Synthesis and Assessment of First-Generation Polyamidoamine

Mar 14, 2007 - School of Pharmacy and Pharmaceutical Sciences, University of Manchester, ... The A f B Papp of the dendrimer prodrugs was significantl...
1 downloads 0 Views 185KB Size
Download PDF
Bioconjugate Chem. 2007, 18, 937−946

937

Synthesis and Assessment of First-Generation Polyamidoamine Dendrimer Prodrugs to Enhance the Cellular Permeability of P-gp Substrates Mohammad Najlah,† Sally Freeman, David Attwood, and Antony D’Emanuele*,† School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK. Received October 19, 2006; Revised Manuscript Received January 13, 2007

The aim of this study is to evaluate the potential use of first-generation (G1) polyamidoamine (PAMAM) dendrimers as drug carriers to enhance the permeability, hence oral absorption, of drugs that are substrates for P-glycoprotein (P-gp) efflux transporters. G1 PAMAM dendrimer-based prodrugs of the water-insoluble P-gp substrate terfenadine (Ter) were synthesized using succinic acid (suc) or succinyl-diethylene glycol (suc-deg) as a linker/spacer (to yield G1-suc-Ter and G1-suc-deg-Ter, respectively). In addition, the permeability of G1-suc-deg-Ter was enhanced by attaching two lauroyl chains (L) to the dendrimer surface (L2-G1-suc-deg-Ter). All of the G1 dendrimer-terfenadine prodrugs were more hydrophilic than the parent drug, as evaluated by drug partitioning between 1-octanol and phosphate buffer at pH 7.4 (log Kapp). The influence of the dendrimer prodrugs on the integrity and viability of human Caucasian colon adenocarcinoma cells (Caco-2) was determined by measuring the transepithelial electrical resistance (TEER) and leakage of lactate dehydrogenase (LDH) enzyme, respectively. The LDH assay indicated that the dendrimer prodrugs had no impact on the viability of Caco-2 cells up to a concentration of 1 mM. However, the IC50 of the prodrugs was lower than that of G1 PAMAM dendrimer because of the high toxicity of terfenadine. Measurements of the transport of dendrimer prodrugs across monolayers of Caco-2 cells showed an increase of the apparent permeability coefficient (Papp) of terfenadine in both apical-tobasolateral (A f B) and basolateral-to-apical (B f A) directions after its conjugation to G1 PAMAM dendrimer. The A f B Papp of the dendrimer prodrugs was significantly greater than B f A Papp. The surface-modified dendrimer prodrug L2-G1-suc-deg-Ter showed the highest A f B permeability among the conjugates.

INTRODUCTION There has been growing interest in polyamidoamine (PAMAM) dendrimers as hyperbranched polymers possessing a well-defined structure that allow precise control of size, shape, and terminal group functionality (1). Dendrimers show potential as carriers for the development of drug delivery systems (2) that can be used in several pharmaceutical fields (2, 3). Dendrimers have the ability to cross cell barriers (4-6) by both paracellular and transcellular pathways (7, 8). The cytotoxicity and permeability of PAMAM dendrimers were found to be concentration, generation, and charge dependent (7). Lowgeneration PAMAM dendrimers (G0 and G1) exhibit significantly less cytotoxicity and higher permeability than higher generations (G2, G3, and G4) (9). However, a significant reduction in cytotoxicity and enhancement in permeability can be achieved by surface-engineered cationic PAMAM dendrimers (7, 10). Recent observations on intestinal absorption of drugs suggest that an intestinal secretion process mediated by P-glycoprotein (P-gp) limits the bioavailability of drugs that are P-gp substrates, such as terfenadine, after peroral administration (11, 12). Several attempts have been reported to enhance the oral absorption of P-gp substrates by (1) inhibiting the P-gp secretion activity using P-gp inhibitors such verapamil and nifedipine (13), (2) coadministrating with excipients such as Tween 80 and acacia which are known to inhibit the P-gp efflux activity (14), (3) improving the paracellular transport by co-administrating modu* To whom correspondence should be addressed. Phone: +44(0) 1772 895801. Fax: +44(0)7092 030763. E-mail: Antony@ DEmanuele.net, www: http://www.dendrimerweb.com. † Present address: School of Pharmacy and Pharmaceutical Sciences, University of Central Lancashire, Preston PR1 2HE, U.K.

lators of tight junctions such as sodium caprate (C10) (15), and (4) changing the physicochemical properties of the drug by synthesis of a prodrug that is able to bypass the P-gp efflux pump, for example, attaching paclitaxel to PEG 5000 resulted in a prodrug with a higher bioavailability than the parent drug (16). Previous work by our group reported potential drug carrier systems based on third-generation (G3) PAMAM dendrimer that may be used to enhance the transport of propranolol across Caco-2 cells. Propranolol has poor water solubility and is a substrate for the P-glycoprotein (P-gp) efflux transporter. When conjugated to surface-modified G3 PAMAM dendrimer, propranolol was shown to bypass the efflux system. Thus, dendrimer prodrugs may be used to enhance the bioavailability of drugs that are substrates of efflux transporters and therefore overcome cellular barriers such as the gastrointestinal epithelium and the blood brain barrier (6, 17). A natural extension of this work is the inclusion of a linker between the drug and the dendrimer to control drug release, use of a lower generation (less toxic) carrier than G3 PAMAM dendrimer, and use of a stronger and more unambiguous P-gp substrate than propranolol to confirm that PAMAM dendrimers have the ability to bypass P-gp efflux transporters. More recently, we explored the nature of the linker between the drug and the dendrimer. It is important to design a linker that is robust enough for the drug-dendrimer prodrug to be absorbed intact yet labile once absorbed. It has been found that a diethylene glycol linker between the drug and dendrimer gave esters with a high chemical stability in buffers, which readily released the drug in plasma. PAMAM dendrimer conjugates incorporating this linker have potential as carriers for low-bioavailability drugs (18). In the present study, the design, characterization, and in vitro evaluation of a novel drug carrier system based on first-

10.1021/bc060325q CCC: $37.00 © 2007 American Chemical Society Published on Web 03/14/2007

938 Bioconjugate Chem., Vol. 18, No. 3, 2007

Figure 1. G1 PAMAM dendrimer (letters on G1 PAMAM dendrimer are included to aid NMR assignments).

generation PAMAM dendrimers (G1) (Figure 1) and utilizing a diethylene glycol linker are described. Terfenadine was selected as an unambiguous P-gp substrate and water-insoluble drug. It was attached to the dendrimer surface using succinyl linker (single ester linker) to give G1-suc-Ter or succinicdiethylene glycol linker (double ester linker) to give G1-sucdeg-Ter. The biological evaluation of both conjugates and the surface-modified conjugate (L2-G1-suc-deg-Ter) involved cytotoxicity studies (LDH assay) and measurement of transport across Caco-2 monolayers.

