Synergism between 5-HT1B/1D and 5-HT1A receptor antagonists on turnover and release of 5-HT in guinea-pig brain in vivo

The effects on 5-HT turnover (5-HIAA/5-HT ratio) and extracellular 5-HT and 5-HIAA levels (in vivo microdialysis in freely moving animals) were analysed in guinea-pig brains following the 5-HT_1B receptor antagonist, GR 127935 {N-[4-methoxy-3-(4-methyl-1-piper_{azinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-}yl) [1,1-biphenyl]-4-carboxamide}, or the 5-HT_1A receptor antagonist, WAY-100635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride), administered alone or in combination. GR 127935, injected alone, increased 5-HT turnover with maximal effects approximately 50% above the control levels in the four brain regions examined (hypothalamus, hippocampus, striatum and frontal cortex). GR 127935 significantly increased extracellular concentrations of 5-HT and 5-HIAA in frontal cortex (40%), whereas 5-HIAA, but not 5-HT, was elevated in striatum (20–30%). WAY-100635 did not significantly change 5-HT turnover but caused a small significant increase in the extracellular 5-HT and 5-HIAA concentrations in both striatum and frontal cortex. The combined treatment with GR 127935 and WAY-100635 resulted in an increased 5-HT turnover reaching maximal effects of 70–90% above the control values in all brain regions tested and produced a significant elevation of striatal and frontal cortex extracellular 5-HT (40% and 60%, respectively) and 5-HIAA (60% and 70%, respectively) concentrations. The synergistic effect of the two receptor antagonists on the 5-HT turnover and the terminal release of 5-HT indicate somatodendritic 5-HT release and stimulation of inhibitory 5-HT_1A receptors at this level. Extracellular 5-HIAA seems to be a better marker than 5-HT itself for the evoked 5-HT release when the reuptake mechanism is intact. Received: 2 September 1998 / Accepted: 19 November 1998     

The 5-HT1A receptor agonist, 8-OH-DPAT,preferentially activates cell body 5-HT autoreceptors in rat brain in vivo

Summary The present study was undertaken in an attempt to assess whether the effects of the potent and selective 5HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin, 8-OH-DPAT, on cerebral 5-hydroxytryptamine (5-HT) neurochemistry in vivo are mediated via 5-HT autoreceptors on the cell bodies or on the terminals, and/or via postsynaptic 5-HT receptors. To this end we determined in vivo indices of 5-HT synthesis and release/turnover rates in a number of prominent 5-HT neuronal projection areas in the CNS i) after systemic administration of 8-OH-DPAT to rats with an acute unilateral axotomy of the ascending mesencephalic monoamine neurones, or ii) after local infusion of the compound into the dorsal raphé (DRN) 5-HT cell body region of intact rats. Transection did not alter 5-HT synthesis per se, but prevented the synthesis-inhibitory effect of 8-OH-DPAT. Thus, the 5-HT synthesis-inhibiting action of 8-OH-DPAT is highly dependent upon intact impulse flow in the central 5-HT neurones. On the other hand, local DRN application of the compound (1 g) resulted in a clearcut reduction of the 5-HT synthesis and release indices measured in 5-HT terminals in, e. g., the striatum. These findings provide direct neurochemical evidence that by preferentially stimulating somatodendritic 5-HT_1A receptors, 8-OH-DPAT inhibits the 5-HT neuronal impulse flow, thereby effectuating decreased terminal 5-HT synthesis and release. Taken together, the data are consistent with the suggestion that 8-OH-DPAT acts as an agonist preferentially at cell body vs. terminal 5-HT autoreceptors, therefore also emphasizing the distinction between terminal and cell body 5-HT autoreceptors. The results obtained may have important implications for the understanding of mechanisms involved in regulating the activity of central serotoninergic neurones.Part of these data were presented at the 6th European Winter Conference on Brain Research, Avoriaz, France, March 9–15, 1985, and at the 18th Annual Meeting, Society for Neuroscience, Washington (DC), USA, Nov. 9–15, 1986 (Hjorth et al. 1986, 1987). Send offprint requests to S. Hjorth at the above address     

