Promising Pre-clinical Validation of Targeted Radionuclide Therapy Using a [131I] Labelled Iodoquinoxaline Derivative for an Effective Melanoma Treatment

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 [I] ICF01012 treatment drastically inhibited growth of B16F0, B16Bl6 and M4Beu tumours whereas [I] 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.


Abstract
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 [ 131 I] ICF01012 treatment drastically inhibited growth of B16F0, B16Bl6 and M4Beu tumours whereas [ 131 I] 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 [ 131 I] labelled iodoquinoxaline derivative for an effective melanoma treatment.

Introduction
Melanoma is a poor prognosis skin cancer that affects about 150000 new patients per year in world. Despite recent advances in prevention and early diagnosis, malignant melanoma remains an increasingly prevalent cancer worldwide (Lorigan et al., 2008; Thompson et al., 2005). It is notably the second most common cancer among patients aged 20-39 years (Garbe and Leiter, 2009;Jennings and Murphy, 2009; Thompson et al., 2005). This aggressive disease displays a strong tendency to metastasize. Although localized disease is frequently curable by surgical removal, there is no effective treatment for metastatic melanoma. Patients with metastatic malignant melanoma have an average median survival rate of 6 months and a 5-year survival rate of less than 15% (Balch et al., 2001;Miller and Mihm, 2006). There are several approved therapies for malignant melanoma treatment as dacarbazine (an alkylating agent) and interleukin-2. However, the response rate obtained with these therapies remains low (less than 20%) and contributes little to overall patient survival (Tarhini and Agarwala, 2006). A higher response rate of up to 40% may be obtained with combined treatment schedules, but without significant prolonged survival in randomized studies (Hauschild et al., 2001;Tarhini and Agarwala, 2006). Globally, the response rate is still rather low and there has been no progress in systemic palliative melanoma treatment for three decades partly due to their high toxicity (Eigentler et al., 2003;Garbe and Eigentler, 2007). In this context, there is an urgency to develop more effective therapy and to find new more specific ways to treat disseminated melanoma. Melanin pigment itself can be a target for melanoma tissue. Melanin is an amorphous, irregular polymer composed of intimate mixtures of two separated but biogenetically related pigments: eumelanins and pheomelanins (Prota, 2000). Recently, radioimmunotherapy using mAb against melanin has been developed and evaluated on nude mice bearing human melanoma. Significant anti-tumoural effect has been obtained but important renal uptake is also detected with this treatment (Dadachova et al., 2004;Dadachova et al., 2008).

Many drugs bind to melanin both in vivo and in vitro:
polycyclic aromatic compounds such methylene blue (MTB), chloroquine or acridine orange. An extended experimental work has been done with 131 I or 211 At labelled MTB giving some promising data but without further clinical development (Link, 1999).
Another class of aromatic compounds with a cationic site such as N-(2-diethylaminoethyl) iodobenzamide was developed as potent melanoma-seeking agents. Two of these compounds named respectively BZA and BZA 2 have been clinically evaluated as suitable imaging agents in nuclear medicine with very encouraging results for metastases imaging

Selection of ICF01012 tracer for targeted radionuclide therapy of melanoma
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 BZA 2 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 125 I 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 131 I utilization were extrapolated using an adaptation of the MIRD program. From this study a quinoxaline deri vat ive m olecule (N-(2-diet hylam inoet hyl )-6iodoquinoxaline-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) Figures 1A and 1B). At the stage 72 h p.i., the tumour concentration was increased up to 11 fold as compared to BZA 2 ( Figure 1D). In term of dosimetry, for a 131 I labelling, the tumour absorbed dose was increased by more than 5 fold. 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 [ 125 I] ICF01012 confirmed this pharmakinetic profile ( Figure 1B). Indeed tumours were easily visualized suggesting a significant [ 125 I] 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) 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 [ 125 I] ICF01012 using a binding assay with synthetic melanin. We showed that [ 125 I] 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 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 [ 123 I] BZA 2, 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.

In vivo anti-tumoural efficacy of TRT
Several radionuclides are developed for the treatment of oncological diseases. Various approaches using 131 I 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 131 I treatment of thyroid cancer and its micrometastases (Nijsen et al., 2007;Verburg et al., 2005). Biodistribution properties of [ 125 I] ICF01012 molecule provide suitable dosimetry parameters using a labelling with 131 I. 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 [ 131 I] 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). [ 131 I] ICF01012 was administered i.v. at days 6 and 10 (2x18.5 MBq). Control groups received [ 131 I] Na or unlabelled ICF01012 (equimolar dose). All groups contained at least 10 animals and experiments were performed twice. The survival of [ 131 I] ICF01012-treated mice, with a median of 39 days, was significantly (p=0.006) better than that of untreated group whereas that of [ 131 I] NaI-treated mice was similar (p=0.3). These results suggested that [ 131 I] 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 [ 131 I] ICF01012 treatment drastically suppressed the growth of both B16F0 (p=0.001) and B16Bl6 (p=0.009) tumours. Figure 2A  In term of proliferation, the tumour volume doubling time (DT) was determined to quantify precisely the tumoural growth. After [ 131 I] 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 in dex an d m icrovessel numbers and an increa se of anisocytosis. This effect was associated with a significant decrease of the tumoural PCNA proliferation marker expression after treatment. All these results suggested a loss of aggressiveness of residual tumour cells after [ 131 I] 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 [ 131 I] ICF01012 treated group ( Figure 2B) suggesting an inhibitory effect of this treatment on lung dissemination (Bonnet-Duquennoy et al.,

Discussion
Our data demonstrated a strong anti-tumoural efficacy associated with low toxicity of [ 131 I] ICF01012 for radionuclide therapy on murine and human pigmented melanoma models whatever their dissemination profiles and their melanin content be. In melanoma, melanin is observed within tumoural cells, into extracellular space and in melanophages (Lazova et al., 2009;Revskaya et al., 2009). In this project we proved that 131 I radionuclide physical proprieties associated with this melanin target distribution was able to deliver efficient cytotoxic irradiation (Bonnet-Duquennoy et al., Chezal et al., 2008). Considering that melanin is detected in more than 90% of melanoma cases, strategy targeting this pigment could be used in the majority of patients.
Optimization of the therapy protocol in mice with lower doses and other schedules of injections will be explored in order to define the better balance between the anti-tumoural effect and the safety on normal pigmented tissues. Interestingly histological evaluation of B16F0 and B16Bl6 tumours in treated mice revealed an increased of extracellular melanin content after radiotherapy providing more abundant target for new injection. [ 131 I] ICF01012 treatments used in this strategy showed low toxicity with an increase of median survival time confirming in vivo pharmacokinetic profile of ICF01012 which demonstrated a rapid clearance of tracer from non-target organs as liver, brain and lung. Additional dosimetric studies are in progress to evaluate the delivery dose in the different organs in these therapeutic conditions by taking into account evolution of the tumoural size. This work should allow to better manage the future protocol and to predict the anti-tumoural response for clinical transfer of this strategy. Furthermore, an imaging clinical trial will be performed to evaluate tumour and possible normally pigmented organ fixation of ICF01012 and to determine dosimetric parameters in human.

JCST/Vol.1 Issue 1
Results presented here support the concept of targeted radionuclide therapy using a [ 131 I] labelled iodoquinoxaline derivative for an effective melanoma treatment.