Praeruptorin A (dl-PA) is a racemic mixture of 3′(S)-angeloyloxy-4′(S)-acetoxy-3′,4′-dihydroseselin (d-PA) and 3′(R)-angeloyloxy-4′(R)-acetoxy-3′,4′-dihydroseselin (l-PA) (Figure 1). Both the racemate and the dextroro-tatory enantiomer are the main pyranocoumarin com-ponents existing in traditional Chinese medicinal herb Peucedani Radix (Chinese name Bai-hua Qian-hu), which is widely used for the treatment of respiratory diseases and pulmonary hypertension in China (Zhao et al., 1999; Rao et al., 2002; Wei et al., 2002). At present,
the Peucedani-based herbal product is also available in Italy (Mei et al., 2001). In China Pharmacopoeia (The state pharmacopoeia commission of P. R. China, 2010), dl-PA was documented as the chemical marker for the quality control of the herb.
Extensive pharmacologic evaluation of dl-PA revealed its cardioprotective and respiratory protection effects. dl-PA has exhibited calcium antagonistic action and potassium channel opening properties (Wang et al., 1995; Feng et al., 1998; Zhang et al., 2001). Furthermore, it inhibited angiotensin-induced cardiomyocytes hypertrophy by decreased c-Jun expression (Wang et al.,
Xenobiotica, 2011; 41(1): 71–81
Address for Correspondence: Prof. Yitao Wang, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China. Tel: +853-83974691; Fax: +853-28841358. E-mail: [email protected] or Dr. Ru Yan, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China. Tel: +853-83974876; Fax: +853-28841358. E-mail: [email protected]
r e s e a r c h a r T I c L e
Transport and metabolism of (±)-praeruptorin A in Caco-2 cell monolayers
W. H. Jing1, Y. L. Song1, R. Yan1, H. C. Bi1,2, P. T. Li3, and Y. T. Wang1,4
1Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau, China, 2Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China, 3Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, China, and 4School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China
abstract1. (±)-Praeruptorin A (dl-PA) is one of the main pyranocoumarins of Peucedani Radix and the chemical marker for quality control of the herb in China. This study investigated the transport and metabolism of dl-PA, for the first time, in Caco-2 cell monolayers.2. PA enantiomers of dl-PA in the transport study were simultaneously determined using a simple and rapid enantio-selective high performance liquid chromatography-UV method.3. Both dextrorotatory (d–PA) and levorotatory (l–PA) enantiomers traversed Caco-2 monolayer rapidly in both directions (absorptive P
app: 2.01–3.03 × 10−5 cm/s; secretory P
app: 1.58–1.96 × 10−5 cm/s) mainly via pas-
sive diffusion. Higher transport rates of dPA were observed in both directions.4. PA enantiomers were incompletely recovered after the transport study. Nonspecific binding to the Transwell inserts, irreversible binding to cellular components and metabolism within the cells accounted for the loss.5. dl-PA was partially hydrolyzed in Caco-2 monolayers and yielded two stereoisomers via removal of the acetyl group from C-4′ position. Both phenylmethylsulphonyl fluoride (a nonspecific esterase inhibitor) and bis(p-nitrophenyl) phosphate sodium salt (a specific inhibitor of carboxylesterases) completely abolished dl-PA hydrolysis.6. In summary, PA enantiomers were rapidly transported across Caco-2 cells and partially hydrolyzed by carboxylesterases during permeation. These findings provide mechanistic understanding of in vivo phar-macokinetic properties of dl-PA.
2006), relieved inflammatory reaction and alleviated apoptosis in I/R myocardium (Chang et al., 2003). dl-PA also showed a remarkable relaxation on rabbit trachea smooth muscles and contraction of pulmonary arteries (Zhao et al., 1999). These beneficial effects support dl-PA a potential candidate for the treatment of cardiovascular and respiratory diseases. However, so far, the absorp-tion, distribution, metabolism and excretion (ADME) properties and hence the in vivo active forms of PA are still unknown.
In China, herbal medicines are traditionally taken orally, thus the extent of absorption along the gastroin-testinal tract is one of the determinant factors governing the entry of drug into the bloodstream and eventually its actions. Therefore, intestinal absorptive property of dl-PA is of particular importance and should be investigated to understand its in vivo fates.
Stereoselectivity has been frequently reported in phar-macokinetic, pharmacodynamic and toxicologic studies of chiral drugs as a consequence of the enantio-selective interaction with chiral biological macromolecules in intestinal absorption, drug–receptor interaction, drug metabolism, serum protein binding, and so on (Drayer, 1986; Ogihara et al., 1996). In general, one of the enan-tiomers of the racemate is more active or less toxic than the other, or sometimes shows distinct pharmacologi-cal performance to its antipode. As a consequence, the demand for the development of enantiomers with higher efficacy yet with lower toxicity has increased and attracted extensive attentions of industries and scien-tists. Enantio-specific pharmacologic properties of PA enantiomers have not been recognized until recently. Compared with its antipode, the dextrorotatory enanti-omer of PA (d-PA) showed more potent relaxation against KCl- and phenylephrine-induced contraction of isolated rat aortic rings with intact endothelium (Xu et al., 2010), indicating unequal contribution of each enantiomer to PA actions. However, the chiral discrimination of the ADME properties of PA enantiomers of the racemate remains unidentified.
Thus, in this study, the absorption property of dl-PA was characterized using Caco-2 cell model and
chiral discrimination was elucidated using a developed enantio-selective analytical method. In addition, the metabolic mechanism of dl-PA during transporting across the cells was also characterized. The results of this study would provide important information on absorp-tive property and metabolic stability of the enantiomers of PA during absorption.
Materials and methods
Materials
dl-PA (purity > 99%) and the dextrorotatory enanti-omer d-PA (purity > 95%) were obtained from Shanghai Traditional Chinese Medicine Research Centre (Shanghai, China).
