John Mehnert Schaus

5-HT1F receptor agonists inhibit neurogenic dural inflammation in guinea pigs.

THE serotonin (5-HT) receptor subtype mediating inhibition of neurogenic dural inflammation in guinea pigs was investigated using a series of serotonin agonists with differing affinities for the 5-HT1B, 5-HT1D and 5-HT1F receptors. When agonist potencies for inhibiting neurogenic inflammation were compared with affinities for these receptor subtypes, a significant positive correlation was seen only with the 5-HT1F receptor. The potency of agonists in inhibiting adenylate cyclase in cells transfected with human 5-HT1F receptor was also highly correlated with their potency in the animal model of migraine. In situ hybridization demonstrated 5-HT1F receptor mRNA in guinea pig trigeminal ganglion neurons. These data suggest that the 5-HT1F receptor is a rational target for migraine therapeutics.

Lu AE58054, a 5-HT6 antagonist, reverses cognitive impairment induced by subchronic phencyclidine in a novel object recognition test in rats.

The in-vitro potency and selectivity, in-vivo binding affinity and effect of the 5-HT(6)R antagonist Lu AE58054 ([2-(6-fluoro-1H-indol-3-yl)-ethyl]-[3-(2,2,3,3-tetrafluoropropoxy)-benzyl]-amine) on impaired cognition were evaluated. Lu AE58054 displayed high affinity to the human 5-HT(6) receptor (5-HT(6)R) with a Ki of 0.83 nm. In a 5-HT(6) GTPgammaS efficacy assay Lu AE58054 showed no agonist activity, but demonstrated potent inhibition of 5-HT-mediated activation. Besides medium affinity to adrenergic alpha(1A)- and alpha(1B)-adrenoreceptors, Lu AE58054 demonstrated >50-fold selectivity for more than 70 targets examined. Orally administered Lu AE58054 potently inhibited striatal in-vivo binding of the 5-HT(6) antagonist radioligand [(3)H]Lu AE60157 ([(3)H]8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline), with an ED(50) of 2.7 mg/kg. Steady-state modelling of an acute pharmacokinetic/5-HT(6)R occupancy time-course experiment indicated a plasma EC(50) value of 20 ng/ml. Administration of Lu AE58054 in a dose range (5-20 mg/kg p.o.) leading to above 65% striatal 5-HT(6)R binding occupancy in vivo, reversed cognitive impairment in a rat novel object recognition task induced after subchronic treatment for 7 d with phencyclidine (PCP 2 mg/kg b.i.d., i.p. for 7 d, followed by 7 d drug free). The results indicate that Lu AE58054 is a selective antagonist of 5-HT(6)Rs with good oral bioavailability and robust efficacy in a rat model of cognitive impairment in schizophrenia. Lu AE58054 may be useful for the pharmacotherapy of cognitive dysfunction in disease states such as schizophrenia and Alzheimers disease.

The PET Radioligand [11C]MePPEP Binds Reversibly and with High Specific Signal to Cannabinoid CB1 Receptors in Nonhuman Primate Brain

The cannabinoid CB1 receptor is one of the most abundant G protein-coupled receptors in the brain and is a promising target of therapeutic drug development. Success of drug development for neuropsychiatric indications is significantly enhanced with the ability to directly measure spatial and temporal binding of compounds to receptors in central compartments. We assessed the utility of a new positron emission tomography (PET) radioligand to image CB1 receptors in monkey brain. [11C]MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) has high CB1 affinity (Kb=0.574±0.207 nM) but also moderately high lipophilicity (measured LogD7.4=4.8). After intravenous injection of [11C]MePPEP, brain activity reached high levels of almost 600% standardized uptake value (SUV) within 10–20 min. The regional uptake was consistent with the distribution of CB1 receptors, with high radioactivity in striatum and cerebellum and low in thalamus and pons. Injection of pharmacological doses of CB1-selective agents confirmed that the tracer doses of [11C]MePPEP reversibly labeled CB1 receptors. Preblockade or displacement with two CB1 selective agents (ISPB; (4-(3-cyclopentyl-indole-1-sulfonyl)-N-(tetrahydro-pyran-4-ylmethyl)-benzamide) and rimonabant) showed that the majority (>89%) of brain uptake in regions with high receptor densities was specific and reversibly bound to CB1 receptors in the high binding regions. [11C]MePPEP was rapidly removed from arterial plasma. Regional brain uptake could be quantified as distribution volume relative to the concentration of parent radiotracer in plasma. The P-glycoprotein (P-gp) inhibitor DCPQ ((R)-4-[(1a,6,10b)-1,1-dichloro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol) did not significantly increase brain uptake of [11C]MePPEP, suggesting it is not a substrate for this efflux transporter at the blood–brain barrier. [11C]MePPEP is a radioligand with high brain uptake, high specific signal to CB1 receptors, and adequately fast washout from brain that allows quantification with 11C (half-life=20 min). These promising results in monkey justify studying this radioligand in human subjects.

