Discovery of DSP-1053, a novel benzylpiperidine derivative with potent serotonin transporter inhibitory activity and partial 5-HT1A receptor agonistic activity

We have previously shown that SMP-304, a serotonin uptake inhibitor with weak 5-HT_1A partial agonistic activity, may act under high serotonin levels as a 5-HT_1A antagonist that improves the onset of paroxetine in the rat swimming test. However, SMP-304 is mostly metabolized by CYP2D6, indicating limited efficacy among individuals and increased side effects. To reduce CYP2D6 metabolic contribution and enhance SERT/5-HT_1A binding affinity, we carried out a series of substitutions at the bromine atom in the left part of the benzene ring of SMP-304 and replaced the right part of SMP-304 with a chroman-4-one. This optimization work led to the identification of the antidepressant candidate DSP-1053 as a potent SERT inhibitor with partial 5-HT_1A receptor agonistic activity. DSP-1053 showed low CYP2D6 metabolic contribution and a robust increase in serotonin levels in the rat frontal cortex.     

Acetaminophen-induced antinociception via central 5-HT2A receptors

Acetaminophen is one of the most widely used analgesic drugs. Although the mechanism of analgesic action of acetaminophen is still not known, the involvement of the central serotonin (5-hydroxytryptamine: 5-HT) system is one possibility. In the present study, we examined the antinociceptive effect of acute and chronic intraperitoneally (i.p.) administered acetaminophen by tail flick latency measurements in the rat. A significantly increased tail flick latency was observed in acute and 15-day acetaminophen-treated rats, but not in 30-day acetaminophen-treated rats, at a dose of 400 mg/kg/day. To investigate the plasticity of receptors at postsynaptic membrane, we conducted a series of experiments by radioligand binding method on frontal cortex and brainstem membrane. The technique involved radioligand binding with [phenyl-4-³H]spiperone and ketanserin for studying 5-HT_2A receptor characteristics. A significant decrease in the maximum number of 5-HT_2A binding sites (B_max) was demonstrated in all treatment groups with acetaminophen 300 and 400 mg/kg on frontal cortex membrane, whereas the value of the dissociation equilibrium constant (K_d) remained unchanged. The down-regulation of 5-HT_2A binding sites in frontal cortex was of a lesser magnitude after 30 days of treatment and the tail flick latency was as in the control animals. These results suggest that down-regulation of 5-HT_2A receptor in response to 5-HT release is a major step in the mechanism underlying analgesia produced by this agent. On the contrary, chronic use of acetaminophen may result in 5-HT depletion, which in turn produces re-adaptation of postsynaptic 5-HT_2A receptors. These data provide further evidence for a central 5-HT-dependent antinociceptive effect of acetaminophen.     

Visualization and quantification of central 5-HT1A receptors with specific antibodies

Polyclonal antibodies were raised by the repeated injection of rabbits with synthetic peptides corresponding to selective portions (peptide 1: aminoacid residues 12–23, and peptide 2: aminoacid residues 243–268) of the aminoacid sequence of the rat 5-HT_1A receptor. Both antisera allowed the immunoprecipitation of 5-HT_1A receptors but not of other 5-HT receptor types and adrenergic receptors solubilized from rat hippocampal membranes. Immunoblots demonstrated that a single protein of 63 kDa, corresponding to the molecular weight of the rat 5-HT_1A receptor binding subunit, was recognized by each antiserum. Immunoautoradiographic labelling of rat brain sections with the anti-peptide 2-antiserum exhibited the same regional distribution as 5-HT_1A sites labelled by selective radioligands such as [³H]8-OH-DPAT and [¹²⁵I]BH-8-MeO-N-PAT. However regional differences apparently existed between the respective intensity of labelling by the agonist radioligands and the antiserum, which might be explained by variations in the degree of coupling of 5-HT_1A receptor binding subunits with G proteins from one brain area to another.     

