Identification of Novel, Selective Ataxia-Telangiectasia Mutated Kinase Inhibitors with the Ability to Penetrate the Blood-Brain Barrier: The Discovery of AZD1390.

The inhibition of ataxia-telangiectasia mutated (ATM) has been shown to chemo- and radio-sensitize human glioma cells in vitro and therefore might provide an exciting new paradigm in the treatment of glioblastoma multiforme (GBM). The effective treatment of GBM will likely require a compound with the potential to efficiently cross the blood-brain barrier (BBB). Starting from clinical candidate AZD0156, 4, we investigated the imidazoquinolin-2-one scaffold with the goal of improving likely CNS exposure in humans. Strategies aimed at reducing hydrogen bonding, basicity, and flexibility of the molecule were explored alongside modulating lipophilicity. These studies identified compound 24 (AZD1390) as an exceptionally potent and selective inhibitor of ATM with a good preclinical pharmacokinetic profile. 24 showed an absence of human transporter efflux in MDCKII-MDR1-BCRP studies (efflux ratio <2), significant BBB penetrance in nonhuman primate PET studies (Kp,uu 0.33) and was deemed suitable for development as a clinical candidate to explore the radiosensitizing effects of ATM in intracranial malignancies.

Preclinical Characterization of AZD9574, a Blood-Brain Barrier Penetrant Inhibitor of PARP1.

PURPOSE: We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with temozolomide (TMZ) in preclinical models. EXPERIMENTAL DESIGN: AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination. RESULTS: AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone. CONCLUSIONS: The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.

Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration.

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Brain exposure of osimertinib in patients with epidermal growth factor receptor mutation non-small cell lung cancer and brain metastases: A positron emission tomography and magnetic resonance imaging study.

Brain metastases (BMs) are associated with poor prognosis in epidermal growth factor receptor mutation-positive (EGFRm) non-small cell lung cancer (NSCLC). Osimertinib is a third-generation, irreversible, EGFR-tyrosine kinase inhibitor that potently and selectively inhibits EGFR-sensitizing and T790M resistance mutations with efficacy in EGFRm NSCLC including central nervous system (CNS) metastases. The open-label phase I positron emission tomography (PET) and magnetic resonance imaging (MRI) study (ODIN-BM) assessed [11 C]osimertinib brain exposure and distribution in patients with EGFRm NSCLC and BMs. Three dynamic 90-min [11 C]osimertinib PET examinations were acquired together with metabolite-corrected arterial plasma input functions at: baseline, after first oral osimertinib 80 mg dose, and after greater than or equal to 21 days of osimertinib 80 mg q.d. treatment. Contrast-enhanced MRI was performed at screening and after 25-35 days of osimertinib 80 mg q.d.; treatment effect was assessed per CNS Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 and per volumetric changes in total BM using a novel analysis approach. Four patients (aged 51-77 years) completed the study. At baseline, ~1.5% injected radioactivity reached the brain (IDmax[brain] ) 22 min (median, Tmax[brain] ) after injection. Total volume of distribution (VT ) in whole brain was numerically higher compared with the BM regions. After a single oral osimertinib 80 mg dose, there was no consistent decrease in VT in whole brain or BMs. After greater than or equal to 21 days' daily treatment, VT in whole brain and BMs were numerically higher versus baseline. MRI revealed 56%-95% reduction in total BMs volume after 25-35 days of osimertinib 80 mg q.d. treatment. The [11 C]osimertinib crossed the blood-brain and brain-tumor barriers and had a high, homogeneous brain distribution in patients with EGFRm NSCLC and BMs.

Synthesis and Preclinical Evaluation of [11C]AZ11895530 for PET Imaging of the Serotonin 1A Receptor.

