Macromolecular prodrugs for use in targeted cancer chemotherapy: melphalan covalently coupled to N- (2-hydroxypropyl) methacrylamide copolymers

Journal of Controlled Release, 16 ( 199 1) 12 1- 136 0 1991 Elsevier Science Publishers B.V. 016%3659/91/$03.50 ADONIS0168365991000506

121

COREL 00588

Macromolecular prodrugs for use in targeted cancer chemotherapy: melphalan covalently coupled to N- ( 2-hydroxypropyl ) methacrylamide copolymers Ruth Duncan’, Isabella C. Hume’ , Harold J. Yardley ‘, Pauline A. Flanagan Karel Ulbrich2, Vladimir Subr2 and Jiri Strohalm2

‘,

‘CancerResearch Campaign’s Polymer Controlled Drug Delivery Group, Universityof Keele, Keele, Staffordshire, U.K. and 21nstituteof Macromolecular Chemistry, CzechoslovakAcademy of Sciences, Prague, Czechoslovakia (Received May 15, 1990; accepted in revised form November

16, 1990)

Although there are approximately thirty antitumour agents in clinical use recent progress has been relatively slow in the identification of novel compounds with improved therapeutic index. Macromolecular drug-carriers afford the potential to permit controlled release and site-specific delivery of those antitumour agents whose activity may be limited by their inherent toxicity and/or lack of tumourspecific deposition. In this study iV-(2-hydroxypropyl )methacrylamide (HPMA) copolymers were synthesised to contain melphalan (ME) or sarcolysin (SE) ( 1 - 10 wt%) linked to the polymer backbone (P ) via peptide side-chains e.g. P-Gly-ME, P-Gly-Gly-ME, P-Gly-Leu-Gly-ME, P-Gly-Phe-GlyME and P-Gly-Phe-Leu-Gly-ME. ME release during incubation with isolated rat liver lysosomal enzymes was dependent on the amino acid composition of the side-chain used. Cytotoxicity measured in vitro towards Walker sarcoma was dependent on the peptide spacer and ME content of the polymer, and likewise those conjugates which showed the fastest rates of cleavage by lysosomal enzymes displayed greatest antitumour activity against Walker sarcoma in vivo. Administration (i.p.) of biodegradable polymer-SE conjugates on day 1 (5 mg/kg) after inoculation of lo6 Walker cells ( S.C. ) reduced or prevented establishment of tumours. Similarly, treatment of established Walker sarcoma (approximately 200 mm* in area) with polymer conjugate of SE or ME caused a reduction in tumour size. However, it was found that free drug at equivalent dose was also effective against Walker sarcoma in vivo and the therapeutic index of polymer conjugated drug was not appreciably greater than that of the free drug and was dependent on the dosing regime. The pharmacokinetics of [ 3H]melphalan and ‘251-labelledHPMA copolymer-ME was followed in animals bearing established Walker tumours. Macromolecular conjugate delivered substantially more radioactivity to the tumour than free drug. Although this altered pharmacokinetics must contribute to tumour specific drug activity, the precise mechanism of action of polymer-bound melphalan remains unclear. Preliminary experiments showed Correspondence to: R. Duncan, CRC Polymer Controlled Drug Delivery Group, Dept. of Biological Sciences, University of Keele, Keele, Staffordshire ST5 5BG, U.K. Abbreviations: ME: melphalan; SE: sarcolysin; P: polymer backbone; s.c.: subcutaneously; i.p.: intraperitoneally; M,: weight average molecular weight; M,: number average molecular weight; GPC: gel permeation chromatography; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

122

a significant increase in the number of macrophages and T and B lymphocytes found in turnouts taken from animals 4 days after administration of polymer conjugate when compared to those from untreated or melphalan treated animals. Keywords: Polymeric drug carriers; Targeting; hydroxypropyl)methacrylamide

Introduction Overall, cancer chemotherapy has progressed dramatically over the last 40 years, but the past decade has seen a slowing in the appearance of new and more effective therapies. Of the thirty antitumour agents used in the clinics (including antimetabolites and alkylating agents) most display poor specificity in their action leading to dose-limiting toxic side-effects [ 11. Covalent conjugation of cytotoxic antitumour agents to macromolecular carriers affords the potential to control their rate of release in the body and to target them, either actively or passively, to their desired site of action - the tumour itself or an organ bearing tumour deposits [ 21. Previously we have shown that N- (2-hydroxypropyl)methacrylamide (HPMA) copolymers can be synthesised to contain anthracycline antibiotics (doxorubicin [ 3 1, daunorubicin [ 41) and alkylating agents (sarcolysin [ 51). Introduction of peptidyl spacers of specific amino acid composition restricts drug liberation to the lysosomal compartment of the cell following pinocytic internalisation of the conjugate [ 6,7]. The resultant change in pharmacokinetics of such macromolecular prodrugs can lead to a significant increase in drug delivered to solid tumours in vivo [ 81. HPMA copolymer - doxorubicin conjugates show a substantially higher therapeutic index than free doxorubicin and they display antitumour activity against a number of model tumours in vivo (L1210 [3], P388 [9], B16 melanoma [ lo] and Walker sarcoma [ 111). Such doxorubicin containing conjugates await clinical evaluation. Melphalan (known also as Alkeran@, L-phenylalanine mustard, L-PAM) (ME) and sarcoly-

Macromolecular

prodrug;

Melphalan;

N- (2-

sin (D,L-phenylalanine mustard) (SE) are alkylating agents which can be administered orally or systemically to treat a variety of cancers including multiple myeloma, ovarian cancer and, by limb perfusion, primary melanoma [ 12 1. Unlike the anthracycline antibiotics, melphalan is relatively unstable in physiological media, due to rapid hydrolysis of the chloroethyl groups, and it has a short plasma half-life of approximately 2 h. In this study, HPMA copolymer conjugates containing either the isopropyl ester derivatives of melphalan or of sarcolysin (to avoid confusion it should be stressed that these are essentially the same compound) were synthesised and their structures and chemical characteristics are shown in Fig. 1 and Table 1). The rate of drug liberation from different peptidyl side-chains was measured in vitro during incubation with isolated lysosomal enzymes or the purified thiol-dependent enzymes cathepsin B (lysosomal) and papain (model enzyme). Subsequently toxicity and antitumour activity of free drug, its isopropyl ester derivative (used for polymer conjugation) and the polymer conjugates was measured in vitro and in vivo using the Walker sarcoma as a model tumour. To monitor pharmacokinetics [ 3H ] melphalan or ‘251-labelled polymer conjugate was administered intravenously to animals bearing established Walker tumours and the fate of radioactivity monitored over the subsequent 48 h. It is known that low doses of alkylating agents such as cyclophosphamide and melphalan can act as immunopotentiators [ 131, therefore in preliminary experiments we examined tumours following treatment with free and polymer-bound ME and using fluorescent labelled antibodies quantified the numbers of macrophages and T and B lymphocytes within the tumour mass.

