Role of NLRP3 Inflammasomes in Monocyte and Microglial Recruitments in Choroidal Neovascularization.

Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1ß protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1ß mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1ß.

Method to Regulate Monocyte Function by Silencing HIF-1α mRNA in a Model of Retinal Neovascularization.

Circulating monocytes migrate into the retina in response to inflammation and neovascularization. Furthermore, under inflammatory conditions such as diabetes, healthy monocytes become activated in the circulation. However, the contribution of activated monocytes to neovascularization is largely unknown. HIF-1α has been shown to contribute to the pathogenesis of neovascularization. We describe here the synthesis of a hybrid nanomaterial for targeted delivery and gene silencing in activated monocytes that are associated with pathological neovascularization. To test the gene silencing ability of AS-shRNA-lipids in vitro, we used the probe to inhibit HIF-1α mRNA induced in mouse monocytes by exposing them to hypoxia. In addition, we tested AS-shRNA-lipids for inhibition of neovascularization in vivo using the mouse model of oxygen-induced retinopathy (OIR). Significant reduction of neovascularization was achieved in mouse OIR by targeting activated monocytes using intraperitoneal injections of AS-shRNA-lipids. Expression of HIF-1α and CD14 mRNA were both inhibited in circulating cells, suggesting normalization of the activated monocytes in P17 OIR animals treated with AS-shRNA-lipids. We hypothesized that inhibition of HIF-1α mRNA in activated monocytes may have a direct impact on VEGF expression in the retinal tissues in vivo. We observed that VEGF mRNA expression was inhibited in P17 retinal tissues after systemic treatment with HIF-1α-targeted AS-shRNA-lipids. These findings may provide a framework for a strategy to inhibit retinal neovascularization by targeting circulating activated monocytes.

A novel optical imaging probe for targeted visualization of NLRP3 inflammasomes in a mouse model of age-related macular degeneration.

Purpose: Wet form of age-related macular degeneration (wet AMD) is a progressive vascular disease that mainly affects older adults and causes severe and irreversible vision loss. A key complication of wet AMD is choroidal neovascularization (CNV), which may be driven in part by NLRP3 inflammasomes that are associated with macrophages migration to CNV lesions. Since activated NLRP3 is correlated with CNV, visualizing NLRP3 inflammasomes and their associated macrophages is of great interest to monitor wet AMD progression and develop effective therapies against it. However, to the best of our knowledge, current ophthalmic imaging systems do not permit such targeted imaging. Therefore, in this study, we developed InflammaProbe-1, an optical imaging probe for targeted visualization of NLRP3 inflammasomes in CNV lesions. Methods: InflammaProbe-1 was synthesized by conjugating a clinically relevant fluorophore, Oregon Green® 488, to the selective NLRP3 inhibitor, CY-09. The ability of InflammaProbe-1 to target NLRP3 was assessed with an enzyme-linked immunosorbent assay by comparing its ability to inhibit NLRP3-mediated secretion of IL-1ß to that of CY-09 in LPS-primed and nigericin-stimulated BMDMs. In vitro confocal imaging of NLRP3 was performed on InflammaProbe-1-stained BMDMs that had been induced to express NLRP3 with LPS. In vivo imaging of NLRP3 was conducted on mouse laser induced choroidal neovascularization (LCNV), a model of AMD, 6 h after an intraperitoneal injection of InflammaProbe-1 at 10 mg/kg on day 4 post-LCNV. Results: InflammaProbe-1 was just as effective as CY-09 at inhibiting IL-1ß secretion (p < 0.01 at 10 µM for both the InflammaProbe-1 and CY-09 groups relative to the control). InflammaProbe-1-stained BMDMs that had been induced to express NLRP3 showed significantly brighter fluorescence than untreated cells (p < 0.0001 for LPS treatment group and p < 0.001 for LPS and nigericin treatment group). Furthermore, in vivo molecular imaging of NLRP3 was achieved in mouse LCNV. Conclusion: We propose that InflammaProbe-1 may be a useful molecular imaging probe to monitor the onset, progression, and therapeutic response of AMD and other NLRP3-mediated diseases.

A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization.

Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA-lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA-lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA-lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.

Visualizing HIF-1α mRNA in a Subpopulation of Bone Marrow-Derived Cells to Predict Retinal Neovascularization.

