Search

The development of alternative therapies for melanoma treatment is of great interest as long-term tumour regression is not achieved with new targeted chemotherapies on selected patients. We previously demonstrated that radioiodinated heteroarylcarboxamide ([131I]ICF01012) induced a strong anti-tumoural effect by inhibiting both primary tumour growth and dissemination process in a B16BL6 melanoma model. In our study, we show that a single injection of [131I]ICF01012 (ranging from 14.8 to 22.2 MBq) was effective and associated with low and transient haematological toxicity. Concerning pigmented organs, cutaneous melanocytes and skin were undamaged. In 30% of treated animals, no histological alteration of retina was observed, and in the remaining 70%, damages were restricted to the optic nerve area. Using the Medical Internal Radiation Dose methodology, we determined that the absorbed dose in major organs is very low (

The increasing incidence of melanoma and the lack of effective therapy on the disseminated form have led to an urgent need for new specific therapies. Several iodobenzamides or analogs are known to possess specific affinity for melanoma tissue. New heteroaromatic derivatives have been designed with a cytotoxic moiety and termed DNA intercalating agents. These compounds could be applied in targeted radionuclide therapy using (125)I, which emits Auger electrons and gives high-energy, localized irradiation. Two iodinated acridine derivatives have been reported to present an in vivo kinetic profile conducive to application in targeted radionuclide therapy. The aim of the present study was to perform a preclinical evaluation of these compounds. The DNA intercalating property was confirmed for both compounds. After radiolabeling with (125)I, the two compounds induced in vitro a significant radiotoxicity to B16F0 melanoma cells. Nevertheless, the acridine compound appeared more radiotoxic than the acridone compound. While cellular uptake was similar for both compounds, SIMS analysis and in vitro protocol showed a stronger affinity for melanin with acridone derivative, which was able to induce a predominant scavenging process in the melanosome and restrict access to the nucleus. In conclusion, the acridine derivative with a higher nuclear localization appeared a better candidate for application in targeted radionuclide therapy using (125)I.

Targeted internal radionuclide therapy (TRT) could be an efficient, specific way to treat disseminated melanoma. Based on a previous pharmacomodulation study, we selected a quinoxaline-derived molecule (ICF01012) for its melanin specificity and kinetic properties suitable for TRT. Here, we determined the efficacy of [(131)I]ICF01012 radiotherapy in vitro and in vivo in relation to melanogenesis using human melanoma models. [(125)I]ICF01012 uptake was first assessed in relation to melanin content. We found that melanin distribution in different models was representative of pathology seen in human tumours: melanin content was high in the extracellular space of SKMel3 tumours, and accumulated primarily in melanophages in M4Beu tumours. Targeted [(131)I]ICF01012 radiotherapy had a strong anti-tumoural efficacy in pigmented versus unpigmented tumours, regardless of target distribution and content. This study supports the use of melanin targeting with (131)I-labelled iodoquinoxaline for effective treatment of melanoma.

This study assessed the 1H HRMAS NMR spectroscopic profile of articular cartilage in both physiological and osteoarthitic situations. One-dimensional and two-dimensional 1H HRMAS NMR spectra were obtained from the tibial plateau cartilage of healthy and operated (unilateral medial meniscectomy and sham surgery) guinea pigs at different stages of disease, over a 6-month period. The major osteoarthritis-induced 1H HRMAS NMR changes were an increase of the N-acetyl peak of proteoglycans (at day 20 after meniscectomy) and a decrease after day 60 as the pathology evolved. These proteoglycan changes revealed by 1H HRMAS NMR analysis were validated by proteoglycan biochemistry assays. 1H HRMAS NMR analysis also evidenced a sharp increase in methylene resonances of chondrocyte membrane lipids from day 90 as a marker of apoptosis. There was an increase of the mobile methyl group of collagen at day 120, which was associated with collagen breakdown. 1H HRMAS NMR analysis provided a multifactorial and sequential picture of cartilage degradation at the extracellular matrix and chondrocyte levels.

N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carbamide (ICF 01012) is a new melanoma imaging agent showing promising properties for application in internal radionuclide therapy. We developed an analytical protocol for detection of ICF 01012 in biological samples using HPLC. The proposed method was first validated using standard of ICF 01012 and four potent metabolites of this compound and then applied to follow the metabolic fate of [(125)I]ICF 01012 after injection in melanoma-bearing mice. The results demonstrate that this method exhibits a good linearity (r(2)=0.9947), specificity and acceptable accuracy. This simple method appears convenient and sufficient for pharmacokinetic studies on [(125)I]ICF 01012.

Proteasome inhibition is a new strategy in cancer therapy. We synthesized three new peptide aldehyde inhibitors linked to the benzamide derivative structure to use their cytotoxic activity against malignant melanoma cells. Of these, 10 displayed the highest cytotoxicity (0.18 +/- 0.16 microM). A radiosynthesis of the iodine aldehyde was performed. Its drug biodistribution showed that some selectivity of the benzamide group toward malignant melanoma tissue was conserved.