EXPERIMENTAL PROCEDURES Materials. First-generation PAMAM dendrimers (G1) with ethylenediamine cores were purchased from Dendritech Inc. (Michigan). Trifluoroacetic acid (TFA), triethylamine (TEA), terfenadine, 4-nitrophenyl chloroformate, succinic anhydride, 1-dodecanol (lauroyl alcohol), trypan blue, 4-dimethylaminopyridine (DMAP), ethylenediamine tetraacetic acid (EDTA), lactate dehydrogenase assay kits, Sephadex 15, and Sephadex LH-20 were purchased from Sigma-Aldrich Co. Ltd. NHydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), and diethylene glycol were purchased from Fluka (Poole, Dorset, U.K.). Cell culture materials were from Gibco BRL Life Technologies (Paisley, Scotland). Polycarbonate cell culture inserts (Transwell 12 mm diameter) and cluster plates (96 well) were purchased from Corning Costar UK (High Wycombe, Bucks, U.K.). Dendrimer-terfenadine conjugates were characterized by 13C and 1H NMR spectroscopy (Bruker Avance 300, Bruker, Coventry, U.K.). 13C NMR spectra were assigned with the aid of DEPT-135. HPLC analyses were carried out using a HewlettPackard Series II 1090 (Germany) instrument equipped with a Luna 5 µm, C18 column (250 × 4.6 mm, Phenomenex, Cheshire, U.K.). For the transport and partition studies the solvent systems were methanol:acetonitrile (ACN):H3PO4 (0.05% w/v) (48:32:20) with phenanthrene as internal standard for G1 conjugates, and ACN:Walpole’s acetate buffer (pH ) 4.5): methanol (40:48:12) with naproxen as internal standard for terfenadine, Ter-suc, and Ter-suc-deg. The flow rate was 1.2 mL/min, and UV detection was at λ ) 230 nm. For characterization of dendrimer-terfenadine conjugates, the solvent system was 10:32:58 methanol:acetonitrile (ACN):H3PO4 (0.05% w/v) for 2 min then 48:32:20 methanol:acetonitrile(ACN):H3PO4 for the remaining elution time. The flow rate was 1.2 mL/min, and UV detection was at λ ) 230 nm. Synthesis of Terfenadine-Succinyl (Ter-suc). Terfenadine (471.7 mg, 1 mmol) was added to a solution of succinic

Najlah et al.

anhydride (200 mg, 2 mmol) and 4-dimethylaminopyridine (DMAP) (12.2 mg, 0.1 mmol) (previously dried under vacuum for 2 h) in DMF (4 mL). Dry pyridine (0.8 mL) was added, and the solution was stirred overnight at room temperature. DMF was evaporated under vacuum, and the residue was washed with petroleum ether and purified by silica gel column chromatography, eluting with chloroform:methanol (90:10) to give Tersuc with a yield of 91% (Rf ) 0.3) (mp ) 198-199 °C). 1H NMR (CDCl3): 1.29 (9H, s, -But) 1.51 (2H, br d, NCH2CH2CH2CH, J ) 13.4), 1.95-1.69 (4H, m, 2 × CHCH2CH2N), 2.21-2.01 (2H, m, NCH2CH2CH2CH), 2.90-2.38 (9H, m, 2 × CHCH2CH2N, CHCH2CH2N, CO-CH2-CH2-CO), 3.39 (H, br d, J1 ) 13.8, NCH2CH2CH2), 3.44 (H, br d, J2 ) 13.8, NCH2CH2CH2), 5.81 (1H, br d, J ) 7.9, CH-O), 7.55-7.10 (14H, m, Ar). 13C NMR (CDCl3): 20.0 (NCH2CH2CH2), 23.4 (2 × CHCH2CH2N), 31.0 (CH2COO), 31.1 (3 × CH3), 31.3 (CH2COOH), 33.7 (NCH2CH2CH2CH), 42.6 (-C(CH3)3), 52.7 (CHCH2CH2N), 53.0 (CHCH2CH2N), 56.1 (CHCH2CH2N), 60.4 (NCH2CH2CH2), 73.8 (CH-O), 78.7 (C-OH), 125.4 (2 × CH, Ar), 125.6 (4 × CH, Ar), 125.9 (2 × CH, Ar), 126.5 (2 × CH, Ar), 128.2 (4 × CH, Ar), 137.2 (C, Ar), 145.9 (2 × C, Ar), 150.8 (C, Ar), 173.1 (COO), 177.2 (COOH). (+)-ESI-MS: 572 [M+ + H]. Accurate MS (C36H46O5N1): 572.3372 (theoretical: 572.3370). Synthesis of Terfenadine-Succinyl-Diethylene Glycol (Ter-suc-deg). CDI (65 mg, 0.4 mmol) was added portion wise to a solution of Ter-suc (200 mg, 0.35 mmol) in DCM (10 mL). The mixture was stirred for 4 h at room temperature. An excess of diethylene glycol (deg) (149 mg, 1.43 mmol) was added and stirred overnight. The reaction mixture was washed with water (2 × 10 mL), aqueous 0.1 M HCl (2 × 10 mL), aqueous 0.1 M NaOH (2 × 10 mL), and water (2 × 10 mL), dried (anhydrous Na2SO4), filtered, and evaporated to dryness in vacuo. The product was purified by size exclusion chromatography using Sephadex LH 20 and eluting with methanol: water (5:1) to give Ter-suc-deg as an oil with a yield of 69%. TLC: Rf ) 0.16 (chloroform:methanol, 90:10). 1H NMR (CDCl3): 1.29 (9H, s, -But), 1.60-1.38 (6H, m, NCH2CH2CH2 and 2 × CHCH2CH2N), 1.99-1.83 (2H, m, NCH2CH2CH2CH), 2.75-2.35 (9H, m, 2 × CHCH2CH2N, CHCH2CH2N, COCH2-CH2-CO), 2.98-2.87 (2H, m, NCH2CH2CH2), 3.693.52 (6H, m, COOCH2CH2OCH2CH2OH), 4.28-4.15 (2H, m, COOCH2CH2OCH2CH2OH), 5.76-5.69 (1H, dd, J ) 6.5 and 13.7, CH-O), 7.55-7.12 (14H, m, Ar). 13C NMR (CDCl3): 22.9 (NCH2CH2CH2), 26.1 (2 × CHCH2CH2N), 29.0 (CH2COO), 29.1 (CH2COO), 31.3 (3 × CH3), 34.2 (NCH2CH2CH2CH), 44.1 (-C(CH3)3), 53.9 (CHCH2CH2N), 54.0 (CHCH2CH2N), 58.2 (CHCH2CH2N), 61.6 (NCH2CH2CH2), 61.7 (OCH2CH2OH), 63.7 (COOCH2CH2O), 67.0 (COOCH2CH2O), 68.8 (OCH2CH2OH), 72.5 (CH-O), 79.4 (C-OH), 125.0 (2 × CH, Ar), 125.6 (4 × CH, Ar), 125.9 (2 × CH, Ar), 126.4 (2 × CH, Ar), 128.1 (4 × CH, Ar), 137.2 (C, Ar), 146.0 (C, Ar), 150.8 (C, Ar), 155.0 (C, Ar), 171.6 (COO), 172.2 (COOH). (+)-ESI-MS: 660 [M+ + H]. Accurate MS (C40H54O7N1): 660.3893 (theoretical: 660.3895). Synthesis of G1 Dendrimer-Succinyl-Terfenadine (G1suc-Ter). DCC (93 mg, 0.45 mmol) was added to a solution of Ter-suc (200 mg, 0.35 mmol) in 3 mL of DMF. The mixture was stirred for 1 h, after which time NHS (52 mg, 0.45 mmol) was added and the mixture was stirred for 24 h. The reaction mixture was filtered, and the filtrate was concentrated. The residue was dissolved in DCM, washed with water (2 × 10 mL), aqueous 0.1 M HCl (2 × 10 mL), aqueous 0.1 M NaOH (2 × 10 mL), and water (2 × 10 mL), dried (anhydrous Na2SO4), filtered, evaporated to dryness in vacuo, and dissolved in DMF (3 mL). The solution was added dropwise (during 6 h) to 2 mL of DMF containing G1 dendrimer (429 mg, 0.3 mmol),

