Synthesis, radiolabeling and preliminary in vivo evaluation of multimodal radiotracers for PET imaging and targeted radionuclide therapy of pigmented melanoma.

Melanin pigment represents an attractive target to address specific treatment to melanoma cells, such as cytotoxic radionuclides. However, less than half of the patients have pigmented metastases. Hence, specific marker is required to stratify this patient population before proceeding with melanin-targeted radionuclide therapy. In such a context, we developed fluorinated analogues of a previously studied melanin-targeting ligand, N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (ICF01012). These latter can be labeled either with (18)F or (131)I/(125)I for positron emission tomography imaging (melanin-positive patient selection) and targeted radionuclide therapy purposes. Here we describe the syntheses, radiosyntheses and preclinical evaluations on melanoma-bearing mice model of several iodo- and fluoro(hetero)aromatic derivatives of the ICF01012 scaffold. After preliminary planar gamma scintigraphic and positron emission tomography imaging evaluations, [(125)I]- and [(18)F]-N-[2-(diethylamino)ethyl]-4-fluoro-3-iodobenzamides ([(125)I]4, [(18)F]4) were found to be chemically and biologically stable with quite similar tumor uptakes at 1 h p.i. (9.7 ± 2.6% ID/g and 6.8 ± 1.9% ID/g, respectively).

Synthesis, radiofluorination, and in vivo evaluation of novel fluorinated and iodinated radiotracers for PET imaging and targeted radionuclide therapy of melanoma.

Our project deals with a multimodal approach using a single fluorinated and iodinated melanin-targeting structure and offering both imaging (positron emission tomography (PET)/fluorine-18) and treatment (targeted radionuclide therapy/iodine-131) of melanoma. Six 6-iodoquinoxaline-2-carboxamide derivatives with various side chains bearing fluorine were synthesized and radiofluorinated, and their in vivo biodistribution was studied by PET imaging in B16Bl6 primary melanoma-bearing mice. Among this series, [(18)F]8 emerged as the most promising compound. [(18)F]8 was obtained by a fully automated radiosynthesis process within 57 min with an overall radiochemical yield of 21%, decay-corrected. PET imaging of [(18)F]8 demonstrated very encouraging results as early as 1 h postinjection with high tumor uptake (14.33% ± 2.11% ID/g), high contrast (11.04 ± 2.87 tumor-to-muscle ratio), and favorable clearance properties. These results, associated with the previously reported pharmacokinetic properties and dosimetry of 8, make it a potential agent for both PET imaging and targeted radionuclide therapy of melanoma.

Synthesis, radioiodination and in vivo screening of novel potent iodinated and fluorinated radiotracers as melanoma imaging and therapeutic probes.

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 [(125)I]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 [(131)I]-labelled analogues were generally higher than 100 cGy/MBq injected (98.9-150.5 cGy/MBq for [(131)I]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.

In vivo scintigraphic imaging of proteoglycans.

In this chapter, we present the methods developed in our lab for the scintigraphic imaging and direct quantitative evaluation of proteoglycan (PG) distribution in vivo. These methods relate to (1) the synthesis and radiolabeling of the NTP 15-5 with (99m)Tc, (2) preclinical scintigraphic imaging using laboratory animals, and (3) quantitative analysis of scintigraphic images.

New aldehyde and vinylsulfone proteasome inhibitors for targeted melanoma therapy.

The proteasome is a promising target in cancer therapy. However, it is ubiquitous and its inhibitors cause side effects. To target melanoma cells we synthesized new peptide aldehyde and vinylsulfone inhibitors of the proteasome conjugated to the melanin-targeting ligand (MTL) derived from radiotracer [(123)I]-N-(2-diethylaminoethyl)benzamide ([(123)I]BZA) or [(125)I]-N-(4-dipropylaminobutyl)-4-iodobenzamide ([(125)I]BZ18). Influence on the cytotoxicity of the benzamide alkyl side chain length and the composition of the amino acid sequence was assessed. Among the conjugates evaluated, compound 16 and 22 presented the highest cytotoxicity (IC(50), 0.71 and 0.64 µM respectively), which persisted in the presence of an MTL derived from N-(dialkylaminoalkylenyl)benzamide residue.

