Metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator[2]

Fenobam (300 µM) significantly reduced the total number of human osteosarcoma LM7 cells (DAPI-positive) and the number of proliferating cells (Ki-67-positive) compared to control, with a p-value < 0.001.[2]
Fenobam treatment (300 µM) induced apoptosis in LM7 cells as measured by TUNEL assay, and the percentage of apoptotic cells was significantly higher compared to control (p < 0.001).[2]
The inhibitory effect of Fenobam on cell proliferation and survival was not rescued by co-treatment with the group I mGluR agonist DHPG (50 µM).[2]
Fenobam showed comparable inhibitory effects to Riluzole on LM7 cell proliferation and apoptosis.[2]

Oral administration of fenobam sulfate (30 or 60 mg/kg) significantly inhibited intravenous cocaine self-administration in rats in a dose-dependent manner. The total number of cocaine infusions and active lever presses were reduced.
Oral fenobam sulfate (30 or 60 mg/kg) dose-dependently shifted the cocaine self-administration dose-response curve downward, particularly at lower cocaine doses (0.06, 0.125, 0.25 mg/kg/infusion), indicating a reduction in cocaine's rewarding efficacy.
Oral fenobam sulfate (60 mg/kg, but not 30 mg/kg) significantly inhibited sucrose-induced reinstatement of sucrose-seeking behavior in rats.
Oral fenobam sulfate (30 or 60 mg/kg) significantly and dose-dependently inhibited cocaine-primed (10 mg/kg, i.p.) reinstatement of drug-seeking behavior in rats.
Oral fenobam sulfate (60 mg/kg) significantly inhibited contextual cue-induced incubation of cocaine craving (extinction responding) after 21 days of withdrawal in rats.
Oral fenobam sulfate (30 or 60 mg/kg) did not produce sedative effects or impair general locomotor activity in rats; a transient increase in locomotion was observed within 10 minutes after 30 mg/kg administration.
Oral fenobam sulfate (30 or 60 mg/kg) significantly reduced the rate (per hour) of oral sucrose self-administration, though it did not alter the total number of sucrose deliveries within a 90-minute test session. [3]

Proliferation assay (immunocytochemistry): LM7 cells were seeded on polylysine-coated coverslips in 24-well plates. After 24 hours, cells were treated with Fenobam (300 µM) or other drugs for 72 hours. Cells were fixed, permeabilized, and stained with anti-Ki-67 antibody and DAPI. Ki-67-positive and DAPI-positive cells were counted using fluorescence microscopy.[2]
TUNEL assay (apoptosis): LM7 cells were treated with Fenobam (300 µM) for 72 hours. Cells were fixed, permeabilized, and incubated with TUNEL reagent containing TMR red-labeled nucleotides. TUNEL-positive and DAPI-positive cells were counted using fluorescence microscopy.[2]
Western blot: Whole cell extracts from LM7 cells were analyzed by SDS-PAGE and immunoblotting using antibodies against mGluR5 to confirm receptor expression.[2]

Animal/Disease Models: Male Long-Evans rat (250-300 g)[3]
Doses: 30-60 mg/kg
Route of Administration: Po
Experimental Results: Self-management is inhibited

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Pharmacokinetic Study:** Two groups of Sprague-Dawley rats (n=6 per group) received a single oral dose (30 mg/kg, based on anhydrous free base equivalents) of either fenobam free base or fenobam sulfate. Fenobam free base was first dissolved in 10% DMSO and then brought to final concentration in 40% hydroxypropyl-β-cyclodextrin (BCD). Fenobam sulfate was dissolved in 40% BCD. Blood samples were collected via jugular vein catheter at multiple time points up to 24 hours post-dose. Plasma was analyzed for fenobam concentration using a validated LC-MS/MS method. [3]
**Cocaine Self-Administration:** Male Long-Evans rats were trained to self-administer intravenous cocaine (0.5 or 1 mg/kg/infusion) under a fixed-ratio (FR) schedule. Once stable self-administration was established, animals received oral fenobam sulfate (0, 30, or 60 mg/kg, dissolved in 40% BCD) 30 minutes prior to the test session. A within-session, multiple-dose cocaine self-administration paradigm was also used to test effects across a range of cocaine doses. [3]
**Sucrose Self-Administration:** Similar to cocaine self-administration, rats were trained to self-administer oral 5% sucrose solution. Oral fenobam sulfate (0, 30, or 60 mg/kg) was administered 30 minutes prior to test sessions. [3]
**Reinstatement Tests:** Animals were first trained to self-administer cocaine or sucrose, then underwent extinction training. On reinstatement test days, animals received oral fenobam sulfate (0, 30, or 60 mg/kg) 30 minutes prior to a priming injection of cocaine (10 mg/kg, i.p.) or non-contingent sucrose deliveries. Lever presses were recorded under extinction conditions (no drug/cue delivery). [3]
**Locomotor Activity:** Naive rats were habituated to locomotor chambers. On test days, baseline activity was recorded for 1 hour, then animals received oral fenobam sulfate (0, 30, or 60 mg/kg, dissolved in 40% BCD). Locomotor activity was recorded for an additional 3 hours. [3]

