Andrew N. Gifford

123I-labeled AM251: a radioiodinated ligand which binds in vivo to mouse brain cannabinoid CB1 receptors

We have investigated the binding of 123I-labeled N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methy l-1 H-pyrazole-3-carboxamide (AM251), an analog of the cannabinoid receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1 H-pyrazole-3-carboxamide] in the mouse brain. Following intravenous injection, the peak whole-brain uptake of about 1% of the administered activity occurred at about 2 h. By 8 h radioactivity in brain had declined to about half its peak value. High-performance liquid chromatographic analysis showed that > 70% of radioactivity extracted from brain at 2 h was still present as [123I]AM251. Co-injection of SR141716A inhibited the in vivo brain binding of [123I]AM251 dose dependently. At 2 mg/kg, the highest dose that could be tested, inhibition was 50% at 2 h post-administration. The ED50 value calculated assuming that 2 mg/kg gave near-maximal inhibition was about 0.1 mg/kg. In contrast to the brain, radioactivity in other major organs (blood, liver, kidney, heart and lung) was little affected by SR141716A. The regional binding of [123I]AM251 in the brain was consistent with the published distribution of cannabinoid receptors in rat brain, in that the order was hippocampus, striatum > cerebellum > brain stem. delta 9-Tetrahydrocannabinol co-administered intravenously at 10 mg/kg, a dose which induced catalepsy and decreased locomotor activity, decreased the 2 h brain uptake of [123I]AM251 by 10%, but this was not significant (P = 0.08). In in vitro binding assays with mouse hippocampal membranes, tetrahydrocannabinol inhibited binding of [123I]AM251 with an IC50 value of about 700 nM, compared with about 0.2 nM for SR141716A.

Methylphenidate and cocaine have a similar in vivo potency to block dopamine transporters in the human brain.

The reinforcing effects of cocaine and methylphenidate have been linked to their ability to block dopamine transporters (DAT). Though cocaine and methylphenidate have similar in vitro affinities for DAT the abuse of methylphenidate in humans is substantially lower than of cocaine. To test if differences in in vivo potency at the DAT between these two drugs could account for the differences in their abuse liability we compared the levels of DAT occupancies that we had previously reported separately for intravenous methylphenidate in controls and for intravenous cocaine in cocaine abusers. DAT occupancies were measured with Positron Emission Tomography using [11C]cocaine, as a DAT ligand, in 8 normal controls for the methylphenidate study and in 17 active cocaine abusers for the cocaine study. The ratio of the distribution volume of [11C]cocaine in striatum to that in cerebellum, which corresponds to Bmax/Kd +1, was used as measure of DAT availability. Parallel measures were obtained to assess the cardiovascular effects of these two drugs. Methylphenidate and cocaine produced comparable dose-dependent blockade of DAT with an estimated ED50 (dose required to block 50% of the DAT) for methylphenidate of 0.07 mg/kg and for cocaine of 0.13 mg/kg. Both drugs induced similar increases in heart rate and blood pressure but the duration of the effects were significantly longer for methylphenidate than for cocaine. The similar in vivo potencies at the DAT for methylphenidate than for cocaine are in agreement with their reported relative in vitro affinities (Ki 390 nM and 640 nM respectively), which is likely to reflect the similar degree of uptake (8-10% of the injected dose) and regional distribution of these two drugs in the human brain. Thus, differences in the in vivo potency of these two drugs at the DAT cannot be responsible for the differences in their rate of abuse in humans. Other variables i.e. longer duration of methylphenidates side effects may counterbalance its reinforcing effects.

Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors

The binding of [123I]AM251 (a radioiodinated analog of the cannabinoid CB1 receptor antagonist SR141716A) was compared to that of [3H]CP 55,940 in mouse and rat brain preparations. Scatchard analysis of the binding of [123I]AM251 and [3H]CP 55,940 to membranes prepared from mouse cerebellum, striatum and hippocampus yielded similar Bmax values (15-41 pmol/g wet wt tissue). Kd values were lower for [123I]AM251 (0.23-0.62 nM) than for [3H]CP 55,940 (1.3-4 nM). CP 55,940 and SR141716A increased dissociation of [123I]AM251 from binding sites in mouse cerebellar homogenates to a similar extent. The structurally dissimilar cannabinoid receptor ligands THC, methanandamide, WIN 55, 212-2, CP 55,940 and SR141716A were each able to fully compete with binding of both [123I]AM251 and [3H]CP 55,940 in mouse cerebellum. In vitro autoradiography demonstrated that the distribution of binding sites for [123I]AM251 in rat brain was very similar to published distributions of binding sites for [3H]CP 55,940. Together, these observations suggest that AM251 binds to the same site (the cannabinoid CB1 receptor) in rodent brains as CP 55,940. However, the binding site domains which interact with AM251 and CP 55,940 may not be identical, since IC50 values for cannabinoid receptor ligands depended on whether [123I]AM251 or [3H]CP 55,940 was used as radioligand.

Ethanol self-administration and ethanol conditioned place preference are reduced in mice lacking cannabinoid CB1 receptors

Cannabinoids are postulated to play a role in modulating the reinforcing effects of abused drugs, including alcohol. Experiment 1 examined alcohol self-administration in cannabinoid CB1 receptor knockout (KO), heterozygous (HT) and wild type (WT) mice in a two-bottle choice paradigm. Mice were trained in a limited 8 h access/day to 10% (v/v) EtOH (EtOH) versus water. After baseline drinking levels (% EtOH preference and total EtOH intake (g/kg)), results indicated that the CB1 knockout mice displayed significantly lower baseline EtOH consumption compared to wild type mice. Subsequently, treatment with SR141716A (5mg/kg) significantly attenuated EtOH intake in the WT and HT mice but had little effect on the knockout mice. Experiment 2 examined the CB1 WT and CB1 KO strains in a conditioned place preference (CPP) procedure between saline and 2g/kg EtOH. The CB1 WT mice spent significantly more time in the EtOH-paired versus saline-paired chambers, whereas no significant preference was observed in the CB1 KO mice. Finally, we observed that CB1 KO mice were significantly lighter than WT and HT and that SR141716A did not significantly alter body weight. These results demonstrate that the cannabinoid CB1 receptor is an essential component of the molecular pathways underlying the reinforcing effects of alcohol. Thus, medications targeting the CB1 receptors may be beneficial for the treatment of alcoholism.

Dopamine-transporter occupancy after intravenous doses of cocaine and methylphenidate in mice and humans

Abstract Objectives: Recent studies using positron emission tomography (PET) have established the relationship between an intravenous dose of cocaine and the percentage occupancy of the dopamine transporter in humans, and have documented the requirement of more than 50% occupancy for perception of the ”high”. The present experiments were conducted to examine dose–occupancy and dose–effect relationships in mice for cocaine and also for methylphenidate, a dopamine uptake blocker used in pediatric psychiatry. Methods: Percentage occupancies of the dopamine transporter by cocaine and methylphenidate were estimated after intravenous injection in mice from the displacement of in vivo binding of [3H]cocaine from the striatum. Locomotor activity was measured in a photocell apparatus. Results: The relationship between drug doses (milligrams of hydrochloride salt per kilogram body weight) and percentage occupancy of the dopamine transporter was indistinguishable for cocaine and methylphenidate, and corresponded to about 50% occupancy at 0.25 mg/kg and about 80% at 1 mg/kg. This was similar to the relationship between drug dose and transporter occupancy, previously measured in human and baboons using [11C]cocaine or [11C]d-threo-methylphenidate and PET. Methylphenidate increased locomotor activity in the mice substantially more than cocaine at the same dose and the same degree of dopamine-transporter receptor occupancy. Conclusions: The range of dopamine-transporter occupancy required for behavioral activation in the mice was thus similar to that previously reported for experience of a cocaine- or methylphenidate-induced ”high” in human subjects. Our results are consistent with other studies in which both cocaine and methylphenidate were evaluated in animal behavioral assays and were found to have very similar psychopharmacological properties.

