[CANCER RESEARCH 50, 1693-1700. March 15. 1990]

Characterization and Anticancer Activity of the Micelle-forming Polymeric

Anticancer Drug Adriamycin-conjugated Poly(ethylene glycol)-Poly(aspartic

acid) Block CopolymerMasayuki Yokoyama,' Mizue Miyauchi, Noriko Yamada, Teruo Okano, Yasuhisa Sakurai, Kazunori Kataoka,

and ShoheiInoueInstitute of BiomédicalEngineering, Tokyo H omen 's Medical College, Kavtada-cho, Shinjuku-ku, Tokyo 162, Japan ¡M.Y., M. M., N. Y., T. O., Y. S.]; Department ofMaterials Science and Technology and Research Institute for Biosciences, Science University of Tokyo, Yama:aki 2641, Noda-shi, Chiba 278, Japan ¡K.A'./; andDepartment of Synthetic Chemistry, Faculty of Engineering, i'ntversity of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan fS. /./

ABSTRACT

Adriamycin (ADR), an anthracycline anticancer drug, was bound tothe poly(aspartic acid) chain of polyfethylene glycol)-poly(aspartic acid)block copolymer by amide bond formation between an amino group ofAdriamycin and the carboxyl groups of the poly(aspartic acid) chain. Thepolymeric drug thus obtained was observed to form a micelle structurepossessing diameter of approximately 50 nm, with a narrow distribution,in phosphate-buffered saline and to show excellent water solubilitydespite a large amount of ADR introduction. Further, it was able to bestored in lyophilized form without losing its water solubility in theredissolving procedure. Increased stability of the bound Adriamycinmolecules in phosphate-buffered saline and elimination of binding affinityfor bovine serum albumin due to the micelle formation were furtheradvantages of this polymeric drug. In vivo high anticancer activity of thismicelle-forming polymeric drug against P 388 mouse leukemia wasobtained with less body weight loss than that seen with free ADR, dueto low toxicity as compared with free ADR.

INTRODUCTION

It is well known that the utility of cancer chemotherapy isconsiderably restricted by toxic side effects of anticancer drugs.This restriction results from the fact that the anticancer drugsused in the present chemotherapy lack efficient selectivity formalignant cells. To suppress the toxic side effects of the anti-cancer drugs to normal cells and to improve their efficiencytoward malignant cells, studies of conjugating anticancer drugsto polymeric carriers have been carried out as one promisingapproach. This drug-polymer conjugate is called a "polymericdrug." Expected advantageous features of polymeric drugs are

preferable tissue distribution of drug given by the character ofthe polymeric carrier, prolonged half-life of drug in plasma,and controlled drug release from the polymeric carrier by adjustment of the chemical properties of the bond between thedrug and the carrier. Several kinds of polymers, naturally occurring and synthetic polymers, have been examined as carriersof anticancer drugs. Among naturally occurring polymers, im-munoglobulins are most widely used as the carrier, due to theirhigh specificity and wide applicability to many kinds of malignant cells. Utility of immunoglobulin as the polymeric carrieris, however, restricted by its chemical and physical properties.For example, modification of immunoglobulins by anticancerdrugs often leads to precipitation due to hydrophobicity of thedrug. Furthermore, modification procedures are limited to onesperformed in mild conditions to avoid denaturation of theimmunoglobulins during the modification. Alternatively, thepolymeric carrier of the drug can be freely designed using manykinds of synthetic polymers available today, and various organic

Received 7/6/89; revised 10/30/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.

reactions can be used to introduce drug to the synthetic polymeric carrier. From this point of view, several kinds of syntheticpolymers have been investigated, such as poly[/V-2-(hydroxy-propyl)methacrylamide] (1), poly(divinyl ether-co-maleic anhydride) (2), poly(styrene-co-maleic anhydride) (3), dextran (4,5), poly(ethylene glycol) (6), poly(L-glutamic acid) (7, 8), poly(t-aspartic acid) (9), and poly(L-lysine) (10, 11). All these examples

are in the category of homopolymers or alternating copolymers.Although considerable improvements in anticancer activityhave been obtained, the polymeric drugs using homopolymeror alternating copolymer often face a problem of water solubility. Introduction of a large quantity of drug to the polymersleads to precipitation of the polymeric drug, since most drugshave a hydrophobic character. One promising approach toovercome this difficulty is utilization of micelle-forming polymeric drug. A hydrophilic outer shell surrounds a drug-conjugated hydrophobic inner core, and this outer shell is consideredto help prevent this conjugate from precipitation.

The basic concept of this paper is the utilization of themicelle-forming polymeric drug as a novel anticancer agent.

The polymeric drug possessing a micellar structure is expectedto have a large diameter, as compared with unbound drug,which is a small molecule. The polymeric drugs with a micellarstructure of the ideal diameter are expected to circulate in theblood stream without embolization at capillaries, to escape fromexcretion in kidney, and to permeate into the target cellsthrough blood vessels. And this micellar form is expected tohelp protect the conjugated drug from enzymatic attack inplasma by concealing the conjugated drug in the hydrophobiccore of the micelles. Furthermore, nonselective uptake of thepolymeric drug by the reticuloendothelial system is thought tobe suppressed by choosing the appropriate character of theouter shell.

