Polymer conjugates of the highly potent cytostatic drug 2-pyrrolinodoxorubicin

European Journal of Pharmaceutical Sciences 42 (2011) 156–163

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Polymer conjugates of the highly potent cytostatic drug 2-pyrrolinodoxorubicin M. Studenovsky a,∗ , K. Ulbrich a , M. Ibrahimova b , B. Rihova b a b

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic

a r t i c l e

i n f o

Article history: Received 16 September 2010 Received in revised form 3 November 2010 Accepted 8 November 2010 Available online 12 November 2010 Keywords: Polymer conjugates Tumor drug delivery pH-controlled release Anticancer agents Multidrug resistance

a b s t r a c t This paper describes the synthesis and biological evaluation of a conjugate of the highly cytotoxic drug 2-pyrrolinodoxorubicin (p-DOX) with an N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) as a water-soluble biocompatible polymer carrier, utilizing the advantageous concept of polymer–drug conjugates. The conjugate of p-DOX with HPMA copolymer (PHPMA/p-DOX) was prepared by reacting the PHPMA/DOX conjugate, where the DOX was bound via a hydrazone bond, with 4-iodobutyraldehyde. The hydrazone bond between the polymer and drug is susceptible to pH-controlled hydrolysis, enabling prolonged stability in circulation and fast p-DOX release under conditions mimicking the intracellular environment. The in vitro cytostatic activity of free p-DOX was in accordance with literature, whereas its PHPMA conjugate exhibited a 1.3- to 5-fold lower cytotoxicity, depending on the cancer cell line, when compared to the free p-DOX. This is in qualitative agreement with the data obtained for DOX and its HPMA copolymer conjugates. On mice bearing T-cell EL4 lymphoma, no tumor suppression was observed from the free p-DOX at a subtoxic dose of 0.1 mg/kg, whereas the PHPMA/p-DOX conjugate significantly inhibited the initial tumor growth at approximately equitoxic doses of 0.4 and 0.8 mg p-DOX eq/kg. However, moderately elevated doses of the p-DOX equivalent in the conjugate caused toxic effects, making accurate dosage setting essential. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The clinical treatment of cancer relies on the complementary effects of surgery, immunotherapy, radiotherapy and chemotherapy. The latter approach involves treatments using a broad variety of synthetic or natural substances that exhibit specific tumorsuppressive properties. The anticancer activity of such compounds is derived from a number of particular effects resulting in the death of tumor cells; their most common mode of action is a direct cytotoxic effect based on the interruption of the tumor cell cycle, inducing cell apoptosis. Anthracyclines are typical representatives of such cytotoxic agents. The anti-prolific activity of anthracyclines arises mainly from their strong interaction with nuclear DNA, disabling its transcription during the cell cycle. Doxorubicin is the most frequently used drug in this family. Despite its high potency and moderate side effects, a strong effort has been made towards the additional improvement of its therapeutic profile. There are several approaches to this optimization, including the modification of drug formulation and the chemical derivation of the parent compound. In response, hundreds of chemical analogs of doxorubicin have been synthesized and evaluated for

∗ Corresponding author. Tel.: +420 296 809 230; fax: +420 296 809 410. E-mail address: [email protected] (M. Studenovsky). 0928-0987/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2010.11.006

their biological activity to date. Despite their partial selectivity to rapidly dividing cells (tumors), in most cases their non-specific toxicity limits their clinical use. In order to reduce some side effects of cytotoxic chemotherapeutics, many strategies have been developed, including a combination of different drugs in one trial, specific time schedule, treatment adapted to the exact diagnosis and modification of drug formulation. In addition, it has been observed that when the parent drug is modified with a natural or synthetic water-soluble polymer, a significant suppression of adverse effects is achieved. Moreover, the solid tumor specificity of such polymer–drug conjugates is dramatically increased thanks to the EPR effect (Maeda et al., 2000; Matsumura and Maeda, 1986; Seymour et al., 1995). Polymer conjugates of anthracyclines have been known for approximately 25 years (Duncan et al., 1987; Kopecek and Duncan, 1987; Rihova and Kopecek, 1985). PHPMA/DOX conjugates with enzymatically degradable spacers were one of the first conjugates synthesized (Duncan, 2009). These conjugates exhibited greatly reduced toxicity, but relatively high doses were required for an effective treatment. We have overcome this drawback by replacing doxorubicin with its more potent anthracycline derivative. A new class of anthracycline analogs has emerged as a result of the strong effort to synthesize more efficient derivatives of doxorubicin (Binaschi et al., 2001). The formation of reactive immonium ions in slightly acidic media is the key feature of these compounds.

