Faculty Bibliography

Objectives: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by selective degeneration of both upper and lower motor neurons, resulting in paralysis of skeletal muscle and respiratory failure, with death occurring within 2-5 years of diagnosis. 90% of cases are sporadic, and of the 10% that are familial more than 20 genes (>150 mutations) have been found to be associated with ALS, most notably copper/zinc superoxide dismutase (SOD1). SOD1 mutant proteins are believed to cause toxicities in degenerating neurons. Studies suggest that the neuronal and non-neuronal cell contributions to the onset and progression of ALS are complex. It was proposed that there are two phases of neuroinflammation in the spinal cord - the first being an early neuroprotective phase followed by a second late neurotoxic phase. One of the challenges in the study of neuroinflammation is that it is difficult to serially track the disease process, as there are no bona fide biomarkers for onset and progression in ALS. For this reason, we use PET with [¹¹C]PBR28 to track microglial neuroinflammation in the brain and spinal cord. We and others have shown that the receptor for advanced glycation end products (RAGE) is highly expressed in human ALS spinal cord, particularly in microglia, and to an increased degree compared to that of age-matched control subjects. Our previous studies with myeloid/microglia deletion of Ager and treatment with sRAGE (soluble RAGE) suggested that RAGE impairs survival and motor function in Sod1G93Amice. The ultimate goal is to test the hypothesis that RAGE inhibition in either initiation or progression phases of the ALS will prolong survival and maintain motor function in adult Sod1G93Amice.
Method(s): MicroPET/CT (Inveon, Siemens) with [¹¹C]PBR28 was used to track and compare microglial neuroinflammation in the brain and spinal cord of WT vs. ALS mice (110-120 day old), also after treatment with RAGE inhibitors (subject identity was blind to study investigator and data analyst). Using IRW (Inveon Research Workplace, Siemens), several ROIs in the thoracic and lumbar spinal cord (T13, L1, L2, L3) were drawn on the fused PET/CT images to obtain the regional SUVs. An automated atlasbased methodology using Firevoxel (https://urldefense.proofpoint.com/v2/url?u=https-3A__wp.nyu.edu_Firevoxel&d=DwIBAg&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=CY_mkeBghQnUPnp2mckgsNSbUXISJaiBQUhM-Uz9W58&m=_uGsTvUTTD_GxqvwK245ZUiiSbzVraIboytFijFDOwU&s=RlC-AQtmqr84rzBwvDmgK_FCVdvbCfsFvuN-dVODTpM&e= ) that we previously developed was used for brain mapping and segmentation to derive regional timeactivity curves (TAC) and SUVs for 20 brain regions.
Result(s): Dynamic regional SUV [¹¹C]PBR28 binding data were obtained and averaged SUVs derived from the last 5 frames (with steady and less variable intensity levels) were compared. Results derived from both spinal cord and brain regions displayed a similar trend with two obvious clusters. Reduced binding was observed for ALS group as compared to WT. RAGE inhibitor-treated ALS mi ce showed increased binding (brain SUV avg. 0.402+/-0.0382 over 20 ROIs) as compared to vehicle-treated (0.157+/-0.0339), suggesting that RAGE inhibition may contribute to the restoration of homeostasis in ALS animals (i.e., their bindings after treatment were closer to those in WT (0.485+/-0.171)). Notably, hypothalamus, brain stem, and olfactory bulb consistently exhibited higher binding, suggesting their role in this regulation.
Conclusion(s): Inconsistent outcomes have been reported in the literature when comparing TSPO ligand binding for imaging neuroinflammation. Our data are consistent with findings from several recent studies; i.e., reduced PBR28 binding was associated with disease state (e.g., in patients with PTSD or alcoholism). A notion that the reduced binding might reflect competition from endogenous TSPO ligands such as cholesterol can't be excluded. The strategies described here will test the hypothesis that pharmacological antagonism of RAGE signal transduction in either initiation or progression phases of the ALS will prolong survival and maintain motor function in adult Sod1G93Amice

