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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

A New O⁶-Alkylguanine-DNA Alkyltransferase Inhibitor Associated with a Nitrosourea (Cystemustine) Validates a Strategy of Melanoma-Targeted Therapy in Murine B16 and Human-Resistant M4Beu Melanoma Xenograft Models

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Recherche 484, Clermont-Ferrand Cedex, France (M.R., J.C.Mau., J.P., P.L., J.C.Mad., E.M.); Université Clermont-Ferrand 1, Clermont-Ferrand Cedex, France (E.M.); Institut Curie, Laboratoire de Microscopie Ionique, Orsay, France (T.-D.W., A.C., J.L.G.-K.); and INSERM Unité 759, Orsay, France (T.-D.W., A.C., J.L.G.-K.)

Received February 6, 2008; accepted April 11, 2008.

	Abstract

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Chemoresistance to O⁶-alkylating agents is a major barrier to successful treatment of melanoma. It is mainly due to a DNA repair suicide protein, O⁶-alkylguanine-DNA alkyltransferase (AGT). Although AGT inactivation is a powerful clinical strategy for restoring tumor chemosensitivity, it was limited by increased toxicity to nontumoral cells resulting from a lack of tumor selectivity. Achieving enhanced chemosensitization via AGT inhibition preferably in the tumor should protect normal tissue. To this end, we have developed a strategy to target AGT inhibitors. In this study, we tested a new potential melanoma-directed AGT inhibitor [2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine; IBgBZ] designed as a conjugate of O⁶-(4-iododbenzyl)guanine (IBg) as the AGT inactivator and a N,N-diethylaminoethylenebenzamido (BZ) moiety as the carrier to the malignant melanocytes. IBgBZ demonstrated AGT inactivation ability and potentiation of O⁶-alkylating agents (cystemustine, a chloroethylnitrosourea) in M4Beu highly chemoresistant human melanoma cells both in vitro and in tumor models. The biodisposition study on mice bearing B16 melanoma, the standard model for the evaluation of melanoma-directed agents, and the secondary ion mass spectrometry imaging confirmed the concentration of IBgBZ in the tumor and in particular in the intracytoplasmic melanosomes. These results validate the potential of IBgBZ as a new, more tumor-selective, AGT inhibitor in a strategy of melanoma-targeted therapy.

Melanoma chemoresistance to O⁶-alkylating agents is mainly mediated by the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT or ATase), also termed O⁶-methylguanine-DNA methyltransferase (Passagne et al., 2006). AGT repairs the O⁶-alkylguanine lesions by a stoichiometric and irreversible transfer of the O⁶-alkyl group to the cysteine 145 residue in the active site (Juillerat and Juillerat-Jeanneret, 2007). The protein is thereby totally inactivated, and restoration of AGT activity then requires de novo synthesis. On the basis of this suicide mechanism of action, several pseudosubstrates derived from 6-substituted guanines were developed to inactivate AGT (McElhinney et al., 2003). Although various highly specific AGT inhibitors are now available [O⁶-benzylguanine (Bg) and O⁶-(4-bromothenyl)guanine (BTg)], clinical trials in association with an O⁶-alkylating agent [chloroethylnitrosourea (CENU) or tetrazine] showed increased side effects, requiring the reduction of a therapeutic agent dose to suboptimal levels (Quinn et al., 2002; Ranson et al., 2006). This emerging clinical limitation led to the development of innovative strategies to reduce the dose-limiting toxicity of the treatments.

