The potential of water-soluble polymeric carriers in targeted and site-specific drug delivery

J~)urnal of'Controlled Release, 11 (1990) 279-290

279

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

THE POTENTIAL OF WATER-SOLUBLE POLYMERIC CARRIERS IN TARGETED A N D SITE-SPECIFIC DRUG DELIVERY* Jindrich Kope6ek Center for Control/ed Chemica/De/ivery and Departments of Pharmaceutics and Bioengineering, University of Utah, 421 Wakara Way, Sa/t Lake City, UT (U.S.A.) Key words: N-(2-hydroxypropyl) methacrylamide copolymers; targetable polymeric drug carriers; photodynamic therapy;

bioadhesive polymers; site-specific 5-aminosalicylic acid delivery

The design of targetable water-soluble polymeric drug carriers based on N-(2-hydroxypropyOmethacrylamide (HPMA) copolymers is described. Three types of conjugates have been studied: (a) H P M A copolymers containing oligopeptide side-chains terminated in anticancer drugs (daunomycin, adriamycin); (b) conjugates of H P M A copolymers with chlorin e6 (photoactivatable drug); and (c) H P M A copolymers for site-specific oral delivery of 5-aminosalicylic acid (5-ASA). Polymer-bound drugs (a) and (b) are lysosomotropic. When conjugated to a targeting moiety (galactosamine or antibody) their concentration in the target tissue is increased. It was shown that binding drugs to H P M A copolymers decreases their toxicity and immunogenicity. Two ways of drug activation in the lysosomal compartment are discussed: release by lysosomal cysteine proteinases (daunomycin, adriamycin) and activation by light (chlorin eJ. Conjugates of chlorin e~ with H P M A copolymers and optionally galactosamine were synthesized and their photodynamic activity was tested in vitro on a human hepatoma cell line (PLC/PRF/5 cells). The conjugate containing galactosamine as the targeting moiety was more active compared to the conjugate without the targeting moiety. A new concept of oral drug delivery was proposed based on a combination of site-specific delivery of 5-ASA to the colon with bioadhesive properties of the carrier. H P M A copolymers containing saccharide units complementary to mucosal lectins of the GI tract are used as carriers. They also contain side-chains terminated in salicylic acid bound via an azo bond. Cleavage experiments were carried out using an isolated strain of bacteria commonly found in the colon. When incubated with Streptococcum faecium in vitro 5-ASA is released. Body distributions in guinea pigs after oral administration have shown that H P M A copolymers containing fucosylamine associate with the colon. Further experiments are necessary to verify the therapeutical importance of these observations and to develop water soluble polymers as carriers for site-specific oral delivery.

INTRODUCTION The potential of water-soluble synthetic *Paper presented at the Fourth International Symposium on Recent Advancesin Drug DeliverySystems, Salt Lake City, UT, U.S.A., February 21-24, 1989.

0168-3659/90/$03.50

polymers used as drug carriers is well established [1-3 ]. Their main advantage compared with other drug delivery systems is their targetability. Soluble macromolecules are handled by the body quite differently t h a n particulate carriers [4 ]. Polymer molecules can pass into m a n y

© 1990 Elsevier Science Publishers B.V.

280 organs by transport across the capillary endothelium. The fact that the uptake of soluble macromolecules into the cells is by pinocytosis and not phagocytosis is very important. This is the main basis of their targeting potential. The only limitation of soluble synthetic macromolecules may be their non-biodegradability. When a non-degradable macromolecule is released upon cell death, its molecular weight distribution must be under the renal threshold to be eliminated from the body. To manipulate the intravascular half-life of macromolecules it is possible to connect short synthetic polymeric chains via oligopeptide crosslinks to high-molecular weight (water-soluble) derivatives [ 1 ]. These crosslinks are cleaved by lysosomal enzymes in secondary lysosomes. Short non-degradable fragments of the carrier are eliminated:from the blood stream by glomerular filtration. Synthetic water-soluble copolymers also have the potential for use in oral drug delivery. Polymeric prodrugs may be synthesized in which the bond between the drug and the carrier macromolecule matches the specificity of enzymes present in a specific region of the gastrointestinal tract. Instead of targeting moieties, bioadhesive moieties are attached to the carrier. In this report our recent results in the use of soluble macromolecules in targeted lysosomotropic drug delivery and in site-specific oral delivery are discussed.