The serotonin 1A (5-HT1A) receptor is a G-protein-coupled receptor implicated in the pathophysiology of several neuropsychiatric and neurodegenerative disorders. We here report the preparation of two candidate 5-HT1A radioligands, [11C]AZ11132132 ([11C]3) and [11C]AZ11895530 ([11C]4), and their subsequent evaluation in vitro using autoradiography and in vivo using positron emission tomography (PET). Compounds 3 and 4 were radiolabeled at high radiochemical purity (>99%) and high molar activity (>38 GBq/µmol) by heteroatom methylation with [11C]methyl iodide. Autoradiography on whole hemispheres from post-mortem human brain revealed substantial nonspecific binding of [11C]3, while the binding of [11C]4 to brain tissue was consistent with the distribution of 5-HT1A receptors and sensitive to co-incubation with the reference 5-HT1A antagonist WAY-100635 (10 µM). Following intravenous injection of [11C]4 into a cynomolgus monkey, brain radioactivity concentration (Cmax ∼ 2.2 SUV) was high whereafter it decreased rapidly. The regional binding potential (BPND) values were calculated using the simplified reference tissue model with cerebellum as reference region. The values varied between 0.2 and 1.0 for temporal cortex, raphe nuclei, frontal cortex, and hippocampus which is consistent with the known 5-HT1A expression pattern. After pretreatment with WAY100635 (0.5 mg/kg), a homogeneous distribution of radioactivity was observed in non-human primate (NHP) brain. Although [11C]4 fulfilled important criteria for successful in vivo neuroimaging, including good blood-brain-barrier permeability and high specific binding in vitro to human brain tissue, the regional BPND values for [11C]4 in NHP brain were low when compared to those obtained with existing radioligands and thus do not merit further investigation of [11C]4. Evaluation of structurally related analogues is underway in our laboratory to identify improved candidates for clinical imaging.

Glia Imaging Differentiates Multiple System Atrophy from Parkinson's Disease: A Positron Emission Tomography Study with [11 C]PBR28 and Machine Learning Analysis.

BACKGROUND: The clinical diagnosis of multiple system atrophy (MSA) is challenged by overlapping features with Parkinson's disease (PD) and late-onset ataxias. Additional biomarkers are needed to confirm MSA and to advance the understanding of pathophysiology. Positron emission tomography (PET) imaging of the translocator protein (TSPO), expressed by glia cells, has shown elevations in MSA. OBJECTIVE: In this multicenter PET study, we assess the performance of TSPO imaging as a diagnostic marker for MSA. METHODS: We analyzed [11 C]PBR28 binding to TSPO using imaging data of 66 patients with MSA and 24 patients with PD. Group comparisons were based on regional analysis of parametric images. The diagnostic readout included visual reading of PET images against clinical diagnosis and machine learning analyses. Sensitivity, specificity, and receiver operating curves were used to discriminate MSA from PD and cerebellar from parkinsonian variant MSA. RESULTS: We observed a conspicuous pattern of elevated regional [11 C]PBR28 binding to TSPO in MSA as compared with PD, with "hotspots" in the lentiform nucleus and cerebellar white matter. Visual reading discriminated MSA from PD with 100% specificity and 83% sensitivity. The machine learning approach improved sensitivity to 96%. We identified MSA subtype-specific TSPO binding patterns. CONCLUSIONS: We found a pattern of significantly increased regional glial TSPO binding in patients with MSA. Intriguingly, our data are in line with severe neuroinflammation in MSA. Glia imaging may have potential to support clinical MSA diagnosis and patient stratification in clinical trials on novel drug therapies for an α-synucleinopathy that remains strikingly incurable. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Effects of sevoflurane anaesthesia on radioligand binding to monoamine oxidase-B in vivo.

BACKGROUND: The molecular actions underlying the clinical effects of inhaled anaesthetics such as sevoflurane and isoflurane are not fully understood. Unexpected observations in positron emission tomography (PET) studies with [11C]AZD9272, a metabotropic glutamate receptor 5 (mGluR5) radioligand with possible affinity for monoamine oxidase-B (MAO-B), suggest that its binding is sensitive to anaesthesia with sevoflurane. The objective of the present study was to assess the effects of sevoflurane anaesthesia on the binding of [11C]AZD9272 and of [11C]L-deprenyl-D2, a radioligand selective for MAO-B in non-human primates (NHPs). METHODS: Altogether, 12 PET measurements were conducted with a high-resolution research tomograph using the ligands [11C]AZD9272 or [11C]L-deprenyl-D2 in six cynomolgus monkeys anaesthetised with sevoflurane or ketamine/xylazine. RESULTS: The specific binding of [11C]AZD9272 and [11C]L-deprenyl-D2 was markedly reduced during anaesthesia with sevoflurane compared with ketamine/xylazine. The reduction was 80-90% (n=3) for [11C]AZD9272 and 77-80% (n=3) for [11C]L-deprenyl-D2. CONCLUSIONS: Sevoflurane anaesthesia inhibited radioligand binding to MAO-B in the primate brain. The observation of lower MAO-B binding at clinically relevant concentrations of sevoflurane warrants further exploration of the potential role of MAO-B related mechanisms in regulation of systemic blood pressure during anaesthesia.