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163

O

O

O

OH

157

O

OH

OH

OH OH

OH O

O

OH O OH N

O

O

O

OH

1.H+

O

2. rearrangement

O

OH + N

2-pyrroline ring

immonium ion

Scheme 1. Formation of reactive immonium cation in p-DOX.

Because the immonium group is a strong electrophile, it has a strong ability to bond covalently with DNA. As the main therapeutic effect of DOX is derived from its interaction with nuclear DNA inside the tumor cell, its cytostatic activity is thus dramatically increased. Moreover, these compounds exhibit much lower cardiotoxicity and multidrug resistances (MDR), which are usually the most serious limitations in therapy with doxorubicin. 2-Pyrrolinodoxorubicin, an anthracycline analog that is approximately 100 times more potent than DOX in vitro, is one of the members of this family (Scheme 1). The aim of this study was to synthesize a PHPMA/p-DOX conjugate with pH-sensitive hydrazone linkage and then evaluate the conjugate’s biological activity in vitro and in vivo. 2. Materials and methods 2.1. Chemicals 1-Aminopropan-2-ol, methacryloyl chloride, 2,2azobisisobutyronitrile (AIBN), N,N-dimethylformamide (DMF), N-ethyldiisopropylamine (DIPEA), dimethyl sulphoxide (DMSO), trifluoroacetic acid (TFA), 6-aminohexanoic acid, methyl 6-aminohexanoate hydrochloride, hydrazine hydrate, 2-(3chloropropyl)-1,3-dioxolane, sodium iodide, sodium thiosulphate, sodium acetate, DOX hydrochloride (DOX·HCl) and silica gel were purchased from Aldrich. Acetonitrile and other common solvents and chemicals were purchased from Lach-Ner, s.r.o. 2.2. Analytical methods HPLC analyses were performed on a HPLC chromatograph (LDC Analytical, USA) using a reverse-phase column (Chromolyth Performance RP-18e 100 × 4.6 mm) with UV detection. A mixture of water–acetonitrile was used as the eluent at a gradient 0–100 vol% and a flow rate of 2 ml/min. Elemental composition was determined using a Perkin Elmer Elemental Analyzer 2400 CHN (Perkin Elmer, USA). Melting point temperatures were determined on a Kofler’s block (VEB Analytik Dresden, Germany). NMR spectra were measured on a Bruker Avance MSL 300 MHz NMR spectrometer (Bruker Daltonik, Germany). MALDI-TOF spectroscopy was carried out using a Bruker Bifex III Mass Spectrometer (Bruker Daltonik, Germany). Molecular weights of the polymers were determined by gel permeation chromatography (GPC) in a mixture of acetate buffer (pH 6.5, 0.3 mol/l) and methanol (20:80, v/v) on a TSK 3000 column (Polymer Laboratories Ltd., UK) using an HPLC System ÄKTA Explorer (Amersham Biosciences, Sweden) equipped with RI, UV

and multi-angle light-scattering DAWN DSP-F detectors (Wyatt, USA). UV/vis spectra were measured on a SPECORD 205 Spectrometer (Analytik Jena AG, Germany). 2.3. Syntheses 2.3.1. Preparation of 2-(3-iodopropyl)-1,3-dioxolane 2-(3-Chloropropyl)-1,3-dioxolane (15.8 g, 0.1 mol) was dissolved in 150 ml of acetone that contained 30 g (0.2 mol) of NaI and 2.5 g of NaHCO3 as a stabilizer. The solution was refluxed for 20 h and then evaporated to dryness. The obtained solid was washed three times with 100 ml of diethyl ether, filtered off and the yellowish filtrate was stirred with 20 g of anhydrous Na2 SO4 , 1 g of Na2 S2 O3 and 1 g of NaHCO3 for 12 h. The mixture was filtered off and 100 mg of Na2 CO3 was added to the filtrate with following removal of diethyl ether under vacuum. The crude oily product was purified by distillation under reduced pressure and stored in the dark at −18 ◦ C with a small amount of fine-grained Na2 CO3 as a stabilizer. The purity of the colorless liquid product was confirmed by HPLC; yield: 23.4 g (92%); elemental analysis: calcd. C, 29.77; H, 4.58; I, 52.43%; found C, 30.04; H, 4.62; I, 52.09%; 1 H NMR 300 MHz (CDCl3 , 297 K): 1.78 m (2H, I–CH2 CH2 CH2 ), 1.97 quint. (2H, I–CH2 CH2 CH2 ), 3.24 t (2H, I–CH2 CH2 CH2 ), 3.89 dt (4H, O–CH2 ), 4.90 t (1H, O–CH–O). 2.3.2. Preparation of 4-iodobutyraldehyde 2-(3-Iodopropyl)-1,3-dioxolane (6 g, 24.8 mmol) was dissolved in 240 ml of tetrahydrofuran, and then a solution of 40 ml of concentrated hydrochloric acid in 800 ml of water was added. An initially cloudy reaction mixture was stirred in the dark at r.t. for 24 h and then extracted five times with 50 ml of dichloromethane. The combined organic layers were dried with anhydrous Na2 SO4 and a small amount of NaHCO3 , the solids were removed via filtration and the solvent was evaporated under reduced pressure with an additional portion of NaHCO3 as a stabilizer. Finally, a small amount of fine-grained Na2 S2 O3 was added as an iodine scavenger. The stabilized 4-iodobutyraldehyde was stored in the dark at −18 ◦ C. Purity of the colorless liquid product was confirmed by HPLC; yield: 4.4 g (90%); elemental analysis: calcd. C, 24.26; H, 3.56; I, 64.09%; found C, 24.06; H, 3.40; I, 63.85%; 1 H NMR 300 MHz (CDCl3 , 297 K): 2.13 quint. (2H, I–CH2 CH2 CH2 ), 2.63 t (2H, I–CH2 CH2 CH2 ), 3.23 t (2H, I–CH2 CH2 CH2 ), 9.80 s (1H, CH O). 2.3.3. Preparation of p-DOX To a solution of doxorubicin hydrochloride (100 mg, 0.17 mmol) in 10 ml of ethanol was added 4-iodobutyraldehyde (270 mg, 1.4 mmol) and NaHCO3 (0.9 g, 10.7 mmol). The reaction mixture

