Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates.

The development of 64Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [64Cu]Cu-15-5 complex prompted us to investigate its potential for the 64Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with 64Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [64Cu]Cu-15-5- and [64Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88-94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition.

ARRONAX Cyclotron: Setting up of In-House Hospital Radiopharmacy.

Whilst radiopharmaceuticals have an important role to play in both imaging and treatment of patients, most notably cancer patients, nuclear medicine and radiopharmacy are currently facing challenges to create innovative new drugs. Traditional radiopharmaceutical manufacture can be considered as either a routine hospital production or a large-scale industrial production. The gap between these two practices has meant that there is an inability to supply innovative radiopharmaceuticals for use at the local level for mono- or multicentric clinical trials with satisfactory quality and safety specifications. This article highlights the regulatory requirements in aseptic pharmaceutical processing and in nuclear medicine to be able to locally produce radiopharmaceuticals. We validate the proof-of-concept for an "in-house" hospital-based radiopharmacy including an on-site cyclotron, that can fulfill the conflicting requirements between radiation safety and aseptic processing. The ARRONAX in-house radiopharmacy is currently able to provide sterile and pyrogenic-free injectable radiopharmaceutical compounds for both industrial and institutional clinical trials.

SPECT-CT Imaging of Dog Spontaneous Diffuse Large B-Cell Lymphoma Targeting CD22 for the Implementation of a Relevant Preclinical Model for Human.

Antibodies directed against CD22 have been used in radioimmunotherapy (RIT) clinical trials to treat patients with diffuse large B-cell lymphoma (DLBCL) with promising results. However, relevant preclinical models are needed to facilitate the evaluation and optimization of new protocols. Spontaneous DLBCL in dogs is a tumor model that may help accelerate the development of new methodologies and therapeutic strategies for RIT targeting CD22. Seven murine monoclonal antibodies specific for canine CD22 were produced by the hybridoma method and characterized. The antibodies' affinity and epitopic maps, their internalization capability and usefulness for diagnosis in immunohistochemistry were determined. Biodistribution and PET imaging on a mouse xenogeneic model of dog DLBCL was used to choose the most promising antibody for our purposes. PET-CT results confirmed biodistribution study observations and allowed tumor localization. The selected antibody, 10C6, was successfully used on a dog with spontaneous DLBCL for SPECT-CT imaging in the context of disease staging, validating its efficacy for diagnosis and the feasibility of future RIT assays. This first attempt at phenotypic imaging on dogs paves the way to implementing quantitative imaging methodologies that would be transposable to humans in a theranostic approach. Taking into account the feedback of existing human radioimmunotherapy clinical trials targeting CD22, animal trials are planned to investigate protocol improvements that are difficult to consider in humans due to ethical concerns.

64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study.

BACKGROUND: Diacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression. METHODS: µPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels. RESULTS: In vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages. CONCLUSION: In the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

Potential for Nuclear Medicine Therapy for Glioblastoma Treatment.

Glioblastoma is the most common malignant adult brain tumor and has a very poor patient prognosis. The mean survival for highly proliferative glioblastoma is only 10 to 14 months despite an aggressive current therapeutic approach known as Stupp's protocol, which consists of debulking surgery followed by radiotherapy and chemotherapy. Despite several clinical trials using anti-angiogenic targeted therapies, glioblastoma medical care remains without major progress in the last decade. Recent progress in nuclear medicine, has been mainly driven by advances in biotechnologies such as radioimmunotherapy, radiopeptide therapy, and radionanoparticles, and these bring a new promising arsenal for glioblastoma therapy. For therapeutic purposes, nuclear medicine practitioners classically use ß- particle emitters like 131I, 90Y, 186/188Re, or 177Lu. In the glioblastoma field, these radioisotopes are coupled with nanoparticles, monoclonal antibodies, or peptides. These radiopharmaceutical compounds have resulted in a stabilization and/or improvement of the neurological status with only transient side effects. In nuclear medicine, the glioblastoma-localized and targeted internal radiotherapy proof-of-concept stage has been successfully demonstrated using ß- emitting isotopes. Similarly, α particle emitters like 213Bi, 211At, or 225Ac appear to be an innovative and interesting alternative. Indeed, α particles deliver a high proportion of their energy inside or at close proximity to the targeted cells (within a few micrometers from the emission point versus several millimeters for ß- particles). This physical property is based on particle-matter interaction differences and results in α particles being highly efficient in killing tumor cells with minimal irradiation of healthy tissues and permits targeting of isolated tumor cells. The first clinical trials confirmed this idea and showed good therapeutic efficacy and less side effects, thus opening a new and promising era for glioblastoma medical care using α therapy. The objective of this literature review is focused on the developing field of nuclear medicine and aims to describe the various parameters such as targets, vectors, isotopes, or injection route (systemic and local) in relation to the clinical and preclinical results in glioblastoma pathology.

