N-(2-Hydroxypropyl)methacrylamide Copolymers of a Glutathione

Bioconjugate Chem. 2005, 16, 598−607

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N-(2-Hydroxypropyl)methacrylamide Copolymers of a Glutathione (GSH)-Activated Glyoxalase I Inhibitor and DNA Alkylating Agent: Synthesis, Reaction Kinetics with GSH, and in Vitro Antitumor Activities Zhe-Bin Zheng,† Guozhang Zhu,† Heekyung Tak,† Erin Joseph,‡ Julie L. Eiseman,‡ and Donald J. Creighton*,† Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, and University of Pittsburgh Cancer Center, Pittsburgh, Pennsylvania 15213. Received February 13, 2004; Revised Manuscript Received March 28, 2005

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called “enhanced permeability and retention” effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k ≈ 2.3 mM-1 min-1) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (Ki ) 46 nM), or with poly-HPMA-7 (k ≈ 0.02 mM-1 min-1) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC50 values of 168 ( 8 and 284 ( 5 µM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.

INTRODUCTION

A conceptually important strategy for improving tumor targeting during cancer chemotherapy is to use high molecular weight copolymer prodrugs to better direct the drug to tumor tissue via the so-called “enhanced permeability and retention” (EPR)1 effect (for reviews, see ref 1). This effect arises, in part, from the tendency of high molecular weight copolymer prodrugs (>70 000 Da) to remain in circulating plasma longer than low molecular weight drugs and from the fact that blood vessels in rapidly growing tumors are more permeable to high molecular weight species than are blood vessels in normal, established tissues. Once concentrated in tumor tissue, the copolymer prodrug can then enter tumor cells by endocytosis. The challenge is to design copolymer prodrugs that will release the antitumor agents into the cell cytosol after endocytosis. A frequently used design strategy is to covalently attach the drug to the polymer via a peptide linkage, which will undergo catalyzed hydrolysis when * To whom correspondence should be addressed. Tel: 410455-2518. Fax: 410-455-2608. E-mail: [email protected]. † University of Maryland. ‡ University of Pittsburgh Cancer Center. 1 Abbreviations: EPR, enhanced permeability and retention; GPC, gel permeation chromatography; AIBN, azobisisobutylnitrile; HPMA, N-2-(hydroxypropyl)methacrylamide; Mn, number average molecular weight; Mw, weight average molecular weight; GlxI, glyoxalase I.

the copolymer prodrug-filled endosomes fuse with the peptidase-filled lysosomes. The free drug is then available to diffuse out of the lysosomes into the cell cytosol. Indeed, prodrug conjugates of poly-N-2-(hydroxypropyl)methacrylamide (HPMA) have been designed to deliver the anticancer drugs doxorubicin (2), daunomycin (3), and 5-fluorouracil (4) and the DNA alkylating agent melphalan (5) into tumor cells. Moreover, some of these conjugates are now entering phase I/II clinical trials (6). Nevertheless, a potential limitation of this approach is that cleavage of the peptide bond in the linker tends to be a slow process taking several hours (2). An alterative strategy that avoids reliance on lysosomal peptidases is to use lysosomal thiols such as GSH and/or cysteine as cleaving agents after endocytotic uptake of appropriately designed copolymer prodrugs (7). This approach is particularly interesting because GSH concentrations are often elevated by as much as 2-fold in tumor tissues (8-10) vs normal tissues, which are in the range of 2-8 mM (10, 11). Moreover, some drug resistant tumors overexpress GSH by as much as 10-fold (12). Thus, these concentration differences could give rise to preferential activation of the copolymer prodrug in tumor cells vs normal cells. The adventitious activation of the copolymer prodrug in circulating human plasma should be minimal, as GSH concentrations are typically 1-2 µM (13). To test the feasibility of this strategy, we have been working to prepare copolymer prodrugs that will react with GSH to give two different classes of GSH

10.1021/bc0499634 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/19/2005

HPMA Copolymers of GSH-Activated Antitumor Agents

Bioconjugate Chem., Vol. 16, No. 3, 2005 599

Scheme 1

Scheme 2

derivatives currently undergoing development as a new generation of antitumor agents (14-17). The first class of antitumor agents are transition state analogue inhibitors of the human methylglyoxal-detoxifying enzyme glyoxalase I (hGlxI) (18); e.g., S-(N-4chlorophenyl-N-hydroxycarbamoyl)glutathione (1, Ki ) 46 nM) (19). Previous work has shown that this compound can be delivered into tumor cells either as a [glycyl, glutamyl] diethyl ester prodrug 1(Et)2 (20) or as the membrane permeable sulfoxide prodrug S-(N-4-chlorophenyl-N-hydroxycarbamoyl)ethyl sulfoxide 2, which undergoes an acyl interchange reaction with GSH to give 1 (Scheme 1) (21). Both prodrugs are cytostatic and cytotoxic to several different tumor cell lines in vitro, apparently because inhibition of GlxI by the enediol analogue 1 results in the accumulation of cytotoxic methylglyoxal. The basis of the exceptional sensitivity of tumors to methylglyoxal toxicity is not well-understood but appears to arise, in part, from extensive covalent modification of heat shock protein 27 (Hsp27), which induces caspase-dependent differentiation and apoptosis (programmed cell death) in tumor cells (22). Methylglyoxal is also potentially genotoxic, as methylglyoxal has been demonstrated to covalently modify nucleotide bases (23, 24). The second class of antitumor agents includes electrophilic endocyclic enones such as 2-crotonyloxymethyl-2cyclohexenone (3), which rapidly diffuses across cell membranes and, once inside the cell, undergoes a Michael addition reaction with intracellular GSH to give the reactive exocyclic enone 4 (Scheme 2) (25, 26). This species can potentially react with either free GSH to give 5 or form covalent adducts with proteins and/or nucleic acids (6) critical to cell viability. Indeed, mass spectral studies show that in the presence of GSH compound 3 alkylates the exocyclic amino groups of nucleotide bases composing single-stranded oligonucleotides (27). However, neither of the prodrug strategies described above is designed to deliver antitumor agents specifically

