65 results about "Liver microsomes" patented technology

The invention provides a cultivation method of Cyp gene knocked-out rats, and a preparation method of liver microsome of the rats. The Cyp gene knock-out herein includes Cyp single gene knock-out and Cyp multiple gene knock-out. In the method, firstly a Cyp gene knocked-out rat is constructed by means of a CRISPR/Cas system, which includes selection of a knocked-out target site, in-vitro synthesis and transcription of sg RNA and Cas9m RNA, preparation of a pseudopregnant female rat, in-vitro micro-injection and transplanting of a single-cell embryo, and cultivation of the rat, and finally, a homozygote Cyp gene knocked-out rat can be cultured. Furthermore, the liver of the Cyp gene knocked-out rat is extracted and is subjected to homogenization and differential centrifugation to prepare the liver microsome of the rat in Cyp gene deletion. The invention also provides an application of the Cyp gene knocked-out rats and the liver microsome thereof in study on drug metabolism.

The invention discloses an oxadiazole-contained cyclic compound, a preparation method, intermediates, compositions and application thereof. The medically acceptable salts, the enantiomer, the diastereoisomer, the tautomer, the solvate, the metabolite or the prodrug of the dthe oxadiazole-contained cyclic compound shown as the formula I is high in inhibitory activity on IDO (dioxygenase) at a molecular level and a cellular level and significant in proliferation inhibition effects on cancer cells related to IDO activity at an animal level and good in liver microsome stability of human, mouse andthe like, has no significant inhibition over metabolic enzymes and achieves good absorption properties and high bioavailability in rats and mice as well as good druggability.

The invention relates to a detection method for simultaneously determining the metabolic products of seven CYP450 enzyme probe substrate in human liver microsomes, and belongs to the technical field of biological detection. The detection method comprises the following steps: performing incubation on specificity probe substrates of CYP450 enzyme and the human liver microsomes for different periods of reaction time to generate corresponding metabolic products, using Zaltoprofen as an interior label, adopting a high performance liquid chromatography-tandem mass spectrometry after protein precipitation pretreatment, and thus simultaneously detecting the concentrations of the metabolic products of seven probe substrates. The method disclosed by the invention is high in specificity, high in sensitivity and simple and convenient in operation, and has already successfully been applied to the research of baicalin on CYP450 enzyme inhibiting action of the human liver microsomes.

The invention relates to a preparation method, a detection method and application of a probe drug composition for determination of metabolic activity of cytochrome P450. The composition mainly comprises a preparation made with a specific probe with major isoforms of CYP450, i.e. CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, as an active component. Cocktail probe drug solution is prepared, the probe drug composition is injected into an animal or liver microsomes for in vitro co-incubation, and the concentration of each probe drug is determined to assess the metabolic activity of the CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. In the early stage of research and development of new drugs, the effects of the drugs on the activity of each isoform of the cytochrome P450 are screened in a high-throughput way, and the interactions of the drugs can be predicted. In the stage of clinical research, the testing can be performed with the probe drug composition in an in-vivo probe method, and the effects of the drugs on the in-vivo metabolic activity of different isoforms of the human liver CYP450 can be examined.

The invention relates to an LC-MC method used for determining NNK metabolites in liver microsomes and specifically to an LC-MC method used for analyzing and determining 4-(methyl nitroso)-1-(3-pyridyl)-1-butanone (NNK) metabolites in liver microsomes, which belongs to the field of biochemical analysis. The method provided in the invention mainly comprises the following steps: (1) preparation of an incubation system for liver microsomes; (2) sample treatment; (3) sample analysis and determination of NNK metabolites in the liver microsomes by using the LC-MC method. The invention has the following advantages of simple pre-treatment, a fast analysis speed, good selectivity, high detection sensitivity and capacity of analyzing and comparing the metabolism of NNK in different kinds of liver microsomes, can lay a methodological foundation for further research on in-vivo and in-vitro metabolism of NNK and other nitrosamine compounds and has critical significance.

The invention relates to an absolute quantitative method for biomass spectra of CYP450 enzyme hypotypes, and belongs to the technical field of proteomics. In the method, on the basis of liquid chromatography (LC)/mass spectrometry (MS)/MS, the absolute quantification of four CYP450 enzyme hypotypes, namely CYP1A2, CYP2B6, CYP3A4 and CYP3A5 in systems such as liver microsome and the like is realized simultaneously by a strategy that CYP450 generates specific peptide quantitative enzymes by pancreatic enzyme hydrolysis. The method mainly comprises the following steps of: preprocessing a proteinsample, preparing a standard curve, measuring the protein sample and preparing a quality control sample. The absolute quantitative method has excellent linear relations, is high in accuracy and repeatability, is sensitive, precise and reliable, and can be used for medicinal metabolism and the evaluation of the mutual effect of medicaments.

