A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor.

The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure-activity relationship (SAR) studies led to the identification of compound 12b with a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.

Discovery and Preclinical Characterization of 5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-(difluoromethyl)pyridin-2-amine (PQR620), a Highly Potent and Selective mTORC1/2 Inhibitor for Cancer and Neurological Disorders.

Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague-Dawley mice, maximum concentration ( Cmax) in plasma and brain was reached after 30 min, with a half-life ( t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.

Evaluation of spectral photon counting computed tomography K-edge imaging for determination of gold nanoparticle biodistribution in vivo.

Spectral photon counting computed tomography (SPCCT) is an emerging medical imaging technology. SPCCT scanners record the energy of incident photons, which allows specific detection of contrast agents due to measurement of their characteristic X-ray attenuation profiles. This approach is known as K-edge imaging. Nanoparticles formed from elements such as gold, bismuth or ytterbium have been reported as potential contrast agents for SPCCT imaging. Furthermore, gold nanoparticles have many applications in medicine, such as adjuvants for radiotherapy and photothermal ablation. In particular, longitudinal imaging of the biodistribution of nanoparticles would be highly attractive for their clinical translation. We therefore studied the capabilities of a novel SPCCT scanner to quantify the biodistribution of gold nanoparticles in vivo. PEGylated gold nanoparticles were used. Phantom imaging showed that concentrations measured on gold images correlated well with known concentrations (slope = 0.94, intercept = 0.18, RMSE = 0.18, R2 = 0.99). The SPCCT system allowed repetitive and quick acquisitions in vivo, and follow-up of changes in the AuNP biodistribution over time. Measurements performed on gold images correlated with the inductively coupled plasma-optical emission spectrometry (ICP-OES) measurements in the organs of interest (slope = 0.77, intercept = 0.47, RMSE = 0.72, R2 = 0.93). TEM results were in agreement with the imaging and ICP-OES in that much higher concentrations of AuNPs were observed in the liver, spleen, bone marrow and lymph nodes (mainly in macrophages). In conclusion, we found that SPCCT can be used for repetitive and non-invasive determination of the biodistribution of gold nanoparticles in vivo.

Telomerase inhibition improves tumor response to radiotherapy in a murine orthotopic model of human glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most frequent and aggressive type of adult brain tumor. Most GBMs express telomerase; a high level of intra-tumoral telomerase activity (TA) is predictive of poor prognosis. Thus, telomerase inhibitors are promising options to treat GBM. These inhibitors increase the response to radiotherapy (RT), in vitro as well as in vivo. Since typical treatments for GBM include RT, our objective was to evaluate the efficiency of Imetelstat (TA inhibitor) combined with RT. FINDINGS: We used a murine orthotopic model of human GBM (N = 8 to11 mice per group) and µMRI imaging to evaluate the efficacy of Imetelstat (delivered by intra-peritoneal injection) alone and combined with RT. Using a clinically established protocol, we demonstrated that Imetelstat significantly: (i) inhibited the TA in the very center of the tumor, (ii) reduced tumor volume as a proportion of TA inhibition, and (iii) increased the response to RT, in terms of tumor volume regression and survival increase. CONCLUSIONS: Imetelstat is currently evaluated in refractory brain tumors in young patients (without RT). Our results support its clinical evaluation combined with RT to treat GBM.

Tif1γ suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway.

Transcriptional intermediary factor 1γ (TIF1γ; alias, TRIM33/RFG7/PTC7/ectodermin) belongs to an evolutionarily conserved family of nuclear factors that have been implicated in stem cell pluripotency, embryonic development, and tumor suppression. TIF1γ expression is markedly down-regulated in human pancreatic tumors, and Pdx1-driven Tif1γ inactivation cooperates with the Kras(G12D) oncogene in the mouse pancreas to induce intraductal papillary mucinous neoplasms. In this study, we report that aged Pdx1-Cre; LSL-Kras(G12D); Tif1γ(lox/lox) mice develop pancreatic ductal adenocarcinomas (PDACs), an aggressive and always fatal neoplasm, demonstrating a Tif1γ tumor-suppressive function in the development of pancreatic carcinogenesis. Deletion of SMAD4/DPC4 (deleted in pancreatic carcinoma locus 4) occurs in approximately 50% of human cases of PDAC. We, therefore, assessed the genetic relationship between Tif1γ and Smad4 signaling in pancreatic tumors and found that Pdx1-Cre; LSL-Kras(G12D); Smad4(lox/lox); Tif1γ(lox/lox) (alias, KSSTT) mutant mice exhibit accelerated tumor progression. Consequently, Tif1γ tumor-suppressor effects during progression from a premalignant to a malignant state in our mouse model of pancreatic cancer are independent of Smad4. These findings establish, for the first time to our knowledge, that Tif1γ and Smad4 both regulate an intraductal papillary mucinous neoplasm-to-PDAC sequence through distinct tumor-suppressor programs.