Lucifer yellow, propranolol, rhodamine-123, vera-pamil, indomethacin, ouabain, phenylmethylsulphonyl fluoride (PMSF), phosphate buffer tablets, collagen type I, sodium pyruvate, and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich Co. (St Louis, MO). Bis(p-nitrophenyl) phosphate sodium salt (BNPP) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Dulbecco’s Modified Eagle’s medium (DMEM), foetal bovine serum (FBS), penicillin–streptomycin and nonessential amino acids were obtained from Gibco BRL Life & Technologies (Grand Island, NY). 3-(4,5-Dimethylthiazole-2-ly)-2,5-diphenyl tetrazolium bro-mide (MTT) was purchased from USB Co. (Cleveland, OH). Ultra-pure water was obtained from a Milli-Q plus water purification system (Millipore, Bedford, MA). Methanol and acetonitrile of HPLC grade were supplied by Merck (Darmstadt, Germany). Transwell® plates (12-well, 0.4-μm pore size, 1.12 cm2, polycarbonate membrane) were purchased from Corning Costar Co. (Cambridge, MA). Caco-2 cells at passage 19 were obtained from the American Type Culture Collection (ATCC, Rockville, MD).
Cell culture
Caco-2 cells were cultured in DMEM supplemented with 10% FBS and 1% nonessential amino acids, in an atmosphere containing 5% CO
2 and 90% relative humid-
ity at 37°C. Cells were sub-cultured at 80–90% conflu-ence by trypsinization with 0.05% trypsin–EDTA. In transport studies, cells at passages 30–40 were seeded in Transwell® polycarbonate in 12-well plates at a den-sity of 1 × 105 cells per insert and supplied with fresh medium every other day. The transport study of dl-PA in the Caco-2 cells was performed when the cells had reached integrated confluence after seeding for 21 days. The transepithelial electrical resistance (TEER) was measured at 37°C using the epithelial voltammeter
O
O O O
d-PA I-PA M1 & M2
O
O O O O O1′1′
4′4′3′3′ O
O O O O
O O O
OOH
Figure 1. The chemical structures of dextrorotatory (d-PA) and levo-rotatory (l-PA) enantiomers of praeruptorin A (dl-PA), and the putative metabolites M1 and M2 formed in Caco-2 cell monolayers during the transport study.
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
Transport and metabolism of (±)-praeruptorin A 73
(World Precision Instruments, Sarasota, FL) and flux of the paracellular marker lucifer yellow was determined in parallel to monitor the integrity of the monolayers. Only cell monolayers with TEER values greater than 300 Ω cm2 and the P
app values of lucifer yellow less than 0.5 × 10−6
cm/s were used for the transport experiments (Walgren et al., 1998; Suzuki and Sugiyama, 2000). Propranolol was used as a marker for the passive transcellular transport (Violini et al., 2002), and the P
app values, 1.23 ± 0.26 × 10−5
cm/s as determined in our laboratory, were compara-ble with those reported previously (Yee, 1997), indi-cating the normal function of the passive transcellular transport on the Caco-2 monolayers formed. The efflux transporter P-glycoprotein (P-gp) functionality in Caco-2 monolayers was validated using the P-gp probe substrate rhodamine-123 and exhibited normal expression of P-gp transporter in the Caco-2 cell monolayer formed.
Cytotoxicity assay
The cytotoxicity of dl-PA on Caco-2 cells was measured by MTT assay. In brief, Caco-2 cells were seeded onto a 96-well plate at a density of 2 × 104 cells/well in DMEM culture medium. After 24-h culture at 37°C, the medium was replaced with 200 μl of dl-PA at different concentra-tions (0.15–19.2 μg/ml) dissolved in HBSS (Hank’s bal-anced salt solution). HBSS alone served as a negative control. Then, the 96-well plate was incubated at 37°C for 4 h. Supernatant was removed and 200 μl of MTT (0.5mg/mL) was added to each well before incubation for another 4 h. Then, the supernatant in the well was removed. The formazan crystals formed in the cells were dissolved in 200 μL of DMSO and absorbance was measured at 570 nm using Multilabel Counter (Tecan Instrument Inc., Research Triangle Park, NC).
Transport studies
The transport of dl-PA across Caco-2 monolayers was investigated using the method reported previously with minor modifications (Ruan et al., 2010). In brief, cells were rinsed twice with HBSS before the transport study, and then the plates were preincubated at 37°C for 30 min. Then, all inserts were distributed evenly between treat-ments according to the TEER values measured. Final concentrations of dl-PA at 4.80, 9.60 and 19.20 μg/ml, corresponding to 12.5, 25 and 50 μM, respectively, were chosen for the transport study based on its solubility (maximum 20 μg/ml in HBSS), results of MTT assays and LOD (limit of deflection) of the analytical method. dl-PA was loaded to the apical (AP) or basolateral (BL) side (donor side), and HBSS alone was loaded to the opposite side (receiver side) before incubation. An aliquot (100 μl) was collected from the donor side (0 min) or receiver side (0, 30, 60, 90 and 120 min), and 70 μl was directly
injected into the HPLC instrument. After each sampling, 100 μl of HBSS was supplemented to maintain a constant volume of each side.
To determine the involvement of efflux transporters in PA transport, the cell monolayers were incubated for 30 min at 37°C in the presence of one of the follow-ing inhibitors at concentrations reported previously: verapamil (100 μM), the P-gp inhibitor (Petri et al., 2004), indomethacin (10 μM), the multidrug resistance-associ-ated proteins (MRPs) inhibitor (Zhu et al., 2006) or oua-bain (1 μM), the Na+/K+ ATPase inhibitor (Dickman et al., 2000), in both AP and BL sides before adding dl-PA.