Dendrimer Prodrugs for Enhanced Bioavailability

stirred for 5 days, and evaporated under vacuum. The residue was dissolved in 3 mL of water and filtered. The filtrate was purified by size exclusion chromatography, using Sephadex 15 and eluting with methanol:water (1:5). The product was further purified using Sephadex LH 20 and eluting with methanol:water (5:1). The yield of G1-suc-Ter was 39%. 1H NMR (d4MeOD): 1.28 (9H, s, -But), 1.95-1.42 (8H, m, NCH2CH2CH2, 2 × CHCH2CH2N, and NCH2CH2CH2CH, Ter), 2.45-2.18 (61H, m, c-G1), 3.05-2.50 (61H, 3 m, a-G1, (2 × CHCH2CH2N, Ter), (CHCH2CH2N, Ter), (CO-CH2-CH2-CO, Suc), b-G1, f-G1), 3.73-3.10 (26H, e-G1 and (NCH2CH2CH2, Ter)), 5.805.60 (1H, m, CH-O), 7.65-7.05 (14H, m, Ar). 13C NMR (d4MeOD): 22.0 (NCH2CH2CH2), 25.9 and 26.0 (2 × CHCH2CH2N), 30.8 (CH2COO), 31.8 (3 × CH3), 34.7 (12 × c-G1), 35.4 (CH2CONH, Suc), 37.0 (NCH2CH2CH2CH), 38.6 (8 × f-G1), 41.4 (12 × e-G1) 43.6 (-C(CH3)3), 51.1 (12 × b-G1), 52.2 (CHCH2CH2N), 53.0 (CHCH2CH2N), 53.4 (6 × a-G1), 54.3 (CHCH2CH2N), 57.8 (NCH2CH2CH2), 75.8 (C-OH), 80.0 (CH-O), 126.4 (2 × CH, Ar), 127.0 (4 × CH, Ar), 127.1 (2 × CH, Ar), 127.5 (2 × CH, Ar), 129.1 (4 × CH, Ar), 138.9 (C, Ar), 147.5 (2 × C, Ar), 152.0 (C, Ar), 174.6 (COO), 175.2 (CONH, 12 × d-G1), 175.7 (CONH, Suc). Synthesis of G1 Dendrimer-Succinyl-Diethylene GlycolTerfenadine (G1-suc-deg-Ter). Ter-suc-deg (139 mg, 0.21 mmol) was dissolved in THF (5 mL), and TEA (43 mg, 0.42 mmol) was added. The mixture was stirred for 10 min, after which 4-nitrophenyl chloroformate (85 mg, 0.42 mmol) was added portion wise and stirred for 24 h at room temperature. THF was evaporated under vacuum, and the residue was dissolved in DCM, filtered, and concentrated. The residue was dissolved in DMF (1 mL) and added dropwise (during 6 h) to a stirred solution of G1 dendrimer (200 mg, 0.14 mmol) in DMF (2 mL). The reaction mixture was stirred for 5 days. DMF was evaporated under vacuum, and the residue was purified by size exclusion chromatography using Sephadex LH 20 and eluting with methanol:water (5:1). The yield of G1-suc-deg-Ter was 72%. 1H NMR (d4-MeOD): 1.29 (9H, s, -But), 2.10-1.38 (8H, m, NCH2CH2CH2, 2 × CHCH2CH2N, and NCH2CH2CH2CH, Ter), 2.44-2.24 (24H, m, c-G1), 3.11-2.47 (61H, 3 m, a-G1, (2 × CHCH2CH2N, Ter), (CHCH2CH2N, Ter), (CO-CH2CH2-CO, Suc), b-G1, f-G1), 3.79-3.11 (30H, m, e-G1, (NCH2CH2CH2, Ter), COOCH2CH2OCH2CH2O-, deg), 4.32-4.10 (4H, m, COOCH2CH2OCH2CH2O-), 5.84-5.73 (1H, t, J ) 6.7, CH-O), 7.55-7.05 (14H, m, Ar). 13C NMR (d4-MeOD): 23.5 (NCH2CH2CH2), 27.1 (2 × CHCH2CH2N), 29.9 (CH2COO), 30.3 (CH2COO), 31.8 (3 × CH3), 34.4 (12 × c-G1), 35.4 (NCH2CH2CH2CH), 38.6 (8 × f-G1), 41.4 (12 × e-G1) 45.2 (-C(CH3)3), 51.2 (12 × b-G1), 52.3 (2 × CHCH2CH2N), 53.5 (6 × a-G1), 55.1 (CHCH2CH2N), 59.3 (NCH2CH2CH2), 62.2 (COOCH2CH2OCH2CH2O-), 65.1 (COOCH2CH2OCH2CH2O-), 68.1 (COOCH2CH2OCH2CH2O-), 70.5 (COOCH2CH2OCH2CH2O-), 77.3 (CH-O), 80.3 (C-OH), 127.0 (2 × CH, Ar), 127.2 (4 × CH, Ar), 127.3 (2 × CH, Ar), 128.3 (2 × CH, Ar), 128.9 (4 × CH, Ar), 137.0 (C, Ar), 138.9 (C, Ar), 148.0 (C, Ar), 152.0 (C, Ar), 156.6 (OCONH-), 173.4 (COO), 174.0 (COO), 175.2 (CONH, 12 × d-G1). Synthesis of Lauroyl 4-Nitrophenyl Carbonate. Lauroyl alcohol (0.93 g, 5 mmol) was dissolved in THF (10 mL), and TEA (1.02 g, 10 mmol) was added. The mixture was stirred for 10 min, after which 4-nitrophenyl chloroformate (2.01 g, 10 mmol) was added portion wise and stirred for 24 h at room temperature. THF was evaporated under vacuum, and the residue was dissolved in hexane:EtOAc (85:15), filtered, and purified by silica gel column chromatography, eluting with the same solvent system to give lauroyl 4-nitrophenyl carbonate (Rf ) 0.4) with a yield of 84%.1H NMR (CDCl3): 0.80 (3H, t, J ) 6.9, CH3), 1.35-1.15 (18H, m, 9 × -CH2-), 1.68 (2H, pentet,

