Macromolecular drug carrier systems in cancer chemotherapy: macromolecular prodrugs

Critical Reviews in

ONCOLOG Y/ HEMA TOLOG Y ELSEVIER

Critical

Reviews in Oncology,‘Hematology

18 (1995) 207-23 I

Macromolecular drug carrier systems in cancer chemotherapy: macromolecular prodrugs Yoshinobu Takakura, Mitsuru Hashida* Department of Drug Delivery Research, Faculty of Pharmaceutical Sciences, Kyoro University, Sakyo-ku. Kyoto 606-01, Japan Accepted I5 April

1094

Contents I.

Introduction

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..___.._..........._................

2.

Anatomy and physiology

3.

Design of macromolecular prodrugs ...................................................... 3.1. Choice of macromolecular carrier .................................................. 3.2. Chemotherapeutic agent .......................................................... 3.3. Design of linkage structure between macromolecule and drug ..........................

209 209 210 210

4.

In vivo fate of macromolecular prodrugs .................................................. 4.1. Disposition after systemic administration ............................................ 4.2. Disposition after local administration ............................................... 4.2.1. Intra-arterial injection and local perfusion .................................. Local injection ........................................................... 4.2.2.

210 211 213 213 213

5.

Action mechanism of macromolecular prodrugs ........................ 5.1. Cellular uptake mechanism .................................... 5.2. Drug release ................................................. 5.2.1. Release in intracellular space ........................... 5.2. I. I. Release in lysosomeli ......................... 5.2.1.2. Release in intracellular space outside lysosomes 5.2.2. Release in extracellular space ........................... 5.3. Mode of action of antibody-drug conjugates .....................

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6.

Therapeutic efftcacy of macromolecular prodrugs ...................... 6. I. Anthracycline antibiotics prodrugs ............................. 6.2. Mitomycin C prodrugs ........................................ 6.3. Methotrexate prodrugs ........................................ 6.4. Neocarzinostatin conjugates ................................... 6.5. Combination of antibody-enzyme conjugates and prodrugs

of tumor tissue

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7.

Clinical applications of macromolecular prodrugs .......................................... 7. I. Current status of clinical applications .............................................. Systemic administration 7.1. I. .................................................. Local administration 7.1.2. ...................................................... 7.2. Future assignment ................................................................

8.

Conclusions

and perspective

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* Corresponding author, Tel.: 81 75 753 4525; Fax: 81 75 753 4575. 1040-8428/95/%29.00 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI lO40-8428(94)0013 I-C

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1. Introduction For an effective therapy with medications, it is necessaryto optimize the delivery of therapeutic agents since most drugs are associatedwith both beneficial effects and toxic actions. Cancer chemotherapy is a good example of this principle becauseof the extreme toxicity of most antitumor agents. Therefore, research in the field of drug delivery has centered on cancer chemotherapy. Among several therapeutic modalities for neoplastic diseases,chemotherapy has played an important role and has been widely applied in the treatment of both hematological malignancies and some solid tumors. Despite efforts to develop novel cytotoxic drugs and treatments, however, progress is being made in small steps and no effective chemotherapy has yet been achieved for certain advanced tumors. In addition, while many chemotherapeutic agents with high cytotoxicity to tumor cells are available, these agents also display very severe toxicities to rapidly proliferating normal cell populations in the bone marrow, gastrointestinal and genitourinary epithelium, etc. Thus, the low therapeutic index of most antitumor agents has prompted us to search not only for novel cytotoxic compounds but also for innovative ways to improve the therapeutic efficacy of existing drugs. In general, the lack of selectivity in the cytotoxicity of most antitumor drugs is closely related to their pharmacokinetic properties. That is, the in vivo fate of a drug given by a certain administration route is determined by both the physicochemical properties of the drug and the anatomical and physiological characteristics of the body, and most cytotoxic agents which diffuse freely throughout the body show essentially even tissue distribution due to their low molecular weight. Therefore, any systemsor techniques which enable us to control the in vivo behavior of antitumor agents would be useful in improving cancer chemotherapy. We can promote the therapeutic effects of an antitumor agent and minimize its toxic effectsby increasing the amount and persistence of the agent in the vicinity of the tumor cells while reducing the exposure of non-target normal cells to the drug. The fundamental rationale for the introduction of site-specilic drug delivery in cancer chemotherapy lies in this straightforward hypothesis [ 11. Prominent among the strategiesfor site-specific drug delivery has been the employment of various drug carrier systems including soluble macromolecules, particulate carriers, and cells [l-7].

231

As a method for site-specific drug delivery, alteration of the physicochemical and biological properties of an antitumor agent, through chemical modification with macromolecular carriers, would be a formidable tool for the optimization of cancer chemotherapy [1,8-lo]. At present, many kinds of natural and synthetic macromolecules are available as antitumor drug carriers. In particular, the advent of monoclonal antibody technology developed in 1975 by Kohler and Milstein [ 1l] has reawakened the ‘magic bullet’ concept of Ehrlich proposed at the beginning of this century, whereby cytotoxic agents can be specifically targeted to tumors [12-161, if designed and used wisely. Although a great deal of effort has been devoted to this area for more than a decade,the primary goal of site-specific delivery has not be realized. The basic and general aspectsof macromolecule-drug conjugatesin cancer chemotherapy have been extensively discussedin our earlier reviews [1,8], so the present article will focus on the current status of this field. In particular, the in vivo behavior and mode of action of antitumor drug-macromolecular conjugates will be discussed, followed by a review of recent advances in clinical research in cancer chemotherapy. 2. Anatomy and physiology of tumor tissue In order to construct a strategy for tumor specific drug delivery using macromolecular carrier systems,it is important, to summarize the anatomical and physiological characteristics of tumor tissues. Neoplastic tissues,as well as most normal tissues, can be divided into three parts: vascular, interstitial, and cellular compartment. However, the anatomical and physiological characteristics of tumor tissuesare significantly different from those of most normal tissues(Fig. 1). Once an anti-

Tumor

Tissue

Fig. 1. Anatomical and physiological characteristics of normal and tumor tissues.

Y. Takakura, hf. Hashida/Crit.

Rev. Oncol. Hemaiol. 18 (1994) 207-231

cancer drug is injected into the bloodstream, it encounters the following processes before reaching the intracellular spacewhere the pharmacological action of the drug occurs: (i) distribution within the vascular space;(ii) penetration acrossthe microvascular wall; (iii) movement through the interstitial space;(iv) interaction with the cell surface; and (v) cellular uptake. In order to achieve effective delivery to tumor cells, one needs to understand the characteristics and mechanisms of these processes,and to control the behavior of the drugs in these processes. The anatomical structure of tumor vessels plays an important role in determining drug distribution within the interstitial space.A tumor has two types of vessels: (i) vesselsrecruited from the pre-existing network of the host vasculature, and (ii) vessels resulting from the angiogenic response induced by cancer cells. Thus, the overall characteristics of tumor vessels depend on the ratio of host vessels to newly formed tumor vessels which would vary from one location to another and from one tumor to another. General characteristics of tumor vessels are summarized as follows [17,18]: (i) micro- and macroscopical heterogeneities; (ii) enhanced microvascular permeability which allows large molecules to penetrate; (iii) vessel collapse as well as flow stasisand reversal due to low blood pressure; and (iv) no definite response of newly formed vessels lacking smooth muscle to physical and chemical stimuli. Once a drug molecule enters the interstitial space, its transport is governed by local physiological properties. In general, tumor interstitum is characterized by [ 19,201: (i) large interstitial volume with high collagen and low proteoglycan and hyaluronate concentrations; (ii) high fluid pressure and flow; (iii) high effective diffusion rate of macromolecules; and (iv) absence of a functioning lymphatic network. The transport of molecules across vessel walls and through the interstitum is generally governed by diffusion and convection. Diffusive transport depends on the concentration gradient of molecules. On the other hand, movement of fluid caused by pressure gradients leads to convective transport of molecules. While diffusion is the primary mechanism of transport of small molecules, transport of macromolecules is determined by both convection and diffusion. Increased pressure gradients in the tumor interstitum suggestthe existence of significant convective flow in the tumor. Butler et al. [21] reported that the fluid loss in rat tumors was 0.14 to 0.22 ml/h/g or 4.5-10.2% of perfusing plasma volume, which is significantly larger than that of the lymph drainage in most normal tissues. Thus, convection should be considered in assessingdisposition of macromolecules in solid tumors. In summary, tumor tissuesare characterized by a high interstitial pressure which may retard the extravasation

209

of macromolecules. On the other hand, large vascular permeability and high interstitial diffusivity of macromolecules seemto facilitate their migration to tumor tissues In addition, the lack of functional lymphatic drainage will result in the accumulation of macromolecules by a ‘passive’ mechanism as has been shown in several reports [22-261. Thus, these anatomical and physiological characteristics of solid tumors would provide a reliable rationale for the use of macromolecular carrier systems in cancer chemotherapy. The classic finding that many tumor cells have high endocytic activity [27,28] would also support this approach. 3. Design of macromolecular prodrugs The prodrug approach has been one of the most promising meansof site-specific drug delivery [29,30]. Here, the active drugs are chemically transformed into inactive derivatives which revert to the parent compounds by virtue of an enzyme or chemical lability, or both, before or after reaching the site of action in the body. Although some studies showed that polymer-bound anthracycline antibiotics exhibited antitumor activities in the conjugated form without liberating the free drugs [31-361, most antitumor agentsconjugated with macromolecular carriers are designedto exhibit their pharmacological efficacy after conversion to the parent compounds. In this sense,antitumor drug-macromolecule conjugates can be regarded as ‘macromolecular prodrugs’ as discussed in our previous review [ 11. 3.1. Choice of macromolecular carrier

A variety of biological and synthetic macromolecules have been employed as carriers of chemotherapeutic agents. The criteria for choice of macromolecular carriers can be summarized as follows [ 181. The carrier should: (i) be biodegradable; (ii) lack intrinsic toxicity and antigenecity; (iii) show no accumulation in the body; (iv) have adequate functional groups for chemical fixation; (v) retain the original specificity for target; and (vi) keep the original activity of the delivered drug, until it reaches the site of action. Table 1 shows some examples of macromolecular carriers classified according to their function and origin. In principle, target-non-specific and target-specific macromoleculeswould correspond to passive and active mechanisms of targeting, respectively. However, nonspecific macromolecules can be used as carriers for active targeting by introducing a special recognition system (homing device). For example, albumin derivatives synthetically modified with sugar moieties called ‘neoglycoproteins’ are applied for drug targeting to certain cell types having receptors specific to the sugar structure [37].

