Evaluation of the hydrolytic and enzymatic stability of macromolecular Mitomycin C derivatives

journal of

ELSEVIER

Journal of Controlled Release31 (1994) 89-97

controlled release

Evaluation of the hydrolytic and enzymatic stability of macromolecular Mitomycin C derivatives Anne De Marre a, Leonard W. Seymour u, Etienne Schacht a'* aDepartment of Organic Chemistry, Biomaterials Research Group, Universityof Gent, Krijgslaan 281 S4-bis, 9000 Gent, Belgium bDepartment of Clinical Oncology, Universityof Birmingham School of Medicine, Birmingham B15 2TH, England, UK

Accepted 24 January94

Abstract

The cytostatic agent Mitomycin C (MMC) was coupled to a polymeric carder, poly-[N-(2-hydroxyethyl)-L-glutamine] (PHEG), via peptide spacers. The influence of the amino acid sequence of the spacer on the hydrolytic and enzymatic stability of the macromolecular drug conjugate was investigated under different conditions. Accordingly the conjugates were incubated at neutral and slightly acid pH, and in presence of lysosomal enzymes or in serum. It was observed that tetrapeptide-based conjugates generally release MMC more effectively than tripeptide derivatives. Conjugates having a terminal glycine in the spacer are less hydrolytically stable than those with a terminal hydrophobic amino acid both in buffer and in serum. Gly-PheLeu-Gly, Gly-Phe-Ala-Leu and Ala-Leu-Ala-Leu derivatives released MMC very rapidly in the presence of tysosomal enzymes. Keywords: Hydrolyticstability;Enzymaticstability;MitomycinC derivative;Macromoleculardrug

1. Introduction

One of the major problems in cancer chemotherapy is the low selectivity of existing antitumor agents. Frequently not only tumor cells but also healthy cells are exposed to cytotoxic concentrations of the drug, resulting in unwanted peripheral toxicities. One way to alter the body distribution and reduce these side effects is by coupling the drug onto a targetable polymeric carrier [1]. Macromolecules and drug conjugates are generally unable to diffuse through membranes; their entry into cells is restricted to endocytosis and they are usually transported to the lysosomal compartment of the cell [2]. After interaction with lysosomal enzymes these polymeric-drug conjugates could release the active agent intracellularly provided the polymer-drug *Correspondingauthor. 0168-3659/94/$07.00 © 1994 ElsevierScienceB.V. All rights reserved SSD10168-3659 (94)00009-J

linkage is susceptible to cleavage by lysosomal enzymes [ 3-6]. Ideally, the macromolecular prodrugs of antitumor agents should be stable and pharmacologically inactive in circulation but, after internalization, degrade in the lysosomes with release of the activated drug. In this study, macromolecular derivatives of Mitomycin C (MMC) were prepared. MMC is a reductive alkylating agent, introduced by Wakaki as antitumor agent, and has been used clinically to treat various tumors, although it is not established as the treatment of choice for any particular disease [7]. Sezaki and co-workers have reported the synthesis and evaluation of a range of dextran-MMC derivatives [ 8-12 ], in order to optimize the pharmacokinetics and the efficacy of the drug. In their approach, a spacer group, 5-amino caproic acid, was first introduced onto the polymeric carrier. Then, MMC was coupled to the

90

A. De Marre et al./ Journal ~'Controlled Release 31 (1994) 89-97

resulting dextran derivative. The effect of the electric charge of the conjugates on cell adhesion, plasma disposition and cellular uptake was tested [ 9-12 ]. Moreover, the effect of the spacer on the release rate of MMC and the resulting antitumor activity has been evaluated [ 13-14]. However, no detailed study of the in vitro hydrolytic or enzymatic stability of the conjugates was reported. Importantly, the characterization of polymer derivatives prepared by a sequence of reactions starting from the parent polymer is not straightforward since most reactions on polymeric side-chain groups are not quantitative. Hence, this strategy is not ideal for preparing polymeric prodrugs with well characterized structure. Therefore, we have worked out an alternative strategy for the preparation of macromolecular MMC conjugates (Fig. 1). In this approach a spacer group was first coupled with MMC. These spacer-MMC derivatives are easily synthesised, purified and characterized. In the next step these derivatives were coupled onto the polymeric carrier. In the present work poly-[N-(2hydroxyethyl)-L-glutamine] (PHEG) was selected as polymeric drug carrier. PHEG has been proposed as plasma expander [15], is soluble in water, not immu-

- - - ~ NH- CH- C O - ~

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nogenic and also biodegradable [16-18]. We have demonstrated before that the hydroxyl groups of the polymer can be easily activated and be used as a site for coupling of bioactive agents [ 19]. A range of oligopeptide spacers were selected to link MMC onto the polymeric carrier [20]. It has been reported in the literature that polymeric-drug conjugates containing anthracyclines or simple alkylating agents can release the drug from proper peptidyl side-chains by lysosomal digestion [3-6,21 ]. So far this has not been reported for MMC conjugates. In order to examine the influence of the spacer composition on the release rate of MMC, the macromolecular MMC derivatives were incubated in buffer, bovine serum and in presence of purified lysosomal enzymes (tritosomes).

