GAT-1 (IC50 = 0.28 μM); GAT-2 (IC50 = 137.34 μM); GAT-3 (IC50 = 202.8 μM)

Weak antiallodynic effects are shown with SKF89976A. SKF89976A mildly inhibits the norepinephrine transporter (NET), dopamine transporter (DAT), and serotonin transporter (SERT) in Chinese hamster ovary (CHO) cells that are stable transducers of each transporter, as determined by a substrate uptake experiment. 3514, 202.13, and 728.8 are the IC50 values, respectively [1]. A GABA transmission blocker is SKF89976A. The inward current generated by GABA (1 mM) was not impacted by 100 μM picrotoxin, but it was totally suppressed by the GABA transport inhibitors tiagabine (10 μM) and SKF89976A (100 μM). It is known that 100 μM SKF 89976-A fully and reversibly blocks GABA-induced currents via blocking GABA transport into cells [2]. GAT-1 is blocked non-transferably by SKF89976A. Additionally, baseline inward currents that may result from background GABA activation of tonic GAT are inhibited by SKF89976-A. In every cell under investigation, SKF89976A (100 μM) reversibly decreased GAT current by 67.9±4.4% (n= 19). GABA-induced GAT currents were gradually decreased and inhibited by intracellular infusion of 20 μM SKF89976-A, whereas GABAAR-mediated currents (n=4) were not prevented [3].

---

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, NNC05-2090 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, NNC05-2090 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 [1].

---

Although glial GABA uptake and release have been studied in vitro, GABA transporters (GATs) have not been characterized in glia in slices. Whole cell patch-clamp recordings were obtained from Bergmann glia in rat cerebellar slices to characterize carrier-mediated GABA influx and efflux. GABA induced inward currents at -70 mV that could be pharmacologically separated into GABA(A) receptor and GAT currents. In the presence of GABA(A/B/C) receptor blockers, mean GABA-induced currents measured -48 pA at -70 mV, were inwardly rectifying between -70 and +50 mV, were inhibited by external Na(+) removal, and were diminished by reduction of external Cl(-). Nontransportable blockers of GAT-1 (SKF89976A and NNC-711) and a transportable blocker of all the GAT subtypes (nipecotic acid) reversibly reduced GABA-induced transport currents by 68 and 100%, respectively. A blocker of BGT-1 (betaine) had no effect. SKF89976-A and NNC-711 also suppressed baseline inward currents that likely result from tonic GAT activation by background GABA. The substrate agonists, nipecotic acid and beta-alanine but not betaine, induced voltage- and Na(+)-dependent currents. With Na(+) and GABA inside the patch pipette or intracellular GABA perfusion during the recording, SKF89976A blocked baseline outward currents that activated at -60 mV and increased with more depolarized potentials. This carrier-mediated GABA efflux induced a local accumulation of extracellular GABA detected by GABA(A) receptor activation on the recorded cell. Overall, these results indicate that Bergmann glia express GAT-1 that are activated by ambient GABA. In addition, GAT-1 in glia can work in reverse and release sufficient GABA to activate nearby GABA receptors [3].

SKF89976A given intravenously (0.3 mg/kg) elicits a mild antiallodynic effect. In PSL model mice, injection of SKF89976A can improve the decrease of withdrawal threshold in a dose-dependent manner [1].

---

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 NNC05-2090 (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 NNC05-2090 also significantly reversed mechanical allodynia in PSL model mice, and a dosedependent response to NNC05-2090 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) [1].

INTRACELLULAR PERFUSION OF A GABA TRANSPORTER BLOCKER DURING THE RECORDING.[3]
Intracellular perfusion of a GABA transporter blocker was performed as previously reported by others for single or multiple drugs application (Tang et al. 1990). We used a straight pipette holder with a perfusion port (EH-U2, E. W. Wright). Through the perfusion port, a polyethylene tube (0.86 mm ID and 1.27 mm OD) was introduced sufficiently far to reach well into the patch pipette solution. A 1-ml syringe containing the LY-filled intracellular solution to be perfused during the recording was connected to the polyethylene tube via an elongated and thinned plastic pipette tip. Before adding the patch pipette, positive pressure was manually applied to fill up the tube all away to the end, remove air bubbles, and visualize efflux of solution. Then, after applying negative pressure to prevent any solution leakage but without adding an air bubble to the end of the tube, the patch pipette was inserted into the holder. To perfuse the LY-filled solution containing either GABA or SKF89976A, a positive pressure was manually applied to add sufficient solution to double the volume in the patch pipette (about 20 μl). The concentrations of GABA and SKF89976A were double to obtain the intended final concentrations in the cell.

Drug preparation and administration [1]
Animals were used in experiments approximately 2 weeks (11 – 16 days) following nerve ligation. Animals were administered NNC05-2090, SKF89976A, (S)- SNAP5114, or amitriptyline. NNC05-2090, SKF89976A, and (S)-SNAP5114 were dissolved in dimethyl sulfoxide (DMSO) and diluted appropriately with ACSF or saline (the final concentration of DMSO was less than 0.5%). The composition of ACSF (in mM) was 142 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1.25 mM NaH2PO4, 10 mM d-glucose, 10 mM HEPES, and 0.05% fatty acid-free bovine serum albumin (pH 7.4). The intraperitoneal (i.p.) injection of drugs was administered in a volume of 0.1 ml/10 g body weight. When given intravenously (i.v.), solutions were injected into the tail vein in a volume of 0.1 ml/10 g body weight. The head of a mouse was placed into a plastic cap and the body was held with one hand for an intrathecal (i.t.) injection. A 27-gauge needle attached to a Hamilton microsyringe was inserted into the subarachnoid space between the L5 and L6 vertebrae of the conscious mouse and 5 ml of the drug solution was slowly injected, as described by Hylden and Wilcox (31). Accurate placement of the needle was confirmed by a quick “flick” of the mouse’s tail, as described by Honda et al.

