Pharmacomodulation study: To discover new molecular derivatives for targeted radionuclide therapy with strong, specific and long-lasting uptake in melanoma tissues, a large pharmacomodulation study was performed (Chezal et al., 2008). This strategy involved the incorporation of aromatic or heteroaromatic structures in place of the phenyl moiety of BZA₂ to take advantage of polycyclic aromatic compounds that display a strong affinity for melanin, while the lipophilic side chain was kept identical. Then, various iodo (hetero) aromatic fused ring systems with a carboxamide part were synthesized and radioiodinated. Biodistribution of these coumpounds radiolabelled with ¹²⁵I was performed in B16F0 melanoma bearing C57BL6 mice after i.v. injection. The radioactivity biodistribution was analysed by quantitative whole body autoradiography using an AMBIS 4000 detector on whole body slices of mice obtained by cryosection. The radioactivity was quantified in different organs including tumour and expressed as percentage of injected dose/g of tissue (%ID/g). Dosimetry parameters for a ¹³¹I utilization were extrapolated using an adaptation of the MIRD program. From this study a quinoxaline der ivat ive molecule (N-(2-diethylaminoethyl )-6- iodoquinoxaline-2-carboxamide dihydrochloride salt named ICF01012) was selected for its high, specific and long-lasting uptake in B16F0 tumour with a rapid clearance from non-target organs (Figure 1) (Chezal et al., 2008). In comparison with [¹²⁵I] BZA₂ (Chezal et al., 2008; Moins et al., 2001), we observed for [¹²⁵I] ICF01012 a more elevated and durable concentration on B16F0 tumour (Figure 1A and Figure 1B). At the stage 72 h p.i., the tumour concentration was increased up to 11 fold as compared to BZA₂ (Figure 1D). In term of dosimetry, for a ¹³¹I labelling, the tumour absorbed dose was increased by more than 5 fold.

Pharmacokinetic pofiles: [¹²⁵I] CF01012 biodistribution in B16F0-bearing C57Bl6 mice to 1 hour from 8 days post-injection was analysed by quantitative whole body autoradiography (AMBIS 4000) (Chezal et al., 2008). Radioact ivi ty quantifications expressed as percentage of injected dose/g of tissue (%ID/g) were reported for different organs including tumour in Table 1. [¹²⁵I] ICF01012 tumoural concentration was significantly observed as early as 1 hour p.i and a significant and durable accumulation until 8 days p.i demonstrating a very high tumour uptake. Moreover the cumulative urinary and fecal excretions collected up to 72 h showed that elimination was almost total (88%) with a mainly urinary route (70%) (Chezal et al., 2008). SPECT imaging of B16F0-bearing C57Bl6 mice injected intravenously with [¹²⁵I] ICF01012 confirmed this pharmakinetic profile (Figure 1B). Indeed tumours were easily visualized suggesting a significant [¹²⁵I] ICF01012 tumoural concentration (Miot-Noirault et al., 2009). Moreover systemic elimination was quite complete from non target organs 24 hours p.i confirming the rapid clearance from non target tissues obtained with autoradiography quantitation (Table 1). 24 hours post-injection, radioactivity concentration was very low within liver (< 1.5% ID/g), and undetectable in spleen, kidneys, lungs, blood (Table 1). This led to high tumour to nontarget organ activity ratios rapidly unquantifiable when organ concentration was within the range of the background value (Table 1). Interestingly, [¹²⁵I] ICF01012 renal uptake is very low (undetectable 24 hours after injection) by comparison with other targeting-melanin molecules as antibodies and α-MSH peptide analogues (Dadachova et al., 2008; Miao et al., 2005).

To trace and quantify the metabolic fate of [¹²⁵I] ICF01012 in B16F0 melanoma bearing mice, high-performance liquid chromatography (HPLC) analysis were performed on tumour (Denoyer et al., 2008). Many HPLC peaks were identified in tumoural extracts with the major peak corresponding to the non-metabolized form of [¹²⁵I] ICF01012. Eight days postinjection, 83.6% of the remaining radioactivity is under [¹²⁵I] ICF01012 unchanged form.

All these promising data made this tracer a strong melanoma seeking agent for both scintigraphic imaging and radionuclide therapy (Chezal et al., 2008).

Melanin specificity: In experiments presented above, we observed B16F0 melanoma targeting using our quinoxaline analogue tracer. We verified the melanin specificity of [¹²⁵I] ICF01012 using a binding assay with synthetic melanin. We showed that [¹²⁵I] ICF01012 displayed a strong affinity for synthetic melanin in water and in PBS (Chezal et al., 2008). Additional study was performed to visualised in vivo ICF01012 melanin specificity by Secondary ion mass spectrometry (SIMS) imaging (Bonnet-Duquennoy et al., 2009). With this technique, we observed a perfect co-localization of ICF01012 signal with melanin confirming the melanin binding properties of this tracer and its internalization in melanoma cells.