1-(5-硝基吲哚-2-羰基)-4-[3-(1-甲基乙胺基)-2-吡啶基]哌嗪的制备

地拉韦定(delavirdine)是由美国Pharmacia&Upjohn公司研制的非核苷HIV-1逆转录酶抑制剂(NNRTIs),临床与其它抗HIV药联合使用,用于治疗获得性免疫缺陷综合征[1].1-(5-硝基吲哚-2-羰基)-4-[3-(1-甲基乙胺基)-2-吡啶基]哌嗪(1)是其重要中间体.文献[2-4]以5-硝基吲哚-2-羧酸(2)和1-[3-(1-甲基乙胺基)-2-吡啶基]哌嗪(3)为原料,在1-(3-二甲胺基丙基)-3-乙基碳二亚胺(EDC)作用下缩合制得.EDC价昂,后处理用氯仿作提取溶剂,且需通过柱色谱纯化,不适于规模化生产.     

Further evidence for the importance of 5-HT1A autoreceptors in the action of selective serotonin reuptake inhibitors

The clinical efficacy of antidepressants that block serotonin (5-hydroxytryptamine, 5-HT) reuptake may be restrained by indirect activation of autoreceptors. In vivo microdialysis in rat hippocampus was used to assess the release-inhibitory properties of the 5-HT reuptake inhibitors citalopram and paroxetine. When reuptake was first blocked by infusing citalopram into the hippocampus, systemic administration of citalopram or paroxetine resulted in a 50–70% decrease in hippocampal 5-HT overflow. This presumably reflected the inhibition of 5-HT release subsequent to reuptake blockade in the raphe nuclei and, in turn, activation of somatodendritic autoreceptors. In support, pretreatment with (±)-pindolol or(+)-WAY100135 ((+)-N-tert-butyl-3-(4- (2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide dihydrochloride), to block 5-HT_1A autoreceptors, abolished the decrease in 5-HT produced by systemic injection of the uptake blockers.     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

A Search for New 5-HT1A/5-HT2A Receptor Ligands. In Vitro and in vivo Studies of 1-[ω-(4-Aryl-1-piperazinyl)alkyl]indolin-2(1H)-ones

A series of 1-[ω-(4-aryl-1-piperazinyl)alkyl]indolin-2(1H)-one derivatives 2–14 was synthesized in order to obtain ligands with a dual 5-HT_1A/5-HT_2A activity. The majority of those compounds ( 2–5, 7, 10–13 ) exhibited a high 5-HT_1A (K_i = 2 – 44 nM) and/or 5-HT_2A affinity (K_i = 51 and 39 for 5 and 7 , respectively). Induction of lower lip retraction (LLR) and behavioral syndrome and inhibition of these efects evoked by 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) were used for determination the agonistic and antagonistic activity, respectively, at 5-HT_1A receptors. The 5-HT_2A antagonistic activity was assessed by the blocking effect on the head twitches induced by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in mice. Two of the tested compounds, 1-{3-[4-(3-chlorophenyl)-1-piperazinyl]propyl}-6-fluoroindolin-2(1H)-one ( 5 ) and 1-{3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl}indolin-2(1H)-one ( 7 ), demonstrated a high 5-HT_1A/5-HT_2A affinity and an in vivo antagonistic activity towards both receptor subtypes.     

In vivo control of 5-hydroxytryptamine release by terminal autoreceptors in rat brain areas differentially innervated by the dorsal and median raphe nuclei