Dopamine receptors labelled by [3H] quinpirole

Since quinpirole (or LY171555) has a high affinity for dopamine D2 receptors, and since the high-affinity state of D2 appears to be the functional state of D2, we prepared [3H]quinpirole to investigate its suitability for labelling the high-affinity state of the D2 receptor. The dissociation constant of [3H]quinpirole binding to canine striatum homogenate was 3.9 nM in the absence of NaCl and 6.8 nM in the presence of NaCl. Only 50% of the total binding was specifically displaced by 10 microM S-sulpiride. The data are consistent with the conclusion that much or most of the [3H]quinpirole binds to the high-affinity state of the D2 receptor, since dopamine D2 agonists and antagonists were the most potent in inhibiting the binding of this ligand, because the density of binding sites was 8-9 pmol/g, about half that for [3H]spiperone, and because the density was reduced by 70% in the presence of guanylylimidodiphosphate. Since quinpirole has a reported Ki value of 5.1 nM for dopamine D3 receptors, similar to the quinpirole Ki value of 4.8 nM for the high-affinity state of the dopamine D2 receptor, it appears that [3H]quinpirole with its Kd of 3.9-6.8 nM could label both these two dopamine receptors. However, since the spiperone and haloperidol Ki values against [3H]quinpirole were the same as their values at dopamine D2 receptors rather than dopamine D3 receptors, it appears that [3H]quinpirole predominantly labels dopamine D2 receptors in the canine striatum. The guanine nucleotide-insensitive component of [3H]quinpirole binding (about 30%) may be to dopamine D3 receptors.

Imaging and Quantitation of Cannabinoid CB1 Receptors in Human and Monkey Brains Using 18F-Labeled Inverse Agonist Radioligands

We recently demonstrated that 11C-MePPEP, a PET ligand for CB1 receptors, has such high uptake in the human brain that it can be imaged for 210 min and that receptor density can be quantified as distribution volume (VT) using the gold standard of compartmental modeling. However, 11C-MePPEP had relatively poor retest and intersubject variabilities, which were likely caused by errors in the measurements of radioligand in plasma at low concentrations by 120 min. We sought to find an analog of 11C-MePPEP that would provide more accurate plasma measurements. We evaluated several promising analogs in the monkey brain and chose the 18F-di-deutero fluoromethoxy analog (18F-FMPEP-d2) to evaluate further in the human brain. Methods: 11C-FMePPEP, 18F-FEPEP, 18F-FMPEP, and 18F-FMPEP-d2 were studied in 5 monkeys with 10 PET scans. We calculated VT using compartmental modeling with serial measurements of unchanged parent radioligand in arterial plasma and radioactivity in the brain. Nonspecific binding was determined by administering a receptor-saturating dose of rimonabant, an inverse agonist at the CB1 receptor. Nine healthy human subjects participated in 17 PET scans using 18F-FMPEP-d2, with 8 subjects having 2 PET scans to assess retest variability. To identify sources of error, we compared intersubject and retest variability of brain uptake, arterial plasma measurements, and VT. Results: 18F-FMPEP-d2 had high uptake in the monkey brain, with greater than 80% specific binding, and yielded less radioactivity uptake in bone than did 18F-FMPEP. High brain uptake with 18F-FMPEP-d2 was also observed in humans, in whom VT was well identified within approximately 60 min. Retest variability of plasma measurements was good (16%); consequently, VT had a good retest variability (14%), intersubject variability (26%), and intraclass correlation coefficient (0.89). VT increased after 120 min, suggesting an accumulation of radiometabolites in the brain. Radioactivity accumulated in the skull throughout the entire scan but was thought to be an insignificant source of data contamination. Conclusion: Studies in monkeys facilitated our development and selection of 18F-FMPEP-d2, compared with 18F-FMPEP, as a radioligand demonstrating high brain uptake, high percentage of specific binding, and reduced uptake in bone. Retest analysis in human subjects showed that 18F-FMPEP-d2 has greater precision and accuracy than 11C-MePPEP, allowing smaller sample sizes to detect a significant difference between groups.