First ex vivo study demonstrating that 99mTc-NTP 15-5 radiotracer binds to human articular cartilage.

PURPOSE: Preclinical data pointed to (99m)Tc-NTP 15-5 as a good candidate for single photon emission computed tomography (SPECT) imaging of cartilaginous disease. We set out to investigate and quantify (99m)Tc-NTP 15-5 ex vivo uptake by human articular cartilage relative to bone (99m)Tc-hydroxymethylene diphosphonate (HMDP) radiotracer. METHODS: Three osteoarthritic human tibial plateaux and four tibiofemoral joints were incubated with (99m)Tc-NTP 15-5 and (99m)Tc-HMDP for 2 h. Affinity of tracers for cartilage was determined by visual analysis of SPECT/CT acquisitions and measurement of cartilage to cortical bone uptake ratios. RESULTS: Cartilage to cortical bone uptake ratios were 3.90 ± 2.35 and 0.76 ± 0.24, respectively, for (99m)Tc-NTP 15-5 and (99m)Tc-HMDP radiotracers. Visual analysis of fused SPECT/CT slices showed selective, intense (99m)Tc-NTP 15-5 accumulation in articular cartilage, whereas (99m)Tc-HMDP binding was low. Interestingly, a cartilage defect visualized on CT was clearly associated with focal decreased uptake of (99m)Tc-NTP 15-5. CONCLUSION: The tracer (99m)Tc-NTP 15-5 is of major interest for human cartilage molecular imaging and could find clinical applications in osteoarthritis staging and monitoring.

Single photon emission computed tomography/positron emission tomography imaging and targeted radionuclide therapy of melanoma: new multimodal fluorinated and iodinated radiotracers.

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.

Evaluation of new iodinated acridine derivatives for targeted radionuclide therapy of melanoma using 125I, an Auger electron emitter.

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.

Preclinical investigation of tolerance and antitumour activity of new fluorodeoxyglucose-coupled chlorambucil alkylating agents.

Our strategy is to increase drug accumulation in target tumour cells using specific "vectors" tailored to neoplastic tissue characteristics, which ideally are not found in healthy tissues. The aim of this work was to use 2-fluoro-2-deoxyglucose (FDG) as a drug carrier, in view of its well-known accumulation by most primary and disseminated human tumours. We had previously selected two FDG-cytotoxic conjugates of chlorambucil (CLB), i.e. compounds 21a and 40a, on the basis of their in vitro profiles. Here we investigated the antitumour profile and tolerance of these compounds in vitro and in vivo in two murine cell lines of solid tumours. In vitro, we found that micromolar concentrations of compounds 21a and 40a inhibited proliferation of B16F0 and CT-26 cell lines. Interestingly, compounds 21a and 40a were found to act at different levels in the cell cycle: S and subG1 accumulation for 21a and G2 accumulation for 40a. In vivo, a single-dose-finding study to select the Maximum Tolerated Dose (MTD) by the intraperitoneal route (IP) showed that the two peracetylated glucoconjugates of CLB were less toxic than CLB itself. When given to tumour-bearing mice (melanoma and colon carcinoma models), according to a "q4d × 3" schedule (i.e., three doses at 4-day intervals) both compounds demonstrated a promising antitumour activity, with Log Cell Kill (LCK) values higher than 1.3 in both B16F0 and CT-26 models. Hence compounds 21a and 40a are good candidates for further works to develop new highly active antineoplastic compounds.

The use of [(125)I] scintigraphic in vivo imaging in melanoma-bearing mice for a rapid prescreening of vectors to melanoma tissue.

