ABSTRACT
The metabolism study of radiolabeled drugs plays an important role in the development of new drugs. It provides information on drug absorption, metabolism, tissue distribution and excretion, and plays an irreplaceable role in the metabolite safety evaluation and mass balance of new drugs. The new guidance draft on clinical trials of radiolabeled drugs recently released by the US FDA puts forward higher standards and has been widely concerned by the industry. In recent years, in the research and development of new drugs in China, 14C labeled drugs have been used to carry out clinical metabolism studies, which has overcome key technical bottlenecks and accumulated experience. This paper summarizes the above research progress, analyzes the existing problems, and preliminarily looks forward to the future technological development and application.
ABSTRACT
Rocuronium bromide is an acetylcholine N2 receptor antagonist, which can be used as an auxiliary drug for general anesthesia. It has been reported that rocuronium has two possible metabolic pathways: N-dealkylation and O-deacetylation, which are mainly taken up by liver and excreted by bile in the form of primary drugs. In this paper, the metabolites of rocuronium in human bile were detected by UHPLC-QE-orbitrap-MS, thirteen metabolites were detected, including eleven phase I metabolites and two phase II metabolites, eleven of which had not been previously reported. At the same time, HEK293 cells overexpressing transporter were used to explore the transmembrane transport mechanism of rocuronium, the results showed that rocuronium was the substrate of MATE1, OCT1, OATP1B1 and OATP1B3. The above research results enrich the metabolic pathway of rocuronium in vivo, and put forward the possible transport mechanism of liver uptake and bile excretion, which can better guide the accurate and safe clinical drug application. The collection of human bile samples in this study was approved by the ethics committee of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Approval Number: 2019-775-130-01).
ABSTRACT
ZSP1601, a novel pan-phosphodiesterase inhibitor is in development for the treatment of nonalcoholic steatohepatitis. A physiologically-based pharmacokinetic (PBPK) model was developed to predict the pharmacokinetics of ZSP1601 in human. The PBPK model following intravenous and oral dose of ZSP1601 in rats and dogs was firstly built using preclinical in vitro and in vivo data. The PBPK model in human was then built based on models in animal. The in vitro-in vivo extrapolation (IVIVE) method and some allometric scaling methods were used to predict the clearance in human, respectively. The PBPK models using IVIVE and allometry of unbound CL plus the rule of exponents methods predicted the pharmacokinetics of ZSP1601 in healthy Chinese subjects successfully. The predicted parameters Cmax and AUC following single oral dose administration were within 0.5-2 folds of the observed data. The model was optimized and the final model was used to predict the pharmacokinetics of ZSP1601 in North European Caucasian, Geriatrics, Obese and Morbidly Obese, respectively. Animal studies were approved by the Animal Management and Use Committee of Suzhou AppTec Inc., and the approved No. is SZ20140916.
ABSTRACT
FGF21-164 is a fusion protein obtained by structural modification and coupling of endogenous FGF21. It is a candidate drug used in the treatment of glucose and lipid metabolic disorders caused by obesity. In this study, the candidate peptide mass spectrometry information of the protein hydrolyzed by trypsin was predicted by Skyline software and verified by high resolution mass spectrometry. The specific surrogate peptide (YLYTDDAQQTEAHLEIR) with the best mass response was selected after optimizing ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Under ESI positive ion mode, the parent ion m/z 689.3 with 3 charge and the product ion m/z 738.4 with single charge can be monitored. After dilution by PBS, the serum samples were denatured under 60 ℃ and alkylated to reduce the matrix effect, then incubated with trypsin at 37 ℃ for 2 h, to obtain the surrogate peptide. The chromatographic separation was carried out on an EclipsePlus C18 column (2.1 mm×50 mm, 1.8 μm) using aqueous solution containing 0.1% formic acid (phase A) and acetonitrile solution containing 0.1% formic acid (phase B). Finally, the concentration of FGF21-164 fusion protein in mouse serum was quantitatively analyzed by external standard method by monitoring the above ion pairs using triple quadrupole mass spectrometer. This method showed a good linearity in the range of 2.50-500 μg·mL-1 (r = 0.998 8), and was successfully applied to the pharmacokinetic study of FGF21-164 fusion protein in mice. This experiment was approved by the Experimental Animal Ethics Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences (batch number: 20180004040450). Compared with the endogenous FGF21, the t1/2 of FGF21-164 fusion protein was prolonged from 0.5 h to 2.6 h, which is expected to prolong the therapeutic efficacy of this protein.