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was diluted with 10 ml of dichloromethane and stirred in the dark at r.t. for 4 h. The suspension was filtered off, the filtrate was diluted with 50 ml of hexane and the precipitate that formed was separated by centrifugation. The obtained solid was subsequently re-dissolved in 5 ml of methanol containing 100 ␮l of trifluoroacetic acid and the solution was diluted with 100 ml of ethyl acetate and concentrated under reduced pressure to approximately one-tenth of the original volume. The obtained p-DOX trifluoroacetate was left to crystallize at −18 ◦ C overnight, separated by centrifugation and then dried under vacuum. Purity of the dark-red crystalline product was confirmed by HPLC; yield: 85 mg (78%); MALDI-TOF MS: calcd. 595, found 595.98 (M+H). 2.3.4. Preparation of N-(2-hydroxypropyl)methacrylamide (HPMA) Synthesis of this monomer was carried out according to the literature procedure (Ulbrich et al., 2000). Yield: 32 g (75%); elemental analysis: calcd. C, 58.72; H, 9.15; N, 9.78%; found C, 58.90; H, 9.10; N, 9.88%; m.p. 69–71 ◦ C. 2.3.5. Preparation of 6-methacrylamidohexanohydrazide (MA-HH) Synthesis of this reactive co-monomer was carried out using a two-step literature procedure (Hruby et al., 2007). Yield: 5.6 g (80%); elemental analysis: calcd. C, 56.32; H, 8.98; N, 19.70%; found C, 56.49; H, 8.63; N, 19.83%; m.p. 77–79 ◦ C. 2.3.6. Preparation of copolymer of N-(2-hydroxypropyl)-methacrylamide with 6-methacrylamidohexanohydrazide (poly(HPMA-co-MA-HH)) HPMA (3.2 g, 22.4 mmol), MA-HH (165 mg, 0.77 mmol) and AIBN (150 mg, 0.91 mmol) were dissolved in 20 ml of methanol and dry nitrogen was bubbled through the solution for 20 min. The solution was then heated in a sealed glass ampoule at 60 ◦ C for 17 h. The copolymer was precipitated into ethyl acetate, filtered off and purified by recrystallization from methanol into ethyl acetate. The suspension was filtered off and the resulting copolymer was dried under vacuum. Yield: 2.7 g (80%); molecular weight (GPC): Mw = 30 kDa, Mw /Mn = 2.2; hydrazide content (TNBSA assay (Etrych et al., 2001)): 0.14 mmol/g; molar ratio of HPMA: MA-HH monomer units (calculated from the hydrazide content) ∼98:2. 2.3.7. Attachment of DOX to poly(HPMA-co-MA-HH) DOX·HCl (98 mg, 0.17 mmol) was added to a solution of poly(HPMA-co-MA-HH) (470 mg, 0.066 mmol of hydrazide groups equivalent) and 200 ␮l of acetic acid in 3 ml of methanol and the reaction mixture was stirred in the dark at r.t. for 48 h. The unreacted DOX was then removed by centrifugation and the resulting solution was diluted with 5 ml of methanol and subjected to separation by chromatography on a Sephadex LH-20 column using methanol as the eluent. The PHPMA/DOX conjugate was isolated as a high-molecular-weight fraction, concentrated under reduced pressure and precipitated into dried diethyl ether. The suspension was centrifuged and the precipitated dark-red powder was dried under vacuum. Yield: 450 mg (88%); molecular weight (GPC): Mw = 40 kDa, Mw /Mn = 2.2; total DOX content (determined spectrophotometrically in methanol solution at 480 nm; 1 ε = 11200 l mol−1 cm−1 ): 6.2 wt%; free DOX content (calculated as a ratio between the corresponding low-molecular-weight and

