Design of Macromolecular Prodrug of Cisplatin Using Dextran with

Department of Applied Chemistry, Faculty of Engineering & High Technology Research Center,. Kansai University, Suita, 564-8680, Japan. Received March ...
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Biomacromolecules 2001, 2, 927-933

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Design of Macromolecular Prodrug of Cisplatin Using Dextran with Branched Galactose Units as Targeting Moieties to Hepatoma Cells Yuichi Ohya,* Hidekazu Oue, Kazuya Nagatomi, and Tatsuro Ouchi Department of Applied Chemistry, Faculty of Engineering & High Technology Research Center, Kansai University, Suita, 564-8680, Japan Received March 13, 2001; Revised Manuscript Received May 28, 2001

We previously reported that a macromolecular prodrug synthesized by immobilizing cisplatin (CDDP) to dextran (Dex) through six-membered chelate-type coordination bond (DCM-Dex/CDDP conjugate) showed a significantly longer half-life in bloodstream and excellent in vivo tumor growth inhibitory effect against mice bearing Colon 26 cancer cells. In this report, to provide DCM-Dex/CDDP conjugate having targetability to hepatoma cells, we designed a new macromolecular prodrug of CDDP using dextran having branched galactose units (Gal4As, four branched galactose residues), DCM-Dex/Gal4A/CDDP conjugate. Galactose was employed as a homing device, because it is well-known that galactose receptors (asialoglycoprotein receptors) were exposed on the surface of liver parenchymal cells. The antennary (branched) structure of Gal4A was designed based on the fact that a saccharide cluster having a branched structure shows highly effective binding with the saccharide receptors, that is a “cluster effect”. The apparent affinity constant per galactose residue against RCA120 lectin for dextran carrying Gal4As was higher than that for dextran carrying monomeric galactose residues. Moreover, the DCM-Dex/Gal4A/CDDP conjugate showed cell-specific cytotoxic activity against HepG2 human hepatoma cells in vitro. The cytotoxic activity of the conjugate was inhibited by the addition of galactose and strongly inhibited by the addition of Gal4A. The results suggest that the DCM-Dex/Gal4A/CDDP conjugate having branched galactose units has a higher affinity to hepatoma cells. Introduction Some antitumor agents, used in cancer chemotherapy at present, have remarkable antitumor activities; however, the distributions of antitumor agents to normal cells cause undesirable side effects. In comparison with a conventional low-molecular-weight drug, macromolecular prodrug, that is a polymer-drug conjugate, can be expected to overcome such serious problems by improving the body distribution of antitumor agent through enhancement permeability and retention (EPR) effect.1 Indeed, Yamaoka et al. studied the in vivo blood half-life and the body distribution of some water-soluble polymers and reported that the higher-molecular-weight polymers showed longer blood half-lives.2,3 Many macromolecular prodrugs using water-soluble polymers as drug carriers have been studied.4-14 Dextran (Dex) is a watersoluble biodegradable polymer and has some advantages as a drug carrier, because it shows low immunogenicity and low toxicity and has many hydroxyl groups which can be easily modified chemically. Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is one of the most potent antitumor platinum complexes15 and is widely used in the treatment of various solid tumors. However, renal clearance of CDDP is quite fast, and the accumulation of CDDP in kidney causes severe renal toxicities.16 Moreover, CDDP is sparingly soluble not only in water but also in lipid. Actually, the clinical application of CDDP has been limited. We previously reported the syn* To whom all correspondence should be addressed. E-mail: yohya@ ipcku.kansai-u.ac.jp.