123

CHz-CQ--Cl

c-o

I

0

I

A

CH, CH. Fig 1. Structure of HPMA copolymers containing metphalan isopropylester or sarcolysin isopropylester. The monomers shown are; x, N- (2-hydroxypropyl)methacrylamide; y, drug bound via a peptidyl side-chain; z, methacryloylated tyrosinamide approximately 1 mol%.

TABLE 1 Characterisation of HPMA copolymers containing melphalan or sarcolysin Code no.

1 2 3 4 5 6 7 8 9 10 11 12 13 “M, and MJM,

Structure

P-GIy-ME P-Gly-Gly-ME P-Gly-Phe-Gly-ME P-Gly-Leu-Gly-ME P-Gly-Phe-Leu-Gly-ME P-Gly-SE P-Gly-Phe-Gly-SE P-Gly-Leu-Gly-SE P-Gly-Phe-Leu-Gly-SE PqGly-Phe-Gly-ME TyrNH* PqGly-Phe-Gly-ME TyrNHz P;\Gly-Phe-Giy-ME TyrNH, PqGly-Phe-Gly-ME TyrNH,

Content of drug/TyrNHz

MWa

MwM,’

Mel%

wt’%,

5.6 5.4 6.1 5.3 3.7 4.5 5.4 3.6 5.8 0.6 -1.0 1.5 -1.0 2.4 -1.0 5.1 u 1.0

10.0 11.5 12.0 10.8 7.6 9.9 10.6 7.3 11.9 1.4

21,000 22,700 19,200 20,000 20,100 22,000 21,000 22,000 20,000 40,000

1.4 1.4 1.4 1.5 1.4 1.4 1.4 1.4 1.4 1.8

3.5

28,000

1.8

5.3

23,000

1.5

10.4

18,000

1.4

of the aminolysed polymer precursor.

124

Materials and methods Chemicals 1-Aminopropan-2-01, methacryloylchloride, glycylglycine, dimethylsulphoxide (DMSO ), papain (3 U/mg) and 4nitrophenol were from Fluka AG, Buchs, Switzerland. Glycylphenylalanine, leucylglycine, tyrosinamide, collagenase type V-5, hyaluronidase Type VI-5 and DNAase were from Sigma Chemical Co., Poole, Dorset, U.K. [ ‘25I]Iodide and [ 3H]melphalan were from Amersham International, U.K. and melphalan was from The Wellcome Foundation Ltd, Dartford, England. Antibodies MAS 258c, RMAS 010~ and MAS 369 were from Sera Lab, Sussex, U.K. Preparation and characterisation of HPMA copolymers containing melphalan

Monomers were prepared by procedures previously described: HPMA [ 14 1, methacryloyltyrosinamide [ 15 1, methacryloylated peptidyl p nitrophenyl esters [ 16 1. Polymer precursors were then prepared by copolymerisation of HPMA with the appropriate methacryloylated monomers. Polymerisation was carried out in acetone (precipitation polymerisation ) at 50’ C, AIBN as initiator (0.6 wt%) and the concentration of monomers in solution was 12 wt%. The polymerisation mixture was placed in ampoules, bubbled through with nitrogen (5 min), then sealed and left to polymerise for 24 h. After this time the precipitated product was isolated by liltration, decanted several times in acetone and dried under vacuum. The polymerisation yield was 50-70%. After aminolysis with aminopropanol the weight average molecular weight (M,) and polydispersity (MJM,) were determined using gel permeation chromatography (GPC ). GPC was carried out using a mixture of Sepharose 4B and 6B ( 1: 1) packed in a column height 100 cmxintemal diameter 1.6 cm with TRIS buffer (pH 8.0) as the eluent, (flow rate 11 ml/ h ). M, and M, were calculated from the relationship between the distribution curves obtained and a calibration curve obtained with

poly (HPMA) fractions having a precisely determined molecular weight (Photo-gonio-diffusometer Wippler-Scheibling ) . Content of incorporated pnitrophenylester ( ONp ) was determined by measurement of the UV absorption of the polymer solution in DMSO at 274 nm. The extinction coefficient of -0Np group= 9500 1 mol- ’ cm-‘. The M, and MJM, are shown in Table 1. To facilitate binding to the polymer precursor, melphalan isopropyl ester was first prepared. Melphalan (8.0 g) was dissolved in a 20% solution of HCl in dry isopropanol ( 150ml) and the solution boiled under reflux for 4 h. The solvent was then evaporated, the remaining oil dissolved in a 20% solution of HCl in isopropanol ( 100 ml) and boiled for a further 2 h. After repeated evaporation of the solvent, the oily residue was dissolved in isopropanol, and the product crystallised from this solution. Yield 75%, m.p. 202-204°C Rf=0.67 (mixture MeOH : CHC13 7 : 3 ) L-configuration by CD spectra measurement. Analysis: C H N Cl C,6H&2N2Cl3 theory 50.08 6.57 7.30 27.72 Mw 383.76 found 49.76 6.59 7.27 27.66 To prepare the free base, the product was suspended in dry diethyl ether, and dry ammonia bubbled through the suspension. After filtration the product was concentrated to dryness. BOC-Gly-melphalan isopropyl ester and BOCLeu-Gly-melphalan isopropyl ester were prepared by reacting glycine or dipeptide (amino group protected by BOC) with melphalan isopropyl ester in tetrahydrofuran in the presence of dicyclohexylcarbodiimide. The protective BOC group was then removed using a 15% solution of HCl in dry methanol. The corresponding hydrochlorides were precipitated into diethyl ether. Analysis of HCl.Gly-ME: C,8H28N303Cl3 C H N theory 49.04 6.40 9.53 found 49.50 6.47 9.62

