HPMA-based polymer conjugates with drug combination

European Journal of Pharmaceutical Sciences 37 (2009) 405–412

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HPMA-based polymer conjugates with drug combination Hana Krakoviˇcová ∗ , Tomáˇs Etrych, Karel Ulbrich Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic v.v.i., Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

a r t i c l e

i n f o

Article history: Received 8 December 2008 Received in revised form 18 March 2009 Accepted 20 March 2009 Available online 1 April 2009 Keywords: HPMA copolymer Drug carriers Doxorubicin Dexamethason Combination therapy Anti-tumor activity Carboxyesterase

a b s t r a c t Synthesis and physico-chemical behavior of new polymer–drug conjugates intended for the treatment of cancer were investigated. In the polymer conjugate with the expected dual therapeutic activity, two drugs, a cytostatic agent doxorubicin (DOX) and anti-inflammatory drug dexamethason (DEX) were covalently attached to the same polymer backbone via hydrolytically labile pH-sensitive hydrazone bonds. The precursor, a copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) bearing hydrazide groups randomly distributed along the polymer chain, was conjugated with DOX (through its C13 keto group) or with a keto ester (DEX). Two derivatives of DEX, 4-oxopentanoate and 4-(2-oxopropyl)benzoate esters, were synthesized and employed for conjugation reaction. As a control, also a few polymer conjugates containing only a single drug (DOX or DEX) attached to the polymer carrier were synthesized. Physicochemical properties of the polymer conjugates strongly depend on the attached drug, spacer structure and the drug content. Polymer–drug conjugates incubated in buffers modeling intracellular environment released the drug (DOX) or a drug derivatives (DEX) at the rate significantly exceeding the release rate observed under conditions mimicking situation in the blood stream. Incubation of the DEX conjugates in a buffer containing carboxyesterase resulted in complete ester hydrolysis thus demonstrating susceptibility of the system to release free active drug in the two-step release profile. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The anti-cancer drugs mostly consist of low-molecular-weight compounds hence they are very quickly excreted from organism by glomerular filtration or entrapped by the reticulo-endothelial system and metabolized (Ringsdorf, 1975). The main disadvantages of such anti-cancer low-molecular-weight drugs are non-specific body distribution, low bioavailability and various, often toxic side effects. With the aim to reduce the side effects, improve the drug distribution in the body, prolong its blood circulation and persistence in the body, the low-molecular-weight drugs were incorporated into polymeric nanoparticles and liposomes or covalently conjugated to water-soluble polymer carriers. Previously, many types of polymers were used as water-soluble drug carriers, e.g. poly(l-glutamic acid) (Li, 2002; Tansey et al., 2004), poly(ethylene glycol) (Veronese and Pasut, 2005) and its biodegradable multiblock polymers (Pechar et al., 2001, 2005), N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers (Etrych et al., 2001; Kopeˇcek and Duncan, 1987; Ulbrich et al., 2003) or poly(vinylpyrrolidone) (Bharali et al., 2003; Yamamoto et al., 2004). HPMA copolymers rank among the most intensively studied

∗ Corresponding author. Tel.: +420 296 809 216; fax: +420 296 809 410. E-mail address: [email protected] (H. Krakoviˇcová). 0928-0987/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2009.03.011

polymer–drug carrier systems. Some of the HPMA copolymer–drug conjugates have been subjected to clinical trials (Julyan et al., 1999; Vasey et al., 1999). Previously, the anti-cancer drug doxorubicin (DOX) was attached to the HPMA copolymers via enzymatically degradable oligopeptide spacer GlyPheLeuGly (GFLG) (Duncan et al., 1983; Ulbrich et al., 1980) or via a hydrolytically labile spacer containing hydrazone bond susceptible to pH-controlled hydrolysis (Etrych et al., 2001, 2002). The oligopeptide spacer GFLG used in the HPMA copolymer–DOX conjugate was tailored for specific enzymatic cleavage by lysosomal enzymes in lysosomes of the target cells, the spacer was stable during blood circulation. The conjugates containing spacer with the pH-sensitive hydrazone linkage were fairly stable in model buffers at pH 7.4, simulating blood pH, and released the drug by chemical hydrolysis at pH modeling the endosomal and lysosomal environment inside the target cell (pH 5–6). It was shown previously that high-molecular-weight polymers (above the limit of the renal threshold) accumulate in tumor tissues at much higher concentrations than in normal tissues or organs. Maeda defined such accumulation of polymers in solid tumors as the enhanced permeability and retention effect (EPR effect) (Maeda and Matsumura, 1989). The EPR effect is caused by the ‘leaky’ endothelium of angiogenic tumor vasculature and lack of effective tumor lymphatic drainage. Therefore, the polymers in solid tumors can easily extravasate but cannot be removed by the lymphatic sys-