The cell cycle is a progression of four distinct phases (G1, S, G2, M), with various cycle proteins being essential in regulating this process. In breast cancer, alterations in the cell cycle and uncontrolled proliferation led to several studies interrogating the relationship between cyclins and their counterpart cyclin-dependent-kinases (CDKs). We aimed to develop a radiolabeled CDK4/6 inhibitor for breast cancer imaging. Our transfluorinated analog ([¹⁸F]-CDKi) was evaluated and validated as a novel PET imaging agent to quantify CDK4/6 expression in ER-positive HER2-negative breast cancer. Methods: [¹⁸F]-CDKi was synthesized and assayed for its inhibitory activities against CDK4/6 kinases. [¹⁸F]-CDKi was prepared with a 2-step automated synthetic strategy that yielded the final product with remarkable purity and molar activity. In vitro and in vivo biologic activity and specificity was assessed in a MCF-7 cell line and in mice bearing MCF-7 breast tumors. Non radioactive Palbociclib (Inbrance, Pfizer®) was used as blocking agent to investigate the binding specificity and selectivity of [¹⁸F]-CDKi. Results: To generate [¹⁸F]-CDKi, we introduced an F-18 prosthetic group (¹⁸F-fluorobenzoic acid, [¹⁸F-FBA]), and transformed palbociclib into a different, PET active functional molecule. [¹⁸F]-CDKi was obtained with an overall radiochemical uncorrected yield of 15% and radiochemical purity > 98 %. The total synthesis time from the start of synthesis to final injectable formulated tracer is 70 minutes. The retention time reported for [¹⁸F]-CDKi and [¹⁹F]-CDKi is 27.4 min as demonstrated by co-injection with [¹⁹F]-CDKi in a HPLC. The first experiments aimed to analyze pharmacokinetics (PK) and in vitro activity of [¹⁸F]-CDKi (figure 5, Blood HL). In vivo blood half-life [t1/2(weighted) = 7.03 minutes], and octanol/water phase partition coefficient (logDO/W = 1.91 ± 0.24) showed a mainly lipophilic behavior. [¹⁸F]-CDKi is stable in vitro and in vivo (>98% at 4h post injection) and maintained its potent targeting affinity to CDK4/6 (~¹³NM for CDK4 and ~15nM for CDK6). Cellular uptake experiments performed in MCF-7 breast cancer cell line (ER-positive/HER2-negative) demonstrated specific uptake with a maximum intracellular concentration of ~65% as early as 10 minutes post incubation. The tracer uptake was reduced to <5% when cells were co-incubated with a molar excess of Palbociclib. In vivo imaging and ex-vivo biodistribution of ER-positive/HER-2 negative MCF-7 breast cancer models showed a ~4%ID/g tumor specific uptake (reduced to ~0.3%ID/g with a 50-fold excess of cold palbociclib). A comprehensive biodistribution analysis revealed also a significantly lower activation of CDK4/6 in non-targeting organs. Conclusion: [¹⁸F]-CDKi represents the first ¹⁸F positron emission tomography (PET) CDK4/6 imaging agent and a promising imaging agent for ER-positive HER2-negative breast cancer.

OBJECTIVES/OBJECTIVE:This study sought to develop an integrative positron emission tomography (PET) with magnetic resonance imaging (MRI) procedure for accurate atherosclerotic plaque phenotyping, facilitated by clinically approved and nanobody radiotracers. BACKGROUND:Noninvasive characterization of atherosclerosis remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated. METHODS:mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography, and histological analyses. RESULTS:F-NaF) PET, respectively. We observed an increase in all the aforementioned measures as disease progressed, and the imaging signatures correlated with histopathological features. CONCLUSIONS:We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MRI protocol that allows noninvasive assessment of different processes relevant to atherosclerosis progression. This approach allows the multiparametric study of atherosclerosis and can aid in early stage anti-atherosclerosis drug trials.