Mw=440.82 Cl 24.13 24.80

125

Analysis of HClLeuGly-ME: Mw= 554.01 C24H&NKl3 C H N Cl theory 52.03 7.10 10.11 19.20 found 52.45 7.18 10.42 20.01 To synthesise the polymer conjugates listed in Table 1 the amino acid derivatives of melphalan isopropyl ester were bound by aminolysis as described at length elsewhere [ 17,18 1. The reprecipitated polymeric conjugates were dissolved in methanol and purified using GPC, Sephadex LH20 with methanol as the eluent. The purified product was dried by lyophilisation from an aqueous solution. Content of free melphalan was determined using FPLC (Pharmacia, column height 30 cm x internal diameter 1.2 cm, packed with Sephacryl S-200, phosphate buffer 0.15 M, pH 6.0 as eluent, flow rate 0.4 ml/min), this was always below 0.5% relative to the bound material. The content of bound melphalan isopropylester was determined by measuring UV absorptionatiZ=260nm (e=22,0001~mo1-‘~cm-’ (MeOH); e=20,000 l*mol-‘*cm-’ (DMSO) and A=302 nm (~=2000 l*mol-‘cm-l (DMSO ) ) . The results obtained were compared with data obtained by elemental analysis of chlorine. HPMA copolymers containing methacryloyltyrosinamide radioiodinated were with [ “‘I]iodide using the chloramine T method as previously described [ 15 ] to give a specific activity of approximately 13 ,Ki/mg and the preparation contained less than 1% free [ 125I]iodide assessed by paper electrophoresis. Hydrolytic stability and enzymatic degradation of HPMA copolymer containing melphalan

The hydrolytic stability of melphalan isopropyl ester HCl was examined in phosphate buffer (0.15 M, pH 6.0 at 37°C) using FPLC (Sephacryl S-200,30 x 1.2 cm column, eluent phosphate buffer 0.15 M, pH 6.0, flow rate 0.4 ml/min with Rl and UV dectors). Hydrolysis of polymerbound drug was studied in phosphate buffer pH 7.4 i.e., physiological pH. (It is known that acid pH can stabilise melphalan ) . In this case release

of Cl- was monitored using a Radiometer Copenhagen pH meter attached to a chloride selective silver electrode and calomel electrode with a double bridge as reference. To study enzymatic degradation of the peptidyl spacers in the HPMA copolymer conjugate samples were incubated with a model enzyme papain (a plant thiol protease ), a mixture of rat liver lysosomal enzymes (Tritosomes) [ 191 or an isolated lysosomal thiol protease cathepsin B [ 20 1. In each case hydrolytic cleavage was determined as described above using FPLC and the incubation conditions were as follows: Papain: HPMA copolymer conjugates were incubated with papain ( 5 x lo-’ mol. 1-l) in phosphate buffer (0.15 M, pH 6.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM reduced glutathione at 37 ‘C. Cathepsin B: Cathepsin B (as previously described [ 2 1 ] ) was incubated with conjugates as above using phosphate buffer (0.15 M, pH 5.5 ) containing 1 mM EDTA and 5 mM reduced glutathione. The enzyme concentration was 1.8x lo-’ mol. 1-r. Tritosomes: A mixture of rat liver lysosomal enzymes was prepared according to the methods of Trouet [ 191. The polymeric substrates were dissolved in 0.7 ml phosphate buffer (0.15 M, pH 5.5 ) containing 1 mM EDTA, 5 mM reduced glutathione and 20 ~1of a 10% solution of Triton X-100. Tritosomes (0.3 ml) were added and the mixture was incubated at 37°C. After a certain time, 100 ~1 of trichloroacetic acid was added, the precipitated fraction removed by centrifugation, the pH of the solution was adjusted to 6.0, before application to the FPLC column as previously described. In all cases a bound melphalan isopropylester concentration of 1x 1Om3 mol. I-’ was used. Cytotoxicity to Walker sarcoma in vitro

Walker sarcoma cells (LLC-WRC 256) were incubated in Medium 199 with Earle’s Salts including 5% foetal bovine serum and glutamine (2 mM) . The cells were split 1: 10 every week. For cytotoxicity assay, cells were grown in microtitre plates (volume 200 ~1) and incubated

126

with increasing concentrations of polymer conjugate, ME or melphalan isopropyl ester for 72 h after which time the tetrazolium dye 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added (20 p/well of a 5 mg/ml solution) and then the plates were allowed to stand at 37°C for 5 h. At the end of this period the culture medium was removed and the insoluble formazan crystals were dissolved in spectrophotometer grade DMSO and the absorbance measured at 550 nm. Viability of cells exposed to conjugate was expressed as a percentage of that seen in untreated control cells. (The MTT cytotoxicity assay is described at length elsewhere [22]). Activity against Walker sarcoma measured in vivo

Walker sarcoma (W256 ), ( 10 6 cells) were injected on day 0 S.C.into the thigh of male Wistar rats (approximately 75 g in weight). Two different treatment regimes were used. In the first, free ME or SE, or polymer conjugate was subsequently administered (at the doses shown) i.p. on day 1. Animals were then weighed daily and the progress of tumour development measured (taking the two maximum diameters). At the end of the experiment tumours were excised and weighed. The second approach was similar, but the S.C. tumours were allowed to progress until they reached palpable size (approximately lo- 15 days). Animals were then treated i.p. as described above and the tumour size and animal weight monitored. Cellular immune response following treatment of Walker sarcoma in vivo

Preliminary experiments were carried out to investigate the ability of ME and polymer-ME conjugates to modify the cellular immune response. Animals bearing established Walker sarcoma were treated i.p. with ME or P-Gly-PheLeu-Gly-ME ( 1 mg/kg). After 4 days the tumours were removed, minced with scissors and digested using collagenase (2 mg/ml), hyaluronidase ( 10 U/ml) and DNAase (0.4 mg/ml)

for approximately 1 h at room temperature. The cells, tumour cells, lymphocytes and macrophages, were separated from debris on a FicollHypaque gradient. Briefly, PBS (5 ml) was added to the cell suspension ( 3 ml), gently mixed and layered (8 ml) onto a Histopaque gradient (3 ml). The gradient was spun (400 xg, 30 min, room temperature) and the upper layers subsequently aspirated to within 0.3 cm of the interface containing the cells. The cells were removed and washed (three times) in PBS and their concentration adjusted to lo5 cells per ml. Samples ( 10 ~1, lo3 cells) were smeared over the surface of a Flow multitest slide and dried at 50°C before fixing in acetone (4’ C, 1 min) . After lixation the cells were exposed to antibodies recognising macrophages (MAS 369)) T lymphocytes (RMAS 0 1Op) and B lymphocytes (MAS 258~) and the number of antibody positive cells was estimated by fluorescence microscopy. Body distribution of melphalan and polymer-melphalan in rats bearing Walker sarcoma