Background and purpose: Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G1/S and G2/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern. Experimental approach: Proteins from B16 cells incubated with [14C-urea]-CCEU and [125I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry. Key results: Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but β-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G2 and G1 phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G1 phase. Conclusions and implications: This study reinforces our findings that CEUs alkylate proteins through an ester linkage with an acidic amino acid and shows that PHB alkylation contributes to G1/S arrest in CCEU treated B16 cells. Modification of PHB status and/or activity is an open route for new cancer therapeutics. British Journal of Pharmacology (2007) 152, 449–455; doi:10.1038/sj.bjp.0707415; published online 20 August 2007

Melanoma is a neoplasia of dramatically increasing incidence that has a propensity to spread rapidly. Early detection is fundamental and patient management requires reliable, sensitive and reproducible staging methods, such as a single examination by planar scintigraphy or single-photon emission tomography (SPET) using a radiopharmaceutical with selectivity for melanoma tissue. Among iodobenzamides reported to possess an affinity for melanoma, a new compound, N-(2-diethylaminoethyl)-2-iodobenzamide (BZA(2)), was selected for a clinical trial in view of its pharmacokinetic experimental profile in melanoma-bearing mice. Planar whole-body scintigraphy using (123)I-BZA(2) was performed in 25 patients with histologically proven cutaneous melanoma. Performance was evaluated in two groups of patients with one or more documented secondary lesions ( n=13) or with no known secondary lesions ( n=12), and results were compared with those of conventional investigation techniques. No adverse clinical or biological events were recorded. Lesions were imaged by increased tracer uptake, and good quality images were obtained 4 h after administration. After a follow-up of more than 1 year, the overall results of (123)I-BZA(2) scintigraphy on a per patient basis showed a sensitivity of 100%, a specificity of 95%, a positive predictive value of 86% and a negative predictive value of 100%. The proven secondary lesions were imaged with a sensitivity of 100% and a specificity of 91%. In seven patients with suspected metastases, the absence of (123)I-BZA(2) uptake was confirmed as true negative, and in one patient without suspected metastases, (123)I-BZA(2) scintigraphy revealed a gastric lesion. Hence eight diagnoses would have been modified by (123)I-BZA(2) scintigraphy data. (123)I-BZA(2) allowed discrimination between benign and malignant lesions and, in the case of malignancies, between those of melanomatous origin and others. This compound, which is selective for melanoma tissue, appears promising for the staging and restaging of melanoma.

International audience; We previously selected two melanin-targeting radioligands [I-125]ICF01035 and [I-125]ICF01040 for melanoma-targeted I-125 radionuclide therapy according to their pharmacological profile in mice bearing B16F0 tumors. Here we demonstrate in vitro that these compounds present different radiotoxicities in relation to melanin and acidic vesicle contents in B16F0, B16F0 PTU and A375 cell lines. ICF01035 is effectively observed in nuclei of achromic (A375) melanoma or in melanosomes of melanized melanoma (B16F0), while ICF01040 stays in cytoplasmic vesicles in both cells. [I-125]ICF01035 induced a similar survival fraction (A(50)) in all cell lines and led to a significant decrease in S-phase cells in amelanotic cell lines. [I-125]ICF01040 induced a higher A(50) in B16 cell lines compared to [I-125]ICF01035 ones. [I-125]ICF01040 induced a G2/M blockade in both A375 and B16F0 PTU, associated with its presence in cytoplasmic acidic vesicles. These results suggest that the radiotoxicity of [I-125]ICF01035 and [I-125]ICF01040 are not exclusively reliant on DNA alterations compatible with gamma rays but likely result from local dose deposition (Auger electrons) leading to toxic compound leaks from acidic vesicles. In vivo, [I-125]ICF01035 significantly reduced the number of B16F0 lung colonies, enabling a significant increase in survival of the treated mice. Targeting melanosomes or acidic vesicles is thus an option for future melanoma therapy.

International audience; The GATE Monte Carlo simulation platform based on the Geant4 toolkit is under constant improvement for dosimetric calculations. In this study, we explore its use for the dosimetry of the preclinical targeted radiotherapy of melanoma using a new specific melanin-targeting radiotracer labeled with iodine 131. Calculated absorbed fractions and S values for spheres and murine models (digital and CT-scan-based mouse phantoms) are compared between GATE and EGSnrc Monte Carlo codes considering monoenergetic electrons and the detailed energy spectrum of iodine 131. The behavior of Geant4 standard and low energy models is also tested. Following the different authors' guidelines concerning the parameterization of electron physics models, this study demonstrates an agreement of 1.2% and 1.5% with EGSnrc, respectively, for the calculation of S values for small spheres and mouse phantoms. S values calculated with GATE are then used to compute the dose distribution in organs of interest using the activity distribution in mouse phantoms. This study gives the dosimetric data required for the translation of the new treatment to the clinic.

[123I]-N-(2-Diethylaminoethyl)-4-iodobenzamide (123I-BZA) has been the best scintigraphic agent described so far for malignant melanoma and ocular melanoma diagnosis. We replaced 123I by the more convenient radioisotope 99mTc and synthesized four bis(aminoethanethiol) derivatives. We describe the synthesis of a new oxo-technetium complex (TcO-Cf), prepared in very high yield (radiochemical yield > 95%), that exhibits an affinity for the pigmented tumor cells. This complex was evaluated in vivo in mice bearing C57B16 murine melanoma. After injection, a rapid decrease in the radioactivity levels was noted for all tissues and organs except for eyes (1.26 %ID/g at 1 h and 2.69 %ID/g at 24 h postinjection) and the tumor (1.19 %ID/g at 1 h and 0.80 %ID/g at 24 h postinjection), suggesting a specific in vivo binding of this complex to the pigmented cells. These results were compared with those already published for three other technetium-99m bis(aminothiol) complexes with benzamide derivatives.

