Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Single-dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

Development of an inflammation imaging tracer, 111In-DOTA-DAPTA, targeting chemokine receptor CCR5 and preliminary evaluation in an ApoE-/- atherosclerosis mouse model.

BACKGROUND: Chemokine receptor 5 (CCR5) plays an important role in atherosclerosis. Our objective was to develop a SPECT tracer targeting CCR5 for imaging plaque inflammation by radiolabeling D-Ala-peptide T-amide (DAPTA), a CCR5 antagonist, with 111In. METHODS: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated DAPTA (DOTA-DAPTA) was labeled with 111In. Cell uptake studies were conducted in U87-CD4-CCR5 and U87-MG cells. Biodistribution was determined in C57BL/6 mice. Autoradiography, en face and Oil Red O (ORO) imaging studies were performed in ApoE-/- mice. RESULTS: DOTA-DAPTA was radiolabeled with 111In with high radiochemical purity (> 98%) and specific activity (70 MBq·nmol). 111In-DOTA-DAPTA exhibited fast blood and renal clearance and high spleen uptake. The U87-CD4-CCR5 cells had significantly higher uptake in comparison to the U87-MG cells. The cell uptake was reduced by three times with DAPTA, indicating the receptor specificity of the uptake. Autoradiographic images showed significantly higher lesion uptake of 111In-DOTA-DAPTA in ApoE-/- mice than that in C57BL/6 mice. The tracer uptake in 4 month old ApoE-/- high fat diet (HFD) mice with blocking agent was twofold lower than the same mice without the blocking agent, demonstrating the specificity of the tracer for the CCR5 receptor. CONCLUSION: 111In-DOTA-DAPTA, specifically targeting chemokine receptor CCR5, is a potential SPECT agent for imaging inflammation in atherosclerosis.

Effects of an endothelin receptor antagonist, Macitentan, on right ventricular substrate utilization and function in a Sugen 5416/hypoxia rat model of severe pulmonary arterial hypertension.

BACKGROUND: Altered myocardial energy metabolism has been linked to worsening of RV function in pulmonary arterial hypertension (PAH). The aim of this study was to evaluate RV glucose and fatty acid metabolism in vivo in a rat model of PAH using positron emission tomography (PET) and investigate the effects of Macitentan on RV substrate utilization. METHODS: PAH was induced in male Sprague-Dawley rats by a single subcutaneous injection of Sugen 5416 (20 mg/kg) followed by 3 weeks of hypoxia (10% oxygen). At week 5 post-injection, the PAH rats were randomized to Macitentan (30 mg/kg daily) treatment or no treatment. Substrate utilization was serially assessed 5 and 8 weeks post-injection with 2-[18F]fluoro-2-deoxyglucose (FDG) and 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid (FTHA) PET for glucose and fatty acid metabolism respectively and correlated with in vivo functional measurements. RESULTS: PAH induction resulted in a 2.5-fold increase in RV FDG uptake (standardized uptake value (SUV) of normal control: 1.6 ± 0.4, week 5: 4.1 ± 1.9, week 8: 4.0 ± 1.6, P < 0.05 for all groups vs. control). RV FTHA showed twofold increased uptake at week 5 (SUV control: 1.50 ± 0.39, week 5: 3.06 ± 1.10, P = 0.03). Macitentan significantly decreased RV FDG uptake at 8 weeks (SUV: 2.5 ± 0.9, P = 0.04), associated with improved RV ejection fraction and reduced RV systolic pressure, while FTHA uptake was maintained. CONCLUSION: PAH is associated with metabolic changes in the RV, characterized by a marked increase in FDG and FTHA uptake. Macitentan treatment reduced PAH severity and was associated with a decrease in RV FDG uptake and improved RV function.

Engineering Rapalog-Inducible Genetic Switches Based on Split-T7 Polymerase to Regulate Oncolytic Virus-Driven Production of Tumour-Localized IL-12 for Anti-Cancer Immunotherapy.