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tem and remain entrapped in the tumor (Matsumura and Maeda, 1986). Recently, in agreement with new findings in oncology, a new approach in designing polymer–drug conjugates intended for treatment of cancer has appeared—the polymer–drug conjugates bearing a combination of two drugs differing in their mechanism of action, both attached to the same carrier via biodegradable spacers (Kopeˇcek et al., 1990; Shiah et al., 2001; Vicent et al., 2005). HPMA copolymer conjugates, with combination of DOX and BSRNase or DOX and RNase A exhibited a synergistic effect in vivo and effectively inhibited growth of melanoma, solid tumors and metastases in mice (Pouˇcková et al., 2004; Souˇcek et al., 2001, 2002). Here we describe the synthesis and physico-chemical characteristics of the new types of conjugates using HPMA copolymers as drug carriers, bearing the anti-inflammatory and anti-cancer drug dexamethasone or a combination of two anti-cancer drugs, DOX and DEX. DEX has been used for treatment of many inflammatory and autoimmune diseases, malignant diseases such as lymphatic leukemia, lymphomas and multiple myeloma. DEX has been also used for treatment of cancer patients undergoing chemotherapy to eliminate side effects of the treatment. It was shown that treatment of multiple myeloma and other types of cancer with DEX combined with anti-cancer drugs, e.g. doxorubicine, vincristine, bortezomid or thalidomide (Alexanian et al., 1992; Hussein et al., 2002; Oakervee et al., 2005; Tosi et al., 2004) represents an effective method that produces more rapid response than other regimens (Hussein, 2003). Unfortunately, long-lasting continuous infusion is required for such treatment. Use of a polymer carrier bearing both drugs and releasing them simultaneously for long period of time can help to overcome this problem and bring improvement into the treatment. Various spacers enabling pH-dependent hydrolytically controlled drug release were described as suitable structures for designing polymer–drug conjugates with anti-cancer activity ˇ (Ulbrich and Subr, 2004). In the conjugates used in this study the drug or its derivative was attached to the polymer carrier via hydrolytically degradable hydrazone bond. In the DEX-containing conjugates the drug was first esterified with 4-oxopentanoic acid (levulic acid, LEV) or 4-(2-oxopropyl)benzoic acid (OPB) and then

the respective ester derivate was attached to the polymer via hydrazone bond in the same way as in the case of DOX. Several polymer conjugates containing either single drug (DOX, DEX derivative), or combination of both drugs were synthesized and their physico-chemical properties were studied. The rates of release of the drugs or their derivatives from the polymer conjugates were studied in vitro in buffers modeling blood circulation or intracellular environment. 2. Experimental 2.1. Chemicals N,N -dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC), dimethylformamide (DMF), 4-(dimethylamino)pyridine (DMAP), dichloromethane, methanol, tetrahydrofuran, 2,2 -azobis(isobutyronitrile) (AIBN), 1-aminopropan-2-ol, methacryloyl chloride, methyl 6-aminohexanoate hydrochloride (ah), hydrazine hydrate, dexamethasone (DEX), doxorubicin hydrochloride (DOX·HCl) and 4-oxopentanoic acid were purchased from Fluka. 4-(2Oxopropyl)benzoic acid was obtained from Rieke Metals. 2,4,6-Trinitrobenzene-1-sulfonic acid (TNBSA) was purchased from Serva, Heidelberg. Germany. Rabbit liver carboxyesterase (EC 3.1.1.1) was obtained from Sigma–Aldrich. 2.2. Synthesis of monomers N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized by methacryloylation of 1-aminopropan-2-ol as described earlier (Ulbrich et al., 2000) using Na2 CO3 as a base. M.p. 64–66 ◦ C; elemental analysis: calculated C 58.74, H 9.0, N 9.79; found: C 58.81, H 9.09, N 9.82. 6-Methacrylamidohexanohydrazide (Ma-ah-NHNH2 ) was prepared as described in the literature (Etrych et al., 2008). M.p. 79–81 ◦ C; elemental analysis: calculated C 56.32, H 8.98, N 19.70; found: C 56.49, H 8.63, N 19.83. Purity of monomers was examined in Shimadzu HPLC system equipped with a reverse-phase column Chromolith Performance RP-18e (100 mm × 4.6 mm) (water–acetonitrile, gradient 0–100%

Fig. 1. Dexamethasone derivatives.

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Fig. 2. Polymer conjugate 6 with drug combination (DEX and DOX).

acetonitrile) with UV–VIS detection (Shimadzu SPD-M10A vp) (220 nm). 2.3. Synthesis of DEX derivatives A DEX ester with levulic acid (DEX–LEV, see Fig. 1) was synthesized by esterification of hydroxy group on C21 of DEX with levulic acid as follows: DCC (39.5 mg, 0.191 mmol) was dissolved in 200 ␮L DMF, levulic acid (16.3 mg, 0.141 mmol) was dissolved in 200 ␮L of DMF, both solutions were mixed and cooled for 20 min to −18 ◦ C. Then a solution of DEX (50 mg, 0.128 mmol) and DMAP (6 mg, 0.05 mmol) in 200 ␮L DMF was added. The reaction proceeded at 4 ◦ C for 26 h, its course being monitored by TLC: Rf (DEX) = 0.62, Rf (DEX–LEV) = 0.74, Rf (DEX–LEV2 , diester) = 0.8, using ethyl acetate as mobile phase. Free DEX and its dilevulate ester were removed from the reaction mixture on a column filled with Silica gel 60 (2 cm × 30 cm, eluent ethyl acetate–dichloromethane 2:1) with UV detection (240 nm). Eluates containing pure DEX–LEV were collected, the solvent was evaporated and a waxy solid was triturated with diethyl ether. The product was finally isolated by filtration. The yield was 20 mg (30%). 1 H NMR 300 MHz (CDCl , 297 K) ppm: 7.19–7.23 d (1H, C–CH–CH); 3

