Glycine transporter type 1 (GlyT1). It is a potent and noncompetitive inhibitor of glycine reuptake at GlyT1.

- Inhibition of [³H]glycine uptake at human GlyT1b: IC₅₀ = 25 ± 2 nM. [1]
- Inhibition of [³H]glycine uptake at mouse GlyT1b: IC₅₀ = 22 ± 5 nM. [1]
- Displacement of [³H]ORG24598 binding at human GlyT1b: Kᵢ = 8.1 nM. [1]
- No effect on human GlyT2-mediated glycine uptake up to 30 μM. [1]
- Inactive (binding inhibition <45%) at 10 μM against 86 other targets, including various receptors, ion channels, and enzymes. [1]

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].

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Bitopertin exhibits potent and selective inhibition of GlyT1 and modulates NMDA receptor function.

1. Glycine Uptake Inhibition: In CHO cells stably expressing human or mouse GlyT1b, Bitopertin potently inhibited [³H]glycine uptake with IC₅₀ values of 25 ± 2 nM and 22 ± 5 nM, respectively. Kinetic analysis revealed that Bitopertin reduces the Vmax of the transporter without affecting the apparent glycine affinity (Km), indicating it is a noncompetitive inhibitor with respect to glycine. [1]

2. [³H]ORG24598 Binding Displacement: In membranes from CHO cells expressing human GlyT1b, Bitopertin displaced [³H]ORG24598 binding with a Ki of 8.1 nM. Saturation binding experiments showed that increasing concentrations of Bitopertin increased the Kd of [³H]ORG24598 without affecting Bmax, confirming a competitive interaction between Bitopertin and ORG24598 at a site distinct from the glycine binding site. This competitive binding profile was also observed with native GlyT1 transporters from rat, mouse, monkey, and dog forebrain membranes. [1]

3. Effect on Long-Term Potentiation (LTP): In rat hippocampal slices, Bitopertin enhanced NMDA-dependent LTP in CA1 pyramidal cells. The potentiation was concentration-dependent, with a significant increase at 100 nM (269 ± 44% of baseline) but not at 30 nM (213 ± 18%) or 300 nM (152 ± 14%, not significant vs. control 186.3 ± 15.9%). The lack of effect at the highest concentration suggests an inverted U-shaped dose-response curve. [1]

4. Selectivity Profile: At a concentration of 10 μM, Bitopertin showed less than 45% inhibition in binding assays against a panel of 86 targets, including glycine, glutamate, dopamine, GABA, serotonin, adrenergic, muscarinic, adenosine, opioid, and histamine receptors, as well as various ion channels. In 21 functional assays, it did not inhibit enzymes such as monoamine oxidase, catechol-O-methyltransferase, acetylcholinesterase, and several phosphodiesterases. [1]

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].

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Bitopertin demonstrates robust in vivo activity by increasing brain glycine levels and showing efficacy in several rodent models related to schizophrenia.

1. Increase in Extracellular Striatal Glycine (Microdialysis in Rats): Oral administration of Bitopertin (1-30 mg/kg, p.o.) to freely moving rats produced a dose-dependent and sustained increase in extracellular glycine levels in the striatum. The effect began 20-40 minutes post-dose, peaked at 80-100 minutes, and lasted for at least 3 hours. At the 30 mg/kg dose, glycine levels increased to 2.5 times baseline. Bitopertin did not affect striatal levels of D-serine. [1]

2. Increase in CSF Glycine (in Rats): Three hours after oral administration, Bitopertin (1-10 mg/kg, p.o.) dose-dependently increased glycine concentration in the cerebrospinal fluid (CSF) of rats, as measured by ¹H NMR. The magnitude of increase was similar to that observed in the striatal microdialysis study at the same time point. [1]

3. Prevention of L-687,414-Induced Hyperlocomotion (in Mice): Bitopertin (1-10 mg/kg, p.o.) dose-dependently and completely prevented the hyperlocomotion induced by the NMDA receptor glycine site antagonist L-687,414 (50 mg/kg, s.c.) in mice, with an ID₅₀ of 0.63 mg/kg. This effect was maintained after 4 days of once-daily dosing (ID₅₀ on day 5 was 0.28 mg/kg vs. 0.73 mg/kg for acute treatment). The inhibitory effect was observed when administered 0.5, 2.5, and 4.5 hours before the test but was no longer significant at 24 hours. Bitopertin alone did not affect baseline locomotor activity. [1]

4. Prevention of Amphetamine-Induced Hyperlocomotion (in Mice): Bitopertin (0.3-3 mg/kg, p.o.) significantly reduced hyperlocomotion induced by D-amphetamine (2 mg/kg, i.p.) in mice. The maximal effect was reached at the lowest dose tested (0.3 mg/kg). In comparison, the antipsychotics clozapine and olanzapine had ID₅₀ values of 5 mg/kg and 0.55 mg/kg, respectively, in this model. [1]