Nanomedicine research produces hundreds of studies every year, yet very few formulations have been approved for clinical use. This is due in part to a reliance on murine studies, which have limited value in accurately predicting translational efficacy in larger animal models and humans. Here, we report the scale-up of a nanoimmunotherapy from mouse to large rabbit and porcine atherosclerosis models, with an emphasis on the solutions we implemented to overcome production and evaluation challenges. Specifically, we integrated translational imaging readouts within our workflow to both analyze the nanoimmunotherapeutic's in vivo behavior and assess treatment response in larger animals. We observed our nanoimmunotherapeutic's anti-inflammatory efficacy in mice, as well as rabbits and pigs. Nanoimmunotherapy-mediated reduction of inflammation in the large animal models halted plaque progression, supporting the approach's translatability and potential to acutely treat atherosclerosis.

Objectives: There is an urgent need for the development of Parkinson's disease (PD) treatments that can slow disease progression. LRRK2 (leucine-rich repeat kinase 2) has recently been identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Several LRRK2 inhibitors have been developed and evaluated in vitro; however, in vivo target engagement has never been characterized. Despite research efforts invested to date, there is no radiotracer available for in vivo imaging of LRRK2 using PET. In our pilot studies, we synthesized and evaluated two tritium-labeled potent and selective kinase inhibitors, [³H]LRRK2-IN-1 (1^st generation) and [³H]GNE-9605 (second-generation LRRK2 inhibitors), via in vitro (IC50, Kd, Bmax) and in vivo/ex vivo methodologies (autoradiography, bio-distribution, and blocking experiments) in rodents and human striatum tissues. Comparative studies indicated that, although LRRK2-IN-1 has a lower DELTAG (lower free binding energy to the target enzyme determined via docking studies) than GNE-9605, GNE-9605 is more CNS permeable due to its higher lipophilicity than LRRK2-IN-1, suggesting more promising properties for ligands derived from second-generation LRRK2 inhibitors. We have, since then, identified candidates that are more potent and selective than current known LRRK2 inhibitors, based on in vitro assays. Radiolabeling and microPET evaluation studies are ongoing, preliminary results will be presented.
Method(s): We have prepared and evaluated several novel LRRK2 inhibitors and compared their in vitro properties (e.g., IC50 values,membrane permeability, and the P-glycoprotein liability) with those previously developed LRRK2 inhibitors, such as GNE compounds. We have also synthesized several corresponding precursors and carried out radiolabeling with either C-11 or F-18 to obtain the desired target molecules for further evaluation of their in vivo properties as PET ligands via microPET/CT studies, including in vivo/ex vivo bio-distribution and blocking studies.
Result(s): Several in vitro assays were conducted to compare IC50 values (for example, in HEK293 cells with transient overexpression of LRRK2 G2019S measuring decrease of phosphoserine 935 with an antibody in a robust Meso Scale Discovery assay) for LRRK2-a and LRRK2-b (novel inhibitors) vs. GNE-7915 and GNE-9605. The results are consistently indicated that LRRK2-b (10 nM) is more potent and selective than GNE-7915 (40 nM), and GNE-9605 (90 nM). The results of the low efflux ratio (<3) from the CNS permeability measurement via MDR assay in the absence or presence of a Pgp inhibitor suggested that LRRK2-b is a promising candidate as its BBB permeability may not be a concern and it is not a good Pgp substrate. Radiolabeling of LRRK2-a and LRRK2-b with F-18 has been accomplished via one-step fluoro-for-tosyl radiosynthesis. In vivo microPET/CT evaluation studies in mice are underway.
Conclusion(s): Potential in vivo imaging of LRRK2 with PET is an exciting, but at the same time uncharted research area, limited thus far by a lack of relevant information, resources and tools. Based on the in vitro and in vivo/ex vivo results, we have identified several candidates and will fine-tune the structure-activity relationship (SAR) to generate promising PET ligand candidates for in vivo imaging of brain LRRK2

