Synthesis, Conformation, Biodistribution, and Hormone-Related in

Bioconjugate Chem. 1996, 7, 642−650

642

Synthesis, Conformation, Biodistribution, and Hormone-Related in Vitro Antitumor Activity of a Gonadotropin-Releasing Hormone Antagonist-Branched Polypeptide Conjugate Ga´bor Mezo¨,† Imre Mezo¨,‡ Malcolm V. Pimm,§ Judit Kajta´r,| Jańos Sepro¨di,‡ Istvań Teplań,‡ Magdolna Kovaćs,⊥ Borba´la Vincze,@ Istvań Pa´lyi,@ Miklo´s Idei,‡ Ma´ria Szekerke,† and Ferenc Hudecz*,† Research Group of Peptide Chemistry, Hungarian Academy of Sciences, H-1518 Budapest, Hungary, 1st Instute of Biochemistry, Semmelweis University Medical School, H-1444 Budapest, Hungary, Cancer Research Laboratory, University of Nottingham, Nottingham NG7 2RD, U.K., Department of Anatomy, University Medical School, H-7643 Pećs, Hungary, National Institute of Oncology, H-1122 Budapest, Hungary, and Department of Organic Chemistry, Eo¨tvo¨s L. University, H-1518 Budapest, Hungary. Received March 11, 1996X

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3,DCpa2,D-Lys6,D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free γ-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

INTRODUCTION

The native gonadotropin-releasing hormone (GnRH)1 and its agonist analogues are capable of either stimulat* Ferenc Hudecz, Ph.D., D.Sc., Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s L. University, Budapest 112, POB 32, H-1518 Hungary. Fax: 36-12090602. Telephone: 36-1-2090555. † Hungarian Academy of Sciences. ‡ Semmelweis University Medical School. § University of Nottingham. | Eo ¨ tvo¨s L. University. ⊥ University Medical School. @ National Institute of Oncology. X Abstract published in Advance ACS Abstracts, October 15, 1996. 1 Abbreviations: AcEAK, poly[Lys(Ac-Glu 0.96-DL-Ala3.1)]; AK, poly[Lys(DL-Alam)]; AUC, area under the curve; BOP, [(benzotriazol-1-yl)oxy]tris(dimethylamino)phosphonium hexafluorophosphate; CD, circular dichroism; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; EAK, poly[Lys(Glu0.96-DL-Ala3.1)]; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; HOBt, 1-hydroxybenzotriazole; LH, luteinizing hormone; MI-1544, Ac(D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)-GnRH antagonist; PBS, phosphate-buffered saline; TEA, triethylamine; XAK, poly[Lys(XiDL-Alam)]; Z, benzyloxycarbonyl.

S1043-1802(96)00057-2 CCC: $12.00

ing or inhibiting secretion of the luteinizing hormone (LH) and the follicle-stimulating hormone (FSH), depending on the mode of their application (1). Knobil (2) published two different dose-dependent effects of GnRH agonists on the hypophysis. When GnRH agonists are given repeatedly, they desensitize the hypophysis, leading to decreases in gonadotropin and steroid release. In this way, they could inhibit the growth of hormonedependent tumors (3). It has been reported that prolonged clinical treatment with high doses of agonists resulted in reversible inhibition of gonadal function. There are differences between GnRH agonist and antagonist analogues in their mechanism of action. It has been demonstrated that a GnRH antagonist prevents GnRH action at the pituitary level by competitive GnRH receptor occupancy (4) rather than by down-regulation of GnRH binding sites. This GnRH antagonist did not affect the de novo synthesis of GnRH receptors in vitro (5). LH/FSH stimulation induced by native GnRH could be dose-dependently blocked by a GnRH antagonist, leading to inhibition of ovulation. Probably due to their different mode of action, GnRH antagonists may have stronger antitumor properties in vitro (6) than the agonists. The GnRH analogues exert their antitumor action through chemical castration (7), a partial hypophysec© 1996 American Chemical Society

GnRH Antagonist−Branched Polypeptide Conjugate

Figure 1. Schematic representation of MI-1544-AcEAK indicating the C- and the N-terminal portions of MI-1544 (OO and bb, respectively), starting from the grafting lysine residue.

