The use of macromolecules as carriers of antitumor drugs

Europ. J. Cancer Vo[. 13, pp. 529-537. Pergamon Press 1977. Printed in Great Britain

The Use of Macromolecules as Carriers of Antitumor Drugs MARIA SZEKERKE* and JOHN S. DRISCOLL~" *Institute of Organic Chemistry, EOtv6s University, Budapest 1088, Hungary ~Laboratory of Medicinal Chemistry and Biology, DR & DP, DCT, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. Abstract~The tntrpose of this investigation is an extension of the use of protein carriers to various types of antitumor drugs, including antimetabolites and antitumor antibiotics. The variety of carriers has also been increased by the use of branched chain synthetic polymers with a poly-L-lysinc backbone. Most drug-protein combinations were non-covalent ~in nature. Murine antitumor results are discussed based on the evaluation of activii~ in the leukemia L1210 and P388 systems. In several cases the therapeutic effcacy qfan antitumor drug was increased by macromolecular complexation. This effect was partly due to reduced toxicity and seems to be dependent on the choice of carrier as well as the choice of drug. Actinomycin D, cytosine arabinoside, 5-azacytidine and dichlorortu'thotrexate appear to be good candidates for a macromolecular complex approach to cancer chemotherapy.

INTRODUCTION SEVERAL approaches have been made to enhance the efficacy of existing antitumor agents through the discovery of better ways to use these drug, s. One successful attempt has been combination therapy, which has been receiving increasing attention. Another recent approach has been the use of large molecular weight materials as carriers of cytotoxic drugs [1, 2]. The high endocytic activity reported for several types of tumor cells might allow a preferential uptake of the drug-macromolecular combinations by the target cell. This device could serve as a means of directing drugs preferentially into a chosen cell type. Since almost any substance can be linked by suitable physical or chemical coupling methods with an appropriate carrier, this field of research offers rich possibilities for a novel kind of drug design. It has been demonstrated that cytotoxic drugs can be covalently coupled to macromolecules without loss of biological activity [1, 3-5]. Our earlier studies of a series of products in which a nitrogen mustard derivative [p-(N, N-bis-2-chloroethyl) aminoaniline] was chemically attached to albumin, fibrinogen, ~-globulin or their poly-alanyl derivatives showed that this type of covalent coupling Accepted 14 October 1976.

(conjugate formation) led to an improvement of up to 8 fold in therapeutic index in the ADJ/PC6A plasma cell mouse tumor screen [1]. It was further shown that covalent bond formation was not a necessity and that complex formation by physical association was adequate in many cases [2]. As a result of several studies it has become clear that the strength or stability of bonds between a carrier molecule and a drug plays a critical role in the determination of its biological properties. While covalent bonds are sufficient, they are not necessary since the cumulative energy of multiple ionic and other non-covalent bonds between two molecules may produce very stable complexes [6]. In order to assess the value of different types of protein carriers, and evaluate complexes with antitumor agents other than nitrogen mustard derivatives, a series of new protein-antitumor agent combinations were prepared and evaluated in the leukemia L1210 system. The structural formulas of the various drugs used in these experiments are shown in Fig. 1. All of the antitumor agents evaluated were of some interest to the Division of Cancer Treatment, National Cancer Institute at the time of the study. Included are a broad spectrum of antitumor agents (antibiotics, nucleosides, intercalating agents, antimetabolites). In most instances non-covalent bonding was employed (complexes). In a few cases an 529

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attempt to form covalent bonds between drug and protein (conjugates) was made. Noncovalent linkages have the great advantage that they can be formed without the help of special chemical technology. The number of carriers studied was increased relative to previous experiments [I, 2] by the use of synthetic multi-chain polyamino acids with a polylysine backbone. This type of material has been extensively used by Sela et al. [7, 8] as model compounds in immunological studies. These structures can be modified easily in many ways (charge, spatial requirements, immunological properties, etc.). There are indications in the literature that the pinocytic activities of different cell types

show some chemical specificity [9]. Our previous results showed that coupling equal amounts of the same drug covalently to different carriers gave rise to considerable variation in the tumor-inhibitory potency of the conjugates. In order to achieve cell-specific effects, additional studies are necessary in order to develop carriers which rely on selective endocytic uptake by the target cells. MATIEIIJAL A_NI) MIETI'IOI)$ Drugs The cytotoxic drugs used are shown in Fig. I. All the drugs were supplied by the Drug Development Branch, National Cancer Institute (Bethesda, Maryland).

