Design, Synthesis, In Vitro Stability and Cytostatic Effect of Design, Synthesis,Anticancer Drug-Bioconjugates Containing GnRH-III

Multifunctional In Vitro Stability and Cytostatic Effect of Multifunctional Anticancer Drug-Bioconjugates Containing GnRH-III as a Targeting Moiety as a Targeting Moiety
¨ ´ ´ ¨ Ulrike Leurs,1 Gabor Mez},2 Erika Orban,2 Peter Ohlschlager,3 Andreas Marquardt,4 o 1,5 Marilena Manea
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Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, 78457 Konstanz, Germany ´ ¨ ¨ Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos Lorand University, 1117 Budapest, Hungary Department of Biology, Laboratory of Immunology, University of Konstanz, 78457 Konstanz, Germany Department of Biology, Proteomics Facility, University of Konstanz, 78457 Konstanz, Germany Zukunftskolleg, University of Konstanz, 78457 Konstanz, Germany

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Received 25 January 2011; revised 16 March 2011; accepted 28 March 2011 Published online 20 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21640

ABSTRACT:
Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule. Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a
Additional Supporting Information may be found in the online version of this article. Correspondence to: Dr. Marilena Manea, University of Konstanz, Zukunftskolleg and Department of Chemistry, Laboratory of Analytical Chemistry and Bio¨ polymer Structure Analysis, Universitatsstrasse 10, 78457 Konstanz, Germany; e-mail: marilena.manea@uni-konstanz.de Contract grant sponsor: University of Konstanz (Zukunftskolleg) Contract grant number: 879/08 Contract grant sponsor: University of Konstanz (AFF and Young Scholar Fund) Contract grant number: 01/09 Contract grant number: 836/09 Contract grant sponsor: Hungarian National Science Fund (OTKA NK) Contract grant number: 77485
C V 2011

chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [4Lys]-GnRH-III (Glp-His-Trp-Lys-His-AspTrp-Lys-Pro-Gly-NH2) containing two lysine residues in positions 4 and 8, whose e-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-ProGly-NH2) was used as a scaffold; an additional lysine residue was coupled to the e-amino group of 8Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines. Compared with a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect. # 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 1–10, 2012. Keywords: gonadotropin-releasing hormone-III; multifunctional anticancer drug-peptide bioconjugates; drug design; targeted cancer chemotherapy; cytostatic effect

Wiley Periodicals, Inc.

PeptideScience Volume 98 / Number 1

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This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com

INTRODUCTION

T

he human decapeptide hormone GnRH-I (Glp-HisTrp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) plays an important role in the regulation of the reproduction by stimulating the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. LH and FSH then stimulate the sex-steroid hormone production and gametogenesis in the gonads to ensure reproductive competence. Various GnRH agonists and antagonists have been developed and some of them are in clinical use for the treatment of hormone-dependent tumors of the reproductive tract. However, a major drawback in the use of agonists is the so-called ‘‘flare-up,’’ an overproduction of steroid sex hormones immediately after administration of the agonist which could aggravate the disease. Furthermore, GnRH agonists cause a down-regulation of GnRH receptors, leading to a profound decrease in gonadotropin secretion and chemical castration in long-term use.1 Because of the fact that GnRH receptors (GnRH-Rs) are not only found in the pituitary, but are also highly expressed on various tumor types, the GnRH derivatives can be used directly for the treatment of cancer and employed as targeting moieties for the attachment of chemotherapeutic agents. GnRH-R positive cancers include breast, ovarian, endometrial, prostate, colon, oral, laryngeal cancers, as well as melanomas and nonHodgkin’s lymphoma.2,3 Bioconjugates containing GnRH-I analogs as targeting moieties for the attachment of cytotoxic agents were initially developed by A. V. Schally’s group, among them AN152 containing [D-6Lys]-GnRH-I as a targeting moiety conjugated to doxorubicin.4 As shown by cellular uptake studies, AN-152 was internalized in a receptor-mediated way by GnRH-R positive cells. No uptake was observed on GnRH-R negative cells or after blocking the receptors with the superagonist Triptorelin.5 Apoptosis was induced in GnRH-R positive human ovarian and endometrial cancer cell lines by AN-152, without activating the MDR-1 (multi-drug resistance-1) efflux pump system.6 Furthermore, in 2002 Krebs et al. could show that AN-152 exhibited higher cytotoxicity on KB human oral carcinoma cells and HEp-2 human laryngeal carcinoma cells than the free drug.7 This finding suggests that the drug resistance of these cancer cell lines can be overcome by drug targeting

