Zhao-Hui Jin

Noninvasive Optical Imaging of Ovarian Metastases Using Cy5-labeled RAFT-c(-RGDfK-)4

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin αvβ3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RβADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for SC tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection.

Activatable fluorescent probes for tumour-targeting imaging in live mice.

The direct and noninvasive in vivo visualisation of molecular processes such as ligand–receptor interaction, enzymatic activity, or gene expression, is the goal of molecular imaging. Optical imaging appears as a new complementary modality to the traditional nuclear or MRI (magnetic resonance imaging) techniques because of its low cost and fewer constraints. 4] Thus, the need for new optical probes with increased targeting and imaging capabilities arises. 6] Tung et al. introduced the concept of “smart” or activatable probes for imaging the activity of proteases over-expressed in mice tumours, such as MMP2 or cathepsin D. In these activatable probes, Cy5.5 dyes are grafted to peptide branching arms of a co-polymer, in such a way that they auto-quench initially. Whenever the proteases specifically cut the peptide sequence, the Cy5.5 fluorescence is recovered. In the present work, the concept of activatable probes is extended to the imaging of different molecular events, in the expectation of improving the image contrast in comparison to that obtained with classical targeting agents. The molecular structures we propose can be used for imaging enzymatic activity, specific targeting, and molecular processes triggered by ligand–receptor interaction. Moreover, their nonpolymeric nature can be very well controlled and characterised for pharmacology and medical applications. These activatable probes are built on a cyclodecapeptide template named RAFT (Regioselectively Addressable Functionalized Template), a new molecular vector for targeted drug delivery and molecular imaging of tumours and metastasis. This new class of molecules differs from other systems by the topological separation of two independent functional domains, which can be addressed in a spatially controlled manner: a cell targeting domain, and a therapeutic or imaging domain (Scheme 1). Clustered RGD-containing markers are used for cell recognition by the aVb3 integrin, [16–19] a vitronectin receptor over-expressed on the surface of endothelial cells of growing blood vessels, and therefore a cardinal feature of many malignant tumours. A multimeric presentation of the RGD motif is essential for integrin-mediated internalisation of the probe. 22–24] In the activatable probes we designed, the imaging function is composed of the cyanine 5 fluorescent dye (Cy5), a cleavable bond, and a quencher (Q). The cleavable bond is used to follow the cellular internalisation of the probe induced by binding to its receptor. Disulfide bridges are known to be reduced enzymatically by thioredoxines in the cytosol, and have been used to improve the efficiency of targeted drug delivery. The redox potential within the endosomal system could also be reducing. Therefore, -Cysteine-S S-Cysteine(abbreviated as S–S below) is chosen as a cleavable bond, to follow the internalisation of the fluorescent probe into the cells after its RGD-mediated binding on their surface. Demonstrating targeting can be crucial for screening and comparing the recognition efficiency of different biological markers. Moreover, demonstrating internalisation is also very important to estimate the ability of a molecule, such as the RAFT, to be used as a vector for targeted drug delivery. The self-quenching of the cyanine dyes allows the use of the Cy5-S S-Cy5 group as an activatable unit (Scheme 1). Alternatively, a diarylrhodamine derivative quencher (QSY21) can be used in place of one of the Cy5 units. Cy5 fluorescence is markedly inhibited in the Cy5-S S-QSY21 group (Table 1). This dynamic and static quenching in aqueous buffer can be accounted for by the hydrophobic nature and opposite charges of the molecules, which favour their stacking. In this context, the RAFT-(cRGD)4 Cy5-S S-Cy5 1, RAFT-(cRGD)4 Cy5-S SQSY21 2, and RAFT-(cRGD)4 QSY21-S S-Cy5 3 have been synthesised (Scheme 1). The peptide moiety of the molecule is prepared by a combination of solid and solution phase synthesis using a Fmoc/tBu strategy, followed by a chemoselective assembling of ligand moieties and template, as reported elsewhere. Npys-protected cysteine is chosen to graft the fluorescent unit to the RAFT. This allows a regioselective disulfide bond formation. Cy5 and QSY21, available in the activated ester form, are then attached successively to the macromolecule, providing, after RP-HPLC purification, the desired compounds in satisfying overall yields (13–18%). The activation of the fluorescent probes is demonstrated first in vitro. Figure 1A displays the absorption spectra of the RAFT-(cRGD)4 Cy5-S S-QSY21 2 before and 2 h after addition of 2-mercaptoethanol (2-MCE). The spectrum after cleavage matches that of the sum of Cy5 and QSY21, whereas for 2, the electronic interaction between the dye and quencher is evidenced by an intense absorption band at 600 nm. Similar results are obtained for 1: the 600 nm absorption band in this case is indicative of the presence of Cy5 dimers, whereas the 647 nm band is attributed to the dye monomer. Fluorescence measurements (Figure 1B) show the initial Cy5 fluorescence quenching and its 100% recovery upon the S S bond [a] Dr. J. Razkin, Dr. D. Boturyn, Prof. P. Dumy LEDSS, UMR CNRS 5616, 301 rue de la chimie, BP 53, 38041 Grenoble Cedex 9 (France) Fax: (+33)476-635-540 E-mail : Didier.Boturyn@ujf-grenoble.fr [b] Dr. I. Texier LETI/DTBS CEA Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9 (France) Fax: (+33)438-785-787 E-mail : isabelle.texier-nogues@cea.fr [c] Dr. Z.-h. Jin, Prof. M. Favrot, Dr. J.-L. Coll INSERM U578, Institut Albert Bonniot, 38706 La Tronche (France) [d] Dr. V. Josserand ANIMAGE—CREATIS, B=timent CERMEP, 59 Boulevard Pinel, 69677 Bron Cedex (France) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.