The Use of Macromolecules as Carriers of Antitumor Drugs Carriers Three groups of carriers were used. (a) Plasma proteins:', bovine serum albumin, bovine 7-globulin, bovine fibrinogen, fraction I (Sigma). (b) Poly-DL-alanyl proteins. These were prepared as reported previously [1, 10]. (e) Synthetic polypeptides. Intermediates: N'-carbobenzyloxy-L-lysine [11], N'-carbobenzyloxy-N'-carboxy,L-lysine anhydride [12], N-carboxy-~L-alanine anhydride [13], 7benzyl e-ghitamate [14], 7-benzyl N-carboxyL-glutamate anhydride [15]. Poly-L-lysine 'was prepared from N'-carbo benzyloxy-e-lysine via the N-carboxy-anhydride [12]. Conditions for polymerisation were chosen to obtain a degree of polymerization of approx. 100-300 [16]. The molecular weight of N~-carbobenzyloxy-poly-e-lysine was estimated by viscosity measurements [12]. The carbobenzyloxy blocking groups were removed by HBr/acetic acid. The purified poly-Llysine had a molcular weight of 30,000 (approx.). Multi-poly-DL-alanyl-poly-e-lysine (pDLAla --pLys) was prepared according to Sela et al. [17] choosing a Lys:Ala ratio 1:3.65. Amino acid analysis of the polymer indicated a ratio Lys:Ala = 1:3-5. For the preparation of multi-poly-e-glutamy 1-poly- DL- alanyl- poly- L- lysine (pGlupDLAla--pLys) the method of Sela et al. [18] was adopted. Input amino acid ratio Lys: Glu--1 : 6.7. Analysis of end product gave Lys:Glu = 1:5.8. Coupling methods Method A [2]. A solution of the macromolecule i n pH 6 phosphate buffer was incubated at 4°C with a solution of the drug. Agents which ihad a poor water solubility were applied in an organic solvent (Table 1). Stirring was continued for 3-4 hr in the cold. The macromolecular complex formed w a s purified in each case by dialysis for 36 hr against several changes of water using Visking tubing with the temperature being maintained below +4°C. In the case of poly-DT-alanyl fibrinogen derivatives, dialysis was carried out against 0.3% sodium citrate solution. Products were isolated by freeze drying. Estimates of the drug content were based either on microanalytical data obtained for elements not present in proteins (e.g., chlorine) or by u.v. absorption measurements. Method B. With drugs of reasonable water solubility, complex formation was achieved by mixing a solution of the macromolecule

531

in water with an aqueous solution of a weighed amount of the drug for 30 min at 4°C followed by freeze-drying (Table 1). An advantage of this method is that no further analytical measurement is needed to estimate the drug content. Method C. Covalent bonds were formed by the aid o f a water soluble carbodiimide [1cyclohexyl-3-(2-morpholinoethyl)-carbodiimide methotoluene-p-sulphonate] in pH 6 phosphate buffer, as described previously [1]. Determination of antitumor activity All in vivo antitumor tests were carried out at Hazleton Laboratories, Vienna, Virginia, U.S.A., using previously published National Cancer Institute protocols [19]. With the exception of cytembena and its protein combinations, all products were tested in the murine leukemia L1210 system. Cytembena and its derivatives (B 900302-B 900304) were screened in the P388 lymphocytic leukemia test system since the parent drug is inactive in the L1210 system. In each instance, the uncomplexed parent drug was tested for comparison in the same experiment. None of the macromolecular carriers by themselves inhibited tumor growth. Combinations formed which were found to have a low drug content and a relatively high parent drug optimum dose (e.g., Myleran) were not tested in the tumor model. Water was used as the injection vehicle. Tumor cells (L1210, 1 x 105; P388, 1 x 106) were injected into the peritoneal cavity of the test animals. Intraperitoneal injection of the test compounds began the next day and continued daily for a total of nine injections (QD 1-9 treatment schedule). Six mice were used to evaluate each dose level. Results are reported in percent survival time of treated animals versus untreated tumored control animals [(treated survival + control survival) x 100% = T/C%]. The percent T/C values greater than 125% indicate statistically significant antitumor effects [ 19]. RESULTS