210 I‘able I Classification

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of macromolecular

Target non-specijic carriers Natural macromolecules Proteins Polysaccharides Nucleic iacids Synthetic macromolecules Polyamino acids Miscellaneous

Target specific, carriers Antibodies Lectins Peptide hormones Glycoproteins

carriers

Albumin, globulin Dextran, pullulan, chitin, chitosan, inulin Deoxyribonucleic acid (DNA) Polylysine, polyaspartic acid, polyglutamic acid Styrene-maleic acid anhydride copolymer (SMA), divinyl ethermaleic anhydride copolymer (DIVEMA), N-(2-hydroxypropyl)methacrylamide copolymers (HPMA), polyethylene glycol (PEG), polyvinyl alcohol (PVA)

Monoclonal antibodies Concanavalin A. wheat germ agglutinin Melanotropin, thyrotropin Asialofetuin, asialoorosomucoid

3.2. Chemotherapeuticagent For the preparation of macromolecular prodrugs, the cytotoxic component should: (i) show enough cytotoxicity at relatively low dosesin order to decreasethe load of a carrier macromolecule; (ii) be chemically stable in the conjugated form until released; and (iii) have adequate functional groups in its molecular structure for chemical fixation [7]. In most macromolecular prodrugs, conventional antitumor drugs are employed. However, if it can be delivered to the target tumor cells with enough specificity and accuracy, the active moiety is not necessarily required to be ‘a drug’ available in clinical medicine. For example, toxins conjugated with antibodies called ‘immunotoxins’ have been eagerly evaluated as possible candidates for drugs in cancer therapy and clinical trials have also been carried out [16]. Similarly, recent work by Chari et al. [38] demonstrated that immunoconjugates of maytasinoids that have lOO- to lOOO-foldhigher cytotoxic potency than conventional anticancer drugs showed remarkable antigen-specific cytotoxicity against human cancer cells by releasing fully active drugs inside the cells. Thus, the macromolecular prodrug approach may open the door for the clinical application of compounds with extreme cytotoxicity.

3.3. Design of linkage structure betweenmacromolecule and drug In designing macromolecular prodrugs, chemical and/or biological stability of the linkage between the drug and the macromolecular carrier should be considered sincethe chemotherapeutic activity of the system requires the releaseof the free drug from the conjugate. Generally, drugs are coupled either directly to macromolecular carriers or to spacers introduced into a macromolecular backbone via covalent bonds, which can be accomplished using various kinds of crosslinking agents, including carbodiimides, glutaraldehyde, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP reagent), etc. (71.Functional groups in the drug and the carrier molecule employed for chemical coupling include amino, carboxyl, hydroxyl, and free thiol groups. Most of the reactions are carried out in aqueous media under mild conditions to avoid any alteration in the parent drug and the macromolecular carrier. For the preparation of macromolecular prodrugs using antibodies, drugs are occasionally conjugated to other nonspecific macromolecular intermediates such as dextran and albumin before they are coupled with antibody molecules in order to increase numbers of drug molecules attached and minimize undesirable change in antibody affinity. In addition to a covalent bond, some non-covalent interactions between drugs and macromolecular carriers have been utilized for the preparation of macromolecular prodrugs, e.g., intercalation between daunorubicin and DNA [39], ionic binding between bleomycin and dextran sulfate [40], metastable complexation of cisplatin and carboxymethyl dextran or poly+glutamic acid (in which mechanism of binding have not been clearly documented) [41-441, and antigen-antibody interaction between methotrexate and an anti-methotrexate antibody [45]. Generally speaking, a linkage between the drug and the carrier must be designed to be cleaved at an appropriate rate for the preparation of macromolecular prodrugs. When the linkage is expected to be cleaved by enzymatic reaction(s), one should pay attention to animal speciesdifferences in types and activities of the enzyme(s). 4. In vivo fate of macromolecular prodrugs The rationale of macromolecular prodrug approach in site-specific drug delivery lies in the altered disposition of a carrier-conjugated drug in the body, which is largely dictated by the properties of the carrier and accordingly differs from that of the free drug administered by the sameroute. Site-specific drug delivery is broadly categorized as passive and active targeting [46,47]. ‘Passivetargeting’ refers to the exploitation of the natural (passive)disposition profiles of a drug carrier, which

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61ev. Oncol. Hemarol. 18 (1994) 207-231

is passively determined by the physicochemical properties of the carrier in relation to the anatomical and physiological properties of the body. On the other hand, ‘active targeting’ alters or intends to alter the natural disposition pattern of a drug or a carrier, directing it to specific cells, tissues, or organs, by employing a special means,e.g., the use of monoclonal antibodies which can recognize the specific antigen on the surface of target tumor cells. Apparently, active targeting appears to be much more attractive than passive targeting; consequently, many efforts have been focused on the development of sophisticated macromolecular prodrugs of antitumor agents with specific recognition potential. After administration of a macromolecular prodrug to the body, a series of events normally occurs before the free active drug finally affects its target tumor cells. Fig. 2 schematically illustrates such pathways. In the caseof systemic administration by which most macromolecular prodrugs are applied, disposition of prodrugs involves interaction with blood components, organ distribution, uptake by reticuloendothelial systems, renal excretion, etc. Among these steps, general elimination processes such as urinary excretion and hepatic uptake are determinants for the fate of the macromolecular prodrug. Some prodrug molecules in the circulation which survive theseevent.swill extravasate out of the capillaries in the tumor tissue. Then they move in the interstitum and then finally encounter the tumor cells. Active free drugs liberated from the conjugates in the intracellular space or in the vicinity of tumor cells exhibit pharmacological action. In the caseof intratumoral injection, movements in the tumor tissues and absorption via capillary and lymphatic routes should be considered. The distribution and elimination patterns of macromolecular prodrugs are dictated mainly by such physicochemical properties of the conjugates as molecular size, electric charge, and lipophilic/hydrophilic balance. Even if tumor specific molecules like antibodies, lectins, and hormones which can function as targetrecognizing devices when facing the target, are an in-

Fig. 2. Schematic representation of the fate of macromolecular prodrug in the body.

211

tegral part of the conjugate, the physicochemical properties still play an important role in deciding the localization of the conjugates in the tumor tissue. Therefore, the understanding of the general relationship between physicochemical properties of macromolecular prodrugs and their disposition behaviors is a major prerequisite for not only passive but also active targeting. 4.1. Disposition after systemic administration

Immediately after an intravenous injection, distribution of macromolecules is basically restricted to the intravascular space due to low capillary permeability in most organs. Then the kidneys play an important role in the disposition of macromolecules circulating in the vasculature [48]. Macromolecules with a molecular weight of less than 30 000 are susceptible to glomerular filtration and may be excreted into the urine. Since the glomerular capillary walls also function as a charge selective barrier having negative charges, positively charged macromolecules show higher glomerular permeation compared with anionic macromolecules with similar molecular weight. For larger macromolecules which escapesieving through the glomerulus, the liver plays an important role. In contrast to most other organs in which the capillary presents a substantial barrier between the vascular and interstitial spaces,the liver has discontinuous endothelial capillaries [49] and this structure brings any circulating macromolecules in the blood into free contact with the surface of parenchymal cells. Due to this anatomical character which offers a wide surface area for electrostatic interaction, cationic macromoleculesare highly distributed to the liver based on adsorption on the negatively charged cell surface [50--531. On the other hand, strongly anionic macromolecules are known to be taken up by the liver nonparenchymal cells by scavenger receptor-mediated endocytosis [54]. Pharmacokinetic properties of macromolecular prodrugs can be analyzed basedon a physiological pharmacokinetic model and are shown in Fig. 3 [55]. Assuming of a unidirectional organ uptake, general disposition characteristics of macromolecules can be characterized using organ uptake clearance (CL,,) as an index. Then targeting efficacy, i.e., the total amount of the macromolecule arrived at the target site at the infinite time, is estimated as a ratio of CL,,, at the target site and the total body clearance (CLtotal). Therefore macromolecules with high CL,,, at the target site but with small %%a1 result in high accumulation in the target site. Pharmacokinetic studies based on the clearance concept [25,26] quantitatively demonstrated the general disposition characteristics of macromolecular carriers and prodrugs in tumor bearing mice. In Figs. 4 and 5, hepatic uptake and urinary excretion clearanceswhich essen-

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/ Crit. Rev. Oncol. Hematol. 18 (1994) 207-231

catBSA

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wnole body

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Fig. 3. Pharmacokinetics for evaluating in vivo disposition of macromolecular prodrugs.

