http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(9): 1150–1157 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.879906
ORIGINAL ARTICLE
Bioavailability of caffeic acid in rats and its absorption properties in the Caco-2 cell model Su-Jun Wang*, Jie Zeng*, Ben-Kun Yang, and Yun-Ming Zhong
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Department of Clinical Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China
Abstract
Keywords
Context: Caffeic acid (CA) is widely distributed in edible plants, and it is beneficial to human health by exerting various biological effects. The potential pharmacological activities of CA are dependent on its absorption in the gastrointestinal tract. Objective: To investigate the bioavailability of CA in rats and its absorption properties in the Caco-2 cell model. Materials and methods: A sensitive LC-MS/MS method was successfully applied to determine CA in rat plasma, perfusate, and Hank’s balanced salt solution (HBSS). The absolute bioavailability (Fabs) of CA was obtained after i.v. (2 mg/kg) or i.g. administration (10 mg/kg) to rats. Blood samples (approximately 250 mL) were collected from the jugular vein catheter. Pharmacokinetic parameters were calculated using the 3P97 software (version 2.0 PK software; Chinese Society of Mathematical Pharmacology, Anhui, China). The intestinal absorption of CA was explored by the in situ vascularly perfused rat intestinal preparation. CA (5 mg/kg) was administered into the duodenum. Samples (250 mL) were collected from reservoir at specific times, and the same volume fresh perfusate was replaced. The Caco-2 cell model was applied to measure the permeability of CA from the apical to basolateral side (A ! B) and from the basolateral to apical side (B ! A). Results: The absolute bioavailability (Fabs) of CA was 14.7%, and its intestinal absorption was 12.4%. The Papp A!B values of CA were ranging from (4.87 ± 1.72) 107 cm/s to (5.05 ± 0.66) 107 cm/s as the concentration varied from 5 to 15 mg/mL. Conclusion: CA was shown to have low oral bioavailability in rats, low intestinal absorption, and poor permeability across Caco-2 cells.
Permeability, pharmacokinetics, vascular perfusion
Introduction Epidemiologic studies have demonstrated that the phenolic acids protect humans against degenerative diseases such as cancer and cardiovascular diseases (Manach et al., 2005). Phenolic acids could be divided in two classes: hydroxybenzoic and hydroxycinnamic acids. The hydroxybenzoic acids, such as gallic acid and protocatechuic acid, are found only in a few plants eaten by humans; this is the reason why they are not currently considered to be of great nutritional interest (Manach et al., 2004). Hydroxycinnamic acids (HCAs) are a group of phenolic compounds characterized by the C6–C3 structure (Cartea et al., 2011). The most common hydrocinnamic acids are ferulic, cinnamic, sinapic and caffeic acid (CA) (EI-Seedi et al., 2012). HCAs are widely distributed in plants and fruits. CA is the most common and accounts for up to 70% of total hydroxycinnamic acids in fruits, and is the major representative hydroxycinnamic acids
*These authors contribute equally to this work. Correspondence: Su-jun Wang, Department of Clinical Pharmacy, Guangdong Pharmaceutical University, Guangzhou Higher Education Mega Center, 280 Wai Huan East Road, Guangzhou 510006, China. Tel: +86 20 3935 2123. E-mail: [email protected]
History Received 4 June 2013 Revised 11 November 2013 Accepted 30 December 2013 Published online 17 March 2014
(D’Archivio et al., 2007). A meta-analysis suggested that an increased consumption of coffee may reduce the risk of liver cancer, and recent studies suggest that CA exerts anticarcinogenic effects such as skin cancer (Kang et al., 2009; Larsson & Wolk, 2007). The pharmacological properties of CA can be summarized as follows: antioxidant (Zhang et al., 2013), antibacterial (Fernandez et al., 1996), and anticarcinogenic activities (Jaqanathan, 2012). CA is also the main constituent of CA tablets used to prevent bleeding during surgical operations (Liu & Xiao, 2011). CA is mainly metabolized by intestinal microflora (Perrercorn & Goldman, 1972), and its metabolites found in rat plasma are in the form of glucuronide, sulfate, and sulfate/glucuronide conjugates of CA or its methylated compounds (Azuma et al., 2000). The biological properties of CA depend on its absorption in the gut and on its metabolism. Although the bioavailabilities of some representative hydroxycinnamic acids such as cinnamic acid and ferulic acids have been investigated in our previous studies (Mo et al., 2012; Yang et al., 2013), little is known about the bioavailability of CA. Therefore, in the present work, the bioavailability of CA was first investigated and then the intestinal absorption as well as the permeability of CA across Caco-2 cell monolayers was studied. In this paper, the in situ
DOI: 10.3109/13880209.2013.879906
Bioavailability and absorption properties of caffeic acid
vascularly perfused rat intestinal preparation was first used to explore the intestinal absorption of CA in rats.
gas 2 (GS2) at 55 psi, ionspray voltage (IS) at 4500 V, and collision-activated dissociation (CAD) at 10 units; declustering potential (DP) at 53 V for CA and 65 for IS, entrance potential (EP) at 10 V, collision energy (CE) at 53 V for CA and 37 for IS, and collision exit potential (CXP) at 9 V.