Male Wistar rats were injected S.C.with lo6 cells (W256) as described above and tumours allowed to develop to palpable size (approximately 200 mm2 in area). Animals were briefly subjected to halothane anaesthesia and [ ‘Hlmelphalan or [ ‘251]labelIedP-Gly-Phe-LeuGly-ME were administered intravenously into the femoral vein. After 5,24 or 48 h the animals were sacrificed and the following assayed for radioactivity: liver, lung, kidney, spleen, heart, tumour, urine, faeces and blood (the animals were maintained in metabolic cages throughout to allow collection of urine and faeces). The radioactivity recovered in each compartment was expressed as a percentage of total radioactivity recovered. Results Polymer synthesis and characterisation

The polymer precursors used in this study had a similar molecular weight average, M, of ap-

127

proximately 20,000 and low polydispersity Mw/ Mn approximately 1.3. However, it should be noted that these parameters are affected by the ratio of the pnitrophenol - containing comonomer in the polymerisation reaction mixture, decreased Mw being observed with increasing content of this comonomer (Table 1, samples 6-9 ) . The rate of binding of melphalan isopropyl ester onto two polymer precursors is shown in Fig. 2. It can be seen that the reaction proceeded quickly (complete within 2 h) and was independent of the structure of the peptidyl side-chains. GPC characterisation of the polymer conjugate containing melphalan isopropyl ester showed that the distribution curve obtained was dependent on ME content of the polymer. At 2.4 and 5.1 mol% the curve had two maxima (Fig. 3) and this was typical for all the other polymer samples prepared with similar drug loading (results not shown). In contrast the elution profile of all polymer precursors containing the hydro-

b

b I

:

/’

:

:

‘1 \ I

1 \

\

‘1 :\

:

I’

’\

:

I’

30

40

!so

e

c

Fractfon

number

Fig. 3. Gel permeation chromatography elution of HPMA copolymers containing different contents of bound melphalan. In each panel the elution profile of polymer is given by the solid line; (a) polymer [ lo], (b) polymer [ 111, (c) polymer [ 121 and (d) polymer [ 131. The broken line represents the elution profile of the corresponding polymeric precursor aminolysed with aminopropanol not melphalan isopropyl ester.

philic substituent aminopropan-2-01 had a normal distribution.

00 0

Hydrolytic and enzymatic degradation of polymer-melphalan 100

200

300

400

Time(min) Fig. 2. Kinetics of melphalan isopropyl ester binding to polymer precursors. Binding of melphalan isopropyl ester to PGly-Leu-Gly-ONp (O-O ) or P-Gly-Phe-L.eu-Gly-ONp ( 0-O ) using reaction conditions described in the Materials and Methods section.

Although measured at different pHs (6.0 and 7.4) it can be seen that the rate of hydrolysis of ME and P-Gly-Phe-Gly-ME was essentially identical (Fig. 4). (Similar rates of hydrolysis were observed for P-Gly-Phe-Leu-Gly-ME, results not shown). Enzymatic release of melphalan from polymer conjugates was highly de-

128

1

0

5

10

15

Time(h)

I

0

20

40

60

60

Time (mln)

Fig. 4. Hydrolytic stability of melphalan isopropylester and polymer bound melphalan isopropylester. Release of Cl- was measured using a chloride selective electrode. Panel (a) shows the hydrolysis of ME.HCl ( 1 x 10m3 M) in phosphate buffer pH 6.0 at 37°C. Panel (b) shows the hydrolysis of P-Gly-Phe-Gly-ME ( 1 x 10m3 M with respect to ME.HCl) measured in phosphate buffer pH 7.4 at 37°C.

pendent on the amino acid composition of the oligopeptide spacer used to attach the drug (Fig. 5). Although the rate of release of drug varied according to the enzyme used in the incubation, in all cases the rate order of degradation was as follows: P-Gly-Leu-ME 2 P-Gly-Phe-Leu-GlyME > P-Gly-Phe-Gly-ME > P-Gly-Gly-ME > P-Gly-ME. (The latter sequence was not degradable in any experiment, results not shown). Antitumour activity of melphalan/sarcolysin and their polymer conjugates measured in vitro and in vivo Chemical characterisation of polymer-bound melphalan suggested that drug content altered the molecular conformation of the conjugate (Fig. 3 ) . Cytotoxicity of HPMA copolymers containing ME bound via a Gly-Phe-Leu-Gly spacer showed differential cytotoxicity towards Walker sarcoma in vitro (Fig. 6). The polymer containing the highest ME substitution ( 5.1 mol%) was the least toxic. Both free sarcolysin isopropylester and polymer conjugates showed ability to inhibit the development of Walker sarcoma in vivo (Fig.7a) and caused regression of established tumours

(Fig. 7b). It is interesting to note in Fig. 7a that the ability of polymer bound SE to prevent tumour growth is closely related to the rate of drug liberation (Fig. 6) from the peptidyl spacer used for conjugate synthesis. P-Gly-Leu-Gly-SE and PGly-Phe-Leu-Gly-SE, the most readily degraded sequences (by all enzymes studied) showed greatest antitumour activity, and the non-degradable P-Gly-SE was not active. However, it should be noted that free sarcolysin isopropyl ester shows good activity in both assays (Fig. 7a,b). The relative efficacy of free and polymer bound drug is discussed later. Animal weight was monitored throughout all experiments undertaken to measure treatment efficacy. Toxicity was characterised by a rapid initial weight loss from which, in severe cases, animals did not recover (results not shown). Table 2 summarises the survival of animals within the first 12 days of treatment following treatment with melphalan isopropyl ester or P-GlyPhe-Leu-Gly-ME. A single i.p. dose of 10 mg/kg melphalan caused acute toxicity in all the animals studied, melphalan isopropyl ester was slightly less toxic and likewise the polymer conjugate (Table 2). However, the differences in toxicities seen for the

129

Time (h)

Melphalan concentration

0

10

20

30

Time (h)

(pgglml)

Fig. 6. Cytotoxicity of P-Gly-Phe-Leu-Gly-ME in vitro. Dependancy on ME content. Walker cells were incubated with melphalan (O-.-O), polymer 13 (O-O), polymer 12 (H), polymer 11 (0-O ) and polymer 10 ( O-O ) for 72 h. Cell viability was assessed using the MTT assay as described in the Materials and Methods section.