Radioiodobenzamides are the best-known agents under study for the diagnosis of cutaneous melanoma and its metastases. We report the synthesis of a new BAT derivative radiopharmaceutical in which radioiodine is replaced by 99m-technetium. The cyclic intermediary methyl 4-[3-(4,4,7,7-tetramethyl-5,6-dithia-2, 9-diazacyclodecyl)-2-oxapropyl]benzoate (5) occurred in two different conformations identified by spectroscopic analysis. The final BAT ligand was radiolabeled using the nitridotechnetium core by a ligand-exchange reaction. Two different complexes were purified. After macroscopic 99-technetium synthesis, syn and anti isomers were identified. The global radiochemical yield was over 80%. The biodistribution of these two complexes was evaluated in mice bearing murine B16 melanoma. Extensive liver and kidney uptake was observed, but the benzamide tropism for the tumor was partially preserved.

International audience; N-(2-diethylaminoethyl)-4-iodobenzamide has been reported to be an excellent agent for malignant melanoma diagnosis by SPECT. To obtain a 99mTc analog, we synthesized a new bis(aminothiol) (BAT) derivative. The benzamide function was conjugated onto a nitrogen of the BAT backbone. This ligand was successfully radiolabelled with both nitrido-technetium and oxo-technetium cores. These complexes were purified by HPLC. Tumour uptake was measured in intravenously injected mice bearing the B16 murine melanoma.

N-(2-diethylaminoethyl)-4-iodobenzamide has been reported to be an excellent agent for malignant melanoma diagnosis by SPECT. To obtain a 99mTc analogue, we synthesized a sodium N-{2-[4-(N-diethylaminoethylcarbamoyl)phenyl]ethyl}-N-methyldithiocarbamate ligand in 7 steps from methyl 4-(bromomethyl)benzoate. The corresponding nitridotechnetium complex formed by a ligand exchange method was purified and its biodistribution in mice bearing the B16 murine melanoma was determined. Copyright © 1999 John Wiley & Sons, Ltd.

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea), a new anticancer chloroethylnitrosourea (CENU) is being tested in a phase II clinical trial of disseminated melanoma. The antitumour effect of this drug is mainly due to DNA damage in malignant melanocytes. Recently, we have shown that this damage can induce apoptosis in some melanoma cell lines. In others, apoptosis is not clearly observed, although there is a strong cytostatic effect. In this paper, we have characterized the cytological effect of cystemustine on murine malignant melanocytes (B16 cell line) which are resistant to apoptosis induced by this CENU. The results show that 3 days after cystemustine treatment, these melanocytes had accumulated in phase G2 of the cell cycle. There was then a strong morphological modification during a long cytostatic phase up to 30 days after treatment. During this cytostatic phase, there was uncontrolled DNA synthesis and marked swelling. Also, tyrosinase activity, melanin content and the number of mature melanosomes were greatly increased. These results suggest that when malignant melanocytes are not able to undergo apoptosis after treatment with CENU, they accumulate in G2 and this is followed by enhancement of melanogenesis.

N-(4-dipropylaminobutyl)-4-iodobenzamide was recently developed in our laboratory as a potentially improved imaging agent for metastatic melanoma. Our purpose was to seek an efficient route to the labelling of this tracer with iodine-125 at a carrier free level. Three different approaches were explored: Wallach triazene reaction, nucleophilic exchange of bromide for iodide, iododestannylation. The most suitable method appeared to be the electrophilic radio-iododestannylation of the tributyl-stannyl precursor using no-carrier added sodium [125I] iodide oxidized with chloramine T. The exchange between iodine-125 and the leaving group occured in 15 min at room temperature. A chemically and radiochemically pure product was obtained after reverse phase HPLC purification, with a radiolabeling yield of about 75%.Copyright © 1998 John Wiley & Sons, Ltd.

Whole-body autoradiography (WBA) and multi-wire proportional counting allow for spatial imaging of the radioactive material present in the tissues and organs of dehydrated animal sections. AMBIS 4000 counting of whole-body cryosections offers a sensitive, accurate and reproducible novel method for the quantitative measurement of the tissue distribution of a [14C] radiopharmaceutical. Intensity of AMBIS 4000 counting (net cpm/mm2) and concentration of radioactivity (nCi/g) were linearly related, yielding a standard curve. Evaluating biodistribution (a) provides pharmacokinetic data for predicting the potential tissue deposition of an absorbed dose of radioactivity in man, and (b) allows visual and quantitative evaluation of radioactivity in small anatomical structures that otherwise could not be detected by conventional tissue combustion technology. This new method of WBA, coupled with AMBIS 4000 counting, should prove a valuable method for pharmacodynamic studies, and afford a predictive tool for nuclear medicine by assessing specific targeting of selected tissues.

A boronated benzamide which accumulates in melanin-rich tissues in mice is presented.

