CN103336128A - Detection method of tissue distribution of small molecule polypeptide drugs - Google Patents
Detection method of tissue distribution of small molecule polypeptide drugs Download PDFInfo
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- CN103336128A CN103336128A CN2013102295915A CN201310229591A CN103336128A CN 103336128 A CN103336128 A CN 103336128A CN 2013102295915 A CN2013102295915 A CN 2013102295915A CN 201310229591 A CN201310229591 A CN 201310229591A CN 103336128 A CN103336128 A CN 103336128A
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Abstract
The invention provides a detection method of tissue distribution of small molecule polypeptide drugs. The detection method comprises the following steps of: preparing homogenate extracting solutions of different organs/tissues of an experimental animal by adopting key technologies such as cell lysate extraction and liquid nitrogen frozen storage; according to the properties and characteristics of drugs to be detected, taking an immunological assay determination technology, namely a radioimmunoassay (RIA) method or an enzyme-linked immunoassay (ELISA) method, as a major quantitative assay means.
Description
Technical field
The invention belongs to the life science technical field, relate to a kind of method that detects micromolecule polypeptide class medicine Tissue distribution, specifically, relate to a kind of cell pyrolysis liquid extraction, liquid nitrogen cryopreservation and immune analysis determination techniques are combined and detect the method for micromolecule polypeptide class medicine Tissue distribution.
Background technology
In recent years along with genomics, protein science and development of biology, various micromolecule polypeptide class medicines with endogenous activated protein or peptide matters structure, functional similarity or function uniqueness continue to bring out, the each side such as prevention, diagnosis and treatment that relate to disease especially have significant curative effect at numerous areas such as cancer, autoimmune disease, the disease treatments of metabolism class.Compare with traditional little molecular chemistry medicine, advantage such as micromolecule polypeptide class medicine biologically active is strong, dosage is little, but based on the molecular structure characteristics, also have poor stability, easily degraded by the body endoproteinase, the half life period is short, diffusivity is poor, the little deficiency that waits of partition factor.It is current research focus that micromolecule polypeptide class medicine and novel medicine-releasing system such as microballoon, micro-capsule, nanoparticle, liposome, emulsion etc. are combined.The interior system of the body delivery technique difficult point that had both solved micromolecule polypeptide class drug half-life weak point, needed long-term frequent drug administration by injection, structural instability, body intrinsic permeability difference etc. to cause also can utilize the drug administration carrier realization to discharge targeting and the slow release of medicine at particular organization and organ.
The Tissue distribution of research micromolecule polypeptide class medicine, the distribution situation of different time medicine in multiple tissue was significant after namely monitoring gave medicine.Aspect pharmacodynamic study, can understand medicine site of action in vivo, provide foundation for exploring possible target organ, target tissue and the mechanism of action; Aspect toxicologic study, in vivo the degree of accumulating and toxicity target organ, target tissue after can understanding overdose and giving, the toxic reaction that prediction may occur is for the explaination medicine poisoning target organ that institute's toxigenicity reacts in the repeat administration toxicity research provides foundation.Especially for the medicine of targeting, carry out the targeting that Tissue distribution research also helps to estimate and check its preparation process, level and effect.
At present, carry out morely at the Tissue distribution research of chemicals and high molecular weight protein, method is also ripe relatively, and detects at the Tissue distribution of micromolecule polypeptide class medicine, does not still have comparatively maturation and the strong method of universality at present.Because micromolecule polypeptide class medicine stability is poor, easily degraded by the body endoproteinase, and immunogenicity is low, therefore the preparation method to tissue extract has higher requirement.Based on the medicine characteristics, method should guarantee to extract to greatest extent the medicine in the tissue on the basis that keeps original biologic activity or immunologic competence, organize the requirement of Chinese traditional medicine content to satisfy quantitative measurement.
