Rat 5-HT1B Receptor (IC50 = 47 nM)

NAS181 exhibits extremely low affinities (Ki>3000 nM) for every other receptor that has been studied, such as dopamine D1 and D2, α1-, α2-, and β-adrenoceptors, 5-HT2A, 5-HT2C, 5-HT6, and 5-HT7, and 5-HT2[1]. In preloaded rat occipital cortex slices, NAS181 (10-1000 nM) dose-dependently increases the K+-stimulated [3H]-5-HT release[1].

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In the search for new 5-hydroxytryptamine (5-HT) receptor antagonists it was found that the compound (R)-(+)-2-[[[3-(morpholinomethyl)-2H-chromen-8-yl]oxy] methyl] morpholine methanesulfonate, (R)-25/NAS-181, is a selective rat 5-hydroxytryptamine1B (r5-HT1B) receptor antagonist. The binding profile showed a 13-fold preference for r5-HT1B (Ki = 47 +/- 5 nM; n = 3) vs bovine 5-HT1B (Ki = 630 nM; n = 1) receptors. The compound had very low affinity for other monoaminergic receptors examined. The r5-HT1B receptor antagonism was demonstrated by the potentiation of the K+-stimulated release of [3H]-5-HT from superfused rat brain slices in vitro, an effect that was antagonized by addition of 5-HT to the superfusion fluid. [1]
The affinity of (R)-25/NAS-181 for the 5-HT1D receptors in calf caudate membranes (mainly constituting the homologous bovine 5-HT1B receptor) was more than 10 times less than that for the rat r5-HT1B receptor. (R)-25 had very low affinities for all other receptors examined, including 5-HT2A, 5-HT2C, 5-HT6, and 5-HT7, α1-, α2-, and β-adrenoceptors, and dopamine D1 and D2 (Table 2). [1]
The r5-HT1B antagonistic property of (R)-25/NAS-181 was characterized in the K+-stimulated release of [3H]-5-HT from preloaded rat occipital cortical slices. As shown in Table 3 (R)-25 dose-dependently potentiated the [3H]-5-HT release in the concentration range 10−1000 nM. Furthermore, the potentiation of the release caused by (R)-25 was antagonized at 1000 nM but not at 100 nM of unlabeled 5-HT into the superfusion fluid (Table 4), which indicates competition between these two compounds. [1]

Acetylcholine (ACh) release is dose-dependently increased in the frontal, ventral hippocampus cortex, and VHipp by NAS181 (1–10 mg/kg; sc)[1]. In four rat brain regions (the striatum, frontal cortex, hippocampus, and hypothalamus), NAS181 (20 mg/kg; sc) increases 5-HT turnover by roughly 40%[1]. Rats that are given NAS181 (3 mg/kg; sc) exhibit a notable increase in the quantity of wet dog shakes[1].

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(R)-25 (NAS-181) at 20 mg/kg sc enhanced the 5-HT turnover in four rat brain regions (hypothalamus, hippocampus, striatum, and frontal cortex) with about 40% measured as the 5-HTP accumulation after decarboxylase inhibition with 3-hydroxybenzylhydrazine. At 3 mg/kg sc (R)-25 produced a significant increase in the number of wet dog shakes in rats, a 5-HT2A/5-HT2C response that was abolished by depletion of 5-HT after pretreatment with the tryptophan hydroxylase inhibitor p-chlorophenylalanine. These observations show that (R)-25, by inhibiting terminal r5-HT1B autoreceptors, increases the 5-HT turnover and the synaptic concentration of 5-HT.[1]

