Discrepancy between in vitro and in vivo antitumor ...

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platinum compounds with a trans geometry, platinum(IV) compounds with four ligands (e.g. satraplatin), multinuclear or platinum-intercalator conjugates [1, 2, 8].
Invest New Drugs DOI 10.1007/s10637-010-9461-z

PRECLINICAL STUDIES

Discrepancy between in vitro and in vivo antitumor effect of a new platinum(II) metallointercalator Johnny Moretto & Bruno Chauffert & François Ghiringhelli & Janice R. Aldrich-Wright & Florence Bouyer

Received: 30 March 2010 / Accepted: 14 May 2010 # Springer Science+Business Media, LLC 2010

Summary Platinum(II) metallointercalators represent a new class of DNA-damaging antitumor complexes active in cisplatin- and oxaliplatin-resistant cell lines. In the first part of our work, we have screened in vitro a serie of 18 metallointercalators with the structure [Pt(AL)(IL)]2+ where AL =ethylenediamine (EN) or diaminocyclohexane in R,R(RR) or S,S- (SS) configuration ; and IL = 1,10-phenanthroline with different degree of methylation : no methylation (PHEN), mono-methylated in position 4 (4ME) or 5 (5ME), or di-methylated in positions 4 and 7 (47ME) or in positions 5 and 6 (56ME) or tetramethylated in positions 3,4,7 and 8 (3478ME). Eight compounds: PHENEN, 56MEEN, 47MERR, 56MERR, 4MESS, 5MESS, 47MESS and 56MESS exhibited significant cytotoxic effect, equivalent or higher than cisplatin, oxaliplatin or carboplatin in the J. Moretto : B. Chauffert : F. Ghiringhelli : F. Bouyer (*) Faculties of Medicine and Pharmacy, INSERM, UMR 866, University of Burgundy, 7 Bd Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France e-mail: [email protected] J. Moretto Department of Haematology, Dijon University Hospital, 2 Bd Maréchal de Lattre de Tassigny, 21000 Dijon, France J. R. Aldrich-Wright School of Biomedical and Health Sciences, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia B. Chauffert : F. Ghiringhelli GF Leclerc Regional Anticancer Centre, 1 rue Pr Marion, BP 77980, 21079 Dijon Cedex, France

human HCT8 colon and IGROV1 ovarian cancer cell lines for both 1 and 24 h incubation time. The high cytotoxicity of the most active compound, the 56MESS, could be related to the hydrophobicity of the phenanthroline ligand that increases cellular uptake in human HCT8, HT29 (colon) and IGROV1 (ovarian) as well as in rat PROb colon cell lines. Unfortunately, intravenous or intraperitoneal administration of 56MESS had no antitumoral activity in BD-IX rats with peritoneal carcinomatosis induced by an intraperitoneal PROb cells inoculation. Moreover, 56MESS displayed nephrotoxicity at pharmacological dose. Thus, these data query the in vivo/in vitro correlation and reconsider the place of the in vivo screening to select adequate candidate drug for further preclinical and clinical developments. Keywords Platinum(II) metallointercalators . 1,10-phenanthroline . Cytotoxicity . Platinum cellular accumulation . Antitumor effect

Introduction Platinum compounds represent a class of anticancer drugs currently used for the treatment of solid tumours in human [1–3]. In clinic, only three compounds, recognized as classical agents, are commercially available all around the world: cisplatin, oxaliplatin, and carboplatin (Fig. 1a,b,c), all exhibit cis conformation and a platinum(II) metal center. However, their clinical effectiveness is restricted by toxicities such as nephrotoxicity or neurotoxicity [1, 4] and by acquired or intrinsic resistances [5, 6]. Such limitations as well as recent elucidations in cell signalling and DNA targeting have stimulated the research for new platinum antitumor compounds, including pharmacomodulation of existing agents [1, 2, 7, 8]. Among the thousands

Invest New Drugs Fig. 1 Chemical structures of platinum(II) compounds assessed in our study. They included classical platinum agents such as (a) cisplatin, (b) oxaliplatin, and (c) carboplatin, and new platinum intercalators (d, e) containing a methyl-substituted 1,10-phenanthroline intercalating ligand and various ancillary ligands : (d) ethylenediamine, (e) diaminacyclohexane in S,S- or R,R- configuration. All metallointercalators tested and their acronyms are listed in (f). (*: asymetric carbon)

a

b H 3N

Pt

H 3N

O

NH2

Cl

c

O Pt

Cl

NH2

d

O

O

O

e

R5

2+

R6

R4

R6

R4

Pt

H 3N

O

2+ R5

O O

H 3N

R7

R7

R3 R3

R8

R8 N

N

Pt

Pt H2N

N

N

H2N

NH2

NH2

*

*

f Intercalating ligand

Ancillary ligand

Methyled carbons of phenantroline

R3, R4, R5, R6, R7, R8 = H (PHEN) R3, R5, R6, R7, R8 = H, R4 = Me (4ME) R3, R4, R6, R7, R8 = H, R5 = Me (5ME) R3, R5, R6, R8 = H, R4, R7 = Me (47ME) R3, R4, R7, R8 = H, R5, R6 = Me (56ME) R5, R6= H, R3, R4, R7, R8 = Me (3478ME)