In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)(4).

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)4 presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin aVb3 with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a “stealth” molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)4-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)4-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)4-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.

Synthesis and biological characterisation of targeted pro-apoptotic peptide.

We report herein the synthesis and in vitro assay of new, multimeric RGD‐peptide conjugates for cell‐targeted drug delivery. We generated a peptide scaffold comprising two functional domains, one a tumour blood vessel “homing” motif and the other a programmed cell‐death‐inducing peptide sequence. RGD peptides were selected to direct the molecular conjugate to αVβ3 integrin‐containing tumour cells. The pro‐apoptotic (Lys‐Leu‐Ala‐Lys‐Leu‐Ala‐Lys)2 peptide was found to be nontoxic outside cells, but toxic when internalized into targeted cells as it disrupted the mitochondrial membrane. The synthesis of these targeted pro‐apoptotic conjugates was carried out by assembling three different units (that is, scaffold, RGD units and pro‐apoptotic peptide) through chemoselective ligations. We show that one compound displays significant biological effect in αVβ3 integrin‐containing tumour cells.

Molecular imaging of ectopic metabotropic glutamate 1 receptor in melanoma with a positron emission tomography radioprobe (18) F-FITM.

Oncoimaging using positron emission tomography (PET) with a specific radioprobe would facilitate individualized cancer management. Evidence indicates that ectopically expressed metabotropic glutamate 1 (mGlu1) receptor independently induces melanocyte carcinogenesis, and it is therefore becoming an important target for personalized diagnosis and treatment strategies for melanomas. Here, we report the development of an oncoprotein‐based PET imaging platform in melanomas for noninvasive visualization and quantification of mGlu1 with a novel mGlu1‐specific radioprobe, 4‐18F‐fluoro‐N‐[4‐[6‐(isopropyl amino)pyrimidin‐4‐yl]‐1,3‐thiazol‐2‐yl]‐N‐methylbenzamide (18F‐FITM). 18F‐FITM shows excellent pharmacokinetics, namely the dense and specific accumulation in mGlu1‐positive melanomas versus mGlu1‐negative hepatoma and normal tissues. Furthermore, the accumulation levels of radioactivity corresponded to the extent of tumor and to levels of mGlu1 protein expression in melanomas and melanoma metastasis. The 18F‐FITM PET imaging platform, as a noninvasive personalized diagnostic tool, is expected to open a new avenue for defining individualized therapeutic strategies, clinical trials, patient management and understanding mGlu1‐triggered oncologic events in melanomas.

