Development of an Oxime Bond Containing Daunorubicin

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Bioconjugate Chem. 2009, 20, 656–665

Development of an Oxime Bond Containing Daunorubicin-Gonadotropin-Releasing Hormone-III Conjugate as a Potential Anticancer Drug Ildiko´ Szabo´,#,† Marilena Manea,#,§,| Erika Orbań,† Antal Csa´mpai,‡ Szilvia Bo˝sze,† Rita Szabo´,† Miguel Tejeda,⊥ Dezso˝ Gaa´l,⊥ Bence Kapuva´ri,⊥ Michael Przybylski,§ Ferenc Hudecz,†,‡ and Ga´bor Mezo˝*,† Research Group of Peptide Chemistry, Hungarian Academy of Sciences, and Institute of Chemistry, Eo¨tvo¨s Lorańd University, 1117 Budapest, Hungary, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry and Zukunftskolleg, University of Konstanz, 78457 Konstanz, Germany, and National Institute of Oncology, 1122 Budapest, Hungary. Received December 14, 2008; Revised Manuscript Received February 9, 2009

Here, we report on the synthesis and biological properties of a conjugate in which daunorubicin (Dau) as chemotherapeutic agent was attached through an oxime bond to gonadotropin-releasing hormone-III (GnRHIII) as targeting moiety. In vitro toxicity and the cytostatic effect of the conjugate on MCF-7 human breast and C26 murine colon cancer cell lines were determined, and the results were compared with those obtained for the free daunorubicin, as well as with the doxorubicin containing derivative. In vivo antitumor effect of daunorubicin-GnRH-III was studied on Balb/c female mice transplanted with C26 tumor. Our data indicate that the daunorubicin-GnRH-III conjugate had a lower toxic effect than the free daunorubicin and it was essentially nontoxic up to 15 mg (Dau content)/kg body weight. The treatment of the C26 tumor bearing mice with the conjugate led to tumor growth inhibition and longer survival time in comparison with the controls and with the administration of the free drug. When mice were treated twice with the conjugate (on days 4 and 7 after tumor transplantation), 46% tumor growth inhibition was obtained. In this case, the increase of the median survival time was 38% compared to the controls.

INTRODUCTION Chemotherapy is still one of the primary modalities for the treatment of cancer. However, the application of free anticancer drugs has several drawbacks. Frequently applied high doses are required for the efficacy because of the fast elimination of the drugs from the circulation. Furthermore, the lack of selectivity generally results in toxic side effects. Also, the aquired or intrinsic multidrug resistance (MDR) of cancer cells may restrict the use of chemotherapy. Targeted chemotherapy has been developed to overcome these disadvantages (1, 2) and it has been suggested as an alternative for differentiating healthy and afflicted cells (3). Tumor targeting is usually achieved by conjugation of the chemotherapeutic agent to a targeting moiety consisting of sugars (4), lectins (5-7), receptor ligands (1, 3, 8, 9), or antibodies (10-12), which is specifically directed to certain types of binding sites on cancer cells. One of the various targeted chemotherapeutic approaches is based on the finding that receptors of several peptide hormones (e.g., gonadotropin-releasing hormone (GnRH1), bombesin, somatostatin) are overexpressed on cancer cells compared to normal cells and organs. GnRH receptor (GnRHR) expression was identified on different tumors (breast, * Correspondence to Ga´bor Mezo˝, Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s L. University, 1117 Budapest, Pa´zmańy P. stny. 1/A, Hungary. Tel.: (+36)-1-2090555/1433; Fax: (+36)-1-372-2620; e-mail: [email protected]. # Ildiko´ Szabo´ and Marilena Manea contributed equally to this work. † Hungarian Academy of Sciences, Eo¨tvo¨s Lorańd University. ‡ Institute of Chemistry, Eo¨tvo¨s Lorańd University. § Department of Chemistry, University of Konstanz. | Zukunftskolleg, University of Konstanz. ⊥ National Institute of Oncology.