To unravel the isozymes catalyzing PA metabolism in Caco-2 monolayers, PMSF, a nonspecific esterase inhibi-tor, or BNPP, a specific inhibitor of carboxylesterases, was added at 500 μM (Tantishaiyakul et al., 2002) to the AP side and preincubated for 15 min. The transport study was initiated by replacing the solution in the AP side with 0.5 ml of fresh HBSS containing PA (4.80 µg/ml). The transport study was terminated at 120 min by removing the inserts from the plates. An aliquot (100 μL) was col-lected from AP or BL side, and 70 μl aliquot was directly injected into the HPLC instrument.
Binding of dl-PA to Caco-2 cell monolayers
dl-PA (9.60 μg/ml in AP or BL side) was incubated with Transwell insert alone or Transwell insert coated with normal or denatured Caco-2 cell monolayers in the same manner as described in the bidirectional transport study under the section Transport Studies. Denatured Caco-2 cell monolayers were obtained by treating the normal Caco-2 cell monolayers with MeOH at −80°C for 1 h to deactivate the transporters and drug-metabolizing enzymes and then washed with HBSS twice before the binding study. Untreated Caco-2 cell monolayers that were incubated in parallel at 37°C served as controls. After incubation for 120 min, the insert membranes were taken out and washed with ice-cold PBS for six times. Then, the membranes were cut separately with a blade and sonicated in 300 μl of MeOH for 60 min at room tem-perature. The resultant sample was centrifuged at 15,000g for 15 min, and MeOH was added to make a final volume of each supernatant to 300 μl. Seventy microlitres of each were subjected for HPLC analysis.
Basic hydrolysis of d-PA
To aid structural identification of the metabolites of dl-PA formed in Caco-2 cells, a wet chemistry study of d-PA was carried out according to a previous report (Wu et al., 2002). In brief, the dextrorotatory enantiomer d-PA (250 μg dissolved in tetrahydrofuran) was treated with 250 μl of KOH (0.5 M), and the mixture was stirred at 60°C for 30 min. The solution was neutralized with 10%
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
74 W. H. Jing et al.
H2SO
4 to around pH 6, stirred at room temperature for
120 min and then extracted with CHCl3. The organic layer
was collected and evaporated using the rotatory evapo-rator at 40°C. The residue was reconstituted with 500 μl of methanol, filtered through a 0.45-μm membrane filter before being subjected to HPLC-MS/MS analysis.
Chiral HPLC-UV separation and determination of PA enantiomers
Sample analysis was performed on an Agilent series 1200 (Agilent Technologies, Santa Clara, CA) liquid chromatography, equipped with a vacuum degasser, a binary pump, an autosampler and a diode array detector system, and controlled with an Agilent ChemStation B 3.0 software. The chiral separation of dl-PA was obtained on a CHIRALPAK® AD-RH column (150 mm × 46 mm i.d., 5 μm; Daicel, Japan), which was connected to a CHIRALPAK® AD-RH guard column (10 × 40 i.d., 5 μm; Daicel, Japan). The column temperature was maintained at 35°C. The authentic racemate PA and samples from transport studies were eluted at a flow rate of 0.5 ml/min with a mobile phase consisting of acetonitrile (ACN) and 0.1% formic acid (65:35, v/v). The sample chamber was kept at 4°C. PA enantiomers and the metabolites were monitored at 323 nm.
Construction of calibration curve of the racemic PA
PA enantiomers were quantified separately in this study of dl-PA to identify potential enantioselectivity. Quantification was carried out using the calibration curve of the racemate due to the following reasons: l-PA is not commercially available, the d/l ratio of the racemate was 1 as evidenced by nil optical rotation of the racemic mixture and the peak areas of enantiomers were equal at each concentration of the racemate tested.
A stock solution of dl-PA was prepared by dissolving a known amount of the compound in DMSO at a final concentration of 3.84 mg/ml. The stock solution was then serially diluted with HBSS to obtain working solutions of PA at 0.30, 0.60, 1.20, 2.40, 4.80, 9.60 and 19.20 μg/ml. An aliquot (100 μl) of each working solution was filtered and subjected to HPLC analysis as described above. Calibration curves were constructed by plotting the peak area of dl-PA as a function of the corresponding concentration.
LC-MS/MS analysis
Samples were separated on the same Agilent series 1200 (Agilent Technologies, Santa Clara, CA) system as described above. The analytical column was ODS RP C
18
column (250 mm × 4.6 mm i.d., particle size 5 μm; Agilent Technologies, Santa Clara, CA). The column temperature was kept at 35°C. The mobile phases consisted of 0.1%
formic acid (A) and methanol (B), and samples were eluted at 1.0 ml/ min using a gradient as follows: 0–4 min, 25–50% B; 4–16 min, 50–53% B; 16–17 min, 53–56%; 17–20 min, 56%; 20–23 min, 56–69%; 23–40 min, 69–76%; 40–41 min, 76–100%; 41–44min, 100%. The injection volume was 70 µl.
The mass spectrometer analysis was performed on an LC/MSD Ion Trap system (Palo Alto, CA) equipped with an ESI (electrospray ionization) interface. A quarter of the eluent was directly introduced into the ESI interface through a 50-cm-long PEEK (polyether ether ketone) tub-ing (0.13-mm i.d.). Nitrogen was used as both the nebuliz-ing gas at 45 psi and the drying gas at 8 l/min and 350°C. The mass spectrometer was operated in the positive ion mode over a mass range of m/z 100–1000. Helium was used as the collision gas for the tandem mass spectromet-ric experiments. Fragmentation was induced with reso-nant excitation amplitude of 0.6 V, followed by an isolation of the precursor ion over a unit mass window of 1 Da.