Materials and methods
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Chemicals and reagents CA (98% purity) and cinnamic acid (internal standard, IS, purity 98%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM), non-essential amino acids, and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Penicillin and streptomycin were supplied by Invitrogen (Auckland, New Zealand). Ultrapure water was supplied by a Milli-Q Water Purification System from Millipore (Bedford, MA). Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). HPLC grade methanol was obtained from Fisher Scientific (Pittsburgh, PA). Ammonium acetate was purchased from Merck (Darmstadt, Germany). Animals The Ethics Committee of Guangdong Pharmaceutical University permitted the use of animals in the present study (GDPUIAEC No. 201002). Sprague–Dawley (SD) rats (6 weeks old, gender in half, 200–250 g) were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China) and acclimated for 3 d in an environmentally controlled room (temperature: 25 ± 2 C, humidity: 50 ± 5%, 12 h dark-light cycle), with free access to tap water and food before the experiments. Chromatographic conditions The HPLC system (Shimadzu, Kyoto, Japan) consisted of LC-20AD solvent delivery module, SPD-20A UV–visible spectrophotometric detector, a CTO-10vp column oven, and a SIL-20A autosampler. Separation was achieved on a C18 column (5 mm, 2.0 50 mm; Luna, Phenomenex, Torrance, CA). The optimized method used a binary gradient mobile phase with methanol–water (5:95, v/v) containing 10 mM ammonium acetate as mobile phase A, and methanol–water (95:5, v/v) containing 10 mM ammonium acetate as mobile phase B. The gradient profile was 0–0.5 min, 0% solvent B; 0.5–0.8 min, 0–100% solvent B; 0.8–3.0 min, 100% solvent B; 3.0–3.2 min, 100–0% solvent B; 3.2–5.0 min, 0% solvent. The flow rate was 0.2 mL/min and the injection volume was 10 mL. Mass spectrometry detection conditions Detection was performed with a 4000Q-trap mass spectrometer from Applied Biosystem (Foster City, CA) with ESI source operated in the negative ion mode. The MS analysis was carried out in multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 178.9 ! 134.8 for CA and m/z 146.8 ! 102.8 for IS. The chemical structures and fragmentation patterns for CA and the internal standard cinnamic acid are presented in Figure 1. The MS/MS conditions were as follows: ion spray source temperature at 550 C, curtain (CUR) gas at 25 psi, gas 1 (GS1) at 55 psi,
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Preparation of standards and calibration curves Separate stock solutions of CA and IS were prepared in DMSO (1 mg/mL) and working standard solutions were prepared by serial dilution in a mixture solution of methanol and water (50:50, v/v). Standard solutions of CA in rat plasma for the calibration curves were prepared by spiking the working standard solution into blank plasma (containing 50 mL aliquot of 40 ng/mL IS in 100 mL of plasma), giving final CA concentrations of 1500, 1000, 500, 200, 100, 50, 10, 2, and 1 ng/mL. Standard solutions of CA in perfusate for the calibration curves were prepared by spiking the working standard solution into blank perfusate (containing 50 mL aliquot of 40 ng/mL IS in 100 mL of perfusate), resulting final CA concentrations of 1500, 1000, 800, 600, 500, 250, 200, 150, and 100 ng/mL. Calibration standards of CA in Hank’s balanced salt solution (HBSS) were prepared by spiking the working standard solutions into blank HBSS (containing 50 mL aliquot of 40 ng/mL IS in 100 mL of HBSS), yielding final concentrations of 15, 10, 5, 1, 0.5, 0.2, 0.1, 0.05, and 0.02 ng/mL. The linearity of each calibration curve was determined by plotting the peak area ratio of CA to IS in plasma (perfusate and HBSS). The least-squares linear regression method (1/2 weight) was used to determine the slope, intercept, and correlation coefficient of the linear regression equation. The calibration curve samples were analyzed along with the quality control (QC) samples in three different matrixes (plasma, perfusate, and HBSS). The calibration curve samples were analyzed along with the quality control (QC) samples in three different matrixes (plasma, perfusate, and HBSS). The QC samples were prepared at three different concentration levels of 1000, 100, and 2 ng/mL for CA in plasma, 1000, 500, and 150 ng/ mL for CA in perfusate, and 10, 0.5, and 0.05 ng/mL for CA in HBSS. As for testing the matrix effects that may impact HPLCMS/MS analysis, the relative peak areas of QC samples after spiking blood (perfusate and HBSS) samples at three concentration levels were comparable to similarly prepared aqueous standard solutions.
Pharmacokinetic study In vivo experiments Rats were fasted overnight for at least 12 h, with free access to water, and randomly divided into two groups for intravenous and oral administration. All rats were fitted with a jugular vein catheter. Rats (n ¼ 6) were administered CA (2 mg/kg) with a vehicle composition consisting of 0.9% physiological saline (pH 7.0) intravenously in a final injection volume of 1.0 mL. Catheters were flushed with 1 mL of heparinized saline after intravenous bolus. An equal volume of heparinized saline was
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Figure 1. Chemical structures and fragmentation patterns for caffeic acid (A) and the internal standard cinnamic acid (B).
injected to replace the removed blood, and blood samples (250 mL) were collected via the jugular vein catheter at 5, 10, and 30 min and 1, 2, 3, 5, 8, and 12 h. CA was also given by oral gavage at 10 mg/kg to evaluate its oral bioavailability. All blood samples (250 mL) after oral administration were
collected via the jugular vein catheter at 5, 10, 20, and 30 min and 1, 2, 3, 4, 6, 8, and 12 h. Plasma samples were prepared by centrifuging the blood samples at 988 g for 10 min. Separated plasma (100 mL) and adding IS methanol solution (40 ng/mL, 300 mL) were centrifuged at 12 100 g
DOI: 10.3109/13880209.2013.879906
Bioavailability and absorption properties of caffeic acid
for 30 min. The supernatant was taken for LC-MS/MS analysis.
where Cn is the concentration of CA at the nth sampling point (ng/mL), Ci is the concentration of CA at the n 1 sampling point (ng/mL), 100 is the total volume of perfusate (mL), 0.25 is the sampling volume (mL), and D is the intraduodenal administration dose (ng).
Pharmacokinetic data analysis
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Pharmacokinetic parameters were calculated using the 3P97 software (version 2.0 PK software, Chinese Society of Mathematical Pharmacology, Anhui, China). The area under the plasma concentration–time curves (AUC) was calculated by the trapezoidal method; the maximum plasma concentration (Cmax) and the time to reach the maximum plasma concentration (Tmax) were obtained by visual inspection of the experimental data; the terminal half-life (t1/2b) was calculated as 0.693/k, and k was the slope of the terminal regression line. The absolute bioavailability (Fabs%) was calculated as the ratio of AUC0–1 (i.g.)/dose (i.g.) to the AUC0–1 (i.v.)/ dose (i.v.).
