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Extracellular vesicles (EVs) have garnered increasing interest as delivery vehicles for multiple classes of therapeutics based on their role as mediators in an important, natural intercellular communication system. We recently described a platform to allow the design, production and in vivo study of human EVs with specific properties (drug or tropism modifiers). This article seeks to compare and expand upon historical biodistribution and kinetic data by comparing systemically and compartmentally administered labeled engineered EVs using in vivo and ex vivo techniques.EVs were surface-labeled to high radiochemical purity and specific activity withOver time, systemic administration of engineered EVs distributed preferentially to the liver and spleen (Intravenous, IV), gastrointestinal tract and lymph nodes (Intraperitoneal, IP) and local/regional lymph nodes (Subcutaneous, SC). Immunostaining of dissected organs displaying PET signal revealed co-localization of an EV marker (PTGFRN) with a subset of macrophage markers (CD206, F4/80, IBA1). Compartmental dosing into NHP cerebrospinal fluid (CSF) resulted in a heterogenous distribution of labeled EVs depending upon whether the route was intrathecal (ITH), intracisterna magna (ICM) or intracerebroventricular (ICV), compared to the homogeneous distribution observed in rodents. Thus anatomically, ITH administration in NHP revealed meningeal distribution along the neuraxis to the base of the skull. In contrast ICM and ICV dosing resulted in meningeal distribution around the skull and to the cervical and thoracic spinal column. Further characterization using IHC shows uptake in a subset of meningeal macrophages.The present studies provide a comprehensive assessment of the fate of robustly and reproducibly labeled engineered EVs across several mammalian species. The in vivo distribution was observed to be both spatially and temporally dependent upon the route of administration providing insight into potential targeting opportunities for engineered EVs carrying a therapeutic payload.

BackgroundEngineered exosomes are emerging as a novel therapeutic modality for cancer immunotherapy. Leveraging cell type specific delivery, tumor restricted pharmacology and compartmental dosing, exosome-based immunotherapy can elicit a tumor specific immune response that may not be achievable with other traditional drugging modalities. Pre-clinical studies have shown that exosomes loaded with a STING agonist (exoSTINGTM) or engineered to express the cytokine interleukin-12 (exoIL-12TM) can substantially improve potency and selectivity resulting in improved therapeutic window [1,2]. Both exoSTING and exoIL-12 are currently in clinical trials in cancer patients. Utilizing a combination strategy involving exoSTING and exoIL-12, we demonstrate the development of potent systemic anti-tumor responses in both injected and non-injected tumors.Methods exoSTING exosomes are engineered to overexpress PTGFRN, an abundant exosome surface protein, and loaded ex vivo with a proprietary STING agonist. exoIL-12 exosomes are engineered to overexpress functional IL-12 attached via fusion to PTGFRN. In these studies, exoSTING and exoIL-12 were dosed intratumorally into one flank tumor into mice bearing dual flank subcutaneous MC38 or B16F10 tumors, or B16F10 single flank subcutaneous tumors with B16F10 lung metastases. T-cell infiltration in the non-injected tumor was monitored by histopathology.ResultsIn the checkpoint therapy refractory B16F10 melanoma dual flank tumor model, exoSTING/exoIL-12 combination provided 93% and 78% tumor growth inhibition (TGI) in both the injected and non-injected tumors, respectively, whereas monotherapy of exoSTING or exoIL-12 provided modest anti-tumor activity (44% and 48% TGI) in the non-injected tumors, respectively. In a MC38 subcutaneous CRC model, the addition of anti-PD-1 checkpoint inhibitor further enhanced anti-tumor activity with 100% TGI (7/7 CR) in injected and non-injected tumors. The tumor free animals were refractory to tumor re-challenge demonstrating immunological memory. A dosing schedule optimization experiment showed that same day dosing of exoSTING and exoIL-12 significantly inhibited the tumor growth in the non-injected tumors. In a lung metastasis model, the triple combination also showed potent anti-tumor effect in decreasing distal lung metastases when dosed intratumorally into the subcutaneous tumors. Subsequent imaging and histology studies demonstrated enhanced T cell infiltration in the non-injected subcutaneous tumor with the combination therapy.ConclusionsBy combining both exosome immunotherapies with a checkpoint blockade, we are able to elicit systemic anti-tumor immune immunity in both injected and non-injected tumors.ReferencesJang SC, Economides KD, Moniz RJ, et al. ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance. Commun Biol. 2021;4(1):497.Lewis ND, Sia CL, Kirwin K, et al. Exosome surface display of IL12 results in tumor-retained pharmacology with superior potency and limited systemic exposure compared with recombinant IL12. Mol Cancer Ther. 2020;20(3):523-534.Ethics ApprovalAll animals were maintained and treated at the animal care facility of Codiak Biosciences in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (CB2020-001).

