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1.
Acta Pharmaceutica Sinica B ; (6): 1607-1616, 2021.
Article in English | WPRIM | ID: wpr-888823

ABSTRACT

Remdesivir (RDV) is the only US Food and Drug Administration (FDA)-approved drug for treating COVID-19. However, RDV can only be given by intravenous route, and there is a pressing medical need for oral antivirals. Significant evidence suggests that the role of the parent nucleoside GS-441524 in the clinical outcomes of RDV could be largely underestimated. We performed an

2.
Article in Chinese | WPRIM | ID: wpr-887992

ABSTRACT

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.


Subject(s)
Animals , Benzofurans , Coumarins , Dogs , Glucuronides , Glucuronosyltransferase/metabolism , Kinetics , Mice , Microsomes, Liver/metabolism , Phenotype , Rats , Species Specificity , Swine , Swine, Miniature/metabolism
3.
China Pharmacy ; (12): 2059-2065, 2021.
Article in Chinese | WPRIM | ID: wpr-886739

ABSTRACT

OBJECTIVE:To i nvestigate the metabolism stabilities of novel hypoglycemic compound LSM- 13 in rat liver microsomes,and to analyze the possible metabolites. METHODS :LSM-13 was dissolved in rat liver microsome incubation system initiated by reduced nicotinamide adenine dinucleotide phosphate ,and was incubated in water at 37 ℃. The reaction was terminated with acetonitrile at 0,5,10,15,30,45 and 60 min,respectively. Using indomethacin as internal standard ,the concentration of LSM-13 in incubation system was determined by HPLC. The residual percentage and enzyme kinetic parameters of LSM- 13 were calculated at different incubation time points with the concentration of LSM- 13 incubated for 0 min as reference. UPLC-Q-TOF/MS was used to analyze and speculate the metabolites of LSM- 13 in rat liver microsomes. RESULTS :After 60 min incubation ,the remaining percentage of LSM- 13 was(56.07±0.95)%,the half-life was 42.78 min,and the intrinsic clearance was 0.032 4 mL/(min·mg). Compared with total ion flow diagram of rat liver microsome blank samples ,three chromatographic peaks were added in the samples incubated for 60 min;the corresponding molecular ion peaks were m/z 505.133 8,417.102 4,293.111 7 [M+H]+;the possible metabolites may be dehydrogenation ,O-debentylation and hydrolysis products of LSM- 13. CONCLUSIONS : The compound LSM- 13 has moderate stability in rat liver microsomes ,and may undergo dehydrogenation ,O-debentylation and hydrolysis.

4.
China Pharmacy ; (12): 1325-1329, 2021.
Article in Chinese | WPRIM | ID: wpr-877253

ABSTRACT

OBJECTIVE:To establish the metho d for the concentration det ermination of foretinib derivative LWK- 126 in liver microsomes,and to study its metabolism stability in liver microsomes of rats ,Beagle dogs and human. METHODS :In the in vitro incubation system of liver microsomes ,LWK-126 was dissolved in liver microsomal incubation systems of rats ,Beagle dog and human initiated by reduced nicotinamide adenine dinucleotide phosphate solution. After incubation in water at 37 ℃ for 0,5,10,20, 30 and 60 min,the reaction was terminated with acetonitrile containing internal standard (1 μg/mL tolbutamide). UPLC-MS/MS method was applied to determine the concentration of LWK- 126 in the incubation systems. The determination was performed on Waters BEH C 18 column with mobile phase consisted of water (containing 0.1% formic acid )-acetonitrile(containing 0.1% formic acid)by gradient elution at the flow rate of 0.4 mL/min. The column temperature was 30 ℃,and the sample size was 2 μL. The mass spectral analysis was performed in a positive electrospray ionization mode ,and the full MS experiment was run with the selective reaction monitoring mode with a scanning range of m/z 50→1 200. Taking the concentration of LWK- 126 at 0 min as reference,the remaining percentage and the enzyme kinetic parameters were calculated. RESULTS :The linear range of LWK- 126 was 0.05-15 μg/mL(R 2=0.999 2);the lower limit of quantification was 0.05 μg/mL,and the lowest detection limit was 0.01 μg/mL. The precision,accuracy,extraction recovery and matrix effect all met the analysis requirements of biological samples. The remaining percentage of LWK- 126 in liver microsomes of human ,rats and Beagle dogs for 60 min were (33.17±4.52)%,(3.14± 6.73)%,(1.38±5.85)%;t1/2 of them were 33.15,11.76,5.62 min;the clearance rates were 38.45,118.81,245.76 μL(/ min·mg), respectively. CONCLUSIONS :The method for the content ; determination of LWK- 126 in liver microsomes is established successfully. The order of metabolic stability of LWK- 126 in 〔2016〕4015) liver microsomes of different species is human >rats>Beagle dogs.