"1 @' W-

TABLE 2 Toxicity of melphalan and its polymeric derivative

10

20

30

Time (h)

Fig. 5. Enzymatic degradation of polymer bound melphalan isopropylester. Release of melphalan from the polymeric carrier was monitored using FPLC as described in the Materials and Methods section. Degradation by (a) papain, (b) cathepsin B and (c) tritosomes is shown. The polymer substrates were P-Gly-Phe-Gly-ME ( n a ), P-Gly-Leu-Gly-ME (0-O ), P-Gly-Phe-Leu-Gly-ME (O-O ) and P-Gly-GlyME (0-O).

Dose administered (mg/kg drug)

0.1 0.3 1.0 3.0 10.0 20.0 30.0 100.0

Treatment”

Melphalan

Melphalan iso-propyl ester

P-Gly-Phe-LeuGly-ME

313 515 15115 5/5 O/5 ND ND o/5

515 515 15/15 515 4115 ND ND o/5

5/5 515 25125 515 418 o/4 O/4 ND

‘Animals surviving at day I2 after i.p. treatment on day 1.

130

61

(a)

1

-

2

3 4 Treatment

5

6

(W ‘2-I

l-

Time (daya atter treatment)

1

4

Latment3

Fig. 7. Effect of sarcolysin isopropylester and polymer bound SE sarcolysin on Walker sarcoma in vivo. Panel (a) shows the ability of free and polymer bound SE (both 5 mg/kg administered on day 1) to inhibit the development of s.c. Walker tumours (weight measured on day 15). Key: ( 1) no treatment; (2) sarcolysin isopropyl ester; (3) P-Gly-Phe-Leu-GlySE; (4) P-Gly-Phe-Gly-SE; (5) P-Gly-Leu-Gly-SE; (6) PGly-SE. Panel (b) shows the regression of established Walker sarcoma treated on day 9 with ( 1) no treatment; (2) P-GlyPhe-Gly-SE (5 mg/kg); (3) P-Gly-Phe-Leu-Gly-SE ( 10 mg/ kg) or (4) sarcolysin isopropyl ester ( 10 mg/ kg).

melphalan derivatives were slight and the polymer conjugate was markedly toxic at doses of 20 and 30 mg/kg (relative to drug content ) . When administered at the lower dose of 1 mg/ kg, P-Gly-Phe-Leu-Gly-ME showed better activity against established Walker sarcoma than free ME (Fig. 8). These preliminary experiments used only 3 animals per group and await repetition. However, the macrophage and lymphocyte (T and B) content of the tumours taken from these animals showed profound and reproduci-

Fig. 8. Effect of low dose ( 1 mg/kg) melphalan and polymermelphalan isopropylester on established Walker sarcoma. Walker sarcoma was allowed to progress until the tumour area was 200-300 mm’. Animals were treated i.p. with melphalan ( 1 mg/kg) (O-O ), P-Gly-Phe-Leu-Gly-ME ( 1 mg/kg drug) (H ) or left untreated ( 0-O ) . Tumour size was followed and on day 4 (as shown) tumours were excised and their cell content analysed. See Fig. 9 for the results.

ble changes (Fig. 9 ) . Four days following treatment with ME the macrophage content of tumours was increased approximately 7-fold, whereas treatment with polymer conjugate produced a 24-fold increase. Free melphalan greatly enhanced the T cell content, but polymer melphalan effectively doubled this response. Similarly the polymer conjugate greatly elevated the number of B cells observed. The pharmacokinetics of free and polymer bound melphalan were quite different (Fig. 10). Within 24 h more than 90% of the radioactivity recovered after administration of [ ‘H ] melphalan was found in the urine and faeces. The tumour level of radioactivity was approximately 0.3% of the recovered dose at 24 h. In contrast, ‘251-labelled polymer-melphalan was excreted more slowly and 20% of the radioactivity was still in the bloodstream at 24 h. An increase in deposi-

131

100 30

60 40 1

2

3

20

Traatmant 0

x20-’

0

10

20

50

40

30

(b)

lOO-

sowm20OA 1

2

3

Treatment

70-

*-*

60so0

4030 -

10

20

30

40

50

Time (h)

2010 0”

I

’ 1

2

3

Fig. 9. Macrophages and lymphocyte content of established Walker sarcoma following treatment with free melphalan and polymer bound melphalan isopropylester. Tumours obtained from animals treated in Fig. 8 were analysed for content of T lymphocytes (a), macrophages (b) or B lymphocytes (c) using fluorescent antibodies as described in the methods section. Data obtained using untreated animals ( 1 ), melphalan treated (2) or polymer-melphalan treated animals (3) are shown.

Fig. 10. Body distribution of [3H] melphalan and 1251-labelled P-Gly-Phe-Gly-ME after intravenous administration to tumour bearing rats. The body distribution of radioactivity is shown following administration of [ 3H] melphalan (a) and ‘251-labelled polymer-melphalan (b ) . Key: blood (O-O ); urine and faeces (0-O ); liver ( A-A ) and tumour (0-O ).

conjugate. All other organs sampled contained less than 5% of recovered radioactivity throughout the experiment.

Discussion tion of radioactivity was observed both in the tumour (approximately 5-fold in peak level) and liver ( 1Zfold) following administration of the

Many polymeric derivatives have been described which contain anthracycline antibiotics, HPMA copolymer-doxorubicin [ 3 1, poly-L-glu-

132

tamic acid-doxorubicin [ 23 1, poly-aspartic aciddoxorubicin [ 25 ] and recently a block copolymer of poly aspartic acid and polyethylene glycol also bound to doxorubicin [ 26 1. Providing such conjugates incorporate a covalent linkage for attachment of drug to carrier that is not rapidly hydrolysed in the circulation they have the ability to produce a marked increase in therapeutic index. For example HPMA copolymer doxorubitin is 5-10 times less toxic than free drug, at equidose shows greater antitumour activity and has the added advantages that the polymer conjugate is approximately 10 times more soluble (relative to drug content) than the anthracycline [31. Drug delivery systems for alkylating agents have been much less widely explored. This might be explained by their, thus far, more limited clinical use, their lack of specificity of action (mechanism of action is usually attributed to DNA interstrand crosslinking [ 271, but alkylation of cell membranes has also been correlated with their toxicity [ 281)) rare but serious side-effects such as teratogenesis and carcinogenesis (reviewed in [ 291) and not least the stability of the compounds themselves. Unlike anthracyclines which are relatively stable [ 301 melphalan undergoes rapid hydrolysis in physiological solutions to yield inactive metabolites [ 3 11. For effective delivery of melphalan, the system used must be amenable to synthesis and storage without hydrolysis of drug and ideally following administration should preserve the drug in active form until it arrives within the target tumour. These are not always easy criteria to fulfill. Low molecular weight and macromolecular prodrugs of alkylating agents have been reported, but they frequently show poor activity. Peptidyl derivatives of acivicin and phenylenediamine mustard were synthesised for selective hydrolysis by the enzyme plasminogen activator reportedly elevated in many tumours [ 32 1. Plasminogen activator was shown to be necessary for prodrug activation; however, although the prodrug of phenylenediamine was active against B 16 melanoma in vivo, it was no more active than the parent drug. Similarly conjugates of mel-