INTRACELLULAR DELIVERY Polymer-drug conjugate pharmacokinetics is certainly very different from that observed for free drug. Low molecular weight pharmaceuticals can penetrate the cell membrane by diffusion. Attaching them to water-soluble macromolecules limits their cellular capture to the pinocytic route. The first intracellular compartment that pinocytized materials enter is calledthe endosome or receptosome [5,6]. This is an intracellular organelle with an acidic in-

terior which probably contains little enzymatic activity. In the next step, the uncoupling of the ligand and receptor may occur. Then the receptor is recycled to the cell surface, and the ligand is routed to its intracellular location. In most cell types it appears that macromolecules that are internalized are transferred to the lysosomal compartment of the cell. Thus, binding drugs to soluble macromolecules renders them lysosomotropic. Targeting can be achieved by conjugating the polymer-bound drug to a targeting moiety having a structure complementary to cell surface antigens or receptors. We have evaluated two independent methods of drug activation in secondary lysosomes; activation by enzymes and by light. Release of anticancer drugs by lysosomal enzymes We have been developing targetable anticancer polymeric drug carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers for several years [1-3,7]. These copolymers contain oligopeptide side-chains terminated in drug. The structures of oligopeptide side-chains are tailor-made to be stable in the blood stream, but susceptible to hydrolysis in secondary lysosomes catalyzed by cysteine (thiol) proteinases. The rate of enzymatically catalyzed hydrolysis depends both on sterical factors {sterical hindrance caused by the polymeric chain on the formation of the enzymesubstrate complex) and on the length and detailed structure of the oligopeptide sequence. It was shown that cysteine proteinases are responsible for the cleavage of H P M A copolymer-drug conjugates. Subsequently, copolymers were tailor made to match the specificity of individual cysteine proteinases, cathepsin B [8], cathepsin H [9], and cathepsin L [9], and the structure-degradability relationship was determined. The stability of these copolymers in blood plasma and serum was also determined [10]. Based on these results it was possible to

281

control the degradability of H P M A copolymers in vivo. These copolymers were modified by attaching targeting moieties such as monosaccharides [11,12] and antibodies [13,14]. It was shown that H P M A copolymers modified with galactosamine interact with asialoglycoprotein receptors on hepatocytes. Subcellular fractionation of the liver cells after administration of galactosamine-containing copolymers to rats [15] confirmed that they were internalized by hepatocytes and that radiolabeled model drug was liberated intralysosomally and passed out of the lysosome across its membrane. Similarly, H P M A copolymers were modified with fucosylamine [16-18] and it was shown that they interact with the receptor on L1210 cells in vitro and in vivo. Subsequent experiments were performed to test the pharmacological activity of anticancer drugs (daunomycin, adriamycin) attached to H P M A copolymers containing fucosylamine against L1210 leukemia in DBA2 mice. Two localizations of tumor were studied: intraperitoneal and subcutaneous. In both cases experimental animals were treated intraperitoneally with free drug or drugH P M A copolymer conjugates. The latter have shown therapeutic effect only when the oligopeptide sequence between the drug and the polymeric carrier was biodegradable (Gly-PheLeu-Gly). Polymer-bound drugs produced increased life span and an increased number of long-term survivors [2,17] depending on the structure of the conjugate, timing of administration and the number of doses [2,17].

