GABAB receptor (IC50 = 85 nM)

The GABAB receptor antagonist CGP52432 has an IC50 of 85 nM, which is 35 and 100 times lower than the receptors that control glutamate efflux and somatostatin, respectively [1].

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As previously reported GABAB receptors are heterogeneous. Three pharmacologically distinct receptor subtypes mediating inhibition of gamma-aminobutyric acid (GABA), glutamate or somatostatin release, respectively, exist on axon terminals of rat cerebral cortex. We investigated the novel GABAB receptor antagonist, [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxy-methyl) phosphinic acid (CGP52432), on the above receptor subtypes. The effects of (-)-baclofen on the K(+)-evoked release of GABA, glutamate or somatostatin from rat cortical synaptosomes were antagonized by CGP52432. The IC50 of the drug at GABA autoreceptors (0.085 microM) was 35- and 100-fold lower than at the receptors regulating somatostatin and glutamate overflow, respectively. At the autoreceptor the calculated pA2 for CGP52432 amounted to 7.70, which makes the drug about 1000-fold more potent than phaclofen at this receptor. The potency and selectivity characteristics of CGP52432 indicate that the drug is by far the most appropriate tool to investigate the terminal GABAB autoreceptors of the rat cerebral cortex [1].

In mice in the elevated plus maze, CGP52432 (10, 30 mg/kg) had no influence on total head tilt or total arm entry [2]. In rats, GABA's renal protection is eliminated when CGP52432 (100 nmol/kg, i.v. or 1 nmol/kg, i.v.) eliminates the inhibitory action of GABA (50 μmol/kg, i.v.) on augmented renal sympathetic nerve activity (RSNA) during ischemia [3].

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To this end, we treated male BALB/c mouse pups with the either the selective GABAB receptor agonist, R-baclofen (2 mg/kg, s.c), the GABAB receptor antagonist CGP52432 (10 mg/kg and 30 mg/kg) or vehicle from postnatal days (P) 14-28. The anxiety behaviour of these mice was then assessed in adulthood (P62 onwards) in a battery of behavioural tests comprising; the stress induced hyperthermia (SIH) test, defensive marble burying (DMB), elevated-plus maze (EPM) and the forced swim test (FST). Postnatal R-baclofen treatment resulted in increased anxiety-like behaviour in the EPM as shown by approach-avoidance and ethological measures. Other behavioural measures were not significantly altered. Interestingly, blockade of GABAB receptors with CGP52432 in early life caused no alterations in emotional behaviour. These data suggest that during early life GABAB receptor signalling can play a functional role in programing anxiety behaviour in adulthood. The underlying neurodevelopmental processes underlying these effects remain to be discovered.[2]

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Effects of i.v. bicuculline or CGP52432 on GABA-induced improvements in ischaemic AKI [3]
Pre-ischaemic treatment of GABA (50 μmol/kg, i.v.) markedly suppressed the enhanced RSNA during the ischaemic period (Fig. 1a,b,d). This suppressive effect was inhibited by the selective GABAB receptor antagonist CGP52432 (10 and 100 nmol/kg, i.v.) in a dose-dependent manner (Fig. 1c,d). Conversely, treatment with the selective GABAA receptor antagonist bicuculline (1 and 10 μmol/kg, i.v.) failed to attenuate the suppressive effect of GABA on RSNA (Fig. 1d).

As shown in Fig. 2, the renal function of rats subjected to 45 min ischaemia showed marked deterioration when measured 29 h after reperfusion. Compared with sham-operated rats, vehicle-treated AKI rats showed significant increases in blood urea nitrogen (BUN), plasma creatinine (PCr) concentrations and urine flow (UF) and a significant decrease in creatinine clearance (CCr), indicating renal dysfunction. Intravenous injection of GABA (50 μmol/kg) to ischaemic AKI rats markedly attenuated the I/R-induced renal dysfunction and this improvement was reversed by 100 nmol/kg, i.v., CGP52432. However, the renoprotective effects of GABA were not affected by bicuculline (1 and 10 μmol/kg) or 10 nmol/kg CGP52432. In addition, we confirmed that 10 μmol/kg, i.v., bicuculline alone had no effect on I/R-induced renal injury (data not shown).

