Pharmacokinetics Of Ganoderma Lucidum A And Potential Drug Interactions Of Ganoderma Lucidum

The traditional Chinese herbal medicine Lingzhi is the body of Ganoderma lucidum and Ganoderma sinense.Triterpenoids and polysaccharides are its two main types of components and considered to be responsible for most of its pharmacological activities.Ganoderic acid A(GAA)is one of the most abundant triterpenoids of Lingzhi,and generally exists in Ganoderma genus.GAA reportedly exhibited antinociceptive,antioxidative,cytotoxic,hepatoprotective and anticancer activities.As the main active component of Lingzhi,there is few published research on the metabolism,pharmacokinetics and bioavailability of GAA when it was given in pure compound.Lingzhi is commonly used as a dietary supplement or as a prescription herb in clinics to cure many diseases,and is often concurrently administered with other conventional prescription herb.However,study on drug drug interaction caused by Lingzhi or its products is limited.In present study,we investigated the pharmacokinetics and absorption mechanism of GAA and potentially drug-drug interaction of Lingzhi that mediated by efflux transporter and CYP450.The main contents of this paper are as follows:1.Study on metabolism and metabolic pathway of GAA Using HPLC-DAD-MS/MS techniques,we studied the metabolism of GAA and the metabolic kinetics of its main metabolites.We identified GAA metabolites,in vivo by analyzing the biological samples after intravenous administration to rats and in vitro by incubating with rat liver microsomes(RLMs)and human liver microsomes(HLMs).In addition,we investigated the metabolic kinetics of main GAA metabolites.A total of 37 metabolites were tentatively characterized from the bile,plasma and urine samples of rats.Nine metabolites were detected in RLMs,and seven metabolites were detected in HLMs,which were also found in rats in vivo.The result indicated the metabolism similarity between human and rat.GAA could undergo extensive metabolism,including reduction,oxidation,and hydroxylation phase I metabolism,and glucuronidation and sulfation phase II metabolism.The main metabolic soft spots of GAA were 3,7,11,15,23-carbonyl groups(or hydroxyl groups)and 12,20,28(29)-carbon atoms.The reduction metabolites of GAA were formated more quickly in RLMs than in HLMs,and C YP3 A catalyzed the reduction metabolism of GAA in both RLMs and HLMs.2.Pharmacokinetics of GAA and its main metabolites in rats In this study,we established a sensitive UFLC-MS/MS method to determine the concentration of GAA in rat plasma,bile,urine and cerebral spinal fluid.The results indicated that the calibration curve showed good linearity with low limits of detection and quantification of 0.25 and 2.00 nmol/L,respectively.The precision,accuracy,extraction recovery and stability was suitable for biological analysis.The developed method was successfully applied to a pharmacokinetic study of GAA and its main metabolites ganoderic acid C2(C1),7β,11,15-trihydroxy-3,23-dioxo-lanost-8-en-26-oic acid(C2),11,15-dihydroxy-3,7,23-trioxo-lanost-8-en-26-oic acid(C3)and ganoderic acid B(C4)in rats.The metabolites were determined with GAA serving as standard.After intravenous(i.v.)administration of GAA at 20 mg/kg in rats,the parent compound eliminated gradually in vivo,and transformed to metabolites C1-C4 in the same time.The metabolites reached the Cmax in about 5 min,with Cmax being 2.61,0.17,2.84 and 0.51 μmol/L,respectively.C1,C2 and C3 showed reabsorption peak at about 3-6 h.The t1/2 of GAA and metabolites C1-C4 were 2.40,13.08,12.35,2.16 and 2.79 h,respectively.They mainly excreted in bile,the excretion rate were 21.37%,18.02%,2.22%,2.33%and 0.70%of dose in 24 h,respectively.Well,in urine,the excretion rate of GAA was only 2.59%,the total excretion rate of the metabolites C1-C4 was 0.076%.After oral administration of GAA at 20 mg/kg,GAA and metabolites C1-C4 reached the first plasma concentration peak in 10-40 min,and showed reabsorption peak at about 6-8 h.The absolute oral bioavailability of GAA was 8.68%.GAA could penetrate the brain rapidly after i.v.dosing(Tmax,0.25 h),and the