ovarian, endometrial, prostate, renal, brain, pancreatic, melanomas, and non-Hodgkin’s lymphomas) with high percentages in specimens (1, 13, 14). The primary biological function of the hormonal decapeptide GnRH (also called LH-RH) is the regulation of gonadal functions by releasing gonadotropic hormones, luteinizing hormone (LH), and follicle stimulating hormone (FSH), and therefore it plays a pivotal role in vertebrate reproduction (15). However, it was demonstrated that high doses of human GnRH (hGnRH) and its superagonist analogues inhibit the growth of hormone-dependent mammary and prostate cancers through chemical castration (16). Some of the GnRH agonists and antagonists not only have an indirect effect on tumors, but also can affect the cancer cells directly by the interaction with GnRH receptors that activate a G-protein-dependent transducing mechanism (17-19). GnRH derivatives not only might be anticancer agents, but also could serve as targeting moieties to deliver chemotherapeutic agents to tumor cells. Chemotherapeutic antineoplastic radicals including alkylating nitrogen mustard derivatives (e.g., D-melphalan), antimetabolites (e.g., methotrexate), and anthracycline derivatives (e.g., doxorubicin and 2-pyrrolinodoxorubicin), were conjugated to [D-Lys6]-GnRH through amide or ester bonds (1, 20-22). However, in the case of the application of this superactive GnRH agonist as the targeting moiety, temporary damage to the pituitary was observed, which was recovered in about two weeks (23). 1 Abbreviations: Aoa, aminooxyacetic acid; Dau, daunorubicin; Dox, doxorubicin; GnRH, gonadotropin-releasing hormone; LH-RH, luteinizing hormone releasing hormone; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; C26 tumor, solid murine colon carcinoma; MCF-7 tumor, solid human estrogen receptor positive breast carcinoma.

10.1021/bc800542u CCC: $40.75  2009 American Chemical Society Published on Web 03/18/2009

Oxime Bond-Linked Daunorubicin-GnRH-III

Bioconjugate Chem., Vol. 20, No. 4, 2009 657

Figure 1. Conjugation sites of daunorubicin (A) and doxorubicin (B).

The antitumor effect of the conjugate Dox-[Lys6]-GnRH (AN152), which is currently in the phase III clinical trial, was studied on many types of tumors. The highest antitumor effect was determined on human epithelial ovarian cancers. The clinical trials are performed in female patients with LH-RH-receptor expressing gynecological tumors (advanced or recurrent ovarian and endometrial tumors). The selectivity of the antitumor effect in targeted chemotherapy can be increased by using GnRH analogues that have lower binding to gonadotropic cells. One of the weak agonistic analogues of GnRH is the variant (Glp-His-Trp-Ser-His-AspTrp-Lys-Pro-Gly-NH2; GnRH-III) isolated from sea lamprey (Petromyzon marinus) (24) that specifically binds to receptors on cancer cells and recognizes not only the high- but also the low-affinity binding sites of GnRH receptors (25). GnRH-III has 500-1000 times less potency in LH and FSH secretion both in vitro and in vivo indicating its selective antitumor activity (26, 27). It was also demonstrated that the modification of 8Lys by conjugation (28), cyclization (29), Ala-scan (30), or dimerization (31) did not result in the loss of the antiproliferative activity of GnRH-III. However, the absence of the free ε-amino group of lysine decreased the endocrine effect of the compounds (27, 31, 32) The aim of our study was to prepare bioconjugates in which daunorubicin (also called daunomycin, Dau) as the chemotherapeutic agent was covalently attached to GnRH-III as a targeting moiety with its own antiproliferative effect. Daunorubicin is an anthracycline derivative which differs from doxorubicin (Dox) in the substitution at the C-14 position (H instead of OH). It has been shown that daunorubicin has less cardiotoxic side effect than doxorubicin (33, 34). However, the lack of the aliphatic OH-group in daunorubicin prevents the possibility of synthesizing conjugates containing ester bonds (Figure 1). Also, the conjugation through the amino group of the aminosugar moiety (daunosamine) might lead to a significant loss of bioavailability of the compound (32, 35). Therefore, daunorubicin is not a common part of the conjugates tested in experiments for targeted chemotherapy. Here, we report on the conjugation of daunorubicin to GnRH-III(GFLG) via an oxime bond. In vitro and in vivo toxicity of the conjugate was studied. The cytostatic effect of the compound on MCF-7 human breast and C26 murine colon tumor cell lines was determined. The results were compared with those obtained for the free daunorubicin, as well as with the doxorubicin containing derivative. The in vivo antitumor effect of the GnRH-III(Dau)Aoa-GFLG) conjugate (throughout the manuscript, the conjugate will be abbreviated as Dau-GnRH-III) was studied on mice transplanted with C26 murine colon tumor xenograft. The oxime bond containing Dau-GnRH-III conjugate had in vitro and in vivo antitumor effects and enhanced the survival of treated mice bearing C26 tumor compared to the untreated

Figure 2. Conjugation of anthracyclines to GnRH-III derivative.

