Ganoderic Acid A Metabolites and Their Metabolic Kinetics

Ganoderic acid A (GAA), a representative active triterpenoid from Ganoderma lucidum, has been reported to exhibit antinociceptive, antioxidative, cytotoxic, hepatoprotective and anticancer activities. The present study aims (1) to identify GAA metabolites, in vivo by analyzing the bile, plasma and urine after intravenous administration to rats (20 mg/kg), and in vitro by incubating with rat liver microsomes (RLMs) and human liver microsomes (HLMs); (2) to investigate the metabolic kinetics of main GAA metabolites. Using HPLC-DAD-MS/MS techniques, a total of 37 metabolites were tentatively characterized from in vivo samples based on their fragmentation behaviors. The metabolites detected in in vitro samples were similar to those found in vivo. GAA underwent extensive phase I and 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. Ganoderic acid C2 (GAC2) and 7β,15-dihydroxy-3,11,23-trioxo-lanost-26-oic acid were two main reduction metabolites of GAA, and their kinetics followed classical hyperbolic kinetics. The specific isoenzyme responsible for the biotransformation of the two metabolites in RLMs and HLMs was CYP3A. This is the first report on the comprehensive metabolism of GAA, as well as the metabolic kinetics of its main metabolites.


INTRODUCTION
Ganoderma lucidum (GL), an ancient remedy, has been used for increasing energy, improving immunity, and promoting health and longevity for over 2,000 years in Asian countries, particularly China, Japan and Korea . GL is commonly used as a dietary supplement or prescription in clinics to cure many diseases. Polysaccharides and triterpenoids are its two main types of components and considered to be responsible for the most of its pharmacological activities (Cheng P.-G. et al., 2013). Triterpenoids are highly oxygenated lanostane. Up to now, more than 140 triterpenoids have been isolated from the fruiting bodies, spores and mycelia of GL (Guo X.-Y. et al., 2013). Ganoderic acid A (GAA), generally exists in Ganoderma genus, is one of the most abundant triterpenoids of GL, and can be viewed as a marker component for evaluating GL quality Abbreviations: EPI, Enhanced product ion; ER, enhanced resolution; HLMs, human liver microsomes; IDA, independent data acquisition; GAA, ganoderic acid A; GLA, glycyrrhizic acid; MRM, multiple reaction monitoring; RLMs, rat liver microsomes; UDPGA, uridine 5 ′ -diphosphoglucuronic acid; NADPH, β-Nicotinamide adenine dinucleotide phosphate. (Zhao et al., 2006;Lu et al., 2012). In a previous study, GAA was chosen as the single reference substance for multiple components determination for quantity control of GL (Da et al., 2015). GAA reportedly exhibited antinociceptive (Koyama et al., 1997), antioxidative (Zhu et al., 1999), cytotoxic (Guan et al., 2008) and hepatoprotective activities (Kim et al., 1999), especially anticancer activity (Jiang et al., 2008;Yao et al., 2012;Das et al., 2015;Radwan et al., 2015;Shao et al., 2015), which is the most attractive character of this compound.
Recently, the anticancer activity of GAA attached the considerable attention of scientists. Recent studies showed that GAA exhibits antitumor activity on human osteosarcoma (Shao et al., 2015), lymphoma (Radwan et al., 2015), meningioma (Das et al., 2015) and breast cancer cells (Jiang et al., 2008) through suppressing growth and invasive behavior and/or inducing apoptosis of cancer cells. GAA could also enhance chemosensitivity of HepG2 cells to Cisplatin (Yao et al., 2012). Radwan et al. firstly studied the anticancer activity of GAA in vivo and revealed that GAA can significantly prolong the survival of EL4 syngeneic mice and decrease tumor metastasis to the liver, and enhance cell-mediated immune responses by attenuating myeloid-derived suppressor cells (Radwan et al., 2015). Thus, GAA can be viewed as a promising anticancer candidate or used in combination with conventional chemotherapeutic agents for treatment of cancer.
So far, there is no any published research on the metabolism of GAA. This study aims to detect and identify GAA metabolites in vivo by analyzing the bile, plasma and urine after intravenous administration to rats and in vitro by incubating with rat liver microsomes (RLMs) and human liver microsomes (HLMs), analyzing by using a simple and accurate HPLC-DAD-MS/MS method. Besides, the metabolic kinetics of the main reduction product of GAA were determined by a sensitive and rapid UFLC-MS/MS method.