of derivates synthesized, several classes of platinum compounds, with structures different from the classical derivates, have exhibited interesting features. They include platinum compounds with a trans geometry, platinum(IV) compounds with four ligands (e.g. satraplatin), multinuclear or platinum-intercalator conjugates [1, 2, 8]. Platinumintercalator conjugates, which are under current investigation, consist of two specific DNA-recognizing groups connected either by a flexible linker chain (usually a polymethylene chain) or directly to the platinum metal center [8, 9]. Such structures were designed to potentially combine two mechanisms of action: platination and intercalation. Metallointercalators represent a particular class of these compounds, characterized by a rigid structure, which may allow their recognition by specific DNA sequences turning on several signaling pathways [9–12]. Platinum(II) metallointercalators consist in square-planar metal complexes associating an intercalating ligand (IL) and a co-planar non-intercalating ancillary ligand (AL) [10, 11, 13, 14]. IL behaves as a DNA anchor for the metal complex via π-π stacking interactions and directs AL orientation [9, 15]. Compounds with substituted 1,10-phenanthroline (phen) as IL have already displayed in vitro cytotoxic

ethylenediamine

R,R-dach

S,S-dach

(EN)

(RR)

(SS)

PHENEN 4MEEN 5MEEN 47MEEN 56MEEN 3478MENN

PHENRR 4MERR 5MERR 47MERR 56MERR 3478MERR

PHENSS 4MESS 5MESS 47MESS 56MESS 3478MESS

activity against different cell lines resistant to cisplatin [10, 12, 13, 16, 17] and one has been investigated in vivo [18]. The resulting antitumor effect has been attributed to intercalation into the DNA minor groove, although other mechanisms such as electrostatic interaction or self-aggregating along the DNA strand cannot be excluded [8–11, 13, 15, 19]. In this study, several platinum(II) metallointercalators were investigated in vitro and in vivo for their cytotoxic and antitumoral activities. Platinum(II) intercalating compounds containing methyl-substituted phen were chosen from preliminary screenings in our laboratory [20], demonstrating their higher potential cytotoxic effect in a cisplatin- and oxaliplatin-resistant cancer cell line in vitro compared to other types of intercalating ligands, and from other laboratories [11]. Thus, we have investigated and compared in vitro, using a clonogenic assay, the cytotoxic effect of [Pt (AL)(IL)]2+ complexes in which AL was ethylenediamine (EN) or diaminocyclohexane (dach) either in R,R (RR) or S,S (SS) configurations, and IL was various methylated phen (Fig. 1d,e,f). From this screening, one of the most active compounds, the 56MESS, was selected for further investigations and its effect was compared to reference molecules such as cisplatin and oxaliplatin. Dose-incubation time study

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(using clonogenic assay) and extent of cellular uptake were carried out in colon and ovarian cancer cell lines in vitro. In vivo experiments were performed on BD-IX rats to evaluate toxicological and antitumor effect in a rat model of colon cancer.

Material and methods Drugs All metallointercalators were synthesized as previously reported [10–14, 18] and generously supplied by Prof. Aldrich-Wright. Metallointercalators, cisplatin (SigmaAldrich, France), oxaliplatin (Sanofi-Aventis, France) and carboplatin (Teva, France) were diluted in sterile physiological serum (Aguettant Co., France). Cell lines Human colon HT29 and HCT8 and ovarian IGROV1 cancer cells were obtained from the American Type Culture Collections (Manassas, VA, United States). HT29 and IGROV1 were cultured in RPMI 1640 medium (Biowhittaker, France), and HCT8 in DMEM containing glucose 4.5 g.L−1 and L–glutamine 2 mM (Biowhittaker, France). The rat PROb cell line, a tumorigenic clone derived from the dimethylhydrazine-induced rat colon tumor DHD/K12 [21] was cultured in Ham F10 medium. All medium were supplemented with 10% fetal bovine serum (Biowhittaker, France). All cell lines were maintained as exponentially growing monolayers in mycoplasma free culture condition checked by PCR analysis (PCR Mycoplasma Test Kit I/C, PromoKine, PromoCell France). Animals Male and female BD-IX rats, 3 to 5 month old, weighing 205 to 405 g, were bred in adequate conditions of temperature, hygrometry, and exposure to artificial light. All experimental protocols were consistent with the “Guidelines on the protection of experimental animals” published by the Council of the European Community (1986). All the procedures were approved by the Animal Care and Use Committee of the University of Burgundy (approval form number: 1108).

