CN104483407B

CN104483407B - Micro-Imatinib assay method and the application in zero clinical trial phase in blood sample

Info

Publication number: CN104483407B
Application number: CN201410800327.7A
Authority: CN
Inventors: 王兴河; 周保利; 陈刚; 王进; 漆璐; 王泽娟; 金辉
Original assignee: Beijing Shijitan Hospital
Current assignee: Beijing Shijitan Hospital
Priority date: 2014-12-18
Filing date: 2014-12-18
Publication date: 2016-01-06
Anticipated expiration: 2034-12-18
Also published as: CN104483407A

Abstract

The present invention relates to micro-Imatinib assay method and the application in zero clinical trial phase in blood sample.Specifically, one aspect of the invention relates to a kind of method measuring Imatinib or its pharmaceutical salts in biological sample, the method uses high performance liquid chromatography-tandem mass to measure, and comprises the following steps and test condition: the process, Specimen Determination, liquid phase chromatogram condition, Mass Spectrometry Conditions etc. of test sample.The invention still further relates to the application of the inventive method in zero clinical trial phase of Imatinib or its pharmaceutical salts; And the application of the inventive method in the pharmacokinetic trial research of Imatinib or its pharmaceutical salts.Have been found that the detection method with feature of the present invention can valuably for detecting Imatinib or the concentration determination of its pharmaceutical salts such as mesylate in its medicine zero clinical trial phase in biological sample such as blood plasma.

Description

Micro-Imatinib assay method and the application in zero clinical trial phase in blood sample

Technical field

The invention belongs to medical art, relate to a kind of assay method of medicine, in particular to the assay method of the medicine in a kind of biological sample such as blood plasma, relate more particularly to a kind of for the cancer therapy drug Imatinib of denier in biological sample such as blood plasma and the assay method of pharmaceutical salts thereof.The invention still further relates to the application of the method in the research of medicine zero clinical trial phase.

Background technology

Imatinib mesylate (ImatinibMesylate) is a kind of selectivity tyrosine kinase inhibitor of Novartis Co., Ltd of Switzerland exploitation, belong to aniline quinazoline compounds, it is the selective depressant of the tyrosine kinase activity for P210 albumen, targeted inhibition Ph chromatin-positive (Philadelphiachromosomepositive, the paraprotein Bcr-Abl of what Ph+) chronic myelocytic leukemia (ChronicMyeloidLeukaemia, CML) produced have tyrosine kinase activity.May calendar year 2001, approval is used for the treatment of chronic myelogenous leukemia to FDA, and in February, 2002, FDA ratified the treatment of imatinib mesylate for gastrointestinal stromal tumors further.Imatinib mesylate all can suppress Bcr-Abl tyrosine kinase in vivo and in vitro on a cellular level, can Selective depression Bcr-Abl positive cell line cell, the chronic myelocytic leukemia of Ph chromatin-positive and the fresh cells of acute lymphoblastic leukemia patient propagation and induce its apoptosis.In addition, imatinib mesylate also can suppress platelet derived growth factor (PDGF) acceptor, stem cell factor (SCF), the tyrosine kinase of c-Kit acceptor, thus suppresses by the cell behavior of PDGF and SCF mediation.Its chemistry is by name: 4-[(4-methyl isophthalic acid-piperazine) methyl]-N-[4-methyl-3-[[4-(3-pyridine)-2-pyrimidine] is amino] phenyl]-aniline mesylate, molecular formula is C29H31N7O-CH4SO3, and molecular weight is 589.7.The chemical constitution of imatinib mesylate is as follows:

Imatinib mesylate is that white-off-white color is to light brown or pale yellow look lookthe crystalline powder adjusted.Imatinib mesylate dissolves in≤aqueous buffer solution of pH5.5 in, and in the aqueous buffer solution of neutrality to alkalescence slightly soluble to insoluble.In non-aqueous solvent, imatinib mesylate is freely dissolved into and is atomicly dissolved in dimethyl sulfoxide, methyl alcohol and ethanol, but is insoluble to n-octyl alcohol, acetone and acetonitrile.

Imatinib mesylate commercially can be called by commodity thin membrane coated tablet obtain, it by NovartisPharmaSteinAG (Stein, Switzerland) prepare, NovartisPharmaceuticalsCorporation (EastHanover, NewJersey) sell. the active component that blade comprises counts 100mg or 400mg with imatinib free alkali.

Imatinib mesylate is applicable to treatment Philadelphia chromosome positive chronic myelocytic leukemia (CML) and the positive unresectable and/or metastatic malignant gastrointestinal stromal tumors (GIST) of Kit (CD117).

usually to the adult patients prescription dosage of 400mg/ days of chronic phase CML and the adult patients prescription dosage of 600mg/ days giving acceleration phase or burst crisis.Adult patients in addition for suffering from unresectable and/or metastatic malignant GIST is recommended 400mg/ days or 600mg/ days dosage . usual oral administration, has a meal and a mug water is taken, and dosage is 400mg or 600mg, once a day, and gives 800mg dosage altogether twice daily with 400mg.

About the concept of zero clinical trial phase.The initial dose of clinical testing is often carried out in traditional clinical research at human body by animal data supposition medicine, but metabolic enzyme kind and quantity, metabolic pathway etc. are all different between humans and animals, medicine absorption in animal body, distribution, metabolism and secretion and human body have very big-difference, therefore, this simple deduction often makes clinical testing, and the clinical testing risk especially for original new drug increases.The catastrophic event that Britain TNG1412 in 2006 causes, for we have beaten alarm bell.

Before zero clinical trial phase refers to that reactive compound does not formally enter clinical testing after completing preclinical test, the drug test that development person uses micro-dosage to carry out a small amount of healthy volunteer or patient's (being generally 6 ~ 15 people), collect necessary relevant safety of medicine and the test figure of pharmacokinetics, whether having to assess research and development medicine the possibility being developed as new drug or biopreparate further, is the intermediate link being transitioned into I clinical trial phase from preclinical test.

The object of carrying out zero clinical trial phase is, by studying compound or formulation, obtain the Pharmacokinetic data that comprise protein binding rate, enzyme inhibition rate etc. and comprise the pharmacodynamics data relevant in conjunction with situation to target spot, and the tissue distribution situation adopting various iconography research means to obtain, to determine that the lead compound having research and development to be worth most carries out I clinical trial phase and follow-up research and development in early days from one group of candidate compound.In addition.Understand the metabolic characteristics of lead compound at human body as early as possible.Animal for non-clinical safety evaluation research selects, improves the predictive value of animal test results also very meaningful.

About the research method of zero clinical trial phase.The research side of zero clinical trial phase method mastercomprise micro-dose study (micro-dosestudies) and pharmacological relevant doses research (pharmacologicallyrelevantdose).

The exploratory drug research that micro-dose study carries out at human body, use the dose value lower than 1/100 animal experimental data to calculate the dosage that human body produces pharmacological effect, object be understand tested medicine in human pharmacokinetics feature, evaluate its biodistribution at human body and Targeting Effect, measure its dosage range and administration number of times and order, medicine generation under clearly two or more medicaments derivative state and drug effect, simultaneously to exploitation noveldevelopment probe or developing technique have very great help.U.S. FDA and the definition of European EMEA to micro-dosage are: the plan derived lower than the animal safety data obtained by clinical front toxicologic study may produce 1/100 of clinical pharmacology effect dose for human body, and maximum dose is no more than the dosage of 100 μ g.For protide product, dosage need≤30nmol.

The exploratory drug research that pharmacological relevant doses research is carried out in animal body, belong to the category of preclinical study, its object is to the pharmacological action evaluating tested medicine, pre-clinical safety data (sensitive species 2 weeks toxicological experiment results) before testing, should be had.Pharmacological relevant doses is studied usual continuous use and is no more than 7d, and initial dose is NOAEL (unobservable spinoff dosage, the mg/m of sensitive species 1/50 ²), maximum dose is the NOAEL of 1/4 or 1/2 or occurs change or the spinoff of target index.Pharmacological relevant doses research not only has great help to the tested medicine of understanding at the mechanism of action of human body, can also provide medicine generation-drug effect related data in human body before the I phase tests, for determining to adopt single preparation or mix preparation, role of evaluation drug effect in the two or more medicine of same target and the human body of derivant provides reference.Lead compound likely is selected to provide scientific basis to researching and developing further.

About the difference of zero clinical trial phase and traditional I clinical trial phase.Because test objective is different, zero clinical trial phase and traditional I clinical trial phase number of subjects, the tested time, Test Drug Dosages, benefited to assess etc. in there is very large difference, refer to table 1.

table 1: the difference of zero clinical trial phase and traditional I clinical trial phase

in upper table, * refers to tumour medicine

About the detection method of zero clinical trial phase.Because 0 clinical trial phase is micro-dosetest, therefore need hypersensitive and accurate detection means realize mensuration, medicine and special receptor to medicine and Metabolites Concentration thereof or enzyme in conjunction with situation, with determine medicine PK/PD feature, the information of medicine and its target spot effect be provided, evaluate the mechanism of action of medicine, by the information etc. of the security that provides medicine preliminary to the chorologic research of medicine and validity, the primary analysis method of application at present has:

(1) quantitative analysis method: mainly comprise accelerator mass spectrometry determination method (zcceleratormassspectrometry, AMS), using high performance liquid chromatography tandem mass spectrum instrument determination method (liquidchromatography-massspectrometry/massspectrometry, and inductively coupled plasma Optical Emission Spectrometer determination method (inductivelycoupledplasma-opticemissionspectrometer, ICP-OES) LC/MS/MS);

(2) semi-quantitative analysis method: mainly comprise Positron Emission Computed Tomography instrument (positronemissiontomography, PET-CT), immunoassay (Immunoassays) and molecular engram (molecularimprintingpolymer, IMP).

AMS is the sensitiveest method, the blood concentration of fg/ml level can be measured, its sensitivity is 1000 times of fluorimetric HPLC technology, and the ray exposure value of experimenter is very low, should as first-selection in the situation of having ready conditions, but need label isotope and number of devices is few, limit it and widely use; PET-CT has higher sensitivity, can carry out Real time Organization distribution and target analysis to compound, meaningful to the target spot pharmacological action specificity disclosing compound, and PET-CT quantity gets more and more, and can be used for extensively carrying out the work; LC/MS/MS analyzes thing to great majority higher sensitivity, and its mensuration can reach pg level, easy and simple to handle, can measure in batches; Immunoassay sensitivity can be higher than LC/MS method, but there is cross reaction and antibody and produce the problems such as retardation time.