Imaging the Brain Marijuana Receptor: Development of a Radioligand that Binds to Cannabinoid CB1 Receptors In Vivo

Abstract: The major active ingredient of marijuana, (−)‐Δ9‐tetrahydrocannabinol, exerts its psychoactive effects via binding to cannabinoid CB1 receptors, which are widely distributed in the brain. Radionuclide imaging of CB1 receptors in living human subjects would help explore the presently unknown physiological roles of this receptor system, as well as the neurochemical consequences of marijuana dependence. Currently available cannabinoid receptor radioligands are exceedingly lipophilic and unsuitable for in vivo use. We report the development of a novel radioligand, [123I]AM281{N‐(morpholin‐4‐yl)‐5‐(4‐[123I]iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide}, that is structurally related to the CB1‐selective antagonist SR141716A [N‐(piperidin‐1‐yl)‐5‐(4‐chlorophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide]. Baboon single photon emission computed tomography studies, mouse brain dissection studies, and ex vivo autoradiography in rat brain demonstrated rapid passage of [123I]AM281 into the brain after intravenous injection, appropriate regional brain specificity of binding, and reduction of binding after treatment with SR141716A. AM281 has an affinity in the low nanomolar range for cerebellar binding sites labeled with [3H]SR141716A in vitro, and binding of [123I]AM281 is inhibited by several structurally distinct cannabinoid receptor ligands. We conclude that [123I]AM281 has appropriate properties for in vivo studies of cannabinoid CB1 receptors and is suitable for imaging these receptors in the living human brain.

A Feedback Regulatory Loop between G3P and Lipid Transfer Proteins DIR1 and AZI1 Mediates Azelaic-Acid-Induced Systemic Immunity

Systemic acquired resistance (SAR), a highly desirable form of plant defense, provides broad-spectrum immunity against diverse pathogens. The recent identification of seemingly unrelated chemical inducers of SAR warrants an investigation of their mutual interrelationships. We show that SAR induced by the dicarboxylic acid azelaic acid (AA) requires the phosphorylated sugar derivative glycerol-3-phosphate (G3P). Pathogen inoculation induced the release of free unsaturated fatty acids (FAs) and thereby triggered AA accumulation, because these FAs serve as precursors for AA. AA accumulation in turn increased the levels of G3P, which is required for AA-conferred SAR. The lipid transfer proteins DIR1 and AZI1, both of which are required for G3P- and AA-induced SAR, were essential for G3P accumulation. Conversely, reduced G3P resulted in decreased AZI1 and DIR1 transcription. Our results demonstrate that an intricate feedback regulatory loop among G3P, DIR1, and AZI1 regulates SAR and that AA functions upstream of G3P in this pathway.

Examination of the effect of the cannabinoid receptor agonist, CP 55,940, on electrically evoked transmitter release from rat brain slices.

In the present study we examined the effect of the cannabinoid receptor agonist, [[1 a,2-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyc lohexyl]-phenol; CP 55,940] on [14C]acetylcholine and [3H]norepinephrine release from hippocampal slices and on [14C]acetylcholine release from striatal slices. CP 55,940 potently inhibited electrically evoked [14C]acetylcholine release from hippocampal slices, with an EC50 of 0.02 microM and a maximal inhibition of 61% at 1 microM. The inhibition of acetylcholine release by CP 55,940 was partially antagonized (60%) by the cannabinoid receptor antagonist, [[N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride; SR 141716A]. Alone, SR 141716A significantly enhanced stimulated [14C]acetylcholine release. In contrast to the effects of CP 55,940 on [14C]acetylcholine release, electrically evoked [3H]norepinephrine release from hippocampal slices and [14C]acetylcholine release from striatal slices were both unaffected by this compound. Similarly, hippocampal [3H]norepinephrine release and striatal [14C]acetylcholine release were not affected by SR 141716A. In conclusion, the results of this study extend our previous data indicating that cannabinoid receptors modulate acetylcholine release in the hippocampus. The effects of cannabinoid receptor activation on [3H]acetylcholine release in the hippocampus does not appear to extend to [3H]norepinephrine release from this region or to acetylcholine release from the striatum.

