Comparison of the pharmacological and biological properties of HPMA copolymer-pirarubicin conjugates: A single-chain copolymer conjugate and its biodegradable tandem-diblock copolymer conjugate

Accepted Manuscript Comparison of the pharmacological and biological properties of HPMA copolymer-pirarubicin conjugates: A single-chain copolymer conjugate and its biodegradable tandem-diblock copolymer conjugate

Tomas Etrych, Kenji Tsukigawa, Hideaki Nakamura, Petr Chytil, Jun Fang, Karel Ulbrich, Masaki Otagiri, Hiroshi Maeda PII: DOI: Reference:

S0928-0987(17)30259-2 doi: 10.1016/j.ejps.2017.05.031 PHASCI 4045

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

20 February 2017 11 May 2017 13 May 2017

Please cite this article as: Tomas Etrych, Kenji Tsukigawa, Hideaki Nakamura, Petr Chytil, Jun Fang, Karel Ulbrich, Masaki Otagiri, Hiroshi Maeda , Comparison of the pharmacological and biological properties of HPMA copolymer-pirarubicin conjugates: A single-chain copolymer conjugate and its biodegradable tandem-diblock copolymer conjugate, European Journal of Pharmaceutical Sciences (2017), doi: 10.1016/ j.ejps.2017.05.031

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Research Article For European Journal of Pharmaceutical Sciences

Comparison of the pharmacological and biological properties of HPMA

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copolymer-pirarubicin conjugates: a single-chain copolymer conjugate

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and its biodegradable tandem-diblock copolymer conjugate

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Tomas Etrycha, Kenji Tsukigawab, Hideaki Nakamurab, Petr Chytila, Jun Fangb,c, Karel

a

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Ulbricha, Masaki Otagirib,c, Hiroshi Maedac*

Institute of Macromolecular Chemistry v.v.i., Academy of Sciences of the Czech

Faculty of Pharmaceutical Sciences, Sojo University, Ikeda 4-22-1, Nishi-ku,

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b

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Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

Kumamoto, 860-0082, Japan

Institute for Drug Delivery Science, Sojo University, Ikeda 4-22-1, Nishi-ku,

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c

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Kumamoto, 860-0082, Japan

*Corresponding author at: Institute for Drug Delivery Science, Sojo University, Ikeda 4-22-1, Nishi-ku, Kumamoto, 860-0082, Japan. Tel.: +81 96 326 4114; fax: +81 96 326 3185E-mail address: [email protected] (H. Maeda).

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ABSTRACT In this study, we compared the enhanced permeability and retention (EPR) effect, toxicity,

and

therapeutic

effect

of

the

conjugate

of

the

linear

polymer

poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) with pirarubicin with an Mw below

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the renal threshold (39 g/mol) (named LINEAR) and the disulfide-linked

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tandem-polymeric dimer of the poly(HPMA)-pirarubicin conjugate with an Mw above

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the renal threshold (93 g/mol) (named DIBLOCK). The DIBLOCK conjugate, which was susceptible to reductive degradation, showed both a better EPR effect (tumor In addition,

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delivery) (2.5 times greater at 24 h) and a prolonged plasma half-life.

DIBLOCK had a better antitumor effect, as judged by percent survival, than did

model.

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LINEAR (80% vs 65% at 150 days), without any apparent toxicity in an S180 tumor However, the LD50 value of LINEAR was slightly higher than that of

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DIBLOCK (50 mg/kg vs 37.5 mg/kg, respectively). DIBLOCK required a longer time

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than LINEAR to reach maximum accumulation in the tumor. DIBLOCK also showed a greater time-dependent increase in the concentration in the tumor compared with the

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plasma concentration.

Keywords: pirarubicin, PHPMA conjugate, tandem-diblock PHPMA conjugate, EPR effect, tumor drug targeting

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1. Introduction Pirarubicin (4'-O-tetrahydropyranyldoxorubicin, THP), which is a derivative of doxorubicin (DOX) (Umezawa et al., 1979)), can be applied to DOX-resistant cell lines (Kunimoto et al., 1984) and shows less cardiac toxicity and more rapid cellular uptake

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compared with DOX (Koh et al., 2002; Kunimoto et al., 1983)). However, both free

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THP and DOX are distributed indiscriminately to both tumor tissues and normal healthy

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tissues, which causes adverse effects and may result in cessation of therapy. To overcome this drawback, improved targeting of drug to tumor is critical.

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Compared with low-molecular-weight drugs, biocompatible macromolecular drugs (>40 g/mol) show prolonged blood circulation and preferentially accumulate in

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tumor tissues for a long time because of the extensive vascular permeability and the lack of effective lymphatic drainage of tumors. This tumor-selective delivery of

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biocompatible macromolecular drugs is driven by the enhanced permeability and

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retention (EPR) effect in tumor tissues (Fang et al., 2011; Maeda, 2015; Maeda et al., 2013; Matsumura and Maeda, 1986)). During the development of drug delivery systems liposomes

and

micelles),

polyethylene

glycol

(PEG)

conjugates

and

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(e.g.,

N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) conjugates have been used

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as carriers of antitumor drugs that exhibit the EPR effect. However, despite the high accumulation of liposomes and polymer-drug conjugates in tumors, some reportedly showed insufficient therapeutic effects because of the low release of active drugs at tumor sites (Malugin et al., 2007; Prabhakar et al., 2013)). Also, some polymer-modified drugs show slower cellular uptake and lower cytotoxicity than non-modified drugs (Hatakeyama et al., 2011)). Therefore, release of active free drugs from polymer-drug conjugates at target tissues is important for 3

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efficient cellular uptake and extended therapeutic activity. Given these circumstances, we recently developed anticancer nanomedicines: PHPMA conjugates with THP that were bound to a carrier via a hydrazone bond that may be cleaved in the mild acidic environment of tumor tissues (Dozono et al., 2016;

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Nakamura et al., 2014; Nakamura et al., 2015)). The hydrazone bond responds to the

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acidic milieu of tumor tissues (Gerweck and Seetharaman, 1996; Tannock and Rotin,

SC

1989)), which results in more effective liberation of free THP in tumor tissues than in normal tissues (Nakamura et al., 2014; Nakamura et al., 2015)).

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Increasing the molecular size of the conjugate is a rational approach to improving accumulation of drugs in tumors by means of the EPR effect (Chytil et al.,

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2008; Etrych et al., 2008a)). We recently reported that the poly(amidoamine) dendrimer-based star-like PHPMA conjugate with THP (SP-THP, 400 g/mol) showed

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superior accumulation in tumors and enhanced antitumor effects in tumor-bearing mice

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compared with the effects of a linear PHPMA-THP conjugate (LINEAR, 39 g/mol) (Nakamura et al., 2015)). However, body weight decreased after an intravenous (i.v.)

