Polymer micelles as novel drug carrier: Adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer

Journal of Controlled Release, 11 (1990) 269-278

269

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

POLYMER MICELLES AS NOVEL DRUG CARRIER: ADRIAMYCIN-CONJUGATED POLY(ETHYLENE GLYCOL)-POLY(ASPARTIC ACID) BLOCK COPOLYMER* Masayuki Yokoyama**, Mizue Miyauchi, Noriko Yamada, Teruo Okano, Yasuhisa Sakurai Institute of Biomedical Engineering, Tokyo Women "s Medical College, Kawada-cho, Shinjuku-ku, Tokyo 162 (Japan)

Kazunori Kataoka** Department of Materials Science and Research Institute for Biosciences, Science University of Tokyo, Yamazaki2641, Noda-shi, Chiba 278 (Japan)

and Shohei Inoue Department of Synthetic Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113 (Japan) Key words: polymeric drugs; micelles; adriamycin; block copolymers; cancer chemotherapy

A miceUe-formingpolymeric drug was synthesized using adriamycin (an anti-cancer drug) and poly(ethylene glycol)-poly(aspartic acid) block copolymers. The micelle had a narrow and unimodal size distribution, and its diameter was observed to be ca. 50 nm, the range corresponding to viruses. This micelle-forming polymeric drug exhibited good water solubility and good stability against precipitation irrespective of the large quantity of incorporated hydrophobic adriamycin. In vivo anti-cancer activity of this polymeric drug against P-388 mouse leukemia was studied by changing the length of the poly(aspartic acid) chain of the block copolymer. The polymeric drug showed excellent in vivo anti-cancer activity, judged from the ratio of the survival period of the treated mice to that o/the control (T/C), with lower toxicity than that of adriamycin. These results point to a promising figure o/polymeric micelles as novel drug carriers.

INTRODUCTION Several types of drug carriers, such as microspheres, liposomes, and polymeric carriers, have been investigated to increase the efficiency of chemotherapy and to decrease side effects of drug. Generally, microspheres are suitable for chemoembolization and local injection, but not very useful for the drugs used in blood circulation. Liposomes are structurally fragile, are *Paper presented at the Fourth International Symposium on Recent Advances in Drug Delivery Systems, Salt Lake City, UT, U.S.A., February 21-24, 1989. **To whom correspondence should be addressed.

0168-3659/90/$03.50

taken up easily by the reticuloendothelial cells, and have a low efficiency of loading of hydrophobic drugs. Therefore, the use of liposomes is restricted. Polymers are promising candidates for drug carriers used in the blood stream for whole body circulation. However, polymeric drugs with homopolymer, statistical copolymer, or natural polymer carriers have problems with their solubility in water. Since most of the drugs have a hydrophobic character, conjugation of polymer and drug easily leads to precipitation due to the concentrated presence of hydrophobic drug molecules along the polymer chain.

© 1990 Elsevier Science Publishers B.V.

270

We here present polymer micelles as a novel drug carrier. Block or graft copolymers composed of hydrophobic and hydrophilic segments can form micellar structures by their amphiphilic character, as shown in Fig. 1 (illustrated by an AB type of block copolymer as an example). Hydrophobic drug-binding segments form the hydrophobic core of the micelle, and the other, hydrophilic segments of the graft or block copolymer surround this core as an outer shell. The superiority of the micelle-type drug carrier over the above-mentioned other three types of carrier is evident from the easy control of particle size, the good structural stability, the good water solubility, and the separated functionality. The size of the particles is the primary factor affecting the interaction with cells as well as the pharmacokinetics in the body. The

: component A (hydrophilie) ~

:

component B mieelle formation

: drug

Fig. 1. Concept of micelle-forming polymeric drug.

