- Email: [email protected]
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation
J. DRUG DEL. SCI. TECH., 19 (1) 37-41 2009
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto1, H. Azuma2, I. Yamamoto1, M. Otagiri2, T. Imai2* 1
School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-machi, Nobeoka, Miyazaki, 882-8508, Japan 2 Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan *Correspondence: [email protected]
The membrane transport properties of 5-FU and its prodrugs were investigated using Caco-2 cell monolayers. To elucidate a membrane transport of prodrug itself, 2-O-alkoxy-5-fluoropyrimidone (2-O-alkoxy-5FP) derivative, which was stable in buffer and Caco-2 cell, was selected as a test compound. The permeation of 2-O-alkoxy-5FP derivatives was markedly greater than that of 5-FU. The dose-dependency of apical to basal (AP-BL) and BL-AP transports showed that 5-FU was dominantly transported by passive diffusion and ethoxy-5FP was transported by passive diffusion and unsaturated efflux transporter in the apical membrane of Caco-2 cell monolayers. Furthermore, treatment of Cap-Na, an enhancer of paracellular transport, demonstrated that 5-FU and 2-O-alkoxy-5FP derivatives permeated through paracellular and transcellular route, respectively. The significantly increased permeability of 2-O-alkoxy-5FP derivatives was mainly related to capping of hydrophilic functional group, and the moderate increase in permeability among 2-O-alkoxy-5FP derivatives was dependent on prolongation of the alkyl chain length. Key words: 5-fluorouracil – Prodrug – Caco-2 cell – Permeability – Paracellular transport – Transcellular transport – Polar surface area.
5-Fluorouracil (5-FU), an anticancer agent, is used in the chemotherapy of solid tumors. Due to the structural similarity of 5-FU to the pyrimidine base uracil, it is incorporated into the metabolic cycles through the same routes as uracil, and exhibits antagonistic action against anabolic metabolism (e.g. inhibition of DNA synthesis and RNA function), and kills tumor cells [1, 2]. However, 5-FU exhibits low lipid solubility and low membrane permeability because of it being a highly polar molecule. Moreover, the efficacy of 5-FU is limited due to its rapid degradation into dihydro-5-fluorouracil catalyzed by dihydropyrimidine dehydrogenase. The first-pass metabolism of 5-FU in the intestine and liver after oral administration resulted in an incomplete absorption and a highly interindividual variability of its bioavailability (0 to 74%; mean 28%). Furthermore, even in the rectal route where fast-pass metabolism is limited, bioavailability of 5-FU is less than the oral administration because of markedly low absorption [3]. Prodrug approach is useful for improving bioavailability of 5-FU through an increase in membrane permeability and protection against first-pass metabolism. Some successful prodrugs (tegafur, carmofur, doxifluridine, capecitabine, etc.) have been developed to improve the safety and efficacy of 5-FU. For example, tegafur is a sustained-release drug that maintains the effective 5-FU concentration over a long period [4]. Doxifluridine increases selectivity toward tumors through its conversion to 5-FU by higher pyrimidine nucleoside phosphorylase in the tumor tissue than in normal tissue [5]. There are some reports about a correlation between structures of prodrugs and their efficacy [6, 7]. However, it is difficult to rationally design an optimum prodrug, because both characteristics of membrane permeation of prodrug and its conversion to parent drug should be considered. Here, we designed the study that clarifies the relationship between structure of 5-FU prodrugs and their membrane permeability in order to develop a highly absorbed prodrug. Therefore, a stable 5-FU prodrug, 2-O-alkoxy-5-fluropyrimidone (2-O-alkoxy-5-FP) derivative, was selected as a model prodrug, and Caco-2 cell monolayer was used for simple evaluation of their membrane permeability. Caco-2 cell derived from human colon adenocarcinoma has been
widely used as a model of human intestinal epithelium for studies of intestinal drug absorption [10] due to morphological and functional similarities to human intestinal enterocytes [8, 9]. The fraction of a dose absorbed in vivo correlates significantly with the permeability of passively transported compounds across Caco-2 cell monolayer [11]. Caco-2 cell expresses several drug-metabolizing enzymes and transporters that are present in the human enterocyte, including cytochrome P450 [12, 13], carboxylesterases [14] and efflux transporters such as P-glycoprotein [15]. In the present study, the physicochemical properties and stability of 2-O-alkoxy-5FP derivatives of 5-FU were studied and their transport across Caco-2 cell monolayer was characterized. Finally, an increase in membrane permeability of 5-FU was achieved drastically by capping the hydrophilic functional group and moderately by prolonging the alkyl chain length.
I. Materials and Methods 1. Materials
Caco-2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium, 0.25% trypsin-EDTA, Dulbecco’s phosphate-buffered saline (PBS), Hanks’ balanced salt solution (HBSS) and Ca2+ and Mg2+ free HBSS (CMF-HBSS) were purchased from Sigma (St. Louis, MO, USA). Nonessential amino acids, L-glutamine, and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum was purchased from Cansera International Inc. (Rexdale, ON, Canada). 5-FU was purchased from Wako Pure Chemical Industries (Osaka, Japan). [14C] mannitol was purchased from Moravek Biochemicals Inc. (Brea, CA, USA). All other chemicals and reagents were of analytical grade.