thesis of a macromolecular prodrug using dextran by immobilizing CDDP or other platinum complexes through six-membered chelate-type coordination bonds to dextran derivative having dicarboxymethyl groups (DCM-Dex/platinum complex conjugate).12-14 It is known that the cytotoxic activity of the CDDP and similar antitumor platinum complexes gradually decreases in the bloodstream because of ligand exchange reactions with substances having amino groups in serum, such as proteins, amino acids, and so on. The DCM-Dex/CDDP conjugates obtained showed comparable in vitro cytotoxic activity with free CDDP and good maintenance of the cytotoxic activity in the presence of serum because of the steric hindrance of Dex chains and stability of the six-membered chelate-type coordination bond.11 The DCM-Dex/CDDP conjugate showed sustained in vitro release behavior of platinum complex in physiological phosphate buffer solution: half of the CDDP immobilized on the conjugate was released in about 48 h.13 Moreover, the DCM-Dex/CDDP conjugate showed a significantly longer half-life in the bloodstream of rats and an excellent in vivo tumor growth inhibitory effect against mice bearing Colon 26 cancer cells.13,14 It is well-known that some kinds of saccharide play important roles in biological recognition on cellular surfaces. Liver parenchymal cells exclusively express large numbers of asialoglycoprotein receptors that strongly bind with galactose. In particular, the “cluster effect” referred to by Lee et al.17,18 is an increase in the binding strength of galactose cluster beyond that expected from an increase in the galactose concentration. The cluster effect depends on the spatial

10.1021/bm010053o CCC: $20.00 © 2001 American Chemical Society Published on Web 08/03/2001

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Figure 1. Structure of DCM-Dex/Gal4A/CDDP conjugate.

distances between galactose residues, the flexibility of the arm connecting galactose residues, and the structure of branch points.19 In our previous paper, a chitosan conjugate having antennary galactose units (four branched galactose residues) was designed as a gene delivery tool, and the cluster effect of the conjugate against hepatoma cells was observed.20 Therefore, a multiantennary saccharide unit can be expected to apply for effective cellular recognition devices. In this study, to achieve effective accumulation of the DCM-Dex/CDDP conjugate to hepatoma cells by receptormediated recognition, we introduced branched galactose units (Gal4As) carrying four galactose residues into the DCMDex/CDDP conjugate to give DCM-Dex/Gal4A/CDDP conjugate (Figure 1). The branched structure of Gal4A was designed to achieve high affinity to hepatoma cells by cluster effect of saccharide residues. The apparent affinity constant of dextran having Gal4As with RCA120 lectin was evaluated. Moreover, the cytotoxic activity and the galactose receptormediated recognition of the DCM-Dex/Gal4A/CDDP conjugate against HepG2 human hepatoma cells having galactose receptors21 were investigated in vitro. Experimental Section Materials. Dextran and CDDP were purchased from Sigma Chemical Co. Ltd. The organic solvents were purified by usual distillation methods. The other materials were of commercial grades and used without further purification. Synthesis of Branched Galactose Unit. The synthesis of the branched galactose unit (Gal4A) was performed as shown in Schemes 1-3. Lactonolactone was prepared from lactose according to the method described in the reference.22 Lactonolactone (890 mg, 2.5 mmol) and 4,7,10-trioxa-1,13tridecanediamine (610 mg, 2.8 mmol) were dissolved in methanol (80 mL) and then refluxed for 16 h. This reaction mixture was reprecipitated by diethyl ether and then the produced Gal-TEG-amine 1 was isolated by a cationexchange resin column (SP-Sephadex C-25, eluent: water

Scheme 1. Synthesis of Gal-TEG-amine 1

Scheme 2. Synthesis of Branch Unit 2

to 0.1 M NH4HCO3(aq) gradient). Yield: 710 mg, 53%. IR (KBr): ν ) 1652 cm-1 (CONH). 1H NMR (DMSO-d6): δ ) 1.62 (4H, CH2CH2CH2), 2.95 (2H, CH2NH2), 3.10-4.30 ppm (CH2NH, CH2O and saccharide). MALDI-TOF-MS: [M + Na]+ ) 583.6 (calcd), [M + Na]+ ) 583.7 (found). [M + K]+ ) 599.7 (calcd), [M + K]+ ) 599.7 (found). Branch unit 2 was typically synthesized by the following procedures. 6-(t-Butoxycarbonylamino)capronic acid (Boccapronic acid) (694 mg, 3.0 mmol) and diethyl glutamate (719 mg, 3.0 mmol) were reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC‚HCl) (690 mg, 3.6 mmol, 1.3 equiv) in the presence of TEA (0.5