Rate

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of fluid-phase

endocytosis

of liver

t

t

t

100

1000

10000

I

1 100000

Urinary clearance (ul/hr)

tially decide the disposition of macromolecules at a whole body level are plotted in a logarithmic scale for a variety of macromolecules examined in this experimental system. Small and positively charged macromolecules had large urinary and hepatic uptake clearances, respectively, and accordingly large CL,,,, values. The relationship between general disposition characteristics of macromolecules in the kidney and liver and their physicochemical properties is thus demonstrated. Similarly, the uptake indexes of these macromolecules in the tumor were determined while these values varied depending on physicochemical pro-

Small macromolecule. I “, urinary excretion I

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Urinary clearance (ul/hr) Fig. 4. Hepatic and urinary clearancesof macromoleculesin mice after intravenous injection: effect of molecular weight. apo NCS, apoprotein of neocarzinostatin; BSA, bovine serum albumin; CMDex, carboxymethyl-dextran; Dex (T-10) or (T-70), dextran with average molecular weight of 10000 or 70 Ooo; Dex-sul, dextran sulfate with average molecular weight of 8ooO;IgG, immunoglobulin G.

Fig. 5. Hepatic and urinary clearancesof macromoleculesin mice after intravenous injection: effect of electric charge. catBSA, cationized BSA; DEAEDex, diethylaminocthyl-dextran; sucBSA, succinylated BSA.

perties of macromolecules, the changesare significantly smaller compared with that in CLtotal which is also affected by the physicochemical properties. From these results, it can be concluded that macromolecular prodrugs with adequate molecular size (mol.wt > 70 000) and weak anionic nature will show prolonged retention in the plasma circulation and then large accumulation in the target, i.e., the tumor. This working hypothesis has beenvalidated by the fact that an anionic macromolecular prodrug of mitomycin C, mitomycin C-dextran conjugate with molecular weight of 70 000, showed a marked accumulation in the solid tumor inoculated subcutaneously in mice [25]. Considerable growth inhibition of the tumor was also recorded in the same system [25]. Thus, these studies have demonstrated a quite simple but important principle that macromolecular prodrugs should have a long plasma circulation time for passive targeting. Actually, this principle is also valid in active targeting to the tumor. That is, a tumor specific carrier must be designed to have adequate physicochemical properties to escapeany undesirable recognition by other tissuesin addition to the introduction of a homing device. Based on these considerations, Noguchi et al. [56,57] developed a mitomycin C-monoclonal antibody conjugate using anionic dextran as an intermediate for active targeting (Fig. 6). The final conjugate exhibited a small CLtotit as expected and accumulated to a great extent in a targeted human colorectal tumor implanted in nude mice (Fig. 7). Intravenous administration of the conjugate also exhibited corresponding growth in-

Y. Takakura. h4. Hashida/Crit.

Rev. Oncol. Hematol. 18 (1994) 2007-231

213

4.2. Disposition after local administration

Fig. 6. Representative structure of mitomycin C-monoclonal antibody conjugate.

hibitory effect on the target tumor inoculated in nude mice [56]. Nishikawa et al. [58] designed a macromolecular prodrug of cytosine /3-D-arabinoside employing dextran as a carrier backbone in an attempt to accomplish active targeting to the normal liver or hepatoma cells utilizing a carbohydrate recognition system. Prior to glycosylation of the backbone with 2-irnino 2methoxyethyl I-thioglycoside [37], i.e., an introduction of a homing device, carboxymethyl groups were introduced to dextran to avoid any tissue interaction by anionization. Following these processes,the drug was conjugated at the last step. After intravenous injection, the conjugate was very rapidly taken up by the liver parenchyrnal cells via receptor-mediated endocytosis. In vitro study showed that the conjugate was also taken up by mouse hepatoma cells and exhibited a superior growth inhibitory effect on the cells, suggesting the potential of the targeting system to hepatoma cells based on carbohydrate recognition mechanism.

Fig. 7. Relationship between total body clearance, organ clearance in the tumor, and resulted tumor accumulation of macromolecular prodrugs of mitomycin C. Prodrugs: A7, mouse monoclonal antibody against human colorectal cancer; MMC-Dan, mitomycin C-dextran conjugate with anionic charges; MMC-Lkat, mitomycin C-dextran conjugate with cationic charges;NSIgG, non-specific IgG. Tumors: S180, sarcoma 180; SW1116, human colorectal cancer (target of A7).

In order to circumvent the side-effects in systemic chemotherapy, local or regional treatment modalities such as intra-arterial infusion, organ perfusion, and intratumoral injection have been applied in clinical trials. Macromolecular prodrugs of antitumor drugs may confer additional advantages in these approaches. In constructing the strategy, disposition characteristics of macromolecular prodrugs after local administration should also be clarified. The tissue-isolated tumor preparation, originally established by Gullino [59,60], is a unique and useful experimental system in cancer research. This system is composedof solid tumor with an artery and a vein, and local disposition events occurring in the tumor tissue such as extravasation, interstitial diffusion, and tissue binding can be independently evaluated. The systemhas been applied to elucidate the physiological properties of solid tumors such as blood flow 1601,interstitial pressure [61,62], and energy metabolism [64]. Recently, Ohkouchi et al. [65] studied the pharmacokinetic properties of anticancer drugs after intra-arterial injection in this system. 4.2.1. Intra-arterial

injection and local perfusion

Imoto et al. [66] analyzed the disposition characteristics of model macromolecules and macromolecular prodrugs of mitomycin C administered by intra-arterial perfusion in tissue-isolated tumor preparations of a Walker 256 carcinoma with the use of a single-passvascular perfusion technique (Fig. 8). During the perfusion, the macromolecules in the per&sate were gradually accumulated in the tumor, but the degree and pattern greatly varied depending on their electric charge. Positively charged macromolecules were markedly accumulated compared with neutral and negatively charged macromolecules (Fig. 9). In addition, concentrations of polycations were significantly higher in viable than in necrotic regions, while neutral and negative compounds were distributed in necrotic rather than in viable regions. Pharrnacokinetic analysis demonstrated that extravasation of cationic compounds was driven by convective fluid flow, and that their high tissue accumulation could be explained by strong binding with the tissue due to electrostatic interaction. For neutral and anionic macromolecules with negligible affinity to the tissue, it was suggestedthat the final concentration was decided by tissue fluid content in viable and necrotic regions. Thesefindings are useful for the development of macromolecular prodrugs aiming at local cancer chemotherapy. 4.2.2, Local injection

Local administration methods, including intratumoral injection and intracoeliac injection into the

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Sampling

Fig. 8. Schemeof the perfusion system(left) and angiogram (right) of tissue-isolatedtumor preparation. (The angiogram is reproduced with permission from Ohkouchi K, Imoto H, Takakura Y, Hashida M, Sezaki H. Cancer Res 50:1640-1644, 1990)

body cavity in which tumor cells have spread, are primitive but effective ways to impose the cytotoxicity of antitumor drugs selectively upon the tumor cells. However, the use of such methods is limited becauselow molecular weight antitumor drugs are rapidly cleared from the injection site while exhibiting severe local damage to normal tissues surrounding the lesion irrespective of their short exposure time. Here, macromolecular prodrugs will have great advantages in these approaches.

Fig. 9. Concentrations of radiolabeled macromolecules in viable and necrotic regions of tissue-isolated tumors after constant infusion for 3 h. Data are expressedas mean f S.D. of the percentage of radioactivity in the tissue to that in the inflow perfusate, assuming the specific gravity of the tissue to be 1.0. See legends of Figs. 4, 5 and 7 for abbreviations for compounds. *, Significant difference (P < 0.05, Itest) between concentrations in viable and necrotic regions. (Reproduced with permission from Imoto H, Sakamura Y, Ohkouchi K et al. Cancer Res 52:4396-4401, 1992)

Pharmacokinetic properties of mitomycin C conjugated with dextran (mol.wt 70 000) after intratumoral injection were studied in tissue-isolated tumor preparation of a Walker 256 carcinosarcoma [67]. In contrast to free mitomycin C, which appeared in the venous outflow perfusate immediately after intratumoral injection, the appearance of cationic and anionic mitomycin Cdextran conjugates was highly restricted, clearly demonstrating that the absorption of mitomycin C from the tumor by blood circulation can be greatly retarded by dextran conjugation. In particular, a pronounced effect was observed in the caseof the cationic conjugate. The venous outflow data were analyzed by the compartment model, in which the tumor tissue is assumedto be divided into two compartments, well- and poorlyperfused regions. The venous appearancerate-time profiles were fitted to equations derived from the model by non-linear regression analysis. Using the calculated parameters,the concentrations of conjugated drug and free drug generated from the conjugate in the tumor were simulated in Fig. 10. It was demonstrated that less than 0.1% of dose remained in the tumor at 40 min after injection of free mitomycin C. By contrast, the simulation clearly showed that a certain level of active free mitomytin C could be maintained in the tissue for a much longer period after the intratumoral injection of the conjugates, especially the cationic one. A cationic mitomycin C-dextran conjugate with an average molecular weight of 500 000 was applied by intratumoral injection in patients with advanced solid tumors and superior

Y. Tukakura, M. Hashida / Crit. Rev. Oncol. Hematol. 18 (1994) 207-231

0

0.5

I

2

6 ‘Time

12

18

24

(hr)

Fig. 10. Simulation of drug disposition in the tissue-isolated tumor after intratumoral injection of mitomycin C (MMC) and mitomycin Cdextran conjugates (MMCDcat and MMCDan).

therapeutic effects have been obtained [68,69]. Together with the finding reported by Kennedy et al. [70] that mitomycin C was selectively toxic to hypoxic tumor cells at low concentrations, the pharmacokinetic study in the tissue-isolated tumors would strongly support the clinical efficacy of the conjugate. The prevention of lymphatic metastasis which occasionally leads to a poor prognosis is a very important problem in cancer chemotherapy. However, it is usually difficult to supply sufficient antitumor agents to the lymphatic system even by local injection, since these agents are absorbed from the interstitial space through the capillaries but not the lymphatic vessels[71]. Based on the finding that the lymphatics readily take up large molecules from the interstitum [72], several macromolecular conjugates aimed at lymphotropic delivery have been reported [39,73-761. Takakura et al. studied the pharmacokinetics of lymphatic transfer of cationic and anionic mitomycin C-dextran conjugates with various molecular sizes after intramuscular injection in rats [74-761. These studies demonstrated that large cationic conjugates accumulated to a great extent in the regional lymph node. Intramuscular injection of these conjugates resulted in an increased effectiveness in suppressing lymph node metastasis of L1210 leukemia cells in a mouse model 1741. Thus, macromolecular prodrugs offer a potentially interesting method of local cancer chemotherapy aimed at the prevention of lymphatic metastasis.