2. Cell culture
Caco-2 cells were grown in 75-cm2 culture flasks in culture medium consisting of Dulbecco’s modified Eagle’s medium with 10% heatinactivated fetal bovine serum, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin
37
J. DRUG DEL. SCI. TECH., 19 (1) 37-41 2009
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto, H. Azuma, I. Yamamoto, M. Otagiri, T. Imai
in an atmosphere of 95% air and 5% CO2 at 37°C. Before reaching confluence, the cells were trypsinized with 0.25% trypsin and 0.53 mM EDTA and plated at a density of 8.0 × 104 cells/cm2 in culture medium on a polycarbonate membrane (Transwell, 3 µm pore size, 24.5 mm in diameter; Costar, Cambridge, MA, USA). Culture medium was replaced [1.5 mL on the apical (AP) side and 2.6 mL on the basolateral (BL) side] every other day for the first week and daily thereafter. After seeding, the cells were cultured for 21 to 28 days to allow them to fully differentiate into confluent enterocyte-like monolayers. Transepithelial electrical resistance was measured using a Millicell ERS ohmmeter (Millipore Corporation, Billerica, MA, USA). All cell monolayers in these studies showed transepithelial electrical resistance values ranging from 900 to 1,300 ohm·cm2. Caco-2 cells used in this study were between passages 41 and 48.
HBSS was added to the receiver compartment. The cell monolayers were incubated at 37°C. Samples (120 µL on the receiver compartment and 20 µL on the donor compartment) were withdrawn at various times up to 90 min. The volume removed from the receiver side was always replaced with fresh HBSS. Samples were analyzed by HPLC. A permeability of paracellular route across Caco-2 cell monolayers was evaluated by [14C] mannitol. Apparent permeability coefficient (Papp) was calculated using the following equation: Papp = dQ/dt/A/C0 (square centimeters per second), where dQ/dt is the rate of appearance of drugs in the receiver compartment (steady-state flux, micromoles per second), A is the surface area of the monolayer (i.e., 4.71 cm2), and C0 is the initial concentration (micromolar) in the donor compartment. In an inhibition experiment of active transport, cultured cells were preincubated with metabolic inhibitor (25 µM rotenone) and/or Na+, K+-ATPase inhibitor (100 µM ouabain) for 30 min at 37°C. In the sodium ion-replacement studies, HBSS buffer free from Na+ was used instead of HBSS, and N-methyl-glucamin was used instead of NaCl. The opening of paracellular route was performed by preincubation of cultured cells in Ca2+, Mg2+ free-HBSS buffer with 0.2% sodium caprate for 20 min at 37°C, according to previous reports [17].
3. Preparation of Caco-2 cell 9,000g supernatant (S9)
Caco-2 cells cultured for 7 days in 75-cm2 culture flasks, and cells cultured for 21 days on Transwell were washed with ice-cold PBS and then removed by a cell scraper. The cells were suspended in SET buffer (0.29 M sucrose, 1 mM EDTA, and 50 mM Tris) and then sonicated with a Sonifier cell disruptor (Branson, Danbury, CT, USA). The sonicated cells were then homogenized in a Potter-Elvehjem glass homogenizer with a Teflon pestle under ice-cold conditions. After centrifugation of the cell homogenate at 9,000g for 20 min at 4°C, the supernatant (S9) was obtained. Protein content was determined by the method described by Bradford [16] with bovine serum albumin used as the standard.
7. HPLC analysis
The HPLC system comprised a Jasco 880-PU pump, a Jasco 875UV detector, a Waters autosampler and a Shimadzu Chromatopack C-R7A. An aliquot of the sample was injected onto a Mightysil RP18 column (5 µm, 4.6 i. d. × 250 mm; Cica Merk Co., Tokyo, Japan) and eluted at a flow rate of 1.0 mL/min with sodium acetate buffer/ acetonitrile [5-FU, 97.5/2.5 (v/v); ethoxy-5FP, 90/10 (v/v); propoxy5FP, 80/20 (v/v); butoxy-5FP, 70/30 (v/v); penthoxy-5FP, 60/40 (v/v)]. UV detection was performed at 266 nm, and the detection limit for both 5-FU and its prodrugs was 1 µM.
4. Synthesis of 2-O-alkoxy-5-fluropyrimidone (2-O-alkoxy-5FP) [7]
N, N- Dimethylaniline and phosphorus oxychloride were added to 5-FU and refluxed at 110-130°C for 2 h. After evaporation of remained phosphorus oxychloride, the residue was slowly poured into a mixture of ice and ether, and vigorously stirred. The ether layer was dried, and then 2, 4-dichloro-5-fluoropyrimidine was obtained in 26% yield. 2, 4-dichloro-5-fluoropyrimidine was added into 1.9 N NaOH aq., and reacted at 45°C for 60 min. After cooling the reaction mixture, conc. HCl was added to give precipitate. It was recrystallized from absolute ethanol to give 2-chloro-5-fluoro-4-pyrimidol in 97% yield. 2-chloro-5fluoro-4-pyrimidol was dissolved in a solution of RONa in a respective alcohol (R: CnH2n+1: n=2-5), prepared by dissolving sodium in alcohol, and the mixture was stirred at 100-160°C for 9 h in a sealed tube. The solvent was concentrated in vacuo after cooling, and the residue was dissolved in water. The solution (pH 4-5, adjusted by 10% HCl) was extracted with chloroform. After evaporation of chloroform, the residue was recrystallized from absolute ethanol to give 2-O-alkoxy-5FP derivatives in 42-71% yield. Structures of products were determined by IR, NMR, mass spectrometry and elemental analysis. Purity of products was confirmed by HPLC, TLC and measuring the melting point.
8. Calculation of polar surface area (PSA)
Polar surface area (PSA) was calculated using MOE 2002 (Chemical Computing Group, Montreal, QB, Canada).
