Pascale Perret

BMP9 and BMP10 mutations were recently identified in patients with pulmonary arterial hypertension, but their specific roles in the pathogenesis of the disease are still unclear. We aimed to study the roles of BMP9 and BMP10 in cardiovascular homeostasis and pulmonary hypertension using transgenic mouse models deficient in Bmp9 and/or Bmp10. Single- and double-knockout mice for Bmp9 (constitutive) and/or Bmp10 (tamoxifen inducible) were generated. Single-knock-out (KO) mice developed no obvious age-dependent phenotype when compared with their wild-type littermates. However, combined deficiency in Bmp9 and Bmp10 led to vascular defects resulting in a decrease in peripheral vascular resistance and blood pressure and the progressive development of high-output heart failure and pulmonary hemosiderosis. RNAseq analysis of the lungs of the double-KO mice revealed differential expression of genes involved in inflammation and vascular homeostasis. We next challenged these mice to chronic hypoxia. After 3 weeks of hypoxic exposure, Bmp10-cKO mice showed an enlarged heart. However, although genetic deletion of Bmp9 in the single- and double-KO mice attenuated the muscularization of pulmonary arterioles induced by chronic hypoxia, we observed no differences in Bmp10-cKO mice. Consistent with these results, endothelin-1 levels were significantly reduced in Bmp9 deficient mice but not Bmp10-cKO mice. Furthermore, the effects of BMP9 on vasoconstriction were inhibited by bosentan, an endothelin receptor antagonist, in a chick chorioallantoic membrane assay. Our data show redundant roles for BMP9 and BMP10 in cardiovascular homeostasis under normoxic conditions (only combined deletion of both Bmp9 and Bmp10 was associated with severe defects) but highlight specific roles under chronic hypoxic conditions. We obtained evidence that BMP9 contributes to chronic hypoxia-induced pulmonary vascular remodelling, whereas BMP10 plays a role in hypoxia-induced cardiac remodelling in mice.

NeoB is a radiotracer targeting the gastrin-releasing peptide receptor (GRPR), a G-protein–coupled receptor expressed in various cancers. The aim of the present study was to evaluate the biodistribution and efficacy of this new therapeutic agent in Gastrointestinal Stromal Tumors (GIST). Eighty-two SCID mice bearing GIST-882 tumors were employed. [¹⁷⁷Lu]Lu-NeoB biodistribution was evaluated up to seven days by organ sampling (200 pmol/0.8 MBq, i.v.). For efficacy evaluation, mice received either saline, 400 pmol or 800 pmol of [¹⁷⁷Lu]Lu-NeoB (37MBq, 1/w, 3 w, i.v.). SPECT/CT imaging was performed at 24 h, and tumor volume was determined up to 100 days. Elevated and specific [¹⁷⁷Lu]Lu-NeoB uptake was found in the GIST tumor, as demonstrated by in vivo competition (19.1 ± 3.9 %ID/g vs. 0.3 ± 0.1 %ID/g at 4h). [¹⁷⁷Lu]Lu-NeoB tumor retention (half-life of 40.2 h) resulted in elevated tumor-to-background ratios. Tumor volumes were significantly reduced in both treated groups (p < 0.01), even leading to complete tumor regression at the 400 pmol dose. [¹⁷⁷Lu]Lu-NeoB exhibited excellent pharmacokinetics with elevated and prolonged tumor uptake and low uptake in non-target organs such as pancreas. The potential of this new theragnostic agent in different indications, including GIST, is under evaluation in the FIH [¹⁷⁷Lu]Lu-NeoB clinical trial.

