Milt Titeler

Evidence for 5-HT2 involvement in the mechanism of action of hallucinogenic agents

The affinities (Ki values) of twenty two psycho-active agents, including LSD, 5-OMe DMT and a series of phenalkylamine derivatives, for cortical 5-HT1 and 5-HT2 binding sites were compared with two measures of behavioral activity. It was found that a significant correlation (r = 0.938) exists between the 5-HT2 binding affinities of these agents and their ED50 values as determined in tests of stimulus generalization using 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) as the training drug. Furthermore, for fifteen of these agents where human data were available, a significant correlation (r = 0.924) also exists between 5-HT2 binding affinities and their human hallucinogenic potencies. The results of this study suggest that the mechanism of action of these agents involves 5-HT2-related events.

Radioligand binding evidence implicates the brain 5-HT2 receptor as a site of action for LSD and phenylisopropylamine hallucinogens

Alterations in brain serotonergic function have been implicated in the mechanism of action of LSD, mescaline, and other similarly acting hallucinogenic drugs of abuse such as STP (2,5-dimethoxyphenylisopropylamine; DOM). In order to test the hypothesis that the mechanism of action of LSD and phenylisopropylamine hallucinogens is through stimulation of a specific brain serotonin receptor sub-type, the affinities of these compounds for radiolabelled 5-HT2, 5-HT1A, 5-HT1B, and 5-HT1C receptors have been determined using recently developed in vitro radioligand binding methodologies. The 5-HT2 receptor was labelled with the agonist/hallucinogen radioligand 3H-DOB (4-bromo-2,5-dimethoxyphenylisopropylamine). The 5-HT1A, 5-HT1B, and 5-HT1C receptors were labelled with 3H-OH-DPAT, 3H-5-HT, and 3H-mesulergine, respectively. In general, the phenylisopropylamines displayed 10–100 fold higher affinities for the 5-HT2 receptor than for the 5-HT1C receptor and 100–1000 fold higher affinities for the 5-HT2 receptor than for the 5-HT1A or 5-HT1B receptor. There was a strong correlation between hallucinogenic potencies and 5-HT2 receptor affinities of the phenylisopropylamines (r=0.90); the correlation coefficients for the 5-HT1A, 5-HT1B, and 5-HT1C were 0.73, 0.85, and 0.78, respectively. Because there is no evidence that 5-HT1A-selective or 5-HT1B-selective agonists are hallucinogenic and because the phenylisopropylamines are potent hallucinogens, a 5-HT2 receptor interaction is implicated and supports our previous suggestions to this effect. A secondary role for 5-HT1C receptors cannot be discounted at this time. These results, when integrated with other receptor pharmacological information, indicate that an important component of the mechanism of action of LSD and the phenylisopropylamine hallucinogens is through stimulation of brain 5-HT2 receptors.

NAN-190: an arylpiperazine analog that antagonizes the stimulus effects of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT)

In order to determine whether NAN 190 might serve as a 5-HT 1A antagonist, it was evaluated in rats trained to discriminate 8-OH-DPAT HBr from saline in a 2-lever operant procedure

Detection of a Novel Serotonin Receptor Subtype (5‐HT1E) in Human Brain: Interaction with a GTP‐Binding Protein