Luminescent probes for optical in vivo imaging

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

Multiple Administrations of 64Cu-ATSM as a Novel Therapeutic Option for Glioblastoma: a Translational Study Using Mice with Xenografts

Glioblastoma is the most aggressive malignant brain tumor in humans and is difficult to cure using current treatment options. Hypoxic regions are frequently found in glioblastoma, and increased levels of hypoxia are associated with poor clinical outcomes of glioblastoma patients. Hypoxia plays important roles in the progression and recurrence of glioblastoma because of drug delivery deficiencies and induction of hypoxia-inducible factor-1α in tumor cells, which lead to poor prognosis. We focused on a promising hypoxia-targeted internal radiotherapy agent, 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM), to address the need for additional treatment for glioblastoma. This compound can target the overreduced state under hypoxic conditions within tumors. Clinical positron emission tomography studies using radiolabeled Cu-ATSM have shown that Cu-ATSM accumulates in glioblastoma and its uptake is associated with high hypoxia-inducible factor-1α expression. To evaluate the therapeutic potential of this agent for glioblastoma, we examined the efficacy of 64Cu-ATSM in mice bearing U87MG glioblastoma tumors. Administration of single dosage (18.5, 37, 74, 111, and 148 MBq) and multiple dosages (37 MBq × 4) of 64Cu-ATSM was investigated. Single administration of 64Cu-ATSM in high-dose groups dose-dependently inhibited tumor growth and prolonged survival, with slight and reverse signs of adverse events. Multiple dosages of 64Cu-ATSM remarkably inhibited tumor growth and prolonged survival. By splitting the dose of 64Cu-ATSM, no adverse effects were observed. Our findings indicate that multiple administrations of 64Cu-ATSM have effective antitumor effects in glioblastoma without side effects, indicating its potential for treating this fatal disease.

Positron emission tomography of cerebral angiogenesis and TSPO expression in a mouse model of chronic hypoxia.

The present study aimed to examine whether positron emission tomography (PET) could evaluate cerebral angiogenesis. Mice were housed in a hypoxic chamber with 8–9% oxygen for 4, 7, and 14 days, and the angiogenic responses were evaluated with a radiotracer, 64Cu-cyclam-RAFT-c(-RGDfK-)4, which targeted αVβ3 integrin and was imaged with PET. The PET imaging results showed little uptake during all of the hypoxic periods. Immunofluorescence staining of the β3 integrin, CD61, revealed weak expression, while the microvessel density assessed by CD31 staining increased with the hypoxic duration. These observations suggest that the increased vascular density originated from other types of vascular remodeling, unlike angiogenic sprouting. We then searched for any signs of vascular remodeling that could be detected using PET. PET imaging of 11C-PK11195, a marker of the 18-kDa translocator protein (TSPO), revealed a transient increase at day 4 of hypoxia. Because the immunofluorescence of glial markers showed unchanged staining over the early phase of hypoxia, the observed upregulation of TSPO expression probably originated from non-glial cells (e.g. vascular cells). In conclusion, a transient increase in TSPO probe uptake was detected with PET at only the early phase of hypoxia, which indicates an early sign of vascular remodeling induced by hypoxia.

67Cu-Radiolabeling of a multimeric RGD peptide for αVβ3 integrin-targeted radionuclide therapy

Objective Copper-67 (67Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of 67Cu. Encouraged by these, we performed 67Cu-radiolabeling of a tetrameric cyclic Arg–Gly–Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets &agr;V&bgr;3 integrin (&agr;V&bgr;3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. Materials and methods 67Cu, produced through the 64Ni(&agr;, p)67Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous &agr;V&bgr;3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: 67Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. Results 67Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of 67Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in 67Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. Conclusion 67Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for &agr;V&bgr;3 integrin-targeted internal radiotherapy.

67: stability, therapeutic efficacy, and safety studies in micecu-radiolabeling of a multimeric Rgd peptide for α: stability, therapeutic efficacy, and safety studies in micev: stability, therapeutic efficacy, and safety studies in miceβ: stability, therapeutic efficacy, and safety studies in mice3: stability, therapeutic efficacy, and safety studies in mice integrin-targeted radionuclide therapy: stability, therapeutic efficacy, and safety studies in mice

Objective Copper-67 (67Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of 67Cu. Encouraged by these, we performed 67Cu-radiolabeling of a tetrameric cyclic Arg–Gly–Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets &agr;V&bgr;3 integrin (&agr;V&bgr;3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. Materials and methods 67Cu, produced through the 64Ni(&agr;, p)67Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous &agr;V&bgr;3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: 67Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. Results 67Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of 67Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in 67Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. Conclusion 67Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for &agr;V&bgr;3 integrin-targeted internal radiotherapy.