Bone marrow-derived progenitor cells and macrophages are known to migrate into the retina in response to inflammation and neovascularization. These migratory cells might play important regulatory roles in the pathogenesis of neovascularization, a common complication observed in diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion. Hypoxia-inducible factor 1α (HIF-1α) has been shown to contribute to the pathogenesis of retinal inflammation and neovascularization. However, contributions of monocyte-derived macrophages to neovascularization are largely unknown. We hypothesized that selective visualization of these microglia/macrophages could be a powerful method for predicting the onset of neovascularization and its progression at the molecular level. In this report, we describe the synthesis of a new hybrid nanoparticle to visualize HIF-1α mRNA selectively in microglia/macrophages in a mouse model of neovascularization. HIF-1α expression was confirmed in MRC-1 positive monocytes/macrophages as well as in CD4 positive T-cells and CD19 positive B-cells using single-cell RNA sequencing data analysis. The imaging probes (AS- or NS-shRNA-lipid) were synthesized by conjugating diacyl-lipids to short hairpin RNA with an antisense sequence complementary to HIF-1α mRNA and a fluorophore that is quenched by a black hole quencher. We believe that imaging mRNA selectively in tissue specific microglia/macrophages could be a powerful method for predicting the onset of neovascularization, its progression, and its response to therapy.

Discovery of Furanone-Based Radiopharmaceuticals for Diagnostic Targeting of COX-1 in Ovarian Cancer.

In vivo targeting and visualization of cyclooxygenase-1 (COX-1) using multimodal positron emission tomography/computed tomography imaging represents a unique opportunity for early detection and/or therapeutic evaluation of ovarian cancer because overexpression of COX-1 has been characterized as a pathologic hallmark of the initiation and progression of this disease. The furanone core is a common building block of many synthetic and natural products that exhibit a wide range of biological activities. We hypothesize that furanone-based COX-1 inhibitors can be designed as imaging agents for the early detection, delineation of tumor margin, and evaluation of treatment response of ovarian cancer. We report the discovery of 3-(4-fluorophenyl)-5,5-dimethyl-4-(p-tolyl)furan-2(5H)-one (FDF), a furanone-based novel COX-1-selective inhibitor that exhibits adequate in vivo stability, plasma half-life, and pharmacokinetic properties for use as an imaging agent. We describe a novel synthetic scheme in which a Lewis acid-catalyzed nucleophilic aromatic deiodo[18F]fluorination reaction is utilized for the radiosynthesis of [18F]FDF. [18F]FDF binds efficiently to COX-1 in vivo and enables sensitive detection of ovarian cancer in subcutaneous and peritoneal xenograft models in mice. These results provide the proof of principle for COX-1-targeted imaging of ovarian cancer and identify [18F]FDF as a promising lead compound for further preclinical and clinical development.

Targeted Imaging of VCAM-1 mRNA in a Mouse Model of Laser-Induced Choroidal Neovascularization Using Antisense Hairpin-DNA-Functionalized Gold-Nanoparticles.

Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.

Real-time imaging of VCAM-1 mRNA in TNF-α activated retinal microvascular endothelial cells using antisense hairpin-DNA functionalized gold nanoparticles.

Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.

In Vivo Imaging of Retinal Hypoxia Using HYPOX-4-Dependent Fluorescence in a Mouse Model of Laser-Induced Retinal Vein Occlusion (RVO).

Purpose: To demonstrate the utility of a novel in vivo molecular imaging probe, HYPOX-4, to detect and image retinal hypoxia in real time, in a mouse model of retinal vein occlusion (RVO). Methods: Retinal vein occlusion was achieved in adult mice by photodynamic retinal vein thrombosis (PRVT). One or two major retinal vein(s) was/were occluded in close proximity to the optic nerve head (ONH). In vivo imaging of retinal hypoxia was performed using, HYPOX-4, an imaging probe developed by our laboratory. Pimonidazole-adduct immunostaining was performed and used as a standard ex vivo method for the detection of retinal hypoxia in this mouse RVO model. The retinal vasculature was imaged using fluorescein angiography (FA) and isolectin B4 staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) analysis. Results: By application of the standard ex vivo pimonidazole-adduct immunostaining technique, retinal hypoxia was observed within 2 hours post-PRVT. The observed hypoxic retinal areas depended on whether one or two retinal vein(s) was/were occluded. Similar areas of hypoxia were imaged in vivo using HYPOX-4. Using OCT, retinal edema was observed immediately post-PRVT induction, resolving 8 days later. Nominal preretinal neovascularization was observed at 10 to 14 days post-RVO. Conclusions: HYPOX-4 is an efficient probe capable of imaging retinal hypoxia in vivo, in RVO mice. Future studies will focus on its use in correlating retinal hypoxia to the onset and progression of ischemic vasculopathies.

