mGlu4 (EC50 = 0.13 μM); mGlu8 (EC50 = 0.29 μM); mGlu6 (EC50 = 1.0 μM); mGlu7 (EC50 = 249 μM)

A group III metabotropic glutamate (mGlu) receptor agonist (PCEP) was identified by virtual HTS. This orthosteric ligand is composed by an L-AP4-derived fragment that mimics glutamate and a chain that binds into a neighboring pocket, offering possibilities to improve affinity and selectivity [1].

In eight nerve-ligated rats, L-AP4 (5-30 μg, intrathecal inhection 4-5 days) dose-dependently and significantly raises the paw withdrawal threshold in response to the administration of von Frey filaments. Different doses of L-AP4 administered intrathecally do not appear to cause any obvious motor dysfunction[2]. The paw withdrawal latency of these normal rats is not significantly affected by an intrathecal injection of 30 μg of L-AP4[2]. The evoked response of neurons to touch, pressure, pinch, and von Frey filaments was dramatically suppressed by topical injection of 5 to 50 μM L-AP4 to the spinal cord in a concentration-dependent manner[2].

---

Increased glutamatergic input to spinal dorsal horn neurons constitutes an important mechanism for neuropathic pain. However, the role of group III metabotropic glutamate receptors (mGluRs) in regulation of nociception and dorsal horn neurons in normal and neuropathic pain conditions is not fully known. In this study, we determined the effect of the group III mGluR specific agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) on nociception and dorsal horn projection neurons in normal rats and a rat model of neuropathic pain. Tactile allodynia was induced by ligation of L5/L6 left spinal nerves in rats. Allodynia was determined by von Frey filaments in nerve-injured rats. The nociceptive threshold was tested using a radiant heat and a Randall-Selitto pressure device in normal rats. Single-unit activity of ascending dorsal horn neurons was recorded from the lumbar spinal cord in anesthetized rats. An intrathecal (5-30 microg) L-AP4 dose-dependently attenuated allodynia in nerve-injured rats but had no antinociceptive effect in normal rats. Topical spinal application of 5 to 50 microM L-AP4 also significantly inhibited the evoked responses of ascending dorsal horn neurons in nerve-ligated but not normal rats. Furthermore, blockade of spinal group III mGluRs significantly decreased the withdrawal threshold and increased the evoked responses of dorsal horn neurons in normal but not nerve-injured rats. These data suggest that group III mGluRs play distinct roles in regulation of nociception and dorsal horn neurons in normal and neuropathic pain states. Activation of spinal group III mGluRs suppresses allodynia and inhibits the hypersensitivity of dorsal horn projection neurons associated with neuropathic pain [2].

Pharmacological Assays on Recombinant mGlu Receptors [1]
Metabotropic glutamate receptors were transiently transfected in HEK293 cells by electroporation as described elsewhere and plated in 96-well microplates. The high affinity glutamate transporter EAAT3 was co-transfected with the receptor to avoid any influence of glutamate released by the cells in the assay medium. In our experiments, group III mGlu receptors were co-transfected with a chimeric G-protein which couples the activation of the receptor to the phospholipase-C (PLC) pathway. Thus, receptor activation induces production of inositol phosphate (IP), which in turn induces intracellular Ca2+ release. Receptor activity was then determined by measuring of the IP production or Ca2+ release as already described. We previously reported that these assays are more easily handled and gave more accurate results than the classical measurement of the inhibition of the forskolin-activated adenylyl-cyclase activity and that the pharmacology of these receptors was not altered. Indeed, over the recent years, we and others have obtained evidence showing that the pharmacological profiles of these receptors are well conserved in these assays. To determine inositol phosphate production, mGlu receptors expressing cells were seeded in 96-well microplates and incubated overnight with 3H-myoinositol (23.4 Ci/mol) in the culture medium. The following day, cells were stimulated by agonists for 30 min, at the end of which the medium was changed and cells incubated for 1 h with cold 0.1 M formic acid which induced cell lysis. The 3H-IP produced following receptor stimulation were recovered by ion exchange chromatography using a Dowex resin. Following elution, radioactivity was counted using a Wallac 1450 Microbeta stintillation and luminescence counter. The radioactivity remaining in the membranes was used to normalize the IP produced. Results are expressed as the ratio between IP and the total radioactivity corresponding to IP plus membrane. All data points represent triplicates. For intracellular calcium measurements, cells expressing mGlu receptors were loaded with Ca2+-sensitive fluorescent dye Fluo-4 AM dissolved in Hanks’ Balanced Salt Solution containing 2.5 mM Probenicid for 1 h at 37 °C, then washed and incubated with HBSS containing probenecid. A drug plate was prepared with the various concentrations of agonist to be tested, and drug solution was added to each well after 20 s of recording. Fluorescence signals (excitation 485 nm, emission 525 nm) were measured by using the fluorescence microplate reader Flexstation III at sampling intervals of 1.5 s for 60 s. All data points represent triplicates. The dose–response curves were fitted using the GraphPad Prism program and the following equation: y = [(ymax −ymin)/(1 + (x/EC50)n)] + ymin where EC50 is the concentration of the compound necessary to obtain the half-maximal effect and n is the Hill coefficient.