The approval of different cytokines as anti-neoplastic agents has been challenged by dose-limiting toxicities. Although reducing dose levels affords improved tolerability, efficacy is precluded at these suboptimal doses. Strategies combining cytokines with oncolytic viruses have proven to elicit potent survival benefits in vivo, despite promoting rapid clearance of the oncolytic virus itself. Herein, we developed an inducible expression system based on a Split-T7 RNA polymerase for oncolytic poxviruses to regulate the spatial and temporal expression of a beneficial transgene. This expression system utilizes approved anti-neoplastic rapamycin analogues for transgene induction. This treatment regimen thus offers a triple anti-tumour effect through the oncolytic virus, the induced transgene, and the pharmacologic inducer itself. More specifically, we designed our therapeutic transgene by fusing a tumour-targeting chlorotoxin (CLTX) peptide to interleukin-12 (IL-12), and demonstrated that the constructs were functional and cancer-selective. We next encoded this construct into the oncolytic vaccinia virus strain Copenhagen (VV-iIL-12mCLTX), and were able to demonstrate significantly improved survival in multiple syngeneic murine tumour models through both localized and systemic virus administration, in combination with rapalogs. In summary, our findings demonstrate that rapalog-inducible genetic switches based on Split-T7 polymerase allow for regulation of the oncolytic virus-driven production of tumour-localized IL-12 for improved anti-cancer immunotherapy.

A T cell-targeted multi-antigen vaccine generates robust cellular and humoral immunity against SARS-CoV-2 infection.

SARS-CoV-2, the etiological agent behind the coronavirus disease 2019 (COVID-19) pandemic, has continued to mutate and create new variants with increased resistance against the WHO-approved spike-based vaccines. With a significant portion of the worldwide population still unvaccinated and with waning immunity against newly emerging variants, there is a pressing need to develop novel vaccines that provide broader and longer-lasting protection. To generate broader protective immunity against COVID-19, we developed our second-generation vaccinia virus-based COVID-19 vaccine, TOH-VAC-2, encoded with modified versions of the spike (S) and nucleocapsid (N) proteins as well as a unique poly-epitope antigen that contains immunodominant T cell epitopes from seven different SARS-CoV-2 proteins. We show that the poly-epitope antigen restimulates T cells from the PBMCs of individuals formerly infected with SARS-CoV-2. In mice, TOH-VAC-2 vaccination produces high titers of S- and N-specific antibodies and generates robust T cell immunity against S, N, and poly-epitope antigens. The immunity generated from TOH-VAC-2 is also capable of protecting mice from heterologous challenge with recombinant VSV viruses that express the same SARS-CoV-2 antigens. Altogether, these findings demonstrate the effectiveness of our versatile vaccine platform as an alternative or complementary approach to current vaccines.

Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies.

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.

Fatty acid transport protein inhibition sensitizes breast and ovarian cancers to oncolytic virus therapy via lipid modulation of the tumor microenvironment.

Introduction: Adipocytes in the tumour microenvironment are highly dynamic cells that have an established role in tumour progression, but their impact on anti-cancer therapy resistance is becoming increasingly difficult to overlook. Methods: We investigated the role of adipose tissue and adipocytes in response to oncolytic virus (OV) therapy in adipose-rich tumours such as breast and ovarian neoplasms. Results: We show that secreted products in adipocyte-conditioned medium significantly impairs productive virus infection and OV-driven cell death. This effect was not due to the direct neutralization of virions or inhibition of OV entry into host cells. Instead, further investigation of adipocyte secreted factors demonstrated that adipocyte-mediated OV resistance is primarily a lipid-driven phenomenon. When lipid moieties are depleted from the adipocyte-conditioned medium, cancer cells are re-sensitized to OV-mediated destruction. We further demonstrated that blocking fatty acid uptake by cancer cells, in a combinatorial strategy with virotherapy, has clinical translational potential to overcome adipocyte-mediated OV resistance. Discussion: Our findings indicate that while adipocyte secreted factors can impede OV infection, the impairment of OV treatment efficacy can be overcome by modulating lipid flux in the tumour milieu.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.

Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy.

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

Personalized oncology and BRAFK601N melanoma: model development, drug discovery, and clinical correlation.

PURPOSE: Mutations in BRAF are the most prominent activating mutations in melanoma and are increasingly recognized in other cancers. There is currently no accepted treatment regimen for patients with mutant BRAFK601N melanoma, and the study of melanoma driven by BRAF mutations at the 601 locus is lacking due to a paucity of cellular model systems. Therefore, we sought to better understand the treatment and clinical approach to patients with mutant BRAFK601N melanoma and subsequently develop a novel personalized oncology platform for rare or treatment-refractory cancers. METHODS: We developed and characterized the first patient-derived, naturally occurring BRAFK601N melanoma model, described herein as OHRI-MEL-13, and assessed efficacy using the Prestwick Chemical Library and select targeted therapeutics. RESULTS: OHRI-MEL-13 exhibits loss of heterozygosity of BRAF, closely mimics the original tumor's gene expression profile, is tumorigenic in immune-deficient murine models, and is available for public accession through American Type Culture Collection. We present in silico modeling data, which illustrates the therapeutic failure of BRAFV600E-targeted therapies in BRAFK601N mutants. Our platform elucidated a unique role for MEK inhibition with cobimetinib, which resulted in short-term clinical success by reducing the metastatic burden. CONCLUSION: Our model of BRAFK601N-activated melanoma was developed, thoroughly characterized, and made available for public accession. This model served to demonstrate the feasibility of a novel personalized oncology platform that could be optimized at an institutional level for rare variant or treatment-refractory cancers. We also demonstrate the clinical utility of monotherapy MEK inhibition in a case of BRAFK601N melanoma.

