5-HT2A/5-HT2C Receptor

Deramciclane is a novel anxiolytic drug with a high affinity for 5-HT2A/2C receptors. Deramcicane's interaction with the serotonin 5-HT2C receptor was further investigated utilizing receptor phosphoinositide hydrolysis assay and receptor autoradiography. Deramciclane inhibits 5-HT2C receptor-mediated 5-HT-induced phosphoinositide hydrolysis with an IC50 of 168 nM. Deramcicane also lowers basal phosphoinositide hydrolysis in the choroid plexus physiological system by up to 33% (EC50=93 nM), showing that it acts as an inverse agonist at this receptor [1].

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5-HT-stimulated phosphoinositide hydrolysis Results of phosphoinositide hydrolysis studies in choroid plexus are summarized in Fig. 1a and b. 100 nM 5-HT stimulated the accumulation of inositol monophosphate (IP) 4- to 5-fold over basal values of about 5000 DPM. Deramciclane antagonized 5-HT (100 nM)-stimulated phosphoinositide hydrolysis, with an IC50 value of 168 nM (Fig. 1a). Deramciclane did not stimulate phosphoinositide hydrolysis itself, but inhibited basal PI hydrolysis by up to 33% with an EC50 value of 90 nM, indicating that deramciclane is a 5-HT2C receptor inverse agonist (Fig. 1b) [1].

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Deramciclane (EGIS-3886) is a putative antiserotonergic compound that reduces 5-HT-induced phosphoinositol hydrolysis and a variety of actions caused by serotonergic agonists. The receptor binding profile of Deramciclane is rather similar to that of ritanserin. Deramciclane has a high affinity for 5-HT2A and 5-HT2C receptors; it acts as an antagonist at both receptor subtypes and has inverse agonist properties at the 5-HT2C receptors without direct stimulatory agonist effects [2].

While 30 mg/kg deramcicane did not substantially modify dopamine levels at 40-100 min and 160-240 min significantly raised dopamine levels (P<0.05), 3 mg/kg and 10 mg/kg deramcicane did not significantly alter dopamine levels at any time point when compared with basal levels. Deramcicane is a suspected antiserotonergic substance that lessens the effects of serotonergic agonists and the hydrolysis of phosphoinositides generated by 5-HT. Deramcicane and ritanserin share many similarities when it comes to receptor binding. With a high affinity for both 5-HT2A and 5-HT2C receptors, deramcicane functions as an antagonist against both subtypes of receptors and exhibits inverse agonist characteristics at 5-HT2C receptors without directly stimulating the effects of agonists. Numerous animal studies have demonstrated deramcicane's anxiolytic-like properties [2].

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Administration of single doses of 0.5 mg/kg and 10 mg/kg resulted in a maximal 5-HT2C receptor occupancy of up to 45% and 79%, respectively, in the choroid plexus. Chronic (14 days) treatment with 0.5 mg/kg or 10 mg/kg Deramciclane did not alter [125I]DOI (agonist) or [3H]mesulergine (antagonist) binding to 5-HT2C receptors in the choroid plexus compared to saline-treated controls, as determined by quantitative receptor autoradiography. In comparison, the effects of deramciclane on 5-HT2A binding characteristics and receptor occupancy were also studied. Deramciclane treatment resulted in 5-HT2A receptor occupancy of up to 78%, but no significant effect of chronic treatment on 5-HT2A receptor agonist binding levels was found. In conclusion, these data indicate that Deramciclane is a 5-HT2C receptor inverse agonist and occupies 5-HT2C receptors during treatment, and that chronic treatment with deramciclane does not lead to 5-HT2C receptor down-regulation [1].