Selective serotonin reuptake inhibitors (SSRIs) reduce the 5-HT release in vivo. This effect is due to the activation of somatodendritic 5-HT_1A receptors and it displays a regional pattern comparable to that of selective 5-HT_1A agonists, i.e., preferentially in forebrain areas innervated by the dorsal raphe nucleus (DRN). However, despite a comparatively lower 5-HT_1A-mediated inhibition of 5-HT release and a greater density of serotonergic uptake sites in hippocampus, the net elevation produced by the systemic administration of SSRIs is similar in various forebrain areas, regardless of the origin of serotonergic fibres. As terminal autoreceptors may also limit the SSRI-induced elevations of 5-HT in the extracellular brain space, we reasoned that a differential control of 5-HT release by terminal autoreceptors in DRN- and median raphe-innervated areas might be accountable. To examine this possibility, we have conducted a regional microdialysis study in the DRN, MRN and four forebrain regions preferentially innervated either by the DRN (frontal cortex, striatum) or the median raphe nucleus (MRN; dorsal and ventral hippocampus) using freely moving rats. Dialysis probes were perfused with 1 μM of the SSRI citalopram to augment the endogenous tone on terminal 5-HT autoreceptors. The non-selective 5-HT₁ antagonist methiothepin (10 and 100 μM, dissolved in the dialysis fluid) increased extracellular 5-HT in frontal cortex and dorsal hippocampus in a concentration-dependent manner. The 5-HT_1B/1D antagonist GR 127935 was ineffective at 10 μM and tended to reduce 5-HT in dorsal hippocampus at 100 μM. The local infusion of 100 μM methiothepin significantly elevated the extracellular 5-HT concentration to 142–173% of baseline (mean values of 260 min post-administration) in the DRN, MRN, frontal cortex, striatum and hippocampus (dorsal and ventral). Comparable elevations were noted in the four forebrain regions examined. As observed in frontal cortex and dorsal hippocampus, the perfusion of 10 μM GR 127935 did not elevate 5-HT in DRN, MRN, striatum or ventral hippocampus. Because the stimulated 5-HT release in the DRN has been suggested to be under control of 5-HT_1B/1D receptors, we examined the possible contribution of these receptor subtypes to the effects of methiothepin in the DRN. The perfusion of sumatriptan (0.01–10 μM) or GR 127935 (0.01–10 μM) did not significantly modify the 5-HT concentration in dialysates from the DRN. Thus, the present data suggest that the comparable effects of SSRIs in DRN- and MRN-innervated forebrain regions are not explained by a preferential attenuation of 5-HT release by terminal 5-HT_1B autoreceptors in hippocampus, an area with a low inhibitory influence of somatodendritic 5-HT_1A receptors. Methiothepin-sensitive autoreceptors (possibly 5-HT_1B) appear to play an important role not only in the projection areas but also with respect to the control of 5-HT release in the DRN and MRN. In addition, our findings indicate that GR 127935 is not an effective antagonist of the actions of 5-HT at rat terminal autoreceptors. Received: 27 February 1998 / Accepted: 12 June 1998     

Extracellular serotonin in the prefrontal cortex is limited through terminal 5-HT<Subscript>1B</Subscript> autoreceptors: a microdialysis study in knockout mice

RATIONALE: Serotonin (5-HT) autoreceptors regulate extracellular 5-HT levels and have been suggested to limit the effects of acute treatment with selective serotonin reuptake inhibitors (SSRIs). OBJECTIVES: The role of terminal 5-HT(1B) autoreceptors was assessed by comparing the effects of a SSRI on extracellular 5-HT in wild-type and 5-HT(1B) receptor knockout (KO) mice and by using a 5-HT(1B) receptor antagonist. Since systemic SSRI administration also activates somatodendritic 5-HT(1A) autoreceptors, a SSRI was administered locally to study the role of terminal 5-HT(1B) autoreceptors. METHODS: In vivo microdialysis in wild-type and 5-HT(1B) receptor KO mice was used to study the effects of the 5-HT(1B) receptor agonist CP93129 (1 micro M), the SSRI fluvoxamine (0.3 micro M and 1.0 micro M) and the 5-HT(1B) receptor antagonist NAS-181 (1 micro M) on extracellular 5-HT in the medial prefrontal cortex. RESULTS: The 5-HT increase induced by local SSRI administration was augmented in 5-HT(1B) KO mice relative to wild-type mice and was augmented by simultaneous administration of a 5-HT(1B) receptor antagonist in the latter genotype. Basal 5-HT levels did not differ between the two genotypes. Activation of 5-HT(1B) receptors by CP93129 decreased extracellular 5-HT, whereas 5-HT levels in wild-type mice were not affected by the 5-HT(1B) receptor antagonist NAS-181. In 5-HT(1B) KO mice, NAS-181 did not affect extracellular 5-HT and did not further increase the effect of fluvoxamine, showing that NAS-181 is a selective 5-HT(1B) receptor antagonist. The greater increase in 5-HT levels following combined administration of a SSRI with NAS-181 in wild-type mice, relative to 5-HT(1B) KO mice, suggests possible adaptive changes in the KO mice. CONCLUSIONS: The present study shows that terminal 5-HT(1B) autoreceptors play a significant role in the regulation of 5-HT release in the prefrontal cortex.     