5.
Article in Chinese | WPRIM | ID: wpr-873325

ABSTRACT

Objective::To investigate in vivo and in vitro metabolites of coptisine and their metabolic pathways. Method::SD rats were given coptisine by single gavage (dose of 25 mg·kg-1). Urine and feces from 0 h to 48 h, bile from 0 h to 24 h, and plasma and brain tissue samples at 0.25, 1, 2 h after administration were collected.In vitro metabolism was incubated with rat liver microsomes and intestinal flora.The metabolites were analyzed and identified by the high-resolution HPLC-MS/MS technique.The liquid chromatography separation was carried out on ZORBAX SB-C18 column (4.6 mm×150 mm, 5 μm) with acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution, the flow rate was 1.0 mL·min-1, and column temperature was 25 ℃.The mass spectra were obtained in positive and negative ion mode with electrospray ionization (ESI), the scanning range was m/z 50-1 200.The relative molecular weight was determined according to the quasi-molecular ion peaks.The structures of metabolites were elucidated by comparing the data with literature data, including main ion peaks, UV spectrum and HPLC retention time information. Result::A total of 17 metabolites were identified in each sample, including 11 phase Ⅰ metabolites and 6 phase Ⅱ metabolites.The pathways to these metabolites were hydroxylation, demethylation, dehydrogenation, sulfation and glucuronide conjugation. Conclusion::Coptisine can produce metabolic reaction of phase Ⅰ and phase Ⅱ in rat, and metabolites are predominantly present in urine, and the main metabolic site is liver.Coptisine is poorly absorbed and rarely metabolized in gastrointestinal tract, so it is mostly excreted through feces by prototype.This experiment can provide material basis for the pharmacodynamics and pharmacology of coptisine.

6.
Acta Pharmaceutica Sinica ; (12): 1484-1492, 2019.
Article in Chinese | WPRIM | ID: wpr-780247

ABSTRACT

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.

7.
Article in Chinese | WPRIM | ID: wpr-777490

ABSTRACT

The paper studies and compares the metabolic difference of active ingredients of lipid-lowering flavonoid extract of Daidai in rat livers and intestinal microsomes,in order to explore the phase Ⅰ metabolism characteristics of active ingredients in livers and intestines. UPLC-MS/MS was used to establish a quantitative analysis method for active ingredients,neohesperidin and narngin,in a phase Ⅰ metabolism incubation system of liver and intestinal microsomes. Differential centrifugation was used to make liver and intestinal microsomes of rats. A phase Ⅰ metabolism incubation system was established,and the concentrations of the residual at different incubation time points were analyzed. Graphs were plotted to calculate the metabolic elimination half-life of the main active parts,with the natural logarithm residual percentage values ln( X) at different time points as the y axis,and time t as the x axis. The metabolism characteristics of the active ingredients were compared. The established UPLC-MS/MS quantitative analysis method has a good specialization,standard curve and linear range,accuracy and precision,with a satisfactory lower quantitative limit. The method allows quantitative detection of the active ingredients in a phase Ⅰ metabolism incubation system of liver and intestinal microsomes of rats. In the rats liver microsomes incubation system,the metabolic elimination half-life of neohesperidin and narngin were( 2. 20 ± 0. 28) h and( 1. 97±0. 28) h respectively. The elimination half-life of neohesperidin was larger than that of narngin,but with no statistically significant difference. In the rats intestinal microsomes incubation system,the metabolic elimination half-lives of neohesperidin and narngin were( 3. 68±0. 54) h and( 2. 26±0. 13) h respectively. The elimination half-life of neohesperidin was larger than that of narngin,with statistically significant differences( P<0. 05). The elimination half-lives of the active ingredients in liver microsomes were smaller than those in intestinal microsomes. The experiment results showed that the active ingredients of lipid-lowering flavonoid extract of Daidai had different elimination half-lives in phase Ⅰ rats liver and intestinal microsomes incubation system. This implied that they had different metabolic characteristics in rats liver and intestine,and liver may be the main metabolism site of the active ingredients. The phaseⅠ metabolism of narngin was stronger than that of neohesperidin. The differences between their metabolic characteristics may be related to the binding sites of B-ring hydroxyl in flavonoid glycosides and the number of methoxyl group. The results provided an important experimental basis for further development and clinical application of lipid-lowering flavonoid extract preparation of Daidai.