Bioconjugate Chem., Vol. 18, No. 3, 2007 939

J ) 6.8, -CH2-), 4.20 (2H, t, J ) 6.8, -CH2-O-CO-), 7.35-7.25 (2H, m, Ar), 8.2-8.15 (2H, m, Ar). 13C NMR (CDCl3): 14.5 (-CH3), 23.1 (-CH2-), 26.0 (-CH2-), 30.028.9 (7 × -CH2-), 32.3 (-CH2-), 70.0 (-CH2-O-CO-), 122.1 (2 × CH, Ar), 125.6 (2 × CH, Ar), 145.7 (CH, Ar), 152.9 (CH, Ar), 156.0 (-CO-). Synthesis of (Lauroyl)2-G1-suc-deg-Ter (L2-G1suc-deg-Ter). Lauroyl 4-nitrophenyl carbonate (25 mg, 0.07 mmol) in DMF (1 mL) was added dropwise to a stirred solution of G1-suc-deg-Ter (51 mg, 0.023 mmol) in DMF (2 mL). The reaction mixture was stirred for 5 days. DMF was evaporated under vacuum, and the residue was purified by size exclusion chromatography using Sephadex LH 20 and eluting with methanol:water (5:1). The resulting product was dissolved in water and filtered; the filtrate was concentrated under vacuum and purified again by size exclusion chromatography (same conditions). The yield of L2-G1-suc-deg-Ter was 57%. 1H NMR (d4-MeOD): 0.89 (6H, t, J ) 6.8, 2 × CH3, L), 2.111.20 (60H, m, (-But, Ter), 2 × (10 × -CH2-, L)), (NCH2CH2CH2, 2 × CHCH2CH2N, and NCH2CH2CH2CH, Ter)), 2.462.22 (24H, 24, c-G1), 3.78-2.48 (91H, 4 m, a-G1, (2 × CHCH2CH2N, Ter), (CHCH2CH2N, Ter), (CO-CH2-CH2CO, suc), b-G1, f-G1, e-G1, (NCH2CH2CH2, Ter), (COOCH2CH2OCH2CH2O-, deg)), 4.32-3.92 (8H, (2 × CH2-O, L), (COOCH2CH2OCH2CH2O-, deg)), 5.76-5.66 (1H, t, J ) 6.7, CHO), 7.55-7.05 (14H, m, Ar). 13C NMR (d4-MeOD): 14.1 (2 × -CH3, L), 22.8 (NCH2CH2CH2), 23.4 (2 × -CH2-, L), 26.6 (2 × CHCH2CH2N), 29.9 (CH2COO), 30.1 (CH2COO), 30.4 (14 × -CH2-, L), 31.4 (3 × CH3), 32.7 (2 × -CH2-, L), 34.3 (12 × c-G1), 35.4 (NCH2CH2CH2CH), 38.2 (8 × f-G1), 41.4 (12 × e-G1), 44.8 (-C(CH3)3), 50.7 (12 × b-G1), 51.8 (2 × CHCH2CH2N), 53.0 (6 × a-G1), 54.7 (CHCH2CH2N), 58.0 (NCH2CH2CH2), 61.8 (2 × -CH2-, L), 64.7 (COOCH2CH2OCH2CH2O-), 65.6 (COOCH2CH2OCH2CH2O-), 69.6 (COOCH2CH2OCH2CH2O-), 70.1 COOCH2-CH2OCH2CH2O-), 76.8 (CH-O), 79.9 (C-OH), 126.0 (2 × CH, Ar), 126.8 (4 × CH, Ar), 126.9 (2 × CH, Ar), 128.5 (2 × CH, Ar), 128.8 (4 × CH, Ar), 135.0 (C, Ar), 138.1 (C, Ar), 147.5 (2 × C, Ar), 156.6 (3 × OCONH-), 174.4 (2 × COO), 174.8 (CONH, 12 × d-G1). Determination of Partition Coefficients. The apparent partition coefficients (Papp) of terfenadine and conjugates between 1-octanol and phosphate buffer (pH 7.4) were determined at 37 °C. Before use, the 1-octanol was saturated with phosphate buffer for 24 h by vigorous stirring. A known concentration of compound in phosphate buffer (pH 7.4, 5 mL) was shaken for 72 h with 1-octanol (5 mL) to achieve equilibrium, and the phases were separated by centrifugation at 9000g for 5 min. All experiments were performed in triplicate. The concentrations of the compounds in the buffer phase before and after partitioning were determined by HPLC. Lactate Dehydrogenase (LDH) Leakage Assay (Cytotoxicity Studies). Human Caucasian colon adenocarcinoma cells (Caco-2) (passage 101-105) were seeded at 10 000 cells/well in 96-well plates and maintained at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% foetal bovine serum, 1% nonessential amino acids, 50 IU/mL penicillin, and 50 mg/mL streptomycin. After 24 h the medium was removed and the cells were washed in phosphate-buffered saline. Hanks Balanced Salt Solution (HBSS, 200 µL) containing G1 PAMAM dendrimer, conjugates, or third-generation (G3) PAMAM dendrimer at concentrations of up to 1 mM was added to Caco-2 cells. Caco-2 cells were also treated with blank HBSS and 1% Triton X-100 as negative and positive controls, respectively. After 3 h of incubation at 37 °C, 100 µL/well supernatant was removed carefully and transferred into corresponding wells of an optically clear 96-well flat bottom microplate.