to tumor tissue. For example, in vivo efficacy studies show that intravenous administration of 1(Et)2 to tumorbearing mice inhibits the growth of B16 melanotic melanoma, PC3 prostate tumors, and HT29 human colon tumors (28). While there were no significant side effects detected in these short-term efficacy studies, intravenous administration of 1(Et)2 results in the appearance of 1 in all major organs of tumor-bearing mice. This could give rise to significant side effects during long-term administration of drug. Here, we describe for the first time the synthesis, physicochemical properties, and in vitro antitumor activities of two lead HPMA copolymer prodrugs, which rapidly release 1 and/or the reactive exocyclic enone 4 in neutral aqueous solutions of GSH. EXPERIMENTAL PROCEDURES

Analytical Instrumentation. NMR spectra were taken on a GE QE-300 NMR spectrometer. IR spectra measured with a ThermoNicolet Avatar 370 FTIR spectrometer using a Pike MIRacle ATR accessory (AMTIR crystal). Mass spectral data were obtained at the Center for Biomedical and Bio-organic Mass Spectrometry, Washington University. UV spectra were recorded using a Beckman DU 640 spectrophotometer. High-performance liquid chromatography (HPLC) was carried out using a Waters HPLC System composed of a 600 Controller, Delta 600 Pumps, and 996 photodiode array detector. Analytical HPLC was performed using a Waters Nova-Pak C18, 4 µm, 3.9 mm × 150 mm column or Symmetry C18, 5 µm, 4.6 mm × 150 mm column. Preparative HPLC was performed using a SymmetryPrep C18, 7 µm, 19 mm × 150 mm column. The molecular weights of the HPMA copolymers were estimated by gel permeation chromatography (GPC) using a high-performance gel permeation column (Tricorn Superose 12 10/ 300 GL) from Amersham Biosciences. Materials. Dextran molecular weight standards were purchased from Sigma Chem. Co. (1000, 5000, and 12 000

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Scheme 3. Mechanism for Generating t.s. Analogue 1 and Exocyclic Enone 4 from Copolymer-Prodrug Conjugate in the Presence of GSH

Da) and Polysciences, Inc. (40 000 Da). Lyophilized human serum and GSH were purchased from Sigma Chem. Co. HPMA was purchased from Polysciences, Inc. Azobisisobutylnitrile (AIBN), methacryloyl chloride, 4-methylmorpholine, and 3-chloroperoxybenzoic acid (8085% pure) were purchased from Aldrich Chem. Co. All other reagents were of the highest purity commercially available. S-(N-4-Chlorophenyl-N-hydroxycarbamoyl)thioethylamine (9). To a solution of S-(N-4-chlorophenylN-hydroxycarbamoyl)ethyl sulfoxide (2) (21) (495 mg, 2 mmol) in a mixture of 12 mL of methanol and 12 mL of phosphate buffer (0.1 M, pH 7.5) was added a solution of cysteamine (990 mg, 12.9 mmol) in 5 mL of phosphate buffer (0.1 M, pH 7.5). The mixture was stirred at 0 °C for 1 h. The precipitate was collected by filtration, washed with water, and dried under vacuum to give the final product as a white solid; yield 86% (424 mg). 1H NMR (300 MHz, methanol-d4/TMS): δ 3.01 (2H, t, J ) 6.6 Hz), 3.50 (2H, t, J ) 6.6 Hz), 7.32 (2H, d, J ) 9.2 Hz), 7.61 (2H, d, J ) 9.2 Hz). HRMS (ESI): m/z 247.0294 (calcd for C9H12N2O2SCl: 247.0308). [Note added in proof: This procedure must form the thiol ester and not the amide because the “sulfoxide”-containing copolymers (synthesized using 9; Scheme 4) react with free GSH to give the enediol analogue 1 at rates similar to those observed for the reaction of GSH with the simple sulfoxide 2 to give 1 (Scheme 1); see Discussion.] S-(N-4-Chlorophenyl-N-hydroxycarbamoylthioethyl)methacrylamide (8). To a stirring solution of 9 (410 mg, 1.66 mmol) in 26 mL of anhydrous dimethyl formamide and 13 mL of pyridine at 0 °C was added slowly over 30 min methacryloyl chloride (318 µL, 3.29 mmol), and the reaction mixture was stirred at room temperature for an additional 20 min. The solvent was removed in vacuo, and the residue was fractionated by reversephase HPLC, using 50% acetonitrile in water, containing 0.1% trifluoroacetic acid, as a running solvent. The product peak was collected and dried under vacuum overnight to give the final product as a white solid; yield 51% (269 mg). 1H NMR (300 MHz, methanol-d4/TMS): δ 1.90 (3H, s), 3.00 (2H, t, J ) 6.6 Hz), 3.45 (2H, t, J ) 6.6 Hz), 5.34 (1H, br s), 5.67 (1H, br s), 7.32 (2H, d, J ) 9.2 Hz), 7.59 (2H, d, J ) 9.2 Hz). HRMS (ESI): m/z 337.0379 (calcd for [M + Na]+, C13H15N2O3NaSCl: 337.0390). 2-Methacryloyloxymethyl-2-cyclohexenone (7). To a stirring solution of 2-hydroxymethyl-2-cyclohexenone

Scheme 4. Synthesis of HPMA Conjugates of 7- and 8-Sulfoxidea

Polyacrylamide

a Reagents and conditions: (i) AIBN/acetone, 55 °C; (ii) 3-chloroperoxybenzoic acid; (iii) mercaptoethylamine/methanolphosphate buffer (pH 7.5); (iv) methacryloyl chloride/pyridine; and (v) methacryloyl chloride/4-methylmorpholine.