This paper deals with the anticancer activity as well as severalchemical and physical properties of a newly designed micelle-forming polymeric drug, poly(ethylene glycol)-poly(asparticacid) block copolymer conjugated with Adriamycin. Adriamycin is one of the most powerful and widely used anticancerdrugs. Poly(ethylene glycol) is known to be a nontoxic andnonimmunogenic water-soluble polymer, which is expected tobe the outer shell of the micelle. Poly(aspartic acid) is a syntheticpoly(amino acid) and, therefore, possible hydrolysis of its mainchain may play a role in releasing Adriamycin quickly from theconjugate after the uptake by the target cells. And it is notedthat the substance resulting from the main chain hydrolysis ofthe poly(aspartic acid) is a naturally occurring amino acid.

MATERIALS AND METHODS

Chemicals

a-Methyl-w-aminopoly(oxyethylene) (1) [CH3-poly(ethylene glycol)-NH2, M, = 4300] was kindly supplied by Nippon Oil & Fats Co., Ltd.,

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MICELLE-FORMING POLYMERIC ANTICANCER DRUG

ADR-HC12 was kindly supplied by Nippon Kayaku Co., Ltd. EDC was

purchased from Peptide Institute, Inc. (Japan). BSA was purchasedfrom Sigma Chemical Co.

HN—C'

Synthesis of Polymeric Drug

The synthetic procedure for the polymeric drug, Adriamycin-boundpoly(ethylene glycol)-poly(aspartic acid) block copolymer, was reportedelsewhere (12, 13). Here, the procedure is stated briefly. The syntheticroute to the polymeric drug is shown in Fig. 1.

The starting material, a-methyl-w-aminopoly(oxyethylene) (1) [CHj-poly(ethylene glycol)-NH2, M, = 4300], is poly(ethylene glycol) with a

methoxy group at one terminal and a primary amino group at the otherterminal. /i-Benzyl L-aspartate 7V-carboxy anhydride (2) was polymerized from the terminal amino group of 1 to obtain poly(ethylene glycol)-poly(/i-benzyl L-aspartate) block copolymer (3). /3-Benzyl L-aspartate/V-carboxy anhydride (2) (7.21 g) was dissolved in 12 ml of doublydistilled MA'-dimethylformamide followed by an addition of 60 ml of

distilled chloroform. <v-Methyl-u)-aminopoly(oxyethylene) (1) (6.00 g)

was dissolved in 60 ml of distilled chloroform and added to the solutionof 2. The reaction mixture was stirred for 70 h at 35°Cin a stream of

dry nitrogen. The product was precipitated with diethyl ether followedby freeze-drying from 1,4-dioxane. Yield was 10.09 g (84.5%). Thenumber of /J-benzyl L-aspartate units of 3 was found to be 17 by a 'H

NMR spectrum of 3.Protective benzyl groups of the 0-benzyl L-aspartate units were

removed by alkaline hydrolysis to obtain PEG-P(Asp) (4). Poly(ethyleneglycol)-poly(/3-benzyl L-aspartate) block copolymer (3) (10.03 g) wasdissolved in 100 ml of chloroform. Then, 78 ml of a solution of NaOH(0.43 N) in a mixture of water, methanol, and 2-propanol (volume ratio,1:2:2) were added. After vigorous stirring at 0°Cfor 10 min, the reaction

mixture was poured into 2000 ml of diethyl ether, and a precipitatewas collected by filtration, dissolved in distilled water, and dialyzedagainst distilled water with Spectrapor 7 dialysis membrane (molecularweight cut-off = 1000), followed by lyophilization. Yield was 3.94 g(49%). A 'H NMR spectrum of 4 revealed that main chain cleavage of

the poly(aspartic acid) chain did not occur with this alkaline hydrolysisprocedure and that 80 mol % of aspartic acid residues of 4 wereconverted to the /3-amide form.

ADR was introduced into the PEG-P(Asp) (4) by amide bond for

mation between an amino group of the Adriamycin molecule and acarboxyl group of an aspartic acid residue in the poly(aspartic acid)chain, using EDC as a coupling agent. ADR •¿�HC1 was dissolved in N,N-

dimethylformamide (guaranteed grade) followed by the addition of 1.3equivalents of triethylamine. PEG-P(Asp) (4) dissolved in a small

amount of distilled water and EDC was subsequently added to thesolution of ADR. The reaction mixture was stirred at 0°Cfor 4 h,

followed by a second addition of EDC. Then, the reaction mixture wasstirred overnight at room temperature. The resulting solution wasdialyzed against 0.1 Msodium acetate-buffered solution (pH 4.5), usingSpectrapor 7 (molecular weight cut-off = 1000), for 3 h, followed bygel filtration using Sephadex G-25 in the same buffer (pH 4.5) or byultrafiltration with Amicon PM-30 membranes to remove unboundADR and low molecular weight contaminants. The content of ADR inthe polymeric drug was determined by measuring absorbance at 485nm, on the assumption that t485ofthe Adriamycin residue bound to thepolymer was the same as that of free ADR. The ADR content wasexpressed in mol % with respect to the aspartic acid residues of PEG-P(Asp) in the reactants. By changing a weight ratio of PEG-P(Asp):ADR-HCl, the polymeric drugs PEG-P(Asp(ADR)) of various

ADR contents were synthesized.