Does acute behavioral testing reflect successful ischemia in rats with transient middle cerebral artery occlusion?

BACKGROUND: Models of intraluminal middle cerebral artery occlusion present an intrinsic variability in infarct size. Behavioral evaluation is frequently performed during arterial occlusion to confirm success of surgery. AIMS AND/OR HYPOTHESIS: We compared the value of behavioral testing and multimodal magnetic resonance imaging performed during arterial occlusion for identifying successfully operated animals. METHODS: Rats were tested with behavioral assessment (using three scoring scales and the adhesive removal test) and multimodal magnetic resonance imaging (including magnetic resonance angiography, diffusion-weighted and perfusion-weighted imaging), both performed during the two-hours of middle cerebral artery occlusion using the intraluminal suture model. Behavioral assessment was repeated 24 h after reperfusion, followed by sacrifice. RESULTS: Acute apparent diffusion coefficient lesion volume was correlated with both 2,3,5-triphenyl tetrazolium chloride infarct size (r = 0·75, P = 0·02) and behavioral status (r = 0·66, P = 0·05) on day one. Conversely, no correlation was found between acute behavioral examination and day one outcomes (2,3,5-triphenyl tetrazolium chloride infarct volume, r = 0·40, P = 0·28; behavioral examination, r = 0·39, P = 0·30). Day zero apparent diffusion coefficient volumes (P = 0·04), but not behavioral assessment (P = 0·60), discriminated animals with day one corticostriatal infarcts from these with subcortical infarcts. CONCLUSIONS: Acute behavioral testing performed during arterial occlusion fails to identify successfully operated animals. Acute diffusion magnetic resonance imaging may be more appropriate to assess and reduce infarct size variability in this model.

Synthesis and luminescence properties of new red-shifted absorption lanthanide(III) chelates suitable for peptide and protein labelling.

The synthesis and photo-physical properties of an original bis-pyridinylpyrazine chromophore efficiently sensitising europium(III) and samarium(III) are described. The corresponding lanthanide(III) complexes display in aqueous solutions a maximum excitation wavelength which is significantly red-shifted compared to the usual terpyridine-based chelates, and a valuable luminescence brightness above 2,000 dm(3) mol(-1) cm(-1) at 345 nm was obtained with a europium(III) derivative. Further functionalisation with three different bioconjugatable handles was also investigated and their ability to efficiently label a model hexapeptide was evaluated and compared. Finally, the best bioconjugatable europium(III) chelate was used in representative labelling experiments involving monoclonal antibodies and the luminescence features of the corresponding bioconjugates remained satisfactory.

MRI monitoring of neuroinflammation in mouse focal ischemia.

BACKGROUND AND PURPOSE: A growing body of evidence suggests that inflammatory processes are involved in the pathophysiology of stroke. Phagocyte cells, involving resident microglia and infiltrating macrophages, secrete both protective and toxic molecules and thus represent a potential therapeutic target. The aim of the present study was to monitor phagocytic activity after focal cerebral ischemia in mice. METHODS: Ultrasmall superparamagnetic particles of iron oxide (USPIO) were intravenously injected after permanent middle cerebral artery occlusion and monitored by high resolution MRI for 72 hours. RESULTS: We here present the first MRI data showing in vivo phagocyte-labeling obtained in mice with focal cerebral ischemia. USPIO-enhanced MRI kinetic analysis disclosed an inflammatory response surrounding the ischemic lesion and in the contralateral hemisphere via the corpus callosum. The imaging data collected during the first 36 hours postinjury suggested a spread of USPIO-related signal from ipsi- to contralateral hemisphere. Imaging data correlated with histochemical analysis showing inflammation remote from the lesion and ingestion of nanoparticles by microglia/macrophages. CONCLUSIONS: The present study shows that MR-tracking of phagocyte cells is feasible in mice, which may have critical therapeutic implications given the potential neurotoxicity of activated microglia/macrophages in central nervous system disorders.