In vivo SPECT imaging of Tc-99 m radiolabeled exosomes from human umbilical-cord derived mesenchymal stem cells in small animals.

Extracellular vesicles derived from human umbilical cord-derived mesenchymal stem cells (UCMSC-EVs) have been postulated to have therapeutic potential for various diseases. However, the biodistribution and pharmacokinetics of these vesicles are still unclear. For a better understanding of the in vivo properties of UCMSC-EVs, in the present study, these vesicles were first radiolabeled with Technetium-99 m (99mTc-UCMSC-EVs) and evaluated using in vivo single photon emission computed tomography (SPECT) imaging and biodistribution experiments. SPECT images demonstrated that the liver and spleen tissues mainly took up the 99mTc-UCMSC-EVs. The biodistribution study observed slight uptake in the thyroid and stomach, indicating that 99mTc-UCMSC-EVs was stable at 24 h in vivo. The pharmacokinetic analyses of the blood half-life demonstrated the quick distribution phase (0.85 ±â€¯0.28 min) and elimination phase (25.22 ±â€¯20.76 min) in mice. This study provides a convenient and efficient method for 99mTc-UCMSC-EVs preparation without disturbing their properties. In conclusion, the biodistribution, quick elimination, and suitable stability in vivo of 99mTc-UCMSC-EVs were quantified by the noninvasive imaging and pharmacokinetic analyses, which provides useful information for indication selection, dosage protocol design, and toxicity assessment in future applications.

Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search.

PURPOSE: Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. METHODS: Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. RESULTS: In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aß42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). CONCLUSION: Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aß and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

Glycolytic Plasticity of Metastatic Lung Cancer Captured by Noninvasive 18F-FDG PET/CT and Serum 1H-NMR Analysis: An Orthotopic Murine Model Study.

We aim to establish a noninvasive diagnostic platform to capture early phenotypic transformation for metastasis using 18F-FDG PET and 1H-NMR-based serum metabolomics. Mice with implantation of NCI-H460 cells grew only primary lung tumors in the localized group and had both primary and metastatic lung tumors in the metastatic group. The serum metabolites were analyzed using 1H-NMR at the time of PET/CT scan. The glycolysis status and cell proliferation were validated by Western blotting and staining. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic accuracy of SUVmean and serum metabolites in metastasis. In the metastatic mice, the SUVmean of metastatic tumors was significantly higher than that of primary lung tumors in PET images, which was supported by elevated glycolytic protein expression of HK2 and PKM2. The serum pyruvate level in the metastatic group was significantly lower than that in the localized group, corresponding to increased pyruvate-catalyzed enzyme and proliferation rates in metastatic tumors. In diagnosing localized or metastatic tumors, the areas under the ROC curves of SUVmean and pyruvate were 0.92 and 0.91, respectively, with p < 0.05. In conclusion, the combination of 18F-FDG PET and 1H-NMR-based serum metabolomics demonstrated the feasibility of a glycolytic platform for diagnosing metastatic lung cancers.

Characterization of Sigma-2 Receptor-Specific Binding Sites Using [3H]DTG and [125I]RHM-4.

The sigma-2 receptor/transmembrane protein 97 (σ2R/TMRM97) is a promising biomarker of tumor proliferation and a target for cancer therapy. [3H]DTG has been used to evaluate σ2R/TMEM97 binding affinity in compound development studies. However, [3H]DTG has equal and moderate binding affinities to both sigma 1 receptor (σ1R) and σ2R/TMEM97. Furthermore, co-administration with the σ1R masking compound (+)-pentazocine may cause bias in σ2R/TMEM97 binding affinity screening experiments. We have developed a radioiodinated ligand, [125I]RHM-4, which has high affinity and selectivity for σ2R/TMEM97 versus σ1R. In this study, a head-to-head comparison between [3H]DTG and [125I]RHM-4 on the binding affinity and their effectiveness in σ2R/TMEM97 compound screening studies was performed. The goal of these studies was to determine if this radioiodinated ligand is a suitable replacement for [3H]DTG for screening new σ2R/TMEM97 compounds. Furthermore, to delineate the binding properties of [125I]RHM-4 to the σ2R/TMEM97, the structure of RHM-4 was split into two fragments. This resulted in the identification of two binding regions in the σ2R, the "DTG" binding site, which is responsible for binding to the σ2R/TMEM97, and the secondary binding site, which is responsible for high affinity and selectivity for the σ2R/TMEM97 versus the σ1R. The results of this study indicate that [125I]RHM-4 is an improved radioligand for in vitro binding studies of the σ2R/TMEM97 versus [3H]DTG.