An Oncolytic Adenovirus Vector Expressing p14 FAST Protein Induces Widespread Syncytium Formation and Reduces Tumor Growth Rate In Vivo.

Intratumoral injection of oncolytic viruses provides a direct means of tumor cell destruction for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this study, we explore the efficacy of a conditionally replicating HAdV vector expressing the p14 Fusion-Associated Small Transmembrane (FAST) protein (CRAdFAST) in both immunocompetent and immunodeficient mouse models of cancer. The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect. In the murine 4T1 model of cancer, treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene, but it did not reduce the tumor growth rate in vivo. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion, which led to the formation of large acellular regions within the tumor and significantly reduced the tumor growth rate compared to control vector. Our results indicate that expression of p14 FAST from an oncolytic HAdV can improve vector efficacy for the treatment of cancer.

Deletion of Apoptosis Inhibitor F1L in Vaccinia Virus Increases Safety and Oncolysis for Cancer Therapy.

Vaccinia virus (VACV) possesses a great safety record as a smallpox vaccine and has been intensively used as an oncolytic virus against various types of cancer over the past decade. Different strategies were developed to make VACV safe and selective to cancer cells. Leading clinical candidates, such as Pexa-Vec, are attenuated through deletion of the viral thymidine kinase (TK) gene, which limits virus growth to replicate in cancer tissue. However, tumors are not the only tissues whose metabolic activity can overcome the lack of viral TK. In this study, we sought to further increase the tumor-specific replication and oncolytic potential of Copenhagen strain VACV ΔTK. We show that deletion of the anti-apoptosis viral gene F1L not only increases the safety of the Copenhagen ΔTK virus but also improves its oncolytic activity in an aggressive glioblastoma model. The additional loss of F1L does not affect VACV replication capacity, yet its ability to induce cancer cell death is significantly increased. Our results also indicate that cell death induced by the Copenhagen ΔTK/F1L mutant releases more immunogenic signals, as indicated by increased levels of IL-1ß production. A cytotoxicity screen in an NCI-60 panel shows that the ΔTK/F1L virus induces faster tumor cell death in different cancer types. Most importantly, we show that, compared to the TK-deleted virus, the ΔTK/F1L virus is attenuated in human normal cells and causes fewer pox lesions in murine models. Collectively, our findings describe a new oncolytic vaccinia deletion strain that improves safety and increases tumor cell killing.

Enhancing Expression of Functional Human Sodium Iodide Symporter and Somatostatin Receptor in Recombinant Oncolytic Vaccinia Virus for In Vivo Imaging of Tumors.

Oncolytic virus (OV) therapy has emerged as a novel tool in our therapeutic arsenals for fighting cancer. As a live biologic agent, OV has the ability to target and selectively amplify at the tumor sites. We have reported that a vaccinia-based OV (Pexa-Vec) has shown good efficacy in preclinical models and in clinical trials. To give an additional tool to clinicians to allow both treatment of the tumor and improved visualization of tumor margins, we developed new viral-based platforms with 2 specific gene reporters. METHODS: We incorporated the human sodium iodide symporter (hNIS) and the human somatostatin receptor 2 (hSSR2) in the vaccinia-based OV and tested viral constructs for their abilities to track and treat tumor development in vivo. RESULTS: Early and high-level expression of hNIS is detrimental to the recombinant virus, leading to the aggregation of hNIS protein and early cell death. Putting hNIS under a late synthetic promoter allowed a higher functional expression of the protein and much stronger 123I or 99Tc uptake. In vivo, the hNIS-containing virus infected and amplified in the tumor site, showing a better efficacy than the parental virus. The hNIS expression at the tumor site allowed for the imaging of viral infection and tumor regression. Similarly, hSSR2-containing OV vaccinia infected and lysed cancer cells. CONCLUSION: When tumor-bearing mice were given hNIS- and hSSR2-containing OV, 99Tc and 111In signals coalesced at the tumor, highlighting the power of using these viruses for tumor diagnosis and treatment.

Formulation, characterization and tissue distribution of a novel pH-sensitive long-circulating liposome-based theranostic suitable for molecular imaging and drug delivery.

