Poly(ethylene glycol)-Poly(aspartic acid) Block

Characterization and Anticancer Activity of the Micelle-forming Polymeric Anticancer Drug Adriamycin-conjugated Poly(ethylene glycol)-Poly(aspartic acid) Block Copolymer Masayuki Yokoyama, Mizue Miyauchi, Noriko Yamada, et al. Cancer Res 1990;50:1693-1700. Published online March 1, 1990.

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[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 Copolymer Masayuki Yokoyama,' Mizue Miyauchi, Noriko Yamada, Teruo Okano, Yasuhisa Sakurai, Kazunori Kataoka, and ShoheiInoue Institute of BiomÃ©dicalEngineering, Tokyo H omen 's Medical College, Kavtada-cho, Shinjuku-ku, Tokyo 162, Japan Â¡M.Y., M. M., N. Y., T. O., Y. S.]; Department of Materials Science and Technology and Research Institute for Biosciences, Science University of Tokyo, Yama:aki 2641, Noda-shi, Chiba 278, Japan Â¡K.A'./; and Department 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 to the poly(aspartic acid) chain of polyfethylene glycol)-poly(aspartic acid) block copolymer by amide bond formation between an amino group of Adriamycin and the carboxyl groups of the poly(aspartic acid) chain. The polymeric drug thus obtained was observed to form a micelle structure possessing diameter of approximately 50 nm, with a narrow distribution, in phosphate-buffered saline and to show excellent water solubility despite a large amount of ADR introduction. Further, it was able to be stored in lyophilized form without losing its water solubility in the redissolving procedure. Increased stability of the bound Adriamycin molecules in phosphate-buffered saline and elimination of binding affinity for bovine serum albumin due to the micelle formation were further advantages of this polymeric drug. In vivo high anticancer activity of this micelle-forming polymeric drug against P 388 mouse leukemia was obtained with less body weight loss than that seen with free ADR, due to low toxicity as compared with free ADR.

INTRODUCTION It is well known that the utility of cancer chemotherapy is considerably restricted by toxic side effects of anticancer drugs. This restriction results from the fact that the anticancer drugs used in the present chemotherapy lack efficient selectivity for malignant cells. To suppress the toxic side effects of the anticancer drugs to normal cells and to improve their efficiency toward malignant cells, studies of conjugating anticancer drugs to polymeric carriers have been carried out as one promising approach. This drug-polymer conjugate is called a "polymeric drug." Expected advantageous features of polymeric drugs are preferable tissue distribution of drug given by the character of the polymeric carrier, prolonged half-life of drug in plasma, and controlled drug release from the polymeric carrier by ad justment of the chemical properties of the bond between the drug and the carrier. Several kinds of polymers, naturally oc curring and synthetic polymers, have been examined as carriers of anticancer drugs. Among naturally occurring polymers, immunoglobulins are most widely used as the carrier, due to their high specificity and wide applicability to many kinds of malig nant cells. Utility of immunoglobulin as the polymeric carrier is, however, restricted by its chemical and physical properties. For example, modification of immunoglobulins by anticancer drugs often leads to precipitation due to hydrophobicity of the drug. Furthermore, modification procedures are limited to ones performed in mild conditions to avoid denaturation of the immunoglobulins during the modification. Alternatively, the polymeric carrier of the drug can be freely designed using many kinds 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 in accordance 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 poly meric carrier. From this point of view, several kinds of synthetic polymers have been investigated, such as poly[/V-2-(hydroxypropyl)methacrylamide] (1), poly(divinyl ether-co-maleic an hydride) (2), poly(styrene-co-maleic anhydride) (3), dextran (4, 5), poly(ethylene glycol) (6), poly(L-glutamic acid) (7, 8), poly(taspartic 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 activity have been obtained, the polymeric drugs using homopolymer or alternating copolymer often face a problem of water solubil ity. Introduction of a large quantity of drug to the polymers leads to precipitation of the polymeric drug, since most drugs have a hydrophobic character. One promising approach to overcome this difficulty is utilization of micelle-forming poly meric drug. A hydrophilic outer shell surrounds a drug-conju gated hydrophobic inner core, and this outer shell is considered to help prevent this conjugate from precipitation. The basic concept of this paper is the utilization of the micelle-forming polymeric drug as a novel anticancer agent. The polymeric drug possessing a micellar structure is expected to have a large diameter, as compared with unbound drug, which is a small molecule. The polymeric drugs with a micellar structure of the ideal diameter are expected to circulate in the blood stream without embolization at capillaries, to escape from excretion in kidney, and to permeate into the target cells through blood vessels. And this micellar form is expected to help protect the conjugated drug from enzymatic attack in plasma by concealing the conjugated drug in the hydrophobic core of the micelles. Furthermore, nonselective uptake of the polymeric drug by the reticuloendothelial system is thought to be suppressed by choosing the appropriate character of the outer shell. This paper deals with the anticancer activity as well as several chemical and physical properties of a newly designed micelleforming polymeric drug, poly(ethylene glycol)-poly(aspartic acid) block copolymer conjugated with Adriamycin. Adriamy cin is one of the most powerful and widely used anticancer drugs. Poly(ethylene glycol) is known to be a nontoxic and nonimmunogenic water-soluble polymer, which is expected to be the outer shell of the micelle. Poly(aspartic acid) is a synthetic poly(amino acid) and, therefore, possible hydrolysis of its main chain may play a role in releasing Adriamycin quickly from the conjugate after the uptake by the target cells. And it is noted that the substance resulting from the main chain hydrolysis of the 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 HNâ€”C'

