5-HT₂ Receptor ( IC50 = 0.9 nM ); H₁ Receptor ( IC50 = 35 nM ); D₂ Receptor ( IC50 = 70 nM ); Adrenergic α1 ( IC50 = 97 nM ); Adrenergic α2 ( IC50 = 150 nM )

Ritanserin (R 55667) is a highly efficient, relatively coupled, long-acting 5-HT2 clamp-coupled antioxidant with an IC50 of 0.9 nM against histamine-H1 (IC50, 35 nM), dopamine-D2 (IC50, 70 nM) , first of all, pretin-α1 (IC50, 97 nM) and first of all, pretin-α2 receptors (IC50, 150 nM) are less active [1]. In in vitro assays, Ritanserin dissociated very slowly from serotonin-S2 (t1/2 = 160 min) and histamine-H1 sites (t1/2 = 77 min) and rapidly from dopamine-D2 sites (t1/2 = 11 min). Half-times of dissociation from adrenergic-alpha 1 and -alpha 2 sites were 18 and 26 min. The inhibition by ritanserin of [3H]ketanserin binding was found to be partially noncompetitive and the inhibitory potency increased with drug preincubation. Due to the slow dissociation of ritanserin from the serotonin-S2 sites, the drug cannot be displaced completely by [3H]ketanserin. In contrast, inhibition by ritanserin of [3H]haloperidol binding to dopamine-D2 sites in rat striatum was fully competitive, in agreement with the rapid dissociation of the drug from the latter sites. [1]

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Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency [2].

In ex vivo binding assays using brain areas of rats and guinea pigs treated subcutaneously with Ritanserin, occupation of serotonin-S2 sites was observed at very low dosage (50% occupation at 0.08-0.1 mg/kg) and sites remained occupied during a prolonged time period (greater than 70% occupation up to 48 hr after 2.5 mg/kg ritanserin). Histamine-H1 receptor sites in guinea pig cerebellum became occupied at dosages 25-fold higher than the dosage producing occupation of frontal cortical serotonin-S2 sites. Dopamine-D2 sites in rat striatum and cortical adrenergic-alpha 1 sites became only slightly occupied (less than 20%) at higher dosages and the effect was not dose-dependent. Adrenergic-alpha 2 sites were not occupied up to doses of 160 mg/kg given subcutaneously. In vivo binding assays using [3H]spiperone confirmed the occupation of frontal cortical serotonin-S2 sites following low dosage of ritanserin and a minor occupation of striatal dopamine-D2 sites. Levels of dopamine and serotonin and their metabolites remained unchanged in brain areas of rats orally treated with ritanserin up to dosages of 40 mg/kg. At 160 mg/kg, there seemed to be a slight reduction in dopamine and serotonin content.[1]

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5HT(2A/2C) and 5HT(3) receptors have an important role in cognitive behavior specially in spatial learning and memory but the literature concerning the role of these receptors in hippocampus in cognition remains controversial. In the present study a 5HT(2A/2C) antagonist Ritanserin (0, 2, 4, 8 microg/0.5 microl) and a 5HT(3) antagonist granisetron (0.0, 0.05, 0.25, 0.5 microg/0.5 microl) were injected bilaterally into the CA1 region of rat hippocampus, 20 min before each training session in Morris Water Maze (MWM) task. Compare with control group, ritanserin (4 microg/0.5 microl) significantly reduced the escape latency and traveled distance of swimming to platform, but granisetron (0.25 microg/0.5 microl) significantly increased those parameters. Both drugs had no effect on escape latency and traveled distance of a non-spatial visual discrimination task. These results suggest a differential role of 5HT(2A/2C) and 5HT(3) receptors during spatial learning that ritanserin improves rat performance in spatial discrimination task whereas granisetron impairs it [3].

Screening [2]
The assay was optimized in 384-well plate format. In brief, TRE-tomato-HEK cells were seeded into 384-well black plate, pre-coated with Poly-d-Lysine following compounds addition at a concentration of 10 μM for 24 h. Recombinant PAX6 protein [8 μg/ml] was used as a positive control, while DMSO [0.1%] was used as a negative control. Following 24 h, cells were stained with Hoechst (nuclear staining) and Calcein AM green (cytoplasm staining) and imaged using InCell2200 High Content Analyser. Images were analyzed using High Content image analysis algorithms calculating the intensities of the tdTomato reporter within the calculated cellular area. Compounds with tomato intensity of > 1.5 standard deviations and less than 40% toxicity were further analyzed for their half maximum effective concentration (EC50). Among 20 hits, Ritanserin was selected for further validations based on low toxicity profile and high efficacy to enhance PAX6 gene expression.

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qRT-PCR analysis [2]
Cells were treated for 24 h with 1 μM of Ritanserin or its DMSO buffer (control) for rescue experiments, and harvested as a dry pellet. RNA was then extracted using RNEasy Mini kit and cDNA were synthesized from 1 μg RNA using iScript cDNA synthesis kit. Quantitative PCR were performed in triplicate using 2X SYBR Green PCR Master Mix. Expression of each gene was calculated using the 2–ΔΔCt method. Results are presented as fold change normalized to B2M house-keeping gene and relative to control (treated with protein buffer or untreated) T-LSCs. Specific primers sequences used are listed in Roux et al.

