Floxuridine (FUdR) was obtained from Lancaster (Windham, NH). The tert-butyloxycarbonyl (Boc) protected amino acids Boc-l-isoleucine, Boc-l-glycine, Boc-l-valine, Boc-l-phenylalanine, Boc-l-leucine, Boc-l-glycyl-l-leucine, Boc-l-phenylalanalyl-l-glycine, Boc-l-leucyl-l-glycine, Boc-l-isoleucyl-l-glycine, Boc-l-valyl-l-phenylalanine, and Boc-l-phenylalanyl-l-tyrosine were obtained from Chem-Impex (Wood Dale, IL). High-performance liquid chromatography (HPLC) grade acetonitrile was obtained from Fisher Scientific (St. Louis, MO). N,N-Dicyclohexylcarbodiimide (DCC), N,N-dimethylaminopyridine (DMAP), trifluoroacetic acid (TFA), and all other reagents and solvents were purchased from Aldrich Chemical Co. (Milwaukee, WI). Cell culture reagents were obtained from Invitrogen (Carlsbad, CA), and cell culture supplies were obtained from Corning (Corning, NY) and Falcon (Lincoln Park, NJ). All chemicals were either analytical or HPLC grade.

The synthesis and characterization of 5′-mono amino acid ester prodrugs of floxuridine have been reported previously.^11,13 Dipeptide prodrugs of floxuridine were synthesized in a similar manner. Briefly, Boc-protected dipeptides Boc-l-glycyl-l-leucine, Boc-l-phenylalanalyl-l-glycine, Boc-l-leucyl-l-glycine, Boc-l-isoleucyl-l-glycine, Boc-l-valyl-l-phenylalanine, and Boc-l-phenylalanyl-l-tyrosine, (1.1 mmol), DCC (1.1 mmol), and DMAP (0.1 mmol) were allowed to react with floxuridine (1 mmol) in 7 mL of dry DMF for 24 h. The reaction progress was monitored by TLC (ethyl acetate). The reaction mixture was filtered and DMF was removed under vacuum at 40 °C. The residue was extracted with ethyl acetate (30 mL) and washed with water (2 × 20 mL), and saturated NaCl (20 mL). The organic layer was dried over MgSO₄ and concentrated under vacuum. The reaction yielded a mixture of 3′-monoester, 5′-monoester, and 3′,5′-diester floxuridine prodrugs. The three spots observed on TLC were separated and purified using column chromatography (dichloromethane (DCM)/methanol, 20:1). Fractions from each spot were concentrated under vacuum separately. The Boc group was cleaved by treating the residues with 5 mL TFA:DCM (1:1). After 4 h, the solvent was removed and the residues were reconstituted with water and lyophilized. The TFA salts of amino acid prodrugs of floxuridine were obtained as white fluffy solids. The combined yield, consisting of 3′-monoester, 5′-monoester, and 3′,5′-diesters, of each floxuridine prodrug was ∼60%. The prodrugs were determined to be 90%−99% pure by reverse-phase HPLC, and were easily separated from their parent compounds by reverse-phase HPLC. The observed molecular weights of all prodrugs, determined by electrospray ionization mass spectra (ESI-MS) obtained on a Micromass LCT ESI-MS, were found to be consistent with those predicted by their structures. The structural identities of the prodrugs were then confirmed using proton nuclear magnetic resonance spectra (¹H NMR), obtained on a 300 MHz Bruker DPX-300 NMR spectrometer.

5′-l-Leucyl-l-glycyl-floxuridine: yield, 15%; percent purity, 93%; ¹H NMR (DMSO-d₆) δ, 0.84−0.92 (6H, m, ^δ(CH₃)₂), 1.50−1.77 (3H, m, ^β,γCH₂), 2.12−2.28 (2H, m, C2′), 3.78−4.35 (7H, m, ^αCH of Leu and ^αCH₂ of Gly, C3′, C4′, C5′), 6.16 (1H, t, C1′, J = 6.0 Hz), 7.94 (1H, d, CHF, J = 6.9 Hz); ESI-MS, 417.1 (M + H)⁺.