D2 dopaminergic and 5-HT1A serotonergic activity of 2-(1-naphthyl)ethyl- and 2-(2-naphthyl)ethyl amines

Several tertiary 2-phenylethyl, 2-(1-naphthyl)ethyl and 2-(2-naphthyl)ethyl amines were synthesized and their binding affinities for dopamine D(1), D(2) and serotonin 5-HT(1A) receptors evaluated in radioligand binding assays. All compounds were inactive in D(1) dopamine radioligand binding assay. The 2-(1-naphthyl)ethyl analogues expressed a low but significant binding affinity for the D(2) and moderate one for the 5-HT(1A) receptor subtypes. Most of the remaining compounds expressed binding affinity at the 5-HT(1A) receptor subtype but were inactive in D(2) receptor binding assay. Based on these results and considering the chemical characteristics of the compounds synthesized and evaluated for dopaminergic and serotonergic activity throughout the present study it can be concluded that hydrophobic type of interaction (stacking or edge-to-face) plays a significant role in the formation of receptor-ligand complexes of 2-(1-naphthyl)ethyl amines. This structural motive can be applied to design and synthesize new, more potent dopaminergic/serotonergic ligands by slight chemical modifications.     

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (K_Aapp 8.6 μM) and inhibited the specific binding of [³H]5-HT to 5-HT₁ sites. The PAT-induced inhibition of [³H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC₅₀ 2 nM) with the 5-HT_1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [³H]5-HT bound to hippocampal membranes was markedly increased by Mn²⁺ (1 nM) and reduced by GTP (0.1 nM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (K_i 1.4 μM) [³H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K⁺-evoked release of [³H]5-HT previously taken up or newly synthesized from [³H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K⁺-induced depolarization on the synthesis of [³H]5-HT from [³H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.     

3-(ω-Aminoalkyl)-5,5-dialkyl(or spirocycloalkyl-1′,5-)hydantoins as New 5-HT1A and 5-HT2A Receptor Ligands

A series of new 3-(ω-aminoalkyl)-5,5-disubstituted hydantoins, containing 1-phenylpiperazine, 1-(o-methoxyphenyl)piperazine or 1,2,3,4-tetrahydroisoquinoline fragments, were synthesized by standard alkylation procedures and their 5-HT_1A and 5-HT_2A receptor affinities were determined. It has been shown that the investigated derivatives are recognized by 5-HT_1A and 5-HT_2A receptors due to the presence of a 1-arylpiperazine fragment; however, the terminal hydantoin moiety plays an important role in stabilization of the receptor-ligand complex. It has also been found that the two 1-phenylpiperazine derivatives 32 and 36 are new, selective 5-HT_2A receptor ligands (K_i = 34 and 37 nM, respectively), whereas the derivative of 1-(o-methoxyphenyl)piperazine ( 38 ) is a new, highly potent 5-HT_1A receptor ligand (K_i = 0.51 nM) with a moderate affinity for 5-HT_2A receptors (K_i = 213 nM).     

Simultaneous blockade of 5-HT<Subscript>1A/B</Subscript> receptors and 5-HT transporters results in acute increases in extracellular 5-HT in both rats and guinea pigs: in vivo characterization of the novel 5-HT<Subscript>1A/B</Subscript> receptor antagonist/5-HT transport inhibitor SB-649915-B