Abstract: [3H]Serotonin (5‐hydroxytryptamine, [3H]5‐HT) was used as a radioligand probe of brain 5‐HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 μM pindolol (to block 5‐HT1Aand 5‐HT1B receptors), and 100 nM mesulergine (to block 5‐HT1c and 5‐HT2 receptors). One of these sites demonstrated high affinity for 5‐carboxyamidotryptamine (5‐CT) and er‐gotamine, consistent with the known pharmacology of the 5‐HT1D receptor; the second site demonstrated low affinity for 5‐CT and ergotamine. Computer‐assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5‐CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5‐CT and ergotamine, respectively) for 45% of the sites. To investigate the non‐5‐HT1D component of the binding, 100 nM 5‐CT (to block 5‐HT1A, 5TTT1B, and 5‐HT1D receptors) was coincubated with [3H]5‐HT, membranes, and mesulergine. The remaining [3H]5‐HT binding (hereafter referred to as “5‐HT1E”) displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5‐CT and ergotamine displayed the highest selectivity for the 5‐HT1D site versus the 5‐HT1E site. The interaction of the 5‐HT1E site with a GTP‐binding protein was demonstrated; the nonhydrolyzable derivatives of GTP, guanosine 5′‐0‐(3‐thiotriphosphate) (GTPγS) and 5′‐guanylylimidodiphosphate [Gpp(NH)p], potently inhibited binding of [3H]5‐HT to the 5‐HT1E site (IC50 values of 16 and 172 nM, respectively) while adenosine 5′‐0‐(3‐thiotriphosphate) (ATPγS) and 5′‐adenylylimidodi‐phosphate [App(NH)p] were without effect. The high affinity of 5‐HT for the site, the unique pharmacological profile of the site, and the interaction of the site with a GTP‐binding protein indicate that this site represents a unique 5‐HT receptor subtype heretofore undescribed, and which we propose to call 5‐HT1E in keeping with the current system of nomenclature in the 5‐HT receptor field.

Detection and Characterization of the Serotonin 5‐HT1D Receptor in Rat and Human Brain

Abstract: In the presence of 1 μM (±)‐pindolol [to block 5‐hydroxytryptamine (5‐HT, serotonin) 5‐HT1A and 5‐HT1B receptors] and 100 nM mesulergine (to block 5‐HT1C receptors), 2.0 nM [3H]5‐HT binding to rat cortical homogenates is specific, saturable, and reversible. Scatchard analysis of [3H]5‐HT binding, in the presence of 1 μM (±)‐pindolol and 100 nM mesulergine, produced a KD of 3.2 nM and Bmax of 43 fmol/mg protein. Distribution studies show this site to be present in most rat brain regions. This site is also detectable in human caudate. The pharmacological profile of this site is distinct from the previously identified 5‐HT receptor subtypes. Compounds with high affinity for 5‐HT1A (8‐hydroxydipropylaminotetralin), 5‐HT1B (trifluoromethylphenylpiperazine), 5‐HT1C (mesulergine), 5‐HT2 (4‐bromo‐2,5‐dimethoxyphenylisopropylamine), and 5‐HT3 (ICS 205–930) receptors have low affinity for this site. These data suggest the presence of an additional, previously unidentified, 5‐HT binding site in rat and human brain tissue. This putative novel 5‐HT receptor has a similar pharmacology to the “5‐HT1D” site detected in bovine brain by Heuring and Peroutka.

Selectivity of serotonergic drugs for multiple brain serotonin receptors: Role of [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB), A 5-HT2 agonist radioligand