Recent progress in breast cancer research has led to the identification of Vascular Cell Adhesion Molecule-1 (VCAM-1) as a key actor of metastatic colonization. VCAM-1 promotes lung-metastases and is associated with clinical early recurrence and poor outcome in triple negative breast cancer (TNBC). Our objective was to perform the in vivo imaging of VCAM-1 in mice models of TNBC. The Cancer Genomic Atlas (TCGA) database was analyzed to evaluate the prognostic role of VCAM-1 in TNBC. MDA-MB-231 (VCAM-1+) and control HCC70 (VCAM-1-) TNBC cells were subcutaneously xenografted in mice and VCAM-1 expression was assessed in vivo by single-photon emission computed tomography (SPECT) imaging using ^99mTc-cAbVCAM1-5. Then, MDA-MB-231 cells were intravenously injected in mice and VCAM-1 expression in lung metastasis was assessed by SPECT imaging after 8 weeks. TCGA analysis showed that VCAM-1 is associated with a poor prognosis in TNBC patients. In subcutaneous tumor models, ^99mTc-cAbVCAM1-5 uptake was 2-fold higher in MDA-MB-231 than in HCC70 (p < 0.01), and 4-fold higher than that of the irrelevant control (p < 0.01). Moreover, ^99mTc-cAbVCAM1-5 uptake in MDA-MB-231 lung metastases was also higher than that of ^99mTc-Ctl (p < 0.05). ^99mTc-cAbVCAM1-5 is therefore a suitable tool to evaluate the role of VCAM-1 as a marker of tumor aggressiveness of TNBC.

Insulin resistance is a key feature of the metabolic syndrome and type 2 diabetes, in which noninvasive assessment is not currently allowed by any methodology. We previously validated an iodinated tracer of glucose transport (6DIG) and a new methodology for the in vivo quantification of cardiac insulin resistance in rodents. The aim of this study was to investigate the safety, biodistribution, and radiation dosimetry of this method using 123I-6DIG in 5 healthy and 6 diabetic volunteers. The collection of adverse effects (AEs) and medical supervision of vital parameters and biological variables allowed the safety evaluation. Biodistribution was studied by sequentially acquiring whole-body images at 1, 2, 4, 8, and 24 hours postinjection. The total number of disintegrations in each organ normalized to the injected activity was calculated as the area under the time-activity curves. Dosimetry calculations were performed using OLINDA/EXM. No major adverse events were observed. The average dose corresponding to the 2 injections of 123I-6DIG used in the protocol was 182.1 ± 7.5 MBq. A fast blood clearance of 123I-6DIG was observed. The main route of elimination was urinary, with greater than 50% of urine activity over 24 hours. No blood or urine metabolite was detected. 123I-6DIG accumulation mostly occurred in elimination organs such as kidneys and liver. Mean radiation dosimetry calculations indicated an effective whole-body absorbed dose of 3.35 ± 0.57 mSv for the whole procedure. 123I-6DIG was well tolerated in human with a dosimetry profile comparable to that of other commonly used iodinated tracers, thereby allowing further clinical development of the tracer.

Background: Mesothelin is a cell-surface glycoprotein restricted to mesothelial cells overexpressed in several cancers, including triple negative breast cancers (TNBC) not responding to trastuzumab and hormonal-based therapies. Mesothelin-targeting therapies are currently being developed. However, the identification of patients potentially eligible to such therapeutic strategy remains challenging. The objective of this study was to perform the radiolabeling and pre-clinical evaluation of ^99mTc-A1 and ^99mTc-C6, two anti-mesothelin single domain antibodies (sdAb)-derived imaging agents. Methods: A1 and C6 were radiolabeled with ^99mTc and evaluated in vitro on recombinant protein and cells, as well as in vivo in xenograft mice models of TNBC HCC70 (mesothelin-positive) and MDA-MB-231 (mesothelin-negative) cell lines. Results: Both ^99mTc-A1 and ^99mTc-C6 bound mesothelin with high affinity in vitro, with ^99mTc-A1 affinity being 2.4-fold higher than that of ^99mTc-C6 (KD= 43.9 ± 4.0 vs 107 ± 16 nM, P80 % at 2h) as well as ex vivo in human blood (>90 % at 6h). In vivo ^99mTc-A1 uptake in HCC70 tumors was 5-fold higher than in MDA-MB-231 tumors and 1.5-fold higher than that of ^99mTc-C6 (2.34 ± 0.36 vs 0.48 ± 0.18 and 1.56 ± 0.43 %ID/g respectively, P99mTc-A1 uptake in HCC70 tumors. Conclusion: Mesothelin-positive tumors were successfully identified by single-photon emission computed tomography (SPECT) using ^99mTc-A1 and ^99mTc-C6. Considering its superior characteristics, ^99mTc-A1 was selected as the most suitable tool for further clinical translation.