10 (29) (504 mg, 4 mmol) and 4-methylmorpholine (1 mL, 9 mmol) in 10 mL of CH2Cl2 at 0 °C was added methacryloyl chloride (0.79 mL, 8 mmol) dropwise over about 30 min. The reaction mixture was allowed to stir for an additional 20 min. The solvent was removed in vacuo, and the crude product was fractionated by preparative reverse phase HPLC using 40% acetonitrile in water, containing 0.1% trifluoroacetic acid, as a running solvent. The product peak was collected and brought to dryness under vacuum to give the final product as a colorless oil; yield 66% (515 mg). 1H NMR (300 MHz, acetone-d6): δ 1.96 (3H, s), 2.03 (2H, p, J ) 6.2), 2.48 (2H, t, J ) 7.33 Hz), 2.42-2.60 (2H, m), 4.84 (2H, s), 5.58 (1H, br s), 6.13 (1H, br s), 7.01 (1H, t, J ) 4.3 Hz). HR-FABMS (3-NBA/ Li): m/z 201.1100 (calcd for [M + Li]+, C11H14O3Li: 201.1103). Copolymer P1. The methacryloyl derivative 8 (41 mg, 0.13 mmol), HPMA (110 mg, 0.77 mmol), and AIBN (6 mg) were dissolved in 0.75 mL of acetone under argon in


a closed vial and incubated at 50-55 °C for 24 h. The white precipitate was recovered by filtration and was dried under vacuum for 30 min. The crude product was dissolved in 0.25 mL of methanol and then precipitated by the slow addition of excess acetone:diethyl ether (3: 1). The precipitation procedure was repeated, and the precipitate was dried under vacuum overnight; yield 63 mg. A portion of the residue (30 mg) was dissolved in 1 mL of methanol at 0 °C, and 3-chloroperoxybenzoic acid (5.6 mg, 0.017 mmol) in 0.05 mL of diethyl ether was slowly added dropwise to this solution. The reaction mixture was allowed to stir for an additional 1 h at 0 °C. The solvent was removed in vacuo, the residue was dissolved in 0.2 mL of methanol, and the desired product precipitated by the slow addition of 5 mL of excess diethyl ether. The precipitation procedure was repeated twice more, and the product was dried under vacuum overnight to give the final product; yield overall from 8 and HPMA, 12 mg. IR (ATR, AMTIR): br 3352, 2973, 2934, 2907, s 1636, s 1526, s 1487 (S-O), 1202 cm-1. Copolymers P2 and P3. These copolymers were prepared by the same general procedure used to prepare P1, with reaction mixtures having the following composition. P2: 8 (21 mg, 0.067 mmol), HPMA (76 mg, 0.53 mmol), and AIBN (2 mg) dissolved in 0.4 mL of acetone. Acetone was used as a precipitant during the precipitation procedures; mass yield 29%. IR (ATR, AMTIR): br 3349, 2972, 2930, 2886, s 1636, s 1527, 1487 (S-O), 1203 cm-1. P3: 8 (42 mg, 0.13 mmol), HPMA (282 mg, 2 mmol), and AIBN (18 mg) dissolved in 1.6 mL of acetone; yield 32 mg. IR (ATR, AMTIR): br 3353, 2972, 2934, 2905, s 1638, s 1527, 1487 (S-O), 1202 cm-1. 1 H NMR (300 MHz, Methanol-d4/TMS) Spectra of P1-P3. The spectra were all very similar to one another, with significant line broadening due to the high molecular weights of the copolymers. The chemical shift assignments were based on comparisons with the NMR spectra of poly-HPMA and 8; relative integrated intensities varied as a function of the mol % of the 8-sulfoxide function: δ 1.01 [s, CH3C(C)3], 1.16 (d, CH3CO-), 1.62.0 [m, -(CH2)-HPMA], 2.9-3.3 (m, -NCHaHbCO-; N-CH2CH2-S), 3.87 (m, -HCO-), 7.40 (d, arom. H, meta to Cl), 7.70 (d, arom. H, ortho to Cl). The mol % of the 8-sulfoxide function in the different polymers was estimated from the integrated intensities of the aromatic ring protons (δ 7.40, 7.70) vs that of H-C-O (δ 10.02): P1, ∼10; P2, ∼9; and P3, ∼5. Copolymers P4-P6. These copolymers were prepared by the same general procedure used to prepare P1 with the exception that the oxidation step with 3-chloroperoxybenzoic acid was not used. P4: 7 (68 mg, 0.351 mmol), HPMA (100 mg, 0.702 mmol), and AIBN (9 mg) dissolved in 0.8 mL of acetone; yield 38 mg. IR (ATR, AMTIR): br 3366, 2970, 2931, m 1723 (CdO, cyclohexenone function), s 1639, s 1527, 1175, 1138 cm-1. P5: 7 (44 mg, 0.227 mmol), HPMA (130 mg, 0.907 mmol), and AIBN (9 mg) dissolved in 0.7 mL of acetone; yield 112 mg. IR (ATR, AMTIR): br 3362, 2970, 2929, m 1724 (CdO, cyclohexenone function), s 1639, s 1528, 1199, 1138 cm-1. P6: 7 (15 mg, 0.076 mmol), HPMA (87 mg, 0.61 mmol), and AIBN (5 mg) dissolved in 0.5 mL of acetone; yield 52 mg. IR (ATR, AMTIR): br 3354, 2971, 2931, m 1721 (CdO, cyclohexenone function), s 1637, s 1527, 1201, 1137 cm-1. 1 H NMR (300 MHz, Methanol-d4/TMS) Spectra of P4-P6. The spectra were all very similar to one another, with significant line broadening due to the high molecular weights of the copolymers (Figure 1). The chemical shift assignments were based on comparisons with the NMR spectra of poly-HPMA, 7, and 8; relative integrated