Introduction: Breast cancer is a worldwide health issue with about 1 million new cases every year. Dysregulated cellular proliferation, a common feature to all human cancers, is a key player of aberrant proliferative signaling and is therefore considered as a "hallmark of cancer. In breast cancer, a lot of attention focuses on particular members of the cell cycle machinery, the D-type cyclins and their partner cyclin-D kinases 4 and 6 (CDK4/6). CDKs are serine/threonine kinases key in regulating cell cycle progression by associating with cyclins. Several studies have identified alterations of cell cycle regulators in human breast cancer and provide a strong rationale for a therapeutic role for CDK4/6 inhibition in this tumor type. Amplification of the cyclin-D1 occurs in about 20% of human breast cancers, while overexpression of the protein is above 60%. The subtype for which CDK4/6 inhibition has the strongest rationale is estrogen receptor (ER)-positive disease. These subtypes almost always retain Rb function, thereby CDK4 and CDK6 targeting agents can block pRb phosphorylation (in low nanomolar concentration) and induce G1 arrest in sensitive cell lines. It is often controversial whether CDK4/6 inhibitors are capable to prolong overall survival. Moreover, it is urgent to find a way to select which patients are most likely to benefit from these drugs and to monitor, non-invasively, the progress of the disease and the overall treatment response. To this aim, we developed a PET imaging agent ([¹⁸F]-CDKi) as an in vivo PET reporter of CDK4/6 status with the final aim to improve efficacy of breast cancer therapy. To generate our fluorine-18 inhibitor, we introduced an F-18 prosthetic group (¹⁸F-fluorobenzoic acid, [¹⁸F-FBA]), on the terminal piperazine and transformed palbociclib (Inbrance, Pfizer) into a different PET active functional molecule. The first in vitro experiment aiming to analyze pharmacokinetics (PK) and in vitro activity revealed that [¹⁸F]-CDKican be a successful PET agent with nearly ideal imaging characteristics. Moreover, we demonstrated that [¹⁸F]-CDKi is stable in vitro and in vivo (>98% at 4h post injection) and maintained a potent targeting affinity to CDK4/6. Cellular uptake experiments performed in MCF-7 breast cancer cell line (ER-positive/HER2-negative) demonstrated specific uptake. Similar significant uptake values were also observed in biodistributed MCF-7 bearing mouse models. The strong activation of CDK4/6 in cancer cells in concert with its low activation in untransformed healthy cells makes [¹⁸F]-CDKi a nearly ideal imaging agent for the early detection of malignant growth of the breast. Moreover, we also hypothesize it could be an excellent PET imaging agent for metastatic breast cancer due to their high proliferative rate. CDKi represents the first of a new generation of PET imaging agents critical to study how cancer cells escape the cell cycle arrest and develop resistance to conventional treatment

The DNA repair enzyme PARP1 is over-expressed in glioblastoma, with overall low expression in healthy brain tissue. Paired with the availability of specific molecularly targeted small molecules for this biomarker, PARP1 is a near-ideal target for novel radiotherapeutics. A successful PARP1-targeted radiotherapeutic would induce DNA damage and apoptosis in cancer cells, while sparing healthy brain tissue. We synthesized a ¹³¹I-labeled poly(ADP-ribose) polymerase 1 (PARP1) therapeutic and investigated its pharmacology using in vitro and in vivo methodologies. A subcutaneous mouse model was used to quantify retention times and therapeutic efficacy. A potential clinical scenario, intratumoral convection-enhanced delivery (CED), was mimicked using an orthotopic glioblastoma model combined with an implanted osmotic pump system to study local administration of [¹³¹I]PARPi. [¹³¹I]PARPi is a 1(2H)-phthalazinone, similar in structure to the FDA-approved PARP inhibitor Olaparib (Lynparza, Astra-Zeneca). In vitro studies have shown that [¹³¹I]PARPi and Olaparib share similar pharmacologic profiles. [¹³¹I]PARPi delivers 134.1 cGy/MBq intratumoral injected activity; doses to non-target tissues, including liver and kidney, were significantly lower. Radiation damage and cell death could be shown by p53 activation via bioluminescence imaging in U87 MG cells transfected with a p53-bioluminescent reporter in treated tumors. Treated mice had significantly longer survival than mice receiving only vehicle (29 vs. 22 days, P < 0.005) in a subcutaneous model. CED demonstrated efficient retention of [¹³¹I]PARPi in orthotopic brain tumors, while quickly clearing from healthy brain tissue. We validated the novel PARP1-targeted radiotherapeutic [¹³¹I]PARPi, studying its performance in mouse models. Our results demonstrate [¹³¹I]PARPi's high potential as a therapeutic, and highlight PARP's relevance as a target for radionuclide therapy. Radiation plays an integral role in brain tumor therapy, and radiolabeled PARP therapeutics could ultimately lead to improvements in the standard of care.