1 ε value was determined previously for conjugated DOX (Pillai and Panchagnula, 2001).

high-molecular-weight peak areas in GPC UV/VIS chromatogram at 480 nm): <0.1 wt%. 2.3.8. Preparation of PHPMA/p-DOX conjugate 4-Iodobutyraldehyde (108 mg, 0.55 mmol) was added to a solution of PHPMA/DOX (103 mg, 11.4 ␮mol of DOX equivalent) and anhydrous sodium acetate (190 mg, 2.3 mmol) in 4 ml of methanol, and the reaction mixture was stirred for 2 h. The resulting solution was diluted with 4 ml of methanol and subjected to separation by chromatography on a Sephadex LH-20 column using methanol as the eluent. The PHPMA/p-DOX conjugate was isolated as a highmolecular-weight fraction, concentrated under reduced pressure and precipitated into dried diethyl ether. The suspension was centrifuged and the precipitated product was dried under vacuum. Yield: 80 mg (76%); molecular weight (GPC): Mw = 48 kDa, Mw /Mn = 2.2; p-DOX/DOX conversion rate (HPLC analysis of completely hydrolyzed conjugate in 5% aqueous TFA): 95%; total p-DOX content (calculated from the p-DOX/DOX conversion rate): 6 wt%; free p-DOX content (calculated as a ratio between the corresponding low-molecular-weight and high-molecular-weight peak areas in GPC UV/VIS chromatogram at 480 nm): <0.1 wt %. 2.4. In vitro and in vivo studies 2.4.1. Cancer cell lines Two cancer cell lines of mouse origin (T cell lymphoma EL 4 and B cell lymphoma 38C13) and two cell lines of human origin (UKF-NB4 and UKF-NB4/Dox) were used in this study. Mice cancer cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in cultivation flasks at 37 ◦ C with 5% CO2 in RPMI 1640 medium (Gibco Laboratories) supplemented with heat-inactivated 10% fetal calf serum (FCS), 2 mM l-glutamine, 50 mM 2-mercaptoethanol, 4.5 g/l glucose, 1.0 mM sodium pyruvate, 100 U/ml penicillin and 100 ␮g/ml streptomycin. Human cancer cell lines were the kind gift of Professor Tomas Eckschlager (Pediatric Clinic, University Hospital Motol, Prague, Czech Republic). They were established from human surgical samples (neuroblastoma) and maintained as doxorubicinsensitive (UKF-NB4) or doxorubicin-resistant (UKF-NB4-DOXO) clones (Bedrnicek et al., 2005). Cancer cell lines were grown in IMDM medium (Gibco Laboratories) supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine, 100 U/ml penicillin and 100 ␮g/ml streptomycin. The doxorubicin-resistant subline was kept at 20 ␮g/ml of doxorubicin. 2.4.2. Experimental animals Inbred strains of C57BL/6 (H-2b ) mice, aged 8–12 weeks, were purchased from the Animal Centre of the Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i. All mice were housed in accordance with approved guidelines and were provided with food and water ad libitum. All studies were approved by the Institutional Review Board. 2.4.3. In vitro cytostatic activity The cytostatic potential of the conjugates was assessed using a [3 H]-thymidine incorporation assay. NUNCLON 96-well, flatbottomed plates were seeded with 5 × 103 /well of EL4 cells, 2 × 103 /well of 38C13 cells, 1 × 104 /well of UKF-NB4 cells and 1 × 104 /well of UKF-NB4-DOXO cells. The samples were tested in triplicate or quadruplicate and then added to the wells to achieve the desired concentrations. The plates were cultured in 5% CO2 for 72 h at 37 ◦ C. For the last 6 h of the incubation, 18.5 kBq of [3 H]thymidine was added per well. The cells were then collected onto glass fiber filters (Filtermat, Wallac, Finland) using a cell harvester (Tomtec, Orange, CT). After drying, the fiber filter was placed into

