68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment

ACS Omega

Neurotensin receptor 2 (NTS) is a well-known mediator of central opioid-independent analgesia. Seminal studies have highlighted NTS overexpression in a variety of tumors including prostate cancer, pancreas adenocarcinoma, and breast cancer. Herein, we describe the first radiometalated neurotensin analogue targeting NTS. JMV 7488 (DOTA-(βAla)-Lys-Lys-Pro-(D)Trp-Ile-TMSAla-OH) was prepared using solid-phase peptide synthesis, then purified, radiolabeled with Ga and In, and investigated on HT-29 cells and MCF-7 cells, respectively, and on HT-29 xenografts. [Ga]Ga-JMV 7488 and [In]In-JMV 7488 were quite hydrophilic (logD = -3.1 ± 0.2 and -2.7 ± 0.2, respectively, < 0.0001). Saturation binding studies showed good affinity toward NTS ( = 38 ± 17 nM for [Ga]Ga-JMV 7488 on HT-29 and 36 ± 10 nM on MCF-7 cells; = 36 ± 4 nM for [In]In-JMV 7488 on HT-29 and 46 ± 1 nM on MCF-7 cells) and good selectivity (no NTS binding up to 500 nM). On cell-based evaluation, [Ga]Ga-JMV 7488 and [In]In-JMV 7488 showed high and fast NTS-mediated internalization of 24 ± 5 and 25 ± 11% at 1 h for [In]In-JMV 7488, respectively, along with low NTS-membrane binding (<8%). Efflux was as high as 66 ± 9% at 45 min for [Ga]Ga-JMV 7488 on HT-29 and increased for [In]In-JMV 7488 up to 73 ± 16% on HT-29 and 78 ± 9% on MCF-7 cells at 2 h. Maximum intracellular calcium mobilization of JMV 7488 was 91 ± 11% to that of levocabastine, a known NTS agonist on HT-29 cells demonstrating the agonist behavior of JMV 7488. In nude mice bearing HT-29 xenograft, [Ga]Ga-JMV 7488 showed a moderate but promising significant tumor uptake in biodistribution studies that competes well with other nonmetalated radiotracers targeting NTS. Significant uptake was also depicted in lungs. Interestingly, mice prostate also demonstrated [Ga]Ga-JMV 7488 uptake although the mechanism was not NTS-mediated

Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1β or TNF-α Release from Human Hepatic Stellate Cells

International audienceInflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver disease

ACS Med Chem Lett

Bivalent ligands, i.e., molecules having two ligands covalently connected by a linker, have been gathering attention since the first description of their pharmacological potential in the early 80s. However, their synthesis, particularly of labeled heterobivalent ligands, can still be cumbersome and time-consuming. We herein report a straightforward procedure for the modular synthesis of labeled heterobivalent ligands (HBLs) using dual reactive 3,6-dichloro-1,2,4,5-tetrazine as a starting material and suitable partners for sequential SAr and inverse electron-demand Diels-Alder (IEDDA) reactions. This assembly method conducted in a stepwise or in a sequential one-pot manner provides quick access to multiple HBLs. A conjugate combining ligands toward the prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPR) was radiolabeled, and its biological activity was assessed and (receptor binding affinity, biodistribution, imaging) as an illustration that the assembly methodology preserves the tumor targeting properties of the ligands.France Life Imagin

Influence of inflammasome pathway activation in macrophages on the matrix metalloproteinase expression of human hepatic stellate cells

International audienceInflammasomes are protein complexes that produce IL-1β in response to damage or pathogens. As such, inflammasomes are involved in several types of hepatic fibrosis. However, the mechanisms by which these complexes drive the liver's fibrogenic status remain unclear. We co-cultured differentiated macrophages (the THP-1 cell line or human monocyte-derived macrophages (MDMs)) with human hepatic fibroblasts (either the LX-2 cell line or primary human hepatic stellate cells (HSCs)). The inflammasome pathway was activated with lipopolysaccharide (LPS) and monosodium urate (MSU) crystals, and the HSCs' responses were analyzed. Our results show that co-culture of HSCs with THP-1 cells upregulated transcription of the genes coding for metalloproteinase (MMP)-3 and MMP-9. After inflammasome pathway activation, the HSCs' phenotype was the same in the presence of THP-1 cells or MDMs (i.e. upregulation of MMP-3, MMP-9, and the pro-inflammatory cytokine IL-1β). We found that two cytokines were involved in these changes IL-1β regulated MMP-3 and IL-1β mRNA expression, whereas TNF-α regulated MMP-9 mRNA expression. Experiments with primary cells revealed that a general inflammatory environment is responsible for the downregulation of pro-fibrotic markers. Our present results suggest that inflammasome pathway activation in macrophages leads to a pro-inflammatory environment for HSCs leading to MMP/TIMP imbalance and enhanced fibrolytic properties

Effects of IL-6 treatment on the fibrolysis balance, fibrotic response and inflammatory response in LX-2 cells.