The in situ vascularly perfused rat intestinal preparation Model preparation and study design The rats were fasted overnight and had free access to water before the experiment. Rats were anesthetized with urethane and the surgical procedure was then performed as previously described (Mo et al., 2012; Pang et al., 1985; Wang et al., 2012). Two incisions were made from the abdominal wall to the level of the diaphragm. The pyloric vein, hepatic, and celiac arteries were ligated. The superior mesenteric artery, right renal artery, and aorta were separated from the connecting tissues. Loose ligatures were placed around the aorta before and after the common juncture of the superior mesenteric artery and right renal artery. A bulldog clamp was placed at the right renal artery just at its point of entry to the aorta. The right renal artery was then tied close to the right kidney. An incision was made on the right renal artery between the ligature and the bulldog clamp. A catheter was inserted into the right renal artery, through the aorta, and then into the superior mesenteric artery. Ligature was used to fasten the catheter, and all other loose ligatures were then tied. Perfusion was started at a flow rate of about 5 mL/min with a peristaltic pump with Krebs–Ringer buffer solution containing (g/L): NaCl 7.8, KCl 0.35, CaCl2 0.37, MgCl2 0.22, NaH2PO4 0.32, glucose 3.0, NaHCO3 1.37, dextran 30, and BSA 50. The flow rate for perfusion was increased to 7.5 mL/ min, and a heating lamp was used to maintain the temperature of the preparation at 37 C. First, the residual blood in the vessel was washed 15 min with the perfusate; next the volume of 100 mL fresh perfusion was replaced. Then CA (5 mg/kg) was administered into the duodenum. Samples (250 mL) were collected from the reservoir at 0, 5, 15, 30, 45, 60, 75, 90, 105, and 120 min, and the same volume of fresh perfusate was replaced. The samples were stored at 20 C until further LC-MS/MS assay was performed.
Caco-2 cells model Cell culture Caco-2 cells (passages 35–40) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% FBS, 1% non-essential amino acids, 1% L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, at 37 C in an environment of 90% humidity, and 5% CO2. For the transport experiments, cells were seeded on a 12-well plate at a density of 6 104 cells/cm2. The culture medium was changed every 2 d for the first 1 week and then daily after 1 week. The transepithelial electrical resistance (TEER) of the monolayer was considered to be the indicator of cell monolayer integrity and was measured using a Millicell-ERS meter (Thermo Fisher Scientific, Hudson, NH). Caco-2 cell monolayers with TEER values above 500 V cm2 after 21 d post-seeding were used in this study. Transport study A 1 mg/mL stock solution of CA was prepared in DMSO and diluted in HBSS to the appropriate concentration (5, 10, and 15 mg/mL) prior to the experiment. Following the removal of culture medium and pre-equilibration with HBSS at 37 C for 15 min, the test compound was loaded at the apical or basolateral side of the Caco-2 cell monolayer. Bidirectional transport of CA was conducted. For apical to basolateral transport study (A ! B), 200 mL of CA solution (5, 10, and 15 mg/mL) was added to the apical side and 800 mL of blank HBSS was added to the basolateral side. Samples of 100 mL were withdrawn from receiver compartments at 120 min and then replaced with an equal volume of fresh HBSS. The profile for CA transport in the opposite direction (B ! A) was also examined in the same manner. The experiment was performed in the shaker incubator at 37 C and 50 rpm. About 50 mL of the internal standard solution (40 ng/mL cinnamic acid in methanol and water solution, v:v, 50:50) was added to 100 mL of samples. After centrifugation at 12 100 g for 30 min, 10 mL of the supernatant was injected into HPLC-MS/ MS for the analysis. Transport data analysis Data from three independent experiments were presented as mean ± SD. Results of bi-directional transport were expressed as permeability coefficient, which was calculated using the following equation: Papp ¼
Data analysis The intestinal absorption of CA was calculated according to the following equation: P 100Cn þ 0:25 n¼1 i¼1 Ci 100% ð1Þ Intestinal absorption % ¼ D
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dQ 1 dt A C0
ð2Þ
where dQ/dt is the slope of the cumulative drug transported versus time curve (mg/s), A is the surface area of the filter (cm2), C0 is the initial concentration of the drug (mg/mL), and Papp is the apparent permeability coefficient (cm/s).
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Figure 2. LC/MS/MS chromatograms of caffeic acid and IS cinnamic acid: (A) blank rat plasma spiked with 2 ng/mL of caffeic acid and the internal standard (40 ng/mL); (B) plasma sample from a rat at 8 h after intragastric administration of a 10 mg/kg dose of caffeic acid; 1-caffeic acid; 2-IS cinnamic acid.
DOI: 10.3109/13880209.2013.879906
Bioavailability and absorption properties of caffeic acid
The efflux ratio (EfR) across the monolayers was calculated using the following equation:
Pharmacokinetic study
PappðB!AÞ efflux ratio ¼ PappðA!BÞ
ð3Þ
where Papp(B!A) is the permeability from the basolateral to apical side, and Papp(A!B) is the permeability from the apical-to-basolateral direction.
Results
The retention time for CA and IS was 0.82 and 3.33 min, respectively. The chromatogram shows baseline separation of CA and IS without any interference from the endogenous plasma components. The representative chromatograms are presented in Figure 2. The calibration curves of CA spiked with blank blood (perfusate and HBSS) were constructed by plotting the ratio of peak areas of CA and IS versus concentration, and the results are given in Table 1. The matrix effect was calculated by comparing the responses of post-extracted spiked sample and non-extracted sample. The peak areas of CA after spiking plasma samples at three concentration levels were comparable to similarly prepared aqueous standard solutions (ranged from 90.1% to 100.6%), suggesting that there was no measurable matrix effect that interfered with CA determination in the rat plasma. Rat intestine perfusate and Caco-2 medium had no significant matrix effect after comparing the standard curve of these two matrixes with their respective aqueous samples. The LC-MS/ MS assay developed in this study was verified to be linear, specific, and suitable for analyzing the CA.
Table 1. The standard curve for caffeic acid in three kinds of matrix.