Radiation dosimetry of [131 I]ICF01012 in rabbits: Application to targeted radionuclide therapy for human melanoma treatment.

PURPOSE: Dosimetry for melanoma-targeted radionuclide therapy (TRT) with [131 I]ICF01012, a melanin ligand, has been previously evaluated in mice bearing melanomas. In this study, activity distribution and dosimetry are performed on healthy rabbits (Fauve de Bourgogne) using SPECT-CT imaging and ex vivo measurements. MATERIAL AND METHODS: Ex vivo biodistribution (i.v. injection: 370 kBq/kg, n = 2 per point) is performed on blood, eyes, brain, lung, liver, kidneys, heart, stomach, and spleen. Dosimetry calculations follow the MIRD formalism: S values are calculated from CT images using the GATE Monte Carlo platform and activity distributions are obtained from SPECT-CT imaging (i.v. injection: 37 MBq/kg n = 3 per point). A specific study is presented to assess dose to human retina. RESULTS: Time-integrated activities based on SPECT-CT are in accordance with ex vivo measurements except for spleen. Doses to liver and eyes are the most significant, with respectively, 6.38 ± 0.50 Gy/GBq (evaluated through SPECT-CT imaging) and 45.8 ± 7.9 Gy/GBq (evaluated through ex vivo measurements). Characterization of ocular [131 I]ICF01012 biodistribution in rabbits and quantification of melanin allowed to assess a dose of 3.07 ± 0.70 Gy/GBq to human retina. CONCLUSION: This study sustains [131 I]ICF01012 as a good candidate for melanoma TRT and open perspectives for personalized dosimetry calculation during phase I clinical transfer.

Doxycycline and its quaternary ammonium derivative for adjuvant therapies of chondrosarcoma.

PURPOSE: This study was conducted during the development of innovative treatment targeting the microenvironment of chondrosarcoma. In this context, MMP inhibitors were conjugated with a quaternary ammonium (QA) function as a targeting ligand to proteoglycans of chondrosarcoma extracellular matrix. Here we report the proof of concept of this strategy applied to the MMP13 inhibitor, doxycycline (Dox). METHODS: A quaternary ammonium derivative of the MMP13 inhibitor doxycycline (QA-Dox) was synthesized, and its anticancer activity was evaluated in the Swarm rat chondrosarcoma (SRC) model compared with the parent drug doxycycline, in vitro and in vivo. In vivo, dox and QA-Dox efficiency was assessed at equimolar doses according to a q4dx4 schedule by monitoring tumour volume by MRI and PG-targeted scintigraphy. Molecular mechanism (MMP13 expression, proteoglycan level) and histology studies were performed on tumours. RESULTS: The link of QA targeting function to Dox maintained the MMP13 inhibitory activity in vitro. Interestingly, the bacteriostatic activity was lost. SRC cells incubated with both drugs were blocked in S and G2 M phases. Tumour growth inhibition (confirmed by histology) was observed for both Dox and QA-Dox. Undesirable blood effects (leukocyte decrease) were reduced when Dox was targeted to tumour tissue using the QA function. CONCLUSIONS: In the SRC model, the MMP13 inhibitor Dox and its QA derivative are promising as adjuvant therapies for chondrosarcoma management.

Theranostic Approach for Metastatic Pigmented Melanoma Using ICF15002, a Multimodal Radiotracer for Both PET Imaging and Targeted Radionuclide Therapy.