Subject(s)
Animals , Chromatography, Liquid , Citrus sinensis , Flavonoids , Intestines , Lipids , Liver , Microsomes, Liver , Rats , Rats, Sprague-Dawley , Tandem Mass Spectrometry
8.
Article in Chinese | WPRIM | ID: wpr-857532

ABSTRACT

OBJECTIVE: To investigate the metabolic characteristics of bakuchiol mediated by cytochrome P450 enzyme (CYP) and UDP-glucuronosyltransferase (UGT) in rat liver microsomes (RLMs) or human liver microsomes (HLMs), and to compare the metabolic gender differences. METHODS: Bakuchiol was incubated at 37? with male and female RLMs or HLMs in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) or uridine 5′-diphosphoglucuronic acid (UDPGA). The residual concentrations of bakuchiol were measured in each incubation system using high performance liquid chromatography (HPLC). The metabolic stability and metabolic gender differences of bakuchiol were evaluated by the remaining percentage of bakuchiol after incubation.RESULTS: When bakuchiol was metabolized by CYP in RLMs, the intrinsic clearance (Clint) value in male RLMs ?(326.6±15.4) mL·min-1·kg-1?was significantly higher than that of female RLMs ?(77.2±4.8) mL·min-1·kg-1? (P<0.01). When bakuchiol was metabolized by UGT in RLMs, female RLMs had a significantly higher Clint value ?(419.1±24.1) mL·min-1·kg-1? than male RLMs ?(164.5±8.4) mL·min-1·kg-1? (P<0.01). When bakuchiol was metabolized by both CYP and UGT in RLMs, male RLMs had a significantly higher Clint value ?(1063.1±27.2) mL·min-1·kg-1? than female RLMs ?(781.2±16.5) mL·min-1·kg-1?(P<0.01). When bakuchiol was metabolized by CYP in HLMs, male HLMs had a significantly higher Clint value ?(24.8±2.1) mL·min-1·kg-1? than female HLMs ?(17.6±1.0) mL·min-1·kg-1? (P<0.01). There were no significant gender differences in the metabolism of bakuchiol when it was metabolized by UGT in HLMs. The Clint values were 176.4±26.5 and (165.9±8.6) mL·min-1·kg-1, respectively. The metabolic parameters of bakuchiol mediated by CYP and UGT in HLMs had no significant gender differences. The Clint values were 262.5±20.9 and (236.2±10.5) mL·min-1·kg-1, respectively. CONCLUSION Bakuchiol can be metabolized by CYP and UGT in RLMs or HLMs, and the metabolic parameters exhibit species differences and gender differences.