transferred into 24-well plates and incubated for 7 days at 37°C in a 5% CO2 atmosphere. This transfer allows the determination of the rate of clonogenic cells, including treatment-resistant and DNA-repaired cells. Two clonogenic protocols were performed: the first assay was conducted with all compounds (metallointercalators and classical agents) in HCT8 and IGROV1 resistant cell lines. Drugs were incubated during a short time incubation of 1 h (to identify compounds active very quickly) and for a long time exposure of 24 h (not to discard active compounds). The second assay was designed for the selected metallointercalator to assess its time dependent cytotoxic effect and to compare to cisplatin and oxaliplatin. It was carried out in human HT29 and IGROV1 cells as well as in rat PROb cells, which was used for in vivo investigations (see Section on antitumoral study), exposed to different incubation times (2, 24 or 72 h). After incubation, cells were washed in PBS 1×, fixed in pure ethanol, stained with 1% crystal violet, and eluted in 33% acetic acid. The intensity of coloration was determined by the measurement of absorbance by spectrophotometry (UVM 340, Bioserv) at λ=570 nm. Results were expressed as dose-response curves and the determination of the Inhibitory Concentration 50 (IC50) of at least three sets of measurements performed on different days. The IC50 represents the concentration which inhibits 50% of cell growth, and was determined after linearization using the Probit method. Accumulation assay Cellular penetration of the selected metallointercalator was evaluated versus cisplatin by quantification of total intracellular platinum content. HT29, HCT8, IGROV1 and PROb cells were seeded at a density of 500,000 per well in 12-well plates. At sub-confluence, cells were treated by drugs for 30, 60, 90 or 120 min at an equivalent dose of platinum (8.5 mg/L), that reflects the in vivo intraperitoneal amount of platinum administrated in BD-IX rats. At the end of the treatment, cells were washed twice in HBSS (Biowhittaker, France), digested in HNO3 and platinum quantified by atomic absorption spectrometry (SpectrAA285Z with Zeeman correction, Varian). Results were expressed as kinetic curves of platinum accumulation according to different incubation time which were performed at least three times on different days. Toxicological assessment of 56MESS in BD-IX rats

Assay of clonogenicity Cells were seeded at a density of 20,000 per well in 96-well plates, and treated at sub-confluence by increasing concentrations (from 0 to 100 μM) with the different platinum derivatives. After different time of incubation, cells were

This study was conducted to determine the dose and the toxicity of the selected metallointercalator. Several groups of BD-IX rats received a bolus intravenous injection of drug at increasing doses (0, 4, 6, 8, 10 and 20 mg per kg) and were observed for one month. Rats from the control group received

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an intravenous injection of sterilized physiologic serum. During the protocol, body weight, rat behaviour and survival rate were regularly assessed. Blood was collected from the tail or by cardiac punctures under anaesthesia by isoflurane at day 0, 3, 7, 14, 21 and 28 for biochemical (Vista, Roche, Biochemistry Laboratory, University Hospital, Dijon) and haematological (Coulter LH750 Analyzer, Beckman, Hematology Laboratory, University Hospital, Dijon) analyses. At day 28, animals were sacrificed and liver, kidney, heart, intestine and lung were removed for histological analyses. Organs were fixed in buffered formalin, embedded in paraffin and stained by hematoxilin/eosin coloration (Leica equipments, IFR100, University of Burgundy). This preclinical study enabled the determination of the Maximal Tolerated Dose (MTD) which corresponds to the maximal dose for which no death occurs. Assessment of 56MESS antitumor effect in a syngeneic rat model of cancer This study evaluated the ability of the selected drug to cure rats bearing a peritoneal carcinomatosis. Syngeneic model of peritoneal carcinomatosis was obtained after intraperitoneal injection of 2 × 106 PROb cells in BD-IX rats. Three days after PROb injection, size tumor was approximately 3×3 mm² and rats received intravenous or intraperitoneal injection of metallointercalator or cisplatin, both given as a single bolus at their respective MTD. Animals were daily observed for 35 days after PROb injection. At the end of the protocol, rats were autopsied and quantification of antitumoral efficacy was made using a tumor scale from CP0 (no peritoneal carcinomatosis left), to CP1 (few millimetric nodules present), to CP2 (many supra-millimetric nodules mainly in the epiploic area) and CP3 (advanced carcinomatosis with multiple peritoneal, parietal and diaphragmatic nodules). Presence or absence of hemmoraghic ascites were indicated by respectively A+and A0. Statistical analyses For all data sets, values were indicated as mean±standard deviation (SD). Associations of data were assessed with Student’s t test or Mann-Whitney-Wilcoxon test, as appropriate. Correlation between two quantitative variables was evaluated by linear Pearson’s correlation coefficient. The level of significance for all tests was 5%.

Results In vitro screening of the different families of platinum(II) intercalators with a substituted phen IL and various AL (EN, RR, SS)

Globally, in a family of metallointercalators bearing the same AL, the scale of cytotoxicity between variously methylated compounds was quite similar to the two human cisplatin-resistant cell lines studied: HCT8 and IGROV1, results from dose-response curves being confirmed by IC50 values. In all families, the tetra-substituted complexes were the less active (equal or inferior to carboplatin). Moreover, the prolongation of incubation from 1 h to 24 h emphasized the cytotoxic effect of many compounds. To compare, carboplatin was poorly active in HCT8 (IC50 not determined) and significantly less effective than cisplatin (p