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The purpose of this study was to investigate the effects of the 5-HT(1B) receptor antagonist NAS-181 ((R)-(+)-2-(3-morpholinomethyl-2H-chromen-8-yl) oxymethyl-morpholine methanesulfonate) on cholinergic, glutamatergic and GABA-ergic neurotransmission in the rat brain in vivo. Extracellular levels of acetylcholine, glutamate and GABA were monitored by microdialysis in the frontal cortex (FC) and ventral hippocampus (VHipp) in separate groups of freely moving rats. NAS-181 (1, 5 or 10 mg/kg, s.c.) caused a dose-dependent increase in ACh levels, reaching the maximal values of 500% (FC) and 230% (VHipp) of controls at 80 min post-injection. On the contrary, NAS-181 injected at doses of 10 or 20 mg/kg s.c. had no effect on basal extracellular levels of Glu and GABA in these areas. The present data suggest that ACh neurotransmission in the FC and VHipp, the brain structures strongly implicated in cognitive function, is under tonic inhibitory control of 5-HT(1B) heteroreceptors localized at the cholinergic terminals in these areas [2].

Radioligand Binding Studies. [1]
Affinities for the following receptors were determined as described by Jackson et al. 27 (the radioligand and tissue in parentheses): 5-HT1A ([3H]OH-DPAT, rat hippocampus), α1-adrenoceptors ([3H]prazosin, rat cortex), α2-adrenoceptors ([3H]RX821002 ((1,4-[6,7-3H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride), rat cortex), β-adrenoceptors ([3H]dihydroalprenolol, rat cortex), dopamine D1 ([3H]SCH-23390, rat striatum); dopamine D2 ([3H]raclopride, cell (LtkhD2A) membranes). The following receptors were determined as described by Johansson et al.: 28 5-HT2A ([3H]ketanserin, rat cortex), 5-HT2C ([3H]mesulergine, rat cortex), 5-HT6 ([3H]-5-HT, cell (CHOr5-HT6) membranes), 5-HT7 ([3H]-5-HT, cell (CHOr5-HT7) membranes). [125I]Iodocyanopindolol was used as the ligand for r5-HT1B receptors in rat cerebral cortical membranes in the presence of 60 μM isoproterenol in order to avoid binding to β-adrenoceptors as described by Hoyer et al. 29 Membranes from calf caudate were used for the assay of 5-HT1D receptors with [3H]-5-HT in the presence of 8-OH-DPAT, 100 nM, and mesulergine, 100 nM, to avoid binding to 5-HT1A and 5-HT2C receptors according to the method of Heuring and Peroutka; 30 5-HT, 10 μM, was used in these three assays to determine the specific binding. Binding to σ recepors in membranes from whole rat brains was assayed with [3H]DTG (N,N‘-di(o-tolyl)guanidine) as described by Ross.

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Potassium-Stimulated [3H] Overflow from Rat Cortical Slices Preloaded with [3H]-5-HT. [1]
The method described by Rényi et al was used. Slices from occipital cortex (0.3 × 0.3 mm) preloaded with [3H]-5-HT were superfused with freshly oxygenated Krebs-Henseleit's buffer, pH 7.4, containing 2.5 μM citalopram at a flow rate of 0.4 mL/min. After a 50 min washing, 4-min fractions were collected, and after 62 min a buffer solution containing 25 mM KCl was administered for 4 min followed by superfusion with the original buffer. The slices were superfused with the test compound at appropriate concentrations in the buffer for 72 min. A second addition of 25 mM KCl and the test compound were administered for 4 min beginning at 98 min, and the superfusion was continued and stopped at 122 min. The radioactivities in the fractions and remaining activities in the slices were counted by liquid scintillation, and the percentage of fractional release for each fraction was determined. The ratio of release in the presence of the test compound after the second stimulation (S2) to that after the first stimulation (S1) was determined and expressed as a percentage of the corresponding ratio in the controls without test compound. To evaluate the intrinsic activity, the test compound and 5-HT were administered in the same solution.

Animal/Disease Models: Adult male SD (Sprague-Dawley) rats (250-300 g)
Doses: 1, 5, 10 mg/kg
Route of Administration: Sc in the scruff of the neck
Experimental Results: Increased the ACh release in the frontal cortex, reaching the maximal value of 500% of the control group within 80 min after the injection of the highest dose. Increased the ACh releases in VHipp with a maximum of 230% of the control values at 80 min after the injection of the highest dose.