Current synaptic vesicle 2A (SV2A) positron emission tomography (PET) imaging agents include the nanomolar affinity probes [11C]UCB-J and [18F]UCB-H derived from the anti-epileptic drug levitaracetam (Keppra®). An industry-utilized “de-risking” approach was used to carry out initial pharmacological characterization and to assess potential next-generation candidates amenable to F-18 radiolabeling for preliminary evaluation. Radioligand binding methods were employed in mammalian brain homogenates to determine the SV2A affinity (Kd) and maximal binding capacity (Bmax) of [3H]UCB-J. Novel leads were then screened to identify compounds minimally with comparable binding affinities with UCB-J in order to select a F-18-labeled candidate for subsequent in vivo assessment in rat. In parallel, mammalian brain tissue section autoradiography was performed to assess specific SV2A distribution. [3H]UCB-J bound with high affinity to a single population of sites in the rat brain (Kd = 2.6 ± 0.25 nM; Bmax = 810 ± 25 fmol/mg protein) and control human cortex (Kd = 2.9 ± 0.54 nM; Bmax = 10,000 ± 640 fmol/mg protein). Distribution of specific SV2A binding was shown to be homogeneous throughout the rodent brain and primarily in gray matter regions of rodent and human brain sections. Analog screening identified MNI-1038, MNI-1126/SDM-8, and SDM-2 as having comparable binding affinities with the currently available PET ligands. Subsequent [18F]MNI-1126/[18F]SDM-8 dynamic micro-PET imaging in rats revealed in vivo uptake and accumulation in the brain with favorable kinetics. Chase studies using 30 mg/kg levetiracetam confirmed that in vivo brain uptake of [18F]MNI-1126/[18F]SDM-8 was reversible. Taken together, these data suggest [18F]MNI-1126/[18F]SDM-8 (since renamed as [18F]SynVesT-1) characterized via an in vitro screening cascade provided a measurable in vivo SV2A specific signal in the rodent brain. This tracer as well as the close analog [18F]SDM-2 (since renamed as [18F]SynVesT-2) is currently undergoing further evaluation in preclinical and clinical studies.

Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [(3)H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer’s disease (AD), suggesting application for these compounds in AD diagnosis and identified [(11)C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3β-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC(50) = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2(T514) and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.

Extracellular matrix (ECsM) bioscaffolds have been successfully used to treat five esophageal adenocarcinoma (EAC) patients following resection of neoplastic mucosal tissue. The present study evaluated the in vitro effect of ECM harvested from nonmalignant, decellularized tissue on EAC cell phenotype to understand the molecular mechanisms underlying the clinical findings. Nonmalignant (Het-1A), metaplastic (CP-A), and neoplastic (SK-GT-4, OE33) esophageal epithelial cells were exposed to ECM degradation products (250 μg/mL) prepared from heterologous urinary bladder tissue or homologous esophageal mucosa tissue, and evaluated for cell morphology, cell function, and EAC signaling pathways. Both the ECM sources downregulated neoplastic cell phenotype, but had distinctive tissue-specific effects. Urinary bladder ECM decreased OE33 and SK-GT-4 metabolism and increased CP-A apoptosis. Esophageal ECM decreased SK-GT-4, CP-A, and Het-1A proliferation; robustly downregulated PI3K-Akt-mTOR, cell cycle/DNA replication signaling, and upregulated autophagy signaling in OE33 cells; and increased cell cycle/DNA replication signaling in Het-1A cells. Both ECM sources decreased OE33 proliferation and phosphorylated AKT in OE33 cells, and in contrast, increased phosphorylated AKT in Het-1A cells. The results support the concept that the biochemical signals in nonmalignant ECM can downregulate neoplastic cell phenotype with minimal, and sometimes opposite, effects on normal cells. PI3K-Akt signaling has been implicated in EAC progression and these ECM-mediated effects may be favorable for an esophageal therapy following cancer resection. IMPACT STATEMENT: Extracellular matrix (ECM) biomaterials were used to treat esophageal cancer patients after cancer resection and promoted regrowth of normal mucosa without recurrence of cancer. The present study investigates the mechanisms by which these materials were successful to prevent the cancerous phenotype. ECM downregulated neoplastic esophageal cell function (proliferation, metabolism), but normal esophageal epithelial cells were unaffected in vitro, and suggests a molecular basis (downregulation of PI3K-Akt, cell cycle) for the promising clinical results. The therapeutic effect appeared to be enhanced using homologous esophageal ECM. This study suggests that ECM can be further investigated to treat cancer patients after resection or in combination with targeted therapy.