intensities varied as a function of the mol % of the cyclohexenone function derived from 7: δ 1.01 [s, CH3C(C)3], 1.16 (d, CH3CO-), 1.6-2.0 [m, -(CH2)-HPMA], 2.0-2.15 [m, -(C(4)H2)-cyclohex-2-enone ring], 2.452.60 [m, -(C(5)H2, C(6)H2)-cyclohex-2-enone ring], 2.93.3 (m, -NCHaHbCO-; N-CH2CH2-S), 3.87 (m, -HCO), 7.2-7.3 (t, vinyl-H). The mol % of the cyclohexenone function was estimated from the integrated intensities of the cyclohexenone ring protons C(5)H2, C(6)H2 (δ 9.85), vs that of H-C-O (δ 10.02): P4, ∼27; P5, ∼20; and P6, ∼12. Copolymer P7. A solution of 7 (15 mg, 0.076 mmol), 8 (24 mg, 0.076 mmol), HPMA (87 mg, 0.61 mmol), and AIBN (6 mg) in 0.6 mL of acetone under argon in a closed vial was heated at 50-55 °C for 24 h. The white precipitate was recovered by filtration and brought to dryness to give 70 mg of crude product. The crude product was dissolved in 2 mL of methanol at 0 °C, and 3-chloroperoxybenzoic acid (9.1 mg, 0.042 mmol) in 0.1 mL of diethyl ether was added dropwise. The reaction mixture was allowed to stir for an additional 1 h, the solvent was removed in vacuo, and the residue was dissolved in 0.4 mL of methanol. The product was precipitated by the dropwise addition of acetone:diethyl ether (3:1). The precipitation procedure was repeated, and the final copolymer product was dried under vacuum overnight; yield 52 mg. IR (ATR, AMTIR): br 3364, 2970, 2930, m 1721(CdO, cyclohexenone function), 1638, 1527, s 1487 (S-O), 1200, 1137 cm-1. Copolymer P8 was prepared by the same general procedure used to prepare P7 starting with a reaction mixture composed of 7 (16 mg, 0.084 mmol), 8 (26 mg, 0.084 mmol), HPMA (191 mg, 1.336 mmol), and AIBN (12 mg) dissolved in 1.2 mL of acetone; yield 91 mg. IR (ATR, AMTIR): br 3365, 2971, 2931, m 1723(CdO, cyclohexenone function), 1639, 1527, s 1482 (S-O), 1201, 1138 cm-1. 1 H NMR (300 MHz, Methanol-d4/TMS) Spectra of P7 and P8. The spectra were both similar to one another, with significant line broadening due to the high molecular weights of the copolymers (Figure 1). The chemical shift assignments were based on comparisons with the NMR spectra of poly-HPMA, 7, and 8; relative integrated intensities varied as a function of the mol % cyclohexenone and 8-sulfoxide functions: δ 1.01 [s, CH3C(C)3], 1.16 (d, CH3CO-), 1.6-2.0 [m, -(CH2)-HPMA], 2.0-2.15 [m, -(C(4)H2)-cyclohex-2-enone ring], 2.45-2.60 [m, -(C(5)H2, C(6)H2)-cyclohex-2-enone ring], 2.9-3.3 (m, -NCHaHbCO-), 3.87 (m, -HCO-), 7.2-7.3 (t, vinyl-H), 7.40 (d, arom. Hs, meta to Cl), 7.70 (d, arom. Hs, ortho to Cl). The mol % of the 8-sulfoxide and the cyclohexenone functions were estimated from the integrated intensities of the aromatic ring protons (δ 7.40, 7.70) and cyclohexenone ring protons C(5)H2, C(6)H2 (δ 2.5), vs that of H-C-O (δ 3.9). For P7: mol % 8-sulfoxide, ∼2%; cyclohexenone function, ∼2%. For P8: mol % 8-sulfoxide, ∼6%; cyclohexenone function, ∼6%. S-(N-4-Chlorophenyl-N-hydroxycarbamoyl)propylsulfoxide (2a). To S-(N-4-chloro-phenyl-N-hydroxycarbamoyl)ethylsulfoxide (2) (21) (150 mg, 0.61 mmol) in 3 mL of pyridine on ice was added thiopropane (138 mg, 1.187 mmol). The reaction mixture was brought to room temperature and stirred for 30 min, and aqueous 1.6 N HCl (15 mL) was slowly added to the stirring reaction mixture. The reaction mixture was extracted with methylene chloride (3 × 10 mL), the organic layer was dried over sodium sulfate, and the solvent was removed in vacuo. The residue was crystallized from hexane to give the synthetic intermediate S-(N-4-chlorophenyl-N-hy-


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Figure 1. Spectral data for HPMA-copolymer P7 containing both the sulfoxide and the 2-methylenecyclohexenone functions: (top) IR (ATR, AMTIR); (bottom) 1H NMR 300 MHz (methanol-d4/TMS) spectra. Spectral lines unique to the three functional groups in the copolymers are indicated in the spectra. Other assignments may be found in the Experimental Procedures.

