Group III metabotropic glutamate receptor; L-AP4-sensitive presynaptic mGluR (KD = 51 μM)

The dose-response curve of L-AP4 showed a parallel shift to the right in the presence of 200 μM MSOP; an apparent KD of 51±6 μM (n=3) was determined. The apparent KD of MSOP interaction with (1S, 3S)-ACPD-sensitive receptors was determined to be larger than 700 μM (n=3), indicating that MSOP is selective for L-APC-sensitive presynaptic mGluRs [1].

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The antagonist selectivity and potency of two novel serine-O-phosphate derivatives (RS)-alpha-methylserine-O-phosphate (MSOP) and the monophenylester (RS)-alpha-methylserine-O-phosphate monophenyl-phosphoryl ester (MSOPPE) was investigated against L-2-amino-4-phosphonobutyrate (L-AP4)- and (1S,3S)-1-aminocyclopentane-1, 3-dicarboxylate (ACPD)-induced depressions of the monosynaptic excitation of neonatal rat motoneurones, mediated via metabotropic glutamate receptors (mGLuRs). MSOP was shown to be a selective antagonist for the L-AP4-sensitive presynaptic mGluR, displaying an apparent KD of 51 microM, compared to > 700 microM for the (1S,3S)-ACPD-sensitive presynaptic mGluR. In contrast, MSOPPE displayed antagonist activity at both presynaptic mGluR, with a three times greater selectivity for the (1S,3S)-ACPD-sensitive receptor over the L-AP4-sensitive mGluR (apparent KD values 73 microM and 221 microM, respectively). Therefore, on addition of an alpha-methyl group to the mGluR agonist serine-O-phosphate, we have developed an mGluR antagonist which is selective for the presynaptic L-AP4-sensitive receptor. In contrast, monoesterification of MSOP to give the monophenylphosphoryl ester (MSOPPE), confers a degree of selectivity for the (1S,3S)-ACPD-over the L-AP4-sensitive presynaptic mGluR. Neither MSOP nor MSOPPE had any activity on either postsynaptic mGLuRs or ionotropic receptors[1].

The results (formalin model second phase: F3,16=30.96, P<0.001; CFA model: F3,16=30.77, P<0.001) demonstrated that intrathecal simultaneous administration of MSOP could significantly block the analgesic effect generated by TBOA. Anticipatedly, a second-stage intrathecal injection of TBOA (10 μg) prevented CFA-induced ipsilateral paw reduction and decreased the amount of formalin-induced tremors and flinches by 47% in the saline-treated group (P<0.001). Compared to the value in the saline treatment group, the withdrawal latency was 60% shorter (P=0.01). In the second stage of the MSOP and TBOA treatment groups, there were 56% more formalin-induced withdrawals than in the TBOA treatment group (P=0.04). When comparing the CFA-induced paw withdrawal delay between the TBOA and MSOP treatment groups and the TBOA treatment group, the difference was 86% (P=0.03)[2].

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In the superficial dorsal horn, excitatory amino acid carrier 1 is localized in presynaptic membrane, postsynaptic membrane, and axonal and dendritic membranes at nonsynaptic sites, whereas glutamate transporter-1 and glutamate/aspartate transporter are prominent in glial membranes. Although expression of these three spinal glutamate transporters was not altered 1 h after formalin injection or 6 h after CFA injection, glutamate uptake activity was decreased at these time points. Intrathecal (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline had no effect on formalin-induced pain behaviors. In contrast, intrathecal TBOA, dihydrokainate, and DL-threo-β-hydroxyaspartate reduced formalin-evoked pain behaviors in the second phase. Intrathecal TBOA also attenuated CFA-induced thermal hyperalgesia at 6 h after CFA injection. The antinociceptive effects of TBOA were blocked by coadministration of MSOP/(RS)-α-methylserine-O-phosphate. Conclusion: Our findings suggest that spinal glutamate transporter inhibition relieves inflammatory pain through activation of inhibitory presynaptic group III metabotropic glutamate receptors.[2]

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Effect of blocking group III metabotropic glutamate receptors on the antinociception of TBOA in formalin and CFA models [2]
Group III mGluRs are expressed in primary afferent terminals in the dorsal horn, and their activation reduces glutamate release from the primary afferent terminals. To determine whether group III mGluRs participate in the antinociception caused by spinal glutamate transporter inhibition, we examined the effect of intrathecal MSOP, a group III mGluR antagonist, on TBOA-produced antinociception in the formalin and CFA models. We found that TBOA-induced antinociceptive effects were significantly blocked by intrathecal co-administration of MSOP (second phase of formalin model: F3,16 = 30.96, P < 0.001; CFA model: F3,16 = 30.77, P < 0.001). As expected, intrathecal TBOA (10 μg) reduced the number of formalin-induced flinches and shakes by 47% of the value in the saline-treated group in the second phase (P < 0.001; Fig. 8A) and blocked the CFA-induced decrease in ipsilateral paw withdrawal latency by 60% of the value in the saline-treated group (P = 0.01; Fig. 8B). The number of formalin-induced flinches in the second phase in the group treated with MSOP and TBOA was increased by 56% (P = 0.04; Fig. 8A) of the value in the TBOA-treated group. CFA-induced paw withdrawal latency in the group treated with MSOP and TBOA was decreased by 86% (P = 0.03; Fig. 8B) of the value in the TBOA-treated group. MSOP (10 μg) alone did not affect formalin-induced pain behaviors in either phase (P = 0.73; Fig. 8A) or CFA-induced thermal hyperalgesia (P = 0.65; Fig. 8B). It also had no effect on contralateral-side basal paw withdrawal (Fig. 8B).