Data analysis
Transport rate of each enantiomer was obtained according to the following equation (1)
V ( Q t) A= d /d /
(1)
The apparent permeability coefficient (Papp
) was calculated according to the following equation (2)
P Q t C Aapp 0 (d d ) ( )= / / ×
(2)
where dQ/dt is the rate of appearance of drug in the receiver chamber (in μg/s), A represents the membrane surface area of Caco-2 monolayer (1.12 cm2) and C
0 is the
initial drug concentration in the donor side (μg/ml).The extent of the polarized transport was assessed by
efflux ratio:
Efflux ratio = /app BL to AP app AP to BLP P
(3)
where Papp BL to AP
and Papp AP to BL
represent the mean perme-ability coefficient of lPA or dPA obtained from BL to AP direction and AP to BL direction, respectively.
All data were presented as mean ± standard deviation (SD). Statistical significance between two groups was determined by a two-tailed Student’s t-test and deemed significant when P < 0.05.
Results
Enantioseparation and determination of PA enantiomers in the racemate
Representative HPLCs of dl-PA on the achiral column (ODS RP C
18) and chiral column (Chiralpak AD-RH) are
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
Transport and metabolism of (±)-praeruptorin A 75
shown in Figure 2. The racemate was eluted at 7 min on the achiral column. On the chiral column, the enantiom-ers achieved baseline separation with retention times of 7.2 and 10.5 min for d-PA and l-PA, respectively. There was no interference from HBSS, and all the inhibitors utilized, including verapamil, indomethacin, ouabain, PMSF and BNPP, were eluted earlier and did not interfere with the analytes during the analysis.
The calibration curve of dl-PA showed good linearity over a concentration range of 0.3–19.2 μg/ml. The regres-sion equation was Y = 107.6X − 68.89 with the coefficient of determination r2 > 0.999. The overall intra– and inter–batch precision and accuracy were acceptable (with coefficient of variation < 5%) as determined at concen-trations of 0.60, 2.40 and 9.60 μg/ml of the racemate. The stability study of dl-PA in HBSS under transport conditions revealed no significant degradation of PA enantiomers, and no additional peaks were detected within 4 h. The limit of detection and the limit of quan-tification were 0.075 and 0.15 μg/ml, respectively, for each enantiomer.
Transport of dl-PA across Caco-2 monolayers
According to the solubility of dl-PA in HBSS and its cytotoxicity towards Caco-2 cells as determined by MTT assays, the bidirectional transport study of dl-PA across Caco-2 cell monolayers was carried out at three concen-trations: 4.80, 9.60 and 19.20 μg/ml.
As shown in Figure 3, both dPA and its antipode in the racemate traversed the Caco-2 monolayers rapidly (transport rates > 5 × 10−5 µg/s/cm2) in both absorp-tive and secretory directions. The transport rates of both enantiomers were remarkably higher than those of propranolol (0.4 ± 0.08 × 10−6 µg/s/cm2), the passive transcellular transport marker. The overall transport rates of each enantiomer increased with concentra-tion and were higher in absorptive (AP to BL) direction than in the secretory (BL to AP) direction. d-PA was transported faster than its antipode in both directions with significant difference observed at 19.20 μg/ml of dl-PA.
When the Papp
values of each enantiomer were cal-culated, an inverse change of P
app with concentration
was observed with each enantiomer in the absorp-tive direction, and the P
app values were significantly
higher than the secretory direction (d-PA: absorptive direction 2.25–3.03 × 10−5 cm/s, secretory direction 1.65–1.97 × 10−5 cm/s; lPA: absorptive direction 2.01–2.82 × 10−5 cm/s, secretory direction 1.58–1.88 × 10−5 cm/s). Similar to the transport rate, the dextrorotatory enantiomer showed higher P
app than its antipode. Yet
no significant difference was identified between the two enantiomers.
In addition, both enantiomers exhibited similar efflux ratios, which were 0.6–0.8, all less than 1, indicating a polarized transport of both compounds in the absorptive direction.
mAUA
BmAU
50
25
20
15
10
5
0
−5
40
20
10
0
0 2
dI-PA
d-PA
I-PA
4 6 8 10 12 14 min
0 2 4 6 8 10 12 14 min
Figure 2. Representative HPLCs of dl-PA on (A) an ODS RP C18
column and (B) a Chiralpak AD-RH column.
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
76 W. H. Jing et al.
Effect of inhibitors on transepithelial transport of PA enantiomers
Three inhibitors, including verapamil (a specific inhibitor of P-gp), indomethacin (a MRP1 inhibitor) and ouabain (a Na+/K+ ATPase inhibitor), were examined to elucidate whether the polarized transport of PA includes energy-dependent efflux process. As shown in Figure 4, the pres-ence of these inhibitors neither affected the permeation rates of racemic PA in both directions nor did the efflux ratios change.
Recoveries of PA enantiomers during the transport study
The amounts of PA enantiomers recovered from both donor and receiver sides at the end of the transport study were calculated and shown in Figure 5. The recov-eries of each enantiomer from both absorptive and
secretory transports showed a tendency of decrease when the concentration of the racemate increased and was only 60–70% at 19.20 μg/ml of the racemate, indicating a loss of 30–40% of the initial amount of PA added to the donor chamber during transport. There was no significant difference between absorptive and secretory transports. Again, there was no significant chiral discrimination.
When dl-PA was incubated with Transwell insert alone, as shown in Figure 6, there was around 10% of each enan-tiomer stayed on the Transwell membrane. The amounts of enantiomers recovered from methanol-treated Caco-2 monolayers, ∼21% and ∼15% from absorptive and secre-tory transport, respectively, were significantly higher than those from the inserts alone yet similar to the untreated Caco-2 monolayers, indicating another ∼10% (absorptive direction) and ∼5% (secretory direction) of the enantiomers binding to the cell membrane.