940 Bioconjugate Chem., Vol. 18, No. 3, 2007

Najlah et al.

Scheme 1. Synthesis of G1-suc-Ter

LDH leakage in the compartment was quantified using an LDH assay kit following the manufacturer’s specifications. Transport Studies of Ter, Ter-suc, Ter-suc-deg, and G1 PAMAM Dendrimer Conjugates. Caco-2 cells (passage 104-112) were seeded onto polycarbonate filters (pore size 3.0 µm) at a density of 1.2 × 105 cells/cm2. Cells were grown at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity in DMEM supplemented with 10% foetal bovine serum, 1% nonessential amino acids, 50 IU/mL penicillin, and 50 mg/mL streptomycin. The medium was changed every other day for 21-22 days. The integrity of each batch of cells was tested by measuring the TEER using a voltohmmeter (EVOM, World Precision Instruments, Sarasota, FL) before and after the experiments. Before the experiments, cells were equilibrated with HBSS for 20 min at 37 °C and the TEER was determined. The TEER value, corrected for the blank filter resistance, was in the range 800-1000 Ω cm2. Only confluent monolayers were used for the transepithelial transport studies. Transport was determined in both A f B and B f A directions. The transport medium (TM) was HBSS with N-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid (HEPES, 25 mM) and placed in the donor and receiver compartments. Terfenadine, terfenadine prodrugs (each equivalent to 50 µM of terfenadine), or a mixture of G1 and terfenadine (50 µM each) was placed in the donor compartment, and cells were incubated in a humidified atmosphere at 37 °C. TEER was measured every 30 min during the experiment; samples (50 µL) were removed from the receiver

compartment at time zero and after 60, 120, and 180 min and from the donor compartment after 180 min. The samples were analyzed by HPLC. The apparent permeability coefficient (Papp) was calculated from

Papp ) dQ/dt × (V/ACo) (cm s-1) where dQ/dt is the change in the donor concentration over time (mol L-1 s-1), V is the volume in the reservoir in the receiver side (cm3), Co is the initial concentration in the donor compartment (mol L-1), and A is the surface area of the membrane (cm2).

RESULTS AND DISCUSSION Synthesis and Characterization of Dendrimer-Ter Conjugates. A prodrug consisting of four components, the dendrimer, the drug, the spacer, and the permeability enhancer group, was synthesized. G1 dendrimer-based prodrugs of terfenadine with a succinyl linker (single ester linker, G1-sucTer) and succinic-diethylene glycol linker (double ester linker, G1-suc-deg-Ter) were evaluated in vitro as drug carriers. G1-suc-Ter. Terfenadine was linked by its secondary hydroxyl (butan-1-ol) group with succinic anhydride (Scheme 1). The resulting product Ter-suc was characterized by 1H NMR, 13C NMR, and mass spectroscopy. Successful formation

Bioconjugate Chem., Vol. 18, No. 3, 2007 941

Dendrimer Prodrugs for Enhanced Bioavailability

Figure 2. 1H NMR spectra (300 MHz) of (a) terfenadine (CDCl3) and (b) Ter-suc (CDCl3).

of the ester bond between the secondary hydroxyl group of terfenadine and the carboxyl group of succinyl linker was demonstrated by a downfield shift in the 1H NMR chemical shift of the methine group (CH-O) from 4.60 ppm in terfenadine to 5.81 ppm in Ter-suc (Figure 2). Accurate mass spectrometry confirmed that only one succinyl group was attached to terfenadine via the secondary hydroxyl group and that the tertiary hydroxyl diphenyl group (Ph2C(OH)-) remained free. The carboxyl group of the succinyl linker was utilized to conjugate Ter-suc to a surface amine group of the G1 dendrimer using the active ester method involving NHS (Scheme 1) as this method of dendrimer conjugation was found to be more efficient than direct condensation using coupling agents such as CDI (18). In order to obtain an approximately 1:1 ratio of dendrimer and terfenadine, Ter-suc or Ter-suc-deg was added to the reaction in quantities slightly higher than the equimolar ratio to dendrimer. G1-suc-deg-Ter. Synthesis of Ter-suc-deg was performed by condensing diethylene glycol to the carboxyl of the succinic linker using CDI as coupling agent (Scheme 2). Adding diethylene glycol to Ter-suc changed its physical state from a solid to an oil without any noticeable enhancement in the water solubility. Ter-suc-deg (Scheme 2) and lauroyl alcohol (Scheme 3) were attached to the primary amines of G1 dendrimer using the 4-nitrophenyl chloroformate activation method. Dendrimer conjugates were purified by size exclusion chromatography, solubilization in water (as terfenadine and lauroyl alcohol are insoluble in water), and dialysis (MW cutoff ) 1000 Da) for 48 h. The resulting conjugates were characterized by 1H and 13C NMR spectroscopy and RP-HPLC. As shown in Figure 3, the RP-HPLC chromatograms of the conjugates confirm that the drug is covalently bound to G1 PAMAM dendrimers; no traces of the free drug or drug linker were detected. The drug payloads in the conjugates were estimated using the relative intensities of the peaks in the 1H NMR