Assessment of formalin-induced pain behaviors [2]
The rats were placed individually in an open Plexiglas chamber (34×30×30 cm) for 1 h before actual experimental sessions. To examine whether altering spinal glutamate transporter uptake activity affected formalin-induced pain behaviors, we intrathecally injected saline (control; 10 μl; n = 10), MS-153 (10, 100, or 1,000 μg/10 μl; n = 5/dose), DL-THA (1.5, 7.5, or 15 μg/10 μl; n = 6/dose), dihydrokainate (2, 10, or 20 μg/10 μl; n = 7/dose), or TBOA (1, 5, or 10 μg/10 μl; n = 10/dose) followed by 10 μl of saline to flush the catheter. Ten minutes later, the experimenter injected 2% formalin (100 μl) into the plantar side of one hind paw and immediately placed the rat into a transparent cage to count the number of paw flinches and shakes over the next 60 min.20,21 The observational session was divided into two phases: 0–10 min and 10–60 min. The mean number of flinches and shakes for each period was calculated for each treatment group.
To examine the role of group III mGluRs in the antinociceptive effect produced by intrathecal TBOA in the formalin model, we intrathecally injected the rats with saline (10 μl; n = 5), MSOP (10 μg/10 μl; n = 5), TBOA (10 μg/10 μl; n = 5), or MSOP plus TBOA (n = 5). Ten minutes later, 2% formalin (100 μl) was injected into the plantar side of a hind paw and formalin-induced pain behaviors were assessed.

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Assessment of CFA-induced thermal pain hypersensitivity [2]
To induce persistent inflammatory pain, CFA (100 μl, 1 mg/ml Mycobacterium tuberculosis) solution was injected into the plantar side of one hind paw. Our previous studies showed that CFA-induced thermal pain hypersensitivity reaches a peak level at 6 h and persists for at least 24 h post-injection.22 We chose these two time points for the pharmacologic and behavioral studies. At 6 h and 24 h after CFA injection, we intrathecally injected saline (10 μl; n = 5/time point) or TBOA (1, 5, or 10 μg/10 μl; n = 5/dose/time point) followed by 10 μl of saline to flush the catheter. Thermal testing was carried out 1 day before CFA injection (baseline) and at 20 min after saline or TBOA administration. Paw withdrawal response to thermal stimulation was measured as described previously.14,18,19 Briefly, each rat was placed in a Plexiglas chamber on a glass plate above a light box. A beam of radiant heat from the apparatus was applied through a hole in the light box to the middle of the plantar surface of each hind paw through the glass plate. When the rat lifted its foot the light beam automatically shut off. The length of time between the start of the light beam and the foot lift was defined as the paw withdrawal latency. Each trial was repeated five times at 5-min intervals for each side. A cut-off time of 20 s was used to avoid tissue damage to the paw.
To examine the role of group III mGluRs in the antinociceptive effect produced by intrathecal TBOA in the CFA model, we intrathecally injected the rats with saline (10 μl; n = 5), MSOP (10 μg/10 μl; n = 5), TBOA (10 μg/10 μl; n = 5), or MSOP plus TBOA (n = 5) at 6 h post-CFA and then measured paw withdrawal latencies.

[1]. alpha-Methyl derivatives of serine-O-phosphate as novel, selective competitive metabotropic glutamate receptor antagonists. Neuropharmacology. 1996 Jun;35(6):637-42.

[2]. Effect of inhibition of spinal cord glutamate transporters on inflammatory pain induced by formalin and complete Freund’s adjuvant. Anesthesiology. 2011 Feb; 114(2): 412–423.

Background: Spinal cord glutamate transporters clear synaptically released glutamate and maintain normal sensory transmission. However, their ultrastructural localization is unknown. Moreover, whether and how they participate in inflammatory pain has not been carefully studied. Methods: Immunogold labeling with electron microscopy was carried out to characterize synaptic and nonsynaptic localization of glutamate transporters in the superficial dorsal horn. Their expression and uptake activity after formalin- and complete Freund's adjuvant (CFA)-induced inflammation were evaluated by Western blot analysis and glutamate uptake assay. Effects of intrathecal glutamate transporter activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline and inhibitors (DL-threo-β-benzyloxyaspartate [TBOA], dihydrokainate, and DL-threo-β-hydroxyaspartate), or TBOA plus group III metabotropic glutamate receptor antagonist (RS)-α-methylserine-O-phosphate, on formalin- and CFA-induced inflammatory pain were examined.[2]

C4H10NO6P

199.09906244278

199.025

66515-29-5

3964633

White to off-white solid powder

-5

4

7

4

12

224

0

P(=O)(O)(O)OCC(C(=O)O)(C)N

GSFCOAGADOGIGE-UHFFFAOYSA-N

InChI=1S/C4H10NO6P/c1-4(5,3(6)7)2-11-12(8,9)10/h2,5H2,1H3,(H,6,7)(H2,8,9,10)

2-amino-2-methyl-3-phosphonooxypropanoic acid

MSOP; 66515-29-5; alpha-MSOP; 2-amino-2-methyl-3-phosphonooxypropanoic acid; alpha-methylserine-O-phosphate;

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)

H2O : ~62.5 mg/mL (~313.91 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.)