Animals
Male Sprague-Dawley rats (200 ± 20 g) were supplied by Beijing Vital Laboratory Animal Technology (Beijing, China). The animal experiment was approved by the Animal Ethics Committee at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. The rats were housed under standard conditions of temperature (23 ± 2 • C), humidity (60 ± 5%) and light (12 h light-dark cycle) in a specific pathogenfree animal room, and had free access to standard rodent diet and water before the experiments. On the day before the experiment, the rats were suffered with light surgeries. Under anesthesia by an intraperitoneal dose of 10% chloral hydrate at 3.50 ml/kg, a polyethylene catheter (0.50 mm ID, 1.00 mm OD, Portex Limited, Hythe, Kent, England) was cannulated into the right jugular vein for intravenous drug administration and blood collection, and another catheter (0.28 mm ID, 0.61 mm OD, Portex Limited, Hythe, Kent, England) was cannulated into the bile fistul for bile collection. After surgery, the rats were placed individually in metabolism cages and allowed to recover for at least 24 h. The rats were fasted over night with free access to water prior to drug administration.

Drug Administration and Sample Preparation
GAA (25 mg) was dissolved in 500 µl of physiological saline containing 2% sodium carbonate and diluted with physiological saline to make the volume of 5 ml . The solution was given to rats (n = 5) intravenously at 20 mg/kg through the jugular vein catheter. After dosing and blood sample collection, 0.2 ml of normal saline with 20 units of heparin was injected into the body through the catheter to flush the catheter and prevent blood coagulation.
Rats were held in metabolism cages, blood was collected into heparinized tubes from the jugular vein before (blank) and at 5 and 30 min post dosing, and then centrifuged at 1,500 g for 10 min for plasma separation. At the same time, bile and urine samples were collected before (blank) and over the period of 0-4 h post dosing. All of the collected blank plasma, blank bile, blank urine, dosing plasma, dosing bile or dosing urine from five rats were pooled together respectively and stored at −20 • C until assay.
For plasma, an aliquot of 200 µl sample was mixed with 20 µl HCl solution (1 M), and then extracted with 1.5 ml of ethyl acetate by vortex-mixing for 10 min. The ethyl acetate layer was collected and concentrated to dryness by a vacuum concentrator. The residue was reconstituted in 100 µl 80% methanol and centrifuged at 20,000 g for 15 min. The supernatant was collected for HPLC-DAD-MS/MS assay. For bile, an aliquot of 150 µl sample was mixed with 150 µl 80% methanol and 10 µl 5% formic acid, and then centrifuged at 20,000 g for 15 min. The supernatant was collected for HPLC-DAD-MS/MS assay. For urine, an aliquot of 2 ml sample was loaded on a pretreated ODS-C18 cartridge (Agilent AccuBOND, 500 mg). After washing with 5 ml of water, the cartridge was eluted with 10 ml of methanol. The methanol eluate was collected and concentrated to dryness by a vacuum concentrator at 37 • C. The residue was reconstituted in 200 µl 80% methanol and centrifuged at 20,000 g for 15 min, and the supernatant was collected for HPLC-DAD-MS/MS assay.

In vitro Metabolite Identification
For in vitro metabolites identification, the enzyme incubation was performed in a medium containing 1.0 mg protein /ml RLMs or HLMs, 100 µM of GAA, 3.3 mM of MgCl 2 , 3.0 mM of NADPH, 3.0 mM of UDPGA and 100 mM of sodium phosphate buffer (pH 7.4). The total volume was 500 µl. The metabolism reaction was initiated by adding NADPH and UDPGA after 10 min preincubation at 37 • C. The reaction was maintained at 37 • C for 1 h and terminated by adding with 500 µl of ice-cold acetonitrile. The mixture was centrifuged at 20,000 g for 15 min, and 10 µl of the supernatant was directly injected for HPLC-DAD-MS/MS assay. Control samples were prepared as described above by using inactivated enzymes in the incubation system. Each of the incubations was performed in duplicate.

