rat mGluR5 ( Ki = 0.67 nM )
Negative allosteric modulator (NAM) of the metabotropic glutamate receptor subtype 5 (mGluR5) (IC50 = 2.4 ± 0.3 nM in functional assay; Ki = 0.00067 ± 0.00001 µM for rat mGluR5 binding)
Off-target binding observed at Prostaglandin Thromboxane A2 receptor (TXA2) (Ki = 2.02 ± 0.85 µM) and peripheral Monoamine oxidase-B enzyme (MAO-B) (Ki = 0.77 ± 0.16 µM) [1]

MFZ 10-7 exhibits affinities for MAO-B (monoamine oxidase-B enzyme) and TXA2 (thromboxane A2 receptor) that are approximately 1150-fold and 3000-fold lower, respectively, in comparison to mGluR5 [1].

---

In an in vitro functional assay using HEK293 cells stably expressing rat mGluR5, MFZ 10-7 inhibited Gq protein-mediated production of the intracellular messenger inositol 1,4,5-trisphosphate (IP3), measured via IP1 accumulation. Its IC50 for inverse agonism was 2.4 ± 0.3 nM, making it approximately 13-, 46-, and 188-fold more potent than MPEP, MTEP, and fenobam, respectively [1].
In competitive radioligand binding assays using rat brain membranes and [3H]MPEP, MFZ 10-7 demonstrated high binding affinity for mGluR5 with a Ki of 0.00067 ± 0.00001 µM, which was approximately 63- and 330-fold higher than MTEP and fenobam, respectively [1].
A broad receptor screen against 64 functional receptor/enzyme proteins revealed that MFZ 10-7 (at 10 µM) bound only to two off-target sites: TXA2 receptor and peripheral MAO-B enzyme. Subsequent Ki determinations confirmed low affinity for these sites relative to mGluR5 (selectivity ratios: ~1150-fold for MAO-B, ~3000-fold for TXA2) [1].

MFZ 10-7 reduces the amplitude of sucrose self-regulation but has no effect on total sucrose intake [1].

---

In male Long-Evans rats, intraperitoneal (i.p.) administration of MFZ 10-7 (3 and 10 mg/kg) dose-dependently inhibited intravenous cocaine self-administration maintained by a single dose (0.5 mg/kg/infusion) under an FR2 schedule [1].
When tested in a multi-dose cocaine self-administration paradigm (0.03-1.0 mg/kg/infusion), i.p. administration of MFZ 10-7 (3 and 10 mg/kg) produced a downward shift in the cocaine dose-response curve, significantly reducing the number of infusions at lower cocaine doses (0.06, 0.125, and 0.25 mg/kg/infusion). This suggests a reduction in cocaine's rewarding efficacy [1].
Pretreatment with MFZ 10-7 (3 and 10 mg/kg, i.p.) dose-dependently inhibited cocaine-primed reinstatement (10 mg/kg cocaine, i.p.) of extinguished drug-seeking behavior [1].
Pretreatment with MFZ 10-7 (3 and 10 mg/kg, i.p.) dose-dependently inhibited contextual cue-induced cocaine-seeking behavior (incubation of craving) assessed after 21 days of forced abstinence [1].
In oral sucrose self-administration experiments, i.p. administration of MFZ 10-7 (10 mg/kg) significantly reduced the rate of sucrose deliveries per hour but did not alter the total number of sucrose rewards received in a 90-minute session capped at 100 deliveries [1].
In contrast to MTEP, pretreatment with MFZ 10-7 (3 and 10 mg/kg, i.p.) did not produce a statistically significant reduction in sucrose-primed reinstatement of sucrose-seeking behavior [1].
Administration of MFZ 10-7 (3 and 10 mg/kg, i.p.) did not significantly alter basal locomotor activity in drug-naïve rats, indicating the effects on drug-seeking are not due to sedation or motor impairment [1].

The binding affinity of test compounds for mGluR5 was determined using a radioligand competition binding assay. Membranes were prepared from whole brains (minus cerebellum) of male Sprague-Dawley rats. Binding reactions were conducted at room temperature for 60 minutes in a buffer containing 50 mM Tris, 120 mM NaCl, 5 mM KCl (pH 7.4). Each reaction contained 4 nM [3H]MPEP, 7.5 mg of brain tissue (original wet weight), and varying concentrations of test compounds. Nonspecific binding was defined using 100 µM MPEP. Incubations were terminated by rapid filtration and washing with cold buffer. Filter-bound radioactivity was measured by liquid scintillation counting. Ki values were determined from full dose-response curves performed in triplicate across at least three independent experiments [1].
To assess off-target activity, the binding affinities of compounds at Monoamine oxidase-B (MAO-B) and Thromboxane A2 receptor (TXA2) were also determined. The experimental details for these specific assays are not provided in the manuscript but were conducted under a contract [1].