Figure 3. Numbering of atoms used for NMR assignments (arbitrary).

control group. The conjugate also prevented the toxic side effects of the free drug.

EXPERIMENTAL SECTION Materials. All amino acid derivatives and MBHA resin were purchased from NovaBiochem (Laüfelfingen, Switzerland) or Reanal (Budapest, Hungary). Scavengers, coupling agents, and cleavage reagents (p-cresol, dithiothreitol (DTT), N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIPCI), 1,2-ethanedithiol (EDT), (benzotriazol-1-yloxy)-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), Ndiisopropyl-ethylamine (DIEA), 1-hydroxybenzotriazole (HOBt), 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), piperidine, trifluoroacetic acid (TFA), and hydrogen fluoride (HF)), as well as daunorubicin (Dau), doxorubicin (Dox), aminooxyacetic acid (Aoa), and Boc-aminooxyacetic acid (Boc-Aoa-OH), were Fluka

658 Bioconjugate Chem., Vol. 20, No. 4, 2009

Szabo´ et al.

Table 1. Characteristics of Conjugates, Dau-Aminooxyacetic Acid Derivative and GnRH-III(GFLG)a RP-HPLCb

ESI-MSc

compound

Rt (min)

[M + H]+calc.

[M + H]+exp.

Dau-GnRH-III Dox-GnRH-III Dau-Aoa-OH GnRH-III(GFLG)

32.3 31.9 31.6 26.3

2217.3 2233.3 601.2 1634.8

2216.8 2232.9 601.2 1634.6

a HPLC chromatograms and mass spectra are shown in the Supporting Information (S3-S5). b Column: Phenomenex Jupiter C18 (250 mm × 4.6 mm) with 5 µm silica (300 Å 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: λ ) 220 nm. c Bruker Daltonics Esquire 3000+ ion trap mass spectrometer.

products (Buchs, Switzerland). Solvents for synthesis (dichloromethane (DCM) and N,N′-dimethylformamide (DMF)) were purchased from Reanal (Budapest, Hungary). HPLC-grade acetonitrile (MeCN) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich Kft (Budapest, Hungary). Synthesis of Conjugates. Branched GnRH-III derivative (100

1.95 ( 0.01 4.51 ( 0.16 22.46 ( 1.74 46.10 ( 6.06 >100

(2 × 102) µM concentration range were dissolved in fresh culture medium and added to the cells. After 6 h incubation at 37 °C, the amount of living cells was determined by MTT assay using 0.367 mg/mL final concentration of MTT in each well. After 3.5 h incubation with MTT, cells were centrifuged at 2000 rpm for 5 min, supernatant was removed, and crystals were dissolved in DMSO. The absorbance was measured using an ELISAreader (Labsystems MS Reader, Finland) at λ ) 540 nm and at λ ) 620 nm as reference wavelengths. Statistical analysis of data was performed by Student’s t-test of Origin7.5 at the 95% confidence level. In the case of the measurement of cytostatic effect, cells were plated into 96-well tissue culture plates in 100 µL culture medium with an initial cell number of 5 × 103 cells/well, 24 h prior to the treatment. The compounds or serum free medium as a control were added to the wells in 100 µL in serum free medium at a 200 µL final volume. After 6 h incubation, cells were washed twice with serum free medium, and then fresh culture medium was added to each well. Cells were maintained at 37 °C for a further 72 h, and then the quantity of living cells was determined by MTT-assay as described above. Four parallel measurements were performed in all cases, and they were repeated twice. Acute Toxicity Measurement. First-generation hybrid BDF1 (C57BL/6 female and DBA/2 male) adult female mice, weighing 22-23 g, specified pathogen free (SPF) from Department of Exprimental Pharmacology, National Institute of Oncology, Budapest, Hungary, were used. Colonies were used for these experiments. The animals were kept in macrolon cages at 22-24 °C (40-50% humidity), with a lighting regimen of 12/12 h light/ dark. The animals had free access to tap water and were fed a sterilized standard diet (Charles River VRF1, autoclavable, Germany) ad libitum. The animals used in these studies were cared for according to the “Guiding Principles for the Care and Use of Animals” based upon the Helsinki declaration, and they were approved by the local ethical committe. Our permission for breeding and performing experiments with laboratory animals is valid up to 10/06/2010.