Our approach is based on O⁶-alkylating agent chemotherapy in combination with a melanoma-selective AGT inhibitor. Our aim was to sensitize tumor cells to the therapeutic O⁶-alkylating agents while preventing toxic side effects by maintaining the DNA repair process (i.e., normal AGT levels) in healthy tissues. We had previously developed a series of AGT inhibitors derived from O⁶-alkyl/aralkyl guanosine or 2'-deoxyguanosine that enhanced resistant malignant melanoma sensitivity to CENUs both in vitro and in animal models (Cussac et al., 1994a; Debiton et al., 1997; Mounetou et al., 1997; Buchdahl et al., 1998). Like others inhibitors, they were widely distributed in the organism and could deplete AGT in various normal tissues (Cussac et al., 1994b). To achieve melanoma-selectivity, we designed new AGT inhibitors [2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine (IBgBZ)], conjugating the O⁶-(4-iododbenzyl)guanine (IBg) and the N,N-diethylaminoethylenebenzamido (BZ) moiety as a carrier to the malignant melanocytes. Indeed, we developed [¹²⁵I]benzamide derivatives as imaging agents, currently under Phase III clinical evaluation as radiopharmaceuticals for the diagnosis of disseminated melanoma (Michelot et al., 1993; Moreau et al., 1993). They form a reversible BZ-melanin complex, resulting in a high concentration in malignant melanocytes (Labarre et al., 2002; Guerquin-Kern et al., 2004). This property suggested that the BZ moiety would be a useful tool as a carrier to melanoma.

The present study was conducted as a proof-of-concept for melanoma-targeted therapy and assessed, in vitro and in vivo, the AGT inhibition, the O⁶-alkylating agent (CENU) antineoplastic activity enhancement, and the tumoral melanic tissue affinity of the new conjugate (IBgBZ) in murine and human-resistant melanoma.

	Materials and Methods

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Drugs. [¹²⁵I]BgBZ was labeled with high specific activity by radioiododestannylation in the presence of 37 MBq (1 mCi) of nocarrier-added sodium [¹²⁵I]iodide (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and peracetic acid in aqueous ethanol adapted from a previously described procedure (Mounetou et al., 1995). [¹²⁵I]BgBZ was obtained in 85% radiochemical yield and 98% purity after high-pressure liquid chromatography (HPLC) purification. The reaction was initiated by nonradioactive sodium iodide, and IBgBZ was fully identified by proton and carbon nuclear magnetic resonance and mass spectrometry. The detailed syntheses will be described in a forthcoming article.

Cystemustine was synthesized by previously described procedures (Madelmont, 1994) and supplied by Orphachem (Clermont-Ferrand, France).

Cell Culture. The human melanoma M4Beu cells (obtained from Dr. J. F. Doré) (Jacubovich and Doré, 1979) and the B16-F0 melanoma (no. CRL-6322; American Type Culture Collection, Manassas, VA) were cultured in GlutaMAX (Fisher Scientific Bioblock, Illkirch, France) (Eagle's minimum essential medium with glutamine) supplemented with 10% fetal calf serum and solutions of vitamins, sodium pyruvate, nonessential amino acids, and gentamicin at 37°C in a 5% CO₂-humidified incubator.

Determination of AGT Activity. The AGT assay was performed on protein extracts of M4Beu cells treated in vitro by IBgBZ, using a previously described procedure (Marchenay et al., 2001). Increasing amounts of M4Beu cell protein extracts were incubated with [³H]N-methyl-N-nitrosourea-methylated calf thymus DNA substrate for 1 h at 37°C. The substrate was then acid hydrolyzed, and the alkylated bases released in the sample were separated by reverse-phase HPLC (HP 1100 series; Hewlett Packard, Les Ulis, France) using a C18 Uptisphere column (5 µm, 25 x 4.6 mm) eluted at a flow rate of 1.5 ml/min by a 20 mM NH₄H₂PO₄ (pH 5.0) mobile phase containing 6% methanol (v/v) for 11 min and 15% methanol (v/v) for 14 min. The radioactivity of column effluents was automatically measured in a Packard flow scintillation analyzer (500TR series; PerkinElmer, Courtabeuf, France). AGT activity was determined by measuring the disappearance of O⁶-[³H]methylguanine from the substrate and was expressed in femtomoles of methyl groups transferred to protein per milligram of total protein present in the sample.

To measure in vivo AGT activity after drug exposure in vivo, a group of mice with tumors that reached approximately 500 mm³ were treated with IBgBZ by i.p. injection. The animals were euthanized by CO₂ inhalation at different times after injection. The tumors were removed and flash frozen in liquid nitrogen and stored at –80°C. Increasing amounts of M4Beu cell protein extracts were analyzed as described above.