Design of Macromolecular Prodrug of Cisplatin

Figure 2. IR spectra for the synthesis of Gal4A-Boc. (a) IR spectrum (KBr) of branch unit 2 before reaction with Gal-TEG-amine 1. (b) IR spectrum (KBr) of Gal4A-Boc obtained after reaction of branch unit 2 with Gal-TEG-amine 1. Scheme 3. Synthesis of Gal4A

mL) in dichloromethane. After 18 h stirring, the reaction mixture was washed with 10% aqueous citric acid, 4% aqueous sodium bicarbonate, and water, three times each. The obtained product, Boc-capronic acid-Glu(OEt)2, was dissolved in MeOH (4.6 mL) and then 1.15 mL of 1 N NaOH(aq) was added to the solution. After 6 h of stirring, 10 mL of water was added to the solution and MeOH was evaporated. The obtained alkali saponification products were acidified to pH ) 3 with 1 N HCl(aq), extracted with dichloromethane, and dried with anhydrous sodium sulfate. The obtained Boc-capronic acid-Glu(OH)2 was coupled with 2 equiv of diethyl glutamate and then alkali saponificated in the same manner descried above. The products in each step were identified 1H NMR spectra. Total yield: 21%. IR (KBr): Figure 2a. 1H NMR (CD3OD): δ ) 1.35 (2H, CH2CH2CH2NH), 1.46 (9H, Boc), 1.51 (2H, CH2CH2NH), 1.64 (2H, CH2CH2CO), 1.81 (2H, CH2CH2NH), 1.90-2.15 (6H, CH2CH), 2.10-2.50 (6H, CH2CH2CH), 3.08 (2H, CH2CO), 4.15 (3H, COCHNH), 4.77 ppm (3H, NHCO). 13C NMR (CD3OD): δ ) 29.0-33.9 (CH2CH2CH, CH2CH2CH2CH2CH2, and CCH3 of Boc), 35.3 (CH2CH2CH2CO), 39.1

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(CH2CH2NH), 55.7-56.5 (COCHNH), 82.3 (CCH3 of Boc), 161.0 (COO of Boc), 176.5-178.8 ppm (CHNHCO and COOH). MALDI-TOF-MS: [M + Na]+ ) 641.6 (calcd), [M + Na]+ ) 641.6 (found). [M + K]+ ) 657.7 (calcd), [M + K]+ ) 656.6 (found). The obtained branch unit 2 (480 mg, 0.78 mmol) was dissolved in N,N-dimethylformamide (DMF) and added to the solution of EDC‚HCl (700 mg, 3.7 mmol) and 1-hydroxybenzotriazole (HOBt) (500 mg, 3.7 mmol) in DMF at 0 °C. After 3 h of stirring, Gal-TEG-amine 1 (2070 mg, 3.7 mmol) was added to the solution and then further stirred at 0 °C for 1 h, following by stirring at room temperature for 25 h. The reaction mixture was subjected to gel-filtration chromatography (Sephadex LH-20; column, o.d. 40 × 550 mm; eluent, DMF) to give protected Gal4A with tert-butoxycarbonyl group (Gal4A-Boc). The reaction steps were repeated until all of the carboxylic acid groups were replaced by Gal-TEG-amine by confirming with IR spectra (Figure 2b). Gal4A-Boc (1420 mg, 0.52 mmol) was dissolved in dioxane (20 mL) and added to 4 M HCl/dioxane (20 mL). The mixture was stirred at room temperature for 2 h. The reaction mixture was reprecipitated by diethyl ether to give Gal4A. The deprotecting reaction was confirmed by disappearance of the Boc group signal in the 1H NMR spectra (CD3OD). The spectral data and their assignments of Gal4A are shown in Figure 3. Yield: 1250 mg, 44%. MALDITOF-MS: [M + Na]+ ) 2623.2 (calcd), [M + Na]+ ) 2622.9 (found). Synthesis of the Conjugate. The preparation of DCMDex/Gal4A/CDDP conjugate was carried out according to Schemes 4-6. Dextran (Mw ) 4.2 × 104, Mw/Mn ) 2.4) (1000 mg, 4.5 mmol in sugar unit) was dissolved in 8.8 M NaOH(aq) (30 mL) at 0 °C. Diethyl bromomalonate (2.5 × 104mg, 100 mmol) in tetrahydrofuran (30 mL) was added to the solution at 0 °C. The solution was stirred at room temperature for 24 h. The reaction mixture was reprecipitated by acetone and then dialyzed in distilled water using cellulose tubing for 7 days. The obtained solution was passed through a column packed with cation-exchange resin (Amberlite 120B H+ type) and freeze-dried to give DCM-Dex. The degree of introduction of dicarboxymethyl group (DDCM) was measured by a neutralization titration method23 to be 25.6 mol % per sugar unit. Yield: 920 mg. IR (KBr): 1723 cm-1 (COOH). The obtained DCM-Dex (500 mg, 3.1 mmol in sugar unit) was dissolved in dimethyl sulfoxide (DMSO). 4-Nitrophenylchloroformate (650 mg, 3.2 mmol) and 4-(dimethylamino)pyridine (DMAP) (350 mg, 2.8 mmol) were added to the ice-cooled solution. The reaction mixture was stirred at 0 °C for 4 h and then reprecipitated by acetone/diethyl ether/ ethanol (1/1/2, v/v/v) to give DCM-Dex activated ester. The DCM-Dex activated ester was dissolved in DMSO, and then Gal4A was added to the solution. The mixture was stirred at room temperature for 36 h. After evaporation, the residue was dissolved in DMF and subjected to gel-filtration chromatography (Sephadex LH-20; column, o.d. 40 × 550 mm; eluent, DMF) to give DCM-Dex/Gal4A. The degree of introduction of Gal4A (DGal4A) per sugar unit was estimated

