Molecules 2008, 13, 1441-1454; DOI: 10.3390/molecules13071441 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.org/molecules Article Enhanced Absorption and Growth Inhibition with Amino Acid Monoester Prodrugs of Floxuridine by Targeting hPEPT1 Transporters Yasuhiro Tsume 1, Balvinder S. Vig 2, Jing Sun 3, Christopher P. Landowski 4, John M. Hilfinger 5, Chandrasekharan Ramachandran 1 and Gordon L. Amidon 1,* 1 Department of Pharmaceutical Science, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109-1065, USA; E-mails: [email protected]; [email protected] 2 Pharmaceutical Research Institute, Bristol-Myers Squibb Company, New Brunswick, NJ 08502; E- mail: [email protected] 3 Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA; Email: [email protected] 4 Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland; Email: [email protected] 5 TSRL, Inc. Ann Arbor, Michigan 48108, USA; Email: [email protected] * Author to whom correspondence should be addressed; E-mail: [email protected]; Phone: +1- 734-764-2440; Fax: +1-734-763-6423. Received: 12 June 2008 / Accepted: 27 June 2008 / Published: 28 June 2008 Abstract: A series of amino acid monoester prodrugs of floxuridine was synthesized and evaluated for the improvement of oral bioavailability and the feasibility of target drug delivery via oligopeptide transporters. All floxuridine 5′-amino acid monoester prodrugs exhibited PEPT1 affinity, with inhibition coefficients of Gly-Sar uptake (IC50) ranging from 0.7 – 2.3 mM in Caco-2 and 2.0 – 4.8 mM in AsPC-1 cells, while that of floxuridine was 7.3 mM and 6.3 mM, respectively. Caco-2 membrane permeabilities of floxuridine prodrugs (1.01 – 5.31 x 10-6 cm/sec) and floxuridine (0.48 x 10-6 cm/sec) were much higher than that of 5-FU (0.038 x 10-6 cm/sec). MDCK cells stably transfected with the human oligopeptide transporter PEPT1 (MDCK/hPEPT1) exhibited enhanced cell growth inhibition in the presence of the prodrugs. This prodrug strategy offers great potential, not Molecules 2008, 13 1442 only for increased drug absorption but also for improved tumor selectivity and drug efficacy. Keywords: 5-FU; floxuridine; prodrugs; Caco-2 permeability; cell proliferation assays; oligopeptide transporter 1 (PEPT1) Introduction The anti-metabolites 5-fluorouracil (5-FU) and floxuridine (5-fluoro-2'-deoxyuridine) continue to be mainstay drugs for colorectal cancer treatment after 50 years [1]. However, the improvement of 5- FU and floxuridine therapeutic efficacy is critical because of the poor response rate of 5-FU, erratic oral absorption of floxuridine, and the adverse effects associated with those chemotherapeutics [2]. The anabolic mechanism of 5-FU and floxuridine is well studied [3]. Floxuridine activity is quite specific for DNA related pathways and results in DNA-directed cytotoxicity with little or no RNA directed cytotoxicity, unlike 5-FU [4-6]. The utilization of the more potent floxuridine is appealing because it inhibits in vitro cell proliferation at 10- to 100-fold lower concentrations compared to 5-FU [7-9]. However, the abundant presence of thymidine phosphorylase (TP) in many tissues, including the liver and intestine, rapidly converts floxuridine to 5-FU [10]. Thus, improving the resistance of floxuridine to enzymatic degradation may also increase its therapeutic efficacy. With the consideration of improved chemical stability, the prodrugs must be converted to active compounds for the desired therapeutic effect. In prodrug development, the activation of the prodrug is an essential step. It has been suggested that the biphenyl hydrolase-like protein recently identified as being responsible for hydrolysis of the prodrug valacyclovir (VACVase), might be involved in the activation of other amino acid prodrugs [11]. Kim et al. suggest that the substrate specificity of this enzyme is largely determined by the amino acid acyl promoiety of prodrug [12]. Another enzyme, carboxylesterase I, has been shown to preferentially hydrolyze phenylalanine containing nucleoside ester prodrugs, while also displaying 100-fold less activity toward aliphatic esters [13]. Amino acid ester prodrugs of floxuridine and the antiviral agent acyclovir have been shown to be substrates of the PEPT1 transporter [14, 15]. PEPT1 has broad substrate specificity for dipeptides, tripeptides, and β-lactam antibiotics [16-23]. Improved oral bioavailability of the valyl ester prodrug of acyclovir has been attributed to the presence of oligopeptide transporters [24, 25]. Two pancreatic cancer cell lines, AsPC-1 and Capan-2, have been reported to have significant expression of oligopeptide transporters, where they might represent possible targets