2The abbreviations used are: ADR-HC1, Adriamycin hydrochloride; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carboduniide; BSA, bovine serum albumin;ADR, Adriamycin; PBS, sodium phosphate-buffered solution; PBSa, phosphate-buffered saline: PEG, polyfethylene glycol); P(Asp), poly(aspartic acid); PEG-P(Asp), poly(ethylene glycol)-poly(aspartic acid) block copolymer; PEG-P(Asp(ADR)), Adriamycin-bound polyfethylene glycol)-poly(aspartic acid) blockcopolymer: SDS, sodium dodecyl sulfate; T/C, treated/control.

l, CH3-PEG-NH2CH2COOCH2<¡

2, BLA-NCA

CH3-(OCH2CH2^ NH-(COCHNH^H

CH2COOCH2<3, PEG-PBLA

CHa-iOCHzCHztNH -<COCHNH)—(COCHzCHNHh-HI I vCH2COOH COOH

4, PEG-P(Asp)

ADRCHs-<OCH2CH2-)iïNH-<COCHNH)r-(COCH2CHNH>7H

CH2COR COR

O OH

OH or

5, PEG-P(Asp(ADR))

Fig. 1. Synthetic route for polymeric drug. BLA-NCA, /3-benzyl L-aspartate N-carboxy anhydride; PEG-PBLA, poly(ethylene glycol)-poly(fi-benzyl L-aspartate)block copolymer.

Laser Scattering

Laser scattering measurement was carried out using a Photon Cor-relater LPA-3000 (Otsuka Electronics) with a He/Ne laser beam, inPBSa (pH 7.4, 0.155 M), at room temperature.

Fluorescence Measurement

Fluorescence was measured using a Hitachi Fluorescence Spectro-photometer 650-60, with excitation at 471 nm, in PBSa (pH 7.4, 0.155M), at room temperature.

Albumin Binding

Binding affinity of ADR and PEG-P(Asp(ADR)) to BSA was investigated using ultrafiltration for ADR and gel filtration for PEG-P(Asp(ADR)).

Binding Assay of Adriamycin to BSA by Ultrafiltration. Two ml of asolution of ADR (at 7-8 X IO"5 M) and BSA (at 5 and 40 mg/ml) in

0.1 M PBS (pH 7.4) were prepared. This mixed solution was incubatedfor 10 min and then was ultrafiltrated with a Centricon-30 tube(equipped with a YM-30 ultrafiltration membrane), followed by washing twice with 1.0 ml of PBS. [Blank tests with a BSA solution (1 mg/ml) and an ADR solution (7.1 x 10~5 M) revealed that 95% of BSA

was filtered out and 91% of ADR was recovered in the filtrate.]Amounts of ADR in the residue on the ultrafiltration membrane andin the filtrate were determined by measuring absorbance at 485 nm.

Binding Assay of PEG-P(Asp(ADR)) to BSA by Gel Filtration. ThirtyMlof solutions of PEG-P(Asp(ADR)) (5) ([ADR] = 1.83 x 10~" M) and

of BSA (20 mg/ml) in 0.1 M PBS (pH 7.4) were mixed. The mixturewas analyzed by high performance liquid chromatography (gel filtrationtype) using a Toyo Soda model HLC-803 in 0.1 M PBS (pH 7.4,containing 0.3 M NaCl), at a flow rate of 1.0 ml/min, equipped with anAsahipak GS-520 column. Detection was performed at 280 nm(JACSO Uvidec 100) and 470 nm (ISCO UA-5).

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MICELLE-FORMING POLYMERIC ANTICANCER DRUG

In Vitro Stability

Samples were incubated in PBSa (pH 7.4, 0.155 M) at 37°Cin the

dark. Absorbance of the samples at 485 nm was measured at appropriatetime intervals.

In Vitro Cytotoxicity

P 388 mouse leukemia cells (1 x IO5 cell/ml) were incubated in

RPMI 1640 medium (containing 10% fetal calf serum and 5000 units/liter penicillin and streptomycin), in a Multiwell tissue culture plate(24-well) (Falcon 3047) for 24 h in 5% CO2, at 37*C. Drug was added

to the cells, and the number of cells was counted with a Coulter CounterZB-1 after 24 and 48 h of incubation.

In Vivo Anticancer Activity

P 388 mouse leukemia cells were maintained by i.p. passage in DBA/2 mice every week. Female CDF, mice were i.p. inoculated with P 388mouse leukemia cells (1 x IO6cells in 0.1 ml) on day 0. The mice were

i.p. inoculated with drug dissolved in a 0.9% NaCI solution on day 1,in a volume of 0.1 ml/10 g of body weight (the average body weight isapproximately 21 g). The controls were i.p. inoculated with a 0.9%NaCI solution on day 1, in a volume of 0.1 ml/10 g of body weight. Sixmice were included in each group (with some exceptions). The mortalitywas monitored daily (until day 60), and body weights were measured atintervals of a few days.

RESULTS

Synthesis of Polymeric Drug. The polymeric drug PEG-P(Asp(ADR) (5) was successfully synthesized without any precipitation in various molar ratios of ADR-HCl:Asp unit in thereactants, as shown in Table 1. Comparing runs 3-5, the contentof ADR bound to the block copolymer was observed to increasewith an increase in a ratio of ADR •¿�HCl:Asp unit in the reactants. As for the purification method, the runs purified byultrafiltration afforded higher ADR content than the runs purified by gel filtration, comparing the ADR content at the samemolar ratio of ADR-HCl:Asp unit in the reactants. Ultrafiltra-tion-purified run 4 (Asp unit:ADR-HCl = 1:0.52) afforded 22mol % of the bound ADR with respect to the aspartic acidresidue of the reactant, whereas run 1, done at almost the samemolar ratio as that of run 4 but purified by gel filtration, had a

smaller value (18 mol %) for the ADR content. Comparing theADR content of the runs done at Asp unit:ADR-HCl =1:1,the difference becomes clearer. Run 5-7 afforded 1.5-1.7 timesas high a content of the conjugated ADR as run 2. Thesedifferences in the ADR content due to the purification methodindicate that the synthesized PEG-P(Asp(ADR)) was adsorbedon Sephadex G-25 gel to some extent in 0.1 M acetate buffer.