Myocardial protection is usually studied in vitro on perfused heart preparations, but never directly on cultured cardiomyocytes. We evaluated a model of cultured newborn rat cardiomyocytes to study both the cytotoxicity and the protective effect against chemical hypoxia of three cardioplegic solutions (St Thomas' I, Bretschneider, St Thomas' II) under normothermic (37°C) and hypothermic (4°C) conditions. Cytotoxicity was evaluated in 50% and 100% concentrations of the cardioplegic solutions with incubation times from 90 to 360 min. Myocardial protection was studied in 50% cardioplegic solution with metabolic inhibitors. Immediate and late viabilities, after 24 h of recovery in the medium, were evaluated by simultaneous staining with fluorescein diacetate and propidium iodide.

In order to develop new iodinated and fluorinated matched-pair radiotracers for Single-Photon Emission Computed Tomography (SPECT)/Positron Emission Tomography (PET) imaging and targeted radionuclide therapy of melanoma, we successfully synthesized and radiolabelled with iodine-125 seven new derivatives, starting from our previously described lead structure 3. The relevance of these radiotracers for gamma scintigraphic imaging of melanoma in rodent was assessed. The tumoural radioactivity uptake was most often high and specific even at early time points (12.1–18.3% ID/g at 3 h p.i. for [125I]39–42) and a fast clearance from the non-target organs was observed. Also, calculated effective doses that could be delivered to tumours when using corresponding [131I]-labelled analogues were generally higher than 100 cGy/MBq injected (98.9–150.5 cGy/MBq for [131I]39–42). These results make compounds 39–42 suitable candidates for (i) PET imaging of melanoma after labelling with fluorine-18 and (ii) targeted radionuclide therapy of disseminated melanoma after labelling with iodine-131.

N-(2-diethylaminoethyl)-4-iodobenzamide (I) and N-(3-dimethylaminopropyl)-4-iodobenzamide (11), recently developed in our laboratory, are promising agents for metastatic melanoma detection in nuclear medicine. In order to study their in vivo metabolism, we labelled them with carbon-14 and carbon-13 on two sites : on the carbonyl group (la, lla), and on the dialkylaminoalkyl moiety (Ib, IIb).The overall yields calculated from sodium [ 14 C] or [ 13 C] cyanide used as precursor are in the range of 55 % for Ia, IIa, llb, and 21 % for Ib.

Nine healthy young men were studied under strict conditions for 48 h. The subjects were selected after a clinical examination and exploration of their rest-activity rhythm by actometry. The circadian rhythms of cortisol (peak at 8 AM) and melatonin (peak at 4 AM) were confirmed. The interleukin 15 (IL-15) was detected in the plasma samples with an Elisa kit (R&D System), but no reproducible variation could be observed during day 1 and day 2. In conclusion, in the conditions of our study, no rhythm was observed for IL-15. Our population will be completed with the inclusion of 6 additional subjects. These results will be specified.

The development of alternative therapies for melanoma treatment is of great interest as long-term tumour regression is not achieved with new targeted chemotherapies on selected patients. We previously demonstrated that radioiodinated heteroarylcarboxamide ([131I]ICF01012) induced a strong anti-tumoural effect by inhibiting both primary tumour growth and dissemination process in a B16BL6 melanoma model. In our study, we show that a single injection of [131I]ICF01012 (ranging from 14.8 to 22.2 MBq) was effective and associated with low and transient haematological toxicity. Concerning pigmented organs, cutaneous melanocytes and skin were undamaged. In 30% of treated animals, no histological alteration of retina was observed, and in the remaining 70%, damages were restricted to the optic nerve area. Using the Medical Internal Radiation Dose methodology, we determined that the absorbed dose in major organs is very low (4 Gy) and that a delivery of 30 Gy to the tumour is sufficient for an effective anti-tumoural response. Molecular analyses of treated tumours showed a strong radiobiological effect with a decrease in proliferation, survival and pro-angiogenic-related markers and an increase in tumour suppressor gene expression, melanogenesis and anti-angiogenic markers. All these features are in accordance with a tumour cell death mechanism that mainly occurs by mitotic catastrophe and provide a better understanding of in vivo anti-tumoural effects of [131I] radionuclide. Our findings raise [131I]ICF01012 a good candidate for disseminated melanoma treatment and strongly support transfer of [131I]ICF01012 to clinical trial.

This study reports a series of 14 new iodinated and fluorinated compounds offering both early imaging ((123)I, (124)I, (18)F) and systemic treatment ((131)I) of melanoma potentialities. The biodistribution of each (125)I-labeled tracer was evaluated in a model of melanoma B16F0-bearing mice, using in vivo serial γ scintigraphic imaging. Among this series, [(125)I]56 emerged as the most promising compound in terms of specific tumoral uptake and in vivo kinetic profile. To validate our multimodality concept, the radiosynthesis of [(18)F]56 was then optimized and this radiotracer has been successfully investigated for in vivo PET imaging of melanoma in B16F0- and B16F10-bearing mouse model. The therapeutic efficacy of [(131)I]56 was then evaluated in mice bearing subcutaneous B16F0 melanoma, and a significant slow down in tumoral growth was demonstrated. These data support further development of 56 for PET imaging ((18)F, (124)I) and targeted radionuclide therapy ((131)I) of melanoma using a single chemical structure.