215

of extracellular solutes into the cell. A macromolecule, when dissolved in the extracellular fluid can enter a cell at a relatively slow rate. This process is called ‘fluidphase endocytosis’. Macromolecular prodrugs using carriers without any special affinity to tumor cells are considered to be endocytosed by this mechanism as shown in Fig. 11. In ‘adsorptive endocytosis’, macromolecules bound to the plasma membrane are internalized at rates usually faster than those by fluid-phase endocytosis. Tumor cells may endocytose cationic macromolecular prodrugs, following adsorption on the plasma membrane by electrostatic force by this process. Actively targeted macromolecular prodrugs with carriers of glycoproteins, hormones, lectins, etc. are rapidly and effectively internalized by adsorptive or receptormediated endocytosis, which occurs via coated pits (Fig. 11). The fate of drugs conjugated to antibodies after binding to the cell surface antigens is important and interesting as will be discussedlater. In conjunction with drug release problems, the rate and extent of endocytosis of macromolecular prodrugs are of particular importance to their pharmacological efficacy. 5.2. Drug release

As mentioned earlier, the pharmacological activity of macromolecular prodrugs requires the release of free drugs by chemical and/or enzymatic reactions from the conjugate. In terms of drug release, the stability of the linkage between the carrier and the drug, and the site of regeneration of the free drug from the conjugate are important factors. Since the site of action of most antitumor drugs, such as nuclei, is located in the intracellular space of tumor cells, the therapeutic eficacy of a macromolecular prodrug greatly depends on where the free drugs are released. 5.2.1. Release in intracellular space S..?.l.l. Release in lysosomes. The most well-known

concept for the mechanism of action of macromolecular

Tumor-Nonspecific Macromolecular

Tumor-Specific Mncromoleculsr Prodrug (Gamer: Homwne, lam, Glycoprorem etc.) Tumor-Specific Mscromolecula! (Carrier An&d)

5. Action mechanism of macromolecular prodrugs 5.1. Cellular uptake mechanism

Since macromolecules normally cannot enter cells by passive diffusion across the plasma membrane, the general mechanism whereby they pass the cell membrane is endocytosis. This is a widespread processof cell surface invagination and subsequent internalization of plasma membrane as vacuoles and is associated with transport

Fig. 11. Cellular uptake and drug releasemechanismsof macromolecular prodrugs.

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prodrugs is the principle of a ‘lysosomotropic’ delivery which was advocated more than two decades ago by Trouet et al. [39] for a DNA-daunorubicin complex. Here, the complex undergoes ‘piggyback’ endocytosis which representsengulfment by an enfolding of the plasma membrane with formation of a cytoplasmic vacuole (phagosome). Following endocytosis, fusion with the lysosome enriched with hydrolytic enzymes yields the digested vacuole. Then the free drug will be regenerated within the lysosome following digestion of the carrier and diffuse into the other cellular compartments such as the nucleus. The advantage of lysosomotropic agents is the fact that the cell membrane transport of the drug molecules attached to the carrier is restricted to the endocytic route. Therefore, the intracellular concentration of the drug dependson the endocytic activity of the cells and the digestive potential of their lysosomes. Thus, lysosomotropic agents would exhibit a larger effect on tumor cells with a high endocytic activity [27,28]. This mechanism originally demonstrated for non-specific macromolecular prodrugs also can be applied to active targeting to the tumor cells employing such a processas receptor-mediated endocytosis. Based on this strategy, Trouet at al. [77] designed a macromolecular prodrug of daunorubicin with a linkage structure which is stable in serum and labile to hydrolases in the lysosome. The drug conjugated to succinylated albumin via various peptide spacer arms gave diverse activities against L1210 leukemia, depending on the lability of the spacer arm to lysosomal enzymes.The best therapeutic effects were obtained with the conjugates having tri- and tetrapeptide spacer arms which exceededthat of free daunorubicin. Based on the same concept, a seriesof studies on anthracyclines coupled to N-(2-hydroxypropyl)methacrylamide copolymers has been carried out by the group of Duncan and Kopecek [78-841. These studies also clearly demonstrated the importance of the spacer structure: that is, conjugates with the lysosomally degradable Gly-Phe-Leu-Gly side chain showed high activity while conjugates with a side chain resistant to lysosomal digestion failed to show activity. The anthracycline macromolecular prodrugs will shortly undergo clinical evaluation [lo]. Another mechanism for the intralysosomal drug releaseinvolves the low pH in the lysosomal milieu. In this approach, free active drugs are generated from the conjugates by a chemical reaction under the acidic condition. Shen and Ryser [85] developed a pH-sensitive linkage for daunorubicin which hydrolyzed spontaneously at pH levels prevalent in lysosomes.N-cis-aconitic derivative of daunomycin prepared by reacting the amino sugar moiety of the drug and cis-aconitic anhydride was conjugated to a non-biodegradable macromolecule, poly(tMysine). In vitro experiments have shown that the conjugate enters cells and reaches the lysosomal compartment where the cis-aconitil spacer is

cleaved to release the drug. The participation of a free cis-carboxylic group in the release of daunomycin was suggestedby the fact that another conjugate synthesized with maleic anhydride did not releaseactive drug in the lysosomes.Similar releasing mechanisms have been applied to a variety of macromolecular prodrugs; i.e., daunomycin or doxorubicin was covalently conjugated to polypeptide carrier [86] and monoclonal antibodies [87-911 using the acid labile linker cis-aconitic anhydride. Furthermore, other types of acid cleavable spacers have been developed for the preparation of macromolecular prodrugs of anthracyclines aimed at selective drug release in acidic endosomal vesicles and lysosomes [92-951. 5.2.1.2. Release in intracellular

space outside lyso-

Shen et al. [96] reported the intracellular release of active drug from a prodrug in compartments other than endosomesor lysosomes.While direct coupling of methotrexate to poly@lysine) yielded a conjugate devoid of cytotoxic effects because the carrier is not digested, the indirect conjugation using a triglycine spaceror a disulflde spacerresulted in strong growth inhibition against methotrexate transport-defective Chinese hamster ovary cells. Cell treatments with lysosomeinhibitors, ammonium chloride and leupeptin, prevented the effect of the conjugate with triglycine spacerbut not of that with disulfide spacer.On the other hand, preincubation with 2-mercaptoethanol abolishes the effect of the drug-disulfide conjugate, but not the effect of the drug-triglycine conjugate. From these results, the authors concluded that the reductive process of disulfide structure through which methotrexate is released occurs inside cells and requires neither acidic pH nor lysosomal enzymes. Furthermore, the drug releasewas not mediated by a glutathione-disulfide exchangereaction which requires high glutathione concentration since two cell types with two-fold difference in intracellular glutathione level responded similarly to the disullide conjugate. Although the cellular compartment in which this reductive process occurs is not yet identified, it is assumed to be prelysosomal. Similar mechanism was reported for drug release from an immune complex composed of methotrexate, trinitrophenyllabeled pOly(D-lySine), heparin, and antibody targeted to Fc receptor-bearing cells [97]. somes.

X2.2. Release in extracellular

space

In the caseof dextran conjugates of mitomycin C, the predominant mechanism of action has been shown to include drug release in the extracellular space. The physicochemical properties of the conjugates, such as the molecular weight, the electric charge, and the drug regeneration rate, can be controlled through the selection of the carrier and the coupling method of the spacer [98,99]. The conjugate liberates active mitomycin C by base-catalyzedhydrolysis, but not by an enzymatic reac-

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tion [loo]. The releaserate can be controlled by changing the length of the spacer [loll; i.e., the half lives are in the range of 11to 50 hours under physiological conditions (pH 7.4, 37°C) in vitro. Pharmacokinetic analysis of plasma concentrations of the conjugated and free forms of mitomycin C after intravenous injection of the conjugate demonstrated that in vivo releaseproceeds at a rate almost identical to that of in vitro hydrolysis [75,102]. In order to elucidate the mechanism of action of macromolecular produrgs cellular interactions and in vitro antitumor activities of mitomycin C-dextran conjugates have been studied in a cell culture system [103]. While the uptake of an anionic conjugate of mitomycin C is less than that of the parent drug, the cationic conjugate is avidly taken up by the tumor cells. The cellular uptake remains almost constant during the course of the incubation and no significant difference is observed between experiments at 4°C and at 37°C. More than 90% of the uptake amount is associated with the plasma membrane. These results indicate that the cationic conjugate is physically adsorbed on the surface of tumor cells and that internalization of the conjugate by endocytosis is likely to be negligible. In continuous drug exposure experiments, both cationic and anionic conjugates show growth inhibition essentially equal to that of free mitomycin C. In a l-h exposure experiment, however, only the cationic conjugate is more active than mitomycin C and a good correlation is observed between the cytotoxic effects and the extent of cellular association. These findings suggest that free mitomycin C generated from the conjugates in the vicinity of tumor cells or on the tumor cell surface induces the cytotoxicity. Macromolecular prodrugs endocytosed by the tumor cells also may have exhibited cytotoxicity, but contribution of this mechanism seemto be minimal because drug release is slower [loo] and mitomycin C is unstable [104] at a low pH in endosomesand lysosomes, in addition to slow internalization rate. 5.3. Mode of

action

of antibody-drug conjugates

Although numerous reports have been published on the pharmacological efficacies of antitumor agentantibody conjugates, the number of studies on the mode of their actions is limited. Ghose et al. [105] studied the in vitro cytotoxic effect on mouse hepatoma cells of Trenimon, an alkylating agent, linked to immunoglobulins. The authors suggested that, unlike the original drug, the target molecules of the conjugate are not intracellular DNA but are located on the surface of hepatoma cells based on the finding that there was no significant endocytosis of the conjugates by the cells. However, this mechanism does not seemto be applicable for other antibody-drug conjugates becausemost studies have suggestedthe involve-