PURPOSE: This work reports, in melanoma models, the theranostic potential of ICF15002 as a single fluorinated and iodinated melanin-targeting compound. METHODS: Studies were conducted in the murine syngeneic B16BL6 model and in the A375 and SK-MEL-3 human xenografts. ICF15002 was radiolabeled with fluorine-18 for positron emission tomography (PET) imaging and biodistribution, with iodine-125 for metabolism study, and iodine-131 for targeted radionuclide therapy (TRT). TRT efficacy was assessed by tumor volume measurement, with mechanistics and dosimetry parameters being determined in the B16BL6 model. Intracellular localization of ICF15002 was characterized by secondary ion mass spectrometry (SIMS). RESULTS: PET imaging with [18F]ICF15002 evidenced tumoral uptake of 14.33±2.11%ID/g and 4.87±0.93%ID/g in pigmented B16BL6 and SK-MEL-3 models, respectively, at 1 hour post inoculation. No accumulation was observed in the unpigmented A375 melanoma. SIMS demonstrated colocalization of ICF15002 signal with melanin polymers in melanosomes of the B16BL6 tumors. TRT with two doses of 20 MBq [131I]ICF15002 delivered an absorbed dose of 102.3 Gy to B16BL6 tumors, leading to a significant tumor growth inhibition [doubling time (DT) of 2.9±0.5 days in treated vs 1.8±0.3 in controls] and a prolonged median survival (27 days vs 21 in controls). P53S15 phosphorylation and P21 induction were associated with a G2/M blockage, suggesting mitotic catastrophe. In the human SK-MEL-3 model, three doses of 25 MBq led also to a DT increase (26.5±7.8 days vs 11.0±3.8 in controls) and improved median survival (111 days vs 74 in controls). CONCLUSION: Results demonstrate that ICF15002 fulfills suitable properties for bimodal imaging/TRT management of patients with pigmented melanoma.

Proteoglycans as Target for an Innovative Therapeutic Approach in Chondrosarcoma: Preclinical Proof of Concept.

To date, surgery remains the only option for the treatment of chondrosarcoma, which is radio- and chemoresistant due in part to its large extracellular matrix (ECM) and poor vascularity. In case of unresectable locally advanced or metastatic diseases with a poor prognosis, improving the management of chondrosarcoma still remains a challenge. Our team developed an attractive approach of improvement of the therapeutic index of chemotherapy by targeting proteoglycan (PG)-rich tissues using a quaternary ammonium (QA) function conjugated to melphalan (Mel). First of all, we demonstrated the crucial role of the QA carrier for binding to aggrecan by surface plasmon resonance. In the orthotopic model of Swarm rat chondrosarcoma, an in vivo biodistribution study of Mel and its QA derivative (Mel-QA), radiolabeled with tritium, showed rapid radioactivity accumulation in healthy cartilaginous tissues and tumor after [3H]-Mel-QA injection. The higher T/M ratio of the QA derivative suggests some advantage of QA-active targeting of chondrosarcoma. The antitumoral effects were characterized by tumor volume assessment, in vivo 99mTc-NTP 15-5 scintigraphic imaging of PGs, 1H-HRMAS NMR spectroscopy, and histology. The conjugation of a QA function to Mel did not hamper its in vivo efficiency and strongly improved the tolerability of Mel leading to a significant decrease of side effects (hematologic analyses and body weight monitoring). Thus, QA conjugation leads to a significant improvement of the therapeutic index, which is essential in oncology and enable repeated cycles of chemotherapy in patients with chondrosarcoma. Mol Cancer Ther; 15(11); 2575-85. ©2016 AACR.

Focus on the Controversial Aspects of (64)Cu-ATSM in Tumoral Hypoxia Mapping by PET Imaging.

Mapping tumor hypoxia is a great challenge in positron emission tomography (PET) imaging as the precise functional information of the biological processes is needed for many effective therapeutic strategies. Tumor hypoxia has been widely reported as a poor prognostic indicator and is often associated with tumor aggressiveness, chemo- and radio-resistance. An accurate diagnosis of hypoxia is a challenge and is crucial for providing accurate treatment for patients' survival benefits. This challenge has led to the emergence of new and novel PET tracers for the functional and metabolic characterization of tumor hypoxia non-invasively. Among these tracers, copper semicarbazone compound [64Cu]-diacetyl-bis(N (4)-methylthiosemicarbazone) (=64Cu-ATSM) has been developed as a tracer for hypoxia imaging. This review focuses on 64Cu-ATSM PET imaging and the concept is presented in two sections. The first section describes its in vitro development and pre-clinical testing and particularly its affinity in different cell lines. The second section describes the controversial reports on its specificity for hypoxia imaging. The review concludes that 64Cu-ATSM - more than a hypoxic tracer, exhibits tracer accumulation in tumor, which is linked to the redox potential and reactive oxygen species. The authors concluded that 64Cu-ATSNM is a marker of over-reduced cell state and thus an indirect marker for hypoxia imaging. The affinity of 64Cu-ATSM for over-reduced cells was observed to be a complex phenomenon. And to provide a definitive and convincing mechanism, more in vivo studies are needed to prove the diagnostic utility of 64Cu-ATSM.

