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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
1
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
2 3 4 5
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 specically to cancer cells expressing GnRH receptors, thereby increasing their local efcacy 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 rst 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: 110, 2012. Keywords: gonadotropin-releasing hormone-III; multifunctional anticancer drug-peptide bioconjugates; drug design; targeted cancer chemotherapy; cytostatic effect
Leurs et al.
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
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 are-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 nonHodgkins lymphoma.2,3 Bioconjugates containing GnRH-I analogs as targeting moieties for the attachment of cytotoxic agents were initially developed by A. V. Schallys 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) efux 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 nding 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 specically 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 insignicant 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 signicant 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 modied, 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 signicant decrease of the antiproliferative effect on cancer cells. Moreover, Kovacs and colleagues demonstrated that [4Lys]-GnRH-III did not exert signicantly higher LH- and
Biopolymers (Peptide Science)
FSH-releasing activity compared with the native GnRH-III.16 Furthermore, the modication 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 rst 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 modication 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 efcient 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 ow 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.
Leurs et al. BCA (bicinchoninic acid) protein assay according to the manufacturers protocol (ThermoFisher Scientic, 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.
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 humidied 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 502500 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 ow rate of 50 lL/min. Spectra were recorded in positive ion mode in the 502500 m/z range.
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
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 (nal 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 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% 1 ODtreated =ODcontrol 3100 where ODtreated and ODcontrol correspond to the optical densities of treated and control cells, respectively. Cytostasis % was plotted as a function of concentration, tted 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.
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 rst 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 puried compounds were characterized by analytical RP-HPLC and mass spectrometry (Table I and Supporting Information S3S4). As previously reported,11,20 the glycosidic bonds in daunorubicin were very labile under ESI-mass spectrometric conditions resulting in the loss of daunosamine (129, 147). These fragments were marked in all mass spectra by an asterisk.
Leurs et al.
FIGURE 1 Structure representation of daunorubicin-GnRH-III derivative bioconjugates. A: GnRH-III sequence and the corresponding modications in positions 4 (R1) and 8 (R2); B: Structure representation of Dau Aoa; C: Structure representation of Dau Aoa-Lys(Dau Aoa).
(Supporting Information, S5S6). In another control experiment, aqueous solutions of bioconjugates (c 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 Aoa)-OH, which has been shown to efciently bind to DNA.18 For compound 2, the smallest metabolite found was H-Lys(Dau Aoa-Lys(Dau Aoa))-OH. This indicates that the free daunorubicin could not be released from the lysine modied 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
Compound 1 2 3
a
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); ow rate: 1 mL/min; detection at k 280 nm. b Bruker Daltonics Esquire 3000+ ion trap mass spectrometer.
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(DauAoa)] Fragment <EHWK(DauAoa)HDWK(DauAoa)-OH <EHWK(DauAoa)HD-OH <EHWK(DauAoa)H-OH <EHWK(DauAoa)-OH H-HDWK(DauAoa)PG-NH2 H-HDWK(DauAoa)-OH H-K(DauAoa)P-OH H-K(DauAoa)-OH H-HDWK(DauAoa-K(DauAoa))PG-NH2 H-K(DauAoa-K(DauAoa))PG-NH2 H-K(DauAoa-K(DauAoa))P-OH H-K(DauAoa-K(DauAoa))-OH
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(DauAoa-Lys(DauAoa))]
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 0.10.4 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 Aoa)], GnRH-III[8Lys(Dau Aoa-Lys(Dau Aoa))] and GnRH-III[8Lys(Dau Aoa)], MCF-7 cells were treated for 6 h with different concentrations of the bioconjugates (0.16100 lM), afterwards the percentage of Dau-positive cells was determined by ow 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 signicant cellular uptake could be observed in the lower concentration range between 0.164 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 efciently 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
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(DauAoa)] 2 GnRH-III[8Lys(DauAoa-Lys(DauAoa))] 3 GnRH-III[8Lys(DauAoa)] 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
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 inuence 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 efciently 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 signicantly 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 Aoa-Lys(Dau Aoa))-OH, the IC50 values determined for compound 2 did not signicantly 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 efcacy. 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 efcient drug delivery systems for targeted cancer chemotherapy. Both drug design approaches, either based on the native GnRH-III sequence or on the modied one, led to multiBiopolymers (Peptide Science)
10
Leurs et al. 14. Neill, J. D.; Musgrove, L. C.; Duck, L. W. Trends Endocrinol Metab 2004, 15, 383392. 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, 33533358. 16. Kovacs, M.; Vincze, B.; Horvath, J. E.; Seprodi, J. Peptides 2007, 28, 821829. 17. Heredi-Szabo, K.; Lubke, J.; Toth, G.; Murphy, R. F.; Lovas, S. Peptides 2005, 26, 419422. 18. Mezo, G.; Manea, M.; Szabi, I.; Vincze, B.; Kovacs, M. Curr Med Chem 2008, 15, 23662379. 19. Emons, G.; Sindermann, H.; Engel, J.; Schally, A. V.; Grundker, C. Neuroendocrinology 2009, 90, 1518. 20. Sleno, L.; Campagna-Slater, V.; Volmer, D. A. Int J Mass Spectrom 2006, 255256, 130138.
7. Krebs, L. J.; Wang, X.; Nagy, A.; Schally, A. V.; Prasad, P. N.; Liebow, C. Oral Oncol 2002, 38, 657663. 8. Sower, S. A.; Chiang, Y. C.; Lovas, S.; Conlon, J. M. Endocrinology 1993, 132, 11251131. 9. Lovas, S.; Palyi, I.; Vincze, B.; Horvath, J.; Kovacs, M.; Mezo, I.; Toth, G.; Teplan, I.; Murphy, R. F. J Pept Res 1998, 52, 384389. 10. Kovacs, M.; Seprodi, J.; Koppan, M.; Horvath, J. E.; Vincze, B.; Teplan, I.; Flerko, B. J Neuroendocrinol 2002, 14, 647655. 11. Orban, E.; Mezo, G.; Schlage, P.; Csik, G.; Kulic, Z.; Ansorge, P.; Fellinger, E.; Moller, H. M.; Manea, M. Amino Acids 2010 (DOI: 10.1007/s00726010-07661). 12. Szabo, I.; Manea, M.; Orban, E.; Csampai, A.; Bosze, S.; Szabo, R.; Tejeda, M.; Gaal, D.; Kapuvari, B.; Przybylski, M.; Hudecz, F.; Mezo, G. Bioconjug Chem 2009, 20, 656665. 13. Schally, A. V.; Nagy, A. Trends Endocrinol Metab 2004, 15, 300 310.