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injection of SP-THP, and a higher toxicity (2-3 times) of SP-THP compared with the LINEAR conjugate was also found after i.v. administration. Therefore, a PHPMA-THP

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conjugate with improved therapeutic effects and lower toxicity is desirable. Previously, a biodegradable diblock PHPMA conjugate with DOX (about 80 g/mol), which consisted of two linear PHPMA-DOX conjugate blocks connected by a disulfide bond, showed much better pharmacokinetic properties and superior antitumor effects than did a linear single-block PHPMA-DOX conjugate (about 40 g/mol) (Etrych et al., 2010a; Etrych et al., 2014; Kostkova et al., 2013)).

The greater accumulation in

tumors and better antitumor effects were ascribed to the higher molecular weight Mw of 4

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the diblock conjugate, being above the kidney threshold (for PHPMA ~ 50 g/mol), which protected the conjugate from renal excretion and allowed a much longer blood circulation. Similarly, diblock PHPMA conjugates with an oligopeptide spacer between the blocks that had been custom-made for lysosomotropic enzymatic degradation were

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described in detail as drug carriers (Luo et al., 2011; Pan et al., 2013; Yang et al., 2015;

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Yang et al., 2013; Zhang et al., 2013).

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Indeed, here as well, the increased Mw enhanced the pharmacokinetic properties of the diblock/multiblock polymer systems. We found the PHPMA-THP

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conjugate, with THP being attached by a pH-sensitive hydrazone bond, to be superior than the PHPMA-DOX conjugate in terms of both uptake by cells in an in vitro system

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and antitumor effects in vivo (Nakamura et al., 2016)).

Because of the superiority of the antitumor effects of the PHPMA-THP

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conjugate in a cellular system and in vivo, we found it of interest to investigate the

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biological properties of the similar tandem-diblock PHPMA conjugate instead of DOX bearing THP (DIBLOCK, 93 g/mol). In contrast to structures of published PHPMA

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conjugates requiring the presence of lysosomal enzymes for drug release and polymer chain degradation, in our system we used a pH-sensitive hydrazone bond for drug

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conjugation and a disulfide linker that connected polymer blocks by using a reductively degradable spacer that did not require entering the lysosomes to achieve drug release and polymer degradation.

2. Materials and methods

2.1. Materials 5

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N,Nʹ-Dicyclohexylcarbodiimide,

N,N-dimethylformamide

(p.a.),

N,N-dimethyl-4-aminopyridine, dichloromethane (p.a.), methanol [for ultra performance liquid chromatography/high-performance liquid chromatography (UPLC/HPLC)], ethyl acetate (p.a.), 2,2ʹ-azobisisobutyronitrile (AIBN), 4,4ʹ-azobis(4-cyanopentanoic acid) 1-aminopropan-2-ol,

methacryloyl

chloride,

methyl-6-aminohexanoate

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(ABIC),

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hydrochloride (ah), hydrazine hydrate, tert-butyl carbazate, N-ethyldiisopropylamine

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(EDPA), dithiothreitol, trifluoroacetic acid (TFA), triisopropylsilane, GSH, and cystamine dihydrochloride were purchased from Sigma-Aldrich (Prague, Czech

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Republic). Pirarubicin (THP) was purchased from Meiji Seika (Tokyo, Japan), and 2,4,6-trinitrobenzene-1-sulfonic acid (TNBS) was purchased from Serva (Heidelberg,

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Germany). RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), and other reagents and solvents were purchased from Wako Pure Chemical (Osaka, Japan). calf

serum

was

obtained

from

GIBCO

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Fetal

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3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

(Grand

Island,

bromide

(MTT)

NY). was

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purchased from Dojindo Chemical Laboratories (Kumamoto, Japan).

2.2. Synthesis of monomers

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N-(2-Hydroxypropyl)methacrylamide methacryloylation

of

1-aminopropan-2-ol

(HPMA) with

was

synthesized

methacryloyl

chloride

by in

dichloromethane carried out in the presence of base as described previously, with Na2CO3 used as the base (Ulbrich et al., 2000)). 6-Methacrylamidohexanoylhydrazine (Ma-ah-NHNH2) was prepared in a two-step procedure as described previously (Etrych et al., 2008b)). Briefly, methyl 6-aminohexanoate hydrochloride dissolved in dichloromethane was methacryloylated 6

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with methacryloyl chloride in the presence of Na2CO3 and an inhibitor. After this reaction, the crude oily methyl 6-methacrylamidohexanoate was hydrazinolyzed with an excess of hydrazine hydrate in methanol. After removal of excess hydrazine hydrate, Ma-ah-NHNH2 was purified by crystallization from ethyl acetate.

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N-(tert-Butoxycarbonyl)-N'-(6-methacrylamidohexanoyl)hydrazine

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(Ma-ah-NHNH-Boc) was synthesized by using a two-step synthesis, as described in

N-methacryloyl-6-aminohexanoic

acid

was

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detail in a previous study (Ulbrich et al., 2004)). Briefly, in the first step, prepared

by methacryloylation

of

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6-aminohexanoic acid with methacryloyl chloride in an aqueous alkaline solution. In the next step, N-methacryloyl-6-aminohexanoic acid was coupled with tert-butyl carbazate

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in tetrahydrofuran by using the dicyclohexylcarbodiimide coupling agent. After removal of dicyclohexylurea and tetrahydrofuran, the crude product was purified by repeated

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crystallization from a mixture of ethyl acetate and hexane.

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The purity of the monomers was examined by means of an HPLC system (Shimadzu, Kyoto, Japan) equipped with a reversed-phase column (Chromolith

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Performance RP-18e, 100 mm × 4.6 mm) and elution with water-acetonitrile (gradient 0-100% acetonitrile), with UV-VIS photodiode array detection (Shimadzu SPD-M10A

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vp) (220-250 nm).

2.3. Synthesis of polymer precursors and polymer conjugates Linear polymer precursor 1 poly(HPMA-co-Ma-ah-NHNH2)

containing

hydrazide groups randomly distributed along the polymer chain (Scheme 1C) was prepared by means of free radical polymerisation with AIBN as an initiator, as described previously (Etrych et al., 2008). The linear polymer conjugate containing THP attached 7

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via a pH-sensitive hydrazone bond (LINEAR, Scheme 1A) was prepared by reacting polymer precursor 1 (2.0 g, 5.6 mol% hydrazide groups) with THP (220 mg) in 16 mL of methanol in the presence of acetic acid (0.65 mL) and kept in the dark for 15 h. LINEAR was then purified of low-molecular-weight impurities (THP or its degradation products)

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by precipitation into ethyl acetate.

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The semitelechelic copolymer of HPMA and Ma-ah-NHNH-Boc with a

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thiazolidine-2-thione (TT) group at the chain terminus (polymer precursor 2, Scheme 1D) was prepared by means of radical solution copolymerisation [HPMA (2.4 g, 16.8 mmol)

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with Ma-ah-NHNH-Boc (444 mg, 2.082 mmol)] in DMSO (17.4 mL) initiated with the azo initiator ABIC-TT (0.972 g) at 55 °C for 6 h.