217

ment of the lysosomotropic concept in the cytotoxic action of antibody-drug conjugates. Hara and co-workers have investigated the mode of action of macromolecular prodrugs of methotrexate utilizing a monoclonal antibody as a homing device [106-1091. In these studies, methotrexate was conjugated in its active ester derivative form with a murine monoclonal antibody to a mouse mammary tumor antigen [106]. The target selective cytotoxicity of the conjugate was verified by the observations that; (i) this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin conjugate; (ii) neither the monoclonal antibody nor the normal immunoglobulin was cytotoxic; and (iii) its cytotoxicity to the non-target cells was less than that to the target cells but was similar to that of the normal immunoglobulin conjugate to the target cells. In the experiments using radiolabeled conjugate in combination with an acidwash treatment which removes cell surface-bound conjugate, the authors confirmed the internalization of the conjugate after binding to cell surface antigen. Furthermore, leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of the conjugate, suggesting that lysosomal degradation is involved in its action. The samemechanism of action was proposed by this group for methotrexate-monoclonal antibody conjugate using human serum albumin as an intermediary [ 1071 or conjugates with disulfide-bondlinkage [108]. The cytotoxicity of the latter conjugate was decreased significantly by ammonium chloride which elevated lysosomal pH but was not decreasedby leupeptin. The authors concluded that the cytotoxicity of the conjugate with disulfide bond was mediated by lysosomal enzymes other than cathepsins B, H, and L. Methotrexate conjugates with anti-stage-specific embryonic antigen- 1 monoclonal antibody (IgM) prepared by Shen et al. [l lo] and with anti-melanoma antibodies prepared by Ghose and co-workers [l 1l] were also shown to include similar mechanism of action. i.e., the lysosomotropic concept. Hara et al. [109] reported an additional mechanism responsible for cytotoxic effect of the methotrexateantibody conjugate prepared with its active ester form of methotrexate. They found that the drug is conjugated not only via an amide bond but also via a less stable bond(s). In contrast to the amide bond-linked methotrexate which is cleaved by lysosomal enzymesafter endocytic internalization, a substantial portion of the drug linked by an ester or other less stable bond(s) is released from the conjugates in an extracellular spaceand enters the cells by an active transport systemfor methotrexate. In addition to these methotrexate conjugates with antibodies, Smyth et al. [ 1121showed selective cytotoxicity of a N-acetyl melphalan-monoclonal antibody conjugate and suggestedthat it enters cells by endocytosis and active molecules are released in lysosomes. On the

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other hand, Trouet et al. [ 1131reported that a conjugate constructed between daunorubicin and a well-characterized monoclonal antibody [ 1141on the basis of the lysosomotropic concept did not exhibit any activity in vitro. This antibody was shown to be an appropriate carrier for active targeting of daunorubicin basedon the specific recognition of a high molecular weight human milk fat globule membrane-associated antigen [ 1141. After specific binding to the glycoprotein present on the plasma membrane of the breast carcinoma cells, the antibody is endocytosed and gains accessto lysosomes where it is broken down. However, the conjugate was found to be inactive [ 1131and the authors concluded that the extrusion of daunorubicin molecules released from the conjugate out of the cells prevented the drug from reaching the intracellular concentration required for exhibiting cytotoxicity. In assessing the mechanism of action of drugantibody conjugates, a dual label fluorescence technique seemsto be useful for visual examination of the fate of the conjugates after binding to the cell surface antigen. Gamett and Baldwin [ 1151utilized this technique to investigate the endocytosis of a monoclonal antibody recognizing a cell surface glycoprotein antigen. They used fluorescent antibody conjugates and demonstrated incomplete endocytosis of the model antibody conjugates by the target cells within a short period. Even after four hours, some conjugates still remained bound to the cell surface, but were not endocytosed. Thus endocytosis in this system was suggested to differ from receptor-mediated endocytosis occurring via coated pits which is more rapid and complete. Starling et al. [ 1161have developed a dual fluorescence technique that allows the simultaneous visualization and quantitative assessmentof both the antibody and the drug portions of a monoclonal antibody-Vinca alkaloid conjugate, after it has bound to target cells in vitro and in vivo. The technique was utilized to examine

Table 2 Therapeutic efficacies of macromolecular

prodrugs of antitumor

Method of conjugation

Daunorubicin Albumin Dextran

Carbodiimide (oligopeptide Periodate oxidation

DNA Poly-o-lysine

Complexation Carbodiimide (cis-acotinyl acid

HPMA Polyclonal antibody Monoclonal antibody (anti-Thy

1.2)

6. Therapeutic eilkacy of macromolecular prodrugs 6.1. Anthracycline antibiotics prodrugs

The anthracycline antibiotics such as daunorubicin and doxorubicin represent one of the most important classesof antitumor drugs and are active against a variety of hematological malignancies and solid tumors in current therapy. However, their clinical efficacy is often restricted by dose-limiting cardiac toxicity and myelosuppression. Therefore, for more than two decades, anthracyclines have been widely studied as candidates for transformation into macromolecular prodrugs (Table 2). Although there is a growing body of evidence to show that anthracyclines also exert effects on the cell surface [36], their primary action is intercalation with nuclear DNA with subsequent inhibition of DNA and RNA synthesis. Basically, therapeutic effects of macromolecular prodrugs of anthracyclines also can be discussedbased on the intercalation mechanism. Most macromolecular prodrugs of daunorubicin and doxorubicin with successfultherapeutic activities appear

agents

Drug and carrier

Poly-L-aspartic

the rate of internalization of the anti-transferrin receptor monoclonal antibody-Vinca alkaloid conjugate by the targeted human lung adenocarcinoma cells. In this case, there was a loss of membrane associated fluorescenceof about 80% for both the antibody and drug components after only a 5-min incubation at 37°C indicating that a rapid endocytosis of intact conjugates occurred. However, the intracellular fate of the conjugates including drug release was not clarified. Thesefindings clearly demonstrated that the behavior of a drug-antibody conjugate at a cellular level greatly dependson the type of target cell and antigen recognized by the antibody. Consequently, attention should be paid to this point and the mechanism of action in designing drug-antibody conjugates.

Results spacer)

spacer)

Ester linkage via methylketone side chain Active ester (Oligopeptide spacer) Periodate oxidation of the drug Carbodiimide (cis-acotinyl spacer)

Enhanced in vivo antitumor activity 1771 Enhanced in vivo and in vitro antitumor activity [I 171, reduced toxicity [ 1181 Reduced cardiac toxicity [I 191 Antitumor activity based on pH-dependent drug release 1851 Enhanced in vivo antitumor activity [I201 Enhanced in vivo and in vttro antitumor activity [78-801 Enhanced in vivo antitumor activity [I211 lmmunosupression by selective cytotoxicity to T cells

1871

Y. Takakura, hi. Hashidn/Crii.

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219

Table 2 (Continued) Drug and carrier

Method of conjugation

Results

Monoclonal antibody (anti-a-fetoprotein) Monoclonal antibody (anti-a-fetoprotein) Concanavalin A Melanotropin Galactosylated albumin Maleylated albumin

Periodate oxidation (dextran intermediate) Carbodiimide and SPDP reagent (poly-tglutamic acid intermediate) Periodate oxidation of the drug Periodate oxidation of the drug Carbodiimide (oligopeptide spacer) Glutaraldehyde

Enhanced in vivo and in vitro antitumor activity [122,123] Enhanced in vivo and in vitro antitumor activity [124,125] Enhanced in vitro antitumor activity [I261 Enhanced in vitro antitumor activity [ 1271 Selective uptake by hepatoma cells [I281 Enhanced in vitro and in vivo antitumor activity [129,130]

Albumin

Glutaraldehyde

DNA DIVEMA

Complexation Reaction with maleic anhydride

HPMA

Active ester (oligopeptide spacer)

PEG-poly(aspartic acid) block copolymer

Carbodiimide

Circumvention of multidrug resistance in vitro and in vivo [131,132] Reduced cardiotoxicity [I 191 Enhanced in vivo antitumor activity and reduced toxicity [133,134] Prolonged circulation in plasma and reduced heart level [81], reduced cardiotoxicity [82] Enhanced in vivo antitumor activity [ 1351,enhanced in vivo antitumor activity by long circulation in blood

Monoclonal antibody (anti-melanoma) Monoclonal antibody (anti-carcincembryonic antigen) Monoclonal antibody (anti-epidermal growth factor receptor) Galactosylated HPMA

Carbodiimide (cis-acotinyl spacer)

Doxorubicin

[I361

Mitomycin

Carbodiimide (amino-dextran intermediate) and periodate oxidation Periodate oxidation (dextran intermediate) Active ester (oligopeptide spacer)

Preferential tumor accumulation and enhanced in vivo and in vitro antitumor activity [88,89] Tumor accumulation, enhanced in vivo antitumor activity and reduced toxicity [I371 Enhanced in vivo antitumor activity [138] Selective uptake by the liver and hepatoma cells [83]