The water solubility of PEG-P(Asp(ADR)) was maintainedeven after the lyophilization (for runs 3, 4, and 5). Its aqueoussolution was able to be concentrated by ultrafiltration, withoutany precipitate, up to 20 mg equivalent of ADR-HCl/ml (forruns 6 and 7). And an addition of 0.9% (w/v) NaCI to thisconcentrated solution did not lead to precipitation nor gelationof the solution.

As shown in runs 6 and 7, PEG-P(Asp(ADR)) containingapproximately 30 mol % bound ADR was synthesized repro-ducibly and was used for assays of in vitro cytotoxicity and invivo anticancer activity. In these runs, the aqueous solutionswere concentrated to 20 mg ADR-HC1 equivalent/ml andwashed with distilled water by repeated ultrafiltration, until theADR concentration of the filtrate was reduced to under VIHOOofthe ADR concentration of the residual solution on PM 30membrane. Finally, 0.9% (w/v) NaCI was added to these solutions.

Laser Scattering. PEG-(P(Asp(ADR)) was analyzed by laserscattering in PBSa. Results are shown in Fig. 2 and Table 2.PEG-P(Asp(ADR)), run 5 of Table 1, at a concentration of 0.1mg equivalent ADR-HCl/ml, was found to show unimodaldistribution in size, and its mean diameter (number average)was found to be 48.5 nm. This value was much larger than thatexpected from its molecular weight (approximately 9000). Thisresult indicates that PEG-P(Asp(ADR)) in run 5 formed amicellar structure in PBSa. An addition of 1% (w/v) SDSbrought about a complete shift of the peak to a smaller sizeand, accordingly, the mean diameter was reduced to 2.7 nm.This decrease in mean diameter with the SDS addition indicatesdisruption of these micelles due to the interaction with thesurfactant. PEG-P(Asp)ADR)) in run 5 formed almost the samesize of micelles in water and at higher (0.4 mg equivalent ADR •¿�HCl/ml) and lower (0.025 mg equivalent ADR-HCl/ml) con-

Table 1 Reaction conditions and results of introduction of ADR into PEG-P(Asp) with amide bond formation between the amino group of ADR and the carboxyl Rroupof Asp units

ADR HCI (mg)/ PEG-P(Asp)Run DMF°(ml)(mg)1

20.7/1527.32

19.9/1512.7Ì

10.6/827.84

11.7/814.75

20.4/1513.26

349.2/260230.37

604.6/450 391.8Asp:ADR

HCImolratio*1:0.491:1.021:0.251:0.521:1.001:0.981:1.00EDC

(„I),reaction lime

(h)<121212121

2122100,4

100.1850,450.

1750,4

50.1850.4

50.19.550,4

50.18886.4

886.191500,4

1500. 12.5Purification''GelGelUltraUltraUltraUltraUltraADR

concent(mol%)'18201522333129

' DMF. /V.A'-dimethylformamide.b Mol ratio of aspartic acid residue to ADR:HCI in the reactants.' First addition of EDC at O'C: second addition at room temperature.d Purification procedure to remove unbound ADR. Gel. gel filtration with Sephadex G-25 (medium) gel: Ultra, ultrafiltration with Amicon PM-30 membrane.' ADR content is shown in mol c'cwith respect to aspartic acid residues of PEG-P(Asp) of the reactant.

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MICELLE-FORMING POLYMERIC ANTICANCER DRUG

(a) 20600 n

L. 15B

10

o4—l 10 100

diameter (nm)

1000

(b)

i_ 15

.0

3

nu

1000

diameter (nm)Fig. 2. Diameter distribution of PEG-P(Asp(ADR)) in run 5 of Table Ileasured by laser scattering) in PBSa without SDS (a} or with \% (w/v) SDS(measi

(*).

Table 2 Mean diameter of PE(i-P(Asp(ADR)) measured by laser scattering inPBSa

Number and »eightaverages are shown as the mean •¿�SD.

Sample"A

AAA

AAB[ADR]

(mg/ml)*0.1o.r0.10.1

0.0250.40.1SDS

additionNone

None1% (w/v)0. 1% (w/v)NoneNoneNoneNumber

average(nm)48.5

±953.4 ±9

2.7 ±16.7 ±1

44.3 ±148.1 ±848.1 ±8Weight

average(nm)57.3

62.93.58.4

51.055.3:55.5 i28

321

121:

2.1t 23

" A, PEG-P(Asp(ADR)) in run 5 of Table 1 (ADR. 33 mol

P(Asp(ADR)) in run 4 of Table 1 (ADR. 22 mol %).* [ADR]. ADR-HC1 equivalent/ml.' Measured in HjO.

B. PEG-

centrations in PBSa. The mean diameter of run 5 with 0.1%(w/v) SDS addition was found to be a little larger than thatwith 1% (w/v) SDS. This fact suggests that the degree ofdestruction of the micellar structure was dependent on theconcentration of the surfactant, SDS. PEG-P(Asp(ADR)) inrun 4 was found to form almost the same size of micelles asrun 5, irrespective of the difference in the ADR content.