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Based on Chinese clinical guidance for COVID-19 pneumonia diagnosis and treatment (7th edition), the metabolism and pharmacokinetics of drugs used in clinical treatment of COVID-19 were reviewed. The antiviral drugs include remdesivir, chloroquine/hydroxychloroquine, lopinavir/ritonavir, favipiravir, arbidol, baicalin, baicalein and forsythin. Among them, the metabolism and pharmacokinetics of arbidol, baicalin and forsythin are the research results of the author's laboratory. This article aims to provide reference for the efficacy evaluation and rational drug use of COVID-19.
ABSTRACT
Liquid chromatography-tandem mass spectrometry (LC-MS) is a promising alternative or complementary method for traditional ligand-binding assays (LBA) in antibody drug bioanalysis. However, issues related to method development, sample preparation, sensitivity and quantitative accuracy need to be addressed. This paper reviews progress in bioanalysis of antibody drugs by LC-MS methods, introduces the principle of the LC-MS method for the analysis of antibody drugs, and describes the challenges faced in quantitative antibody analysis by the LC-MS method. New strategies that can be used to deal with these challenges include: selection of surrogate peptides, purification and enrichment of samples, improvement in enzymatic digest efficiency, enrichment of peptides, and use of low rate LC. We review the application of LC-MS technology in the biological analysis of antibody drugs and discuss the prospect of using the LC-MS method for the analysis of antibody drugs.
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The purpose of current study is to investigate the metabolic profile of a triptolide derivative (5R)-5-hydroxytriptolide in vitro. (5R)-5-Hydroxytriptolide was incubated with the hepatocytes of human, monkey, dog, rat or mouse, respectively. Compared with inactivated hepatocytes, four metabolites were identified in hepatocytes from all five species: oxidative ring-opening metabolite (M1), glutathione-conjugating metabolite (M2), and monooxidative combined with glutathione-conjugating metabolites (M3-1 and M3-2), respectively. In human or rat liver microsomes, seven metabolites of (5R)-5-hydroxytriptolide were found, dehydrogenated metabolite (M4) and monooxidative metabolites (M5-1–M5-6), respectively. Reference standards for the metabolites were obtained either through chemical semisynthesis or biotransformation through rat primary hepatocytes. The structures of five metabolites were confirmed, which were 12,13-epoxy ring-opening metabolite M1, 12-glutathione-conjugating metabolite M2, (16S)-, (2R)- and (19R)-monohydroxylated metabolites M5-1, M5-4, and M5-5, respectively. In vitro activity assay revealed that only (2R)-hydroxylated metabolite exhibited weak immunosuppressive activity with less than one-tenth the activity of its parent drug, and a significant decrease in toxicity was observed. It is suggested that (5R)-5-hydroxytriptolide might undergo metabolic inactivation and detoxification in vivo.
ABSTRACT
Covalent tyrosine kinase inhibitors (TKIs) can inhibit the signaling pathway of tumor cells by covalent binding with cysteine residues of target proteins, which has the advantages of high potency, extended duration of action and overcoming drug resistance. In this article, we will review the metabolism and pharmacokinetics of some covalent TKIs. Currently, the covalent TKIs approved by US food and drug administration (FDA) are afatinib, neratinib, dacomitinib, osimertinib, ibrutinib and acalabrutinib. Pyrotinib have been approved by National Medical Products Administration (NMPA) to reach the market recently. Covalent TKIs can covalently bind with plasma proteins, especially human serum albumin, thus effected the pharmacokinetics of these drugs.
ABSTRACT
Nifedipine, a calcium channel antagonist, is metabolized mainly by CYP3A4 to dehydronifedipine. A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine nifedipine and dehydronifedipine in human plasma using d6-nifedipine/d6-dehydronifedipine as internal standards. After extraction from the plasma by protein precipitation, the analytes and internal standard were separated on a Hypersil Gold C18 (50 mm×2.1 mm, 1.9 μm). The mobile phase consisted of methanol and 5 mmol·L-1ammonium acetate aqueous solution (0.1% formic acid). Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 347.3→254.1 for nifedipine, m/z 345.2→283.9 for dehydronifedipine, m/z 353.3→257.1 for d6-nifedipine, m/z 351.2→286.9 for d6-dehydronifedipine. The method had a linear calibration curves over the concentrations of 0.10-80.0 ng·mL-1 for nifedipine and 0.050-40.0 ng·mL-1 for dehydronifedipine. The validated LC-MS/MS method has been successfully used study pharmacokinetic interactions of apatinib (CYP3A4 inhibitor) and nifedipine (CYP3A4 substrate) in human. This clinical trial was approved by the society of ethics and conducted in the first hospital of China medical university.
