Blood Pharmacokinetics Imaging by Noninvasive Heart Fluorescence Tomography and Application to Kidney Glomerular Filtration Rate Assessment.

In mouse pharmacokinetic (PK) studies, current standard methods often require large numbers of animals to support collection of blood samples serially over a defined time range. We have developed and validated a noninvasive fluorescence molecular tomography (FMT) heart imaging approach for blood PK quantification that uses small numbers of mice and has the advantage of repeated, longitudinal live imaging. This method was validated using a variety of near infrared (NIR) fluorescent-labeled molecules, ranging in size from 1.3 to 150 kDa, that were assessed by microplate blood assays as well as by noninvasive FMT 4000 imaging. Excellent agreement in kinetic profiles and calculated PK metrics was seen for the two methods, establishing the robustness of this noninvasive optical imaging approach. FMT heart imaging was further assessed in the challenging application of inulin-based glomerular filtration rate (GFR) measurement. After a single bolus injection of an NIR fluorescent-labeled inulin probe in small cohorts of mice (n = 5 per group), 2-minute heart scans (at 2, 6, 15, 30, and 45 minutes) were performed by FMT imaging. GFR was calculated using two-compartment PK modeling, determining an average rate of 240 ± 21 µl/min in normal mice, in agreement with published mouse GFR ranges. Validation of GFR assessment in unilaterally nephrectomized mice and cyclosporin A-treated mice both measured ∼50% decreases in GFR. Imaging results correlated well with ex vivo plasma microplate assays for inulin blood kinetics, and the decreases in GFR were accompanied by increases in plasma creatinine and blood urea nitrogen.

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.

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.

Synthesis and evaluation of a gamma-lactam as a highly selective EP2 and EP4 receptor agonist.

Gamma-lactam analogs (2) of EP(4) receptor agonists were identified by substitution of the pyrazolidinone ring (1) with a pyrrolidinone ring. Several compounds (such as 2a, 2h) with high potency, selectivity and acceptable PK profiles were discovered. These were assessed in animal models of ovulation induction and bronchoconstriction.

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.

Pharmacological inhibition of phosphodiesterase 4 triggers ovulation in follicle-stimulating hormone-primed rats.

Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides to render them biologically inactive. As such, these enzymes are critical regulators of signal transduction pathways that use cyclic nucleotides as second messengers. PDE4 is one such member that has been identified in ovarian tissue and purported to have a role in the regulation of gonadotropin action. In the present study, selective PDE4 inhibitors enhanced intracellular signaling in a human LH receptor-expressing granulosa cell line. In vivo, PDE4 inhibition in FSH-primed rats resulted in ovulation, indicating that the PDE4 inhibitors can substitute for LH and human chorionic gonadotropin (hCG) in this process. Moreover, when coadministered with a subeffective dose of hCG, PDE4 inhibitors acted synergistically to enhance the ovulation response. Inhibitors of PDE3 or PDE5 had no ovulatory effect under similar conditions. Oocytes that were ovulated after PDE4 inhibition could be fertilized in vitro at a rate similar to that of oocytes from hCG-induced ovulation. Moreover, such oocytes were fully capable of being fertilized in vivo and developing into normal live pups. These results indicate that small molecule PDE4 inhibitors may be orally active alternatives to hCG as part of a fertility treatment regimen.

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.

Quantitative Longitudinal Imaging of Vascular Inflammation and Treatment by Ezetimibe in apoE Mice by FMT Using New Optical Imaging Biomarkers of Cathepsin Activity and α(v)ß(3) Integrin.

Inflammation as a core pathological event of atherosclerotic lesions is associated with the secretion of cathepsin proteases and the expression of α(v)ß(3) integrin. We employed fluorescence molecular tomographic (FMT) noninvasive imaging of these molecular activities using cathepsin sensing (ProSense, CatB FAST) and α(v)ß(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)ß(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.

Discovery and evaluation of a non-Zn chelating, selective matrix metalloproteinase 13 (MMP-13) inhibitor for potential intra-articular treatment of osteoarthritis.

Osteoarthritis (OA) is a nonsystemic disease for which no oral or parenteral disease-modifying osteoarthritic drug (DMOAD) is currently available. Matrix metalloproteinase 13 (MMP-13) has attracted attention as a target with disease-modifying potential because of its major role in tissue destruction associated with OA. Being localized to one or a few joints, OA is amenable to intra-articular (IA) therapy, which has distinct advantages over oral therapies in terms of increasing therapeutic index, by maximizing drug delivery to cartilage and minimizing systemic exposure. Here we report on the synthesis and biological evaluation of a non-zinc binding MMP-13 selective inhibitor, 4-methyl-1-(S)-({5-[(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyrimidine-7-carbonyl}amino)indan-5-carboxylic acid (1), that is uniquely suited as a potential IA-DMOAD: it has long durability in the joint, penetrates cartilage effectively, exhibits nearly no detectable systemic exposure, and has remarkable efficacy.