Rationale The delay in onset and treatment resistance of subpopulations of depressed patients to conventional serotonin reuptake inhibitors has lead to new drug development strategies to produce agents with improved antidepressant efficacy. Objectives We report the in vivo characterization of the novel 5-HT_1A/1B autoreceptor antagonist/5-HT transporter inhibitor (6-[(1-{2-[(2-methyl-5-quinolinyl)oxy]ethyl}-4-piperidinyl)methyl]-2H-1,4-benzoxazin-3(4H)-one), SB-649915-B. Materials and methods Ex vivo binding was used to ascertain 5-HT_1A receptor and serotonin transporter occupancy. 8-OH-DPAT-induced hyperlocomotion and SKF-99101-induced elevation of seizure threshold were used as markers of central blockade of 5-HT_1A and 5-HT_1B receptors, respectively. In vivo electrophysiology in the rat dorsal raphe and microdialysis in freely moving guinea pigs and rats were used to evaluate the functional outcome of SB-649915-B. Results SB-649915-B (1–10 mg/kg p.o.) produced a dose-related inhibition of 5-HT_1A receptor radioligand binding and inhibited ex vivo [³H]5-HT uptake in both guinea pig and rat cortex. SB-649915-B (0.1–10 mg/kg p.o.) reversed both 8-OH-DPAT-induced hyperlocomotor activity and SKF-99101-induced elevation of seizure threshold in the rat, demonstrating in vivo blockade of both 5-HT_1A and 5-HT_1B receptors, respectively. SB-649915-B (0.1–3 mg/kg i.v.) produced no change in raphe 5-HT neuronal cell firing per se but attenuated the inhibitory effect of 8-OH-DPAT. Acute administration of SB-649915-B resulted in increases (approximately two- to threefold) in extracellular 5-HT in the cortex of rats and the dentate gyrus and cortex of guinea pigs. Conclusions Based on these data, one may speculate that the 5-HT autoreceptor antagonist/5-HT transport inhibitor SB-649915-B will have therapeutic efficacy in the treatment of affective disorders with the potential for a faster onset of action compared to current selective serotonin reuptake inhibitors.     

Effects of a selective 5-HT reuptake blocker,citalopram, on the sensitivity of 5-HT autoreceptors: Electrophysiological studies in the rat brain

Summary Citalopram (CIT), is a selective serotonin (5-HT) reuptake blocker and a clinically effective antidepressant. The present electrophysiological studies were undertaken to investigate in vivo the acute and long-term effects of CIT administration on 5-HT neurotransmission. In a first series of experiments, a single dose of CIT (0.05–0.5 mg/kg) was administered intravenously to naive rats while recording the activity of a 5-HT-containing neuron in the nucleus raphe dorsalis. A dose-response relationship of the inhibitory effect of CIT on the firing activity of 5-HT neurons was obtained with an ED₅₀ of 0.23±0.03 mg/kg. In a second series of experiments, rats were treated with CIT (20 mg/kg/day, i.p.) for 2, 7 and 14 days. In rats treated for 2 days, there was a marked reduction in the firing activity of 5-HT neurons in the nucleus raphe dorsalis; there was a partial recovery after 7 days and a complete recovery after 14 days of treatment. The response of 5-HT neurons to intravenously administered LSD was decreased in rats treated for 14 days with CIT, indicating a desensitization of the somatodendritic 5-HT autoreceptor. In a third series of experiments, carried out in rats treated with CIT (20 mg/kg/day, i.p.) for 14 days, the suppression of firing activity of CA₃ hippocampal pyramidal neurons produced by microiontophoretically-applied 5-HT and by the electrical activation of the ascending 5-HT pathway was measured. Long-term treatment with CIT did not modify the responsiveness of these neurons to microiontophoretically-applied 5-HT; however, the effect of the electrical activation of the ascending 5-HT pathway on these same neurons was enhanced. To determine if 5-HT reuptake blockade could be responsible for this enhancement, CIT (1 mg/kg) was injected intravenously in naive rats while stimulating the ascending 5-HT pathway; it failed to modify the effectiveness of the stimulation. To assess the involvement of the 5-HT terminal autoreceptor, methiothepin, a 5-HT autoreceptor antagonist, was injected intravenously (1 mg/kg) in naive rats and in rats treated for 14 days with CIT while stimulating the ascending 5-HT pathway. Methiothepin enhanced the effect of the stimulation in naive rats but failed to do so in the CIT-treated rats. It is concluded that long-term CIT treatment enhances 5-HT neurotransmission by desensitizing both the somatodendritic and terminal 5-HT autoreceptors.     