size of the polymer micelle can be easily controlled by changing the chain length of the polymer. The stability of the polymer micelle is thought to be higher than that of liposomes since the hydrophobic segments interwind with each other in the polymer micelle through inter- and intramolecular hydrophobic interactions, whereas the hydrophobic interaction in liposomes is limited to that between adjacent lipids. Since the hydrophilic outer shell is expected to prevent the hydrophobic core from forming aggregates which precipitate, the micelle-forming polymeric drug is thought to possess excellent water solubility irrespective of hydrophobic drug loading. The ideal drug carrier should satisfy many requirements: binding of drug, controlled drug release, preservation of drug activity during delivery to the target, and desired interaction with proteins, cells, and tissues. The micellar structure with hydrophobic core and hydrophilic outer shell can satisfy these requirements due to the highly distinctive character of the constituent polymer segments. Three mechanisms for drug action of the micelle-forming polymeric drug can be proposed, as illustrated in Fig. 2. The first mechanism is that micelles directly interact with cells. Secondly, drug which is released from the micelle brings about drug action, the micelle itself not taking part in drug action. Bader et al. [ 1 ] proposed this type of micelle-forming polymeric drug. However, they have not shown any direct evidence for micelle formation in their system. The third mechanism is equilibrium control, in which only a single polymer chain, which exists in equilibrium with the micellar form, gives drug action. These various modes of actions allow the application of the micelle-forming polymeric drug to many kinds of drugs because each drug has a characteristic ideal pattern of action. Some of these three mechanisms may be combined in actual applications. We have been studying micelle-forming polymeric drugs using poly(ethylene glycol)poly (aspartic acid) block copolymers and adriamycin (anti-cancer drug). The poly (aspartic

271 (1) direct interaction with cells

(2) drug release from micelle ~

l

__

release

~ 0 drug

.

(3) equlibrium control l :

ulibrium

drug action

Fig. 2. Mechanismsof action of micelle-formingpolymeric drugs.

modification [9] and surface modification of microspheres [10] to decrease hepatic uptake. Considering these properties, PEG is suitable for the outer shell of the micelle. As reported previously [11], the conjugates of adriamycin and poly(ethylene glycol)poly(aspartic acid) block copolymer were observed to form micelles of ca. 50 nm diameter. This size approximately corresponds to that of viruses. An ideal model for drug carriers possessing high specificity without non-specific uptake by the reticuloendothelial system are viruses. This study aims to demonstrate that virus-mimetic drug carriers can be constructed using this polymer micelle. This paper reports micelle formation and in vivo anti-cancer activity of the adriamycinconjugated poly (ethylene glycol )-poly (aspartic acid) block copolymer. Special focus is placed on the effect of poly (aspartic acid) chain length on the micelle structure as well as on anti-cancer activity as compared with the previous report [11]. EXPERIMENTAL PART

acid) segment is hydrolyzable and possesses carboxyl groups for binding adriamycin with its amino group to form biodegradable amide linkages. The main chain of the poly (aspartic acid) is hydrolyzable. Though there is a possibility that the polymer-bound adriamycin molecule exhibits its cytotoxic activity through direct contact with cellular plasma membranes without being released from the polymer chain [ 2 ], it has been accepted generally that adriamycin expresses its activity only after its release from the poly(aspartic acid) chain. The other constituent of our polymeric drug is poly (ethylene glycol) (PEG). Poly (ethylene glycol) is known to be a non-toxic and non-immunogenic watersoluble polymer. It has been used in protein modification to decrease antigenicity [3,4] of the intact protein and to prolong its half life in the blood stream [5-9]. Furthermore, poly (ethylene glycol) has been used for protein

Chemicals Adriamycin hydrochloride (ADR. HCI) was kindly supplied by Nippon Kayaku Co., Ltd. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Peptide Institute, Inc. (Japan). A sample of c~-methyl-coaminopoly (oxyethylene) (1, CH3-PEG-NH2, M.W. -- 5,100 determined by vapor pressure osmometry in benzene) was kindly supplied by Nippon Oil & Fats Co., Ltd. Fifty-eight percent of the molecules of this sample were estimated by acid-base back titration to have amino groups at one terminal. Synthesis of polymeric drug The synthetic procedure of the polymeric drug, adriamycin-bound poly(ethylene glycol)-poly (aspartic acid) block copolymer, was reported elsewhere [ 12,13 ]. The synthetic route

272

of the polymeric drug is shown in Scheme 1.