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6.32–6.36 d (1H, CH–CH–(C O)); 6.12 s (1H, C–CH–(C O)); 4.67 s (2H, O–CH2 –(C O)) (Fig. 1). 13 C NMR 300 MHz (DMSO-d , 297 K) ppm: 206.51 (C-21); 204.63 6 (C-26); 185.17 (C-3); 171.69 (C-23); 166.96 (C-5); 152.64 (C-1); 128.89 (C-2); 124.00 (C-4); 101.17 (d, J = 175.8 Hz, C-9); 90.34 (C-17); 70.38 (d, J = 37 Hz, C-11); 67.87 (C-22); 47.83 (d, J = 22.8 Hz, C-10); 47.82 (C-13); 43.73 (C-14); 37.28 (C-25); 35.54 (C-12); 35.24 (C-16); 33.47 (d, J = 19.3 Hz, C-8); 31.80 (C-15); 30.16 (C-6); 29.45 (C-27); 27.22 (C-7 and C-24); 22.86 (C-19); 16.13 (C-20); 15.01 (C-18). Purity of DEX–LEV (97.4%) was determined by HPLC as described above for monomers (peak maximum at 11.2 min (240 nm)). DEX 4-(2-oxopropyl)benzoate (DEX–OPB, see Fig. 1) was synthesized according to DEX–LEV: OPB (99.8 mg, 0.56 mmol) together with EDC (146.7 mg, 0.77 mmol) were dissolved in a mixture of 1.5 mL DMF and 0.5 mL dichloromethane and kept at −18 ◦ C for 20 min. Then a solution of DEX (200 mg, 0.51 mmol) and DMAP (62.33 mg, 0.51 mmol) in 1.5 mL DMF was added and the reaction mixture was kept at 4 ◦ C for 24 h. The reaction course was monitored by TLC (ethyl acetate–hexane, 2:1) (Rf (DEX) = 0.34, Rf (DEX–OPB) = 0.61). The reaction mixture was purified on a column filled with Silica gel 60 (2 cm × 30 cm, ethyl acetate–dichloromethane, 2:1) using UV detector (240 nm). DEX–OPB was obtained after evaporation of the solvent, washing with ethyl acetate, filtration and drying in vacuum. The yield was 101.5 mg (42%). HPLC showed 97% purity (peak maximum at 11.7 min). 1 H NMR 300 MHz (DMSO-d + D O, 297 K): 7.93–7.96 d and 6 2 7.35–7.38 d (2H, C–CH–CH–C); 7.32–7.35 d (1H, C–CH–CH); 6.23–6.27 d (1H, CH–CH–(C O)); 6.03 s (1H, C–CH–(C O)); 5.04–5.3 dd (2H, O–CH2 –(C O)). 13 C NMR 300 MHz (DMSO-d , 297 K) ppm: 205.28 (C-21); 204.82 6 (C-29); 185.26 (C-3); 167.05 (C-5); 165.10 (C-23); 140.89 (C-27); 130.16 (C-25); 129.25 (C-26); 128.99 (C-2); 127.57 (C-24); 124.09 (C-4); 99.01 (d, J = 167.5 Hz, C-9); 90.55 (C-17); 70.52 (d, J = 37 Hz, C11); 68.66 (C-22); 49.301 (C-28); 48.06 (C-13); 47.79 (d, J = 20.6 Hz, C-10); 43.31 (C-14); 35.69 (C-12); 35.46 (C-16); 33.58 (d, J = 19.17 Hz, C-8); 30.47 (C-6); 29.73 (C-30); 27.29 (C-7); 22.97 (C-19); 16.25 (C20); 15.13 (C-18). 2.4. Synthesis of polymer precursor Polymer precursor poly(HPMA-co-Ma-ah-NHNH2 ) (1, Table 1) was prepared by radical copolymerization of HPMA and Ma-ahNHNH2 in methanol (monomer concentration 18 wt.%, molar ratio 93:7) using AIBN (0.8 wt.%) as initiator. HPMA (1.27 g, 8.88 mmol), Ma-ah-NHNH2 (143 mg, 0.67 mmol) and AIBN (63 mg, 0.38 mmol) were dissolved in methanol (8 mL). The solution in an ampule was bubbled with nitrogen and then sealed. The polymerization was carried out at 60 ◦ C for 17 h. The polymer was isolated by precipitation into 160 mL of ethyl acetate and purified by reprecipitation from methanol into ethyl acetate. The polymer was filtered off, washed with ethyl acetate and dried. The yield was 1.12 g (79.2%).

Table 1 Characteristics of polymer precursor and polymer conjugates. Polymer precursor and conjugates

Polymer precursor

DEX esters

Mw

Mw /Mn

DEXa (wt.%)

DOX (wt.%)

Rh (nm)

1 2 3 4 5 6 7

– 1 1 1 1 3 4

– DEX–LEV DEX–OPB DEX–OPB – DEX–OPB DEX–OPB

27,000 41,000 34,000 30,000 28,500 56,000 43,000

1.9 2.1 2.2 2.1 2.0 2.2 2.0

– 5.0 6.6 2.3 – 6.6 2.3

– – – – 9.9 8.4 9.5

4.3 5.3 9.3 5.6 4.7 8.5 5.0

a

Content of DEX calculated from content of DEX derivatives.

