β-GABA transporter (BGT-1) (IC50 = 10.6 μM)

NNC05-2090 hydrochloride's IC50 values for binding to spiperone and prazosin are 1632 nM and 266 nM, respectively[1]. With an IC50 value of 4.4 μM, NNC 05-2090 hydrochloride (0.1-100 μM) inhibits the uptake of [3H]GABA in rat cortical synaptosomes. With an IC50 value of 2.5 μM, NNC05-2090 hydrochloride (0.1-100 μM) inhibits [3H]GABA absorption in inferior colliculus synaptosomes [1]. Serotonin, norepinephrine, dopamine transporter, and BGT-1 are all inhibited by NNC05-2090 hydrochloride, with IC50 values of 5.29, 7.91, 4.08, and 10.6 μM, respectively[1]. GAT-1, GAT-2, and GAT-3 are inhibited by NNC05-2090 hydrochloride, with IC50 values of 29.62, 45.29, and 22.51 μM, respectively[1].

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Comparison of the inhibitory potency of GAT inhibitors To analyze the effects of GAT inhibitors on the uptake of [3 H]GABA, we previously established cell lines stably expressing GAT subtypes using CHO cells. Using these cell lines together with CHO cells stably expressing rat monoamine transporters SERT, NET, and DAT to compare the potencies in inhibiting GAT subtypes, we analyzed the effect of GAT inhibitors on the uptake of GABA and monoamines. The inhibitors SKF89976A and (S)-SNAP5114 exhibited subtype selectivity for GAT-1 (IC50: 0.28 mM) and GAT-3 (IC50: 5.31 mM), respectively (Table 1). However, NNC 05-2090 potently inhibited GAT-1 (IC50: 29.62 mM), with IC50 values more than 2.5-fold lower being observed for BGT-1 (10.60 mM). NNC05-2090 also showed markedly higher IC50 values for GAT-2 (45.29 mM) and GAT-3 (22.51 mM). These results indicate that of all the GAT inhibitors examined, NNC 05-2090 has highest potency inhibiting BGT-1 between GAT inhibitors. NNC05-2090 also inhibited SERT, NET, and DAT (IC50: 5.29 mM, 7.91 mM, and 4.08 mM, respectively), and these IC50 values were similar to that for BGT-1 [2].

NNC 05-2090 hydrochloride (ip) protects DBA/2 mice against tonic and clonic convulsions with an ED50 of 19 μmol/kg and 26 μmol/kg, respectively, and from maximum electric shock (MES) with an ED50 of 73 μmol/kg[1]. In sciatic nerve ligation mice, NNC05-2090 hydrochloride (0.01, 0.1, 0.3 mg/kg; intraperitoneal injection or intrathecal injection, once) can correct some mechanical allodynia.

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Two novel nipecotic acid derivatives, 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(4-methoxyphenyl)-4-piperidino l (NNC 05-2045) and 1-(3-(9H-Carbazol-9-yl)-l-propyl)-4-(2-methoxyphenyl)-4-piperidino l (NNC 05-2090) have been tested for inhibition of gamma-amino butyric acid (GABA) transporters in synaptosomal preparations of rat cerebral cortex and inferior colliculus and found to differ markedly from gabitril (tiagabine), a selective GAT-1 inhibitor. IC50 values for inhibition of [3H]GABA uptake into synaptosomes from cerebral cortex for NNC 05-2045 and NNC 05-2090 were 12 +/- 2 and 4.4 +/- 0.8 microM, respectively. In synaptosomes from inferior colliculus in the presence of 1 microM 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid (NNC 05-0711), a highly potent and selective GAT-1 inhibitor, IC50 values for inhibition of [3H]GABA uptake were 1.0 +/- 0.1 and 2.5 +/- 0.7 microM, respectively. A receptor profile showed that NNC 05-2045 has binding affinities to sigma-, alpha 1- and D2-receptors of 113, 550 and 122 nM, respectively. NNC 05-2090 displayed alpha 1- and D2-receptor affinity of 266 and 1632 nM, respectively. The anticonvulsant action of both compounds was tested in four rodent models after intra peritoneal (i.p.) injection. Both NNC 05-2090 dose-dependently inhibited sound-induced tonic and clonic convulsions in DBA/2 mice with ED50 values of 6 and 19 mumol/kg, respectively. NNC 05-2045 also antagonized sound-induced seizures in genetic epilepsy prone rats (GEP rats) with ED50 values against wild running, clonic and tonic convulsions of 33, 39 and 39 mumol/kg, respectively (NNC 05-2090 was not tested in GEP rats). Both NNC 05-2045 and NNC 05-2090 dose-dependently antagonized tonic hindlimb extension in the maximal electroshock (MES) test with ED50 values of 29 and 73 mumol/kg, respectively. In amygdala kindled rats NNC 05-2045 and NNC 05-2090 significantly (P < 0.05) reduced generalized seizure severity (seizure grade 3-5) at highest doses (72-242 mumol/kg) and NNC 05-2090 also significantly reduced afterdischarge duration at these doses (P < 0.05). These data show that inhibition of GABA uptake through non-GAT-1 transporters has different anticonvulsant effects than selective GAT-1 inhibitors (e.g. tiagabine) in that enhanced efficacy against MES and reduced efficacy against kindled seizures is observed. Although a contribution of adrenergic agonistic effects cannot be entirely ruled out, it is proposed that inhibition of GAT-3 (mouse GAT4) is primarily responsible for the anticonvulsant action of these two nipecotic acid derivatives in MES, amygdala kindled rats and in sound-induced seizures in GEP-rats and DBA/2 mice.[1]