CH3-(OCH~CH2-)ENH2 +

m

HC--C( CH~COOCH2 O I, CH3-PEG-NH2 2, BLA-NCA CH3 -( OCH2CH2-)'FNH-(COCHNH)%"mH I CH2COOCH2O 3, PEG-PBLA CH3-'(OCH2CH2-)FNH -(COCHNH)~--(COCH2OHNH)T H I

I

CH2COOH

COOH

product was precipitated with diethyl ether. Yield: 23.79 g (59%).

Purification procedure to remove PEG homopolymer P E G - P B L A block copolymer (3) was dissolved in chloroform, and this solution was poured into alcohol (methanol, ethanol, 1-propanol, or 1-butanol ) at 40 ° C. A precipitate was collected by centrifugation followed by drying i n vacuo. This precipitate was dissolved in chloroform, and the solution was poured into a large excess of diethyl ether. A precipitate was collected and dried in vacuo.

4, PEG-P(Asp) ADR CH3 -(OCH2CH2")ENH -'(COCHNH)~---(COCH2CHNH~ H I I CH2COR COR 0 ~ R = OH

or

h

LJ~ CH30

~

OH ~

£

.~

0

cOCH2OH ~J ~OH

OH

"O

OH~

5. PEG-P(Asp(ADR)) --

O~

NH

Scheme 1. Synthesis of polymeric drug.

Synthesis of poly(ethylene glycol)-poly~benzyl L-aspartate) block copolymer (PEGPBLA, 3) fl-Benzyl L-aspartate N-carboxy anhydride (BLA-NCA, 2 ) was polymerized from the terminal amino group of c~-methyl-o)-aminopolyoxyethylene (1, CH3-PEG-NH2, M.W. =5100) to obtain poly(ethylene glycol)poly (fl-benzyl L-aspartate) block copolymer (PEG-PBLA, 3 ). fl-Benzyl L-aspartate N-carboxy anhydride (2, 28.8 g) was dissolved in 43 ml of doubly distilled N,N-dimethylformamide (DMF) followed by the addition of 380 ml of distilled chloroform. ~-Methyl-o)-aminopolyoxyethylene (1, 12.00 g) was dissolved in 50 ml of distilled chloroform and added to the solution of 2. The reaction mixture was stirred for 17 h at 35 ° C in a stream of dry nitrogen. The

Synthesis of poly(ethylene glycol)poly(aspartic acid) block copolymer (PEGP(Asp), 4) Protective benzyl groups of the fl-benzyl Laspartate units were removed by alkaline hydrolysis to obtain poly(ethylene glycol)poly(aspartic acid) block copolymer (PEGP (Asp), 4 ). PEG-PBLA block copolymer purified by precipitation (3, 9.00 g) was dissolved in 90 ml of chloroform. Then, 115 ml of a solution of NaOH (0.43 N) in a mixture of water, methanol, and 2-propanol (vol. ratio 1: 2: 2) was added. After vigorous stirring at 0 ° C for 10 min, the reaction mixture was neutralized with acetic acid and was poured into a large excess of diethyl ether, and a precipitate was collected. This precipitate was dissolved in distilled water, and dialyzed against distilled water with a Spectrapor 7 dialysis membrane (molecular weight cutoff= 1000) for 4 h, followed by lyophilization. Yield: 5.48 g (91%). Introduction of adriamycin to PEG-P(Asp) block copolymer Adriamycin (ADR) was introduced to P E G P(Asp) block copolymer (4) by amide bond formation between the amino group of the adriamycin molecule and the carboxyl group of an aspartic acid residue in the poly (aspartic acid) chain using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a coupling agent.

273

Adriamycin hydrochloride (ADR.HC1) was dissolved in DMF (guaranteed grade) followed by an addition of 1.3 eq of triethylamine. P E G P(Asp) block copolymer (4) dissolved in a small amount of distilled water and EDC were subsequently added to the ADR solution, and the reaction mixture was stirred at 0 °C for 4 h followed by a second addition of EDC. Then, the reaction mixture was stirred overnight at room temperature. The resulting solution was dialyzed against 0.1 M sodium acetate buffered solution (pH 4.5) using Spectrapor 7 (M.W. cut-off= 1000) for 3 h, followed by ultrafiltration with an Amicon PM-30 membrane to remove unbound ADR and low molecular weight contaminants. By changing the weight ratio PEG-P(Asp)/ADR-HC1, polymeric P E G P(Asp(ADR)) drugs with various ADR contents were synthesized.