C

Dextran

Carbodiimide (w-aminoaliphatic acid spacer) Carbodiimide (o-haroaliphatic acid spacer)

Enhanced in vivo and in vitro antitumor activity [ 1451,enhanced lymphatic transfer and prevention of lymph node metastasis [74] Prolonged plasma concentration [76], enhanced in vivo antitumor activity [99], accumulation and antitumor activity in solid tumor [25] Tumor accumulation and enhanced in vivo antitumor activity [I461 Enhanced in vivo and in vitro antitumor activity

Poly-tglutamic acid

Carbodiimide (glutaric anhydride spacer) Carbodiimide (glutaric and succinic anhydride spacer) Carbodiimide

Poly-L-aspartic acid

Carbodiimide

Monoclonal antibody (anti-colon cancer)

SPDP reagent and carbodiimide (anionic dextran intermediate)

Monoclonal antibody (anti-major histocompatibility antigen) Monoclonal antibody (anti-gastric cancer) Asialofetuin

Periodate oxidation (dextran intermediate) Periodate oxidation (dextran intermediate) Carbodiimide (succinic anhydride spacer)

Enhanced in vitro antitumor activity [I481

Albumin

Carbodiimide

Poly-L-lysine Monoclonal antibody (anti-transferrin receptor)

Carbodiimide Active ester

Enhanced in vivo and in vitro antitumor activity [l52-1541 Circumvention of drug resistance [ 155- 1571 Enhanced in vivo and in vitro antitumor activity

SPDP reagent Periodate oxidation

Antitumor effect with ionophore [ 1591 Enhanced transfer into the brain [I601

Albumin Poly-L-lysine

11471

Enhanced in vivo and in vitro antitumor activity 11471

Enhanced in vivo and in vitro antitumor activity H471

Enhanced in vitro antitumor activity 1571,enhanced tumor accumulation and in vivo antitumor activity 1561

Enhanced in vivo and in vitro antitumor activity 11491

Accumulation in the liver [I501

Methotrexate

11581

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Table 2 (Gmtinued) Drug and carrier -~

Method of conjugation

Results

Monoclonal antibody (anticarcinoembryonic antigen; IgM) Monoclonal antibody (anticarcinoembrionic antigen) Monoclonal antibody (anti-mammary cancer) Monoclonal antibody (antiosteogenic sarcoma) Concanavalin A Mannosylated albumin

Carbodiimide

Enhanced in vivo antitumor activity [I lO,l61]

Active ester and periodate oxidation (dextran intermediate) Active ester Active ester (albumin intemtediate) Carbodiimide and active ester (albumin intermediate) Carbodiimide Carbodiimide

Enhanced in vivo antitumor

Maleylated

Carbodiimide

albumin

Cytosine &o-arabinoside Dextran Poly-L-glutamic acid Polyclonal antibody Galactosylated

carboxymethyldextran

Periodate oxidation Mixed anhydride Periodate oxidation intermediate) Mixed anhydride

(dextran

activity [I621

Target selective cytotoxicity [106,109] Target selective cytotoxicity [IO71 Selective in vitro efficacy against resistant cell lines 11631 Enhanced in vitro antitumor activity [I641 Selective in vitro and in vivo antileishmanial effect in macrophages [165,166] Enhanced in vitro and in vivo antitleishmanial activity [167,168]

Immunosuppresive effect [ 1701 Enhanced in vivo and in vitro antitumor activity [I711 Enhanced in vivo and in vitro antitumor activity u721 Selective delivery to the liver parenchymal cells [58]

5-Fluorourucil Poly(malic acid) Vinyl polymer

Carbodiimide, etc. Polymerization

S-Fluorowdine Monoclonal antibody (anti-colon cancer)

Periodate oxidation intermediate)

2’-Deoxy-:i-ji’uorouridine Monoclonal antibody (anti-Ly-2. I.)

Active ester

In vitro cell-specific antitumor activity [176]

Chlorambucil Polyclonal antibody (anti-melanoma)

Complexation

Clinical therapeutic effect [I771

Melpharan HPMA

Active ester (oligopeptide

Enhanced in vivo antitumor activity [I731 Enhanced in vivo antitumor activity [174]

(poly+lysine

spacer)

Enhanced in vivo antitumor activity [I751

Enhanced in vivo antitumor activity [I781

N-acetylmelpharan Monoclonal antibody (anti-Ly-2. I and transferrin receptor)

Active ester

Vindesine Monoclonal antibody (anticarcinoembrionic antigen)

Acid hydrazide

Tumor accumulation

Complexation

Enhanced in vivo and in vitro antitumor activity

Cisplatin Carboxymethyl

dextran

Enhanced in vivo and in vitro antitumor activity

[I121 in patients [ 1791

PI,421 Poly-t+iutamic acid Polyclonal antibody

Neocarzinostatin Styrene-maleic acid anhydride copolymer Monoclonal antibody (anti-colon cancer)

Complexation Complexation and carbodiimide (carboxymethyl dextran intermediate)

Enhanced in vivo and in vitro antitumor activity [44] Enhanced in vitro antitumor activity [42]

Reaction with succinic anhydride

Enhanced in vivo antitumor activity [181,182]

SPDP reagent

Specific antitumor effect after intratumoral injection [183], enhanced in vitro and in vivo antitumor efficacy [ I841

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to belong to the category of lysosomotropic agents. In some investigations in the late 1970s [121,126,127], periodate oxidation of the sugar moiety of the drug was used for conjugation to macromolecules. However, the sites of the anthracycline molecule commonly used for macromolecular conjugation were at Cl4 position [120] and an amino group of sugar residue. The amino sugar residue has been widely used by covalently linking to the carboxyl group of the macromolecular carrier or the spacer arm. In principle, the resultant amide bond is chemically quite stable without a special neighboring group such as a. free carboxyl group in the case of cisaconityl spacer. Therefore, digestion with lysosomal enzymes is required for the release of an active drug from the conjugate. Thus, this type of prodrug is expected to exhibit its activity based on the lysosomotropic concept, i.e., the drug will be selectively broken down by enzymatic reactions in lysosomes but not by chemical hydrolysis after endocytosis in the target cells while being stable in the extracellular space.In addition, the anthracyclines are stable in acidic milieu and the optimal condition for daunorubicin and doxorubicin is around pH 4 in terms of their stability [ 1391. This also offers an advantage for the lysosomotropic mechanism. Recently, Yokoyama et al. [ 135,136] have developed micelle-forming polymer-doxorubicin conjugates (Fig. 12). Doxorubicin is directly bound to a poly(ethylene glycol)-poly(aspartic acid) block polymer by amide bond formation between the amino group of the drug and the carboxyl group of aspartic acid residue in the polymer chain, resulting in amphiphilic character. The drug-binding segment forms the hydrophobic core of the micelle while the hydrophilic segment composed of poly(ethylene glycol) surrounds this core as a hydrated outer shell. The micelle-forming conjugate with a diameter of approximately 50 nm has a long circulation time in the blood after intravenous injection [ 1361and

biodistributio". pharmaeokineties

Fig. 12. Schematic chemical structure of micelle-forming polymeric drug. (Reproduced with permission from Yokoyama M, Kwon GS, Okano T, Sakurai Y, Seto T, Kataoka K. Bioconjugate Chem 3:295-301, 1992)

221

exhibits an improved therapeutic effect against leukemia [ 1351‘and solid tumors [ 1361in mice compared with the free drug. Although the mechanism of action remains to be elucidated, the authors postulated that the conjugate may show cytotoxic activity without releasing intact doxorubicin molecules becausethe linkage between the drug and the polymer backbone is very stable [ 1351. Poly(ethylene glycol), an inert synthetic polymer, has been utilized as a modifier of proteins [140] and the surface of liposomes [141], which enable these substances to avoid rapid clearance from the systemic circulation by decreasing urinary excretion and/or reticuloendothelial uptake. The therapeutic potential of monoclonal antibodyconjugates was also reported for other anthracyclines including morpholinodoxorubicin [93] and idarubicin [142-1441, which have higher cytotoxicities than daunorubicin and doxorubicin. 6.2. Mitomycin

C prodrugs

Mitomycin C is a highly active antitumor antibiotic but its therapeutic utility has been limited primarily by severecumulative myelosuppression. Mitomycin C is a bioreductive alkylating agent that requires reductive biotransformation in order to exhibit cytotoxic activity. Hypoxic tumor cells which offer reductive environments suitable for activation, are susceptible to mitomycin C [70]. In particular, the drug is selectively toxic to hypoxic tumor cells at a low concentration. Therefore, it would be of significance from a therapeutic view point to develop macromolecular prodrugs of mitomycin C which can attain sustained release of the parent drug. The therapeutic potential of various types of macromolecular conjugates of mitomycin C has been reported (Table 2). In most cases,the aziridine group of mitomycin C is used for the conjugation; i.e., the coupling is performed to a carboxyl group of the carrier or the spacerat the laN position [loo]. The resultant imide bond is relatively unstable and the active mitomycin C molecule is spontaneously regenerated by chemical hydrolysis. Most macromolecular conjugates of mitomycin C listed in Table 2 are classified to this type of prodrug which, unlike lysosomotropic agents, do not require enzymatic reactions for activation. Instead, mitomycin C cannot be activated on the basis of the lysosomotropic mechanism since it is labile to enzymatic degradation [ 1511and also unstable under acidic conditions [104]. As discussed previously, the mechanism of action of these prodrugs involves drug releasein the extracellular spaceby chemical hydrolysis. This would be preferable in the clinical application of a prodrug since no attention should be paid to the problem of interspeciesdifferences in its activation. Clinical efficacy of mitomycin C-dextran conjugate [68,69] supported this postulation.