Histological examination revealed severe lesions in the kidney of vehicle-treated AKI rats 29 h after reperfusion. These changes were characterized by proteinaceous casts in the tubules of the inner medulla (Fig. 3b), medullary congestion and haemorrhage in the outer zone of the inner medullary stripe (Fig. 3g) and tubular necrosis in the outer zone of the outer medullary stripe (Fig. 3l) compared with kidneys from sham-operated rats (Fig. 3a,f,k). Intravenous injection of GABA to ischaemic AKI rats significantly attenuated the development of all lesions (Table 1; Fig. 3c,h,m). In addition, 100 nmol/kg CGP52432 (Fig. 3e,j,o) abolished these GABA-induced improvements, whereas 10 μmol/kg, i.v., bicuculline had no effect on the effects of GABA (Fig. 3d,i,n).

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Effects of i.c.v. CGP52432 on GABA-induced improvements in ischaemic AKI [3]
As shown in Fig. 4, the suppressive effect of GABA (50 μmol/kg) on RSNA was partially attenuated by 0.1 nmol/kg, i.c.v., CGP52432, whereas 1 nmol/kg, i.c.v., CGP52432 almost abolished the effects of GABA. Similarly, the GABA-induced improvements in renal dysfunction were partially attenuated by 0.1 nmol/kg, i.c.v., CGP52432, but were almost abolished by 1 nmol/kg, i.c.v., CGP52432 (Fig. 5). Bicuculline (10 nmol/kg, i.c.v.) failed to affect the GABA-induced renoprotective effects (data not shown).

Treatment with the GABAB receptor antagonist CGP52432 (100 nmol/kg, i.v., or 1 nmol/kg, i.c.v.) abolished the suppressive effects of 50 μmol/kg, i.v., GABA on enhanced renal sympathetic nerve activity (RSNA) during ischaemia, leading to elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with 0.5 μmol/kg GABA or i.v. treatment with 1 μmol/kg baclofen, a selective GABAB receptor agonist, prevented the I/R-induced renal injury equivalent to i.v. treatment with GABA. Conversely, i.v. treatment with 10 μmol/kg bicuculline, a GABAA receptor antagonist, failed to affect the preventive effects of GABA against ischaemic AKI. We therefore concluded that GABAB receptor stimulation in the central nervous system, rather than peripheral GABAB receptor stimulation, mediates the preventive effect of GABA against ischaemic AKI by suppressing the enhanced RSNA induced by renal ischaemia [3].

Two separate cohorts of male pups were used for the study. One cohort was treated with either R(+)baclofen HCl (2 mg/kg; Sigma; n = 10) or with vehicle (phosphate buffered saline, PBS; n = 13). The second cohort was treated with the GABAB receptor antagonist CGP52432 (10, 30 mg/kg; n = 10) or vehicle (PBS; n = 10). Drugs were freshly prepared for injection each day, by dissolution in PBS with vortexing and brief sonication. Doses of R(+)baclofen and CGP52432 were chosen based on those previously shown to be well tolerated in adult mice (Colombo et al., 2001, Voigt et al., 2011). All drug treatments were given via subcutaneous injection, once daily from P14-28 in a volume of 0.05 ml. This treatment regime was chosen as P14-28 has been demonstrated to be a period of vulnerability to the developmental effects of drugs acting on the GABAergic system [2].