INTRODUCTION: The use of radiolabeled molecules allows the study of in vivo biodistribution, target organs, and kinetic profile after systemic administration by 1) radioactive organ counting and 2) quantitative autoradiographic analysis of whole-body slices (WBA). However, these techniques are time- and animal consuming for several times studied. So, in vivo scintigraphic imaging should appear of interest for a first screening of compounds, as it is able to rapidly demonstrate tumoral uptake and kinetics by serial examinations in the same mice. MATERIALS AND METHODS: In this study, the tumoral distribution and kinetics of six molecules considered as potential melanoma tracers radiolabeled with (125)I were analyzed by gamma-scintigraphy comparatively to the results obtained by WBA. Tumoral uptake has been quantified and expressed by: 1) tumor-to-background ratios and 2) standardized tumoral uptake (STU) in percent injected dose per gram, with tumor weight being extrapolated from the measurement of the two diameters. RESULTS: Results from STU analysis showed good agreement (correlation coefficient = 0.92) with those of WBA, and the same classification of compounds (on the basis of their melanoma affinity) was obtained, with two compounds (of six) being rejected. CONCLUSIONS: [(125)I] scintigraphic imaging appeared as a relevant, easy-going method for a first pharmacologic selection in mice.

Targeted radionuclide therapy of melanoma: anti-tumoural efficacy studies of a new 131I labelled potential agent.

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 [(131)I]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 [(131)I]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 non-target organs.

Longitudinal profiling of articular cartilage degradation in osteoarthritis by high-resolution magic angle spinning 1H NMR spectroscopy: experimental study in the meniscectomized guinea pig model.

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.

Phase II trial of the association of a methionine-free diet with cystemustine therapy in melanoma and glioma.

BACKGROUND: In a previous phase I clinical trial of dietary methionine (MET) restriction with cystemustine treatment for melanoma or glioma, we determined the optimal MET-free diet duration to be 1 day. On this basis, a phase II clinical trial was initiated to evaluate safety and efficacy of this combination. PATIENTS AND METHODS: Twenty-two patients (20 with metastatic melanoma and 2 with recurrent gloma) received a median of 4 cycles of the association of a 1-day MET-free diet with cystemustine (60 mg/m(2)) every two weeks. RESULTS: This association was well tolerated (toxicity and nutritional status). Toxicity remained mainly hematological and consisted of WHO grade 3-4 thrombocytopenia, leucopenia and neutropenia in 36, 27 and 27% of patients respectively. The median disease-free survival was 1.8 months and the median survival was 4.6 months, with 2 long-duration stabilizations. The plasmatic MET depletion obtained was of 40 + or - 31%.

Melanoma affinity in mice and immunosuppressed sheep of [(125)I]N-(4-dipropylaminobutyl)-4-iodobenzamide, a new targeting agent.

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, [(125)I]-N-(4-dipropylaminobutyl)-4-iodobenzamide or [(125)I]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 [(125)I]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, [(125)I]BZ18 appeared a promising melanoma targeting agent in the nuclear medicine field.

Development of a high-performance liquid chromatographic method for the determination of a new potent radioiodinated melanoma imaging and therapeutic agent.

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.

Design, synthesis and preliminary biological evaluation of acridine compounds as potential agents for a combined targeted chemo-radionuclide therapy approach to melanoma.

Various iodo-acridone and acridine carboxamides have been prepared and evaluated as agents for targeted radionuclide and/or chemotherapy for melanoma, due to their structural similarity to benzamides which are known to possess specific affinity for melanin. Three of these carboxamides selected for their in vitro cytotoxic properties were radioiodinated with [(125)I]NaI at high specific activity. Biodistribution studies carried out in B16F0 murine melanoma tumour-bearing mice highlighted that acridone 8f and acridine 9d, presented high, long-lasting tumour concentrations together with an in vivo kinetic profile favourable to application in targeted radionuclide therapy.