ABSTRACT
The study was designed to establish an LC-MS/MS method for the simultaneous determination of scutellarin and its major metabolite isoscutellarin in rat tissues and plasma, and to investigate the effect of different route of administration on the tissue distribution of scutellarin and its metabolite in rats. Rats were treated both intravenously and intragastrically with 20 and 80 mg·kg−1 scutellarin, respectively. Blood and tissues were collected at predetermined intervals. The concentrations of scutellarin and isoscutellarin were determined by a validated LC-MS/MS method. The method was linear in concentration ranges of 10.0/5.00 − 5 000/2 500 ng·mL−1 for scutellarin/isoscutellarin in the rat plasma and 30.0/15.0 − 10 000/5 000 ng·g−1 in tissues with acceptable accuracy and precision. Data obtained after an intravenous administration of scutellarin to rats showed that the drug was distributed predominantly into the small intestine, bladder and kidney. The exposures of the metabolite isoscutellarin in plasma and tissue were both less than 5%of the parent drug. After an intragastric administration, stomach wall and small intestine were the preferred sites for scutellarin disposition, followed by bladder, adrenal gland and lung at concentrations significantly higher than its plasma concentration. The plasma exposure of isoscutellarin was higher than that of the parent drug, but its tissue exposure was significantly lower than that of scutellarin. The method established in this study was successfully applied to characterization of the tissue profiles of scutellarin and its metabolite in rats. The route of administration has a marked impact on the disposition of scutellarin and its metabolite in rats. Ratios of the tissue to plasma concentrations after intragastric administration were obviously higher than those after intravenous administration. Scutellarin could pass the blood-brain barrier in a marked extent, but isoscutellarin was not detected in the rat brain, which may be attributed to the fact that scutellarin is a higher-affinity substrate for OATP than isoscutellarin.
ABSTRACT
Flavonol glycoside is in clinical trials for treatment of hyperlipidemia. An accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of flavonol glycoside (M0), aglycone (M1) and glucuronide conjugate (M2) in rat plasma. d6-Flavonol glycoside was used as internal standard (IS). After extraction from the plasma by protein precipitation, the analytes and internal standard were separated on a XDB C18 column (50 mm×4.6 mm, 1.8 μm) using a gradient elution procedure. The mobile phase consisted of methanol and water (0.2% formic acid) at a flow rate of 0.6 mL·min−1. The total run time was 4.5 min. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 461.3 → m/z 299.1 for M0, m/z 299.1 → m/z 283.1 for M1, m/z 475.0 → m/z 299.1 for M2, and m/z 467.3 → m/z 305.1 for d6-flavonol glycoside. The method was validated and successfully applied to the pharmacokinetics study of flavonol glycoside in SD rats which were given flavonol glycoside (30 mg·kg−1) by gavage. The Cmaxof M0 is (341 ±106) ng·mL−1 and AUC0−t is (1 960 ±725) h·ng·mL−1, while the Cmaxof M2 is (1 720 ±843) ng·mL−1and AUC0−t is (8 510 ±2 920) h·ng·mL−1. The results suggest that flavonol glycoside existed mainly in the form of M0 and M2 in rats. After flavonol glycoside being hydrolyzed by the intestinal flora, it was absorbed in the form of aglycone and further metabolized to M2 after the first-pass effect. In this paper, the main metabolites of flavonol glycoside in rat plasma were determined for the first time, which provided a basis for the design of clinical pharmacokinetic experiment.
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Human carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are important numbers of the serine esterase superfamily. They are involved in hydrolytic procedure of human endogenous cholesteryl esters, as well as drug metabolism, activation and detoxication. They are closely related to the personalized medication of drugs, especially for prodrugs. This review summarizes their structure and distribution, metabolic characteristics and research progress in recent years, which will provide a reference for new drug development and rational drug design.