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163

159

CH3

CH3 CH2

CH2 O

O

a

NH

b

NH (CH2)5

CH2 CH OH

O

CH3

NH

O

N

OH

OH OH O

O

OH O O OH NH2

H2O, pH 7.4 (slowly)

H2O, pH 5.5 (quickly)

CH3

O

CH3 CH2

CH2 O NH

NH

CH3

b

(CH2)5

CH2 CH OH

O OH

O

a

OH

O NH NH2

+

OH O

O

OH O O OH NH2

Scheme 2. Release of DOX from the polymer conjugate. At pH 7.4, only 10% of free DOX was released vs. 90% at pH 5.5 after 24 h. a:b ∼ 98:2.

a sample bag, a solid scintillator-Meltilex (Wallac) was applied and the bags were sealed (Microsealer, Wallac). Counting was performed in a 1450 MicroBeta TriLux (Wallac). Cells cultivated in fresh medium were used as controls.

2.4.4. In vivo tumor model C57BL/6 (B/6; H-2b ) females were subcutaneously transplanted once with a lethal dose (1 × 105 ) of EL4 T cell lymphoma cells, a lethal dose being defined as that which induces tumor growth in all controls. The mice that developed palpable tumors reaching 5–8 mm in diameter within 8–9 days after the implantation were intravenously treated with polymer conjugates diluted in PBS, as described in Section 3. Those surviving at least 60 days without any signs of tumor were considered long-term survivors (LTS), re-transplanted with a lethal dose of the same tumor cells and left without treatment to determine therapy-induced tumor resistance.

2.4.5. Acute toxicity A symptom of acute toxicity was determined as a decrease of body weight in the experimental animals in the days following treatment.

2.4.6. Statistical analysis All in vivo experiments were repeated at least once, and similar results were obtained. The Studentˇıs t-test was used for evaluating statistical significance. P < 0.05 was considered to be a significant value.

3. Results and discussion In this report, we show the possibility of utilizing a watersoluble synthetic polymer as a carrier for 2-pyrrolinodoxorubicin (p-DOX), a highly potent cytotoxic analog of doxorubicin. The benefits of the concept of polymer–drug conjugates have been proven extensively, encompassing a wide range of therapeutics conjugated with synthetic polymers (Pillai and Panchagnula, 2001). In our group, HPMA copolymer-based conjugates have been investigated in detail, and doxorubicin has been particularly studied as a drug. The hydrazone-type PHPMA/DOX conjugate strategy provides the usual profits of polymer conjugates as well as the advantageous drug pharmacokinetics: the hydrazone linkage between DOX and the polymer is sufficiently stable at pH ∼ 7.4 (blood stream) but quickly hydrolyzed at pH ∼ 5–6 (cell endosomes and secondary lysosomes) (Scheme 2). Consequently, the circulation period of the conjugated DOX in the body is dramatically increased compared with its free form, but the free drug is rapidly released inside the tumor. Therefore, this scheme was applied as a design for a polymer p-DOX conjugate.2

2 The kinetics of hydrolysis of the PHPMA conjugate could not be easily determined in in vitro model system because of freed p-DOX re-attaching to the polymer via coupling of p-DOX immonium groups (Scheme 1) with the polymer hydrazide groups (data not shown). As stated below, chemical modification of the daunosamine amino group in DOX molecule can only negligibly affect reactivity of the outlying hydrazone bond. Thus, we expect the same release profile for DOX and p-DOX from the polymer carrier, i.e. 10% release at pH 7.4 and 90% release at pH 5.5 within 24 h.

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2-(3-chloropropyl)1,3-dioxolane O

Cl O

2-(3-iodoropropyl)1,3-dioxolane KI acetone

O

I

4-iodobutyraldehyde H2O , H+

O

I

O

Scheme 3. Synthesis of 4-iodobutyraldehyde.

