mGlu7

In Chinese hamster ovary (CHO) cells coexpressing rat mGluR7 with Gα15 (IC50=26 nM), MMPIP reduces the intracellular Ca2+ mobilization elicited by L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; 0.5 mM) [1]. MMPIP blocks L-AP4-induced suppression of forskolin-stimulated cAMP buildup (IC50 220 nM) in CHO cells expressing rat mGluR7[1]. Additionally, MMPIP counteracts the suppression of cAMP buildup mediated by L-AP4 in CHO-human mGluR7/Gα15, with an IC50 of 610 nM[1].

MMPIP (10 mg/kg) significantly increases the prepulse-induced suppression of the auditory startle response (up to 137% of control) while attenuating the intensity of the acoustic startle reaction[2]. By increasing decision accuracy, MMPIP (10 mg/kg) mitigates the cognitive deficits caused by MK -801 (0.1 mg/kg)[2]. After being injected intraperitoneally (10 mg/kg), zamifenacin shows brief elimination half-lives (plasma 1.16 h, brain 1.75 h) in mice[ 2].

Novel isoxazolopyridone derivatives that are metabotropic glutamate receptor (mGluR) 7 antagonists were discovered and pharmacologically characterized. 5-Methyl-3,6-diphenylisoxazolo[4,5-c]pyridin-4(5H)-one (MDIP) was identified by random screening, and 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP) was produced by chemical modification of MDIP. MDIP and MMPIP inhibited L-(+)-2-amino-4-phosphonobutyric acid (L-AP4)-induced intracellular Ca2+ mobilization in Chinese hamster ovary (CHO) cells coexpressing rat mGluR7 with Galpha(15) (IC50 = 20 and 26 nM). The maximal response in agonist concentration-response curves was reduced in the presence of MMPIP, and its antagonism is reversible. MMPIP did not displace [3H](2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) bound to mGluR7. These results suggested that these isoxazolopyridone derivatives are allosteric antagonists. In CHO cells expressing rat mGluR7, MDIP and MMPIP inhibited l-AP4-induced inhibition of forskolin-stimulated cAMP accumulation (IC50 = 99 and 220 nM). In CHO cells coexpressing human mGluR7 with Galpha(15), MDIP and MMPIP also inhibited the l-AP4-induced cAMP response. The maximal degree of inhibition by MMPIP was higher than that by MDIP in a cAMP assay. MMPIP was able to antagonize an allosteric agonist, the N,N'-dibenzhydryl-ethane-1,2-diamine dihydrochloride (AMN082)-induced inhibition of cAMP accumulation. In the absence of these agonists, MMPIP caused a further increase in forskolin-stimulated cAMP levels in CHO cells expressing mGluR7, whereas a competitive antagonist, LY341495, did not. This result indicates that MMPIP has an inverse agonistic activity. The intrinsic activity of MMPIP was pertussis toxin-sensitive and mGluR7-dependent. MMPIP at concentrations of at least 1 microM had no significant effect on mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, and mGluR8. MMPIP is the first allosteric mGluR7-selective antagonist that could potentially be useful as a pharmacological tool for elucidating the roles of mGluR7 on central nervous system functions[1].

cAMP[2]
A homogeneous time-resolved fluorescence (HTRF) cAMP dynamic 2 assay was performed as previously described (Chruścicka et al., 2015) with recombinant cell lines. Briefly, HEK 293 T-REx cells stably expressing mGlu7 receptor, were collected and suspended in Hanks-HEPES buffer. The cell suspension was added to compounds solution with 5 μM of forskolin (final concentration). After 5 min incubation in 37°C, 5 μl of cAMP-d2 conjugate in lysis buffer was added and mixed with the 10 μl cell suspension by means of an automated pipetting system. Next, 5 μl anti-cAMP cryptate conjugate was added and the fluorescence at 620 and 665 nm was read after 1 h . The results are shown as the 665 nm/620 nm ratio multiplied by 104. The detected signal was inversely proportional to the concentration of cAMP in the sample. Antagonist activity of ADX71743 or MMPIP are shown as a percentage of the inhibition of L-Glu activity at its EC80 concentration. Dose response data from ADX71743 or MMPIP were analyzed with Prism Version 7.03. Each experiment was performed three times (n = 3), and each data point was in triplicate.