Matrix Plasma Perfusate HBSS
Linear range LOD (ng/mL) (ng/mL) 1–1500 100–1500 0.02–15
1 100 0.02
Linear equation Y ¼ 0.00844X 0.0163, r ¼ 0.9999 Y ¼ 0.0806X 0.118, r ¼ 0.9992 Y ¼ 0.000554X 0.000195, r ¼ 0.9984
The concentration–time profiles of CA were best described by a 2-compartment model after i.v. and i.g. administration of CA to rats (Figure 3). The pharmacokinetic parameters of CA are summarized in Table 2. Following intravenous dosing, the t1/2 and total body clearance of CA were 1.54 ± 0.13 h and 4.64 ± 0.75 L/h/kg, respectively. After oral dose of CA, the t1/2 and total body clearance of CA were 2.13 ± 0.75 h and 3.35 ± 1.46 L/h/kg, respectively. The absolute bioavailability of CA in rat was found to be 14.7%. The in situ vascularly perfused rat intestinal preparation After an intraduodenal injection of a dose of CA (5 mg/kg), the appearance of CA in the reservoir was rapid (Figure 4). The concentration of CA in reservoir was 255.0 ± 58.89 ng/ mL at 5 min. After 60 min, the concentration of CA in perfusate changed gently, and the concentration was 1211.52 ± 41.97 ng/mL at the end of perfusion. The intestinal absorption of CA was 12.4%. Transport studies TEER measurement was continued for 21 d, throughout the period of culturing. Detectable values emerged from the 5th day of culture and continued to increase until Table 2. Caffeic acid pharmacokinetic parameters following a single i.v. (2 mg/kg) or i.g. (10 mg/kg) administration (mean ± SD, n ¼ 6). Parameter
Unit
i.g. group
i.v. group
t1/2b V CL MRT0!t MRT0!1 AUC0!t AUC0!1 Tmax Cmax Fabs%
H L kg1 L h1 kg1 h h ng h mL1 ng h mL1 h ng mL1
2.13 ± 0.75 2.41 ± 4.89 3.35 ± 1.46 2.17 ± 0.27 2.96 ± 0.61 302.9 ± 24.4 355.4 ± 32.5 0.33 ± 0.12 250.4 ± 37.6 14.70
1.54 ± 0.13 1.60 ± 0.41 4.64 ± 0.75 0.88 ± 0.12 4.44 ± 4.38 442.0 ± 44.8 483.6 ± 51.0 – – –
t1/2b, half-life; V, volume of distribution; CL, the clearance; MRT, mean residence time; AUC, area under concentration–time curve; Tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration; Fabs, absolute bioavailability.
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for CA
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Figure 3. Plasma concentration–time curves of caffeic acid after a single intravenous (2 mg/kg, g) and oral (10 mg/kg, m) administration. Each point represents an average of six determinations and the error bars are standard deviations of the mean.
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Figure 4. The absorption of caffeic acid by the recirculating intestinal preparation after an intraduodenal injection of a dose of 5 mg/kg of caffeic acid. Each data point is the average of six determinations and the error bar is the standard deviation of the mean.
Table 3. Papp of caffeic acid at different concentrations in Caco-2 cells (mean ± SD, n ¼ 3). Papp (107) cms1 Caffeic acid/mgmL1 5 10 15
A!B
B!A
Efflux ratio
5.05 ± 0.66 5.00 ± 0.32 4.87 ± 1.72
13.40 ± 1.98 13.02 ± 1.75 13.16 ± 2.19
2.65 2.60 2.70
Papp is the apparent permeability coefficient; A–B is apicalto-basolateral transport; B–A is basolateral-to-apical transport; efflux ratio is calculated as the ratio of Papp(B ! A) to Papp(A ! B).
reaching a plateau on the 20th day of culture and values above 500 V cm2. Different concentrations of CA transport across the Caco-2 cell monolayers were measured (Table 3). The Papp A!B values of CA were ranging from (4.87 ± 1.72) 107 cm/s to (5.05 ± 0.66) 107 cm/s as the concentration varied from 5 to 15 mg/mL, and the bi-direction transport of the drug was almost constant in the concentration range of 5–15 mg/mL.
Discussion The pharmacokinetic profile of CA was fitted in a twocompartment model after the i.v. injection that was in accordance with the previous report (Uang & Hsu, 1997). The absolute bioavailability of CA was 14.7%. In order to define the mechanisms responsible for poor bioavailability of CA, the intestinal absorption of CA and its permeability were explored. The in situ vascularly perfused rat intestinal preparation was originated in 1985 and was successfully utilized to assess the rate and extent of the intestinal absorption of enalapril (Pang et al., 1985). This in situ preparation is advantageous in that the circulation and morphology of the tissues remain intact. Moreover, it is precise and in direct assessment of the contribution of intestinal absorption of drug. The in situ vascularly perfused rat intestinal preparation was used to assess the extent of absorption of CA after its intraduodenal administration. The finding was that the intestinal absorption of CA was 12.4%, which matched up with the absolute bioavailability obtained above. Compared to the in vivo pharmacokinetic study, the in situ vascularly perfused rat intestinal preparation can properly change the experimental conditions (drug
concentration, perfusate flow, or composition of perfusate) to survey the effect on the absorption of test drug. Caco-2 cell model is publicly known to simulate drug intestinal absorption. The apparent permeability coefficient of compound ranking with Papp A!B51 106 cm/s, between (1–10) 106 cm/s and410 106 cm/s can be classified as poorly (0–20%), moderately (20–70%), and well (70–100%) absorbed compound, respectively (Yee, 1997). The Papp A!B values of CA were ranging from (4.87 ± 1.72) 107 cm/s to (5.05 ± 0.66) 107 cm/s as the concentration varied from 5 to 15 mg/mL indicating that CA was not easy to transport through intestinal wall epithelial cells, and it should be classified as poorly absorbed compound. The Papp B!A values of CA were significantly higher than the Papp A!B values, indicating that CA was easy to exhaust out from the intestinal wall epithelial. All results indicating the poor bioavailability of CA were mostly due to the low intestinal absorption and low permeability across Caco-2 cells.
Conclusions This study validated that the absolute bioavailability (Fabs) of CA was 14.7%, and its intestinal absorption was 12.4%. In the Caco-2 cells model, the Papp A!B values of CA were 51 106 cm/s as the concentration varied from 5 to 15 mg/ mL. The data suggested that CA was not easy to transport through intestinal wall epithelial cells. CA was shown to have poor permeability across the Caco-2 cell monolayer, and was easy to exhaust out from the intestinal wall epithelial. Therefore, the poor bioavailability of CA was attributable to the low intestinal absorption and low permeability across Caco-2 cells.
Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by National Natural Science Foundation of China (81073141), Guangdong Provincial Natural Science Foundation (9152402301000007) and Guangdong Province ‘‘12-5’’ medical key subject.
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Perrercorn MA, Goldman P. (1972). Caffeic acid metabolism by gnotobiotic rats and their intestinal bacteria. Proc Natl Acad Sci USA 69:1413–15. Uang YS, Hsu KY. (1997). A dose-dependent pharmacokinetic study on caffeic acid in rabbits after intravenous administration. Biopharm Drug Dispos 18:727–36. Wang SJ, Mo LL, Yang BK, et al. (2012). Optimization of in situ perfused rat intestine-live model. Chin J Clin Pharmacol Ther 17:361–6. Yang BK, Wang SJ, Mo LL, et al. (2013). Studies on the absolute bioavailability of cinnamic acid in rats and its absorption properties. Pharm J Chin PLA 29:185–8. 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 14:763–6. Zhang R, Zeng Q, Deng Y, et al. (2013). Phenolic profiles and antioxidant activity of litchi pulp of different cultivars cultivated in Southern China. Food Chem 136:1169–76.