3.1. Synthesis An improved synthesis of 4-iodobutyraldehyde, the key substance needed for the synthesis of p-DOX, is described. Although a two-step preparation procedure of this synthesis was published (Nagy et al., 1996), its difficult reproducibility and the massive formation of various side-products using this procedure were major obstacles. These complications were observed predominantly during the isolation of 4-iodobutyraldehyde in the second synthetic step, but also to a lesser extent after the conversion of 2(3-chloropropyl)-1,3-dioxolane to 2-(3-iodopropyl)-1,3-dioxolane (Scheme 3). In an attempt to synthesize the 4-iodobutyraldehyde in sufficient yield and purity, the synthesis was conducted repeatedly under variable, well-controlled conditions. While doing these experiments, we observed a self-acidification of freshly prepared 4-iodobutyraldehyde, where the pH value of its aqueous emulsion was ∼4.5. Since aldehydes are potential substrates for an acid-catalyzed aldol reaction (Smith and March, 2001), we considered this as a possible dominant source of side-reactions. Traces of hydrogen iodide, formed by an elimination reaction from 4iodobutyraldehyde, could be a source of acid needed for the initialization of aldol condensation. Therefore, a small amount of NaHCO3 was added to a reaction mixture in order to neutralize this acid. This modification led to a proper reaction course with excellent yields in both reaction steps and was, in turn, proposed as a final synthesis of 4-iodobutyraldehyde. p-DOX, in its free form as a trifluoroacetate, was prepared by the modified synthesis described in literature (Nagy et al., 1996). The original procedure involved the use of N-ethyldiisopropylamine (DIPEA) as a base during the alkylation step of DOX with 4-iodobutyraldehyde. According our observations, replacement of DIPEA with a milder alkali, NaHCO3 , significantly lowered the amount of byproducts observed. This was probably due to its low basicity and, therefore, low reactivity towards the 4iodobutyraldehyde (base catalyzed aldol condensation). The latter reaction could be the source of undesired byproducts. The above mentioned modification simplified the original procedure, as there was no need to purify the crude product by preparative HPLC. Syntheses of HPMA and MA-HH co-monomers, used for the preparation of the reactive polymer precursor bearing the hydrazide groups, were carried out as described previously (Hruby et al., 2007; Ulbrich et al., 2000). Both the procedures were reproducible; similarly, all characteristics of the products obtained were in a good accordance with the literature. The substance of the conversion of DOX to p-DOX is an alkylation of a primary amino group on the saccharide moiety of DOX. Because of the substantially long distance between the amino and carbonyl groups in the DOX molecule, the chemical reactivity of the latter remains unchanged. This fact made possible the synthesis of the p-DOX/polymer conjugate based on the same strategy as that used for DOX, which was linked to a polymer via a hydrazone bond derived from its carbonyl group. The respective polymer precursor was therefore synthesized by the same procedure that was previously described (Etrych et al., 2008). Its chemical composition corresponded to a copolymer of HPMA with MA-HH in an appropriate molar ratio with respect to a desired final content of p-DOX.

HPMA

MA-HH

CH3

CH3

CH2

CH2 O

O

NH

NH

+

CH2

(CH2)5

CH OH O

CH3

NHNH2

polymerization AIBN, 60°C CH3

CH3 CH2

CH2 O NH

O a

NH

CH2

b

(CH2)5

CH OH CH3

O

NHNH2

poly(HPMA-co-MA-HH) Scheme 4. Synthesis of the polymer precursor by the copolymerization of monomers. a:b ∼ 98:2.

The synthesis consisted of a free radical random copolymerization of the co-monomers carried out in methanol at 60 ◦ C in a presence of AIBN as the initiator (Scheme 4). A different strategy than that used for DOX had to be developed to attach the p-DOX to the polymer precursor. This was because an N-alkylation of the polymer hydrazide groups with the protonated pyrroline ring of p-DOX (see Scheme 1) under acidic conditions necessary for successful condensation (acetic acid in methanol). Consequently, an uncontrolled increase in molecular weight and in the polydispersity of the formed PHPMA/p-DOX conjugate was observed. Therefore, the transformation of DOX to p-DOX was accomplished by derivation of DOX that was already attached to a polymer. The reaction procedure consisted in a direct alkylation of the previously prepared PHPMA/DOX conjugate with an excess of 4-iodobutyraldehyde under mildly alkaline conditions (sodium acetate) in methanol (Scheme 5). This technique represented an effective, immediate way to synthesize the PHPMA/p-DOX conjugate with nearly quantitative conversion of DOX to p-DOX and no recordable undesired side-reactions. 3.2. Inhibition of in vitro cell proliferation Table 1 summarizes IC50 values (the drug concentration inducing 50% cell death) that were obtained for the free p-DOX and its PHPMA conjugate and for the doxorubicin and its PHPMA conjugate. The data show similar cytostatic activities of p-DOX and PHPMA/p-DOX, even in DOX resistant neuroblastoma cancer

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163

DOX O

poly(HPMA-co-MA-HH) O

OH

CH3 OH

O

CH3

CH2

OH O

161

O

+

OH O O

CH2 O

a

NH

(CH2)5

CH2 CH OH

O

CH3

OH NH2

NH

acetic acid methanol

NH2

CH3

CH3 CH2

CH2 O

O

a

NH

b

NH (CH2)5

CH2 CH OH

PHPMA/DOX

b

NH

I

CH3

NH

O

O

O

OH

CH3COONa

N OH OH

O

O

OH O O CH3

CH3

OH NH2

CH2

CH2 O

O

a

NH CH2

(CH2)5

CH OH

O

CH3

PHPMA/p-DOX

b

NH

NH

O

OH

N OH OH

O

O

OH O O OH

N

Scheme 5. A two-step synthesis of PHPMA/p-DOX conjugate. a:b ∼ 98:2.