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Figure 3.

1H

Ohya et al.

NMR spectrum of Gal4A in CD3OD and the assignments.

Scheme 4. Synthesis of DCM-Dex

Scheme 6. Synthetic Route of DCM-Dex/Gal4A/CDDP Conjugate

Scheme 5. Synthesis of DCM-Dex/Gal4A

to be 2.9 mol % from the N/C ratio of the elemental analysis. Yield: 540 mg. CDDP (500 mg, 1.6 mmol) dissolved in water was added to 0.1 M AgNO3 solution. The reaction mixture was stirred at 60 °C for 6 h. The silver chloride precipitate was removed

by filtration. The filtrate containing CDDP (nitrato) was passed through a column packed with anion-exchange resin (Diaion SA-10A OH- type) to convert to CDDP (hydroxo). The obtained solution was added to the DCM-Dex/Gal4A (540 mg, 3.3 mmol in sugar unit) in water. The reaction mixture was stirred at 60 °C for 24 h. The product was purified by gel-filtration chromatography (Sephadex G-25; column, o.d. 30 × 350 mm; eluent, water). The highmolecular-weight fraction was corrected and freeze-dried to give the DCM-Dex/Ga4A/CDDP conjugate. The degree of

Design of Macromolecular Prodrug of Cisplatin

introduction of platinum (DPt) per sugar unit was estimated to be 4.6 mol % by o-phenylenediamine (OPDA) method.24 Yield: 450 mg. DCM-Dex/Gal was synthesized from DCM-Dex and Gal-TEG-amine using 4-nitrophenylchloroformate and DMAP as condensation reagents by similar procedures described above. The degree of introduction of galactose residue per sugar unit (DGal) was estimated to be 8.0 mol % by N/C ratio of elemental analysis. DCM-Dex/Gal4A having 8.0 mol % of DGal4A was also synthesized by the same procedures described above. These DCM-Dex and DCM/ Gal4A were used for the investigations of interactions with lectin. DCM-Dex/CDDP conjugate having no galactose residue was synthesized using DCM-Dex and CDDP by the same procedures described above and used as a control for in vitro cytotoxic acitvity measurement. The DPt was estimated to be 5.9 mol % by OPDA method. Interaction of Dextran Derivatives with RCA120 Lectin. The interactions of dextran derivatives, DCM-Dex/Gal4A carrying branched galactose unit (DGal4A ) 5.0 mol % per sugar unit), DCM-Dex/Gal carrying single galactose residue (DGal ) 8.0 mol % per sugar unit), or DCM-Dex carrying no galactose residue, with RCA120 lectin, which recognizes β-D-galactose and β-D-N-acetylgalactosamine residues, were evaluated by surface plasmon resonance (BIAcore system, Pharmacia Biotech).25 The biotin-labeled RCA120 lectin solution was injected onto the streptoavidine preimmobilized surface of a sensor chip, and various concentrations of the dextran derivatives were injected to measure sensorgrams. The obtained sensorgrams were used for kinetic analyses of the interactions between RCA120 lectin and dextran derivatives to determine the apparent affinity constants of dextran derivatives. Cytotoxic Activity Measurement. The cytotoxic activity of the DCM-Dex/Gal4A/CDDP conjugate was measured against HepG2 human hepatoma cells in vitro. The tumor cells suspension containing 5 × 104cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) was distributed in a 96-wells microplate and incubated with each drug in a humidified atmosphere containing 5% CO2 at 37 °C for 48 h. The number of viable cells was determined by means of the MTT (3-(4,5dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay using a microplate reader. The cytotoxic activity was estimated by the following equation. cytotoxic activity (%) ) (C - T)/C × 100 C ) number of viable cells of control T ) number of viable cells of treated The galactose receptor-mediated cytotoxic activity against HepG2 human hepatoma cells in vitro was evaluated by the addition of inhibitor (galactose or Gal4A). The tumor cells suspension containing 5 × 104cells in DMEM containing 10% FCS was distributed in a 96-wells microplate. The tumor cells were preincubated in culture medium containing certain amount of galactose or Gal4A at 4 °C for 2.5 h and then incubated with each drug at 4 °C for 2 h. The concentration of platinum of drugs was 1.4 × 10-4 or 2.8 ×