2. Materials and methods

2.1. Chemicals MMC was a kind gift of Kyowa Hakko Ltd (Tokyo, Japan). All aminoacids, peptides and Fmoc-Cl were obtained from Bachem Chem. Co. (Bubendorf, Switzerland). 4-Nitrophenyl chloroformate was obtained from Merck (Darmstadt, Germany). Reduced glutathione and EDTA were obtained from Sigma Chem. Co. (St. Louis, MO, USA). All other chemicals were purchased from Janssen Chimica (Beerse, Belgium).

C=O

I

2.2. Synthesis of polymeric-MMC conjugates

NH

I spacer

I

C=O I

N MMC

0

0II

~T~h_~j~CH2

- 0 - C - NH2

=

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The synthesis of the PHEG-spacer-MMC derivatives has been described previously [20] .In summary,in the first step MMC was coupled to the Fmoc-protected oligopeptide pentafluorophenyl ester. After deprotection the amine-containing spacer-MMC derivatives were coupled with 4-nitrophenyl carbonate containing PHEG. After purification of the conjugates, the content of polymer-bound MMC was determined by UV analysis (eM = 22000 1 mo1- 1 cm- J, Amax= 364 nm).

HN~MMC

Fig. I. Structure of the PHEG-oligopeptide-MMC conjugates.

2.3. Degradation of the drug conjugates Incubation in buffer The release of MMC from polymer conjugates was

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investigated at 37°C in phosphate buffer, pH 7.4 (0.1 M KH2PO4) and pH 5.5 (0.1 M Na2HPO4/citric acid). One mg of PHEG-spacer-MMC was dissolved in 1 ml buffer. At regular times 150 /zl samples were withdrawn and analysed by HPLC SEC.

Quantitation of drug release The amount of MMC released from the polymer conjugates was determined using an HPLC SEC method (column, Biorad Biosil 125 HPLC SEC; mobile phase, citrate buffer, pH 6; flow, 0.8 ml/min; injection volume, 20/zl; detection, UV 364 nm). By comparing the integration of the peaks corre50

sponding to the polymer-bound and free MMC, the amount of released MMC was calculated.

Degradation in presence of tritosomes The rate of MMC release of the conjugates during incubation with tritosomes [22] was studied at 37°C in buffer, pH 5.5. One mg of conjugate was dissolved in 400 /xl phosphate buffer (0.2 M Na2HPO4/citric acid, 0.2 Triton X-100 w/v%), 100 /zl of a l-mM EDTA solution, 100/zl of a 5-raM reduced glutathione solution and finally 400/zl tritosomes were added [ 23 ]. Samples were taken at time 0, 1, 3 and 5 h and analysed for free MMC by HPLC. 80

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Fig. 2. Time course of hydrolysis of PHEG-peptide-MMC conjugates in buffer. (a) ( 1 ) Giy-Phe-Gly, ([]) Gly-Phe-Leu, (e) Gly-Phe-Phe, (©) Gly-Gly-Phe. (b) (ll) Gly-Phe-Leu-Gly, (•) Gly-Gly-Phe-Leu, (o) Ala-Leu-Ala-Leu, (O) Gly-Phe-Ala-Leu. (c) ( 1 ) Gly-Phe-Gly, (lq) Gly-Phe-Leu, (e) Gly-Phe-Phe, (O) Gly-Gly-Phe. (d) (ll) Gly-Phe-Leu-Gly, (D) Gly-Gly-Phe-Leu, (e) Ala-Leu-Ala-Leu, (O) GlyPhe-Ala-Leu Conditions of hydrolysis: (a-b) phosphate buffer (KH2PO4 0.1 M), pH 7.4, 37°C; (c-d) phosphate buffer (Na2HPO,, 0.1 M/ citric acid), pH 5.5, 37°C.

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Degradation in bovine serum For the determination of the stability of the conjugates in serum 1 mg of the PHEG-MMC conjugates was dissolved in 1 ml of bovine serum at 37°C. At regular time intervals samples were withdrawn for HPLC analysis.