Microinjection procedure [3]
Drugs and vehicles were administered intrahippocampally through guide cannulas (21-gauge) using injection needles (27-gauge) connected by polyethylene tubing to 5.0 μl Hamilton microsyringe. The injection needle was inserted 0.5 mm beyond the tip of the cannula and 0.5 μl vehicle (saline or Dimethyl Sulfoxide, DMSO) or different doses of drugs (Ritanserin or granisetron) were injected during 3–4 min. Ritanserin, 6-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidinin-5-one, (RBI)was dissolved in DMSO and granisetron hydrochloride (kytril), endo-N-(9-methyl-9-azabiocyclo [3.3.1]non-3-yl)-1-methyl-1H-indazol-3-carboxamide hydrochloride was dissolved in saline. All injections were done 20 min before training session each day.

[1]. Receptor-binding properties in vitro and in vivo of ritanserin: A very potent and long acting serotonin-S2 antagonist. Mol Pharmacol. 1985 Jun;27(6):600-11.

[2]. Ritanserin, a potent serotonin 2A receptor antagonist, represses MEK/ERK signalling pathway to restore PAX6 production and function in aniridia-like cellular model. Biochem Biophys Res Commun. 2021 Dec 10:582:100-104.

[3]. The effect of intrahippocampal injections of ritanserin (5HT2A/2C antagonist) and granisetron (5HT3 antagonist) on learning as assessed in the spatial version of the water maze. Behav Brain Res. 2005 Feb 28;157(2):205-10.

Ritanserin is a thiazolopyrimidine that is 5H-[1,3]thiazolo[3,2-a]pyrimidin-5-one which is substituted at position 7 by a methyl group and at position 6 by a 2-{4-[bis(4-fluorophenyl)methylidene]piperidin-1-yl}ethyl group. A potent and long-acting seratonin (5-hydroxytryptamine, 5-HT) antagonist of the subtype 5-HT2 (Ki = 0.39 nM), it is used in the treatment of a variety of disorders including anxiety, depression and schizophrenia. It has little sedative action. It has a role as a dopaminergic antagonist, a serotonergic antagonist, an antipsychotic agent, an anxiolytic drug, an antidepressant and an EC 3.4.21.26 (prolyl oligopeptidase) inhibitor. It is an organofluorine compound, a member of piperidines and a thiazolopyrimidine.
Ritanserin has been used in trials studying the treatment of Cocaine-Related Disorders.
A selective and potent serotonin-2 antagonist that is effective in the treatment of a variety of syndromes related to anxiety and depression. The drug also improves the subjective quality of sleep and decreases portal pressure.

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This is the first report indicating that 5HT3 antagonism produces a deficit in cognitive processes. However, it should be considered that the precise mechanism(s) through which granisetron causes impairment in spatial learning is not clear for us. In conclusion, our results show that blockade of hippocampal 5HT2A/2C receptors by ritanserin improves but hippocampal 5HT3 receptors blockade by granisetron in same region (CA1) impairs learning in spatial version of Morris water maze task. In accordance with several previous studies in which 5HT system has been manipulated, these findings suggest that various 5HT receptor subtypes implicate in learning and memory differently, and indicate that 5HT2A/2C and 5HT3 receptors are importantly involved in spatial learning.[3]

C27H25F2N3OS

477.57

477.169

C, 67.91; H, 5.28; F, 7.96; N, 8.80; O, 3.35; S, 6.71

87051-43-2

93076-39-2 (tartrate); 87051-43-2

5074

White to yellow solid powder

1.3g/cm3

618.7ºC at 760 mmHg

328ºC

1.646

5.42

0

6

5

34

877

0

O=C1N2C(SC=C2)=NC(C)=C1CCN1CC/C(=C(\C2C=CC(F)=CC=2)/C2C=CC(F)=CC=2)/CC1

JUQLTPCYUFPYKE-UHFFFAOYSA-N

InChI=1S/C27H25F2N3OS/c1-18-24(26(33)32-16-17-34-27(32)30-18)12-15-31-13-10-21(11-14-31)25(19-2-6-22(28)7-3-19)20-4-8-23(29)9-5-20/h2-9,16-17H,10-15H2,1H3

6-[2-[4-[bis(4-fluorophenyl)methylidene]piperidin-1-yl]ethyl]-7-methyl-[1,3]thiazolo[3,2-a]pyrimidin-5-one

Ritanserin; R55667; R-55667; ritanserin; 87051-43-2; Tiserton; Ritanserina; Ritanserine; Ritanserinum; R-55,667; Ritanserine [French]; R 55667

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.

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

DMSO: ~25 mg/mL (~52.4 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.23 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: ≥ 2.08 mg/mL (4.36 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 20.8 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.)