Measurements Fluorescence measurements were carried out using a Hitachi Fluorescence Spectrophotometer 650-60 with excitation at 471 nm. 1H-NMR spectra were measured in CDC13 and D20 using a J N M - N M R at 400 MHz. Spectra in CDC13 were measured at 3 % w/v, and the pD for measurements in D20 was adjusted to 9.2 with D20 solutions of DC1 and NaOD using a pH meter (HORIBA F-7ss, uncorrected for deuterium substitution). Vapor pressure osmometry (VPO) was carried out with a Corona Molecular Weight Apparatus 117 using benzene as a solvent. Laser scattering was carried out using a Photon Corelater LPA-3000 (Otsuka Electronics) with a H e / N e laser beam in phosphate-buffered saline (pH 7.4, 0.155 M) unless otherwise stated. In vivo anti-cancer activity: P 388 mouse leukemia cells were maintained by intraperitoneal (IP) passage in DBA/2 mice every week. Female CDF, mice were intraperitoneally inoculated with P 388 mouse leukemia (1 × 106 cells in 0.1 ml) on day 0. Drug in 0.9% NaCl aqueous

solution was intraperitoneally inoculated on day 1 in a volume of 0.1 ml/10 g body weight (the average body weight is ca. 21 g). The control mice were inoculated with 0.9% NaC1 aqueous solution. Six mice were included in each group. The mortality was monitored daily, and anticancer activity was evaluated by the ratio of the median survival period of the treated mice to that of the control mice (T/C).

RESULTS AND DISCUSSION Synthesis of polymeric drug The polymerization of BLA-NCA initiated with CH3-PEG-NH2 proceeded homogeneously, and a white-powdery polymer was obtained after reprecipitation in diethyl ether followed by washing with diethyl ether and drying in vacuo. The amino group functionality of 1 reveals that only 58% of poly (ethylene glycol) chains initiated the polymerization of BLANCA, and that 42% of the PEG chains remained as homopolymer in the obtained polymer. Since the absence of contaminating PEG homopolymer is desirable for the accurate molecular design of the polymeric drug, the PEG homopolymer was removed using precipitation from chloroform solution into various alcohols. The results are shown in Table 1. In all the experimental conditions examined, CH3-PEGTABLE 1 Purification of PEG-PBLA block copolymer Run Crude Precipitation solvent polymer (g) CHC13 ratio~ alcohol (ml)

Yield (g)

Wt contentb of BLA units (%)

1 2 3 4

0.38 (76%) 0.35 (69%) 2.15 (72%) 9.89 (82%)

77 76 78 76

0.50 0.51 3.00 12.00

5 5 30 100

1:5 1:5 1:5 1:5

1-propanol 1-butanol 1-butanol 1-butanol

aVolume ratio of CHC13:alcohol. bObtained from 1H-NMR spectra: calculated value (explained in the text) after purification is 76%.

274 NH2 was found to be still dissolved after the precipitation procedure into the alcohols. The compositions of the products were determined by 1H-NMR spectroscopy. The unit ratio of poly (fl-benzyl L-aspartate) (PBLA) / poly(ethylene glycol) (PEG) was obtained from the peak intensities of the methylene proton of the PEG chain and the benzyl proton of the PBLA chain (PEG: -OCH2CH2, J=3.6; PBLA:-COOCH2C~Hs, J = 5 . 0 ) . The weight content of BLA units of the product before the precipitation procedure was found to be 64%. Assuming that only the PEG homopolymer is selectively removed by the purification procedure, the weight content of BLA units after precipitation is calculated to be 76%. For run 1, using 1-propanol as the precipitation solvent, a good yield of the polymer was obtained, and the weight content of BLA units was found to be 77%, which is nearly equal to the calculated value described above. Precipitation with 1-butanol also brought about good yields, ranging from 69% to 82 %; the calculated yield is 85%, for the case that only PEG homopolymer is selectively removed by the purification procedure. The weight contents of BLA units were also found to be nearly equal to the calculated value for all three runs; 76% for runs 2 and 4, 78% for run 3. For run 4, the number of BLA units in a molecule of the block copolymer chain was calculated to be 77 from the BLA units content and the molecular weight of the PEG chain. Precipitation using methanol and ethanol was also examined, but afforded only small amounts of precipitate. Purified P E G - P B L A (run 4) was deprotected by alkaline hydrolysis. The obtained polymer was analyzed by 1H-NMR spectroscopy at pD 9.2 in D20. The absence of residual benzyl groups (benzyl methylene proton at 5.0 ppm) proved the complete debenzylation of the poly(fl-benzyl L-aspartate) chain of P E G PBLA. From the intensity ratio of peaks due to methylene protons of poly(ethylene glycol) (J=3.7 ppm) and poly(aspartic acid) (6=2.7 ppm), the number of aspartic acid units per