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6.3. Methotrexate prodrugs

Methotrexate, a folic antagonist which potently inhibits dihydrofolate reductase, has been used as the active component of macromolecular prodrugs. Table 2 summarizes pharmacological efficacies of these prodrugs. Macromolecular prodrugs of methotrexate, which continuously release the free drug, appear to be advantageous since the cytotoxic activity of methotrexate is time-dependent. In addition, methotrexate is stable in the acidic milieu of the lysosomal compartment [169], suggesting the possibility of activation on the basis of a lysosomotropic mechanism. However, a large number of methotrexate molecules should be conjugated to the carrier in order to supply enough of the drug to compete with endogenous folic acid. In the design of monoclonal antibody-drug conjugate, an inert macromolecule is occasionally used as an intermediate to increase the amount of drugs attached. A high in vitro cytotoxic effect has been reported for methotrexate-monoclonal antibody conjugates synthesized by employing albumin 11071and dextran [162] as intermediary, which carry 25-28 and 30-50 molecules of the drug, respectively. On the other hand, Persiani et al. [161] demonstrated that methotrexate conjugated to IgM antibody, at an extent of 97 molecules per one IgM molecule, shows a superior in vivo antitumor effect against solid tumor in mice. IgM is also considered to have advantages such as numbers of antigen-binding sites and functional groups for chemical fixation of drugs. Recently, Friden et al. [160] have demonstrated that brain uptake of methotrexate after intravenous injection is greatly enhanced when conjugated to a monoclonal antibody specific to the transferrin receptor. In general, the transport of hydrophilic drugs to the brain is extremely hindered by the tightly opposed capillary endothelial cells which make up the blood-brain barrier. The methotrexate-monoclonal antibody conjugate is designed to take advantage of the high density of transferrin receptors on brain endothelial cells which shuttle molecules across the blood-brain barrier. The therapeutic potential of these conjugates remains to be clarified. In addition to cytotoxic activity, methotrexate is also found to have a strong antileishmanial effect presumably by virtue of its inhibitory action against dihydrofolate reductase. Das and co-workers [ 165,166] have indicated that methotrexate coupled to mannosyl bovine serum albumin strongly inhibits the growth of Leishmania parasites inside macrophages; i.e., the conjugate is 100 times more active than free methotrexate. Moreover, the conjugate reduced the spleen parasite burden to a great extent in a murine model of experimental visceral leishmaniasis. These results indicate that the conjugate binds specifically to the mannose receptor on macrophages,and is internalized and degraded in ly-

sosomesreleasing the active drug to act on the parasites. Similar antileishmanial effects on parasitized macrophages has been reported for methotrexate conjugated with maleylated albumin, which is targeted to macrophagesvia scavengerreceptors for polyanions [ 167,168]. Pharmacological activities have also been evaluated for macromolecular prodrugs of other antimetabolites such as cytosine arabinoside, 5fluorouraci1, and 5deoxyuridine (Table 2). 6.4. Neocarzinostatin conjugates

Neocarzinostatin is a proteinous antitumor antibiotic with a molecular weight of about 12 000. It is a unique drug composed of two parts, a low molecular weight chromophore which is non-proteinous and biologically active and an apoprotein which contributes the stability of the chromophore but is irrelevant to the drug activity. The molecular mechanism of neocarzinostatin is known to be the inhibition of DNA synthesis by direct DNA strand scission. Following release from the apoprotein, the chromophore diffuses through membranesof nearby cells, causesDNA breaks, and leads to cell death. In a sense, therefore, neocarzinostatin is a natural macromolecular prodrug. Neocarzinostatin is highly cytotoxic and its therapeutic use is hampered due to non-specific toxicity to normal tissues as well as rapid urinary excretion. Maeda and co-workers [180] prepared the conjugate of neocarzinostatin with a synthetic copolymer of styrene-maleicacid anhydride, having a mean molecular weight of 1500, utilizing two free amino groups (Ala-l and Lys-20) in the apoprotein. The conjugate can be synthesized with either partially hydrolyzed or halfesterified copolymer resulting in varied physicochemical properties [ 1811.The obtained conjugate with a molecular weight of approximately 15 000 can be considered to be a macromolecular prodrug composed of an active chromophore and a polymer-modified apoprotein. The conjugate is more stable as compared with neocarzinostatin. It is soluble in neutral-alkaline aqueous solution and slightly soluble in a number of organic solvents [ 1801.The in vivo plasma half-life is more than ten times longer than that of the original drug [24], and it binds to plasma proteins. This prolonged vascular circulation resulted in accumulation in the peripheral tumor tissues and lymph nodes [24]. Increased activities against primary and metastatic tumors have been demonstrated [ 181,182]. As will be discussed later, intra-arterial administration with the lymphographic agent Lipiodol@ can produce good therapeutic results. 6.5. Combination of antibody-enzyme prodrugs

conjugates and

Recently, a great effort has been made to develop a

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novel active targeting systemcombining an tumor specific antibody-enzyme conjugate and a low molecular weight prodrug of antitumor agent [185]. As shown in Fig. 13, the strategy involves a two-step procedure in which the antibody coupled with the enzyme capable of generating active drug from a prodrug is first bound to cell-surface antigens and then a prodrug is administered. The drug releasedin the extracellular spacewould then diffuse into tumor cells. Key features of this approach are that the released drug can act not only on the antigen-positive tumor cells but also on nearby tumor cells to which the conjugate fails to bind due to insufficient antigen expression and that a number of active drug molecules that can be generated by numerous substrate turnovers with the enzyme on the cell surface. In contrast to most of antitumor drug-antibody conjugates, the antibody used in this approach should not be internalized by the cells. Senteret al, [186] have provided the first evidence that etoposide phosphate, a relatively non-cytotoxic prodrug of etoposide, combined with a monoclonal antibodyalkaline phosphatase conjugate shows antitumor effect. The conjugate was intraperitoneally administered to mice with subcutaneous tumor at 18-24 h before treatment with the prodrug. A profound antitumor response was observed in animals receiving this therapy. The activity was superior to that of free etoposide or the prodrug alone, or the combination of a non-specific antibody-enzyme conjugate and the prodrug. They also reported enhanced in vivo antitumor activities of phosphate prodrugs of mitomycin C [ 1871 and phenol mustard [188] in combination with a monoclonal antibody-alkaline phosphatase conjugate. However, the authors have proposed some questions for the use of alkaline phosphatase, a ubiquitous enzyme present in many biological tissue, which may lead to non-selective activation of the prodrug in the body. On the other hand, Bagshawe and co-workers [189] investigated the use of a bacterial enzyme, carboxypeptidase G2, for antibody-directed enzyme prodrug therapy. They observed significant increase in survival of

Fig. 13. Basic concept of selective activation of prodrugs by antibody directed enzyme.

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nude mice with palpable transplanted human tumor xenografts which are resistant to conventional chemotherapy, by the treatment with the enzyme targeted to the tumor cells by monoclonal antibodies followed by the administration of a prodrug with an interval of 72 h. Following a seriesof basic studies, pilot scale clinical trials with this strategy have been initiated by their group [190]. Very recently, the in vivo therapeutic potential of another type of site-specific prodrug activation in animal models has been reported [ 1911. Theoretically, this approach appears to have a great advantage in targeted chemotherapy. However, due to the complexity of the prodrug-enzyme systems,it is likely that many aspects of this approach should be elucidated in order to construct an optimal strategy. The therapeutic efficacy of this methodology depends on various factors such as the pharmacokinetic behaviors of the antibody-enzyme conjugate, the prodrug, and its parent drug, the interval of administration of the two compounds, the stability of the conjugate and prodrug, etc. In particular, the pharmacokinetic behavior of the conjugate is the most critical one, i.e., a specific tumoral localization of antibody-enzyme conjugate should be attained. One apparent primary problem is that of relatively slow clearance of the conjugate from the circulation. In order to achieve rapid elimination of the conjugate, Bagshawe et al. [190,192] have carried out intravenous injection of a galactosylated anti-enzyme antibody following administration of the conjugate. The galactosylated antibody bound to the conjugate will undergo a rapid uptake by the liver based on galactosereceptor mediated endocytosis resulting in a low plasma level of the conjugate. 7. Clinical applications of macromolecular prodrugs 7.1. Current status of clinical applications 7.1.I. Systemic administration Early clinical trials of antitumor drug-macromolecule complexeswere carried out for daunorubicin-DNA and doxorubicin-DNA by systemic administration [ 193, 1941. Clinical data of hundreds of leukemia patients treated with thesecomplexessuggestedthat they have an activity at least equal or superior to those of free drugs. Mounting evidence also suggeststhat the anthracyclineDNA complexesare lesscardiotoxic than corresponding free drugs. Very recently, doxorubicin conjugated to dextran has been tested in phase I clinical trials: the conjugate was administered as intravenous single dose every 21-28 days and the maximal tolerated dose was determined [195]. In addition to these conjugates with tumor nonspecific carriers, clinical applications of the conjugates with tumor specific antibodies have been reported since an early trial of Ghose et al. [ 1771on chlorambucil non-