PURPOSE: When designing liposome formulas for treatment and diagnostic purposes, two of the most common challenges are 1) the lack of a specific release mechanism for the encapsulated contents and 2) a short circulation time due to poor resistance to biological fluids. This study aimed to create a liposome formula with prolonged in vivo longevity and pH-sensitivity for cytoplasmic drug delivery. MATERIALS AND METHODS: Liposomal particles were generated using hydrogenated soy (HS) phosphatidylcholine, cholesteryl hemisuccinate (CHEM), polyethylene glycol (PEG) and diethylenetriaminepentaacetic acid-modified phosphatidylethanolamine with film hydration and extrusion methods. The physicochemical properties of the different formulas were characterized. pH-sensitivity was evaluated through monitoring release of encapsulated calcein. Stability of the radiolabeled liposomes was assessed in vitro through incubation with human serum. The best formula was selected and injected into healthy rats to assess tissue uptake and pharmacokinetics. RESULTS: Liposomal particles were between 88 and 102 nm in diameter and negatively charged on the surface. Radiolabeling of all formulas with indium-111 was successful with good efficiency. 1%PEG-HS-CHEM not only responded to acidification very quickly but also underwent heavy degradation with serum. The 4%PEG-HS-CHEM, which exhibited both comparatively good pH-sensitivity (up to 20% release) and satisfactory stability (stability >70% after 24 h), was considered the best candidate for in vivo evaluation. Tissue distribution of 4%PEG-HS-CHEM was comparable to that of 4%PEG-HS-Chol, a long-circulating but pH-insensitive control, showing major accumulation in liver, spleen, intestine and kidneys. Analysis of blood clearance showed favorable half-life values: 0.6 and 14 h in fast and slow clearance phases, respectively. CONCLUSION: 4%PEG-HS-CHEM showed promising results in pH-sensitivity, serum stability, tissue uptake and kinetics and is a novel liposome formulation for multifunctional theranostic applications.

A modified TEW approach to scatter correction for In-111 and Tc-99m dual-isotope small-animal SPECT.

PURPOSE: In dual-isotope (Tc-99m/In-111) small-animal single-photon emission computed tomography (SPECT), quantitative accuracy of Tc-99m activity measurements is degraded due to the detection of Compton-scattered photons in the Tc-99m photopeak window, which originate from the In-111 emissions (cross talk) and from the Tc-99m emission (self-scatter). The standard triple-energy window (TEW) estimates the total scatter (self-scatter and cross talk) using one scatter window on either side of the Tc-99m photopeak window, but the estimate is biased due to the presence of unscattered photons in the scatter windows. The authors present a modified TEW method to correct for total scatter that compensates for this bias and evaluate the method in phantoms and in vivo. METHODS: The number of unscattered Tc-99m and In-111 photons present in each scatter-window projection is estimated based on the number of photons detected in the photopeak of each isotope, using the isotope-dependent energy resolution of the detector. The camera-head-specific energy resolutions for the 140 keV Tc-99m and 171 keV In-111 emissions were determined experimentally by separately sampling the energy spectra of each isotope. Each sampled spectrum was fit with a Linear + Gaussian function. The fitted Gaussian functions were integrated across each energy window to determine the proportion of unscattered photons from each emission detected in the scatter windows. The method was first tested and compared to the standard TEW in phantoms containing Tc-99m:In-111 activity ratios between 0.15 and 6.90. True activities were determined using a dose calibrator, and SPECT activities were estimated from CT-attenuation-corrected images with and without scatter-correction. The method was then tested in vivo in six rats using In-111-liposome and Tc-99m-tetrofosmin to generate cross talk in the area of the myocardium. The myocardium was manually segmented using the SPECT and CT images, and partial-volume correction was performed using a template-based approach. The rat heart was counted in a well-counter to determine the true activity. RESULTS: In the phantoms without correction for Compton-scatter, Tc-99m activity quantification errors as high as 85% were observed. The standard TEW method quantified Tc-99m activity with an average accuracy of -9.0% ± 0.7%, while the modified TEW was accurate within 5% of truth in phantoms with Tc-99m:In-111 activity ratios ≥0.52. Without scatter-correction, In-111 activity was quantified with an average accuracy of 4.1%, and there was no dependence of accuracy on the activity ratio. In rat myocardia, uncorrected images were overestimated by an average of 23% ± 5%, and the standard TEW had an accuracy of -13.8% ± 1.6%, while the modified TEW yielded an accuracy of -4.0% ± 1.6%. CONCLUSIONS: Cross talk and self-scatter were shown to produce quantification errors in phantoms as well as in vivo. The standard TEW provided inaccurate results due to the inclusion of unscattered photons in the scatter windows. The modified TEW improved the scatter estimate and reduced the quantification errors in phantoms and in vivo.