---

Pharmacological Assay on Recombinant NMDA Receptors [1]
Recombinant NMDA receptors were expressed in Xenopus laevis oocytes after coinjection of 30 nL of a mixture of cDNAs (at 10–30 ng/μL; nuclear injection) coding for rat GluN1-a and rat GluN2A or mouse GluN2B subunits (ratio 1:1). Oocytes were prepared, injected, voltage-clamped, and superfused as described previously. Data were collected and analyzed using pClamp 9.2. They were fitted using Sigmaplot 8.0. The standard external solution used for recordings at pH 7.3 contained (in mM): 100 NaCl, 0.3 BaCl2, 5 HEPES, 2.5 KOH. The pH was adjusted to 7.3 with HCl. Then 10 μM DTPA was added to all the solutions to chelate contaminating zinc, which acts as a very potent allosteric inhibitor of GluN1/GluN2A NMDA receptors. NMDA currents were induced by simultaneous application of l-glutamate and glycine and recorded at a holding potential of −60 mV and at room temperature. Their inhibition by 17m was measured by adding various amounts of the compound during an application of a saturating concentration of glycine (100 μM) and a concentration of glutamate close to its EC50 (5 μM). The solutions of different concentrations of 17m were obtained from the dilution in the agonist solution of a 50 mM stock solution of the compound in water additionned with 1 equiv of NaOH. Inhibition dose–response curves were fitted with the following Hill equation: I17m/I0 = 1–1/(1 + (IC50/[17m])nH), where I17m/I0 is the relative current, and IC50 is the concentration of 17m producing 50% of the maximal potentiation and nH is the Hill coefficient.

Cerebellar Slices Preparations [1]
Rats were stunned and then decapited. Coronal and sagittal cerebellar slices (250 μm thick) were prepared with a vibrosclicer, Microm HM 650 from the vermis of 18–29-day-old rats. Slices were prepared in an ice-cold (3 °C) sucrose-based solution saturated with 95% O2–5% CO2, containing (mM): sucrose 230, KCl 2.5, KH2PO4 1.25, MgCl2 8, glucose 25, NaHCO3 26, CaCl2 0.8 (osmolarity 330 mOsm l–1). The slices were kept at room temperature for at least 1 h before recording in saline solution gassed with 95% O2–5% CO2. This solution contained (in mM): NaCl, 124; KCl, 3; NaHCO3, 24; KH2PO4, 1.15; MgSO4, 1.15; CaCl2, 2; glucose, 10; osmolarity 330 mOsm l–1 and pH 7.35 at 25 °C. The recording chamber was perfused at a rate of 2 mL per minute with this same oxygenated saline solution, supplemented with the GABAA receptor antagonist bicuculline methiodide (10 μM).

---

Electrophysiology [1]
Whole-cell patch-clamp recordings of Purkinje cells (PC) were performed in sagittal slices with an Axopatch-1D amplifier. PC somas were directly visualized with Nomarski optics through the ×60 water-immersion objective of an upright microscope. All recordings were made at 28–30 °C. Patch pipettes (3.5–5 MΩ, borosilicate glass) were filled with an internal solution of the following composition (mM): KGlu, 140; KCl, 6; HEPES, 10; EGTA, 0.75; MgCl2, 1, Na-GTP, 0.4; Na2-ATP, 4; pH 7.3 with KOH; 300 mOsmol l–1. PCs were clamped at −70 mV, and parallel fibers (PFs) were stimulated once every 6 s through a glass saline filled monopolar electrode placed at the surface of the slice, in the lower half of the molecular layer, to elicit PF-evoked excitatory postsynaptic currents (EPSCs). PF-EPSCs were evoked with pairs of stimuli of the same intensity applied to the cell with an interstimulus interval of 40 ms. The paired-pulse facilitation (PPF) was calculated online as the ratio of the amplitude of the second PF-EPSC over the first one. Mean PPF values were obtained by averaging PPFs in individual traces for each cell. In the cells conserved for analysis, access resistance (usually 5–10 MΩ) was partially compensated (50–70%) according to the procedure described by Llano et al. Throughout the experiment, PF-EPSCs were elicited on a 10 mV hyperpolarizing voltage step, which allowed monitoring of passive membrane properties. PF-EPSC were analyzed online and offline with Acquis1 software.