block copolymer chain was determined to be 84. This value was almost the same as the number of BLA units per block copolymer chain (77) of P E G - P B L A block copolymer (run 4). This indicates that loss ofpoly (aspartic acid) chains by main-chain cleavage during alkaline hydrolysis was negligible. As reported previously [ 13 ], this alkaline hydrolysis procedure converted 80 mol% of aspartic acid residues to fl-amide form, as shown in Fig. 3. The results of the introduction of ADR to the P E G - P (Asp) block copolymer (obtained by alkaline hydrolysis in run 4 of Table 1) are summarized in Table 2. All experiments except run 6 resulted in the successful introduction of ADR to the block copolymer without any precipitation. The ADR content in the obtained P E G - P (Asp (ADR)) was found to be primarily controlled by the Asp:ADR ratio of the reactants as shown in runs 1-4. When the amount of solvent (DMF) relative to ADR- HC1 was decreased, the &DR content of the obtained P E G P (Asp (ADR)) was found to be almost the same for an Asp: ADR ratio of 1 : 0.25 (runs 3 and 5 ); however, precipitation occurred at 1 : 1.00 (run 6). When the relative quantity of DMF was increased, the ADR content decreased also, as indicated in runs 7 and 8. A large batch for the preparation of the sample for in vivo assay (run 9 of Table 2) was per-

OH- (D --NH--CH--C --N. --

I

~

"-

i cV_o

_ CH2-- IIC-., ~ --

o

(2~)

.o.

-

(2)

B :base

-- NH-- CH ~ -amid¢ l "COOH CH2--CO--NH--- NH--CH--CO-- NH-I

a -amidc

( : ~ 2 - - {:X:)OH

Fig. 3. Formationof fl-amidein debenzylationof PBLA.

275 TABLE 2 Introduction of ADR to P E G - P (Asp) block copolymer Run

ADR. HC1 (mg)

DMF (ml) 7.5

P E G - P (Asp) a (mg)

Asp : A D R b mole ratio

EDC c, reaction time

A D R content d ( mol % )

6.1

1:0.50

15

6.8

1:1.00

(1) 50pl, 4 h (2) 50~1, 17 h (1) 100pl, 4 h (2) 100]d, 14 h (1) 50 ttl, 4 h (2) 50zl, 14 h (1) 50 ~tl, 4 h (2) 50pl, 17 h (1) 150/d, 4 h (2) 150zl, 17 h (1) 150~tl, 4 h (2) 150]d, 15 h (1) 150#1, 4 h (2) 150 ~tl, 17.5 h (1) 150/d, 4 h (2) 150 Ftl, 18 h (1) 1250 #l, 4 h (2) 1250]~1, 16.5 h

1

9.0

2

20.0

3

10.0

7.5

13.6

1:0.25

4

10.0

7.5

6.8

1:0.50

5

30.2

6

40.9

1:0.25

6

30.2

6

10.2

1:1.00

7

30.0

90

20.4

1:0.50

8

30.0

90

10.2

1:1.00

9

500.1

375

170.1

1 : 1.00

15

30 14 13 12 _e 9 13 37

aObtained from P E G - P B L A run 4 of Table 1. bMole ratio; aspartic acid residue : ADR. CFirst addition of EDC at 0 ° C, second at room temperature. dADR content is expressed in mole% with respect to aspartic acid residue of P (Asp) chain. eA precipitate formed.