Bone Morphogenetic Protein-, Antimicrobial Agent-, and Analgesic-Incorporated Nanofibrous Scaffolds for the Therapy of Alveolar Clefts.

An alveolar cleft is a bone defect in the maxillary arch. Although the use of autologous iliac bone grafts to repair alveolar clefts is the preferred treatment method, donor-site morbidity remains a concern. In this study, we incorporated bone morphogenetic protein (BMP), an antimicrobial agent, and an analgesic into nanofibrous scaffolds for alveolar cleft therapy. Three-dimensional (3D) printing and coaxial electrospinning techniques were used to fabricate the scaffolds. BMP-2, ketorolac, and amoxicillin were used as the growth factor, analgesic, and antimicrobial agent, respectively. The in vitro properties of the nanofibrous scaffolds were characterized, and in vivo efficacy was evaluated in a rat alveolar-cleft model. The empirical data indicated that the biomolecule-incorporated scaffolds offered extended discharge of BMP-2, amoxicillin, and ketorolac for >4 weeks. The animal test outcomes also demonstrated favorable bone healing at the cleft site. Biomolecule- and drug-incorporated nanofibrous scaffolds demonstrated their efficacy in alveolar cleft treatment.

The Sigma-2 Receptor/TMEM97, PGRMC1, and LDL Receptor Complex Are Responsible for the Cellular Uptake of Aß42 and Its Protein Aggregates.

Our lab has recently shown that the Sigma-2 Receptor/Transmembrane Protein 97 (TMEM97) and Progesterone Receptor Membrane Component 1 (PGRMC1) form a complex with the Low Density Lipoprotein Receptor (LDLR), and this intact complex is required for efficient uptake of lipoproteins such as LDL and apolipoprotein E (apoE). These receptors are expressed in the nervous system where they have implications in neurodegenerative diseases such as Alzheimer's disease (AD), where apoE is involved in neuronal uptake and accumulation of Aß42, eventually cascading into neurodegeneration, synaptic dysfunction, and ultimately, dementia. We hypothesize that the intact Sigma-2 receptor complex-TMEM97, PGRMC1, and LDLR-is necessary for internalization of apoE and Aß42 monomers (mAß42) and oligomers (oAß42), and the disruption of the receptor complex inhibits uptake. The results of this study suggest that the intact Sigma-2 receptor complex is a binding site for mAß42 and oAß42, in the presence or absence of apoE2, apoE3, and apoE4. The loss or pharmacological inhibition of one or both of these proteins results in the disruption of the complex leading to decreased uptake of mAß42 and oAß42 and apoE in primary neurons. The TMEM97, PGRMC1, and LDLR complex is a pathway for the cellular uptake of Aß42 via apoE dependent and independent mechanisms. This study suggests that the complex may potentially be a novel pharmacological target to decrease neuronal Aß42 internalization and accumulation, which may represent a new strategy for inhibiting the rate of neurotoxicity, neurodegeneration, and progression of AD.

Characterization of 18F-PM-PBB3 (18F-APN-1607) Uptake in the rTg4510 Mouse Model of Tauopathy.

Misfolding, aggregation, and cerebral accumulation of tau deposits are hallmark features of Alzheimer's disease. Positron emission tomography study of tau can facilitate the development of anti-tau treatment. Here, we investigated a novel tau tracer 18F-PM-PBB3 (18F-APN-1607) in a mouse model of tauopathy. Dynamic PET scans were collected in groups of rTg4510 transgenic mice at 2-11 months of age. Associations between distribution volume ratios (DVR) and standardized uptake value ratios (SUVR) with cerebellum reference were used to determine the optimal scanning time and uptake pattern for each age. Immunohistochemistry staining of neurofibrillary tangles and autoradiography study was performed for ex vivo validation. An SUVR 40-70 min was most consistently correlated with DVR and was used in further analyses. Significant increased 18F-PM-PBB3 uptake in the brain cortex was found in six-month-old mice (+28.9%, p < 0.05), and increased further in the nine-month-old group (+38.8%, p < 0.01). The trend of increased SUVR value remained evident in the hippocampus and striatum regions except for cortex where uptake becomes slightly reduced in 11-month-old animals (+37.3%, p < 0.05). Radioactivity distributions from autoradiography correlate well to the presence of human tau (HT7 antibody) and hyperphosphorylated tau (antibody AT8) from the immunohistochemistry study of the adjacent brain sections. These findings supported that the 40-70 min 18F-PM-PBB3 PET scan with SUVR measurement can detect significantly increased tau deposits in a living rTg4510 transgenic mouse models as early as six-months-old. The result exhibited promising dynamic imaging capability of this novel tau tracer, and the above image characteristics should be considered in the design of longitudinal preclinical tau image studies.