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3,3´-[Azobis(4-cyano-4-methyl-1-oxobutane-4,1-diyl)]bis(thiazolidine2-thione) (ABIC-T) was synthesized as previously described (Subr and Ulbrich, 2006).

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The polymer was isolated by means of precipitation into a mixture of acetone and diethyl

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ether (1:3), filtration, and drying in a vacuum. The content of end-chain TT groups was determined spectrophotometrically with a Helios α (Thermochrom) spectrophotometer

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with ε305 = 10,700 L mol-1 cm-1 (methanol) (Subr and Ulbrich, 2006)). Polymer precursor 3 (Scheme 1E), the linear tandem-diblock copolymer, was

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prepared by reacting TT groups of polymer precursor 2 with cystamine dihydrochloride. Cystamine dihydrochloride (12.4 mg, 55 mol) was dissolved in 7.75 mL of methanol, and EDPA (18 L, 110 mol) was added under stirring. Polymer precursor 2 (2.2 g; 106 mol polymer chains) was dissolved in 16.1 mL of methanol, and a solution of cystamine was added to the solution of polymer, which was stirred continuously, within 15 min. After 2 h of reaction, 4.6 L of 1-aminopropan-2-ol was added, and the reaction mixture was left for 5 min. After the mixture was more than 95% pure (HPLC Shimadzu, TSKgel 8

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G3000SWXL column), no further purification from the unimer was needed. Polymer precursor 3 was separated by precipitation into ethyl acetate. Linear tandem-diblock copolymer with free hydrazide groups (polymer precursor 4, Scheme 1F) was prepared by deprotection of Boc of polymer precursor 3 in

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concentrated TFA.

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The diblock copolymer conjugate containing THP attached via the hydrazone

SC

bond (DIBLOCK, Scheme 1B) was prepared by THP conjugation with polymer precursor 4 as described above. Scheme 1 provides the structures of polymer conjugates,

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LINEAR and DIBLOCK, whose characteristics are summarized in Table 1.

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Scheme 1. Structures of (A) LINEAR, (B) DIBLOCK, (C) polymer precursor 1

(poly(HPMA-co-Ma-ah-NHNH2)),

(D)

polymer

precursor

2

[poly(HPMA-co-Ma-ah-NHNH-Boc) with a terminal thiazolidine-2-thione

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(TT)], (E) polymer precursor 3 [linear tandem-diblock homo-copolymer

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(polymer precursor 2)], and (F) polymer precursor 4 [linear tandem-diblock

SC

homo-copolymer (polymer precursor 3) with free hydrazide groups].

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2.4. Purification and characterization of polymer precursors and polymer conjugates The polymer conjugates were characterized and tested for the content of free THP

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and the absence of unimer by using the Shimadzu HPLC with TSKgel G3000SWXL or TSKgel G4000SWXL column (300 mm × 7.8 mm, 5 μm) equipped with a refractive

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index and UV-VIS detector (488 nm) and by thin-layer chromatography (aluminum

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sheets, Kieselgel 60 F254) over the course of the reactions. The average Mw and the polydispersity index PĐ of the polymer precursors and

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the conjugates were determined with the Shimadzu HPLC system equipped with a multiangle light scattering DAWN 8 photometer with Optilab rEX (Wyatt Co., CA, USA)

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and refractive index detectors and using the TSKgel column as described above, with the mobile phase consisting of 20% acetate buffer (0.3 M, pH 6.5) and 80% methanol at a flow rate of 0.5 mL/min. The dynamic light scattering of the conjugate solutions (0.5 wt%) in phosphate buffer, pH 7.4, was measured at a 173° scattering angle on a Zetasizer ZEN3600 instrument (Malvern Instruments, Malvern, UK). The hydrodynamic radius (Rh) was determined by using the DTS (Nano) program. 10

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The content of the hydrazide groups in the polymer precursor and in the polymer conjugates was determined by means of a modified TNBS assay, as described previously (Etrych et al., 2001)), with 500 nm = 17,200 L mol-1 cm-1 for estimation of the -NHNH2 group.

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The total content of THP in the polymer conjugates was determined

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spectrophotometrically via a Helios  spectrophotometer, with ε488 nm = 11,500 L mol-1

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cm-1 in water.

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2.5. In vitro drug release from polymer conjugates

Each PHPMA-THP conjugate was dissolved, at a concentration of 1.0 mg/mL, in

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0.1 M sodium acetate buffer (pH 5.5) or 0.1 M phosphate buffer (pH 6.0, pH 6.8, pH 7.0, or pH 8.0) and was incubated at 37 °C. After specific times, an aliquot of the solutions

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was mixed with an equal volume of 0.2 M sodium bicarbonate buffer (pH 9.8) and three

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times the volume of chloroform to extract released free THP in chloroform. The chloroform phase was evaporated to dryness, and the pellet was dissolved in the mobile

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phase for HPLC and analyzed by using the Shimadzu HPLC system with an RF-20AXS fluorescence detector (excitation at 488 nm, emission at 590 nm). The column was

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COSMOSIL 5C8-MS (4.6 mm × 150 mm) (Nacalai Tesque, Kyoto, Japan), and the column temperature was maintained at 40 °C. The mobile phase consisted of 33% acetonitrile and 67% sodium acetate buffer (0.1 M, pH 5.0) at flow rate of 1.2 mg/mL. A standard curve of free THP was used to determine the amount of released free THP.

2.6. Degradation of tandem-diblock copolymer conjugates In vitro cleavage of the disulfide bonds in diblock polymer precursor 4 (10 11

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mg/mL) and DIBLOCK (10 mg/mL) was performed with 0.1 M phosphate buffer and 0.05 M NaCl (pH 5.0) at 37 °C (for indicated time period). The degradation was initiated by adding 20 µL of GSH stock solution (1.5 mg/mL) to the solution of diblock copolymer to achieve the indicated concentrations. At selected time intervals, an aliquot

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was withdrawn, and the Mw of polymer degradation products was determined by size

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exclusion chromatography via a Superose 6 column with a DAWN 8 photometer and

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Optilab rEX detectors (acetate buffer, pH 6.5). All experiments were performed in

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triplicate.

2.7. In vitro cytotoxicity assay of THP, LINEAR, and DIBLOCK

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HeLa cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin G, and 100 μg/mL streptomycin under 5% CO2/95% air at 37 °C.

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Two types of DMEM with different pH values (pH 6.9 and pH 7.4), prepared by adding

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of different amounts of NaHCO3 (1.0 g/L and 3.7 g/L, respectively), were used in this study. Cells (3000 cells/well) were plated in 96-well plates (Corning, Corning, NY,

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USA). After overnight incubation, free THP, LINEAR, or DIBLOCK was added, followed by the MTT assay after 72 h of culture to quantify the viable cells, with

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absorbance at 570 nm, by using the Infinite M200 PRO microplate reader (Tecan Japan Co., Ltd., Kanagawa, Japan).