5′-l-Glycyl-l-leucyl-floxuridine: yield, 15%; percent purity, 96%; ¹H NMR (DMSO-d₆) δ, 0.85 (3H, d, ^δCH₃, J = 6.5 Hz), 0.90 (3H, d, ^δCH₃, J = 6.5 Hz), 1.51−1.68 (3H, m, ^β,γCH₂), 2.12−2.26 (2H, m, C2′), 3.54−4.40 (7H, m, ^αCH of Leu and ^αCH₂ of Gly, C3′, C4′, C5′), 6.16 (1H, t, C1′, J = 6.0 Hz), 7.90 (1H, d, CHF, J = 6.8 Hz); ESI-MS, 417.1 (M + H)⁺.

5′-l-Valyl-l-phenylalanyl-floxuridine: yield, 30%; percent purity, 95%; ¹H NMR (DMSO-d₆) δ, 0.90 (3H, d, ^γCH₃, J = 6.9 Hz), 0.95 (3H, d, ^γCH₃, J = 6.9 Hz), 2.08−2.20 (3H, m, C2′, ^βCH of Val), 3.02 (2H, m, ^βCH₂ of Phe), 3.62−4.60 (6H, m, ^αCH of Val and Phe, C3′, C4′, C5′), 6.14 (1H, t, C1′, J = 6.1 Hz), 7.22−7.31 (5H, m, aromatic protons), 7.91 (1H, d, CHF, J = 6.9 Hz); ESI-MS, 494.1 (M + H)⁺.

5′-l-Isoleucyl-l-glycyl-floxuridine: yield, 15%; percent purity, 93%; ¹H NMR (DMSO-d₆) δ, 0.87 (3H, t, ^δCH₃, J=7.3 Hz), 0.94 (3H, d, ^γCH₃, J = 6.8 Hz), 1.17 (1H, m, ^γCH₂), 1.52 (1H, m, ^γCH₂), 1.81 (1H, m, ^βCH), 2.11−2.28 (2H, m, C2′), 3.90−4.31 (7H, m, ^αCH of Ile and ^αCH₂ of Gly, C3′, C4′, C5′), 6.15 (1H, t, C1′, J = 6.5 Hz), 7.94 (1H, d, CHF, J = 6.9 Hz); ESI-MS, 416.9 (M + H)⁺.

5′-l-Phenylalanyl-l-glycyl-floxuridine: yield, 16%; percent purity, 98%; ¹H NMR (DMSO-d₆) δ, 2.12−2.28 (2H, m, C2′), 2.92−3.16 (2H, m, ^βCH₂ of Phe), 3.90−4.34 (7H, m, ^αCH of Phe and ^αCH₂ of Gly, C3′, C4′, C5′), 6.15 (1H, t, C1′, J = 6.5 Hz), 7.27−7.36 (5H, m, aromatic protons), 7.94 (1H, d, CHF, J = 6.5 Hz); ESI-MS, 451.1 (M + H)⁺.

5′-l-Phenylalanyl-l-tyrosyl-floxuridine: yield, 3.2%; percent purity, 99%; ¹H NMR (DMSO-d₆) δ, 2.10−2.32 (2H, m, C2′), 2.72−2.92 (4H, m, ^βCH₂ of Phe and Tyr), 3.90−4.29 (6H, m, ^αCH of Phe and Tyr, C3′, C4′, C5′), 6.15 (1H, m, C1′), 6.65−7.32 (9H, m, aromatic protons), 7.93 (1H, m, CHF); ESI-MS, 557.2 (M + H)⁺.