Fluorescence Measurement. Fluorescence measurements supported the formation of a micellar structure of PEG-P(Asp(ADR)). Fluorescence of the ADR bound to PEG-P(Asp(ADR)) (runs 3-5 of Table 1) was found to be stronglyquenched in PBSa, as shown in Fig. 3. This quenching issupported to result from local high concentrations of the boundADR. due to the micelle formation in PBSa, since the fluorescence intensity of runs 3-5 was observed to increase with anaddition of SDS. [The same fluorescence measurement resultswere obtained in 0.1 M sodium acetate-buffered solution at pH

a§

-

[ADR] / MFig. 3. Plot of fluorescence intensity of ADR and PEG-P(Asp(ADR)). .run

3 of Table 1: *. run 3 plus 1% (w/v) SDS: D. run 4; •¿�run 4 plus 1% (w/v) SDS;A. run 5: A. run 5 plus Ie", (w/v) SDS: x. ADR: +. ADR plus 1% (w/v) SDS.

4.5 (13).] For Adriamycin itself, the same quenching behavior,which was reduced by the SDS addition, was also observed.Martin (14) reported dimerization of daunomycin (an analogueof Adriamycin) by circular dichroism measurements. Considering his result, the quenching of the fluorescence intensity ofAdriamycin itself results from the dimerization or oligomeri-zation of ADR molecules by hydrophobic interaction. Thedifference in the degree of quenching of the fluorescence intensity between PEG-P(Asp(ADR)) in run 4 and ADR suggeststhe enhanced quenching is due to the micelle formation for run4. PEG-P(Asp(ADR)) in run 4 showed 14.9-fold quenching ofthe fluorescence intensity (without SDS addition) at an ADRconcentration of 6.0 x 10~4 M, as compared with the intensity

with SDS addition, whereas ADR showed only 3.5-fold quenching at 5.7 x 10~4M. More significant quenching of the fluores

cence intensity for PEG-P(Asp(ADR)) than for ADR supportsthe micelle formation of PEG-P(Asp(ADR)).

Plots of fluorescence intensity against concentration of thebound ADR of runs 3-5 with and without the addition of SDSwere found to be almost identical but showed a little difference.A run with a lower content of ADR showed a little higherfluorescence intensity. This fact indicates that the content ofthe bound ADR reflected the packing degree of the bound ADRmolecules in the micelles as well as the degree of resistance tothe destruction of the micellar structure by the SDS addition.

Albumin Binding. Binding affinity of ADR and l'I dP(Asp(ADR)) (ADR content = 18 mol %; run 1 in Table 1) tobovine serum albumin, which plays a central role in drugbinding in plasma, was measured by two different methods,ultrafiltration for ADR and gel filtration for PEG-P(Asp(ADR)). ADR was observed to bind to BSA. The bindingaffinity was dependent on the concentration of BSA; 45% ofADR (at 8.45 x 10~5M) was bound to BSA at a concentrationof 40 mg/ml, and 8% of ADR (at 7.38 x 10~5M) was bound to

BSA at a concentration of 5 mg/ml.In contrast, the Adriamycin introduced into PEG-P(Asp) did

not bind to BSA at all, as shown in Fig. 4. PEG-P(Asp(ADR))(30 n\; [ADR] = 1.83 x 10~5M) was eluted at the gel exclusion

volume (8.4 ml), due to the micelle formation of this blockcopolymer. BSA (30 »;[BSA] = 20 mg/ml) was eluted at 13.8ml. A 1:1 mixture (60 ßl)of PEG-P(Asp(ADR)) and BSAsolutions had elution peaks at both 8.4 and 13.8 ml, and theintensities of these two peaks were quantitatively the same asthose obtained from the separate injections.

In Vitro Stability. The change in absorbance at 485 nm for1696

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280 nm

•¿�13.8ml

elution-

470 nm

PEG-P(Asp(ADR))

13.8ml

USA PEG-P(Asp(ADR))

+ BSA

Fig. 4. Binding behavior of PEG-P(Asp(ADR)) to BSA analyzed by highperformance liquid chromatography; column. Asahipak GS-520; eluent, 0.1 MPBS (pH 7.4, containing 0.3 M NaCl). [BSA] = 20 mg/ml [ADR] of PEG-P(Asp(ADR)) = 1.83 x IO"1 M. BSA. 30 p\; PEG-P(Asp(ADR)). 30 n\; BSA plus

PEG-P(Asp(ADR)), 30 */l + 30 /il.

8C-ÃŽ

time (h)Fig. 5. Stability of ADR. PEG-P(Asp) plus ADR (mixture), and PEG-

P(Asp(ADR)) in PBSa at 37'C in the dark, estimated by absorbance at 485 nm.O, ADR ([ADR] = 6.7 X 10~! M); •¿�PEG-P(Asp) plus ADR ([ADR] = 5.9 X10~s M); •¿�.PEG-P(Asp(ADR)) ([ADR] = 4.9 X 10~5M).

1), ADR, and a mixture of ADR and PEG-P(Asp) in PBSa wasmonitored to elucidate the stability of ADR molecules. Asshown in Fig. 5, absorbance at 485 nm of these three sampleswas found to decrease as the samples were incubated in PBSa.These decreases mean destruction of an anthracycline ring ofan Adriamycin molecule, possibly by nucleophilic attack ofhydroxyl ion, since shift of the peak at 485 nm for all threesamples was not observed. Stabilization of Adriamycin by co-valent bonding to PEG-P(Asp) was remarkable. For example,ADR retained only 42% of its initial absorbance at 485 nmafter a 168-h incubation, whereas the ADR introduce into theblock copolymer with covalent bonding [PEG-P(Asp(ADR))]maintained 91% of its initial absorbance after a 168-h incubation. A mixture of ADR and the block copolymer [in the sameproportion as that of PEG-P(Asp(ADR))] was also found toimprove its stability, as compared with ADR. This stabilizationin the mixture is considered to result from localization of ADRmolecules around the block copolymer by ionic interaction

between amino groups of Adriamycin molecules and carboxylgroups of the block copolymer. Localized hydrophobic ADRmolecules are expected to restrict the access of hydrophilichydroxyl ion. ADR molecules in the mixture, however, werestill less stable than the bound ADR of PEG-P(Asp(ADR)).

