Milind Rajopadhye

Hybrid In Vivo FMT-CT Imaging of Protease Activity in Atherosclerosis With Customized Nanosensors

Objective—Proteases are emerging biomarkers of inflammatory diseases. In atherosclerosis, these enzymes are often secreted by inflammatory macrophages, digest the extracellular matrix of the fibrous cap, and destabilize atheromata. Protease function can be monitored with protease activatable imaging probes and quantitated in vivo by fluorescence molecular tomography (FMT). To address 2 major constraints currently associated with imaging of murine atherosclerosis (lack of highly sensitive probes and absence of anatomic information), we compared protease sensors (PS) of variable size and pharmacokinetics and coregistered FMT datasets with computed tomography (FMT-CT). Methods and Results—Coregistration of FMT and CT was achieved with a multimodal imaging cartridge containing fiducial markers detectable by both modalities. A high-resolution CT angiography protocol accurately localized fluorescence to the aortic root of atherosclerotic apoE−/− mice. To identify suitable sensors, we first modeled signal kinetics in-silico and then compared 3 probes with oligo-l-lysine cleavage sequences: PS-5, 5 nm in diameter containing 2 fluorochromes, PS-25, a 25-nm version with an elongated lysine chain and PS-40, a polymeric nanoparticle. Serial FMT-CT showed fastest kinetics for PS-5 but, surprisingly, highest fluorescence in lesions of the aortic root for PS-40. PS-40 robustly reported therapeutic effects of atorvastatin, corroborated by ex vivo imaging and qPCR for the model protease cathepsin B. Conclusions—FMT-CT is a robust and observer-independent tool for noninvasive assessment of inflammatory murine atherosclerosis. Reporter-containing nanomaterials may have unique advantages over small molecule agents for in vivo imaging.

Dual In Vivo Quantification of Integrin-targeted and Protease-activated Agents in Cancer Using Fluorescence Molecular Tomography (FMT)

PurposeIntegrins, especially αvβ3 and αvβ5, are upregulated in tumor cells and activated endothelial cells and as such, serve as cancer biomarkers. We developed a novel near-infrared-labeled optical agent for the in vivo detection and quantification of αvβ3/αvβ5.ProceduresA small peptidomimetic αvβ3 antagonist was synthesized, coupled to a near-infrared fluorescent (NIRF) dye, and tested for binding specificity using integrin-overexpressing cells, inhibition of vitronectin-mediated cell attachment, binding to tumor and endothelial cells in vitro, and competition studies. Pharmacokinetics, biodistribution, specificity of tumor targeting, and the effect of an antiangiogenic treatment were assessed in vivo.ResultsThe integrin NIRF agent showed strong selectivity towards αvβ3/αvβ5in vitro and predominant tumor distribution in vivo, allowing noninvasive and real-time quantification of integrin signal in tumors. Antiangiogenic treatment significantly inhibited integrin signal in vivo but had no effect on a cathepsin-cleavable NIR agent. Simultaneous imaging revealed different patterns of distribution reflecting the underlying differences in integrin and cathepsin biology during tumor progression.ConclusionsNIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors.

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.

Noninvasive In Vivo Quantification of Neutrophil Elastase Activity in Acute Experimental Mouse Lung Injury

We developed a neutrophil elastase-specific near-infrared fluorescence imaging agent, which, combined with fluorescence molecular tomographic imaging, allowed us to detect and quantify neutrophil elastase activity in vivo, in real time, and noninvasively in an acute model of lung injury (ALI). Significantly higher fluorescent signal was quantified in mice with LPS/fMLP-induced ALI as compared to healthy controls, correlating with increases in the number of bronchoalveolar lavage cells, neutrophils, and elastase activity. The agent was significantly activated ex vivo in lung sections from ALI but not from control mice, and this activation was ablated by the specific inhibitor sivelestat. Treatment with the specific inhibitor sivelestat significantly reduced lung signal in mice with ALI. These results underscore the unique ability of fluorescence molecular imaging to quantify specific molecular processes in vivo, crucial for understanding the mechanisms underlying disease progression and for assessing and monitoring novel pharmacological interventions.