Preclinical Comparison of the Blood-brain barrier Permeability of Osimertinib with Other EGFR TKIs.

PURPOSE: Osimertinib is a potent and selective EGFR tyrosine kinase inhibitor (EGFR-TKI) of both sensitizing and T790M resistance mutations. To treat metastatic brain disease, blood-brain barrier (BBB) permeability is considered desirable for increasing clinical efficacy. EXPERIMENTAL DESIGN: We examined the level of brain penetration for 16 irreversible and reversible EGFR-TKIs using multiple in vitro and in vivo BBB preclinical models. RESULTS: In vitro osimertinib was the weakest substrate for human BBB efflux transporters (efflux ratio 3.2). In vivo rat free brain to free plasma ratios (Kpuu) show osimertinib has the most BBB penetrance (0.21), compared with the other TKIs (Kpuu ≤ 0.12). PET imaging in Cynomolgus macaques demonstrated osimertinib was the only TKI among those tested to achieve significant brain penetrance (C max %ID 1.5, brain/blood Kp 2.6). Desorption electrospray ionization mass spectroscopy images of brains from mouse PC9 macrometastases models showed osimertinib readily distributes across both healthy brain and tumor tissue. Comparison of osimertinib with the poorly BBB penetrant afatinib in a mouse PC9 model of subclinical brain metastases showed only osimertinib has a significant effect on rate of brain tumor growth. CONCLUSIONS: These preclinical studies indicate that osimertinib can achieve significant exposure in the brain compared with the other EGFR-TKIs tested and supports the ongoing clinical evaluation of osimertinib for the treatment of EGFR-mutant brain metastasis. This work also demonstrates the link between low in vitro transporter efflux ratios and increased brain penetrance in vivo supporting the use of in vitro transporter assays as an early screen in drug discovery.

Brain exposure of the ATM inhibitor AZD1390 in humans-a positron emission tomography study.

BACKGROUND: The protein kinase ataxia telangiectasia mutated (ATM) mediates cellular response to DNA damage induced by radiation. ATM inhibition decreases DNA damage repair in tumor cells and affects tumor growth. AZD1390 is a novel, highly potent, selective ATM inhibitor designed to cross the blood-brain barrier (BBB) and currently evaluated with radiotherapy in a phase I study in patients with brain malignancies. In the present study, PET was used to measure brain exposure of 11C-labeled AZD1390 after intravenous (i.v.) bolus administration in healthy subjects with an intact BBB. METHODS: AZD1390 was radiolabeled with carbon-11 and a microdose (mean injected mass 1.21 µg) was injected in 8 male subjects (21-65 y). The radioactivity concentration of [11C]AZD1390 in brain was measured using a high-resolution PET system. Radioactivity in arterial blood was measured to obtain a metabolite corrected arterial input function for quantitative image analysis. Participants were monitored by laboratory examinations, vital signs, electrocardiogram, adverse events. RESULTS: The brain radioactivity concentration of [11C]AZD1390 was 0.64 SUV (standard uptake value) and reached maximum 1.00% of injected dose at Tmax[brain] of 21 min (time of maximum brain radioactivity concentration) after i.v. injection. The whole brain total distribution volume was 5.20 mL*cm-3. No adverse events related to [11C]AZD1390 were reported. CONCLUSIONS: This study demonstrates that [11C]AZD1390 crosses the intact BBB and supports development of AZD1390 for the treatment of glioblastoma multiforme or other brain malignancies. Moreover, it illustrates the potential of PET microdosing in predicting and guiding dose range and schedule for subsequent clinical studies.

Quantification and reliability of [11C]VC - 002 binding to muscarinic acetylcholine receptors in the human lung - a test-retest PET study in control subjects.