Moreover, we noted on biodistribution study (Table 1) an important radioactivity concentration on uvea confirming the high ICF01012 specificity for pigmented structures and so for melanin (Chezal et al., 2008). However fixation of tracer on normal pigmented tissues in these pre-clinical experiments should not be considered as a limit for our strategy. Indeed these experiments were performed on the heavily pigmented C57BL/6J mice associated with an important uveal melanin content which is not predictive for human status of uvea. On a clinical imaging study with [¹²³I] BZA₂, the parental benzamide analogue of ICF01012, no significant uveal uptake was imaged excepted for an ocular melanoma case while important uveal uptake was observed with the same tracer on this mouse model (Moins et al., 2002; Sillaire-Houtmann et al., 2004). Because of murine and human differences on ocular geometry and melanin content, ICF01012 accumulation within C57Bl6 mice eyes should not be considered as hampering for clinical transfer of this radiotherapy concept. Nevertheless this point has to be taking into account in our studies.

Several radionuclides are developed for the treatment of oncological diseases. Various approaches using ¹³¹I for TRT were developed to treat different cancers because of its relatively low beta energy with a maximum range of 3 mm in tissue which is effective for treatment (Kassis and Adelstein, 2005). Some of them have resulted in very good curative responses like ¹³¹I treatment of thyroid cancer and its micrometastases (Nijsen et al., 2007; Verburg et al., 2005). Biodistribution properties of [¹²⁵I] ICF01012 molecule provide suitable dosimetry parameters using a labelling with ¹³¹I. Indeed an administered dose of 37 MBq will allow the delivery of 49 Gy to the tumour, a usefully high dose for a good therapeutic efficacy (Chezal et al., 2008).

We investigated the in vivo therapeutic efficacy of [¹³¹I] ICF01012 as a radionuclide therapeutic agent (Bonnet-Duquennoy et al., 2009) on murine melanoma models B16F0 and on the metastatic B16Bl6 cell lines grafted s.c. into syngenic C57Bl/6J mice (Poste et al., 1980). [¹³¹I] ICF01012 was administered i.v. at days 6 and 10 (2×18.5 MBq). Control groups received [¹³¹I] Na or unlabelled ICF01012 (equimolar dose). All groups contained at least 10 animals and experiments were performed twice. The survival of [¹³¹I] ICF01012-treated mice, with a median of 39 days, was significantly (p=0.006) better than that of untreated group whereas that of [131I] NaI-treated mice was similar (p=0.3). These results suggested that [¹³¹I] ICF01012 treatments showed low toxicity confirming precedent pharmacokinetic experiments and rapid systemic elimination.

We observed that the treatment with unlabelled ICF01012 was without any significant effect whereas [¹³¹I] ICF01012 treatment drastically suppressed the growth of both B16F0 (p=0.001) and B16Bl6 (p=0.009) tumours. Figure 2A illustrates the strong difference on B16Bl6 tumour size at day 19 between control and treated animals. [¹³¹I] NaI alone did not have any efficacy against tumour growth showing interest of targeting strategy with [¹³¹I] ICF01012 for an efficient therapeutic effect (Bonnet-Duquennoy et al., 2009).

In term of proliferation, the tumour volume doubling time (DT) was determined to quantify precisely the tumoural growth. After [131I] ICF01012 treatment, DT was significantly increased (range +44 to +147%) confirming inhibition of tumoural growth. Histological analysis showed that after targeted radiotherapy treatment, residual tumoural cells exhibited a decrease of mitotic index and microvessel numbers and an increase of anisocytosis. This effect was associated with a significant decrease of the tumoural PCNA proliferation marker expression after treatment. Al l these results suggested a loss of aggressiveness of residual tumour cells after [¹³¹I] ICF01012 treatment (Bonnet-Duquennoy et al., 2009).

After macroscopic analysis, we observed that 55% of the untreated mice bearing B16Bl6 tumours had lung metastases whereas no metastasis was counted on [131I] ICF01012 treated group (Figure 2B) suggesting an inhibitory effect of this treatment on lung dissemination (Bonnet-Duquennoy et al., 2009). We demonstrated a strong and similar anti-tumoural effect of [¹³¹I] ICF01012 on both nonmetastatic B16F0 tumour and the more aggressive metastatic B16Bl6 model and a complete inhibition of B16Bl6 dissemination towards lungs. These results provide useful data for support our approach to treating all melanoma forms including disseminated disease.

Because B16F0 and B16Bl6 tumours were highly pigmented, we assessed radiotherapy experiment on human melanoma cells which exhibit lower melanin content (Bonnet-Duquennoy et al., 2009). Tumour distribution of radiotracer was evaluated on nude mice bearing M4Beu melanoma which contain a lower melanin concentration than B16F0 tumour (ratio: 1/2.8) (Bonnet-Duquennoy et al., 2009). We observed that M4Beu tumour uptake of [¹²⁵I] ICF01012 was consistent with 6.3±1.3% and 2.3±0.5% ID/g 6 and 24 hours after injection respectively but lower than B16F0 tumour uptake (Bonnet-Duquennoy et al., 2009). Then targeted radiotherapy was evaluated using 2 injections of 18.5 MBq [¹³¹I] ICF01012 into nude mice bearing M4Beu tumours. In term of proliferation, the tumor DT were significantly increased (range +46%) after [¹³¹I] ICF01012 treatment. These results showed that treatment significantly slowed the M4Beu tumoural growth (p=0.003) confirming strong efficacy of this TRT strategy even in melanoma containing lower target concentration.