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In the present study, we assessed the effect of single graded doses of a putative anxiolytic compound, the 5-HT(2A/C )antagonist, Deramciclane fumarate (EGIS-3886), on the dopamine efflux and metabolism in nucleus accumbens and striatum and thus evaluated the dose window for Deramciclane to cause adverse effects related to the brain dopaminergic system. Dual probe in vivo microdialysis in freely moving rats was used to compare the effects of graded doses of deramciclane fumarate (3, 10 and 30 mg/kg), 5-HT(2A/C )antagonist ritanserin (1 mg/kg) and a partial 5-HT(1A) agonist buspirone hydrochloride (5 mg/kg) on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in nucleus accumbens and striatum assayed by high performance liquid chromatography with electrochemical detection. The indirect dopamine agonist, D-amphetamine sulfate (2 mg/kg), was used as a positive control. Ritanserin, buspirone and deramciclane 3 and 10 mg/kg had no significant effects on the extracellular dopamine levels in either brain area but Deramciclane 30 mg/kg significantly increased accumbal dopamine as well as DOPAC and HVA in both brain areas. As expected, the positive control D-amphetamine significantly increased both striatal and accumbal dopamine levels. The effects of buspirone or the highest deramciclane dose and D-amphetamine on DOPAC and HVA levels were opposite; buspirone and deramciclane increased while D-amphetamine decreased the metabolite levels in both brain areas. The results indicate that a single high dose of deramciclane has the neuroleptic- or buspirone-like effect, particularly in mesolimbic regions. There is at least a 5-fold margin between the anxiolytic and neuroleptic doses of deramciclane in the rat [2].

5-HT2C receptor mediated phosphoinositide hydrolysis assay [1]
5-HT-induced phosphoinositide hydrolysis was measured in rat choroid plexus as previously described (Kuoppamäki et al. 1993). Choroid plexi were rapidly dissected out after decapitation and a single choroid plexus (about 0.5–1.0 mg wet weight) was placed into 5 ml Krebs-bicarbonate (KRB) bu¤er (118 mM NaCl, 5.0 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3) containing 10 mM glucose. KRB bu¤er was changed once before incubation for 1 h at 37°C in a shaking water bath with two intermediate changes of bu¤er. The samples were subsequently labeled with 1 mCi of [3H]myo-inositol for 90 min in the presence of O2/CO2(95: 5). 10 mM pargyline and 10 mM lithium were added and the incubation was continued for 15 min. The antagonists, if present, were added at the same time with pargyline and lithium. Thereafter, 5-HT or an equal volume of bu¤er was added to give a Þnal volume of 300 ml and the samples were incubated for 30 min. The reaction was stopped by addition of 940 ml chloroform/methanol (1 :2 v/v). After vortexing and standing for 15 min, 300 ml chloroform and 300 ml deionized H2O were added and the tubes were vortexed and centrifuged. A portion (750 ml) of the upper aqueous phase containing water soluble inositol phosphates was removed and applied to a column of Dowex-1 anion-exchange resin in the formate form. Free inositol was eluted with 10 ml of 10 mM myoinositol, followed by 10 ml of 5 mM sodium tetraborate/60 mM ammonium formate to elute glyceroinositol phosphate. Inositol monophosphate (IP) was eluted with 10 ml of 200 mM ammonium formate/0.1 M formic acid. The Þrst 5 ml of inositol monophosphate was eluted directly into scintillation vials, while the additional 5 ml was discarded. OptiPhase “Hisafe” 3 was used as a scintillation ßuid and the IC50 values were calculated with the software GraphPad InPlot 4.1.

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Receptor autoradiography [1]
For determination of 5-HT2C antagonist binding (chronic treatment experiments), slides were Þrst preincubated in 170 mM TRIS-HCl bu¤er at room temperature for 15 min, then incubated in 170 mM TRIS-HCl plus 5 nM [3H]mesulergine and 100 nM spiperone at room temperature for 120 min. Nonspeci Þc binding was determined in the presence of 5 mM methysergide. Slides were subsequently washed in 0°C 170 mM TRIS-HCl (2 ´ 10 min) and dipped in ice-cold deionized water. For determination of 5-HT2C receptor agonist binding, dried, room-temperature slides were Þrst preincubated in a 50 mM TRIS-HCl bu¤er containing 10 mM MgSO4 and 0.1% (w/v) bovine serum albumin (BSA) at room temperature for 10 min to remove endogenous serotonin. Slides were then incubated in a similar bu¤er plus 200 pM (±)-1-(2,5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane ([125I]DOI) and 100 nM spiperone (to block 5-HT2A receptors) at room temperature for 60 min. Non-speciÞc binding was deÞned in the presence of 1 mM methysergide. After incubation, the slides were rinsed in a similar ice-cold bu¤er (2 ´ 10 min) and dipped in ice-cold deionized water to remove salt. For determination of 5-HT2A receptor binding, the 5-HT2C agonist binding protocol was employed with the following exceptions : spiperone was excluded from the incubation bu¤er, and non-speciÞc binding was determined in the presence of 1 mM ketanserin instead of 5 mM methysergide. Thoroughly dried slides and standards were apposed to X-ray Þlm for 30 h ([125I]DOI autoradiography) or 21 days ([3H]mesulergine autoradiography) at +4°C in an X-ray Þlm cassette wrapped in plastic for protection from humidity.