through the GnRH receptor. Taken together, these results show that tumor targeting through the GnRH-R can deliver chemotherapeutic agents specifically to cancer cells, while reducing their peripheral toxicity and overcoming drug resistance. However, it is important to note that the administration of GnRH-I based bioconjugates might be accompanied by endocrine side effects. GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-GlyNH2) is a native isoform of the human GnRH-I isolated from the brain of the sea lamprey (Petromyzon marinus) in 1993.8 It is a weak GnRH agonist which exerts a direct antiproliferative effect on cancer cells9; furthermore, the LH and FSH releasing potency of GnRH-III is insignificant in mammals.10 These observations reveal the advantages of GnRH-III to be used as a targeting moiety for drug delivery. In our previous work, bioconjugates containing the GnRH-III peptide as a targeting moiety and daunorubicin (also called daunomycin) as a chemotherapeutic agent were prepared as drug delivery systems for targeted cancer chemotherapy. These daunorubicin-GnRH-III bioconjugates had both in vitro and in vivo cytostatic activity without significant toxic side effects.11,12 As the GnRH-Rs are known to desensitize under continuous hormone treatment,13 this could lead to a resistance of the cancer cells towards the anticancer drug-GnRH bioconjugates. Furthermore, the number of GnRH-Rs on cancer cells is limited and they internalize slowly (especially GnRH-IR).14 A possible approach to enhance the treatment potency and prevent drug resistance of cancer cells towards GnRH-III bioconjugates would be the application of multifunctional drug delivery systems containing more than one molecule of anticancer drug(s). A bioconjugate carrying more than one chemotherapeutic agent might be more effective due to its ability to exert an elevated cytotoxicity compared with a monofunctional compound. Ideally, the anticancer drugs attached to one GnRH-III peptide should act synergistically or additively. The initial step in the development of the multifunctional GnRH-III containing bioconjugates was the synthesis of GnRH-III peptides that allow the attachment of two anticancer drugs. When considering that the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2) only contains one lysine residue in position 8 which can be chemically modified, a second conjugation site needed to be introduced. To this end, two different drug design and synthetic strategies were pursued. As shown in 1997 by Mez} et al.,15 the replaceo ment of 4Ser by a lysine residue did not lead to a significant decrease of the antiproliferative effect on cancer cells. ´ Moreover, Kovacs and colleagues demonstrated that [4Lys]-GnRH-III did not exert significantly higher LH- and
Biopolymers (Peptide Science)

Cytostatic Effect of Multifunctional Anticancer Drug-Bioconjugates

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FSH-releasing activity compared with the native GnRH-III.16 Furthermore, the modification of the side chain of 8Lys was well tolerated regarding the receptor binding properties.17,18 Taking into account these features of GnRH-III, the first drug design was based on the bifunctional [4Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH2) containing two lysine residues in positions 4 and 8; their e-amino groups were used for the coupling of daunorubicin. Because it could not be predicted whether the replacement of 4Ser by lysine and its side chain modification would lead to an effective and functional drug delivery system, a second approach to prepare a multifunctional bioconjugate was employed. The native GnRH-III peptide (Glp-His-Trp-Ser-His-AspTrp-Lys-Pro-Gly-NH2) was used as a scaffold for the anticancer drug attachment. To obtain two free amino groups available for daunorubicin conjugation, an additional lysine residue was coupled to the e-amino group of 8Lys. The aim of pursuing these two different synthetic strategies was to ascertain which drug design leads to multifunctional bioconjugates with improved biological activity compared with a drug delivery system containing only one chemotherapeutic agent. Conjugation of daunorubicin was achieved in both cases via oxime bond formation, which is a simple and efficient chemoselective ligation method. The advantage of oxime over, for example, ester bond (applied for the preparation of AN-152) is its chemical and enzymatic stability, thus preventing premature drug release from the bioconjugates in human serum. The in vitro stability/degradation of the compounds was assessed in human serum, as well as in the presence of rat liver lysosomal homogenate by liquid chromatography in combination with mass spectrometry. Their cellular uptake was determined by flow cytometry on MCF-7 human breast cancer cells. Furthermore, the in vitro cytostatic effect was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on HT-29 human colon, MCF7 human breast and LNCaP human prostate cancer cell lines.

and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Ltd. (St. Louis, MO). N,N-dimethylformamide (DMF) and acetonitrile were purchased from Acros Organics (Geel, Belgium), while ethanol and diethyl ¨ ether were from Riedel deHaen (Seelze, Germany). All reagents and solvents were of analytical grade or highest available purity.