Identification of a nanomolar affinity α-synuclein fibril imaging probe by ultra-high throughput in silico screening.

Small molecules that bind with high affinity and specificity to fibrils of the α-synuclein (αS) protein have the potential to serve as positron emission tomography (PET) imaging probes to aid in the diagnosis of Parkinson's disease and related synucleinopathies. To identify such molecules, we employed an ultra-high throughput in silico screening strategy using idealized pseudo-ligands termed exemplars to identify compounds for experimental binding studies. For the top hit from this screen, we used photo-crosslinking to confirm its binding site and studied the structure-activity relationship of its analogs to develop multiple molecules with nanomolar affinity for αS fibrils and moderate specificity for αS over Aß fibrils. Lastly, we demonstrated the potential of the lead analog as an imaging probe by measuring binding to αS-enriched homogenates from mouse brain tissue using a radiolabeled analog of the identified molecule. This study demonstrates the validity of our powerful new approach to the discovery of PET probes for challenging molecular targets.

Imaging CAR T Cell Trafficking with eDHFR as a PET Reporter Gene.

Cell-based therapeutics have considerable promise across diverse medical specialties; however, reliable human imaging of the distribution and trafficking of genetically engineered cells remains a challenge. We developed positron emission tomography (PET) probes based on the small-molecule antibiotic trimethoprim (TMP) that can be used to image the expression of the Escherichia coli dihydrofolate reductase enzyme (eDHFR) and tested the ability of [18F]-TMP, a fluorine-18 probe, to image primary human chimeric antigen receptor (CAR) T cells expressing the PET reporter gene eDHFR, yellow fluorescent protein (YFP), and Renilla luciferase (rLuc). Engineered T cells showed an approximately 50-fold increased bioluminescent imaging signal and 10-fold increased [18F]-TMP uptake compared to controls in vitro. eDHFR-expressing anti-GD2 CAR T cells were then injected into mice bearing control GD2- and GD2+ tumors. PET/computed tomography (CT) images acquired on days 7 and 13 demonstrated early residency of CAR T cells in the spleen followed by on-target redistribution to the GD2+ tumors. This was corroborated by autoradiography and anti-human CD8 immunohistochemistry. We found a high sensitivity of detection for identifying tumor-infiltrating CD8 CAR T cells, ∼11,000 cells per mm3. These data suggest that the [18F]-TMP/eDHFR PET pair offers important advantages that could better allow investigators to monitor immune cell trafficking to tumors in patients.

Correlation analysis of [18F]ROStrace using ex vivo autoradiography and dihydroethidium fluorescent imaging in lipopolysaccharide-treated animals.

Reactive oxygen species (ROS) are believed to play an important role in the proinflammatory form of neuroinflammation. Therefore, the availability of a radiotracer labeled with a positron-emitting radionuclide that can measure levels of ROS in tissue could provide a valuable method for imaging neuroinflammation in vivo with the functional imaging technique positron emission tomography (PET). We previously reported the synthesis and in vivo evaluation of [18F]ROStrace, a radiotracer for imaging ROS in vivo with PET, in an LPS model of neuroinflammation. In the current study, we conducted additional validation studies aimed at determining the cellular localization of this radiotracer in the same model. Our results indicate that [18F]ROStrace is primarily localized in microglia/macrophages and neurons in LPS-treated animals, and provide further support in the use of this radiotracer as a PET-based probe for imaging the proinflammatory form of neuroinflammation.

Targeting PARP-1 with Alpha-Particles Is Potently Cytotoxic to Human Neuroblastoma in Preclinical Models.