Daunorubicin and Dau-GnRH-III conjugate were dissolved in distilled water at a concentration that allowed the dose to be given at a volume of 0.1 mL/10 g body weight. In this experiment, 7-7 mice/group were used. The mice were treated by i.p. administration. Toxicity in mice was characterized on the basis of lifespan and body weight. Survival time was followed over 21 days. In Vivo Antitumor Effect of Daunorubicin and Dau-GnRHIII Conjugate on Colon-26 Carcinoma. Balb/c female mice, weighing 22-24 g, kept under the same conditions described above were used in this experiment. Tumor tissue fragments (Colon-26 mouse carcinoma, SRI, Birmingham, AL) were transplanted (3-4 mm, ∼25 mg weight) subcutaneously (s.c.) into the intrascapulare region of the mice using a tweezers. In the first trial, the treatments started on day 7 after tumor transplantation by i.p. administration of the compounds dissolved in d.i. water. In this experiment, 5-5 mice/group were used. Except for the control group, the mice were treated with either free daunorubicin (once 5 mg (8.86 µmol)/kg body weight or 2 mg (3.55 µmol)/kg 5 times on every second day) or Dau-GnRHIII conjugate (once 62.5 mg/kg (26.6 µmol; 15 mg Dau content) or 20.8 mg/kg (8.86 µmol; 5 mg Dau content) 5 times on every second day. In the second experiment, tumor bearing mice (7 mice in each group) were treated twice with the conjugate using 62.5 mg (26.6 µmol)/kg. Treatments were carried out either on days 4 and 7 or days 7 and 10. In this case, the antitumor activity of the free GnRH-III(GFLG) was also studied. The hormone derivative was injected i.p. on days 4 and 7 using 45 mg (26.6 µmol)/kg body weight. The tumor growth inhibitory effect of various treatments was controlled 7 times within three weeks following the tumor transplantation using a digital caliper. Tumor volume was calculated using the following formula: V ) a2 × b × π/6 (where “a” and “b” mean the shortest and the longest diameter, respectively of a given tumor) (37). Changes of tumor volume and body weight were controlled and evaluated. Mean values and standard deviations were calculated. The evaluation of antitumor activity on the basis of survival time was evaluated as well. The survival probability was presented according to the Kaplan-Meier curve calculated/drawn by the MedCalc9 program.

RESULTS AND DISCUSSION Due to the limited conjugation sites of daunorubicin (Figure 1), the drug was attached to GnRH-III derivative as homing device via oxime bond formation. The in vitro antitumor effect of the conjugate was determined on MCF-7 and C26 tumor cell lines. Similar experiments were performed with a conjugate containing doxorubicin instead of daunorubicin, as well as with the free drugs. In spite of the higher in vitro activity of the conjugate on MCF-7 tumor cells, the C26 tumor bearing mice was a suitable animal model for in vivo experiments, because C26 colon tumor is a fast-growing and aggressive tumor type. It is worth noting that colon carcinomas are rarely used for the study of in vivo antitumor activity. However, the first results of the treatment of colon cancer bearing mice with Dox and Dox-[Lys6]-GnRH (AN-152) were recently reported (38). The treatment of nude mice bearing HT29 tumor started 14 days after tumor transplantation and continued for 55 days. The conjugate was administered intraperitoneally (i.p.) on days 1 and 28, whereas Dox (20.7 µmol/ kg; 13.6 mg/kg) was injected only once on day 1. AN-152 (20.7 µmol/kg) inhibited the tumor growth by 41% (735 ( 158 mm3 tumor volume), while Dox only by 28% (885 ( 201 mm3) compared to the controls (1219 ( 372 mm3). All animals were alive at the end of the experiment in the group treated with AN-152, but 6/9 mice Dox treated and 2/9 from the controls died. Some other colon tumors, such as HCT-116, HCT-15,



Figure 6. In vivo antitumor effect of daunorubicin and Dau-GnRH-III conjugate on mice bearing C26 colon tumor. Table 3. Antitumor Effect of Daunorubicin and Dau-GnRH-III Conjugate on Mice Bearing C26 Tumor (Experiment 1) tumor volume (cm3) ( SD days after tumor transplant

number of mice

control

Dau 2 mg/kg 5 × q2d

T/C (%)a

Dau 5 mg/kg 1 ×

T/C (%)

Dau-GnRH-III conjugate 5 mg/kg Dau 5 × q2d

T/C (%)

Dau-GnRH-III conjugate 15 mg/kg Dau 1 ×

T/C (%)