Is there an interest to use deuteron beams to produce non-conventional radionuclides?

With the recent interest on the theranostic approach, there has been a renewed interest for alternative radionuclides in nuclear medicine. They can be produced using common production routes, i.e., using protons accelerated by biomedical cyclotrons or neutrons produced in research reactors. However, in some cases, it can be more valuable to use deuterons as projectiles. In the case of Cu-64, smaller quantities of the expensive target material, Ni-64, are used with deuterons as compared with protons for the same produced activity. For the Sc-44m/Sc-44g generator, deuterons afford a higher Sc-44m production yield than with protons. Finally, in the case of Re-186g, deuterons lead to a production yield five times higher than protons. These three examples show that it is of interest to consider not only protons or neutrons but also deuterons to produce alternative radionuclides.

99mTc-NTP 15-5 Imaging for Cartilage Involvement in Experimental Rheumatoid Arthritis: Comparison with Routinely Used Molecular Imaging Methods and Sensitivity to Chronic Nonsteroidal Antiinflammatory Drug Treatment.

UNLABELLED: This study determined, using the intraarticular complete Freund adjuvant arthritis mice model, whether the radiotracer (99m)Tc-N-(triethylammonium)-3-propyl-[15]ane-N5 ((99m)Tc-NTP 15-5) targeting proteoglycans has a pathophysiologic validity for in vivo imaging of rheumatoid arthritis (RA) and its response to chronic nonsteroidal antiinflammatory drugs. METHODS: We investigated the time course of cartilage remodeling by (99m)Tc-NTP 15-5 scintigraphy, bone damages by (99m)Tc-hydroxymethylene diphosphonate imaging, inflammation by (18)F-FDG PET, and joint proteoglycan content and pain behavior in animals, without and with meloxicam treatment. Paw circumference, thermal pain behavior, and histology as well as proteoglycan content of the whole joint were determined. RESULTS: (99m)Tc-NTP 15-5 showed specific tracer accumulation within RA joints, with a significant increase in scintigraphic ratio observed in RA versus shams from day 3 to day 28. (18)F-FDG evidenced uptake in RA joints from day 15 to day 29. Animals treated with meloxicam (5 mg/kg) exhibited a dose-dependent decrease in both (99m)Tc-NTP 15-5 and (18)F-FDG uptake ratios versus saline-treated animals. (99m)Tc-hydroxymethylene diphosphonate bone scans were only positive at day 14 in RA versus shams, with a significant effect of meloxicam. An increase in proteoglycans of RA joint and thermal pain behavior were observed and were dose-dependently reduced by meloxicam. CONCLUSION: These experimental results bring data in favor of the (99m)Tc-NTP 15-5 radiotracer for assessing, in vivo, cartilage remodeling in RA that could be used to monitor therapy.

Development of a freeze-dried kit formulation for the preparation of 99mTc-NTP 15-5, a radiotracer for scintigraphic imaging of proteoglycans.

The cartilage-targeting strategy is based on the strong affinity of quaternary ammonium (QA) functions for cartilage proteoglycans. We use a bifunctional agent containing QA moiety and a polyazamacrocycle structure able to complex technetium-99m. (99m)Tc-NTP 15-5 was selected for its high stability and its high affinity for proteoglycans in vivo. Labeling conditions of NTP 15-5 were optimized, and a lyophilized kit was developed for radiolabeling of (99m)Tc-NTP 15-5 (radiochemical yields 94.6±1.8%). (99m)Tc-NTP 15-5 was stable and resulted in favorable biological evaluations.

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).

Development and Preliminary Evaluation of TFIB, a New Bimodal Prosthetic Group for Bioactive Molecule Labeling.

