| Picrasma quassiodes(D.Don)Benn.is a traditional herbal medicine for the treatment of gastroenteritis,snakebite,infection and hypertension in China,so it is an important composition of many Chinese patent medicines(such as Kumu injection,Kumu tablet,Kumu mixture)which have been used to treat enteritis,bacillary dysentery,hypertension,influenza,upper respiratory tract infection and acute tonsillitis in clinic.4,5-dimethoxycanthin-6-one is the main active ingredient of P.quassioides,many reports show that it has the activities of anti-inflammation,anticancer,inhibition on cAMP phosphodiesterase,anti-virus,anti-hypertension and so on.Comparing to the pharmacological study,there are few reports about its pharmacokinetics.In this paper,high resolution and sensitive liquid chromatography-mass spectrometry technique(such as UPLC-Q-TOF-MS/MS and UPLC-MS/MS)were used for the following pharmacokinetic study of 4,5-dimethoxycanthin-6-one in vivo and in vitro.1.Identification of in vivo and in vitro metabolites of 4,5-dimethoxycanthin-6-oneIn the present study,the in vitro metabolites of 4,5-dimethoxycanthin-6-one in rat,mouse,dog and human liver microsomes,as well as the in vivo metabolites in rat plasma and urine following a single dose intramuscular administration of 4,5-dimethoxycanthin-6-one,were identified by ultra-performance liquid chromatography coupled with triple TOF mass spectrometry(UPLC-TOF/MS/MS).The results showed:,After incubation in liver mcrosomes for 50 min,4,5-dimethoxycanthin-6-one produced 8 phase I metabolites including 2 mono-demethylated metabolites(M1,M2),3 mono-hydroxylated metabolites(M3-M5),and 3 mono-demethylated and mono-hydroxylated metabolites(M6-M8)in rat and mouse liver microsomes;7 phase I metabolites(without M7)in dog and human liver microsomes.②After a single dose intramuscular administration of 4,5-dimethoxycanthin-6-one to rats,there were 3 phase I metabolites(M1,M2 and M5)detected in rat plasma and 5 phase I metabolites(M1-M5)in rat urine.Phase II metabolites were not detected in rat plasma and urine.③Among these metabolites,mono-demethylated metabolites(M1 and M2)were the major metabolites of 4,5-dimethoxycanthin-6-one,mono-hydroxylated metabolites(M3-M5)were the minor metabolites.Therefore,there were some similarities(the primary metabolic behaviour is methylation and hydroxylation is the secondary)in vivo and in vitro.2.Identification of metabolic enzymes responsible for the formation of metabolites of 4,5-dimethoxycanthin-6-one Based on the identification of in vitro metabolites,the specific chemical inhibitors a-naphthoflavone(for CYP1A2),clopidogrel(for CYP2B6),fluconazole(for CYP2C9),ticlopidine(for CYP2C19),quinidine(for CYP2D6),diallyldisulfide(for CYP2E1),ketoconazole(for CYP3A4),methoxsalen(for CYP2A6)and pioglitazone(for CYP2C8)were co-incubated with 4,5-dimethoxycanthin-6-one in HLM,respectively;UPLC-MS/MS was used for the detection of metabolites production,so as to screen the metabolic enzymes responsible for 4,5-dimethoxycanthin-6-one;after that,the recombinant CYP450 enzyme incubation experiment was performed to confirm the metabolic enzymes of 4,5-dimethoxycanthin-6-one in HLM.The results indicated that the in vitro metabolic enzyme of 4,5-dimethoxycanthin-6-one was identified to be CYP3A4(mediated the formation of metabolites M1 to M5),CYP2C9(mediated the formation of metabolites Ml to M3,M6 and M8)and CYP2D6(mediated the formation of metabolite M3)in HLM.3.Interactions of 4,5-dimethoxycanthin-6-one and the substrates of cytochrome P450(CYP450)in vitroTo determine the inhibitory effect of 4,5-dimethoxycanthin-6-one on the activity of