Microglia play a role in several central nervous system (CNS) diseases and are a highly sought target for positron emission tomography (PET) imaging and therapeutic intervention. 5-Cyano-N-(4-(4-[11C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([11C]CPPC) is a radiopharmaceutical designed to selectively target microglia via macrophage colony stimulating factor-1 receptor (CSF-1R) in the CNS. Herein, we report the first preclinical evaluation of [3H]CPPC using radioligand binding methods for the evaluation of putative CSF-1R inhibitors in rodent models of neuroinflammation. The distribution of [3H]CPPC by autoradiography did not align with 18 kDa translocator protein (TSPO) distribution using [3H]PBR28 and IBA-1 staining for microglia. In the CNS, [3H]CPPC had considerable nonspecific binding, as indicated by a low displacement of the tritiated ligand by unlabeled CPPC and the known CSF1R inhibitors BLZ-945 and PLX3397. Spleen was identified as a tissue that provided an adequate signal-to-noise ratio to enable screening with [3H]CPPC and a library of 20 novel PLX3397 derivatives. However, unlabeled CPPC lacked selectivity and showed off-target binding to a substantial number of kinase targets (204 out of 403 tested) at a concentration relevant to in vitro radioligand binding assays (10 μM). These findings suggest that, while [3H]CPPC may have utility as a radioligand tool for the evaluation of peripheral targets and screening of CSF-1R inhibitors, it may have limited utility as an in vivo CNS imaging probe on the basis of the current evaluation.

Oral delivery of esophageal extracellular matrix hydrogel mitigates mucosal inflammation and metaplasia in a dog model of reflux., Chronic inflammatory gastric reflux alters the esophageal microenvironment and induces metaplastic transformation of the epithelium, a precancerous condition termed Barrett’s esophagus (BE). The microenvironmental niche, which includes the extracellular matrix (ECM), substantially influences cell phenotype. ECM harvested from normal porcine esophageal mucosa (eECM) was formulated as a mucoadhesive hydrogel, and shown to largely retain basement membrane and matrix-cell adhesion proteins. Dogs with BE were treated orally with eECM hydrogel and omeprazole (n = 6) or omeprazole alone (n = 2) for 30 days. eECM treatment resolved esophagitis, reverted metaplasia to a normal, squamous epithelium in four of six animals, and downregulated the pro-inflammatory tumor necrosis factor–α+ cell infiltrate compared to control animals. The metaplastic tissue in control animals (n = 2) did not regress. The results suggest that in vivo alteration of the microenvironment with a site-appropriate, mucoadhesive ECM hydrogel can mitigate the inflammatory and metaplastic response in a dog model of BE.

BackgroundLeptomeningeal disease (LMD) occurs when cells from primary tumors metastasize to the cerebrospinal fluid (CSF) space leading to multifocal neurological deficits. LMD has an overall prevalence of ~5% in cancer patients, but is most commonly observed in breast, lung and melanoma patients. With improved therapies emerging for several primary tumor types, the incidence of LMD is rising, and with treatment options limited to radiotherapy and chemotherapy, the median survival of LMD patients remains poor at 3–6 months. Thus, there is high unmet medical need for development of effective therapeutic strategies for LMD.The STING (Stimulator of Interferon Genes) pathway has been shown to play a critical role in activating anti-tumor immunity through initiation of a tumor antigen-specific T cell response. exoSTING is an engineered extracellular vesicle exogenously loaded with a CDN (cyclic dinucleotide) STING agonist. We have previously demonstrated that it enhances the potency of the CDN, preferentially activates antigen presenting cells in the tumor microenvironment and increases CNS retention of the drug without systemic inflammatory cytokine stimulation. Histological data in LMD is scarce, however high levels of inhibitory macrophages and low T cell infiltration have recently been described, providing support for the therapeutic potential of exoSTING in LMD.MethodsA mouse model of LMD was generated by intracerebral inoculation of B16F10-Luc melanoma cells and was used to assess the efficacy of intracranial administration of exoSTING.ResultsTumor growth was monitored by bioluminescence imaging during course of each study, with rapid loss of signal post treatment observed in exoSTING treated groups compared to steady tumor growth in vehicle treated groups. Animals within vehicle treated groups demonstrated survival less than 30 days, whereas exoSTING treated mice survived 50+ days with a high complete response rate (over 85%), confirmed by ex vivo histopathological analysis. Peripheral immunological responses were demonstrated by lack of tumor growth following flank rechallenge in exoSTING treated mice. Strong anti-tumor response and tumor-specific immune activation in the absence of systemic inflammation was demonstrated. The presentation will summaries the immunological changes in the tumor microenvironment following exoSTING administration.ConclusionsexoSTING, which previously showed strong efficacy against primary melanoma in mouse models, has been demonstrated in this study to also suppress tumor growth and improve survival in the LMD context. Our study supports the therapeutic rationale for using exoSTING for the treatment of LMD.