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Receptor occupancy measurement [1]
For 5-HT2C receptor occupancy experiments, the above-described autoradiography protocol was used with the following exceptions to achieve optimal conditions for this type of assay: preincubation was omitted to maintain the in vivo drug concentrations, the slides were incubated for 15 min and subsequently wiped o¤ into scintillation vials. For 5-HT2A receptor occupancy experiments, 4 nM [3H]ketanserin was used as antagonist radioligand and the assays were carried out similarly to the 5-HT2C occupancy experiments, with the exception of the incubation time which, in the 5-HT2A assay, was 20 min. In both receptor occupancy assays, for each section, non-speciÞc binding was determined for an adjacent section and then subtracted from total binding. The radioactivity in the scintillation vials was measured by beta-counting using OptiFluor-O scintillation ßuid at 50% e¦ciency, except in the 5-HT2A receptor binding assay where OptiPhase “Hisafe” 3 scintillation ßuid was used. Computer processing of the binding data [EBDA (equilibrium binding data analysis)/LIGAND] was performed as previously described (Hietala et al. 1990).

Drug treatments [1]
Deramciclane was diluted in 0.9% saline and the solution was emulsiÞed with a drop of Tween 80. Clozapine (25 mg/ml) was used as commercially available ampoules. In the chronic treatment experiment, four groups of rats (n = 6 in each group) received SC injections of clozapine (25 mg/kg), Deramciclane (0.5 or 10 mg/kg) or an equal volume (1 ml /kg) of saline (with Tween 80 added) once a day for 14 days. In the receptor occupancy experiment, rats were given a single dose of deramciclane (0.5 or 10 mg/kg), clozapine (25 mg/kg), or an equal volume (1 ml/kg) of saline. Doses refer to the free base of a given drug.

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Treatments. [2]
Samples for determination of basal levels of dopamine, DOPAC and HVA were collected for 60 min. and after that the drugs (doses refer to the salts) were given intraperitoneally in a volume of 5 ml/kg of body weight. Treatments were Deramciclane fumarate 3 mg/kg (= 7.2 µmol/kg), 10 mg/kg (= 24 µmol/kg) and 30 mg/kg (= 72 µmol/kg); D-amphetamine sulfate 2 mg/kg (= 5.4 µmol/kg); ritanserin 1 mg/kg (= 2.1 µmol/kg) and buspirone hydrochloride 5 mg/kg (= 12 µmol/kg). All drugs were suspended in 0.5% carboxymethylcellulose (CMC) dissolved in 0.9% saline. Vehicle control group (n = 5) were injected intraperitoneally with 5 ml/kg of 0.5% CMC solution. There were nine rats in each treatment group.
The doses of Deramciclane fumarate were considered to produce plasma levels comparable to therapeutic plasma levels in human beings (3 mg/kg) or about three times higher (10 mg/kg) 1–3 hr after the administration of the drug.Behavioural data from earlier deramciclane studies in rats indicates that Deramciclane has some antidopaminergic activity at high doses (20–40 mg/kg). The highest dose of deramciclane (30 mg/kg) was selected in this dose range. Fairly high doses of the reference drugs were chosen based on the literature and our own experience to detect the ability of selected drugs to modify extracellular dopamine levels in either the striatum or the nucleus accumbens. After administration of each drug, samples were collected for 240 min. and then divided into two aliquots (35 µl/15 µl). The first aliquot of the samples was stored at 4°C and assayed for dopamine within 24 hr. The other aliquot was frozen and stored at –70°C until assayed for DOPAC and HVA.

Metabolism / Metabolites
Deramciclane has known human metabolites that include N-Methyl-2-[(1,7,7-trimethyl-2-phenyl-2-bicyclo[2.2.1]heptanyl)oxy]ethanamine.

