Tolerance to the Diuretic Effects of Cannabinoids and Cross-Tolerance to a κ-Opioid Agonist in THC-Treated Mice.

Daily treatment with cannabinoids results in tolerance to many, but not all, of their behavioral and physiologic effects. The present studies investigated the effects of 7-day exposure to 10 mg/kg daily of Δ(9)-tetrahydrocannabinol (THC) on the diuretic and antinociceptive effects of THC and the synthetic cannabinoid AM4054. Comparison studies determined diuretic responses to the κ-opioid agonist U50,488 and furosemide.

Intranasal administration of glial-derived neurotrophic factor (GDNF) rapidly and significantly increases whole-brain GDNF level in rats.

Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF) exerts significant neuroprotective effects on substantia nigra (SN) neurons in the rat 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD). In this study we used enzyme-linked immunosorbent assay (ELISA) to determine GDNF brain levels and distribution to target regions (i.e.

Synthesis and preliminary evaluation of N-(16-18F-fluorohexadecanoyl)ethanolamine (18F-FHEA) as a PET probe of N-acylethanolamine metabolism in mouse brain.

N-Acylethanolamines are lipid signaling molecules found throughout the plant and animal kingdoms. The best-known mammalian compound of this class is anandamide, N-arachidonoylethanolamine, one of the endogenous ligands of cannabinoid CB1 and CB2 receptors. Signaling by N-acylethanolamines is terminated by release of the ethanolamine moiety by hydrolyzing enzymes such as fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing amidase (NAAA).

Assay and inhibition of diacylglycerol lipase activity.

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.

A methodology for radiolabeling of the endocannabinoid 2-arachidonoylglycerol (2-AG).

The metabolic intermediate and endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) has not been readily labeled, primarily because of its instability toward rearrangement. We now detail a synthetic method that easily gives tritiated 2-AG from [5,6,8,9,11,12,14,15-(3)H(N)]arachidonic acid in two steps. We utilized a short chain 1,3-diacylglycerol and proceeded through the "structured lipid" [5'',6'',8'',9'',11'',12'',14'',15''-(3)H(N)]2-arachidonoyl-1,3-dibutyrylglycerol, a triacylglycerol that was conveniently deprotected in ethanol with acrylic beads containing Candida antarctica lipase B to give [5'',6'',8'',9'',11'',12'',14'',15''-(3)H(N)]2-arachidonoylglycerol ([(3)H]2-AG).

The preparation of 2-O-[1'-C]arachidonoyl-1-O-stearoyl-sn-glycerol.

2-O-Arachidonoyl-1-O-stearoyl-sn-glycerol is the most abundant molecular species of the 1,2-diacyl-sn-glycerol signaling lipids in neural tissue. The facile preparation of 2-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycerol from 2-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine at a hexane and phosphate buffer interface with phospholipase C was demonstrated on a 20 µCi scale in 83% radiochemical yield. The specific activity of the product 2-O-[1'-(14)C]arachidonoyl-1-O-stearoyl-sn-glycerol was 57.

Biodistribution of radiolabeled ethanol in rodents.

The biodistribution of [1-(14)C]ethanol in rodents was examined to determine sites of concentration of ethanol or its metabolites that may contribute to its toxicological and pharmacokinetic characteristics. After i.v.

Applications of Clinical Dopamine Imaging.

Recent technologic advances make it increasingly possible to image neurotransmitter systems in living human brain. The dopamine system has been most intensively studied owing to its involvement in several brain disorders, including motor disorders such as Parkinson's disease and Huntington's disease, as well as psychiatric disorders such as schizophrenia, depression, and compulsive behavioral disorders of multiple types. A variety of aspects of dopamine receptor density, function, and dopaminergic terminal status can now be assessed using the minimally invasive neuroimaging techniques of positron emission tomography and single-photon emission computed tomography.

Applications of clinical dopamine imaging.

Recent technologic advances make it increasingly possible to image neurotransmitter systems in living human brain, The dopamine system has been most intensively studied owing to its involvement in several brain disorders such as Parkinson's disease and Huntington's disease, as well as psychiatric disorders such as schizophrenia, depression, and compulsive behavioral disorders of multiple types. A variety of aspects of dopamine receptor density, function, and dopaminergic terminal status can now be assessed using the minimally invasive neuroimaging techniques of positron emission tomography and single-photon emission computed tomography. Although these techniques are currently used most often in the context of research, clinical applications are rapidly emerging.