II. Results and Discussion 1. Physicochemical properties of 5-FU and its prodrugs
The tested 5-FU prodrug, 2-O-alkoxy-5FP, is structured by binding alkyl group to 5-FU through ether linkage. The physicochemical properties of 5-FU and its prodrugs are listed in Table I. The logP value of 2-O-alkoxy-5FP increased with elongation of the alkyl chain. Yamashita et al. [7] have reported that 5-FU derived from 2-O-alkoxy-5FP was slowly released in plasma by its metabolism after oral administration in rat. However, 2-O-alkoxy-5FP was not degraded in pH 7.4 phosphate buffer and Caco-2 cell homogenate that included some enzymes such as cytochrome P450 and esterase. Since 2-O-alkoxy-5FP is stable in Caco-2 cell, Caco-2 cell monolayer is a suitable system for evaluating membrane transport of 2-O-alkoxy-5FP under non-degraded condition.
5. Physicochemical properties of 5-FU and its prodrugs
LogP values were measured by equilibrating compounds in noctanol saturated with HBSS at 25°C. The fraction of the compounds in aqueous layer was measured by UV detection (266 nm). Hydrolysis of 5-FU prodrugs in pH 7.4 phosphate buffer at 37°C was initiated by the addition of prodrugs (final concentration: 200 µM), and the 5-FU formed was determined by HPLC.
2. 5-FU and ethoxy-5FP transport
Figure 1 shows transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayers. When ethoxy-5FP was applied in donor side, 5-FU was not detected in the receiver side, suggesting that no degradation of ethoxy-5FP in Caco-2 cell occurred during transport. Transport of ethoxy-5FP across Caco-2 cell monolayers was significantly greater than that of 5-FU in both AP to BL and BL to AP direction. Interestingly, BL to AP transport of ethoxy-5FP was greater than that of the opposite directions, suggesting that ethoxy-5FP was transported by efflux transporter on the apical membrane. In contrast, the amount of 5-FU transported in the direction of AP to BL was equal to that in the BL to AP direction. Moreover, the Papp value of 5-FU (3.67 × 10-6 cm/s)
6. Transport experiments
Caco-2 cell monolayers were gently washed with HBSS, followed by preincubation of both the AP and BL sides for 30 min with HBSS. After preincubation, 5-FU or its prodrugs solution (100 µM) was added to the donor compartment of the cell monolayer and fresh 38
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto, H. Azuma, I. Yamamoto, M. Otagiri, T. Imai
J. DRUG DEL. SCI. TECH., 19 (1) 37-41 2009
Table I - Physicochemical properties of 5-fluorouracil (5-FU) and its 2-O-alkoxy-5FP derivatives. Compound
H N
RO
N F O
Log P*
Half-life for hydrolysis (min)** pH 7.4 phosphate buffer
Caco-2 cell homogenate
5-Fluorouracil (5-FU)
-0.77
-
-
-C2H5: 2-O-ethoxy-5-fluoropyrimidone (ethoxy-5FP) -C3H7: 2-O-propoxy-5-fluoropyrimidone (propoxy-5FP) -C4H9: 2-O-butoxy-5-fluoropyrimidone (butoxy-5FP) -C5H11: 2-O-pentoxy-5-fluoropyrimidone (pentoxy-5FP)
-0.58 -0.09 0.40 0.83
NH NH NH NH
NH NH NH NH
*Log P was determined by distribution of drug between n-octanol and pH 7.4 HBSS at 25ºC. **Reaction conditions: substrate concentration, 100 µM; protein concentration, 300 µg/mL; temperature, 37ºC. NH: not hydrolyzed.
5-FU
5-FU
100 AP-to-BL BL-to-AP
AP-to-BL BL-to-AP
80 60
4 40 20 0
0 0
15
30 Time ( min)
45
60
7
AP-to-BL BL-to-AP
0.6
6
2
Ethoxy-5FP
0.7
Permeation rate (nmol/min)
8 Amount transported (nmol)
Ethoxy-5FP
0.5
5
0.4
4
0.3
3
0.2
2
0.1
1
0 0
15
30
45
0 0
60
100 200 300 400 500
0
Applied concentration ( µM)
Time ( min)
Figure 1 - Transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayers (mean ± SD, n = 3). The applied concentration of each compound was 100 µM.
AP-to-BL BL-to-AP
6
200
400
600
800
Applied concentration ( µM)
Figure 2 - Dose dependency of permeation rate on 5-FU and ethoxy-5FP transport across Caco-2 cell monolayers (mean ± SD, n = 3).
was as low as that of the paracellular transport marker mannitol (approximately 1.03 × 10-6 cm/s). Thus, 5-FU is passively transported through paracellular and transcellular route in Caco-2 cell monolayers. It has been reported that 5-FU is transported by Na+-dependent carrier presented in rat intestinal apical membrane in in situ intestinal perfusion experiments [18] and its transport is inhibited by several pyrimidines and metabolic inhibitors in rat intestine [19]. However, the Na+-dependent transport of 5-FU was not observed in blush border membrane vesicles (BBMVs) of rat intestine [18]. Caco-2 cell and rat intestinal BBMVs showed the passive diffusion of 5-FU. In fact, the treatment with rotenone (metabolic inhibitor) and displacement of Na+ did not affect the transport of 5-FU (data not shown). Furthermore, a dose dependency of the permeation rate of 5-FU and ethoxy-5FP was studied. As shown in Figure 2, the permeation rate was linearly increased with applied concentration of both 5-FU and ethoxy-5FP. A passive transport of 5-FU was further confirmed by the present data. Interestingly, BL to AP transport of ethoxy-5FP was greater than its AP to BL transport in the 50-600 µM range, indicating that ethoxy-5FP was transported by passive diffusion and unsaturated efflux transporter in apical membrane of Caco-2 monolayers. Similar results were obtained in other 2-O-alkoxy-5FP derivatives.