Quantitative Longitudinal Imaging of Vascular Inflammation and Treatment by Ezetimibe in apoE Mice by FMT Using New Optical Imaging Biomarkers of Cathepsin Activity and αvβ3 Integrin

Inflammation as a core pathological event of atherosclerotic lesions is associated with the secretion of cathepsin proteases and the expression of α v β 3 integrin. We employed fluorescence molecular tomographic (FMT) noninvasive imaging of these molecular activities using cathepsin sensing (ProSense, CatB FAST) and α v β 3 integrin (IntegriSense) near-infrared fluorescence (NIRF) agents. A statistically significant increase in the ProSense and IntegriSense signal was observed within the chest region of apoE−/− mice (P < 0.05) versus C57BL/6 mice starting 25 and 22 weeks on high cholesterol diet, respectively. In a treatment study using ezetimibe (7 mg/kg), there was a statistically significant reduction in the ProSense and CatB FAST chest signal of treated (P < 0.05) versus untreated apoE−/− mice at 31 and 21 weeks on high cholesterol diet, respectively. The signal of ProSense and CatB FAST correlated with macrophage counts and was found associated with inflammatory cells by fluorescence microscopy and flow cytometry of cells dissociated from aortas. This report demonstrates that cathepsin and α v β 3 integrin NIRF agents can be used as molecular imaging biomarkers for longitudinal detection of atherosclerosis, and cathepsin agents can monitor anti-inflammatory effects of ezetimibe with applications in preclinical testing of therapeutics and potentially for early diagnosis of atherosclerosis in patients.

An Albumin-Activated Far-Red Fluorochrome for In Vivo Imaging

A variety of far red (FR) and near infrared (NIR) fluorochromes have been described recently for in vivo fluorescence imaging of disease. These fluorochromes have been classified generally into nonspecific enhancers (such as ICG), molecularly targeted fluorochromes (such as RGD-Cy), and enzyme-activated agents (such as cathepsin-sensitive NIRF probes). Collectively, these agents have shown tremendous potential for the in vivo imaging of specific molecular targets, biological processes and cells. For example, specific fluorochromes have been used to image angiogenesis, apoptosis, vascular permeability, protease activity, receptor status, macrophage activity and to track cells. 9] Most of the affinity agents developed thus far have molecular weight (kDa) values in excess of several thousand to millions to carry affinity ligands and/or to allow efficient quenching/de-quenching in high molecular weight constructs. We hypothesized that small-molecule fluorochromes with affinity for specific targets could potentially exhibit an increase in fluorescence upon target binding. Thus, a family of “activatable” fluorophores could be developed that consequently would provide superior in vivo target-to-background ratios in live animals. As proof-of-principal, we developed and tested VM315, a far-red indocyanine-based fluorophore, which contains an organic aromatic moiety that serves as a “handle” for noncovalent binding to albumin. Herein, we show that the model compound exhibits a remarkable increase in fluorescence upon binding to its target. We anticipate that other small molecules with high affinity for specific proteins (for example, therapeutic drugs) could be synthesized in ways that are similar to the prototypic “activatable” albumin agent. Herein, we show how such a molecule can be used to improve the detection of small cancers in vivo. VM315, a small-molecule NIR fluorophore (MALDI MS: calculated 1231.3, found 1231.3) was fully soluble in water, with an octanol/water partition coefficient of 11% (Cy5.5 partition coefficient: 5%). The absorption maximum is at 685 nm and the emission maximum is at 705 nm. Figure 1 a shows the fluorescence emission of the compound in saline solution and serum; there is an increase in fluorescence of >210% upon albumin binding. Additional experiments at variable concentrations confirmed that the fluorescence increase was not a result of quenching effects at the baseline state which can occur with less water-soluble indocyanine compounds such as ICG. VM315 bound albumin selectively, with >90% fluorochrome attachment (determined by HPLC) at diagnostic concentrations. The compound did not bind to any other plasma proteins (such as globulins) as determined by gel chromatography (Figure 1). We next performed a set of in vivo experiments to determine the potential utility of the compound in preclinical settings. Intravital confocal microscopy was used to measure the half-life of VM315 in the bloodstream upon intravenous injection (Figure 2). The compound exhibited interesting kinetics ; initially a rapid (1 min) increase in vascular fluorescence was observed, followed by a further steady but slower increase over the subsequent 30 min, presumably the result of “albumin activation”. Beyond 30 min, microvascular fluorescence slowly decreased by elimination (t1/2=76 min). The apparent com[a] Dr. X. Montet, Dr. R. Weissleder Center for Molecular Imaging Research Massachusetts General Hospital Building 149, 13th St. , Room 5403 Charlestown, MA 02129 (USA) Fax: (+1)617-726-8226 E-mail : weissleder@helix.mgh.harvard.edu [b] Dr. M. Rajopadhye VisEn Medical, Woburn MA (USA) Figure 1. Characterization of VM315: a) fluorescence spectroscopy (lexcitation=685 nm) shows that binding of the compound to albumin is 90% at diagnostic concentrations; note the marked increase in fluorescence upon albumin binding (blue=unbound, red=bound); b) gel chromatography confirms selective binding to albumin but not other proteins (lanes 1=bovine serum albumin, lanes 2=human serum albumin with Cy5.5, lanes 3=human serum albumin with VM315).