Male Albino Swiss mice (20–25 g) were used in most behavioral tests. Male Wistar rats (200–250 g) were used in spatial delayed alternation test and PPI of the acoustic startle response test. Male C57BL/6J WT and mGlu7 KO mice were used in electrophysiological studies. The animals were kept in a room with 12:12 light–dark cycle at a temperature of 21–22°C. Food and water were provided ad libitum. The animals were used only once, none of the animals has been run multiple experiments.[2]

MMPIP was formulated in 0.5% methylcellulose[2]

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Pharmacokinetic Studies[2]
The method described below was successfully applied to a pharmacokinetic study of ADX71743 and MMPIP in mouse (Albino Swiss) after i.p. injection. Compound ADX71743 and MMPIP were administered to mice at 10 mg/kg i.p. At 0.25, 0.50, 1.0, 2.0, 4.0, 6.0 h, the mice were anesthetized, and the blood was collected from the portal vein to the tubes containing 5% EDTA. The mice were then perfused with 0.1M PBS to remove remaining blood from the body, and the brains were taken out for the analysis. Blood was centrifuged at 2000 rpm for 10 min at 4°C, and the plasma was collected and frozen at -80°C for further analysis. [2]
Plasma and tissue samples from all drug-treated animals were thawed at room temperature prior to use. Standard protocol of sample preparation: 200 μl acetonitrile was added to the eppendorfs with 50 μl of studied plasma samples or tissue homogenate. Samples were mixed for 5 min on a mixer at 25°C and 1400 rpm. Tubes were then centrifuged at 2000 × g for 15 min at 4°C. About 180 μl of each supernatant was transferred into a plate well. Finally, each sample was injected into the column.

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MK-801-Induced Hyperactivity[2]
The locomotor activity was recorded individually for each animal in OPTO-M3 locomotor activity cages linked online to a compatible PC activity, as described previously by Woźniak et al., 2016b. Each cage (13 cm × 23 cm × 15 cm) was surrounded with an array of photocell beams. Interruptions of these photobeams resulted in horizontal activity defined as ambulation counts. The mice were placed in the locomotor activity cages for acclimatization for 30 min Then, MMPIP (10, 15 mg/kg) or ADX71743 (5, 10 mg/kg) were administered i.p. Both drugs were given 30 min prior to MK-801 injection (0.35 mg/kg, i.p.). The locomotor activity was measured for 60 min immediately after MK-801 administration.

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DOI-Induced Head Twitches[2]
The experiment was performed according to previously described procedure (Wierońska et al., 2012, 2013). Immediately after a 30 min acclimatization period, DOI (2.5 mg/kg, i.p.) was administered in order to induce head twitches. The number of head twitches was then counted for 20 min. MMPIP (5, 10, and 15 mg/kg) or ADX71743 (2.5, 5, and 10 mg/kg) were administered i.p. 30 min before DOI. Subsequently the compounds were administered chronically (for 10 days) each at the two active doses (MMPIP 5 and 10 mg/kg and ADX71743 2.5 and 5 mg/kg). The test was performed on 11th day, 30 min after the last administration.

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Modified Forced Swim Test[2]
The modified forced swim test was performed according to the method introduced by Noda (Noda et al., 1995, 1997; Wierońska et al., 2015a; Woźniak et al., 2016a). The swim tests were performed in a glass cylinder (height, 20 cm; internal diameter, 15 cm) containing 11 cm of water maintained at 23–24°C. After the acclimation period, the animals underwent the first swim test, where the immobility time was measured during a 3 min period (T1). On the next day, chronic (13 days) MK-801 administration (0.4 mg/kg, i.p.) was started. After a 1-day break, on the 15th day of experiment, the second swim session was performed and the immobility time during 3-min test was measured again (T2). The T2 - T1 difference was reported as the result of the experiment. MMPIP (1, 5, and 15 mg/kg, i.p.) or ADX71743 (5, 10, and 15 mg/kg, i.p.) were administered acutely 30 min before the T2 session.

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Social Interaction Test[2]
The method was adapted from de Moura Linck et al., 2008 and Woźniak et al., 2016b. After the 2-day habituation trial (10 min/day) a pair of mice was placed in the open field for 5 min. The social interactions between two mice were determined based on the total time spent participating in social behavior such as genital investigation, sniffing, chasing, and fighting each other. The total number of social episodes was also measured. The test was video-recorded and viewed by a trained observer. MMPIP (5, 10, and 20 mg/kg, i.p) or ADX71743 (1, 5, and 15 mg/kg, i.p.) were administered 30 min before MK-801 (0.3 mg/kg, i.p.), which was administered 30 min before the test.