Summary Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Preclinical research has long been at the forefront of software and methodological innovation for multimodal imaging. In vivo imaging, ex vivo imaging, and the combination of the two offer a wide variety of complementary image modalities for deriving biological insight. It is increasingly common for research applications to use images from hybrid modality scanners, images from multiple scanners, and derived images in tandem for advanced analysis and quantitation. A long-standing example of the value of multimodal imaging comes from positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging, where it is a common practice to acquire a computed tomography (CT) image for attenuation correction (AC) of the reconstructed signal [1]. It demonstrates how combining image modalities can not only provide complementary information but also enhance the quality and reliability of one or more of the modalities. In the subsequent analysis of PET and SPECT images, it is common for a researcher to quantify the sum or concentration of signal within a region of interest. As “functional” modalities, they may only exhibit contrast to background in regions where the targeted function takes place; however, more regions than those visible may be desired a priori for quantification. The quantitation of such regions greatly benefits from fusing an anatomical modality with a functional one for use in region segmentation.

Supplementary_Data for The Binding of BF-227-Like Benzoxazoles to Human α-Synuclein and Amyloid β Peptide Fibrils by Lee Josephson, Nancy Stratman, YuTing Liu, Fang Qian, Steven H. Liang, Neil Vasdev, and Shil Patel in Molecular Imaging

Multi-modality molecular imaging techniques have expanded the role of imaging biomarkers in the pharmaceutical industry and are beginning to streamline the drug discovery and development process. The World Molecular Imaging Society (WMIS) serves as a forum for discussing innovative and exploratory multi-modal, interdisciplinary molecular imaging research with a mission of bridging the gap between pathology and in vivo imaging. To formalize the role of the WMIS in pharmaceutical research efforts, members of the society have formed an interest group entitled Advancing Drug Discovery and Development using Molecular Imaging (ADDMI). The ADDMI interest group launched their efforts at the 2016 World Molecular Imaging Congress by hosting a session of invited lectures on translational positron emission tomography (PET) imaging in the central nervous system. This article provides a synopsis of those lectures and frames the role of translational imaging biomarker strategies in the drug discovery and development process.

Primary cardiac tumors are extremely rare and constitute only about 5% of all cardiac tumors. Cardiac myxomas are noncancerous primary tumors of the heart and constitute about of 50% of all primary heart tumors. Left-sided atrial myxomas are more common than right-sided atrial myxomas. Atrial myxomas can lead to a triad of complications. The most common symptoms are associated with obstruction due to the size and location of the tumor. The next most common symptoms are associated with pulmonary and systemic embolization. Patients may also present with constitutional symptoms. Diagnosis is made via means of transesophageal echocardiography and magnetic resonance imaging. Early diagnosis and surgical resection remain the treatment of choice to prevent complications. Patients usually have a good prognosis after resection.

A 71-year-old man with a medical history significant for high-grade pleomorphic undifferentiated soft tissue sarcoma of the neck and multiple lung metastases presented to the emergency room, with exertional dyspnoea. He was actively receiving palliative radiation at the time of presentation as his tumour was deemed untreatable by his oncologist. Physical examination revealed a cachectic man in mild respiratory distress, at rest. A firm, non-tender 8×8 cm mass was palpated in the suboccipital area of his neck. On auscultation, there were bilateral scattered rhonchi, but no crackles. Cardiovascular examination revealed a grade 3/6 ejection systolic murmur, heard loudest in the pulmonic area, which worsened on inspiration but did …

Objective: With healthcare focusing towards quality based healthcare it has created significant interest among physicians in measuring and defining appropriateness in the treatment of medical conditions. We compared the indications for which the Intravenacaval (IVC) filter was placed in our hospital with the current guidelines by three of the major medical societies the American Heart Association (AHA), American College of Chest Physicians (ACCP) and Society for Interventional Radiology (SIR) to compare the measure of appropriateness in the placement of IVC filters among the guidelines Methods: We conducted a retrospective review of all charts with the ICD 9 code for placement of IVC filter from January, 2010 to January, 2015. Each patient chart was reviewed for patient demographics, complications and indications which were compared with the guidelines as defined by AHA, ACCP and SIR Results: During the 5 years, 592 patients underwent the procedure which included 233 men and 359 women who had a mean age of 67.2 +/- 17.4. 75.8% filters were inserted by vascular surgery and 24.2% filters were inserted by interventional radiology. 1.8% of the patients had some form of complication either during insertion or retrieval. On comparing the indications, we found that 35.5%(AHA), 30.9%(ACCP), 35.5%(SIR) were appropriate. While 4.4%(AHA), 4.4%(ACCP), 47.4%(SIR) were relatively appropriate and 60.1%(AHA), 64.7%(ACCP), 17.1%(SIR) were not appropriate. Commonly used indications not defined clearly included prophylaxis for bariatric surgery (14.4%) and high risk for fall (11%). Statistical analysis with the Chi-square test showed there was no statistical difference between AHA and ACCP (p =0.243), while statistical difference was present between AHA and SIR (p Conclusion: This study demonstrates that there are significant differences between the guidelines from major medical societies for the placement of IVC filter which leads to compelling discrepancies amongst physicians in their decision for the placement of IVC filters