About the medicament categories being applicable to zero clinical trial phase.Beautiful country of statefirst ICR and Abbott have carried out zero clinical trial phase of antineoplastic ABT-888 in 2007, be intended to find the biomarker change of taking medicine in patient body, evaluate pharmacokinetics and the antitumous effect of ABT-888.Study from approving to complete and obtain critical biological chemistry and human body medicine dynamics data, the time of 5 months has only been used in the test of ABT-888 zero phase, and researcher no longer needs to carry out the individually dosed formal I phase after this test tests, can directly carry out testing with the I phase of other anticarcinogen conbined usage at least saving for 1 year for researcher.But the candidate compound of not all type is all applicable to the test of zero phase.Internationally recognized zero phase test is at present mainly used in the research and development of malignant tumour new drug.Usually, there is the compound of following characteristics, the antitumor original new drug that especially toxicity is larger, can consider to carry out this kind of exploratory new drug research: 1, there is target trusty (change of target index can bring about the desired effect); 2, window is treated wider; 3, expect short open-assembly time adjustment drug effect (administration time≤7d) of low dosage; 4, relatively small sample when (6 ~ 15) can the Targeting Effect of evaluate efficacy.

About the superiority and shortage of zero clinical trial phase.The clinical research of zero phase is still in the starting stage, and its starting point is cost-saving, improves new drug development efficiency, for the exploitation of medicine of promoting innovation provides opportunity.The advantage of zero phase clinical research is to try: do not need the toxicological study expanded before testing, and dosage is very low, the quick approval of supervision department can be obtained, therefore can shorten cycle development time significantly, non-clinical study required time is foreshortened to 3 ~ 6 months; Experimenter's quantity is few, and administration time is short, expense is low; The I phase that the clinical testing risk that may bring is more traditional tests little; More early can obtain the activity of candidate compound to target spot, be beneficial to and earlier carry out R & D Decision; There is provided early stage human body PK data accurately, prediction exact test dosage accurately estimates that the dosage escalation in clinical testing accurately estimated by clinical research sample; For toxic side effect problem new drug energy Timeliness coverage, stop early.Therefore, the clinical research of zero phase, to original new drug, is particularly very helpful to the research and development of innovation antineoplastic.

But as new drug pilot study, zero clinical trial phase remains to be needed to inquire into further in following problem; Domestic current nothing zero clinical trial phase governing principle, although have, minority new drug product is granted carries out small-scale test a small amount of experimenter before carrying out clinical testing, but belong to I clinical trial phase on a small scale, object is research safety and maximum tolerated dose, there are the different of essence from zero clinical trial phase; Lack the researchist of rational research and design and specialty; Lack and possess the research equipment of high sensitivity and accuracy and the researchist of correlation experience; Lack the confidence level that test method and interpretation of result are confirmed, as studied dosage well below therapeutic dose, examining whether can pharmacokinetics parameter exactly under predicted treatment dosage, and whether the drug metabolism namely within the scope of micro-dosage and therapeutic dose can the problem of retention wire sexual intercourse; Test number of subjects is little, and continue to use traditional clinical Test model synthetic techniques, whether its evaluation result accurately can judge the quality of candidate, and error in judgement has much, and in group, variation between laboratories has great problem; Reaction medicine and target spot interactional iconography data conclusion whether fully problem is obtained by test.

In a word, the test of zero phase, as pilot study, has certain advantage, also there is certain limitation.Promotion attitude is held to pilot study in the international market being representative with Europe and the U.S. at present, especially after the zero phase research of ABT-888 obtains significant result.In " 12 " period, will there be more than a 100 kind new medicine listing in China, according to me state of statefeelings set up related guidance principle, and the original new drug R&D mode improving and develop rapid screening, reduce costs, reduce risks, promotes that more active compound goes on the market, and promote that China's original new drug research and development are significant to real.

Although Imatinib or its pharmaceutical salts such as its mesylate has gone on the market and for clinical, carried out medicine zero clinical trial phase be still significant to it.But routine dose 400mg every day, once takes clinically.According to the general norm of medicine zero clinical trial phase, in this medicine zero clinical trial phase, the dosage once a day of experimenter is 4mg, after this very low dose is taken, drug dose in experimenter's body fluid such as blood plasma will be lower, the direct problem having caused determination of plasma concentration difficulty, because existing Imatinib plasma drug level detection method is not applicable to the detection of this very low dose, may can not detect out and/or detected value differs greatly with actual value, and other test problems.

Therefore, the assay method having very low dose Imatinib or its pharmaceutical salts such as mesylate in a kind of biological sample such as blood plasma is badly in need of in this area.

Summary of the invention

The object of the present invention is to provide the assay method of very low dose Imatinib or its pharmaceutical salts such as mesylate in a kind of biological sample such as blood plasma, expect that this method is enough to be used in the detection of medicine zero clinical trial phase.The present inventor have been surprisingly found that, the detection method with feature of the present invention can valuably for detecting Imatinib or the concentration determination of its pharmaceutical salts such as mesylate in its medicine zero clinical trial phase in biological sample such as blood plasma.The present invention is based on this find and be accomplished.

For this reason, first aspect present invention provides the method measuring Imatinib or its pharmaceutical salts in biological sample, and the method uses high performance liquid chromatography-tandem mass to measure, and comprises the following steps and test condition:

(1) process of sample is tested:

Get 100 μ L pastille biological samples in 1.5mL pipe, add 20 μ L methyl alcohol, add 20 μ L inner mark solutions (300ng/mL), vortex mixes, add 100 μ L methyl alcohol and 200mL acetonitrile, vortex 50 seconds, in 4 DEG C of centrifugal 5min of 14000rpm, getting supernatant 200 μ l is transferred in 1.5mL sample injection bottle, to be detected as test sample;

(2) Specimen Determination:

Get 20 μ L and test sample injection high performance liquid chromatography-tandem mass instrument, detect the Imatinib in sample and interior target chromatographic peak, and calculate the concentration of the Imatinib in described biological sample accordingly;

(3) liquid phase chromatogram condition:

Chromatographic column: PhenomeneC18100A post (3.0 × 50mm); Mobile phase: acetonitrile (containing 0.1% formic acid): water (containing 2mmol/L ammonium acetate and 0.1% formic acid), gradient elution (5:95,1min; 90:10,3.5min; 90:10,4min; 5:95,4.1min; 5:95,6min), flow velocity: 0.4mL/min, sample size: 20 μ L;

(4) Mass Spectrometry Conditions:

Ion gun: ion spray ionisation source; Ion injection electric: 5500V; Temperature: 550 DEG C; Gas 1 (GS1, N2) pressure: 50psi in source; Gas 2 (GS2, N2) pressure: 50ps in source; Curtain gas (N2) pressure: 20psi; Positive ion mode detects; Scan mode: Selective ion mode reaction monitoring (MRM) mode; Ionic reaction for quantitative test is respectively m/z494.3 → m/z394.1 (Imatinib) and m/z408.2 → m/z235.0 (MK0431, interior mark).

The method of arbitrary embodiment according to a first aspect of the present invention, the pharmaceutical salts of wherein said Imatinib is selected from imatinib mesylate, hydrochloric acid Imatinib, phosphoric acid Imatinib, toluenesulfonic acid Imatinib etc.In view of these different salt forms Imatinib after oral administration, all exist with imatinib free alkali form in vivo but not the existence of its pharmaceutical salt form, and be also be as the criterion with Imatinib when in fact measuring blood plasma, therefore the inventive method is applicable to take imatinib free alkali and above-mentioned various different salt form completely.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said biological sample is selected from blood plasma, urine, saliva etc.

The method of arbitrary embodiment according to a first aspect of the present invention, the concentration of the Imatinib comprised in wherein said pastille biological sample lower than 1000ng/ml, such as, lower than 500ng/ml, such as, lower than 100ng/ml, such as lower than 75ng/ml, such as, lower than 50ng/ml.

The method of arbitrary embodiment according to a first aspect of the present invention, the concentration of the Imatinib in the pastille biological sample that wherein said high performance liquid chromatography-tandem mass can detect is higher than 0.1ng/ml, such as higher than 0.2ng/ml, such as, higher than 0.3ng/ml, such as, higher than 0.4ng/ml.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said interior mark is Xi Gelieting.

The method of arbitrary embodiment according to a first aspect of the present invention, in wherein said the process of sample " (1) test ", add in 20 μ L methyl alcohol in comprise organic acid.In one embodiment, described organic acid is selected from tartrate, citric acid, maleic acid, malic acid, fumaric acid.In one embodiment, the concentration of described organic acid in methyl alcohol is 20 ~ 200ug/ml, such as 25 ~ 150ug/ml, such as 50 ~ 100ug/ml.Have been surprisingly found that, when use comprises the organic acid of particular types and specified quantitative in this step, described high performance liquid chromatography-tandem mass can be measured to the concentration of wherein Imatinib quantitatively, exactly; And when not adding this organic acid, recording concentration and always present lower than the concentration of theory interpolation and show unacceptable result.

The method of arbitrary embodiment according to a first aspect of the present invention, in wherein said " (2) Specimen Determination ", the concentration calculating the Imatinib in described biological sample adopts Internal standard curve method to calculate.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said Internal standard curve method also comprises the step of Criterion curve.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said Criterion curve comprises the following steps:

Get the blank biological sample 8 parts of 100 μ L not pastille, be placed in 1.5mL pipe respectively, each pipe adds 20 μ L methyl alcohol respectively, add 20 μ L inner mark solutions (300ng/mL) respectively, Imatinib or its pharmaceutical salts is added respectively with Imatinib 0.04ng in the form of stock solution, 0.1ng, 0.2ng, 0.5ng, 1ng, 2ng, 3ng, 4ng, by each pipe vortex mixing, add 100 μ L methyl alcohol and 200mL acetonitrile respectively, vortex 50 seconds, in 4 DEG C of centrifugal 5min of 14000rpm, getting supernatant 200 μ l is transferred in 1.5mL sample injection bottle, as 8 parts of standard models, to be detected,

Get 20 μ L standard models respectively and inject high performance liquid chromatography-tandem mass instrument, detect the Imatinib in sample and interior target chromatographic peak, and obtain typical curve accordingly, for the concentration of the Imatinib calculated in described biological sample.