A boronated benzamide which accumulates in melanin-rich tissues in mice is presented.

Radioiodobenzamides are the best-known agents under study for the diagnosis of cutaneous melanoma and its metastases. We report the synthesis of a new BAT derivative radiopharmaceutical in which radioiodine is replaced by 99m-technetium. The cyclic intermediary methyl 4-[3-(4,4,7,7-tetramethyl-5,6-dithia-2,9-diazacyclodecyl)-2-oxapropyl]benzoate (5) occurred in two different conformations identified by spectroscopic analysis. The final BAT ligand was radiolabeled using the nitridotechnetium core by a ligand-exchange reaction. Two different complexes were purified. After macroscopic 99-technetium synthesis, syn and anti isomers were identified. The global radiochemical yield was over 80%. The biodistribution of these two complexes was evaluated in mice bearing murine B16 melanoma. Extensive liver and kidney uptake was observed, but the benzamide tropism for the tumor was partially preserved.

In recent years, there has been dramatic worldwide increase in incidence of malignant melanoma. Although localised disease is often curable by surgical excision, metastatic melanoma is inherently resistant to most treatments. In this context, targeted radionuclide therapy could be an efficient alternative. After pharmacomodulation study, we selected a quinoxaline derivative molecule (ICF01012) for its high, specific and long-lasting uptake in melanoma with rapid clearance from nontarget organs providing suitable dosimetry parameters for targeted radiotherapy. Aim of this study was to investigate, in vivo, efficacy of [131I]ICF01012 on nonmetastatic B16F0, metastatic B16Bl6 or human M4Beu melanoma tumours. First, colocalisation of ICF01012 with melanin by SIMS imaging was observed. Second, we showed that treatment drastically inhibited growth of B16F0, B16Bl6 and M4beu tumours whereas [ 131 I]NaI or unlabelled ICF01012 treatment was without significant effect. Histological analysis and measure of PCNA proliferation marker expression showed that residual B16 tumour cells exhibit a significant loss of aggressiveness after treatment. This effect is associated with a lengthening of the treated-mice survival time. Moreover, with B16Bl6 model, 55% of the untreated mice had lung metastases whereas no metastasis was counted on treated group. Our data demonstrated a strong anti-tumoural effect of [131I]ICF01012 for radionuclide therapy on murine and human in vivo pigmented melanoma models, whatever their dissemination profiles and their melanin content be. Further studies will attempt to optimise therapy protocol by increasing the balance between the anti-tumoural effect and the safety on nontarget organs. ' 2009 UICC

Targeted internal radionuclide therapy (TRT) would be an effective alternative to current therapies for disseminated melanoma treatment. At our institution, a class of iodobenzamides has been developed as potent melanomaseeking agents. This review described the preclinical validations of a quinoxaline derivative molecule (ICF01012) as tracer for TRT application. It was selected for its high, specific and long-lasting uptake in tumour with rapid clearance from non-target organs providing suitable dosimetry parameters for TRT. Extended in vivo study of metabolic profiles confirmed durable tumoural concentration of the unchanged molecule form. Moreover melanin specificity of ICF01012 was determined by binding assay with synthetic melanin and in vivo by SIMS imaging. Then, we showed in vivo that [131I] ICF01012 treatment drastically inhibited growth of B16F0, B16Bl6 and M4Beu tumours whereas [131I] NaI or unlabelled ICF01012 treatment was without significant effect. Histological analysis showed that residual tumour cells exhibit a significant loss of aggressiveness after treatment. This anti-tumoural effect was associated with a lengthening of the treated-mice survival time and an inhibition of lung dissemination for B16Bl6 model. Results presented here support the concept of TRT using a [131I] labelled iodoquinoxaline derivative for an effective melanoma treatment.

The increasing incidence of melanoma and the lack of effective therapy have prompted the development of new vectors, more specific to the pigmented tumor, for early detection and treatment. Targeted agents have to exhibit a rapid, high tumor uptake, long tumor retention and rapid clearance from nontarget organs. This joint work presents results obtained with a new melanoma targeting agent, [125I]-N-(4-dipropylaminobutyl)-4-iodobenzamide or [125I]BZ18. After labeling with a high specific activity, the biodistribution of the compound was investigated in two animal models, the mouse and the sheep. Melanotic tumor retention was observed lasting several days. We visualized the internalization of the agent inside the melanosomes by secondary ion mass spectroscopy imaging, we measured the affinity constants of [125I]BZ18 on a synthetic melanin model and we demonstrated a radiotoxic effect of this labeled agent on B16F0 melanoma cell culture due to its cellular internalization. From this work, [125I]BZ18 appeared a promising melanoma targeting agent in the nuclear medicine field.