Cytotoxicity Tests. For colony-forming assays, M4Beu cells were plated in six-well multidishes (150 cells/dish) and allowed to adhere for 20 h before treatment. The cells were treated with a culture medium containing increasing concentrations of IBgBZ (1–25 µM) dissolved in dimethyl sulfoxide (DMSO) before dilution in fresh culture medium, and they were incubated for various times. For combination treatment tests, cystemustine (50 µM) diluted in fresh culture medium was added 4 h after IBgBZ (2.5 or 5 µM) and incubated for 2 h. After the last treatment, the treated medium was replaced by fresh medium, and the cells were grown for 14 days at 37°C in a CO₂ incubator. Dishes were rinsed with phosphate-buffered saline, and the cells were fixed with methanol and stained with 0.2% crystal violet solution. The plating efficiency of cells not treated with drugs was approximately 70%. Colonies (>50 cells) were counted. The surviving fraction (percentage of survival) was calculated as the ratio of treated to untreated cells (control) x 100.

In Vivo Biodistribution Study. For tissue distribution analyses, 6-week male C57BL/6Jco mice (Charles River Laboratories, L'Arbresle, France) were inoculated with 5 x 10⁵ melanoma B16 cells by dorsal s.c. injection. When the tumor diameter reached approximately 0.5 cm, the animals received an i.v. injection of ¹²⁵IB-gBZ (0.74 MBq, 20 µCi, 10 mg/kg) dissolved in DMSO and 0.9% sodium chloride. Mice were euthanized by CO₂ inhalation at 1 h, 3 h, 5 h 30 min, 6 h, and 48 h after drug administration and were immediately frozen by immersion in liquid nitrogen. They were then embedded in a 2% carboxymethylcellulose gel, and the block was sagittally sectioned at –22°C with a Reichert-Jung Cryopolycut cryomicrotome (Reichert Jung, Heidelberg, Germany). The selected 40-µm-thick slices were taken using no. 810 Scotch band tape (3M, St. Paul, MN), dried for 48 h at –22°C, fixed on a hard-bound sheet, and introduced into an Ambis 4000 detector (Scanalytics/CSPI, San Diego, CA) to quantify the radioactivity in selected organs (n = 6 for each time studied) (Labarre et al., 1999). The final results are expressed as percentages of injected dose (ID) per gram of tissue.

Excretion Study. The excretion study was conducted using 6-week-old male C57BL/6Jco mice given [¹²⁵I]BgBZ as described above and housed in metabolic cages (Charles River Laboratories), allowing separate collection of feces and urine at 24, 48, and 72 h after administration. Radioactivity was measured in a gamma counter (Minaxi 5530; Packard, Rungis, France).

In Vivo Stability. Plasma, urine, rehydrated feces, and organs homogenized in a Potter blender (Fisher Scientific Bioblock) were sampled, extracted three times with methanol, and centrifuged at 2000g for 10 min at 4°C. The recovery of radioactivity ranged from 80 to 85%. After evaporation, the dry residues were dissolved in the mobile phase. Chromatography (HPLC) analysis was performed on an HP 1100 system with a UV/visible detector (Hewlett Packard) equipped with a reverse-phase Kromasil column (C18, 5 µm, 250 x 4.6 mm; Hewlett Packard) and connected to a 500TR flow scintillation analyzer (Packard Instrument S.A., Rungis, France). The flow rate was 1 ml/min with a gradient mobile phase starting from a methanol/0.4% NH₄OH mixture:70/30 (v/v) to 100/0 at 10 min to 15 min.

Secondary Ion Mass Spectrometry Analysis. Secondary ion mass spectrometry (SIMS) imaging was performed using a Nano-SIMS-50 Ion Microprobe (CAMECA, Gennevilliers, France). B16 murine melanoma cells were intravenously injected into male C57BL/6Jco mice to obtain, within 12 days, tumor cell colonies in lungs mimicking pulmonary micrometastases. After IBgBZ administration, mice were euthanized at 1 h, 3 h, and 5 h 30 min. Typical sample preparation for SIMS analysis was described previously (Guerquin-Kern JL et al., 2005). Image processing was performed using ImageJ (W. S. Rasband, United States National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/), a public-domain Java image-processing program, to obtain proper colocalization of the observed structures on the processed maps for all of the ion species, and allowed further correlation with light microscope images.