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

Characterisation of the selective 5-HT1B receptor antagonist SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): in vivo neurochemical and behavioural evidence of anxiolytic/antidepressant activity

The 5-HT_1B receptor has attracted significant interest as a potential target for the development of therapeutics for the treatment of affective disorders such as anxiety and depression. Here we present the in vivo characterisation of a novel, selective and orally bioavailable 5-HT_1B receptor antagonist, SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride). SB-616234-A reversed the 5-HT_1/7 receptor agonist, SKF-99101H-induced hypothermia in guinea pigs in a dose related manner with an ED₅₀ of 2.4 mg/kg p.o. Using in vivo microdialysis in freely moving guinea pigs, SB-616234-A (3–30 mg/kg p.o.) caused a dose-related increase in extracellular 5-HT in the dentate gyrus. Evaluation of antidepressant- and anxiolytic-like effects of this 5-HT_1B receptor antagonist was performed in a variety of models and species. SB-616234-A produced a decrease in immobility time in the mouse forced swim test; an effect suggestive of antidepressant activity. Furthermore, SB-616234-A produced dose-related anxiolytic effects in both rat and guinea pig maternal separation-induced vocalisation models with an ED₅₀ of 1.0 and 3.3 mg/kg i.p., respectively (vs fluoxetine treatment ED₅₀ = 2.2 mg/kg i.p. in both species). Also a significant reduction in posturing behaviours was observed in the human threat test in marmosets; an effect indicative of anxiolytic activity. In summary, SB-616234-A is a novel, potent and orally bioavailable 5-HT_1B receptor antagonist which exhibits a neurochemical and behavioural profile that is consistent with both anxiolytic- and antidepressant-like activity in a variety of species. Taken together these data suggest that SB-616234-A may have therapeutic efficacy in the treatment of affective disorders.     

Cis-(+)-8-OH-1-CH3-DPAT, (+)ALK-3, a novel stereoselective pharmacological probe for characterizing 5-HT release-controlling 5-HT1A autoreceptors

Summary The somatodendritic 5-HT_1A autoreceptor regulating 5-HT neuronal activity is currently poorly defined pharmacologically because there are no specific antagonists, but also because potent and stereoselective agonists are scarce. Moreover, there have been few, if any, attempts to specifically investigate structure-activity relationships for agonists acting at this site. Employing brain microdialysis techniques, we have examined the effects of the enantiomers of cis-8-hydroxy-1-methyl-2-(di-n-propylamino)tetralin (ALK-3; 0.01-0.3 mg/kg s.c.), its trans-1-methyl analogue (ALK-4; 0.3 mg/kg s.c.) and the pure enantiomers of the parent compound - 8-OH-DPAT (0.3 mg/kg s.c.) — in an attempt to address stereochemical agonist structure-activity requirements of 5-HT release-controlling 5-HT_1A autoreceptors in brain. The cis-1-methylated 8-OH-DPAT analogue (+)ALK-3 was comparable to the parent compound in reducing the 5-HT output from rat ventral hippocampus. In comparison, both (–)ALK-3 and the racemic rans-diastereomer to ALK-3, ALK-4, were inactive, while the two stereoisomers of 8-OH-DPAT strongly reduced 5-HT release. Pretreatment with (–)pindolol (8 mg/kg s.c.), which has high affinity for 5-HT_1A radioligand binding sites, blocked the reduction of hippocampal 5-HT release induced by a submaximally effective dose of (+)ALK-3. The direct intrahippocampal administration of (+)ALK3 (10 M) via the perfusion medium did not affect 5-HT output.In summary, the data indicate that (+)ALK-3, like 8-OH-DPAT, is a very potent 5-HT receptor agonist which inhibits terminal 5-HT release in rat hippocampus, probably via activation of somatodendritic 5-HT_1A autoreceptors. However, unlike 8-OH-DPAT, (+)ALK-3 is highly stereoselective and may therefore represent a useful probe in the further characterization of 5-HT_1A receptor-mediated mechanisms and function. The present study defines some of the stereochemical requirements for 5-HT_1A receptor interaction, emphasizing the importance of the receptor region complementary to the C₁ and C₂ carbons of the 8-OH-DPAT molecule. These findings contribute to the establishment of structure-activity relationships for the cell body 5-HT_1A autoreceptors and might be of value in resolving structural features that determine agonist/antagonist activity at central 5-HT_1A receptors. Finally, in conjunction with our recent finding that (+)ALK-3 is a partial agonist at postsynaptic 5HT_1A receptors, the present study extends previous observations suggesting that pre- and postsynaptic 5-HT_1A receptor populations differ in their characteristics. Send offprint requests to S. Hjorth at the above address     

