Method for determining main metabolite of amine silver mesylate B based on isotope labeling

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a method for determining main metabolites of amine silver mesylate B based on isotope labeling. And identifying the plasma protein binding rate, metabolic pathway, drug distribution and metabolite by adopting a biological sample LC-MS (Liquid Chromatography-Mass Spectrometry) determination method. The isotope labeling tracing overcomes the difference in individuals in the experiment, and the test precision and the credibility of experimental data are improved; and the demand quantity of samples such as plasma, urine and tissues is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for determining the main metabolites of silver amine mesylate lactone B based on isotope labeling. Background Art

[0002] Ischemic cardiovascular and cerebrovascular disease is one of the 10 major diseases in the guidelines for the creation of major new drugs. Currently, the most effective treatment is antiplatelet aggregation, but it can easily cause bleeding. The development of new antiplatelet aggregation drugs that can avoid the risk of bleeding is a global research hotspot.

[0003] Silver biloba lactone mesylate B is a platelet activating factor (PAF) antagonist, which is made by etherification and salt refinement of ginkgolide B. PAF is one of the strongest platelet aggregation inducing factors discovered so far. PAF antagonists can effectively inhibit platelet aggregation, and this mechanism of action is closely related to the treatment of ischemic stroke. So far, my country has approved 116 ginkgo leaf preparations, including tablets, capsules, soft capsules, injections, granules, dispersible tablets, pills, oral liquids and other dosage forms. As a traditional Chinese medicine, it has many shortcomings that are difficult to overcome, such as: unclear ingredients, many side effects, and low bioavailability.

[0004] Ginkgolide B is an optimized and modified drug based on the main anti-cerebral ischemia component of Ginkgo biloba, and is the world's first innovative drug that antagonizes PAF receptors to treat cerebral ischemia. As a new compound, it has clear ingredients, high purity (>99%), controllable quality (single impurity content is less than 0.5%), good safety, and antiplatelet activity is nearly 10 times that of the same dose of Ginkgolide B. The median lethal dose of intravenous injection in mice is 50.9 times that of Ginkgolide B. It has significant advantages in both pharmacodynamics and toxicology.

[0005] So far, my country has approved 116 ginkgo leaf preparations, including tablets, capsules, soft capsules, injections, granules, dispersible tablets, pills, oral liquids and other dosage forms. As a traditional Chinese medicine, it has many shortcomings that are difficult to overcome, such as: unclear ingredients, many side effects, and low bioavailability.

[0006] Ginkgolide B is an optimized and modified drug based on the main anti-cerebral ischemia component of Ginkgo biloba, and is the world's first innovative drug that antagonizes PAF receptors to treat cerebral ischemia. As a new compound, it has clear ingredients, high purity (>99%), controllable quality (single impurity content is less than 0.5%), good safety, and antiplatelet activity is nearly 10 times that of the same dose of Ginkgolide B. The median lethal dose of intravenous injection in mice is 50.9 times that of Ginkgolide B. It has significant advantages in both pharmacodynamics and toxicology.

[0007] The design concept and mechanism of action of silver amine lactone mesylate B are unique. The drug is a new chemical entity molecule and the first innovative drug to treat cerebral ischemia by antagonizing PAF receptors. Silver amine lactone mesylate B is safe and effective in the treatment of cerebrovascular diseases. It is suitable for a wide range of people and has a large clinical demand. Its efficacy is better than that of ordinary ginkgo preparations.

[0008] Isotope tracer pharmacokinetic studies, including the synthesis of C14-labeled drugs, material balance, quantitative whole body autoradiography (QWBA) tissue distribution, plasma protein binding rate and metabolism; identification of major metabolites, and preparation using micro-reaction technology to complete activity studies. Summary of the invention

[0009] The object of the present invention is to provide a method for determining the main metabolites of silver amine mesylate lactone B based on isotope labeling.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] A method for determining the major metabolites of silver amine mesylate lactone B based on isotope labeling, using biological sample LC-MS determination to perform plasma protein binding rate, metabolic pathways, drug distribution, and metabolite identification.