Alpha-emitters can be pharmacologically delivered for irradiation of single cancer cells, but cellular lethality could be further enhanced by targeting alpha-emitters directly to the nucleus. PARP-1 is a druggable protein in the nucleus that is overexpressed in neuroblastoma compared with normal tissues and is associated with decreased survival in high-risk patients. To exploit this, we have functionalized a PARP inhibitor (PARPi) with an alpha-emitter astatine-211. This approach offers enhanced cytotoxicity from conventional PARPis by not requiring enzymatic inhibition of PARP-1 to elicit DNA damage; instead, the alpha-particle directly induces multiple double-strand DNA breaks across the particle track. Here, we explored the efficacy of [211At]MM4 in multiple cancers and found neuroblastoma to be highly sensitive in vitro and in vivo Furthermore, alpha-particles delivered to neuroblastoma show antitumor effects and durable responses in a neuroblastoma xenograft model, especially when administered in a fractionated regimen. This work provides the preclinical proof of concept for an alpha-emitting drug conjugate that directly targets cancer chromatin as a therapeutic approach for neuroblastoma and perhaps other cancers.

Leveraging a Low-Affinity Diazaspiro Orthosteric Fragment to Reduce Dopamine D3 Receptor (D3R) Ligand Promiscuity across Highly Conserved Aminergic G-Protein-Coupled Receptors (GPCRs).

Previously, we reported a 3-(2-methoxyphenyl)-9-(3-((4-methyl-5-phenyl-4 H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (1) compound with excellent dopamine D3 receptor (D3R) affinity (D3R Ki = 12.0 nM) and selectivity (D2R/D3R ratio = 905). Herein, we present derivatives of 1 with comparable D3R affinity (32, D3R Ki = 3.2 nM, D2R/D3R ratio = 60) and selectivity (30, D3R Ki = 21.0 nM, D2R/D3R ratio = 934). Fragmentation of 1 revealed orthosteric fragment 5a to express an unusually low D3R affinity ( Ki = 2.7 µM). Compared to piperazine congener 31, which retains a high-affinity orthosteric fragment (5d, D3R Ki = 23.9 nM), 1 was found to be more selective for the D3R among D1- and D2-like receptors and exhibited negligible off-target interactions at serotoninergic and adrenergic G-protein-coupled receptors (GPCRs), common off-target sites for piperazine-containing D3R scaffolds. This study provides a unique rationale for implementing weakly potent orthosteric fragments into D3R ligand systems to minimize drug promiscuity at other aminergic GPCR sites.

TMEM97 and PGRMC1 do not mediate sigma-2 ligand-induced cell death.

Sigma-2 receptors have been implicated in both tumor proliferation and neurodegenerative diseases. Recently the sigma-2 receptor was identified as transmembrane protein 97 (TMEM97). Progesterone receptor membrane component 1 (PGRMC1) was also recently reported to form a complex with TMEM97 and the low density lipoprotein (LDL) receptor, and this trimeric complex is responsible for the rapid internalization of LDL. Sigma-2 receptor ligands with various structures have been shown to induce cell death in cancer cells. In the current study, we examined the role of TMEM97 and PGRMC1 in mediating sigma-2 ligand-induced cell death. Cell viability and caspase-3 assays were performed in control, TMEM97 knockout (KO), PGRMC1 KO, and TMEM97/PGRMC1 double KO cell lines treated with several sigma-2 ligands. The data showed that knockout of TMEM97, PGRMC1, or both did not affect the concentrations of sigma-2 ligands that induced 50% of cell death (EC50), suggesting that cytotoxic effects of these compounds are not mediated by TMEM97 or PGRMC1. Sigma-1 receptor ligands, (+)-pentazocine and NE-100, did not block sigma-2 ligand cytotoxicity, suggesting that sigma-1 receptor was not responsible for sigma-2 ligand cytotoxicity. We also examined whether the alternative, residual binding site (RBS) of 1,3-Di-o-tolylguanidine (DTG) could be responsible for sigma-2 ligand cytotoxicity. Our data showed that the binding affinities (K i) of sigma-2 ligands on the DTG RBS did not correlate with the cytotoxicity potency (EC50) of these ligands, suggesting that the DTG RBS was not fully responsible for sigma-2 ligand cytotoxicity. In addition, we showed that knocking out TMEM97, PGRMC1, or both reduced the initial internalization rate of a sigma-2 fluorescent ligand, SW120. However, concentrations of internalized SW120 became identical later in the control and knockout cells. These data suggest that the initial internalization process of sigma-2 ligands does not appear to mediate the cell-killing effect of sigma-2 ligands. In summary, we have provided evidence that sigma-2 receptor/TMEM97 and PGRMC1 do not mediate sigma-2 ligand cytotoxicity. Our work will facilitate elucidating mechanisms of sigma-2 ligand cytotoxicity.

