Fluselenamyl: Evaluation of radiation dosimetry in mice and pharmacokinetics in brains of non-human primate.

INTRODUCTION: To allow quantitative assessment of therapeutic efficacy for therapeutic interventions (either approved or undergoing FDA approvals) for either inhibiting or reducing development of Aß pathophysiology in vivo, 18F-labelled tracers, such as Florbetapir, Florbetaben, and Flutemetamol have been approved. Previously, we have reported on development and preclinical validation of 18F-Fluselenamyl, comprising traits of translatable Aß imaging agents. Herein, we report the dosimetry data for 18F-Fluselenamyl to provide radiation dose deposited within organs and determine effective dose (ED) for human studies, while also evaluating its pharmacokinetics in the nonhuman primate brains. METHODS: To evaluate safety profiles of 18F-Fluselenamyl for enabling its deployment as a PET imaging agent for monitoring Aß pathophysiology in vivo, we estimated the human radiation dosimetry extrapolated from rodent biodistribution data obtained by standard method of organ dissection. Animal biodistribution studies were performed in FVB/NCR mice (20 males, 20 females), following tail-vein injection of the tracer. Following euthanasia of mice, organs were harvested, counted, radiation dose to each organ and whole body was determined using the standard MIRD methodology. For evaluation of pharmacokinetics in non-human primates, following intravenous injection of the tracer, dynamic PET scan of rhesus monkey brains were performed, and co-registered with MR for anatomical reference. Parametric images of tracer transport rate constant and distribution volume relative to cerebellum were generated using a simplified reference tissue model and a spatially-constraint linear regression algorithm. RESULTS: The critical organ in humans has been determined to be the gall bladder with a gender average radiation absorbed dose of 0.079 mGy/MBq with an effective dose of 0.017 mSv/MBq and 0.020 mSv/MBq, in males and females, respectively. Therefore, these data provide preliminary projections on human dosimetry derived from rodent estimates, thereby defining safe imaging conditions for further validations in human subjects. Additionally, the tracer penetrated the non-human primate brain and excreted to background levels at later-time points thus pointing to the potential for high signal/noise ratios during noninvasive imaging. Tissue time activity curves (TACs) also show fast initial uptake with maximum projection of activity at 2-6 min post administration followed by clearance of activity at later time-points from cortex, cerebellum, and white matter of nonhuman primate brain. Parametric images confirmed that the 18F-Fluselenamyl has relative high transport rate constant at striatum, thalamus, and cortex. CONCLUSIONS: The data obtained from radiation dosimetry studies in mice indicate that 18F-Fluselenamyl can be safely used for further evaluation in humans. Additionally, 18F-Fluselenamyl demonstrated ability to traverse the blood brain barrier (BBB) and indicated high initial influx, followed by clearance to background levels in non-human primate brains. Combined information indicates that 18F-Fluselenamyl would be a potential candidate for detecting amyloid plaques in the living human brain.

Live-cell fluorescence imaging: assessment of thioflavin T uptake in human epidermal carcinoma cells.

Thioflavin T (ThT), a positively charged heterocyclic small molecule, is a widely used fluorescent marker of amyloid pathophysiology to confirm the cause of death in post mortem brain tissue of Alzheimer's disease (AD) patients. Literature precedents indicate that current positron emission tomography (PET) agents, such as 11C-PIB and 18F-flutemetamol, share significant structural similarity with ThT, a lipophilic dye which does not traverse the blood-brain barrier (BBB) to enable the detection of Aß plaques in vivo. While vital for maintaining normal physiology and healthy brain function, the BBB comprises brain endothelial cells sealed via paracellular protein complexes, bound by an extracellular matrix forming tight junctions thus controlling the delivery of molecules into the brain. The human P-glycoprotein (Pgp/ABCB1, 170 kD plasma membrane protein), belonging to the ABC family of efflux transporter proteins, also lines the luminal surface of brain endothelial cells thus poised to secrete its recognized substrates into the blood. Herein, we postulate that thioflavin T (ThT), due to its physico-chemical attributes, such as moderate lipophilicity and protonated nitrogen, could very well be recognized as a transport substrate of Pgp (P-glycoprotein, ABCB1) thus restricting its permeation into the brain. To evaluate whether or not ThT is indeed recognized by Pgp as its transport substrate thus limiting its BBB permeability, herein, we evaluate cellular accumulation profiles of ThT and PiB (a similar structural uncharged mimetic) in human epidermal carcinoma KB-3-1 (Pgp-) and MDR KB-8-5 (Pgp+) cells, using live-cell fluorescence imaging. While ThT penetrates KB-3-1 cells, it gets excluded from KB-8-5 cells, and also indicates LY335979-induced uptake in Pgp-expressing cells. Furthermore, the cellular uptake profiles of PiB are not impacted by the expression of Pgp under identical conditions. These data show that uptake profiles of ThT have been modified by the expression of Pgp in these cells, and are inversely proportional to the expression of the transporter protein located on the plasma membrane of these cells. Combined data demonstrate that ThT is efficiently recognized by Pgp as its transport substrate.