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An LC-MS/MS method was developed for the simultaneous determination of fosaprepitant and aprepitant in human plasma, and applied to a pharmacokinetic study of 150 mg fosaprepitant dimeglumine injection to 12 Chinese healthy volunteers. The analytes and internal standards were extracted from plasma by protein precipitation with acetonitrile and separated on a Cortex C18+ (50 mm×2.1 mm, 2.7 μm) column using a gradient elution procedure. Mass spectrometry was performed in negative MRM mode, and parent-to-produce transitions were as follows:m/z 613.1→78.9 for fosaprepitant, m/z 617.0→78.9 for d4-fosaprepitant, m/z 533.2→275.1 for aprepitant and m/z 537.2→279.1 for d4-aprepitant. Plasma sample was basified to stabilize fosaprepitant. The standard curves were demonstrated to be liner in the range of 15.0 to 6 000 ng·mL-1 for fosaprepitant and 10.0 to 4 000 ng·mL-1 for aprepitant. The intra-day precisions and inter-day precisions and accuracy were within the acceptable limits for all concentrations.
ABSTRACT
The major non-P450 enzymes involved in the oxidative metabolism of drugs are:the flavincontaining monooxygenase (FMO), the monoamine oxidase (MAO), the aldehyde oxidase (AO), the xanthine oxidase (XO), the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). In recent years, the role of non-P450 enzymes in drug oxidative metabolism has garnered increasing attention. However, the contribution of non-P450 enzymes to the drug oxidative metabolism is possibly underestimated in many cases, as most metabolism studies in drug discovery and lead optimization are conducted using in vitro test systems related to P450 enzymes. In this article, these non-P450 enzymes in terms of catalyzed reaction types, common substrates, gene polymorphism and drug interaction are reviewed, and the in vitro models and factors for non-P450-mediated oxidative metabolism are summarized. Similar to P450 enzymes, non-P450 enzymes can directly catalyze the oxidation of drugs, yielding therapeutically active metabolites or toxic metabolites. These enzymes can also oxidize the toxic metabolites, generated from P450-catalyzed reaction, to nontoxic metabolites. In general, most non-P450 enzymes (such as FMO and MAO) appear to be much less inducible than P450 enzymes.
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Mogrol is the aglycone of seven kinds of mogrosides and siamenoside I. Mogrol has drawn more attention in recent years for its anti-leukemia and anti-diabetes activities. An ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was applied to identify the main metabolites of mogrol in rat plasma. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for determination of the main components in rat plasma. After an oral administration of 100 mg·kg-1 mogrol in rats, 13 metabolites were detected along the main component of parent drug in the plasma. The major metabolites were oxidated and dehydrogenated products. In this study, mogrol was quantitative analyzed using lithium carbonate reagent with high sensitivity. The assay was linear in concentration range 5.00-1 000 ng·mL-1 with intra-and inter-day precision within 9.3% and accuracy in range of -4.5% to 2.9%. Mogrol was absorbed into the blood very fast after oral administration, and the time to reach maximum concentration (tmax) was 1.67 h. The half-life (t1/2) of mogrol in rats was 2.34 h, and the oral absolute bioavailability was 3.5%.
ABSTRACT
Donafenib is the deuterium derivative of sorafenib, and is an anti-tumor drug in clinical trials. An accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of donafenib and its N-oxide metabolite in human plasma. The analytes and internal standards (sorafenib and sorafenib N-oxide) were extracted from plasma by protein precipitation with acetonitrile, and separated on a Gemini C18 (50 mm×2.0 mm, 5 μm) column using a gradient elution procedure. The mobile phase consisted of acetonitrile and 5 mmol·L-1 ammonium acetate (0.2% formic acid) at a flow rate of 0.7 mL·min-1. The total run time was 5.0 min. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 468.2→273.2 for donafenib and m/z 465.2→270.2 for its internal standard sorafenib, m/z 484.2→289.2 for donafenib N-oxide and m/z 481.2→286.2 for its internal standard sorafenib N-oxide. The standard curves were linear in the range of 5.00-5 000 ng·mL-1 for donafenib, and 1.00-1 000 ng·mL-1 for donafenib N-oxide. The method was validated and successfully applied to the pharmacokinetics study of donafenib tosylate tablets in volunteers.