A B20
15
10
p = 0.99
p = 0.52
p = 0.13
p = 0.41p = 0.13
d-PA in racemate I-PA in racemate*p = 0.02
Tran
spor
tate
(mg/
s/cm
2 ×
10−5
)
5
0
20
15
10
5
04.80 9.60
Concentration of dI-PA (µg/mL)19.20 4.80 9.60
Concentration of dI-PA (µg/mL)19.20
Figure 3. Effects of the concentration of dl-PA on the transport rate of d–PA and l–PA from (A) apical (AL) to basolateral (BL) and (B) BL to AP side in a transport study of the racemate. dl-PA was loaded on either AP or BL side and incubated at 37°C for up to 120 min. Data are the mean ± SD (n = 3); *P < 0.05.
p = 0.09p = 0.20
p = 0.89p = 0.81
p = 0.67
p = 0.44p = 0.18
p = 0.79
d-PA in racemate I-PA in racemate
Pap
p (c
m/s
× 10
−5)
4
3
2
1
0
4
3
2
1
0Control Indomethacin
10 µMOuabain
1 µMVerapamil
100 µMControl Indomethacin
10 µMOuabain
1 µMVerapamil
100 µM
A B
Figure 4. Effects of transporter inhibitors on dl-PA (9.60 μg/ml) transport across Caco-2 cell monolayers from (A) apical (AP) to basolateral (BL) side and (B) BL to AP side. Indomethacin, ouabain or verapamil was added to both AP and BL sides 30 min before adding dl-PA. Data are the mean ± SD (n = 3).
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
Transport and metabolism of (±)-praeruptorin A 77
Metabolism of dl-PA in Caco-2 monolayers
Two additional peaks (M1 and M2), which were absent in samples collected from the donor side at 0 min or from incubations with methanol-pretreated Caco-2 monolayers, were observed in the HPLC chromatograms of samples collected from either donor side or receiver side after 120-min incubation (Figure 7). These two peaks
were eluted earlier than the enantiomers with retention times of 6.20 min (M1) and 6.82 min (M2), respectively, suggesting that metabolism of dl-PA occurred in Caco-2 cell monolayers during transport. The percentages of metabolism, as calculated based on the ratios of the sum of peak areas of the two metabolites to the sum of the enantiomers initially added to the donor side, were around 14% in the absorptive transport and 9% in the secretory transport at 4.80 μg/ml of the racemate and decreased with concentration, indicating that metabo-lism also accounted for the loss of dl-PA during transport and the metabolic process might be saturable at higher concentrations.
Structural identification of dl-PA and its metabolites
To get better separation and to facilitate structural iden-tification of the two metabolites M1 and M2, a gradient elution was adopted for HPLC-MS/MS analysis of the samples from the transport study. M1, M2 and dl-PA were eluted at retention times of 24.1, 27.0 and 31.0 min, respectively (Figure 8A).
The mass spectra of dl-PA and its metabolites and their proposed fragmentation mechanism are shown in Figure 9. The MS1 spectrum of dl-PA exhibited adduct ions at m/z 409 ([M+Na]+) and m/z 425 ([M+K]+) and dimer adduct ion at m/z 795 ([2M+Na]+), corresponding to the molecular weight of m/z 386 for dl-PA. MS2 of the sodiated ion showed the presence of a major product ion at m/z 327, which was 82 mass units less than the parent ion, indicating the loss of the CH
3COONa moiety from the
C-4′ sodiated adduct of PA.Both metabolites M1 and M2 showed similar char-
acteristic ions in their mass spectra, including sodiated adduct ion at m/z 367 and potassium adduct ion at m/z 383, indicating that these two metabolites could be iso-mers and the molecular weights are m/z 344, correspond-ing to a loss of an acetyl group from dl-PA (MW = 386). MS2 of the sodiated ion at m/z 367 yielded the predominant product ion at m/z 267, corresponding to the cleavage
d-PA in racemate I-PA in racemate
Rec
over
y (%
)
100
80
60
40
20
0
100
80
60
40
20
04.80 9.60 19.20 4.80 9.60 19.20Concentration of dI-PA (µg/mL) Concentration of dI-PA (µg/mL)
A B
Figure 5. Recoveries of d-PA and l-PA from (A) absorptive and (B) secretory transport of the racemate dl-PA. Data are the mean ± SD (n = 3).
Per
cent
age
of b
indi
ng (%
)P
erce
ntag
e of
bin
ding
(%)
30Control MeOH- treated cell Transwell membrane
****
****
****
****
25
20
15
10
5
0
30
25
20
15
10
5
0
AP to BL BL to AP
AP to BL BL to AP
A
B
Figure 6. Percentages of (A) d–PA and (B) l–PA recovered from Transwell inserts coated with normal Caco-2 cells (control) or meth-anol-treated Caco-2 cells, or Transwell inserts alone, after incuba-tion of the racemate (9.60 μg/ml) for 120 min. Methanol-treated cells were Caco-2 cell monolayers incubated with methanol at −80°C for 1 h to deactivate the transporters and enzymes. Percentage of binding was calculated by comparing the amount of drug recovered from the Transwell membrane at 120 min with the initial amount of drug added in the donor side. Data are the mean ± SD (n = 3); *P < 0.05, **P < 0.01.
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
78 W. H. Jing et al.
of the angeloyl group from the C-3′ position. Thus, M1 and M2 were tentatively identified as stereoisomers of 3′-O-angeloyl khallectone with different arrangements of the hydroxyl group exposed as a result of hydrolysis of the acetyl group from the C-3′ position.