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10-4 mol/L. The tumor cells were washed with culture medium several times and then incubated in fresh culture medium in a humidified atmosphere containing 5% CO2 at 37 °C for 48 h. The cytotoxic activities were measured by the same methods described above. Results and Discussion Synthesis of the Conjugate. To provide a macromolecular prodrug of CDDP using dextran having high targetability to hapatoma cells, DCM-Dex/Gal4A/CDDP conjugate having branched galactose units was synthesized. Triethylene glycol spacer was employed as a flexible connecting arm between galactose and the branch unit to get good mobility of four galactose residues. Moreover, glutamic acid was chosen for a branch point of the branch unit because a high recognition ability against galactose receptor was reported for the galactose cluster using glutamic acids as branch points.19 Gal4A was synthesized according to the reaction steps shown in Schemes 1-3. The reaction steps of the branch unit 2 with Gal-TEG-amine 1 to prepare Gal4A-Boc were repeated until all of the carboxylic acid groups were replaced by Gal-TEG-amine, because it was difficult to isolate Gal4A-Boc from the partially reacted products (branched unit carrying 0-3 residues of galactose). Parts a and b of Figure 2 show the IR spectra of branch unit 2 before reaction with Gal-TEG-amine 1 and Gal4A-Boc obtained after reaction of the branch unit 2 with Gal-TEG-amine 1, respectively. Although a peak at 1720 cm-1 attributed to a carboxylic acid group was observed in Figure 2a, such a peak was not observed in Figure 2b. These results show that the introduction of Gal-TEG-amine 1 into branch unit 2 proceeded quantitatively. This was also confirmed by integration ratios in the 1H NMR spectra (Figure 3). The degree of introduction of Gal-TEG-amine was estimated to be over 99 mol % per carboxylic acid group by 1H NMR spectra. The conjugates having Gal4A were synthesized by the reaction steps shown in Schemes 4-6. The preparation of DCM-Dex/Gal4A was carried out by the coupling reaction of the primary amino group and the hydroxyl group of DCM-dextran activated with 4-nitrophenylchloroformate. After the coupling reaction, low-molecular-weight compounds, the coupling reagents, and unreacted Gal4A were successfully removed by gel-filtration chromatography. The DCM-Dex/Gal4A/CDDP conjugate was prepared by the ligand exchange reaction of DCM-Dex/Gal4A and CDDP (hydroxo) in water. The conjugate was purified by gelfiltration chromatography to remove unreacted CDDP derivatives. The introduction of platinum for DCM-Dex/ Gal4A/CDDP conjugate per sugar unit was estimated to be 4.6 mol %. The obtained conjugates showed high watersolubility. Interaction of Polysaccharides with RCA120 Lectin. The affinities of dextran derivatives carrying branched galactose unit, monomeric galactose or no galactose (DCM-Dex/ Gal4A, DCM-Dex/Gal, or DCM-Dex) with RCA120 lectin were investigated by surface plasmon resonance method. The results are summarized in Table 1. The apparent affinity constant was obtained as a ratio of apparent association rate constant kassn to apparent dissociation rate constant kdis and