To identify proteins involved in melanoma metastasis mechanisms, comparative proteomic studies were undertaken on B16F10 and B16Bl6 melanoma cell lines and their subsequent syngenic primary tumours as pulmonary metastases were present only in the mice bearing a B16Bl6 tumour. 2DE analyses followed by MALDI-TOF identification showed variations of 6 proteins in vitro and 13 proteins in vivo. Differential expressed proteins in tumours were related to energy production and storage. Two differentially expressed proteins which had not been previously associated to melanoma progression, annexin A1 (ANXA1) and creatine kinase B (CKB), were found both in cells and in tumours. To characterize ANXA1 involvement in melanoma B16 dissemination, we reduced ANXA1 protein level by siRNA and observed a significant decrease of B16Bl6 cell invasion through Matrigel coated chambers. We further demonstrated that the presence of several formyl peptide receptors (FPR1, FPRrs1 and 2) revealed by qRT-PCR, played a role in B16 invasion: incubation of B16Bl6 cells with the FPR agonist (fMLP) or antagonist (tBOC) enhanced or decreased Matrigel coated chamber invasion respectively, with a correlation of ANXA1 levels in both treatments. As ANXA1 could bind to FPRs, this should amplify invasion and enhance melanoma dissemination.

Chemoresistance to O(6)-alkylating agents is a major barrier to successful treatment of melanoma. It is mainly due to a DNA repair suicide protein, O(6)-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 O(6)-(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 O(6)-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.

Further development of nuclear medicine for imaging and internal radiotherapy demands a precise knowledge of the tissue and cellular distribution of radiopharmaceuticals. Ion microscopy (secondary ion mass spectrometry [SIMS]) may be particularly useful in this respect. We used SIMS to study the biodistribution of the melanoma-targeting molecule N-(2-diethylaminoethyl)-4-iodobenzamide (I-BZA), both in its native state and radiolabeled with (14)C.C57BL6/J1/co mice bearing pulmonary colonies of B16 melanoma cells were injected with I-BZA or (14)C-I-BZA. Appropriate tissues were fixed and included in epoxy embedding resin for SIMS studies. The distribution of unlabeled I-BZA was studied by detecting its stable iodine atom ((127)I). (14)C-I-BZA distribution was studied by dual detection of (127)I and (14)C. The time course of I-BZA concentrations at sites of tissue fixation was studied by measuring the signal ratio of (14)C and the naturally occurring isotope (13)C.SIMS showed that I-BZA concentrated in the cytoplasm of tumoral melanocytes (melanoma cells) and in the cytoplasm of tumor-infiltrating macrophages (melanophages). I-BZA was also detected in the cytoplasm of normal melanocytes in the pigmented structures of skin and eye. Interpretation of I-BZA distribution by using electron micrographs of adjacent sections showed that the intracytoplasmic melanin-rich organelles (melanosomes) were responsible for I-BZA retention. The distributions of (127)I and (14)C after (14)C-I-BZA injection were identical, even when I-BZA was separately labeled with (14)C at 2 different positions, indicating the stability of the amide bond of I-BZA. The time course of the (14)C/(13)C ratio in the melanosomes of melanoma cells suggested a retention half-life of about 38 h.Contrary to previous suggestions that I-BZA fixes principally to sigma-1 membrane receptors, our results strongly indicate that I-BZA associates with intracytoplasmic melanin pigments. Early I-BZA accumulation, in both melanocytes and melanophages, suggests that this compound fixes to preformed melanin rather than being incorporated during de novo melanin synthesis. These quantitative and qualitative data obtained with I-BZA illustrate the excellent potential of SIMS for studying the biologic fate of radiopharmaceuticals.

N -(2-diethylaminoethyl)-2-iodobenzamide (BZA 2 ) has been singled out as the most efficacious melanoma scintigraphy imaging agent. Our work was designed to assess the mechanisms of the specific affinity of the radioiodinated iodobenzamide for melanoma tissue. We studied the cellular uptake and retention of [ 125 I]-BZA 2 on various cell lines. In vitro, cellular [ 125 I]-BZA 2 uptake was related to the pigmentation status of the cells: higher in pigmented melanoma cell lines (M4 Beu, IPC 227, B 16) than in a nonpigmented one (M3 Dau) and nonmelanoma cell lines (MCF 7 and L 929). Two mechanisms were assessed: binding of the tracer to melanin or to sigma receptors of melanoma cells. First, the uptake of [ 125 I]-BZA 2 after melanogenesis stimulation by α-melanocyte-stimulating hormone and l-tyrosine increased in the B 16 melanoma cell line both in vitro and in vivo according to melanin concentration. Moreover, the binding of [ 125 I]-BZA 2 to synthetic melanin was dependent on melanin concentration and could be saturated. Second, no competition was evidenced on M4 Beu cells between [ 125 I]-BZA 2 and haloperidol, a sigma ligand, at concentrations ≤10 −6 M. We show that the specificity and sensibility of BZA 2 as a melanoma scintigraphic imaging agent are mostly due to interactions with melanic pigments.