Capan-2 cells (passages 50−54), and AsPC-1 cells (passages 63−65) from American type Culture Collection (Rockville, MD) were routinely maintained in RPMI-1640 containing 10% fetal bovine serum. Caco-2 cells (passages 30−35) from American type Culture Collection (Rockville, MD) were routinely maintained in DMEM containing 10% fetal bovine serum, 1% nonessential amino acids, 1 mmol/L sodium pyruvate, and 1% l-glutamine at 5% CO₂ and 90% relative humidity at 37 °C. Cells were grown in antibiotic-free media to avoid the possible transport interference by antibiotics.

Confluent Caco-2, Capan-2, and AsPC-1 cells were rinsed twice with saline. The cells were washed with 5 mL of pH 7.4 phosphate buffer (10 mmol/L), lysed by ultrasonication (Micro ultrasonic cell disrupter model KT40, Kontes, Vineland, NJ), and pelleted by centrifugation for 5 min at 1000g. Protein amount was quantified with Bio-Rad (Hercules, CA) DC Protein Assay using bovine serum albumin as a standard. The protein amount was adjusted to 500 μg/mL, and the hydrolysis reactions were carried out in 96-well plates (Corning). Caco-2, AsPC-1, and Capan-2 cell suspensions (250 μL) were placed in triplicate wells, the reactions started with the addition of substrate, and cells were incubated at 37 °C for 120 min. At the desired time point, sample aliquots (35 μL) were removed and added to 150 μL of acetonitrile (ACN) containing 0.1% TFA. The mixtures were filtered with a 0.45 μm filter at 1000g for 10 min at 4 °C. The filtrate was then analyzed via reverse-phase HPLC.

The stability of the prodrugs in human plasma was determined using the procedure below. Undiluted plasma (250 μL) was added to each well in triplicate, and substrate was added to initiate the reactions that were conducted at 37 °C for 2 h. At various time points, aliquots (35 μL) were removed and added to 150 μL of ACN containing 0.1% TFA. The mixtures were filtered with a 0.45 μm filter at 1000g for 10 min at 4 °C. The filtrate was then analyzed via reverse-phase HPLC.

The nonenzymatic hydrolysis of the prodrugs was determined as described above, except that each well contained pH 7.4 phosphate buffer (10 mmol/L) instead of cell homogenate or human plasma.

The stability of floxuridine and its prodrugs in the presence of thymidine phosphorylase (TP) was assessed by incubating the desired substrates (200 μM) with TP (2.0 ng/μL) in phosphate buffer (pH 7.0) at 37 °C. Aliquots of the incubation mixture were sampled at 0, 1, 3, 5, 10, 30, 60, and 120 min, and quenched with cold acetonitrile (ACN) with 0.1% TFA, filtered through a 0.45 μm membrane, and analyzed for the concentrations of prodrug, floxuridine, and 5-FU by HPLC.

Caco-2 cells at nine days postseeding, and AsPC-1 and Capan-2 cells, both at four days postseeding, were incubated with 10 μmol/L Gly-Sar (9.98 μmol/L Gly-Sar and 0.02 μmol/L [³H]Gly-Sar) along with various prodrug concentrations (5−0.05 mmol/L) for 30 min. The cells were washed three times with ice-cold PBS and solubilized with 10 mL of scintillation cocktail (ScintiVerse, Fisher Scientific, St.Louis, MO), and the amount of cell-associated radioactivity was determined by scintillation counting (Beckman LS-9000, Beckman Instruments, Fullerton, CA). IC₅₀ values were determined using nonlinear data fitting (GraphPad Prism version 3.0).

Caco-2 cell monolayers were grown on collagen-coated polytetrafluoroethylene membranes for 21 to 24 days, and Capan-2 cell monolayers were grown on the same type of membrane for 14 days. Transepithelial electrical resistance (TEER) was monitored, and values of 240−280 Ω/cm² in Caco-2 and 380−420 Ω/cm² in Capan-2 (total area for both cells was 4.67 cm²) were used in the study. Apical side and basolateral sides of transwell inserts were washed with MES (pH 6.0) and HEPES (pH 7.4), respectively. Fresh MES and HEPES buffers were reapplied to transwell inserts and incubated at 37 °C for 15 min. Freshly prepared 0.1 mM drug solution in MES buffer (total 1.5 mL) was placed in the donor chamber, and the receiver chamber was filled with HEPES buffer (total 2.5 mL) Sampling from the receiver chamber (200 μL) was conducted up to a period of 2 h at time intervals of 15, 30, 45, 60, 75, 90, and 120 min, at 37 °C and replaced with an equal volume of fresh HEPES buffer to maintain sink conditions in the receiver chamber. All samples were immediately acidified with 0.1% TFA and analyzed by reverse-phase HPLC.