7 9 11 14 16

5 5 5 5 5

0.60 ( 0.10 1.91 ( 0.35 3.33 ( 0.51 6.04 ( 0.53 8.47 ( 1.33

0.64 ( 0.10 1.24 ( 0.26 2.75 ( 0.44 4.78 ( 0.98 6.56 ( 0.80

64.9 82.6 79.1 77.4

0.67 ( 0.11 1.00 ( 0.23 2.40 ( 0.23 3.84 ( 0.93 ntb

52.4c 72.1d 63.6c ---

0.58 ( 0.16 1.18 ( 0.32 2.95 ( 0.47 5.54 ( 0.78 7.14 ( 1.01

61.3d 88.6 91.7 84.3

0.59 ( 0,05 1.04 ( 0.21 2.63 ( 0.45 4.58 ( 0.96 6.59 ( 1.41

54.4c 79.0 75.8 77.7

a

Treated/control in percentage. b Not tested because of the death of mice. c p < 0.01. d p < 0.05.

LoVo, and Colo320DM, were examined. A lower amount of compounds (6.9 µmol/kg) but more frequent treatments (days 1, 7, 11, 15, 22, 28, 49) were applied. The tumor growth inhibition of AN-152 was 20%, 75%, 67%, and 74%, respectively. These are the only reported data on the treatment of colon cancer bearing mice to which our results can be compared. Conjugates. Daunorubicin, an antineoplastic agent widely used in tumor therapy, was attached to GnRH-III as a targeting moiety, which has itself an antiproliferative effect on cancer cells. The oxo group at the C-13 position was used as a conjugation site, because the acylation of the amino group of daunosamine by GnRH peptide led to the loss of biological activity (Figure 1). An oxime bond was formed between the keto group of daunorubicin and the aminooxyacetyl group of the GnRH-III derivative (Figure 2). As the oxime bond is relatively stable between pH 3 and 8 (39), as well as under in vitro and in vivo biological experimental conditions (40), an enzyme-labile GFLG tetrapeptide spacer was incorporated between the decapeptide and daunorubicin. This spacer sequence can be cleaved by lysosomal enzymes (e.g., Cathepsin B) (41, 42), and it was successfully applied in methotrexate and doxorubicin containing conjugates (42, 43). For comparative studies, the doxorubicin containing conjugate (Dox-GnRH-III) was also prepared under the same conditions. The purified conjugates were characterized by RP-HPLC and ESI-MS (Table 1; Supporting Information S3-S5). 1 H and 13C NMR Analyses of the Carboxymethyloxime Derivative of Daunorubicin (Dau-Aoa-OH). A simple model compound was prepared in order to analyze the oxime bond structure of Dau conjugate. The structure of O-carboxymethyloxime is unambiguously evidenced by the 1H and 13C NMR data assigned on the basis of DEPT, 2D-COSY, 2D-HMQC, 2D-HMBC, and DNOE measurements (Figure 3). The relative configuration of the daunoseamine motif is clearly reflected from

the split of the signals originated from the skeletal hydrogens and the mutual NOEs (3.7-4.2%) measured between the axial 3′-H and 5′-H protons. The broad singlets of 1′-H and 4′-H associated with weak vicinal couplings refer to their equatorial position, and consequently to the axial position of 1′-cycloalkoxy- and 4′-OH groups, respectively. The relatively small split (4.9 Hz) of the triplet from the 7-H cyclohexene proton points to its pseudoequatorial orientation. In the 1H-13C-HMBC spectrum, 7-H exhibits a cross peak with the C-13 signal originated from their 4J coupling along a special “W”arrangement of the bonds transmitting the interaction. Accordingly, the cyclohexene ring condensed to antraquinone carries the pseudoequatorial oxime moiety in the trans position relative to the pseudoaxial daunosamine unit. The weak NOEs (1.6-1.8%) detected between the 14-H methyl- and 15-H O-carboxymethyl protons in the oxime moiety with “E” configuration can be ascribed to the increased lifetime of the rotamer stabilized by a quasi six-membered chelate ring involving the imino nitrogen and the carboxyl proton [δ · (CO2H) ∼ 14.0 ppm]. This downfield-shifted signal is considerably broadened due to exchange. The “E”-oxime configuration is also supported by the upfield-shifted carbon line of C-14 (10.8 ppm), of the which signal would be expected at around 16-17 ppm in a “Z”-oxime isomer. Enzymatic Digestion of Dau-GnRH-III Conjugate by Cathepsin B. Cathepsin B, known to be overexpressed in cancer cells (44), is one of the lysosomal enzymes involved in the intracellular digestion of extracellular proteins taken up by endocytosis. The cleavage specificity of Cathepsin B can be used as a basis for controlled intracellular drug release. In the present work, oxime bond linked Dau-GnRH-III conjugate containing a GFLG tetrapeptide spacer between the anticancer drug and the GnRHIII decapeptide was subjected to Cathepsin B catalyzed hydrolysis. Liquid chromatography in combination with mass