At micromolecule polypeptide class medicine biologically active height, dosage is little, blood concentration is low, technological difficulties such as endogenous material interference, how setting up sensitivity, special, the fast and convenient multiple tissue of quantitative detection, the method for organ Chinese traditional medicine concentration also is the difficult point place of micromolecule polypeptide class medicine Tissue distribution research.Immune analysis (radio-immunity and enzyme linked immunological) determination techniques is one of main means of micromolecule polypeptide class medicine biological sample analysis, have sensitivity, fast, be suitable for characteristics such as batch processing.Its ultimate principle is based on medicine has selectivity and saturability when corresponding antibodies is combined characteristics, utilize that tested medicine is emulative between enzyme or labelled with radioisotope medicine is combined with antibody, and tested medicine principle such as is combined with the enzymic-labelled antibody specificity, adopts the quantitative measurement technology of means such as radioactive isotope counting method or colourimetry.Therefore, the immune analysis technology is that research micromolecule polypeptide class medicine tissue target is to the comparatively suitable method that distributes.
Summary of the invention
Relate to a kind of method that detects micromolecule polypeptide class medicine Tissue distribution, specifically, relate to a kind of cell pyrolysis liquid extraction, liquid nitrogen cryopreservation and immune analysis determination techniques are combined and detect the method for micromolecule polypeptide class medicine Tissue distribution.
Therefore, the purpose of this invention is to provide a kind of method that detects micromolecule polypeptide class medicine Tissue distribution, this method organically combines cell pyrolysis liquid extraction, liquid nitrogen cryopreservation and immune analysis determination techniques, successfully applies to detect in the micromolecule polypeptide class medicine Tissue distribution.Experiment shows, this is the tissue extraction liquid and preparation method thereof of gordian technique with cell pyrolysis liquid extraction, liquid nitrogen cryopreservation, be the method for the detection micromolecule polypeptide class medicine Tissue distribution of main quantitative means with the immunology determination techniques, can successfully be applied to detect in the Tissue distribution of micromolecule polypeptide class medicines such as injection triptorelin acetate microballoon.
Method for mensuration micromolecule polypeptide class medicine Tissue distribution provided by the invention may further comprise the steps:
1. tissue extract preparation: animal used as test is particular point in time enforcement euthanasia before administration or after the administration, pharmacologically active and mechanism of action according to tested medicine, choose multiple tissue/internal organs of animal, after one's own heart, liver, spleen, lung, kidney, stomach, intestines, thymus gland, muscle, brain, fat, ovary, uterus, testis, prostate etc., behind the residual blood of ice bath 0.9% normal saline flushing tissue surface, use the filter paper suck dry moisture, weigh, in the tapping nitrogen frozen 2 hours.Take out the back and add the cracking of certain proportion cell pyrolysis liquid, homogenate under ice bath, centrifugal, get supernatant, freezing preservation is to be measured.
2. immunologic assay technology: according to the character of tested medicine, can adopt radiommunoassay (RIA) method or enzyme-linked immuno assay (ELISA) method.
The RIA method:
(1) adopts the labelled with radioisotope micromolecule polypeptide; Radioactive isotope is selected from
125I, 99mTc,
3H,
14C or
35S is preferably
125I, labeling method is chloramine-t method or Iodogen method.Behind the separation and purification labelled antigen, investigate its radiochemical purity, radioactivity, biologically active and immunocompetence etc.
(2) blank tissue homogenate extracts such as the heart for preparing with step 1, liver, kidney, muscle, brain, and blank serum is mixed with tested medicine the standard solution of a series of concentration known.
(3) the tested medicine with a certain amount of labelled with radioisotope is added in standard solution or the tested medicine tissue extract, makes itself and a certain amount of competing property of specific antibody immunity association reaction.Tested content of medicines is certain functional relation in the tested medicine of labelled with radioisotope and the binding capacity of antibody and standard solution or the sample tissue extract.
(4) with immunity separate (PR) reagent with the Ag-Ab bound fraction with after free antigen partly separates, measure the radioactive intensity (B of Ag-Ab bound fraction, usually represent with radiocounting cpm), and/or the radioactive intensity (F) of free antigen part, and the preparation standard curve.Wherein ordinate is the radioaction parameter, comprises B/B
0(zero standard pipe), B/(B+F), F/B, B/F etc.; Horizontal ordinate is the concentration of step (2) standard solution.Automatically get tested drug concentrations in the excellent method calculation sample of degree of fitting after the match.
The ELISA method:
(1) adopts enzyme labeling micromolecule polypeptide class medicine (antigen) or specific antibody, enzyme commonly used comprises horseradish peroxidase (horseradish peroxidase, HRP), alkaline phosphatase (alkalinephosohatase, AP) and glucose oxidase (glucose oxidase, GOX) etc., wherein be most widely used with HRP.