9.
Article in Chinese | WPRIM | ID: wpr-850669

ABSTRACT

Objective: To investigate the metabolic stability, the main CYP450 enzymes phenotypes and metabolites of Diosbulbin B based on in vitro metabolism model. Methods: For metabolic stability study, UPLC-MS/MS was used to detect the remaining Diosbulbin B content in the incubation solution after being incubated with human and rat liver microsomes, respectively. Ten recombinant human CYP450 enzymes (1A1, 1A2, 1B1, 2A13, 2A6, 2B6, 2D6, 2C9, 2C19, 3A4) were used for identifying the metabolic enzyme phenotypes of Diosbulbin B. Moreover, the major metabolic enzyme phenotype for the metabolism of Diosbulbin B was confirmed and verified by the rat isolated hepatic perfusion model. The metabolites of Diosbulbin B in human and rat liver microsomes were determined by LC-MS/MS. Results: The metabolic percentage of Diosbulbin B in human and rat liver microsomes were 37% and 59%, respectively. Its half-lives t1/2 in human and rat liver microsomes were 97.4 and 52.3 min, respectively. The intrinsic clearance rates CLint in human and rat livers were 8.23 and 23.9 mL/(min•kg), and liver clearance CLh in human and rat livers were 5.89 and 16.8 mL/(min•kg). It can be found that the metabolic rate of Diosbulbin B in rat liver microsomes was faster than in human liver microsomes. There were five CYP enzymes, including 3A4, 2C19, 2C9, 1A13 and 1A1, related to the metabolism of Diosbulbin B, especially CYP3A4. The hepatic perfusion experimental results showed that the metabolism of Diosbulbin B was inhibited by ketoconazole, and the inhibitory effect was enhanced along with the increasing dosage of ketoconazole, which confirmed that CYP3A4 played an important role in metabolism of Diosbulbin B. There was one metabolite (M1) of Diosbulbin B has been found in both human and rat liver microsomes incubation. Conclusion: The metabolic rate of Diosbulbin B in rat liver microsomes was faster than human liver microsomes. The CYP3A4 plays a leading role in the metabolism of Diosbulbin B. And a demethylated metabolite of Diosbulbin B was appeared in both human and rat liver microsomes incubation.

10.
China Pharmacy ; (12): 2497-2502, 2019.
Article in Chinese | WPRIM | ID: wpr-817266

ABSTRACT

OBJECTIVE: To establish a determination method for the concentration of cajanonic acid A (CAA) in liver microsome incubation system, and to compare the metabolism characteristics of it in different species of liver microsomes. METHODS: CAA was dissolved in liver microsome incubation system of rat, Beagle dog and human initiated by reduced nicotinamide adenine dinucleotide phosphate (NADPH), and was incubated in water at 37 ℃. The reaction was terminated with acetonitrile at 0, 5, 10, 15, 30, 45 and 60 min, respectively. Using genistein as internal standard, the concentration of CAA in  different incubation systems was determined by UPLC-MS/MS. The determination was performed on Waters BEH C18 column with mobile phase consisted of water (containing 0.1% formic acid)-acetonitrile (containing 0.1% formic acid) (45 ∶ 55, V/V) at the flow rate of 0.25 mL/min. The column temperature was 30 ℃, and the sample size was 2 μL. The electrospray ionization source was used to the select reaction monitoring mode for negative ion scanning. The ion pairs for quantitative analysis were m/z 353.14→309.11 (CAA), m/z 269.86→224.11 (internal standard) respectively. The residual percentage and enzymatic kinetic parameters of CAA in different incubation systems were calculated according to the mass concentration of CAA at 0 min. RESULTS: The linear range of CAA was 0.05-20 μg/mL; the limit of quanti- tation was 0.05 μg/mL, and the lowest detection limit was 0.01 μg/mL. RSDs of intra-day and inter-day were lower than 10%; relative errors ranged -4.83%-8.94%; extraction method and matrix effect did not affect the determination of the substance to be measured. At 60 min of incubation, residual percentages of CAA in rat, Beagle dog and human liver microsomes were(62.79±9.99)%,(64.07±11.59)%,(96.66±5.71)%, respectively. The half-life period (72.19, 68.61 min) of CAA in rat and Beagle dog liver microsomes were significantly shorter than human liver microsome (364.74 min). The clearance rates [0.019 2, 0.020 2 mL/(min·mg)] were significantly higher than human liver microsome [0.003 8 mL/(min·mg)] (P<0.05). CONCLUSIONS: Established UPLC-MS/MS method is simple, rapid, specific and sensitive, and can be used for the determination of CAA concentration in liver microsome incubation system and the study of metabolism stability in vitro. The stability of CAA metabolism in rat and Beagle dog liver microsomes are poorer than human liver microsome.