droxycarbamoyl)thiopropyl ester as light brown crystals (yield 41%). The ester (0.252 mmol) was dissolved in 2 mL of diethyl ether to which was added dropwise 3-chloroperoxybenzoic acid (0.248 mmol) in 2 mL of diethyl ether. The white precipitate was collected and washed with diethyl ether to give the final product; the yield from the intermediate ester was 48%. 1H NMR (300 MHz, methanol-d4/TMS): δ 0.99 (3 H, t, J ) 7.3 Hz), 1.73 (2 H, m), 3.04 (2 H, m), 7.37 (2 H, d, J ) 9.2 Hz), 7.68 (2 H, d, J ) 9.2 Hz). S-(N-4-Chlorophenyl-N-hydroxycarbamoyl)butylsulfoxide (2b). This compound was prepared by a procedure analogous to that used to prepare 2a; yield 29%. 1H NMR (300 MHz, CDCl3/TMS): δ 0.87 (3 H, t, J ) 7.3 Hz), 1.40 (2 H, m), 1.70 (2 H, m), 3.08 (2 H, m), 7.37 (2 H, d, J ) 8.8 Hz), 7.69 (2 H, d, J ) 8.8 Hz).

Determination of the Molecular Weights of the Copolymer Prodrugs. The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw/Mn) of the HPMA copolymers were obtained by high-performance GPC (30). The gel permeation column was eluted with 50 mM sodium phosphate buffer (pH 7.0) containing 0.25 M NaCl at a flow rate of 1 mL/min. Molecular weights were interpolated from standard curves (log Mn and log Mw vs retention time) obtained using polydextran molecular weight standards. Determination of mol % Sulfoxide and Cyclohexenone Functions in the Copolymers. Ethanolic solutions of copolymer were prepared, and 20 µL aliquots were mixed with 80 µL of 10 mM GSH in potassium phosphate buffer (0.1 M, pH 7.5) and incubated at room temperature for 10 min to convert the 8-sulfoxide and/


Figure 2. Elution profile from a reverse phase HPLC column (Waters Symmetry C18 5 µm column, 4.6 mm × 150 mm) of a reaction mixture obtained after incubating P8 (Table 1) with 10 mM GSH in potassium phosphate buffer (0.1 M, pH 6.5) for 15 min. Abscissa: percent total absorbency (200-400 nm). Peaks at 1.02, 1.42, and 3.72 min correspond to GSH, the GSH2-methylcyclohexenone adduct 5, and the transition state analogue 1, respectively. Running solvent: 25% acetonitrile in water containing 0.1% TFA.

or cyclohexenyl functions to 1 and the GSH adduct 5, respectively. A 10 µL aliquot of this solution was then fractionated by reverse phase HPLC (Nova-Pak C18, 4 µm, 3.9 mm × 150 mm column). For the analysis of 1, the running solvent was 25% acetonitrile in water, containing 0.1% trifluoroacetic acid; for 5, the running solvent was 13% acetonitrile in water, containing 0.1% trifluoroacetic acid. The areas under the peaks corresponding to 1 and 5 were converted to mole quantities by comparison with standard curves of peak areas vs moles of authentic 1


and 5 injected onto the same column. The amounts of 1 and 5 were then used to calculate mol fractions of 8-sulfoxide and cyclohexenyl groups in the original copolymer. Kinetics of Drug Release. Reactions were initiated by the introduction of ethanolic solutions of copolymer into cuvettes containing at least a 20-fold excess of GSH over the equivalents of 8-sulfoxide and/or cyclohexenyl groups in degassed/N2-saturated potassium phosphate buffer (0.1 M), pH 6.5, 25 °C. Rate constants were calculated from the first-order decrease in absorbency at 305 and 235 nm resulting from the loss of 8-sulfoxide and cyclohexenyl groups, respectively (Figure 3). Stability of Copolymers in Human Serum. To 0.45 mL of human serum at 37 °C was added P8 to an initial concentration of approximately 1 mM in 8-sulfoxide and cyclohexenyl groups. As a function of time, 30 µL aliquots of the incubation mixture were transferred to 20 µL of potassium phosphate buffer (0.1 M, pH 7.5) containing 1.6 mM GSH to convert the 8-sulfoxide and cyclohexenyl functions to 1 and 5, respectively. After incubation at room temperature for 5 min, the samples were deproteinized by the addition of 100 µL of ethanol. The protein precipitate was sedimented by centrifugation at 13000g, and the supernatant was fractionated by reverse phase

Figure 3. Time-dependent change in the UV spectrum of (A) P2 (0.025 mM in 8-sulfoxide) in potassium phosphate buffer (0.1 M, pH 6.5), GSH (0.5 mM), EDTA (0.05 mM), and 2.5 vol % ethanol at 25 °C (spectral scans taken every 25 s) and of (B) P6 (0.05 mM in 7) in potassium phosphate buffer (0.1 M, pH 6.5), GSH (1.0 mM), and 5 vol % ethanol at 25 °C (spectral scans taken every 10 min).