---

Calcium Sensitive Fluorometric Measurements [1]
Using coronal slices, PF tracts were loaded by focal application of a saline solution containing the low affinity calcium sensitive dye Fluo-4FF-AM (100 μM), as previously described. At least 45 min after loading, a confined region of labeled PFs was illuminated at a single excitation wavelength (485 ± 22 nm). Excitation light obtained from a 100 W mercury lamp was gated with an electromechanical shutter. PFs located in the recording window were stimulated every minute, with a single 100 Hz train of five electrical stimuli, through a saline-filled glass electrode placed in the molecular layer between the loading site and the recording site. Evoked fluorescent transients from labeled PFs were recorded in a 20 μm × 50 μm window placed above the molecular layer, approximately 500–800 μm away from the loading site and 100 μm above PC layer, collected through a ×60 water-immersion objective of an upright microscope, filtered by a barrier filter at 530 ± 30 nm, and converted into an electric signal by a photometer. Fluorescence signals corrected for dye bleaching were expressed as relative fluorescence changes ΔF/F, where F is the baseline fluorescence intensity, and ΔF is the change induced by PF stimulation. When background fluorescence of the tissue in unlabeled regions of the slice was greater than 5% of the basal fluorescence intensity of the indicator, the data were corrected for background fluorescence. All the experiments were performed at 28–30 °C. Fluorometric measurements were analyzed online and offline with Acquis1 software.

Animal/Disease Models: Rats.[2]
Doses: 5-30 μg.
Route of Administration: Intrathecal inhection 4-5 days.
Experimental Results: Dose-dependently increased paw withdrawal threshold.

[1]. Increased Potency and Selectivity for Group III Metabotropic Glutamate Receptor Agonists Binding at Dual sites. J Med Chem. 2018 Mar 8;61(5):1969-1989.

[2]. Distinct roles of group III metabotropic glutamate receptors in control of nociception and dorsal horn neurons in normal and nerve-injured Rats. J Pharmacol Exp Ther. 2005 Jan;312(1):120-6.

(2S)-2-amino-4-phosphonobutanoic acid is a non-proteinogenc L-alpha-amino acid that is L-alpha-aminobutyric acid in which one of the hydrogens of the terminal methyl group has been replaced by a dihydroxy(oxido)-lambda(5)-phosphanyl group. It is a potent and selective agonist for the group III metabotropic glutamate receptors (mGluR4/6/7/8). It has a role as a metabotropic glutamate receptor agonist. It is a non-proteinogenic L-alpha-amino acid and a member of phosphonic acids.

---

A group III metabotropic glutamate (mGlu) receptor agonist (PCEP) was identified by virtual HTS. This orthosteric ligand is composed by an l-AP4-derived fragment that mimics glutamate and a chain that binds into a neighboring pocket, offering possibilities to improve affinity and selectivity. Herein we describe a series of derivatives where the distal chain is replaced by an aromatic or heteroaromatic group. Potent agonists were identified, including some with a mGlu4 subtype preference, e.g., 17m (LSP1-2111) and 16g (LSP4-2022). Molecular modeling suggests that aromatic functional groups may bind at either one of the two chloride regulatory sites. These agonists may thus be considered as particular bitopic/dualsteric ligands. 17m was shown to reduce GABAergic synaptic transmission at striatopallidal synapses. We now demonstrate its inhibitory effect at glutamatergic parallel fiber-Purkinje cell synapses in the cerebellar cortex. Although these ligands have physicochemical properties that are markedly different from typical CNS drugs, they hold significant therapeutic potential.[1]

C4H12NO6P

201.11

201.04

2247534-79-6

D-AP4;78739-01-2;L-AP4;23052-81-5

146401139

White to off-white solid powder

5

7

4

12

187

1

P(O)(O)(=O)CC[C@H](N)C(=O)O.O

ZAXJAZYNVPZMRL-DFWYDOINSA-N

InChI=1S/C4H10NO5P.H2O/c5-3(4(6)7)1-2-11(8,9)10;/h3H,1-2,5H2,(H,6,7)(H2,8,9,10);1H2/t3-;/m0./s1

(2S)-2-amino-4-phosphonobutanoic acid;hydrate

L-AP4 monohydrate; L-AP4 (monohydrate); 2247534-79-6; (2S)-2-amino-4-phosphonobutanoic acid;hydrate; SCHEMBL21747432;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)