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covalently bound to globulins. Biodistribution and tumor localization of 1311-labeledvindesine-antibody conjugate [ 1791and methotrexate-monoclonal antibody conjugate [196] after intravenous injection have been studied in patients by means of gamma camera imaging technique. Thesestudies demonstrated that the behavior of theseconjugates was similar to those of unconjugated antibodies after systemic administration. A phase I clinical trial was undertaken by Elias et al. [ 1971 for a methotrexate-monoclonal antibody conjugate in patients with non-small cell lung carcinoma. Five patients received the conjugate in escalating doses via a 4-b intravenous infusion. Mild to moderate sideeffects including fever, chills, vomiting, and diarrhea were observed and there was only one possible clinical response. Other clinical trials by systemic administration have been reported for monoclonal antibodies conjugated with doxorubicin [ 1981,mitomycin C [ 1991,and a Vinca alkaloid [200], and enzyme-antibody in combination with prodrug [ 1901. In general, only a few highly successful results have been obtained by systemic administration of antitumor drug-macromolecule conjugates. 1.2. Local administration So far, topical administration of macromolecular prodrugs of antitumor agents appears to give more promising clinical efficacy than systemic administration. Local injection therapy has been carried out using polycationic mitomycin C-dextran conjugate [68,69] which is retained in the tumor tissue for a long period after intratumoral injection based on electrostatic interaction. In patients with advanced tumors, the conjugate was injected directly into the tumor under laparotomy or under guidance by ultrasonography. Intraperitoneal injection against cancerous peritonitis and local injection into the submucosa around stomach cancer using endoscopehave been also carried out. More than 50 patients have been treated with the conjugate and objective tumor responseshave been observed in more than 50% of the patients with no serious side-effects other than temporary fever, localized pain, and mild leukopenia. Intra-arterial administration of a drug-macromolecule conjugate also seemsto be an effective method for its clinical application. Takahashi et al. [201,202] evaluated the therapeutic potential of neocarzinostatin conjugated with anti-human colon cancer monoclonal antibody. Patients with advanced colorectal cancer, including postoperative patients with liver, lung, and peritoneal metastaseswere treated with single or multiple injection of the conjugate. Intra-arterial injection was carried out mainly through a catheter inserted from the femoral or subclavian artery to the feeding artery of the tumor or intra-operatively from an artery proximal to the tumor. Some promising clinical results such as reduction in tumor size, decreasedtumor marker levels, and pain relief have been obtained without serious sideI.

effects in any of the patients given the conjugate. A recent follow-up study of 77 patients demonstrated that this modality is superior to other systemic chemotherapy resulting in a longer survival time of patients with liver metastasesof colorectal cancer [202]. Tjandra et al. [203] reported preliminary results of phaseI clinical trial of N-acetylmelphalan conjugated to three types of anticolon cancer monoclonal antibodies in advanced colorectal carcinoma patients with extensive hepatic metastases.After the selection of an appropriate monoclonal antibody based on the immunoperoxidase staining of the primary colon cancer tissue, the conjugates were administered via the hepatic artery up to a dose of 20 mg/m’ as free drug. Minor antitumor responseswere obtained in three of nine patients with no significant side-effects. Thus some successfulresults have been attained with the intra-arterial injection therapy with tumor specific antibodies. On the other hand, successful results have been obtained with tumor non-specific macromoleculedrug conjugate when combined with a special formulation. Maeda and co-workers [204] have performed clinical trials of a conjugate of neocarzinostatin with poly(styrene-co-maleic acid), designated as SMANCS. They employed SMANCS synthesized using poly(styrene-co-maleic acid) in which about 30-50% of the maleic acid is in reactive anhydride form and half of the free carboxyl groups are butylated. Aqueous SMANCS formulations have been tested in pilot studies in patients with solid tumors of the ovary, esophagus, lung, stomach, and adrenal gland, and in the brain [204]. In addition, the same group has done extensive clinical trials of SMANCS suspended in Lipiodol@ (SMANCS/Lipiodol@). Lipiodol@ , a lipid contrast medium of an ethyl ester of iodinated poppy seed oil, is used for lymphography because of selective recovery into the lymphatics and detection by X-ray (Fig. 14).

Fig. 14. Selective depositing of LipiodoP in the tumor tissue demonstrated by X-ray CT scan of a patient with hepatoma. (From Dr. Toshimitsu Konno, Department of Surgery, Kumamoto University Medical School, Japan.)

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The formulation is usually injected via the tumor feeding arteries and SMANCS rapidly leaves the bloodstream into the tumor tissue with LipiodoP and shows prolonged retention there becauseof the absence of lymphatic drainage. This formulation has been shown to be effective for both diagnosis and therapy of solid tumors where the formulation is given arterially via a catheter [205,206]. In a pilot study of primary unresectable hepatoma initiated in 1982, a reduction in tumor size was observed in about 90% of patients who have receivedan adequate amount of the conjugate. A patient receiving such treatment with no active liver cirrhosis and tumor nodules/lesion confined within one liver segment is expected to have a 90% chance of survival after treatment for at least five years [204]. The SMANCS/LipiodoP formulation was officially approved in Japan as a chemotherapeutic agent for hepatoma in October, 1993. 7.2. Future assignment A variety of problems must be circumvented before the clinical application of antitumor drug-macromolecule conjugates can be realised. One essential problem is related to their chemistry. A conjugate should have a well-defined molecular structure and should be able to be synthesized in a reproducible manner. The characteristics relating to chemical structure such as the number of drug molecules attached, the position of attachment in the carrier, and the density of electric chargesdetermine the physicochemical properties of the conjugate, and their pharmacokinetic and subsequent pharmacological and toxicological characteristics. Another important problem involves the pharmacokinetic aspect of drug-macromolecule conjugates. The fundamental fate of macromolecules and macromolecular prodrugs in animals including tumor localization has been clarified to some extent in relation to their physicochemical and biological properties. However, there is scant quantitative data on in vivo behavior of macromolecules in humans. Animal scale-up approach developedin the field of physiological pharmacokinetics may be a helpful tool [207]: that is, pharmacokinetic behavior of macromolecules in humans could be theoretically estimated from the data obtained in experimental animals in conjunction with physiological and/or biochemical parameters for humans. On the other hand, the useof someexternal scintigraphic techniques such as gamma scintigraphy, positron emission tomography, and single photon emission computed tomography will offer important information about the fate of macromolecules in patients in a quantitative and non-invasive manner. Using these approaches, the pharmacokinetics of macromolecules in man can be clarified. One of the most critical problems inherent in the use of a macromolecular carrier system is the occurrence of adverse immunological reactions which is most impor-

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tant when proteinous carriers like antibodies are employed. The majority of human clinical trials utilizing murine monoclonal antibody has faced the somewhat universal problem of production of human anti-mouse antibody in the host. For example, serum anti-mouse antibody responses were noted in all patients having monoclonal antibody conjugates of neocarzinostatin [ 1981and N-acetylmelpharan [ 1991.In the former case, serum sickness, a fever, and a rash developed in one patient who was given a second course of treatment. This problem can be overcome by the use of less immunogenic antibodies prepared by genetic engineering technologies, i.e., chimeric, humanized or totally human antibodies [ 161. Chemical modification with poly(ethylene glycol) might be an alternative way to reduce the immunogenicity of antibodies [204]. However, Johnson et al. [209] have suggestedthat drug conjugation may cause the otherwise non-immunogenic antibody to become significantly immunogenic. The authors demonstrated in a rat model that a Vinca alkaloid derivativemonoclonal antibody conjugate induced anticonjugate antibody which was directed primarily against the linker and drug moiety of the conjugate. Although it is not clear whether such modification of less immunogenic humanized proteins will render them more immunogenic in humans, care must be taken for the immunological problem. Further studies are required on the relationship between the fine chemical structure of drugmacromolecule conjugates and their immunogenicity. 8. Conclusions and perspective

This review has attempted to analyze the current status of the targeted cancer chemotherapy using macromolecular carrier systems.Undoubtedly, the concept of macromolecular prodrug with site specificity is an attractive approach [1,8]. However recent results in clinical trials does not warrant optimism in applying this novel modality of cancer chemotherapy. The key to success of macromolecular prodrugs would involve: (i) rational design of anticancer drug-macromolecule conjugates; (ii) precise understanding of physiological and anatomical characteristics of tumors, especially those of human tumors; (iii) assessmentof pharmacokinetic properties of macromolecular prodrugs including tumor localization; (iv) elucidation of mechanisms of cellular association of macromolecular prodrugs and release of active component from them; and (v) estimation of toxicity and antigenicity of macromolecular prodrugs. Thus, the development of macromolecular prodrug researchdepends on a variety of research fields such as biochemistry, immunology, cell biology, bioengineering, pharmaceutics, pharmacology and oncology. Work currently in progress in these fields should provide insight into the complex problems of macromolecular prodrugs and eventually give answers to some of the vital ques-

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tions posed here. It is hoped that the integration of multidisciplinary researchesover all the life scienceswill acceleratethe emergenceof novel macromolecular prodrugs that are commercially available in clinical cancer chemotherapy.

21 Butler TP, Grantham FH, Gullino PM. Bulk transfer of fluid in the interstitial compartment of mammary tumors. Cancer Res 35:3034-3088, 1975. 22 Song CW, Levitt SH. Quantitative study of vascular&y in Walker

carcinoma 256. Cancer Res 31:587-589, 1971. 23 O’Connor SW, Bale WF. Accessibility of circulating immuno-

Reviewer 24

This paper was reviewed by Wei-Chiang Shen, Ph.D., School of Pharmacy, USC, Los Angeles, CA, USA. 25

References 26

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Biographies Yoshinobu Takakura received a Ph.D in Pharmaceutical Sciencesfrom Kyoto University in 1987. He pursued his postdoctoral studies in the Department of Pharmaceutical Chemistry, University of Kansas, from 1989to 1990.He is currently Associate Professor in the Faculty of Pharmaceutical Sciences,Kyoto University. Mitsuru Hashida received a Ph.D in Pharmaceutical Sciences,from Kyoto University in 1979. From 1979to 1980he pursued his postdoctoral studies in the Department of Pharmaceutical Chemistry, University of Kansas. He is currently Professor in the Faculty of Pharmaceutical Sciences,Kyoto University.