Biological properties of HPMA copolymeranthracycline conjugates Pharmacokinetics of HPMA copolymerbound adriamycin. H P M A copolymer-adriamycin conjugates show a therapeutic effect in rat and mouse models much greater than that seen for the same dose of free adriamycin [2,17,18]. This is true despite the fact that a part of polymer-bound

adriamycin is excreted from the body rapidly [19], whereas the free drug has a half-life of about 18 hours [20]. Conjugation of adriamycin to HPMA copolymers eliminated the high initial level of free drug detected in plasma following the administration in free form. The circulating half-life of polymer-bound adriamycin was about fifteen times higher compared with free drug. When the conjugate was applied, a decreased concentration of adriamycin in the heart was also observed. There are at least two plausible explanations [19] for this increased therapeutic activity: (a) Prolonged circulation of the polymer-adriamycin complex acts as a slow-release depot whose release kinetics are particularly well suited to the eradication of the animal tumor models studied so far; (b) the physico-chemical properties of the H P M A copolymer conjugates may cause enhanced deposition in tumor endothelium, perhaps as a result of especially wide interendothelial cell junctions [21 ]. Finally, there are occasional reports in the literature that tumor cells in vivo have exceptionally high rates of endocytosis [22] and therefore the prolonged plasma circulation time of the adriamycin-HPMA copolymer conjugate may give rise to increased pinocytic accumulation by tumor cells.

Treatment of solid tumor (Walker 256) with HPMA copolymer-daunomycin conjugate To verify the possibility of using HPMAbound anticancer drugs to treat a solid tumor, preliminary in vivo experiments on Wistar rats bearing subcutaneous Walker 256 tumor were performed [23,24]. Five animals in four groups were given intravenously: (1) saline as control; (2) 5 mg/kg of free daunomycin; (3) 5 mg/kg of daunomycin bound to an HPMA copolymer via a biodegradable oligopeptide sequence (GlyPhe-Leu-Gly); (4) 5 m g / k g of daunomycin bound to an H P M A copolymer via a non-degradable sequence (Gly-Gly). The injections were performed the same day as the subcutaneous implantation of 0.5 cm 3 of Walker tumor.

282 Subsequent tumor growth was measured with calipers every 2-3 days. The only group showing a statistically significant growth delay was the group (3). In fact 4/ 5 animals in this group were long-term survivors ( > 30 days) with no evidence of tumor. Only one other animal from any group survived and this was in the free daunomycin group. These results are consistent with the observation that the polymer-bound daunomycin was found in the tumor in higher concentrations than the free drug at all time points. There was also a reduction in the cardiac concentration of daunomycin with H P M A copolymer-bound preparations, suggesting an improved therapeutic index in this model system [24]. The biological reason for selective delivery of H P M A copolymer-bound daunomycin to solid tumor is unclear. It is possible [ 24 ] that tumor cells have increased endocytic activity resulting in enhanced uptake of macromolecules [25 ]. Also it has been suggested t h a t Walker 256 tumor contains fenestrated capillaries [ 20 ] which may allow preferential transport of the conjugate into the tumor vascular bed.

Body distribution and T-cell accumulation of free [12Sl]daunomyci n and [12Sl]dauno. mycin bound to an HPMA copolymer-antiThy 1.2 antibodies conjugate [26] Coupling [ 12~I] daunomycin to H P M A copoly m e r - a n t i - T h y 1.2 antibody conjugates considerably alters its body distribution [26].

[12~I]Daunomycin bound to H P M A copolymers is detectable in the blood and other tissues in higher concentrations for a prolonged period of time. An increased concentration of the targetable conjugate in the target tissues, spleen and thymus, was observed [26]. However, the increase is not dramatic (Table 1 ). In spite of only the 2-4 times increased concentration in the target tissues, compared with the free drug, a nontargetable conjugate or a conjugate with a nonspecific rabbit gamma globulin [26], the targetable conjugate, significantly improves the therapeutic efficacy [ 14 ].