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Table 1. Apparent Affinity Constants of Dextran Delivatives against RCA120 Lectin apparent affinity constant Kc dextran delivative

kassna × 10-3

kdisb × 104

DCM-Dex/Gal4A 2.18 4.41 DCM-Dex/Gal 0.423 6.57 DCM-Dex -0.00598 166

per conjugate × 10-6 (M-1)

per galactose × 10-5 (M-1)

4.95 0.644 -0.000360

2.3 0.74

a Apparent association rate constant. b Apparent dissociation rate constant. c Apparent affinity constant K ) kassn/kdis.

evaluated per conjugate or per galactose residue. DCMDex carrying no galactose showed almost no affinity against the lectin. DCM-Dex/Gal and DCM-Dex/Gal4A showed effective values of apparent affinity constant. The apparent affinity constant of DCM-Dex/Gal4A was higher than that of DCM-Dex/Gal in both per conjugate and per galactose residue. DGal and DGal4A for DCM-Dex/Gal and DCM-Dex/ Gal4A was 5.0 and 8.0 mol %/sugar unit, respectively. These mean that about 8.7 residues of galactose was on a DCMDex/Gal and about 5.4 units of Gla4A () 21.6 residue of galactose) was on a DCM-Dex/Gal4A, which were calculated based on the number-average molecular weight. Both of DCM-Dex/Gal and DCM-Dex/Gal4A were polymeric ligands. The multivalent effects might be observed in the binding both of DCM-Dex/Gal and DCM-Dex/Gal4A with RCA120 lectin. Although the average distance between Gal4A units in DCM-Dex/Gal4A is smaller than the distance between galactoses in DCM-Dex/Gal, Dex-Dex/Gal4A showed a higher apparent affinity constant than DCM-Dex per galactose. These results suggest that a cluster effect contributed to the recognition of RCA120 lectin with DCMDex/Gal4A and the introduction of Gal4A unit is effective to obtain polymeric carrier having high affinity to hepatocytes. In Vitro Cytotoxic Activity of the Conjugate. The cytotoxic activity for DCM-Dex/Gal4A/CDDP conjugate was investigated against HepG2 human hepatoma cells in vitro for 48 h compared with free CDDP, DCM-Dex/CDDP conjugate having no galactose residue, and the mixture of DCM-Dex and free CDDP (Figure 4). Table 2 summarizes the IC50 values, the drug concentration at which the cytotoxic activity is 50%, estimated from the results shown in Figure 4. Although the cytotoxic activity of DCM-Dex/CDDP conjugate was lower than that of free CDDP, the cytotoxic activity of DCM-Dex/Gal4A/CDDP conjugate was the same level as that of free CDDP. The IC50 value for DCM-Dex/ Gal4A/CDDP conjugate was slightly smaller than that of free CDDP or the mixture of free CDDP and DCM-Dex (not significant). Normally the cytotoxic activity of the polymerdrug conjugate is smaller than that of parent drug, because the drug attached on the polymer is not active and it takes certain period to release the active parent drug. We had previously reported the in vitro release behavior of platinum complex from DCM-Dex/CDDP conjugate under physiological condition: about the half of CDDP immobilized on the conjugate was released in 48 h. Indeed, DCM-Dex/ CDDP conjugate showed larger IC50 value than free CDDP. The platinum complex release behavior of DCM-Dex/ Gal4A/CDDP conjugate must be similar that of DCM-Dex/ CDDP conjugate. It is an unusual and interesting result that

Figure 4. Cytotoxic activities of DCM-Dex/Gal4A/CDDP conjugate, DCM-Dex/CDDP conjugate, free CDDP, and the mixture of DCMDex/Gal4A and free CDDP against HepG2 human hepatoma cells at 37 °C for 48 h in vitro. Key (b) DCM-Dex/Gal4A/CDDP conjugate; ([) DCM-Dex/CDDP conjugate; (2) free CDDP; (9) mixture of DCM-Dex/Gal4A and free CDDP. Table 2. Cytotoxic Activity of Dextran Delivatives/CDDP Conjugates and Free CDDP against HepG2 Human Hepatoma Cells in Vitro sample DCM-Dex/Gal4A/CDDP DCM-Dex/Gal/CDDP free CDDP mixtureb

