Oleg Sadovski

Radiosynthesis and Evaluation of [11C-Carbonyl]-Labeled Carbamates as Fatty Acid Amide Hydrolase Radiotracers for Positron Emission Tomography

Fatty acid amide hydrolase (FAAH) plays a key role in regulating the tone of the endocannabinoid system. Radiotracers are required to image and quantify FAAH activity in vivo. We have synthesized a series of potent FAAH inhibitors encompassing two classes of N-alkyl-O-arylcarbamates and radiolabeled eight of them with carbon-11. The [11C-carbonyl]-radiotracers were evaluated in vitro and ex vivo in rats as potential FAAH imaging agents for positron emission tomography (PET). Both sets of [11C]O-arylcarbamates showed good to excellent brain penetration and an appropriate regional distribution. Pretreatments with a FAAH inhibitor demonstrated that 80–95% of brain uptake of radioactivity constituted binding of the radiotracers to FAAH. Brain extraction measurements showed that binding to FAAH was irreversible and kinetically different for the two classes of carbamates. These promising results are discussed in terms of the requirements of a suitable radiotracer for the in vivo imaging of FAAH using PET.

Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

INTRODUCTION Fatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [(11)C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor. METHODS PF-04457845 was radiolabeled at the carbonyl position via automated [(11)C]CO(2)-fixation. Ex vivo brain biodistribution of [(11)C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [(11)C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma. RESULTS The title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [(11)C]CO(2) (decay uncorrected) within 25 min from end-of-bombardment in >98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/μmol at end-of-synthesis. Uptake of [(11)C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2-4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71-81%, demonstrating specificity for FAAH. The binding of [(11)C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted. CONCLUSIONS [(11)C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in conscious rodents demonstrate that [11C PF-04457845 is a promising candidate radiotracer for imaging FAAH in the brain with PET. These results coupled with the known pharmacology and toxicology of PF-04457845 should facilitate clinical translation of this radiotracer.

Development and characterization of a promising fluorine-18 labelled radiopharmaceutical for in vivo imaging of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH), the enzyme responsible for terminating signaling by the endocannabinoid anandamide, plays an important role in the endocannabinoid system, and FAAH inhibitors are attractive drugs for pain, addiction, and neurological disorders. The synthesis, radiosynthesis, and evaluation, in vitro and ex vivo in rat, of an (18)F-radiotracer designed to image FAAH using positron emission tomography (PET) is described. Fluorine-18 labelled 3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate, [(18)F]5, was synthesized at high specific activity in a one-pot three step reaction using a commercial module with a radiochemical yield of 17-22% (from [(18)F]fluoride). In vitro assay using rat brain homogenates showed that 5 inhibited FAAH in a time-dependent manner, with an IC50 value of 0.82nM after a preincubation of 60min. Ex vivo biodistribution studies and ex vivo autoradiography in rat brain demonstrated that [(18)F]5 had high brain penetration with standard uptake values of up to 4.6 and had a regional distribution which correlated with reported regional FAAH enzyme activity. Specificity of binding to FAAH with [(18)F]5 was high (>90%) as demonstrated by pharmacological challenges with potent and selective FAAH inhibitors and was irreversible as demonstrated by radioactivity measurements on homogenized brain tissue extracts. We infer from these results that [(18)F]5 is a highly promising candidate radiotracer with which to image FAAH in human subjects using PET and clinical studies are proceeding.

Development of new carbon-11 labelled radiotracers for imaging GABAA- and GABAB-benzodiazepine receptors.

Two quinolines identified as positive allosteric modulators of γ-aminobutyric acid (GABA)(A) receptors containing the α(2) subunit, 9-amino-2-cyclobutyl-5-(6-methoxy-2-methylpyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (4) and 9-amino-2-cyclobutyl-5-(2-methoxypyridin-3-yl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one (5), were radiolabelled at the methoxy position with carbon-11 (half-life=20.4 min). These quinolines represent a new class of potential radiotracers for imaging the benzodiazepine site of GABA(A) receptors with positron emission tomography (PET). Both radiotracers were reliably isolated following reaction of their respective pyridinone/pyridinol tautomeric precursors with [(11)C]CH(3)I in clinically useful, formulated quantities (2.9% and 2.7% uncorrected radiochemical yield, respectively, relative to [(11)C]CO(2)) with high specific activities (>70 GBq μ mol(-1); >2 Ci μ mol(-1)) and high radiochemical purities (>95%). The radiosyntheses reported herein represent rare examples of selectively isolating radiolabelled compounds bearing [(11)C]2-methoxypyridine moieties. Although both radiotracers demonstrated promising imaging characteristics based on preliminary ex vivo biodistribution studies in conscious rodents, higher brain uptake was observed with [(11)C]5 and therefore this radiotracer was further evaluated. Carbon-11 labelled 5 readily penetrated the brain (>1 standard uptake value in cortical regions at 15 min post-injection of the radiotracer), had an appropriate regional brain distribution for GABA(A) receptors that appeared to be reversible, and did not show any appreciable radiometabolites in rat brain homogenates up to 15 min post-injection. Preadministration of flumazenil (1, 10 mg kg(-1)) or 5 (5 mg kg(-1)) effectively blocked >50% of [(11)C]5 binding to the GABA(A) receptor-rich regions, thereby suggesting that this radiotracer is worthy of further evaluation for imaging GABA(A) receptors. Additionally (R,S)-N-(1-(3-chloro-4-methoxyphenyl)ethyl)-3,3-diphenylpropan-1-amine, 6, an allosteric modulator of GABA(B) receptors, was efficiently labelled in one step using [(11)C]methyl iodide. Ex vivo biodistribution studies in conscious rats showed low brain uptake, therefore, efforts are underway to discover alternative radiotracers to image GABA(B). In conclusion, [(11)C]5 is worthy of further evaluation in higher species for imaging GABA(A) receptors in the central nervous system.