5-FU Amount transported (nmol)
80
Ethoxy-5FP 80
Without Cap -Na With 0. 2 % Cap -Na
60
60
40
40
20
20
Without Cap -Na With 0.2 % Cap -Na
0
0 0
15
30
45
Time ( min)
60
0
15
30
45
60
Time ( min)
Figure 3 - Effects of sodium caprate (Cap-Na) on the apical-to-basolateral transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayers (mean ± SD, n = 3). 5-FU and ethoxy-5FP (100 µM) were applied to the apical side following the treatment of 0.2 % Cap-Na for 20 min.
to BL transports of 5-FU and ethoxy-5-FP (100 µM) across Caco-2 cell monolayer after treatment with 0.2% Cap-Na. Transport of 5-FU significantly increased by treatment with Cap-Na, while any differences in the AP to BL transport of ethoxy-5-FP were not observed in the absence and presence of 0.2% Cap-Na. These data suggest that 5-FU and ethoxy-5FP dominantly permeate paracellular and transcellular route at 100 µM, respectively. Thus, prodrug formation of 5-FU improves its permeation via a shift from paracellular to transcellular transport.
3. Effects of sodium caprate (Cap-Na) on transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayer
In order to determine the major transporting route of 5-FU and ethoxy-5FP, Caco-2 cell monolayers were treated with Cap-Na, an absorption enhancer. Cap-Na has been reported to facilitate transport via the paracellular route through the opening of the tight junction, which is caused by an increase in intracellular calcium concentration followed by alteration of actin filament [19, 20]. Figure 3 shows AP 39
J. DRUG DEL. SCI. TECH., 19 (1) 37-41 2009
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto, H. Azuma, I. Yamamoto, M. Otagiri, T. Imai
3. 4. Comparison of transport of 2-O-alkoxy-5FP with 5-FU
Amount transported (% of dose)
50
Transport of several 2-O-alkoxy-5FP derivatives was compared with 5-FU using Caco-2 cell monolayers. As shown in Figure 4, the transported amount of 2-O-alkoxy-5FP derivatives was about 10-fold greater than 5-FU, but their transported amount was nearly the same among prodrugs with alkoxy group of ethoxy to pentoxy. Table II lists Papp and logP of 5-FU and its prodrugs. The logP of 5-FU and ethoxy5FP were -0.77 and -0.58, respectively. The difference between the logP of 5-FU and ethoxy-5FP was only 0.2, but Papp of ethoxy-5FP was 10 times greater than that of 5-FU. The marked increase in Papp is due to shift from paracellular transport for 5-FU to transcellular transport for ethoxy-5FP. The polar surface area (PSA) was calculated as a factor to explain the change of transport route. As shown in Table II, PSA of 5-FU was 59.6, but all prodrugs showed the same value of 49.2. The structural difference between 5-FU and 2-O-alkoxy-5FP derivatives is the presence or absence of 3-N-hydrogen that can behave as proton donor. Therefore, 5-FU is able to form an intermolecular hydrogen bond with water molecules. 5-FU can therefore penetrate through the paracellular route because it is highly hydrophilic and a relatively small molecule. On the other hand, a lack of 3-N-hydrogen on 2-O-alkoxy-5FP led to low PSA value that was same for all 2-O-alkoxy-5FP derivatives. 2-O-substituted derivative is unable to form a hydrogen bond with water at 3-N-position, and shows increasing logP with prolongation of the alkyl group. Generally, permeability of a compound transported through transcellular route depends on its hydrophobicity. Among 2-O-alkoxy derivatives, their Papp was increased with logP that was proportional to the alkyl chain length. Similarly, Burr found that logP (octanol/buffer) could be correlated with the permeability of thirteen 5-FU prodrugs in Caco-2 cell monolayers [6]. However, the increase in permeability is succeeded by ethoxy-5FP, and other prodrugs with longer alkyl chains show nearly the same permeability. These data suggest that PSA is an important factor for the transport of 5-FU derivatives rather than hydrophobicity. In this study, the transport of 5-FU prodrug was characterized using Caco-2 cell monolayers. The results show the shift from paracellular transport to transcellular transport by prodrug formation, due to a lack of 3-N-hydrogen that behaves as a proton donor. In the case of hydrophilic and small parent drug such as 5-FU, prodrug formation might change transport route in absorption process, and preparation of prodrug with various alkyl chain lengths is not profitable to obtain a candidate with improved bioavailability. In such a case, a prodrug design will be considered on the basis of molecular structure such as hydrogen bond rather than hydrophobicity.
2. 3.
4. 5.
6.
5-FU Ethoxy-5FP
30
Propoxy -5FP Butoxy-5FP
20
Pentoxy -5FP
10 0 0
15
30
45
Time (min)
Figure 4 - The apical-to-basolateral transport of 5-FU and 2-O-alkoxy5FP across Caco-2 cell monolayers (mean ± SD, n = 3). 5-FU and its prodrugs (100 µM) were applied to the apical side. Table II - Papp for the apical-to-basolateral transport and physicochemical parameters of 5-FU and O-alkoxy-5FP (mean ± SD, n = 3). Compounds
Papp (x 10-6 cm/s)
Log P*
PS A**
5-FU Ethoxy-5FP Propoxy-5FP Butoxy-5FP Pentoxy-5FP
3.67 ± 0.19 38.8 ± 0.65 43.2 ± 3.64 45.6 ± 6.84 54.3 ± 1.12
-0.77 -0.58 -0.09 0.40 0.83
59.6 49.2 49.2 49.2 49.2
*Log P was determined by distribution of drug between n-octanol and pH 7.4 HBSS at 25ºC. **PSA was defined as the area occupied by nitrogen and oxygen atoms, and hydrogen atoms attached to these heteroatoms.
7.
8.
9.
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Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto, H. Azuma, I. Yamamoto, M. Otagiri, T. Imai
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Manuscript Received 17 June 2008, accepted for publication 2 September 2008.