11.
China Pharmacy ; (12): 1316-1320, 2019.
Article in Chinese | WPRIM | ID: wpr-816933

ABSTRACT

OBJECTIVE: To study in vitro metabolism pathway of effective component of Bletilla striata as Militarine in liver microsomes and kinetics characteristics of enzyme-catalyzed reactions. METHODS: The in vitro incubation system of rat and human liver microsomes was established, and incubation reaction of Militarine was performed. UPLC-QTOF-MS was used to identify the structure of its metabolites in combination with UNIFI database and references. Using puerarin as internal standard, UPLC-Triple Quad-MS was used to quantitatively analyze metabolic transformation of Militarine in rat liver microsomes. The kinetic parameters (vmax, km, CLint) of Militarine enzyme-catalyzed reactions with/without reducing coenzyme Ⅱ (NADPH) were calculated by fitting the curves with GraphPad Prism 5.0 software. RESULTS: After incubation in rat and human liver microsomes, Militarine produced a chemical formula C21H29O11, which was presumed to be a metabolite of Militarine ester bond hydrolysis. The kinetic study of enzyme-catalyzed reactions showed that vmax of Militarine enzyme-catalyzed reactions with/without NADPH were 1.955, 2.129 nmol/(h·mg); km were 8.601, 9.854 nmol/mL; CLint were 0.227 3, 0.216 1 mL/(h·mg); there was no significant difference between with NADPH and without NADPH. CONCLUSIONS: The main metabolic pathway of Militarine in liver microsomes is the hydrolysis of C1 and C4 ester bonds. Its metabolism does not depend on the pathway of cytochrome P450 enzymes initiated by NADPH.

12.
China Pharmacy ; (12): 170-175, 2019.
Article in Chinese | WPRIM | ID: wpr-816715

ABSTRACT

OBJECTIVE: To establish a method for the determination of piperitylmagnolol in the incubation system of liver microsomes, and to investigate the metabolic characteristics of it in different species of liver microsomes. METHODS: The piperitylmagnolol were respectively dissolved in NADPH activated liver microsome incubation systems of human, rat, mouse, monkey and dog, and then incubated in water at 37 ℃. The reaction was terminated with methanol at 0, 2, 5, 10, 15, 20, 30, 45 and 60 minutes of incubation, respectively. Using magnolol as internal standard, UPLC-MS/MS method was used to determine the concentration of piperitylmagnolol in the incubation system. The determination was performed on Acquity UPLCTM CSH C18 column with mobile phase consisted of 0.1% formic acid-methanol (gradient elution) at the flow rate of 0.3 mL/min. The column temperature was set at 30 ℃, and the sample size was 2 μL. The ion source was electrospray ion source, and the positive ion scanning was carried out in the multiple reaction monitoring mode. The ion pairs used for quantitative analysis were m/z 401.2→331.1 (piperitylmagnolol) and m/z 265.1→247.0 (internal standard), respectively. Using the concentration of piperitylmagnolol at 0 min of incubation as a reference, the residual percentage, metabolism half-life in vitro (t1/2) and intrinsic clearance (CLint) were calculated for different incubation systems. The metabolic pathway of piperitylmagnolol was studied by chemical inhibitor method. Under the above chromatographic conditions, the metabolites in vitro were preliminarily analyzed by first-order full scanning and positive ion detection. RESULTS: The linear range of piperitylmagnolol was 3.91-500.00 ng/mL. The limit of quantitation was 3.91 ng/mL. RSDs of intra-day and inter-day were less than 10%. The accuracy ranged 87.40%-103.75%. Matrix effect didn’t affect the determination of the substance to be measured. The piperitylmagnolol was metabolized significantly in human, rat, mouse and dog liver microsomes, but not in monkey liver microsomes. After incubating for 30 min, residual percentage of piperitylmagnolol kept stable in different species of liver microsomes. The t1/2 of piperitylmagnolol were 12.07, 17.68, 17.59, 216.56 and 61.88 min in human, rat, mouse, monkey and dog liver microsomes; CLint were 0.115, 0.078, 0.079, 0.006, 0.022 mL/(min·mg), respectively. Inhibitory rates of CYP2A6, CYP2D6, CYP2C19, CYP3A4, CYP2C9, CYP2E1 and CYP1A2 to compound metabolism were 55.76%, 93.94%, 96.01%, 93.69%, 71.81%, 23.25%, 28.04%, respectively. Quasi-molecular ion peaks of the two main metabolites of piperitylmagnolol in human liver microsomes were m/z 441.2([M+Na]+) and m/z 337.2([M+H]+), respectively. CONCLUSIONS: Established UPLC-MS/MS method is simple, rapid and specific, and can be used for the determination of piperitylmagnolol concentration in the incubation system of liver microsomes and pharmacokinetic study. The metabolic characteristics of the compound are different among liver microsomes of human, rat, mouse, monkey and dog. Its metabolism process may be associated with CYP2D6, CYP2C19, CYP3A4, CYP2C9, etc.