Synthesis, Ex Vivo Evaluation, and Radiolabeling of Potent 1,5-Diphenylpyrrolidin-2-one Cannabinoid Subtype-1 Receptor Ligands as Candidates for In Vivo Imaging

We have reported that [methyl- (11)C] (3 R,5 R)-5-(3-methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one ([(11)C] 8, [(11)C]MePPEP) binds with high selectivity to cannabinoid type-1 (CB 1) receptors in monkey brain in vivo. We now describe the synthesis of 8 and four analogues, namely, the 4-fluorophenyl (16, FMePPEP), 3-fluoromethoxy (20, FMPEP), 3-fluoromethoxy- d 2 (21, FMPEP- d 2), and 3-fluoroethoxy analogues (22, FEPEP), and report their activity in an ex vivo model designed to identify compounds suitable for use as positron emission tomography (PET) ligands. These ligands exhibited high, selective potency at CB 1 receptors in vitro (K b < 1 nM). Each ligand (30 microg/kg, iv) was injected into rats under baseline and pretreatment conditions (3, rimonabant, 10 mg/kg, iv) and quantified at later times in frontal cortex ex vivo with liquid chromatography-mass spectrometry (LC-MS) detection. Maximal ligand uptakes were high (22.6-48.0 ng/g). Under pretreatment, maximal brain uptakes were greatly reduced (6.5-17.3 ng/g). Since each ligand readily entered brain and bound with high selectivity to CB 1 receptors, we then established and here describe methods for producing [(11)C] 8, [(11)C] 16, and [(18)F] 20- 22 in adequate activities for evaluation as candidate PET radioligands in vivo.

Positron Emission Tomography Imaging Using an Inverse Agonist Radioligand to Assess Cannabinoid CB1 Receptors in Rodents

[11C]MePPEP is an inverse agonist and a radioligand developed to image cannabinoid CB1 receptors with positron emission tomography (PET). It provides reversible, high specific signal in monkey brain. We assessed [11C]MePPEP in rodent brain with regard to receptor selectivity, susceptibility to transport by P-glycoprotein (P-gp), sensitivity to displacement by agonists, and accumulation of radiometabolites. We used CB1 receptor knockout mice and P-gp knockout mice to assess receptor selectivity and sensitivity to efflux transport, respectively. Using serial measurements of PET brain activity and plasma concentrations of [11C]MePPEP, we estimated CB1 receptor density in rat brain as distribution volume. CB1 knockout mice showed only nonspecific brain uptake, and [11C]MePPEP was not a substrate for P-gp. Direct acting agonists anandamide (10 mg/kg), methanandamide (10 mg/kg), CP 55,940 (1 mg/kg), and indirect agonist URB597 (0.3 and 0.6 mg/kg) failed to displace [11C]MePPEP, while the inverse agonist rimonabant (3 and 10 mg/kg) displaced >65% of [11C]MePPEP. Radiometabolites represented ~13% of total radioactivity in brain between 30 and 120 min. [11C]MePPEP was selective for the CB1 receptor, was not a substrate for P-gp, and was more potently displaced by inverse agonists than agonists. The low potency of agonists suggests either a large receptor reserve or non-overlapping binding sites for agonists and inverse agonists. Radiometabolites of [11C]MePPEP in brain caused distribution volume to be overestimated by approximately 13%.