41
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto1, H. Azuma2, I. Yamamoto1, M. Otagiri2, T. Imai2* 1
School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-machi, Nobeoka, Miyazaki, 882-8508, Japan 2 Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan *Correspondence: [email protected]
The membrane transport properties of 5-FU and its prodrugs were investigated using Caco-2 cell monolayers. To elucidate a membrane transport of prodrug itself, 2-O-alkoxy-5-fluoropyrimidone (2-O-alkoxy-5FP) derivative, which was stable in buffer and Caco-2 cell, was selected as a test compound. The permeation of 2-O-alkoxy-5FP derivatives was markedly greater than that of 5-FU. The dose-dependency of apical to basal (AP-BL) and BL-AP transports showed that 5-FU was dominantly transported by passive diffusion and ethoxy-5FP was transported by passive diffusion and unsaturated efflux transporter in the apical membrane of Caco-2 cell monolayers. Furthermore, treatment of Cap-Na, an enhancer of paracellular transport, demonstrated that 5-FU and 2-O-alkoxy-5FP derivatives permeated through paracellular and transcellular route, respectively. The significantly increased permeability of 2-O-alkoxy-5FP derivatives was mainly related to capping of hydrophilic functional group, and the moderate increase in permeability among 2-O-alkoxy-5FP derivatives was dependent on prolongation of the alkyl chain length. Key words: 5-fluorouracil – Prodrug – Caco-2 cell – Permeability – Paracellular transport – Transcellular transport – Polar surface area.
5-Fluorouracil (5-FU), an anticancer agent, is used in the chemotherapy of solid tumors. Due to the structural similarity of 5-FU to the pyrimidine base uracil, it is incorporated into the metabolic cycles through the same routes as uracil, and exhibits antagonistic action against anabolic metabolism (e.g. inhibition of DNA synthesis and RNA function), and kills tumor cells [1, 2]. However, 5-FU exhibits low lipid solubility and low membrane permeability because of it being a highly polar molecule. Moreover, the efficacy of 5-FU is limited due to its rapid degradation into dihydro-5-fluorouracil catalyzed by dihydropyrimidine dehydrogenase. The first-pass metabolism of 5-FU in the intestine and liver after oral administration resulted in an incomplete absorption and a highly interindividual variability of its bioavailability (0 to 74%; mean 28%). Furthermore, even in the rectal route where fast-pass metabolism is limited, bioavailability of 5-FU is less than the oral administration because of markedly low absorption [3]. Prodrug approach is useful for improving bioavailability of 5-FU through an increase in membrane permeability and protection against first-pass metabolism. Some successful prodrugs (tegafur, carmofur, doxifluridine, capecitabine, etc.) have been developed to improve the safety and efficacy of 5-FU. For example, tegafur is a sustained-release drug that maintains the effective 5-FU concentration over a long period [4]. Doxifluridine increases selectivity toward tumors through its conversion to 5-FU by higher pyrimidine nucleoside phosphorylase in the tumor tissue than in normal tissue [5]. There are some reports about a correlation between structures of prodrugs and their efficacy [6, 7]. However, it is difficult to rationally design an optimum prodrug, because both characteristics of membrane permeation of prodrug and its conversion to parent drug should be considered. Here, we designed the study that clarifies the relationship between structure of 5-FU prodrugs and their membrane permeability in order to develop a highly absorbed prodrug. Therefore, a stable 5-FU prodrug, 2-O-alkoxy-5-fluropyrimidone (2-O-alkoxy-5-FP) derivative, was selected as a model prodrug, and Caco-2 cell monolayer was used for simple evaluation of their membrane permeability. Caco-2 cell derived from human colon adenocarcinoma has been
widely used as a model of human intestinal epithelium for studies of intestinal drug absorption [10] due to morphological and functional similarities to human intestinal enterocytes [8, 9]. The fraction of a dose absorbed in vivo correlates significantly with the permeability of passively transported compounds across Caco-2 cell monolayer [11]. Caco-2 cell expresses several drug-metabolizing enzymes and transporters that are present in the human enterocyte, including cytochrome P450 [12, 13], carboxylesterases [14] and efflux transporters such as P-glycoprotein [15]. In the present study, the physicochemical properties and stability of 2-O-alkoxy-5FP derivatives of 5-FU were studied and their transport across Caco-2 cell monolayer was characterized. Finally, an increase in membrane permeability of 5-FU was achieved drastically by capping the hydrophilic functional group and moderately by prolonging the alkyl chain length.
I. Materials and Methods 1. Materials
Caco-2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium, 0.25% trypsin-EDTA, Dulbecco’s phosphate-buffered saline (PBS), Hanks’ balanced salt solution (HBSS) and Ca2+ and Mg2+ free HBSS (CMF-HBSS) were purchased from Sigma (St. Louis, MO, USA). Nonessential amino acids, L-glutamine, and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum was purchased from Cansera International Inc. (Rexdale, ON, Canada). 5-FU was purchased from Wako Pure Chemical Industries (Osaka, Japan). [14C] mannitol was purchased from Moravek Biochemicals Inc. (Brea, CA, USA). All other chemicals and reagents were of analytical grade.