13.
Article in English | WPRIM | ID: wpr-776924

ABSTRACT

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.


Subject(s)
Animals , Cells, Cultured , Cytochrome P-450 CYP1A2 , Metabolism , Cytochrome P-450 CYP3A , Metabolism , Ginkgo biloba , Chemistry , Glucuronosyltransferase , Metabolism , Hepatocytes , Chemistry , Metabolism , Humans , Kinetics , Liver , Chemistry , Metabolism , Microsomes, Liver , Chemistry , Metabolism , Plant Extracts , Chemistry , Metabolism , Toxicity , Rats , Rats, Sprague-Dawley , Salicylates , Chemistry , Metabolism , Toxicity
14.
Article in English | WPRIM | ID: wpr-812346

ABSTRACT

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.


Subject(s)
Animals , Cells, Cultured , Cytochrome P-450 CYP1A2 , Metabolism , Cytochrome P-450 CYP3A , Metabolism , Ginkgo biloba , Chemistry , Glucuronosyltransferase , Metabolism , Hepatocytes , Chemistry , Metabolism , Humans , Kinetics , Liver , Chemistry , Metabolism , Microsomes, Liver , Chemistry , Metabolism , Plant Extracts , Chemistry , Metabolism , Toxicity , Rats , Rats, Sprague-Dawley , Salicylates , Chemistry , Metabolism , Toxicity
15.
Acta Pharmaceutica Sinica ; (12): 1743-1747, 2017.
Article in Chinese | WPRIM | ID: wpr-779784

ABSTRACT

This study was designed to investigate the metabolites of 5F-AMB by human liver microsomes model in vitro. 5F-AMB was added in the reaction mixture to simulate the metabolic process in human hepatocytes in vivo, and then to determine the reaction points and pathways of metabolism by ultra performance liquid chromatography (UPLC) coupled to high resolution mass spectrum (HR-MS). 5F-AMB generated 9 metabolites in total in the human liver microsomes model. Ester hydrolysis, combination of ester hydrolysis and oxidative defluorination, combination of ester hydrolysis and hydroxylation on pentyl chain moiety and combination of ester hydrolysis and hydroxylation on indazole ring moiety reactions were its main metabolic pathways. The method is fast and efficient so that the ester hydrolysis, combination of ester hydrolysis and oxidative defluorination, combination of ester hydrolysis and hydroxylation on pentyl chain moiety and combination of ester hydrolysis and hydroxylation on indazole ring moiety metabolites of 5F-AMB can be used as the suitable and potential biomarkers in the urine samples.