Cell-Proliferation Imaging for Monitoring Response to CDK4/6 Inhibition Combined with Endocrine-Therapy in Breast Cancer: Comparison of [18F]FLT and [18F]ISO-1 PET/CT.

PURPOSE: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors in combination with endocrine-therapy have emerged as an important regimen of care for estrogen receptor (ER)-positive metastatic breast cancer, although identifying predictive biomarkers remains a challenge. We assessed the ability of two PET-proliferation tracers, [18F]FLT and [18F]ISO-1, for evaluating response to CDK4/6-inhibitor (palbociclib) and ER-antagonist (fulvestrant). EXPERIMENTAL DESIGN: To determine the effect of CDK4/6 inhibition combined with estrogen-blockade, we assessed cell proliferation in six breast cancer cell lines after 1, 3, and 6 days of treatment with palbociclib and/or fulvestrant. These data were correlated to in vitro radiotracer assays and results were verified by longitudinal [18F]FLT and [18F]ISO-1 micro-PET imaging performed in MCF7 tumor-bearing mice. RESULTS: All palbociclib-sensitive cell lines showed decreased [18F]FLT accumulation and S-phase depletion after treatment, with both measures augmented by combination therapy. In contrast, these cells showed changes in [18F]ISO-1 analogue-binding and G0 arrest only after prolonged treatment. MicroPET imaging of MCF7 xenografts showed a significant decrease in [18F]FLT but no changes in [18F]ISO-1 uptake in all treated mice on day 3. On day 14, however, mice treated with combination therapy showed a significant decrease in [18F]ISO-1, corresponding to G0 arrest, while maintaining reduced [18F]FLT uptake, which corresponded to S-phase depletion. CONCLUSIONS: Our data suggest complementary roles of [18F]FLT and [18F]ISO-1 PET in evaluating tumor-proliferation after combined CDK4/6 inhibitor and endocrine therapy in breast cancer. [18F]FLT is more sensitive to immediate changes in S-phase, whereas [18F]ISO-1 can assess more delayed changes related to cell-cycle arrest and transition to G0 quiescence from combination therapy. These data suggest a potential role for early prediction of long-term response using these imaging biomarkers.

Synthesis and evaluation of an AZD2461 [18F]PET probe in non-human primates reveals the PARP-1 inhibitor to be non-blood-brain barrier penetrant.

Poly(ADP-ribose)polymerase-1 inhibitor (PARPi) AZD2461 was designed to be a weak P-glycoprotein (P-gp) analogue of FDA approved olaparib. With this chemical property in mind, we utilized the AZD2461 ligand architecture to develop a CNS penetrant and PARP-1 selective imaging probe, in order to investigate PARP-1 mediated neuroinflammation and neurodegenerative diseases, such as Alzheimer's and Parkinson's. Our work led to the identification of several high-affinity PARPi, including AZD2461 congener 9e (PARP-1 IC50 = 3.9 ±â€¯1.2 nM), which was further evaluated as a potential 18F-PET brain imaging probe. However, despite the similar molecular scaffolds of 9e and AZD2461, our studies revealed non-appreciable brain-uptake of [18F]9e in non-human primates, suggesting AZD2461 to be non-CNS penetrant.

Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.

CRISPR/Cas gene studies were conducted in HeLa cells where either PGRMC1, TMEM97 or both proteins were removed via gene editing. A series of radioligand binding studies, confocal microscopy studies, and internalization of radiolabeled or fluorescently tagged LDL particles were then conducted in these cells. The results indicate that PGRMC1 knockout (KO) did not reduce the density of binding sites for the sigma-2 receptor (σ2R) radioligands, [125I]RHM-4 or [3H]DTG, but a reduction in the receptor affinity of both radioligands was observed. TMEM97 KO resulted in a complete loss of binding of [125I]RHM-4 and a significant reduction in binding of [3H]DTG. TMEM97 KO and PGRMC1 KO resulted in an equal reduction in the rate of uptake of fluorescently-tagged or 3H-labeled LDL, and knocking out both proteins did not result in a further rate of reduction of LDL uptake. Confocal microscopy and Proximity Ligation Assay studies indicated a clear co-localization of LDLR, PGRMC1 and TMEM97. These data indicate that the formation of a ternary complex of LDLR-PGRMC1-TMEM97 is necessary for the rapid internalization of LDL by LDLR.

Examination of Diazaspiro Cores as Piperazine Bioisosteres in the Olaparib Framework Shows Reduced DNA Damage and Cytotoxicity.