Formation Kinetics of the Metabolites M2 and M4
The formation kinetics of the two main reduction metabolites M2 (GAC 2 ) and M4 (7β,15-dihydroxy-3,11,23-trioxo-lanost-26oic acid) were determined. The incubations were performed in a medium (100 µl) containing 0.25 mg protein /ml RLMs or HLMs, different concentrations of GAA (1-50 µM), 3.3 mM of MgCl 2 , 3.0 mM of NADPH and 100 mM of sodium phosphate buffer (pH 7.4). After 10 min of preincubation, GAA at each concentration was incubated with RLMs and HLMs for 5 and 30 min, respectively. The reactions were terminated by adding with 100 µl of ice-cold acetonitrile containing 2 µM GLA used as internal standard (IS). After centrifugation, metabolites in the mixture were analyzed by UFLC-MS/MS detection as described below. The metabolite formation rates vs. GAA concentrations were plotted to obtain the Michaelis-Menten constant (K m ) and maximum velocity (V max ) values of the metabolic reactions. Each of the incubations was performed in triplicate.

Participation of CYP Enzymes in Formation of the Metabolites M2 and M4
GAA (10 µM) was incubated with 0.25 mg/ml RLMs or HLMs in the incubation system as described in Section Formation Kinetics of the Metabolites M2 and M4, in the present or absent of the following CYP enzymes inhibitors, 10 µM α-naphthoflavone (for CYP1A2), 100 µM ticlopidine (for CYP2C19), 10 µM quinidine (for CYP2D), 10 µM ketoconazole (for CYP3A), 100 µM fluconazole (for CYP2C9) and 100 µM diethyldithiocarbamate (for CYP2E1), respectively. The M2 and M4 formation rates in the present and absent of inhibitors were compared. The percentage of inhibition was calculated. Each of the incubations was performed in triplicate.

HPLC-DAD-MS/MS Conditions for Metabolite Identification
The identification of metabolites were conducted by a HPLC-DAD-MS/MS system equipped with an Agilent 1,260 HPLC system (Agilent Technologies, Santa Clara, CA, USA), 1,260 diode array detector (DAD) and 4,500 Q-Trap mass spectrometer with electrospray ionization source (AB SCIEX, Framingham, MA, USA). Chromatographic separation was performed on a C 18 column (100 × 4.6 mm, 2.4 µm, BDS hypersil, Thermo, PA) maintained at 40 • C. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid aqueous solution (B) with following gradient elution at a flow rate of 0.40 ml/min: 0-10 min, 20-35% A; 10-20 min, 35% A; 20-25 min, 35-65% A; 25-30 min, 65% A; 30-35 min, 65-20% A; and 35-45 min, 20% A. The DAD spectral data was collected from 190 to 400 nm. The injection volume of all the tested samples was 10 µl. Mass data were acquired in negative mode under the following conditions, ion spray voltage −4,500 V, ion source temperature 450 • C, curtain gas 10 psi, nebulizer gas 60 psi and auxiliary gas 60 psi. Data were collected using Analyst 1.6.2 (Applied Biosystems) in both first quadrupole (Q1) mass scan mode and multiple reaction monitoring (MRM) independent data acquisition (IDA) mode using enhanced product ion scans (EPI) and enhanced resolution (ER) in the ion trap mode.

Metabolic Kinetic Analysis
The kinetic parameters of GAA metabolism by RLMs or HLMs were calculated by fitting the data to the hyperbolic Michaelis-Menten model: Where V max is the maximal velocity of formation, S is the concentration of substrate, K m is the substrate concentration of half maximal velocity, and the intrinsic clearance (CL int ) is calculated as V max /K m .

Statistical Analysis
The statistical difference was performed by Student's t-test, and p < 0.05 was considered statistically significant.