In the present invention, term " blank biological sample " typically refers to the biological sample that experimenter gathered before taking medicine.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein when Criterion curve, comprises organic acid in the 20 μ L methyl alcohol that each pipe adds respectively.In one embodiment, described organic acid is selected from tartrate, citric acid, maleic acid, malic acid, fumaric acid.In one embodiment, the concentration of described organic acid in methyl alcohol is 20 ~ 200ug/ml, such as 25 ~ 150ug/ml, such as 50 ~ 100ug/ml.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein when Criterion curve, the Imatinib added in blank biological sample or its pharmaceutical salts are added with the form of stock solution.In one embodiment, in this stock solution Imatinib or its pharmaceutical salts in Imatinib concentration for 1 ~ 1000ng/ml, such as 2 ~ 500ng/ml, such as 4 ~ 400ng/ml; Adopt the stock solution of these concentration ranges, when adding in the blank biological sample to described 100 μ L not pastille, the stock solution of appropriate volume can be added, such as 5 ~ 50 μ L, such as 5 ~ 20 μ L are feasible, that is, when defining the concentration of Imatinib addition and stock solution when at Criterion curve, the interpolation volume of this stock solution just need not make special agreement.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein when Criterion curve, blood plasma drug concentration in Imatinib in 0.4 ~ 40ng/ml concentration range, by internal standard method with calculated by peak area, linear between drug concentration and chromatogram response intensity (i.e. active substance and interior target peak area ratio), and r value >0.9990, such as r value >0.9992, such as r value >0.9995, such as r value >0.9998.

The method of arbitrary embodiment according to a first aspect of the present invention, this stock solution methyl alcohol is solvent preparation.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said inner mark solution methyl alcohol is solvent preparation.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said biological sample (comprise pastille biological sample and the not blank biological sample of pastille, all have similar meaning in this article) takes from mammiferous biological sample.Described mammal is such as people.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said biological sample is the biological sample taking from people.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said pastille biological sample is the biological sample taking from people, and the oral Imatinib of this people or its pharmaceutical salts count 1 ~ 10mg with Imatinib.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said pastille biological sample gathers 0 ~ 48 hour period after oral Imatinib or its pharmaceutical salts count 1 ~ 10mg with Imatinib described mammal such as people.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said pastille biological sample gathers 0 ~ 36 hour period after oral Imatinib or its pharmaceutical salts count 2 ~ 8mg with Imatinib described mammal such as people.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said pastille biological sample gathers 0 ~ 24 hour period after oral Imatinib or its pharmaceutical salts count 3 ~ 6mg with Imatinib described mammal such as people.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said pastille biological sample gathers 0 ~ 24 hour period after oral Imatinib or its pharmaceutical salts count 4mg with Imatinib described mammal such as people.In this article, in phrase " described pastille biological sample gathers 0 ~ 24 hour period after oral Imatinib or its pharmaceutical salts count 4mg with Imatinib described mammal such as people " and other similar statement, " 0 ~ 24 hour period " comprises 0 hour and 24 hours two end points, and for 0 hour, as well known to those skilled in the art, usually can blood sampling immediately obtain after the tablet has been ingested, also can be that blood sampling of (such as taking medicine first 10 minutes) before taking medicine obtains usually, these situations can be used as the blood sample of 0 hour.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said pastille biological sample be described mammal such as people after oral Imatinib or its pharmaceutical salts count 4mg with Imatinib 0,0.5,1,1.5,2,2.5,3,4,6,8,10,12,24h time gather.

The method of arbitrary embodiment according to a first aspect of the present invention, wherein said pastille biological sample is the biological sample taking from people, and this body weight for humans is 50 ~ 80kg.

The method of arbitrary embodiment according to a first aspect of the present invention, it is the pharmacokinetic in mammal such as human body for Imatinib or its pharmaceutical salts.

Further, second aspect present invention provides the application of method described in any one of first aspect present invention in zero clinical trial phase of Imatinib or its pharmaceutical salts.

The application of arbitrary embodiment according to a second aspect of the present invention, wherein said zero clinical trial phase is men's health experimenter single oral Imatinib or its pharmaceutical salts such as, in carried out the afterwards research of Imatinib 1 ~ 10mg (such as 2 ~ 8mg, 3 ~ 6mg).

The application of arbitrary embodiment according to a second aspect of the present invention, wherein said zero clinical trial phase is that men's health experimenter single oral Imatinib or its pharmaceutical salts are in Imatinib 1 ~ 10mg (such as 2 ~ 8mg, such as 3 ~ 6mg) carried out afterwards research, this research comprises pharmacokinetic trial.

Further, third aspect present invention provides the application of method described in any one of first aspect present invention in the pharmacokinetic trial research of Imatinib or its pharmaceutical salts.

The application of arbitrary embodiment according to a third aspect of the present invention, the research of wherein said pharmacokinetic trial is men's health experimenter single oral Imatinib or its pharmaceutical salts such as, in carried out the afterwards research of Imatinib 1 ~ 10mg (such as 2 ~ 8mg, 3 ~ 6mg).

Arbitrary technical characteristic that arbitrary embodiment of either side of the present invention or this either side has is suitable for arbitrary embodiment of other arbitrary embodiment or other either side equally, as long as they can not be conflicting, certainly at where applicable each other, necessary words can be done suitably to modify to individual features.Be further described with feature to various aspects of the present invention below.

All documents that the present invention quotes from, their full content is incorporated to herein by reference, and if the implication expressed by these documents and the present invention inconsistent time, be as the criterion with statement of the present invention.In addition, the various term that the present invention uses and phrase have and well known to a person skilled in the art general sense, nonetheless, the present invention still wishes to be described in more detail at this these terms and phrase and to explain, the term mentioned and phrase, if any inconsistent with common art-recognized meanings, are as the criterion with the implication that the present invention states.

Be further described to various aspects of the present invention below.

Have been surprisingly found that, the inventive method valuably for clinical research particularly its zero clinical trial phase research of Imatinib or its pharmaceutical salts, such as, can study relevant pharmacokinetic with its zero clinical trial phase.

Accompanying drawing explanation

figure1 is the exemplary clear HPLC-MS/MS chromatogram obtained with Imatinib figure( figurein, ordinate is intensity, and unit is cps; Horizontal ordinate is the time, and unit is min; Lower same);

figure2 be within mark the exemplary clear HPLC-MS/MS chromatogram obtained figure;

figure3 is the typical matrices HPLC-MS/MS chromatograms obtained with Imatinib figure;

figure4 be within mark the typical matrices HPLC-MS/MS chromatogram obtained figure;

figure5 is HPLC-MS/MS of the typical LLOQ obtained with Imatinib figure;

figure6 be within mark the HPLC-MS/MS of the typical LLOQ obtained figure;

figure7 is HPLC-MS/MS of No. 1 experimenter the 5th sampling spot plasma sample (Imatinib) figure;

figure8 is HPLC-MS/MS of No. 1 experimenter the 5th sampling spot plasma sample (interior mark) figure;

figure9 is HPLC-MS/MS of No. 1 experimenter the 6th sampling spot plasma sample (Imatinib) figure;

figure10 is HPLC-MS/MS of No. 1 experimenter the 6th sampling spot plasma sample (interior mark) figure.

figurepharmacokinetic curve after the oral Imatinib capsule (4mg) of 11:8 name experimenter figure(horizontal ordinate is the time (hour), and ordinate is Imatinib plasma concentration (ng/ml), lower same).

figure12: the pharmacokinetic curve after the oral Imatinib capsule (400mg) of experimenter figure.

Embodiment

Can be conducted further description the present invention by the following examples, but scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite not deviating from the spirit and scope of the present invention, can carry out various change and modification to the present invention.The present invention carries out generality and/or concrete description to the material used in test and test method.Although for realizing many materials that the object of the invention uses and method of operating is well known in the art, the present invention still describes in detail as far as possible at this.

In further describing hereafter, if not otherwise specified, the Imatinib used or its pharmaceutical salts refer to imatinib mesylate, and the imatinib mesylate formulations used is commercially available capsule H20100238.Hereinafter, use interior timestamp, if not otherwise specified, refer to Xi Gelieting, interior mark also can represent with English abbreviation IS.When hereafter mentioning biological sample, if not otherwise indicated, all blood plasma is referred to.

one, the generality of zero clinical trial phase research describes

The generality of the medicine zero clinical trial phase research provided below describes, and is only determined for instructing from zero clinical trial phase research methodology well known in the art.One important in the research of this medicine zero clinical trial phase is measure the pharmacokinetic parameter of medicine.

1, research purpose:

Determine the Pharmacokinetic Characteristics of Chinese male health volunteer single oral very low dose imatinib mesylate; Explore and set up the pharmacokinetic method of imatinib mesylate under very low dose, for CFDA foundation " Chinese zero clinical trial phase guide " provides foundation.

2, experiment process comprises: screening, experimental observation phase and off-test phase.Wherein,

Screening comprises: signature Informed Consent Form, every inspection Pass Test require, screening about 20 people;

The experimental observation phase comprises: enter group and be 8 people, by testing program administration, gather medicine for sample, full safety assessment;

The off-test phase comprises: leave and take laboratory sample and enter row peacefull property is assessed, complete research case history, medicine for test sample, statistical study, final report.

3, inclusion criteria (meeting whole inclusion criteria can be selected in):

(1), medical history before test, health check-up, Laboratory project and the relevant every inspection of test, detect all normal or without the mile abnormality of clinical meaning, Clinical Research Physician judges to think the qualified Health China male sex;

(2), 18 to 40 years old ages;

(3), BMI was 19 to 24kg/m2 (comprising), body weight >=50kg;

(4), before entering group research, sign written informed consent according to GCP and local law and date.

4, exclusion standard (meeting an exclusion standard namely to get rid of):

(1), any abnormal and have the medical inspection of clinical meaning to find (comprising BP, PR and ECG);

(2), the evidence of any clinical relevant accompanying diseases;

(3), stomach and intestine, liver, kidney, respiratory system, cardiovascular, metabolism, immunity or hormone disturbance;

(4), gastrointestinal surgery history (except appendectomy);

(5), central nervous system disease (as epilepsy) or phrenoblabia or the nervous system disease;

(6), relevant postural hypotension, dizziness or dizzy history;

(7), chronic or relevant acute infection;

(8), associated allergic history/hypersensitivity history (comprising medicine or its excipient allergy);

(9), (24 hours) medicine before research administration or the long half-lift of taking at least one moon of duration of test, or relative medicine is less than 10 half life period, or use taboo medicine (CYP3A4 inhibitor and derivant comprise ketoconazole, rifampin, carbamazepine, phenytoinum naticum, phenobarbital, dexamethasone, catarrh miaow piperazine etc.);

(10), in research first 3 months of administration or duration of test participated in the test of other drugs;

(11), smoker's (every day 10 cigarettes or 3 cigar or 3 pipe tobaccos);

(12), can not ban on opium-smoking and the opium trade at duration of test;

(13), alcohol abuse or drug abuse (hemp, Benzodiazepine, group of barbiturates, opiates, ***e, amphetamine, methadone);

(14), to donate blood in 3 months before administration history;

(15), excessive physical exertion (before research administration in 1 week or during research);

(16), any laboratory examination value with clinical meaning beyond term of reference;

(17) diet program in research centre can not, be observed;

(18), baseline QT/QTc interval significant prolongation (QTc interval is greater than 450 milliseconds as repeatedly occurred);

(19), there is TdP additional risks factor history (as heart failure, hypopotassaemia, long QT syndrome family history);

(20), fertility person in six months is planned.