BACKGROUND: The radioligand [11C]VC-002 was introduced in a small initial study long ago for imaging of muscarinic acetylcholine receptors (mAChRs) in human lungs using positron emission tomography (PET). The objectives of the present study in control subjects were to advance the methodology for quantification of [11C]VC-002 binding in lung and to examine the reliability using a test-retest paradigm. This work constituted a self-standing preparatory step in a larger clinical trial aiming at estimating mAChR occupancy in the human lungs following inhalation of mAChR antagonists. METHODS: PET measurements using [11C]VC-002 and the GE Discovery 710 PET/CT system were performed in seven control subjects at two separate occasions, 2-19 days apart. One subject discontinued the study after the first measurement. Radioligand binding to mAChRs in lung was quantified using an image-derived arterial input function. The total distribution volume (VT) values were obtained on a regional and voxel-by-voxel basis. Kinetic one-tissue and two-tissue compartment models (1TCM, 2TCM), analysis based on linearization of the compartment models (multilinear Logan) and image analysis by data-driven estimation of parametric images based on compartmental theory (DEPICT) were applied. The test-retest repeatability of VT estimates was evaluated by absolute variability (VAR) and intraclass correlation coefficients (ICCs). RESULTS: The 1TCM was the statistically preferred model for description of [11C]VC-002 binding in the lungs. Low VAR (< 10%) across analysis methods indicated good reliability of the PET measurements. The VT estimates were stable after 60 min. CONCLUSIONS: The kinetic behaviour and good repeatability of [11C]VC-002 as well as the novel lung image analysis methodology support its application in applied studies on drug-induced mAChR receptor occupancy and the pathophysiology of pulmonary disorders. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03097380, registered: 31 March 2017.

First Radiolabeling of a Ganglioside with a Positron Emitting Radionuclide: In Vivo PET Demonstrates Low Exposure of Radiofluorinated GM1 in Non-human Primate Brain.

Gangliosides are biologically important glycolipids widely distributed in vertebrate cells. An important member of the ganglioside family is the monosialylganglioside GM1, which has been suggested as a potential therapeutic for Parkinson's disease. In the current study, a late-stage radiofluorination protocol was developed, in which fluorine-18 was introduced by substitution of a terminal tosyl group in the fatty acid backbone of GM1. The radiofluorination procedure was remarkably simple and furnished the radiofluorinated ganglioside, [18F]F-GM1, in sufficient quantity and quality without protection of the glycosyl moiety. A positron emission tomography measurement in cynomolgus monkey revealed high uptake of [18F]F-GM1 in heart, bone marrow, and lungs but low (<0.4% of injected dose) distribution to the brain. Thus, choosing administration route of GM1 for therapy of central nervous system disorders poses further challenges. The present study demonstrates the importance of application of positron emission tomography microdosing studies in guiding early clinical drug development.

Synthesis and Preclinical Evaluation of 6-[18F]Fluorine-α-methyl-l-tryptophan, a Novel PET Tracer for Measuring Tryptophan Uptake.

The positron emission tomography (PET) radioligand α-[11C]methyl-l-tryptophan ([11C]AMT) has been used to assess tryptophan metabolism in cancer, epilepsy, migraine, and autism. Despite its extensive application, the utility of this tracer is currently hampered by the short half-life of the radionuclide used for its labeling (11C, t1/2 = 20.4 min). We herein report the design, synthesis, radiolabeling, and initial in vivo evaluation of a fluorine-18 (18F, t1/2 = 109.7 min) labeled analogue that is fluorinated in the 6-position of the aromatic ring ([18F]6-F-AMTr). In a head-to-head comparison between [18F]6-F-AMTr and [11C]AMT in mice using PET, peak brain radioactivity, regional brain distribution, and kinetic profiles were similar between the two tracers. [18F]6-F-AMTr was however not a substrate for IDO1 or TPH as determined in in vitro enzymatic assays. The brain uptake of the tracer is thus more likely related to LAT1 transport over the blood-brain barrier than metabolism along the serotonin or kynurenine pathways.

A PET study in healthy subjects of brain exposure of 11C-labelled osimertinib - A drug intended for treatment of brain metastases in non-small cell lung cancer.

Osimertinib is a tyrosine kinase inhibitor (TKI) of the mutated epidermal growth factor receptor (EGFRm) with observed efficacy in patients with brain metastases. Brain exposure and drug distribution in tumor regions are important criteria for evaluation and confirmation of CNS efficacy. The aim of this PET study was therefore to determine brain distribution and exposure of 11C-labelled osimertinib administered intravenously in subjects with an intact blood-brain barrier. Eight male healthy subjects (age 52 ± 8 years) underwent one PET measurement with 11C-osimertinib. The pharmacokinetic parameters Cmax(brain) (standardized uptake value), Tmax(brain) and AUC0-90 minbrain/blood ratio were calculated. The outcome measure for 11C-osimertinib brain exposure was the total distribution volume (VT). 11C-osimertinib distributed rapidly to the brain, with higher uptake in grey than in white matter. Mean Cmax, Tmax and AUC0-90 minbrain/blood ratio were 1.5 (range 1-1.8), 13 min (range 5-30 min), and 3.8 (range 3.3-4.1). Whole brain and white matter VT were 14 mL×cm-3 (range 11-18) and 7 mL×cm-3 (range 5-12). This study in healthy volunteers shows that 11C-osimertinib penetrates the intact blood-brain barrier. The approach used further illustrates the role of molecular imaging in facilitating the development of novel drugs for the treatment of malignancies affecting the brain.