(2) blank tissue homogenate extracts such as the heart for preparing with step 1, liver, kidney, muscle, brain, and blank serum is mixed with tested medicine the standard solution of a series of concentration known.
(3) double antibody sandwich method: by the tissue extract of 96 microwell plates of insolubilized antibody adding standard solution or tested medicine, form solid phase antigen-antibody complex, unconjugated other material of flush away behind the reaction certain hour at pre-bag.The enzyme-labeled secondary antibody that adds step (1) preparation makes it be combined with antigen behind the reaction certain hour, and the unconjugated enzyme labeling two of flush away resists, and adds chromogenic enzyme substrate, stops enzyme reaction with stop buffer at last.Adopt colorimetric method for determining absorbance (with the OD value representation).The OD value is directly proportional with the concentration of tested antigen.Competition law: wrapping the tissue extract that is added standard solution or tested medicine by 96 microwell plates of insolubilized antibody in advance, and the enzyme-labelled antigen of preparation in the step (1), the two and antibody competition specific binding site.Behind the reaction certain hour, unconjugated other material of flush away adds chromogenic enzyme substrate, stops enzyme reaction with stop buffer at last.Adopt colorimetric method for determining absorbance (with the OD value representation).The concentration of OD value and tested antigen is inversely proportional to.
(4) according to the distinct methods requirement, choose tested drug concentrations in the excellent method calculation sample of degree of fitting.
3. the Tissue distribution of medicine: according to the measured concentration of step 2, the contained tested medication amount of unit of account quality organization as follows: (tested drug concentrations * cell pyrolysis liquid volume)/tested tissue quality * 100%.By measuring the concentration of different time organ-/ tissue Chinese traditional medicine, the Tissue distribution that can get medicine is variation tendency in time.
At present, at the early-stage for the Tissue distribution research of micromolecule polypeptide class medicine, the pertinent literature report is less both at home and abroad, does not still have ripe and strong method and the technology of universality.The principle of micromolecule polypeptide class medicine Tissue distribution research is to keep on its original biologic activity or the immunocompetent basis, extracts the medicine in tissue, the organ to greatest extent.Molecular characterization based on this type of medicine, we optimize the preparation method of tissue extract repeatedly, at first investigated the broken histocyte of 3 kinds of distinct methods such as cell pyrolysis liquid, ultrasonic, 4 ℃ of soaked overnight, preparation homogenate to detect the extraction efficiency of tested medicine, the result is with the tissue homogenate extraction effect optimum of cell pyrolysis liquid in conjunction with liquid nitrogen cryopreservation; Secondly also means such as centrifugal condition are explored.Along with improving constantly of labelling technique and Antibody Preparation level, the immunologic assay technical development is swift and violent, can realize the quantitative measurement research of multiple micromolecule polypeptide class medicine.The bio-matrix of its mensuration mainly still is blood plasma, serum and cell culture fluid etc. at present.Characteristics and ultimate principle at the immunologic assay technology, we are by repeatedly practising, success is applied to it in Tissue distribution research field, and first it is combined with the extraction of cellular lysate liquid and liquid nitrogen cryopreservation method, set up at the strong Tissue distribution detection method of the universality of micromolecule polypeptide class medicine.
In sum, the method that the present invention combines cell pyrolysis liquid extraction, liquid nitrogen cryopreservation and the immune analysis determination techniques of setting up first is applied to detect micromolecule polypeptide class medicine Tissue distribution research field, filled up the blank that such medicine Tissue distribution detects, perfect experimental technique and the method for pharmacokinetic.The present invention is quick, sensitive, high specificity, is applicable to the Tissue distribution that detects micromolecule polypeptide class medicine especially.
Description of drawings
Below, describe embodiment of the present invention by reference to the accompanying drawings in detail, wherein:
Fig. 1 is the typical curve (the blank tissue of liver) of triptorelin acetate;
Fig. 2 gives average triptorelin acetate content in the different tissues of (i.m.) injection triptorelin acetate microballoon (0.34mg/kg) back for rat muscle
N=2);
The behave typical curve (liver blank tissue) of reorganization thymic peptide of Fig. 3;
Fig. 4 is that rat skin lower injection (s.c.) people recombinates after the thymic peptide (rh-T α 1) (300 μ g/kg), average rh-T α 1 content in the different tissues.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail, the embodiment that provides is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.