Synthesis of Oxime Bond-Linked Daunorubicin-GnRH-III Derivative Bioconjugates
Aminooxyacetic acid (Aoa) derivatives of GnRH-III (<EHWK(Aoa) HDWK(Aoa)PG-NH2, <EHWSDWK(Aoa-K(Aoa))PG-NH2 and <EHWSHDWK(Aoa)PG-NH2, where <E is pyroglutamic acid) were prepared manually by solid phase peptide synthesis according to Fmoc/tBu chemistry on a Rink-Amide MBHA resin (0.38 mmol/g coupling capacity). The following Fmoc-protected amino acid derivatives were used: Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH and Fmoc-Ser(tBu)-OH. Pyroglutamic acid (Glp) was attached to the peptide chain without any protection. The protocol of the synthesis was as follows: (i) DMF washing (4 3 1 min), (ii) Fmoc deprotection with 2% DBU, 2% piperidine in DMF (four times; 2 + 2 + 5 + 10 min), (iii) DMF washing (10 3 1 min), (iv) coupling of 5 equiv a-Fmoc-protected amino acid derivative : PyBOP : NMM (1 : 1 : 2) in DMF (60 min), (v) DMF washing (4 3 1 min). For the preparation of <EHWSDWK(Aoa-K(Aoa))PG-NH2, an additional lysine residue was coupled to the e-amino group of 8Lys. Therefore, its Mtt-protecting group was removed with 2% TFA in DCM (6 3 5 min) and then Fmoc-Lys(Mtt)-OH was coupled according to the above mentioned protocol. To synthesize <EHWK(Aoa)HDWK(Aoa)PG-NH2 and <EHWSHDWK(Aoa)PG-NH2, the Mtt-protecting group(s) was removed from the e-NH2 group(s) of 8Lys and 4Lys with 2% TFA in DCM (6 3 5 min) after completion of the synthesis of the protected decapeptides. After that, bis-Boc-Aoa-OH was attached to the free e-NH2 group(s) after preactivation with PyBOP in the presence of NMM (10 or 5 eq to the resin capacity depending on the number of free amino groups; coupling time 45 min). In case of <EHWSDWK(Aoa-K(Aoa))PG-NH2, prior to the coupling of Aoaderivative, both Fmoc and Mtt protecting groups were removed from the additional Lys coupled to 8Lys as described above. Aminooxyacetylated peptides were cleaved from the resin using a mixture of 95% TFA, 2.5% TIS, and 2.5% water (v/v/v) for 2.5 h at room temperature and then precipitated with ice-cold diethyl ether, washed three times with diethyl ether and solubilized in 100% acetic acid prior to freeze drying. The crude products were purified by semipreparative RP-HPLC and analyzed by mass spectrometry. The conjugation of daunorubicin to the aminooxyacetylated GnRH-III derivatives was carried out in 0.2 M sodium acetate buffer (pH 5), at a peptide concentration of 10 mg/mL. Daunorubicin was used in 30% excess compared with the aminooxyacetylated derivatives of GnRH-III. The reaction mixtures were stirred at room temperature for 24 h and then subjected to RP-HPLC purification. The purified bioconjugates, GnRH-III[4,8Lys(Dau ¼ Aoa)] (1), GnRH-III[8Lys(Dau ¼ Aoa-Lys(Dau ¼ Aoa)) (2) and GnRH-III [8Lys(Dau ¼ Aoa)] (3) were characterized by analytical RP-HPLC and mass spectrometry.

MATERIALS AND METHODS
Materials
All amino acid derivatives, benzotriazol-1-yloxytrispyrrolidinophosphonium-hexafluoro-phosphate (PyBOP), bis-Boc-aminooxyacetic acid (bis-Boc-Aoa-OH) and Rink-Amide MBHA resin were ¨ purchased from NovaBiochem (Laufelfingen, Switzerland) and GL Biochem Shanghai Ltd (Shanghai, China). Scavengers, coupling agents and cleavage reagents (triisopropylsilane (TIS), 4-methylmorpholine (NMM), piperidine, 1,8-diazabicyclo[5.4.0]undec-7ene (DBU), trifluoroacetic acid (TFA)), as well as daunorubicin (Dau), N-diisopropylethylamine (DIPEA), acetic anhydride (Ac2O)

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Leurs et al. BCA (bicinchoninic acid) protein assay according to the manufacturer’s protocol (ThermoFisher Scientific, Rockford, IL). The degradation of the bioconjugates in the presence of rat liver lysosomal homogenate was determined as follows: bioconjugates were dissolved in 0.2 M NaOAc (pH 5.0) at a concentration of 0.1 lg/lL and then the rat liver lysosomal homogenate was added at a 1:1 (w/w) ratio. The reaction mixtures were incubated at 378C and aliquots of 50 lL were taken after 5 min, 1, 2, 4, 6, 8, and 24 h. The reactions were quenched by adding 5 lL of acetic acid and followed by LC-MS analysis. Control experiments were performed with 0.1 lg/lL solutions of bioconjugates in 0.2 M sodium acetate buffer (pH 5.0), which were incubated at 378C for 24 h and then analyzed by LC-MS.