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Effects of GAT inhibitors on mechanical allodynia in a mouse model of PSL The mechanical withdrawal threshold was significantly lower on the ipsilateral side in partial sciatic nerve ligation (PSL) model mice than in sham-operated animals (Fig. 1). A reduction was not observed in the mechanical withdrawal threshold on the contralateral side (Figs. 2B, 3B, 4, and 5). Mechanical allodynia in the mouse model of PSL was not affected by the saline treatments (Fig. 1B). An increase was observed in the mechanical withdrawal threshold in PSL model mice 3 h after the administration i.p. of 0.1 mg/kg NNC 05-2090 (Fig. 1B). On the other hand, a 0.3 mg/kg, i.p. injection of SKF89976A did not induce any significant effect on the withdrawal threshold in PSL model mice (Fig. 1B). Intravenous (i.v.) injection of the BGT-1 inhibitor NNC05-2090 significantly reversed mechanical allodynia in PSL model mice (Fig. 2). A dose-dependent response to NNC05-2090 was shown in the range of 0.01 – 0.1 mg/kg by an i.v. injection. i.t. injection of the BGT-1 inhibitor NNC 05-2090 also significantly reversed mechanical allodynia in PSL model mice, and a dosedependent response to NNC 05-2090 was also shown in the range of 15 – 150 pmoles by an i.t. injection to PSL model mice (Fig. 3). Antiallodynic effects peaked within 1 h after the injection of NNC05-2090, with the exception of the 0.01 mg/kg, i.v. administration (Figs. 2 and 3). No significant difference was observed in the withdrawal threshold on the contralateral side after the administration of NNC05-2090. SKF89976A produced a weak antiallodynic response when administered i.v. (0.3 mg/kg) (Fig. 4A). As is shown in Fig. 5A, the i.t. injection of SKF89976A dose-dependently ameliorated the reduction in the withdrawal threshold in PSL model mice. We also injected the GAT-3 inhibitor (S)-SNAP5114 to examine its antiallodynic effect on mechanical allodynia in PSL mice. Neither the i.v. nor i.t. injection of (S)-SNAP5114 had a significant effect on the reduction in the withdrawal threshold in PSL model mice (Fig. 4B and 5B) [2].

Cell culture and expression [2]
Chinese hamster ovary (CHO) cells were cultured at 37°C under 5% CO2 / 95% air in Minimum Essential Medium-alpha (a-MEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 mg/ml streptomycin, and 0.25 mg/ml fungisone. Cell lines stably expressing the transporters were designated as CHO/ mouse GAT-1, CHO/mouse GAT-2, CHO/mouse GAT-3, CHO/mouse BGT-1, CHO/rat noradrenaline transporter (NET), CHO/rat serotonin transporter (SERT), and CHO/rat dopamine transporter (DAT), respectively, and had been cloned previously (35 – 38). Cells were diluted in the culture medium, plated in 48-well culture plates, and cultured for 24 h.

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Uptake assay [2]
Cells were washed 3 times with oxygenated Krebs Ringer HEPES-buffered solution (KRH; 125 mM NaCl, 5.2 mM KCl, 1.2 mM CaCl2, 1.4 mM MgSO4, 1.2 mM KH2PO4, 5 mM glucose, and 20 mM HEPES, pH 7.3) and incubated for 10 min at 37°C with 10 nM [3 H]GABA or other radiolabeled ligands, as described previously. Aminooxyacetic acid (100 mM) and ascorbic acid (100 mM) plus pargyline (50 mM) were added to the incubation solution during GABA and monoamine (5-HT, NA, and dopamine) uptake assays, respectively. After the removal of excess radioligands by aspiration, the cells were washed rapidly 3 times with ice-cold KRH, and any radioactivity remaining in the cells was extracted with NaOH (1M) and measured by liquid scintillation spectrometry. Nonspecific uptake was determined in the presence of 10 mM cold GABA for GABA uptake or 10 mM cocaine for each monoamine uptake. The inhibitory potency of GAT inhibitors (IC50 values) was analyzed using Prism 5。

Animal/Disease Models: Partial sciatic nerve ligation (PSL) mice with mechanical allodynia[2]
Doses: 0.01, 0.1 and 0.3 mg/kg
Route of Administration: intraperitoneal (ip) injection or intrathecal injection; 0.1 mg/kg, once
Experimental Results: Dose-dependently reversed mechanical allodynia in PSL model mice by both intraperitoneal (ip) injection and intrathecal injection.