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2.5. Synthesis of polymer conjugates Polymer conjugates containing only DEX (2–4, Table 1) were prepared by the reaction of the polymer precursor with corresponding derivative of DEX in methanol in the presence of acetic acid. Example of synthesis: 3.6 mg of DEX–OPB ester and 100 mg of polymer precursor were dissolved in 2.5 mL of methanol. Afterwards, 100 ␮L of acetic acid was added and the solution was stirred at room temperature for 2 h. Low-molecular-weight impurities were removed from polymer by gel filtration using Sephadex LH-20 column and methanol as eluent. The polymer fractions were combined and the polymer conjugate was isolated by precipitation into ethyl acetate. The polymer was filtered off, washed with ethyl acetate and dried in vacuum. The yield was 83 mg. Polymer conjugates with DOX attached via hydrazone bond (5–7; Table 1; Fig. 2) were prepared by the reaction of polymer precursor hydrazide or polymer conjugates 3 or 4 with DOX·HCl in methanol as described earlier (Etrych et al., 2001). The polymer–drug conjugates were purified from low-molecularweight impurities as above. 2.6. Characterization of polymer precursor and polymer conjugates All the polymer conjugates were characterized and tested for the content of free polymer or free drug using a HPLC Shimadzu with a GPC TSKgel G3000SWXL column and TLC (Aluminium sheets Kieselgel 60 F254 ). In addition, the content of free DEX was determined by HPLC Shimadzu after chloroform extraction of DEX from aqueous solution. Molecular weights and polydispersities of polymer precursor and the conjugates were determined with the HPLC Shimadzu system equipped with RI, UV and multiangle light scattering DAWN EOS (Wyatt Co., USA) detectors using a TSKgel G3000SWXL column (300 mm × 7.8 mm, 5 ␮m) and the mobile phase consisting of 20% 0.3 M acetate buffer (pH 6.5; 0.5 g/L NaN3 ) and 80% methanol at a flow rate of 0.5 mL/min. The dynamic light scattering (DLS) of conjugates solutions (0.5 wt.%) in a phosphate buffer of pH 7.4 was measured at the scattering angle 173◦ on a Nano-ZS, Model ZEN3600 zetasizer (Malvern, UK). The hydrodynamic radius (Rh ) was determined by the DTS (Nano) program. The content of free hydrazide groups in side chains of the polymer precursor and in polymer conjugates was determined by a modified TNBSA assay as described earlier (Etrych et al., 2001). Molar absorption coefficient ε500 = 17,200 L mol−1 cm−1 ( = 500 nm) estimated for the model reaction of Ma-ah-NHNH2 with TNBSA was used. 1 H NMR and 13 C NMR spectra were measured with a Bruker DPX300 in DMSO-d6 or DMSO-d6 + D2 O. HMBC was measured with a Bruker DPX600 in DMSO-d6 . The total content of DOX in polymer conjugates was determined spectrophotometrically on a Helios ␣ spectrophotometer. Molar absorption coefficients of the free drug (ε488 = 11,500 L mol−1 cm−1 in water) and of the modified DOX (attached via hydrazone bond to Ma-ah-NHNH2 units; ε488 (water) = 9800 L mol−1 cm−1 ) (Etrych et al., 2008) were used for calculation of the DOX content. The total content of DEX derivatives in polymer conjugates was determined with the HPLC Shimadzu system after incubation of the polymer conjugates in HCl solution (pH 2) for 1 h at 37 ◦ C and chloroform extraction of DEX derivatives. 2.7. Carboxyesterase activity The activity of carboxyesterase (EC 3.1.1.1) was determined by incubating the enzyme with the 4-methylumbelliferyl acetate

