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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 non-invasive 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 non-invasive FMT®4000 imaging. Excellent agreement in kinetic profiles and calculated PK metrics was seen for the two methods, establishing the robustness of this non-invasive 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), two-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 (CsA)-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. SIGNIFICANCE STATEMENT N/A

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.

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.

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.

Changes in mRNA expression for estrogen receptor (ER beta) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in granulosa and theca cells from proestrous rat ovarian follicles. Of the 30 ovaries harvested from 15 adult rats, 24 were processed for in situ hybridization, and the remaining were used for reverse transcription-polymerase chain reaction. Messenger RNAs for ER beta, LHr, cytochrome P450 side-chain cleavage enzyme (P450(scc)), 17 alpha-hydroxylase (P450(c17)), aromatase (P450(arom)), and steroidogenic acute regulatory protein (StAR) were localized in cross sections of ovaries by in situ hybridization and quantified in granulosa and theca cell layers by a computer-image analyzing system. Ovarian follicles were classified as healthy or atretic. Healthy follicles were divided into four size groups: very small (40-100 microm), small (101-275 microm), medium (276-450 microm), and large (451-850 microm). Atretic follicles were divided into medium (276-450 microm) or large follicles (451-850 microm). A low level of ER beta mRNA expression was first detected in granulosa cells of very small healthy follicles, and the expression increased progressively up to medium-sized follicles. The expression of ER beta mRNA was highest (P < 0.01) in medium-sized follicles that was followed by a decrease (P < 0.01) in large follicles. Messenger RNAs for LHr, P450(scc), and P450(arom) were first detected in granulosa cells of medium-sized healthy follicles, while mRNAs for LHr, P450(scc), P450(c17), and StAR were first detected in theca cells associated with very small follicles. The highest expression of LHr, P450(scc), P450(c17), P450(arom), and StAR was seen in granulosa and/or theca cells of large healthy follicles. In atretic follicles, level of gene expression was relatively low in both granulosa and theca cells. In conclusion, stage-specific expression of ER beta mRNA was observed in granulosa cells during follicular development. The increased expression of ER beta and a concomitant initiation of LHr, P450(scc), and P450(arom) expression in granulosa cells of medium follicles may signify a role for estrogen in follicular development. Also, a strong correlation between ER beta mRNA expression in granulosa cells, and the expression of mRNAs for LHr, P450(scc), P450(c17), and StAR in theca cells associated with growing follicles suggests a possible role for estrogen in steroidogenesis.

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.

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.

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.

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.

Folate receptors (FR) are overexpressed on the surface of many different human tumor cells, including ovarian, breast, cervical, renal, colorectal and nasopharyngeal cancer cells, with little expression in normal tissues. As such, folic acid has been successfully exploited as a cancer specific targeting moiety for the efficient delivery of chemotherapeutic agents, drug carriers, photosensitizers and diagnostic reporters. Critical to the success of such agents is the determination of the level of FR expression for a given tumor, since weak FR-expressing cancers will not respond well to folate-targeted therapies. There is therefore a need for specific and quantitative imaging agents and methods for the determination of FR expression in vivo. Optical imaging in the near-infrared (NIR) range allows efficient penetration of photons through living tissue and minimizes interference from tissue autofluorescence. Combined with quantitative fluorescence molecular tomography (FMT), NIR fluorescent agents have emerged as invaluable tools for quantitative, deep tissue imaging across a range of important areas of disease research including oncology. We have developed a new, near-infrared fluorescent folate targeted agent, VM3244, and used it to noninvasively quantify FR expression in vivo by FMT of mouse tumor xenografts. Two cell types, KB and HeLa with different degrees of FR expression were employed in the present study. In vitro, FR expression levels of each cell type were visualized with anti-FR antibody by microscopy, showing high FR expression in KB cells and lower expression in HeLa cells. The binding of VM3244 to FR was quantified by flow cytometry, confirming high and low FR expression in KB and HeLa cells, with good agreement between VM3244 and the antibody. Specificity of the agent was demonstrated by blocking of the signal by addition of an excess of unlabeled folic acid. In vivo quantification was performed by injection of 2 nmol of VM3244 into nude mice mice bearing KB or HeLa tumor xenografts and imaging by FMT at 4 to 24 h post injection. KB tumors showed a high level of FR expression with approximately 200 pmol (10% injected dose) quantified in the tumors, with little fluorescence in other tissues except the kidneys at 4 h post injection biodistribution study. Consistent with the in vitro profile, HeLa tumors had less tumor fluorescence, with about 75 pmol (3.8% injected dose) quantified in the tumors. Both tumor cell lines showed significant (∼80%) knockdown of signal by co-injection of the mice with unlabeled folic acid and ex vivo colocalization with FR antibody on tumor slices, confirming the in vivo specificity of the agent. Thus, we have demonstrated the ability of VM3244 to quantify FR expression both in vitro and in vivo and, combined with FMT, to noninvasively distinguish between high and lower FR expressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2444. doi:1538-7445.AM2012-2444