2. Cell culture
Caco-2 cells were grown in 75-cm2 culture flasks in culture medium consisting of Dulbecco’s modified Eagle’s medium with 10% heatinactivated fetal bovine serum, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin
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J. DRUG DEL. SCI. TECH., 19 (1) 37-41 2009
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto, H. Azuma, I. Yamamoto, M. Otagiri, T. Imai
in an atmosphere of 95% air and 5% CO2 at 37°C. Before reaching confluence, the cells were trypsinized with 0.25% trypsin and 0.53 mM EDTA and plated at a density of 8.0 × 104 cells/cm2 in culture medium on a polycarbonate membrane (Transwell, 3 µm pore size, 24.5 mm in diameter; Costar, Cambridge, MA, USA). Culture medium was replaced [1.5 mL on the apical (AP) side and 2.6 mL on the basolateral (BL) side] every other day for the first week and daily thereafter. After seeding, the cells were cultured for 21 to 28 days to allow them to fully differentiate into confluent enterocyte-like monolayers. Transepithelial electrical resistance was measured using a Millicell ERS ohmmeter (Millipore Corporation, Billerica, MA, USA). All cell monolayers in these studies showed transepithelial electrical resistance values ranging from 900 to 1,300 ohm·cm2. Caco-2 cells used in this study were between passages 41 and 48.
HBSS was added to the receiver compartment. The cell monolayers were incubated at 37°C. Samples (120 µL on the receiver compartment and 20 µL on the donor compartment) were withdrawn at various times up to 90 min. The volume removed from the receiver side was always replaced with fresh HBSS. Samples were analyzed by HPLC. A permeability of paracellular route across Caco-2 cell monolayers was evaluated by [14C] mannitol. Apparent permeability coefficient (Papp) was calculated using the following equation: Papp = dQ/dt/A/C0 (square centimeters per second), where dQ/dt is the rate of appearance of drugs in the receiver compartment (steady-state flux, micromoles per second), A is the surface area of the monolayer (i.e., 4.71 cm2), and C0 is the initial concentration (micromolar) in the donor compartment. In an inhibition experiment of active transport, cultured cells were preincubated with metabolic inhibitor (25 µM rotenone) and/or Na+, K+-ATPase inhibitor (100 µM ouabain) for 30 min at 37°C. In the sodium ion-replacement studies, HBSS buffer free from Na+ was used instead of HBSS, and N-methyl-glucamin was used instead of NaCl. The opening of paracellular route was performed by preincubation of cultured cells in Ca2+, Mg2+ free-HBSS buffer with 0.2% sodium caprate for 20 min at 37°C, according to previous reports [17].
3. Preparation of Caco-2 cell 9,000g supernatant (S9)
Caco-2 cells cultured for 7 days in 75-cm2 culture flasks, and cells cultured for 21 days on Transwell were washed with ice-cold PBS and then removed by a cell scraper. The cells were suspended in SET buffer (0.29 M sucrose, 1 mM EDTA, and 50 mM Tris) and then sonicated with a Sonifier cell disruptor (Branson, Danbury, CT, USA). The sonicated cells were then homogenized in a Potter-Elvehjem glass homogenizer with a Teflon pestle under ice-cold conditions. After centrifugation of the cell homogenate at 9,000g for 20 min at 4°C, the supernatant (S9) was obtained. Protein content was determined by the method described by Bradford [16] with bovine serum albumin used as the standard.
7. HPLC analysis
The HPLC system comprised a Jasco 880-PU pump, a Jasco 875UV detector, a Waters autosampler and a Shimadzu Chromatopack C-R7A. An aliquot of the sample was injected onto a Mightysil RP18 column (5 µm, 4.6 i. d. × 250 mm; Cica Merk Co., Tokyo, Japan) and eluted at a flow rate of 1.0 mL/min with sodium acetate buffer/ acetonitrile [5-FU, 97.5/2.5 (v/v); ethoxy-5FP, 90/10 (v/v); propoxy5FP, 80/20 (v/v); butoxy-5FP, 70/30 (v/v); penthoxy-5FP, 60/40 (v/v)]. UV detection was performed at 266 nm, and the detection limit for both 5-FU and its prodrugs was 1 µM.
4. Synthesis of 2-O-alkoxy-5-fluropyrimidone (2-O-alkoxy-5FP) [7]
N, N- Dimethylaniline and phosphorus oxychloride were added to 5-FU and refluxed at 110-130°C for 2 h. After evaporation of remained phosphorus oxychloride, the residue was slowly poured into a mixture of ice and ether, and vigorously stirred. The ether layer was dried, and then 2, 4-dichloro-5-fluoropyrimidine was obtained in 26% yield. 2, 4-dichloro-5-fluoropyrimidine was added into 1.9 N NaOH aq., and reacted at 45°C for 60 min. After cooling the reaction mixture, conc. HCl was added to give precipitate. It was recrystallized from absolute ethanol to give 2-chloro-5-fluoro-4-pyrimidol in 97% yield. 2-chloro-5fluoro-4-pyrimidol was dissolved in a solution of RONa in a respective alcohol (R: CnH2n+1: n=2-5), prepared by dissolving sodium in alcohol, and the mixture was stirred at 100-160°C for 9 h in a sealed tube. The solvent was concentrated in vacuo after cooling, and the residue was dissolved in water. The solution (pH 4-5, adjusted by 10% HCl) was extracted with chloroform. After evaporation of chloroform, the residue was recrystallized from absolute ethanol to give 2-O-alkoxy-5FP derivatives in 42-71% yield. Structures of products were determined by IR, NMR, mass spectrometry and elemental analysis. Purity of products was confirmed by HPLC, TLC and measuring the melting point.
8. Calculation of polar surface area (PSA)
Polar surface area (PSA) was calculated using MOE 2002 (Chemical Computing Group, Montreal, QB, Canada).
II. Results and Discussion 1. Physicochemical properties of 5-FU and its prodrugs
The tested 5-FU prodrug, 2-O-alkoxy-5FP, is structured by binding alkyl group to 5-FU through ether linkage. The physicochemical properties of 5-FU and its prodrugs are listed in Table I. The logP value of 2-O-alkoxy-5FP increased with elongation of the alkyl chain. Yamashita et al. [7] have reported that 5-FU derived from 2-O-alkoxy-5FP was slowly released in plasma by its metabolism after oral administration in rat. However, 2-O-alkoxy-5FP was not degraded in pH 7.4 phosphate buffer and Caco-2 cell homogenate that included some enzymes such as cytochrome P450 and esterase. Since 2-O-alkoxy-5FP is stable in Caco-2 cell, Caco-2 cell monolayer is a suitable system for evaluating membrane transport of 2-O-alkoxy-5FP under non-degraded condition.