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Pharmacokinetic Study: Two groups of Sprague-Dawley rats (n=6 per group) received a single oral dose (30 mg/kg, based on anhydrous free base equivalents) of either fenobam free base or fenobam sulfate. Fenobam free base was first dissolved in 10% DMSO and then brought to final concentration in 40% hydroxypropyl-β-cyclodextrin (BCD). Fenobam sulfate was dissolved in 40% BCD. Blood samples were collected via jugular vein catheter at multiple time points up to 24 hours post-dose. Plasma was analyzed for fenobam concentration using a validated LC-MS/MS method. [3]
Cocaine Self-Administration: Male Long-Evans rats were trained to self-administer intravenous cocaine (0.5 or 1 mg/kg/infusion) under a fixed-ratio (FR) schedule. Once stable self-administration was established, animals received oral fenobam sulfate (0, 30, or 60 mg/kg, dissolved in 40% BCD) 30 minutes prior to the test session. A within-session, multiple-dose cocaine self-administration paradigm was also used to test effects across a range of cocaine doses. [3]
Sucrose Self-Administration: Similar to cocaine self-administration, rats were trained to self-administer oral 5% sucrose solution. Oral fenobam sulfate (0, 30, or 60 mg/kg) was administered 30 minutes prior to test sessions. [3]
Reinstatement Tests: Animals were first trained to self-administer cocaine or sucrose, then underwent extinction training. On reinstatement test days, animals received oral fenobam sulfate (0, 30, or 60 mg/kg) 30 minutes prior to a priming injection of cocaine (10 mg/kg, i.p.) or non-contingent sucrose deliveries. Lever presses were recorded under extinction conditions (no drug/cue delivery). [3]
Locomotor Activity: Naive rats were habituated to locomotor chambers. On test days, baseline activity was recorded for 1 hour, then animals received oral fenobam sulfate (0, 30, or 60 mg/kg, dissolved in 40% BCD). Locomotor activity was recorded for an additional 3 hours. [3]

Compared to fenobam free base, oral administration of fenobam sulfate (30 mg/kg) in rats resulted in a higher maximal plasma concentration (Cmax), longer average half-life (T1/2), and a significantly larger area under the plasma concentration-time curve (AUC). The sulfate formulation showed approximately two- to three-fold higher oral bioavailability than the free base.
Cmax and AUC were compared using unpaired, two-tailed t-tests. [3]

In the locomotor activity test, oral fenobam sulfate (30 or 60 mg/kg) did not cause sedation or motor impairment. A transient increase in locomotion was observed within 10 minutes after 30 mg/kg administration, but no significant inhibitory effect was seen thereafter. This suggests a lack of acute CNS-depressant toxicity at these doses in rats. [3]

[1]. Fenobam: a clinically validated nonbenzodiazepine anxiolytic is a potent, selective, and noncompetitive mGlu5 receptor antagonist with inverse agonist activity. J Pharmacol Exp Ther. 2005 Nov;315(2):711-21.

[2]. Osteosarcoma cell proliferation and survival requires mGluR5 receptor activity and is blocked by Riluzole. PLoS One. 2017 Feb 23;12(2):e0171256.

[3]. Fenobam sulfate inhibits cocaine-taking and cocaine-seeking behavior in rats: implications for addiction treatment in humans. Psychopharmacology (Berl). 2013;229(2):253-265.

1-(3-Chlorophenyl)-3-(3-methyl-5-oxo-4H-imidazol-2-yl)urea is a type of urea compound. Fennobam is being investigated in the clinical trial NCT00637221 (an open-label study investigating the safety and efficacy of anhydrous fennobam 50 mg to 150 mg in adult patients with Fragile X syndrome for pre-pulse inhibition testing and continuous operational tasks).

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Fenobam is described as a specific negative allosteric modulator of mGluR5.[2]
Blocking mGluR5 with Fenobam significantly inhibits proliferation and induces apoptosis in human osteosarcoma LM7 cells, indicating that mGluR5 activity is required for their growth and survival.[2]
The effects of Fenobam are similar to those of Riluzole, which inhibits glutamate release.[2]
mGluR5 expression was confirmed in LM7 cells by western blot.[2]
The study suggests that targeting mGluR5 with allosteric modulators like Fenobam could be a potential therapeutic strategy for osteosarcoma.[2]

266.057

57653-26-6

135659063

White to off-white solid powder

1.47 g/cm3

1.668

1.126

2

2

1

18

385

0

CN1CC(=O)NC1=NC(=O)NC2=CC(=CC=C2)Cl

DWPQODZAOSWNHB-UHFFFAOYSA-N

InChI=1S/C11H11ClN4O2/c1-16-6-9(17)14-10(16)15-11(18)13-8-4-2-3-7(12)5-8/h2-5H,6H2,1H3,(H2,13,14,15,17,18)

(3Z)-1-(3-chlorophenyl)-3-(1-methyl-4-oxoimidazolidin-2-ylidene)urea

Fenobam NPL2009 NPL-2009

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 : ~100 mg/mL (~374.98 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.)