ORIGINAL ARTICLE
Bioavailability of caffeic acid in rats and its absorption properties in the Caco-2 cell model Su-Jun Wang*, Jie Zeng*, Ben-Kun Yang, and Yun-Ming Zhong
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Department of Clinical Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China
Abstract
Keywords
Context: Caffeic acid (CA) is widely distributed in edible plants, and it is beneficial to human health by exerting various biological effects. The potential pharmacological activities of CA are dependent on its absorption in the gastrointestinal tract. Objective: To investigate the bioavailability of CA in rats and its absorption properties in the Caco-2 cell model. Materials and methods: A sensitive LC-MS/MS method was successfully applied to determine CA in rat plasma, perfusate, and Hank’s balanced salt solution (HBSS). The absolute bioavailability (Fabs) of CA was obtained after i.v. (2 mg/kg) or i.g. administration (10 mg/kg) to rats. Blood samples (approximately 250 mL) were collected from the jugular vein catheter. Pharmacokinetic parameters were calculated using the 3P97 software (version 2.0 PK software; Chinese Society of Mathematical Pharmacology, Anhui, China). The intestinal absorption of CA was explored by the in situ vascularly perfused rat intestinal preparation. CA (5 mg/kg) was administered into the duodenum. Samples (250 mL) were collected from reservoir at specific times, and the same volume fresh perfusate was replaced. The Caco-2 cell model was applied to measure the permeability of CA from the apical to basolateral side (A ! B) and from the basolateral to apical side (B ! A). Results: The absolute bioavailability (Fabs) of CA was 14.7%, and its intestinal absorption was 12.4%. The Papp A!B values of CA were ranging from (4.87 ± 1.72) 107 cm/s to (5.05 ± 0.66) 107 cm/s as the concentration varied from 5 to 15 mg/mL. Conclusion: CA was shown to have low oral bioavailability in rats, low intestinal absorption, and poor permeability across Caco-2 cells.
Permeability, pharmacokinetics, vascular perfusion
Introduction Epidemiologic studies have demonstrated that the phenolic acids protect humans against degenerative diseases such as cancer and cardiovascular diseases (Manach et al., 2005). Phenolic acids could be divided in two classes: hydroxybenzoic and hydroxycinnamic acids. The hydroxybenzoic acids, such as gallic acid and protocatechuic acid, are found only in a few plants eaten by humans; this is the reason why they are not currently considered to be of great nutritional interest (Manach et al., 2004). Hydroxycinnamic acids (HCAs) are a group of phenolic compounds characterized by the C6–C3 structure (Cartea et al., 2011). The most common hydrocinnamic acids are ferulic, cinnamic, sinapic and caffeic acid (CA) (EI-Seedi et al., 2012). HCAs are widely distributed in plants and fruits. CA is the most common and accounts for up to 70% of total hydroxycinnamic acids in fruits, and is the major representative hydroxycinnamic acids
*These authors contribute equally to this work. Correspondence: Su-jun Wang, Department of Clinical Pharmacy, Guangdong Pharmaceutical University, Guangzhou Higher Education Mega Center, 280 Wai Huan East Road, Guangzhou 510006, China. Tel: +86 20 3935 2123. E-mail: [email protected]
History Received 4 June 2013 Revised 11 November 2013 Accepted 30 December 2013 Published online 17 March 2014
(D’Archivio et al., 2007). A meta-analysis suggested that an increased consumption of coffee may reduce the risk of liver cancer, and recent studies suggest that CA exerts anticarcinogenic effects such as skin cancer (Kang et al., 2009; Larsson & Wolk, 2007). The pharmacological properties of CA can be summarized as follows: antioxidant (Zhang et al., 2013), antibacterial (Fernandez et al., 1996), and anticarcinogenic activities (Jaqanathan, 2012). CA is also the main constituent of CA tablets used to prevent bleeding during surgical operations (Liu & Xiao, 2011). CA is mainly metabolized by intestinal microflora (Perrercorn & Goldman, 1972), and its metabolites found in rat plasma are in the form of glucuronide, sulfate, and sulfate/glucuronide conjugates of CA or its methylated compounds (Azuma et al., 2000). The biological properties of CA depend on its absorption in the gut and on its metabolism. Although the bioavailabilities of some representative hydroxycinnamic acids such as cinnamic acid and ferulic acids have been investigated in our previous studies (Mo et al., 2012; Yang et al., 2013), little is known about the bioavailability of CA. Therefore, in the present work, the bioavailability of CA was first investigated and then the intestinal absorption as well as the permeability of CA across Caco-2 cell monolayers was studied. In this paper, the in situ
DOI: 10.3109/13880209.2013.879906
Bioavailability and absorption properties of caffeic acid
vascularly perfused rat intestinal preparation was first used to explore the intestinal absorption of CA in rats.
gas 2 (GS2) at 55 psi, ionspray voltage (IS) at 4500 V, and collision-activated dissociation (CAD) at 10 units; declustering potential (DP) at 53 V for CA and 65 for IS, entrance potential (EP) at 10 V, collision energy (CE) at 53 V for CA and 37 for IS, and collision exit potential (CXP) at 9 V.