Cardiovascular effects of methylphenidate in humans are associated with increases of dopamine in brain and of epinephrine in plasma

RationaleThe cardiovascular effects of psychostimulant drugs (methylphenidate, amphetamine, cocaine) have been mostly associated with their noradrenergic effects. However, there is some evidence that dopaminergic effects are involved in the cardiovascular actions of these drugs. Here, we evaluated this association in humans.MethodsPositron emission tomography (PET) and [11C]raclopride, a dopamine (DA) D2 receptor radioligand that competes with endogenous DA for occupancy of the D2 receptors, were used to measure changes in brain DA after different doses of intravenous methylphenidate in 14 healthy subjects. Cardiovascular (heart rate and blood pressure) and catecholamine (plasma epinephrine and norepineprhine) responses were determined in parallel to assess their relationships to methylphenidate-induced changes in brain DA.ResultsMethylphenidate administration significantly increased heart rate, systolic and diastolic blood pressures and epinephrine concentration in plasma. The increases in blood pressure were significantly correlated with methylphenidate-induced increases of DA in striatum (r>0.78, P<0.001) and of plasma epinephrine levels (r>0.82, P<0.0005). In turn methylphenidate-induced DA increases in striatum were correlated with increases of epinephrine in plasma (r=0.85, P<0.0001). Subjects in whom methylphenidate did not increase DA had no change in blood pressure or in plasma epinephrine concentration.DiscussionThese results are consistent with the hypothesis that methylphenidate-induced increases in blood pressure are in part due to its central dopaminergic effects. They also suggest that methylphenidates pressor effects may be in part mediated by DA-induced increases in peripheral epinephrine.

Comparable changes in synaptic dopamine induced by methylphenidate and by cocaine in the baboon brain.

Though the blockade of dopamine transporters (DAT) is associated with cocaines and methylphenidates reinforcing effects, it is the stimulation of dopamine (DA) receptors, achieved by increases in synaptic DA, that enables these effects to occur. Positron emission tomography (PET) and [11C]raclopride were used to assess the levels of occupancy of DA D2 receptors by dopamine achieved by doses of cocaine or methylphenidate previously documented to block over 70% of DAT. Studies were performed in five baboons using a paired scan protocol designed to measure DA D2 receptor availability (Bmax/Kd) at baseline conditions and after intravenous administration of either cocaine or methylphenidate. Cocaine (1–2 mg/kg) or methylphenidate (0.5 mg/kg) administered 5 min prior to [11C]raclopride decreased Bmax/Kd by 29 ± 3% and 32 ± 4%, respectively. Smaller reductions in Bmax/Kd (13% for cocaine given 30 min before [11C]raclopride and 25 ± 10% for methylphenidate given 40 min before [11C]raclopride) were seen with longer periods between drug and radioligand. These observations are consistent with the slower striatal clearance kinetics of [11C]methylphenidate than [11C]cocaine observed in previous PET experiments and with the approximately twofold higher potency of methylphenidate than cocaine in in vitro experiments. Though the elevation of synaptic DA induced by >70% occupancy of DAT by these drugs lead to a modest increase in occupancy of D2 receptors (25–30%), further studies are required to assess if this is an underestimation because of differences in D2 receptor binding kinetics between raclopride and DA. Synapse 31:59–66, 1999.