brain permeability of GAA was 2.96%.The metabolites C1-C4 were not detected in rat cerebral spinal fluid.3.Intestinal transport mechanism of GAA The intestinal absorption mechanism of GAA was investigated using Caco-2 cell monolayer model and UFLC-MS/MS method for the first time.The result indicated that the amount of transported GAA increased in a concentration and time-dependent manner in both the apical-to-basolateral(AP→BL)and the basolateral-to-apical(BL→AP)direction.The apparent permeability PappA→B of GAA were(4.30-4.99)×10-7cm/s,the Papp B→A of GAA were(33.52-37.77)×10-7cm/s,and the efflux ratio of GAA were 6.72-8.79.The PappA→B of GAA increased to 5.79×10-7,12.14×10-7 and 4.62×10-7 cm/s,respectively,Papp B→A decreased to 30.17×10-7,23.55×10-7 and 25.57×10-7 cm/s,respectively,and efflux ratio decreased significantly in the presence of P-glycoprotein(P-gp)inhibitor verapamil,multidrug resistance-associated protein(MRP)inhibitor MK571 and breast cancer resistance protein(BCRP)inhibitor dipyridamole.The results indicated that P-gp,MRP and BCRP might all govern the GAA secretion,which might be the main cause of the poor bioavailability of GAA.4.Drug-drug interaction of Ganoderma lucidum mediated by efflux transporter The effects of Ganoderma lucidum extract and GAA on the function of P-gp,MRP and BCRP were investigated by Caco-2 cell cellular uptake and transcellular transport experiments.The result of cellular uptake experiment indicated that Ganoderma lucidum extract(GLE)and Ganoderma lucidum triterpenoid extract(GLT)at concentration of 100μg/mL could significantly increase the cellular accumulation amount of P-gp substrate Rhodamine 123(Rho)and MRP substrate Calcein(Cal),and inhibited the function of P-gp and MRP.They could significantly decrease the cellular accumulation amount of BCRP substrate Hoechst 33342(Hoe),and induced the function of BCRP.Ganoderma lucidum polysaccharide extract(GLP)at concentration of 100 μg/mL did not show any significant effects on function of P-gp and MRP,well,could induce the function of BCRP.GAA at experimental concentration did not show any significant effects on function of all the three efflux transporters.In the Caco-2 cell transcellular transport experiments,the amount of transported Rho in AP-—BL direction and Papp A→B of Rho were both increased significantly in the present of different concentration of GLE and GLT,well,in the BL→AP direction,the corresponding values were both decreased significantly.These results suggested that GLE and GLT could inhibit the efflux transport of Rho by P-gp.In the present of different concentration of GLE and GLT,the amount of transported Cal on apical side was decreased,and the amount of accumulated Cal in the Caco-2 cell monolayer was increased,which indicating that GLE and GLT could inhibit the efflux transport of Cal by MRP.All these results indicated that triterpenoid is the main active component of Ganoderma lucidum responsible for the inhibition effects on efflux transporters.There are potential drug interactions between Ganoderma lucidum and efflux transporters substrate drugs.5.Drug-drug interaction of Ganoderma lucidum mediated by CYP450 An in vitro cocktail method was established for simultaneous determination of six CYP450 isoenzymes activities by monitoring the metabolites of probe substrates,including phenacetin(CYP1A2),omeprazole(CYP2C19),dextromethorphan(C YP2D6),testosterone(CYP3A4),tolbutamide(CYP2C9),chlorzoxazone(CYP2E1).Inhibition activities of Ganoderma lucidum extract and GAA on the six isoenzymes were evaluated using established method.In HLMs,GLE weakly inhibited the CYP2C19,CYP2D6,CYP3A4 and CYP2C9 activities with IC50 of 131.2,164.4,150.5 and 142.2 μg/mL,respectively.GLT possessed relative potent inhibition effects on these four isoenzymes than GLE with IC50 of 102.5,116.1,136.4 and 82.2 μg/mL,respectively.GLP showed no inhibitory effects on CYP450 enzymes.In RLMs,GLT weakly inhibited the CYP2C9 activity with IC50 of 163.1 μg/mL.GLE and GLP showed no inhibitory effects on CYP450 enzymes.GAA at 1-50 μmol/L showed no inhibitory effects on the six isoenzymes in either RLMs or HLMs.