tomy resulting in decreased levels of gonadotropin and prolaction (8), which consequently have a direct effect on mammary tumors (9-11). However, permanently high levels of GnRH agonist and antagonist analogues are necessary to reach direct and/or indirect antitumor effect (10). Therefore, much emphasis was put on the development of retarded-release devices for GnRH analogues and derivatives (12, 13). It has been found that the maintenance of a constant hormone analogue level can be efficiently achieved by formulation of microcapsules filled with GnRH agonists or antagonists (14-17). Alternatively, GnRH agonists or antagonists can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) potential prodrugs producing prolonged release or (b) macromolecular therapeutics with beneficially altered receptor binding or blood clearance. It should be noted that a similar strategy was successfully applied in passive tumor targeting of soluble macromolecules and drug conjugates (see reviews in refs 18-21). In order to evaluate the feasibility of this approach in relation to a GnRH antagonist, a prototype construct has been prepared with a potent GnRH antagonist Ac-D-Trp-D-CpaD-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH2 [Ac(D-Trp1,3,DCpa2,D-Lys6,D-Ala10)-GnRH, MI-1544] (22). As a carrier, a synthetic, biodegradable branched polypeptide with poly(L-lysine) backbone has been used (23-25). This polypeptide represents a new generation of the poly[Lys(Xi-DL-Alam)] (XAK) type poly-R-amino acids developed in our laboratory (21, 23) in which X is an acetylated derivative of glutamic acid (AcEAK) (26, 27). This modification of the carrier structure resulted in a polyanionic polypeptide in which predominantly free γ-carboxyl groups are present. Consequently, this molecule seems to be ideal for conjugation to MI-1544, which has only a single amino group at position 6 (Figure 1). In this paper, we describe (i) the synthesis and structural (primary structure, solution conformation) characterization of the MI-1544-AcEAK conjugate, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the MI1544-AcEAK conjugate studied by in vitro assays. MATERIALS AND METHODS DL-Alanine, glutamic acid, N-Z-lysine, imidazole, and acetic anhydride were purchased from Reanal (Budapest, Hungary). N,N-Diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBt), and bis(trichloromethyl) car-

Bioconjugate Chem., Vol. 7, No. 6, 1996 643

bonate were Fluka products (Buchs, Switzerland), while [(benzotriazol-1-yl)oxy]tris(dimethylamino)phospho-nium hexafluorophosphate (BOP) was from Bachem (Bubendorf, Switzerland). Dimethylformamide (DMF) was stored over molecular sieves. The human GnRH antagonist Ac(D-Trp1,3,D-Cpa2,DLys6,D-Ala10)-GnRH (MI-1544) was synthesized in our laboratory (MI) by the solid phase method on benzhydrylamine resin using NR-tert-butyloxycarbonyl amino acids as described earlier (22). The average degree of polymerization (DPn) of the polylysine used for the synthesis of AcEAK was estimated by sedimentation analysis and gel permeation chromatography (28). Sedimentation analysis was carried out in a MOM 3170 ultracentrifuge. The average relative molar mass of the branched polypeptides (AK, EAK, and AcEAK) was calculated from the DPn of the poly(Lys) backbone and from the amino acid ratios Ala:Lys or Glu: Ala:Lys as described earlier (23, 28). Coupling reactions between the GnRH antagonist and AcEAK were monitored by thin layer chromatography (TLC) on precoated silica gel plates purchased from Merck (Darmstadt, Germany) using ninhydrine, tolidine, and Ehrlich reagents. Abbreviations for amino acids and their derivatives follow the revised recommendation of the IUPAC-IUB Committee on Biochemical Nomenclature, entitled Nomenclature and Symbolism for Amino Acids and Peptides (29). The nomenclature of branched polypeptides is used in accordance with the recommended nomenclature of graft polymers (30). For the sake of brevity, codes of branched polypeptides were constructed by us using the one-letter symbols of amino acids. All amino acids are of the L configuration unless otherwise stated. Visking tubes with a 12000-14000 Da cutoff (Fisons, Loughborough, U.K.) were used for the dialysis of polymer products. Synthesis of Branched Polypeptides. (a) Poly[Lys(Glui-DL-Alam)] (EAK), where i ) 0.96 and m ) 3.1, was synthesized as previously described (31) with minor modification using HOBt as a catalyst in the active ester coupling method for the attachment of the glutamic acid residue to the end of the side chains of the branched polypeptide poly[Lys(DL-Ala3.1)] (AK) (32). Briefly, poly(L-Lys) (DPn ) 155) was prepared by the polymerization of NR-carboxy-N-(benzyloxycarbonyl)lysine anhydride. After cleavage of the protecting groups with HBr in glacial acetic acid, poly[Lys(DL-Ala3.1)] (AK) was formed by grafting of short oligomeric DL-alanine side chains onto the -amino groups of polylysine using N-carboxy-DLalanine anhydride (31). Attachment of suitably protected and activated glutamic acid residues to AK was carried out by the active ester method using a 50% molar excess of Z-Glu(OBzl)-OPcp and HOBt over AK in a 1:5 water/ DMF (v/v) solvent mixture in the presence of 4-methylmorpholine (at pH 7-8). The overnight reaction at room temperature was followed by removal of the solvent in vacuo, and the residue was triturated several times with ether containing 10% DCM. The protecting groups were cleaved with a HBr/acetic acid mixture afterward. Finally, EAK was purified by extensive dialysis using Visking tubing (molecular mass cutoff ) 12000-14000 Da) followed by lyophilization. The removal of protecting groups was verified by UV spectroscopy (31). Amino acid analysis yielded Lys:Ala:Glu ) 1:3.1:0.98. (b) Acetylation of Poly[Lys(Glu0.96-DL-Ala3.1)]. EAK (85 mg, 174 µmol) was dissolved in 2 mL of deionized water, and then the solution was diluted with 10 mL of DMF under cooling. TEA (24 µL, 174 µmol) was added to the