In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy.

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

Nanoengineering of therapeutics for retinal vascular disease.

Retinal vascular diseases, including diabetic retinopathy, neovascular age related macular degeneration, and retinal vein occlusion, are leading causes of blindness in the Western world. These diseases share several common disease mechanisms, including vascular endothelial growth factor (VEGF) signaling, hypoxia, and inflammation, which provide opportunities for common therapeutic strategies. Treatment of these diseases using laser therapy, anti-VEGF injections, and/or steroids has significantly improved clinical outcomes. However, these strategies do not address the underlying root causes of pathology, and may have deleterious side effects. Furthermore, many patients continue to progress toward legal blindness despite receiving regular therapy. Nanomedicine, the engineering of therapeutics at the 1-100 nm scale, is a promising approach for improving clinical management of retinal vascular diseases. Nanomedicine-based technologies have the potential to revolutionize the treatment of ophthalmology, through enabling sustained release of drugs over several months, reducing side effects due to specific targeting of dysfunctional cells, and interfacing with currently "undruggable" targets. We will discuss emerging nanomedicine-based applications for the treatment of complications associated with retinal vascular diseases, including angiogenesis and inflammation.

Applications of azo-based probes for imaging retinal hypoxia.

We report the design and synthesis of an activatable molecular imaging probe to detect hypoxia in mouse models of retinal vascular diseases. Hypoxia of the retina has been associated with the initiation and progression of blinding retinal vascular diseases including age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. In vivo retinal imaging of hypoxia may be useful for early detection and timely treatment of retinal diseases. To achieve this goal, we synthesized HYPOX-3, a near-infrared (NIR) imaging agent coupled to a dark quencher, Black Hole Quencher 3 (BHQ3), which has been previously reported to contain a hypoxia-sensitive cleavable azo-bond. HYPOX-3 was cleaved in hypoxic retinal cell culture and animal models, enabling detection of hypoxia with high signal-to-noise ratios without acute toxicity. HYPOX-3 fluorescences in hypoxic cells and tissues and was undetectable under normoxia. These imaging agents are promising candidates for imaging retinal hypoxia in preclinical disease models and patients.

Molecular probes for imaging of hypoxia in the retina.

Hypoxia has been associated with retinal diseases which lead the causes of irreversible vision loss, including diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. Therefore, technologies for imaging hypoxia in the retina are needed for early disease detection, monitoring of disease progression, and assessment of therapeutic responses in the patient. Toward this goal, we developed two hypoxia-sensitive imaging agents based on nitroimidazoles which are capable of accumulating in hypoxic cells in vivo. 2-nitroimidazole or Pimonidazole was conjugated to fluorescent dyes to yield the imaging agents HYPOX-1 and HYPOX-2. Imaging agents were characterized in cell culture and animal models of retinal vascular diseases which exhibit hypoxia. Both HYPOX-1 and -2 were capable of detecting hypoxia in cell culture models with >10:1 signal-to-noise ratios without acute toxicity. Furthermore, intraocular administration of contrast agents in mouse models of retinal hypoxia enabled ex vivo detection of hypoxic tissue. These imaging agents are a promising step toward translation of hypoxia-sensitive molecular imaging agents in preclinical animal models and patients.

Synthesis and antimalarial activity of prodigiosenes.

Several analogues of the natural compound prodigiosin with modified A- and C-rings were synthesised as were some of their tin, cobalt, boron and zinc complexes. The antimalarial activity of these prodigiosenes was evaluated in vitro using the 3D7 Plasmodium falciparum strain. The presence of a nitrogen atom in the A-ring is needed for antimalarial activity but the presence of an alkyl group at the ß'-position of the C-ring seems detrimental. Dibutyl tin complexes exhibit IC50 values mostly in the nanomolar range with equal or improved activity compared to the free-base prodigiosene ligand, despite the fact that the general toxicity of such tin complexes is demonstrably lower than that of the free-bases.

