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Enhanced Enzymatic Stability and Antitumor Activity of Daunorubicin-GnRH-III Bioconjugates Modified in Position 4 € Marilena Manea,*,§,† Ulrike Leurs,§ Erika Orban,‡ Zsuzsa Baranyai,‡ Peter Ohlschl€ ager,3,^ || # # # kos Schulcz, Miguel Tejeda, Bence Kapuvari, Jozsef Tovari,# and Gabor Mezo00 ‡ Andreas Marquardt, A §
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry Zukunftskolleg ^ Laboratory of Immunology, Department of Biology Proteomics Facility University of Konstanz, 78457 Konstanz, Germany ‡ Research Group of Peptide Chemistry, Hungarian Academy of Sciences, E€otv€os Lorand University, 1117 Budapest, Hungary # National Institute of Oncology, 1122 Budapest, Hungary
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bS Supporting Information ABSTRACT: Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropinreleasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), 4 Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which 4Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/ degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the 4Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which 4Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.
’ INTRODUCTION The hormonal decapeptide gonadotropin-releasing hormone-I (GnRH-I; Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) plays a major role in the regulation of the reproduction by influencing the secretion of pituitary gonadotropin hormones.1 Moreover, GnRH-I and many of its derivatives have an antiproliferative effect on various cancer cells,2,3 which can be exerted by indirect or direct ways.4,5 In the first case, the continuous exposure to GnRH-I results in the suppression of gonadotropin and sex-steroid secretion due to the desensitization of gonadotropic cells and the downregulation of pituitary GnRH receptors (GnRH-Rs). The observed chemical castration is a therapeutic approach of hormonedependent tumors such as prostate and breast cancers. However, it has been shown that GnRH analogues also have direct r 2011 American Chemical Society
antitumor activity on both hormone-dependent and -independent tumors, an effect which could be mediated through the GnRH receptors highly expressed on many types of tumors. Thousands of synthetic peptide analogues of GnRH have been prepared since its discovery in order to increase, e.g., the antiproliferative effect. Interestingly, one of the less modified amino acids in the sequence is Ser in position 4. It seems that 4Ser is not directly involved in the receptor binding;6,7 however, the secondary structure is stabilized by a backbone hydrogen bond between 4 SerNH and 1GlpCO or by a side chain to backbone hydrogen bond Received: December 3, 2010 Revised: May 7, 2011 Published: June 13, 2011 1320
dx.doi.org/10.1021/bc100547p | Bioconjugate Chem. 2011, 22, 1320–1329
Bioconjugate Chemistry of 4SerOH and 1GlpCO.8 These interactions might influence the biologically active conformation of the N-terminal part of the peptide which is important for binding to the receptor. Moreover, it has been shown that in a GnRH-tandem-dimer peptide used as an antigen for immunocastration, Ser could be replaced by Ala without negatively affecting the efficacy.9 The stability of peptide-based drugs against enzyme-catalyzed hydrolysis is crucial for their therapeutic applications; for instance, in the case of oral administration, the compounds should be stable in the presence of digestive enzymes present in stomach and intestine. GnRH and its derivatives are highly susceptible to degradation by chymotrypsin, the main cleavage site being the -3Trp-4Ser- peptide bond. Slower degradation rates of GnRH derivatives were observed in the case of elastase, while most of them were stable to trypsin catalyzed hydrolysis. The -3Trp-4Ser- bond was also cleaved by angiotensin-converting enzyme. Furthermore, it was found that peptide bonds involving 4Ser were cleaved in different cell homogenates.10 Haviv et al. reported the complete stabilization of leuprolide against chymotrypsin and lumenal degradation by substituting the 4Ser by N-Me-Ser.11 Most recently, Pappa et al. indicated the incorporation of N-Me-Ser in position 4 in leuprolide analogues as a good strategy to enhance the stability against degradation by R-chymotrypsin and subtilisin, while maintaining their antiproliferative bioequivalence to leuprolide.12 In addition to human GnRH, intensive research is currently being conducted on GnRH-III (Glp-His-Trp-Ser-His-Asp-TrpLys-Pro-Gly-NH2), a native GnRH analogue isolated from sea lamprey13 which acts as a weak GnRH agonist in the pituitary. Compared to GnRH-I, GnRH-III causes 23 orders of magnitude lower release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in mammals.14 Moreover, GnRH-III directly and specifically inhibits the growth of cancer cells expressing GnRHRs,15 and it seems to be the most potent analogue containing only native amino acids on MDA-MB 231 human breast cancer cells.16 Only a few GnRH-III analogues have been prepared so far. The in vitro evaluation of their antitumor activity indicated that slight modifications of the amino acid sequence did not result in a loss of the antitumor activity. It has been shown that two analogues containing either Lys or Lys(Ac) instead of Ser in position 4 were as active as GnRH-III on different tumor types.17 However, tryptophane residues in positions 3 and 7 were essential for the biological activity. The modification of 8Lys by cyclization with 6Asp,17 Ala-scan,18 conjugation to a carrier,19 or dimerization through disulfide bond20 was also tolerated. The LH- and FSH-releasing activity of [4Lys]-GnRH-III was slightly higher than that of GnRH-III, while the modification of the ε-amino group of 8Lys by cyclization, conjugation, or dimerization decreased the endocrine effect of the peptide hormone.20,21 In our previous work, the side chain of 8Lys was used for the attachment of anticancer drugs (e.g., anthracycline derivatives) leading to the formation of drug delivery systems for targeted cancer chemotherapy.10 Some of the anthracycline derivativeGnRH-III bioconjugates had in vitro cytostatic activity with IC50 values in the low μM range. Moreover, two oxime bond-linked daunorubicin-GnRH-III bioconjugates had significant tumor growth inhibitory effect on colon carcinoma bearing mice.22,23 Both bioconjugates were administered intraperitoneally. Oral administration is considered to be the most convenient route for the delivery of drugs and might be very efficient in the case of colorectal cancer. In order to be orally administered, anticancer drug-GnRH-III derivative bioconjugates must have increased stability in the presence of digestive enzymes present in the
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gastrointestinal tract, such as trypsin, chymotrypsin, and pepsin. We have previously reported that GnRH-III and its dimer derivatives were not substrates for trypsin catalyzed hydrolysis; however, they were cleaved by chymotrypsin, the main cleavage site being the -3Trp-4Ser- peptide bond.20 In the case of an oxime bondlinked daunorubicin-GFLG-GnRH-III bioconjugate (