To further distinguish the spatial arrangement of the hydroxyl group of the two metabolites, the sample from the transport study of dl-PA was analyzed under the same conditions as reported previously for analysis of samples from basic hydrolysis of d-PA. A total of three products P1, P2 and P3 (Figure 8B) were generated when d-PA was treated with KOH. The retention time and mass spectra of M2 were the same as those of P3 of which the absolute structure has been unambigu-ously identified as 3′(S)-angeloyl-4′(R)-hydroxyl-3′,4′-dihydrokhellactone based on IR, NMR and MS analyses (Wu et al., 2002). Thus, the absolute configuration
of M1 (3′S, 4′S) and M2 (3′S, 4′R) was preliminarily proposed. Further study is warranted to confirm this speculation.
Effects of esterase inhibitors on hydrolysis of dl-PA in Caco-2 cell monolayers
PMSF, a broad specificity inhibitor of esterases, and BNPP, a selective inhibitor of carboxylesterases (Tanishaiyakul et al., 2002) were chosen to identify the enzymes catalyz-ing dl-PA metabolism in Caco-2 monolayers. As shown in Figure 7, both BNPP and PMSF could abolish M1 and M2 production from dl-PA. The percentage of metabolism (12.73%) as calculated from the ratio of the total peak areas of both metabolites determined in both donor and receiver sides to the peak area of dl-PA initially added in the donor side was close to those calculated based on the percentage
mAU mAU
mAU
mAU
mAU
mAU
10.0
4.5
M1 M1M2 M2
3.0
1.5
0.0 0.0
mAU
mAU
4.5
4.0
0 2 4 6 8 10 12 14 min 0 2 4 6 8 10 12 14 min
3.02.01.00.0
−1.0 −0.5
3.0
1.5
0.0
d-PA
d-PA
d-PA
d-PA d-PA
d-PA
d-PA
I-PA
I-PA
I-PA
I-PA I-PA
I-PA
I-PA
8.06.04.02.00.0
8.0
6.0
4.0
2.0
0.0
1.2
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
0.8
0.4
A
C
E
G
B
D
F
H
Figure 7. Representative HPLCs of samples from an absorptive transport study of dl-PA (4.80 μg/ml). For inhibition study, PMSF (0.5 mM, a non-specific esterases inhibitor) or BNPP (0.5 mM, a specific carboxylesterases inhibitor) was added into the AP side and incubated for 15 min before replacing the solution with fresh medium containing no inhibitor. Left: samples collected from AP side (A) no inhibitor, 0 min (C) no inhibitor, 120 min (E) PMSF pretreatment, 120 min (G) BNPP pretreatment, 120 min; right: samples collected from BL side (B) no inhibitor, 0 min (D) no inhibitor, 120 min (F) PMSF, 120 min, (H) BNPP 120 min.
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
Transport and metabolism of (±)-praeruptorin A 79
difference of dl-PA recovered in the absence/ presence of the inhibitors (PMSF:13.4%; BNPP: 7.4%).
Discussion
As one of the most abundant constituents in Peucedani Radix and the chemical marker for quality control of the herb, dl-PA was usually determined or evaluated in the form of the racemic mixture. In this study, a simple and rapid HPLC-UV method has been developed, for the first time, to determine the dextrorotatory and levorotatory forms in the racemate simultaneously during the bidirec-tional transport study of dl-PA on a Caco-2 cell model.
PA enantiomers exhibited rapid transport across the Caco-2 monolayers in both absorptive and secretory directions. The P
app values of both enantiomers were
all above 1 × 10−5 cm/s, predicting an oral absorption of >70% when taken orally in human body (Yee, 1997).
mAU
mAU
504030
M1
P3
P2P1
M2
dI-PA
d-PA
20100
506070
403020100
5 10 15 20 25 30 35 40 min
−10
A
B
Figure 8. Representative HPLC of (A) sample collected from the receiver side at 120 min in a transport study of dl-PA and (B) sample collected from basic hydrolysis of d-PA on an ODS RP C
Figure 9. MS1 and MS2 spectra of dl-PA (A, B), M1 (C, D) and M2 (E, F).
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
80 W. H. Jing et al.
Furthermore, the Papp
values of AP to BL transport of PA enantiomers was approximately 3.0 × 10−5 cm/s, more than 100 times that of the paracellular transport marker lucifer yellow, suggesting that transcellular route domi-nates the absorptive transport of PA enantiomers. The transport rates for both directions increased with con-centration, whereas the P
app values decreased with con-
centration. Both the transport rates and the Papp
values obtained for the absorptive transport were significantly higher than the secretory transport with efflux ratios much lesser than 1. In addition, the P
app values of both
enantiomers were comparable with that of the transcel-lular marker propranolol (P
app 1.23 ± 0.26 × 10−5 cm/s) in
the absorptive direction, but two to three times higher than that of propranolol in the secretory direction. These findings indicate a polarized transport of dl-PA, which might include a transporter-mediated process and be saturable. d-PA in the racemate showed faster transport than its antipode, demonstrating the existence of an enantioselectivity in PA enantiomers transport.
Caco-2 cell monolayer is a human colon cancer cell line recommended by the Food and Drug Administration for investigation and prediction of intestinal permeabil-ity and transport of compounds, has been demonstrated successfully in most cases. Caco-2 cells express several transporters that are present in the human enterocytes, including transport proteins belonging to the ATP-binding cassette (ABC) superfamily, P-gp and MRPs (Hunter et al., 1993; Engman et al., 2001; Taipalensuu et al., 2001). P-gp and MRPs function as energy-dependent efflux pumps and implicate in active efflux of numerous drugs and xenobiotics. In this study, three inhibitors were chosen to examine the involvement of ABC transporters to dl-PA transport. The results showed that both the energy block-age and inhibition of P-gp and MRP transporters failed to alter the polarized transport and the transport rates of PA enantiomers, suggesting that the transport of PA enantiomers is energy independent, and P-gp and MRP transporters were not involved in the process. Thus, the passive transcellular diffusion might dominate the transport behaviour of the two enantiomers. The polar-ized transport of PA enantiomers observed in this study may involve carrier-mediated mechanism and transport proteins from the solute carrier group such as the organic anion transporters can mediate energy-independent drug transport in absorptive direction (Sun et al., 2002). On the other hand, the significant larger surface area of the brush border membranes of the AP side than that of the BL side might be another reason for asymmetric transport of dl-PA. Because dl-PA was lipophilic com-pound with the log P 3.75 and log D 3.5 (calculated via in silico), it can distribute readily into the cell membranes of the intestinal epithelium.