cell line UKF-NB4-DOXO. The PHPMA/p-DOX conjugate exhibited 1.3- to 5-fold lower cytotoxicy, depending on the cancer cell line, than free p-DOX. This is in agreement with the data already obtained for DOX and its HPMA copolymer conjugates. A remarkable difference, up to four orders of magnitude, could be seen comparing the pharmacological efficacy of the HPMA copolymer

Table 1 IC50 values in EL4 murine T cell lymphoma, 38C13 murine B cell lymphoma, UKF-NB4 neuroblastoma and its DOX resistant subline. Sample

p-DOX PHPMA/p-DOX DOX PHPMA/DOX a b

IC50 concentration (ng/ml)a , b EL4

38C13

UKF-NB4

UKF-NB4-DOXO

0.07 0.38 7 70

0.25 0.33 2.7 13

0.18 0.33 8 95

0.11 0.31 362 2 080

Related to the parent drug content. Concentration which inhibits incorporation of [3 H]thymidine by cells to 50%.

derivatives of doxorubicin and p-doxorubicin (UKF-NB4-DOXO cell line).

3.3. Evaluation of in vivo acute toxicity Acute systemic toxicities of free p-DOX and its polymer conjugate were assessed on the basis of changes in the body weight of treated mice after drug administration. The data for various doses are shown in Figs. 1 and 2. Our results show significantly suppressed toxicity of p-DOX when attached to the polymer. Whereas a single dose of 1 mg/kg of free p-DOX was lethal for the animals, the same dose of its polymer form was only slightly toxic and non-lethal. These data are in accordance with previous findings demonstrating a reduction of toxicity of low-molecular-weight compounds after their attachment to the polymer (Rihova, 2003). This suggests considerably improved tolerance of the polymer form of drug in possible cancer treatment.

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23

0.6 mg/kg g g

22 20 19

0.01 mg/kg

18

0.1 mg/kg

17

1 mg/kg

Survival (%)

Weiight (g)

21

16 15

0

5

10

100

1.2 mg/kg

80

1.8 mg/kg

40 20 0

15

Day

Weight (g)

24 23 22 21 20 19 18 17 16 15 14

0.01 mg/kg 0.1 mg/kg 1 mg/kg 5 mg/kg

5

10

15

20

Day Fig. 2. Average weight of mice after the treatment with a single i.v. dose of PHPMA/p-DOX conjugate at day 0. The doses are related to the parent drug.

3.4. Suppression of tumor growth in vivo The in vivo therapeutic effect of the PHPMA/p-DOX conjugate was assessed on the EL4 T cell lymphoma-bearing C57BL/6 male mice. Tumor growth, weight of animals and overall survival of animals were recorded. Fig. 3 shows the initial tumor growth of mice bearing T-cell lymphoma EL4 treated with the PHPMA/p-DOX conjugate and with free p-DOX at approximately equitoxic doses. Whereas the free p-DOX (0.1 mg/kg) exhibited no tumor growth suppression, the PHPMA/p-DOX conjugate induced a significant retardation of tumor development at doses of 0.4 and 0.8 mg eq/kg. The data clearly showed that free p-DOX was completely inactive contrary

20

40

60

80

Fig. 4. Survival of mice bearing T-cell lymphoma EL4 treated with doses of 0.6, 1.2 and 1.8 mg p-DOX eq/kg of PHPMA/p-DOX conjugates. A single dose was administered intravenously at day 9 after tumor inoculation.

to its polymer conjugates; therefore, the experiment was finished at day 25. The overall survival of mice bearing T-cell lymphoma EL4 (group of 7) at three different doses of the PHPMA/p-DOX conjugate (related to the parent drug content) is shown in Fig. 4. A dose of 0.6 mg/kg represented a therapeutically efficient amount of the conjugate, where 3 of 7 mice were cured with complete tumor regression (Fig. 5). In contrast, the higher doses, 1.2 mg/kg and 1.8 mg/kg, exhibited a critical toxicity where all treated mice died by day 24 and day 16, respectively, while the average survival of mice in the control group was 42 days. The clearly fatal effect of these elevated doses can be attributed solely to the systemic toxicity of p-DOX, as only a marginal development of the average tumor volume in the respective animals was observed (Fig. 5). The first significant shrinkage of cancer was detectable in some experimental animals on day 3 after the treatment. Mice that did not respond by day 10 after the treatment were considered treatment-resistant. Changes in average body weight of mice in each group are shown in Fig. 6, and this information supports the acute toxicity of the PHPMA/p-DOX conjugate. The data obtained showed an immediate loss of average body weight of mice treated with doses of 1.2 and 1.8 mg p-DOX eq/kg, indicating their strong toxic effect. In contrast, the dose of 0.6 mg/kg caused only non-significant loss of body weight, peaking two weeks after drug administration, followed by a course usual for undamaged animals. These findings are in accordance with the above mentioned overall survival data, thus indicating the dose 0.6 mg/kg as a therapeutical optimum for mice bearing T-cell EL4 lymphoma.

