| Background and objectiveFlavonoids are low-molecular-weight phenolic compounds widely distributed in natural plants and frequently consumed in daily diet.For decades,scientists have maintained a strong interest in their potential health benefits,such as anti-aging and cancer-preventive activities.However,only a few inconclusive clinical trials regarding the role of flavonoids in cancer prevention have been conducted.The druggability of flavonoids is limited by two major problems:unclear mechanisms behind their benefits and low bioavailability that is largely attributed to drug metabolism.Flavonoids containing multiple unconjugated phenolic hydroxyls undergo extensive conjugative metabolism performed by phase II enzymes,such as UDP-glucuronosyltransferases(UGT)and sulfotransferase(SULT).Phase II conjugation has been demonstrated to be a major metabolic pathway for flavonoids.Many flavonoid phase II conjugates are substrates of membrane-bound efflux transporters including breast cancer resistance protein(BCRP)and multidrug resistance proteins(MRPs),which are abundantly expressed in the intestine and liver These proteins transport drug molecules out of the cells and perform an important role in drug elimination.Kaempferol,3,4’,5,7-trihydroxyflavone,is a typical flavonol distributed in many edible plants and beverages frequently consumed(e.g.,tomato,grapefruit,broccoli,honey,and tea).Kaempferol provides many promising pharmacological activities,such as anti-oxidation,anti-inflammation,and antitumor.Epidemiological studies have revealed the preventive effects of dietary kaempferol intake on pancreatic cancer,lung cancer,ovarian cancer,and colorectal adenoma.Kaempferol exhibit anticarcinogenic activities occur by impairing cancer angiogenesis,disrupting cancer metastasis,and inducing cell apoptosis.Compared with the large amounts of pharmacological studies,the in vivo exposure of kaempferol is less discussed.In vitro metabolism showed that rat cytochrome P450 1A1 catalyzes the oxidative metabolism of kaempferol;nevertheless,the tendency for UDPGA-dependent conjugation is evidently higher.Kaempferol-7-glucuronide(K-7-G)is the major product of conjugative metabolism using rat liver microsomes(RLMs),whereas small intestine is more likely to produce kaempferol-3-glucuronide(K-3-G).Cell experiments reported independently that kaempferol may be a substrate and inhibitor of P-gp and BCRP.Kaempferol can be conjugated inside the Caco-2 cells and subsequently transported across the monolayer or excreted back to the apical(AP)side;however,the transporters responsible for conjugates efflux remain unknown.The bioavailability of kaempferol is lower than 2%.Though a validated LC-MS/MS method is applied to determine plasma kaempferol,an indirect method is needed to quantify the total glucuronidated kaempferol through hydrolysis using glucuronidases.Moreover,the lack of sensitivity leads to a large plasma usage,which requires excessive blood collection.Kaempferol is easily metabolized,but limited studies have investigated the in vivo disposition and exposure of kaempferol and corresponding regulatory mechanisms.Therefore,to understand the pharmacological properties of kaempferol better,a comprehensive and thorough study of its in vivo disposition is important,which will also be suggestive for the investigation of its drug-like properties.In this study,we developed a sensitive and reliable LC-MS/MS method to determine kaempferol and measurable phase II metabolites in various matrices simultaneously and directly.The pharmacokinetic study of kaempferol was first performed in rats for a general overview of its in vivo exposure.Subsequently,the pharmacokinetics in knockout mice provided an insight into the effect of efflux transporters on the exposure level of the conjugates.Intestinal perfusion in rats was conducted to investigate the metabolism of kaempferol in four intestinal segments,which was used to illustrate the role of intestinal disposition in the in vivo exposure of kaempferol.The Caco-2 cell model combined with selective inhibitors was used as an in vitro approach to confirm the role of efflux transporters.The disposition of kaempferol in UGT1A9 overexpressing and UGT1A1 overexpressing HeLa cells illustrated the mechanism by which UGT-BCRP coupling regulated kaempferol metabolism.Methods1.Development of a LC-MS/MS method for simultaneous quantitation of kaempferol and the metabolites.In vitro glucuronidation by RLMs and sulfation by rat hepatic S9 of kaempferol were conducted.The metabolites were identified by multiple methods using UPLC-DAD-QTOF including accurate mass measurements,analyzing shift in UV spectra compared with the spectra of aglycone,and comparing retention times with the standards.Then a sensitive