International audience; We have shown that beta-tubulin was alkylated by a microtubule disrupter, N-4-iodophenyl-N'-(2-chloroethyl)urea (ICEU), on a glutamic acid residue at position 198 and not on the previously proposed reactive cysteine 239. ICEU belongs to the 4-substituted-phenyl- N'-(2-chloroethyl) urea class that alkylates mainly cellular proteins. Previous studies have shown that the tertbutyl (tBCEU) and iodo (ICEU) derivatives induce microtubule disruption because of beta-tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of beta-tubulin, but this binding site was not clearly confirmed (Cancer Res 60: 985-992, 2000). We have isolated and analyzed beta-tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated beta-tubulin had a lower apparent molecular weight and a more basic isoelectric point than the unmodified protein. Labeled N-4-[I-125]CEU was effectively bound to the modified beta-tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of beta-tubulin was linked to the alkylating agent. In contrast, peptide masses at m/z 4883 and 1792 in trypsin or Asp-N digestions of beta-tubulin confirmed binding of iodophenylethylureido moiety to peptides [175-213] or [197-208] respectively. Fragmentation analyses by electrospray mass spectrometry using triply charged ions of peptide [175-213] identified a glutamic acid at position 198 as target for alkylation via an ester bond with ICEU. This amino acid located in the intermediate domain of the beta-tubulin should play an essential role in the conformational structure necessary for the interaction between dimers in the protofilament.

In the course of our investigations aimed at improving the biological characteristics of iodobenzamides for melanoma therapeutic applications, four new derivatives containing a spermidine chain have been prepared and radiolabeled with 125 I. In vitro studies showed that all compounds displayed high affinity for melanin superior to the reference compound BZA, thus validating our experimental approach. In vivo biodistribution was investigated in B16 melanoma-bearing mice. All four compounds, particularly benzamide 3 , showed accumulation in the tumor, but lower, however, than that of BZA. Moreover, high concentrations of radioactivity in other organs, namely, the liver and lung, demonstrated nonspecific tumoral uptake. In view of these results, compounds 1 2 3 4 do not appear to be suitable radiopharmaceuticals for melanoma radionuclide therapy.

Background Analytical imaging by secondary ion mass spectrometry (SIMS) provides images representative of the distribution of a specific ion within a sample surface. For the last fifteen years, concerted collaborative research to design a new ion microprobe with high technical standards in both mass and lateral resolution as well as in sensitivity has led to the CAMECA NanoSims 50, recently introduced onto the market. This instrument has decisive capabilities, which allow biological applications of SIMS microscopy at a level previously inaccessible. Its potential is illustrated here by the demonstration of the specific affinity of a melanoma marker for melanin. This finding is of great importance for the diagnosis and/or treatment of malignant melanoma, a tumour whose worldwide incidence is continuously growing. Methods The characteristics of the instrument are briefly described and an example of application is given. This example deals with the intracellular localization of an iodo-benzamide used as a diagnostic tool for the scintigraphic detection of melanic cells (e.g. metastasis of malignant melanoma). B16 melanoma cells were injected intravenously to C57BL6/J1/co mice. Multiple B16 melanoma colonies developed in the lungs of treated animals within three weeks. Iodobenzamide was injected intravenously in tumour bearing mice six hours before sacrifice. Small pieces of lung were prepared for SIMS analysis. Results Mouse lung B16 melanoma colonies were observed with high lateral resolution. Cyanide ions gave "histological" images of the cell, representative of the distribution of C and N containing molecules (e.g. proteins, nucleic acids, melanin, etc.) while phosphorus ions are mainly produced by nucleic acids. Iodine was detected only in melanosomes, confirming the specific affinity of the drug for melanin. No drug was found in normal lung tissue. Conclusion This study demonstrates the potential of SIMS microscopy, which allows the study of ultra structural distribution of a drug within a cell. On the basis of our observations, drug internalization via membrane sigma receptors can be excluded.

Recent NMR spectroscopy developments, such as high-resolution magic angle spinning (HRMAS) probes and correlation-enhanced 2D sequences, now allow improved investigations of phospholipid (Plp) metabolism. Using these modalities we previously demonstrated that a mouse-bearing melanoma tumor responded to chloroethyl nitrosourea (CENU) treatment in vivo by altering its Plp metabolism. The aims of the present study were to investigate whether HRMAS proton total correlation spectroscopy (TOCSY) could be used as a quantitative technique to probe Plp metabolism, and to determine the Plp metabolism response of cultured B16 melanoma cells to CENU treatment in vitro. The exploited TOCSY signals of Plp derivatives arose from scalar coupling among the protons of neighbor methylene groups within base headgroups (choline and ethanolamine). For strongly expressed Plp derivatives, TOCSY signals were compared to saturation recovery signals and demonstrated a linear relationship. HRMAS proton TOCSY was thus used to provide concentrations of Plp derivatives during long-term follow-up of CENU-treated cell cultures. Strong Plp metabolism alteration was observed in treated cultured cells in vitro involving a down-regulation of phosphocholine, and a dramatic and irreversible increase of phosphoethanolamine. These findings are discussed in relation to previous in vivo data, and to Plp metabolism enzymatic involvement.

The cellular uptake and incorporation in macromolecules of iodine-125 labelled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), a melanoma imaging agent, was studied using human melanoma cells M3Dau (amelanotic) and M4Beu (melanotic). The interaction between [125I]BZA and synthetic melanin was examined in various conditions of incubation. The results showed that uptake was high only for M4Beu, whereas the incorporation in trichloroacetic acid-precipitable proteins was very low for both model cell lines, with no correlation with melanin content. Experiments with synthetic melanin showed that BZA binding to melanin was saturable and reversible, and involved several types of interaction. The influence of the ionic environment indicated that electrostatic forces play a role in the affinity, and the decrease in binding produced by the presence of an alcohol in the medium suggested that hydrophobic interactions may be involved in the binding mechanism. This was supported by the Scatchard analysis, which revealed two classes of binding sites, and the determination of two association constants (K1 = 3.9 +/- 1.9 x 106/M and K2 = 2.9 +/- 0.9 x 104/M). The affinity of BZA for melanin might explain the good results obtained in a phase II clinical trial for the diagnosis of malignant melanoma metastases, in which the specificity was 100%.