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Drugs or vehicle, except bicuculline and CGP52432, were injected 5 min before the start of ischaemia, whereas bicuculline and CGP52432 were administered 10 min before ischaemia to examine the effects of these drugs on GABA-induced renal protection. In sham-operated control rats, the left kidney was treated as above, but without clamping. Rats exposed to 45 min ischaemia were housed in metabolic cages for 24 h after reperfusion and 5 h urine samples were collected. At the end of urine collection, blood samples were drawn from the thoracic aorta and then the left kidney was excised under pentobarbital anaesthesia (50 mg/kg, i.p.). Plasma was isolated from the blood by centrifugation (1630 g, 15 min, 4°C) and used to measure renal function, as described below, whereas the kidneys were examined by light microscopy for histological analysis. CGP52432 was dissolved in saline (0.9%). [3]

[1]. CGP 52432: a novel potent and selective GABAB autoreceptor antagonist in rat cerebral cortex. Eur J Pharmacol. 1993 Jun 24;237(2-3):191-5.

[2]. Activation but not blockade of GABAB receptors during early-life alters anxiety in adulthood in BALB/c mice. Neuropharmacology. 2014 Jun;81:303-10.

[3]. Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion-induced renal injury in rats. Clin Exp Pharmacol Physiol. 2015 Mar;42(3):278-86.

3-[(3,4-dichlorophenyl)methylamino]propyl-(diethoxymethyl)phosphinic acid is a dichlorobenzene.

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Several mechanisms have been proposed for the effects of GABA in suppressing the peripheral sympathetic nervous system, including ganglionic blockade and/or inhibition of transmitter release from the nerve terminals.9, 23, 24 In the present study, we did not examine whether GABA suppressed NA overflow from peripheral sympathetic nerves because we did not use isolated tissues. Indeed, in the peripheral sympathetic nerves of AKI rats, systemically applied GABA may prevent I/R-induced renal injury by inhibiting NA release from nerve terminals, even after abrogating the GABA effect using CGP52432 However, the present study revealed that systemically applied GABA failed to prevent I/R-induced renal injury in rats administered i.c.v. CGP52432. These findings suggest that the renoprotective effect of GABA appears to be much more dependent on CNS neurotransmission than that mediated through peripheral sympathetic nerves.
In conclusion, GABA suppressed the enhanced RSNA during ischaemia and increased NA overflow after I/R through activation of GABAB, but not GABAA, receptors and this effect was particularly focused on CNS activity rather than on peripheral nerve activity in the sympathetic nervous system. These inhibitory effects are presumably responsible for renoprotection against I/R-induced renal injury.[3]

C15H24CL2NO4P

384.2351

383.082

C, 46.89; H, 6.30; Cl, 18.45; N, 3.65; O, 16.66; P, 8.06

139667-74-6

139667-74-6

132252

White to off-white solid powder

1.258g/cm3

544.4ºC at 760mmHg

283.1ºC

1.1E-12mmHg at 25°C

1.524

4.491

2

5

11

23

369

0

CCOC(OCC)P(=O)(CCCNCC1=CC(=C(C=C1)Cl)Cl)O

GJZVQXWEIYRHBE-UHFFFAOYSA-N

InChI=1S/C15H24Cl2NO4P/c1-3-21-15(22-4-2)23(19,20)9-5-8-18-11-12-6-7-13(16)14(17)10-12/h6-7,10,15,18H,3-5,8-9,11H2,1-2H3,(H,19,20)

Phosphinic acid, (3-(((3,4-dichlorophenyl)methyl)amino)propyl)(diethoxymethyl)-

Cgp 52432; Cgp52432;Cgp 52,432; Cgp-52,432; Phosphinic acid, P-[3-[[(3,4-dichlorophenyl)methyl]amino]propyl]-P-(diethoxymethyl)-; 4ZH667RFW5; DTXSID20161147; Phosphinic acid, (3-(((3,4-dichlorophenyl)methyl)amino)propyl)(diethoxymethyl)-; (3-(((3,4-Dichlorophenyl)methyl)amino)propyl)(diethoxymethyl) phosphinic acid; ...; 139667-74-6; Cgp-52432.

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 : ~5 mg/mL (~13.01 mM)
H2O : ~4 mg/mL (~10.41 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.)