spectrum originating from attached compound compared to those of dendrimer (Table 1). In the 13C NMR spectrum, the covalent bond between the succinyl linker and dendrimer was confirmed by the appearance of a new amide carbonyl peak at 175.7 ppm (CONH, Suc) for G1-suc-Ter. Similarly, the carbamate bond between diethylene glycol linker and/or lauroyl chain and G1 were represented by carbamate carbonyl peaks at 156.6 ppm for G1-suc-deg-Ter and L2-G1-suc-deg-Ter. Partition Coefficients of Dendrimer Conjugates. Terfenadine, as an insoluble drug (19, 20), is attached to the surface of a highly water-soluble dendrimer. Determination of solubility would require the synthesis of large amounts of the conjugates; therefore, the apparent partition coefficients (Kapp) of terfenadine and conjugates between 1-octanol and phosphate buffer (pH 7.4) were determined as a parameter reflecting the change in the lipophilicity of terfenadine after attaching to G1 PAMAM dendrimer. Table 2 shows a significant decrease in the lipophilicity of terfenadine following its conjugation to the highly water-soluble G1 PAMAM dendrimer. L2-G1-suc-deg-Ter shows the highest lipophilicity among the G1-terfenadine conjugates (but lipophilicity is still significantly lower than that of terfenadine) as a result of the presence of two lipophilic lauroyl chains on the surface of the G1 PAMAM dendrimer. Effect of PAMAM Dendrimer Prodrugs on Caco-2 Cell Viability (LDH Assay). The influence of PAMAM dendrimer prodrugs on the viability of Caco-2 cells was determined using the LDH assay. LDH, a cytosolic enzyme, has been found to leak into the culture medium upon damage to cell membranes (21). As shown in Figure 4, G1 PAMAM dendrimer was relatively nontoxic to Caco-2 cells, with less than 10% cytotoxicity at concentrations of 1 mM after 180 min incubation. A comparison between the IC50 of G1 and that of G3 PAMAM dendrimer shows that G1 is significantly less toxic than G3 (Table 3). This result is in agreement with previous observations which showed that the cytotoxicity of PAMAM dendrimers was concentration, generation, and charge dependent (7, 9). Attach-

942 Bioconjugate Chem., Vol. 18, No. 3, 2007

Najlah et al.

Scheme 2. Synthesis of G1-suc-deg-Ter

ing terfenadine via a linker to the G1 PAMAM dendrimer decreased the viability of Caco-2 cells as indicated by IC50 results (Table 3). G1 dendrimer-terfenadine conjugates showed concentration-dependent toxicity with a leakage of approximately 100% LDH for concentrations of 1 mM (Figure 4). These results are explained by the high toxicity of terfenadine, which has been reported to be both time and dose dependent as indicated by leakage of LDH from rat myocardial cells (in Vitro) (22). However, G1 dendrimer-terfenadine conjugates were nontoxic toward Caco-2 cells at the concentration used in the transport study (50 µM).

Transport Studies of Ter, Ter-suc, Ter-suc-deg, and G1 PAMAM Dendrimer Conjugates Across Caco-2 Monolayers. The transport of terfenadine and prodrugs across Caco-2 monolayers was investigated in the A f B and B f A directions at nontoxic concentrations (as determined by the LDH assay). The stability of Ter-dendrimer conjugates was evaluated at pH 1.2, 7.4, and 8.5. After 24 h of incubation, the conjugates remained intact and no traces of drug were detected using HPLC (data not shown). Following transport studies, no free drug was detected in the receiver compartment.

Dendrimer Prodrugs for Enhanced Bioavailability

Bioconjugate Chem., Vol. 18, No. 3, 2007 943

Scheme 3. Synthesis of L2-G1-suc-deg-Ter

The apparent permeability coefficient (Papp) of terfenadine and prodrugs in both directions at 3 h is shown in Figure 5. Terfenadine, known as a strong substrate for the intestinal P-gp efflux transporter, showed B f A Papp significantly greater than A f B Papp, confirming previous reports showing the transport profile of terfenadine across Caco-2 monolayers (23). No significant difference can be observed on the permeability of terfenadine after attaching succinyl or succinicdiethylene glycol chains as shown in Figure 5 for Ter-suc or Ter-suc-deg. This suggests that attaching a linker to terfenadine had no impact on the permeability of the resulting prodrug. In the presence of G1 there was no change in the transport profile of terfenadine in both directions (Figure 5). However,

the permeability of G1-suc-Ter and G1-suc-deg-Ter across Caco-2 monolayers, especially in the A f B direction, was significantly higher than that of terfenadine, suggesting the ability of G1 dendrimer conjugates to bypass the P-gp efflux transporter. These results are in agreement with a previous report showing that propranolol (P-gp substrate) conjugated to G3 PAMAM dendrimer was also able to bypass the P-gp efflux system in Caco-2 monolayers (17). In the present study, terfenadine, an unambiguous, stronger P-gp substrate (12), was attached to G1 PAMAM dendrimer, a less toxic dendrimer than G3 PAMAM (9), via a linker/spacer. In addition, the A f B permeability of G1 PAMAM dendrimers was found to be several-fold higher than the A f B permeability of higher generations (G3 and G4) (9).

944 Bioconjugate Chem., Vol. 18, No. 3, 2007

Najlah et al.

Figure 3. RP-HPLC chromatograms of (a) G1 PAMAM dendrimer, (b) G1-suc-deg-Ter, (c) G1-suc-Ter, (d) L2-G1-suc-deg-Ter, and (e) terfenadine. Table 1. Properties and Concentrations of G1-Terfenadine Conjugates Used in Transport Studies

G1 G1-suc-Ter G1-suc-deg-Ter L2-G1-suc-deg-Ter a

conjugation ratio G1:terfenadinea

conjugation ratio G1:lauroyl chaina

average mol wt (g/mol)

conjugate concentration (µM)

equivalent concentration of terfenadine (µM)

N/A 1:1.14 1:1.11 1:0.95

N/A N/A N/A 1:2.16

1429.9 1983.6 2115.7 2540.4

N/A 43.9 45.0 52.6

N/A 50 50 50

Determined by 1H NMR spectroscopy.