5, test method:

(1), test method: single centre, opening, single dose;

(2), experimenter selects: 8 Chinese healthy male volunteers;

(3) single oral 4mg imatinib mesylate (namely very low dose commonly uses 1/100, clinical RD 400mg/ days, this test dose 4mg/ days of clinical dosage), is given.

6, medication:

(1), by professional pharmacists be responsible for, use electronic balance, after packing of imatinib mesylate capsule being carried out weigh, again insert in new medical capsule, each newly encapsulated containing active component imatinib mesylate 4mg;

(2), every experimenter gives single oral 4mg imatinib mesylate (namely very low dose commonly uses 1/100 of clinical metering) capsule.Morning, 8:00 started by test number administration, and 240ml warm water is taken.

7, medicine is for collection of specimens and mensuration:

(1), every experimenter need gather the pharmacokinetics sample of 13 time points, each venous blood collection 5ml, 65ml altogether;

(2), before administration, snibject's offside fore-arm lays a remaining needle sample of blood, drawn;

(3), blood sampling point: before administration after (first 10 minutes of administration) and administration 0.5,1,1.5,2,2.5,3,4,6,8,10,12,24h, totally 13 points of taking a blood sample;

(4), specimen centrifuge, be in charge of preservation, to be measured;

(5) plasma concentration of imatinib mesylate (Gleevec), is measured by liquid chromatography-tandem mass spectrum (LC-MS/MS) method verified.

8, medicine is for parameter:

(1), plasma drug concentration data adopts WinNolin and SAS software to analyze, and calculates main pharmacokinetic parameter, to reflect the feature of medicine in people's body absorption, distribution, metabolism and excretion comprehensively;

(2), main pharmacokinetic parameter has: Tmax, Cmax, AUC0-t, AUC0-∞, t1/2, Vd/F, CL/F etc.

9, safety evaluation:

Carry out clinical safety assessment by following content in process of the test: any spontaneous report carry out clinical safety assessment with all adverse events observed directly; Any abnormal change in vital sign, physical examination; Duration of test laboratory examination is abnormal.Wherein, adverse events refers to from subjects signed Informed Consent Form and is selected in on-test to off-test, and whether any bad event of generation, have cause-effect relationship with trial drug.

10, statistical method:

(1), each pharmacokinetic parameter represents with average and standard deviation and comprises Cmax, Tmax, AUC0-t, AUC0-∞, t1/2 etc., Cmax and Tmax represents with measured value, calculates AUC0-t, by formulae discovery AUC0-∞ and t1/2 with trapezoidal method;

(2), safety observations index and baseline check the value compare, and adopt difference before and after in paired t-test comparative group, enumeration data adopts all kinds of number of cases and percentage to describe.

In above-mentioned zero clinical trial phase research, usually need the vital sign, ECG, laboratory examination etc. that obtain or detect experimenter.Provided below is some typical consequence that the present inventor obtains in typical zero clinical trial phase research, these results are general and normal results, and the pharmacokinetic that the present invention relates to can use the experimenter obtained from these results completely.

With following table 1provide the clinical effectiveness about vital sign, display is all in the usual acceptable scope in this area.

table 1:

With following table 2provide the clinical effectiveness about ECG, display is all in the usual acceptable scope in this area.

table 2:

With following table 3-1, table 3-2 Hes table 3-3 provide the clinical effectiveness about laboratory examination, and display is all in the usual acceptable scope in this area.

table 3-1:

table 3-2:

table 3-3:

two, the generality of pharmacokinetic describes

Describe the research of medicine zero clinical trial phase above in general manner, one important in the research of this medicine zero clinical trial phase is, the pharmacokinetics of drugs.

1, Method validation

Pharmacokinetic provided by the invention is classical test method, the project investigated normally is needed to have in this pharmacokinetic: specificity, lower limit of quantitation (LLOQ), haemolysis affects, matrix effect, typical curve (adopts the scope of 0.4-40ng/mL in this test, linear good), in a few days and in the daytime stability, residual effect, relative recovery, stability (standard reserving solution 16 days stability, interior mark storing solution 14 days stability, 12h aftertreatment stability placed by sample, after sample process, room temperature places 24h stability, sample freeze thawing 3 stability etc.).

2, the instrument/condition of following HPLC-MS/MS is employed in this test:

ABSCIEX5500QTrap type high performance liquid chromatography-tandem mass combined instrument, containing electro-spray ionization source and series connection triple quadrupole bar mass detector, AnalystSoftware1.61 data handling system;

Shimadzu DGU-20A highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven;

Vortex mixed instrument, its woods Bel instrument manufacturing company limited of Haimen City;

ML104 and XP-205 electronic analytical balance, METTLERTOLEDO company of Switzerland;

SIGMA3-18K type high speed low temperature centrifugal machine, the U.S.;

Milli-QPlus water purifior etc.

3, following reference substance and reagent is employed in this test:

Imatinib mesylate standard items: Lianyungang Hongchuang Pharmaceutical Co., Ltd. of Jiangsu Hansoh Pharmaceutical Group provides, content 99.5%, lot number YM-605-101201;

MK-0431 reference substance (Xi Gelieting, as interior mark, is abbreviated as IS): Hengrui Medicine Co., Ltd., Jiangsu Prov. provides, content 99.61%, lot number: 20140602;

Blood plasma is originated: gather from health volunteer, anti-coagulants is heparin lithium.

4, following mass spectrum, chromatographic condition is employed in this test:

Chromatographic condition: chromatographic column: PhenomeneC18100A post (3.0 × 50mm); Mobile phase: acetonitrile (0.1% formic acid): water (containing 2mmol/L ammonium acetate 0.1% formic acid), gradient elution (5:95,1min; 9:1,3.5min; 9:1,4min; 5:95,4.1min; 5:95,6min), flow velocity: 0.4mL/min, sample size: 20 μ L.

Mass Spectrometry Conditions: ion gun: ion spray ionisation source; Ion injection electric: 5500V; Temperature: 550 DEG C; Gas 1 (GS1, N2) pressure: 50psi in source; Gas 2 (GS2, N2) pressure: 50ps in source; Curtain gas (N2) pressure: 20psi; Positive ion mode detects; Scan mode: Selective ion mode reaction monitoring (MRM) mode; Ionic reaction for quantitative test is respectively m/z494.3 → m/z394.1 (Imatinib) and m/z408.2 → m/z235.0 (MK0431, interior mark).

5, the processing mode of following typical test sample is employed in this test:

Get 100 μ L pastilles biological sample (blood plasma) in 1.5mL pipe, add 20 μ L methyl alcohol [comprising 75ug/ml citric acid in this methyl alcohol], add 20 μ L inner mark solutions (300ng/mL), vortex mixes, add 100 μ L methyl alcohol and 200mL acetonitrile, vortex 50 seconds, in 4 DEG C of centrifugal 5min of 14000rpm, getting supernatant 200 μ l is transferred in 1.5mL sample injection bottle, to be detected as test sample.

6, the operation steps of following typical Criterion curve is employed in this test

Get the blank biological sample 8 parts of 100 μ L not pastille, be placed in 1.5mL pipe respectively, each pipe adds 20 μ L methyl alcohol [comprising 75ug/ml citric acid in this methyl alcohol] respectively, add 20 μ L inner mark solutions (300ng/mL) respectively, add Imatinib or its pharmaceutical salts respectively with Imatinib 0.04ng in the form of stock solution, 0.1ng, 0.2ng, 0.5ng, 1ng, 2ng, 3ng, (in stock solution, Imatinib or its pharmaceutical salts are in Imatinib concentration for 4 ~ 400ng/ml, specifically employ the stock solution of three concentration: 4ng/ml for 4ng, 40ng/ml, 400ng/ml, Imatinib is respectively 0.4ng/ml relative to the concentration of blood plasma thus, 1ng/ml, 2/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml), by each pipe vortex mixing, add 100 μ L methyl alcohol and 200mL acetonitrile respectively, vortex 50 seconds, in 4 DEG C of centrifugal 5min of 14000rpm, get supernatant 200 μ l and be transferred in 1.5mL sample injection bottle, as 8 parts of standard models, to be detected.

Above 8 parts of standard models measure through HPLC-MS/MS mentioned above, its typical curve is calculated with Internal standard curve method, there is extremely excellent linear dependence, r=0.9997 between result chromatogram response intensity (active substance and interior target peak area ratio) and concentration.

three, the Typical experimental results of pharmacokinetic

Obtain and take 4mg Imatinib and the blood sample gathered at stipulated time point from according in it " generality of one, zero clinical trial phase research describes " method of the present invention by some experimenters, according to the method in the present invention they " two, the generality of pharmacokinetic describe ", the Imatinib blood concentration in each blood sample is measured.

The following describes some typical consequence obtained thus:

figurepharmacokinetic curve after the oral Imatinib capsule (4mg) of 11:8 name experimenter figure(horizontal ordinate is the time (hour), and ordinate is Imatinib plasma concentration (ng/ml)).

figure12: the pharmacokinetic curve after the oral Imatinib capsule (400mg) of experimenter figure(horizontal ordinate is the time (hour), and ordinate is Imatinib plasma concentration (ng/ml)).