2.8. Tissue distribution of LINEAR and DIBLOCK All animal experiments were carried out according to the Laboratory Protocol for Animal Handling of Sojo University. S180 tumor cells (2 × 106 cells) were implanted subcutaneously in the dorsal 12

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skin of male ddY mice (Kyudo Co., Ltd., Saga, Japan). When the diameter of the tumor was about 8-10 mm, a 10 mg/kg of THP equivalent drug in saline was administered by an i.v. injection into the tail vein. At indicated times after this injection, mice were killed and perfused with saline to remove drugs in the blood compartment, followed by

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removal of each tissue. Physiological buffer consisting of 0.01 M phosphate and 0.15 M

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NaCl (pH 7.4) was added to tissue samples (900 μL/100 mg tissue), followed by

SC

homogenization. The homogenate was incubated at 50 °C for 1 h in 1 M HCl to yield the aglycone of THP from released free THP and polymer-bound THP. The aglycone

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was extracted by chloroform. The chloroform phase was evaporated to dryness and the residue was then dissolved in the solvent of the mobile phase of the HPLC.

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The Shimadzu HPLC system with an RF-20AXS fluorescence detector (excitation at 488 nm, emission at 590 nm) was used, with a COSMOSIL 5C8-MS

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column (150 mm × 4.6 mm) (Nacalai Tesque) at 40 °C. The mobile phase consisted of

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33% acetonitrile and 67% sodium acetate buffer (0.1 M, pH 5.0) at a flow rate of 1.2 mL/min. A standard curve of free THP treated with 1 M HCl at 50 °C for 1 h was used

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to quantify the amount of THP in each tissue.

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2.9. In vivo antitumor effects and toxicity of THP, LINEAR, and DIBLOCK The in vivo antitumor effects of free THP, LINEAR, and DIBLOCK were investigated with S180 tumor-bearing male ddY mice. When tumors had a diameter of about 5-7 mm, the desired amounts of free THP, LINEAR, or DIBLOCK were administered by i.v. injections. The tumor volume and body weight of the mice were measured during the study period. The tumor volume (mm3) was calculated as (W2  L)/2 by measuring the width (W) and length (L) of the tumor. 13

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Healthy male ddY mice (6 weeks old) were used to evaluate the toxicity and effects on normal tissues of these THP moieties. After LINEAR or DIBLOCK at 20-80 mg/kg of THP equivalent was injected, the body weight and mortality of the mice were evaluated during 14 days. LD50 values were calculated by using the Reed and Muench

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method. To investigate the comparative influence of free THP, LINEAR, or DIBLOCK

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on normal tissues, free THP at 5 mg/kg (near the maximum tolerable dose) and

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LINEAR or DIBLOCK at 15 mg/kg of THP equivalent were administered by i.v. injection. At days 1, 5, and 10 after drug administration, mice were killed and blood

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samples were obtained to evaluate the effect of the drugs on the liver, heart, and kidney by determining the aspartate aminotransferase, alanine aminotransferase, lactate

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dehydrogenase, creatine kinase, blood urea nitrogen, and total creatinine values.

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2.10. Statistical analysis

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Data are presented as means ± S.D. To determine the significance of the results obtained, two-tailed unpaired Student’s t-test was applied. Results were considered

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statistically significant when p was <0.05.

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3. Results 3.1. Synthesis of polymer precursors The Mw values of the polymer precursors 1 and 2 were 27,000 and 38,300 g/mol, respectively, and they were sufficiently below the glomerular filtration limit (Table 1) to

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ensure safe excretion of the polymer in urine. The final product of the tandem-diblock

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polymer formation reaction showed no signs of unreacted semitelechelic polymer

SC

precursor (Scheme 1) (the yield of the tandem-diblock polymer being more than 95%). Moreover, although the functionality of the terminal TT groups was higher than 1

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(F=1.28, Table 1), selected reaction conditions allowed synthesis of the tandem-diblock polymer without apparent signs of the formation of multiple blocks. Deprotection of

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hydrazide groups by TFA had no significant impact on the physicochemical characteristics of the tandem-diblock polymer precursor. The increase in Mw from

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38,300 g/mol for polymer precursor 2 to 78,200 g/mol for tandem-diblock polymer

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precursor 4 was accompanied by an increase in the Rh from 4.1 nm for polymer

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precursor 2 to 6.3 nm for polymer precursor 4 (Table 1).

3.2. Synthesis of polymer-drug conjugates

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LINEAR (Scheme 1A) and DIBLOCK (Scheme 1B) were prepared by using conjugation of THP with respective polymer precursors in dry methanol. The reaction was completed after 16-17 h, with a 98-99% yield. The Mw values and sizes of the conjugates in aqueous solution showed slight increases when compared with those of the respective polymer precursors (Table 1), which may be attributed to the increased Mw caused by the attached THP. The hydrodynamic radius Rh of DIBLOCK in aqueous solution was 1.5 times larger than that of LINEAR, and the corresponding size of the 15

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polymer coil in solution should be sufficient for effective accumulation of the polymer conjugate in solid tumors as a result of the EPR effect. The polymer conjugates, purified by precipitation into ethyl acetate, contained less than 0.2% of free THP. This content of free THP in a conjugate is sufficiently low as to not affect final biological behavior of

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the conjugates.

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3.3. Release of drugs from polymer conjugates

As Scheme 1 shows, THP was conjugated to PHPMA via an acid-labile

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hydrazone bond. As expected, free THP was released more quickly at acidic pH. The release rate of free THP from its polymer conjugates was practically the same for

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LINEAR and DIBLOCK (Fig. 1). The THP release from LINEAR at 24 h of incubation was 2.5% at pH 8.0, 16.9% at pH 7.0, 32.9% at pH 6.8, 52.8% at pH 6.0, and 60.2% at

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pH 5.5. The THP release from DIBLOCK at 24 h was 2.5% at pH 8.0, 17.4% at pH 7.0,

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32.2% at pH 6.8, 51.1% at pH 6.0, and 62.8% at pH 5.5.

Fig. 1. Release of free THP from polymer conjugates at different pH values.

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Incubation was at 37 °C for the indicated time periods. The amount of released free THP was measured by means of HPLC. Values are means ± S.D. (n = 3).

3.4. In vitro degradation of DIBLOCK conjugates

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Cleavage of the disulfide bonds of diblock polymer precursor 4 and DIBLOCK

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conjugate in buffer solutions containing GSH in concentrations corresponding to the

SC

reported concentration in the cytoplasm of human cells (3 mM GSH) (Saito et al., 2003)) or the concentration in circulating blood (0.06 mM GSH) was investigated;

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cleavage in human blood plasma was also studied. Results showed no significant changes in the Mw of diblock polymer precursor 4 after incubation in 0.06 mM GSH

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(Fig. 2). Only a very small decrease in the Mw of diblock polymer precursor 4 was detected after 48 h of incubation in human plasma at 37 °C, and this change was not

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significant (Fig. 2).