The pro-psychotic metabotropic glutamate receptor compounds fenobam and AZD9272 share binding sites with monoamine oxidase-B inhibitors in humans.

The metabotropic glutamate receptor 5 (mGluR5) ligands fenobam and AZD9272 have been reported to induce psychosis-like adverse events and to bind at unknown, non-GluR5-related, sites. Based on similarities of the regional binding patterns for [11C]AZD9272 and the monoamine oxidase-B (MAO-B) radioligand [11C]L-deprenyl-D2 in PET studies of the human brain we tested the hypothesis that the unique binding of fenobam and AZD9272 may represent specific binding to the MAO-B. PET data previously acquired for subjects examined using [11C]AZD9272 or [11C]L-deprenyl-D2 were re-evaluated to assess the correlations between radioligand binding parameters in human brain. In addition, the pharmacology of AZD9272 binding sites was characterized using competition binding studies carried out in vivo in non-human primates (NHPs) and in vitro using autoradiography in selected human brain regions. The regional binding of [11C]AZD9272 in human brain was closely correlated with that of [11C]L-deprenyl-D2. In PET studies of NHP brain administration of the MAO-B ligand L-deprenyl inhibited binding of radiolabeled AZD9272 and administration of fenobam inhibited binding of [11C]L-deprenyl-D2. Binding of radiolabeled AZD9272 in vitro was potently inhibited by fenobam or MAO-B compounds, and [11C]L-deprenyl-D2 binding was inhibited by fenobam or AZD9272. The findings are consistent with the hypothesis that both fenobam and AZD9272 bind to the MAO-B, which may be of relevance for understanding the mechanism of the psychosis-like adverse events reported for these compounds. Such understanding may serve as a lead to generate new models for the pathophysiology of psychosis.

Building on the success of osimertinib: achieving CNS exposure in oncology drug discovery.

Due to the blood-brain barrier (BBB) limiting the exposure of therapeutics to the central nervous system (CNS), patients with brain malignancies are challenging to treat, typically have poor prognoses, and represent a significant unmet medical need. Preclinical data report osimertinib to have significant BBB penetration and emerging clinical data demonstrate encouraging activity against CNS malignancies. Here, we discuss the oncology drug candidates AZD3759 and AZD1390 as case examples of discovery projects designing in BBB penetrance. We demonstrate how these innovative kinase inhibitors were recognized as brain penetrant and outline our view of experimental approaches and strategies that can facilitate the discovery of new brain-penetrant therapies for the treatment of primary and secondary CNS malignancies as well as other CNS disorders.

The development of a GPR44 targeting radioligand [11C]AZ12204657 for in vivo assessment of beta cell mass.

BACKGROUND: The G-protein-coupled receptor 44 (GPR44) is a beta cell-restricted target that may serve as a marker for beta cell mass (BCM) given the development of a suitable PET ligand. METHODS: The binding characteristics of the selected candidate, AZ12204657, at human GPR44 were determined using in vitro ligand binding assays. AZ12204657 was radiolabeled using 11C- or 3H-labeled methyl iodide ([11C/3H]CH3I) in one step, and the conversion of [11C/3H]CH3I to the radiolabeled product [11C/3H]AZ12204657 was quantitative. The specificity of radioligand binding to GPR44 and the selectivity for beta cells were evaluated by in vitro binding studies on pancreatic sections from human and non-human primates as well as on homogenates from endocrine and exocrine pancreatic compartments. RESULTS: The radiochemical purity of the resulting radioligand [11C]AZ12204657 was > 98%, with high molar activity (MA), 1351 ± 575 GBq/µmol (n = 18). The radiochemical purity of [3H]AZ12204657 was > 99% with MA of 2 GBq/µmol. Pancreatic binding of [11C/3H]AZ12204657 was co-localized with insulin-positive islets of Langerhans in non-diabetic individuals and individuals with type 2 diabetes (T2D). The binding of [11C]AZ12204657 to GPR44 was > 10 times higher in islet homogenates compared to exocrine homogenates. In human islets of Langerhans GPR44 was co-expressed with insulin, but not glucagon as assessed by co-staining and confocal microscopy. CONCLUSION: We radiolabeled [11C]AZ12204657, a potential PET radioligand for the beta cell-restricted protein GPR44. In vitro evaluation demonstrated that [3H]AZ12204657 and [11C]AZ12204657 selectively target pancreatic beta cells. [11C]AZ12204657 has promising properties as a marker for human BCM.