Materials and methods
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Chemicals and reagents CA (98% purity) and cinnamic acid (internal standard, IS, purity 98%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM), non-essential amino acids, and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Penicillin and streptomycin were supplied by Invitrogen (Auckland, New Zealand). Ultrapure water was supplied by a Milli-Q Water Purification System from Millipore (Bedford, MA). Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). HPLC grade methanol was obtained from Fisher Scientific (Pittsburgh, PA). Ammonium acetate was purchased from Merck (Darmstadt, Germany). Animals The Ethics Committee of Guangdong Pharmaceutical University permitted the use of animals in the present study (GDPUIAEC No. 201002). Sprague–Dawley (SD) rats (6 weeks old, gender in half, 200–250 g) were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China) and acclimated for 3 d in an environmentally controlled room (temperature: 25 ± 2 C, humidity: 50 ± 5%, 12 h dark-light cycle), with free access to tap water and food before the experiments. Chromatographic conditions The HPLC system (Shimadzu, Kyoto, Japan) consisted of LC-20AD solvent delivery module, SPD-20A UV–visible spectrophotometric detector, a CTO-10vp column oven, and a SIL-20A autosampler. Separation was achieved on a C18 column (5 mm, 2.0 50 mm; Luna, Phenomenex, Torrance, CA). The optimized method used a binary gradient mobile phase with methanol–water (5:95, v/v) containing 10 mM ammonium acetate as mobile phase A, and methanol–water (95:5, v/v) containing 10 mM ammonium acetate as mobile phase B. The gradient profile was 0–0.5 min, 0% solvent B; 0.5–0.8 min, 0–100% solvent B; 0.8–3.0 min, 100% solvent B; 3.0–3.2 min, 100–0% solvent B; 3.2–5.0 min, 0% solvent. The flow rate was 0.2 mL/min and the injection volume was 10 mL. Mass spectrometry detection conditions Detection was performed with a 4000Q-trap mass spectrometer from Applied Biosystem (Foster City, CA) with ESI source operated in the negative ion mode. The MS analysis was carried out in multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 178.9 ! 134.8 for CA and m/z 146.8 ! 102.8 for IS. The chemical structures and fragmentation patterns for CA and the internal standard cinnamic acid are presented in Figure 1. The MS/MS conditions were as follows: ion spray source temperature at 550 C, curtain (CUR) gas at 25 psi, gas 1 (GS1) at 55 psi,
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Preparation of standards and calibration curves Separate stock solutions of CA and IS were prepared in DMSO (1 mg/mL) and working standard solutions were prepared by serial dilution in a mixture solution of methanol and water (50:50, v/v). Standard solutions of CA in rat plasma for the calibration curves were prepared by spiking the working standard solution into blank plasma (containing 50 mL aliquot of 40 ng/mL IS in 100 mL of plasma), giving final CA concentrations of 1500, 1000, 500, 200, 100, 50, 10, 2, and 1 ng/mL. Standard solutions of CA in perfusate for the calibration curves were prepared by spiking the working standard solution into blank perfusate (containing 50 mL aliquot of 40 ng/mL IS in 100 mL of perfusate), resulting final CA concentrations of 1500, 1000, 800, 600, 500, 250, 200, 150, and 100 ng/mL. Calibration standards of CA in Hank’s balanced salt solution (HBSS) were prepared by spiking the working standard solutions into blank HBSS (containing 50 mL aliquot of 40 ng/mL IS in 100 mL of HBSS), yielding final concentrations of 15, 10, 5, 1, 0.5, 0.2, 0.1, 0.05, and 0.02 ng/mL. The linearity of each calibration curve was determined by plotting the peak area ratio of CA to IS in plasma (perfusate and HBSS). The least-squares linear regression method (1/2 weight) was used to determine the slope, intercept, and correlation coefficient of the linear regression equation. The calibration curve samples were analyzed along with the quality control (QC) samples in three different matrixes (plasma, perfusate, and HBSS). The calibration curve samples were analyzed along with the quality control (QC) samples in three different matrixes (plasma, perfusate, and HBSS). The QC samples were prepared at three different concentration levels of 1000, 100, and 2 ng/mL for CA in plasma, 1000, 500, and 150 ng/ mL for CA in perfusate, and 10, 0.5, and 0.05 ng/mL for CA in HBSS. As for testing the matrix effects that may impact HPLCMS/MS analysis, the relative peak areas of QC samples after spiking blood (perfusate and HBSS) samples at three concentration levels were comparable to similarly prepared aqueous standard solutions.
Pharmacokinetic study In vivo experiments Rats were fasted overnight for at least 12 h, with free access to water, and randomly divided into two groups for intravenous and oral administration. All rats were fitted with a jugular vein catheter. Rats (n ¼ 6) were administered CA (2 mg/kg) with a vehicle composition consisting of 0.9% physiological saline (pH 7.0) intravenously in a final injection volume of 1.0 mL. Catheters were flushed with 1 mL of heparinized saline after intravenous bolus. An equal volume of heparinized saline was
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Figure 1. Chemical structures and fragmentation patterns for caffeic acid (A) and the internal standard cinnamic acid (B).
injected to replace the removed blood, and blood samples (250 mL) were collected via the jugular vein catheter at 5, 10, and 30 min and 1, 2, 3, 5, 8, and 12 h. CA was also given by oral gavage at 10 mg/kg to evaluate its oral bioavailability. All blood samples (250 mL) after oral administration were
collected via the jugular vein catheter at 5, 10, 20, and 30 min and 1, 2, 3, 4, 6, 8, and 12 h. Plasma samples were prepared by centrifuging the blood samples at 988 g for 10 min. Separated plasma (100 mL) and adding IS methanol solution (40 ng/mL, 300 mL) were centrifuged at 12 100 g
DOI: 10.3109/13880209.2013.879906
Bioavailability and absorption properties of caffeic acid
for 30 min. The supernatant was taken for LC-MS/MS analysis.
where Cn is the concentration of CA at the nth sampling point (ng/mL), Ci is the concentration of CA at the n 1 sampling point (ng/mL), 100 is the total volume of perfusate (mL), 0.25 is the sampling volume (mL), and D is the intraduodenal administration dose (ng).
Pharmacokinetic data analysis
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Pharmacokinetic parameters were calculated using the 3P97 software (version 2.0 PK software, Chinese Society of Mathematical Pharmacology, Anhui, China). The area under the plasma concentration–time curves (AUC) was calculated by the trapezoidal method; the maximum plasma concentration (Cmax) and the time to reach the maximum plasma concentration (Tmax) were obtained by visual inspection of the experimental data; the terminal half-life (t1/2b) was calculated as 0.693/k, and k was the slope of the terminal regression line. The absolute bioavailability (Fabs%) was calculated as the ratio of AUC0–1 (i.g.)/dose (i.g.) to the AUC0–1 (i.v.)/ dose (i.v.).