RATIONALE: Ataxia telangiectasia and Rad3-related (ATR) threonine serine kinase is one of the key elements in orchestrating the DNA damage response (DDR). As such, inhibition of ATR can amplify the effects of chemo- and radiation-therapy, and several ATR inhibitors (ATRi) have already undergone clinical testing in cancer. For more accurate patient selection, monitoring and staging, real-time in vivo imaging of ATR could be invaluable; the development of appropriate imaging agents has remained a major challenge. METHODS: 3-amino-N-(4-[18F]phenyl)-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carboxamide ([18F]-ATRi), a close analogue of Ve-821, (a clinical ATRi candidate), was readily accomplished similarly to already established synthetic procedures. Structurally, 18F was introduced at the 4-position of the aromatic ring of Ve-821 for generating a labeled ATR inhibitor. In vitro experiments were conducted in U251 MG glioblastoma cell lines and ex vivo biodistribution were performed in subcutaneous U251 MG xenograft bearing athymic nude mice following microPET imaging. RESULTS: [18F]-ATRi has a similar pharmacokinetic profile to that of Ve-821. Using an U251 MG glioblastoma mouse model, we evaluated the in vivo binding efficiency of [18F]-ATRi. Blood and tumor showed a statistically significant difference between mice injected with only the probe or following blocking experiment with Ve-821 (1.48+/-0.40%ID/g vs. 0.46+/-0.12%ID/g in tumor and 1.85+/-0.47%ID/g vs. 0.84+/-0.3%ID/g in blood respectively). CONCLUSIONS: [18F]-ATRi represents the first 18F positron emission tomography (PET) ATR imaging agent, and is designed on a low nanomolar and clinically relevant ATR inhibitor.

Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a universally poor prognosis. Here, we characterize on a positron emission tomography (PET) probe for imaging DIPG in vivo. In human histological tissues, the probes target, poly(ADP)ribose polymerase 1 (PARP1), was highly expressed in DIPG compared to normal brain. PET imaging allowed for the sensitive detection of DIPG in a genetically engineered mouse model (GEMM), and probe uptake correlated to histologically determined tumor infiltration. Imaging with the sister fluorescence agent revealed that uptake was confined to proliferating, PARP1 expressing cells. Comparison to other imaging technologies revealed remarkable accuracy of our biomarker approach. We subsequently demonstrated that serial imaging of DIPG in mouse models enables monitoring of tumor growth, as shown in modeling of tumor progression. Overall, this validated method for quantifying DIPG burden would serve useful in monitoring treatment response in early phase clinical trials.

Tissue macrophage numbers vary during health versus disease. Abundant inflammatory macrophages destruct tissues, leading to atherosclerosis, myocardial infarction and heart failure. Emerging therapeutic options create interest in monitoring macrophages in patients. Here we describe positron emission tomography (PET) imaging with ¹⁸F-Macroflor, a modified polyglucose nanoparticle with high avidity for macrophages. Due to its small size, Macroflor is excreted renally, a prerequisite for imaging with the isotope flourine-18. The particle's short blood half-life, measured in three species, including a primate, enables macrophage imaging in inflamed cardiovascular tissues. Macroflor enriches in cardiac and plaque macrophages, thereby increasing PET signal in murine infarcts and both mouse and rabbit atherosclerotic plaques. In PET/magnetic resonance imaging (MRI) experiments, Macroflor PET imaging detects changes in macrophage population size while molecular MRI reports on increasing or resolving inflammation. These data suggest that Macroflor PET/MRI could be a clinical tool to non-invasively monitor macrophage biology.