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In contrast, when the GSH concentration was increased to that common in the cytoplasm (3 mM), the Mw values of diblock polymer precursor 4 and DIBLOCK

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rapidly decreased (Fig. 2), which indicated that reductive cleavage of the disulfide bond was successful. After 2 h of incubation, the Mw of DIBLOCK decreased to below the

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renal threshold. After 4-10 h of incubation, the Mw of the resulting polymer fragments became similar to that of semitelechelic polymer precursor 2. These results agree with results of our recent studies of in vitro degradation of disulphide spacer-linked diblock HPMA copolymers incubated with EL4 T-cell lymphoma cells (Etrych et al., 2010b)).

17

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100

diblock polymer precursor 4, 3 mM GSH DIBLOCK, 3 mM GSH diblock polymer precursor 4, plasma diblock polymer precursor 4, 0.06 mM GSH

90

70

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60 50________________________________________________________________________________ 40

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Mw*10-3 (g/mol)

80

30

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20

0 0

10

20

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10

30

40

50

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Incubation time (h) Fig. 2. Time course of Mw of diblock polymer precursor 4 and DIBLOCK during

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incubation with GSH. Diblock polymer precursor 4 and DIBLOCK were

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incubated (at 37 oC) in buffers containing a cytoplasmic concentration of GSH (3 mM) or a plasma concentration of GSH (0.06 mM) or were incubated in human

CE

plasma. Values are means ± S.D. (n = 3).

AC

3.5. In vitro cytotoxicity of LINEAR and DIBLOCK The cytotoxicity of LINEAR and DIBLOCK was investigated by using HeLa cells at physiological pH (pH 7.4) and at the pH occurring in tumor tissue (pH 6.9). After 72 h of incubation at pH 7.4, the IC50 values for free THP, LINEAR, and DIBLOCK were 0.07, 0.52, and 0.56 μg/mL, respectively (Fig. 3A). At pH 6.9, IC50 values for free THP, LINEAR, and DIBLOCK were 0.08, 0.44, and 0.35 μg/mL, respectively (Fig. 3B). No apparent difference occurred in the cytotoxicity of free THP

18

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at pH 7.4 and pH 6.9, whereas both polymer conjugates showed higher cytotoxicity values at pH 6.9 than at pH 7.4 (Fig. 3). No marked difference in cytotoxicity was

NU

SC

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observed for LINEAR and DIBLOCK at pH 7.4 and pH 6.9.

Fig. 3. In vitro cytotoxicity of free THP, LINEAR, and DIBLOCK against

MA

HeLa cells at different pH values. (A) pH 7.4. (B) pH 6.9. Cells were treated with THP derivatives for 72 h, and then the MTT assay was performed to

PT E

D

quantify the viable cells. Values are means ± S.D. (n = 7-8).

3.6. Pharmacokinetic properties of LINEAR and DIBLOCK in mice

CE

We studied the pharmacokinetic properties of LINEAR and DIBLOCK in S180 tumor-bearing mice after i.v. injection. In this study, because LINEAR and DIBLOCK

AC

demonstrated a similar release of free THP, we determined the concentration of total THP (i.e., the sum of released free THP and polymer-bound THP) in each tissue. Figure 4A and B shows the tissue distribution of LINEAR and DIBLOCK, respectively, at 5, 24, 48, and 72 h after i.v. administration. At 24 h after injection, the highest concentrations of LINEAR and DIBLOCK occurred in tumor tissue. As Fig. 4C illustrates, the plasma concentrations of DIBLOCK were significantly higher than those of LINEAR at all time points, with the greatest difference found at 72 h after i.v. 19

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injection, and the area under the curve of the plasma concentration of DIBLOCK was 1.7 times higher than that of LINEAR when integrated between 5 and 72 h. Concentrations of DIBLOCK in tumor tissue were 2.5-5.0 times higher than those of

AC

CE

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D

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SC

RI

PT

LINEAR found at 24, 48, and 72 h after i.v. injection (Fig. 4D).

Fig. 4. Pharmacokinetic properties of LINEAR and DIBLOCK after i.v. injection. After i.v. administration of LINEAR and DIBLOCK, at 10 mg/kg of THP equivalent, to tumor-bearing mice, properties of LINEAR and DIBLOCK 20

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were evaluated. Values are means ± S.D. (n = 3). Tissue distributions of (A) LINEAR and (B) DIBLOCK. Comparison of (C) plasma level and (D) tumor accumulation for LINEAR and DIBLOCK. *p < 0.05, significantly different

RI

3.7. In vivo antitumor activity of LINEAR and DIBLOCK

PT

from LINEAR, according to Student's t-test.

SC

The in vivo antitumor effect was evaluated in S180 tumor-bearing mice after i.v. administration of free THP or polymeric THP at 2 or 5 mg/kg of THP equivalent. As Fig.

NU

5A illustrates, both LINEAR and DIBLOCK suppressed tumor growth in a dose-dependent manner, with the suppression being much greater after treatment with

MA

both LINEAR and DIBLOCK than that after treatment with free THP. We found it surprising that DIBLOCK at 2 mg/kg of THP equivalent had a higher antitumor effect

D

than LINEAR at 5 mg/kg of THP equivalent (Fig. 5A). No loss of body weight was

PT E

observed after treatment with LINEAR and DIBLOCK at both doses (Fig. 5B). In addition, after treatment with DIBLOCK at 5 mg/kg of THP equivalent four

CE

of five mice survived for longer than 150 days after tumor inoculation, which may

AC

indicate a complete cure (Fig. 5C).

21

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Fig. 5. Antitumor effects, body weight changes, and survival after treatment

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with free THP, LINEAR, or DIBLOCK. (A) Antitumor effects of free THP, LINEAR, and DIBLOCK against S180 tumors in male ddY mice. Drugs, at a

AC

dose of 2 or 5 mg/kg of THP equivalent, were administered by i.v. injection on day 12 (vertical arrows). Values are means ± S.D. (n = 5). (B) Body weight changes of ddY mice after the treatments given in (A). When tumor growth is not suppressed, tumor weight adds to the body weight. (C) Survival rate of S180 tumor-bearing male ddY mice after the treatments given in (A). Symbols in (B) and (C) are the same as those in (A).

22

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3.8. Toxicity of LINEAR and DIBLOCK after i.v. administration LINEAR and DIBLOCK, given by i.v. injection at a dose of 20, 40, 60, or 80 mg/kg of THP equivalent to healthy male ddY mice, caused a loss of body weight (Fig.

PT

6). These doses were higher than the doses used for S180 tumor therapy described

RI

above. Table 2 gives the LD50 values of LINEAR and DIBLOCK as 50.0 and 37.3

SC

mg/kg of THP equivalent, respectively, whereas the LD50 value of free THP was 14.2 mg/kg (Nakamura et al., 2015)).

NU

Table 3 summarizes the effects of these polymer-THP conjugates on normal tissues. The i.v. injection of polymer-THP conjugates led to no apparent changes in liver

MA

and kidney functions, whereas the creatine kinase value at 10 days after drug injection increased significantly in all treated groups. However, the increase in CK did not differ

D

significantly between LINEAR and DIBLOCK. No drug suppressed liver and kidney

AC

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functions even at the 15 mg/kg equivalent dose.