[1]. Deramciclane, a putative anxiolytic drug, is a serotonin 5-HT2C receptor inverse agonist but fails to induce 5-HT2C receptor down-regulation. Psychopharmacology (Berl). 1998 Mar;136(2):99-104.
[2]. Comparison of the effects of deramciclane, ritanserin and buspirone on extracellular dopamine and its metabolites in striatum and nucleus accumbens of freely moving rats. Basic Clin Pharmacol Toxicol. 2008 Jan;102(1):50-8

Deramciclane (EGIS-3886) is used for the treatment of a number of anxiety disorders. Deramciclane differs from other anti anxiety medications in that it is not a benzodiazepine and so has a different structure and target. It antagonizes 5-HT2A receptors, agonizes 5-HT2C receptors, and functions as a GABA reuptake inhibitor.

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Drug Indication
Investigated for use/treatment in anxiety disorders.

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Mechanism of Action
Deramciclane is a new putative non-benzodiazepine-type anxiolytic compound. It is a selective serotonin 5-HT(2A) and 5-HT(2C) receptor antagonist and has also inverse agonist properties.

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In summary, the principle Þndings of the present investigation are the following: 1) The putative anxiolytic Deramciclane is a 5-HT2C receptor antagonist with inverse agonist properties; 2) single-dose administration of deramciclane at anxiolytic doses results in marked in vivo occupancy of 5-HT2C (and 5-HT2A) receptors in the rat brain; 3) chronic treatment (14 days, once daily, SC) with deramciclane (10 mg/kg and 0.5 mg/ kg) does not alter the quantity of 5-HT2C receptor agonist or antagonist radioligand binding in the rat choroid plexus.[1]

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The high dose of Deramciclane fumarate effectively increased the extracellular levels of dopamine and metabolites in nucleus accumbens, but also the levels of dopamine metabolites in striatum were significantly increased as judged from the AUC0–240 min. of DOPAC and HVA. Both buspirone hydrochloride and deramciclane fumarate 30 mg/kg increased extracellular dopamine levels significantly more in nucleus accumbens than in striatum. In nucleus accumbens, deramciclane fumarate 30 mg/kg also increased extracellular HVA levels significantly more than in striatum. These changes may be considered relevant for their anxiolytic activities. D-amphetamine sulfate was significantly more effective in increasing extracellular dopamine levels and decreasing dopamine metabolite levels in striatum than in nucleus accumbens. This is in line with the stimulation of locomotor activity evoked by amphetamine.
The single anxiolytic doses of Deramciclane fumarate, buspirone hydrochloride or ritanserin had no significant effect while 30 mg/kg of Deramciclane fumarate significantly increased the levels of extracellular dopamine levels in the rat nucleus accumbens shell. The extracellular levels of DOPAC and HVA were generally increased by buspirone hydrochloride and deramciclane fumarate 30 mg/kg. As to the main aim of this study, we conclude that the highest single dose of deramciclane fumarate studied has the neuroleptic- or buspirone-like effect, particularly in the mesolimbic system. There is at least a 5-fold margin between the anxiolytic and neuroleptic doses of deramciclane in the rat.[2]

C20H31NO

301.46624

301.241

C, 79.68; H, 10.37; N, 4.65; O, 5.31

120444-71-5

120444-71-5;120444-72-6 (fumarate);

119590

Light yellow to yellow oil

1.01g/cm3

375.2ºC at 760mmHg

110.6ºC

7.93E-06mmHg at 25°C

1.541

4.306

0

2

5

22

399

3

CC1(C)[C@]2(C)[C@@](OCCN(C)C)(C3=CC=CC=C3)C[C@@]1([H])CC2

QOBGWWQAMAPULA-RLLQIKCJSA-N

InChI=1S/C20H31NO/c1-18(2)17-11-12-19(18,3)20(15-17,22-14-13-21(4)5)16-9-7-6-8-10-16/h6-10,17H,11-15H2,1-5H3/t17-,19-,20+/m1/s1

N,N-dimethyl-2-[[(1R,2S,4R)-1,7,7-trimethyl-2-phenyl-2-bicyclo[2.2.1]heptanyl]oxy]ethanamine

Deramciclane; 120444-71-5; Deramciclane [INN]; deramciclano; Ethanamine, N,N-dimethyl-2-(((1R,2S,4R)-1,7,7-trimethyl-2-phenylbicyclo(2.2.1)hept-2-yl)oxy)-; UNII-O5KFK61E74; O5KFK61E74; EGIS-3886;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~331.71 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)