High Performance Liquid Chromatography
The crude products were purified on an UltiMate 3000 HPLC system (Dionex, Idstein, Germany) using a semipreparative Vydac C18 ˚ column (250 mm 3 10 mm) with 10 lm silica (300 A pore size). Linear gradient elution (0 min 20% B; 5 min 20% B; 55 min 70% B) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile : H2O (80:20, v/v)) was used at a flow rate of 4 mL/min. Peaks were detected at 220 nm and 280 nm. Analytical RP-HPLC was performed on an UltiMate 3000 system (Dionex, Idstein, Germany) using a Vydac C18 column (250 mm 3 ˚ 4.6 mm) with 5 lm silica (300 A pore size) as a stationary phase. Linear gradient elution (0 min 0% B; 5 min 0% B; 50 min 90% B) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile : H2O (80:20, v/v)) was used at a flow rate of 1 mL/min. Peaks were detected at 280 nm.

Cells
MCF-7 human breast cancer cell line was maintained in DMEM GlutaMAX-I (Sigma Ltd., St. Louis, MO) medium containing 10% FCS (fetal calf serum, Sigma) and gentamicine (160 lg/mL). HT-29 human colon cancer cell line was maintained in RPMI 1640 (GIBCO Invitrogen, Germany) supplemented with 10% FCS and 1% penicillin/streptomycin. LNCaP human prostate cancer cell line was maintained in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin and 10 nM testosterone. Cell cultures were maintained at 378C in a humidified atmosphere with 5% CO2.

Mass Spectrometry
Electrospray (ESI)-mass spectrometric analyses were carried out on an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). Spectra were acquired in the 50–2500 m/z range. Samples were dissolved in a mixture of 50% methanol, 48% water, and 2% acetic acid. Liquid chromatography-mass spectrometry (LC-MS) was carried out on an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an Agilent 1100 HPLC system (Agilent, Waldbronn, Germany) and a diode array detector. Peptides were separated on a Vydac MS C18 column (150 mm 3 ˚ 1 mm; 300 A, 3 lm) using a linear gradient from 90% solvent A [0.1% formic acid in water (v/v)] and 10% solvent B [0.1% formic acid in acetonitrile (v/v)] to 70% solvent B over 60 min and a flow rate of 50 lL/min. Spectra were recorded in positive ion mode in the 50–2500 m/z range.

Cellular Uptake of Bioconjugates Determined by Flow Cytometry
To examine the cellular uptake of the bioconjugates by MCF-7 human breast cancer cell line, cells were plated at a number of 1 3 105 cells per well on 24-well plates in 1 mL complete medium. After 24 h incubation at 378C, cells were centrifuged for 5 min at 1000 rpm and the supernatant was removed. Thereafter, 250 lL of either free daunorubicin or bioconjugate solutions (in serum-free medium) were added onto the cells in the 0.16–100 lM concentration range. Control cells were treated with serum-free medium. Cells were incubated with the free daunorubicin or bioconjugate solutions at 378C for 6 h. After that, the solutions were removed from the cells, 100 lL trypsin were added per well and incubated for 10 min at 378C. After adding 700 lL of 10% FCS in HPMI, cells were moved to FACS tubes and centrifuged for 5 min at 1000 rpm After removing the supernatant, the cells were resuspended in 500 lL FCS-free HPMI. Fluorescence intensity of the cells was determined by flow cytometry (BD LSR II, BD Bioscience, San Jose, CA). Data were analyzed with the FACSDiVa software and the percentage of daunorubicin positive cells was calculated.