Evaluation of the in vivo receptor occupancy for the behavioral effects of cannabinoids using a radiolabeled cannabinoid receptor agonist, R-[125/131I]AM2233.

G-protein coupled receptors exist in both high and low agonist affinity conformations, with tracer levels of agonist radioligands preferentially binding to the former. The goal of the present study was to characterize the in vivo binding of the aminoalkyindole-based, CB1 receptor agonist, R-[125/131I]AM2233 ((2-[125/131I]iodo-phenyl)-[1-(1-methyl-piperidin-2-yl-methyl)-1H-indol-3-yl]-methanone), and to use this radiotracer to selectively measure the receptor occupancy by the related CB1 receptor agonist, WIN55212-2, to the agonist-preferring affinity state of the receptor. In mouse locomotor assays, both WIN55212-2 and AM2233 (racemic) produced an approximately 60% reduction in activity at 1 mg/kg, (i.

Ex vivo imaging of fatty acid amide hydrolase activity and its inhibition in the mouse brain.

There is recent behavioral evidence that fatty acid amide hydrolase (FAAH) inhibitors produce a subset of cannabinoid receptor agonist effects, suggesting both anandamide-specific behavioral functions and possible regional differences in FAAH inhibitory effects. Here, we introduce a novel imaging method to quantify regional differences in brain FAAH activity. Upon intravenous [3H]anandamide administration, brain FAAH activity generates [3H]arachidonic acid, which is promptly trapped in membrane phospholipids.

PET imaging in clinical drug abuse research.

Over the last two decades, SPECT (single photon emission computed tomography) and especially PET (positron emission tomography) have proven increasingly effective imaging modalities in the study of human psychopharmacology. Abusing populations can be studied at multiple times after abstinence begins, to give information about neurochemical and physiological adaptations of the brain during recovery from addiction. Individual human subjects can be studied using multiple positron labeled radiotracers, so as to probe more than one facet of brain function.

Potent cannabinergic indole analogues as radioiodinatable brain imaging agents for the CB1 cannabinoid receptor.

A series of novel aminoalkylindoles was synthesized in an effort to develop compounds that are potent agonists at the CB1 cannabinoid receptor and that are also easily labeled with radioisotopes of iodine for biochemical and imaging studies. 2-Iodophenyl-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]methanone (8, AM2233) had a very high affinity for the rat CB1 receptor, with most of the affinity residing with the (R)-enantiomer. Radioiodinated 8, (R)-8, and (S)-8 were prepared by radioiododestannylation of the tributyltin analogues in high yields, radiochemical purities, and specific radioactivities.

Low doses of alcohol substantially decrease glucose metabolism in the human brain.

Moderate doses of alcohol decrease glucose metabolism in the human brain, which has been interpreted to reflect alcohol-induced decreases in brain activity. Here, we measure the effects of two relatively low doses of alcohol (0.25 g/kg and 0.

Candidate PET radioligands for cannabinoid CB1 receptors: [18F]AM5144 and related pyrazole compounds.

Introduction: The mammalian brain contains abundant G protein-coupled cannabinoid CB(1) receptors that respond to Delta(9)-tetrahydrocannabinol, the active ingredient of cannabis. The availability of a positron emission tomography (PET) radioligand would facilitate studies of the addictive and medicinal properties of compounds that bind to this receptor. Among the known classes of ligands for CB(1) receptors, the pyrazoles are attractive targets for radiopharmaceutical development because they are antagonists and are generally less lipophilic than the other classes.

The slow and long-lasting blockade of dopamine transporters in human brain induced by the new antidepressant drug radafaxine predict poor reinforcing effects.

Background: (2S,3S)-2-(3-Chlorophenyl)-3,5,5,-trimethyl-2-morpholinol hydrochloride (radafaxine) is a new antidepressant that blocks dopamine transporters (DAT). A concern with drugs that block (DAT) is their potential reinforcing effects and abuse liability. Using positron emission tomography (PET) we have shown that for DAT-blocking drugs to produce reinforcing effects they must induce >50% DAT blockade and the blockade has to be fast (within 15 minutes).

Maternal and fetal 11C-cocaine uptake and kinetics measured in vivo by combined PET and MRI in pregnant nonhuman primates.