8,000

free p-DOX poly(HPMA)/p-DOX at 0.4 mg/kg poly(HPMA)/p-DOX at 0.8 mg/kg control

6,000

Average tumor volume (mm3)

Average tumor volume v (mm3)

0

Day

Fig. 1. Average weight of the mice after the treatment with a single i.v. dose of p-DOX at day 0.

0

control

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4,000

2,000

0 0

5

10

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20

25

Day Fig. 3. Initial tumor growth of mice bearing T-cell lymphoma EL4 (group of 8) treated with free p-DOX at dose 0.1 mg/kg and PHPMA/p-DOX conjugate at doses of 0.4 and 0.8 mg p-DOX eq/kg. A single dose was administered intravenously at day 8 after tumor inoculation.

8,000

0.6 mg/kg 1.2 mg/kg

6,000

1.8 mg/kg control

4,000 2,000 0 0

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40

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Day Fig. 5. Tumor growth in mice bearing T-cell lymphoma EL4 treated with doses 0.6, 1.2 and 1.8 mg drug eq/kg of PHPMA/p-DOX conjugate. A single dose was administered intravenously at day 9 after tumor inoculation.

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163

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0 6 mg/kg 0.6 1.2 mg/kg

Weight (g)

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1.8 mg/kg control

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drug, but its narrow “therapeutic window” emphasizes great care required for the accurate dosage setting. Acknowledgements Financial support from the Grant Agency of the Academy of Sciences of the Czech Republic (grant no. IAAX 00500803) is gratefully acknowledged. We also kindly thank Helena Misurcova and Pavla Jungrova for maintaining tissue cultures, proliferation assays and testing polymer drugs in vivo.

20 15 10 0

20

40

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Day Fig. 6. Average body weight of mice bearing T-cell lymphoma EL4 treated with doses 0.6, 1.2 and 1.8 mg p-DOX eq/kg of PHPMA/p-DOX conjugate. A single dose was administered intravenously at day 9 after tumor inoculation.

4. Conclusions In this paper, synthesis and results evaluating the antitumor activity of a polymer conjugate of 2-pyrrolinodoxorubicin, an exceptionally cytotoxic analog of the commonly used anticancer drug doxorubicin, is described. An improved synthesis of free p-DOX and its hydrazone-type conjugate with a copolymer of N-(2-hydroxypropyl)methacrylamide is presented. Whereas the synthesis of p-DOX was optimized mainly with the intent of increasing of its yield and purity, the PHPMA/p-DOX conjugate was synthesized by a tailored, two-step procedure. The substance of this unique procedure was a formation of the p-DOX itself in the last synthetic step, thus protecting the polymer conjugate from branching or even crosslinking. The ultimate cytotoxicity of free p-DOX was confirmed in vitro in several cancer cell lines, including those with developed multidrug resistance. Only a small decrease in cytostatic activity was recorded when p-DOX was attached to a polymer carrier. On the other hand, the PHPMA/p-DOX conjugate exhibited more than five times lower acute systemic toxicity in vivo compared to the free drug. The effective therapeutic dose of the conjugate for mice bearing T-cell EL4 lymphoma was as low as 0.6 mg p-DOX eq/kg with a 43% survival rate, in comparison with the approximately 30 mg DOX eq/kg necessary if the PHPMA/DOX conjugate was used for treatment. Since p-DOX is linked to its carrier via a hydrolytically labile bond, the free drug is responsible for the therapeutic effect after its release inside the tumor. In addition, the PHPMA/p-DOX conjugate has the high potential to bypass multidrug resistance and exhibit no cardiotoxicity at the therapeutic dose as it is in case of the free p-DOX. In spite of the above-mentioned advantageous features, PHPMA/p-DOX exhibits serious systemic toxicity at moderately elevated doses compared to its therapeutic optimum. Therefore, the HPMA conjugate of 2pyrrolinodoxorubicin represents a very potent polymer anticancer

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