substrate (4-MUA) in 0.05 M phosphate buffer (pH 7.5) at 37 ◦ C. 10 ␮L of a stock solution of the enzyme in phosphate buffer (equivalent to 3.5 ␮g enzyme), 20 ␮L of a 25 nM stock solution of 4-MUA in DMSO and 970 ␮L of phosphate buffer in a 1-mL cuvette were incubated at 37 ◦ C. The formation of 4-methylumbelliferone (4-MU) was monitored on a Helios ␣ spectrophotometer at 350 nm for 1 h. Molar absorption coefficient ε350 (phosphate buffer) = 12,200 cm−1 mol−1 L for 4-MU was used (Brzezinski et al., 1994, 1997; Dean et al., 1991). The specific enzyme activity was calculated from the linear part of the time dependence of 4-MU formation. The specific activity of carboxyesterase was 1.9 ␮mol 4-MU/min/mg protein. 2.8. In vitro release of drugs from polymer–drug conjugates The rates of DOX and DEX release, free and/or esters, from polymer conjugates were investigated by incubation of the conjugate in phosphate buffers at pH 5.0 or 7.4 (0.1 M phosphate buffer with 0.05 M NaCl) at 37 ◦ C. The concentration of the conjugate in stock solution was equivalent to 3 × 10−5 mM DOX and 5 × 10−5 mM DEX. 200 ␮L of the solution was placed into 1.5 mL vial (one for each time interval). The amounts of released drugs and their esters were determined, after extraction of the vial content into organic solvent, by HPLC analysis as described previously (Etrych et al., 2002). Analysis was performed on the HPLC analyzer (Shimadzu, Japan) using a reverse-phase column Chromolith Performance RP18e (100 × 4.6, eluent water–acetonitrile with acetonitrile gradient 0–100 vol.%, flow rate 0.5 mL/min) with UV detection at 240 nm for DEX and fluorescence detection for DOX (excitation at 488 nm and emission at 560 nm). All drug-release data are expressed as the percentage of free drug relative to the total drug content in the conjugates. 2.9. In vitro release of DEX from conjugates incubated in the presence of carboxyesterase The release rates of DEX and its esters from polymer–drug conjugates (equivalent to 5 × 10−5 mM DEX) were investigated in phosphate buffers containing carboxyesterase at 37 ◦ C (0.1 M phosphate buffer with 0.05 M NaCl, pH 5 and 7.4). General procedure: an enzyme stock solution (0.3 mg/mL phosphate buffer) was added to a solution of polymer conjugate dissolved in phosphate buffer to obtain the final concentration of the enzyme 0.015 mg/mL and substrate concentration equivalent to 5 × 10−5 mM DEX. The solutions containing both the polymer conjugate and enzyme were incubated at 37 ◦ C. At selected time intervals the amounts of released DEX and its esters were determined using their extraction into organic solvent followed by HPLC analysis as described above. 3. Results and discussion Recently, we have shown (Etrych et al., 2001, 2002) that watersoluble HPMA-based copolymers containing the anti-cancer drug doxorubicin bound to the polymer via a pH-labile hydrazone linkage show a high cytotoxic activity in several cancer cell lines and exhibit a significant therapeutic effect in mice bearing an experimental model of EL4 T-cell lymphoma. These hydrazone conjugates are quite stable in a phosphate buffer of pH 7.4, modeling conditions in the blood stream transport, but releasing rapidly free DOX in buffers modeling mild acid environment in endosomes of target cells (pH 5–6). Here, we present the synthesis and physico-chemical characterization of new structures of water-soluble anti-cancer polymer prodrugs based on HPMA copolymers. This study focuses on the

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synthesis and study of physico-chemical behavior of new polymer conjugates based on HPMA copolymers bearing DOX and DEX derivatives attached to the polymer carrier separately or in combination. Both drugs were attached to the polymer carrier via spacers containing pH-labile hydrazone bond enabling controlled release of DOX, DEX or derivatives of DEX. DEX esters were prepared by esterification of C21 hydroxy group with two keto acids, levulic acid or 4-(2-oxopropyl)benzoic acid. Both esters of DEX were attached to the polymer carrier via a spacer containing 6-aminohexanoic acid residue and hydrazone bond. DOX was attached to the same polymer carrier through the hydrazone bond formed in the condensation of its C13 carbonyl group with the hydrazide group of the polymer precursor. Physico-chemical properties of these polymer conjugates, namely the radius of a polymer coil in aqueous solution, molecular weight of the polymer precursor and polymer conjugates, stability of the conjugates incubated in aqueous solutions of different pH and the rates of in vitro drug release from the conjugates incubated in the presence of carboxyesterase were studied. 3.1. Synthesis of DEX esters The polymer precursor was prepared by copolymerization of HPMA with a comonomer containing hydrazide groups suitable for attachment of compounds containing aldehyde or keto group via pH-sensitive hydrazone bonds. First attempts at conjugation of DEX with the polymer precursor were carried out directly with DEX. The yield of the conjugation reaction carried out at 50 ◦ C was fairly high (93%). Unfortunately, the hydrazone bond formed from the polymer carrier and the C3 keto group of DEX was rather stable and the rate of in vitro drug release measured in phosphate buffer at pH 5 was very low (2% of the drug released after 48 h of incubation). The rate of DEX release at pH 7.4 was, as expected, even lower (0.5% in 48 h). Such rate of the drug release is too low to achieve an effective free drug concentration in target cells and any remarkable anti-tumor or anti-inflammatory effect in treated tumors. Because similar results were published also by Wang et al. (2007) we decided to use another strategy for the synthesis of polymer–DEX conjugates which would be based on a less stable pH-sensitive spacer. We prepared hydrolysable DEX esters with the keto group enabling attachment of such drug derivative to a polymer hydrazide precursor via hydrolytically labile hydrazone bonds. Two keto acids, an aliphatic (levulic acid) and an aromatic (4-(2oxopropyl)benzoic acid), were selected as promising candidates for such synthesis. The first derivative, DEX–LEV, was prepared by esterification of primary hydroxy group on C21 of DEX with levulic acid. The lower yield of reaction (30%) was caused by a competing reaction, double esterification of DEX with levulic acid at C11- and C21-OH and by the necessity of careful purification. Free DEX, its diester and other impurities were removed by preparative chromatography on silica. The 97.4% purity of the product was determined by HPLC analysis with UV detection. Its structure was confirmed by 1 H NMR, 13 C NMR and hetero-correlation spectrum C–H (HMBC). The HMBC showed two cross-peaks between C-23 (originated from LEV) and two hydrogen atoms on the C-22 (originated from DEX) demonstrating that both carbons are located on the same molecule. The other ester, DEX–OPB, was prepared by the same method as the levulate ester. Also in this case purification on a silica column was necessary to obtain the pure compound. The yield of DEX–OPB was 42% (97% purity; HPLC). The structure was confirmed by 1 H NMR, 13 C NMR and HMBC. Also in this case the HMBC showed two cross-peaks between C-23 (originated from OPB) and two hydrogen atoms on the C-22 (originated from DEX).