Unlabelled: Cocaine use during pregnancy has been shown to be deleterious to the infant. This may reflect reduction of flow to placenta or effects on the fetal brain. Methods to assess pharmacokinetics of drugs of abuse in vivo would be useful to investigate the mechanisms underlying the fetal adverse effects.

Brain kinetics of methylphenidate (Ritalin) enantiomers after oral administration.

Methylphenidate (MP) (Ritalin) is widely used for the treatment of attention deficit hyperactivity disorder (ADHD). It is a chiral drug, marketed as the racemic mixture of d- and l-threo enantiomers. Our previous studies (PET and microdialysis) in humans, baboons, and rats confirm the notion that pharmacological specificity of MP resides predominantly in the d-isomer.

DRD2 gene transfer into the nucleus accumbens core of the alcohol preferring and nonpreferring rats attenuates alcohol drinking.

Background: Transient overexpression of the dopamine D2 receptor (DRD2) gene in the nucleus accumbens (NAc) using an adenoviral vector has been associated with a significant decrease in alcohol intake in Sprague Dawley rats. This overexpression of DRD2 reduced alcohol consumption in a two-bottle-choice paradigm and supported the view that high levels of DRD2 may be protective against alcohol abuse.

Methods: Using a limited access (1 hr) two-bottle-choice (water versus 10% ethanol) drinking paradigm, we examined the effects of the DRD2 vector in alcohol intake in the genetically inbred alcohol-preferring (P) and -nonpreferring (NP) rats.

Comparison of the binding of the irreversible monoamine oxidase tracers, [(11)C]clorgyline and [(11)C]l-deprenyl in brain and peripheral organs in humans.

The monoamine oxidase A and B (MAO A and B) radiotracers [(11)C]clorgyline (CLG) and [(11)C]L-deprenyl (DEP) and their deuterium labeled counterparts (CLG-D and DEP-D) were compared to determine whether their distribution and kinetics in humans are consistent with their physical, chemical and pharmacological properties and the reported ratios of MAO A:MAO B in post-mortem human tissues. Irreversible binding was consistently higher for DEP in brain, heart, kidneys and spleen but not lung where CLG >DEP and not in thyroid where there is no DEP binding. The generally higher DEP binding is consistent with its higher enzyme affinity and larger free fraction in plasma while differences in regional distribution for CLG and DEP in brain, heart, thyroid and lungs are consistent with different relative ratios of MAO A and B in humans.

Neuroimaging in drug abuse.

Neuroimaging techniques, including positron emission tomography (PET), are ideally suited for studies of addiction. These minimally invasive modalities yield information about acute and long-term drug-induced structural and functional changes in the brain over time. Changes can be observed in the brains of human and animal subjects during drug self-administration.

Low monoamine oxidase B in peripheral organs in smokers.

One of the major mechanisms for terminating the actions of catecholamines and vasoactive dietary amines is oxidation by monoamine oxidase (MAO). Smokers have been shown to have reduced levels of brain MAO, leading to speculation that MAO inhibition by tobacco smoke may underlie some of the behavioral and epidemiological features of smoking. Because smoking exposes peripheral organs as well as the brain to MAO-inhibitory compounds, we questioned whether smokers would also have reduced MAO levels in peripheral organs.

Monoamine oxidase A imaging in peripheral organs in healthy human subjects.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of many biogenic and dietary amines. Though studies of MAO have focused mainly on its regulatory role in the brain, MAO in peripheral organs also represents a vast mechanism for detoxifying vasoactive compounds as well as for terminating the action of physiologically active amines, which can cross the blood brain barrier. Indeed, robust central and peripheral MAO activity is a major requirement in the safe use of many CNS drugs, particularly antidepressants, and thus an awareness of the MAO inhibitory potential of drugs is essential in therapeutics.

Synthesis of structurally identical fluorine-18 and iodine isotope labeling compounds for comparative imaging.

The synthesis of a benzophenone-based labeling compound designed for comparative imaging studies with both in vivo positron emission tomograph (PET) and single-photon computed tomography (SPECT) and ex vivo autoradiography is described. The new compound can be labeled with either F-18 or iodine radioisotopes to give two different radioisotopmers: N-[2-fluoro-5-(3-[I-131]iodobenzoyl)benzyl]-2-bromoacetamide (1) and N-[2-[F-18]fluoro-5-(3-iodobenzoyl)benzyl]-2-bromoacetamide (2). Compound 1 and 2 have a 2-bromoacetyl group, which can be used to conjugate with biomolecules through a nucleophilic substitution reaction.