644 Bioconjugate Chem., Vol. 7, No. 6, 1996

solution for the neutralization of the polymer. During this time, 90 µL (0.95 mmol) of acetic anhydride and 63 mg (0.93 mmol) of imidazole were mixed in 2 mL of DMF at 0 °C. The preactivation was continued for 10 min, and then the mixture was added to the solution of EAK. The acetylation was continued for 1 h at 0 °C and then overnight at room temperature. The polymer was purified by extensive dialysis against water using Visking tubing (molecular mass cutoff ) 12000-14000 Da) and finally freeze-dried. No ninhydrine positive reaction was observed in the Kaiser test (33) (yield, 87 mg, 94%). Amino acid analysis yielded Lys:Ala:Glu ) 1:3.0:0.96. AcEAK which was to be labeled with 111In was conjugated to diethylenetriaminepentaacetic acid (DTPA) anhydride first (26, 27) and than acetylated as described above. Synthesis of Branched Polypeptide-Hormone Analogue Peptide Conjugates. AcEAK (49 mg, 95 µmol) was dissolved in 1 mL of deionized water, and its solution was diluted with 3 mL of DMF. Ac(D-Trp1,3,DCpa2,D-Lys6,D-Ala10)-GnRH (MI-1544) (60 mg, 47.5 µmol) (0.5 mol calculated for side chains of the polymer) dissolved in 3 mL of DMF was added to the solution of AcEAK. Following the addition of 42 mg (95 µmol) of BOP reagent in 1 mL of DMF, the pH of the reaction mixture was adjusted to pH ∼7.5 with N,N-diisopropylethylamine (DIEA) (65 µL, 382 µmol). The mixture was stirred overnight at room temperature. The solution was placed in Visking tubing (molecular mass cutoff ) 12000-14000 Da) and dialyzed extensively against deionized water for 3 days afterward. Finally, the conjugate, MI-1544-AcEAK was isolated by lyophilization (yield, 66 mg). Amino acid analysis yielded Lys:Glu:Ala:Ser: Tyr:Leu:Arg:Pro:Trp ) 1.7:0.98:3.35:0.35:0.29:0.35:0.33: 0.34:0.69. MI-1544 conjugate of AcEAK-DTPA for labeling with 111In was prepared as described above using AcEAKDTPA instead of AcEAK. Amino Acid Analysis. The amino acid composition of branched polypeptides (AK, EAK, and AcEAK) and MI1544-AcEAK conjugates was investigated by amino acid analysis using a Beckman (Fullerton, CA) model 6300 amino acid analyzer. Under these conditions, Cpa could be determined at the position of the Lys residue. Prior to analysis, samples were hydrolyzed in 6 M HCl in sealed and evacuated tubes at 110 °C for 24 h. Trp content was measured by UV spectroscopy at λ ) 280 nm ( ) 10 000) in a solution of 0.1 M NaOH. GPC-HPLC (Gel Permeation ChromatographyHigh-Performance Liquid Chromatography) Analysis of the MI-1544-AcEAK Conjugate. Instrumentation. The HPLC system consisted of a VARIAN 9021 solvent delivery system and of a Varian 9065 dioda array detector (all from Varian, Zug, Switzerland) and a Model 7125 injector valve (Rheodyne, Cotati, CA). A Biosil TSK 125 type 30 cm × 7.5 mm column and a 5 cm × 7.5 mm guard column (Biorad, Richmond, CA) were used. Conditions. The mobile phase was 0.25 M triethylammonium phosphate buffer (pH 2.25) prepared daily using high-purity (Analar) components and distilled, deionized water. The injection volume was 10 µL containing 10 µg of MI-1544-AcEAK, AcEAK, or MI-1544 dissolved in eluent immediately before application and filtered through 0.45 µm Spartan 13 (Schleicher and Schuell, Dassel, Germany) filters. Free MI-1544 or AcEAK samples were run as standards, and their retention times were determined. UV absorbance was monitored at wavelengths of 215 and 280 nm. All analyses were carried out at ambient temperature with a flow rate of 0.5 mL/min.

Mezo¨ et al.

Figure 2. CD spectra of MI-1544 (- -), AcEAK (-‚-‚-), and their conjugate MI-1544-AcEAK (s) at pH 7.3 in 0.02 M NaCl.