In Vitro Cytotoxicity. Results are shown in Fig. 6. ADR anda mixture of ADR and PEG-P(Asp) [mixed in the same ratioas that of PEG-P(Asp(ADR))j showed high cytotoxicity, and

under these experimental conditions there was no concentrationdependence on ADR and the mixture, since even concentrationsof these two samples of 0.1 ng ADR-HCl/ml attained almostthe maximum cytotoxicity (approximately 50% of the cell number of the control in a 24-h incubation, approximately 20% ina 48-h incubation).

PEG-P(Asp(ADR)) (ADR content = 31 mol %; run 6 inTable 1) showed concentration-dependent cytotoxicity, and theconcentration required for maximum cytotoxicity was found tobe higher than that for ADR, for both 24-h and 48-h incubations. Cytotoxicity of PEG-P(Asp) is shown in the same figurewhere the scale of PEG-P(Asp) concentration is adjusted to theADR concentration in the composition of PEG-P(Asp(ADR)).The cytotoxicity of PEG-P(Asp) itself was negligible for 24 hof incubation and quite low even for 48 h of incubation. Thisfact indicates that the cytotoxicity of PEG-P(Asp(ADR)) obtained was not based on that of PEG-P(Asp) but based on thecytotoxicity of the bound ADR.

In Vivo Anticancer Activity. Results are summarized in Table(a) 24 h incubation

|PEG-KAsp)| Mg/ml0 10 20

(b) 48 h incubation|PEG-P(Asp)] Mg/ml

[ADR] ug/mlFig. 6. In vitro cytotoxicity against P 388 mouse leukemia. Cells (I x 10'cell/

ml) were incubated in RPMI 1640 (containing \0c,i fetal calf scrum and 5000unit/liter penicillin and streptomycin) for 24 h in 5% CO2, at 37'C. Drug wasadded to the cells, and the number of cells was counted after a 24-h incubation(a) or 48-h incubation (A). [ADR] is plotted in ADR-HCI equivalents. Eachexperiment was performed in triplicate, and results are plotted as the mean ±SE.In plots without error bars, the SE were within the si/e of the symbols.) A. PEG-P(Asp): •¿�.PEG-P(Asp(ADR)); •¿�PEG-P(Asp) + ADR: D. ADR.

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MICELLE-FORMING POLYMERIC ANTICANCER DRUG

3. Fig. 7 shows plots of T/C versus dose. For Adriamycin,prolonged survival was observed in doses from 1 to 15 mgADR •¿�HCl/kg, and the maximum value of T/C was obtained ata dose of 15 mg/kg. A dose of 30 mg/kg resulted in a shortersurvival time than that of the control because of its acutetoxicity. All three runs at 15 mg/kg afforded very high antican-cer activity with values of T/C over 300%. These three runs,however, showed a remarkable decrease in body weight on day5, over 12% of the initial body weights of the mice. This factindicates that very high anticancer activity against P 388 leukemia was obtained only in the dose range with serious sideeffects, judged from a significant decrease in body weight.

For PEG-P(Asp(ADR)), doses are expressed in mg of ADR-HC1 equivalent contained in PEG-P(Asp(ADR))/kg of mousebody weight. A dose of 15 mg/kg, which brought about themaximum value of T/C for ADR, brought about only 141% T/C. However, the value of T/C increased with an increase indose up to 200 mg/kg. Although a dose of 400 mg/kg showeda slightly smaller value of T/C than that of 200 mg/kg, it stillheld very high anticancer activity, over 300% T/C. A dose of600 mg/kg resulted in toxic deaths, which were represented ina value of T/C under 100%. These results indicate that PEG-P(Asp(ADR)) expressed very high anticancer activity, similarto ADR, at higher doses than those of ADR. As for the sideeffects judged from body weight change, PEG-P(Asp(ADR))gave fewer side effects than ADR. Comparing the result ofPEG-P(Asp(ADR)) at 200 mg/kg with that of ADR at 15 mg/kg (both are the optimum doses concerning T/C), PEG-P(Asp(ADR)) showed a smaller decrease in body weight (7.4%)than ADR at 15 mg/kg (over 12%). This difference in bodyweight change becomes clearer when the body weight change ismonitored at an interval of a few days. Fig. 8 shows the timecourse of the average body weight of the mice for PEG-

P(Asp(ADR)) at 200 mg/kg and ADR at 15 mg/kg (of controlB).

For ADR, body weight showed a minimum on day 5, and itthen recovered a little to —¿�7.3%on day 9. After day 9, however,body weight decreased to around 15% and never recovered tothe initial value. PEG-P(Asp(ADR)) first showed a decrease,with a minimum body weight on day 2, and recovered to theinitial weight on day 9. Then, the body weights of mice treated

with PEG-P(Asp(ADR)) never decreased after day 9. Thisdifference concerning body weight change proved that PEG-P(Asp(ADR)) could afford very high anticancer activity withmuch smaller side effects than ADR.