for cancer therapy [26]. Selective growth inhibition studies in in vitro cell systems exogenously expressing PEPT1 have demonstrated more accumulation of cancer drug in tumor cells for an enhanced therapeutic effect [15, 27]. Those results support the notion that the promoieties that incorporate amino acids, dipeptides, and tripeptides are well recognized by PEPT1, PEPT2, and ATB0+ transporters [17, 28-32]. Thus, amino acid modification of cancer drugs represents a potential drug delivery strategy to target cells via transporters. Molecules 2008, 13 1443 In this report, we briefly describe the synthesis and characterization of mono amino acid ester prodrugs of floxuridine. We evaluate the prodrug stability, Caco-2 membrane permeability and the feasibility of selective tumor growth inhibitory effect in MDCK and MDCK/hPEPT1 cells by cell proliferation assays. Results and Discussion Prodrug approaches with amino acid modification have been widely employed to improve intestinal absorption of poorly permeant drugs [33]. The antiviral drug valacyclovir is an example of a successful amino acid ester prodrug strategy [34]. The improved oral bioavailabiliy of valacyclovir has been attributed to the enhanced transport by intestinal oligopeptide transporters [14, 24, 35]. Dipeptide and tripeptide compounds, along with mono amino acid derivatives, have been investigated for their suitability as substrates for the oligopeptide transporter [16-18, 21, 28, 36-39]. Mono amino acid ester prodrugs of antiviral and anticancer drugs such as gemcitabine, acyclovir, and 2-bromo-5,6-dichloro- 1-(β-D-ribofuranosyl)benzimidazole (BDCRB) have been synthesized and evaluated for their suitability as transporter substrates in our previous reports [15, 24, 40-43]. Mono amino acid floxuridine prodrugs reported herein were synthesized as shown in Scheme 1. Scheme 1. Synthesis of amino acid ester prodrugs of floxuridine. O O F F NH NH Boc N O O N O HO i HN C O O O H H H H H H H H OH H OH H R Floxuridine ii L-leucine L-isoleucine O F NH O N O L-valine L-phenylalanine H2N C O O H H H H OH H R H2 D-valine L-glycine Reagents: (i) N-t-BOC-protected amino acids, DCC, DMAP, DMF; (ii) TFA, CH2Cl2 Molecules 2008, 13 1444 The total prodrug yields for each amino acid ranged between 2 and 18 % and the purity for all prodrugs was > 95 %, as determined by HPLC. The impurities were easily separated from their parent compounds by reverse-phase HPLC. All prodrug structures and identities were confirmed by ESI-MS and NMR. The prodrug purity and mass spectral data are shown in Table 1. Table 1. Analytical data for amino acid ester prodrugs of floxuridine. + Molecular % Purity ESI-MS (M + H) Weight log P* (HPLC) Required Observed (TFA salt) 5'-O-L-leucyl-floxuridine 96.77 360.2 360.4 473.4 -0.95 5'-O-L-phenylalanyl-floxuridine 96.10 394.2 394.0 507.4 -0.51 5'-O-L-valyl-floxuridine 98.51 346.2 346.0 459.4 -1.30 5'-O-D-valyl-floxuridine 99.52 346.2 346.0 459.4 -1.30 5'-O-L-isoleucyl-floxuridine 95.96 360.2 360.4 473.4 -0.78 5'-O-L-glycyl-floxuridine 95.23 304.2 303.9 417.4 -2.68 * Calculated using ChemDraw 7.0. The experiments concerning prodrug stability were performed at 37°C in pH 7.4 phosphate buffers and Caco-2, AsPC-1, and MDCK cell homogenates. Table 2 displays the estimated half-lives (t1/2) obtained from linear regression of pseudo-first-order plots of prodrug concentration vs. time for the floxuridine prodrugs in pH 7.4 phosphate buffers alone and in Caco-2, AsPC-1, and MDCK cell homogenates. Table 2. Half-lives of the hydrolytic degradation of floxuridine prodrugs in pH 7.4 buffer, and in homogenates from Caco-2 cells, AsPC-1 cells, and MDCK cells. Half Life (min) Homogenates Homogenates Homogenates Prodrug Buffer pH 7.4 from Caco-2 from AsPC-1 from MDCK cells cells cells Floxuridine nd 5.7 ± 0.3 6.4 ± 3.2 68.9 ± 12.8 5'-O-L-valyl-floxuridine 303.9 ± 17.8 9.4 ± 0.6 18.7 ± 6.7 74.7 ± 5.3 5'-O-D-valyl-floxuridine 344.9 ± 10.2 342.6 ± 120.2 290.9 ± 48.9 311.6 ± 45.4 5'-O-L-phenylalanyl-floxuridine 221.7 ± 56.7 11.1 ± 9.9 11.8 ± 1.7 6.0 ± 0.6 5'-O-L-leucyl-floxuridine 77.3 ± 1.2 4.8 ± 0.2 2.0 ± 0.1 9.2 ± 1.1 5'-O-L-isoleucyl-floxuridine 323.5 ± 1.5a 192.3 ± 31.8 198.0 ± 34.1 244.9 ± 18.3 5'-O-L-glycyl-floxuridine 85.5 ± 3.2 24.1 ± 2.0 27.6 ± 5.8 11.2 ± 2.0 Values are presented as mean ± S.D.; nd = not determined; afrom Ref. [40]. Molecules 2008, 13 1445 The 5′-O-D-valyl-floxuridine prodrug exhibited the highest stability in all conditions and thus confirmed previous reported results [44, 45]. These results indicate that there does not appear to be any significant enzymatic component in the breakdown of the 5′-O-D-valyl ester prodrug in cell homogenates.