Stability of Daunorubicin-GnRH-III Derivative Bioconjugates in 90% Human Serum
After dissolving the bioconjugates in water at a concentration of 100 lM, human serum was added to a final peptide concentration of 10 lM. The mixtures were incubated at 378C and aliquots of 100 lL were taken after 5 min, 8 h, and 24 h (the reactions were quenched by adding 10 lL of acetic acid). Prior to mass spectrometric analysis, the larger human serum proteins were removed using Microcon centrifugal devices, cut-off 10 kDa (Millipore Corporation, Bedford, MA) and the lower molecular weight fraction was analyzed by LC-MS. Two control experiments were performed: (1) compounds with molecular weight lower than 10 kDa from human serum were separated and analyzed by LC-MS and (2) aqueous solutions of bioconjugates (c ¼ 10 lM) were incubated at 378C for 24 h and then analyzed by LC-MS.

In Vitro Cytostatic Effect of the Bioconjugates Determined by MTT Assay
The in vitro cytostatic effect of the bioconjugates was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). One day before the treatment with the compounds, 5 3 103 cells per well were plated on 96-well plates. After 24-h incubation at 378C, cells were treated for 6 h either with the bioconjugates (used in the 0.4–50 lM concentration range) or with the free daunorubicin (used in the 0.0003–20 lM concentration range). The solutions were prepared in serum-free

Degradation of Daunorubicin-GnRH-III Derivative Bioconjugates in the Presence of Rat Liver Lysosomal Homogenate
The rat liver lysosomal homogenate was prepared as previously described11 and the protein concentration was determined by Pierce

Biopolymers (Peptide Science)

Cytostatic Effect of Multifunctional Anticancer Drug-Bioconjugates medium. Cells treated for 6 h with serum-free medium were used as a control. After treatment and incubation, cells were washed twice with serum-free medium and cultured in serum containing medium for 72 h. On the fourth day, the MTT assay was performed. Therefore, MTT was added to each well (final concentration: 367 lg/mL) and during 3.5 h incubation at 378C purple crystals were formed by mitochondrial dehydrogenase enzyme present in the living cells. After that, cells were centrifuged for 5 min at 2500 rpm and the supernatant was removed. The crystals were dissolved in 100 lL DMSO and the optical density (OD) was determined at k ¼ 540 and 620 nm using an ELISA Reader (SpectraFluor Plus, Tecan, Switzerland). OD620 was substracted from OD540 and the percentage of cytostasis was calculated using the following equation: Cytostasis% ¼ ½1 À ðODtreated =ODcontrol ފ3100 where ODtreated and ODcontrol correspond to the optical densities of treated and control cells, respectively. Cytostasis % was plotted as a function of concentration, fitted to a sigmoid curve and the 50% inhibitory concentration (IC50) value was determined from these curves. For each compound, eight parallels were used in one experiment which was repeated twice.

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RESULTS AND DISCUSSION
Oxime Bond-Linked Daunorubicin-GnRH-III Derivative Bioconjugates
Bioconjugates with receptor mediated tumor targeting functions are able to deliver chemotherapeutic agents solely to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. Several bioconjugates employing analogs of the GnRH peptide as targeting moieties have already been synthesized and characterized. AN-152 is a bioconjugate containing the [D-Lys6]-GnRH-I as a targeting moiety and doxorubicin as an anticancer drug.4 It showed high antitumor activity on numerous tumor types and recently entered phase II clinical trial on ovarian and endometrial cancer (http://clinicaltrials.gov). Side effects of AN-152 include moderate hematological toxicity due to the premature drug release.19 Therefore, in our work, GnRH-III was used as a targeting moiety; its endocrine effect was shown to be insignificant in mammals. Previously reported data indicated that oxime bond-linked daunorubicin-GnRHIII bioconjugates had significant in vitro and in vivo antitumor activity with marginal toxicity at a dose of 15 mg Dau content in bioconjugate/kg body weight in mice. Furthermore, the oxime bond between GnRH-III and daunorubicin was stable in human serum.11,12 When considering that the number of GnRH receptors on cancer cells is limited, a possible approach to enhance the potency of anticancer drugs containing GnRH-III as a
Biopolymers (Peptide Science)

targeting moiety could be to increase the number of the attached chemotherapeutic agents. To this end, two different drug design approaches were pursued in this work. Taking into account that the replacement of 4Ser by Lys did not affect the cytostatic effect or receptor recognition,15,18 the first drug design was based on [4Lys]-GnRH-III. In the second approach, the native GnRH-III sequence was employed and an additional lysine residue was attached to the e-amino group of 8Lys. Both drug design approaches yielded two sites for daunorubicin conjugation. The aim of pursuing these two different synthetic strategies was to ascertain which drug design leads to multifunctional bioconjugates with improved biological activity compared with a drug delivery system containing only one chemotherapeutic agent. The structures of the synthesized compounds are schematically represented in Figure 1. All bioconjugates were synthesized by a combination of solid phase peptide synthesis (Fmoc-chemistry) and chemical ligation (oxime bond formation). The purified compounds were characterized by analytical RP-HPLC and mass spectrometry (Table I and Supporting Information S3–S4). As previously reported,11,20 the glycosidic bonds in daunorubicin were very labile under ESI-mass spectrometric conditions resulting in the loss of daunosamine (À129, À147). These fragments were marked in all mass spectra by an asterisk.