The initial rates of hydrolysis were used to obtain the apparent first-order rate constants and to calculate the half-lives. The apparent first-order degradation rate constants of various floxuridine prodrugs at 37 °C were determined by plotting the logarithm of prodrug remaining as a function of time. The slopes of these plots are related to the rate constant k and given by The degradation half-lives were then calculated by the equation Statistical significance was evaluated with GraphPad Prism v. 3.0 by performing one-way analysis of variance with posthoc Tukey’s test to compare means.

The apparent permeability (P_app) for the prodrugs was calculated using the following equations: where J_ss is the steady state flux, M is the cumulative amount of prodrug, and regenerated mono amino acid prodrug, drug and 5-FU in the receiver compartment. The apparent permeability was calculated from steady state flux as follows: where A is the surface area of monolayer exposed to the permeant and C₀ is the concentration of the prodrug in the donor solution. The concentrations of floxuridine and its prodrugs in the receiver and donor compartments were analyzed using HPLC.

The concentrations of prodrugs and their metabolites were determined on a Waters HPLC system (Waters, Inc., Milford, MA). The HPLC system consisted of two Waters pumps (model 515), a Waters autosampler (WISP model 712), and a Waters UV detector (996 photodiode array detector) controlled by Waters Millennium 32 software (version 3.0.1). Samples were resolved in a Waters Xterra C₁₈ reverse-phase column (5 μm, 4.6 × 250 mm) equipped with a guard column. The mobile phase consisted of 1% HFBA/water (solvent A) and 1% HFBA/acetonitrile (solvent B) with the solvent B gradient changing from 0−56% at a rate of 2%/min during a 28 min run. Standard curves generated for each prodrug, and their parent drugs were utilized for quantitation of integrated area under peaks. The detection wavelength was 254 nm, and spectra were acquired in the 220−380 nm range.

Cell proliferation studies were conducted with AsPC-1 and Capan-2 cell lines. The cells were seeded onto 96-well plates at 125,000 cells per well and allowed to attach/grow for 24 h before drug solutions were added. The culture medium (RPMI-1640 + 10% fetal bovine serum) was removed, and the cells were gently washed once with sterile pH 6.0 uptake buffer. Floxuridine and floxuridine prodrugs were 2-fold serially diluted in pH 6.0 uptake buffer from 4 to 0.25 mmol/L. Buffer alone was used as 100% viability control. The wash buffer was removed, and 25 μL of drug solution per well was added and incubated at 37 °C for 2 h with AsPC-1 cells and 4 h with Capan-2 cells in the cell incubator. After this time period, the drug solutions were removed and the cells were gently washed twice with sterile uptake buffer. Fresh culture medium was then added to each well after washing, and the cells were allowed to recover for 24 h before evaluating cell viability via 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assays. A mixture (30 μL) containing XTT (1 mg/mL) in sterile RPMI-1640 without phenol red and phenazine methosulfate (N-methyl dibenzopyrazine methyl sulfate in sterile PBS, 0.383 mg/mL) reagents was added to the cells and incubated at 37 °C for 1 h, after which the absorbance at 450 nm was read. GI₅₀ values were calculated using GraphPad Prism version 3.0 by nonlinear data fitting.