formed at the same ADR to DMF ratio as those of runs 2-4. The maximum ADR content in P E G - P ( A s p ( A D R ) ) was obtained in run 9 with 37 mol% substitution of carboxyl groups of the poly(aspartic acid) chain. The value of 37 mol% is quite large considering the strongly hydrophobic character of ADR. Hoes et al. [14] reported the synthesis of ADR-conjugated poly(glutamic acid) homopolymer with maximum substitution of 10 mol% by ADR with respect to glutamic acid units. Tsukada et al. [ 15 ] also reported 10 mol% substitution of poly(Lglutamic acid) homopolymer by daunomycin (an adriamycin analogue) as the maximum value. In these two examples, drugs were introduced by amide bond formation between amino groups of the drugs and carboxyl groups of the poly(a-glutamic acid) chains. The P E G P(Asp(ADR)) synthesized here exceeds the

maximum percent substitution of these two studies. Besides, P E G - P ( A s p ( A R ) ) with 37 mol% substitution by ADR remained water soluble even after concentrating the solution by ultrafiltration to give a concentration of 20 mg equivalent ADR. HC1 per milliliter. Duncan et al. [16] reported that 2 mg equivalent dauomycin (an adriamycin analogue) per milliliter was the critical concentration for precipitation in their conjugate of daunomycin and poly [N(2-hydroxypropyl) methacrylamide ]. Furthermore, the products in runs 2 and 3 were found to be water soluble even after lyophilization. The excellent stability, water solubility and preservability are provided by the linking of the poly (ethylene glycol) chain to the poly (aspartic acid) chain, since a precipitate was produced where poly (aspartic acid) homopolymer was used instead of P E G - P (Asp) under the same

276

reaction conditions as in the synthesis for P E G P (Asp (ADR)). These facts demonstrate one of the advantageous features of the block copolymer drug carrier. It is considered that the good water solubility given by the linked PEG chain is strongly associated with the micelle formation of P E G - P (Asp (ADR)), since intermicellar aggregation of the hydrophobic P(Asp(ADR) ) chain is expected to be inhibited by the hydrophilic PEG chain which forms the outer shell of the micelle.

Micelle formation of polymeric drug (5) Micelle formation of the polymeric drug (5) was observed by laser scattering. Figure 4 shows the particle size distribution of the micelle of 3O (~o) 2O

l0

l0

100

1000

Diameter (nm)

run 3 of Table 2 at 0.1 mg ADR" HC1 equivalent per milliliter. Obviously, the distribution is narrow and unimodal, and this fact indicates that all the block copolymer chains exist as uniform micelles without any aggregates. We obtained previously a value of 48.5 + 8 nm [ 11 ] as the micelle size of P E G - P (Asp (ADR)) with a smaller number (n--17) of Asp units. The number-average diameter of run 3 in this study (the number of Asp units: n = 84) is 46.5 + 9 nm, and this value is almost the same as that of P E G - P (Asp (ADR) ) with the shorter P (Asp) chain ( n = 1 7 ) . This indicates that size of the micelles was not changed by the number of Asp units in the range from 17 to 84. Further, Table 3 shows that particle size of the micelle was not dependent on ADR content. These results indicate that the block copolymers exist as micelles, with sizes in the same range as viruses which are much larger than proteins. Viruses can be regarded as a naturally existing model of drug carrier, considering their ability to penetrate to specific cells. Further, the size of a virus is lower than the threshold of reticuloendothelial recognition. The micelle consisting of P E G - P (Asp (ADR)) may also penetrate through the sinusoidal and fenestrated capillaries which have pores of ca. 100 nm in diameter. Therefore, the micelle-forming polymeric drug is an ideal drug carrier considering its size.

Fig. 4. Particle size d i s t r i b u t i o n o f P E G - P ( A s p ( A D R ) ) ( r u n 3 o f T a b l e 2, [ A D R ] - 0 . 1 m g / m l ) .