The method that cell pyrolysis liquid extraction, liquid nitrogen cryopreservation and radiommunoassay (RIA) method is combined that present embodiment utilizes the present invention to set up, the Tissue distribution of having carried out injection triptorelin acetate microballoon rat detects research.Concrete detection method is as follows:
1. injection triptorelin acetate microballoon is provided by Changchun Genesci Pharmaceuticals Co., Ltd..Specification: 3.75mg/ props up, and includes freeze-dried powder (28 days slowly-releasings) and dedicated solvent (2mL).Lot number is respectively freeze-dried powder 20091104, valid until 20111103; Dedicated solvent 20090601 is valid until 20110531.Store below 25 ℃.Adopt chloramine-t method (Ch-T) mark triptorelin acetate, preparation
125The triptorelin acetate dried frozen aquatic products of I mark.
Adopt chloramine-t method (Ch-T method) mark triptorelin acetate, preparation
125The triptorelin acetate dried frozen aquatic products of I mark.Triptorelin acetate is produced by Changchun Genesci Pharmaceuticals Co., Ltd., and lot number is 20070206, and sample is white powder, purity＞98%, and sample is in 4 ℃ of preservations before the test.Respectively with 20 μ L(0.25 μ g/ μ L) triptorelin acetate, 5 μ L(2mci) Na
125I and 20 μ L(0.4 μ g/ μ L) Ch-T adds in the phosphate buffer of 80 μ L0.05M pH7.4, behind the reaction 2min, adds 20 μ L(0.8 μ g/ μ L immediately) Sodium Metabisulfite (Na
2S
2O
5) cessation reaction.The separation and purification reaction mixture obtains on the post of C18(40 * 0.39cm)
125The I-label, flowing is the A phase mutually: 0.1% trifluoroacetic acid aqueous solution (TFA), the B phase: 80% acetonitrile water gradient elution (flow velocity 1mL/min), program is as follows: 0-10min20%B phase, 10-20min30%B phase, 20-30min40%B phase, 30-40min50%B phase.Collect single iodine labeling
125I-triptorelin acetate peak.Measuring its emission ratio activity is 560mCi/mg(20720MBq/mg), radiochemicsl purity is 98.4%.In the label of collecting, add the 0.05M pH7.4 phosphate buffer freeze-drying that contains 1% bovine serum albumin(BSA) (BSA), standby in-20 ℃ of preservations.
2. present embodiment adopts 16 SD rats, SPF level, body weight 250-270g, male and female half and half.Science and Technology Ltd. provides by the red animal used as test in mountains and rivers, Tianjin.Production unit licence numbering: SCXK(Tianjin) 2009-0001.Animal quality conformity certification: 0002971.Except 2 preparations that are used for organizing before the administration, 14 intramuscular injection (i.m.) 0.34mg/kg injection triptorelin acetate microballoon wherein.Respectively at 0.5h after the administration, 1h, 4h, 24h, 72h, 336h and 672h ether are slightly anaesthetized, and the vena ophthalmica clump is implemented euthanasia after getting blood, wins the different organs tissue, and each time point is put to death 2 animals (1 female 1 hero).
3. get the tissues such as liver, kidney, brain, testis, ovary, prostate, muscle of rat, with behind the residual blood of normal saline flushing tissue surface of ice bath, use the filter paper suck dry moisture, weigh frozen 2h in the tapping nitrogen.Add cell pyrolysis liquid (3mL/0.5g tissue) cracking, homogenate under ice bath, centrifugal 5000rpm, 20min gets its supernatant, and-20 ℃ of preservations are until mensuration.
4. adopt the RIA method to measure triptorelin acetate concentration in the Rats Organs and Tissues tissue.Measuring principle is as follows: use homogeneous phase competition principle, the triptorelin acetate in standard items or the sample and adding
125Common and a certain amount of competing property of specific antibody immunity of I-triptorelin acetate association reaction.
125The content of triptorelin acetate is certain funtcional relationship in the binding capacity of I-triptorelin acetate and antibody and standard items or the sample.With immune separation agent (PR) with bound fraction (B) with after free fraction (F) separates, measure the radioactive intensity of bound fraction, and calculate corresponding to rate B/B
0Adopt bundled software in the GAMMA-C12 calculating instrument machine, get the content that the excellent method of degree of fitting is calculated triptorelin acetate in the testing sample after the match automatically.