Dipeptide floxuridine prodrugs were synthesized using a method similar to that described earlier.^11,13 The total prodrug yield for each dipeptide was >15%, and the purity for all prodrugs was >90% as determined by HPLC, where the impurity was the parent drug, floxuridine. All prodrug structures and their identities were confirmed by ESI-MS and NMR. The data for prodrug purity and mass are shown in Table 1.

The experiments concerning prodrug stability were performed at 37 °C in pH 7.4 phosphate buffer. The estimated half-lives (t_1/2) obtained from linear regression of pseudo-first-order plots of prodrug concentration vs time for floxuridine prodrugs in pH 7.4 phosphate buffers alone and in Caco-2, AsPC-1, and Capan-2 cell homogenates are listed in Table 2. Prodrug metabolites such as floxuridine and 5-FU were monitored along with prodrug disappearance in this experiment. However, mass balance was not achieved because 5-FU was metabolized even further and those metabolites beyond 5-FU were not quantified (Figure 1). 5′-l-Isoleucyl-floxuridine exhibited the highest stability in all media tested. Dipeptide prodrugs with at least one aromatic amino acid moiety were next-best with regard to stability in enzyme-containing solutions such as plasma and cell homogenates, and those with two aromatic amino acids also exhibited chemical stability that was similar to that of 5′-l-isoleucyl-floxuridine. All prodrugs exhibited 3- to 30-fold shorter half-lives in cell homogenates than in pH 7.4 phosphate buffer suggesting enzyme-catalyzed hydrolysis. With a few exceptions, the half-lives of all monoester and dipeptide prodrugs tested exhibited similar trends in the three cell homogenates and showed good linear correlations (r² = 0.87−0.93). The composition of the amino acids in the dipeptide moiety exerted a profound effect on the stability of the ester bond regardless of the mode of attachment. Thus, glycyl-containing dipeptide prodrugs were less stable in buffer alone compared to dipeptide prodrugs containing one or two aromatic amino acids. Glycyl-containing dipeptide prodrugs were also less stable than the 5′-l-glycyl ester prodrug. A comparison of the stability of 5′-l-isoleucyl-floxuridine in various media with 5′-l-isoleucyl-l-glycyl-floxuridine dramatically illustrates this effect (Table 2).

The metabolic stability of floxuridine and its amino ester prodrugs was assessed using thymidine phosphorylase. The results shown in Table 3 indicate that floxuridine was rapidly degraded to the less active metabolite, 5-fluorouracil, by thymidine phosphorylase. The amino acid ester prodrugs of floxuridine were found to be quite resistant to degradation by thymidine phosphorylase. Prodrugs containing the glycyl moiety, 5′-l-glycyl, 5′-l-leucyl-l-glycyl, 5′-l-glycyl-l-leucyl, 5′-l-phenylalanyl-l-glycyl, and 5′-l-isoleucyl-l-glycyl floxuridine were 20- to 40-fold more stable than floxuridine to degradation by thymidine phosphorylase. The half-lives of 5′-l-phenylalanyl-floxuridine, 5′-l-isoleucyl-floxuridine, 5′-l-valyl-l-phenylalanyl-floxuridine, and 5′-l-phenylalanyl-l-tyrosyl-floxuridine were in excess of 500 min reflecting their superior resistance to metabolic degradation by thymidine phosphorylase. The results are consistent with the stability of the prodrugs in buffer systems.