16.
Article in Chinese | WPRIM | ID: wpr-614460

ABSTRACT

Objective To characterize the metabolic kinetics of aloe emodin in human liver microsomes(HLM)and rat liver microsomes(RLM)and identify the CYP phenotyping of phaseⅠmetabolism. Methods Aloe emodin was incubated at 37℃ with HLM and RLM in the presence or absence of NADPH,UDGPA or NADPH+UDGPA. The remaining aloe emodin was determined with a validated LC-MS/MS method to assess the metabolic stability and enzymatic kinetics. A panel of rCYP isoforms(CYP1A2,2B6,2C8, 2C9,2C19,2D6 and 3A4)and HLM with specific inhibitors of CYP isoforms were used to identify the CYP phenotyping of aloe emo?din. Results In HLM and RLM,aloe emodin was metabolically eliminated in the presence of NADPH,with 85.8%and 81.7%of the parent compounds eliminated in 30 min,respectively. The t1/2 were(10.3±0.3)and(11.5±3.3)min,and the CLint were(420.1±10.9) and(573.4±188.2)ml/(min·kg),respectively. The apparent Km and Vmax for HLM and RLM were obtained and found to be(2.4±0.9) and(3.9±1.4)μmol/L,(1492±170.5)and(2783±595.8)nmol/(min·g protein),respectively. In RLM with UDPGA,38.5%of aloe emodin was metabolized in 30 min with t1/2 of 31.6 min and CLint of(197.1±15.5)ml/(min·kg). The results of CYP phenotyping indi?cated that CYP1A2,2B6,2C19 and 3A4 were the major enzymes involved in the metabolism of aloe emodin. By using the method of total normalized rate,the contributions of the major enzymes were assessed to be 35.4%,6.6%,2.2%and 21.9%,respectively. Con?clusion Aloe emodin is mainly eliminated by CYP mediated metabolism in HLM and RLM. CYP1A2 and 3A4 are the major responsi?ble enzymes of aloe emodin,and the contributions are above 20%. Species differences in liver metabolism of aloe emodin are observed. It undergoes notable glucuronidation in RLM only.

17.
Article in Chinese | WPRIM | ID: wpr-853037

ABSTRACT

Objective: To compare in vitro metabolic differences of liquiritigenin, an aglycone of liquiritin, among liver microsomes of different species, which would provide reference for further research and development of liquiritin. Methods: Metabolic stability and metabolic biotransformation were investigated after liquiritigenin was incubated with rat, mouse, human, dog, and monkey liver microsomes. Metabolic stability was evaluated using a substrate depletion approach, and the results were reported as "% liquiritigenin remaining", which was then used to calculate the in vitro half-life (t1/2). Metabolic biotransformation was characterized by metabolite profiling. Results: In liver microsomal incubation systems of five species, the t1/2 values of liquiritigenin for phase I were as follows: rat < mouse < human < monkey < dog, whereas for phase II, the metabolic rates were all fast and the remaining of liquiritigenin were all below 50% in 5 min except mouse. In addition, phase II metabolite profiling in monkey liver microsomes was identical to that of human, but marked differences were found between other species and human. Conclusion: The metabolic characteristics of liquiritigenin in monkey liver microsomes is most similar to that of human, then followed by dog, and marked differences existed between rat, mouse and human. Therefore, monkey or dog could be the animal model for further preclinical pharmacokinetic and toxicological studies.

18.
China Pharmacist ; (12): 1517-1520, 2017.
Article in Chinese | WPRIM | ID: wpr-607295

ABSTRACT

Objective:To study the effect of tannins from Pericarpium Granati on hepatic microsomal enzyme in rats. Methods:Thirty Wistar rats were randomly divided into five groups:the blank control group, high, medium and low dose groups of tannins from Pericarpium Granati, the positive control group of phenobarbital sodium. The blank control group was given physiological saline. The three different doses groups were respectively given tannins from Pericarpium Granati orally at the dose of 150,100 and 75 mg·kg-1 · d-1 for 7 days. The positive control group was given phenobarbital sodium 80 mg·kg-1 ·d-1 with intramuscular injection for 5 days. At the end of the experiment, the activity ofⅠandⅡphase metabolic enzymes in liver microsomes of each group was determined by UV. Results:Compared with the blank control group, the high, medium and low dose groups of tannins from Pericarpium Granati could sig-nificantly decrease the content of CYP 450 and CYPb5, and inhabit the activity of ADM (P<0. 01);the high and medium dose group could significantly inhibit ERD enzyme activity (P<0. 01);the high dose group could significantly reduce GST enzyme activity (P<0. 01). Conclusion:Tannins from Pericarpium Granati has notably inhibitory effect on hepatic microsomal enzyme in rats, which can reduce the expression of CYP3A and CYP2E1 in a dose-dependent manner.