Circular Dichroism Spectroscopy. Circular dichroism (CD) was measured with a Roussel-Jouan model III dichrograph (Jobin-Yvon, Longjumean, France). CD spectra from 195 to 260 nm were recorded in a quartz cell with an optical path of 0.02 cm at room temperature. The dichrograph was calibrated with D-(-)-pantoyllactone at 220 nm (34). CD spectra of free MI-1544, AcEAK, and their conjugate MI-1544-AcEAK were recorded in 0.02 M NaCl solution at pH 7.2, the concentrations of solutions being about 0.5 mg/mL (Figure 2). The data of CD in the figure are expressed in terms of ∆, calculated for one lysine residue in the polypeptide backbone carrying an average side chain. The interpretation of CD spectra was based on the well-characterized CD curves of polylysine (35), from which the branched polypeptides have been derived. Labeling with 125I. 125I labeling of MI-1544 and MI1544-AcEAK-DTPA was carried out with [125I]sodium iodide [125I for protein iodination (IMS.30) in NaOH at pH 7-11, Amersham International plc, Amersham, U.K.], by oxidative incorporation into the tyrosine of the MI-1544 using Iodogen (1,3,4,6-tetrachloro-3R,6R-diphenylglycoluril) as the oxidizing agent. One hundred micrograms of Iodogen [Pierce and Warriner (U.K.) Ltd., Chester, U.K.] was coated onto the inner surface of polypropylene conical microfuge tubes by evaporation under nitrogen from solution in methylene chloride. Two hundred micrograms of MI-1544 or MI-1544-AcEAKDTPA in 200 µL of PBS (pH 7.2) and ∼5 MBq of radioiodine solution were added to the tubes, and the solution was mixed with a Pasteur pipette. After incubation at room temperature for 10 min, the reaction was stopped by removal of the mixture from the reaction tube. Labeled MI-1544-AcEAK-DTPA was purified of unreacted [125I]sodium iodide by passage through Sephadex G-25, elution being in PBS at pH 7.2, using prepacked PD-10 columns (Pharmacia, Milton Keynes, U.K.). Labeled MI-1544 was purified by dialysis against PBS (3 × 1000 volumes over 24 h at 4 °C) in 500 molecular mass cutoff dialysis tubing [Spectrum Spectra/Por CE, molecular mass cutoff ) 500 Da, Pierce and Warriner (U.K.) Ltd.]. Specific activities of the final products were ∼2 MBq/mg. Labeling with 111In. 111In labeling by chelation to the DTPA of AcEAK-DTPA and MI-1544-AcEAK-DTPA was carried out by addition of 5 MBq of [111In]InCl3 (indium chloride in 0.04 M HCl, INS.1P, Amersham International) to solutions of 200 µg in 200 µL of pH 6.0

GnRH Antagonist−Branched Polypeptide Conjugate

0.3 M sodium citrate/citric acid buffer. After 10 min at room temperature, the reaction mixtures were purified of unbound 111In by passage through Sephadex G-25, elution being in PBS. Specific activities of the final products were ∼2-10 MBq/mg. Blood Clearance and Biodistribution Studies. All in vivo studies were carried out in adult (∼20 g) female Balb/c mice (Biomedical Services Unit, University of Nottingham) with appropriate licences from the U.K. Home Office and with due consideration for animal welfare. Groups of mice (n ) 4) were injected intravenously via a tail vein with ∼20 µg of labeled materials in 0.2 mL of PBS. Serial blood samples (10 µL) were taken from the tail tip at 1, 10, and 30 min and at 1, 2, and 3 h after injection directly into microcapillary pipettes (Drummond Microcaps, Drummond Scientific Co., Broomhall, PA). Blood clearance curves were constructed of the percent of injected count rates in the total blood volumes against time [the total intravascular blood volumes were calculated assuming the blood volume of the mice (in milliliters) to be 11.2% of the body weight (in grams)]. Areas under curves (AUC), as percent dose per hour, were calculated using the trapezoidal rule. At 3 h after injection, mice were killed, and weighed samples of blood, visceral organs, and residual carcass were assayed for radioactivity. Results of the tissue distribution analysis were expressed as a percentage of the initially injected count rate recovered in the animal as a whole, and per gram of individual tissue. In Vivo Ovulation Inhibition Assay. Adult female rats (Wistar-R-Amsterdam), 200-250 g, were housed under controlled lighting (light period from 5:00 to 19: 00) with free access to pelleted food and tap water. MI-1544 or its AcEAK conjugate was dissolved in a 4060% mixture of propylene glycol and of 0.9% sodium chloride solution. Aliquots of the solutions containing 10, 50, 75, and 100 µg of MI-1544-AcEAK and 1.5, 3, and 30 µg of MI-1544 were injected subcutaneously (sc) to female rats on the day of proestrous at noon. Only those rats that previously showed three consecutive regular 4-day cycles as tested from vaginal smears were used for these experiments. Animals were ovariectomized on the day of the first diestrous after the injection. Oviducts were prepared under a stereomicroscope and tested for ova as described earlier (22). In Vitro Inhibition of LH Release. The GnRH antagonistic activity of MI-1544 and the MI-1544AcEAK conjugate was assayed by using the superfused rat pituitary cell system (36, 37). Mixed cell populations of four regularly cycling female rats at random stages of the estrous cycle were used in each chamber of the superfusion apparatus. The cells were perfused overnight with Medium 199 (Sigma, St. Loius, MO) at a flow rate of 20 mL/h, and administration of the compounds was started the next morning. Pituitary cells were exposed to 9 min pulses of MI-1544 and the MI-1544AcEAK conjugate in concentrations of 1, 5, 10, 50, and 100 nM prior to 1 nM GnRH. The peptide-containing solutions were prepared from the stock solutions immediately before use. At the beginning of each experiment, cells were challenged with 50 mM K+ and two or three pulses of 1 nM GnRH for 3 min. At the end of the experiments, cells were rechallenged with K+ to check the hormone receptor-independent LH secretion. One milliliter fractions per 3 min were collected, and the concentration of LH released in the system was determined by radioimmunoassay (RIA). Two independent experiments were performed for each concentration, and