The affinities of putative serotonin receptor agonists and antagonists for 5-HT1A, 5-HT1B, 5-HT1C, and 5-HT2 receptors were assayed using radioligand binding assays. The 5-HT1 sites were labeled with the agonist radioligands [3H]-8-hydroxy-2-(di-n-propylamino)-tetralin [3H]-8-OH-DPAT, [3H]-5-HT, and [3H]mesulergine. The 5-HT2 receptor was labeled with the antagonist radioligand [3H]ketanserin or the agonist radioligand [3H]-4-bromo-2,5-dimethoxyphenylisopropylamine ([3H]DOB). The apparent 5-HT1 receptor selectivity of agonist compounds was found to be 50- to 100-fold higher when the 5-HT2 receptor affinity was determined using the antagonist radioligand [3H]ketanserin than when the agonist radioligand [3H]DOB was used. Quipazine, a putative specific 5-HT2 agonist, appeared to be only 3-fold more potent at 5-HT2 than at 5-HT1A receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, quipazine was determined to be 100-fold more potent at 5-HT2 receptors than at 5-HT1A receptors. 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a putative specific 5-HT1B receptor agonist was apparently 10-fold more potent at 5-HT1B receptors than at 5-HT2 receptors when [3H]ketanserin was used as the 5-HT2 radioligand. When [3H]DOB was used as the 5-HT2 radioligand, TFMPP was found to be equipotent at 5-HT1B and 5-HT2 receptors. Using the 5-HT2 antagonist radioligand [3H]ketanserin, a similar pattern of underestimating 5-HT2 receptor selectivity and/or overestimating 5-HT1A or 5-HT1B receptor selectivity was observed for a series of serotonin receptor agonists. Antagonist receptor selectivity was not affected significantly by the nature of the 5-HT2 receptor assay used. These data indicate that, by using an antagonist radioligand to label 5-HT2 receptors and agonist radioligands to label 5-HT1 receptors, the 5-HT1 receptor selectivity may be overestimated. This may be an especially severe problem in serotonin drug development as drugs that interact potently with 5-HT2 receptors have been reported to be psychoactive and/or hallucinogenic.

3,4-Methylenedioxymethamphetamine (MDMA): stereoselective interactions at brain 5-HT1 and 5-HT2 receptors

Abstract3,4-Methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxy-amphetamine (MDA), and their optical isomers, were assayed for their affinities at radiolabeled brain serotonin (5-HT1, 5-HT2) and dopamine (D2) binding sites. (R(−)-MDA and R(−)-MDMA) displayed moderate affinities for 3H-ketanserin-labeled 5-HT2 sites (Ki=3425 and 3310 nM, respectively) whereas the affinities for their S(+)-enantiomers were lower (Ki=13,000 and 15,800 nM, respectively). Similar absolute and relative affinities were obtained at 3H-serotonin-labeled 5-HT1 sites; binding at D2 sites was very low (Ki>25,000 nM in each case). The (−)>(+) order of potency at 5-HT2 sites is consistent with the observation that R(−)-MDA is a more potent psychoactive agent than its S(+)-enantiomer, but contrasts with the reported finding that S(+)-MDMA is more potent than R(−)-MDMA in humans. These results suggest that MDMA, unlike MDA and other hallucinogenic phenylisopropylamines, does not work primarily through a direct interaction at 5-HT sites.

Hallucinogenic drug interactions at human brain 5-HT2 receptors: implications for treating LSD-induced hallucinogenesis.

It has been shown that the hallucinogenic potencies of LSD, the phenylisopropylamines, such as DOB (4-bromo-2,5-dimethoxyphenylisopropylamine) and DOI (4-iodo-2,5-dimethoxyphenylisopropylamine), and the indoleaklylamines, such as DMT (dimethyltryptamine) and 5-OMe-DMT (5-methoxy-dimethyltryptamine), strongly correlate with their in vitro 5-HT2 receptor binding affinities in rat cortical homogenates. In order to ascertain if this correlation applies to human 5-HT2 receptors as well, we examined the affinities of 13 psychoactive compounds at 3H-ketanserin-labelled 5-HT2 receptors in human cortical samples. Both radioligand binding and autoradiographical procedures were used. As in rat brain d-LSD was the most potent displacer of 3H-ketanserin specific binding with a Ki of 0.9 nM. The phenylisopropylamine DOI also displayed high affinity (Ki of 6 nM). Stereospecific interactions were found with DOB; (-_ DOB had a Ki of 17 nM while (+) DOB had a Ki of 55 nM. The behaviorally active compound DOM (4-methyl-2,5-phenylisopropylamine) had an affinity of 162 nM while its behaviorally less active congener iso-DOM had an affinity of 6299 nM. The indolealkylamines 5-OMe-DMT and DMT competed with moderate affinities (207 and 462 nM, respectively). In general, Hill coefficients were significantly less than unity which is consistent with an agonist interaction with 5-HT2 receptors. MDMA, a substituted amphetamine analog was inactive with a Ki of greater than 10 μM. A strong correlation was found for the hallucinogen affinities and human hallucinogenic potencies (r=0.97). Also, human and rat brain 5-HT2 receptor affinities were strongly correlated (r=0.99). These results strongly support the hypothesis that the hallucinogenic effects of these drugs in humans are mediated in whole or in part via 5-HT2 receptors. Furthermore, these studies imply that treatment with 5-HT2 receptor antagonists may be effective in reversing the hallucinogenic effects caused by the ingestion, of LSD and LSD-like drugs.