Fluselenamyl: A Novel Benzoselenazole Derivative for PET Detection of Amyloid Plaques (Aß) in Alzheimer's Disease.

Fluselenamyl (5), a novel planar benzoselenazole shows traits desirable of enabling noninvasive imaging of Aß pathophysiology in vivo; labeling of both diffuse (an earlier manifestation of neuritic plaques) and fibrillar plaques in Alzheimer's disease (AD) brain sections, and remarkable specificity for mapping Aß compared with biomarker proteins of other neurodegenerative diseases. Employing AD homogenates, [18F]-9, a PET tracer demonstrates superior (2-10 fold higher) binding affinity than approved FDA tracers, while also indicating binding to high affinity site on Aß plaques. Pharmacokinetic studies indicate high initial influx of [18F]-9 in normal mice brains accompanied by rapid clearance in the absence of targeted plaques. Following incubation in human serum, [18F]-9 indicates presence of parental compound up to 3h thus indicating its stability. Furthermore, in vitro autoradiography studies of [18F]-9 with AD brain tissue sections and ex vivo autoradiography studies in transgenic mouse brain sections show cortical Aß binding, and a fair correlation with Aß immunostaining. Finally, multiphoton- and microPET/CT imaging indicate its ability to penetrate brain and label parenchymal plaques in transgenic mice. Following further validation of its performance in other AD rodent models and nonhuman primates, Fluselenamyl could offer a platform technology for monitoring earliest stages of Aß pathophysiology in vivo.

Metalloprobes: Fluorescence imaging of multidrug resistance (MDR1) P-Glycoprotein (Pgp)-mediated functional transport activity in cellulo.

Radiolabeled metalloprobes offer sensitive tools for evaluating quantitative accumulation of chemical entities within pooled cell populations. Although beneficial in translational nuclear imaging, this method precludes interrogation of effects resulting from variations at a single cell level, within the same segment of cell population. Compared with radiotracer bioassays, fluorescence imaging offers a cost-efficient technique to assess accumulation of metalloprobes at a single cell level, and determine their intracellular localization under live cell conditions. To evaluate, whether or not radiotracer assay and fluorescence imaging provide complementary information on utility of metalloprobes to assess functional expression of P-glycoprotein (Pgp) on plasma membrane of tumor cells, imaging studies of fluorescent cationic Ga(III)-ENBDMPI (bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-amino-propyl)ethylenediamine) and its neutral counterpart Zn(II)-ENBDMPI are performed. While the uptake profiles of the cationic metalloprobe are inversely proportional to expression of Pgp in tumor cells, the accumulation profiles of the neutral Zn(II)-ENBDMPI in non-MDR and MDR cells are not significantly impacted. The cationic Ga(III)-ENBDMPI maps with Mito-Tracker Red, thereby confirming localization within mitochondria of non-MDR (Pgp-) cells. Depolarization of both plasmalemmal and mitochondrial potentials decreased retention of the cationic Ga(III)-ENBDMPI within the mitochondria. Additionally, LY335979, an antagonist-induced accumulation of the cationic Ga(III) metalloprobe in MDR (Pgp+) cells indicated specificity of the agent. Compared with traits of Ga(III)-ENBDMPI as a Pgp recognized substrate, Zn(II)-ENBDMPI demonstrated uptake in both MDR and non-MDR cells thus indicating the significance of overall molecular charge in mediating Pgp recognition profiles. Combined data indicate that live cell imaging can offer a cost-effective methodology for monitoring functional Pgp expression.

Characterization of a brain permeant fluorescent molecule and visualization of Aß parenchymal plaques, using real-time multiphoton imaging in transgenic mice.