the mean of data is summarized. The interassay variation was less than 10%. NIAMD RIA kits (National Hormone and Pituitary Program) were used for the LH assay (LH-S-9 standard, LH-RP-2 reference preparation, and LH-1-6 hormone for iodination). The superfusion data were analyzed by a special computer program (37). The amount of LH secreted was calculated from the peaks above the baseline after stimulation. Clonogenic Assay. The human mammary carcinoma cell lines MCF-7 (38) and MDA-MB-231 (39) were used throughout. The cells were grown in Dulbecco’s modified Eagle MEM (D-MEM) medium (GIBCO) supplemented with 5% estrogen-depleted (dextran-charcoal-treated) fetal calf serum (FCS-DCC). MCF-7 cells are estradiol (E2) responsive, and MDA-MB-231 cells are E2 unresponsive. Three hundred cells were plated in 35 mm plastic petri dishes in 2.5 mL of medium. The next day, the cells were treated with doses of MI-1544, AcEAK, the MI-1544AcEAK conjugate, and the MI-1544/AcEAK mixture. Compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with complete medium to reach 10, 30, and 50 µM final concentrations related to MI-1544 content. The cultures were incubated at 37 °C in a humidified CO2 incubator (Heraeus) for 8-12 days. The cultures were then stained with crystal violet, and the colonies were counted under a stereomicroscope. Relative cloning efficiency of the treated cultures was expressed as a percentage of untreated controls (40). RESULTS AND DISCUSSION

Preparation of the Hormone Analogue-Branched Polypeptide Conjugate. In order to prepare a structurally well-defined conjugate of the GnRH antagonist, Ac(D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)-GnRH (MI-1544) with a single amino group at the lysine residue in position 6 and branched polypeptide AcEAK with free γ-carboxyl groups were produced from the amphoteric poly[Lys(Glu0.96-DL-Ala3.1)] (EAK) for coupling. Acetylation of EAK was performed in water/DMF (1:5 v/v) after neutralization of the R-amino groups of the polymer with imidazolyl acetate. The efficiency of the reaction was checked by ninhydrine after the isolation of the polymer product (AcEAK), and no detectable amount of free amino groups was observed. This procedure represents a new alternative to the previously described reaction scheme, where acetic anhydride was used as the acetylation agent (25, 26). The coupling of MI-1544 to AcEAK was achieved by the BOP reagent-based activation method in which the γ-carboxyl group of glutamic acid of AcEAK was linked to the -amino group of lysine residue in the hormone analogue peptide to provide covalent isopeptide (γ, type amide) bonding. The carboxyl groups of the polymeric polypeptide were activated by equimolar BOP in situ using a tertiary amine (DIEA). It should be noted that no precipitate was observed during the synthesis of the conjugate under these conditions. GPC-HPLC studies at two wavelengths were carried out to access free GnRH analogue in the MI-1544AcEAK conjugate preparation. The retention time for MI-1544, absorbing at both 215 and 280 nm, was found to be 38.1 min. Free AcEAK was detected at 215 nm at the void volume, whereas the tR value for MI-1544AcEAK was 19.5 min at both 215 and 280 nm, indicating the presence of covalently bound MI-1544. Quantitative


Mezo¨ et al. Table 1. Biodistribution Parameters of MI-1544, Polypeptide AcEAK, and Their Conjugatesa radiolabeled material

area under curve (% dose/h ( SD)

retention (% ( SD)

[125I]MI-1544 [125I]MI-1544-AcEAK [111In]AcEAK-MI-1544 [111In]AcEAK

17.8 ( 1.1 50.3 ( 1.6 57.6 ( 9.6 188.2 ( 0.8

38.8 ( 4.9 60.2 ( 1.8 80.3 ( 4.0 41.0 ( 2.1

a

Mean of four mice per group to 3 h after injection.

Figure 3. Blood clearance curves of 125I-labeled MI-1544, its conjugate with AcEAK labeled with 125I or 111In, and 111Inlabeled AcEAK, with four mice per group. The areas under these time-activity curves are shown in Table 1.

analysis indicated less than 50 ppb of free MI-1544 in the conjugate samples. The amino acid composition of purified conjugates was determined by amino acid analysis. Considering the amino acid composition of the free carrier (AcEAK) and of the free peptide, the average degree of molar substitution (DS) was calculated, and it was found that DS ) MI-1544:AcEAK ) 51:1 mol/mol. This value indicates that 33% of the free carboxyl groups of AcEAK was modified by MI-1544. Consequently, the MI-1544-AcEAK conjugate still possesses polyanionic character. Conformation of the MI-1544-AcEAK Conjugate. The chiroptical properties of the conjugate in solution were studied by CD spectroscopy in the 190-250 nm wavelength region. CD spectra were recorded in water solution at physiological pH (pH 7.3) and 0.02 M NaCl ionic strength. The CD curves of free AcEAK, GnRH antagonist, and their conjugate are shown in Figure 2. In this wavelength region, the circular dichroic spectra indicate an essentially disordered conformation for the unsubstituted AcEAK. The CD signals in the 220-240 nm region of Ac(D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)-GnRH are indicative of the presence of aromatic amino acid residues. In the case of the MI-1544-AcEAK conjugate, with this degree of substitution (33%), the shape of the CD spectrum in the 190-215 nm range was similar to the spectrum of AcEAK and corresponds to the unordered steric arrangement of the polypeptide backbone. Interestingly enough, a strong CD band was observed in the 220-240 nm wavelength region. This signal originated from the optical activity of the attached MI-1544 moiety. The red shift observed in the position of the CD bands (λ ) 202 nm of AcEAK and λ ) 226 nm of MI-1544) in the conjugate could verify the presence of MI-1544 peptide. Previous studies provided evidence (41-44) that, besides the choice of carrier polypeptide, the number of hapten molecules attached to the carrier polypeptide influences markedly the spatial arrangement of the conjugate. Biodistribution of Free and Conjugated MI-1544. 125 I labeling of GnRH and its derivatives into tyrosine by the chloramine T method is an established way of labeling the hormone-related peptides (44, 45). For our studies, radioiodinated MI-1544 and its AcEAK conjugate (MI-1544-AcEAK) were prepared with a similar strategy using Iodogen as the oxidizing agent. Similar to data reported on the pharmacokinetics of GnRH and its analogues after iv injection (47, 48), we found that [125I]MI-1544 was cleared rapidly from circulation (Figure 3), with an AUC of only 17.8% dose/h (Table 1). The major sites of retention of 125I at the 3 h dissection time were