With following table 4provide the typical data that the present invention's HPLC-MS/MS method mentioned above measures the lower limit of quantitation of Imatinib in blood plasma (LLOQ), result shows that the inventive method still has excellent accuracy (89.3% ~ 94.8%) under this extremely low concentration of lower limit of quantitation, meets the general requirement of this area to this type of assay method completely.

table 4:

With following table 5provide batch interior betweenrun precision and the accuracy of the present invention's HPLC-MS/MS method mentioned above, result shows that the inventive method has excellent batch interior betweenrun precision and accuracy, meets the general requirement of this area to this type of assay method completely.

table 5:

With following table 6-1 He table 6-2 provide the present invention's HPLC-MS/MS method mentioned above, measure the result of the matrix effect of Imatinib in blood plasma, result shows that the inventive method meets the general requirement of this area to this type of assay method completely.

table 6-1:

table 6-2:

With following table 7provide the residual confirmation result of the present invention HPLC-MS/MS mentioned above method in sample test, result shows that the inventive method meets the general requirement of this area to this type of assay method completely.

table 7:

With following table 8provide the present invention's HPLC-MS/MS method mentioned above, after Imatinib sample process, room temperature places 24h test findings, and result shows that the inventive method meets the general requirement of this area to this type of assay method completely.

table 8:

With following table 9provide and use the present invention's HPLC-MS/MS method mentioned above, Imatinib sample room temperature places 12h aftertreatment test findings, and result shows that the inventive method meets the general requirement of this area to this type of assay method completely.

table 9:

With following table 10 provides use the present invention HPLC-MS/MS method mentioned above, and Imatinib sample multigelation 3 aftertreatment test findings, result shows that the inventive method meets the general requirement of this area to this type of assay method completely.

table 10:

With following table 11 provide and use the present invention HPLC-MS/MS mentioned above method to measure 8 oral Imatinib capsules (4mg) of experimenter after, the blood concentration-time data of Imatinib; With following table 12 provide and use the present invention HPLC-MS/MS mentioned above method to measure 8 oral Imatinib capsules (4mg) of experimenter after, the pharmacokinetic parameter statistics of Imatinib; figure11 show the pharmacokinetic curve after the oral Imatinib capsule (4mg) of these 8 experimenters figure; figure12 additionally provide the pharmacokinetic curve after 2 groups of experimenters (each 8) oral Imatinib capsule (400mg) figure, this curve figureit is the expection that meets prior art.These results show, still can reflect typical pharmacokinetic curve after oral Imatinib capsule (4mg), and this curve has excellent comparability with the test of taking 400mg.

table 11:

table 12:

four, methodological study

1, with reference to above " one, the generality of zero clinical trial phase research describes ", " two, the generality of pharmacokinetic describes " and " three, the Typical experimental results of pharmacokinetic " method, but " 5, the processing mode of following typical test sample is employed in this test " and " 6, employ the operation steps of following typical Criterion curve in this test " in different, that is: in the 20 μ L methyl alcohol added, comprise the citric acid of 50ug/ml or 100ug/ml, under the test condition of two kinds of citric acid additions, result is all with above " three, the Typical experimental results of pharmacokinetic " substantially identical, such as figure1 ~ figure12 provide with the Typical experimental results of pharmacokinetic " three, " above figuresubstantially identical, example as table 4~ table 1the list data that the Typical experimental results of pharmacokinetic " three, " provide 2 with is above substantially identical, such as table 4in result, the accuracy of 50ug/ml citric acid or 100ug/ml citric acid two kinds of situations is comprised respectively in 90.2 ~ 93.3% scopes and in 90.7 ~ 92.2% scopes in use 20 μ L methyl alcohol, display has the inventive method and has splendid accuracy, and practical measurement result is close with theoretical result of adding.The typical curve set up in two kinds of citric acid consumption situations, its r value is respectively 0.9996 and 0.9995.

2, with reference to above " one, the generality of zero clinical trial phase research describes ", " two, the generality of pharmacokinetic describes " and " three, the Typical experimental results of pharmacokinetic " method, but " 5, the processing mode of following typical test sample is employed in this test " and " 6, employ the operation steps of following typical Criterion curve in this test " in different, that is: in the 20 μ L methyl alcohol added, citric acid is changed into isocyatic tartrate, maleic acid, malic acid, or fumaric acid these there are four kinds of organic acids of similarity or do not add citric acid, regrettably, gained table 4- table 1the result of 0 can not make us accepting completely, such as, and above-mentioned four kinds of organic acids or do not add citric acid gained table 4shown accuracy result is respectively 57 ~ 71%, 43 ~ 58%, 61 ~ 74%, 54 ~ 66%, 49 ~ 61%, and after these acid are used in display instead generally, measured results are well below the result of adding in theory.In addition, using the typical curve set up in above-mentioned four kinds of organic acid situations instead, its r value is respectively 0.993,0.986,0.991,0.994 and 0.985.Find in addition, when citric acid concentration drops to 30ug/ml or 10ug/ml, accuracy all lower than 85%, linearly all lower than 0.995; But when citric acid concentration is increased to 200ug/ml or 300ug/ml, exactness is all greater than 95%, but be linearly all less than 0.998; The amount of visible citric acid is too low or too high is all unfavorable.

3, the method for " two, the generality of pharmacokinetic describe " they " 6, employ the operation steps of following typical Criterion curve in this test " with reference to above, change methyl alcohol into unlike by the blank biological sample of pastille " the 100 μ L not " wherein and do not add blank plasma, 8 parts of standard models are obtained with method operation, the present invention HPLC-MS/MS mentioned above is used to measure, calculate the Imatinib chromatographic peak area of each standard model and the ratio (represent with R0n, wherein n represents the concentration of Imatinib relative to blood plasma) of interior mark peak area.In addition, calculate interpolation blank plasma process in " two, the generality of pharmacokinetic describe " above they " 6, employ the operation steps of following typical Criterion curve in this test " and obtain the Imatinib chromatographic peak area of 8 parts of standard models and the ratio (represent with R1n, wherein n represents the concentration of Imatinib relative to blood plasma) of interior mark peak area.For the sample of same concentration, be calculated as follows relative ratio R:

Relative ratio R=(R1n ÷ R0n) × 100%

Close to 100%, above-mentioned relative ratio R more shows that blood plasma is less on the impact measured.

Result shows, and according to the inventive method gained 8 concentration samples relative ratio R in 96.7 ~ 99.4% scopes, show that in the inventive method, blood plasma is minimum on the impact measured, and the R value of 8 concentration samples closely.

But, similarly, when the 75ug/ml citric acid added by 20 μ L methyl alcohol wherein changes isocyatic similar organic acid tartrate, maleic acid, malic acid or fumaric acid into or does not add citric acid, gained relative ratio R respectively in 76.3 ~ 81.6% scopes, in 62.7 ~ 76.5% scopes, in 67.8 ~ 81.3% scopes, in 69.4 ~ 80.5% scopes, in 65.6 ~ 73.1% scopes, even show similar organic acid, showing blood plasma when using other organic acid has obvious impact to mensuration; And when not adding organic acid under the very low dose medication condition of application claims result be also unsafty.

Visible according to above result, in the blood plasma using the inventive method to survey, Imatinib concentration and theoretical concentration are closely.

4, expressly (Wang Chenming etc. are offered with reference to Wang Chen, HPLC detects concentration and the pharmacokinetic studies thereof of Imatinib in rat plasma, Chinese Pharmaceutical Journal, 2013, 48 (4): 293) method of it " 2.3 sample preparation ", to replace the present invention " two above, the generality of pharmacokinetic describes " it " 5, the processing mode of following typical test sample is employed in this test " and " 6, employ the operation steps of following typical Criterion curve in this test " method process the present invention it " one, the generality of zero clinical trial phase research describes " in the blood sample (pole low content plasma drug) that obtains, other measures with the present invention it " ", " two, the generality of pharmacokinetic describes ".Result shows, table 4in the accuracy result of gained, the accuracy of 6 parts of samples is in 68 ~ 77% scopes, and the r value of the typical curve set up accordingly is 0.997, and method measures and asks the relative ratio R of 8 concentration samples of calculation in 79.6 ~ 80.4% scopes accordingly.Show to use document method can not meet the requirement of the extremely low determination of drug concentration of the present invention.

In addition, use above-mentioned Wang Chen expressly to offer " chromatographic condition " that it " 2.1 chromatographic condition " replace in the present invention " two, the generality of pharmacokinetic describe " they " 4, this test in employ following mass spectrum, chromatographic condition " time, can not effectively Imatinib and internal standard compound be separated.

5, with reference to Qiu's phase monarch document (Qiu Xiangjun etc., reversed-phased high performace liquid chromatographic detects the concentration of human plasma Imatinib, The Chinese Journal of Clinical Pharmacology, 2013, 29 (10): 777) method of it " 4.2 plasma sample process ", to replace the present invention " two above, the generality of pharmacokinetic describes " it " 5, the processing mode of following typical test sample is employed in this test " and " 6, employ the operation steps of following typical Criterion curve in this test " method process the present invention it " one, the generality of zero clinical trial phase research describes " in the blood sample (pole low content plasma drug) that obtains, other measures with the present invention it " ", " two, the generality of pharmacokinetic describes ".Result shows, table 4in the accuracy result of gained, the accuracy of 6 parts of samples is in 71 ~ 76% scopes, and the r value of the typical curve set up accordingly is 0.991, and method measures and asks the relative ratio R of 8 concentration samples of calculation in 68.4 ~ 77.6% scopes accordingly.Show to use document method can not meet the requirement of the extremely low determination of drug concentration of the present invention.

In addition, time " chromatographic condition " that use above-mentioned Qiu's phase monarch document it " 4.1 chromatographic condition " to replace in the present invention " two, the generality of pharmacokinetic describe " they " 4, this test in employ following mass spectrum, chromatographic condition ", can not effectively Imatinib and internal standard compound be separated.

6, with reference to Chinese high dawn offer (Gao Xiaohua etc., the Bioequivalence of two kinds of Imatinib preparations, in state is newmedicine and clinical journals, 2013, 32 (2): 145) method of it " process of plasma sample ", to replace the present invention " two above, the generality of pharmacokinetic describes " it " 5, the processing mode of following typical test sample is employed in this test " and " 6, employ the operation steps of following typical Criterion curve in this test " method process the present invention it " one, the generality of zero clinical trial phase research describes " in the blood sample (pole low content plasma drug) that obtains, other measures with the present invention it " ", " two, the generality of pharmacokinetic describes ".Result shows, table 4in the accuracy result of gained, the accuracy of 6 parts of samples is in 74 ~ 81% scopes, and the r value of the typical curve set up accordingly is 0.989, and method measures and asks the relative ratio R of 8 concentration samples of calculation in 65.7 ~ 78.5% scopes accordingly.Show to use document method can not meet the requirement of the extremely low determination of drug concentration of the present invention.

In addition, use above-mentioned high Chinese dawn to offer " chromatographic condition " that it " chromatographic condition " replace in the present invention " two, the generality of pharmacokinetic describe " they " 4, this test in employ following mass spectrum, chromatographic condition " time, can not effectively Imatinib and internal standard compound be separated.

It is below the generality description of Imatinib clinical medicine information.Imatinib, Imatinib, typically uses its mesylate clinically, and typical commercial dosage forms is the capsule provided by Novartis company, and trade name Gleevec (Glivec) every can be typically 100mg.Such as it is H20100238 at the import drugs registration certificate number of China.