5. pick and place the pipe numbering of being excused from an examination, operate by step shown in the table 1, record at the γ calculating instrument at last and respectively manage radiocounting, calculate each and manage the ratio B/B that counts with zero standard pipe counting
0(%).The calculating of sample concentration: can be by logC and logit (B/B
0) linear fit asks the calculation sample concentration; Also the calculation sample concentration is asked in available quadratic fit, function fundamental equation Y=(D-C)/[1+ (X/A) B]+C, wherein X is testing sample concentration, Y is the cpm value of sample determination, A, B, C, D are that (A is the sample concentration of 50% zero standard pipe cpm correspondence for four parameters of function, B is slope of standard curve, and C, D are respectively the estimated values of asymptote and upper asymptote under the anti-S type curve.The subsidiary software of γ calculating instrument is adopted in this test, gets the excellent method of degree of fitting after the match automatically and calculates the result.
Table 1.RIA method is measured the operation steps of Triptorelin
6. prepare the triptorelin acetate standard solution of variable concentrations respectively with different blank tissue homogenate extracts such as blank serum, liver, kidney, brain, testis, ovary, prostate, muscle, make that its final concentration is respectively 10,30,100,300,1000,3000pg/mL, measure the cpm value of each concentration, make typical curve.Calculate the content of triptorelin acetate in respective organization/organ respectively with each typical curve.Present embodiment is the example explanation with LH liquid.
Get blank liver tissues of rats, with behind the residual blood of normal saline flushing tissue surface of ice bath, use the filter paper suck dry moisture, weigh frozen 2h in the tapping nitrogen.Add cell pyrolysis liquid (3mL/0.5g tissue) cracking, homogenate under ice bath, centrifugal 5000rpm, 20min, get the triptorelin acetate standard solution of its supernatant preparation variable concentrations, make that its final concentration is respectively 10,30,100,300,1000,3000pg/mL, measure the cpm value of each concentration, make typical curve (see Table 2 and accompanying drawing 1).In this concentration range (10～3000pg/mL), drug concentration logarithm and B/B
0Be the Logistic linear dependence.
The typical curve of table 2. triptorelin acetate (the blank tissue homogenate extract preparation of liver)
7. after rat muscle gave injection triptorelin acetate microballoon (0.34mg/kg), the content of triptorelin acetate saw Table 3 during different time points was respectively organized behind the medicine, and the Tissue distribution of medicine variation tendency is in time seen accompanying drawing 2.
The content of average Triptorelin in the different tissues of table 3. rat i.m. triptorelin acetate microballoon (0.34mg/kg) back (the pg/g tissue,
N=2)
a: n=1;
b: concentration is pg/mL
Embodiment 2
The method that cell pyrolysis liquid extraction, liquid nitrogen cryopreservation and enzyme-linked immunosorbent assay (ELISA) are combined that present embodiment utilizes the present invention to set up has been carried out the Tissue distribution research of recombination human thymus peptide beta 4 α 1 rat.Concrete research method is as follows:
1. recombination human thymus peptide beta 4 α 1(rh-T α 1), lot number is provided by Shenzhen Haiwang Pharmaceutical Co., Ltd: 20050301, sample is white freeze-dried powder, purity 98.51%, content: 1.5mg/ bottle; Sample is in 4 ℃ of preservations before the test.
2.ELISA detect rh-T α 1 kit, 2～8 ℃ of preservations be provided by the Beijing Wanger Bioisystech Co., Ltd.550 type enzymes mark microplate reader (Model550Micro-plate reader) is U.S. Bio-red company product.
3. experimental animal: the Wistar rat, 24, the male and female dual-purpose, body weight 180 ± 10 grams are provided by the department of the Chinese Academy of Sciences of Department Of Medicine, Peking University animal used as test section, and animal produces conformity certification: the SCXK(capital) 2002-0001.Rat s.c.rh-T α 1300 μ g/kg(administration volumes are 1.0mL/kg), after administration 0.25,1,4 and 12h put to death animal, get internal organs/tissues such as brain, liver, muscle, the heart, spleen, lung, liver, kidney, thymus gland and uterus, each time point is got 6 animals, male and female half and half.Behind the residual blood of normal saline flushing tissue surface with ice bath, use the filter paper suck dry moisture, weigh frozen 2h in the tapping nitrogen.Add cell pyrolysis liquid (2.5mL/0.5g tissue) cracking, homogenate under ice bath, centrifugal 5000rpm, 20min gets its supernatant, and-20 ℃ of preservations are until mensuration.