In Vitro Stability/Degradation of the Bioconjugates
The stability of the anticancer drug-GnRH-III bioconjugates in human serum and lysosomal homogenates is of remarkable importance for their therapeutic applications. As the main principle of targeted cancer chemotherapy is the delivery of a chemotherapeutic agent solely to cancer cells, the chemical bond between the GnRH-III and the anticancer drug should exhibit high stability in human serum (to prevent the decomposition in the blood stream). In contrast, the compounds taken up by the cancer cells should be degraded in the lysosomes, so as to favor the release of the free chemotherapeutic agent or the formation of an active metabolite. The stability of daunorubicin-GnRH-III derivative bioconjugates in 90% human serum was determined by LC-MS. All bioconjugates were stable in human serum for at least 24 h. LC-MS analyses of the compounds incubated for 24 h at 378C with human serum revealed the presence of intact compounds and did not result in the identification of any degradation products (the detailed mass spectrometric analyses are shown in the supporting information, S5–S6). In order to identify the ions originating from human serum, the components with molecular weight lower than 10 kDa from human serum were separated and analyzed by LC-MS

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FIGURE 1 Structure representation of daunorubicin-GnRH-III derivative bioconjugates. A: GnRH-III sequence and the corresponding modifications in positions 4 (R1) and 8 (R2); B: Structure representation of Dau ¼ Aoa; C: Structure representation of Dau ¼ Aoa-Lys(Dau ¼ Aoa).

(Supporting Information, S5–S6). In another control experiment, aqueous solutions of bioconjugates (c ¼ 10 lM) were incubated at 378C for 24 h and then subjected to LC-MS analysis. Mass spectrometric data indicated that the bioconjugates were chemically stable under these conditions (data not shown). The degradation of the bioconjugates in the presence of rat liver lysosomal homogenate was investigated by LC-MS, as well. The compounds demonstrated high lability in the lysosomal homogenate, resulting in various peptide fragments presented in Table II and Figure 2. In case of com-

pounds 1 and 3, the smallest drug containing metabolite was H-Lys(Dau ¼ Aoa)-OH, which has been shown to efficiently bind to DNA.18 For compound 2, the smallest metabolite found was H-Lys(Dau ¼ Aoa-Lys(Dau ¼ Aoa))-OH. This indicates that the free daunorubicin could not be released from the lysine modified by aminooxyacetic acid and that the isopeptide bond between 8Lys and the additional Lys in the branch was not cleaved by any of the lysosomal enzymes. The detailed LC-MS analyses are shown in the Supporting Information (S7). The analysis of compound 3 has previously been published.11

Table I

Chemical Characterstics of Daunorubicin-GnRH-III Derivative Bioconjugates Code GnRH-III[4,8Lys(Dau¼Aoa)] GnRH-III[8Lys(Dau¼Aoa-Lys(Dau¼Aoa))] GnRH-III[8Lys(Dau¼Aoa)]
a

Compound 1 2 3
a

RP-HPLC Rt [min] 30.3 29.0 26.5

b

ESI-MS MWcalc/MWexp 2465.57/2465.07 2553.66/2553.53 1840.75/1841.05

˚ Column: Vydac C18 (250 mm 3 4.6 mm) with 5 lm silica (300 A pore size); gradient: 0 min 0% B, 5 min 0% B, 50 min 90% B; eluents: 0.1% TFA in water (A) and 0.1% TFA in acetonitrile:water (80:20, v/v) (B); flow rate: 1 mL/min; detection at k ¼ 280 nm. b Bruker Daltonics Esquire 3000+ ion trap mass spectrometer.