The new readily available prosthetic group, tetrafluorophenyl 4-fluoro-3-iodobenzoate (TFIB), designed for both molecular imaging and targeted radionuclide therapy purposes was radiolabeled either with fluorine or iodine radionuclides with excellent radiochemical yields and purities. These radiolabeled tags were conjugated to N,N-diethylethylenediamine to give melanin-targeting radiotracers [ (125) I]9 and [ (18) F]9, which were successfully evaluated by PET and gamma scintigraphic imaging in B16F0 pigmented melanoma-bearing C57BL/6J mice. Then, radiolabeled [ (125) I]/[ (18) F]TFIB was used to tag tumor-targeting peptides (i.e., PEG3[c(RGDyK)]2 and NDP-MSH targeting αvß3 integrin and MC1R receptors, respectively) in mild conditions and with good radiochemical yields (47-83% d.c.) and purities (>99%). The resulting radiolabeled peptides were assessed both in vitro and by PET imaging in animal models.

Small rigid platforms functionalization with quaternary ammonium: targeting extracellular matrix of chondrosarcoma.

This work takes place in the "cartilage targeting strategy", consisting in using the quaternary ammonium (QA) function as a vector to proteoglycans (PGs) of extracellular matrix (ECM). The objective was to demonstrate that QA could address gadolinium based small rigid platforms (SRP) to PG-rich tumors. SRP were functionalized with QA, radiolabeled with (111)Indium and evaluated for biodistribution in vivo, respectively to non functionalized SRP, in two experimental models: (i) the HEMCSS human xenograft model; (ii) the Swarm rat chondrosarcoma (SRC) orthotopic model. The contribution of cellular uptake to tumoral accumulation of nano-objects was also determined from in vitro binding. In the SRC model expressing a highly and homogeneously distributed PG content, tumor accumulation and retention of SRP@QA were increased by 40% as compared to non-functionalized SRP. When considering the radiosensitizing potential of gadolinium based SRP, these results provide hopes for the radiobiological approach of highly resistant tumor such as chondrosarcoma. FROM THE CLINICAL EDITOR: In this study, gadolinium-based complexing DOTA-surfaced small polysiloxane nanoparticles were functionalized with quaternary ammonium derivatives that target the extracellular matrix of chondrosarcoma. The authors demonstrate in a rat model that the use of these constructs results in a 40% increase of tumor accumulation and retention compared to non-functionalized (and otherwise same) platforms. Similar approaches would be welcome additions to the clinical armamentarium addressing chondrosarcoma.

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.

99mTc-NTP 15-5 assessment of the early therapeutic response of chondrosarcoma to zoledronic acid in the Swarm rat orthotopic model.

BACKGROUND: Since proteoglycans (PGs) appear as key partners in chondrosarcoma biology, PG-targeted imaging using the radiotracer 99mTc-N-(triethylammonium)-3-propyl-[15]ane-N5 (99mTc-NTP 15-5) developed by our group was previously demonstrated to be a good single-photon emission computed tomography tracer for cartilage neoplasms. We therefore initiated this new preclinical study to evaluate the relevance of 99mTc-NTP 15-5 imaging for the in vivo monitoring and quantitative assessment of chondrosarcoma response to zoledronic acid (ZOL) in the Swarm rat orthotopic model. FINDINGS: Rats bearing chondrosarcoma in the orthotopic paratibial location were treated by ZOL (100 µg/kg, subcutaneously) or phosphate-buffered saline, twice a week, from day 4 to day 48 post-tumor implantation. 99mTc-NTP 15-5 imaging was performed at regular intervals with the target-to-background ratio (TBR) determined. Tumor volume was monitored using a calliper, and histology was performed at the end of the study. From day 11 to day 48, mean TBR values ranged from 1.7 ± 0.6 to 2.3 ± 0.6 in ZOL-treated rats and from 2.1 ± 1.0 to 4.9 ± 0.9 in controls. Tumor growth inhibition was evidenced using a calliper from day 24 and associated to a decrease in PG content in treated tumor tissues (confirmed by histology). CONCLUSIONS: This work demonstrated two proofs of concept: (1) biphosphonate therapy could be a promising therapeutic approach for chondrosarcoma; (2) 99mTc-NTP 15-5 is expected to offer a novel imaging modality for the in vivo evaluation of the extracellular matrix features of chondrosarcoma, which could be useful for the follow-up and quantitative assessment of proteoglycan 'downregulation' associated to the response to therapeutic attempts.

Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment.

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.

In vivo evidence of the targeting of cartilaginous tissue by pyridinium functionalized nanoparticles.

Ultrasmall gadolinium based particles have been functionalized with positively charged pyridinium quaternary ammonium and labelled with (111)In. Evidence of their active targeting properties towards proteoglycans has been demonstrated in vivo after intravenous injection into rats opening thus a route to cancer imaging and therapy.