Microwave-assisted, one-pot reaction of 7-azaindoles and aldehydes: a facile route to novel di-7-azaindolylmethanes.

A novel and highly efficient synthetic method leveraging microwave-assisted organic synthesis (MAOS) to yield di-7-azaindolylmethanes (DAIMs) is reported. Under MAOS conditions, reaction of 7-azaindole with aldehydes resulted predominantly in DAIMs, as opposed to the expected 7-azaindole addition products that form at ambient temperature. Based upon studies of different indoles and azaindoles with various aromatic and aliphatic aldehydes, we herein propose a mechanism where rapid and efficient microwave heating promotes nucleophilicity of 7-azaindoles towards the corresponding alkylidene-azaindolene intermediate to form the DAIM. This sequence provides a versatile approach to efficiently synthesize novel DAIMs that may be useful pharmaceuticals.

Synthesis and biological evaluation of prodigiosene conjugates of porphyrin, estrone and 4-hydroxytamoxifen.

To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene-tamoxifen conjugates.

Synthesis and structure-activity relationships of 5,6,7-substituted pyrazolopyrimidines: discovery of a novel TSPO PET ligand for cancer imaging.

Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 ((18)F) facilitated production of 2-(5,7-diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ((18)F-6b) in high radiochemical yield and specific activity. In vivo studies of (18)F-6b were performed which illuminated this agent as an improved probe for molecular imaging of TSPO-expressing cancers.

3'-Deoxy-3'-18F-fluorothymidine PET predicts response to (V600E)BRAF-targeted therapy in preclinical models of colorectal cancer.

UNLABELLED: Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of (V600E)BRAF inhibition in melanoma, coupled with the emergence of acquired resistance, underscores the importance of rigorously validating quantitative biomarkers of treatment response in this and similar settings. Because constitutive activation of BRAF leads to proliferation in tumors, we explored 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET to noninvasively quantify changes in tumor proliferation that are associated with pharmacologic inhibition of (V600E)BRAF downstream effectors and that precede changes in tumor volume. METHODS: Human colorectal cancer (CRC) cell lines expressing (V600E)BRAF were used to explore relationships between upregulation of p27 and phosphorylation of BRAF downstream effectors on small-molecule (V600E)BRAF inhibitor exposure. Athymic nude mice bearing (V600E)BRAF-expressing human CRC cell line xenografts were treated with a small-molecule (V600E)BRAF inhibitor (or vehicle) daily for 10 d. Predictive (18)F-FLT PET was conducted before changes in tumor volume occurred. Correlations were evaluated among PET, inhibition of phosphorylated MEK (p-MEK) and phosphorylated-ERK (p-ERK) by Western blot, tumor proliferation by histology, and small-molecule exposure by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). RESULTS: Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and upregulation of p27. In vivo, PLX4720 treatment reduced (18)F-FLT uptake, but not (18)F-FDG uptake, in Lim2405 xenografts before quantifiable differences in xenograft volume. Reduced (18)F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both (18)F-FLT PET and (18)F-FDG PET accurately reflected a lack of response in HT-29 xenografts, which MALDI imaging mass spectrometry suggested may have stemmed from limited PLX4720 exposure. CONCLUSION: We used preclinical models of CRC to demonstrate (18)F-FLT PET as a sensitive predictor of response to (V600E)BRAF inhibitors. Because (18)F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet (18)F-FDG PET did not, these data suggest that (18)F-FLT PET may represent an alternative to (18)F-FDG PET for quantifying clinical responses to BRAF inhibitors.

Rapid, Microwave-Assisted Organic Synthesis of Selective (V600E)BRAF Inhibitors for Preclinical Cancer Research.

We report a dramatically improved total synthesis of two highly selective (V600E)BRAF inhibitors, PLX4720 and PLX4032, that leverages microwave-assisted organic synthesis (MAOS). Compared with previously reported approaches, our novel MAOS method significantly reduces overall reaction time without compromising yield. In addition to providing a gram-scale route to these compounds for preclinical oncology research, we anticipate this approach could accelerate the synthesis of azaindoles in high-throughput, library-based formats.