Evaluation of radiolabeled (hetero)aromatic analogues of N-(2-diethylaminoethyl)-4-iodobenzamide for imaging and targeted radionuclide therapy of melanoma.

Targeted radionuclide therapy using radioiodinated compounds with a specific affinity for melanoma tissue is a promising treatment for disseminated melanoma, but the candidate with the ideal kinetic profile remains to be discovered. Targeted radionuclide therapy concentrates the effects on tumor cells, thereby increasing the efficacy and decreasing the morbidity of radiotherapy. In this context, analogues of N-(2-diethylaminoethyl)-4-iodobenzamide (BZA) are of interest. Various (hetero)aromatic analogues 5 of BZA were synthesized and radioiodinated with (125)I, and their biodistribution in melanoma-bearing mice was studied after i.v. administration. Most [ (125)I] 5-labeled compounds appeared to bind specifically and with moderate-to-high affinity to melanoma tumor. Two compounds, 5h and 5k, stood out with high specific and long-lasting uptake in the tumor, with a 7- and 16-fold higher value than BZA at 72 h, respectively, and kinetic profiles that makes them promising agents for internal targeted radionuclide therapy of melanoma.

Synthesis and cytotoxic properties of new fluorodeoxyglucose-coupled chlorambucil derivatives.

Frequently used in the treatment of malignant cells, alkylating agents, like most anticancer substances, produce adverse side effects caused by the toxicity of the agents toward normal tissues and lose efficiency through poor distribution to target sites. Our approach to developing more selective drugs with low systemic toxicity is based on the premise that the body distribution and cell uptake of a drug can be altered by attaching a neoplastic cell-specific uptake enhancer, such as 2-fluoro-2-deoxyglucose (FDG), the radiotracer most frequently used in PET for tumor imaging. Two properties of deoxyglucose, namely preferential accumulation in neoplastic cells and inhibition of glycolysis, underpin this targeting approach. Here, we report the synthesis of 19 new chlorambucil glycoconjugates in which the alkylating drug is attached to the C-1 position of FDG, directly or via different linkages. This set of compounds was evaluated for in vitro cytotoxicity against different human normal and tumor cell lines. There was a significant improvement in the in vitro cytotoxicity of peracetylated glucoconjugates compared with the free substance. Four compounds were finally selected for further in vivo studies owing to their lack of oxidative stress-inducing properties.

Synthesis, radiosynthesis, and biological evaluation of new proteasome inhibitors in a tumor targeting approach.

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.

First In Vivo SPECT Imaging of Mouse Femorotibial Cartilage Using 99mTc-NTP 15-5.

This study aimed to report the first single-photon emission computed tomographic (SPECT) imaging of articular cartilage in mice using 99mTc-NTP 15-5 radiotracer. Mice intravenously injected with 99mTc-NTP 15-5 were submitted to (1) dynamic planar imaging, (2) static planar imaging, (3) 1 mm pinhole SPECT acquisition, and (4) dissection. Tomographic reconstruction of SPECT data was performed with a three-dimensional ordered subset expectation maximization algorithm, and slices were reconstructed in three axes. 99mTc-NTP 15-5 rapidly accumulated in the joint, with a peak of radioactivity being reached from 5 minutes postinjection and maintained for at least 90 minutes. Given that bone and muscle did not show any accumulation of the tracer, highly contrasted joint imaging was obtained from 15 minutes postinjection. When 1 mm pinhole SPECT acquisition was focused on the knee, the medial and lateral compartments of both the femoral condyle and tibial plateau were highly delineated, allowing a separate quantitation of tracer accumulation within each component of the femorotibial joint. A good correlation was found between tracer uptake determined by region of interest analysis of both planar and SPECT scans and dissection. This new approach to imaging of cartilage in mice provides joint functionality assessment in vivo, giving a unique opportunity to achieve a greater understanding of cartilage physiology in health and disease.