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3.2. Synthesis of polymer precursor and polymer–drug conjugates The polymer precursor 1 was prepared by radical copolymerization of HPMA and MA-ah-NHNH2 in solution, with AIBN as initiator. Molecular weight of the polymer (27,000) was below the limit of the renal threshold and, hence, this polymer is expected to be excreted from the organism by glomerular filtration after fulfilling its function. The polymer precursor 1 contained 5.3 mol% of comonomer units terminating in hydrazide groups (MA-ah-NHNH2 units), this content was sufficient for attachment of both drugs to the polymer precursor. DOX or DEX esters were attached to the polymer precursor by the reaction in methanol in the presence of acetic acid (40 ␮L/mL). The heterogeneous reaction of poorly soluble DOX with polymer precursor proceeded for 24 h giving the soluble product. Both DEX esters were well soluble in methanol and their attachment to the polymer precursor proceeded faster than attachment of DOX. The polymer conjugates purified by gel filtration contained ca. 0.2% of free DOX or DEX esters. The yield of the conjugation reaction reached 92% for DOX, 89% for DEX–OPB and 66% for DEX–LEV. Polymer conjugates with the DEX content ranging from 2.3 to 6.6 wt.% were prepared, while the content of DOX in polymer conjugates was the same. Molecular weight, polydispersity, hydrodynamic radius and the drug content in polymer conjugates 2–7 are shown in Table 1. Molecular weight of the polymer precursor slightly increased after conjugation with drugs. The highest increase in molecular weight was observed after conjugation with both drugs (conjugates 6 and 7); nevertheless, even in this case molecular weight remained below or close to the renal threshold for HPMA polymers (50 kDa). Polydispersity of polymers was not significantly influenced by conjugation. Attachment of DOX to the polymer precursor did not markedly influence the hydrodynamic radius of polymer chain, Rh , increased only slightly from 4.3 to 4.7 nm (see Table 1). This is in good agreement with our earlier results (Etrych et al., 2008). A small increase in Rh was also observed after conjugation of the precursor with DEX–LEV. If one compare, approximately the same molar content of attached DEX–LEV in polymer conjugate 2 induced much higher increase in Rh (ca. 2.5 times) than attachment of DOX in conjugate 5. Increasing the loading of hydrophobic DEX–OPB in the conjugate resulted in a strong increase in the Rh of copolymer. This finding indicates susceptibility of the DEX–OPB-containing conjugate to aggregation in aqueous solution. The difference in physico-chemical behavior between polymer conjugates with DOX and DEX esters could be ascribed to the different hydrophobic/hydrophilic character of attached drugs. While DEX was after attachment a highly hydrophobic substituent, DOX was bound to the polymer carrier through its hydrophobic part (carbonyl group at C13) leaving amino group of daunosamine free for protonization (hydrophilic). Possible protonization leads to decreasing inclination of DOX to hydrophobic interactions. Surprisingly, attachment of both drugs to the same polymer carrier did not contribute to an increase in its Rh . In this case probably the presence of protonized DOX in the polymer structure decreased the tendency of polymer chains to hydrophobic interactions and avoided aggregation of polymer chains and formation of larger polymer coils. 3.3. Release of drugs from polymer conjugates The in vitro release profiles of DEX and its ester from polymer conjugate 2 showed that their rates of release at pH 7.4 (37 ◦ C) are much lower than those at pH 5 (Fig. 3). After 2 h of incubation of polymer conjugate 2 at pH 7.4 only 30% of the sum of DEX and DEX–LEV was released. After 10 min at pH 5 more than 80% of liberated DEX–LEV was found. At pH 7.4 the hydrazone bond was

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Fig. 3. Release of DEX and DEX–LEV from polymer conjugate 2 incubated in phosphate buffers at 37 ◦ C. () DEX–LEV, pH 5; () DEX and DEX–LEV, pH 7.4; () DEX–LEV, pH 7.4; () DEX, pH 7.4.