and reliable LC-MS/MS method was developed to determine kaempferol and measurable phase II metabolites in various matrices simultaneously and directly.2.Pharmacokinetics in SD rats and efflux transporter knockout FVB miceAfter the oral kaempferol administration in SD rats(10 mg/kg and 20 mg/kg),blood samples were collected at the predetermined periods from the postorbital venous plexus veins.After the oral kaempferol administration in wild-type and knockout FVB mice(10 mg/kg),blood samples were collected from the caudal veins.Plasma sample were prepared according to a validated method.The pharmacokinetic parameters were calculated using noncompartmental analysis of WinNonlin(?)3.3 software.3.In situ perfusion in rats and in vitro metabolism of kaempferolAn in situ rat intestinal perfusion model was used and two kaempferol concentrations were perfused.Perfusate,bile,and blood samples were collected.In vitro glucuronidation and sulfation by hepatic and intestinal microsomes/S9 fractions were conducted.4.The metabolism of kaempferol and efflux of the metabolites in Caco-2 and HeLalA9 cell modelKaempferol was loaded on the AP or BL side of the Caco-2 monolayer.The P-gp inhibitor verapamil,BCRP inhibitor Ko143,and MRP2 inhibitor LTC4,respectively,were loaded in the AP side and co-incubated with kaempferol in Caco-2 cell model.The BL-side MRPs inhibitor MK571 was loaded in the BL side.Samples were collected from both sides of each transwell.Different concentrations of kaempferol with or without Ko 143 were incubated with UGT1A9 overexpressing and UGT1A1 overexpressing HeLa cells,respectively.Extracellular and intracellular samples were collected and analyzed.Results and discussion1.Identification of the phase II conjugates of kaempferolBy using UPLC-DAD-QTOF,four mono-glucuronides,namely,K-5-G,K-3-G,K-7-G,and K-4’-G were identified.For sulfation,one mono-sulfate was identified as K-7-S.The standard substance of K-3-G,K-7-G,and K-7-S was obtained through different ways.A sensitive LC-MS/MS method was then developed for simultaneous determination of the three metabolites and kaempferol.2.In vivo exposure of kaempferol and the effect of efflux transportersAfter the oral administration in rats,kaempferol was quickly absorbed through the gastrointestinal tract and metabolized to phase II conjugates,including K-3-G,K-7-G,and K-7-S.Ion currents for diglucuronide,disulfate,or glucurono-sulfate were not detected in plasma.Compared with the amount of metabolites,the proportion of free kaempferol was extremely small.Kaempferol and glucuronides reached the maximum concentration(Cmax)within 30 min and were completely eliminated in 12 h;sulfate had evidently longer peak time(15-19 h)and elimination and was still detected in plasma on 36 h post dose.Notably,K-3-G had higher plasma concentration than K-7-G,which was reflected by approximately 3-5-fold higher Cmax and AUC.The Cmax of K-7-S was much lower than that of K-3-G and K-7-G;however,due to relatively long elimination,the AUC of K-7-S was largely sufficient to attract attention,which indicates that sulfation is also an important metabolic way for kaempferol.Pharmacokinetic study in four types of knockout mice and FVB wild-type mice was conducted to determine which efflux transporter influenced the in vivo exposure of kaempferol.Kaempferol could not be detected in plasma.When Mrp1,expressed at BL membranes,was knocked out,systemic parameters(Cmax,AUC0-t,and MRT)of the three metabolites were dramatically reduced compared with those of the wild-type.The absence of AP-side Bcrp and Mrp2 resulted in much higher plasma concentration of the three metabolites as reflected by significantly higher Cmax,and AUC0_t.The lack of P-gp caused the Cmax and AUC0-t of K-3-G significantly decreased,but did not alter the C max and AUC0-t of K-7-G and K-7-S significantly.Pharmacokinetic study in knockout mice suggested the important positions of Mrpl,Mrp2,and Bcrp in restricting the plasma concentration of kaempferol conjugates.3.Intestinal disposition and in vitro metabolism of kaempferolAfter perfusion with 5 or 20 μM kaempferol,its absorption was fast in colon,which was significantly higher than those in jejunum and ileum at both concentrations.Significant difference was not observed in the P*eff values between 5 and 20 μM concentrations.Kaempferol was converted into glucuronides and sulfate,and large amounts of K-3-G and K-7-G were found in the perfusate,with the highest excretion in duodenum and jejunum whereas lowest excretion in colon.On the contrary,K-7-S had the highest excretion in colon and lowest excretion in ileum.Moreover,K-7-G in the perfusate was slightly higher than K-3-G in duodenum and jejunum but significantly higher in ileum and colon.These glucuronides in the bile