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HPLC, as described above. The transition state analogue 1 and the GSH adduct 5 were quantitated, as described above. The rate constants were calculated from the firstorder rate of loss of 8-sulfoxide or cyclohexenyl groups as a function of the time. In Vitro Cytotoxicity Studies. Murine B16 melanotic melanoma was obtained from the DCT Tumor Repository (NCIsCancer Research and Development Center, Frederick, MD) and was maintained in RPMI 1640 medium containing L-glutamate (Gibco, BRL, Gaithersburg, MD), supplemented with 10% heat-inactivated fetal calf serum and gentamycin (10 µg/mL), under 37 °C humidified air containing 5% CO2. Under these conditions, B16 cells have a doubling time of about 26 h. For the toxicity studies, cells in logarithmic growth were introduced into 24 well plates at a density of 2 × 104 cells/ mL in the absence and presence of drug spanning the IC50 values. After 72 h, the cells were washed with Hank’s balanced salt solution without Ca2+, treated with trypsin for 10 min at 37 °C, and concentrated by centrifugation, and cell densities were determined with a Coulter Counter. Cell viabilities were determined by the trypan blue exclusion method (31). Reported IC50 values (mean ( standard deviation of triplicate determinations carried out in three separate assays on different days) were calculated using the Hill equation and the program Adapt (32). RESULTS

Synthetic Methods. Two new classes of HPMA copolymer prodrugs have been prepared, which employ the same prodrug chemistry previously used to generate the transition state analogue inhibitor of GlxI (1) and exocyclic enone 4 in the presence of GSH (Scheme 3). The prodrugs were prepared by radical polymerization of variable amounts of 2-methacryloylmethyl-2-cyclohexenone 7 and N-(4-chlorophenyl-N-hydroxycarbamoylthioethyl)methacrylamide 8 with HPMA, followed by treatment with 3-chloro-peroxybenzoic acid to oxidize the thioester function to a sulfoxide (Scheme 4). Repeated precipitation from methanol/diethyl ether solution gave copolymer preparations that were free of unreacted 8-sulfoxide and/or 7 by HPLC. The yields were in the range of 28-64%. The polyacrylamide monomers were prepared by an extension of published procedures (21, 25). IR and NMR spectroscopy (e.g., Figure 1) confirmed the chemical identities of the polymers. The 1487 and 1721 cm-1 bands were assigned to the S-O and CdO stretching frequencies of the 8-sulfoxide and cyclohexenenone functions, respectively, on the basis of comparisons with the IR spectra of 2 and 10. The integrated intensities of the resonances shown in the NMR spectrum could be used to calculate approximate values for the mol % compositions of the copolymers. However, more precise values were obtained by reacting the copolymers with GSH and quantitating the GSH adducts 1 and 5 after isolation by HPLC (see the Experimental Procedures). Kinetics of Drug Release. Incubation of the HPMA copolymers with GSH produced a time-dependent increase in product species, which comigrated with authentic samples of transition state analogue 1 and GSH-2methylcyclohexenone adduct 5 monitored by reverse phase HPLC; e.g., Figure 2. The rate constants for formation of these species correlated well with the first-order rate of decrease in absorbency at 305 and 235 nm, corresponding to the loss

Figure 4. Rate profiles for the reaction of (A) copolymer P6 (0.05 mM in cyclohexenyl equivalents) with GSH (1 mM) and of (B) copolymer P3 (0.025 mM in 8-sulfoxide equivalents) with GSH (0.5 mM). Conditions: Potassium phosphate buffer (0.1 M, pH 6.5), 25 °C. The rate constants given in the figures are the best-fit values to the expression for a first-order exponential decay (solid line through the data).

of the sulfoxide and cyclohexenone functions of the copolymers, respectively (Figure 3). These wavelengths were selected for the kinetic analyses, as they permit the independent assessment of the rates of loss of each functional group in copolymers containing both functional groups. The rates of formation of 5 and 1 from copolymers containing variable amounts of either the cyclohexenyl and/or the 8-sulfoxide groups conform to first-order kinetics over 4-5 half-lives (e.g., Figure 4) (Table 1). Stabilities in Human Plasma. The likely chemical stabilities of the 8-sulfoxide and cyclohexenyl groups in the copolymers under physiological conditions were estimated by determining the time-dependent loss of these groups from P8 during incubation human serum at 37 °C. The half-lives for loss of the 8-sulfoxide and cyclohexenyl groups were determined to be 6.6 ( 0.7 and 37.5 ( 7.5 min (n ) 3), respectively. In Vitro Efficacy Studies. Both the HPMA-8-sulfoxide copolymer and the HPMA-7 copolymer inhibit the growth of B16 in a concentration range where the HPMA polymer alone shows no activity (Figure 5). Therefore, growth inhibition must result from the prodrug component of the copolymer. Not unexpectedly, the in vitro efficacies of the unpolymerized prodrugs exceed those of the copolymer prodrugs (Table 2). The simple ethyl, propyl, and butyl sulfoxide prodrugs (2-2b) have IC50 values that are roughly 5-10-fold lower than that of HPMA-8-sulfoxide (P1), while the IC50 value of 3 is over 7000-fold lower than that of HPMA-7 (P4).



Table 1. Mol % Compositions and Spectrophotometrically Determined Second-Order Rate Constants (k2) for the Reaction of GSH with the Sulfoxide and Cyclohexenone Functions of Different Copolymer Prodrugsa copolymer designation

sulfoxide (mol %)

P1 P2 P3 P4 P5 P6 P7 P8

8.7 ( 0.30 5.2 ( 0.03 1.5 ( 0.04

4.3 ( 0.03 3.6 ( 0.04

cyclohexenone (mol %)

Mn (Da) × 10-3

Mw/ Mn

k2 (-sulfoxide) (mM-1 min-1)

19.4 ( 0.7 12.0 ( 0.4 6.4 ( 0.1 4.6 ( 0.2 1.9 ( 0.01

2.80 7.44 26.5 13.5 30.0 31.5 15.4 26.9

1.41 1.47 1.55 1.51 1.56 1.57 1.52 1.55

1.85 ( 0.12 2.54 ( 0.04 2.38 ( 0.07

k2 (-cyclohexenone) (mM-1 min-1)

2.49 ( 0.051 2.11 ( 0.074

0.015 ( 0.001 0.015 ( 0.002 0.021 ( 0.003 0.021 ( 0.001 0.021 ( 0.001

a Kinetic constants were calculated from the first-order rate of loss of the functional group under pseudo-first-order conditions (>20fold excess of GSH) and are the average of triplicate determinations ( standard deviation. Conditions: potassium phosphate buffer (0.1 M, pH 6.5), 25 °C.