Table 4 shows a comparison of in vivo anticancer activitybetween PEG-P(Asp), a mixture of ADR and PEG-P(Asp), andPEG-P(Asp(ADR)). The dose of PEG-P(Asp(ADR)) was 200mg equivalent ADR-HCl/kg, which afforded the maximumanticancer activity as shown in Table 3. Doses of PEG-P(Asp)and a mixture of PEG-P(Asp) and ADR were adjusted to theequivalent quantities of the components of PEG-P(Asp(ADR))at 200 mg/kg. Both PEG-P(Asp) and the mixture brought aboutvalues of T/C under 100%. This fact indicates that the highanticancer activity obtained in PEG-P(Asp(ADR)) resultedfrom the anticancer activity of the bound ADR, not from theactivity of PEG-P(Asp) or the combined activity of PEG-P(Asp)and unbound ADR.

DISCUSSION

The maximum content of bound ADR in PEG-P(Asp(ADR))was obtained in run 5, where 33 mol % of carboxyl groups inthe poly(aspartic acid) chain were substituted with Adriamycinmolecules. The value of 33 mol % is remarkably large, considering the strongly hydrophobic character of ADR. Hoes et al.(15) reported a synthesis of ADR-conjugated poly(glutamicacid) homopolymer with maximum substitution of 10 mol %by ADR with respect to glutamic acid units. Tsukada et al. (8)also reported 10 mol % substitution of poly(L-glutamic acid)homopolymer by daunomycin (an analogue of Adriamycin) asa maximum value. PEG-P(Asp(ADR)) synthesized here exceedsthe maximum substitution percentages of these two studies.

PEG-P(Asp(ADR)) of runs 6 and 7 with approximately 30

mol % substitution by ADR remained water soluble even afterconcentrating these solutions by ultrafiltration to 20 mg equivalent ADR-HCl/ml. Duncan et al. (16) reported that 2 mgequivalent daunomycin/ml was the critical concentration forprecipitation in their conjugate of daunomycin and poly[/V-(2-hydroxypropyl)methacrylamide]. On the other hand, anaqueous solution of ADR-HC1 at 20 mg/ml became a solid gelafter the addition of 0.9% (w/v) NaCl. This gelation was con-

Table 3 In vivoanticancer activity against P 388 mouse leukemia of ADR and PEG-P(Asp(ADR))Female CDF! mice were inoculated with 1 X 10*cells ¡.p.and inoculated with drug [ADR or PEG-P(Asp(ADR))] on day 1. Six mice were included in each group.

SampleADRADRADRADRADRADRADRADRADRPEG-P(Asp(ADR))PEG-P(Asp(ADR))PEG-P(Asp(ADR))PEG-P(Asp(ADR))PEG-P(Asp(ADR))PEG-P(Asp(ADR))PEG-P(Asp(ADR))PEG-P(Asp(ADR))Dose(mg/kg)°12447.515IS15307.5153080120200400600Control*EDDEAABCAEAAABBCCMediansurvival(days)15.514.014.815.515.330.5>59.427.56.511.711.313.418.032.5>42.033.07.5T/C(%Y178163175178191381>69130682135141168225378>48836783Longsurvivors0/60/60/60/50/71/75/70/60/60/60/70/60/62/63/61/60/6Body

weightchange at day 5

<%)'-0.2

±1.4+2.9±0.8+4.8

±-2.3±+4.4

±-12.5±-16.4±-15.2

±-18.7±+

1.9±+8.9±+6.2±+6.2±-5.5±-7.5±-4.6

±-17.5±1.10.90.82.11.41.30.41.01.41.11.20.80.91.51.0

" Expressed in ADR •¿�HCI equivalent/kg of body weight.* Median survival: A, 8.0: B. 8.6; C, 9.0; D, 8.6; E. 8.7 days.

,. Median survival dav of samplec = —¿�x 100(%).Median survival day of control

d 60-day survivors are included as long survivors.' Expressed in the mean ±SE; initial mean body weight = 20-21 g.

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MICELLE-FORMING POLYMERIC ANTICANCER DRUG

1 10 1000

Dose (mg/kg)Fig. 7. In vivoanticancer activity against P 388 mouse leukemia. Female CDF,

mice were i.p. inoculated with P 388 mouse leukemia cells (1 x 10* cells in 0.1ml) on day 0. The mice were i.p. inoculated with drug dissolved in a 0.9cc NaCIsolution, on day 1, in a volume of O.I ml/10 g of body weight. Doses are plottedin ADR-HCI equivalents. G, ADR; •¿�,PEG-P(Asp(ADR)). Doses with multipleruns, shown in Table 3, are plotted as the mean value of all the runs.

20 40 60

DayFig. 8. Body weight change of the mice inoculated with ADR and PEG-

P(Asp(ADR)) at the doses which brought about the maximum T/C values. •¿�.PEG-P(Asp(ADR)). 200 mg/kg; D. ADR, 15 mg/kg (control B in Table 3).

Table 4 Comparison of in vivo anticancer activity against P 388 mouse leukemiaofPEC-P(Asp), PEC-P(Asp) plus ADR (mixture of block copolymer and drug),

and PEC-P(Asp(ADR)) (drug-conjugated block copolymer)

SampleMediansurvival

(days)0PEG-P(Asp)

7.7PEG-P(Asp) + ADR¿ 2.7PEG-P(Asp(ADR))>47.0"

Control, 8.6 days.