5. Physicochemical properties of 5-FU and its prodrugs
LogP values were measured by equilibrating compounds in noctanol saturated with HBSS at 25°C. The fraction of the compounds in aqueous layer was measured by UV detection (266 nm). Hydrolysis of 5-FU prodrugs in pH 7.4 phosphate buffer at 37°C was initiated by the addition of prodrugs (final concentration: 200 µM), and the 5-FU formed was determined by HPLC.
2. 5-FU and ethoxy-5FP transport
Figure 1 shows transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayers. When ethoxy-5FP was applied in donor side, 5-FU was not detected in the receiver side, suggesting that no degradation of ethoxy-5FP in Caco-2 cell occurred during transport. Transport of ethoxy-5FP across Caco-2 cell monolayers was significantly greater than that of 5-FU in both AP to BL and BL to AP direction. Interestingly, BL to AP transport of ethoxy-5FP was greater than that of the opposite directions, suggesting that ethoxy-5FP was transported by efflux transporter on the apical membrane. In contrast, the amount of 5-FU transported in the direction of AP to BL was equal to that in the BL to AP direction. Moreover, the Papp value of 5-FU (3.67 × 10-6 cm/s)
6. Transport experiments
Caco-2 cell monolayers were gently washed with HBSS, followed by preincubation of both the AP and BL sides for 30 min with HBSS. After preincubation, 5-FU or its prodrugs solution (100 µM) was added to the donor compartment of the cell monolayer and fresh 38
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto, H. Azuma, I. Yamamoto, M. Otagiri, T. Imai
J. DRUG DEL. SCI. TECH., 19 (1) 37-41 2009
Table I - Physicochemical properties of 5-fluorouracil (5-FU) and its 2-O-alkoxy-5FP derivatives. Compound
H N
RO
N F O
Log P*
Half-life for hydrolysis (min)** pH 7.4 phosphate buffer
Caco-2 cell homogenate
5-Fluorouracil (5-FU)
-0.77
-
-
-C2H5: 2-O-ethoxy-5-fluoropyrimidone (ethoxy-5FP) -C3H7: 2-O-propoxy-5-fluoropyrimidone (propoxy-5FP) -C4H9: 2-O-butoxy-5-fluoropyrimidone (butoxy-5FP) -C5H11: 2-O-pentoxy-5-fluoropyrimidone (pentoxy-5FP)
-0.58 -0.09 0.40 0.83
NH NH NH NH
NH NH NH NH
*Log P was determined by distribution of drug between n-octanol and pH 7.4 HBSS at 25ºC. **Reaction conditions: substrate concentration, 100 µM; protein concentration, 300 µg/mL; temperature, 37ºC. NH: not hydrolyzed.
5-FU
5-FU
100 AP-to-BL BL-to-AP
AP-to-BL BL-to-AP
80 60
4 40 20 0
0 0
15
30 Time ( min)
45
60
7
AP-to-BL BL-to-AP
0.6
6
2
Ethoxy-5FP
0.7
Permeation rate (nmol/min)
8 Amount transported (nmol)
Ethoxy-5FP
0.5
5
0.4
4
0.3
3
0.2
2
0.1
1
0 0
15
30
45
0 0
60
100 200 300 400 500
0
Applied concentration ( µM)
Time ( min)
Figure 1 - Transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayers (mean ± SD, n = 3). The applied concentration of each compound was 100 µM.
AP-to-BL BL-to-AP
6
200
400
600
800
Applied concentration ( µM)
Figure 2 - Dose dependency of permeation rate on 5-FU and ethoxy-5FP transport across Caco-2 cell monolayers (mean ± SD, n = 3).
was as low as that of the paracellular transport marker mannitol (approximately 1.03 × 10-6 cm/s). Thus, 5-FU is passively transported through paracellular and transcellular route in Caco-2 cell monolayers. It has been reported that 5-FU is transported by Na+-dependent carrier presented in rat intestinal apical membrane in in situ intestinal perfusion experiments [18] and its transport is inhibited by several pyrimidines and metabolic inhibitors in rat intestine [19]. However, the Na+-dependent transport of 5-FU was not observed in blush border membrane vesicles (BBMVs) of rat intestine [18]. Caco-2 cell and rat intestinal BBMVs showed the passive diffusion of 5-FU. In fact, the treatment with rotenone (metabolic inhibitor) and displacement of Na+ did not affect the transport of 5-FU (data not shown). Furthermore, a dose dependency of the permeation rate of 5-FU and ethoxy-5FP was studied. As shown in Figure 2, the permeation rate was linearly increased with applied concentration of both 5-FU and ethoxy-5FP. A passive transport of 5-FU was further confirmed by the present data. Interestingly, BL to AP transport of ethoxy-5FP was greater than its AP to BL transport in the 50-600 µM range, indicating that ethoxy-5FP was transported by passive diffusion and unsaturated efflux transporter in apical membrane of Caco-2 monolayers. Similar results were obtained in other 2-O-alkoxy-5FP derivatives.