In Vivo Antitumor Tests. All experiments were carried out in accordance with the recommendations of the Institute of Laboratory Animal Resources (1996). Swiss nu/nu male mice (Charles River Laboratories) that were anesthetized by isoflurane inhalation received inoculations with 5 x 10⁶ M4Beu cells by abdominal s.c. injection. When tumors reached approximately 150 mm³ in size, the mice were randomly assigned to groups of six and were treated three times every 4 days with IBgBZ by i.p. injection and/or with cystemustine by i.v. injection. Cystemustine was dissolved in 0.9% sterile sodium chloride. IBgBZ was dissolved in DMSO and added to 0.9% sodium chloride. The day before the beginning of treatment was denoted as day 0. Each group of six mice was treated on day 1, 5, and 9. Twice a week, tumor dimensions were recorded by caliper measurement, and tumor volume was calculated using the formula: length x width² x 0.5. Twenty-four days after the beginning of the treatment (day 24), the mice were euthanized. Tumor volumes were heterogeneous. Accordingly, the results are expressed as the factor of tumor volume increase and calculated as follows: (tumor volume on day x – tumor volume on day 0)/tumor volume on day 0.

View larger version (8K): [in this window] [in a new window]   Fig. 1. In vitro IBgBZ evaluation. A, AGT activity in M4Beu cells after treatment with IBgBZ (2.5 µM for 2 h, 5 µM for 2 h, 5 µM for 4 h) or no treatment (control). Values are expressed as the mean of four independent experiments ± S.D. *, p < 0.05 versus control; **, p < 0.05, by Mann-Whitney rank test. B, potentiation of cystemustine cytotoxicity by IBgBZ. M4Beu cells were treated by IBgBZ (2.5 or 5 µM for 4 h) or cystemustine (50 µM for 2 h) or cystemustine in combination with IBgBZ. *, p < 0.05 versus cystemustine alone treatment, by Mann-Whitney rank test.

	Results

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Figure 1A shows the AGT activity that we observed in M4Beu cells by assay of methylated DNA substrate after IBgBZ treatments at 2.5 or 5 µM for 2 or 4 h. AGT levels of treated cells decreased compared with control, depending on the treatment duration rather than the dose of IBgBZ used over the range tested. IBgBZ deactivated AGT from 50 to 60% after 2 h to more than 80% after 4 h. These results demonstrate that IBgBZ is an active substrate of AGT and allows cellular AGT inactivation.

Cytotoxicity was determined in colony-forming assays, a very sensitive method used to detect the killing effects at cell levels. Potentiation of the antiproliferative activity of O⁶-alkylating agents was tested with a CENU: cystemustine (Madelmont, 1994; Cure et al., 1999). The concentration needed to inhibit 50% of cell growth compared with untreated cells (IC₅₀) was first evaluated for IBgBZ alone. The values calculated from dose-response curves were 17.7 ± 3.4 and 9.4 ± 1.8 µM after 2 h and 4 h of treatment, respectively. The two doses selected for the in vitro evaluation (i.e., 2.5 and 5 µM), where IBgBZ exerts a strong inhibitory effect, were confirmed as low toxic. As shown in Fig. 1B, no significant decrease of survival rate was observed for the M4Beu cells treated by IBgBZ alone at these doses. The cytotoxicity evaluation of cystemustine alone (50 µM) or in combination with IBgBZ (2.5 or 5 µM) is shown in Fig. 1B. The antiproliferative activity of cystemustine was significantly increased when the cells were pre-exposed to IBgBZ at 2.5 or 5 µM for 4 h. These results were consistent with the marked reduction in AGT cellular levels observed at the same times (Fig. 1A). In addition, as observed for the AGT inactivation, cystemustine activity potentiation was not IBgBZ dose-dependent in the range tested. Indeed, increasing the IBgBZ dose from 2.5 to 5 µM did not cause a significant decrease in M4Beu cell survival (Fig. 1B). Taken together, these results show that IBgBZ was effective in vitro for AGT inhibition and CENU activity potentiation.