Carbonic anhydrase IX (CA IX), a cell surface enzyme involved in tumor cell acidification, is induced during tumor hypoxia (lack of oxygen) in many different types of tumors. CA IX expression is correlated to tumor cell proliferation and metastasis, poor prognosis, and resistance to therapeutic intervention, making CA IX an important biomarker in the study of hypoxia, tumor cell proliferation, and therapy. Therefore, determination of CAIX expression and identification of hypoxic tumors in vivo are critical for cancer research and treatments. We have designed and synthesized a near-infrared (NIR) fluorescent agent (VM3219) for in vivo detection and quantification of a cell surface biomarker of hypoxia, CA IX based on a well-known CA IX inhibitor, acetazolamide. VM3219 was characterized extensively in HeLa cells and in mouse HeLa tumor xenografts. Preliminary chemical and biochemical characterization of VM3219 showed excellent CA IX selectivity and affinity. In vitro, VM3219 detected 4-7-fold up-regulation of CA IX in live hypoxic HeLa cells as visualized by fluorescence microscopy and quantified by flow cytometry. VM3219 fluorescence signals were blocked by 80% when cells were pre-incubated with unlabeled acetazolamide. Fluorescent signal of VM3219 was low in normoxic HeLa cells and was comparable to the signal that were detected by using a non-binding control agent (VM3182) incubated with normoxic or hypoxic cells. In vivo imaging by fluorescence molecular tomography (FMTTM) showed that approximately 5% of the injected dose (2 nmol VM3219) was retained in the tumor tissues after 24 h (100 -120 pmol), 10-20-fold more than measured with a non-binding control agent. Tumor fluorescence was blocked >65% by prophylactic acetazolamide treatment. Maintenance of tumor-bearing mice on low oxygen (8%), as compared to normally housed tumor bearing mice (20% oxygen), induced a >2X (∼250 pmol) increase in VM3219 tumor fluorescence signal suggesting that CAIX expression was up-regulated within tumors by low oxygen. In tissue sections, VM3219 signal was shown to be specifically co-localized in the tumor hypoxic regions with CA IX antibody and Hypoxyprobe (pimonidazole), and the signal was away from non-hypoxic regions as determined by Hoechst stain. In conclusion, VM3219 is a new non-invasive tumor hypoxia imaging agent that offers a tool for specific imaging of a biologically important hypoxia biomarker, CA IX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3949. doi:1538-7445.AM2012-3949

Cathepsin (Cat) B, a lysosomal cysteine protease, is strongly overexpressed in cancers and premalignant lesions and associated with cancer growth, neo-vascularization and metastasis. Cat B is also expressed in tumor-associated macrophages (TAM), inflammatory cells that comprise a significant proportion of tumor mass and are reported to be important for cancer progression. We evaluated a novel, highly selective, near-infrared fluorescence (NIRF) imaging agent, Cat B 750 FAST, for specific and rapid detection of tumor-associated Cat B activity. The agent consists of a pair of quenched fluorophores separated by a highly selective Cat B substrate, which is further conjugated to a pharmacokinetic modifier to achieve ∼2h plasma half-life. Upon in vitro proteolysis the agent de-quenches and becomes highly fluorescent (35-fold) with em=770 nm. To assess selectivity of activation, a variety of cathepsins and other tumor associated proteases were incubated with the agent under appropriate conditions for each enzyme. Cat B FAST was selectively cleaved and activated in vitro by Cat B. At 5 h, the fluorescence released by Cat B was 3-fold greater than by Cat S, 15-fold greater than by Cat L and >40-fold greater than by Cat K, Cat D or Legumain (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4556.

Drug-induced ex vivo apoptotic tests are widely used pre-clinically to monitor chemotherapeutic efficacy. Real-time non-invasive in vivo imaging and quantification of therapy-induced tumor apoptosis would impact drug discovery significantly. As a key apoptotic marker, phosphatidylserine (PS) translocated to the outer leaflet of the plasma membrane can be detected by fluorescent-labeled Annexin A5 specifically with high affinity. We synthesized an in vivo imaging contrast agent Annexin-Vivo 750 by conjugating a human recombinant Annexin A5 to a near-infrared fluorophore with em 770 nm. Fluorescence microscopy showed 4h anti-FAS Ab treatment significantly increased the number of Annexin-Vivo 750 positive Jurkat cells, confirming that the agent binds to PS on apoptotic cells. Similarly, flow cytometry showed anti-FAS Ab increased Annexin-Vivo 750 positive cells from 15% to 55%. TUNEL positive cells were increased from 0% to 65.14 ± 3.93% (p=0.0004) by anti-FAS antibody. The TUNEL data correlate very well with flow cytometry data and are consistent with the fluorescence microscopy data. In vivo imaging of Annexin-Vivo 750 was performed in nude mice implanted with human colorectal carcinoma HT-29 cells. Once tumors reached a volume of 142.36 + 8.3 mm3 mice were randomized and treated intraperitoneally with cyclophosphamide (170 mg/kg) or vehicle at time 0h. The agent was injected at 24h with a dose of 2.4 nmol/animal and the tumors were imaged at 26h with an FMT2500 quantitative tomography imager for in vivo real-time 3D Annexin-Vivo 750 quantification. Tumor fluorescence signal was 2.55 fold higher with than without cyclophosphamide (p=0.0002), while tumor volumes were the same (p=0.35). Fluorescent signal decreased significantly by 24h and returned to baseline at 48h post agent injection. Likewise, intratumoral injection of TNFα (2.5 μg) plus IFNγ subcutaneous injection (2500 units) also significantly increased HT29 tumor Annexin-Vivo 750 fluorescence (3 fold compared to control, p=0.0099, 24h after treatment, 2h post-agent injection). TUNEL positive HT-29 cells were increased from 11.16 ± 7.05 % to 41.77 ± 8.78 % (p=0.0004) by cyclophosphomide, indicating apoptosis. These results demonstrate that Annexin-Vivo 750 in vivo imaging can be used for detecting, quantifying and monitoring apoptosis following cancer treatments, underscoring its value in drug discovery and development in cancer and potentially in other clinically important areas, such as cardiovascular-, autoimmune-, inflammatory- and neurodegenerative diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4557.