Table 2. In Vitro Inhibition of B16 Melanotic Melanoma by Different HPMA Copolymer-Prodrug Conjugates vs the Monomeric Prodrugs compound Copolymer Prodrugs HPMA-8 sulfoxide (P1) HPMA-7 (P4) HPMA-(8-sulfoxide/7) (P7) Monomer Prodrugs cyclohexenone 3b (Scheme 2) ethyl sulfoxide 2 (Scheme 1) propyl sulfoxide 2a butyl sulfoxide 2b

IC50a (µM) 168 ( 8 284 ( 5 107 ( 9 0.04 ( 0.01 35 ( 8 29 ( 5 15 ( 2

a Values are means (( SD) of three experiments each of which was carried out in triplicate. b Value taken from ref 26.

Figure 5. Growth inhibition of B16 melanoma in the presence of (A) P1 or (B) P4. Data for copolymer drug conjugates are indicated by squares; data for polymer controls with no appended drugs are indicated by triangles. The drug concentration is calculated, on the basis of equivalents of drug/L. For conditions, see the Experimental Procedures.

In contrast, the IC50 values for P1, P4, and the copolymer containing both prodrugs (P7) differ by no more than a factor of 3. DISCUSSION

The central outcome of this work is that the kinetic properties and antitumor activities of the lead compounds described here support the feasibility of using HPMA copolymers to deliver cytotoxic GSH derivatives into tumor cells. Kinetic Properties of the HPMA Copolymers. HPMA copolymers are well-designed to serve as platforms for delivering GSH-activated prodrugs into cells via endocytosis. The reaction between excess GSH and the copolymers to form the GlxI inhibitor 1 or the

alkylating agent 4 follows simple first-order kinetic behavior over several half-lives (e.g., Figure 4). This indicates that the copolymers exist primarily in an open or extended conformation in solution, which allows free access of GSH to the reactive groups appended to the copolymers. Moreover, the polyacrylamide backbone does not interfere significantly with the reaction between the GSH and the 8-sulfoxide function, as the rate constants for reaction of GSH with copolymers P1-P3 (Table 1) are at least as large as that reported for the reaction of GSH with the simple sulfoxide derivative 2 (Scheme 1): k ) 1.84 ( 0.07 mM-1 min-1, potassium phosphate buffer (0.1 M, pH 7.5), 25 °C (21). Indicative of a small steric effect on the reaction of GSH with the cyclohexenone functions of P4-P6, the rate constants are about 3-fold smaller than that reported for the reaction of GSH with the crotonate ester 3 (Scheme 2): k ) 0.068 ( 0.001 mM-1 min-1, potassium phosphate buffer (0.1 M, pH 6.5), 25 °C (17). Therefore, the ∼100-fold greater reactivities of the sulfoxide- vs cyclohexenone-containing copolymers reflect primarily the different intrinsic chemical reactivities of the functional groups with GSH and not the steric properties of the polymer backbone. Finally, there is little difference in the kinetic properties of the copolymers having either different molecular weights or different mole fractions of the appended sulfoxide or cyclohexenone functions (Table 1). Therefore, loading of the HPMA polymer with high levels of prodrug will not adversely affect the kinetic properties of the copolymers. Not surprisingly, the sulfoxide and cyclohexenone functions in the mixed function copolymers P7 and P8 independently react with free GSH, as the rate constants for the individual reactive groups are similar in magnitude to those for the copolymers containing only one of the reactive groups (Table 1). Model for Endocytotic Drug Delivery. There is a substantial body of evidence indicating that endocytosis is the dominant mechanism for delivering HPMA copoly-