Biodisposition and In Vivo Stability. To test the affinity of IBgBZ for melanoma tumors, a tissue distribution study was first conducted in C57BL/6 male mice bearing B16 murine melanoma xenograft. It is the standard model used in our laboratory to evaluate tumor affinity for melanoma-imaging agents (Moreau et al., 1993; Labarre et al., 2002). In addition, it allows formation of the pulmonary colonies needed for subsequent SIMS experiments (Guerquin-Kern et al., 2004). The radioactive tissue concentrations of mice treated with [¹²⁵I]BgBZ (0.74 MBq, 20 µCi, 10 mg/kg) determined at 5 min, 30 min, and 1, 3, 6, and 48 h postinjection are given in Table 1. In addition, a mouse whole-body autoradiography of tissue distribution of the radioactivity at 1 h postadministration of [¹²⁵I]BgBZ is shown in Fig. 2. These data indicate that the radioactivity was not widely distributed in the body. It was mainly found in metabolizing/eliminating organs (liver, kidney, lung) and in the tumor. Concentrations in other organs were very low (brain and muscle) or below the limits of detection. Tumor uptake increased up to 5.05 and 5.87% ID/g between 1 and 3 h after administration, respectively (Table 1). The maximal tumor/blood ratio (3.4) was reached at 3 h after injection. However, radioactivity was still detected in the tumor up to 48 h postinjection. As shown in Fig. 3, the radioactivity was not distributed homogeneously in the tumor. Accordingly, low uptake was observed in tumor necrosed areas. Most of the radioactivity (85%) was cleared by fecal excretion within 24 h, and only 10% was recovered in the urine (Table 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Representative whole-body autoradiography of a mouth bearing B16 melanoma cell xenograft, 1 h after i.v. injection of [125I]BgBZ.

View larger version (53K): [in this window] [in a new window]   Fig. 3. SIMS analysis images and histological sections of a 40 x 40-µm field focusing on a pulmonary colony of B16 melanoma cells, at 3 h after IBgBZ injection. A, distribution of 31P (nuclei). B, distribution of 127I (showing IBgBZ accumulation in melanosomes). C, image shows the histological section (melanosomes appear in black).

In vivo stability of IBgBZ was then evaluated. The percentage of unchanged IBgBZ was determined after HPLC analysis of urine, feces, plasma, liver, and tumor samples. The unchanged IBgBZ represented 75 and 22%, respectively, of the radioactivity in the fecal extract and urine samples, 24 h after administration. In the plasma, liver, and tumor, intact IBgBZ accounted for >70% of the radioactivity up to 3 h postinjection. Moreover, IBgBZ was particularly stable in the tumor (>60% detected at 6 h).

These results clearly indicate that IBgBZ reached the melanoma tumor mainly in its intact form, and it was particularly concentrated in this target tissue between 1 and 3 h postinjection.

SIMS Analysis. SIMS was used to determine the subcellular distribution of IBgBZ (Guerquin-Kern JL et al., 2004). Images showing one B16 melanoma cell colony 3 h after IBgBZ injection and histological sections in the same optical field are shown in Fig. 3. SIMS allows direct identification of chemical elements with high sensitivity and specificity and can be applied to visualize elemental distribution (chemical mapping). Figure 3A shows the distribution of phosphorus (mass, 31), and the condensed chromatin near the nuclear envelope was clearly identified. Figure 3B represents the distribution of iodine (mass, 27), i.e., IBgBZ and/or iodinated-derived compounds. The histological image (Fig. 3C) reveals that melanosomes, the specialized organelles containing melanin small grains and clusters, were largely distributed in the cellular cytoplasm. Superimposition of the melanosome and the iodine signal was observed, indicating a perfect colocalization of IBgBZ and/or iodinated-derived compounds with melanin polymers (Fig. 3, B and C). These data provide further evidence that IBgBZ was rapidly internalized in the melanocytes.