phalan linked via polyglutamic acid to antilymphocyte serum failed to increase immunosuppression in CBA mice bearing skin grafts [ 33 1. Improved antitumour activity of polymer (poly-L-lysine or poly+glutamic acid) [ 341 and immunoconjugates of melphalan [ 35 ] have been claimed, but we feel that there is still a need for systematic studies of the chemical synthesis, chemical and pharmacological activity of drug delivery systems incorporating alkylating agents in order to allow rational design of conjugates with a significantly improved therapeutic index. Binding melphalan isopropyl ester to HPMA copolymers did not alter significantly the rate of drug hydrolysis (Fig. 4). The GPC elution profiles obtained (Fig. 3) did however show that sample containing more than 2.4 mol% of drug underwent complex conformational changes. Distribution curves obtained for polymer melphalan with higher drug loading had two maxima whereas the same polymer precursor aminolysed with the hydrophilic aminopropanol produced a normal distribution. The ratio of the two peaks changed relative to drug loading. The higher the drug content, the larger the higher molecular weight peak, and this phenomenon was probably attributable to the presence of intermolecular aggregates formed as a result of hydrophobic interactions. If the high molecular weight peak was isolated and rechromatographed the elution profile obtained again displayed two peaks identical to the GPC record before fractionation suggested an equilibrium between individual polymer molecules and polymer aggregates, without chemical crosslinking. It is open to speculation whether such polymer aggregates would dissociate easily in physiological solutions when administered at optimum drug doses. Certainly in vitro experiments reported here indicate that the conjugates with higher melphalan substitution were less active against Walker sarcoma (Fig. 7). Unfortunately the difficulty in standardisation of M, of polymer precursors with different pnitrophenol ester content makes the interpretation of these data uncertain (samples 10-l 3 show M, in the range 40,000 to 18,000). Pinocytic capture of polymers has previously

133

been shown to be molecular weight dependent [ 36 ] and polymer aggregation may preclude access to the cell’s interior for lysosomal cleavage of drug thus diminishing the release of melphalan. Enzymatic release of melphalan from HPMA copolymers was clearly dependent on the peptide sequence used to link drug to polymer, an observation consistent with many previous reports describing release of drugs [ 341 and model compounds [ 6,7] from these polymers. It is important to note the tetrapeptide, Gly-Phe-LeuGly and tripeptide, Gly-Leu-Gly were most effective in melphalan liberation, and the dipeptide, Gly-Gly, was degraded to yield free drug more slowly. The latter observation contrasts with previous reports [ 341 that P-Gly-Gly-doxorubicin is not degraded by thiol-proteases. However, it is consistent with the observation that P-Gly-Gly-TyrNH* is degraded intralysosomally [ 371. These data serve to emphasise that the degradation of peptidyl spacers is dependent on the chemical nature of the terminal substituent, making it difficult to select one particular sequence that will yield an optimum rate of release of a wide range of different pharmacological agents. It is noteworthy that the nature of the polymeric backbone also serves to influence the rate and specificity of enzymatic cleavage of pendent peptidyl spacers [ 38 1. Ability of polymer-sarcolysin conjugates to prevent establishment of Walker sarcoma (Fig. 7a) showed good correlation with the in vitro rate of enzymatic degradation of the peptidyl sidechains. The non-degradable P-Gly-SE was completely inactive. However, at doses of 5 and 10 mg/kg (Figs. 7a,b), the optimal tolerated dose for both free and polymer bound drug, the biodegradable conjugates showed activity but little increase in therapeutic index. Preliminary experiments, carried out with a low drug dose ( 1 mg/kg) did indicate increased activity of the conjugate (Fig. 8 ). Although it is known that alkylating agents such as melphalan act in part by DNA interstrand crosslinking with some degree of tumour specificity, their dose-dependent mechanism of

action in humans is still debated. The effect of polymer conjugation on mechanism of drug action is worthy of consideration particularly for future design of more effective drug conjugates. Synthesis of the macromolecular derivative altered the drug pharmacokinetics. In Fig. IOa,b, it can se seen that more than 90% of the [3H] melphalan administered is excreted within 24 h and the blood clearance is very rapid - the tl ,Z (Y of melphalan in humans has previously been reported as 7.7 min [ 391. Free [ 3H] melphalan recovered in the tumour was always less than 1%. In contrast the ‘251-labelled polymer conjugate showed a much slower plasma clearance ( > 50% radioactivity remaining at 5 h), slower excretion and significantly higher levels in the tumour (peak level > 4%). The prolonged half-life of polymer-melphalan in the bloodstream is somewhat surprising as the aminolysed polymer precursor had an M, of 18,000, and we have shown previously that HPMA copolymers of this M, are subject to rapid ultrafiltration through the kidney [40]. Two explanations may account for this discrepancy, either the polymer reaches the kidney in the form of polymer aggregates, or the reactive chloroethyl groups of the pendent drug are able to react nonspecifically with plasma proteins and/or the surface of circulating blood cells. Both would decrease the rate of ultrafiltration. The relatively high levels of radioactivity recovered in the liver would be consistent with phagocytic capture of polymer aggregates. The enhanced tumour deposition versus free drug would suggest the prolonged circulation of a macromolecular form (rather than cell association), permitting polymer access to the tumour via the discontinuous endothelial barrier known to be present in Walker sarcoma [ 4 11. We have shown previously that intravenous administration of PGly-Phe-Leu-Gly-daunorubicin facilitates higher drug deposition in Walker sarcoma than free drug (4-fold increase in the area under the curve) [ 8 1. Taking into account the limitations of radiotracer studies (HPLC analysis of melphalan in tissues is not possible) administration of polymermelphalan would seem to increase greatly the