Emerging paradigms mandate discovery of imaging agents for diagnosing Alzheimer's disease (AD) prior to appearance of clinical symptoms. To accomplish this objective, a novel heterocyclic molecule (4) was synthesized and validated as Aß targeted probe. The agent shows labeling of numerous diffuse Aß plaques in confirmed AD human brain tissues and traverses the blood-brain barrier to enable labeling of parenchymal Aß plaques in live mice (APP(±)/PS1(±)) brains.

A new nucleoside analogue with potent activity against mutant sr39 herpes simplex virus-1 (HSV-1) thymidine kinase (TK).

Nucleoside analogues, such as penciclovir, ganciclovir, acyclovir, and their fluoro-substituted derivatives, have wide utility as antivirals. Among these analogues, FHBG ((18)F-Fluorohydroxybutylguanine) is a well-validated PET (positron emission tomography) probe for monitoring reporter gene expression. To evaluate whether or not imposing rigidity into the flexible side chain of FHBG 4 could also impact its interaction, with amino acid residues within the binding site of HSV1-TK (Herpes Simplex Virus-1 Thymidine Kinase), thus influencing its cytotoxic activity. Herein, the synthesis of a new fluorinated nucleoside analogue 6 (conceived via ligand-docking studies) is reported. Agent 6 demonstrates selective activity against HeLa cells stably transfected with mutant HSV1-sr39TK and is also 47-fold more potent than FHBG.

Weinreb amides in carbene chemistry: a time-resolved IR investigation into a potential intramolecular stabilization mechanism.

A chloromethylhydroxamiccarbene was generated photochemically in an attempt to form an intramolecularly stabilized carbene. A rapidly formed intermediate at 1645 cm(-1) decayed with an observed rate of 1.99 × 10(6) s(-1). Other intermediates were also observed. These also decayed, albeit much more slowly (k(obs) = 3.47 × 10(3) and 1.98 × 10(4) s(-1)). Multiple intermediates are apparently a function of both the proximal N,O-dimethylhydroxamic ester and multiple conformers of both the carbene and precursor.

Synthesis of bioorthogonal and crosslinking amino acids for use in peptide synthesis.

The ability to incorporate non-canonical amino acids into proteins by genetic or chemical methods allows one to introduce novel chemical properties into a protein at a defined residue. Such a residue may then be modified using common organic transformations. In this way, the structure or function of the peptide may be altered without perturbing any of the other neighbouring amino acids in the peptide chain. Here, we describe the syntheses and potential applications of multiple para-substituted phenylalanine derivatives comprising an isothiocyanate, α-diazoketone, or nitrone functionality. In all, three novel amino acids were synthesized in good overall yields. These non-canonical amino acids permit the further development of in vitro and in vivo chemoselective and regioselective bioconjugate reactions not possible with other reagents.

Dipolar cycloaddition of ethyl isocyanoacetate to 3-chloro-2-(methylthio)/2-(methylsulfonyl)quinoxalines: highly regio- and chemoselective synthesis of substituted imidazo[1,5-a]quinoxaline-3-carboxylates.

An efficient route for regio- and chemoselective synthesis of substituted 3-(carboethoxy)imidazo[1,5-a]quinoxalines and novel diimidazo[1,5-a:5',1'-c]quinoxalines via base-induced cycloaddition of ethyl isocyanoacetate to unsymmetrically substituted 3-chloro-2-(methylthio)/2-(methylsulfonyl)quinoxalines has been reported.

Palladium-catalyzed intramolecular N-arylation of heteroarenes: a novel and efficient route to benzimidazo[1,2-a]quinolines.

An efficient new route for the synthesis of benzimidazo[1,2-a]quinolines has been developed via the palladium-catalyzed intramolecular Buchwald-Harwtig aryl amination of newly synthesized 2-(2'-bromoanilino)quinolines.

A concise formal synthesis of alkaloid cryptotackiene and substituted 6H-indolo[2,3-b]quinolines.

A five-step formal synthesis of alkaloid cryptotackiene and its 2-formyl, 11-methyl/phenyl derivatives involving conjugate addition of enolate anion from cyclohexanone (or 4-methylcyclohexanone) to bis[(methylsulfanyl)methylene]-2-oxindole followed by heterocyclization in the presence of ammonium acetate as the key step has been developed. The 11-methylsulfanyl group in the initial precursor can be either desulfurized (Raney Ni) or replaced by methyl/phenyl groups via nickel-catalyzed cross-coupling reaction with appropriate Grignard reagents.