.,,Median survival dayof sample" v inn /c:.\T/C(%)"90

31>547Long

survivors'0/6

0/63/6

Median survival day of controlc 60-day survivors are included as long survivors.d ADR was dissolved in H2O.

siderea to result from self-association of Adriamycin moleculesunder a high ionic strength, as reported for daunomycin byMartin (14). No such gelation was observed for our PEG-P(Asp(ADR)) system even at the ADR concentration of 20 mgADR-HCI equivalent/ml. Furthermore, runs 3-5 were able tobe redissolved in water after lyophilization. All these resultsindicate that PEG-P(Asp(ADR)) has excellent water solubilityand stability against precipitation. These properties were provided by the poly(ethylene glycol) chain linked to thepoly(aspartic acid) chain, since a precipitate was producedduring the introduction reaction of ADR when poly(asparticacid) homopolymer was used instead of PEG-P(Asp) under thesame reaction conditions as those of a synthesis of PEG-

P(Asp( ADR)). These facts demonstrate one of the advantageousfeatures of the block copolymer drug carrier. And this excellentwater solubility and stability against precipitate are thought notto result only from the increased hydrophilicity by the linkedPEG chain but to result from the micelle formation. The PEGchain is expected to exist as the outer shell of the micelle, andthis hydrophilic outer shell is supposed to suppress the inter-micellar aggregation of the hydrophobia P(Asp(ADR)) chain.

Bader et al. (17) proposed a micelle-forming polymeric drugcomposed of cyclophosphamide-conjugated poly(ethylene gly-col)-poly(L-lysine) block copolymer derivative. They obtainedsustained release of cyclophosphamide from the block copolymer. In their system, however, introduction of long alkyl chainsto the poly(t-lysine) chain was needed to add hydrophobicityfor micelle formation. Although they suggested possible micelleformation, there was no direct evidence for micelle formation.Our study is the first example of a micelle-forming polymericdrug with definite diameter. The diameter of the micelle (approximately 50 nm) corresponds to a range of the size of singleunilamellar vesicles of liposomes. Although liposomes havebeen studied as a drug carrier for many years, most examinations of targeting using liposomes have been not very successful,owing to the structural fragility of liposomes (especially forsingle unilamellar vesicles) and nonselective capture of liposomes by the reticuloendothelial cells. Because polymeric micelles are considered to be structurally stronger than liposomes,PEG-P(Asp(ADR)) can be a promising alternative candidatefor the carrier used in drug targeting. The unimodal distributionof diameter of the micelles indicates that the micelles of PEG-P(Asp(ADR)) were free from intermicellar aggregation, andthis distribution is very narrow, possibly owing to the use ofthe well designed block copolymer. Furthermore, linking of thePEG chain is a promising way to inhibit the nonselective uptakeby the reticuloendothelial cells, as speculated from the study onPEG-modified enzymes (18).

The micelle-forming character of PEG-P(Asp(ADR)) is considered to contribute to the drastic changes in binding affinityfor BSA as well as in stability in PBSa, since the micellestructure is considered to inhibit the access of BSA (completeinhibition) and OH ion (partial inhibition) to the bound ADRmolecules by packing the ADR molecules tightly inside a hy-drophobic core of the micelles. Considering the importance ofalbumin binding for pharmacokinetics of drugs, it is very interesting to diminish the binding affinity of ADR for BSA drastically by binding ADR to the block copolymer. And this is thefirst example of ADR stabilization through binding to polymeric carriers. Stability of drugs in plasma is an importantfactor for effective chemotherapy, especially for Adriamycin.The stability is a critical problem for ADR since Adriamycinwas reported (19) to be metabolized very rapidly to inactivederivatives mainly by liver after an injection into the bloodstream. Lisi et al. (18) reported that accumulation of/i-glucu-ronidase in hepatic tissues was reduced by PEG modification.Therefore, the macellar structure of PEG-P(Asp(ADR)) withthe PEG chain in the outer shell is a promising form to bringabout resistance of Adriamycin to metabolism.

The results of in vitro cytotoxicity and in vivo anticanceractivity against P 388 mouse leukemia showed that the polymeric drug required a greater amount of ADR molecules thanintact ADR to obtain the activity equal to intact ADR and thatin vivo very high anticancer activity was obtained for PEG-P(Asp(ADR)) with a smaller body weight loss than that of freeADR. The release rate of ADR from the micelle drug is expected to be very slow, because there is no spacer such as

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MICELLE-FORMING POLYMERIC ANTICANCER DRUG

oligopeptides (20) between ADR and the poly(aspartic acid)chain and because main chain cleavage of poly(aspartic acid)by proteolytic enzymes is thought to be very slow, as reported(21, 22). There is a possibility that the requirement for a largequantity of bound ADR molecules reflected the slow action ofthe polymeric drug due to slow release of ADR molecules. Onthe other hand, Wingard et al. (23) reported that ADR chemically bound to an insoluble gel could exhibit cytotoxicity bydirectly interacting with cell membranes without being takenup by the cells and, therefore, there is another possibility, thatthe polymeric drug PEG-P(Asp(ADR)) showed cytotoxic activity to P 388 without releasing intact ADR molecules.

Although the mechanism of action of the polymeric drug isstill unknown, this paper reports the important example of thepolymeric drug showing high in vivo anticancer activity withgreatly improved body weight loss, as compared with intactADR.

ACKNOWLEDGMENTS

The authors express their gratitude to Professor Tamotsu Kondo ofScience University of Tokyo for his kind support and to TomokoHashimoto for her technical assistance.

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1990;50:1693-1700. Cancer Res   Masayuki Yokoyama, Mizue Miyauchi, Noriko Yamada, et al.   Poly(ethylene glycol)-Poly(aspartic acid) Block CopolymerPolymeric Anticancer Drug Adriamycin-conjugated Characterization and Anticancer Activity of the Micelle-forming

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