4. typical curve: get homogenates such as blank serum, brain, liver, muscle, the heart, kidney with the standard solution of preparation variable concentrations after 5 times of the sample diluting liquid dilutions, make final concentration be respectively 0,1.56,6.25,25,100,400 and 1600ng/mL.
5. assay method: on wrapping by 96 microwell plates of first antibody in advance, add sample (standard items or testing sample) and diluted each 50 μ L of good rh-T α 1 antibody, react 1h in 25 ℃ behind the mixing 20s gently.Remove liquid in the hole, wash plate 5 times, pat dry, add 100 μ L ELIAS secondary antibody, 25 ℃ of reaction 0.5h remove liquid in the hole, wash plate 5 times, pat dry.Add each 50 μ L of substrate solution A and B, 25 ℃ of reactions of lucifuge 30min adds 50 μ L sulfuric acid cessation reactions, reads the light absorption value in each hole at microplate reader 450nm place.Through Logistic four parametric line model matches (Origin Version7.5), can obtain the typical curve that drug concentration logarithm and light absorption value are anti-S type.Accompanying drawing 3 is average curve map (with the blank homogenate preparations of liver), and the curve fit equation is: Abs=0.1095+1.4547/[1+ (logC/11.077)
0.899], r
2=0.9988.
6. rat s.c.rh-T α 1(300 μ g/kg), behind the medicine in each tissue of different time points the content of rh-T α 1 in time variation tendency see accompanying drawing 4.
Conclusion: The above results shows that the method that cell pyrolysis liquid extraction, liquid nitrogen cryopreservation and immune analysis determination techniques are combined of foundation of the present invention can successfully detect the Tissue distribution of micromolecule polypeptide class medicine.
Claims (9)
1. method that detects micromolecule polypeptide class medicine Tissue distribution, this method may further comprise the steps:
(1) preparation internal organs/tissue extract;
(2) adopt the immunologic assay technology to measure the concentration of internal organs/tissue extract Chinese traditional medicine;
(3) according to the measured concentration of step (2), calculate different time internal organs/organize the concentration of Chinese traditional medicine, the Tissue distribution that can get medicine is variation tendency in time.
2. detection method according to claim 1 is characterized in that, in step (1) liquid nitrogen cryopreservation and cell pyrolysis liquid extraction is combined, and is used for the preparation of micromolecule polypeptide class medicine tissue extract.
3. detection method according to claim 2 is characterized in that, the liquid nitrogen cryopreservation time is 0.5h～3.5h in step (1), is preferably 2h.
4. detection method according to claim 2 is characterized in that, the additional proportion of cell pyrolysis liquid is 1mL～4mL/0.5g tissue in step (1), is preferably 2.5mL～3mL/0.5g tissue.
5. detection method according to claim 2 is characterized in that, the centrifugal speed of tissue homogenate and time are in step (1): 5000rpm and 20min.
6. detection method according to claim 1 is characterized in that, according to the character of micromolecule polypeptide class medicine, adopts radiommunoassay (RIA) method and enzyme-linked immuno assay (ELISA) method to measure drug concentration in step (2).
7. detection method according to claim 6 is characterized in that, in the RIA method of step (2), the radioactive isotope of micromolecule polypeptide class medicine is selected from
125I, 99mTc,
3H,
14C or
35S, preferred
125I.
8. detection method according to claim 6, it is characterized in that, in the preparation of the standard solution of step (2), with the homogenate extract of corresponding blank tissue preparations such as the heart, liver, kidney, muscle, brain but not general sample diluting liquid (containing the PBS damping fluid of animal protein etc.) preparation typical curve.
9. detection method according to claim 1 is characterized in that, the contained tested medication amount of unit of account quality organization as follows in step (3): (tested drug concentrations * cell pyrolysis liquid volume)/tested tissue quality * 100%.
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Application publication date: 20131002 |