3. R e s u l t s

3.1. Hydrolytic degradation o f the P H E G - M M C conjugates in buffer

In a first set of experiments the release of MMC by chemical hydrolysis was tested both at pH 7.4 and 5.5 (lysosomal pH). Fig. 2 illustrates that all conjugates are less stable at pH 7.4 than at 5.5. The relationship between side-chain structure and hydrolytic stability is similar in both buffers: release of MMC is much faster from polymeric conjugates having a C-terminal glycine in the spacer (cf. Fig. 2a, pH 7.4: Gly-Phe-Gly 42%, and Gly-Gly-Phe 12% of free MMC after 24 h of incubation. 3.2. Degradation in presence o f lysosomal enzymes

Fig. 3 illustrates the release of MMC in presence of lysosomal proteinases. Very little MMC was released from the conjugates with terminal Phe and Phe-Leu in a

3.3. Stability o f the conjugates in bovine serum

The degradation experiments of the Gly-Phe-Gly and Gly-Phe-Leu-Gly conjugates in serum showed a rapid release of free MMC which was faster than that observed in buffer, pH 7.4. As indicated in Fig. 5 the hydrolysis of the conjugates in heat denaturated serum is comparable to that observed in a buffer, pH 7.4, medium. Fig. 6 demonstrates that conjugates with terminal glycine group in the spacer are less stable in serum than those with a hydrophobic terminal aminoacid: e.g., Gly-Phe-Leu-Gly 65%, and Gly-Gly-Phe-Leu 28% of free MMC after 8 h of incubation in serum.

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the oligopeptide spacer. MMC was rapidly released from the Gly-Phe-Leu-Gly, Gly-Phe-Ala-Leu and AlaLeu-Ala-Leu conjugates. The total release of MMC, the release of free MMC and aminoacid-MMC fragment from these conjugates is shown in Fig. 4. Furthermore the hydrolytic stability of Gly- and Leu-MMC was tested at pH 5.5: 80-90% of free MMC was detected after 3 h of incubation. Moreover, enzymatic degradation of the PHEGbackbone was observed in presence of lysosomal enzymes. The hydrolysis of amide linkages in this polyaminoacid derivative in presence of enzymes (papain, chymotrypsin, tritosomes) is reported extensively in the literature [ 16-18,241.

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Fig. 3. Timecourseof enzymatichydrolysisof PHEG-peptide-MMCwith tritosomes.(a) ( • ) Gly-Phe-Gly,([]) Gly-Phe-Leu,(•) Gly-PhePhe, (©) Gly-Gly-Phe. (b) ( l ) Gly-Phe-Leu-Gly,([~) Gly-Gly-Phe-Leu,(0) Ala-Leu-Ala-Leu,(©) Gly-Phe-Ala-Leu.Conditions of hydrolysis: phosphate buffer (Na2HPO40.2 M/citric acid), pH 5.5, 1 mM EDTA, 5 mM reduced glutathione, 37°C.

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A. De Marre et al. / Journal of Controlled Release 31 (1994) 89-97

12

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Fig. 4. Time course of enzymatic hydrolysisof PHEG-peptide-MMCwith tritosomes. (a) Gly-Phe-Leu-Gly:( • ) total amount of released MMC, ([]) Giy-MMC,(o) MMC. (b) Gly-Phe-Gly:( • ) totalamountof released MMC, ([3) GIy-MMC,(e) MMC. (c) Gly-Phe-Ala-Leu: ( • ) total amountof released MMC, ([]) Leu-MMC,(•) MMC. (d) Ala-Leu-Ala-Leu,(•). Totalamountof releasedMMC ([]); Leu-MMC, (o). MMC Conditions of hydrolysis:see Fig. 3.

4. Discussion

It has been demonstated in the literature that binding of antitumor agents to a polymeric carrier via a proteolytically degradable oligopeptide sequence can lead to a polymeric prodrug with a high therapeutic efficiency when tested against tumor models in vivo [25]. The aim of the present work was to prepare polymeric prodrugs of Mitomycin C (MMC) that are hydrolytically stable but should be susceptible to degradation by lysosomal proteases. A number of conjugates were prepared by binding the cytotoxic agent

MMC via different oligopeptide spacers onto poly- [N(2-hydroxyethyl)-L-glutamine] (PHEG). The structures of the conjugates are given in Fig. 1. The MMC content (molar content, weight content) in the various conjugates is summarized in Table 1. The hydrolytic stability of the PHEG-tripeptideMMC and the PHEG-tetrapeptide-MMC conjugates was tested both at physiological and lysosomal pH. The results of these studies, shown in Fig. 2, indicate that the rate of MMC hydrolysis can be controlled by the selection of the appropriate terminal aminoacid in the oligopeptide spacer. The drug was released more rapidly from conjugates having a terminal glycine in the