The invention provides 2, 3, 5, 7-tetrasubstituted dihydro-pyrazolo piperidine derivative and a preparation method and application thereof. The derivative is 2, 3-bis(substituted phenyl)-5-subsituted arylmethyl-7-substituted benzylidene dihydro-pyrazolo piperidine derivative, having the following formula (I). The preparation method includes using substituted arylmethyl amine and methyl acrylate as raw materials; subjecting the materials to Michael addition, Dieckmann condensation and hydrolysis-decarboxylation sequentially; allowing for Aldol reaction with substituted benzaldehyde to obtain intermediate N-substituted arylmethyl-3, 5-bis(substituted benzylidene)-4-piperidone; allowing for condensation with substituted phenylhydrazine to obtain a compound according to the formula (I). The derivative is efficient in inhibiting multiplication of various carcinoma cell lines such as leukemia, esophagus cancer, ovarian cancer and breast cancer in human, is well stably metabolic in liver microsomes of human and rat, is free of direct and competitive inhibition on five enzymes of liver microsomes, such as CYP3A4, CYP2D6, CYP2C9, CYP1A2 and CYP2C19, is highly bioavailable, is low in toxicity to normal cells, and is available for the preparation of drugs for the cancers.

The invention discloses liver microsome metabolism analysis of koumine (KM) and toxicokinetics of related animals. The method disclosed by the invention mainly comprises the following steps: 1, performing KM liver microsome metabolism analysis comprising the following sub-steps: (1) establishing a KM incubation system; (2) performing sample treatment; and (3) performing sample analysis, namely selecting KM human related animals by comparing the liver microsome metabolism product distribution characteristics of KM in various experimental animals and the kinetic characteristics of various kinds of KM on KM metabolism; 2, performing KM macaca rhesus acute toxicity associated toxicokinetics comprising the following sub-steps: (1) performing blood sample collection and plasma sample treatment on infected experimental animals; (2) performing quantitative determination; and (3) performing blood concentration data analysis. The method has the advantages that due to the in-vitro liver microsome metabolism analysis of KM in various experimental animnals, the human related animals are selected for performing associated toxicokinetics study, so that the predictability of toxicity assessment of the experimental animals on clinical study is improved.

The invention provides application of tetrahydropalmatine enantiomers in preparation of P-glycoprotein inhibitor. The tetrahydropalmatine is one of main active ingredients of the Chinese herbal medicine rhizoma corydalis and comprises L-tetrahydropalmatine and D-tetrahydropalmatine. The research shows that the tetrahydropalmatine enantiomers can obviously inhibit the P-gp function, but the tetrahydropalmatine enantiomers are not the P-gp substrates; the tetrahydropalmatine enantiomers can obviously increase the concentration of anti-tumor medicine adriamycins in medicament-resistant tumor cells and reverse the multi-medicament resistance of tumor cells and do not have obvious effects of inhibiting and inducing main medicament metabolic enzymes of liver microsomes, so that the racemes, laevo isomers or dextro isomers of the tetrahydropalmatine enantiomers can be used as auxiliary medicaments for tumor chemotherapy, and the range of indications of the tetrahydropalmatine is expanded; and simultaneously, the invention provides the medicament for the assistant treatment for tumors, which alleviates pain, improves curative effect and is not addictive, for tumor patients.

The invention belongs to the technical field of medicine detection, discloses an LC-MS/MS (liquid chromatography-tandem mass spectrometry) method for determining cordycepin metabolite in liver microsome, and particularly relates to the LC-MS/MS method for analyzing and determining the cordycepin metabolite in the liver microsome. The LC-MS/MS method mainly includes the steps of (1) incubation of the liver microsome containing cordycepin; (2) preprocessing of a sample; (3) analysis of the sample, thereby determining the cordycepin metabolite in the liver microsome. The LC-MS/MS has the advantages of convenience in operation, sample processing simplicity, high analysis speed, high specificity and high flexibility, provides technical support for researching extrametabolites of the cordycepin in the liver microsome while laying a methodological foundation for researching in-vivo and in-vitro metabolites of the cordycepin, and accordingly has a great significance.

The invention discloses a method for detecting the metabolic performance of a new chemical entity in different species of liver microsomes, which comprises the following steps: in a reaction system containing a buffer solution, liver microsomes, a substrate, MgCl2 and NADPH, screening out an optimal reaction condition by taking the metabolic rate of the substrate as a reference to obtain a human liver microsome incubation system. According to the establishment method of the human liver microsome incubation system, rat, mouse, dog and monkey liver microsome incubation systems are sequentially established. The method comprises the following steps: performing in-vitro metabolism test on a new chemical entity in incubation systems of different species of liver microsomes, performing positive group reaction by using a substrate, and setting a negative group without adding NADPH and a blank group without adding liver microsomes; and identifying a metabolism result after testing. According to the method for detecting the metabolic performance of the new chemical entity in different species of liver microsomes, false positive and false negative results can be avoided, the reliability of an identification result is improved, and a reliable basis is provided for promoting research and development of new drugs.