Biopolymers (Peptide Science)

Cytostatic Effect of Multifunctional Anticancer Drug-Bioconjugates Table II Fragments Produced by the Cleavage of Daunorubicin-GnRH-III Derivative Bioconjugates in the Presence of Rat Liver Lysosomal Homogenate Compound 1 GnRH-III[4,8Lys(Dau¼Aoa)] Fragment <EHWK(Dau¼Aoa)HDWK(Dau¼Aoa)-OH <EHWK(Dau¼Aoa)HD-OH <EHWK(Dau¼Aoa)H-OH <EHWK(Dau¼Aoa)-OH H-HDWK(Dau¼Aoa)PG-NH2 H-HDWK(Dau¼Aoa)-OH H-K(Dau¼Aoa)P-OH H-K(Dau¼Aoa)-OH H-HDWK(Dau¼Aoa-K(Dau¼Aoa))PG-NH2 H-K(Dau¼Aoa-K(Dau¼Aoa))PG-NH2 H-K(Dau¼Aoa-K(Dau¼Aoa))P-OH H-K(Dau¼Aoa-K(Dau¼Aoa))-OH

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MWcalc/MWexp 2312.4/2312.3 1414.6/1414.8 1299.5/1299.6 1162.5/1162.6 1319.6/1320.0 1167.2/1167.4 825.3/825.3 728.3/728.4 2030.8/2031.0 1592.7/1592.2 1536.6/1536.8 1438.6/1438.5

2 GnRH-III[8Lys(Dau¼Aoa-Lys(Dau¼Aoa))]

In Vitro Cytostatic Effect
The in vitro cytostatic effect of daunorubicin-GnRH-III derivative bioconjugates was determined on MCF-7 human breast, HT-29 human colon and LNCaP human prostate cancer cells. The compounds showed overall high cytostatic effects with IC50 values in the low micromolar range on all tested human cancer cell lines (Table III). On HT-29 cell line, the activity of the multifunctional compounds was significantly higher than that of the monofunctional bioconjugate. The cytostatic effect of compounds 1 and 2 which contain two daunorubicin residues exceeded that of the GnRH-III[8Lys(Dau ¼ Aoa)] bioconjugate, confirming our assumption that the therapeutic efficacy can be increased by the attachment of a second anticancer drug to one GnRH-III molecule. Interestingly, despite of the large metabolite produced in the presence of the lysosomal homogenate, compound 2 had the same cytostatic effect as compound 1. This could indicate that the large H-Lys(Dau ¼ Aoa-Lys(Dau ¼ Aoa))-OH fragment might be either further processed inside the cells or that both daunorubicin residues are able to interact with their intracellular targets. A similar pattern of the cytostatic effect was determined on LNCaP human prostate cancer cells, the multifunctional bioconjugates being more active than the monofunctional one. On MCF-7 human breast cancer cells, all bioconjugates had comparable cytostatic effect. For all compounds, the IC50 values were lower on MCF-7 cell line; however, they were not significantly different on LNCaP cells. Interestingly, the multifunctional bioconjugates showed similar cytostatic effect on all three cell lines. Only the GnRH-III[8Lys(Dau ¼ Aoa)] bioconjugate exerted a different effect on various cancer cell lines.
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As a further comparison, also the cytostatic effect of daunorubicin was determined on all three cancer cell lines. The free anticancer drug showed overall the highest activity with IC50 values in the range of 0.1–0.4 lM. As free daunorubicin is taken up by passive diffusion and does not require any intracellular processing, the slightly higher IC50 values obtained for the daunorubicin-GnRH-III derivative bioconjugates are most probably due to their uptake by receptor mediated endocytosis.

Cellular Uptake
To investigate the cellular uptake of compounds GnRHIII[4,8Lys(Dau ¼ Aoa)], GnRH-III[8Lys(Dau ¼ Aoa-Lys(Dau ¼ Aoa))] and GnRH-III[8Lys(Dau ¼ Aoa)], MCF-7 cells were treated for 6 h with different concentrations of the bioconjugates (0.16–100 lM), afterwards the percentage of Dau-positive cells was determined by flow cytometry (see Figure 3). The MCF-7 cell line was selected for the cellular uptake study, because the bioconjugates showed the lowest IC50 values on this cell line. No significant cellular uptake could be observed in the lower concentration range between 0.16–4 lM; therefore, only the values for 20 and 100 lM, as well as a control, are shown in Figure 3. Compound 1 showed the highest cellular uptake (*70% and 100% of the cells contain Dau at 20 and 100 lM concentrations, respectively), followed by compound 3 (*8% and 95%). Compound 3 seems to be taken up less efficiently than compound 1, most probably due to the replacement of serine by lysine in position 4 in compound 1. As previously shown (Manea et al., unpublished results), the replacement of 4Ser by a lysine residue led to increased cellular uptake. Compound 2 showed the lowest cellular uptake at

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Leurs et al.

FIGURE 2 Degradation of daunorubicin-GnRH-III derivative bioconjugates in the presence of rat liver lysosomal homogenate. Cleavage sites are indicated by arrows.