94

A l)e Marre et at,/Journal ~f C'ontrolled Release 3I (1994) 89-97

60

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Time ( hours ) Fig. 5. Timecourse of PHEG-peptide-MMCconjugatesin serumand heat denaturatedserum: ( • ) Gly-Phe-Leu-Glyserum, ([]) Gly-Phe-LeuGly denat, serum, (o) Gly-Phe-Glyserum, (O) Gly-Phe-Glydenat, serum. 50

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Fig. 6. Time course of hydrolysisof PHEG-peptide-MMCin serum: (a) ( • ) Gly-Phe-Gly,(U]) Gly-Phe-Leu,(o) Gly-Phe-Phe,((3)) GlyGly-Phe; (b) ( • ) Gly-Phe-Leu-Gly,([]) Gly-Gly-Phe-Leu,(o) Ala-Leu-Ala-Leu,(C)) Gly-Phe-Ala-Leu. Table 1 Molar and weight percent substitution of MMC in the different PHEG-peptide-MMCconjugates Peptide

mol%

wt%

Gly-Phe-Gly Gly-Phe-Leu Gly-Phe-Phe Gly-Gly-Phe Gly-Phe-Leu-Gly Gly-Gly-Phe-Leu Gly-Phe-Ala-Leu Ala-Leu-Ala-Leu

1.7 2.9 3.2 3.4 2.2 3.0 3.6 2.9

3 5.1 5.5 5.9 3.9 5.2 6.0 5.0

side-chain compared to those with a terminal hydrophobic aminoacid. One of the key factors determining the biological activity of macromolecular antitumor prodrugs is their ability to deliver the drug intracellularly following pinocytic uptake by lysosomal digestion. The effect of incorporation of appropriate spacers in between the carrier and the bioactive moiety on the release of the drug by model and lysosomal enzymes has been studied by several authors [ 26-28 ]. Trouet et al. [27,29] have shown that optimal spacers for lysosomotropic delivery of daunomycin have a terminal

A. De Marre et al. / Journal of Controlled Release 31 (1994) 89-97

Ala-Leu sequence. In their early work Kopecek and Duncan [30-31] studied the release of p-nitroaniline, selected as a model compound, from poly[N-(2hydroxypropyl)-methacrylamide] (HPMA) copolymers with variable oligopeptide side-chains using thiolproteinases (Cathepsin B, L and H). The latter are important representatives of the lysosomal enzyme population. These studies revealed that both the length and the composition of the peptide spacers are crucial for the rate of drug release. Further studies have indicated that the spacer should consist of at least three aminoacids for the lysosomotropic (Cathepsin mediated) release of adriamycin, daunomycin and melphalan from HPMA copolymers [32-33]. The differential rates of degradation can be explained by the relative ease with which the polymeric substrates can form enzyme-substrate complexes. The aminoacids present in the oligopeptide spacer have discreet ability to interact with the individual subsites (P1-P4) of the active site of the enzymes [34]. Cathepsin D, a well-documented lysosomal endoproteinase, shows a clear specificity towards bonds between hydrophobic aminoacids [ 35 ]. Feijen reported on the synthesis and evaluation of poly-glutamic acid prodrugs of adriamycin. These studies demonstrated that conjugates having a peptide spacer with a terminal leucine are cleavable by lysosomal enzymes and show antitumor activity in vivo [36-37]. Since both the length and the composition of the spacer are of major importance, we have synthesized and characterized a number of conjugates with spacers with different aminoacid compositions. The release of MMC by lysosomal enzymes was investigated after incubation of the conjugates in presence of tritosomes. PHEG-GIy-Phe-Leu-Gly, PHEG-GIy-Phe-Ala-Leu and PHEG-Ala-Leu-Ala-Leu conjugates were rapidly hydrolysed by lysosomal enzymes yielding the free drug as well as GIy-MMC resp. Leu-MMC (Figs. 34). It is anticipated that the enzymatic degradation is propably mediated by iysosomal thiolproteinases (Cathepsin B, H and L) [30,38]. The release of GIyMMC from the PHEG-Gly-Phe-Gly- and PHEG-GlyPhe-Leu-Gly-MMC conjugates (Fig. 4) is in contrast with the results obtained by Duncan and Kopecek. In their study only free adriamycin and daunomycin could be observed during degradation of the corresponding