The invention discloses a method for in-vitro metabolism of chlorinated paraffin by liver microsomes, which comprises the following steps: (1) dissolving chlorinated paraffin in an organic solvent to obtain a chlorinated paraffin solution, adding serum into the chlorinated paraffin solution, and standing for balancing to obtain a substrate system; (2) sequentially adding a potassium phosphate buffer solution, liver microsome and an NADPH enzyme regeneration system into the substrate system, uniformly shaking to obtain a final reaction system, and then putting the final reaction system into a water bath at 37 DEG C for oscillating reaction; and (3) adding an ice-colded organic solvent into the reaction system in the step (2) to terminate the reaction, then centrifuging the solution, taking a supernatant liquid, and measuring the content of chlorinated paraffin. The serum is adopted as a carrier of the chlorinated paraffin, and reaction of the chlorinated paraffin and the liver microsome is promoted, so that the metabolic clearance rate of the chlorinated paraffin is remarkably increased, and the measurement of the metabolic clearance rate of the liver microsome for in-vitro metabolism of the chlorinated paraffin is realized.

The present invention is directed to a 2,4-bis(trifluoroethoxy)pyridine compound represented by formula (1):

(wherein X¹ represents a fluorine atom or a hydrogen atom) or a salt thereof, and to a drug containing the compound or the salt as an active ingredient.

The compound has metabolic resistance in human liver microsome, good absorbability upon oral administration, and excellent ACAT inhibitory activity.

The invention discloses a nitrogen-containing fused heterocyclic compound, and a preparation method, an intermediate, a composition and an application thereof. The compound has a high inhibitory activity against different subtypes of CDK at the molecular level, has a good inhibitory activity against breast cancer cells at the cellular level, has a significant proliferation inhibition effect on tumor cells associated with the cyclin-dependent kinase activity at the animal level, and has the advantages of good stability of liver microsomes in humans and mice, no significant inhibition effect onmetabolic enzymes, good absorption properties in rats and mice, high bioavailability, and good drug-forming property.

The invention discloses a UPLC/MS/MS detection method of the 1-hydroxymidazolam concentration in a liver microsome. The UPLC/MS/MS detection method comprises the following steps that 1, a liver microsome incubation system is established, serial concentrations of 1-hydroxymidazolam comparison products are added to prepare a standard curve sample; 2, the standard curve sample is injected into a UPLC/MS/MS liquid chromatograph/mass spectrometer for detection, and a standard 1-hydroxymidazolam curve is obtained according to the detection result; 3, a sample to be detected is prepared by adopting the same method as the step 1 and is injected into the UPLC/MS/MS liquid chromatograph/mass spectrometer, detection is performed under the same conditions as the step 2, and the 1-hydroxymidazolam concentration in the sample to be detected is obtained according to the standard curve. The UPLC/MS/MS detection method is short in analysis time, high in sensitivity and small in sample size, meets the methodological validation requirement, is suitable for determination of the 1-hydroxymidazolam concentration in the liver microsome incubation sample and has wide application prospect.

Reusable microsomal biocatalytic systems (bioreactors) constructed on carbon nanostructure modified electrodes are provided. The bioreactors comprise stable, biologically active immobilized enzymes such as human cytochromes P 450 (CYPs) and their redox partner proteins, e.g. CYP-NADPH (reduced nicotinamide adenine dinucleotide phosphate) reductases (CPR), on the carbon nanostructure surface. The immobilized enzymes may be present in liver microsomes, such as human liver microsomes (HLM) or as bactosomes, S9 fractions, etc. The bioreactors are used, for example, for synthesizing metabolites of interest from compounds such as drugs that are catabolized by the enzymes.

The invention discloses a cocktail method for detecting activities of five UGT enzymes. The method comprises the following steps: selecting substrates with high specificity to the five UGT enzymes, setting a proper concentration, performing substrate interaction survey, mixing and incubating substrates of enzymes UGT1A1/Ugt1a1 and UGT1A6/Ugt1a6 with small interactions, mixing and incubating substrates of UGT1A3/Ugt1a3, UGT1A9/Ugt1a9 and UGT2B7/AZTG, and finally measuring the activities of the UGT enzymes. The cocktail method for detecting the activities of the five UGT enzymes disclosed by the invention can be used for detecting the activities of UGT enzymes in rat and human liver microsome, has the advantages of high efficiency, rapidness and comprehensiveness and can be used for rapidly evaluating the influences of compounds on the activities of UGT enzymes.

The invention provides novel application of a qi-tonifying and heart-rate-restoring preparation for injection and especially to application of the qi-tonifying and heart-rate-restoring preparation for injection in preparing medicines exerting induction action on the activity of the liver microsomes, CYP1A2 and CYP3A4.

The invention discloses a heterocyclic compound and application thereof. The invention specifically discloses a heterocyclic compound as shown in a formula I, and a tautomer or pharmaceutically acceptable salt thereof. The compound has better inhibitory activity on TRPC5, has better metabolic stability in liver microsome, and has better clinical pharmacokinetic properties.