<p>The cells were treated with at 3 ng/mL rhIL-6 for 30 minutes or 24 hours. <b>(A)</b> mRNA expression of metalloproteinases (MMP-1, MMP-3, MMP-2, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. <b>(B)</b> mRNA expression of fibrogenesis factors (COL1A1/collagen I α1, COL4A1/collagen IV α1), myofibroblast differentiation factors (ACTA2/α-smooth muscle actin) and myofibroblast activation factors (EDN1/endothelin-1, PDGFB/platelet-derived growth factor-BB), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. <b>(C)</b> mRNA expression of proinflammatory cytokines (IL1B/interleukin-1β, IL6/interleukin-6) and chemokines (CXCL8/interleukin-8, CXCL1/GROα, CCL2/MCP1), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. <b>(D)</b> IL6R protein expression was determined by western blot on HepaRG and LX-2 cells lysates after 24 hours treatment and relative quantification was evaluated using densitometry. <b>(E)</b> p-STAT3 protein expression was determined by western blot on HepaRG (positive control) and LX-2 cells lysates after 24 hours treatment and relative quantification was evaluated using densitometry. Results are expressed as the mean ± SEM of three independent experiments.</p

Effects of CXCL8/IL-8 treatment on the fibrolysis balance, fibrotic response and inflammatory response in LX-2 cells.

<p>The cells were treated with 3 ng/mL rhIL-8 for 24 hours or 48 hours. <b>(A)</b> mRNA expression of metalloproteinases (MMP-1, MMP-3, MMP-2, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. <b>(B)</b> mRNA expression of fibrogenesis factors (COL1A1/collagen I α1, COL4A1/collagen IV α1), myofibroblast differentiation factors (ACTA2/α-smooth muscle actin) and myofibroblast activation factors (EDN1/endothelin-1, PDGFB/platelet-derived growth factor-BB), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. <b>(C)</b> mRNA expression of proinflammatory cytokines (IL1B/interleukin-1β, IL6/interleukin-6) and chemokines (CXCL8/interleukin-8, CXCL1/GROα, CCL2/MCP1), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. Results are expressed as the mean ± SEM of three independent experiments. * p<0.05, ** p<0.01, relative to a control.</p

Effects of TGF-β1 treatment on the fibrolysis balance, fibrotic response and inflammatory response in LX-2 cells.

<p>The cells were treated with 3 ng/mL rhTGF-β1 for 24 hours or 48 hours. <b>(A)</b> mRNA expression levels of metalloproteinases (MMP-1, MMP-3, MMP-2, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1) were measured and normalized against that of GAPDH (using real-time PCR) after 24 hours of treatment. <b>(B)</b> mRNA expression of fibrogenesis factors (COL1A1/collagen I α1, COL4A1/collagen IV α1), myofibroblast differentiation factors (ACTA2/α-smooth muscle actin) and myofibroblast activation factors (EDN1/endothelin-1, PDGFB/platelet-derived growth factor-BB), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. <b>(C)</b> mRNA expression of proinflammatory cytokines (IL1B/interleukin-1β, TNFA/tumor necrosis factor-α, IL6/interleukin-6) and chemokines (CXCL8/interleukin-8, CXCL1/GROα, CCL2/MCP1), as measured with real-time PCR and normalized against that of GAPDH after 24 hours of treatment. <b>(D)</b> IL-1β, TNF-α, IL-6 and IL-8 secretions into the supernatant by LX-2 cells were detected with an ELISA after 24 hours of treatment. <b>(E)</b> Pro-collagen I α1 secretion into the supernatant by LX-2 cells was detected with an ELISA after 48 hours of treatment. <b>(F)</b> Collagen type I α1 protein expression was determined by western blot of LX-2 cell lysates after 24 hours treatment and relative quantification was evaluated using densitometry. <b>(G)</b> Gelatinase activities of MMP-9 and MMP-2 released by LX-2 cells into the supernatant after 48 hours of treatment were evaluated by zymography and normalized densitometry. Results are expressed as the mean ± SEM of three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, relative to a control.</p

68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

International audienceBackground: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients.Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature.Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min.Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment

IL-1β production is dependent of the activation of purinergic receptors and NLRP3 pathway in human macrophages

International audienceThe Nod-like receptor family protein 3 (NLRP3)-inflammasome pathway is known to be activated by danger signals such as monosodium urate (MSU). We investigated the role of P2 purinergic receptors in the activation of NLRP3-inflammasome pathway after MSU treatment of primary human monocyte-derived macrophages (MDMs). After initial stimulation with a low concentration of LPS (0.1 µg/ml), a 6 h treatment with MSU crystals (250, 500, and 1000 µg/ml) induced the MDMs to release IL-1β, IL-1α, and IL-6 in a dose-dependent manner. Moreover, the caspase 1 inhibitor Z-YVAD-FMK and the cathepsin B inhibitor CA-074Me reduced production of IL-1β in a dose-dependent manner after LPS + MSU treatment. We used real-time reverse transcription-quantitative PCR to show that treatment with LPS and MSU (500 µg/ml) induced significantly greater expression of NLRP3 and IL-1β than after treatment with LPS. We also found that MSU treatment induced P2X purinergic receptor 7 (P2X7R) mRNA and protein expression. Furthermore, addition of the P2X7 purinergic receptor antagonist A-740003 significantly impeded IL-1β production and pro-IL-1β cleavage after treatment with LPS + MSU. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited the release of IL-1β and other M1 macrophage cytokines (such as IL-1α, IL-6, and TNF-α) from MDMs stimulated with LPS + MSU. Taken as a whole, our results show that P2 purinergic receptors and the NLRP3 inflammasome pathway are involved in the secretion of IL-1β from MSU-stimulated human macrophages. This pathway may constitute a novel therapeutic target for controlling the inflammatory process in several associated pathologies.-Gicquel, T., Robert, S., Loyer, P., Victoni, T., Bodin, A., Ribault, C., Gleonnec, F., Couillin, I., Boichot, E., Lagente, V. IL-1β production is dependent of the activation of purinergic receptors and NLRP3 pathway in human macrophage