Decreased toxicity The aim in using targetable polymer-drug conjugates is not only to accumulate the drug at the site of the pathological process, but also to minimize its toxic effect on normal tissue [27]. To evaluate the difference in toxicity of free and polymer-bound daunomycin we have studied the depletion of hematopoietic stem cells from bone marrow [ 14 ]. It was found that free daunomycin is highly toxic and t h a t a dose of 6-24 m g / k g divided into three consecutive IP injections induces 70% depletion of bone marrow precursors. The toxicity of H P M A copolymer-daunomycin conjugate is considerably lower. Only 13% of stem cells were depleted in the latter case (same concentration of drug). These results correlate well with the histological examinations of the thymus, liver,

TABLE 1 Body distribution of free and polymer-bound [125I]daunomycin2 hours after intravenous administration into C57L/J mice [26] Substrate [lz~I]DNM pz G-F-L-G- [125I]DNM \G-F-L-G-anti-Thy 1.2

Blood

Liver

Spleen

Thymus

5.9 _+1.9

2.9 _+0.7

0.16 _+0.09

0.13 _+0.05

40.1__9.1

10.4+2.7

0.5 +0.1

0.29_+0.1

Results represent % of injected radioactivity (37 kBq per mouse) per wholeorgan; 5 mice per group. P, HPMA copolymer backbone; G, glycine;F, phenylalanine;L, leucine;DNM, daunomycin.

283 spleen, heart and kidney [14]. Both free daunomycin and daunomycin bound to an HPMA copolymer-anti-Thy 1.2 antibody conjugate (via degradable oligopeptide sequences) induce a pronounced depletion of thymus lymphocytes. In liver a pronounced effect on Kupffer cells was visible only when free daunomycin was administered. Polymer-bound daunomycin was without pathological effect. The main problem associated with the use of anthracycline antibiotics is their cumulative cardiotoxicity. Free daunomycin did produce changes in heart parenchyma detectable by electron microscopy, whereas polymer bound daunomycin was without detectable effect [28].

Immunogenicity Adriamycin bound to HPMA copolymers does not behave as a hapten [29]. The immunogenicity of antibodies (generally proteins) bound to HPMA copolymers is also reduced [30]. Human IgG and human transferrin were conjugated to HPMA copolymers. The antibody titres elicited, after SC or IP administration to A/J and B10 mice were measured using the ELISA technique. The measured IgG titer against protein-HPMA copolymer conjugates was always higher than the IgM titer. The titer (IgG) measured against native protein was up to 250 fold greater than that raised against protein-HPMA copolymer conjugates. These results indicate that repeated administration of IgG conjugated to HPMA copolymers may not result in an immediate immune response to the conjugate since the majority of memory cells are specific for unconjugated protein. To determine the mechanism of reduction in protein immunogenicity further experiments are necessary. Work is underway [31 ] to determine if the HPMA copolymer is simply masking antigenic determinants and therefore making them unavailable to the immune system [32-35 ] or if the presence of HPMA copolymer is suppressing the immune system by specific interaction with either T-helper, T-suppressor, or B-cells as described by Sehon [36].

Activation of polymer-bound drugs by light When using photosensitizers (as anticancer drugs) bound to lysosomotropic polymeric carriers there is no need to release them from the carrier by the action of lysosomal enzymes. Photosensitizers (porphyrins, phthalocyanines, purpurins, chlorins, naphthalocyanines, cationic dyes, tetracyclines, etc.) are molecules that are activated with light of a characteristic wavelength, ultimately resulting in the formation of singlet oxygen, a highly reactive species [37]. The mechanism of photosensitized reactions has been exploited for use in cancer therapy. Photodynamic therapy (PDT) is the term coined for using a photosensitizer plus light in the destruction of cancer cells. An advantage of using therapy of this type is that the photosensitizer remains inert until it is activated with light. The main advantage of using photosensitizers bound to targetable polymeric carriers is the potential for double targeting. No targeting system works adequately. Usually the concentration of the drug is increased in the target tissue, but a part of the administered dose still reaches other tissues. Photosensitizers are not active without light. Irradiation of only the target tissue activates just the drug which reached its target [38]. We have synthesized [39] HPMA copolymer-galactosamine-chlorin e6 (photosensitizer) and HPMA copolymer-chlorin e6 conjugates and studied their biological properties. The structures of these conjugates are shown in Fig. 1. The photodynamic activity of these conjugates was tested in vitro on a human hepatoma cell line: P L C / P R F / 5 (Alexander cells). These cells contain the asialoglycoprotein receptor [40 ]. The biological activity of the conjugates and proper controls was evaluated [39] in 96-well culture plates seeded with 105 cells per well in minimal essential medium (MEM). After one hour incubation at 37°C (5% CO2) the cells were centrifuged and washed twice with cold

284

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Fig. 1. Structure of HPMA copolymer-chlorin e~ conjugates.