Gleevec (Glivec) is used for the treatment of the chronic phase of the chronic myelogenous leukemia (Ph+CML) of Philadelphia Chromosome Positive, accelerated period or CML-BC clinically; Be used for the treatment of the adult patient that can not excise and/or occur the malignant gastrointestinal stromal tumors (GIST) shifted; Safety and effectiveness information spinner for following indication will from foreign study data, in compatriotsgroup's data are limited: the acute lymphoblastic leukemia (Ph+ALL) being used for the treatment of Philadelphia Chromosome Positive that is that adult is recurred or refractory.Be used for the treatment of the too much syndrome of acidophil (HES) and/or leukemia chronic eosinophilic (CEL) merges kinase whose adult patients with FIPILI-PDGFR α.Be used for the treatment of the adult patients of myelodysplastic syndrome/myeloproliferative disease (MDS/MPD) with platelet derived growth factor receptor (PDGFR) gene rearrangement.Be used for the treatment of aggressive systemic mastocytosis (ASM), without the adult patient of D816Vc-Kit gene mutation or unknown c-Kit gene mutation.Be used for the treatment of and can not excise, recurrence or occur transfer dermatofibrosarcoma protuberans (DFSP).For having the supplemental treatment of the adult patient of obvious risk of recurrence after Kit (CD117) positive GIST excision.The patient of extremely low and low risk of recurrence should not accept this supplemental treatment.

Usage and dosage aspect, imatinib mesylate should be taken at table, and drinks a mug water.Usual adult every mouthful once, each 400mg or 600mg, and oral consumption 800mg and 400mg dosage every day 2 times (in the morning and evening).Children and teenager is taken once a day or at twice (morning and evening).Can not patient's (comprising children) of swallowable capsule, medicine in capsule can be scattered in water or cider.Suggestion period of pregnancy and women breast-feeding their children, when opening capsule, avoid medicine and skin or eye contact, or suck, and should wash one's hands immediately after the capsule that contact is opened.The therapeutic dose adult of Ph+CML patient is 400mg/ day to the RD of chronic phase patient imatinib mesylate, and CML-BC and accelerated period patient are 600mg/ day.For the first-line treatment of the CML patient of WBC > 50000/ μ l, Couple herbs is only limitted to the patient once accepting the treatment of perhydroxyl radical urea.This treatment starts may need to add imatinib mesylate treatment.As long as effectively, just answer SM.If do not have severe drug bad reaction and blood picture license, dosage can be considered to be increased to 600mg/ day from 400mg/ day in situations, or is increased to 800mg/ day from 600mg/ day; Occur that any time progression of disease, treatment fail after at least 3 months to obtain satisfied hematology reaction, treated and within 12 months, do not obtain any cytogenetic response, acquired hematology and/or cytogenetic response and again disappear.More than 3 years old children and the teenager efficacy and saferry that children's clinical data is limited both at home and abroad a few days ago, child patient tightly monitored by need, adjusts dosage if desired in time.This product is used for more than 3 years old children and teen-age safety and effectiveness information spinner will from external clinical data.According to the dosage of adult, recommendation mouth dosage is: chronic phase 260mg/m2 (maximum dose: 400mg), accelerated period and CML-BC 340mg/m2 (maximum dose: 600mg) work out RD every day of child patient, calculate gained dosage and generally should be adjusted to whole hundred milligrams up and down, the dosage of less than 12 years old children generally should be adjusted to whole 50 milligrams up and down.There is no the experience of less than 3 years old children therapy.The therapeutic dose of Ph+ALL patient is to recurrent intractable adult Ph+ALL patient, and the RD of imatinib mesylate is 600mg/ day.The therapeutic dose of GIST patient is to the malignant GIST patient that can not excise and/or shift, and the RD of imatinib mesylate is 400mg/ day.Fail after the treatment to obtain satisfied reaction, if do not have serious adverse drug reaction, dosage can be considered from being increased to 600mg/ day or 800mg/ day 400mg/ day.For GIST patient, imatinib mesylate answers continued treatment, unless disease progression.It is 400mg/ day to the RD of adult patient supplemental treatment after GIST complete excision.In clinical research, Imatinib administration time is 1 year.The best duration of Imatinib supplemental treatment it be unclear that.Dosage this product of HES/CEL patient is used for HES/CEL and treats RD Main Basis foreign study report dosage.FIPILI-PDGFR-α is existed for proof and merges kinase whose HES/CEL, recommend initial dose to be 100mg/ day.If confirm not obtain enough alleviations through suitable detection after treatment, and have no adverse reaction. 100mg/ daily dose can be considered to increase to 400mg/ day.Dosage this product of ASM patient is used for ASM and treats RD Main Basis foreign study report dosage.Without the ASM adult patient of D816Vc-Kit sudden change, imatinib mesylate treatment RD foot 400mg/ day.If c-Kit catastrophe the unknown of ASM patient maybe cannot record, when using other therapies can not obtain satisfied alleviation, should consider that giving imatinib mesylate 400mg/ day treats.With the ASM patient of eosinophilia (a kind of merge the relevant Clonal disease in the blood system of kinases with FIP1L1-PDGFR-α), imatinib mesylate recommends initial dose to be 100mg/ day.If confirm not obtain enough alleviations through suitable detection after treatment, and have no adverse reaction, 100mg dosage can be considered to increase to 400mg.Dosage this product of MDS/MPD patient is used for MDS/MPD and treats RD Main Basis foreign study report dosage.The imatinib mesylate dosage that the atypia MDS/MPD patient of adult's Hypereosinophilic syndrome and PDGFR-α or-β gene rearrangement recommends is 400mg/ day.Therapeutic dose this product of DFSP patient is used for DFSP and treats RD Main Basis foreign study report dosage.The RD of adult DFSP patient imatinib mesylate treatment is 400mg/ day.When needing, dosage can rise to 800mg every day.If occur that the adjustment of bad reaction post dose accepts to occur serious non-blood bad reaction (as serious water retention) in imatinib mesylate treatment process, answer drug withdrawal, until bad reaction disappears, and then adjust dosage according to the order of severity of this bad reaction.During major Liver toxicity, adjustment such as the cholerythrin of dosage raises the > normal range upper limit 3 times or the aminopherase rising > normal range upper limit 5 times, imatinib mesylate should be cut out, until These parameters drops to less than 1.5 or 2.5 times of the normal range upper limit respectively.Later imatinib mesylate treatment can continue to take after decrement.Every daily dose of being grown up should reduce to 300mg from 400mg, or reduces to 400mg from 600mg or be reduced to 600mg from 800mg; Children and teenager reduces sword 200mg/m2 from 260mg/m2 or reduces to 260mg/m2 from 340mg/m2.The adjustment Ph+CML accelerated period of dosage or CML-BC when Neutrophilic granulocytopenia or decrease of platelet, Ph+ALL (initial dose 600mg/ day, or Children and teenager 340mg/m2/ day): if there is serious neutrophil leucocyte and decrease of platelet (neutrophil leucocyte <0.5 × 109/L and/or blood platelet <10 × 109/L), cytopenia relevant with leukaemia (bone marrow extraction or biopsy) should be determined whether, if cytopenia is not caused by leukaemia, recommended doses reduces to 400mg/ day or Children and teenager 260mg/m2/ day.If haemocyte reduces continue 2 weeks, then reduce dosage further to 300mg/ day or Children and teenager 200mg/m2/ day, continue 4 weeks as haemocyte reduces, answer drug withdrawal, until neutrophil leucocyte >=1 × 109/L and blood platelet >=20 × 109/L.Used time dosage is 300mg/ day again; Or Children and teenager 200mg/m2/ day.CML chronic phase and GIST patient's (initial dose 400mg/ day or Children and teenager 260mg/m2/ day): answer drug withdrawal as neutrophil leucocyte <1.0 × 109/L and/or blood platelet <50 × 109/L, in neutrophil leucocyte >=1.5 × 109/L and blood platelet >=75 × 109/L time just can recover medication, treatment can revert to dosage 400mg/ day or Children and teenager 260mg/m2/ day.If again there is critical numerical value (neutrophil leucocyte <1.0 × 109/L and/or blood platelet <50 × 109/L), the therapeutic dose again of having no progeny in treatment reduces to 300mg/ day or Children and teenager 200mg/m2/ day.HES/CEL (initial dose is 100mg/ day): answer drug withdrawal as neutral absolute granulocyte count (ANC) <1.0 × 109/L and/or blood platelet <50 × 109/L, just can recover medication when neutrophil leucocyte ANC >=1.5 × 109/L and blood platelet >=75 × 109/L.Again administration can be started with dosage (namely the dosage before serious adverse events occur) before.ASM (initial dose 100mg/ day): answer drug withdrawal as neutrophil leucocyte ANC<1.0 × 109/L and/or blood platelet <50 × 109/L, just can recover medication when neutrophil leucocyte ANC >=1.5 × 109/L and blood platelet >=75 × 109/L.Again administration can be started with dosage (namely the dosage before serious adverse events occur) before.HES/CEL, ASM, MDS/MPD (initial dose is 400mg/ day): answer drug withdrawal as neutrophil leucocyte <1.0 × 109/L and/or blood platelet <50 × 109/L, in neutrophil leucocyte >=1.5 × 109/L and blood platelet >=75 × 109/L time just should recover medication, therapeutic dose 400mg/ day again.If again there is critical numerical value (as neutrophil leucocyte <1.0 × 109/L and/or blood platelet <50 × 109/L), therapeutic dose should be reduced to 300mg again.DFSP (dosage 800mg/ day) answers drug withdrawal as neutrophil leucocyte <1.0 × 109/L and/or blood platelet <50 × 109/L, in neutrophil leucocyte >=1.5 × 109/L and blood platelet >=75 × 109/L time just can recover medication, therapeutic dose 600mg/ day again.If again there is critical numerical value (as neutrophil leucocyte <1.0 × 109/L and/or blood platelet <50 × 109/L), therapeutic dose should be reduced to 400mg again.The dosage of hepatic disorder patient is light, moderate hepatic disorder person recommendation minimum dose 400mg/ days.There is no the data information that serious hepatic disorder patient (3 times of cholerythrin > normal range) using dosage is 400mg/ days at present.These patients after conscientiously weighing risk assessment, should re-use this product.The kidney of the dosage Imatinib of patients with renal failure is removed and can be ignored.For this reason, estimate not reduce the systemic clearance of kidney function damage patient.But, still need to pay special attention to the patient of severe renal functional lesion.The dosage of gerontal patient does not adjust dosage especially to gerontal patient.The adult patients of plate derived growth factor receptor (PDGFR) gene rearrangement.