Figure 4. Biodistribution of 125I-labeled MI-1544, its conjugate with AcEAK labeled with 125I or 111In, and 111In-labeled AcEAK 3 h after injection, with four mice per group. The whole body retentions of the radiolabels are shown in Table 1.

kidney and liver, with about 15% of the injected dose/g in each of these organs, and the lung with about 7% (Figure 4). However, the whole body retention of 125I was only 38.8% (Table 1), showing that much of the [125I]MI1544 had been excreted unchanged (via the kidneys) and/ or 125I had been removed from its site(s) of clearance following its metabolism and excreted as [125I]iodotyrosine or as free [125I]sodium iodide. In contrast, [125I]MI-1544 conjugated to AcEAK ([125I]MI-1544-AcEAK) showed longer blood survival (Figure 3), with an AUC of 50.3% dose/h (Table 1). There was also a different pattern of biodistribution, with reduced levels in the lung but higher levels in spleen and liver (Figure 4). The higher levels in these organs, and the greater blood survival, resulted in an increased whole body retention of 125I, at about 60%. The blood clearance curve and AUC of [111In]AcEAK conjugated to MI-1544 ([111In]AcEAK-MI-1544) was virtually identical to that of the conjugate labeled in the MI-1544 with 125I (Figure 3, Table 1). The main differences between their biodistributions were a reduced level in the spleen and a higher level in the liver with the 111In-labeled conjugate (Figure 4), which was reflected in a greater whole body retention of about 80% (Table 1). 111In-labeled AcEAK showed the greatest blood survival (Figure 3, Table 1), but with reduced levels in spleen, kidney, and liver compared with MI-1544-AcEAK labeled in either the MI-1544 or AcEAK parts of the conjugate. The results of this comparative study suggest that the survival of free MI-1544 is prolonged and its tissue distribution is altered by conjugation to AcEAK. AcEAK in the conjugate elevated significantly the presence of MI1544 in the circulation (AUC for the conjugate was 3 times higher than that for MI-1544) and in the whole body (1.5-2-fold increase for MI-1544-AcEAK compared to that of free MI-1544). Interestingly enough, the blood clearance profile for both 125I- or 111In-labeled conjugates was almost identical, indicating the lack of free labeled


GnRH Antagonist−Branched Polypeptide Conjugate Table 2. Short-Term and Long-Term Antiovulatory Effect of MI-1544 and the MI-1544-AcEAK Conjugate in Cycling Rats effect inhibition at (h)

1.5 µg 35%a (4/6) ntb

3 µg

30 µg

MI-1544 100% (0/5) nt

50 µg

75 µg

nt

nt

nt

nt

nt

nt

24

100% (0/6) 90% (1/6) nt nt 30% (4/6) MI-1544-AcEAK nt nt nt

48

nt

nt

nt

0% (4/4) nt

72

nt

nt

nt

nt

24 48 72

90% (1/10) 60% (3/7) 40% (5/8)

Table 3. GnRH Inhibitory Effect of MI-1544 and Its Branched Polypeptide Conjugate (MI-1544-AcEAK) in an in Vitro Cell Perfusion System GnRH:antagonist MI-1544 MI-1544-AcEAK molar ratio (nmol/nmol) NI/Ra inhibition (%) NI/Ra inhibition (%) 1:0 1:1 1:5 1:10 1:50 1:100

1.00 0.05 0.02

95 98

1.00 1.00 0.89 0.57 0.04 0.02

0 11 43 96 98

a Net integrated LH released by GnRH plus antagonist/net integrated LH released by GnRH alone (i.e. reference).

a Antiovulatory activity % (number of rats ovulating/total number of rats). b nt ) not tested. Compounds were administered subcutaneously on the day of proestrus at noon.