134

possibility for specific tumour deposition. However, it would seem unlikely, in the case of melphalan, that this would lead to improved effrcacy. Assuming that drug bound to polymer will be hydrolysed during transit in extracellular fluids, and that extravasation must be subsequently followed by pinocytic internalisation into tumour cells to permit subsequent intralysosoma1 liberation of free drug, it seems unlikely that a very high dose of active drug could be released from the conjugate intratumourally. The increased tumour deposition observed for the polymer conjugate may ensure a supply, albeit at a low level, of active drug into the tumour which accounts for the observed limited antitumour activity. However, it is possible that other mechanisms contribute to the observed activity. Low dose chemotherapy is arguably no less effective in the treatment of certain tumours and it has been suggested that this may make tumour cells more immunogenic, impair suppressor cell activity and/or enhance helper cell activity and render tumour cells more susceptible to lysis [ 42 1. A polymer conjugate which releases drug in a controlled and slow fashion might be an ideal candidate to cause immunopotentiation. Preliminary data shown here indicate that equidose of free and polymer bound melphalan ( 1 mg/kg) both cause tumour regression (Fig. 8 ) and do indeed increase the number of macrophages and B and T lymphocytes detected in Walker sarcoma (Fig. 9). Using these indicators the polymer conjugate was much more active than an equivalent dose of melphalan. Further experiments are necessary to examine the dose dependency of this response, and also to examine the effect of combinations of HPMA copolymers and non-conjugated drug, but the results obtained in this preliminary experiment were so striking they are considered worthy of inclusion here. In summary, we have shown that HPMA copolymers can be synthesised to contain melphalan (L) or sarcolysin ( D,L) . Such conjugates tend to form aggregates in solution depending on the drug loading of the polymer and solution concentration and this may affect their pharmacokinetics and mechanism of action in vivo.

Polymers with high drug loading are less cytotoxic in vitro. Following intravenous administration the polymer conjugate showed a much greater deposition in tumour than free drug, but whether this contributes to the mechanism of antitumour activity remains unclear. Direct action against the tumour is possible if non-hydrolysed drug is liberated intralysosomally within the tumour, but preliminary evidence suggests that conjugate is also able to stimulate an antitumour immune response. Whichever mechanism predominates, it is clearly shown that a biodegradable peptidyl spacer linking drug to polymer is an essential requirement for antitumour activity, and those conjugates shown to degrade most rapidly in vitro produce the highest antitumour activity in vivo. Acknowledgements We would like to thank the British Cancer Research Campaign and the Wellcome Foundation Ltd. for supporting this work and the Royal Society for supporting the international collaboration. References D. Schmahl and M.R. Berger, Possibilities and limitations of antineoplastic chemotherapy: experimental and clinical aspects, Int. J. Exp. Clin. Chemotherap., 1 (1988) l-11. J. Kopecek and R. Duncan, Targetable polymeric prodrugs, J. Controlled Release, 6 (1987) 315-327. R. Duncan, I.C. Hume, P. Kopeckova, K. Ulbrich, J. Strohalm and J. Kopecek, Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. 3. Evaluation of adriamycin conjugates against mouse leukaemia Ll210 in vivo, J. ControlledRelease, 10 (1989) 51-63. R. Duncan, P. Rejmanova-Kopeckova, J. Strohalm, I.C. Hume, J.B. Lloyd and J. Kopecek, Anticancer agents coupled to N- (2-hydroxypropyl)methacrylamide copolymers. 2. Evaluation of daunomycin conjugates in vivo against Ll210 leukaemia, Brit. J. Cancer, 57 (1988) 147-156. K. Ulbrich, E.I. Zacharieva, J. Kopecek, I.C. Hume and R. Duncan, Polymer bound derivatives of sarcolysin and their antitumour activity against mouse and human leukaemia in vim, Makromol. Chem., 188 (1987) 24972509.

135

6

8

9

IO il

12

f3

14

15

16

17

IS

19

R. Duncan, H.C. Cable, J.B. Lloyd, P. Rejmanova and J. Kopecek, Polymers containing enzymatically degradable bonds. 7. K&&n of oiigopeptide side chains in poly N- ( 2-hydrox~~p~ )m~thac~la~de copolymers to promote efficient degradation by lysosomal enzymes, Makromol. Chem., 184 ( 1984) 1997-2008. P. Rejmanova, J. Kopecek. R. Duncan and J.B. Lloyd, Stability in plasma and serum of lysosomally degradable oligopeptide sequences in N-( 2-hydroxypropyi ~me~ac~iamide copolymers, Biomaterials, 6 ( 198.5) 45-48. J. Cassidy, R. Duncan, G.J. Morrison, J. Strohalm, D. Plocova, J. Kopecek and S.B. Kaye, Activity of N-(2hydroxypropyl )methacrylamide copolymers containing daunomycin against a rat tumour model, Biochem. Pharmacol., 38 ( 1989) 875-879. R, Duncan, LC. Hume, KB, O’Hare, f. Strohalm, J_ Kopecek and K. Ufbrich, Antitumour activity of N-(2hydroxypropyl )methacrylamide copolymers containing anthracyclines, abstract from the BACR Meeting, Glasgow, Scotland ( 1989). K.B. O’Hare et al., unpublished results. S.R. Wedge, R. Duncan, J. Strohalm and K Ulbrich, Comparison of the therapeutic efRcacy of free and poiymer bound doxorubicin, in the treatment of a murine leukaemia model, The 634th Meeting of the Biochemical Society, Bath, U.K. ( 1990). D.S. Alberts, Alkeran” (Melphalan), A Perspective on Current Clinical Issues, Burroughs Wellcome Co., Research Triangle Park ( 1987 f . S. Dray and M. Mokyr, Cyclophospham~de and melphalan as immunopotentiating agents in cancer therapy, Med. Qncol. Tumour Pharmacother., 6 ( 1989) 7785. J. Strohalm and J. Kopecek, Poly [N-(2-hydroxypropyl ~methac~lamide ] IV. Heterogeneous copolymerisation, Angew. Makromol. Chem. 70 ( 1978 ) 109- 118. R. Duncan, H.C. Cable, P. Rejmanova, J. Kopecek and J.B. Lloyd, Tyrosinamide residues enhance pinocytic capture of N-( 2-hydroxypropyl)methacrylamide copolymers, Biochim. Biophys. Acta, 799 ( 1984) l-8. P. Rejmanova, J. Labsky and J. Kopecek, Aminolysis of monomeric and polyme~c~nit~phenyi esters of methacryloylated amino acids, Makromol. Chem., 178 (1977) 2159-2168. J. Kopecek, Biodegradation of polymers for biomedical use, in: H. Benoit and P. Lempp (Eds. ), IUPAC Macromolecules, Pergamon, Oxford, 1982, pp. 305-320. B. Rihova, J. Kopecek, P. Kopeckova-Rejmanova, J. Strohalm, D. P&ova and H. Semomdova. BioalEnity therapy with antibodies and drugs bound to soluble synthetic polymers, J. Chromatogr. Biomed. Appl., 376 (1986) 221-233. A. Trouet, Isolation of rat liver lysosomal enzymes, Methods Enzymol., 31 ( 1974) 323.