Characterization of GAA Metabolites in Rats
All of the biological samples were analyzed by HPLC-DAD-MS/MS for their retention times, UV spectra and mass fragmentation pathways. Comparing the chromatograms of MRM survey scan and Q1 full scan of the dosing samples with their corresponding blank samples, a total of 37 metabolites of GAA as well as parent GAA (M0) were detected and identified or tentatively characterized from the bile (34), plasma (22) and urine (12) samples. Table 1 lists the chromatographic, spectra and fragment ions data of GAA and its metabolites. Total ion chromatograms and extracted ion chromatograms of the tested samples are showed in Figures 1-3.
As shown in Figure (Table 1) with different retention times. Their fragmentation were very similar to that of GAA and had the product ions at m/z 303 (b), 302 (b−1), 301 (b−2) and 287 (b−H−CH 3 ), all 2 Da heavier than those of GAA, which were generated from ring D cleavage. This indicates that the reduction occurred on the A-, B-or C-rings of M0, that might be 3-carbonyl, 11-carbonyl or double bond between C-8 and C-9. Among these metabolites, M2 was found to be the most abundant in vivo (based on the chromatogram by DAD detector) and identified as GAC 2 by comparing its retention time, UV spectrum and EPI spectrum with those of GAC 2 authentic standard in HPLC-DAD-MS/MS experiments. With shorter retention time than that of M2, M1 could be inferred as a M2 reduction product, formed by that the 23-carbonyl group in the side chain of M2 was reduced into a hydroxyl group and a double bond was simultaneously formed between C-24 and C-25 . Therefore, M1 was identified as 3,7β,15,23-tetrahydroxy-11-oxo-lanost-8,24-dien-26-oic acid. With longer retention times, M3 and M4 might be inferred to be reduction products of GAA, at 11-carbonyl and double bond between C-8 and C-9, respectively, and named 7β,11,15-trihydroxy-3,23-dioxo-lanost-8-en-26-oic acid and 7β,15-dihydroxy-3,11,23-trioxo-lanost-26-oic acid, respectively, which are reported for the first time. In addition, the peak response of the metabolites with side chain modification was found lower than that of the corresponding metabolites without side chain modification, such as M1<M2, M5<M6, M17<M18, and M22<M23. It seems that the metabolites with side chain modification were more unstable than the metabolites without side chain modification. The peak response of M3 was higher than that of M4, therefore, M3 might not be side chain modification metabolite of M4.

Characterization of GAA Metabolites Produced by RLMs and HLMs
The metabolites found in RLMs incubations were similar with those detected in the bile after i.v. dose. Except for the monohydroxylated metabolites M9 and M11, the phase II metabolite M28 formed by glucuronidation was also found. The metabolite M2 was the most abundant metabolite in RLMs incubations, therefore, the relevant M2 metabolites were detected, including two mono-hydroxylated derivatives (M13 and M15) and one glucuronide conjugated derivative (M25).
The metabolites detected in HLMs incubations were fewer than those found in RLMs incubations, including two reduced metabolites (M2 and M4), three monohydroxylated metabolites (M9, M11, and M35) and one glucuronide conjugated metabolite (M28). Both M2 and M4 were abundant in HLMs incubations. However, M4 was very minor in rats and undetected in RLMs incubations.

