ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in ...intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1β, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50=10nM), mouse (IC50=9nM) and human (IC50=11nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1β and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated (3HA-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. 3HA-804598 labeled a single class of high affinity binding sites (Kd=2.4nM and apparent Bmax=0.56pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit 3HA-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and 3HA-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.
Purpose
Bruton's tyrosine kinase (BTK) is a key component of B cell receptor (BCR) signaling, and as such a critical regulator of cell proliferation and survival. Aberrant BCR signaling is important ...in the pathogenesis of various B cell malignancies and autoimmune disorders. Here, we describe the development of a novel positron emission tomography (PET) tracer for imaging BTK expression and/or occupancy by small molecule therapeutics.
Methods
Radiochemistry was carried out by reacting the precursor with
18
Ffluoride on a GE FX-FN TracerLab synthesis module to produce
18
FBTK-1 with a 6% decay-corrected radiochemical yield, 100 ± 6 GBq/µmol molar activity, and a radiochemical purity of 99%. Following intravenous administration of
18
FBTK-1 (3.63 ± 0.59 MBq, 0.084 ± 0.05 µg), 60-min dynamic images were acquired in two xenograft models: REC-1, an efficacious mantle cell lymphoma model, and U87MG, a non-efficacious glioblastoma model. Subsequent studies included vehicle, pretreatment (10 min prior to tracer injection), and displacement (30 min post-tracer injection) studies with different reversible BTK inhibitors to examine BTK binding. Human radiation dosimetry was estimated based on PET imaging in healthy rats.
Results
Uptake of
18
FBTK-1 was significantly higher in BTK expressing REC-1 tumors than non-BTK expressing U87MG tumors. Administration of BTK inhibitors prior to tracer administration blocked
18
FBTK-1 binding in the REC-1 tumor model consistent with
18
FBTK-1 binding to BTK. The predicted effective dose in humans was 0.0199 ± 0.0007 mSv/MBq.
Conclusion
18
FBTK-1 is a promising PET tracer for imaging of BTK, which could provide valuable information for patient selection, drug dose determination, and improving our understanding of BTK biology in humans.
The long-lived radionuclides tritium and carbon-14 have been used for many years in pharmaceutical research and development for making key efficacy and toxicological decisions. Early discovery ...utilizes radiolabels for compound selection through radioligand binding assays and autoradiography. In preclinical safety evaluation, the use of labeled compounds for adsorption, distribution, metabolism, and excretion studies is often preferred for the added detection sensitivity. As the drug substance proceeds to the clinic, human metabolism studies are reliant on the use of labeled materials to fulfill required regulatory applications.
Veliparib, a potent PARP inhibitor used against multiple cancerous cells such as metastatic melanoma, breast, non-small cell lung cancer (NSCLC) and prostatic cancer, is required for absorption, ...distribution, metabolism and excretion (ADME) and environmental toxicology assessments studies. Tritium-labeled Veliparib was prepared via the tritiodebromination using tritium gas (T
) in two steps.
CVeliparib was achieved through an asymmetric synthetic route in seven radioactive steps starting from
C (S)-benzyl 2-aminopropanoate.
Venetoclax (ABT-199), a B-cell lymphoma-2 (Bcl-2) protein inhibitor, is currently in clinical development for the treatment of hematologic malignancies. We characterized the absorption, metabolism, ...and excretion of venetoclax in humans. After a single oral dose of
Cvenetoclax to healthy volunteers, the recovery of total radioactive dose was 100%, with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (∼66% of the administered dose). ∼33% of the administered dose was recovered as the parent drug and its nitro reduction metabolite M30 2-((1H-pyrrolo2,3-bpyridin-5-yl)oxy)-N-((3-amino-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-1,1'-biphenyl-2-yl)methyl)piperazin-1-yl)benzamide (13%) in feces. Biotransformation of venetoclax in humans primarily involves enzymatic oxidation on the dimethyl cyclohexenyl moiety, followed by sulfation and/or nitro reduction. Nitro reduction metabolites were likely formed by gut bacteria. Unchanged venetoclax was the major drug-related material in circulation, representing 72.8% of total plasma radioactivity. M27 (oxidation at the 6 position of cyclohexenyl ring followed by cyclization at the α-carbon of piperazine ring; 4-(10aR,11aS)-7-(4-chlorophenyl)-9,9-dimethyl-1,3,4,6,8,10,10a,11a-octahydropyrazino2,1-b1,3benzoxazin-2-yl-N-3-nitro-4-(tetrahydropyran-4-ylmethylamino)phenylsulfonyl-2-(1H-pyrrolo2,3-bpyridin-5-yloxy)benzamide) was identified as a major metabolite, representing 12% of total drug-related material. M27 was primarily formed by cytochrome P450 isoform 3A4 (CYP3A4). Steady-state plasma concentrations of M27 in human and preclinical species used for safety testing suggested that M27 is a disproportionate human metabolite. M27 is not expected to have clinically relevant on- or off-target pharmacologic activities.
PROGRESS TOWARDS AN A‐SYNUCLEIN PET TRACER Tempest, Paul; Tai, Chin‐Yin; Ono, Maiko ...
Alzheimer's & dementia,
December 2022, 2022-12-00, Volume:
18, Issue:
S6
Journal Article
Peer reviewed
Background
The high value for Parkinson’s disease and related disorders of the availability of a potent, selective, brain penetrant α‐synuclein (α‐Syn) PET tracer is widely acknowledged. We aimed to ...develop such an agent by applying an established radiotracer development strategy that has been successfully used with another misfolded CNS protein target.
Method
We used a proprietary collection of CNS‐focused aggregated protein binding agents that APRINOIA had previously created to map the structure‐activity relationship of its tau PET tracer programs, including 18FAPN‐1607. This tracer has a unique binding site on aggregated tau and has recently been characterized by cryo‐EM.
The collection was first screened for selective binding of α‐Syn aggregates vs. tau aggregates using recombinant fibrils. This was followed by fluorescence‐based binding to DLB and MSA tissues. These assays identified 16 promising compounds for progression into autoradiography and homogenate binding assays.
Result
Tritiation was successful for 13 of the hits. Self‐blocking studies confirmed specific binding in human brain tissue sections and homogenates. A lead compound was selected, and preliminary PK experiments in wild type mice indicated that brain uptake and clearance properties were compatible with a desirable PET tracer profile in a non‐diseased state. Further studies to evaluate the binding properties in the diseased state and optimize the lead are ongoing.
Conclusion
The compound collection and tracer development strategy have led to the development of an advanced lead or potentially a viable α‐synuclein PET tracer.
Aromatic deuterium compounds were prepared from the unlabeled parent compounds under microwave irradiation. This novel methodology was applied to the deuterium substituted benzimidazole derivative.
...Deuterium aromatic substituted compounds were prepared by microwave irradiation of the parent unlabeled compounds in the presence of deuterium oxide and deuterium chloride. The percentage of deuterium incorporation was investigated under various reaction conditions. A rapid synthesis of
2H-substituted benzimidazole under microwave irradiation is described.
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