purchased from Peptide Institute, Inc. (Japan). BSA was purchased from Sigma Chemical Co. Synthesis of Polymeric Drug

CH2COOCH259.427.56.511.711.313.418.032.5>42.033.07.5T/C(%Y17816317517819138 Â±1.4+2.9 0.8+4.8 Â± Â±-2.3 Â±+4.4 Â±-12.5 Â±-16.4Â±-15.2

Â±-18.7 Â±+ Â±+8.9 1.9 Â±+6.2 Â±+6.2 Â±-5.5 Â±-7.5 Â±-4.6 Â±-17.5 Â±1.10.90.82.11.41.30.41.01.41.11.2 " Expressed in ADR â€¢¿ HCI equivalent/kg of body weight. * Median survival: A, 8.0: B. 8.6; C, 9.0; D, 8.6; E. 8.7 days. c,. Median survival dav= of sample â€”¿ x 100(%). Median survival day of control d 60-day survivors are included as long survivors. ' Expressed in the mean Â±SE; initial mean body weight = 20-21 g.

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MICELLE-FORMING

1

POLYMERIC ANTICANCER DRUG

1000

10

Dose (mg/kg) Fig. 7. In vivo anticancer activity against P 388 mouse leukemia. Female CDF, mice were i.p. inoculated with P 388 mouse leukemia cells (1 x 10* cells in 0.1 ml) on day 0. The mice were i.p. inoculated with drug dissolved in a 0.9cc NaCI solution, on day 1, in a volume of O.I ml/10 g of body weight. Doses are plotted in ADR-HCI equivalents. G, ADR; â€¢¿, PEG-P(Asp(ADR)). Doses with multiple runs, shown in Table 3, are plotted as the mean value of all the runs.

40

20

60

Day Fig. 8. Body weight change of the mice inoculated with ADR and PEGP(Asp(ADR)) at the doses which brought about the maximum T/C values. â€¢¿. 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 leukemia ofPEC-P(Asp), PEC-P(Asp) plus ADR (mixture of block copolymer and drug), and PEC-P(Asp(ADR)) (drug-conjugated block copolymer)

(days)0PEG-P(Asp) SampleMedian

survival 7.7 2.7

PEG-P(Asp) + ADRÂ¿ 31>547Long >47.0" PEG-P(Asp(ADR)) Control, 8.6 days. .,,Median survival dayof sample " v inn /c:.\T/C(%)"90 Median survival day of control c 60-day survivors are included as long survivors. d ADR was dissolved in H2O.

survivors'0/6 0/63/6

siderea to result from self-association of Adriamycin molecules under a high ionic strength, as reported for daunomycin by Martin (14). No such gelation was observed for our PEGP(Asp(ADR)) system even at the ADR concentration of 20 mg ADR-HCI equivalent/ml. Furthermore, runs 3-5 were able to be redissolved in water after lyophilization. All these results indicate that PEG-P(Asp(ADR)) has excellent water solubility and stability against precipitation. These properties were pro vided by the poly(ethylene glycol) chain linked to the poly(aspartic acid) chain, since a precipitate was produced during the introduction reaction of ADR when poly(aspartic acid) homopolymer was used instead of PEG-P(Asp) under the same reaction conditions as those of a synthesis of PEG-