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Novel Object Recognition Test[2]
The experiment was performed according to Nilsson et al., 2007 with minor modifications (Woźniak et al., 2016b). Following a 2-day habituation period (10 min/day), a training trial was performed, where mice were allowed to explore two identical objects for 5 min. About 1 h later, a test trial was conducted, where one of the familiar object was replaced by a novel object. The animals were then allowed to explore the objects for 5 min. MMPIP (5, 10, and 15 mg/kg, i.p.) and ADX71743 (1, 5, and 10 mg/kg; i.p.) were administered 30 min before MK-801 (0.3 mg/kg, i.p.), which was administered 30 min before the training trial. Time spend exploring (i.e., sniffing or touching) the familiar (Tfamiliar) or novel object (Tnovel) was measured by a trained observer and then the recognition index was calculated for each mouse [(Tnovel - Tfamiliar)/(Tfamiliar + Tnovel)] × 100.

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Rotarod Test[2]
The animals were trained for 3 consecutive days at the speed of 18 rpm, one session per day for 3 min. If a mice fell during the habituation period, it was placed back on the apparatus. On the following day, the test trial was performed. After the mice were placed on the apparatus (Mouse Rota-Rod NG, UGO BASILE S.R.L.) moving at the speed of 12 rpm, the accelerating mode was started (maximum speed – 24 rpm). The latency to fall was measured during 3-min test session. Mice were injected with MMPIP (5, 15, and 30 mg/kg, i.p.) or ADX71743 (5, 15, and 30 mg/kg, i.p.) 30 min before the test trial.

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Spatial Delayed Alternation Test[2]
The spatial delayed alternation test was performed using a wooden T-maze, according to Sławińska et al., 2013 and Wierońska et al., 2015b. During the adaptation phase, lasting 3 days, the animals were allowed to freely explore the maze for 10 min. For the next 2 days, rats were confined to either of the two end-arms and allowed to drink a 10 % sucrose solution there for 10 min twice daily. On the following day, a 2-week training phase was started. The animals performed one training session per day, which consisted of one forced trial (i.e., one of the end-arms was closed) followed by ten free choice trials. During the free choice trial the animal was placed in the starting arm and after the guillotine door was raised, it was allowed to choose to enter one of the end-arms. After the response, the rat was placed back to the starting arm, where it stayed for 10 s. If the chosen end-arm was the opposite to the previously visited one, a correct response was scored, and the animal was closed in the compartment where it was allowed to drink the sucrose solution for 5 s. After an incorrect response, the animal was gently returned to the starting arm. The training phase was carried out until the animals scored 7 correct responses in a training session in 2 consecutive days. On the day of the test, the animals were injected with MMPIP and/or MK-801, and the aforementioned 10-trial session was repeated. MMPIP was administered at a dose of 5 or 10 mg/kg 30 min prior to MK-801 (0.1 mg/kg) administration. The test was started 30 min after the MK-801 injection.

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Prepulse Inhibition[2]
The procedure was performed according to Czyrak et al., 2003. On the day before the experiment, the animals were subjected to a single startle session consisting of two trials, each presented 20 times during the session. During the first trial, a 120 dB, 40 ms pulse was presented, and on the second trial this pulse was preceded by a 75 dB, 20 ms prepulse. On the day of the experiment, the animals were habituated to the background white noise (65 dB) for 5 min (which continued throughout the test), after that the startle session was carried out as described above. Startle response amplitude was defined as the difference between the maximum force detected during a recording window and the force measured immediately before the stimulus onset (the threshold was set at 10 g). For each animal, the amplitudes were averaged separately for each type of trial. The PPI was calculated as the difference between the amplitudes of the pulse (P) and the prepulse + pulse (PP+P), divided by the amplitude of the pulse alone [([P - (PP + P)]/P)×100]. MMPIP (5, 10, and 15 mg/kg) and ADX71743 (2.5, 5, and 10 mg/kg) were administered 30 min prior to MK-801 (0.3 mg/kg), which was administered 30 min before the habituation phase.