A 62-year-old woman with a past medical history significant only for hypertension and hypothyroidism presented to the emergency department (ER), with right lower quadrant pain. She had, 1 day prior to the day of admission, been treated for bronchitis as an outpatient. She continued to experience multiple episodes of cough at home and eventually developed abdominal pain, which brought her to the ER. Her vitals were within normal limits and physical examination was significant for tenderness on palpation in the right lower quadrant of her abdomen, with guarding. Her blood work was significant for haemoglobin of 6.5 g/dL. Our …

The preclinical pharmacodynamic and pharmacokinetic properties of 4-methylbenzyl (3S, 4R)-3-fluoro-4-[(Pyrimidin-2-ylamino) methyl] piperidine-1-carboxylate (CERC-301), an orally bioavailable selective N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) antagonist, were characterized to develop a translational approach based on receptor occupancy (RO) to guide CERC-301 dose selection in clinical trials of major depressive disorder. CERC-301 demonstrated high-binding affinity (K i, 8.1 nmol L(-1)) specific to GluN2B with an IC 50 of 3.6 nmol L(-1) and no off-target activity. CERC-301 efficacy was demonstrated in the forced swim test with an efficacy dose (ED 50) of 0.3-0.7 mg kg(-1) (RO, 30-50%); increase in locomotor activity was observed at ED 50 of 2 mg kg(-1), corresponding to an RO of 75%. The predicted 50% RO concentration (Occ50) in humans was 400 nmol L(-1), similar to that predicted for rat, dog, and monkey (300, 200, and 400 nmol L(-1), respectively). Safety pharmacology and neurotoxicity studies raised no specific safety concerns. A first-in-human study in healthy males demonstrated a dose-proportional pharmacokinetic profile, with T max of ~1 h and t 1/2 of 12-17 h. Based on the preclinical and pharmacodynamic data, doses of ≥8 mg in humans are hypothesized to have an acceptable safety profile and result in clinically relevant peak plasma exposure.

Two positron emission tomography radiotracers for the glycine transporter 1 (GlyT1) are reported here. Each radiotracer is a propylsulfonamide-containing benzamide and was labeled with either carbon-11 or fluorine-18. [¹¹C]CMPyPB was synthesized by the alkylation of a 3-hydroxypyridine precursor using [¹¹C]MeI, and [¹⁸F]MK-6577 was synthesized by a nucleophilic aromatic substitution reaction using a 2-chloropyridine precursor. Each tracer shows good uptake into rhesus monkey brain with the expected distribution of highest uptake in the pons, thalamus, and cerebellum and lower uptake in the striatum and gray matter of the frontal cortex. In vivo blockade and chase studies of [¹⁸F]MK-6577 showed a large specific signal and reversible binding. In vitro autoradiographic studies with [¹⁸F]MK-6577 showed a large specific signal in both rhesus monkey and human brain slices and a distribution consistent with the in vivo results and those reported in the literature. In vivo metabolism studies in rhesus monkeys demonstrated that only more-polar metabolites are formed for each tracer. Of these two tracers, [¹⁸F]MK-6577 was more extensively characterized and is a promising clinical positron emission tomography tracer for imaging GlyT1 and for measuring GlyT1 occupancy of therapeutic compounds.

Two moderately lipophilic, high affinity ligands for metabotropic glutamate receptor subtype 1 (mGluR1) were radiolabeled with a positron-emitting radioisotope and evaluated in rhesus monkey as potential PET tracers. Both ligands were radiolabeled with fluorine-18 via nucleophilic displacement of the corresponding 2-chloropyridine precursor with [18F]potassium fluoride. [18F]MK-1312 was found to have a suitable signal for quantification of mGluR1 receptors in nonhuman primates and was more thoroughly characterized. In vitro autoradiographic studies with [18F]MK-1312 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the known distribution of mGluR1, with the highest uptake in the cerebellum, moderate uptake in the hippocampus, thalamus, and cortical regions, and lowest uptake in the caudate and putamen. In vitro saturation binding studies in rhesus monkey and human cerebellum homogenates confirmed that [18F]MK-1312 binds to a single site with a Bmax/Kd ratio of 132 and 98, respectively. PET studies in rhesus monkey with [18F]MK-1312 showed high brain uptake and a regional distribution consistent with in vitro autoradiography results. Blockade of [18F]MK-1312 uptake with mGluR1 allosteric antagonist MK-5435 dose-dependently reduced tracer uptake in all regions of gray matter to a similarly low level of tracer uptake. This revealed a large specific signal useful for determination of mGluR1 receptor occupancy in rhesus monkey. Taken together, these results are promising for clinical PET studies with [18F]MK-1312 to determine mGluR1 occupancy of MK-5435. Synapse 2011. © 2010 Wiley-Liss, Inc.