Development of poly(ADP-ribose) polymerase inhibitors (PARPi's) continues to be an attractive area of research due to synthetic lethality in DNA repair deficient cancers; however, PARPi's also have potential as therapeutics to prevent harmful inflammation. We investigated the pharmacological impact of incorporating spirodiamine motifs into the phthalazine architecture of FDA approved PARPi olaparib. Synthesized analogues were screened for PARP-1 affinity, enzyme specificity, catalytic inhibition, DNA damage, and cytotoxicity. This work led to the identification of 10e (12.6 ± 1.1 nM), which did not induce DNA damage at similar drug concentrations as olaparib. Interestingly, several worst in class compounds with low PARP-1 affinity, including 15b (4397 ± 1.1 nM), induced DNA damage at micromolar concentrations, which can explain the cytotoxicity observed in vitro. This work provides further evidence that high affinity PARPi's can be developed without DNA damaging properties offering potential new drugs for treating inflammatory related diseases.

A PET imaging agent for evaluating PARP-1 expression in ovarian cancer.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors are effective in a broad population of patients with ovarian cancer; however, resistance caused by low enzyme expression of the drug target PARP-1 remains to be clinically evaluated in this context. We hypothesize that PARP-1 expression is variable in ovarian cancer and can be quantified in primary and metastatic disease using a novel PET imaging agent. METHODS: We used a translational approach to describe the significance of PET imaging of PARP-1 in ovarian cancer. First, we produced PARP1-KO ovarian cancer cell lines using CRISPR/Cas9 gene editing to test the loss of PARP-1 as a resistance mechanism to all clinically used PARP inhibitors. Next, we performed preclinical microPET imaging studies using ovarian cancer patient-derived xenografts in mouse models. Finally, in a phase I PET imaging clinical trial we explored PET imaging as a regional marker of PARP-1 expression in primary and metastatic disease through correlative tissue histology. RESULTS: We found that deletion of PARP1 causes resistance to all PARP inhibitors in vitro, and microPET imaging provides proof of concept as an approach to quantify PARP-1 in vivo. Clinically, we observed a spectrum of standard uptake values (SUVs) ranging from 2-12 for PARP-1 in tumors. In addition, we found a positive correlation between PET SUVs and fluorescent immunohistochemistry for PARP-1 (r2 = 0.60). CONCLUSION: This work confirms the translational potential of a PARP-1 PET imaging agent and supports future clinical trials to test PARP-1 expression as a method to stratify patients for PARP inhibitor therapy. TRIAL REGISTRATION: Clinicaltrials.gov NCT02637934. FUNDING: Research reported in this publication was supported by the Department of Defense OC160269, a Basser Center team science grant, NIH National Cancer Institute R01CA174904, a Department of Energy training grant DE-SC0012476, Abramson Cancer Center Radiation Oncology pilot grants, the Marsha Rivkin Foundation, Kaleidoscope of Hope Foundation, and Paul Calabresi K12 Career Development Award 5K12CA076931.

Development of a Positron Emission Tomography Radiotracer for Imaging Elevated Levels of Superoxide in Neuroinflammation.

Reactive oxygen species (ROS) are believed to play a major role in the proinflammatory, M1-polarized form of neuroinflammation. However, it has been difficult to assess the role of ROS and their role in neuroinflammation in animal models of disease because of the absence of probes capable of measuring their presence with the functional imaging technique positron emission tomography (PET). This study describes the synthesis and in vivo evaluation of [18F]ROStrace, a radiotracer for imaging superoxide in vivo with PET, in an LPS model of neuroinflammation. [18F]ROStrace was found to rapidly cross the blood-brain barrier (BBB) and was trapped in the brain of LPS-treated animals but not the control group. [18F] ox-ROStrace, the oxidized form of [18F]ROStrace, did not cross the BBB. These data suggest that [18F]ROStrace is a suitable radiotracer for imaging superoxide levels in the central nervous system with PET.

Highly Selective Dopamine D3 Receptor Antagonists with Arylated Diazaspiro Alkane Cores.

A series of potent and selective D3 receptor (D3R) analogues with diazaspiro alkane cores were synthesized. Radioligand binding of compounds 11, 14, 15a, and 15c revealed favorable D3R affinity (Ki = 12-25.6 nM) and were highly selective for D3R vs D3R (ranging from 264- to 905-fold). Variation of these novel ligand architectures can be achieved using our previously reported 10-20 min benchtop C-N cross-coupling methodology, affording a broad range of arylated diazaspiro precursors.