mers into tumor cells (1). In the past, HPMA copolymers were designed to depend on intralysosomal peptidases to cleave the linker between the HPMA and the drug to allow the drug to diffuse into the cytoplasm of the tumor cell. In the present study, thiols rather than peptidases function to liberate the drugs into the cytoplasm of the tumor cell. While there are no good estimates of the steady state concentrations of specific thiols inside the lysosomes of mammalian cells, there is ample evidence to indicate that lysosomes contain highly specific transporter systems for importing cysteine and cysteinyl dipeptides such as cysteinyl-glycine into lysosomes (33). For example, a highly specific cysteine-dependent transporter has been found in human fibroblast lysosomes with a Km ) 0.5 mM, and evidence has been presented for the efflux of cystine and cysteine from these lysosomes (34). An important role of cysteine is to activate intralysosomal thiol-dependent proteases that function to breakdown proteins taken up by endocytosis. Early evidence that GSH also stimulates intralysosomal protein breakdown was attributed to a GSH transporter in the lysosomal membrane (35). However, subsequent studies indicate that some or perhaps all of the activation is due to breakdown of GSH to cysteinylglycine, which is subsequently transported into the lysosomes (36). Therefore, the presence of a GSH transport system in lysosomes has not been firmly established at this time. In Vitro Efficacy. The antitumor activities of the HPMA copolymer prodrugs listed in Table 2 are most likely due to the copolymers themselves and not to toxic contaminants in the copolymer preparations. Antitumor activity is unlikely to arise from unreacted sulfoxide-8 and/or 7 (Scheme 4), as neither NMR nor HPLC analysis of the copolymer preparations indicates that these species are present. Conceivably, 1 and/or 4 might form in the growth medium during the efficacy studies, due to reaction of the copolymers with contaminating GSH (arising from cell lysis). However, this is an unlikely source of antitumor activity, because highly charged S-substituted GSH derivatives such as 1 and 4 do not readily diffuse across cell membranes near neutral pH (20). Thus, the observed antitumor activities most likely result from processing of the HPMA copolymers inside tumor cells, during endocytosis. The in vitro antitumor activity of sulfoxide copolymer P1 (Table 2) implies the presence of intralysosomal GSH to react with P1 to give the cytotoxic transition state analogue inhibitor 1. This follows from reports that yeast GlxI is highly specific for GSH and will not use cysteine or cysteinylglycine as cofactors (37). Conceivably, the cysteine and/or cysteinylglycine homologues of 1 might form inside the lysosomes, diffuse across the lysosomal membrane into the cytosol, and then form 1 via acyl interchange with cytosolic GSH. This kind of cofactor specificity is not required to explain the cytotoxicity of the cyclohexenone-containing copolymer P7, as intralysosomal GSH, cysteine, or cysteinylglycine could all react with P7 to give cytotoxic exocyclic enones. Both the sulfoxide- and the cyclohexenone-containing copolymer prodrugs are significantly less potent than the low molecular weight prodrugs designed to enter cells by diffusion across the cell membrane. HPMA-8 sulfoxide P1 is about 6.5-fold less potent than sulfoxides 2, 2a, and 2b, and HPMA-7 (P4) is about 7 × 103-fold less potent that the simple substituted cyclohexenone 3. This is not a new phenomenon. For example, the HPMA copolymer of 6-(3-aminopropyl)ellipticine (APE) is >75-fold less potent than APE with B16F10 melanoma in vitro, although the copolymer is significantly more potent in

Zheng et al.

tumor-bearing mice (38). This has been attributed to the slow rate of endocytotic uptake of the copolymer and the slow rate of peptide hydrolysis of the linker, which can have half-lives on the order of many hours. Likewise, endocytotic uptake of the copolymers described in the current study might be a slow process; the rate constants for formation of 1 and 4 at pH 5.3 will be approximately 50-fold smaller than the rate constants that apply in the cytosol with a pH of 7. Moreover, efficacy might also be limited by the rate of expulsion of the prodrugs from the lysosomes into the cytoplasm of the cell. Thus, the chemotherapeutic potential of the copolymer prodrugs is difficult to appreciate prior to completion of in vivo efficacy studies. Drug Delivery Strategies. The stability of the copolymer prodrugs in circulating human plasma is another important aspect of drug efficacy in humans. The approximate half-lives for the reaction of the sulfoxide- and cyclohexenone-containing copolymers with free GSH (∼2 µM) (13) that applies in circulating human plasma at pH 7.5 are estimated to be about 175 and 17400 min, respectively, using the rate constants in Table 1. However, these half-lives are significantly longer than the half-lives of 7 and 38 min determined for the loss of the sulfoxide and cyclohexenone functions, respectively, when the copolymers are incubated in noncirculating human plasma. Therefore, the chemistry associated with the latter process is most significant in determining the bioavailability of the copolymer prodrugs to tumor cells. Compounds with such short half-lives in circulating plasma would be poor candidates for optimizing drug pharmacokinetics via controlled release. The HPMA copolymer prodrugs undergoing clinical evaluation at this time have half-lives in circulating plasma on the order of hours (39). However, continuous infusion might still be used to optimize the plasma pharmacokinetics of less stable copolymer prodrug conjugates and allow tumor targeting via the EPR effect. Finally, the lead compounds containing mixed functional groups (P7, P8) provide a precedent for the application of copolymer prodrugs in combination chemotherapy. ACKNOWLEDGMENT

This work was supported by a grant from the NCI (CA 59612) and a grant from Maryland Technology Development Corporation. We thank Diana S. Hamilton and Ellyn Sharkey of this laboratory for preparing the starting materials for the syntheses of 8 and 7. LITERATURE CITED (1) (a) Brocchini, S., and Duncan, R. (1999) Pendent drugs, release from polymers. In Encyclopedia of Controlled Drug Delivery (Mathiowitz, E., Ed.) pp 786-816, John Wiley and Sons: New York. (b) Duncan, R. (2003) The dawning era of polymer therapeutics. Nat. Rev. Drug Discovery 2, 347-360. (c) Thanou, M., and Duncan, R. (2003) Polymer-protein and polymer-drug conjugates in cancer therapy. Curr. Opin. Invest. Drugs 4, 701-709. (d) Duncan, R. (1992) Drug-polymer conjugates for improving chemotherapy. Anti-Cancer Drugs 3, 175-210. (2) Seymour, L. W., Ulbrich, K., Wedge, S. R., Hume, I. C., Strohalm, J., and Duncan, R. (1991) N-(2-Hydroxypropyl)methacrylamide copolymers targeted to the hepatocyte galactose-receptor: Pharmacokinetics in DBA mice. Br. J. Cancer 63, 859-866. (3) Duncan, R., Kopeckova, P., Strohalm, J., Hume, I. C., Lloyd, J. B., and Kopecek, J. (1988) Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. II. Evaluation of daunomycin conjugates in vivo against L1210 leukaemia. Br. J. Cancer 57, 147-156.


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BC0499634

N-(2-Hydroxypropyl)methacrylamide Copolymers of a Glutathione

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