Figure 7. (A) Survival according to the Kaplan-Meier analysis. Control group compared to the groups treated with different doses of Dau or Dau-GnRH-III conjugate (B) Survival according to the Kaplan-Meier analysis. Control group compared to the groups treated twice with DauGnRH-III conjugate on different days or with GnRH-III(GFLG).

spectrometric analysis of the reaction mixture indicated several Cathepsin B cleavage sites, namely, the peptide bonds -GlyPhe-, -Phe-Leu-, -Leu-Gly- inside the spacer sequence and the isopeptide bond -Lys(Gly)-. In addition, the cleavage of the peptide bond -7Trp-8Lys- in the GnRH-III sequence was determined (Figure 4A,C). Total ion chromatogram and mass spectra of each fragment produced by Cathepsin B digestion are presented in the Supporting Information S6 and S7. LC/ MS analysis of a solution of Dau-GnRH-III which was incubated at 37 °C for 24 h revealed no degradation of the conjugate in the absence of Cathepsin B (Figure 4B). In Vitro Cytotoxicity and Cytostatic Effect. In vitro cytotoxicity of free drugs and their GnRH-III conjugates was measured on MCF-7 tumor cell lines by MTT assay. The cells were incubated with the compounds in the (5.1 × 10-4) to (2 × 102) µM concentration range for 6 h at 37 °C. Daunorubicin and doxorubicin elicited similar cytotoxicity with LC50 values of 0.67 and 0.68 µM, respectively (Table 2, Supporting Information S8). The conjugates were toxic at a higher concentration, with Dau-GnRH-III having significant higher toxicity than Dox-GnRH-III (LC50 values are 40.11 ( 25.31 µM and 85.18 ( 3.85 µM, respectively). Long-term tumor cell growth inhibition was also studied on MCF-7 and C26 tumor cell lines that express GnRH receptors (Supporting Information S1). In this case, cells were incubated with Dau-GnRH-III or

Szabo´ et al.

Dox-GnRH-III conjugates for 6 h; then, the treated cells were maintained in fresh culture medium at 37 °C for a further 72 h. The Dau or Dox conjugates showed a higher cytostatic effect on MCF-7 tumor cells than on C26. The difference between the IC50 values of Dau-GnRH-III (3.94 ( 1.18 µM) and DoxGnRH-III (5.38 ( 1.86 µM) was not significant on MCF-7 cells (Table 2, Supporting Information S8). On C26 tumor cells, the conjugates were less potent, and the Dau-GnRH-III conjugate had a lower IC50 value (22.46 ( 1.74 µM) than Dox-GnRH-III (46.10 ( 6.06 µM). The GnRH-III(GFLG) hormone derivative had no cytostatic effect in the 10-4-102 µM concentration range (IC50 > 100 µM). In Vivo Toxicity. In vivo toxicity of the free daunorubicin and Dau-GnRH-III conjugate was studied on healthy BDF-1 female mice (7 mice in each group) in our preliminary toxicological experiments. The BDF-1 inbred mouse strain is internationally accepted for maintenance of the most transplantable tumors. Mice were treated i.p. once with 15 mg (26.6 µmol)/kg body weight free daunorubicin or equivalent daunorubicin containing conjugate (62.5 mg/kg, 24% Dau content). The experiment lasted for 21 days. All mice treated with the free drug died between days 10 and 12 (11.3 ( 0.76 days). However, all mice treated with the Dau-GnRH-III conjugate survived until the end of the experiment (day 21). The body weight of the Dau-GnRH-III treated mice slightly decreased during the first three days, and then it increased up to the body weight values observed in the case of the control group (Figure 5). In Vivo Antitumor Effect. For the study of the in vivo antitumor effect, Balb/c mice were transplanted with C26 tumor (5 mice in each group), because the maintenance of colon-26 solid carcinoma tumor is realizable exclusively in Balb/c inbred mouse strain. Both free daunorubicin and Dau-GnRH-III conjugate showed a moderate in vivo antitumor activity on C26 colon tumor bearing mice. The first treatment in all cases was performed on day 7 after tumor trasplantation. The compounds were administered once or 5 times on every second day. Measurement of the tumor volume was continued until day 16 after tumor transplantation, because one mouse from the nontreated control group died on day 17. The application of the free drug at 5 mg (8.86 µmol)/kg body weight (only one injection) resulted in the most effective inhibition of the tumor growth (36.4% on average to the control) in one week after treatment (Figure 6A, Table 3). However, 4 of 5 mice died between days 15 and 17 (median survival day is 16; p ) 0.0009, log-rank test) because of the toxicity of daunorubicin (Figure 7A). Using a lower dose of free daunorubicin, 5 × 2 mg (3.55 µmol)/kg, resulted in average 22.6% inhibition of tumor growth as compared to the control group before terminating the experiment (Figure 6A, Table 3). The toxic side effect of daunorubicin was also observed in this case, because the median survival time calculated from the Kaplan-Meier survival curve (45) was 21 days (p ) 0.0342, log-rank test) compared with 28 days for control population (Figure 7A). A similar level of inhibition (22.3% on average) was determined in the case of Dau-GnRH-III conjugate treatment when 62.5 mg (26.6 µmol)/ kg body weight dose was used (Figure 6A, Table 3). However, a lower dose (20.8 mg (8.86 µmol/kg) of conjugate was less effective even in the case of 5× repeated treatments of tumor bearing mice (15.7% on average) (Figure 6A, Table 3). Using the conjugate (62.5 mg/kg), the data of median survival time showed a moderate increase to the control (32 days (14.3%), p ) 0.1650 log-rank test), while the increase was significant in comparison to the group of mice treated with free drug (>52%) (Figure 7A). In the second experiment, the treatments with Dau-GnRH conjugate (62.5 mg (26.6 µmol)/kg) were carried out either on