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Anaprazole is a proton pump inhibitor clinically used for curing peptic ulcer. A rapid, sensitive and convenient LC-MS/MS method was first established for the determination of anaprazole in human plasma. d3, 13C-anaprazole was used as internal standard (IS). After extraction from human plasma by protein precipitation with acetonitrile, all components were separated on an Extend C18 column (100 mm×4.6 mm, 3.5 μm). The assay was linear over the concentration range of 5.00-3 000 ng·mL-1 (r2 > 0.995). The method was successfully applied to a pharmacokinetic study of 40 mg anaprazole enteric-coated tablets in 14 Chinese healthy volunteers under fasting or high fat diet conditions. Cmax was (1 020±435) ng·mL-1 and AUC0-t was (2 370±754) h·ng·mL-1 under fasting condition. And Cmax was (538±395) ng·mL-1 and AUC0-t was (1 610±650) h·ng·mL-1 under high fat diet condition. The plasma results suggest that the exposure of anaprazole is reduced by the high fat diet.
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Antibody-drug conjugates (ADCs) are complex molecules with cytotoxic small molecular drugs covalently bound to monoclonal antibodies via a linker and can improve the targeted drug delivery with minimizing the systemic toxicity. ADCs are heterogeneous mixtures with different drug-to-antibody ratios (DARs) and the DAR distribution is dynamically changing in vivo, therefore the bioanalysis of the ADCs is challenging. Enzyme-linked immunosorbent assay (ELISA) and LC-MS have been widely used in the ADCs bioanalytical assays. Just like other biotherapeutics, ADCs may elicit the host immune response and produce the anti-therapeutic antibody (ATA), which could affect its efficacy, pharmacokinetics, and safety. It is thereby important to investigate its immunogenicity in the ADC development. In this review, we summarized the ELISA- and LC-MS-based bioanalysis strategies for the development of ADCs, including DAR distribution, the determination of total antibody, conjugated antibody, conjugated drug, free drug, and ATA, with the expectation of providing insights and reference for the ADC development in China.
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Tapentadol is a novel drug of opioid pain reliever, which is extensively metabolized primarily through conjugation. Tapentadol glucuronide and tapentadol sulfate are major drug-related metabolites in circulation. The objectives of this study were to develop a simple and rapid method to determine tapentadol and evaluate the effects of conjugated metabolites on tapentadol quantification using liquid chromatography with tandem mass spectrometry in dog plasma. The analyte and tramadol (IS) were extracted from plasma by protein precipitation with methanol, and chromatographied on a XDB C18 (50 mm×4.6 mm, 1.8 μm) column using a mobile phase of methanol and 5 mmol·L-1 ammonium acetate (0.01% ammonia). Mass spectrometric detection was performed using the m/z 222→121 transition for tapentadol and the m/z 264→58 transition for the internal standard tramadol, the m/z 398→m/z 121 transition for glucuronides conjugate and the m/z 302→m/z 222 transition for sulfate conjugate. Conjugated metabolites could undergo in-source conversion to generate an ion that interfered the quantification of tapentadol. Chromatographic separation was achieved to elimination interferences due to in-source conversion of the conjugated metabolites. The standard curves were demonstrated to be linear in the range of 0.100 to 20.0 ng·mL-1 for tapentadol. The intra-and inter-day precisions were within 5.1%, and accuracy ranged from -3.2% to 0. This method was successfully applied to the pharmacokinetics of tapentadol hydrochloride sustained release tablets in Beagle dogs.
ABSTRACT
Drug metabolism research plays an essential role in drug discovery and development. Great efforts have been made domestically to be line with the international standardized research on drug metabolism. In this article, we will review new-generation of tyrosine kinase inhibitors (TKIs), these TKIs include icotinib, apatinib, famitinib, flumatinb, allitinib, fruquintinib, and selatinib, among which icotinib and apatinib have been approved by China food and drug administration (CFDA) to reach the market, while others are in clinical trials. For these TKIs, the structural modified sites are active metabolic centers and CYP3A4 is identified as the primary metabolic enzyme. Considering the active intermediates, the crown ether ring of icotinib is oxidated to open to form an aldehyde; the indolylidene ring of famitinib is oxidated followed by rearrangement to form a quinone-imine; the α, β-unsaturated carbonyl group of allitinib is oxidated to form an epoxide, these intermediates are capable of covalently binding biomolecules and generating toxicity. In addition, human 14C radioactive trials of most of these TKIs have not been conducted, and the data of drug-drug interactions in clinic are also absent, which indicate our deficiency compared to the international regular approaches in metabolic research.