one of claim 1-3, which is characterized in that further include diluted step after step (1)
Suddenly, specifically include: the protein concentration for being diluted to be diluted to lung subcellular organization is 0.5-4mg/mL；
Preferably, the lung subcellular organization includes lung S9 or lung microsome；
Preferably, the albumen quality concentration of the lung S9 is 2-4mg/mL；
Preferably, the albumen quality concentration of the lung microsome is 0.5-2mg/mL, preferably 0.5-1mg/mL.
5. method according to any of claims 1-4, which is characterized in that step (2) the positive quality control system packet
Include positive substrate, lung subcellular organization and NADPH coenzyme；
Preferably, the final concentration of 1-2mM of the NADPH coenzyme, preferably 1mM；
Preferably, step (2) the negative Quality Control includes positive substrate, lung subcellular organization and Tris buffer.
6. method according to any one of claims 1-5, which is characterized in that step (2) the control group reaction system
Including determinand, lung subcellular organization and Tris buffer；
Preferably, the test group reaction system includes determinand, lung subcellular organization and NADPH coenzyme；
Preferably, final concentration of 0.1-10 μM of the determinand；
Preferably, the time of step (2) described incubation is 60-240min, preferably 120min.
7. method according to claim 1 to 6, which is characterized in that positive quality control system, negative quality control system,
Step including preincubate before NADPH coenzyme described in control group reaction system and experimental group reaction system or Tris buffer are added
Suddenly；
Preferably, the time of the preincubate is 3-8min, preferably 5min.
8. method according to any one of claims 1-7, which is characterized in that the method specifically comprises the following steps:
(1) lung subcellular organization is prepared, protein content is measured, lung subcellular organization is diluted to egg with 0.1mM Tris buffer
White concentration is 0.5-4mg/mL；
(2) lung subcellular organization obtained by step (1) is used to prepare positive quality control system, negative quality control system, control group respectively anti-
System and test group reaction system are answered, wherein positive quality control system includes that positive substrate and lung subcellular organization are mixed preincubate
3-8min adds NADPH coenzyme and mixes, and final concentration of 0.05-0.2 μM of the positive substrate, the final concentration of NADPH coenzyme
For 1-2mM；Negative quality control system includes that positive substrate and lung subcellular organization are mixed preincubate 3-8min, and it is slow to add Tris
Fliud flushing mixes, and final concentration of 0.05-0.2 μM of the positive substrate；Control group reaction system includes that determinand and lung is sub- thin
Born of the same parents organize to mix preincubate 3-8min, add Tris buffer and mix, and final concentration of 0.1-10 μM of determinand；Test group is anti-
Answering system includes that determinand and lung subcellular organization are mixed preincubate 3-8min, adds the mixing of NADPH coenzyme, and NADPH is auxiliary
The final concentration of 1-2mM of enzyme, final concentration of 0.1-10 μM of determinand；By positive quality control system, negative quality control system, control group
Reaction system and test group reaction system are incubated for 60-240min respectively；The methanol of pre-cooling is added after incubation, terminates each system
Metabolic response, and sedimentation cell albumen, after sample vortex is mixed 15-25s, 12000rpm is centrifuged 3-8min, takes supernatant；
(3) LC-MS instrument LC-MS/MS relative quantification detecting step (2) described supernatant is used, detecting step (2) are described respectively
The parent content and control group reaction system and examination of positive quality control system and the positive substrate in negative quality control system supernatant
Test the parent content of the determinand in group reaction system supernatant；
The positive substrate of the positive control includes 2- aminofluorene, 4- methoxyl group -1,8-naphthalimide, phenacetin or 4-
Any one of isopropanol or at least two combination.
9. method according to claim 1 to 8, which is characterized in that the evaluation criterion of the method are as follows: (1) to
Survey object parent content after incubation than 0min when have significant difference, indicate that the determinand can be metabolized in lung；
(2) the parent content of determinand after incubation than 0min when there was no significant difference, indicate the determinand lung stablize, not by
Metabolism；The significant difference is p < 0.05.
10. a kind of be used to evaluate drug in the application of pulmonary metabolism feature using lung subcellular organization positive substrate, feature exists
In the application uses such as the described in any item methods of claim 1-9；
Preferably, the positive substrate includes 2- aminofluorene.
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