Results of animal screening are summarized in Figs. 2-9. Graphical presentations were constructed by plotting antitumor activity (percentage T/C) against units of drug dosage. Attention should be called to the following points: (a) It can be seen from the graphical presentations that if the actual drug content of the macromolecular combination is calculated, the optimal dose levels are almost

532

Maria Szekerke and John S. Driscoll Table I. Survey of the ,OrqOarationand comlOositionof drug-macromolecular combinations i

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identical for the complexes and the parent compounds. However, a number of the complexes show an increased degree of tumor inhibition; (b) O n l y a few covalent conjugates ( M e t h o d C, T a b l e 1) were formed. I n these cases, a b e h a v i o u r similar to t h a t r e p o r t e d for the nitrogen m u s t a r d derivatives [1] c a n be recognised, i.e., the o p t i m a l dose levels are shifted to higher doses; (c) T h e effect o f the various carrier proteins o n the a n t i t u m o r activity o f a d r u g is different.

The use of poly-alanyl proteins seems to offer a consistent advantage in most cases. A similar observation has been made with respect to nitrogen mustard combinations [1,2]. The various polylysine combinations appear to exert different effects, b u t the p a t t e r n is n o t identical for each drug. T h e carrier must be c a p a b l e o f b i n d i n g the d r u g in a m a n n e r t h a t is unaffected b y b o d y fluids d u r i n g the transport process b u t is reversible in the t a r g e t cell. A separate s t u d y is p l a n n e d to evaluate the b i n d i n g p a r a m e t e r s o f these complexes as

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well as the pH and temperature dependence on complex stability. (d) In the case of Actinomycin D (Fig. 2), there is an indication that complex formation can result in recluced toxicity and an increase in the therapeutic index. If therapeutic index (TI) is defined as [the highest dose producing a T/C of 125% divided by the lowest dose producing the same response], then the parent compound, Acfinomycin D (Fig. 2, B900325) has a TI of 4.3 while the Actinomycin D-

Fig. 5. Effect of dicldoromethotrexate (NSC 29630)macromolecular combinations on the growth of the L 1210 system in BDFI mice; activitycorrelatedto actual drug content. (a) NSC 29630 (B 900317) (b) NSC 29630-BSA, complex (B 900318) (c) NSC29630-BSA, complexB 900319).

pDLAla-BSA complex (Fig. 2, B900328) has a TI of 10.8. The BSA conjugate (B900326) and complex (B900327) of Actinomycin D are also superior giving TI values of 7.3 and 6.5, respectively. Further experiments are needed to evaluate whether this aspect is general. It appears that in favorable cases, a pharmacological effect can be obtained at distinctly lower drug concentrations. Such derivatives might possibly allow the more extensive use of a number of very active drugs that are presently used with difficulty due to their excessive toxicity. Besides a decrease in toxicity, other advantages might be expected from macromolecular complexation. It has been reported [20] that the presence of maeromolecules has a pro-

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Fig. 9. Effect of ellipticine (NSC 71795)-macromolecular combinations on the growth of the L 1210 system in BDFI mire; activity correlated to actual drug content. (a) NSC 71795.(HCl) (B 900307) (b) NSC 71795-BSA, complex (B 900308) (c) NSC 71795-pDLAIa--BSA, complex (B

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tective effect and slows down the rate of hydrolysis of some types of antitumor active nitrogen mustards in aqueous solution. This suggested studies with 5-azacytidine, an antitumor agent given by continuous infusion but which is rapidly decomposed in aqueous solution [21].