23

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Fig. 6. Body weight changes of normal healthy male ddY mice after LINEAR

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or DIBLOCK treatment. (A) LINEAR and (B) DIBLOCK were given by i.v. administration at 20, 40, 60, or 80 mg/kg of THP equivalent (eq.). Vertical

CE

arrows indicate the times of drug injection. Values are means ± S.D. (n = 5). †

AC

indicates that all mice died.

4. Discussion In this study, we compared the biological and pharmacological properties of (a) the linear copolymer-drug conjugate (LINEAR) and (b) the same two linear copolymer-drug conjugates linked in tandem via a disulfide bond (DIBLOCK) that was 24

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susceptible to reductive degradation. The renal threshold of linear PHPMA copolymers is approximately Mw 50,000-70,000 g/mol (Etrych et al., 2012)). DIBLOCK consists of two linear polymers with an Mw slightly higher than 35,000 g/mol each, so the Mw of the conjugate exceeds this threshold. The disulfide bond of DIBLOCK is biodegradable,

PT

and the products of degradation of this copolymer carrier (i.e., the single blocks) would

In addition, the disulfide bond was stable in

SC

carriers in the body (Etrych et al., 2014)).

RI

be subject to excretion, thus avoiding the undesirable accumulation of large drug

blood plasma, which has GSH level of 0.06 mM (Fig. 2), and after i.v. injection

NU

DIBLOCK exhibited a much longer residence time in blood circulation compared with LINEAR, which indicates that the disulfide bond was relatively stable in blood

MA

circulation in vivo. Therefore, a disulfide linkage may be useful for future drug design and development.

D

We utilized an acid-cleavable hydrazone bond as a spacer between THP and the

PT E

PHPMA chain. Polymer-conjugated drugs are generally known to demonstrate slower cellular uptake and lower cytotoxicity than free drugs. For example, PEG-conjugated

CE

drugs reportedly had slower cellular uptake than did native drugs (Hatakeyama et al., 2011; Hatakeyama et al., 2007)). Liberation of a free active pharmacological ingredient

AC

at target tissues is indispensable for efficient cellular uptake and therapeutic activity. We showed in a previous study that free THP was released from LINEAR in tumor tissue outside the tumor cells, where pH is slightly acidic, after which it was taken up by tumor cells quite efficiently (Nakamura et al., 2014)). Our present study also demonstrated that the liberation of free THP from both conjugates studied was rapid at acidic pH (pH 5.5-6.8) but not at pH 7.0 or pH 8.0 (Fig. 1), which indicates that effective THP release should occur not only in endosomes and 25

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lysosomes of tumor cells but also in the mild acidic environment of tumor tissues. We also found no significant difference in THP release between LINEAR and DIBLOCK (Fig. 1). High-molecular-weight poly(amidoamine) dendrimer-based SP-THP, in which THP was also conjugated to the PHPMA chain via a hydrazone bond, showed THP

PT

release profiles that were similar to those of LINEAR (Nakamura et al., 2015)).

In all

RI

water-soluble conjugates, THP was covalently bound to the fully solvated PHPMA

SC

chain of the same composition, and the results show that the net Mw of the PHPMA conjugates may not affect the sensitivity of the hydrazone bond to interaction with a

NU

small molecule of water and to hydrolysis.

We recently found that the HPMA copolymer-THP conjugate showed a much

MA

higher cellular uptake compared with the HPMA copolymer-DOX conjugate (Nakamura et al., 2014)). In this study, both LINEAR and DIBLOCK exhibited much higher

D

cytotoxicity at acidic pH values than at neutral pH (Fig. 3), which agrees with the

PT E

effective release of free THP from the polymer carrier under acidic conditions (Fig. 1). Important differences in pharmacokinetic properties and antitumor effect were

CE

found between LINEAR and DIBLOCK. Compared with LINEAR, DIBLOCK had a prolonged blood circulation time (Fig. 4C) and a higher tumor accumulation as a result

AC

of the EPR effect (Fig. 4D) in S180 tumor-bearing mice. Consequently, DIBLOCK showed much better tumor growth suppression than did LINEAR (Fig. 5A). We found that the LD50 values of LINEAR, DIBLOCK, and SP-THP (Nakamura et al., 2015) were 50.0, 37.3 (Table 2), and 23.4 mg/kg of THP equivalent, respectively, which indicates that the toxicity of DIBLOCK was slightly higher than that of LINEAR but lower than that of SP-THP. Our investigation of the effects of free THP, LINEAR, and DIBLOCK—with doses of LINEAR and DIBLOCK three times higher than that of 26

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free THP—on cardiac, liver, and kidney functions by using blood biochemistry demonstrated that, although more detailed study is needed, increased CK values obtained for free THP (5 mg/kg) indicated cardiac toxicity. Results for both conjugates (at 15 mg/kg of THP equivalent) were about the same (Table 3). This finding may

PT

indicate that both conjugates at 15 mg of THP equivalent had lower cardiac toxicity

RI

than did free THP (at 5 mg). Thus, both conjugates were less toxic than free THP at the

SC

same THP equivalent dose. We also found no significant differences in cardiac, liver, and kidney toxicity in vivo between LINEAR and DIBLOCK at lower doses (e.g., 15

NU

mg/kg of THP equivalent or less), which were similar to the therapeutic dose. At higher doses, 20-80 mg/kg of THP equivalent, which exceeded the therapeutic dose, we found

MA

that DIBLOCK was somewhat more toxic than LINEAR (LD50 37.3 vs 50 mg/kg of THP equivalent).

D

A noteworthy finding was that even the low dose of DIBLOCK (2 mg/kg of

PT E

THP equivalent) had a greater antitumor effect than the high dose of LINEAR (5 mg/kg of THP equivalent) (Fig. 5A). That is, at a dose of about 1/19 of the LD50, DIBLOCK

CE

had a marked therapeutic effect compared with LINEAR at a dose of 1/10 of the LD50, which indicated that DIBLOCK may have a more potent therapeutic effect at a lower

AC

dosage, one at which no apparent adverse effects were seen (Fig. 5). A more important finding was that the antitumor activity of DIBLOCK was comparable to that of SP-THP and yielded significant suppression of S180 tumor growth in mice (Nakamura et al., 2015)).