In Vivo Antitumor Tests and Intratumoral AGT Inhibition. Because AGT expression in B16 cells was very weak, the chemoresistant M4Beu line was used. To evaluate the antitumor efficacy of the cystemustine/IBgBZ combination and intratumoral AGT activity in vivo, M4Beu melanoma-resistant cells were established as xenografts in nude mice. Tumors appeared within 8 to 10 days. In vivo, intratumoral AGT activity of mice from 1 to 24 h after treatment with IBgBZ at the therapeutic dose (50 mg/kg) are shown in Fig. 4B. An initial 30% decrease in the AGT tumor level was observed at 1 to 3 h after treatment followed by an additional drop from 50 to 60% after 3 to 6 h. Initial activity was recovered at 24 h after treatment, consistent with the required de novo AGT resynthesis delay (Passagne et al., 2006). These data show that IBgBZ reached its molecular target (i.e., AGT) in the tumor in an active form.

View larger version (11K): [in this window] [in a new window]   Fig. 4. In vivo IBgBZ evaluation. A, the tumor volume increase of nude mice bearing a M4Beu melanoma cell xenograft after treatment compared with initial volume (at day 0 of treatment). Mice were treated three times for 4 days (beginning on day 1) with 10% DMSO in sterile water (vehicle) or IBgBZ (50 mg/kg) and/or cystemustine (15 mg/kg). *, p < 0.05 versus mice treated with cystemustine alone from day 8 to day 24, by Mann-Whitney rank test. B, AGT activity in tumors of nude mice bearing M4Beu melanoma cell xenograft after administration of IBgBZ (50 mg/kg). Values are expressed as the mean of four independent experiments ± S.D. *, p < 0.05 versus control, by Mann-Whitney rank test.

	Discussion

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New melanoma treatments with both improved tumor selectivity and lower toxicity are urgently needed. To address this issue, strategies based on the selective suppression of AGT activity in malignant cells were considered.

To date, various approaches have been developed. Genetic chemoprotection of hematopoietic and progenitor stem cells with mutant AGT has been explored (Southgate et al., 2006). However, this approach does not consider extra-hematopoietic toxicities, and it needs to be optimized before any clinical application can be envisioned. New generations of AGT inhibitors with improved tumor selectivity have also been developed (Juillerat and Juillerat-Jeannerat, 2007). They were designed as conjugates of a first-generation AGT inhibitor (Bg, BTg, and 2-amino-O⁴-benzylpteridine) and a ligand of a high-expression, tumor-specific receptor or transporter (D-glucose, glucuronic acid, and folic acid) (Kaina et al., 2004; Nelson et al., 2004; Wei et al., 2005). Finally, to date, none of these conjugation approaches has demonstrated any successful in vivo tumor selectivity.

In this study, our goal was to develop a melanoma-targeted therapy combining an O⁶-alkylating agent (cystemustine) and a new guanine conjugate, IBgBZ, able to achieve AGT inactivation more selectively in melanoma tumors. The three essential constituent parts of this compound are as follows: 1) a Bg moiety as the AGT inhibitor; 2) a BZ moiety at the N-9 position of Bg as a carrier to the melanoma tumor; and 3) an iodine atom at the 4-position of the O⁶-benzyl group of Bg as a potential radiation emitter when replaced by a suitable radioisotope (e.g., iodine 131). It would provide an opportunity to combine the effects of radionuclide therapy (Larson and Krenning, 2005) and chemotherapy (i.e., radiochemotherapy), where DNA lesions caused by chemotherapeutic agents would be synergized by local radiation damage induced by the radioiodine atom, preferentially in the melanoma tumor. To validate our targeting strategy, two melanoma models were used. For biodisposition and SIMS studies, the B16 murine melanoma model validated by our previous work on melanoma-imaging agents was used (Moreau et al., 1993; Labarre et al., 2002; Guerquin-Kern et al., 2004). Because AGT levels in the B16 murine melanoma cell line were very low, pharmacological evaluation was performed using M4Beu human melanoma, a model that exhibited a high AGT activity and was chemoresistant to O⁶-alkylating agents such as CENUs (Cussac et al., 1994a; Mounetou et al., 1997).