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On the day of the experiment, the animals were placed in the CMA/120 system for freely moving animals (CMA/Microdialysis) for an initial 2 h in order to habituate to the new environment. A CMA/12 microdialysis probe (8 mm shaft, 2 mm membrane length) was inserted into the guide cannula of the operated animals. The probes were perfused at a constant flow rate of 1 μl/min with the artificial cerebrospinal fluid (aCSF) containing 148 mM NaCl, 4 mM KCl, 0.8 mM MgCl2, 1.4 CaCl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.2 and 0.5 μM neostigmine. Following a 120-min stabilization period, the samples were collected every 20 min using a CMA/170 refrigerated fraction collector (CMA/Microdialysis). The first 4 samples were taken for determination of basal extracellular levels of ACh, Glu and GABA, thereafter, the drug (NAS-181) was injected subcutaneously (s.c.) in the scruff of the neck. The fractions were collected for an additional 3 h. [2]

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5-HT Turnover in Various Brain Regions. [1]
The rate of 5-HT turnover in hypothalamus, hippocampus, frontal cortex, and striatum in rats was determined as the accumulation of 5-hydroxytryptophan (5-HTP) after treatment with the 5-HTP decarboxylase inhibitor 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015), 100 mg/kg sc. The test compound was injected sc 30 min before NSD 1015, and the rats were killed 30 min after. The various brain regions were rapidly dissected out, frozen on dry ice, and stored at −70 °C. 5-HTP, 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) were analyzed by HPLC.

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Determination of Wet Dog Shake Behavior. [1]
The number of wet dog shakes, including whole body shake and head shake, was determined during 60 min, starting 5 min after the injection of the test compound. Groups of 8−10 rats were used and compared with saline-treated rats (n = 30).

[1]. (R)-(+)-2-[[[3-(Morpholinomethyl)-2H-chromen-8-yl]oxy]methyl] morpholine methanesulfonate: a new selective rat 5-hydroxytryptamine1B receptor antagonist. J Med Chem. 1998 May 21;41(11):1934-42.

[2]. Effects of the 5-HT1B receptor antagonist NAS-181 on extracellular levels of acetylcholine, glutamate and GABA in the frontal cortex and ventral hippocampus of awake rats: a microdialysis study. Eur Neuropsychopharmacol. 2007 Sep;17(9):580-6.

The receptor profile of (R)-25 shows that this compound is a selective r5-HT1B receptor ligand with moderate to high potency. Since (R)-25 has low affinity for the 5-HT1D receptor in calf caudate, which mainly appears to consist of the bovine 5-HT1B receptor, the receptor profile of (R)-25 differs from that of 2‘-methyl-4‘-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127,935) which has high affinity for the two homologous forms of the 5-HT1B receptor and also has high affinity for the 5-HT1D receptor. The affinity of (R)-25 for the latter receptor type remains to be elucidated.
The potentiation of the K+-stimulated [3H]-5-HT release from superfused slices of rat occipital cortex and the competition between (R)-25 and 5-HT in this in vitro model showed that (R)-25 is an antagonist at this receptor. Moreover, (R)-25 markedly increased the 5-HTP accumulation in all four brain regions examined in 3-hydroxybenzylhydrazine-treated rats and increased the 5-HIAA/5-HT ratio in the brain even more. These findings suggest that the 5-HT turnover was increased in these 5-HT terminal regions, supposedly due to the increased 5-HT release from the terminals. The induction of wet dog shake behavior by (R)-25 is in strong accordance with this notion since the response was abolished by depletion of 5-HT in the brain with pCPA. These observations indicate that, under normal conditions, the terminal 5-HT1B receptors have a functional role in determining the amount of 5-HT released from the terminals.
Since (R)-25 is a selective r5-HT1B receptor antagonist, this compound may become a valuable tool for studies of the functional role of the r5-HT1B receptors in rodents.[1]