5. Conclusions We described herein the synthesis of the polymer-drug conjugates LINEAR and 27

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DIBLOCK and results of investigations of their properties, including free drug release, in vitro cytotoxicity, in vivo pharmacokinetic properties and toxicity, and in vivo antitumor effects in mice. The release profile of free THP and in vitro cytotoxicity of DIBLOCK were similar to those of LINEAR, but more important, the larger DIBLOCK

PT

had superior pharmacokinetic properties and much improved in vivo antitumor effects

RI

compared with LINEAR. The toxicity of DIBLOCK was lower than that of the

SC

earlier-studied SP-THP, although DIBLOCK was slightly more toxic than LINEAR. Nevertheless, DIBLOCK showed promising antitumor activity and no toxicity at

NU

therapeutically effective doses in vivo, which may allow use of increased doses and improved activity of the conjugate. We also found that the reductively degradable

MA

disulfide bond that connected the two shorter linear copolymers into one diblock copolymer with a higher (double) Mw (above the renal threshold) was quite stable in the

D

blood circulation but susceptible to reductive degradation in tumor tissues. This bond

PT E

may thus be applied to the development of drug delivery carriers with an apparent site specificity for solid tumors. These findings therefore suggest that the DIBLOCK

CE

conjugate manifests improved tumor selectivity and therapeutic efficacy compared with the earlier-developed LINEAR conjugate and that its enhanced accumulation in solid

AC

tumors is accompanied by subsequent biodegradation and elimination of the polymer carrier from the living body.

Acknowledgements The authors acknowledge support from the Czech Science Foundation GA CR (grant no. 15-02986S), from the Academy of Sciences of the Czech Republic (grant no. JSPS-16-05), and from the National Sustainability Program II (Biocev-FAR LQ1604) 28

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and project ―BIOCEV‖ (CZ.1.05/1.1.00/02.0109). We also acknowledge support from the Ministry of Health, Labour and Welfare (MHLW), Japan for Cancer Specialty Grant (2011–2014); from the Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology (MECSST), Japan, and

PT

the Adaptable and Seamless Technology Transfer Program (A-STEP) through

RI

target-driven R&D from Japan Science and Technology Agency (JST); and from the

SC

2016-2017 Japan-Czech Republic Research Cooperative Program (JSPS) for H. Maeda.

AC

CE

PT E

D

MA

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We acknowledge the valuable technical help of Mr. W. Islam and Ms. A. Yamashiro.

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PT

Copolymer-Conjugated Pirarubicin in Multimodal Treatment of a Patient with Stage IV Prostate Cancer and Extensive Lung and Bone Metastases. Target Oncol 11, 101-106.

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Etrych, T., Chytil, P., Mrkvan, T., Sirova, M., Rihova, B., Ulbrich, K., 2008a. Conjugates of doxorubicin with graft HPMA copolymers for passive tumor targeting. J Control Release 132,

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Etrych, T., Jelinkova, M., Rihova, B., Ulbrich, K., 2001. New HPMA copolymers containing

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doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties. J Control Release 73, 89-102.

Etrych, T., Kovar, L., Subr, V., Braunova, A., Pechar, M., Chytil, P., Rihova, B., Ulbrich, K.,

MA

2010a. High-molecular-weight Polymers Containing Biodegradable Disulfide Bonds: Sunthesis and In Vitro Verification of Intracellular Degradation. . J. Bioact. Compat. Polymers 25, 5-26. T.,

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PT E

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AC

Etrych, T., Subr, V., Laga, R., Rihova, B., Ulbrich, K., 2014. Polymer conjugates of doxorubicin bound through an amide and hydrazone bond: Impact of the carrier structure onto synergistic action in the treatment of solid tumours. Eur J Pharm Sci 58, 1-12. Etrych, T., Subr, V., Strohalm, J., Sirova, M., Rihova, B., Ulbrich, K., 2012. HPMA copolymer-doxorubicin conjugates: The effects of molecular weight and architecture on biodistribution and in vivo activity. J Control Release 164, 346-354. Fang, J., Nakamura, H., Maeda, H., 2011. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev 63, 136-151. Gerweck, L.E., Seetharaman, K., 1996. Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer. Cancer Res 56, 1194-1198.

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Hatakeyama, H., Akita, H., Harashima, H., 2011. A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. Adv Drug Deliv Rev 63, 152-160. Hatakeyama, H., Akita, H., Kogure, K., Oishi, M., Nagasaki, Y., Kihira, Y., Ueno, M., Kobayashi, H., Kikuchi, H., Harashima, H., 2007. Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid. Gene Ther 14,

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Koh, E., Ueda, Y., Nakamura, T., Kobayashi, A., Katsuta, S., Takahashi, H., 2002. Apoptosis

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Kostkova, H., Etrych, T., Rihova, B., Kostka, L., Starovoytova, L., Kovar, M., Ulbrich, K., 2013. HPMA copolymer conjugates of DOX and mitomycin C for combination therapy: physicochemical characterization, cytotoxic effects, combination index analysis, and

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anti-tumor efficacy. Macromol Biosci 13, 1648-1660.

Kunimoto, S., Miura, K., Takahashi, Y., Takeuchi, T., Umezawa, H., 1983. Rapid uptake by

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cultured tumor cells and intracellular behavior of 4'-O-tetrahydropyranyladriamycin. J Antibiot (Tokyo) 36, 312-317.

Kunimoto, S., Miura, K., Umezawa, K., Xu, C.Z., Masuda, T., Takeuchi, T., Umezawa, H., 1984.

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PT E

Antibiot (Tokyo) 37, 1697-1702.

D

4'-O-tetrahydropyranyladriamycin in adriamycin-sensitive and resistant tumor cell lines. J Luo, K., Yang, J., Kopeckova, P., Kopecek, J., 2011. Biodegradable Multiblock Poly[N-(2-hydroxypropyl)methacrylamide] via Reversible Addition-Fragmentation Chain

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Transfer Polymerization and Click Chemistry. Macromolecules 44, 2481-2488. Maeda, H., 2015. Toward a full understanding of the EPR effect in primary and metastatic tumors as well as issues related to its heterogeneity. Adv Drug Deliv Rev 91, 3-6.

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Maeda, H., Nakamura, H., Fang, J., 2013. The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev 65, 71-79. Malugin, A., Kopeckova, P., Kopecek, J., 2007. Liberation of doxorubicin from HPMA copolymer conjugate is essential for the induction of cell cycle arrest and nuclear fragmentation in ovarian carcinoma cells. J Control Release 124, 6-10. Matsumura, Y., Maeda, H., 1986. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 46, 6387-6392. Nakamura, H., Etrych, T., Chytil, P., Ohkubo, M., Fang, J., Ulbrich, K., Maeda, H., 2014.

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Two step mechanisms of tumor selective delivery of N-(2-hydroxypropyl)methacrylamide copolymer conjugated with pirarubicin via an acid-cleavable linkage. J Control Release 174, 81-87. Nakamura, H., Koziolova, E., Chytil, P., Tsukigawa, K., Fang, J., Haratake, M., Ulbrich, K., Etrych, T., Maeda, H., 2016. Pronounced Cellular Uptake of Pirarubicin versus That of Other Anthracyclines: Comparison of HPMA Copolymer Conjugates of Pirarubicin and

PT

Doxorubicin. Mol Pharm 13, 4106-4115.

Nakamura, H., Koziolova, E., Etrych, T., Chytil, P., Fang, J., Ulbrich, K., Maeda, H., 2015.