The concentration of MMPIP in mouse plasma and brain are shown in Table ​Table11. Cmax was evident in brain and plasma 0.25 h after injection of ADX71743, and 0.5 h after MMPIP administration. Figure ​Figure33 represents comparison between ADX71743 and MMPIP concentrations in the brain in selected time points after administration.[2]
Data presented in Table ​Table22 showed that ADX71743 and MMPIP had different cytochrome P450 inhibition profile. Weak inhibition (IC50 > 10μM) of cytochrome P450 was observed in case of 1A2, 2B6, 2C9, 2D6 isoforms for both NAM mGluR7 standards. Mild inhibition (3.3 < IC50 < 10) of isoform 2C19 was determined for ADX71743 standard, while strong inhibition (IC50 < 1.1) was observed only for MMPIP in case of isoform 3A4 as well as 2C19.[2]

[1]. In vitro pharmacological characterization of novel isoxazolopyridone derivatives as allosteric metabotropic glutamate receptor 7 antagonists. J Pharmacol Exp Ther. 2007 Oct;323(1):147-56.

[2]. Negative Allosteric Modulators of mGlu 7 Receptor as Putative Antipsychotic Drugs. Front Mol Neurosci. 2018 Sep 20;11:316.

The data concerning antipsychotic-like activity of negative allosteric modulators (NAMs)/antagonists of mGlu7 receptors are limited. The only available ligands for this receptor are MMPIP and ADX71743. In the present studies, we used stable cell line expressing mGlu7 receptor and it was shown that both compounds dose-dependently potentiated forskolin elevated cAMP concentration in the T-REx 293 cells, showing their inverse agonist properties. Subsequently, pharmacokinetic studies were performed. Both compounds were given intraperitoneally (i.p.) at the dose of 10 mg/kg and reached Cmax 0.25-0.5 h after administration, and then they declined rapidly, ADX71743 being almost undetectable 2 h after administration, while the concentration of MMPIP was still observed, suggesting that the concentration of MMPIP was more stable. Finally, we investigated the role of both mGlu7 receptor NAMs in animal models of schizophrenia. Behavioral tests commonly used in antipsychotic drug discovery were conducted. Both tested compounds dose-dependently inhibited MK-801-induced hyperactivity (MMPIP at 15 mg/kg; ADX at 5 and 15 mg/kg) and DOI-induced head twitches (MMPIP at 5, 10, 15 mg/kg; ADX at 2.5, 5, 10 mg/kg). Moreover, the same effects were noticed in novel object recognition test, where MMPIP (5, 10, 15 mg/kg) and ADX71743 (1, 5, 15 mg/kg) reversed MK-801-induced disturbances. In the social interaction test, antipsychotic activity was observed only for ADX71743 (5, 15 mg/kg). ADX71743 at the dose 2.5 mg/kg reversed MK-801-induced disruption in prepulse inhibition while MMPIP at 10 mg/kg reversed MK-801-induced disruption in spatial delayed alternation. The present studies showed that mGlu7 receptor may be considered as a putative target for antipsychotic drugs, though more studies are needed due to limited number of available ligands.[2]

C19H16CLN3O3

369.80

466.072

C, 61.71; H, 4.36; Cl, 9.59; N, 11.36; O, 12.98

1215566-78-1

MMPIP;479077-02-6

52974245

Light yellow to green yellow solid powder

1

5

3

26

524

0

HXHQNERSIJXMTQ-UHFFFAOYSA-N

InChI=1S/C19H15N3O3.ClH/c1-22-15(12-3-5-14(24-2)6-4-12)11-16-17(19(22)23)18(21-25-16)13-7-9-20-10-8-13;/h3-11H,1-2H3;1H

6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-yl-[1,2]oxazolo[4,5-c]pyridin-4-one;hydrochloride

MMPIPHYDROCHLORIDE; MMPIP (hydrochloride); 1215566-78-1; 6-(4-METHOXYPHENYL)-5-METHYL-3-(4-PYRIDINYL)-ISOXAZOLO[4,5-C]PYRIDIN-4(5H)-ONE HYDROCHLORIDE; 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-yl-[1,2]oxazolo[4,5-c]pyridin-4-one;hydrochloride; SCHEMBL20836388;

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, 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: 10 mg/mL (27.04 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (2.70 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 10.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)