BMY 7378: Partial agonist at spinal cord 5-HT1A receptors

Recent data indicate that BMY 7378 demonstrates high affinity, selectivity and low intrinsic activity at hippocampal 5-HT_1A receptors, suggesting that BMY 7378 may represent the first selective 5-HT_1A functional antagonist. The present study examined the agonist and antagonist properties of BMY 7378 at spinal cord 5-HT_1A receptors. In electrophysiological studies, iontophoretic administration of either the 5-HT_1A agonist 8-OH-DPAT (43.8 ± 5.4 nA) or BMY 7378 (46.3 ± 5.2 nA) significantly inhibited the firing rate of wide-dynamic-range dorsal horn units indicating that BMY 7378 demonstrates significant intrinsic activity at spinal cord 5-HT_1A receptors. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 ejection current (20–100 nA) which antagonized the 8-OH-DPAT induced inhibition of dorsal horn unit activity. In behavioral studies in the spinal rat, 8-OH-DPAT increased the animals' sensitivity to noxious levels of mechanical stimulation (ED₅₀ = 269 ± 24 nmol/kg) as did BMY 7378 (ED₅₀ = 295 ± 70 nmol/kg) with no statistically significant difference in the maximal response (Y_max) observed. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 dose (10–1100 nmol/kg) which blocked the hyperalgesic effect of 8-OH-DPAT, rather, each drug produced similar effects which were additive. Further, the 5-HT_1A agonist effects of BMY 7378 were blocked by the 5-HT_1A antagonist, spiperone. Therefore, both the electrophysiologic and reflex data indicate that BMY 7378 possesses significant intrinsic activity at spinal cord 5-HT_1A receptors, and like 8-OH-DPAT is a partial agonist at these receptors. Differences in BMY 7378 intrinsic activity at spinal cord as opposed to hippocampal 5-HT_1A receptors are discussed in terms of regional differences in G-proteins coupled to 5-HT_1A receptors in these two CNS regions.     

Mechanisms responsible for progesterone's protection against lordosis-inhibiting effects of restraint I. Role of progesterone receptors

Progestins and antiprogestins are widely used therapeutic agents in humans. In many cases, these are indicated for the treatment of reproductive activities. However, progesterone has widespread physiological effects including a reduction of the response to stress. We have reported that 5 min of restraint reduced lordosis behavior of ovariectomized rats hormonally primed with estradiol benzoate. When ovariectomized rats received both estradiol benzoate and progesterone priming, restraint had minimal effects on lordosis. Progesterone influences behavior through classical intracellular progesterone receptor-mediated nuclear events as well as extranuclear events. How these multiple events contribute to the response to stress is unclear. The current project was designed to initiate examination of the mechanisms responsible for progesterone's ability to protect against the effects of the restraint. In the first experiment, ovariectomized rats, primed with 10 μg estradiol benzoate, received 500 μg progesterone 4 h, 1 h, or 30 min before restraint. When progesterone was injected 4 h before restraint, progesterone eliminated the effects of restraint. In contrast, progesterone 30 min before restraint offered no protection. Effects of progesterone 1 h before restraint were equivocal allowing the suggestion that less than 4 h of progesterone priming might be sufficient. In the second experiment, the synthetic progestin, medroxyprogesterone, was shown to mimic effects of progesterone in preventing effects of restraint. Finally, the progesterone receptor antagonist, RU486, attenuated progesterone's protection against restraint. These findings offer evidence that ligand-activated progesterone receptor mechanisms contribute to the maintenance of lordosis behavior in the presence of mild stress.     

5-HT1A and 5-HT2A receptors affinity,docking studies and pharmacological evaluation of a series of 8-acetyl-7-hydroxy-4-methylcoumarin derivatives

In this work we describe the synthesis, docking studies and biological evaluation of a focused library of novel arylpiperazinyl derivatives of 8-acetyl-7-hydroxy-4-methylcoumarin. The new compounds were screened for their 5-HT_1A and 5-HT_2A receptor affinity. Among the evaluated compounds, six displayed high affinities to 5-HT_1A receptors (4a-0.9?nM, 6a-0.5?nM, 10a-0.6?nM, 3b-0.9?nM, 6b-1.5?nM, 10b-1?nM). Compound 6a and 10a bearing a bromo- or methoxy- substituent in ortho position of the piperazine phenyl ring, were identified as potent antagonists of the 5-HT_1A receptors. In the tail suspension test, mice injected with 6a showed a dose-dependent increase in depressive-like behavior that was related to a decrease in locomotor activity. Compound 10a did not decrease or prolong immobility time nor did it affect home cage activity. Molecular docking studies using 5-HT_1A and 5-HT_2A homology models revealed structural basis of the high affinity of ortho-substituted derivatives and subtle changes in amino acid interactions patterns depending on the length of the alkyl linker.     

Synthesis of metabolically stable arylpiperazine 5-HT1A receptor agonists

Although N-alkylarylpiperazines as a class are finding use as anxiolytics and antidepressants, many of these arylpiperazines are highly metabolically labile at the n-alkyl-piperazine bond. We have found that cyclopropanating the n-butyl chain contained in the 5-HT_1A receptor agonist ipsapirone (2) instills a resistance to this metabolism as well as providing information about the geometrical requirements of the 5-HT_1A receptor.     