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E^−/− mice. Methods: Apolipoprotein E^−/− mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice (n = 15), we intravenously injected ³H-cholesteryl oleate–labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set (n = 20), we used the imaging agent ^99mTc-cAbVCAM1–5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set (n = 21), we compared VCAM-1 expression with ^99mTc-cAbVCAM1–5 uptake in various tissues. Results: Mice treated with ezetimibe showed a 173% higher LDL–cholesteryl ester plasma disappearance rate (P < 0.001 vs. control) after ³H-cholesteryl oleate–labeled LDL injection. At 96 h after injection, the hepatic fraction of ³H-tracer was 61% lower in mice treated with ezetimibe (P < 0.001). Meanwhile, LDL-derived ³H-cholesterol excretion in the feces was 107% higher (P < 0.001). The antiatherogenic effect of ezetimibe monitored by ^99mTc-cAbVCAM1–5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; P < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with ^99mTc-cAbVCAM1–5 uptake (r = 0.75; P < 0.05). Conclusion: Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy.

New strategies allowing the transfer of molecules, especially peptides, through the blood‐brain barriers are a major pharmacological challenge for the treatment of brain diseases. The present study aims at evaluating in vivo the cerebral bioavailability of carrier systems, based on small and functionalizable 2,5‐diketopiperazine (DKP) motifs. We studied 2 different cyclo(Lys‐Lys) DKP scaffolds alone and a cyclo(Lys‐Gly) DKP carrier bearing as peptide model, the tau protein hexapeptide VQIVYK sequence.The different carrier systems were synthesized and radiolabeled using one of the free domains. The stability, biodistribution, and ability to cross blood‐brain barrier were investigated in vivo in mice for ^99mTc‐DKP scaffolds, ^99mTc‐HVQIVYK peptide alone, and ^99mTc‐DKP‐VQIVYK. ¹²⁵I‐labelled bovine serum albumin was used as negative control for brain uptake.Both radiolabeled DKPs scaffolds and ^99mTc‐DKP‐VQIVYK showed a high stability, while peptide ^99mTc‐HVQIVYK alone was quickly degraded in vivo. The presence of ^99mTc‐DKPs scaffolds and ^99mTc‐DKP‐VQIVYK was observed in the ventricular and subarachnoid spaces and to a lower extent in the brain parenchyma up to 45 minutes post‐injection in mice.This work highlights the potentiality of DKP scaffolds as vectors to transport peptides into the brain by limiting proteolysis and favoring cerebral bioavailability.

Maurocalcine (MCa) is the first natural cell penetrating peptide to be discovered in animal venom. In addition to the fact that it represents a potent vector for the cell penetration of structurally diverse therapeutic compounds, MCa also displays several distinguishing features that make it a potential peptide of choice for clinical and biotechnological applications. The aim of the present study was to gain new information about the properties of MCa in vivo in order to delineate the future potential applications of this vector. For this purpose, two analogues of this peptide with (Tyr-MCa) and without (Lin-Tyr-MCa) disulfide bridges were synthesized, radiolabeled with ¹²⁵I, and their in vitro stabilities were first evaluated in mouse blood. The results indicated that ¹²⁵I-Tyr-MCa was stable in vitro and that the disulfide bridges conferred a competitive advantage for the stability of peptide. Following in vivo injection in mice, ¹²⁵I-Tyr-MCa targeted peripheral organs with interesting quantitative differences and the main route of peptide elimination was renal.