IC₅₀ values of the amino acid/dipeptide monoester prodrugs of floxuridine for PEPT1 determined using inhibition of Gly-Sar uptake in Caco-2, AsPC-1, and Capan-2 cells are summarized in Table 4. All ester prodrugs of floxuridine exhibited greater affinity for PEPT1 than the parent drug. Floxuridine showed minimal inhibition of Gly-Sar uptake, and IC₅₀ values were 8.43 ± 2.66 mM, 6.63 ± 1.52 mM, and 16.06 ± 6.71 mM in Caco-2, AsPC-1, and Capan-2 cells, respectively. 5′-l-Isoleucyl-l-glycyl-floxuridine displayed the highest affinity for oligopeptide transporters in Caco-2 cells (IC₅₀ = 0.39 ± 0.01 mM) and in AsPC-1 cells (IC₅₀ = 0.29 ± 0.00 mM) and was second-best in Capan-2 cells (IC₅₀ = 0.44 ± 0.02 mM). With the exception of 5′-l-leucyl and 5′-l-glycyl dipeptide analogs with Caco-2 cells, all dipeptide prodrugs of floxuridine exhibited 2- to 14-fold higher affinity for oligopeptide transporters than the corresponding 5′-l-monoester prodrug. In particular, dipeptide prodrugs with at least one aromatic amino acid component consistently exhibited 3- to 5-fold higher affinity for the oligopeptide transporters than the corresponding monoester prodrug in all three cell lines tested.

The apical-to-basolateral permeability of mono amino acid/dipeptide monoester prodrugs of floxuridine and parent floxuridine were determined at 37 °C in Caco-2 and Capan-2 cell monolayers. Table 5 shows the permeability values in the two cell systems. All ester prodrugs of floxuridine exhibited 4- to 20-fold higher permeability than floxuridine in Caco-2 cells. With the exception of 5′-l-valyl-l-phenylalanyl-floxuridine, the permeability of all other dipeptide prodrugs of floxuridine across Caco-2 monolayers was 2- to 4-fold higher than the corresponding 5′-l-mono amino acid ester floxuridine prodrug. The permeability trends in Caco-2 monolayers are consistent with the affinity data observed in uptake inhibition studies; however, no simple correlations between permeability and affinity were evident. Floxuridine was impermeant with Capan-2 cell monolayers; neither floxuridine nor its metabolite 5-FU could be detected suggesting the possibility of upregulation of metabolic enzymes such as TP and dihydropyrimidine dehydrogenase (DPD) in Capan-2 cells. DPD is the rate-limiting enzyme involved in the catabolism of pyrimidines and is also the main enzyme involved in the degradation of structurally related compounds like 5-FU (Figure 1). Floxuridine permeability was indeed observed in the presence of DPD inhibitors, dipyridamole and cisplatin; however, it was 1,000- or 10,000-fold lower than that obtained with Caco-2 cells (data not shown). The permeability of 5′-l-valyl-l-phenylalanyl-floxuridine across Caco-2 monolayers was quite low. 5′-l-Phenylalanyl-l-tyrosyl-floxuridine, however, exhibited dramatically lower permeability in Capan-2 cells compared to its permeability in Caco-2 cells. With the exception of 5′-l-phenylalanyl-l-tyrosyl-floxuridine, the permeability of the floxuridine ester prodrugs in Caco-2 cells was about 2-fold higher than the corresponding value in Capan-2 cells (r² = 0.83). Unlike Caco-2 monolayers, general permeability enhancement effects with dipeptide prodrugs compared to the corresponding 5′-l-mono amino ester prodrug were not apparent with Capan-2 cell monolayers.

GI₅₀ values for floxuridine and its 5′-mono amino acid/dipeptide monoester prodrugs determined in cell proliferation studies with the pancreatic cancer cell lines, AsPC-1 and Capan-2, are shown in Table 6. All prodrugs exhibit 4- to 8-fold enhanced antiproliferative activity in the two cell lines compared to parent floxuridine. Thus, the GI₅₀ values of all floxuridine prodrugs were in the range of 1.65−3.36 mM in AsPC-1 and Capan-2 cells as opposed to GI₅₀ values of 22.85 mM and 17.63 mM for floxuridine in AsPC-1 and Capan-2 cells, respectively. These results are consistent with trends observed in Gly-Sar uptake inhibition studies. Gly-Sar and Gly-Pro did not inhibit the growth rate of Capan-2 cells. Additionally, 5′-d-valyl-floxuridine exhibited antiproliferative activity similar to that of floxuridine and Gly-Sar, despite an affinity for PEPT1 that was similar to that of other mono amino acid/dipeptide monoester prodrugs of floxuridine.