95

HPMA copolymers in presence of tritosomes [ 30,33,38]. From our in vitro degradation studies it follows that PHEG-GIy-Phe-Leu-Gly-MMC releases MMC very rapidly in the presence of tritosomes. Due to its low hydrolytic stability, it would be useful to design a conjugate with a comparable proteolytic lability but having a better hydrolytic stability. PHEG-tri- and PHEGtetrapeptide-MMC conjugates were prepared having a hydrophobic terminal amino acid in the spacer. Fig. 3a shows the results of the degradation studies of the PHEG-tripeptide-MMC conjugates. The release of MMC in presence of tritosomes is not significantly higher than that observed in the same buffer without enzymes. This indicates that tripeptide spacers are not a good substrate for lysosomal enzymes. Therefore, the Gly-Phe-Leu spacer was extended with one additional amino acid and again introduced onto the activated PHEG. Moreover, two polymeric tetrapeptide-MMC conjugates with a terminal Ala-Leu sequence were evaluated as well. The results of the degradation of the PHEG-tetrapeptide-MMC derivatives in presence of lysosomal enzymes is given in Fig. 3b. These data clearly indicate that a spacer with terminal Phe-Leu is not a good substrate for the lysosomal enzymes. Chain lengthening of the Gly-Phe-Leu spacer with one glycine has no influence on the enzymatic release rate of the corresponding polymeric tetrapeptide-MMC conjugate. Tetrapeptides with a terminal Ala-Leu in the spacer are very sensitive towards lysosomal hydrolases and release MMC very rapidly (80% after 3 h of incubation, Figs. 3-4). It should be noted that during degradation not only native MMC but also Leu-MMC was released. These data correspond with the results obtained by Trouet [27,29], investigating the release of daunomycin from prodrugs with Ala-Leu spacers by lysosomal hydrolases. Stability studies of Gly-MMC and Leu-MMC in buffer, pH 5.5, demonstrated that the aminoacid-drug fragments very rapidly release MMC (80-90% after 3 h of incubation). From the degradation experiments we can conclude that the PHEG-Gly-Phe-Leu-Gly-, PHEGGly-Phe-Ala-Leu- and PHEG-Ala-Leu-Ala-LeuMMC conjugates are promising candidates for the lysosomotropic delivery of MMC. For lysosomotropic delivery of polymeric prodrugs it is important to use conjugates that are stable during

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plasma circulation [ 39]. Therefore the stability of all PHEG-oligopeptide-MMC conjugates was investigated in serum. A rapid release of MMC in serum, even faster than in buffer, pH 7.4, is observed. In order to investigate the possible influence of enzymatic activity in serum, the stability of the Gly-Phe-Gly and Gly-PheLeu-Gly conjugates was determined in heat denaturated serum (Fig. 5). It was found that the drug release in denaturated serum is comparable to that in buffer, pH 7.4. This may indicate the presence of serum peptidases that can affect the release of MMC. Further studies will be necessary to elucidate these findings. The polymeric tripeptide-MMC conjugates having a terminal hydrophobic amino acid in the spacer show a higher stability in serum compared to those with a terminal glycine (Fig. 6a). Analogous data were obtained during incubation experiments of the tetrapeptide conjugates in serum. The derivatives having a terminal leucine in the spacer showed a higher stability in serum compared to those with a terminal glycine (Fig. 6b). However, for all conjugates the stability in serum is lower than in buffer, pH 7.4.

5. Conclusions It has been shown that polymeric prodrugs of MMC linked via oligopeptide spacers to PHEG are easily accesible. An appropriate strategy for their synthesis was worked out so that the sensitive parts of the MMC molecule are not touched during the chemical modifications. The influence of the oligopeptide spacer of PHEG-MMC conjugates on both the hydrolytic and the enzymatic stability was investigated. The results of this study demonstrate that conjugates with a terminal hydrophobic amino acid in the spacer are hydrolytically more stable than those with a terminal glycine. Moreover, conjugates with the tetrapeptides Gly-Phe-LeuGly, Gly-Phe-Ala-Leu and Ala-Leu-Ala-Leu are very rapidly degraded by lysosomal proteases and are good candidates for further in vitro and in vivo biological evaluation. The results of these biological studies will be reported in a forthcoming paper.

Ackowledgement This research project has been supported by the Belgian Institute for Encouragement of Research in Indus-

try and Agriculture (I.W.O.N.L.) and by the European Community, Concerted Action Programme. Thc authors are grateful to Kyowa Hakko Co., for the supply of MMC.

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