CYP isoforms,the probe substrates phenacetin(for CYP1A2),bupropion(for CYP2B6),tolbutamide(for CYP2C9),dextromethorphan(for CYP2D6),chlorzoxazone(for CYP2E1),testosterone(for CYP3A4),coumarin(for CYP2A6)and taxol(for CYP2C8)were co-incubated with 4,5-dimethoxycanthin-6-one in HLM.After the incubation,the production of metabolites was measured to determine the effect of the original drug on the enzyme activity.The results showed that 4,5-dimethoxycanthin-6-one could uncompetitively inhibit CYP1A2 mediated phenacetin O-deethylation with an IC50 value of 1.7 μM and a Ki value of 2.6 μM,suggesting that the clinical risk based on metabolic interaction would be existed when the substrate drugs of CYP1A2 are co-administered with 4,5-dimethoxycanthin-6-one or traditional Chinese medicine containing 4,5-dimethoxycanthin-6-one.4.Pharmacokinetic study of 4,5-dimethoxycanthin-6-one and its major metabolites in rats3-methylcanthin-2,6-dione was used as internal standard(IS),protein precipitation by methyl alcohol was used for plasma sample preparation and liquid-liquid extraction by ethyl acetate was used for tissue sample.A UPLC-MS/MS method for the determination of 4,5-dimethoxycanthin-6-one,its metabolites M1(5-hydroxy-4-methoxycanthin-6-one)and M2(isomer of M1)in rat plasma and tissues was developed and applied to pharmacokinetics and tissue distribution study.Chromatographic conditions:The separation was carried out on a Shim-pack ODS column(4.6 μm,150 mm×2.0 mm i.d.)at a column temperature of 40℃,the injection volume was 10 μL.A mixture of solvent A(0.1%formic acid in water)and solvent B(methanol)was used as the mobile phase at a flow rate of 0.4 mL/min,and the gradient elution program was as follows:0-14.0 min,40%B;14.01-19.0 min,40%B to 85%B;19.01-24.0 min,40%B.MS conditions:It was set in positive multiple reaction monitoring(MRM)mode.The MS transitions were m/z 281.0→167.1(4,5-dimethoxycanthin-6-one),m/z 267.0→168.1(M1 and M2),m/z 251.0→195.1(IS).The method validation of sensitivity,precision,accuracy,stability,matrix effect and recovery met FDA guidelines for the in vivo sample analysis.After rats were administered intramuscularly with a single dose of 4,5-dimethoxycanthin-6-one at 10,20 and 40 μg/kg3 respectively.The data illustrated that 4,5-dimethoxycanthin-6-one was absorbed rapidly(Tmax=5.4-6.4 min)and eliminated quickly(t1/2z=64.9-77.7 min);in addition,the AUC0-t and Cmax of 4,5-dimethoxycanthin-6-one increased in a dose-dependent manner,but the t1/2z remained unchanged,indicating that the pharmacokinetic profiles of 4,5-dimethoxycanthin-6-one is characterized by the first order kinetics.Compared to the AUC0-t of 4,5-dimethoxycanthin-6-one,M1 was only 15.2-19.9%of it,but M2 was 194.1-218.4%of it,revealing that M2 was the major metabolite of 4,5-dimethoxycanthin-6-one.The tissue distribution results showed that 4,5-dimethoxycanthin-6-one was distributed rapidly and widely in the selected tissues,it could not be detected at 64 min after post-administration indicated that 4,5-dimethoxycanthin-6-one was eliminated quickly as well.Additionally,the highest concentration of 4,5-dimethoxycanthin-6-one was found in liver,but no significant difference was found in other tested tissues.Furthermore,M1 and M2 were detected only in liver at the indicated times.The results of this paper play an active role in the study of metabolism,distribution and metabolic interactions for 4,5-dimethoxycanthin-6-one,making a contribution to the further research on the new drug development and clinical rational drug use of picrasma quassiodes alkaloids. |
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