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GEP rats of either sex were kept in groups of 3–6 in cages (53×35×18 cm) placed in a room maintained at 19–22°C with a relative humidity of 55±3% and a light/dark cycle of 14 h/10 h with ad libitum access to food and water. Male Sprague–Dawley rats for amygdala kindling were held individually in cages (42×26×18 cm) at a reversed light/dark cycle of 12 h/12 h, but otherwise as described for GEP rats. Male NMRI mice for MES test and DBA/2 mice of either sex were housed in groups of 40 in cages (59×38×20 cm) at light/dark cycle of 12 h/12 h, but otherwise as described for GEP rats. All animal experiments were performed between 09:00 and 12:00, except for the study in GEP-rats which was performed between 09:00 and 16:00.
Vehicle for NNC 05-2045 and NNC 05-2090 in seizure tests were saline (0.9% w/v NaCl in MiliQ water) with 5% Cremophor® and 10% dimethyl sulfoxide (DMSO), pH adjusted to 7 with NaOH. In NMRI and DBA/2 mice drug or vehicle administration was performed i.p. at 0.5–1 h prior to seizure test. Kindled rats received i.p. drug or vehicle administration 2 h before seizure test.[1]

[1]. Anticonvulsant properties of two GABA uptake inhibitors NNC 05-2045 and NNC 05-2090, not acting preferentially on GAT-1. Epilepsy Res. 1997 Jul;28(1):51-61.

[2]. Antiallodynic action of 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), a betaine/GABA transporter inhibitor. J Pharmacol Sci. 2014;125(2):217-26.

NNC 05-2090 hydrochloride is a GABA uptake inhibitor and inhibitor of the beta-GABA transporter (BGT-1).

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A final comment which deserves mention is the fact that NNC 05-2045 is equipotent to lamotrigine in antagonizing MES and audiogenic induced seizures in DBA/2 mice, although it is less potent than lamotrigine in reducing severity of secondarily generalized seizures in amygdala kindled rats and audiogenic seizures in GEP rats. This suggests that drugs potently acting at non-GAT-1 GABA transporters have a significant potential as anticonvulsant drugs, with an in vivo profile differing from selective GAT-1 inhibitors. Taken together, the data obtained in the present study demonstrate anticonvulsant effects of two GABA uptake inhibitors in several animal models. Although we cannot reject the possibility that the observed anticonvulsant effects occur through a synergism of the GABAergic system with other transmitter systems (e.g. adrenergic), we suggest that the anticonvulsant effects of NNC 05-2045 and NNC 05-2090 are due primarily to an inhibition of GAT-3 mediated GABA transport with a possible minor contribution of inhibition of BGT-1 mediated GABA transport.[1]

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The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC 05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC 05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC50: 5.29, 7.91, and 4.08 μM, respectively. These values were similar to the IC50 value at BGT-1 (10.6 μM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.[2]

C27H31CLN2O2

451.000246286392

450.207

C, 71.91; H, 6.93; Cl, 7.86; N, 6.21; O, 7.09

184845-18-9

NNC 05-2090;184845-43-0

18782760

Light yellow to yellow solid powder

2

3

6

32

554

0

Cl.OC1(C2C=CC=CC=2OC)CCN(CCCN2C3C=CC=CC=3C3=CC=CC=C23)CC1

WDFXPKKFSNMGOO-UHFFFAOYSA-N

InChI=1S/C27H30N2O2.ClH/c1-31-26-14-7-4-11-23(26)27(30)15-19-28(20-16-27)17-8-18-29-24-12-5-2-9-21(24)22-10-3-6-13-25(22)29;/h2-7,9-14,30H,8,15-20H2,1H3;1H

1-(3-carbazol-9-ylpropyl)-4-(2-methoxyphenyl)piperidin-4-ol;hydrochloride

NNC052090; NNC 05-2090; 1-(3-(9H-Carbazol-9-yl)propyl)-4-(2-methoxyphenyl)piperidin-4-ol; 1-[3-(9H-carbazol-9-yl)propyl]-4-(2-methoxyphenyl)piperidin-4-ol; 1-(3-(9H-carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol; CHEMBL1446457; GTPL4610; SCHEMBL4293184;

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)

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.

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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.)