Evaluation of Apoptosis with 99mTc-rhAnnexin V-128 and Inflammation with 18F-FDG in a Low-Dose Irradiation Model of Atherosclerosis in Apolipoprotein E-Deficient Mice.

Low-dose radiation in apolipoprotein E-deficient (ApoE-/-) mice has a protective effect with less subsequent atherosclerosis. Inflammation and apoptosis play major roles in the development of atherosclerosis. We evaluated the temporal pattern of the development of histologic atherosclerosis, inflammation with 18F-FDG, and apoptosis with 99mTc-rhAnnexin V-128 at 3 time points. METHODS: ApoE-/- mice were fed a high-fat diet, exposed to low-dose 60Co γ-radiation of 25 mGy at 2 mo of age, and evaluated within 1 wk (2-mo group), 1 mo (3-mo group), and 2 mo (4-mo group) from the time of radiation. Mice were divided into 3 subgroups and each received 18F-FDG, 99mTc-rhAnnexin V-128, or no radiotracer for autoradiography. Mice underwent euthanasia and aortic root dissection. The extent of atherosclerosis was determined by en face and Oil red O imaging. Aortic arch inflammation (18F-FDG) and apoptosis (99mTc-rhAnnexin V-128) were determined with digital autoradiography. Aortic sinus sections were stained with Sudan IV for assessment of lesion area and stage, antiCD68 antibody for inflammation and anti-cleaved-caspase 3 antibody for apoptosis. RESULTS: The extent of aortic atherosclerosis increased from 2 to 3 mo and from 3 to 4 mo. Inflammation (CD68) decreased and apoptosis (anti-cleaved-caspase 3 antibody) increased in aortic sinus slices measured as percentage of lesion by 4 mo. With increasing lesion stage, lesion inflammation decreased and lesion apoptosis increased. Aortic arch inflammation (18F-FDG uptake) did not differ over time and did not correlate with average lesion stage. However, aortic arch apoptosis (99mTc-rhAnnexin V-128) increased significantly by 4 mo and correlated with average lesion stage. There were no differences between the treatment subgroups (18F-FDG, 99mTc-rhAnnexin V-128, or no radiotracer). CONCLUSION: The temporal pattern of development of inflammation and apoptosis differ during the development of atherosclerosis in ApoE-/- mice treated with low-dose radiation. Advanced lesions are characterized by increased apoptosis and either less or similar amounts of inflammation, shown on immunohistochemistry and autoradiography. Treatment with radiotracers had no significant effects on extent of atherosclerosis, inflammation, or apoptosis.

Reduced dose measurement of absolute myocardial blood flow using dynamic SPECT imaging in a porcine model.

PURPOSE: Absolute myocardial blood flow (MBF) and myocardial flow reserve (MFR) measurements provide important additional information over traditional relative perfusion imaging. Recent advances in camera technology have made this possible with single-photon emission tomography (SPECT). Low dose protocols are desirable to reduce the patient radiation risk; however, increased noise may reduce the accuracy of MBF measurements. The authors studied the effect of reducing dose on the accuracy of dynamic SPECT MBF measurements. METHODS: Nineteen 30-40 kg pigs were injected with 370 + 1110 MBq of Tc-99m sestamibi or tetrofosmin or 37 + 111 MBq of Tl-201 at rest + stress. Microspheres were injected simultaneously to measure MBF. The pigs were imaged in list-mode for 11 min starting at the time of injection using a Discovery NM 530c camera (GE Healthcare). Each list file was modified so that 3/4, 1/2, 1/4, 1/8, 1/16, and 1/32 of the original counts were included in the projections. Modified projections were reconstructed with CT-based attenuation correction and an energy window-based scatter correction and analyzed with FlowQuant kinetic modeling software using a 1-compartment model. A modified Renkin-Crone extraction function was used to convert the tracer uptake rate K1 to MBF values. The SPECT results were compared to those from microspheres. RESULTS: Correlation between SPECT and microsphere MBF values for the full injected activity was r ≥ 0.75 for all 3 tracers and did not significantly degrade over all count levels. The mean MBF and MFR and the standard errors in the estimates were not significantly worse than the full-count data at 1/4-counts (Tc99m-tracers) and 1/2-counts (Tl-201). CONCLUSIONS: Dynamic SPECT measurement of MBF and MFR in pigs can be performed with 1/4 (Tc99m-tracers) or 1/2 (Tl-201) of the standard injected activity without significantly reducing accuracy and precision.