Table 4. Antitumor Effect of Dau-GnRH-III Conjugate and GnRH-III(GFLG) on Mice Bearing C26 Tumor (Experiment 2) tumor volume (cm3) ( SD days after tumor transplant.

number of mice

control

Dau-GnRH-III conjugate treatment on days 4, 7

T/C (%)a

Dau-GnRH-III conjugate treatment on days 7, 10

T/C (%)

GnRH-III(GFLG) treatment on days 4, 7

T/C (%)

7 9 11 14 16 18 21

7 7 7 7 7 7 7

0.203 ( 0.03 0.387 ( 0.20 0.569 ( 0.14 1.010 ( 0.36 2.094 ( 0.70 3.888 ( 1.20 5.677 ( 1.71

0.172 ( 0.06 0.118 ( 0.03 0.125 ( 0.04 0.586 ( 0.54 1.191 ( 0.85 2.078 ( 1.01 3.044 ( 1.07

30.5b 22.0b 58.0c 56.9c 53.3c 53.6c

0.182 ( 0.06 0.144 ( 0.04 0.127 ( 0.09 0.597 ( 0.48 1.281 ( 0.68 2.344 ( 0.53 3.866 ( 1.50

37.2b 22.3b 59.1c 61.2c 60.3 68.1

0.193 ( 0.06 0.273 ( 0.05 0.344 ( 0.20 0.861 ( 0.48 1.544 ( 0.97 2.630 ( 0.74 4.665 ( 1.63

70.5c 60.5c 85.4 73.7 67.6 82.2

a

Treated/control in percentage. b p < 0.01. c p < 0.05.

days 4 and 7 or on days 7 and 10 after the transplantation of tumor xenograft. Significantly higher tumor growth inhibition was observed in both cases at the end of the experiment (46% and 32% inhibition, respectively, measured on day 21) (Figure 6B, Table 4). Even if the tumor growth was slower in this experiment, we could conclude that until day 11 the tumor volume decreased due to the treatments, with 70-80% tumor growth inhibition being detected during this time. The antitumor effect of the free GnRH-III(GFLG) hormone derivative was also studied. The data indicated slight but not significant inhibition (18%) of tumor growth compared to the control group at the end of the experiment. However, during the treatment period (until day 11) 30-40% tumor growth inhibition induced by the hormone peptide alone was observed using the same amount (26.6 µmol; 45 mg/kg) of peptide as was in conjugate (Figure 6B, Table 4). These data indicate that GnRH-III(GFLG) hormone derivative had its own in vivo antitumor effect on C26 colon cancer, which was only 40-50% of the inhibition determined in the case of treatment with the conjugate. In this experiment, the median survival probability of the control and treated animals (on days 7 and 10 or 4 and 7) was 29, 35, and 40 days, respectively. The increase of the survival time of treated animals was 21% and 38% compared to the control group. The group of mice treated only with the GnRHIII derivative also showed increased survival time, but the last 4 of 7 mice died on day 37 after tumor transplantation; in this way, the median survival time cannot be used properly for comparison. However, the average survival time of the control group (29 days) and the group treated with GnRH-III derivative (33 days) shows 14% increase. According to the Kaplan-Meier curves, the longest survival (40 days) was found in the case of the group treated with Dau-GnRH-III conjugate on days 4 and 7 (compared to the control group, p ) 0.0009, log-rank test). The results show that the animals survived the lethal dose of daunorubicin if it was conjugated to GnRH-III derivative as a targeting moiety even in the case of repeated treatments. A single treatment with Dau-GnRH-III conjugate using 15 mg Dau content/kg body weight provided tumor growth inhibition similar to the treatment with daunorubicin 5 × 2 mg, but the lifetime was lengthened by using the conjugate instead of the free drug. Furthermore, the elevation of the number as well as the earlier start of treatments increased the efficiency of the conjugate. Significant tumor growth inhibition and survival of mice were observed in the case of treatment on days 4 and 7 using 62.5 mg (26.6 µmol)/kg body weight. Further optimization of the treatment schedule and doses (determination of MTD; maximal tolerated dose) is in progress.