Osteosarcoma is the most common bone cancer in children and young adults and has a strong propensity to develop lung metastases. E-selectin is known to be involved in the focal adhesion of tumor cells to cytokine exposed endothelial cells and we postulated may play a central role in osteosarcoma progression. Previously we identified that SDF-1, the main ligand for CXCR4, was upregulated in the pre-metastatic niche (Kaplan et al Nature 2005). Many tumor cells express CXCR4 and may use this signaling pathway to direct disseminated tumor cells to pre- and early metastatic sites in the lung. Based on these findings we examined human osteosarcoma cell lines and primary patient derived xenografts (PDXs) for the expression of CXCR4 and E-selectin ligands by flow cytometry. We found robust expression of these ligands in the majority of both the human osteosarcoma cell lines and PDXs examined. We therefore, investigated the impact of targeting these two axes on metastatic progression of orthotopic osteosarcoma using a small molecule, glycomimetic compound with dual inhibitory activity against E-selectin and CXCR4, GMI-1359. Five days post paratibial injection the HOS cell line, female NMRI-nu mice (n=12/group) were treated with saline; GMI-1359 alone (40 mg/kg IP BID x 25 days); doxorubicin (DOX) alone (5 mg/kg IV days 5, 15 and 25), or the combination of GMI-1359 and DOX. All treatments were well tolerated. The % tumor volume in treatment/control on day 27 of mice treated with GMI-1359, DOX or the combination was 35.5, 36.7 and 32.5, respectively. At study conclusion the incidence of lung metastases was approximately 60% and 50% in mice treated with saline or DOX and 15% in mice treated with GMI-1369 alone or in combination with DOX. Moreover, the extent of ectopic bone formation and/or osteolytic lesions was lower in mice treated with GMI-1359 compared to saline and DOX. These results indicate that the E-selectin and CXCR4 axes are important for the progression of osteosarcoma, and further, that inhibition of these two pro-tumor growth components by GMI-1359 has a therapeutic advantage over chemotherapy alone. Furthermore, studies in the adjuvant setting can provide proof of concept of utility of targeting CXCR4 and E- selectin ligands in the metastatic niche as a therapeutic strategy to limit metastatic progression in high risk patients. Citation Format: Wei Ju, Choh L. Yeung, Arnulfo Mendoza, Meera Murgai, Sabina Kaczanowska, Jennifer Zhu, Shil Patel, David A. Stewart, William E. Fogler, John L. Magnani, Rosandra N. Kaplan. Dual E-selectin and CXCR4 inhibition reduces tumor growth and metastatic progression in an orthotopic model of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5211.

Development of an α-synuclein (α-Syn) positron emission tomography agent for the diagnosis and evaluation of Parkinson disease therapy is a key goal of neurodegenerative disease research. BF-227 has been described as an α-Syn binder and hence was employed as a lead to generate a library of α-Syn-binding compounds. [3H]BF-227 bound to α-Syn and amyloid β peptide (Aβ) fibrils with affinities (KD) of 46.0 nM and 15.7 nM, respectively. Affinities of BF-227-like compounds (expressed as Ki) for α-Syn and Aβ fibrils were determined, along with 5 reference compounds (flutafuranol, flutemetamol, florbetapir, BF-227, and PiB). Selectivity for α-Syn binding, defined as the Ki(Aβ)/Ki(α-Syn) ratio, was 0.23 for BF-227. A similar or lower ratio was measured for analogues decorated with alkyl or oxyethylene chains attached to the oxygen at the 6 position of BF-227, suggesting a lack of involvement of the side chain in fibril binding. BF-227-like iodobenzoxazoles had lower affinities and poor α-Syn selectivity. However, BF-227-like fluorobenzoxazoles had improved α-Syn selectively having Ki(Aβ)/Ki(α-Syn) ranging from 2.2 to 5.1 with appreciable fibril affinity, although not sufficient to warrant further investigation. Compounds based on fluorobenzoxazoles might offer an approach to obtaining an α-Syn imaging agent with an appropriate affinity and selectivity.