Fig. 5. Release of DEX–OPB and DOX from polymer conjugate 6 incubated in phosphate buffers at 37 ◦ C. () DOX, pH 5; () DEX–OPB, pH 5; () DEX–OPB, pH 7.4; () DOX, pH 7.4.

cleaved quite rapidly mainly in the first 5 h of incubation with the rate decreasing with decreasing concentration of hydrazone bonds in solution. Subsequently, free DEX was released slowly by spontaneous hydrolysis of ester bond in DEX–LEV by the zero order kinetics. At pH 5 the hydrazone bond was cleaved very quickly and ca. 90% of DEX–LEV was released within 1 h incubation of polymer conjugate. No evidence of hydrolysis of ester bond in DEX–LEV was observed during 24 h incubation at pH 5. The in vitro release experiment carried out with polymer conjugates 3 and 4 showed that the rate of release of DEX and DEX–OPB is lower than in the case of polymer conjugate 2 at both pH (Fig. 4). Also for polymer conjugates 3 and 4 we have proven that the rate of release of drugs at pH 7.4 is much lower than at pH 5. After 2 h incubation in buffer pH 7.4 only 10% of DEX–OPB was released, three times less than DEX–LEV released from conjugate 2. At pH 5 ca. 80% of released DEX–OPB was found after 8 h, which is a much longer time of incubation than in the case of DEX–LEV. No significant difference in the rate of release was observed for polymer conjugates 3 and 4 differing in the amount of bound DEX–OPB. We assume that the difference in the release rates of DEX–LEV and DEX–OPB is influenced by the presence of benzene ring in the spacer between the drug and polymer precursor. Most likely, the presence of aromatic ring stabilised not only the hydrazone bond, but also the ester bond in the DEX ester, because no free DEX was found during incubation at both pH. The necessary requirement for an effectively functioning of drug delivery system is its stability during transport in the body (blood circulation) and release of the drug at the site of action. This requirement was better fulfilled in polymer conjugates 3 and 4 containing DEX–OPB because of their higher stability in solution at pH 7.4. This

was the reason why we have used polymer conjugates 3 and 4 as precursors for the synthesis of polymer conjugates 6 and 7 bearing both drugs (DEX–OPB and DOX). The rate of release of both drugs from polymer conjugates 6 and 7 (Fig. 5) was similar to that observed for conjugates 3 and 4 with DEX–OPB (Fig. 4) or conjugate 5 with DOX (data not shown). This means that attachment of a second drug to the polymer conjugate bearing a drug did not significantly influence the release profiles of both drugs. The conjugates are quite stable in solution at pH 7.4 (blood stream model) releasing up to 30% of DEX–OPB and 4% of DOX after 8 h of incubation. In contrast, more than 65% of DEX–OPB and 50% of DOX was released at pH 5 (pH in cells) during 2 h. The effect of loading of the conjugate with DEX–OPB (conjugates 6 and 7) on the drug release profile was not significant. System described above is a new type of polymer conjugate bearing two drugs differing in their pharmacological activity. The advantage of this system, compared with a mixture of two respective polymer conjugates bearing single drug is that both drugs in the ratio given by the drug loading can be simultaneously distributed in the body, accumulated in tumor and released in the same tissue (tumor) thus increasing the possibility of their synergistic effect.

Fig. 4. Release of DEX–OPB from polymer conjugate 3 incubated in phosphate buffers at 37 ◦ C. () DEX–OPB, pH 5; () DEX–OPB, pH 7.4.

3.4. Drug release from conjugates in the presence of carboxyesterase The above discussed results of drug release experiments confirmed susceptibility of the hydrazone bond to pH-controlled hydrolysis if used as a spacer between the drug and polymer carrier. The conjugates with DEX–OPB and DEX–LEV exhibited high hydrolytic stability of the ester bond to hydrolysis in mild acid environment while the hydrazone bond was cleaved rapidly. This means that DEX was released mainly as its ester. With the aim to verify susceptibility of DEX esters to subsequent enzymatic degradation after their release from a polymer carrier we studied the rate of drug release from polymer–drug conjugates incubated in buffers containing model enzyme rabbit liver carboxyesterase. This enzyme is only one of various types of carboxyesterases found in many cells and tissues of mammals (Leinweber, 1987), especially in cytosol, endoplasmatic reticulum and membranes as well as in tumor tissue (Guang et al., 2002) and they were also found in serum but no carboxyesterase was found in human serum (Li et al., 2005). Detailed mechanism of action of carboxyesterases is not completely known and this is why we used the rabbit liver esterase with pH optimum close to that of liver human esterase (pH 6.5) as a model of the enzyme with esterase activity. The drug release studies proceeded both in a buffer solution of pH 5 (modelling intracellular environment) and of pH 7.4 mimicking conditions in blood stream.

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Fig. 6. Release of DEX and DEX–LEV from linear polymer conjugate 2 incubated in phosphate buffers at 37 ◦ C at pH 5. () Cumulative spontaneous release of DEX (DEX and DEX–LEV); () cumulative release of DEX (DEX and DEX–LEV) in the presence of carboxyesterase; () DEX release in the presence of carboxyesterase; () DEX–LEV release in the presence of carboxyesterase.

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hydrolyzed faster and the conjugate with DEX–OPB releases the free drug at a higher rate than that containing levulic acid in a spacer. It is clear that hydrolytic activity of the enzyme is lower at pH 7.4 than at pH 5. As a result of this, release of free active drug from the conjugates 2 and 3 and subsequently DEX derivative incubated in the presence of carboxyesterase is higher in mild acidic environment (model for endosomes) than in a buffer modeling environment during transport of the conjugate in blood circulation (as shown in Li et al., 2005, no carboxyesterase was found in human serum). Moreover, it is clear that release of DEX directly from the polymer conjugate is very limited, especially at pH 7.4. The in vitro release study carried out in the presence of carboxyesterase confirmed susceptibility of both spacers to enzymatic and pH-controlled hydrolysis enabling the release of free DEX from the polymer conjugates. The present study confirmed feasibility of the synthesis of a safe drug carrier system enabling delivery of a cytotoxic drug in blood with minimum loss of the transported drug and release of the active drug (DOX and DEX) in a solution modeling the environment inside target cells. 4. Conclusions