In drug interaction, can change the medicine CYP3A4 inhibitor of Imatinib plasma concentration: after health volunteer takes single dose ketoconazole (CYP3A4 inhibitor) simultaneously, the drug exposure of Imatinib significantly increases (average maximum plasma concentration (Cmax) and Imatinib area under curve (AUC) can increase by 26% and 40% respectively).There is no the experience simultaneously taken with other CYP3A4 inhibitor (as: Itraconazole, erythromycin and CLA).CYP3A4 derivant: after healthy volunteer takes rifampin, the removing of Imatinib increases by 3.8 times (90% credibility interval=3.5-4.3 is doubly), but Cmax, AUC (0-24), with AUC (0-8), decline 54%, 68% and 74% respectively.Find in clinical studies, after giving phenytoinum naticum medicine, the plasma concentration of Imatinib reduces simultaneously, thus causes curative effect to lower.Taking the antiepileptic (enzyme-inducinganti-epilepticdrugs of enzyme induction, EIAEDs) as carbamazepine, Oxcarbazepine, phenytoinum naticum, Fosphenytoin, phenobarbital and primoline, accept also to observe similar result in the glioblastoma patient of this product treatment simultaneously.With take compared with EIAEDs time different, the AUC of Imatinib is down to 73%, and other CYP3A4 derivant, as dexamethasone, catarrh miaow piperazine, phenobarbital etc., may have Similar Problems, Imatinib and CYP3A4 derivant therefore should be avoided simultaneously to take.In two researchs delivered, Imatinib and the AUC decline 30-32% that can cause this product when share containing StJohn wheat juice medicinal extract preparation.Imatinib mesylate can make agents change plasma concentration Imatinib make the mean Cmax of simvastatin (CYP3A4 substrate) and AUC increase by 2 times and 3.5 times respectively.The plasma concentration that Imatinib can increase the other drug (as Benzodiazepines, two pyridinium hydroxide, calcium-channel antagonists and other HMG-CoA reductase inhibitor etc.) through CYP3A4 metabolism should be kept in mind.Should be careful when therefore taking this medicine and treatment narrow CYP3A4 substrate (as cyclosporine, the Pimozide) of window simultaneously.Under the concentration similar to suppressing CYP3A4 activity, Imatinib also can suppress the activity of CYP2D6 in vitro, therefore when taking with imatinib mesylate simultaneously, likely increases system to the exposed amount of CYP2D6 substrate, although not yet specialize in, suggestion is cautious use of.Imatinib also can suppress the activity of CYP2C9 and CYP2C19 in vitro, can see prolonged prothrombin after taking warfarin simultaneously.Therefore when the whole story or the change dosage of imatinib mesylate treatment, if simultaneously with bicoumarin, answer Short-Term Monitoring prothrombin time.Imatinib 400mg dosage every day 2 times is very weak to the inhibiting effect of the metoprolol metabolism of CYP2D6 induction, Cmax and AUC of metoprolol approximately increases by 23%.Imatinib and CYP2D6 derivant, as Metoprolol In Combination, seem not exist the hazards of drug drug interaction, can adjust dosage.Experiment in vitro shows, Imatinib can suppress the O-glucuronic acid of paracetamol.Patient should be warned to avoid using the non-prescribed medicine containing paracetamol and prescription medicine.

Pharmacological action aspect, Imatinib all can suppress Bcr-Ab1 tyrosine kinase in vivo and in vitro on a cellular level, can Selective depression Bcr-Ab1 positive cell line cell, the chronic myelogenous leukemia (CML) of Philadelphia Chromosome Positive (Ph+) and the fresh cells of Patients With Acute Lymphoblastic Leukemia propagation and induce its apoptosis.In addition, Imatinib also can suppress platelet derived growth factor (PDGF) acceptor, stem cell factor (SCF), the tyrosine kinase of c-Kit acceptor, thus suppresses the cell behavior by PDGF and stem cell factor mediation.The active kit of gastrointestinal stromal tumors (GIST) cellular expression owes to become, and experiment in vitro display Imatinib suppresses the propagation of GIST cell and induces its apoptosis.Have seldom at the report of clinical generation resistance, about the generation of imatinib-resistant, Initial drug resistence (from treatment and invalid) and the difference of secondary resistance be show in the exposure process of whole Imatinib invalid, Bcr-Ab1 tyrosine kinase, increase in disease process, be the molecular mechanism producing resistance.In the patient for the treatment of, produce resistance incidence very low, or do not take medicine as requested.Therefore, treatment should start as early as possible, and dosage should strictly be taken on request simultaneously.

Toxicological study aspect, after Imatinib long-term treatment, the incidence of rat opportunistic infections increases, and in monkey body, the usual repressed malaria infection state of an illness increases the weight of.Genetoxic is tested an external bacterium (Amcstcst), in a mammalian cells in vitro analysis (mouse lymph lymphoma experiment) and a body in people mouse micronucleus test, Imatinib does not all show any genetoxic.In external mammalian cell clastogenisity (clastogenicity) test (Cinese hamster ovary cell chromosome aberration), when the present age is activated, Imatinib can cause positive findings.Produce because of production run two intermediate products appeared in finished product show mutagenicity in Ames experiment, and one of them intermediate product is also positive in mouse lymphoma assay.In genotoxicity fertility test, within continuous 70 days, give male rat 60mg/kg (being about equivalent to maximum clinical dosage 800mg/ days), the weight saving of testis and epididymis, the mobility of sperm reduces simultaneously.During dog oral dose > 30mg/kg, the generation also observing its sperm has light to moderate reduction.In the fertility research of a female rats, the quantity of mating and pregnant mouse does not change, but when dosage 60mg/kg instead of≤20mg/kg, after implanting, the death of fetus obviously increases, and tire number of simultaneously living reduces.In the perinatal growth research of rat, orally give 45mg/kg/ days, after the quantity of stillborn foetus and birth, between the 0th day to the mat woven of fine bamboo strips 4 days, dead quantity increases.F1 generation newborn mouse gives same dosage, dissects from birth to terminal, and average weight reduces.The fertility of F1 generation is not affected, but notices the quantity increase that dosage group absorbed in 45mg/kg/ days, and the quantity of the fetus that simultaneously can give birth to reduces.Mother gives 45mg/kg/ days for animal, and F1 generation gives 15mg/kg/ days (1/4 of clinical maximum dose 800mg), is the dosage level of free of toxic effects.Give rat Imatinib >=100mg/kg in organogenetic period and have teratogenesis, this dosage is about equivalent to 1.5 times of clinical maximum dose 800mg days.Teratogenesis comprises exencephalia and Naoning tablet, and disappearance/defect frontal bone and/or disappearance parietal bone.Above-mentioned effect is not observed in≤30mg/kg group.Carcinogenicity is in the rat carcinogenicity research of 2 years by a definite date, Imatinib dosage regimen is 15,30 and 60mg/kg/ days, the life-span of the male rat of result 60mg/kg/ days groups and the female rats of >=30mg/kg/ days group significantly shortens, and shows obvious statistical significance.The histopathological findings of rat cadavers shows that cardiomyopathy (male and female), chronic progressive renal disease (female) and preputial gland papilloma are the main causes of the death.Occur that the target organ of tumour change has kidney, bladder, urethra, preputial gland and cloudy band gland, small intestine, parathyroid gland, adrenal gland and stomach Non-gland body district.With not observing poisonous effect level in the target organ of tumoral lesion (without visible amounts of reactants, NOEL) dosage is: kidney, bladder, urethra, small intestine, parathyroid gland, adrenal gland and stomach Non-gland body district 30mg/kg/ days, preputial gland and glans clitoridis 15mg/kg/ days.Preputial gland/glans clitoridis/incidence of papilloma/cancer knurl is more obvious when 30 and 60mg/kg/ days dosage levels, be equivalent to 0.5-4 or 0.3-2.4 of people's exposed amount every day 400mg/ days or 800mg/ days dosage levels doubly (evaluate according to AUC), the dosage level of 340mg/m2 is then equivalent to the 0.4-3.0 of children's (evaluating according to AUC) exposed amount every day doubly.During 60mg/kg/ days dosage levels, kidney adenoma/cancer knurl, bladder and papilloma of urethra, intestinal glands, parathyroid adenoma, adrenal optimum and pernicious medullary substance tumour and Non-gland body stomach papilloma/cancer knurl easily occur.Above rat carcinogenicity result of study. not quite clear at present with the relevance of the mankind, the data of safety analysis coming from clinical testing and the report of spontaneous adverse events not yet proves that the Cancer Mortality of the patient accepting treatment with imatinib is higher than general population.The non-tumoral lesion of cardiovascular system, pancreas, endocrine organ and tooth is not yet confirmed before early clinic in test.In some animal, the most important sign of cardiac insufficiency is caused to comprise myocardial hypertrophy and Heart enlargement.