The in vitro functional potency (IC50) of mGluR5 NAMs was assessed using HEK293 cells stably expressing rat mGluR5. The assay measured agonist-independent (inverse agonism) inhibition of Gq protein-mediated signaling. Cells were incubated with serially diluted test compounds (final concentrations from 10 pM to 10 µM) for 1 hour at 37°C in the presence of LiCl to trap inositol phosphates. After incubation, cells were lysed. The accumulation of D-myo-inositol 1 phosphate (IP1), a degradation product of IP3, was quantified using a competitive immunoassay (IP One ELISA). This assay employs an anti-IP1 monoclonal antibody and an IP1-horseradish peroxidase conjugate. The optical density was measured, and IP1 levels for each treatment were normalized to vehicle-only control values. IC50 values were calculated from at least three independent experiments [1].

For cocaine and sucrose self-administration studies, male Long-Evans rats were housed individually on a reverse light-dark cycle. For cocaine studies, rats were surgically implanted with an intravenous catheter into the right external jugular vein under anesthesia. Catheters were flushed daily with an antibiotic/heparin solution. Self-administration training and testing occurred in operant chambers. For cocaine, rats learned to press a lever for intravenous cocaine infusions (delivered over 4.65 sec) paired with a light+tone cue. Training progressed from FR1 to FR2 schedules until stable responding was achieved. For multi-dose testing, a session consisted of sequential components with different cocaine doses (0, 0.03, 0.06, 0.125, 0.25, 0.5, 1.0 mg/kg/infusion). For single-dose testing, sessions lasted 3 hours with a maximum of 50 infusions. Sucrose self-administration followed similar procedures, except no surgery was involved, and lever presses delivered 0.1 mL of 5% sucrose solution into a food tray along with the cue. Sucrose sessions were 90-min long and capped at 100 deliveries. Test compounds (MFZ 10-7 or MTEP) or vehicle were administered intraperitoneally 15 minutes prior to the start of a self-administration session [1].
For reinstatement experiments, after stable self-administration, animals underwent extinction training where lever presses no longer resulted in drug/sucrose or cue presentation. Extinction continued until seeking behavior was extinguished. On test days, animals were pretreated with vehicle or test compound (i.p.) and then given a non-contingent "priming" injection of cocaine (10 mg/kg, i.p.) or non-contingent deliveries of sucrose (x5) to trigger reinstatement. Animals were then placed back into the operant chamber, and lever presses (under extinction conditions) were recorded for a set period [1].
For the incubation of craving (cue-induced seeking) experiment, after stable cocaine self-administration, rats underwent 21 days of forced abstinence in their home cages. On test days, they were pretreated with vehicle or test compound (i.p.) and then placed into the former self-administration context. Lever presses (under extinction conditions, with no drug or discrete cues available) were recorded for 3 hours as a measure of cue-induced seeking [1].
For locomotor activity tests, drug-naïve rats were habituated to locomotor chambers. Baseline activity was recorded for 1 hour before i.p. injection of vehicle or test compound. Locomotor activity (distance traveled) was then recorded for an additional 3 hours [1].
MFZ 10-7 was suspended in 1% Tween 80 and water for intraperitoneal administration in all behavioral experiments [1].

[1]. A novel mGluR5 antagonist, MFZ 10-7, inhibits cocaine-taking and cocaine-seeking behavior in rats. Addict Biol.

MFZ 10-7 (3-fluoro-5-((6-methylpyridin-2-yl)ethynyl)benzonitrile) is a novel, highly effective and selective mGluR5 receptor negative allosteric modulator (NAM). It was developed as an analogue of MPEP to overcome the limitations of early mGluR5 NAMs (such as MPEP and MTEP) in terms of off-target effects and short half-life, which limited their potential for translational application [1]. This study provides evidence supporting the role of mGluR5 in cocaine reward and relapse. MFZ 10-7 showed significant efficacy in reducing cocaine intake, drug-seeking behavior and cue-induced craving in rats without inhibiting general motor function, suggesting that mGluR5 remains a viable target for the development of addictive drugs [1].
Compared to MTEP, MFZ 10-7 showed a wider therapeutic window in selectively inhibiting cocaine-seeking behavior, as it failed to significantly attenuate sucrose-induced relapse at doses that effectively inhibited cocaine-seeking behavior [1].
The authors believe that MFZ 10-7 could serve as a valuable novel in vitro and in vivo tool compound for further research into the role of mGluR5 in addiction and related diseases [1].

C15H9FN2

236.24

236.075

1224431-15-5

MFZ 10-7 hydrochloride; 1779796-36-9

90488944

Light yellow to yellow solid powder

2.8

1

3

2

19

397

0

WRHOKQFFLQKKNL-UHFFFAOYSA-N

InChI=1S/C15H9FN2.ClH/c1-11-3-2-4-15(18-11)6-5-12-7-13(10-17)9-14(16)8-12;/h2-4,7-9H,1H3;1H

3-fluoro-5-[2-(6-methylpyridin-2-yl)ethynyl]benzonitrile;hydrochloride

MFZ 10-7; MFZ10-7

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: ~17.9 mg/mL (~75.6 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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