PBS. Next, 100/A of MEM with 10% of bovine calf serum were added to each well. The plates were irradiated in a C02 environment at 37 °C for 7.5 or 24 hours. An Enmat slide projector functioned as the light source, it was equipped with a water layer filter (to absorb heat) and a red filter to provide light at a maximum of 660 nm. After irradiation the plates were incubated for 18 hours (5% C02; 37°C) to be certain that any photodynamic damage to the cells was irreversible. Simultaneously, control plates were kept in the dark. The MTT colorimetric assay was used to measure cell viability [41 ]. Typical results are summarized in Table 2. Free chlorin e6 is toxic even when used in dark, whereas polymer-bound chlorin e6 is toxic only

when exposed to light. The reason could be either a higher rate of diffusion of free drug into the cell interior compared with the rate of pinocytic uptake of polymer-bound drug, or the difference in hydrophobicity. Chlorin e6 is a hydrophobic molecule which easily associates with the cell membrane, probably resulting in the alteration of cell functions. Binding chlorin to a hydrophilic copolymer may mask the hydrophobic effect of the former. It appears that the targetable conjugate is more biologically active compared with the nontargetable one (Table 2). The results suggest that the targetable (galactosamine containing) conjugate interacts with the asialoglycoprotein receptor present on the surface of

285 TABLE2 Photodynamic activity of free and HPMA copolymer-bound chlorin e6 towards a human hepatoma cell line P L C / P R F / 5 in vitro [39] Drug

Conc. of drug (#g/ml)

A~7o Irradiated

Non-irradiated

P-chlorin + free galactosamine

13 1.3 64 13 64 13

1.32 0.06 0.12 0.14 1.38 0.10 1.13

1.35 0.08 0.40 1.37 1.32 1.35 1.21

chlorin P\galactosamine

64 13

0.08 0.35

1.40 1.28

Control Chlorin P-chlorin

The results are expressed as absorbancy (A) at 570 nm, which is proportional to the number of live cells. The values shown are the average value from 3-6 experiments. Conditions of experiment: 1 hour incubation at 37 ° C, 7.5 hours of irradiation, 2 day culture [39]. P, HPMA copolymer backbone.

the PLC cells. The results are consistent with the hypothesis that after irradiation the singlet oxygen produced damages the lysosomal membrane, resulting in the release of lysosomal enzymes into the cytoplasm and ultimately cell death. It is of interest to use other targeting moieties and to study the targeting of chlorin e6 in vivo. Conjugates with anti-Thy 1.2 antibodies have been synthesized and their biological evaluation is under way [42 ].

SITE-SPECIFIC ORAL DRUG DELIVERY We are investigating the feasibility of a new concept in oral drug delivery based on a combination ofbioadhesive properties of water-soluble polymeric carriers with site-specific drug delivery. N- (2-hydroxypropyl)methacrylamide copolymers containing sugar moieties complementary to mucous lectins of the gastrointestinal tract are used as carriers. Such copolymers should have better access to the adhesion sites and permit localized release. Depending on the detailed structure of the carrier (content of

bioadhesive moieties and molecular weight) the residence time in the GI tract can be manipulated. As a model drug we are using sulfasalazine, 5- (p- [2-pyridylsulfamoyl]-phenylazo) salicylic acid. It is a standard drug for treatment of ulcerative colitis. It is poorly absorbed from the small intestine, so that it is passed into the colon, where bacterial enzymes release both 5aminosalicylic acid (5-ASA) and sulfapyridine. The former is responsible for the therapeutic effect. Sulfapyridine is also absorbed from the colon and is responsible for the majority of side effects associated with the therapy. Thus, if 5ASA was to be linked to a bioadhesive carrier by an azo bond, the problems with the sulfapyridine could be avoided. Moreover, the bioadhesive properties of the polymeric carrier could improve the therapeutic effect. There have already been several studies where linear and crosslinked polymers containing azo bonds were used to deliver drugs to the colon [43-47]. These studies have shown that bacterial enzymes can cleave an azo bond attached to a polymeric chain. The rationale of bioadhesion is based on our