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These reports provided a rationale to examine the effects of the 5-HT1B receptor antagonist NAS-181 on extracellular Glu and GABA levels by microdialysis sampling in awake rats. Systemic administration of NAS-181 at doses up to 20 mg/kg s.c. did not affect extracellular hippocampal and cortical Glu and GABA concentrations as shown in Figure 3, Figure 4. A possible explanation of this finding is that, under in vivo conditions, glutamatergic and GABA-ergic neurons are only under weak control of inhibitory 5-HT1B heteroreceptors. Another possible explanation is related to technical limitations of microdialysis to monitor neuronally released pools of Glu and GABA. The sources of extracellular Glu and GABA are both of neuronal and glial origin and a probability of sampling neuronally derived Glu and GABA by microdialysis was questioned recently (Timmerman and Westerink, 1997). To our knowledge, there are only few microdialysis studies addressing the modulatory role of 5-HT1B receptors on Glu and GABA release. Thus, Srkalovic et al. (1994) observed reduced basal Glu levels following administration of the 5-HT1B receptor agonist TMFPP, whereas a non-selective 5-HT receptor antagonist metergoline significantly increased extracellular Glu levels in the suprachiasmatic nucleus. In another study, veratridine-evoked increase in extracellular Glu and Asp levels in the rat FC were attenuated by coperfusion with CP-93,129 (Golembiowska and Dziubina, 2002). However, this microdialysis study, as well as, the above mentioned studies involving electrophysiological recording was carried-out at conditions of extensive stimulation and with 5-HT1B receptor agonists, which makes it difficult to draw conclusions on possible tonic modulatory activity of 5-HT1B receptors over the glutamatergic and GABA-ergic neurons in the rat brain. Nevertheless, the results of the present study indicate that the potent stimulatory effect of NAS-181 on ACh release is unlikely to be mediated via disinhibition of GABA-ergic afferents or interneurons in the FC and VHipp areas, rather there is a direct inhibitory control of ACh neurotransmission via 5-HT1B heteroreceptors at the cholinergic terminals.
In conclusion, the present microdialysis study shows that systemic administration of the 5-HT1B receptor antagonist NAS-181 causes marked and dose-dependent increases in extracellular ACh levels both in the frontal cortex and ventral hippocampus of awake rats. The concentrations of Glu and GABA are unaffected. These results suggest that ACh neurotransmission is under tonic inhibitory control by 5-HT1B heteroreceptors. Information on the specific involvement of the 5-HT1B heteroreceptors located at cholinergic terminals or at cell body levels in the rat basal forebrain requires further studies using a dual-probe microdialysis approach. [2]

C21H34N2O10S2

538.63

538.165

1217474-40-2

NAS-181;205242-62-2; 205242-61-1

45073445

Off-white to light yellow solid powder

3

12

5

35

545

1

CS(=O)(=O)O.CS(=O)(=O)O.C1CO[C@H](CN1)COC2=CC=CC3=C2OCC(=C3)CN4CCOCC4

WMRMIRRDPBKNMY-ZEECNFPPSA-N

InChI=1S/C19H26N2O4.2CH4O3S/c1-2-16-10-15(12-21-5-8-22-9-6-21)13-25-19(16)18(3-1)24-14-17-11-20-4-7-23-17;2*1-5(2,3)4/h1-3,10,17,20H,4-9,11-14H2;2*1H3,(H,2,3,4)/t17-;;/m1../s1

methanesulfonic acid;(2R)-2-[[3-(morpholin-4-ylmethyl)-2H-chromen-8-yl]oxymethyl]morpholine

NAS-181; 1217474-40-2; 205242-62-2; (2R)-2-[[[3-(4-Morpholinylmethyl)-2H-1-benzopyran-8-yl]oxy]methyl]morpholine dimethanesulfonate; NAS-181 dimesylate; methanesulfonic acid;(2R)-2-[[3-(morpholin-4-ylmethyl)-2H-chromen-8-yl]oxymethyl]morpholine; NAS181; (2R)-2-[[[3-(4-Morpholinylmethyl)-2H-1-benzopyran-8-yl]oxy]methyl]morpholinedimethanesulfonate;

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, avoid exposure to moisture.

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

DMSO: 62.5 mg/mL (116.04 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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