basically increased with time,and the amount of K-3-G was 3-5 times higher than that of K-7-G at each time point.Only K-3-G was determined in plasma because the other analytes were below the corresponding LLOQ.This indicated that K-3-G was the main metabolite in vivo.In vitro metabolism of kaempferol in rat hepatic and intestinal segments was investigated.High and low glucuronidation rates were observed in the small intestine and colon,respectively.Jejunum exhibited maximal formation rates for the two glucuronides under three kaempferol concentrations.Liver microsomes produced more K-7-G than K-3-G.Sulfation was relatively different from glucuronidation and achieved the highest reaction rates in liver.Among the four intestinal segments,colon showed the highest reaction rates and demonstrated statistically significant differences from duodenum and ileum.Based on the above experiments,we found that glucuronidation mainly occurred in the small intestine and sulfate was mainly produced in colon among the four intestinal segments.The production of K-7-S is lagged compared with that of glucuronides,which may contribute to the atypical concentration-time profile of K-7-S.4.The effects of efflux transporters and UGT-BCRP coupling on kaempferol metabolism.Kaempferol could undergo glucuronidation and sulfation in Caco-2 monolayer.In the BL side,the efflux rate of K-3-G was significantly higher than that of K-7-G whether kaempferol was loaded on the AP or BL side;while in the AP side,the efflux rate of K-3-G was significantly lower than that of K-7-G,thereby indicating that K-3-G was more inclined to be transported into the BL side.This phenomenon,in line with the results from rat intestinal perfusion,illustrated that efflux transporters exhibited regioselectivity to the positional isoforms of kaempferol-glucuronides,from which we drew a conclusion that UGT and efflux transporter coupling determines the in vivo exposure of kaempferol as the large amount of K-3-G.Verapamil did not make any statistical difference in the efflux rate,intracellular concentration,and Fmet in the bidirectional transport of glucuronides.However,the bidirectional efflux rates of K-7-S and Fmet(BL to AP)were significantly decreased,which implicated that verapamil potentially suppresses kaempferol sulfation.Ko143 reduced the excreted amounts of K-3-G and K-7-G in two sides to varying degrees and significantly decreased the bidirectional efflux rates and Fmet of two glucuronides,which implicated that Ko143 potentially suppresses kaempferol glucuronidation.Statistical differences were not observed in the efflux rate and Fmet of K-7-S with Ko143.LTC4 reduced the amounts of two glucuronides in the AP side,and also significantly increased the intracellular amounts of K-3-G and K-7-G(AP to BL),whereas decreased the Fmet(BL to AP)of the three metabolites.In the presence of 10μM MK571,the three metabolites transported into BL side were dramatically reduced,whereas in the AP side,MK571 resulted in higher K-3-G and K-7-S;this observation indicated that the BL-side loading MK571 barely blocked MRP2 in the AP side.As anticipated,MK571 significantly lowered the efflux rates of three metabolites toward BL side,but did not significantly influence their intracellular amounts.These results demonstrated that transport of kaempferol is attributed to efflux transporter MRP2,BCRP and BL-side MRPs.In HeLalA9 and HeLalAl cells,kaempferol was first metabolized to glucuronides(i.e.K-3-G and K-7-G)and then excreted outside.The clearance and Fmet for two glucuronides dramatically decreased with the increase of kaempferol concentrations.Accordingly,the intracellular concentrations of glucuronides increased with the increase of kaempferol concentrations.The excretion rates and clearance of K-3-G and K-7-G were significantly reduced in a concentration-dependent manner in the presence of BCRP selective inhibitor Ko143.Though Fmet was altered by Ko143,the intracellular concentration of two glucuronides was significantly increased.These results suggest that BCRP is responsible for the K-3-G and K-7-G efflux,and ascertain the important pathway for kaempferol disposition mediated by UGT-BCRP coupling.ConclusionsIn conclusion,we adopted a combined strategy to investigate the metabolism of kaempferol,and a good correlation was observed among in vitro,in situ,and in vivo data.Kaempferol is mostly exposed as phase II conjugates in plasma,including two glucuronides and one sulfate,and K-3-G is the principal metabolite.Efflux transporters,namely,BCRP,MRP1,and MRP2,regulate the in vivo exposure level of kaempferol conjugates.Comprehensive pharmacokinetic study may offer a new perspective or implication to subsequent pharmacological research of kaempferol. |
PDF Full Text Request