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Comparison between linear and star-like HPMA conjugated pirarubicin (THP) in 90-96.

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pharmacokinetics and antitumor activity in tumor bearing mice. Eur J Pharm Biopharm 90, Pan, H., Sima, M., Yang, J., Kopecek, J., 2013. Synthesis of long-circulating, backbone degradable

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copolymer-doxorubicin

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molecular-weight-dependent antitumor efficacy. Macromol Biosci 13, 155-160. Prabhakar, U., Maeda, H., Jain, R.K., Sevick-Muraca, E.M., Zamboni, W., Farokhzad, O.C.,

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Barry, S.T., Gabizon, A., Grodzinski, P., Blakey, D.C., 2013. Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res 73, 2412-2417.

Saito, G., Swanson, J.A., Lee, K.D., 2003. Drug delivery strategy utilizing conjugation via

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reversible disulfide linkages: role and site of cellular reducing activities. Adv Drug Deliv Rev Subr,

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55, 199-215.

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N-(2-hydroxypropyl)methacrylamide copolymers containing thiazolidine-2-thione reactive

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groups. Reactive and Functional Polymers 66, 1525-1538. Tannock, I.F., Rotin, D., 1989. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res 49, 4373-4384.

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Ulbrich, K., Etrych, T., Chytil, P., Pechar, M., Jelinkova, M., Rihova, B., 2004. Polymeric anticancer drugs with pH-controlled activation. Int J Pharm 277, 63-72. Ulbrich, K., Subr, V., Strohalm, J., Plocova, D., Jelinkova, M., Rihova, B., 2000. Polymeric drugs based on conjugates of synthetic and natural macromolecules. I. Synthesis and physico-chemical characterisation. J Control Release 64, 63-79. Umezawa, H., Takahashi, Y., Kinoshita, M., Naganawa, H., Masuda, T., Ishizuka, M., Tatsuta, K., Takeuchi, T., 1979. Tetrahydropyranyl derivatives of daunomycin and adriamycin. J Antibiot (Tokyo) 32, 1082-1084. Yang, J., Zhang, R., Radford, D.C., Kopecek, J., 2015. FRET-trackable biodegradable HPMA copolymer-epirubicin conjugates for ovarian carcinoma therapy. J Control Release 218,

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36-44. Yang, Y., Pan, D., Luo, K., Li, L., Gu, Z., 2013. Biodegradable and amphiphilic block copolymer-doxorubicin conjugate as polymeric nanoscale drug delivery vehicle for breast cancer therapy. Biomaterials 34, 8430-8443. Zhang, R., Luo, K., Yang, J., Sima, M., Sun, Y., Janat-Amsbury, M.M., Kopecek, J., 2013. Synthesis and evaluation of a backbone biodegradable multiblock HPMA copolymer

AC

CE

PT E

D

MA

NU

SC

RI

PT

nanocarrier for the systemic delivery of paclitaxel. J Control Release 166, 66-74.

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Table 1 Characteristics of the polymer precursors and polymer conjugates.

(g/mol)

PĐ

End group

Side chain

b

a

F

groups

-NHNH2

THP

Rh

(wt%)

(nm)

-

3.8

5.3

-

4.1

content

PT

Polymer

Mw

(mol%)

1

27,000

1.91

-

-

-NHNH2

2

38,300

1.85

TT

1.28

-NHNH-Boc

3

75,800

1.83

-

-

-NHNH-Boc

5.3

-

5.9

4

78,200

1.85

-

-

-NHNH2

5.3

-

6.3

LINEAR

39,000

1.85

-

-

-NHNH=THP

-

9.9

4.2

DIBLOCK

93,000

1.91

-

-

10.5

6.6

RI

SC

NU

MA -

5.6

-NHNH=THP

c

Abbreviations: Mw, molecular weight; PĐ, polydispersity index; THP, pirarubicin.

b

D

The end group of the semitelechelic polymer precursor. The functionality of the semitelechelic polymer precursor determined by UV-VIS

PT E

a

spectroscopy.

CE

The hydrodynamic radius determined by dynamic light scattering;

AC

c

34

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Table 2 Mortality of mice and LD50 values after i.v. administration of LINEAR or DIBLOCK. Drug dose (mg/kg of THP equivalent)

0/6

40

0/6

LINEAR 6/6

80

6/6

20

1/6

NU

40

3/6

MA

DIBLOCK 60 80

37.3

6/6

D

6/6

PT E

a

50.0

SC

60

a

PT

equivalent)

20

THP, pirarubicin.

LD50 (mg/kg of THP

Mortality

RI

Polymer

LD50 values were calculated by using the Reed and Muench method.

CE

Table 3

AC

Effects of free THP, LINEAR, and DIBLOCK on the liver, heart, and kidney. Time

CK

AST

LDH

BUN

CRE

(U/L)

(U/L)

(U/L)

(mg/dL)

(mg/dL)

18.8 ± 4.6

34.0 ± 4.5

163.0 ± 32.4

27.7 ± 1.7

0.10 ± 0.01

1 day

62.0 ± 39.8

34.5 ± 1.9

193.5 ± 25.5

20.7 ± 3.6

0.10 ± 0.01

5 days

Not tested

30.3 ± 3.3

173.8 ± 27.0

27.3 ± 2.2

0.11 ± 0.01

10 days

71.8 ± 19.3*

33.3 ± 4.6

186.5 ± 42.3

31.6 ± 6.3

0.11 ± 0.02

LINEAR

1 day

68.3 ± 41.0

34.5 ± 5.1

215.5 ± 28.3

21.8 ± 2.1

0.11 ± 0.02

(15 mg/kg of THP

5 days

Not tested

30.3 ± 3.2

176.5 ± 26.2

25.3 ± 4.7

0.10 ± 0.01

equivalent)

10 days

73.5 ± 29.3*

32.3 ± 3.3

172.3 ± 48.6

34.6 ± 5.4

0.11 ± 0.02

Dose of drugs (i.v.)

after

injection

Control (no drug) Free THP

a

(5 mg/kg)

35

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DIBLOCK

1 day

65.5 ± 38.2

31.8 ± 1.5

165.5 ± 18.4

24.4 ± 4.0

0.10 ± 0.01

(15 mg/kg of THP

5 days

Not tested

30.0 ± 2.3

159.0 ± 21.3

25.1 ± 3.1

0.11 ± 0.01

equivalent)

10 days

60.8 ± 10.7*

30.5 ± 1.7

164.0 ± 12.2

28.7 ± 3.3

0.10 ± 0.01

THP, pirarubicin; CK, creatine kinase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; CRE, creatinine.

PT

Analyses were performed on days 1, 5, and 10 after drug administration. Values are

RI

means ± S.D. (n = 4).

SC

*p < 0.05, significantly different from the untreated control group, according to Student's t-test.

CE

PT E

D

MA

NU

THP at 5 mg/kg is about maximum tolerable dose.

AC

a

36

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Graphical abstract

37