IC50a × 10-5 (M) 2.5 13.0 4.8 4.9

a IC ) concentration of Pt at which the cytotoxic activity reaches 50%. 50 Mixture containing the same amount of DCM-Dex/Gal4A and free CDDP as the DCM-Dex/GalA/CDDP conjugate.

b

Figure 5. Effects of galactose addition on the cytotoxic activities of DCM-Dex/Gal4A/CDDP conjugate and free CDDP against HepG2 human hepatoma cells in vitro. [CDDP] ) [Pt] for the conjugate ) 1.4 × 10-4mol/L. (/) P < 0.025 by Student’s t-test, compared with the data without galactose.

the cytotoxic activity of DCM-Dex/Gal4A/CDDP conjugate was no less than that of free CDDP. These results suggest that the DCM-Dex/Gal4A/CDDP conjugate having branched galactose units showed some acceleration effects on the cytotoxic activity against HepG2 human hepatoma cells. To investigate the possibility of galactose receptormediated cytotoxic activity for the DCM-Dex/Gal4A/CDDP conjugate, the effect of the addition of inhibitor (free galactose or Gal4A) on the cytotoxic activity was measured against HepG2 human hepatoma cells (Figures 5 and 6). As shown in Figure 5, the cytotoxic activity of free CDDP was not inhibited by the addition of galactose as an inhibitor.

Design of Macromolecular Prodrug of Cisplatin

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Conclusions

Figure 6. Effect of Gal4A addition on the cytotoxic activity of DCMDex/Gal4A/CDDP conjugate against HepG2 human hepatoma cells in vitro. [Pt] for the conjugate ) 1.4 × 10-4mol/L. (//) P < 0.01 by Student’s t-test, compared with the data without Gal4A.

A DCM-Dex/Gal4A/CDDP conjugate having branched galactose units was synthesized. The in vitro cytotoxic activity of the obtained conjugate against hepatoma cells was higher than that of the similar conjugate having no galactose residue. The cytotoxic activity of the conjugate against HepG2 cells was inhibited by the addition of inhibitor (galactose or Gal4A). Stronger inhibition effects were observed when Gal4A was used as an inhibitor. The higher potential of branched galactose unit, Gal4A, as a targeting moiety for hepatoma was revealed. Galactose receptormediated uptake of the DCM-Dex/Gal4A/CDDP conjugate into hepatoma cells was suggested. The obtained DCMDex/Gal4A/CDDP conjugate carrying CDDPs and branched galactose units can be expected to show high accumulation in hepatoma cells and show good in vivo therapeutic effects. Acknowledgment. This research was financially supported by a Grant-in-Aid for Scientific Research (09240103) from the Ministry of Education, Science, Culture, and Sports, Japan. References and Notes

Figure 7. Effects of Gal4A or galactose on the cytotoxic activity of DCM-Dex/Gal4A/CDDP conjugate against HepG2 human hepatoma cells in vitro. [Pt] ) 2.8 × 10-4mol/L. The conjugate showed 57% of cytotoxic activity. Key: (0) Gal4A; (O) galactose.

On the other hand, the cytotoxic activity of DCM-Dex/ Gal4A/CDDP conjugate was inhibited by the addition of excess amount of galactose. These results show that galactose receptor-mediated recognition for DCM-Dex/Gal4A/CDDP conjugate occurs. Figure 6 shows the effect of the addition of Gal4A on the cytotoxic activity of DCM-Dex/Gal4A/ CDDP conjugate. Comparing the results shown in Figures 5 and 6, the addition of 12 mg/mL of Gal4A showed higher inhibitory effect than the case of 4 mg/mL of galactose addition, where the total galactose concentration in inhibitor was the same. This is because Gal4A, branched galactose unit having multivalent galactose residues, has higher binding ability to the galactose receptor than free galactose. The inhibitory effects for these inhibitors were investigated on various concentrations of inhibitors (Figure 7). Gal4A showed the same level of inhibitory effect as 10 times higher amount of galactose showed. These results suggest that the introduction of Gal4A into the conjugate can accelerate the galactose receptor-mediated uptake into HepG2 cells by cluster effect. This is in good agreement with the previous reports about cluster effect of saccharide recognition on hepatocyte.16,17

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