1—（2—吡啶基）哌嗪的合成

用工业品2－氨基吡啶在Br2-HBr体系中重氮化,制得2－溴吡啶,收率73．5％;2－溴吡啶与工业无水哌秦直接反应,制得1－（2－吡啶基）哌嗪,收率60％。     

Chronic fluoxetine-induced desensitization of 5-HT1A and 5-HT1B autoreceptors: regional differences and effects of WAY-100635

Desensitization of 5-HT(1A) and 5-HT(1B) autoreceptors is thought to be the mechanism underlying the therapeutic effects of fluoxetine and other selective serotonin reuptake inhibitors when these are administered chronically. The blockade of 5-HT(1A) autoreceptors occurring on administration of a selective serotonin reuptake inhibitor together with a 5-HT(1A) autoreceptor antagonist is responsible for the acute increase in 5-hydroxytryptamine (serotonin, 5-HT) levels observed under these circumstances. The effects of repeated administration of selective serotonin reuptake inhibitors together with 5-HT(1A) receptor antagonists have not been widely studied. In this work, we found that the effects of fluoxetine (5 mg/kg, i.p., daily for 12 days) to desensitize 5-HT(1B) autoreceptors in the frontal cortex, as measured by the effect of the locally administered 5-HT(1B) receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129), and to desensitize 5-HT(1A) autoreceptors as measured by the action of the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 50 microg/kg, s.c.) to reduce 5-HT levels in cortex, were prevented by concomitant administration of the 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635; 0.3 mg/kg, s.c.). 5-HT(1B) receptor activity in the hypothalamus, as measured by the effects of locally administered CP 93129, and 5-HT(1A) autoreceptor activity, as determined by the effects of subcutaneous 8-OH-DPAT to reduce 5-HT levels in hypothalamus, were not altered either by fluoxetine alone or by fluoxetine in the presence of WAY-100635. The data suggest that the regulation of extracellular levels of 5-HT in the cortex and hypothalamus is subject to different autoregulatory mechanisms.     

Effect of chronic administration of the selective serotonin (5-HT) uptake inhibitor citalopram on extracellular 5-HT and apparent autoreceptor sensitivity in rat forebrain in vivo

Rats were administered the selective serotonin (5-HT) uptake blocker citalopram or saline for 14 days to determine if prolonged treatment would lead to changes in extracellular 5-HT or autoreceptor sensitivity. One day after drug withdrawal, dialysis probes were implanted in the frontal cortex and dorsal hippocampus. Dialysis experiments were carried out using chloral hydrate anesthetized rats. The experimental protocol comprised the administration of three consecutive drug challenges: (1) After stable baseline levels were obtained, citalopram was infused through the dialysis probes to locally block uptake in the forebrain. (2) Subsequently, a 5-HT_1B receptor agonist (RU24969 or CP93,129) was infused through the probe to test for changes in terminal autoreceptor sensitivity. (3) Last, citalopram was administered systemically to test the effect of indirect activation of somatodendritic autoreceptors. Under these conditions, with uptake already blocked locally in the forebrain, systemic citalopram produces a decrease in extracellular 5-HT, an effect that can be inhibited by pretreatment with antagonists of 5-HT_1A receptors. The results indicate that during local infusion of citalopram extracellular 5-HT was significantly higher in the dorsal hippocampus of the chronic citalopram as compared to saline treatment group. This difference persisted throughout the full time course of the experiment. However, the decreases in 5-HT levels produced by local infusion of a 5-HT_1B receptor agonist or after systemic citalopram administration were not significantly different between the chronic citalopram and saline treated groups. There were no significant differences between chronic citalopram and saline treated animals in frontal cortex. These results suggest that prolonged inhibition of 5-HT uptake may produce a selective change in the regulation of release from median raphe 5-HT neurons, but this change could not be clearly linked to a change in nerve terminal or somatodendritic autoreceptor sensitivity.     

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.