Use of mGluR5 receptor radiotracers to determine whether an in vitro binding assay is able to predict how good a radiotracer is likely to be in imaging receptor in the central nervous system (CNS) via positron emission tomography (PET).Saturation and equilibrium competition studies in rat and rhesus membranes were used to determine receptor concentrations and tracer affinities. In addition, specific binding of metabotropic receptor subtype 5 (mGluR5) radioligands in rhesus and rat brain sections was determined using a "no-wash protocol," and the in vivo binding signal in rats was determined using micro-PET.Affinity values were determined for a series of mGluR5 antagonists (1-5) and ranged from 0.1 to 11 nM in rat. A previously reported "no-wash protocol" was then employed to determine specific binding in tissue sections following a 20-min incubation, and the regional distribution of these mGluR5 radiotracers determined in rat brain via autoradiography. The analogs 1b, 2b, 3b, and 4b, but not 5b, displayed good signal-to-noise ratios under these conditions with high density of binding in caudate, cortex, and hippocampus and lower density in cerebellum. With this information it was predicted that 1c, 2c, 3b, and 4b would display measurable signal-to-noise ratios in vivo, and that the larger in vitro signals for 3b and 4b would translate to 3b and 4b yielding the best in vivo signals. These predictions were investigated using micro-PET imaging in rat. Compound 1c showed a rapid wash-in and rapid wash-out profile in rat brain. Compound 2c showed similar signal-to-noise ratio as 1b, but slower washout. Compounds 3b and 4b showed the best signal-to-noise ratio in vivo, while 5b did not provide a significant signal, as predicted. In vivo occupancy estimates for 2-methyl-6-(phenylethynyl)-pyridine (MPEP) following intravenous administration were determined using radiolabeled compounds 1c, 2c, and 3b; they were essentially the same and were on the order of 1 mg kg(-1) (ID(50)).An in vitro screen of several mGluR5 tracers was used to rapidly predict whether radiolabeled mGluR5 analogs would be useful as PET radiotracers. Results provided an extension to previously reported data. Two of the four radiotracers with the best in vitro "no-wash" results also showed the best potential as measured noninvasively using micro-PET.

The present study examined the potential role of tachykinin NK1 receptors in modulating immobilisation stress-induced increase of dopamine metabolism in rat medial prefrontal cortex. In agreement with previous studies, 20 min immobilisation stress significantly increased medial prefrontal cortex dopamine metabolism as reflected by the concentration of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC). Pretreatment with the high affinity, selective, tachykinin NK1 receptor antagonist (3(S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethyl amino)-2(S)-phenylpiperidine) ((S)-GR205171, 10 mg/kg, s.c.), a dose that in ex vivo binding studies extensively occupied rat brain tachykinin NK1 receptors for approximately 60 min, significantly attenuated the stress-induced increase of mesocortical DOPAC concentration without affecting cortical DOPAC levels per se. In contrast, pretreatment of animals with the less active enantiomer (R)-GR205171 (10 mg/kg, s.c.), which demonstrated negligible tachykinin NK1 receptor occupancy ex vivo, failed to affect either basal or stress-induced DOPAC concentration in medial prefrontal cortex. Furthermore, pretreatment of animals with the benzodiazepine/GABAA receptor antagonist, flumazenil (15 mg/kg, i.p.), did not affect the ability of (S)-GR205171 to attenuate the increase of medial prefrontal cortex DOPAC concentration by acute stress. Results demonstrate that the selective tachykinin NK1 receptor antagonist, (S)-GR205171, attenuated the stress-induced activation of mesocortical dopamine neurones by a mechanism independent of the benzodiazepine modulatory site of the GABAA receptor.

We have characterized the in vitro properties of 3-[3H]methoxy-5-(pyridin-2-ylethynyl)pyridine ([3H]MethoxyPyEP), an analogue of the mGluR(5) receptor subtype antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], in rat tissue preparations using tissue homogenates and autoradiography. Binding of [3H]MethoxyPyEP to rat cortex, hippocampus, thalamus and cerebellum membrane preparations revealed saturable, high affinity binding (3.4 +/- 0.4 nM, n = 4 in rat cortex) to a single population of receptors in all regions studied except for cerebellum. Binding was found to be relatively insensitive to pH and insensitive to DTT. High concentrations of NEM both reduce receptor concentration and binding affinity for the radioligand. In time-course studies at room temperature k(on) and k(off) were determined as 2.9 x 10(7) M(-1) min(-1) and 0.11 min(-1) respectively. The rank order of affinities, as assessed by equilibrium competition studies, of a variety of ligands suggested binding of the radioligand selectively to mGluR5 (MPEPtrans-azetidine-2,4-dicarboxylic acid congruent with (S)-4-carboxyphenylglycine congruent with (+)MK801 congruent with CP-101,606 congruent with clozapine congruent with atropine congruent with ketanserin congruent with yohimbine congruent with benoxathian). Autoradiographic studies with [3H]MethoxyPyEP showed that binding was regioselective, with high density of binding in caudate and hippocampus, intermediate binding in thalamus and very low density in the cerebellum. These data show that [3H]MethoxyPyEP is a high affinity radioligand useful for the in vitro study of mGluR5 receptor distribution and pharmacologic properties in brain.