5. Effect on Amphetamine Challenge after Subchronic PCP (in Rats): In rats treated with PCP (5 mg/kg, i.p.) for 14 days to model NMDA receptor hypofunction, a subsequent amphetamine (1 mg/kg, i.p.) challenge resulted in a significantly exaggerated hyperlocomotor response compared to controls. Bitopertin (3 and 10 mg/kg, p.o.) dose-dependently and significantly abolished this exaggerated amphetamine-induced hyperlocomotion. In non-PCP-treated animals, only the 10 mg/kg dose of Bitopertin significantly decreased amphetamine-induced hyperlocomotion. [1]

6. Ex Vivo [³H]Raclopride Binding (in Rats): To investigate dopaminergic mechanisms, rats subchronically treated with PCP showed a larger decrease in striatal [³H]raclopride (D₂ receptor antagonist) binding following an amphetamine challenge compared to controls. This indicates an exaggerated dopamine release. The study suggests Bitopertin can normalize this altered dopaminergic response. [1]

1. [³H]Glycine Uptake Assay: CHO cells stably expressing GlyT1 or GlyT2 were plated in 96-well plates. Uptake was initiated by adding 25 μM nonradioactive glycine and 60 nM [³H]glycine (for GlyT1) or 200 nM [³H]glycine (for GlyT2) in uptake buffer at 22°C. Nonspecific uptake was determined using 10 μM ORG24598 (for GlyT1) or 5 μM ORG25543 (for GlyT2). After incubation (15 min for hGlyT1b, 30 min for mGlyT1b and hGlyT2), plates were washed, and radioactivity was measured by liquid scintillation. IC₅₀ values were derived using curve-fitting software. [1]

2. [³H]ORG24598 Binding Assay: Membranes from CHO cells expressing hGlyT1b or from rat, mouse, monkey, and dog forebrains were incubated with [³H]ORG24598. For saturation experiments, increasing concentrations of [³H]ORG24598 were used. For inhibition experiments, membranes were incubated with 3 nM [³H]ORG24598 and various concentrations of Bitopertin for 1 hour at room temperature. For Schild analysis, the displacement was examined in the presence of increasing concentrations of [³H]ORG24598 (1-300 nM). Bound radioactivity was separated by filtration and measured. Kd and Bmax were calculated from saturation isotherms. Ki values were calculated from IC₅₀ values. [1]

1. Cell Lines: Flp-In™ CHO cells were stably transfected with cDNAs for human GlyT1b, mouse GlyT1b, and human GlyT2. Cells were maintained in F-12 nutrient mixture with 10% fetal calf serum, 1% penicillin-streptomycin, 600 μg/ml hygromycin, and 1 mM glutamine at 37°C in 5% CO₂. For assays, cells were plated at 40,000 cells/well in 96-well plates 24 hours before the experiment. [1]

2. [³H]Glycine Uptake Assay in Cells: This assay, described in the "Enzyme Assay" section, was performed on these cell lines to determine the inhibitory potency and mechanism of Bitopertin on glycine transport. [1]

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]

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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]

The study mentions that the effects of Bitopertin in microdialysis experiments were long-lasting and consistent with its previously observed pharmacokinetic profile in rats. The time course of efficacy in the L-687,414 model (active at 0.5, 2.5, and 4.5 hours, but not 24 hours) also aligns with its pharmacokinetic properties. However, no specific PK parameters (e.g., half-life, bioavailability, Cmax) are reported in this paper. [1]

hERG Channel Inhibition: Bitopertin shows weak inhibition of the hERG potassium channel, with an IC₅₀ of 17 μM (measured in a patch-clamp assay), translating to >500-fold selectivity over its GlyT1 activity. [2]
- CYP450 Inhibition: No significant inhibition of major CYP enzymes (IC₅₀ > 24 μM). [2]
- Genotoxicity: Bitopertin was without activity in genotoxicity assays (Ames test and micronucleus test, MNT). [2]
- General Safety: No overt adverse effects were noted in the in vivo studies at the doses tested. [2]

[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

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Bitopertin (RG1678; RO-4917838) is an investigational agent for the treatment of schizophrenia. It is a potent, selective, and noncompetitive inhibitor of the glycine transporter type 1 (GlyT1). By inhibiting GlyT1, it increases extracellular glycine levels in the brain, which in turn enhances NMDA receptor function. This mechanism is based on the glutamate hypothesis of schizophrenia, which posits that NMDA receptor hypofunction contributes to the symptoms of the disease. Preclinical studies described here show that Bitopertin modulates both glutamatergic and dopaminergic neurotransmission in animal models relevant to schizophrenia. It was under investigation in multiple clinical trials at the time of this publication. [1]

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.)