First, we verified the in vitro AGT-inhibitory activity of BgBZ in M4Beu human-chemoresistant melanoma cells. Although the conjugation of the BZ vector at the N-9 position of Bg decreased AGT inhibition, as described in previous structure-activity relationship studies (McElhinney et al., 2003), IBgBZA was still an efficient inhibitor (70–80%), and it enhanced M4Beu melanoma cell sensitivity to cystemustine with no intrinsic toxicity. The activity was affected by N-9 substitution more than by O⁶-benzyl para-substitution by halogen atom (McElhinney et al., 2003). However, N-9 and, to a lesser extent, N-2 are the only positions in Bg that are tolerated for conjugation without total loss of activity. AGT inactivation was also observed in the tumor xenograft of the treated animal, showing that IBgBZ reached the targeted tissue in vivo in an active form.

Biodisposition studies in mice bearing B16 murine melanoma demonstrated that IBgBZ concentrated in the tumor up to 5 to 6% of the injected dose per gram of tissue at 1 to 3 h postinjection. The maximal tumor/blood ratio (3.4) was reached at 3 h postinjection. At this time, 70% of the dose was detected as the intact IBgBZ. SIMS analysis at the same time yielded further evidence of IBgBZ concentration in the intracytoplasmic melanin-rich organelles (melanosomes). This result is consistent with our previous experiments showing that BZ derivatives concentrate in the melanosome and form a reversible melanin-BZ complex (Guerquin-Kern et al., 2004). Because nonconjugated parental inhibitors did not display any tissue selectivity (Cussac et al., 1994b; Vaidyanathan et al., 2004), these observations prove that the BZ moiety allows melanoma cell targeting.

Pharmacological studies in nude mice bearing the established M4Beu tumor confirmed that IBgBZ potentiated the cystemustine antitumor action in vivo. Among the protocols tested, the most effective was a 3-h IBgBZ pretreatment followed by a cystemustine injection at days 1, 5, and 9. The tumoral growth of the mice treated by the IBgBZ/cystemustine combination was significantly lower than those treated by cystemustine alone at the same dose. This result was correlated with a decrease in intratumoral AGT levels in mice treated with the combination. The in vivo 3-h pretreatment was probably more efficient because the optimum activity of the cystemustine and the lowest AGT levels were concomitant. When cystemustine was injected 6 h after the inhibitor, the antitumoral activity may have occurred when AGT levels start to increase again, resulting in a limited efficacy. However, the regimen schedules could be optimized. First, the significant efficacy of the inhibitor 3 h pretreatment versus the 6-h pretreatment suggests that cystemustine could be injected earlier. Second, the tumor regrowth observed from day 18, i.e., after a 10-day cessation of the treatment, suggests that an additional treatment cycle starting between days 13 to 15 may improve the efficacy of the combination.

In conclusion, this study describes the first melanoma-targeted AGT inhibitor, IBgBZ. The results confirm the interest of our approach based on the combination of a tumor-specific AGT inhibitor to an O⁶-alkylating agent for a melanoma-targeted therapy. Future development will focus on the following: 1) optimization of inhibitor/cystemustine treatment schedules in correlation with tumoral AGT assays; 2) improvement of IBgBZ properties (selectivity and AGT inhibition) by structural modulation of the inhibitor and/or the vector moieties; and 3) validation of the combined radio- and chemotherapy action after replacement of the iodine 127 atom by the iodine 131 radioisotope in IBgBZ.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.137737.

ABBREVIATIONS: AGT, O⁶-alkylguanine-DNA alkyltransferase; Bg, O⁶-benzylguanine; BTg, O⁶-(4-bromothenyl)guanine; CENU, chloroethylnitrosourea; IBgBZ, 2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine; IBg, O⁶-(4-iododbenzyl)guanine; BZ, N,N-diethylaminoethylenebenzamido; HPLC, high-pressure liquid chromatography; cystemustine, N'-[2-chloroethyl]-N-[2-(methylsulfonyl) ethyl]-N'-nitrosourea; DMSO, dimethyl sulfoxide; ID, injected dose; SIMS, secondary ion mass spectrometry.

Address correspondence to: Dr. Emmanuelle Mounetou, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 484, BP 184, F-63005 Clermont-Ferrand Cedex, France. E-mail: mounetou{at}inserm484.u-clermont.fr

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