Cortical 5-HT1A receptor downregulation by antidepressants in rat brain

Total 5-HT binding sites and 5-HT_1A receptor density was measured in brain regions of rats treated with imipramine (5 mg/kg body wt), desipramine (10 mg/kg body wt) and clomipramine (10 mg/kg body wt), for 40 days, using [³H]5-HT and [³H]8-OH-DPAT, respectively. It was observed that chronic exposure to tricyclic antidepressants (TCAs) results in significant downregulation of total [³H]5-HT binding sites in cortex (42–76%) and hippocampus (35–67%). The 5-HT_1A receptor density was, however, decreased significantly (32–60%) only in cortex with all the three drugs. Interestingly, in hippocampus imipramine treatment increased the 5-HT_1A receptor density (14%). The affinity of [³H]8-OH-DPAT was increased only with imipramine treatment both in cortex and hippocampus. The affinity of [³H]5-HT to 5-HT binding sites in cortex was increased with imipramine treatment and decreased with desipramine and clomipramine treatment. 5-HT sensitive adenylyl cyclase (AC) activity was significantly increased in cortex with imipramine (72%) and clomipramine (17%) treatment, whereas in hippocampus only imipramine treatment significantly increased AC activity (50%). In conclusion, chronic treatment with TCAs results in downregulation of cortical 5-HT_1A receptors along with concomitant increase in 5-HT stimulated AC activity suggesting the involvement of cortical 5-HT_1A receptors in the mechanism of action of TCAs.     

Dipeptide Tyr-Leu (YL) exhibits anxiolytic-like activity after oral administration via activating serotonin 5-HT1A, dopamine D1 and GABAA receptors in mice

We found that Tyr-Leu (YL) dose-dependently exhibits potent anxiolytic-like activity (0.1-1 mg/kg, i.p.) comparable to diazepam in the elevated plus-maze test in mice. YL was orally active (0.3-3 mg/kg). A retro-sequence peptide or a mixture of Tyr and Leu was inactive. The anxiolytic-like activity of YL was inhibited by antagonists for serotonin 5-HT_1A, dopamine D₁ and GABA_A receptors; however, YL had no affinity for them. We also determined the order of their activation is 5-HT_1A, D₁ and GABA_A receptors using selective agonists and antagonists. Taken together, YL may exhibit anxiolytic-like activity via activation of 5-HT_1A, D₁ and GABA_A receptors.     

Competitive interaction of 5-HT1A receptors with G-protein subtypes in CHO cells demonstrated by RNA interference

Following agonist action, G-protein-coupled receptors may exhibit differential coupling to G-proteins or second messenger pathways, supporting the notion of agonist-directed trafficking. To explore these mechanisms, we have designed and transfected synthetic siRNA duplexes to knockdown different G_α subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine 1A receptors (CHO-h5-HT_1A). siRNAs against G_αi2 and G_αi3 transfected alone or in combination caused a large decrease in the corresponding mRNA level (64-80%) and also at the protein level for G_αi3 (60-70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT_1A, 5-HT stimulated guanosine-5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding was differentially affected by transfection of siRNAs against G_αi protein, siRNAs against G_αi2 inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against G_αi3. The high potency component was abolished after transfection of siRNAs against G_αi3 and the lower potency component was suppressed after transfection of siRNAs against G_αi2. To directly investigate G_αi3 activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for G_αi3 activation, a response that was abolished after transfection of siRNAs against G_αi3 protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only G_αi3 protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT_1A receptors, a change occurs that induces coupling to G_αi2 protein and suppresses signalling through G_αi3 subunits.     

Role of progesterone receptors during postpartum estrus in rats

We studied the role of progesterone receptor (PR) in the display of female sexual behavior during postpartum estrus in rats. Adult female rats were treated with the PR antagonist, RU486 (1.25 and 5 mg), 3 h after parturition and sexual behavior was evaluated throughout the first postpartum day. Estradiol and progesterone serum levels changed during the first 24 h postpartum. The highest estradiol and progesterone levels were found at 9 and 12 h postpartum, respectively. The predominant PR isoform in the hypothalamus and the preoptic area was PR-A during postpartum day. The content of PR-A increased at 6 h postpartum in the hypothalamus and the preoptic area, and decreased in both regions at 9 h. PR-B content only increased in the preoptic area at 12 h postpartum. The highest display of lordotic and proceptive behaviors were found at 12 h postpartum. The treatment with 1.25 and 5 mg of RU486 respectively reduced lordosis by 61% and 92% at 12 h postpartum. These results suggest that PR is essential in the display of postpartum estrus in rats.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Effect of chronic l-DOPA treatment on 5-HT1A receptors in parkinsonian monkey brain