The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models.

Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase-related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.

The metabotropic glutamate receptor 5 radioligand [11C]AZD9272 identifies unique binding sites in primate brain.

The metabotropic glutamate receptor 5 (mGluR5) is a target for drug development and for imaging studies of the glutamate system in neurological and psychiatric disorders. [11C]AZD9272 is a selective mGluR5 PET radioligand that is structurally different from hitherto applied mGluR5 radioligands. In the present investigation we compared the binding patterns of radiolabeled AZD9272 and other mGluR5 radioligands in the non-human primate (NHP) brain. PET studies were undertaken using [11C]AZD9272 and the commonly applied mGluR5 radioligand [11C]ABP688. Autoradiography studies were performed in vitro using [3H]AZD9272 and the standard mGluR5 radioligands [3H]M-MTEP and [3H]ABP688 in NHP tissue. Competition binding studies were undertaken in vivo and in vitro using different mGluR5 selective compounds as inhibitors. In comparison to other mGluR5 radioligands radiolabeled AZD9272 displayed a distinct regional distribution pattern with high binding in ventral striatum, midbrain, thalamus and cerebellum. While the binding of [11C]AZD9272 was almost completely inhibited by the structurally unique mGluR5 compound fenobam (2.0 mg/kg; 98% occupancy), it was only partially inhibited (46% and 20%, respectively) by the mGluR5 selective compounds ABP688 and MTEP, at a dose (2.0 mg/kg) expected to saturate the mGluR5. Autoradiography studies using [3H]AZD9272 confirmed a distinct pharmacologic profile characterized by preferential sensitivity to fenobam. The distinctive binding in ventral striato-pallido-thalamic circuits and shared pharmacologic profile with the pro-psychotic compound fenobam warrants further examination of [11C]AZD9272 for potential application in psychiatric neuroimaging studies.

In Vivo Visualization of ß-Cells by Targeting of GPR44.

GPR44 expression has recently been described as highly ß-cell selective in the human pancreas and constitutes a tentative surrogate imaging biomarker in diabetes. A radiolabeled small-molecule GPR44 antagonist, [11C]AZ12204657, was evaluated for visualization of ß-cells in pigs and nonhuman primates by positron emission tomography as well as in immunodeficient mice transplanted with human islets under the kidney capsule. In vitro autoradiography of human and animal pancreatic sections from subjects without and with diabetes, in combination with insulin staining, was performed to assess ß-cell selectivity of the radiotracer. Proof of principle of in vivo targeting of human islets by [11C]AZ12204657 was shown in the immunodeficient mouse transplantation model. Furthermore, [11C]AZ12204657 bound by a GPR44-mediated mechanism in pancreatic sections from humans and pigs without diabetes, but not those with diabetes. In vivo [11C]AZ12204657 bound specifically to GPR44 in pancreas and spleen and could be competed away dose-dependently in nondiabetic pigs and nonhuman primates. [11C]AZ12204657 is a first-in-class surrogate imaging biomarker for pancreatic ß-cells by targeting the protein GPR44.

Toward molecular imaging of the free fatty acid receptor 1.

AIMS: Molecular imaging of the free fatty acid receptor 1 (FFAR1) would be a valuable tool for drug development by enabling in vivo target engagement studies in human. It has also been suggested as a putative target for beta cell imaging, but the inherent lipophilicity of most FFAR1 binders produces high off-target binding, which has hampered progress in this area. The aim of this study was to generate a suitable lead compound for further PET labeling. METHODS: In order to identify a lead compound for future PET labeling for quantitative imaging of FFAR1 in human, we evaluated tritiated small molecule FFAR1 binding probes ([3H]AZ1, [3H]AZ2 and [3H]TAK-875) for their off-target binding, receptor density and affinity in human pancreatic tissue (islets and exocrine) and rodent insulinoma. RESULTS: [3H]AZ1 showed improved specificity to FFAR1, with decreased off-target binding compared to [3H]AZ2 and [3H]TAK-875, while retaining high affinity in the nanomolar range. FFAR1 density in human islets was approximately 50% higher than in exocrine tissue. CONCLUSIONS: AZ1 is a suitable lead compound for PET labeling for molecular imaging of FFAR1 in humans, due to high affinity and reduced off-target binding.