In vivo anti-cancer activity

TABLE 3

D o s e - T / C (ratio of median survival day of the treated mice/the median survival day of the control mice) plots are shown in Fig. 5. Adriamycin itself (ADR) shows high anti-cancer activity against P-388 leukemia. A dose at 15 mg ADR-HC1 per kg gave the maximum value of T / C (305%) and 30 m g / k g was a toxic dose which resulted in a smaller value of T / C than the control. For P E G - P ( A s p / ( A D R ) ) (run 9 of Table 2, n--84), the toxic level shifted to 400 mg/kg, which is somewhat lower than that (600 mg/kg) of P E G - P (Asp (ADR)) with shorter

Particle size of polymeric drug measured by laser scattering~ Sample b

run2 run 3

ADR content [ADR] (mol % ) (mg/ml)

30 14

0.1 0.1

aIn phosphate-buffered saline, pH 7.4.

bRuns from Table 2. CExpressed in ADR. HCI equivalents.

Particle size (mean -+ S.D. (nm)) weight average

number average

52.7_+27 56.8_+36

45.5_+7 46.5_+9

277 500

(~) 400"

300-

1

200"

100

10

100

Dose

1000

(mg/kg)

Fig. 5. In vivo anti-cancer activity of P E G - P (Asp (ADR)) (run 9 of Table 2) against P 388 mouse leukemia, @: ADR, [2: P E G - P (Asp (ADR)) (number of Asp units: n = 17 (Ref. [ 11 ] ), and I1: P E G - P (Asp (ADR)) (n -- 84, run 9 of Table 2).

drug carriers. Their advantages include high water solubility, excellent stability in a proteinaceous environment like plasma, non-recognizable by the reticuloendothelial system, and possible penetration through blood vessel walls. High anti-cancer activity against P 388 mouse leukemia was obtained with low toxic side effects, reflecting the advantageous features of polymeric micelles described above. We would like to emphasize the superiority of the polymeric micelles to conventional carrier systems including liposomes and polymers of synthetic or natural origin.

REFERENCES

1

P(Asp) chain ( n = 1 7 ) [11]. Doses from 50 to 200 mg/kg showed dose-dependent anti-cancer activity, and 200 mg/kg gave the maximum T / C value (490%), which was considerably higher than the maximum value obtained with ADR itself. Although this polymer drug showed higher anti-cancer activity against P-388 mouse leukemia than ADR, a more important effect of conjugation of ADR molecules to this block copolymer consists of lowered toxic side effects judged from body weight changes. Body weight of the mice was measured on day 5 for ADR and PEG-P (Asp (ADR)) with the doses giving the maximum T/C value. For ADR, body weight underwent a 13.2% decrease from its initial value. PEG-P (Asp (ADR)) resulted in lower decrease (9.1%).

2

3

4

5

6

CONCLUSION

The present study demonstrates the successful preparation of micelle-forming polymeric drugs. The size of the micelle is similar to that of viruses, which suggests a promising future for these polymeric micelles as virus-mimetic

7

H. Bader, H. Ringsdorfand B. Schmidt, Water soluble polymers in medicine, Angew. Chem., 123/124 (1984) 457-485. L.B. Wingard, Jr., T.R. Tritton and K.A. Eager, Cell surface effects of adriamycin and carminomycin immobilized on cross-linked polyvinyl alcohol, Cancer Res., 45 (1985) 3529-3536. A. Matsushima, H. Nishimura, Y. Ashihara, Y. Yokota and Y. Inada, Modification of E. coli asparaginase with 2,4-bis (o-methoxypolyethylene glycol)6-chloro-s-triazine (activated PEG2); disappearance of binding ability towards anti-serum and retention of enzymatic activity, Chem. Lett., (1980) 773-776. Y. Ashihara, T. Kono, S. Yamazaki and Y. Inada, Modification of E. coli L-asparaginase with polyethylene glycol: disappearance of binding ability to antiasparaginase serum, Biochem. Biophys. Res. Commun., 83 (1978) 385-391. N.V. Kartre, M.J. Knauf and W.J. Kaird, Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 1487-1491. Y. Kamisaki, H. Wada, T. Yagura, A. Matsushima and Y. Inada, Reduction in immunogenicity and clearance rate of Escherichia coli L-asparaginase by modification with monomethoxypolyethylene glycol, J. Pharmacol. Exp. Ther., 216 (1981) 410-414. A. Abuchowski, T. van Es, N.C. Palczuk and F.F. Davis, Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol, J. Biol. Chem., 252 (1977) 3578-3581.

278 8

9

10

11

12

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