PA enantiomers were not fully recovered during transport across Caco-2 cell monolayers. Nonspecific
binding to Transwell membrane and irreversible binding to cellular components were two main reasons for the low recoveries. The binding to cell membrane was more significant in the absorptive direction. This finding agrees well with our above speculation that dl-PA has high lipophilicity and distribute readily into the brush-border membrane in the AP side. Metabolism of dl-PA in Caco-2 cell monolayer was demonstrated to also contribute to the loss during transport.
The two metabolites formed in Caco-2 cells were proved to be stereoisomers resulting from hydrolysis of PA at the C-4′ position. It has been reported that Caco-2 cells possess hydrolase activity and esterases, espe-cially carboxylesterases are main enzymes that catalyze hydrolysis of xenobiotics in Caco-2 cells (Takai et al., 1997; Imai et al., 2005, 2006; Teruko et al., 2005; Tang et al., 2006). In this study, both PMSF (a nonspecific inhibitor of esterases) and BNPP (a specific inhibitor of carboxylesterases) could abolish the formations of M1 and M2 from PA enantiomers in Caco-2 cells, indicating that carboxylesterases are responsible for PA hydroly-sis in Caco-2 cell line. BNPP inhibits both hCE-1 and hCE-2, the two major carboxylesterase isozymes in human. It is interesting to note that hCE-2 is the pre-dominant carboxylesterase expressed in enterocytes of human small intestine while the hydrolytic activity in Caco-2 cells is mainly attributed to hCE-1 (Imai et al., 2005). Moreover, hCE-1 and hCE-2 possess distinct sub-strate specificity and hCE-1 preferably hydrolyzes esters with a smaller alcohol moiety, whereas hCE-2 prefers to hydrolyze esters with a smaller acyl moiety (Imai et al., 2006; Tang et al., 2006). dl-PA has an acetyl group substituted at the C-4′ position, thus similar hydroly-sis, but to a much significant extent, of dl-PA might be expected during its absorption along small intestine. In contrast to human small intestine, human liver exhibits a similar expression pattern of carboxylesterases to that of Caco-2 cells (Imai et al., 2005; Tantishaiyakul et al., 2002). Therefore, first-pass hepatic metabolism of dl-PA via hydrolysis at the C-4′ position could be expected before it enters the systemic circulation. Further stud-ies are warranted to determine the species difference in hydrolysis of dl-PA and the extent of intestinal and hepatic first-pass effects on an animal model after oral administration.
In conclusion, this study examined the transport property and metabolism of dl-PA in Caco-2 cells through determination of its enantiomers. dl-PA exhib-ited a rapid transport across the Caco-2 monolayers, partially bound to cell membranes and underwent hydrolysis during transport. The hydrolysis of dl-PA catalyzed by carboxylesterases was demonstrated, and it implicates extensive first-pass intestinal and hepatic hydrolysis of the racemate. Slight enantioselectivity was observed in the transport process. The contribution of
Xen
obio
tica
Dow
nloa
ded
from
info
rmah
ealth
care
.com
by
Impe
rial
Col
lege
on
04/3
0/13
For
pers
onal
use
onl
y.
Transport and metabolism of (±)-praeruptorin A 81
each enantiomer to the metabolism of the racemate is yet to be addressed. Further study with individual enantiomers is warranted for better understanding of the ADME properties of the racemate and to identify chiral discrimination if any.
Declaration of interest
This work was supported by the National Basic Research Program of China (973 programme, Grant No. 2009CB522707) and the Research Committee of University of Macau (Project No.: UL016/09-Y1/ CMSWYT01/ICMS).
References
Chang TH, Liu XY, Zhang XH, Xing J, Wang HL. (2003). Effects of Peucedanum Praeruptorum Dunn and dl-Praeruptorin A on IL-6 Level and Fas, bax, bcl-2 protein expressions in ischemia-reperfusion myocardium of rats. Zhongguo Yi Ke Da Xue Xue Bao 32: 1–6.
Drayer DE. (1986). Pharmacodynamic and pharmacokinetic differ-ences between drug enantiomers in humans: an overview. Clin Pharmacol Ther 40:125–133.
Dickman KG, Hempson SJ, Anderson J, Lippe S, Zhao L, Burakoff R, Shaw RD. (2000). Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 279:G757–G766.
Engman HA, Lennernäs H, Taipalensuu J, Otter C, Leidvik B, Artursson P. (2001). CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies. J Pharm Sci 90:1736–1751.
Feng WY, Li JM, Zhang KY. (1998). Effect of Pd-Ia on elctrophysiologi-cal proterties of guinea pig wenticular cell. Zhongguo Yao Xue Za Zhi 33: 660–662.
Hunter J, Hirst BH, Simmons NL. (1993). Drug absorption limited by P-glycoprotein-mediated secretory drug transport in human intestinal epithelial Caco-2 cell layers. Pharm Res 10:743–749.
Imai T, Imoto M, Sakamoto H, Hashimoto M. (2005). Identification of esterases expressed in Caco-2 cells and effects of their hydro-lyzing activity in predicting human intestinal absorption. Drug Metab Dispos 33:1185–1190.
Imai T, Taketani M, Shii M, Hosokawa M, Chiba K. (2006). Substrate specificity of carboxylesterase isozymes and their contribution to hydrolase activity in human liver and small intestine. Drug Metab Dispos 34:1734–1741.