P(Asp( ADR)). These facts demonstrate one of the advantageous features of the block copolymer drug carrier. And this excellent water solubility and stability against precipitate are thought not to result only from the increased hydrophilicity by the linked PEG chain but to result from the micelle formation. The PEG chain is expected to exist as the outer shell of the micelle, and this hydrophilic outer shell is supposed to suppress the intermicellar aggregation of the hydrophobia P(Asp(ADR)) chain. Bader et al. (17) proposed a micelle-forming polymeric drug composed of cyclophosphamide-conjugated poly(ethylene glycol)-poly(L-lysine) block copolymer derivative. They obtained sustained release of cyclophosphamide from the block copoly mer. In their system, however, introduction of long alkyl chains to the poly(t-lysine) chain was needed to add hydrophobicity for micelle formation. Although they suggested possible micelle formation, there was no direct evidence for micelle formation. Our study is the first example of a micelle-forming polymeric drug with definite diameter. The diameter of the micelle (ap proximately 50 nm) corresponds to a range of the size of single unilamellar vesicles of liposomes. Although liposomes have been studied as a drug carrier for many years, most examina tions of targeting using liposomes have been not very successful, owing to the structural fragility of liposomes (especially for single unilamellar vesicles) and nonselective capture of lipo somes by the reticuloendothelial cells. Because polymeric mi celles are considered to be structurally stronger than liposomes, PEG-P(Asp(ADR)) can be a promising alternative candidate for the carrier used in drug targeting. The unimodal distribution of diameter of the micelles indicates that the micelles of PEGP(Asp(ADR)) were free from intermicellar aggregation, and this distribution is very narrow, possibly owing to the use of the well designed block copolymer. Furthermore, linking of the PEG chain is a promising way to inhibit the nonselective uptake by the reticuloendothelial cells, as speculated from the study on PEG-modified enzymes (18). The micelle-forming character of PEG-P(Asp(ADR)) is con sidered to contribute to the drastic changes in binding affinity for BSA as well as in stability in PBSa, since the micelle structure is considered to inhibit the access of BSA (complete inhibition) and OH ion (partial inhibition) to the bound ADR molecules by packing the ADR molecules tightly inside a hydrophobic core of the micelles. Considering the importance of albumin binding for pharmacokinetics of drugs, it is very inter esting to diminish the binding affinity of ADR for BSA drasti cally by binding ADR to the block copolymer. And this is the first example of ADR stabilization through binding to poly meric carriers. Stability of drugs in plasma is an important factor for effective chemotherapy, especially for Adriamycin. The stability is a critical problem for ADR since Adriamycin was reported (19) to be metabolized very rapidly to inactive derivatives mainly by liver after an injection into the blood stream. Lisi et al. (18) reported that accumulation of/i-glucuronidase in hepatic tissues was reduced by PEG modification. Therefore, the macellar structure of PEG-P(Asp(ADR)) with the PEG chain in the outer shell is a promising form to bring about resistance of Adriamycin to metabolism. The results of in vitro cytotoxicity and in vivo anticancer activity against P 388 mouse leukemia showed that the poly meric drug required a greater amount of ADR molecules than intact ADR to obtain the activity equal to intact ADR and that in vivo very high anticancer activity was obtained for PEGP(Asp(ADR)) with a smaller body weight loss than that of free ADR. The release rate of ADR from the micelle drug is ex pected to be very slow, because there is no spacer such as

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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 large quantity of bound ADR molecules reflected the slow action of the polymeric drug due to slow release of ADR molecules. On the other hand, Wingard et al. (23) reported that ADR chemi cally bound to an insoluble gel could exhibit cytotoxicity by directly interacting with cell membranes without being taken up by the cells and, therefore, there is another possibility, that the polymeric drug PEG-P(Asp(ADR)) showed cytotoxic activ ity to P 388 without releasing intact ADR molecules. Although the mechanism of action of the polymeric drug is still unknown, this paper reports the important example of the polymeric drug showing high in vivo anticancer activity with greatly improved body weight loss, as compared with intact ADR. ACKNOWLEDGMENTS The authors express their gratitude to Professor Tamotsu Kondo of Science University of Tokyo for his kind support and to Tomoko Hashimoto for her technical assistance.

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