5-FU Amount transported (nmol)
80
Ethoxy-5FP 80
Without Cap -Na With 0. 2 % Cap -Na
60
60
40
40
20
20
Without Cap -Na With 0.2 % Cap -Na
0
0 0
15
30
45
Time ( min)
60
0
15
30
45
60
Time ( min)
Figure 3 - Effects of sodium caprate (Cap-Na) on the apical-to-basolateral transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayers (mean ± SD, n = 3). 5-FU and ethoxy-5FP (100 µM) were applied to the apical side following the treatment of 0.2 % Cap-Na for 20 min.
to BL transports of 5-FU and ethoxy-5-FP (100 µM) across Caco-2 cell monolayer after treatment with 0.2% Cap-Na. Transport of 5-FU significantly increased by treatment with Cap-Na, while any differences in the AP to BL transport of ethoxy-5-FP were not observed in the absence and presence of 0.2% Cap-Na. These data suggest that 5-FU and ethoxy-5FP dominantly permeate paracellular and transcellular route at 100 µM, respectively. Thus, prodrug formation of 5-FU improves its permeation via a shift from paracellular to transcellular transport.
3. Effects of sodium caprate (Cap-Na) on transport of 5-FU and ethoxy-5FP across Caco-2 cell monolayer
In order to determine the major transporting route of 5-FU and ethoxy-5FP, Caco-2 cell monolayers were treated with Cap-Na, an absorption enhancer. Cap-Na has been reported to facilitate transport via the paracellular route through the opening of the tight junction, which is caused by an increase in intracellular calcium concentration followed by alteration of actin filament [19, 20]. Figure 3 shows AP 39
J. DRUG DEL. SCI. TECH., 19 (1) 37-41 2009
Permeability of 5-fluorouracil and its prodrugs in Caco-2 cell monolayers: evidence for shift from paracellular to transcellular transport by prodrug formation M. Imoto, H. Azuma, I. Yamamoto, M. Otagiri, T. Imai
3. 4. Comparison of transport of 2-O-alkoxy-5FP with 5-FU
Amount transported (% of dose)
50
Transport of several 2-O-alkoxy-5FP derivatives was compared with 5-FU using Caco-2 cell monolayers. As shown in Figure 4, the transported amount of 2-O-alkoxy-5FP derivatives was about 10-fold greater than 5-FU, but their transported amount was nearly the same among prodrugs with alkoxy group of ethoxy to pentoxy. Table II lists Papp and logP of 5-FU and its prodrugs. The logP of 5-FU and ethoxy5FP were -0.77 and -0.58, respectively. The difference between the logP of 5-FU and ethoxy-5FP was only 0.2, but Papp of ethoxy-5FP was 10 times greater than that of 5-FU. The marked increase in Papp is due to shift from paracellular transport for 5-FU to transcellular transport for ethoxy-5FP. The polar surface area (PSA) was calculated as a factor to explain the change of transport route. As shown in Table II, PSA of 5-FU was 59.6, but all prodrugs showed the same value of 49.2. The structural difference between 5-FU and 2-O-alkoxy-5FP derivatives is the presence or absence of 3-N-hydrogen that can behave as proton donor. Therefore, 5-FU is able to form an intermolecular hydrogen bond with water molecules. 5-FU can therefore penetrate through the paracellular route because it is highly hydrophilic and a relatively small molecule. On the other hand, a lack of 3-N-hydrogen on 2-O-alkoxy-5FP led to low PSA value that was same for all 2-O-alkoxy-5FP derivatives. 2-O-substituted derivative is unable to form a hydrogen bond with water at 3-N-position, and shows increasing logP with prolongation of the alkyl group. Generally, permeability of a compound transported through transcellular route depends on its hydrophobicity. Among 2-O-alkoxy derivatives, their Papp was increased with logP that was proportional to the alkyl chain length. Similarly, Burr found that logP (octanol/buffer) could be correlated with the permeability of thirteen 5-FU prodrugs in Caco-2 cell monolayers [6]. However, the increase in permeability is succeeded by ethoxy-5FP, and other prodrugs with longer alkyl chains show nearly the same permeability. These data suggest that PSA is an important factor for the transport of 5-FU derivatives rather than hydrophobicity. In this study, the transport of 5-FU prodrug was characterized using Caco-2 cell monolayers. The results show the shift from paracellular transport to transcellular transport by prodrug formation, due to a lack of 3-N-hydrogen that behaves as a proton donor. In the case of hydrophilic and small parent drug such as 5-FU, prodrug formation might change transport route in absorption process, and preparation of prodrug with various alkyl chain lengths is not profitable to obtain a candidate with improved bioavailability. In such a case, a prodrug design will be considered on the basis of molecular structure such as hydrogen bond rather than hydrophobicity.
2. 3.
4. 5.
6.
5-FU Ethoxy-5FP
30
Propoxy -5FP Butoxy-5FP
20
Pentoxy -5FP
10 0 0
15
30
45
Time (min)
Figure 4 - The apical-to-basolateral transport of 5-FU and 2-O-alkoxy5FP across Caco-2 cell monolayers (mean ± SD, n = 3). 5-FU and its prodrugs (100 µM) were applied to the apical side. Table II - Papp for the apical-to-basolateral transport and physicochemical parameters of 5-FU and O-alkoxy-5FP (mean ± SD, n = 3). Compounds
Papp (x 10-6 cm/s)
Log P*
PS A**
5-FU Ethoxy-5FP Propoxy-5FP Butoxy-5FP Pentoxy-5FP
3.67 ± 0.19 38.8 ± 0.65 43.2 ± 3.64 45.6 ± 6.84 54.3 ± 1.12
-0.77 -0.58 -0.09 0.40 0.83
59.6 49.2 49.2 49.2 49.2
*Log P was determined by distribution of drug between n-octanol and pH 7.4 HBSS at 25ºC. **PSA was defined as the area occupied by nitrogen and oxygen atoms, and hydrogen atoms attached to these heteroatoms.
7.
8.
9.
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Manuscript Received 17 June 2008, accepted for publication 2 September 2008.
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