Mei L, Nicoletti M, Battinelli L, Mazzanti G. (2001). Isolation of praer-uptorins A and B from Peucedanum praeruptorum Dunn., and their general pharmacological evaluation in comparison with extracts of drug. Il Farmaco 56: 417–420.
Ogihara T, Tamai I, Takanaga H, Sai Y, Tsuji A. (1996). Stereoselective and carrier-mediated transport of monocarboxylic acids across Caco-2 cells. Pharm Res 13:1828–1832.
Petri N, Tannergren C, Rungstad D, Lennernäs H. (2004). Transport characteristics of fexofenadine in the Caco-2 cell model. Pharm Res 21:1398–1404.
Rao MR, Sun L, Zhang XW. (2002). Effect of praeruptorum caumarin on cardiac mass, myocardial [Ca2+]i and Na+, K(+)-ATPase, Ca2+, Mg(2+)-ATPase activity in renovascular hypertensive rats. Yao Xue Xue Bao 37:401–404.
Ruan JQ, Leong WI, Yan R, Wang YT. (2010). Characterization of metabolism and in vitro permeability study of notoginsenoside R1 from Radix notoginseng. J Agric Food Chem 58:5770–5776.
Sun D, Lennernas H, Welage LS, Barnett JL, Landowski CP, Foster D, Fleisher D, Lee KD, Amidon GL. (2002). Comparison of human
duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm Res 19:1400–1416.
Suzuki H, Sugiyama Y. (2000). Role of metabolic enzymes and efflux transporters in the absorption of drugs from the small intestine. Eur J Pharm Sci 12:3–12.
Taipalensuu J, Törnblom H, Lindberg G, Einarsson C, Sjöqvist F, Melhus H, Garberg P, Sjöström B, Lundgren B, Artursson P. (2001). Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and in human intestinal epithelial Caco-2 cell monolay-ers. J Pharmacol Exp Ther 299:164–170.
Tantishaiyakul V, Wiwattanawongsa K, Pinsuwan S, Kasiwong S, Phadoongsombut N, Kaewnopparat S, Kaewnopparat N, Rojanasakul Y. (2002). Characterization of mefenamic acid-guai-acol ester: stability and transport across Caco-2 cell monolayers. Pharm Res 19:1013–1018.
Takai S, Matsuda A, Usami Y, Adachi T, Sugiyama T, Katagiri Y, Tatematsu M, Hirano K. (1997). Hydrolytic profile for ester- or amide-linkage by carboxylesterases pI 5.3 and 4.5 from human liver. Biol Pharm Bull 20:869–873.
Tang M, Mukundan M, Yang J, Charpentier N, LeCluyse EL, Black C, Yang D, Shi D, Yan B. (2006). Antiplatelet agents aspirin and clopidogrel are hydrolyzed by distinct carboxylesterases, and clopidogrel is transesterificated in the presence of ethyl alcohol. J Pharmacol Exp Ther 319:1467–1476.
Teruko I, Masumi L, Hisae S, Mitsuru H. (2005). Identification of este-rases expressed in Caco-2 cells and effects of their hydrolyzing activity in predicting human intestinal absorption. Drug Metab Dispos 33: 1185-1190.
The state pharmacopoeia commission of P. R. China. (2010). Peucedani Radix. In: Pharmacopoeia of people’s republic of China, Beijing, China: Chemical industry press. Vol I:187.
Violini S, Sharma V, Prior JL, Dyszlewski M, Worms DP. (2002). Evidence for a plasma membrane-mediated permeability bar-rier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate. BioChemistry 41: 12652–12661.
Walgren RA, Walle UK, Walle T. (1998). Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells. Biochem Pharmacol 55:1721–1727.
Wang LJ, Li JM, Chang TH, Hao LY. (1995). Effect of dl-praeruptorin A on delayed outward potassium current in single ventricular cell of guunea pig. Chinese Journal of pharmacology and toxicology 9:192–195.
Wang Y, Yan SX, Jia L, Tian L, Tu X, Lu JX. (2006). Effect of Pd-Ia on angiotensin II induced c-Jun protein expression in cadiocytes. China Pharmacist 9: 685–687.
Wei EH, Rao MR, Chen XY, Fan LM, Chen Q. (2002). Inhibitory effects of praeruptorin C on cattle aortic smooth muscle cell prolifera-tion. Acta Pharmacol Sin 23:129–132.
Wu XL, Kong LY, Min ZD. (2002). Studies of structure modification of (+)-Praeruptorin A. Acta Pharm Sin 37: 527–534.
Yee S. (1997). In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man–fact or myth. Pharm Res 14:763–766.
Xu Z, Wang X, Dai Y, Kong L, Wang F, Xu H, Lu D, Song J, Hou Z. (2010). (+/-)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis. Chem Biol Interact 186:239–246.
Zhang SL, Li JM, Xiao QH, Wu AH, Zhao Q, Yang GR, Zhang KY. (2001). Effect of dl-praeruptorin A on ATP sensitive potas-sium channels in human cortical neurons. Acta Pharmacol Sin 22:813–816.
Zhao NC, Jin WB, Zhang XH, Guan FL, Sun YB, Adachi H, Okuyama T. (1999). Relaxant effects of pyranocoumarin compounds iso-lated from a Chinese medical plant, Bai-Hua Qian-Hu, on iso-lated rabbit tracheas and pulmonary arteries. Biol Pharm Bull 22:984–987.
Zhu HJ, Wang JS, Markowitz JS, Donovan JL, Gibson BB, Gefroh HA, Devane CL. (2006). Characterization of P-glycoprotein inhibition by major cannabinoids from marijuana. J Pharmacol Exp Ther 317: 850–857.