Table III In Vitro Cytostatic Effect of Free Daunorubicin and Daunorubicin-GnRH-III Derivative Bioconjugates on Various Human Cancer Cell Lines Compound Daunorubicin 1 GnRH-III[4,8Lys(Dau¼Aoa)] 2 GnRH-III[8Lys(Dau¼Aoa-Lys(Dau¼Aoa))] 3 GnRH-III[8Lys(Dau¼Aoa)] MCF-7 IC50 (lM) 0.4 6 0.1 2.9 6 0.9 3.0 6 0.4 6.7 6 1.9 HT-29 IC50 (lM) 0.2 6 0.2 6.8 6 1.0 5.6 6 2.0 29.9 6 0.6 LNCaP IC50 (lM) 0.1 6 0.1 4.9 6 0.3 3.8 6 1.6 15.2 6 2.4

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Cytostatic Effect of Multifunctional Anticancer Drug-Bioconjugates

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FIGURE 3 Cellular uptake of free daunorubicin and daunorubicin-GnRH-III derivative bioconjugates by MCF-7 human breast cancer cells. The percentage of Dau-positive cells after 6 h of incubation with different concentrations of the compounds is shown.

both concentrations, only reaching 72.3% at the highest bioconjugate concentration. In comparison with compound 3, this decrease might be due to the second lysine residue attached to the e-amino group of 8Lys which could alter the structure of the peptide and thereby diminish the receptor binding. The similar IC50 values of compounds 1 and 2 indicate that cellular uptake might play only a minor role for the cytostatic effect and that further intracellular processes (e.g., additional metabolic pathways and/or the DNA binding properties of the metabolites) have a higher influence on it. This would mean that compound 2, while having a diminished cellular uptake, has a high cytostatic effect due to either its intracellular metabolism or better DNA interaction properties of the formed metabolite. In contrast to the bioconjugates, the free daunorubicin was taken up very efficiently by MCF-7 cells. At 20 lM concentration, 100% of the cells were Dau positive (see Figure 3). As already mentioned, the difference in the cellular uptake of the compounds could account for their different cytostatic effects.

functional GnRH-III bioconjugates with significantly increased cytostatic effect on HT-29 and LNCaP cancer cell lines. Even if the degradation of compound 2 in the presence of the lysosomal homogenate resulted in the large metabolite H-Lys(Dau ¼ Aoa-Lys(Dau ¼ Aoa))-OH, the IC50 values determined for compound 2 did not significantly differ from those of compound 1, probably due to further intracellular processing of this fragment. When considering all these data, we conclude that the attachment of a second anticancer drug to the GnRH-III peptide can result in promising multifunctional drug delivery systems with increased therapeutic efficacy. A future perspective for multifunctional bioconjugates containing GnRH-III as a targeting moiety could be the attachment of other types of anticancer drugs which might act synergistically on the cancer cells and thereby could further enhance the bioconjugates’ potency.

REFERENCES
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CONCLUSIONS
Our results indicate that the attachment of a second anticancer drug to one GnRH-III molecule can lead to efficient drug delivery systems for targeted cancer chemotherapy. Both drug design approaches, either based on the native GnRH-III sequence or on the modified one, led to multiBiopolymers (Peptide Science)

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Leurs et al. 14. Neill, J. D.; Musgrove, L. C.; Duck, L. W. Trends Endocrinol Metab 2004, 15, 383–392. 15. Mezo, I.; Lovas, S.; Palyi, I.; Vincze, B.; Kalnay, A.; Turi, G.; Vadasz, Z.; Seprodi, J.; Idei, M.; Toth, G.; Gulyas, E.; Otvos, F.; Mak, M.; Horvath, J. E.; Teplan, I.; Murphy, R. F. J Med Chem 1997, 40, 3353–3358. 16. Kovacs, M.; Vincze, B.; Horvath, J. E.; Seprodi, J. Peptides 2007, 28, 821–829. 17. Heredi-Szabo, K.; Lubke, J.; Toth, G.; Murphy, R. F.; Lovas, S. Peptides 2005, 26, 419–422. 18. Mezo, G.; Manea, M.; Szabi, I.; Vincze, B.; Kovacs, M. Curr Med Chem 2008, 15, 2366–2379. 19. Emons, G.; Sindermann, H.; Engel, J.; Schally, A. V.; Grundker, C. Neuroendocrinology 2009, 90, 15–18. 20. Sleno, L.; Campagna-Slater, V.; Volmer, D. A. Int J Mass Spectrom 2006, 255–256, 130–138.

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