μ Opiate receptors are selectively labelled by [3H]carfentanil in human and rat brain

[11C]Carfentanil is a potent opioid agonist currently in use as a specific PET (position emission tomography) scan radioligand for brain μ opioid receptors. In order to investigate the receptor interactions of carfentanil in detail [3H]carfentanil was used as a radioligand for labelling receptors in rat and human brain tissue homogenates. [3H]Carfentanil was found to bind saturably and with high affinity (KD = 0.08 ± 0.01 nM) to membranes prepared from human cortical (Bmax = 42 ± 3 fmol/mg) and thalamic (Bmax = 84 ± 3 fmol/mg) tissues and rat cortex (Bmax = 82 ± 4 fmol/mg) and deincephalon (Bmax = 105 ± 5 fmol/mg). Association (1.23 ± 0.19 × 1010Mol−1 × min−1 and dissociation rate (0.19 ± 0.03 min−1) constants were determined in human cortical tissues; results from studies in rat cortical, and rat diencephalon tissue homogenates produced similar kinetic rate constants. Competition studies with a variety of drugs indicated that [3H]carfentanil interacts primarily with μ opioid receptors in the four tissues studied; the affinities of a series of non-radioactive opioid ligands were essentially identical in the four tissues (correlation coefficients = 0.88−0.93). Naloxone, morphine, DAGO ([D-Ala2-MePhe4-Gly-ol5]enkephalin), DADL (D-Ala2-D- Leu5]enkaphalin) and EKC (ethylketazocine) potently displaced specific [3H]carfentanil binding with nM potency while the κ agonist U-69593, the σ agonists (+)-SKF 10047, (+)-3-PPP ((3-hydroxyphenyl)-N-propylpiperidine) and haloperidol and PCP (phencyclidine) were less potent displacing agents. The higher affinities of DAGO and morphine versus DADL for the [3H]carfentanil binding site indicates that δ opioid receptors are not being labelled. These data indicate that [3H]carfentanil is a high affinity, specific μ opioid receptor radioligand that may be of use in vitro for studying μ opioid receptors and supports the PET scanning data indicating the μ opioid receptor specificity of [11C]carfentanil. The molecular kinetic rate constants determined in vitro will be of assistance in using modelling procedures to interpret PET scanning information.

Serotonin 5‐HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein

Abstract: Radioligand binding studies were performed to characterize serotonin 5‐HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5‐HT binding, in the presence of pindolol (to block 5‐HT1A and 5‐HT1B receptors) and mesulergine (to block 5‐HTlc receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5‐HT‐labeled 5‐HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8‐hydroxydipropylaminotetralin, trifluoromethylphenylpiper‐azine, mesulergine, 4‐bromo‐2,5‐dimethoxyphenyliso‐propylamine, and ICS 205–930 had low affinity for [3H]5‐HT‐labeled 5‐HT1D sites, indicating that the pharmacology of the 5‐HT1D site is distinct from that of previously identified 5‐HT1A, 5‐HT1B, 5‐HTlc, 5‐HT2, and 5‐HT3 sites. 5‐HT1D sites in human brain have a similar pharmacology to the 5‐HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S) and guanosine 5′‐(β,γ‐imido)‐triphosphate (Gpp(NH)p), modulated the binding of [3H]5‐HT to 5‐HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5‐HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.