20

21 22

23

24

25

26

27

28

29

30

31

32

J. Pohl, M. Baudys, V, Tomasek and V. Kostka, Identification of the active site cysteine and of the disulphide in the N-terminal part of the molecule of bovine spleen cathepsin B, Febs. L&t. f 42 ( 1982 ) 23- = P. Rejmanova, J. Pohl, M. Baudys, V, Kostka and J. Kopecek, Makromol. Chem., 184 (1983) 2009-2020. D. Sgouras and R. Duncan, Methods for the evaluation of biocompatibility ofsoluble synthetic polymers which have potential for biomedical use. 1. Use of the tetrazolium-based calorimetric assay (MTT) as a preliminary screen for evaluation of in vitro ~otoxic~ty, J. Mater. Sci.: Mater. Med. 2 f f990) 61-68. C.J.T. Hoes, W. Potman, WA. Von Heeswijk, R.J. Mud, B.G. De Mooth, J. Greve and J. Feijen, Gptimisation of macromolecular prodrugs of the antitumour antibiotic adriamycin, J. Controlled Release, 2 ( 1985) 205-2 13. F. Zunino, G. Savi, F. Giuliani, R. Gambetta, R. Supino? S. Tine% and G. Pezzoni, Comparison of the antitumour effects of daunorubicin covalently linked to poly+amino acid carriers, Eur. J. Cancer Clin. Oncol., 20(1984)421-425. F. Zunino, G. Pratesi and A. Micheloni, Poly (carboxylic acid) polymers as carriers for anthracyclines, J. Controlled Release, 10 f 1989) 65-73, M. Yokoyama, M. Miyauchi, N. Yarnada, T. Ukano, Y. Sakurai, K. Kataska and S. Inoue, Cbaracterisation and anticancer activity of the micelle-forming polymeric anticancer drug adriamycin-conjugated poly (ethylene glycol)-poly (aspartic acid) block copolymer, Cancer Res,, 50(1990)1693-1700. T. Connors, M~b~isms of action of ~~hlo~e~ylamine derivatives, sulphar mustards, epoxides and aziridines, in: AC. Sartorelli and D.G. Johns (Eds.), Antineoplastic and Immunosuppressive Agents, Part II, Handbuch der Experimentellen Pharmakologie, Vol. 3% Springer Verlag, Berlin, 1975, pp. 18-34. W. Doppler, 3. Hogmann, H. Oberhuber, K. Maly and J. Grunicke, Nitrogen mustard interference with potassium transport in Ehrlich ascites tumour cells, J. Cancer ’ Res. Clin Ocol., t 10 (1985) 35- . T.A. Connors, Alkylating agents, nitrosoureas and alkyltinazenes, in: WM. Pinedo and B.A. Chabner, (Eds.), Cancer Chemotherapy, Vol8, Elsevier Science Publishers, BV, Amsterdam, 1986,28-5 1. A.G. Bosanquet, Stability of solutions of antineoptastic agents during preparation and storage for in vitro assays. II Assay methods, adriamycin and other antitumour antibiotics, Cancer Chemother. Pharmacol., 17 (1986) l-10. A.G. Bosanquet, Stability of melphalan solutions during preparation and storage? J. Pharm. Sci,, 74 ( 1985) 348-351. P.K. Chakravarty, P.L. Carl, M.J. Weber and J.A. Katzenellenbogen, Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidyl acivicin and peptidylphenylenediamine mustard, J. Med. Chem., 26 (1983) 633-638.

136 33

34

35

36

37

L. Brent, T. Horsburgh, G. Rowland and P. Wood, Failure to increase the in vivo immunosuppressive activity of antilymphocyte globulin by conjugation with melphalan, Transplantation, 29 ( 1990) 280-282. Y. Morimoto, K. Sugibayashi, S. Sugihara, K.I. Hosoya, S. Nozaki and Y. Ogawa, Antitumour agent poly (amino acid) conjugates as a drug carrier in cancer chemotherapy, J. Pharm. Dyn., 7 (1984) 688-698. N. Sugita, K. Niimura, M. Fujii, K. Matsunaga, T. Fujii, T. Furusho, C. Yashikumi, Y. Kawai and T. Taguchi, In vivoantineoplastic activity of mouse or human IgG conjugated with melphalan, In vivo, 1 ( 1987) 205-2 14. R. Duncan, M.K. Pratten, H.C. Cable, H. Ringsdorfand J.B. Lloyd, Effect of molecular size of 12SI-labelled poly (vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and rat peritoneal macrophages, Biochem. J., 196 (1980) 49-55. R. Duncan, P. Rejmanova, J. Kopecek and J.B. Lloyd, Pinocytic uptake and intracellular degradation of N-( 2hydroxypropyl)methacrylamide copolymers. A poten-

38

39

40

41 42

tial drug delivery system, Biochim. Biophys. Acta, 678 (1981) 143-150. K. Ulbrich, J. Strohalm and J. Kopecek, Polyethyleneglycols containing enzymatically degradable bonds, Makromol. Chem., 187 (1986) 1131-l 144. D.S. Alberts, S.Y. Chang, H.S.G. Chen, T.E. Moon,T.L. Evans, R.L. Furrier, K. Himmelstein and J.F. Gross, Kinetics of intravenous melphalan, Clin. Pharmacol. Ther., 26 (1979) 73-80. L.W. Seymour, R. Duncan, J. Strohalm and J. Kopecek, Effect of molecular weight (M,) of N- (2-hydroxypropyl)methacrylamide copolymers on body distributions and rate of excretion after subcutaneous, intraperitoneal and intravenous administration to rats, J. Biomed. Mater. Res., 21 (1987) 1341-1358. R.K. Jain, Determination of tumour blood flow: a review, Cancer Res., 48 (1988) 2641-2658. M.B. Mokyr and S. Dray, Interplay between the toxic effects of anticancer drugs and host antitumour immunity in cancer therapy, Cancer Invest., 5 (1987) 31-38.