GnRH-III conjugates had less but significant in vitro antitumor effects on MCF-7 human breast and C26 murine colon carcinoma cells compared to the free drugs. It is worth mentioning that daunorubicin conjugates previously prepared in our laboratory using branched-chain polypeptides with polylysine backbone (EAK and SAK) as carriers and cis-aconitic acid (cA) pH labile spacer had significantly less antiproliferative effect in vitro on C26 tumor cells (IC50 ) 117.4 µM (cADSAK) and 629.9 µM (cAD-EAK)) and MDA-MB 435 P human breast cancer cells (IC50 ) 195.2 µM for both) (46). The in vivo antitumor effect of the new Dau-GnRH conjugate was determined on mice transplanted with a fast-growing, aggressive tumor type (C26 colon tumor). We found that higher daunorubicin content than the lethal dose was well-tolerated in the case of Dau-GnRH-III conjugate administration. Dau-GnRHIII could decrease the tumor growth similarly to daunorubicin (∼23%) in the case of a single treatment. The animals treated with Dau-GnRH-III had longer survival times compared to the untreated control group, while mice treated with the free drug died early. The repeated treatment with the conjugate (15 mg daunorubicin content/kg body weight) increased significantly the tumor growth inhibition (∼32%). We also found that the earlier start of the treatment had a positive effect on the efficiency (∼46% inhibition). In conclusion, the Dau-GnRHIII conjugate in which the components are connected by an oxime bond exhibited antitumor effect both in vitro and in vivo. To our knowledge, this is the first Dau-peptide conjugate containing an oxime bond that has in vivo antitumor activity. The conjugate was well-tolerated and did not show significant toxic side effects even in high doses. In spite of the significant tumor inhibition of Dau, the effect on survival was considerably decreased by the late toxic effect. In contrast to Dau, in addition to the tumor inhibitory effect, the prolongation of the lifetime was observed in the case of conjugate-treated tumor bearing mice. The pharmacological optimization of the treatment using the Dau-GnRH-III conjugate as well as the detailed study of the mechanism of action of the conjugate are in progress. Furthermore, in vitro and in vivo antitumor activity of DauGnRH-III conjugate will be tested on other types of tumors.

ACKNOWLEDGMENT

CONCLUSIONS

This work was supported by grants from the Hungarian National Science Fund (OTKA T 049814 and K-68285), the Ministy of Education (“Medichem 2” 1/A/005/2004), the Ministry of Health (ETT 202/2006), GVOP-3.2.1.-2004-040005/3.0, GVOP-3.2.1-2004-04-0352/3.0, and the Zukunftskolleg, University of Konstanz. The authors thank Aniko´ Horva´th for her excellent technical support.

In the present study, we report on the conjugation of daunorubicin to GnRH-III through the oxime bond. Considering the chemical and biological stability of the oxime bond, a tetrapeptide spacer cleavable by Cathepsin B was incorporated between the drug and the hormone. Dau-GnRH-III and Dox-

Supporting Information Available: (i) Western blot analysis of solubilized membrane proteins from C26 (mouse colon), HT29 (human colon), and MCF-7 (human breast) cells; (ii) HPLC chromatograms and mass spectra of the compounds; (iii) enzymatic digestion of Dau-GnRH-III conjugate by Cathepsin


B: LC/MS analysis of the reaction mixture (24 h reaction time). (iv) Cytostatic effect of the compounds on MCF-7 human breast cancer cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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Development of an Oxime Bond Containing Daunorubicin

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