MI-1544 and free AcEAK and the stability of the γ, amide bond in the circulation. In contrast, the whole body retention and the spleen and kidney uptake were not the same for the two conjugates. For the interpretation of these findings, it should be borne in mind that the retention of 111In tends to be different from that of 125 I at the site of clearance (27). This means that the tissue biodistribution results with 111In-labeled and 125Ilabeled MI-1544-AcEAK are unlikely to be identical. Our recent comparative study with branched polypeptides indicated that generally kidney, spleen, and liver levels of radiometals (111In or 51Cr) were higher than those of radioiodine, while levels in the gastrointestinal tract were higher with radioiodine (49). It is important to note that the covalent attachment of MI-1544 to branched polypeptide AcEAK resulted in some alterations (e.g. reduction in blood survival and increased liver, kidney, and spleen uptake) in the biodistribution of the free carrier. This observation has to be considered for the design of future conjugates of MI1544. Inhibition of Ovulation. The inhibitory effect of the GnRH analogue, MI-1544, and the MI-1544-AcEAK conjugate is presented in Table 2. A marked difference was found in the short-term (24 h) antiovulatory potency of the analogue and the conjugate. The minimal antiovulatory dose (ED100) of MI-1544 was found to be 3 µg, while its AcEAK conjugate was only partially (90%) inhibitory even with a 75 µg dose. Interestingly, in the long-term (72 h) antiovulatory study, there was 30% inhibition with free MI-1544 with a 30 µg dose. A similar effect was observed with the MI-1544-AcEAK conjugate at a higher dose (40% inhibition at 75 µg). These data suggest that the MI-1544-AcEAK conjugate was able to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single sc injection. The necessity for high-dose conjugate administration to achieve an antiovulatory effect comparable to that of free antagonist indicates differences in the bioavailability and/or pharmacokinetic properties (e.g. absorption and/or transport) of the free and conjugated MI-1544. GnRH Antagonistic Effects. The in vitro GnRH inhibitory effect of MI-1544 and the MI-1544-AcEAK conjugate is presented in Table 3. MI-1544 prevented the LH-releasing effect of 1 nM GnRH even at 1 nM (95% inhibition), while MI-1544-AcEAK showed this effect (96% inhibition) only at a 50 nM concentration. At

Figure 5. Effect of free MI-1544 (0), its AcEAK conjugate (2), free AcEAK (b), and the mixture of MI-1544 and AcEAK (×) on the colony formation capacity of estradiol responsive (MCF7) (A) and of estradiol unresponsive (MDA-231) (B) human mammary carcinoma cell lines. Relative cloning efficiencies of the treated cultures are expressed as a percentage of untreated controls.

concentrations of 1, 5, and 10 nM, it produced 0, 11, and 43% inhibition, respectively. These results show that MI1544-AcEAK is capable of inhibiting LH release with high efficacy, but for this, the molar ratio of antagonist: GnRH must be 50-100. In contrast, the same level of inhibition can be achieved by antagonist:GnRH ratios of 1-5 with the free MI-1544. Consequently, the MI-1544AcEAK conjugate is 20-50 times less inhibitory for GnRH than MI-1544 in the in vitro cell superfusion system. These findings might reflect the differences between the interaction of pituitary cells with free or covalently coupled MI-1544. Clonogenic Assay. MCF-7 cells were essentially insensitive to the effect of the GnRH antagonist MI-1544. At a 50 µM mg dose, the cell survival was reduced only by 10%. The cells were also resistant to free AcEAK (Figure 5A). MDA-MB-231 cells were somewhat more sensitive to the antagonist (Figure 5B), the relative plating efficiency being reduced to 78% compared to the controls. AcEAK did not reduce cell survival at all


(Figure 5B). The MI-1544-AcEAK conjugate, however, decreased cell survival by 70% (MCF-7) and 50% (MDA231), respectively (Figure 5A,B). It should be noted that, in control experiments, when the mixture of free peptide and free carrier was applied, the survival profile in both cell lines was similar to that when free peptide was administered (Figure 5). These data show that the survival of both cell lines could be only slightly reduced by the treatment with the antagonist MI-1544 in the concentration range studied. Conjugation but not mixing of this peptide with AcEAK however increased its effect significantly. The difference between the effect of the free peptide and the conjugate was especially considerable in the case of MCF-7 cells. The damaging effect of the MI-1544-AcEAK conjugate observed in the clonogenic assay is supposed to be the result of a direct antitumor effect, which can be explained by the presence of GnRH-binding sites on both MCF-7 and MDA-MB-231 mammary carcinoma cells (10). According to the literature, the GnRH antagonists inhibit the GnRH receptor-mediated signal transduction by blocking the GnRH receptors (50, 51). As the compounds were given to the cultures only once during the 8-12 days of incubation, it is thought that the moderate effect of the free peptide compared to that of the conjugate can be accounted for by its limited stability. This observation was supported by a comparative study in which the effects of these compounds were investigated on cell proliferation (52). Conclusion. These studies have indicated that it is possible to prepare a synthetic branched polypeptide conjugate of a potent GnRH antagonist Ac(D-Trp1,3,DCpa2,D-Lys6,D-Ala10)-GnRH (MI-1544) with a relatively high molar substitution ratio. In this construct, a stable γ, type amide bonding was introduced between the carboxyl groups of the carrier AcEAK and the amino group at position 6 of MI-1544 to achieve prolonged body retention of the antagonist with maintained hormonerelated antitumor activity. This conjugate, as revealed by CD spectroscopy, adopts a disordered conformation under physiological condition, indicating the sterically random arrangement of the carrier backbone and allowing potential accessibility of the GnRH antagonist to recognition by relevant receptors. Data presented in this paper have demonstrated that in fact (a) the blood/body presence of MI-1544 can be significantly prolonged and (b) the direct in vitro antitumor effect of MI-1544 can be maintained or even enhanced by its coupling to an anionic branched polypeptide, AcEAK. On the other hand, in this study, we have shown that this prototype conjugate was able (a) to antagonize the effect of GnRH in vitro and (b) to prevent ovulation, but its efficiency was markedly decreased. Although the precise mechanism of conjugate action is unclear, our in vitro data indicate that the presence of biodegradable polymeric carrier could provide new route(s) of cellular uptake and/or cell-antagonist interaction, which are not available for the free GnRH antagonist. This speculation is in accord with recently published in vitro data on MI-1544 conjugated to a nonbiodegradable polymer, poly(N-vinylpyrrolidonecomaleic anhydride) (53). For the reliable explanation of these results, more experimentation is needed to clarify potential aspects of polymer activity. Taken together, these results can be considered as a proper basis for further development of the conjugate structure by e.g. selecting a different carrier, an attachment site, or a spacer for producing a functionally further improved bioconjugate.