The in situ vascularly perfused rat intestinal preparation Model preparation and study design The rats were fasted overnight and had free access to water before the experiment. Rats were anesthetized with urethane and the surgical procedure was then performed as previously described (Mo et al., 2012; Pang et al., 1985; Wang et al., 2012). Two incisions were made from the abdominal wall to the level of the diaphragm. The pyloric vein, hepatic, and celiac arteries were ligated. The superior mesenteric artery, right renal artery, and aorta were separated from the connecting tissues. Loose ligatures were placed around the aorta before and after the common juncture of the superior mesenteric artery and right renal artery. A bulldog clamp was placed at the right renal artery just at its point of entry to the aorta. The right renal artery was then tied close to the right kidney. An incision was made on the right renal artery between the ligature and the bulldog clamp. A catheter was inserted into the right renal artery, through the aorta, and then into the superior mesenteric artery. Ligature was used to fasten the catheter, and all other loose ligatures were then tied. Perfusion was started at a flow rate of about 5 mL/min with a peristaltic pump with Krebs–Ringer buffer solution containing (g/L): NaCl 7.8, KCl 0.35, CaCl2 0.37, MgCl2 0.22, NaH2PO4 0.32, glucose 3.0, NaHCO3 1.37, dextran 30, and BSA 50. The flow rate for perfusion was increased to 7.5 mL/ min, and a heating lamp was used to maintain the temperature of the preparation at 37 C. First, the residual blood in the vessel was washed 15 min with the perfusate; next the volume of 100 mL fresh perfusion was replaced. Then CA (5 mg/kg) was administered into the duodenum. Samples (250 mL) were collected from the reservoir at 0, 5, 15, 30, 45, 60, 75, 90, 105, and 120 min, and the same volume of fresh perfusate was replaced. The samples were stored at 20 C until further LC-MS/MS assay was performed.
Caco-2 cells model Cell culture Caco-2 cells (passages 35–40) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% FBS, 1% non-essential amino acids, 1% L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, at 37 C in an environment of 90% humidity, and 5% CO2. For the transport experiments, cells were seeded on a 12-well plate at a density of 6 104 cells/cm2. The culture medium was changed every 2 d for the first 1 week and then daily after 1 week. The transepithelial electrical resistance (TEER) of the monolayer was considered to be the indicator of cell monolayer integrity and was measured using a Millicell-ERS meter (Thermo Fisher Scientific, Hudson, NH). Caco-2 cell monolayers with TEER values above 500 V cm2 after 21 d post-seeding were used in this study. Transport study A 1 mg/mL stock solution of CA was prepared in DMSO and diluted in HBSS to the appropriate concentration (5, 10, and 15 mg/mL) prior to the experiment. Following the removal of culture medium and pre-equilibration with HBSS at 37 C for 15 min, the test compound was loaded at the apical or basolateral side of the Caco-2 cell monolayer. Bidirectional transport of CA was conducted. For apical to basolateral transport study (A ! B), 200 mL of CA solution (5, 10, and 15 mg/mL) was added to the apical side and 800 mL of blank HBSS was added to the basolateral side. Samples of 100 mL were withdrawn from receiver compartments at 120 min and then replaced with an equal volume of fresh HBSS. The profile for CA transport in the opposite direction (B ! A) was also examined in the same manner. The experiment was performed in the shaker incubator at 37 C and 50 rpm. About 50 mL of the internal standard solution (40 ng/mL cinnamic acid in methanol and water solution, v:v, 50:50) was added to 100 mL of samples. After centrifugation at 12 100 g for 30 min, 10 mL of the supernatant was injected into HPLC-MS/ MS for the analysis. Transport data analysis Data from three independent experiments were presented as mean ± SD. Results of bi-directional transport were expressed as permeability coefficient, which was calculated using the following equation: Papp ¼
Data analysis The intestinal absorption of CA was calculated according to the following equation: P 100Cn þ 0:25 n¼1 i¼1 Ci 100% ð1Þ Intestinal absorption % ¼ D
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dQ 1 dt A C0
ð2Þ
where dQ/dt is the slope of the cumulative drug transported versus time curve (mg/s), A is the surface area of the filter (cm2), C0 is the initial concentration of the drug (mg/mL), and Papp is the apparent permeability coefficient (cm/s).
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Figure 2. LC/MS/MS chromatograms of caffeic acid and IS cinnamic acid: (A) blank rat plasma spiked with 2 ng/mL of caffeic acid and the internal standard (40 ng/mL); (B) plasma sample from a rat at 8 h after intragastric administration of a 10 mg/kg dose of caffeic acid; 1-caffeic acid; 2-IS cinnamic acid.
DOI: 10.3109/13880209.2013.879906
Bioavailability and absorption properties of caffeic acid
The efflux ratio (EfR) across the monolayers was calculated using the following equation:
Pharmacokinetic study
PappðB!AÞ efflux ratio ¼ PappðA!BÞ
ð3Þ
where Papp(B!A) is the permeability from the basolateral to apical side, and Papp(A!B) is the permeability from the apical-to-basolateral direction.
Results
The retention time for CA and IS was 0.82 and 3.33 min, respectively. The chromatogram shows baseline separation of CA and IS without any interference from the endogenous plasma components. The representative chromatograms are presented in Figure 2. The calibration curves of CA spiked with blank blood (perfusate and HBSS) were constructed by plotting the ratio of peak areas of CA and IS versus concentration, and the results are given in Table 1. The matrix effect was calculated by comparing the responses of post-extracted spiked sample and non-extracted sample. The peak areas of CA after spiking plasma samples at three concentration levels were comparable to similarly prepared aqueous standard solutions (ranged from 90.1% to 100.6%), suggesting that there was no measurable matrix effect that interfered with CA determination in the rat plasma. Rat intestine perfusate and Caco-2 medium had no significant matrix effect after comparing the standard curve of these two matrixes with their respective aqueous samples. The LC-MS/ MS assay developed in this study was verified to be linear, specific, and suitable for analyzing the CA.
Table 1. The standard curve for caffeic acid in three kinds of matrix.