Evaluation of a novel radiotracer for positron emission tomography imaging of reactive oxygen species in the central nervous system

INTRODUCTION Few, if any, radiotracers are available for the in vivo imaging of reactive oxygen species (ROS) in the central nervous system. ROS play a critical role in normal cell processes such as signaling and homeostasis but overproduction of ROS is implicated in several disorders. We describe here the radiosynthesis and initial ex vivo and in vivo evaluation of [11C]hydromethidine ([11C]HM) as a radiotracer to image ROS using positron emission tomography (PET). METHODS [11C]HM and its deuterated isotopologue [11C](4) were produced using [11C]methyl triflate in a one-pot, two-step reaction and purified by high performance liquid chromatography. Ex vivo biodistribution studies were performed after tail vein injections of both radiotracers. To demonstrate sensitivity of uptake to ROS, [11C]HM was administered to rats treated systemically with lipopolysaccharide (LPS). In addition, ex vivo autoradiography and in vivo PET imaging were performed using [11C]HM on rats which had been microinjected with sodium nitroprusside (SNP) to induce ROS. RESULTS [11C]HM and [11C](4) radiosyntheses were reliable and produced the radiotracers at high specific activities and radiochemical purities. Both radiotracers demonstrated good brain uptake and fast washout of radioactivity, but [11C](4) washout was faster. Pretreatment with LPS resulted in a significant increase in brain retention of radioactivity. Ex vivo autoradiography and PET imaging of rats unilaterally treated with microinjections of SNP demonstrated increased retention of radioactivity in the treated side of the brain. CONCLUSIONS [11C]HM has the attributes of a radiotracer for PET imaging of ROS in the brain including good brain penetration and increased retention of radioactivity in animal models of oxidative stress.

Radiosynthesis and ex vivo evaluation of [18F]-(S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one for imaging MAO-B with PET

Carbon-11 labeled SL25.1188 ((S)-5-(methoxymethyl)-3-(6-(4,4,4-trifluorobutoxy)benzo[d]isoxazol-3-yl)oxazolidin-2-one) is a reversible radiotracer for monoamine oxidase B that was recently evaluated in healthy volunteers by positron emission tomography (PET). Herein we report the preparation and ex vivo evaluation of a fluorinated SL25.1188 derivative as a candidate (18)F-labeled PET radiotracer. (S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxy methyl)oxazolidin-2-one (1) was labeled with fluorine-18 in 51% uncorrected radiochemical yield having high radiochemical purity (>98%) and specific activity (109±26GBq/μmol). Ex vivo biodistribution studies demonstrated low radioactivity retention, specific binding and metabolic stability within rat brains. High uptake of radioactivity in bone is consistent with metabolic defluorination. In vitro binding assays of longer chain fluoroalkoxy derivatives revealed that the length of the carbon chain is an integral feature in MAO-B inhibitor potency and selectivity within this scaffold.

Development of New PET radiopharmaceuticals for imaging monoamine oxidase-B

Introduction: Imaging monoamine oxidase-B (MAO-B) in the CNS with PET is an important goal for psychiatric studies of high socioeconomic burden including major depressive disorder and Parkinsons disease, as well as epilepsy and addiction to tobacco. The most clinically established radiotracer for this target is the irreversible binding ligand, [C]-D2-L-Deprenyl (Methods, 2002, 27, 263). Our goal was to develop new PET radiopharmaceuticals for imaging MAO-B with superior pharmacological and imaging profiles. We here report the synthesis, radiosynthesis, and cerebral ex vivo biodistribution studies of two potent and selective candidates for imaging cerebral MAO-B, namely a carbon-11 labelled 3-phenylcoumarin and pyrrole.