19.
Drug Evaluation Research ; (6): 627-632, 2017.
Article in Chinese | WPRIM | ID: wpr-619529

ABSTRACT

Objective To study the inhibitory effects ofisorhamnetin on six kinds of CYPs of liver in vitro,and the toxic effect on rat hepatocytes Methods This report uses warm incubation of human liver microsomes in vitro to investigate the inhibition of isorhamnetin on 6 kinds of CYPs (CYP2C19,CYP2D6,CYP3A4,CYP2E1,CYP1A2 and CYP2C9),and using HPLC-MS/MS to detect product of metabolism as well as analysing of the pathways of metabolic.At the same time,using rat primary hepatocytes which has low CYPs activity in vitro to explore whether the use of isorhamnetin will cause effects on the ALT,AST and LDH of hepatocytes.Results Isorhamnetin has inhibition effects on CYP2E1 and CYP1A2,the inhibition rate were 59.48% and 39.91%,respectively.Methylated metabolite is produced after incubating of isorhamnetin and HLMs.The isorhmnetin becomes high polarity and water solubility metabolite 3,3',4',5,7-hydroxyflavone.Isorhamnetin of 30,100 and 300 μmol/L cause a significant rise of ALT and LDH in primary cultured rat hepatocytes cultured (P < 0.01).isorharnnetin of 100 μmol/L cause a rise of AST in primary cultured rat hepatocytes cultured (P < 0.05) and 300 μmol/L cause a significant rise (P < 0.01).It was a dose-dependent manner.Conclusion Isorhamnetin in vitro mainly metabolized by HLMs,and at the same time have a certain inhibitory effect on CYP2E1 and CYP1A2,which may cause the drugs which are metabolized by CYP2E1 and CYP1A2 in vivo accumulation that lead to a series of drug interactions.The results also indicate that heavy use of isorhamnetin cause some adverse effects on hepatocytes,and it was a dose-dependent manner.Individuals need to pay attention to the dose ofisorhamnetin and the potential drug interactions.

20.
Article in Chinese | WPRIM | ID: wpr-667836

ABSTRACT

OBJECTIVE To compare the species difference of T-2 toxin metabolism in liver micro-somes of different animals. METHODS T-2 toxin was incubated with liver microsomes from mice, rats,Beagle dogs, monkeys and humans, respectively, at 37℃ for some time. Then, the incubation liquid was detected by high liquid chromatography-mass spectrometry method after albumen precipitation. RESULTS The half-life (t1/2) of T-2 toxin was less than 1 min, 2-4 min in mouse and monkey liver microsomes, 13 min in dog liver microsomes, and 39 min in rat liver microsomes. The hepatic clear-ance (Clh) of T-2 toxin was divided into three groups among the five species of animals:humans, dogs and rats were in one group, monkeys a second group, and mice in another group. The Clh of mouse group was 3-4 times that of the human, dog and rat group. The affinity to T-2 toxin was different between the liver microsomes of these five species. The affinity of mouse liver microsomes was the strongest, followed by that of humans, dogs, rats and monkeys. The enzyme transfer rate of T-2 toxin was the highest in monkey liver microsomes followed by that of rats and dogs. It was one million times higher in monkey liver microsomes than in human and mouse liver microsomes. The major metabolites were 3′-hydroxyl-T-2 toxin and neosolaniol. T-2 triol and HT-2 toxins were the major metabolites in human and rat liver microsomes. HT-2 toxin and 3′-OH-T-2 toxin were the dominating metabolites in dog liver microsomes and T-2 triol and 3′-OH-T-2 toxin in mouse liver microsomes. T-2 toxin metabolited by hydrolysis effect in mouse, rat, dog and human liver microsomes, but through hydroxylation in monkey liver microsomes. CONCLUSION There are species differences in metabolic parameters, metabolites, amounts of metabolites, metabolic pathways of T-2 toxin in mouse, rat, dog, monkey and human liver microsomes.

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