N'-[2-chloroethyl]-N[2-(methylsulfonyl) ethyl]-N'-nitrosourea (cystemustine),a chloroethylnitrosourea antineoplastic drug, provokes cellular proliferation inhibition and redifferentiation, but there was no cell death in B16 melanoma tumors. Because the phospholipid (Plp) metabolism is tightly involved in tumor growth regulation and tumor cell survival, we tested the hypothesis that melanoma tumors undergo adaptive Plp metabolism changes to survive treatment. Measurements of Plp derivatives were performed using a novel proton nuclear magnetic resonance Spectroscopy application using magic angle spinning on intact tumor tissue samples. Phosphatidylcholine levels were obtained from one-dimensional spectra, and relative levels of choline- and ethanolamine-containing compounds were derived from two-dimensional spectra (total correlation spectroscopy sequence). Two major findings emerged from this study: (a) during tumor growth inhibition, there was a transient accumulation of choline, glycerophosphocholine, and glycerophosphoethanolamine and a sustained increase in phosphocholine and phosphoethanolamine, whereas phosphatidylcholine levels remained unchanged; and (b) during tumor growth recovery, only phosphocholine and phosphoethanolamine remained elevated. Therefore, cystemustine-treated B16 melanoma tumors acquire a new Plp metabolism phenotype, a mechanism that could participate in tumor cell redifferentiation and/or survival.

Iodobenzamides are reported to possess some affinity for melanoma. In order to identify the compound having the most appropriate pharmacokinetic properties as a potential melanoma imaging agent, thirteen new [125I]radioiodobenzamides with a butylene amide-amine spacer and various substituents on the terminal amino group were investigated. Their synthesis, radioiodination and biodistribution in B16 melanoma bearing C57BL6 mice are described and compared to [125I] labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), our reference compound. Changes in the terminal amino constituents induced modifications of lipophilicity, tumor uptake and organ distribution. The dimethylaminobutyl iodobenzamide appeared to be the most promising radiopharmaceutical imaging agent for the detection of melanoma and its metastases.

This study was designed in order to evaluate the feasibility of establishing an animal model of human uveal melanoma. IPC227, a cell line established from the biopsy of a patient with a spindle cell ciliary body melanoma, was transplanted into the anterior chamber of the eye in immunosuppressed New Zealand rabbits. In a second step, a tumour fragment from the anterior chamber was implanted transclerally into the posterior choroid. Complete ophthalmological examinations were then performed on the animals. Characteristic growth patterns were noted depending on the location of implantation. In the anterior chamber, diffuse, flat, heavily pigmented tumours appeared 8 days after the injection of the cell suspension that covered the iris and the angle by day 25, with a success rate of 100%. Nodular, lightly pigmented tumours were obtained 6-7 weeks after subchoroidal implantation, with a 25% success rate. Clinical examination, including fundus photography, ultrasound and magnetic resonance imaging, demonstrated the same characteristics as those of human uveal melanoma, confirming the value of this model for the evaluation of new therapeutic and diagnostic methods in the management of uveal melanoma.

AMBIS 4000, a multi-wire proportional counter, was calibrated for iodine-125 measurements. The detector displayed a linear response over a wide dynamic range. Using whole-body mice cryosections, a linear relationship could be established between count rate per area (cpm/mm2) measured with the AMBIS 4000 detector and the count rate per gram (dpm/g) determined with an NaI(Tl) scintillation detector. A calibration curve could, thus be constructed. This new method allowed direct visual and quantitative evaluation of the biodistribution of a short series of 125I-labelled benzamides in melanoma-bearing mice. All the compounds studied showed good tumoral targeting ability. For one of them, ortho-N-(2-diethylaminoethyl)-4-iodobenzamide, liver and lung uptake decreased rapidly after dosing, making it a suitable tracer for scintigraphic detection of malignant melanoma.

N -(2-Diethylaminoethyl)-4-iodobenzamide (BZA) is a radiopharmaceutical recently developed in our laboratory for the scintigraphic detection of melanoma and metastases. Optimal time for imaging was between 18–24 h p.i. of [ 123 I] BZA. With a view to selecting compounds able to provide quality images shortly after the injection, synthesis of an initial series of BZA derivatives and their evaluation in B16 melanoma bearing mice have been carried out. The [ 125 I] radiolabeled products were obtained by a simple isotopic exchange procedure with high radiochemical yields (85–95%). After i.v. administration of the compounds we observed a good tumoral targeting ability. Tumoral activity peaked at 2.6 to 7.70% injected dose per g within 1 h post-injection. One of the benzamides with a blood clearance faster than that of BZA—0.06 vs. 0.2% I D/g—6 h p.i. gave the same tumor to blood and to organ ratios as BZA at 12–18 h p.i. Based on these preclinical data we hope to obtain good tumoral images 6 h p.i. in scintigraphic studies in man.