Mezo¨ et al. ACKNOWLEDGMENT

This work was supported by grants from the Hungarian Academy of Sciences (OTKA 2994 and T 4217), from the Ministry of Health (ETT T-569/90 and T 405), and from the Association for International Cancer Research. We thank Ms. Eniko¨ Nagy, Ms. Eva Fleischman, and Ms. Sandra Gribben for the experimental assistance. The authors are greatly indebted to Ms. Judit Ma´the´ for excellent editorial assistance. We are grateful to the National Hormone and Pituitary Program (NHPP) for providing the RIA kits. M.V.P. is supported by the Cancer Research Campaign, London, U.K. LITERATURE CITED (1) Hazum, E. (1988) Molecular mechanism of gonadotropin releasing hormone (GnRH) action. The GnRH receptor. Endocr. Rev. 9, 379-386. (2) Knobil, E. (1980) The neuroendocrine control of the menstrual cycle. Recent Prog. Horm. Res. 36, 53-88. (3) Lissoni, P., Barni, S., Crispino, S., Cattaeno, G., and Tancini, G. (1988) Endocrine and clinical effects of LHRH analogue in pretreated advanced breast cancer. Tumori 74, 303-308. (4) Folkers, K., Bowers, C. Y., Sieh, H. M., Yin-Zeng, L., SaoBo, X., Tang, P. F. L., and Ji-Yu, C. (1984) Antagonists of the luteinizing hormone releasing hormone (LHRH) with emphasis on the Trp7 of the salmon and chicken II LHRH’s. Biochem. Biophys. Commun. 123, 1221-1226. (5) Braden, T. D., and Conn, P. M. (1990) Altered rate of synthesis of gonadotropin releasing hormone receptors: effects of homologous hormone appear independent of extracellular calcium. Endocrinology 126, 2577-2582. (6) Bajusz, S., Kovaćs, M., Gazdag, M., Bokser, L., Karashima, T., Csernus, V. J., Jana´ky, T., Guot, J., and Schally, A. V. (1988) Highly potent antagonists of LHRH free edematogenic effects. Proc. Natl. Acad. Sci. U.S.A. 85, 1637-1641. (7) Pedrosa, E., Vilchez-Martinez, J. A., Coy, D. H., Arimura, A., and Schally, A. V. (1980) Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analogue of luteinizing hormone-releasing hormone. Biochem. Biophys. Res. Commun. 95, 1056-1062. (8) Lamberts, S. W. J., Vitterlinden, P., Zuiderwijk-Roest, J. M., Bons-van Evelingen, E. G., and De Jong, F. H. (1981) Effects of a luteinizing hormone-releasing hormone analog and tamoxifen in the growth of an estrogen-induced prolactinsecreting rat pituitary tumor and its influence on pituitary gonadotropins. Endocrinology 108, 1878-1884. (9) Miller, W. R., Scott, W. N., Morris, R., Frase, H. M., and Sharpe, R. M. (1985) Growth of human breast cancer cells inhibited by a luteinizing hormone-releasing hormone agonist. Nature 313, 231-233. (10) Vincze, B., Pa´lyi, I., Daubner, D., Kremmer, T., Sza´mel, I., Bodrogi, I., Suga´r, J., Sepro¨di, J., Mezo¨, I., Teplań, I., and Eckhardt, S. (1991) Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts. J. Steroid Biochem. Mol. Biol. 38, 119-126. (11) Pa´lyi, I., Vincze, B., Ka´lnay, A., Gaa´l, D., Mezo¨, I., Sepro¨di, J., and Teplań, I. (1994) Antitumor effect of new chicken GnRH analogues in mammary cell lines. Ann. Oncol. 5 (Suppl. 5), 85. (12) Karten, M. J., and Rivier, J. E. (1986) Gonadotropinreleasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspectives. Endocr. Rev. 7, 44-66. (13) Danforth, D. R., Williams, R. F., Gordon, K., Leal, J. A., and Hodgen, G. D. (1991) Inhibition of pituitary gonadotropin secretion by the gonadotropin-releasing hormone antagonist antide. Endocrinology 128, 2041-2044.

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BC9600574

Synthesis, Conformation, Biodistribution, and Hormone-Related in

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