In the membrane of Chinese hamster ovary cells, bitopertin (RG1678) competitively blocks the [3H]ORG24598 binding site on human GlyT1b. Cells stably expressing hGlyT1b and mGlyT1b are efficiently inhibited from uptaking [3H]glycine by bitopertin, with IC50 values of 25±2 nM and 22±5 nM (n=6), respectively. Conversely, at doses as high as 30 μM, bitopertin exhibited no influence on hGlyT2-mediated glycine absorption. Bitopertin binds to the recombinant hGlyT1b transporter with a strong affinity. Bitopertin dispenses with [3H]ORG24598 binding under equilibrium conditions (one hour at room temperature) with a Ki of 8.1 nM. Bitopertin improved NMDA-dependent long-term potentiation in hippocampal CA1 pyramidal cells at 100 nM, but not at 300 nM [1]. Further investigation revealed that Bitopertin (RG1678) possesses exceptional selectivity for a panel of 86 targets, which includes soluble and transmembrane receptors, enzymes, ion channels, and monoamine transporters (IC50>30 μM) (Measurements for all targets are 10 μM) [2].

Bitopertin (RG1678) elevates glycine levels in rat striatum and cerebrospinal fluid in a dose-dependent manner as determined by microdialysis. Furthermore, in mice stimulated with D-amphetamine or the NMDA receptor glycine site antagonist L-687,414, Bitopertin can reduce excessive movement. In rats receiving long-term phencyclidine treatment—an NMDA receptor open channel blocker—bitopertin also inhibits hyperresponsiveness to D-amphetamine stimulation. The extracellular levels of striatal glycine were unaffected by the vehicle and stayed unchanged during the experiment. Extracellular glycine levels, on the other hand, increased in a dose-dependent manner upon oral administration of Bitopertin (1–30 mg/kg). Glycine levels were 2.5 times greater after 30 mg/kg of biseptertin than they were before treatment. When comparing the CSF of rats given oral bitopertin (1–10 mg/kg) three hours after dosage to rats given a vehicle, a similar dose-dependent rise in glycine concentration was seen. Remarkably, the rise in glycine levels in the cerebrospinal fluid three hours following the administration of bitopertin was strikingly similar to the rise observed at the same time in microdialysis trials [1]. Bitopertin (RG1678) has been shown in in vivo pharmacokinetic tests conducted in rats and monkeys to exhibit low plasma clearance, a moderate distribution volume, and good oral bioavailability in both species (78% in rats and 56% in monkeys). Its terminal half-life (5.8 hours in rats and 6.4 hours in monkeys) is really good. In humans, 98%, and two preclinical species, 97%, plasma protein binding was observed. Rats (brain/plasma=0.7) have a higher rate of intrathecal penetration of betapertin than mice (brain/plasma=0.5) [2].

Several distinct animal protocols were used to evaluate the in vivo effects of Bitopertin.

1. **Drug Formulation and Administration:** For all in vivo studies, Bitopertin was dissolved in water with 0.3% Tween 80 and administered orally (p.o.) at a volume of 10 ml/kg body weight. [1]

2. **Microdialysis in Rats:** Male Sprague-Dawley rats were implanted with a microdialysis probe in the striatum (coordinates: A 0.2 mm, L 2.9 mm, V 7 mm). After 3-4 days recovery, perfusate samples were collected. Following baseline collection, rats received Bitopertin (1-30 mg/kg, p.o.) or vehicle. Glycine levels in the perfusate were analyzed by HPLC-EC. [1]

3. **CSF Collection in Rats:** Male Wistar rats received Bitopertin (1-10 mg/kg, p.o.) or vehicle. Three hours later, under isoflurane anesthesia, cerebrospinal fluid was collected from the cisterna magna. Glycine concentration was quantified by ¹H NMR spectroscopy. [1]

4. **L-687,414-Induced Hyperlocomotion in Mice:** Male NMRI mice were treated with Bitopertin (0.3-10 mg/kg, p.o.) or vehicle. One hour later, they received L-687,414 (50 mg/kg, s.c.) or vehicle. After 15 minutes of habituation, horizontal activity was recorded for 60 minutes in an activity monitoring system. For the time-course study, L-687,414 was administered at different intervals after Bitopertin. For the subchronic study, mice received Bitopertin (1 mg/kg, p.o.) daily for 4 days and were tested on day 5. [1]

5. **Amphetamine-Induced Hyperlocomotion in Mice:** Male NMRI mice were habituated to activity chambers for 60 minutes. They then received Bitopertin (0.3-3 mg/kg, p.o.), clozapine (0.3-3 mg/kg, p.o.), olanzapine (0.003-1 mg/kg, p.o.), or vehicle. Immediately after, they were injected with D-amphetamine (2 mg/kg, i.p.), and horizontal activity was recorded for another 60 minutes. [1]

6. **Subchronic PCP and Amphetamine Challenge in Rats:** Male Wistar rats received PCP HCl (5 mg/kg, i.p.) or vehicle daily for 14 days. Twenty-four hours after the last injection, they were habituated to test boxes for 30 minutes. Rats then received Bitopertin (1, 3, 10 mg/kg, p.o.) or vehicle, followed 1 hour later by D-amphetamine (1 mg/kg, i.p.) or vehicle. Horizontal activity was recorded for 120 minutes post-amphetamine. [1]

7. **Ex Vivo [³H]Raclopride Binding in Rats:** Rats (naive or after 14-day PCP/saline treatment) received 10 mg/kg unlabeled raclopride ± 1 mg/kg D-amphetamine, followed after 15 minutes by [³H]raclopride. After 20 minutes, rats were sacrificed, and radioactivity was measured in the striatum and frontal cortex. Specific binding was calculated. [1]

[1]. Glycine reuptake inhibitor RG1678: A pharmacologic characterization of an investigational agent for the treatment of schizophrenia. Neuropharmacology. 2012 Feb;62(2):1152-61.

[2]. Selective GlyT1 Inhibitors: Discovery of [4-(3-Fluoro-5-trifluoromethylpyridin-2-yl)piperazin-1-yl][5-methanesulfonyl-2-((S)-2,2,2-trifluoro-1-methylethoxy)phenyl]methanone (RG1678), a Promising Novel Medicine To Treat Schizophrenia. J Me.

Bitopertin has been used in clinical trials investigating its use in the treatment of schizophrenia and obsessive-compulsive disorder.

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Drug Indications
Treatment of Schizophrenia

C21H20F7N3O4S

543.4550

543.106

845614-11-1

Bitopertin (R enantiomer);845614-12-2

24946690

White to off-white solid powder

5.018

0

13

5

36

872

1

C[C@@H](C(F)(F)F)OC1=C(C=C(C=C1)S(=O)(=O)C)C(=O)N2CCN(CC2)C3=C(C=C(C=N3)C(F)(F)F)F

YUUGYIUSCYNSQR-LBPRGKRZSA-N

InChI=1S/C21H20F7N3O4S/c1-12(20(23,24)25)35-17-4-3-14(36(2,33)34)10-15(17)19(32)31-7-5-30(6-8-31)18-16(22)9-13(11-29-18)21(26,27)28/h3-4,9-12H,5-8H2,1-2H3/t12-/m0/s1

[4-[3-fluoro-5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl]-[5-methylsulfonyl-2-[(2S)-1,1,1-trifluoropropan-2-yl]oxyphenyl]methanone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ≥ 50 mg/mL (~92.00 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.60 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 2: ≥ 2.5 mg/mL (4.60 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.)