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.

Detection and quantification of enzymatically active prostate-specific antigen in vivo

Abstract. Assays for blood levels of prostate-specific antigen (PSA), performed in prostate cancer detection, measure mostly inactive/complexed PSA and do not provide information regarding enzymatically active PSA, which is biologically more relevant. Thus, we designed and synthesized an enzymatically cleavable peptide sequence labeled with near-infrared (NIR) fluorophores (ex/em 740/770  nm) and coupled it to a pharmacokinetic modifier designed to improve its plasma kinetics. In its native state, the agent, PSA750 FAST™ (PSA750), is optically quenched (>95%) and only becomes fluorescent upon cleavage by active PSA, yielding a significant increase in signal. This activation is highly selective for PSA relative to a large panel of disease-relevant enzymes. Active PSA was detected in tumor frozen sections using PSA750 and this activity was abolished in the presence of the inhibitor, alpha-1 anti-chymotrypsin. In vivo imaging of tumor-bearing mice using fluorescence molecular tomography demonstrated a significantly higher fluorescent signal in PSA+ LNCaP tumors as compared to PSA− prostate cancer 3 tumors (13.0±3.7 versus 2.8±0.8  pmol, p=0.023). Ex vivo imaging of tumor sections confirms PSA750-derived NIR signal localization in nonvascular tissue. This is the first report that demonstrates the feasibility and effectiveness of noninvasive, real time, fluorescence molecular imaging of PSA enzymatic activity in prostate cancer.

Fluorescence imaging of bombesin and transferrin receptor expression is comparable to 18F-FDG PET in early detection of sorafenib-induced changes in tumor metabolism

Physical measurement of tumor volume reduction is the most commonly used approach to assess tumor progression and treatment efficacy in mouse tumor models. However, it is relatively insensitive, and often requires long treatment courses to achieve gross physical tumor destruction. As alternatives, several non-invasive imaging methods such as bioluminescence imaging (BLI), fluorescence imaging (FLI) and positron emission tomography (PET) have been developed for more accurate measurement. As tumors have elevated glucose metabolism, 18F-fludeoxyglucose (18F-FDG) has become a sensitive PET imaging tracer for cancer detection, diagnosis, and efficacy assessment by measuring alterations in glucose metabolism. In particular, the ability of 18F-FDG imaging to detect drug-induced effects on tumor metabolism at a very early phase has dramatically improved the speed of decision-making regarding treatment efficacy. Here we demonstrated an approach with FLI that offers not only comparable performance to PET imaging, but also provides additional benefits, including ease of use, imaging throughput, probe stability, and the potential for multiplex imaging. In this report, we used sorafenib, a tyrosine kinase inhibitor clinically approved for cancer therapy, for treatment of a mouse tumor xenograft model. The drug is known to block several key signaling pathways involved in tumor metabolism. We first identified an appropriate sorafenib dose, 40 mg/kg (daily on days 0–4 and 7–10), that retained ultimate therapeutic efficacy yet provided a 2–3 day window post-treatment for imaging early, subtle metabolic changes prior to gross tumor regression. We then used 18F-FDG PET as the gold standard for assessing the effects of sorafenib treatment on tumor metabolism and compared this to results obtained by measurement of tumor size, tumor BLI, and tumor FLI changes. PET imaging showed ~55–60% inhibition of tumor uptake of 18F-FDG as early as days 2 and 3 post-treatment, without noticeable changes in tumor size. For comparison, two FLI probes, BombesinRSense™ 680 (BRS-680) and Transferrin-Vivo™ 750 (TfV-750), were assessed for their potential in metabolic imaging. Metabolically active cancer cells are known to have elevated bombesin and transferrin receptor levels on the surface. In excellent agreement with PET imaging, the BRS-680 imaging showed 40% and 79% inhibition on days 2 and 3, respectively, and the TfV-750 imaging showed 65% inhibition on day 3. In both cases, no significant reduction in tumor volume or BLI signal was observed during the first 3 days of treatment. These results suggest that metabolic FLI has potential preclinical application as an additional method for detecting drug-induced metabolic changes in tumors.