Matrix Plasma Perfusate HBSS
Linear range LOD (ng/mL) (ng/mL) 1–1500 100–1500 0.02–15
1 100 0.02
Linear equation Y ¼ 0.00844X 0.0163, r ¼ 0.9999 Y ¼ 0.0806X 0.118, r ¼ 0.9992 Y ¼ 0.000554X 0.000195, r ¼ 0.9984
The concentration–time profiles of CA were best described by a 2-compartment model after i.v. and i.g. administration of CA to rats (Figure 3). The pharmacokinetic parameters of CA are summarized in Table 2. Following intravenous dosing, the t1/2 and total body clearance of CA were 1.54 ± 0.13 h and 4.64 ± 0.75 L/h/kg, respectively. After oral dose of CA, the t1/2 and total body clearance of CA were 2.13 ± 0.75 h and 3.35 ± 1.46 L/h/kg, respectively. The absolute bioavailability of CA in rat was found to be 14.7%. The in situ vascularly perfused rat intestinal preparation After an intraduodenal injection of a dose of CA (5 mg/kg), the appearance of CA in the reservoir was rapid (Figure 4). The concentration of CA in reservoir was 255.0 ± 58.89 ng/ mL at 5 min. After 60 min, the concentration of CA in perfusate changed gently, and the concentration was 1211.52 ± 41.97 ng/mL at the end of perfusion. The intestinal absorption of CA was 12.4%. Transport studies TEER measurement was continued for 21 d, throughout the period of culturing. Detectable values emerged from the 5th day of culture and continued to increase until Table 2. Caffeic acid pharmacokinetic parameters following a single i.v. (2 mg/kg) or i.g. (10 mg/kg) administration (mean ± SD, n ¼ 6). Parameter
Unit
i.g. group
i.v. group
t1/2b V CL MRT0!t MRT0!1 AUC0!t AUC0!1 Tmax Cmax Fabs%
H L kg1 L h1 kg1 h h ng h mL1 ng h mL1 h ng mL1
2.13 ± 0.75 2.41 ± 4.89 3.35 ± 1.46 2.17 ± 0.27 2.96 ± 0.61 302.9 ± 24.4 355.4 ± 32.5 0.33 ± 0.12 250.4 ± 37.6 14.70
1.54 ± 0.13 1.60 ± 0.41 4.64 ± 0.75 0.88 ± 0.12 4.44 ± 4.38 442.0 ± 44.8 483.6 ± 51.0 – – –
t1/2b, half-life; V, volume of distribution; CL, the clearance; MRT, mean residence time; AUC, area under concentration–time curve; Tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration; Fabs, absolute bioavailability.
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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for CA
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Figure 3. Plasma concentration–time curves of caffeic acid after a single intravenous (2 mg/kg, g) and oral (10 mg/kg, m) administration. Each point represents an average of six determinations and the error bars are standard deviations of the mean.
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Figure 4. The absorption of caffeic acid by the recirculating intestinal preparation after an intraduodenal injection of a dose of 5 mg/kg of caffeic acid. Each data point is the average of six determinations and the error bar is the standard deviation of the mean.
Table 3. Papp of caffeic acid at different concentrations in Caco-2 cells (mean ± SD, n ¼ 3). Papp (107) cms1 Caffeic acid/mgmL1 5 10 15
A!B
B!A
Efflux ratio
5.05 ± 0.66 5.00 ± 0.32 4.87 ± 1.72
13.40 ± 1.98 13.02 ± 1.75 13.16 ± 2.19
2.65 2.60 2.70
Papp is the apparent permeability coefficient; A–B is apicalto-basolateral transport; B–A is basolateral-to-apical transport; efflux ratio is calculated as the ratio of Papp(B ! A) to Papp(A ! B).
reaching a plateau on the 20th day of culture and values above 500 V cm2. Different concentrations of CA transport across the Caco-2 cell monolayers were measured (Table 3). The Papp A!B values of CA were ranging from (4.87 ± 1.72) 107 cm/s to (5.05 ± 0.66) 107 cm/s as the concentration varied from 5 to 15 mg/mL, and the bi-direction transport of the drug was almost constant in the concentration range of 5–15 mg/mL.
Discussion The pharmacokinetic profile of CA was fitted in a twocompartment model after the i.v. injection that was in accordance with the previous report (Uang & Hsu, 1997). The absolute bioavailability of CA was 14.7%. In order to define the mechanisms responsible for poor bioavailability of CA, the intestinal absorption of CA and its permeability were explored. The in situ vascularly perfused rat intestinal preparation was originated in 1985 and was successfully utilized to assess the rate and extent of the intestinal absorption of enalapril (Pang et al., 1985). This in situ preparation is advantageous in that the circulation and morphology of the tissues remain intact. Moreover, it is precise and in direct assessment of the contribution of intestinal absorption of drug. The in situ vascularly perfused rat intestinal preparation was used to assess the extent of absorption of CA after its intraduodenal administration. The finding was that the intestinal absorption of CA was 12.4%, which matched up with the absolute bioavailability obtained above. Compared to the in vivo pharmacokinetic study, the in situ vascularly perfused rat intestinal preparation can properly change the experimental conditions (drug
concentration, perfusate flow, or composition of perfusate) to survey the effect on the absorption of test drug. Caco-2 cell model is publicly known to simulate drug intestinal absorption. The apparent permeability coefficient of compound ranking with Papp A!B51 106 cm/s, between (1–10) 106 cm/s and410 106 cm/s can be classified as poorly (0–20%), moderately (20–70%), and well (70–100%) absorbed compound, respectively (Yee, 1997). The Papp A!B values of CA were ranging from (4.87 ± 1.72) 107 cm/s to (5.05 ± 0.66) 107 cm/s as the concentration varied from 5 to 15 mg/mL indicating that CA was not easy to transport through intestinal wall epithelial cells, and it should be classified as poorly absorbed compound. The Papp B!A values of CA were significantly higher than the Papp A!B values, indicating that CA was easy to exhaust out from the intestinal wall epithelial. All results indicating the poor bioavailability of CA were mostly due to the low intestinal absorption and low permeability across Caco-2 cells.
Conclusions This study validated that the absolute bioavailability (Fabs) of CA was 14.7%, and its intestinal absorption was 12.4%. In the Caco-2 cells model, the Papp A!B values of CA were 51 106 cm/s as the concentration varied from 5 to 15 mg/ mL. The data suggested that CA was not easy to transport through intestinal wall epithelial cells. CA was shown to have poor permeability across the Caco-2 cell monolayer, and was easy to exhaust out from the intestinal wall epithelial. Therefore, the poor bioavailability of CA was attributable to the low intestinal absorption and low permeability across Caco-2 cells.
Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by National Natural Science Foundation of China (81073141), Guangdong Provincial Natural Science Foundation (9152402301000007) and Guangdong Province ‘‘12-5’’ medical key subject.
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DOI: 10.3109/13880209.2013.879906
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