Proposed GAA Metabolic Pathway
By using HPLC-DAD-MS/MS techniques, a total of 37 metabolites were identified or tentatively characterized from the bile (34), plasma (22) and urine (12) samples of rats after i.v. dose of GAA. The results reveal that hepatocyte metabolism is the major route of clearance for GAA. The metabolic pathways of the in vitro samples were in consistent with those of the in vivo samples for rats. Nine metabolites ( Table 1) were detected in RLMs reaction medium. The metabolites detected in RLMs were also found in the in vivo samples, indicating the metabolism consistency of GAA in vitro and in vivo for rats. For predicting GAA metabolism in humans and understanding the enzymes involved in drug metabolism differing between humans and rats, the identification of metabolites in HLMs was conducted. The GAA metabolites generated in HLMs were also observed in those in vitro and in vivo samples from rats, indicating the presence of the similar pathway of GAA metabolism in humans and rats.
Proposed metabolic pathways of GAA are showed in Figure 5. Both phase I and phase II metabolites were observed. The phase I metabolism in rats involved reduction, oxidation, oxidoreduction and hydroxylation. The reduction product, ganoderic acid C 2 , was the most abundant. Extensive hydroxylation products of GAA or its reduction/oxidation metabolites were observed in the bile and plasma. The phase II biotransformation observed included glucuronidation, and sulfation. There were a group of glucuronidation and sulfation metabolites of parent GAA and its reduction/oxidation metabolites in bile, and abundant glucuronidation metabolites of GAA and its reduction/oxidation metabolite in urine. Few glucuronidation metabolites were detected in plasma. These data indicated that the phase II metabolites were primarily excreted into the bile and urine.
GAA is composed of a highly oxygenated tetracyclic ring skeleton and an acidic side chain. The hydroxyl groups at C-3, C-7, and C-15 in GAA and its metabolites could be oxidized into carbonyl groups or conjugated with glucuronic acid and sulfuric acid. The carbonyl groups at C-3 and C-11 could be reduced to hydroxyl groups. The C-12, C-20, and C-28 (29) of GAA and its oxidoreduction metabolites were easily hydroxylated. The side chains at C-17 of GAA and its metabolites could be isomerized. Therefore, the main metabolic soft spots in the chemical structure of GAA were the 3, 7, 11, 15, 23-carbonyl groups (or hydroxyl groups) and 12, 20, 28 (29)-carbon atoms.

Formation Kinetics of the Metabolites M2 and M4
The reduction metabolites were found to be the most abundant both in vivo and in vitro. Their formation kinetics in RLMs and HLMs were examined to further explore the differences in reduction metabolism between RLMs and HLMs. The major reduction metabolite was M2 in RLMs, whereas M2 and M4 in HLMs. The kinetics of both M2 and M4 in RLMs and HLMs were fitted to the Michaelis-Menten equation. The kinetic parameters of M2 in RLMs were estimated as K m of 28.80 µM, V max of 185.81 pmol/min/mg, and Cl int of 6.45 µl/min/mg. The kinetic parameters of M2 and M4 in HLMs were estimated as K m of 91.81 and 6.12 µM, V max of 30.18 and 29.70 pmol/min/mg, and Cl int of 0.33 and 4.85 µl/min/mg, respectively. The Lineweaver-Burk transformation revealed a monophasic plot (Figure 6). The K m value of M2 generated by HLMs was approximately 3-fold higher than that by RLMs. The V max and CL int values of M2 formation by RLMs were 6-fold and 20-fold higher than those by HLMs, respectively, suggesting that M2 was formated faster in RLMs than in HLMs. In HLMs, The K m value of M2 was 15-fold higher than that of M4. The V max values of M2 and M4 were similar. The CL int of M4 formation was 15-fold higher than that of M2, suggesting that M4 was formated faster than M2 in HLMs.

Participation of CYP Enzymes in Formation of the Metabolites M2 and M4
The effects of six selective inhibitors of CYP enzymes on the formation of M2 and M4 were evaluated in vitro, and the results are showed in Figure 7. Ketoconazole (10 µM), a CYP3A inhibitor, could significantly inhibit the formation of M2 in RLMs by 19.91%, and strongly inhibit the formation of M2 and M4 in HLMs by 55.99 and 38.25%, respectively. Other inhibitors could not produce significant effects on the formation of the two metabolites. These data suggested that the formation of M2 and M4 might be mainly catalyzed by CYP3A enzyme.

CONCLUSIONS
GAA could undergo extensive metabolism, including reduction, oxidation, and hydroxylation phase I metabolism, and glucuronidation and sulfation phase II metabolism. Its main metabolic soft spots were 3, 7, 11, 15, 23-carbonyl groups (or hydroxyl groups) and 12, 20, 28 (29)-carbon atoms. The reduction metabolism were catalyzed by CYP3A isoenzyme both in RLMs and HLMs, but with different kinetics. These results will be valuable for understanding the mechanism of pharmacological activities and further pharmacokinetic studies of GAA.