Table 2. Log Ko/w (pH 7.4) Values of Terfenadine and its Conjugates at 37 °C compound

log Ko/w (pH 7.4)

Ter Ter-suc Ter-suc-deg G1-suc-Ter G1-suc-deg-Ter L2-G1-suc-deg-Ter

1.25 ( 0.17 1.38 ( 0.22 1.15 ( 0.22 -0.11 ( 0.08 -0.16 ( 0.07 0.15 ( 0.06

Table 3. Effect of G1 PAMAM Dendrimer, G1-Terfenadine Conjugates, and G3 PAMAM Dendrimer on the Viability of Caco-2 Cells As Determined by IC50 (mean ( SD, n ) 4) compound

IC50 (mM)

G1 G1-suc-Ter G1-suc-deg-Ter L2-G1-suc-deg-Ter G3

6.23 ( 1.20 0.52 ( 0.08 0.55 ( 0.10 0.48 ( 0.04 0.25 ( 0.09

The high permeability of G1 PAMAM-terfenadine conjugates may be attributed to the positively charged amine groups of the G1 PAMAM dendrimers. It has been reported that positively charged molecules permeate at a higher rate across Caco-2 monolayers compared to neutral or anionic molecules because of the favorable electrostatic interaction with the negatively charged epithelial surfaces (24). In addition, Jevprasesphant and co-workers found that the permeability of positively charged PAMAM dendrimers was higher than that of anionic PAMAM dendrimers (7). The apparent lack of impact of the G1 dendrimer on the transport profile of terfenadine may be a consequence of the transcellular rather than paracellular transport of terfenadine; G3 dendrimer was reported to enhance paracellular transport by opening up the tight junctions (7). Moreover, the significant increase in the permeability of terfenadine was only shown when terfenadine was covalently bound to G1 PAMAM dendrimers. It is proposed that G1 PAMAM dendrimer enhances the permeability of the conjugated drug by acting as an efficient carrier across the cellular membrane rather than influencing the transport mechanism of the drug. This suggestion is confirmed by reports showing that

Figure 4. Effect of G1 PAMAM dendrimer, G1-terfenadine conjugates, and G3 PAMAM dendrimer on the viability of Caco-2 cells (LDH assay) (mean ( SD, n ) 4).

PAMAM dendrimers are neither P-gp inhibitors nor P-gp substrates (8, 17). Lauroyl alcohol, known to function as a permeability enhancer (25), was used as a surface modifier of G1 PAMAM dendrimers to enhance the permeability of the resulting conjugates. Surfacemodified dendrimers with medium chain fatty acids were also found to be significantly less cytotoxic and exhibited enhanced permeation through Caco-2 monolayers (7). Two lauroyl chains were attached to the surface amine group of G1-suc-degTer to give the conjugate L2-G1-suc-deg-Ter. This conjugate showed the highest permeability in both directions across Caco-2 cell monolayers of all the G1 conjugates of this study with an A f B permeability over 25-fold higher than that of terfenadine alone. Transepithelial electrical resistance (TEER) measurements were used to assess the influence of G1-terfenadine conjugates on the integrity of Caco-2 monolayers. TEER values were measured following apical incubation with Ter, Ter-suc, Ter-

Bioconjugate Chem., Vol. 18, No. 3, 2007 945

Dendrimer Prodrugs for Enhanced Bioavailability

the observed increase in the permeability of L2-G1-suc-degTer (Figure 6) results from dilation of tight junctions, which suggests that transport of L2-G1-suc-deg-Ter may also involve a transcellular pathway. Previous work has demonstrated that transepithelial transport of dendrimer conjugates involves both paracellular and transcellular pathways (6).

CONCLUSIONS

Figure 5. A f B (0) and B f A (9) permeability after 3 h across Caco-2 cell monolayers at 37 °C of free terfenadine, conjugates, and terfenadine in the presence of G1 PAMAM dendrimer (mean ( SD, n ) 4).

The design, synthesis, characterization, and transport studies of terfenadine conjugates of G1 PAMAM dendrimer using biodegradable spacer/linkers are reported. Partition coefficient results indicate that the G1-terfenadine conjugates were significantly more hydrophilic than the parent drug. Cytotoxicity studies showed that G1 PAMAM dendrimer was only slightly toxic to Caco-2 monolayers with a significantly lower toxicity than that of G3 PAMAM dendrimer. The presence of G1 PAMAM dendrimer in the transport medium had no impact on the transport profile of terfenadine, but attaching terfenadine to the surface of G1 PAMAM dendrimers via a covalent bond significantly increased its A f B permeability across Caco-2 cell monolayers. A more pronounced increase of A f B terfenadine permeability was reported when lauroyl-modified dendrimer prodrug was used as a carrier. The transport mechanism of G1-terfenadine conjugates is thought to involve both transcellular and paracellular pathways. Our results suggest that G1 PAMAM dendrimers demonstrate potential as nanocarriers for enhancement of oral bioavailability of terfenadine as a model for low-solubility drugs that are P-glycoprotein substrates.

ACKNOWLEDGMENT We thank Dr. Jeff Penny for use of Caco-2 cell facilities and Al-Baath University for financial support.

LITERATURE CITED

Figure 6. Effect of terfenadine and terfenadine prodrugs (each equivalent to 50 µM terfenadine) and EDTA (1 mM) on the TEER of Caco-2 cell monolayers after apical incubation (mean ( SD, n ) 4).

suc-deg, and G1 PAMAM dendrimer conjugates (each equivalent to 50 µM of Ter) and EDTA (1 mM). EDTA (anionic permeation enhancer) was used as a positive control, modulating the tight junction by chelating the extracellular Ca2+ and Mg2+ ions required to maintain the integrity of tight junctions (26). Figure 6 shows that EDTA decreased the TEER values by approximately 65% compared to the control (HBSS with 25 mM HEPES) after 3 h of incubation. Ter, Ter-suc, and Tersuc-deg had no significant impact on TEER values, which may suggest transcellular transport for these compounds. However, TEER values decreased by approximately 25% when exposed to G1-suc-Ter and G1-suc-deg-Ter and by approximately 35% for L2-G1-suc-deg-Ter. This suggests that the enhancement of permeability may be partly due to modulation of the tight junction. A previous report showed that transport of propranolol was increased when conjugated to lauroylmodified G3 PAMAM and that this increase might involve endocytosis-mediated transport (17). However, as no significant differences were observed between the TEER values for L2G1-suc-deg-Ter and other G1 conjugates, it is unlikely that