286

previous results on the bioadhesion of sugar containing H P M A copolymers in the GI tract of Wistar rats [48-50] and on the observation that some pathogenic microorganisms bind to the mucosa. For example, in guinea pig, Shigella flexneri bacteria avidly adhere to colonic mucosal cells. Their binding capacity was shown to reside in the host cells and be fucose and glucose specific [51]. A similar observation has been made in vivo and in vitro for E. coli 0/24 [52]. Thus, there was enough evidence supporting both main assumptions of our concept: bioadhesion and controlled release.

--

To verify the feasibility of our concept copolymers of the structure shown in Fig. 2 were synthesized [53] and both their bioadhesive properties in guinea pigs and the release of 5-ASA were studied. Release of 5-ASA from HPMA copolymers Cleavage experiments were carried out [53] using Streptococcum faecium (ATCC-19434), a strain of bacteria commonly found in the colon. An experimental procedure described previously [44 ] was used. The release of [ 14C] 5-ASA

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NH

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287

was determined using a scintillation counter after separating the copolymer and the low molecular weight products on a Sephadex G-25 column. The rate of release appeared to be a function of bacterial and substrate concentration. The presence of benzylviologen (redox mediator) increases the rate of 5-ASA release [53].

the bioadhesive copolymer 2 in the feces. This suggests that copolymer 2 is selectively retained in the colon. After 48 hours, however, the amount of both copolymers associated with the colon decreased, but there was still a higher amount of copolymer 2 present. The decrease of radioactivity in colon between 24 and 48 hours is probably due to rapid renewal of the mucous layer.

Bioadhesion

We have studied the body distribution of H P M A copolymers containing bioadhesive moieties in guinea pigs 1, 5, 24, and 48 hours after oral administration [54 ]. Typical results are shown in Fig. 3. Two copolymers were compared: copolymer 1, HPMA without bioadhesive moieties; and copolymer 2, fucosylamine-containing H P M A copolymer. Both copolymers contained about 1 mol% of methacryloyltyrosinamide units to permit radioiodination [55]. There is a clear difference in the distribution of the two copolymers studied. The association of copolymers with the colon was time dependent. 24 hours after oral administration the amount of copolymer 2 was two times higher compared with copolymer 1 (without bioadhesive moieties). Correspondingly, we found [54] a lower level of 20

o

,2

lo

n..

o

CONCLUSIONS N- ( 2-hydroxypropyl ) methacrylamide copolymers have a potential in targeted and sitespecific delivery. Although their potential as lysosomotropic carriers of anticancer drugs is well established, much more work is necessary to develop them as bioadhesive carriers for sitespecific oral delivery. However, the results obtained so far are encouraging and it is probable that they will also be suitable in this area of delivery. ACKNOWLEDGEMENTS The work reported herein was performed in cooperation with Drs. K. Ulbrich, B. l ~ o v e l and coworkers (Czechoslovak Academy of Sciences), Drs. R. Duncan, L.W. Seymour, J.F. Woodley, P.A. Flanagan and coworkers (University of Keele, U.K. ) and Dr. P. Kope6kov~i, Dr. L. Fornfisek, N.L. Krinick and Y.A. Grim (University of Utah). This study was supported in part by TheraTech, Inc., and by NIH grant DK 39544. REFERENCES

10

20

30

40

50

Time (h)

Fig. 3. Amount of radioactivity associated with the colon of guinea pigs (n--5) after administration of 125I-labeled HPMA copolymer solutions in the stomach [54]. ([]) copolymer 1; HPMA copolymer containing 1 mol% of methacryloyltyrosinamide units; ( . ) copolymer 2; HPMA copolymer containing 1 mol% of methacryloyltyrosinamide units and approximately 3 mol% of side-chains terminated in fucosylamine.

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