Pharmacokinetics aspect, the pharmacokinetics of Imatinib is at 25-1000mg dosage range, at single dose with reach stable state postevaluation.Imatinib dosage is within the scope of 25-1000mg, and under its averaged curve, the increase of area (AUC) and dosage exist proportionate relationship.The drug accumulation amount of repeat administration when reaching stable state is 1.5 ~ 2.5 times.The mean absolute bioavailability absorbing Imatinib is 98%, and the coefficient of variation fluctuation of oral rear blood plasma Imatinib AUC is between 40-60%.With compare on an empty stomach, after high fat diet, this medicine absorptivity slightly reduces (Cmax reduce 11%, tmax delay 1.5 hours), and AUC slightly reduces (7.4%).Distribution about 95% is combined with plasma proteins, and the overwhelming majority is combined with albumin, and small part is combined with acid seromucoid, only has few part to be combined with lipoprotein.Population distribution concentration in whole body is higher, and distribution volume is 4.9L/kg body weight, but in red blood cell, distribution ratio is lower.In in-vivo tissue, relevant drug distribution situation only derives from preclinical data.Absorb in adrenal gland and sexual gland level is high, absorb level in central nervous system low.In metabolism human body, main cyclic metabolism product is N-demethyl bridged piperazine derivatives, and its drug effect is similar to former medicine in vitro.The plasma A UC of this metabolin is 16% of former medicine imatinib mesylate AUC.Imatinib is the substrate of CYP3A4, is again the inhibitor of CYP3A4, CYP2D6, CYP2C9 and CYP2C19, therefore, can affect the metabolism (see [drug interaction]) of combination with medication.The elimination half life period of eliminating Imatinib is 18 hours, and its active metabolite half life period is 40 hours, about can drain 81% of institute's drug dosage in 7 days, wherein from ight soil, drains 68%, drains 13% in urine.About 25% is former medicine (in urine 5%, in stool 20%), and all the other are metabolic product, and in ight soil and urine, active metabolite and former medicine is in similar proportion.The pharmacokinetics Adult group pharmacokinetic of particular patients ' group shows, gender on pharmacokinetics is without impact, and the impact of body weight also can be ignored.Give same dosage (400mg/ days), drug exposure during its stable state of GIST patient is 1.5 times of CML patient.Population pharmacokinetics according to preliminary GIST patient is studied, and the pharmacokinetics of Imatinib has the change of 3 indexs (albumin, WBC and cholerythrin) to have a significant impact statistically.Low albumin level reduces to be removed, as higher WBC level.But these impacts are not sufficient to conclude that dosage needs adjustment.The using dosage of children Children and teenager 260mg/m2 and 340mg/m2 can produce same drug exposure, is equivalent to 400mg and 600mg be grown up respectively.With the dosage of 340mg/m2/ days after repeat administration once a day, the AUC (0-24) of the 8th day and the 1st day) than the drug accumulation demonstrating 1.7 times.Old medication it is reported the clinical study results being greater than 20% a patient more than 65 years old, and the age does not significantly affect pharmacokinetics.Organ replication Imatinib and metabolic product thereof f hardly pass through renal excretion.Gently, the plasma exposure amount of moderate renal insufficiency patient a little more than the patient of normal renal function, increase about 1.5-2 doubly, increase by 1.5 times with plasma A GP level and conform to, AGP can with Imatinib strong bonded.Because Imatinib is hardly through renal excretion, therefore the Imatinib of renal insufficiency and normal renal function patient person former medicine clearance rate basic simlarity.Although pharmacokinetic results shows individual difference, compared with the normal patient of liver function, the mean exposure measurement of patient to Imatinib with dyshepatia in various degree has no increase.

Claims

1. measure the method for Imatinib or its pharmaceutical salts in biological sample, the method uses high performance liquid chromatography-tandem mass to measure, and comprises the following steps and test condition:

(1) process of sample is tested:

Get 100 μ L pastille biological samples in 1.5mL pipe, add the methanol solution 20 μ L containing 50 ~ 100ug/ml citric acid, add the interior mark Xi Gelieting solution that 20 μ L concentration are 300ng/mL, vortex mixes, add 100 μ L methyl alcohol and 200mL acetonitrile, vortex 50 seconds, in 4 DEG C with the centrifugal 5min of 14000rpm, getting supernatant 200 μ l is transferred in 1.5mL sample injection bottle, to be detected as test sample;

(2) Specimen Determination:

(3) liquid phase chromatogram condition:

Chromatographic column: PhenomeneC18100A post, post specification is 3.0 × 50mm; Mobile phase: the acetonitrile solution containing 0.1% formic acid: containing 2mmol/L ammonium acetate and 0.1% first aqueous acid, gradient elution, flow velocity: 0.4mL/min, sample size: 20 μ L;

Described gradient elution program is: 5:95,1min; 90:10,3.5min; 90:10,4min; 5:95,4.1min; 5:95,6min;

(4) Mass Spectrometry Conditions:

Ion gun: ion spray ionisation source; Ion injection electric: 5500V; Temperature: 550 DEG C; Gas 1N in source ₂pressure: 50psi; Gas 2N in source ₂pressure: 50psi; Curtain gas N2 pressure: 20psi; Positive ion mode detects; Scan mode: Selective ion mode reaction monitoring mode;

Ionic reaction for quantitative test is respectively: m/z494.3 → m/z394.1, and it is Imatinib; With m/z408.2 → m/z235.0, it is interior mark.

2. method according to claim 1, the pharmaceutical salts of described Imatinib is selected from imatinib mesylate, hydrochloric acid Imatinib, phosphoric acid Imatinib, toluenesulfonic acid Imatinib.

3. method according to claim 1, described biological sample is selected from blood plasma, urine, saliva.

4. method according to claim 1, the concentration of the Imatinib comprised in described pastille biological sample is lower than 100ng/ml.

5. method according to claim 1, the concentration of the Imatinib comprised in described pastille biological sample is lower than 75ng/ml.

6. method according to claim 1, the concentration of the Imatinib comprised in described pastille biological sample is lower than 50ng/ml.

7. method according to claim 1, the concentration of the Imatinib in the pastille biological sample that described high performance liquid chromatography-tandem mass can detect is higher than 0.1ng/ml.

8. method according to claim 1, the concentration of the Imatinib in the pastille biological sample that described high performance liquid chromatography-tandem mass can detect is higher than 0.2ng/ml.

9. method according to claim 1, the concentration of the Imatinib in the pastille biological sample that described high performance liquid chromatography-tandem mass can detect is higher than 0.3ng/ml.

10. method according to claim 1, the concentration of the Imatinib in the pastille biological sample that described high performance liquid chromatography-tandem mass can detect is higher than 0.4ng/ml.

11. methods according to claim 1, in described (2) Specimen Determination, the concentration calculating the Imatinib in described biological sample adopts Internal standard curve method to calculate.

12. methods according to claim 11, described Internal standard curve method also comprises the step of Criterion curve.

13. methods according to claim 12, described Criterion curve comprises the following steps:

Get the blank biological sample 8 parts of 100 μ L not pastille, be placed in 1.5mL pipe respectively, each pipe adds the methanol solution 20 μ L containing 50 ~ 100ug/ml citric acid respectively, add the interior mark Xi Gelieting solution that 20 μ L concentration are 300ng/mL respectively, Imatinib or its pharmaceutical salts is added respectively with Imatinib 0.04ng in the form of stock solution, 0.1ng, 0.2ng, 0.5ng, 1ng, 2ng, 3ng, 4ng, by each pipe vortex mixing, add 100 μ L methyl alcohol and 200mL acetonitrile respectively, vortex 50 seconds, in 4 DEG C of centrifugal 5min of 14000rpm, getting supernatant 200 μ l is transferred in 1.5mL sample injection bottle, as 8 parts of standard models, to be detected,

14. methods according to claim 13, in described stock solution Imatinib or its pharmaceutical salts in Imatinib concentration for 1 ~ 1000ng/ml.

15. methods according to claim 13, in described stock solution Imatinib or its pharmaceutical salts in Imatinib concentration for 2 ~ 500ng/ml.

16. methods according to claim 13, in described stock solution Imatinib or its pharmaceutical salts in Imatinib concentration for 4 ~ 400ng/ml.

17. methods according to claim 13, when Criterion curve, blood plasma drug concentration in Imatinib in 0.4 ~ 40ng/ml concentration range, by internal standard method with calculated by peak area, drug concentration and chromatogram response intensity and linear between active substance and interior target peak area ratio, and r value >0.9990.

18. methods according to claim 17, r value >0.9992.

19. methods according to claim 17, r value >0.9995.

20. methods according to claim 17, r value >0.9998.

21. methods according to claim 13, described stock solution methyl alcohol is solvent preparation.

22. methods according to claim 13, described inner mark solution methyl alcohol is solvent preparation.

23. methods according to claim 1, described biological sample takes from mammiferous biological sample.

24. methods according to claim 23, described mammal is people.

25. methods according to claim 1, described pastille biological sample is the biological sample taking from people, and the oral Imatinib of this people or its pharmaceutical salts count 1 ~ 10mg with Imatinib.

26. methods according to claim 24, described pastille biological sample gathers 0 ~ 48 hour period after oral Imatinib or its pharmaceutical salts count 1 ~ 10mg with Imatinib described mammal people.

27. methods according to claim 24, described pastille biological sample gathers 0 ~ 36 hour period after oral Imatinib or its pharmaceutical salts count 2 ~ 8mg with Imatinib described mammal people.

28. methods according to claim 24, described pastille biological sample gathers 0 ~ 24 hour period after oral Imatinib or its pharmaceutical salts count 3 ~ 6mg with Imatinib described mammal people.

29. methods according to claim 24, described pastille biological sample gathers 0 ~ 24 hour period after oral Imatinib or its pharmaceutical salts count 4mg with Imatinib described mammal people.

30. methods according to claim 24, described pastille biological sample be described mammal people after oral Imatinib or its pharmaceutical salts count 4mg with Imatinib 0,0.5,1,1.5,2,2.5,3,4,6,8,10,12,24h time gather.

31. methods according to claim 1, described pastille biological sample is the biological sample taking from people, and this body weight for humans is 50 ~ 80kg.

32. according to the method for any one of claims 1 to 31, and it is the pharmacokinetic in mammalian body for Imatinib or its pharmaceutical salts.

The application of method described in 33. any one of claims 1 to 31 in zero clinical trial phase of Imatinib or its pharmaceutical salts.

34. according to the application of claim 33, and wherein said zero clinical trial phase is that men's health experimenter single oral Imatinib or its pharmaceutical salts are in the research carried out after Imatinib 1 ~ 10mg.

35. according to the application of claim 33, and wherein said zero clinical trial phase is that men's health experimenter single oral Imatinib or its pharmaceutical salts are in the research carried out after Imatinib 2 ~ 8mg.

36. according to the application of claim 33, and wherein said zero clinical trial phase is that men's health experimenter single oral Imatinib or its pharmaceutical salts are in the research carried out after Imatinib 3 ~ 6mg.

37. according to the application of claim 33, described zero clinical trial phase be men's health experimenter single oral Imatinib or its pharmaceutical salts in the research carried out after Imatinib 1 ~ 10mg, this research comprises pharmacokinetic trial.

38. according to the application of claim 33, described zero clinical trial phase be men's health experimenter single oral Imatinib or its pharmaceutical salts in the research carried out after Imatinib 2 ~ 8mg, this research comprises pharmacokinetic trial.

39. according to the application of claim 33, described zero clinical trial phase be men's health experimenter single oral Imatinib or its pharmaceutical salts in the research carried out after Imatinib 3 ~ 6mg, this research comprises pharmacokinetic trial.

The application of method described in 40. any one of claims 1 to 31 in the pharmacokinetic trial research of Imatinib or its pharmaceutical salts.

41. according to the application of claim 40, and described pharmacokinetic trial research is that men's health experimenter single oral Imatinib or its pharmaceutical salts are in the research carried out after Imatinib 1 ~ 10mg.

42. according to the application of claim 40, and described pharmacokinetic trial research is that men's health experimenter single oral Imatinib or its pharmaceutical salts are in the research carried out after Imatinib 2 ~ 8mg.

43. according to the application of claim 40, and described pharmacokinetic trial research is that men's health experimenter single oral Imatinib or its pharmaceutical salts are in the research carried out after Imatinib 3 ~ 6mg.