Another possible advantage of complexation is related to the pharmaceutical formulation o f water insoluble compounds. Complexation of these materials with water soluble proteins might enhance the solubility of the parent compound. An active, but relatively insoluble acridine derivative, NSC 141549 (Fig. 1) was used for this purpose. The effect of complexation on the decomposition of 5-azacytidine was followed with u.v. spectroscopy. The change in u.v. absorption of 5-azacytidine at 243 nm was measured in the presence of different proteins in pH 7.3 phosphate buffer for periods of 30 min to 75 hr after incubation of 4 ° using a drugprotein ratio 1:9. Retardation of decomposition was largest in the first two hours (up to 30-40%) but with some of the carriers an effect could be demonstrated even 5 hours after incubation. These preliminary results are somewhat difficult to interpret since the binding characteristics of the decomposition products and the proteins are not yet available. Protein complexes containing 5-9% of the acridine derivative (NSC 141549) retained activity and had better water solubility characteristics than the parent drug. The improvement, however, was not great enough in this instance to provide parenteral formulation possibilities. DISCUSSION A reason for an improved therapeutic efficacy with the use of macromolecular carriers may be related to the findings that the administration of radioactive albumin or fibrinogen leads to a relatively high incorpor-

The Use of Macromolecules as Carriers of Antitumor Drugs

ation of radioactivity into tumors [22-24]. De Duve suggeslted [9] the term "lysosomotropism" for drug-macromolecular combinations taken up by cellular endocytosis. According to this hypothesis the carrier is transported into the cell and there it is digested by lysosomal enzymes and the drug is released in its active form. The daunorubicin-DNA and adriamycin--DNA complexes were prepared based on this principle. Encouraging animal screening results [25, 26] resulted in clinical trials with these materials in the treatment of leukemia [25, 27]. Lysosomotropism was shown to be operative for the adriamycinDNA complex [91[. A lysosome-mediated effect should be considered for drug-protein complexes. However, other mechanisms are also possible. Rubens and Dulbecco [28] have shown that chlorambucil and a tumor specific globulin are synergistic, but there is no requirement for physical absorption of the: two materials since separate addition of the two components can still bring about the observed effect. The effect of simultaneous injection of separate solutions of the protein carriers and antitumor drugs described here is in progress. This will assess whether a similar synergistic action occurs with these materials. It is hoped that the investigation of complex stability and the nature of the bonds formed between the protein and cytotoxic drugs might offer important information for structure--activity relationships. There is certainly a need for further research work to establish the actual mechanism of antitumor action of the macromolecular derivatives. With the development of tumor immunology, many investigators have sought to use antibodies to antigenic determinants expressed preferentially on tumor cells as carriers of cytotoxic agents in the hope of increasing the specificity of the drug and reducing its

535

systemic toxicity. Several reports have appeared in which drug-antibody complexes [28-33] and conjugates, i.e.,drugs covalently linked to immunoglobulins [3-5, 34] have bccn studied. For this approach to succeed, both the antibody and the toxic agent must retain activity when the two arc linked together. The most powerful antitumor propcrties wcrc achieved by firstloading a polyL-glutamic acid chain with the drug, followed by coupling of some of the chains to immuno' globulin [34]. This method also seems applicable in other cases, e.g., when hormone activity is to bc saved, or when the covalent coupling reaction requires a chemical technique which would extensively denature the protein. W c arc presently involved in trying in such cases the application of the concept of the "inertintermediate carrier". Although the idea of using tumor-specific antibodies as carriersis attractive,the isolation and purification of tumor-specific antibodies is presently still rather tedious, preventing general access to larger amounts of carriers of this type. W c therefore fccl that the exploration of readily obtainable non-specific macromolccular carriers might lead to combinations rnorc easily exploitable for practical application. Art augmenting effect of macromolcculcs on cytotoxic drug action has bccn demonstrated and is supported by a number of experimental examples. It is hoped that the results obtained in these preliminary attempts to apply the principle of macromolccular carriers to antitumor drugs will stimulate further efforts in this area. Aclmowledgemeats--M. Szekerke gratefully acknowledges the Hungarian Institute of Cultural Relations for an I1LEX (International Research and Exchanges Board) Fellowship for a period of five months.

REFERENCES

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Maria Szekerke and John S. Driscoll

6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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