After chronic use of l-3,4-dihydroxyphenylalanine (l-DOPA), most Parkinson’s disease (PD) patients suffer from its side effects, especially motor complications called l-DOPA-induced dyskinesia (LID). 5-HT_1A agonists were tested to treat LID but many were reported to worsen parkinsonism. In this study, we evaluated changes in concentration of serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) and of 5-HT_1A receptors in control monkeys, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys, dyskinetic MPTP monkeys treated chronically with l-DOPA, low dyskinetic MPTP monkeys treated with l-DOPA and drugs of various pharmacological activities: Ro 61-8048 (an inhibitor of kynurenine hydroxylase) or docosahexaenoic acid (DHA) and dyskinetic MPTP monkeys treated with l-DOPA + naltrexone (an opioid receptor antagonist). Striatal serotonin concentrations were reduced in MPTP monkeys compared to controls. Higher striatal 5-HIAA/serotonin concentration ratios in l-DOPA-treated monkeys compared to untreated monkeys suggest an intense activity of serotonin axon terminals but this value was similar in dyskinetic and nondyskinetic animals treated with or without adjunct treatment with l-DOPA. As measured by autoradiography with [³H]8-hydroxy-2-(di-n-propyl) aminotetralin (8-OH-DPAT), a decrease of 5-HT_1A receptor specific binding was observed in the posterior/dorsal region of the anterior cingulate gyrus and posterior/ventral area of the superior frontal gyrus of MPTP monkeys compared to controls. An increase of 5-HT_1A receptor specific binding was observed in the hippocampus of MPTP monkeys treated with l-DOPA regardless to their adjunct treatment. Cortical 5-HT_1A receptor specific binding was increased in the l-DOPA-treated MPTP monkeys alone or with DHA or naltrexone and this increase was prevented in low dyskinetic MPTP monkeys treated with l-DOPA and Ro 61-8048. These results highlight the importance of 5-HT_1A receptor alterations in treatment of PD with l-DOPA.     

Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT₂ receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT_2A and 5-HT_2C receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT_2A receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT_2A receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.     

Primary Structure of the Human Platelet Serotonin 5-HT2A Receptor: Identity with Frontal Cortex Serotonin 5-HT2A Receptor

Abstract: Previous radioligand binding studies have demonstrated human platelet serotonin_2A (5-HT_2A) receptor binding sites. Pharmacological similarities between platelet and frontal cortex 5-HT_2A receptor binding parameters have been demonstrated. However, it is not clear whether the platelet 5-HT_2A receptor primary structure is identical to that of the brain receptor. Three overlapping cDNAs were obtained to span completely the coding region of the 5-HT_2A receptor. These clones were sequenced with external and internal primers. The nucleotide sequence of human platelet 5-HT_2A cDNA was identical to that reported for the human frontal cortex 5-HT_2A receptor, except for nucleotide 102 (T → C), which has been reported to represent a normal DNA polymorphism that does not alter the amino acid sequence. This finding may have implications in the study of neuropsychiatric disorders for which altered platelet 5-HT_2A receptor binding has been demonstrated.     

Butyrophenone analogues in the carbazole series: Synthesis and determination of affinities at D2 and 5-HT2A receptors

We describe a practical and efficient route for synthesis of 2-aminomethyl-1,2,3,9-tetrahydro-4H-carbazol-4-ones using an effective Fisher indole methodology. The most active compounds, 4b (QF 2003B) and 4c (QF 2004B), with pKi (5-HT_2A/D₂) ratio of 1.28 show an antipsychotic profile according to Meltzer's classification.     

Regulation of Serotonin2A Receptors in Heterologous Expression Systems

Abstract: The serotonin_2A and serotonin_2C receptors are unique among receptors coupled to guanine nucleotide binding proteins in that chronic treatment in vivo with agonists as well as antagonists decreases receptor density. In an attempt to uncover molecular events involved in down-regulation of the serotonin_2A receptor, the ability of agonists and antagonists to alter receptor density was examined in three heterologous expression systems, i.e., transfected NIH 3T3, transfected Madin-Darby canine kidney, and transfected AtT-20 cells. All three transfected cell lines exhibited pharmacological properties consistent with that predicted for cells expressing the serotonin_2A receptor. However, the three cell lines displayed different receptor regulation properties in response to drugs acting at the serotonin_2A receptor. In transfected NIH 3T3 cells, neither agonist nor antagonist treatment altered receptor density. Treatment with agonist as well as antagonist led to up-regulation of the serotonin_2A receptor in transfected Madin-Darby canine kidney cells. In transfected AtT-20 cells, treatment with agonist led to receptor down-regulation, whereas antagonist treatment increased receptor density. Thus, the cellular background in which the serotonin_2A receptor is expressed appears to determine the regulation properties of the receptor.     

Differential Membrane Targeting and Pharmacological Characterization of Chimeras of Rat Serotonin 5-HT1A and 5-HT1B Receptors Expressed in Epithelial LLC-PK1 Cells

Abstract: The serotonin 5-HT_1A and 5-HT_1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT_1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT_1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT_1A and 5-HT_1B receptors still able to bind specifically [³H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT_1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT_1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT_1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT_1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).