(1) Tomalia, D. A., Baker, H., Dewald, J., Hall, M., Kallos, G., Martin, S., Roeck, J., Ryder, J., and Smith, P. (1985) A new class of polymers: Starburst-dendritic macromolecules. Polym. J. (Tokyo, Jpn.) 17, 117-32. (2) D’Emanuele, A., and Attwood, D. (2005) Dendrimer-drug interactions. AdV. Drug DeliVery ReV. 15, 2147-2162. (3) D’Emanuele, A., Attwood, D., and Abu-Rmaileh, R. (2003) Dendrimers. In Encyclopedia of Pharmaceutical Technology (Swarbrick, B. J. C., Ed.) pp 1-21, Marcel Dekker, New York. (4) Florence, A. T., Sakthivel, T., and Toth, I. (2000) Oral uptake and translocation of a polylysine dendrimer with a lipid surface. J. Controlled Release 65, 253-259. (5) Wiwattanapatapee, R., Carreno-Gomez, B., Malik, N., and Duncan, R. (2000) Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system? Pharm. Res. 17, 991-998. (6) Najlah, M., and D’Emanuele, A. (2006) Crossing cellular barriers using dendrimer nanotechnologies. Curr. Opin. Pharmacol. 6, 522527. (7) Jevprasesphant, R., Penny, J., Attwood, D., McKeown, N. B., and D’Emanuele, A. (2003) Engineering of dendrimer surfaces to enhance transepithelial transport and reduce cytotoxicity. Pharm. Res. 20, 1543-1550. (8) El-Sayed, M., Rhodes, C. A., Ginski, M., and Ghandehari, H. (2003) Transport mechanism(s) of poly (amidoamine) dendrimers across Caco-2 cell monolayers. Int. J. Pharm. 265, 151-7. (9) El-Sayed, M., Ginski, M., Rhodes, C., and Ghandehari, H. (2002) Transepithelial transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers. J. Controlled Release 81, 355-365. (10) Jevprasesphant, R., Penny, J., Jalal, R., Attwood, D., McKeown, N. B., and D’Emanuele, A. (2003) The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int. J. Pharm. 252, 263-266. (11) Saitoh, H., and Aungst, B. J. (1995) Possible involvement of multiple P-glycoprotein-mediated efflux systems in the transport of

946 Bioconjugate Chem., Vol. 18, No. 3, 2007 verapamil and other organic cations across rat intestine. Pharm. Res. 12, 1304-10. (12) Polli, J. W., Wring, S. A., Humphreys, J. E., Huang, L., Morgan, J. B., Webster, L. O., and Serabjit-Singh, C. S. (2001) Rational use of in vitro P-glycoprotein assays in drug discovery. J. Pharmacol. Exp. Ther. 299, 620-628. (13) Trambas, C. M., Muller, H. K., and Woods, G. M. (1997) P-glycoprotein mediated multidrug resistance and its implications for pathology. Pathology 29, 122-130. (14) Lo, Y. L., Hsu, C. Y., and Huang, J. D. (2005) Comparison of effects of surfactants with other MDR reversing agents on intracellular uptake of epirubicin in Caco-2 cell line. Anticancer Res. 18, 3005-9. (15) Tomita, M., and Hayashi, M. (2005) Improvement of intestinal drug absorption based on structural changes of tight junction and functional changes of P-glycoprotein. Drug DeliVery Syst. 20, 416423. (16) Choi, J. S., Jo, B. W., and Kim, Y. C. (2004) Enhanced paclitaxel bioavailability after oral administration of paclitaxel or prodrug to rats pretreated with quercetin. Eur. J. Pharm. Biopharm. 57, 3138. (17) D’Emanuele, A., Jevprasesphant, R., Penny, J., and Attwood, D. (2004) The use of a dendrimer-propranolol prodrug to bypass efflux transporters and enhance oral bioavailability. J. Controlled Release 95, 447-453. (18) Najlah, M., Freeman, S., Attwood, D., and D’Emanuele, A. (2006) Synthesis, characterization and stability of dendrimer prodrugs. Int. J. Pharm. 308, 175-182. (19) Loftsson, T., Hreinsdottir, D., and Masson, M. (2005) Evaluation of cyclodextrin solubilization of drugs. Int. J. Pharm. 302, 18-28.

Najlah et al. (20) Choi, H.-G., Lee, B.-J., Han, J.-H., Lee, M.-K., Park, K.-M., Yong, C. S., Rhee, J.-D., Kim, Y.-B., and Kim, C.-K. (2001) Terfenadineβ-cyclodextrin inclusion complex with antihistaminic activity enhancement. Drug. DeV. Ind. Pharm. 27, 857-862. (21) Decker, T., and Lohmann-Matthes, M. L. (1998) A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (tnf) activity. J. Immunol. Methods 115, 61-69. (22) Melchert, R. B., Levin, P. S., and Acosta, D., Jr. (1996) Cardiotoxicity of nonsedating antihistamines in vitro. In Vitro Toxicol. 9, 431-440. (23) Raeissi, S. D., Hidalgo, I. J., Segura-Aguilar, J., and Artursson, P. (1999) Interplay between CYP3A-mediated metabolism and polarized efflux of terfenadine and its metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers. Pharm. Res. 16, 625632. (24) Knipp, G. T., Ho, N. F. H., Barsuhn, C. L., and Borchardt, R. R. (1997) Paracellular diffusion in Caco-2 cell monolayers: effect of perturbation on the transport of hydrophilic compounds that vary in charge and size. J. Pharm. Sci. 86, 1105-1110. (25) Aungst, B. J. (2000) Intestinal Permeation Enhancers. J. Pharm. Sci. 89, 429-442. (26) Noach, A. B. J., Kurosaki, Y., Blom-Roosemalen, M. C. M., de Boer, A. G., and Breimer, D. D. (1993) Cell-polarity dependent effect of chelation on the paracellular permeability of confluent Caco-2 cell monolayers. Int. J. Pharm. 90, 229-237. BC060325Q