Pharmacological Characterization of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Inhibitors

 1. The mechanism of anandamide uptake and disposal has been an issue of considerable debate in the cannabinoid field. Several compounds have been reported to inhibit anandamide uptake or fatty acid amide hydrolase (FAAH; the primary catabolic enzyme of anandamide) activity with varying degrees of potency and selectivity. We recently reported the first evidence of a binding site involved in the uptake of endocannabinoids that is independent from FAAH. There are no direct comparisons of purported selective inhibitory compounds in common assay conditions measuring anandamide uptake, FAAH activity and binding activity.2. A subset of compounds reported in the literature were tested in our laboratory under common assay conditions to measure their ability to (a) inhibit [14C]-anandamide uptake in cells containing (RBL-2H3) or cells lacking (HeLa) FAAH, (b) inhibit purified FAAH hydrolytic activity, and (c) inhibit binding to a putative binding site involved in endocannabinoid transport in both RBL and HeLa cell membranes.3. Under these conditions, nearly all compounds tested inhibited (a) uptake of [14C]-anandamide, (b) enzyme activity in purified FAAH preparations, and (c) radioligand binding of [3H]-LY2183240 in RBL and HeLa plasma membrane preparations. General rank order potency was preserved within the three assays. However, concentration response curves were right-shifted for functional [14C]-anandamide uptake in HeLa (FAAH−/−) cells.4. A more direct comparison of multiple inhibitors could be made in these three assay systems performed in the same laboratory, revealing more information about the selectivity of these compounds and the relationship between the putative endocannabinoid transport protein and FAAH. At least two separate proteins appear to be involved in uptake and degradation of anandamide. The most potent inhibitory compounds were right-shifted when transport was measured in HeLa (FAAH−/−) cells suggesting a requirement for a direct interaction with the FAAH protein to maintain high affinity binding of anandamide or inhibitors to the putative anandamide transport protein.

Human 5-HT1F receptor-stimulated [35S]GTPγS binding: correlation with inhibition of guinea pig dural plasma protein extravasation

To determine the potency and efficacy of 5-HT1F receptor ligands, a [35S]GTPgammaS binding assay was developed and optimized for the human 5-HT1F receptor. Compounds which are known to be effective in the abortive treatment of migraine were tested for efficacy and potency in this assay. Naratriptan, sumatriptan, zolmitriptan, and rizatriptan all had agonist activity. The 5-HT1F receptor ligand LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]-benzamide) was the most potent compound tested with an EC50 of 2.13 +/- 0.15 nM. LY302148 (5-fluoro-3-[1-[2-(1-methyl-1H-pyrazol-4-yl)ethyl]-4-piperidinyl]-1H-ind ole), methysergide, LY306258 (3-dimethylamino-2,3,4,9-tetrahydro-1H-carbazol-6-ol), dihydroergotamine (DHE), L-694,247 and CP-122,288 were also investigated for potency and efficacy. There was a statistically significant correlation between the pEC50 for the stimulation of [35S]GTPgammaS binding and the pID50 for the inhibition of trigeminal nerve-stimulated dural plasma protein extravasation in the guinea pig. In the course of these studies, it was found that the purportedly selective 5-HT1D receptor antagonist GR127935 inhibited 5-HT1F receptor-stimulated [35S]GTPgammaS binding with a Ki of 39.6 +/- 9.5 nM. These studies demonstrate that 5-HT1F receptor-mediated stimulation of [35S]GTPgammaS binding in a clonal cell system is a reproducible, high throughput assay that is predictive of an in vivo model of 5-HT1F receptor activation.

Synthesis of the Dopamine Agonist (-)-Quinpirole

Abstract A convenient synthesis and resolution of (-)-trans-4aR,8aR-1-propyl-6-oxo-decahydroquinoline (4) is described. This compound is then converted into the D-2 selective dopamine agonist, (-)-quinpirole hydrochloride (3).