[0012] The method also includes the following steps: using quantitative whole body autoradiography (QWBA) to develop images using phosphor imaging plates, detect and store ion rays, and quantitatively generate images of radioactive samples.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] Isotope labeling and tracing overcomes intra-individual differences in experiments, improves test precision and the credibility of experimental data, and reduces the demand for samples: plasma, urine, and tissue.

[0015] Drug distribution research is an important part of pharmacokinetic research. Traditional distribution quantitative research mainly uses online separation and analysis techniques such as high-performance liquid chromatography and liquid scintillation counting. Both methods have steps such as dissecting organs and sample processing, which are cumbersome to operate and have a large coefficient of variation in results. It is difficult to determine the distribution of drugs in the brain and bile, and it is also impossible to monitor the blood-brain barrier and liver-biliary-blood circulation transport of drugs. Quantitative Whole-Body Autoradiography (QWBA), which has been developed in the past decade, can accurately, precisely and simply determine the distribution of drugs in the body, and can make breakthroughs in the distribution of drugs in the nervous system, cell targeting and intracellular distribution of drugs, and transport processes that are difficult to involve with traditional methods. It has an irreplaceable advantage in explaining the accumulation of drugs in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the technical roadmap of the present invention.

[0017] Figure 2 Representative whole-body autoradiographic images show the tissue distribution of radioactivity in LE (colored) rats after oral administration of carbon-14 labeled drugs (10 mg / kg) in the first and fourth weeks. DETAILED DESCRIPTION

[0018] The following will be combined with the accompanying drawings of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Embodiment 1.

[0020] A method for determining the major metabolites of silver amine mesylate lactone B based on isotope labeling, using biological sample LC-MS determination to perform plasma protein binding rate, metabolic pathways, drug distribution, and metabolite identification.

[0021] The method also includes the following steps: using quantitative whole body autoradiography (QWBA) to develop images using phosphor imaging plates, detect and store ion rays, and quantitatively generate images of radioactive samples.

[0022] The Bruker Avance series 500MHz NMR spectrometer can be used to perform detailed NMR spectroscopy analysis on isotope-labeled (C and 3H) samples below the milligram level.

[0023] The quantitative whole body autoradiography (QWBA) technology of XenoBiotic Laboratories, Inc., equipped with the ultra-low temperature cryostat Vibratome 9800 and Fujifilm BAS-5000 Phosphor Imager, combined with the tissue confirmation technology of specific radioactivity (HVSSR), can provide the highest quality autoradiography data and derive the most accurate analytical map of radioactivity distribution.

[0024] This technology uses phosphor imaging plates for imaging, which can detect and store ion rays, so it is also called phosphor imaging technology. After the animal sample is placed on the phosphor imaging plate, it is irradiated with a laser beam, and the radiation energy on the plate is converted into light energy. The luminous intensity is proportional to the radiation intensity, thereby quantitatively generating an image of the radioactive sample. The image can be quantitatively analyzed by the system software to obtain the pharmacokinetic data of the drug distribution in various animal tissues, which can be used to predict the distribution of C-labeled drugs in the human body. The linear range of QWBA measurement can reach 5 orders of magnitude, and it can simultaneously measure high and low intensity radioactivity; the sample exposure time is shortened from several months to several hours, which greatly improves the analysis speed and quality, and is expected to replace the traditional method of measuring distribution after tissue dissection.

Claims

1. A method for determining the main metabolites of silver amine mesylate lactone B based on isotope labeling, characterized in that The method comprises the following steps: using biological sample LC-MS determination method to carry out plasma protein binding rate, metabolic pathway, drug distribution and metabolite identification.

2. A method according to claim 1, characterized in that: The method also includes the following steps: using quantitative whole body autoradiography (QWBA) to develop images using phosphor imaging plates, detect and store ion rays, and quantitatively generate images of radioactive samples.

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