An alpha(1) adrenoceptor (alpha(1)-AdR) assay using [(3)H]prazosin binding in mouse brain is described which allows in vivo determination of central alpha(1)-AdR occupancy for ligands with alpha(1)-AdR affinity. Binding of [3H]prazosin in rat and mouse brain membranes in vitro was used to characterise the pharmacological profile of alpha(1)-AdRs in order to determine any potential species variations. Saturation and displacement studies yielded comparable affinity and pharmacological profile for [(3)H]prazosin binding in mouse and rat brain homogenates. These studies confirmed the absence of species variation for ligands in central alpha(1)-AdR pharmacology which is in good agreement with previous studies in rat brain. Subsequently, in vivo binding of [(3)H]prazosin in mouse whole brain was used to measure the occupancy of a number of AdR ligands. Timecourse studies revealed that a [3H]prazosin (5 mu Ci/mouse) pretreatment time of at least 20 min following intravenous (i.v.) administration was required for optimal specific binding. Ligands were administered systemically 40 min prior to i.v. administration of radiolabel. The alpha(1)-adrenoceptor ligands prazosin (ED(50)=0.15 mg/kg i.p.), benoxathian (0.52 mg/kg i.p.) and phentolamine (51 mg/kg i.p.) were all able to block in vivo [(3)H]prazosin binding from mouse brain. In addition, receptor occupancy values for a number of compounds including haloperidol (ED(50)=0.83 mg/kg s.c.), clozapine (2.2 mg/kg s.c.) and MDL-100907 [R(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol], (10 mg/kg s.c.)], which possess high to moderate affinity at alpha(1)-adrenoceptors, were also determined. These results suggest that in the mouse, [(3)H]prazosin binding can be used to measure in vivo receptor occupancy of ligands with affinity at central alpha(1)-adrenoceptors.

After the requirement of pseudocycle formation in the ureas 3 and 7 for hD(4) binding and selectivity was confirmed, structural hybridization with the known hD(4) ligand 2 led to the design and identification of the lead 4-(2-oxo-1, 3-dihydroimidazol-2-yl)piperidine 8. Optimization studies were carried out on 8 with the aim of achieving 1000-fold selectivity for hD(4) over all other receptors while retaining the good pharmacokinetic properties of the lead. After initial preparation of 8 as a minor component in a low-yielding reaction, a novel and regioselective "four-step/one-pot" procedure was developed which proved to be applicable to rapid investigation of the SAR of the 1, 3-dihydroimidazol-2-one ring. Various changes to substituents attached to the 3-, 4-, or 5-position of the 1, 3-dihydroimidazol-2-one core of 8 did not significantly improve selectivity for hD(4) over hD(2) and hD(3). Greater selectivity (1000-fold) was ultimately achieved by meta substitution of the benzyl group of 8 with various substituents. Compounds 28, 31, and 32 all possess the required selectivity for hD(4) over the other dopamine subtypes, but only 32 has1000-fold selectivity over all the key counterscreens we tested against. Compound 32 is an antagonist at hD(4) and has a good pharmacokinetic profile in the rat, with excellent estimated in vivo receptor occupancy, thus making it a potentially useful pharmacological tool to investigate the role of the D(4) receptor.

The syntheses of a number of different N-linked heterocyclic pyrazole replacements based on the structure 1 are described (compounds 3-12) as hD4 ligands. After further optimisation the best compound identified was 13 which has high affinity for hD4 (5.2 nM) and >300-fold selectivity for hD4 receptors over hD2 and hD3 receptors.

5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening of the Merck sample collection as a human dopamine D4 (hD4) receptor ligand with moderate affinity (61 nM) and 4-fold selectivity over human D2 (hD2) receptors. Four separate parts of the molecule have been examined systematically to explore structure-activity relationships with respect to hD4 affinity and selectivity over other dopamine receptors. It was found that the 4-chlorophenyl group attached to the pyrazole is optimal, as is the 4-substituted piperidine. The lipophilic group on the basic nitrogen is more amenable to change, with the optimal group found to be a phenethyl. The aromatic heterocyle can be altered to a number of different groups, with isoxazoles and pyrimidines showing improved affinities. This heterocycle can also be advantageously alkylated, improving the selectivity of the compounds over D2 receptors. It is hypothesized that the conformation around the bond joining the aromatic heterocycle to the piperidine is important for D4 affinity, based on crystal structures of isoxazoles (29 and 30) and on a conformationally constrained compound (28). Putting all the favorable changes together led to the discovery that 5-(4-chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)iso xazole (36) is a nanomolar antagonist at human dopamine D4 receptors with500-fold selectivity over hD2 and200-fold selectivity over hD3. Compound 36 is an antagonist of hD4 receptors with good oral bioavailability of 38%, a half life of 2 h, and brain levels 10-fold higher than plasma levels.