Comparison of the rates of DEX release from polymer conjugate 2 incubated in a buffer of pH 5 and in the same buffer containing 0.015 mg/mL carboxyesterase is shown in Fig. 6. As mentioned above, the hydrazone bond was promptly hydrolyzed and only DEX–LEV was released in the absence of the enzyme. In contrast, in the presence of carboxyesterase in addition to DEX–LEV, also free DEX was slowly released reaching 60% after 8 h of incubation. After primary burst release of the DEX ester due to hydrolysis of the hydrazone bond, slower enzymatic hydrolysis of the ester occurred and free DEX was released. Incubation of polymer conjugate 3 (DEX–OPB) in the buffer containing carboxyesterase (Fig. 7) led to similar findings as in the case of conjugate 2 with DEX–LEV. Whereas only DEX–OPB was released during incubation in a buffer of pH 5, the presence of carboxyesterase led to almost complete release of free DEX within 6 h of incubation. Enzymatic hydrolysis of DEX–OPB ester proceeded quite rapidly both in the polymer conjugate incubated in solution and in the already released DEX–OPB ester. In the first hour of incubation of the conjugate in buffer of pH 5 ca. 40% of DEX–OPB and 50% of DEX was released. In the course of subsequent 5 h incubation, the amount of DEX–OPB decreased and after 6 h of incubation ca. 95% of free DEX was found in incubation media. Comparing the drug release from polymer conjugates with DEX–OPB or DEX–LEV, i.e. the hydrolysis of both esters, the ester group of DEX–OPB is

Fig. 7. Release of DEX and DEX–OPB from linear polymer conjugate 3 incubated in phosphate buffers at 37 ◦ C at pH 5. () Cumulative spontaneous release of DEX (DEX and DEX–OPB); () cumulative release of DEX (DEX and DEX–OPB) in the presence of carboxyesterase; () DEX release in the presence of carboxyesterase; () DEX–OPB release in the presence of carboxyesterase.

A new water-soluble HPMA copolymer-based drug delivery system intended for simultaneous delivery of a combination of anticancer and anti-inflammatory drugs (DOX and DEX) was designed and synthesized. In this system both drugs were attached to the polymer carrier via spacers containing pH-labile hydrazone bonds and, in the case of DEX, ester bond. Such system enables controlled release of active DOX and DEX or the release in a two-step process in buffers mimicking intracellular environment, while in buffers modeling transport conditions in the organism they are rather stable. A detailed study of drug release rates in mild acid environment (intracellular space) showed that the drug release is almost complete after 6 h of incubation. The attachment of a second drug to the carrier does not influence the release profile of both the polymerbound drugs. Biological evaluation of the conjugates is under way; a synergistic effect of drugs bound to the polymer carrier is expected. Acknowledgements This work was supported by the Academy of Sciences of the Czech Republic (Grants Nos. KAN 200200651 and IAAX 00500803) and Grant Agency of Academy of Sciences of the Czech Republic (Grant No. IAA 400500806). References Alexanian, R., Dimopoulos, M.A., Delasalle, K., Barlogie, B., 1992. Primary dexamethasone treatment of multiple myeloma. Blood 80, 887–890. Bharali, D.J., Sahoo, S.K., Mozumdar, S., Maitra, A., 2003. Cross-linked polyvinylpyrrolidone nanoparticles: a potential carrier for hydrophilic drugs. J. Colloid Interface Sci. 258, 415–423. Brzezinski, M.R., Abraham, T.L., Stone, C.L., Dean, R.A., Bosron, W.F., 1994. Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine. Biochem. Pharmacol. 48, 1747–1755. Brzezinski, M.R., Spink, B.J., Dean, R.A., Berkman, C.E., Cashman, J.R., Bosron, W.F., 1997. Human liver carboxylesterase hCE-1: binding specificity for cocaine, heroin, and their metabolites and analogs. Drug. Metab. Dispos. 25, 1089–1096. Dean, R.A., Christian, C.D., Sample, R.H.B., Bosron, W.F., 1991. Human liver cocaine esterases: ethanol-mediated formation of ethylcocaine. FASEB J. 5, 2735–2739. Duncan, R., Cable, H.C., Lloyd, J.B., Rejmanová, P., Kopeˇcek, J., 1983. Polymers containing enzymatically degradable bonds. 7. Design of oligopeptide side-chains in N-(2-hydroxypropyl)methacrylamide copolymers to promote efficient degradation by lysosomal enzymes. Makromol. Chem. 184, 1997–2008. ˇ Etrych, T., Chytil, P., Jelínková, M., Ríhová, B., Ulbrich, K., 2002. Synthesis of HPMA copolymers containing doxorubicin bound via a hydrazone linkage. effect of spacer on drug release and in vitro cytotoxicity. Macromol. Biosci. 2, 43–52. ˇ Etrych, T., Jelínková, M., Ríhová, B., Ulbrich, K., 2001. New HPMA copolymers containing doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties. J. Control. Rel. 73, 89–102.

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