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Pharmacokinetic analysis method for covalent drug and metabolite thereof

A technology of pharmacokinetics and metabolites, which is applied in the field of pharmacokinetic analysis of covalent drugs and their metabolites, can solve problems such as complicated and difficult operation processes, hindering the detection of adducts, and achieve simplified operation processes, Increased abundance, accurate separation, and detection

Pending Publication Date: 2022-02-08
MACAU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its operation process is quite complicated and difficult, and the low abundance of the drug and its metabolite-protein adduct also hinders the detection of the adduct

Method used

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  • Pharmacokinetic analysis method for covalent drug and metabolite thereof
  • Pharmacokinetic analysis method for covalent drug and metabolite thereof
  • Pharmacokinetic analysis method for covalent drug and metabolite thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Establishment and verification of UHPLC-QQQ-MS quantitative analysis method based on amino acid level

[0059] 1. In vitro incubation of covalent drugs and identification of adducts

[0060] Osimertinib stock solution was prepared with DMSO as solvent, the concentration was 4 mg / mL. The stock solution of osimertinib was diluted to a final concentration of 10.0 mmol / L with potassium phosphate buffer solution (PBS, pH7.4, 0.1 mol / L) containing NAPDH generating solution A, B and cysteine. Pre-incubated at 37°C for 2 min, then added 1.0 mg / mL human liver microsomes to start the metabolic reaction, with a total incubation volume of 400 μL. An equal amount of 50 μL incubation solution was collected at 0, 10, 30, 60, 120, 180, and 240 min, respectively. The samples were centrifuged at 15000 g for 10 min, and the supernatant was collected and dried with nitrogen. The residue was redissolved with 50 μL of 50% aqueous methanol, and the supernatant was collected afte...

Embodiment 2

[0082] Example 2. Quantitative analysis of in vivo samples in rats after covalent drug administration

[0083] 1. Optimization of complete enzymatic hydrolysis method

[0084] By comparing the peak areas of AC1 obtained under different enzyme mixing ratios and enzymatic hydrolysis time, it was concluded that the optimal enzyme ratio required to detect 1500 μg protein was 8.4 units of pronase E and 16 units of chymotrypsin, and the optimal enzymatic hydrolysis time was 20 hours.

[0085] 2. Animal administration and sample processing

[0086] 16 SD rats, weighing about 200g (male) and 180g (female), were randomly divided into 4 groups (n=4, half male and half male). Groups 2, 3, and 4 were given osimertinib with 1% polysorbate 80 suspension (36 mg / kg), and group 1 was given 1% polysorbate 80 as a control. The rats in groups 1 and 2 were killed 1 hour after administration, and the rats in groups 3 and 4 were killed 6 hours and 24 hours after administration and blood was collec...

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Abstract

The invention discloses a method for quantitatively analyzing the modification level of a covalent drug and a metabolite of the covalent drug on protein from the amino acid level, and is applied to the research of pharmacokinetics. The method comprises the following steps: (1) adding a covalent drug and a capture reagent into an in-vitro incubation system for incubation, taking incubation liquid for analysis, identifying an adduct formed by the covalent drug and a metabolite thereof and the capture reagent, and determining a metabolite structure with covalent modification capacity and modified target amino acid; (2) preparing a standard substance of the adduct in the step (1), detecting chromatographic and mass spectrometric data of the standard substance through UHPLC-QQQ-MS, and establishing a quantitative analysis method; and (3) carrying out enzymolysis on a biological drug delivery sample of the covalent drug, detecting the obtained enzymolysis product by adopting UHPLC-QQQ-MS, and determining the content of the adduct in the biological drug delivery sample according to the detection result in combination with the chromatographic and mass spectrometric data in the step (2).

Description

technical field [0001] The invention belongs to the field of pharmaceutical analytical chemistry, and in particular relates to a pharmacokinetic analysis method for covalent drugs and their metabolites. Background technique [0002] Covalent drugs are a class of drugs that interact with target protein residues through covalent bonds, thereby changing the protein conformation and inhibiting protein activity. This mechanism of action makes the pharmacokinetics (PK) of covalent drugs specific and induces pharmacodynamic responses beyond the predicted range of PK or drug elimination time. Therefore, if the PK study of covalent drugs is performed using free drug and / or metabolites like conventional studies, the difference in the mechanism of action of covalent drugs and conventional non-covalent drugs will be ignored, and the covalent drugs cannot be accurately analyzed. Evaluate. In recent years, researchers have also begun to realize that alternative methods of free drug equi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88
CPCG01N30/06G01N30/88
Inventor 李娜伍建林宫诗林卓越胡晓兰
Owner MACAU UNIV OF SCI & TECH
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