[1]. 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.

[2]. Glutamate modulation of GABA transport in retinal horizontal cells of the skate. J Physiol. 2003 Feb 1;546(Pt 3):717-31.

[3]. GAT-1 and reversible GABA transport in Bergmann glia in slices. J Neurophysiol. 2002 Sep;88(3):1407-19.

1-(4,4-Diphenylbut-3-enyl)-3-piperidinic acid is a diarylmethane. The GABAergic system in the spinal cord has been shown to be involved in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In this study, we used a sciatic nerve ligation mouse model to compare betaine/GABA transporter (BGT-1) with other GAT isoforms to determine its role in neuropathic pain. 1-(3-(9H-carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol (NNC05-2090) is a moderately selective inhibitor of BGT-1 that showed anti-hyperalgesic effects in model mice via both intrathecal and intravenous administration. On the other hand, the selective GAT-1 inhibitor SKF89976A showed weak anti-hyperalgesic activity, while the selective GAT-3 inhibitor (S)-SNAP5114 had no anti-hyperalgesic activity. A systematic analysis of GABA uptake by these compounds in CHO cells stably expressing BGT-1 revealed that NNC05-2090 inhibited not only BGT-1 but also serotonin, norepinephrine, and dopamine transporters. Substrate uptake assays in CHO cells stably expressing each transporter showed IC50 values of 5.29, 7.91, and 4.08 μM for NNC05-2090, respectively. These values are similar to the IC50 value of BGT-1 (10.6 μM). These results indicate that the anti-hyperalgesic effect of NNC05-2090 is due to its inhibitory effect on BGT-1 and monoamine transporters. [1] The transport of the amino acid GABA to neurons and glial cells plays a key role in regulating the effects of GABA on the vertebrate retina. We investigated the regulatory effect of glutamate (a potential neurotransmitter in vertebrate photoreceptor cells) on GABA-induced transport currents in retinal level cells. We used whole-cell voltage clamp technology to detect the effects of enzymatically isolated ray lateral level cells. The inward current induced by GABA (1 mM) was completely inhibited by the GABA transport inhibitors tiagabin (10 μM) and SKF89976A (100 μM), but was not affected by 100 μM cucurbitacin. Pre-application of 100 μM glutamate significantly reduced the GABA-induced current. Glutamate inhibited the dose-response curve of GABA, but did not cause a transverse shift in the curve or change the voltage dependence of the current. The ionotropic glutamate receptor agonists fucoidan and AMPA also reduced GABA-induced currents, the effects of which could be blocked by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline. NMDA neither induced nor altered GABA-induced currents, and metabolized glutamate analogs had no effect. The inhibitory effects of glutamate and fucoidan on GABA-induced currents were weakened when extracellular calcium ions were removed or when a high concentration of the calcium chelator BAPTA was added to the recording electrode. Caffeine (5 mM) and carotenoids (2 nM), which are known to alter intracellular calcium ion levels, also reduced GABA-induced currents, but simple depolarization-induced increases in calcium ion concentration did not have this effect. Our data suggest that glutamate regulates GABA transport in retinal cells through a calcium-dependent process, implying a close physical link between calcium-permeable glutamate receptors and GABA transporters in these cells. [2]

---

Although the uptake and release of GABA by glial cells has been studied in vitro, GABA transporters (GATs) have not yet been characterized in glial cells in brain slices. We used whole-cell patch-clamp technique to record the electrophysiological activity of Bergmann glial cells in rat cerebellar slices to characterize carrier-mediated GABA influx and efflux. GABA induces an inward current at -70 mV, which can be pharmacologically separated into GABA(A) receptor current and GAT current. In the presence of GABA(A/B/C) receptor blockers, the mean GABA-induced current at -70 mV was -48 pA, exhibiting inward rectifying characteristics between -70 and +50 mV. This current was inhibited by extracellular Na⁺ removal and attenuated by extracellular Cl⁻ reduction. Nontransporter inhibitors of GAT-1 (SKF89976A and NNC-711) and transporter inhibitors of all GAT isoforms (nipoic acid) reversibly reduced GABA-induced transport currents by 68% and 100%, respectively. BGT-1 inhibitors (betaine) did not have this effect. SKF89976-A and NNC-711 also inhibited baseline inward currents that could be caused by persistent activation of GAT by background GABA. The substrate agonists nipoic acid and β-alanine (but not betaine) induced voltage-dependent and sodium-dependent currents. SKF89976A blocked baseline outward currents activated at -60 mV and enhanced with increasing depolarization potential when sodium ions and GABA were added to the patch-clamp electrode or when intracellular GABA was perfused during recording. This carrier-mediated GABA efflux leads to the local accumulation of extracellular GABA, which can be detected by recording the activation of GABA_A receptors on the cell. Overall, these results indicate that Bergman's glial cells express GAT-1 and that GAT-1 can be activated by surrounding GABA. Furthermore, GAT-1 in glial cells can also have a reverse effect, releasing enough GABA to activate nearby GABA receptors. [3]

C22H25NO2.HCL

371.90034

371.165

C, 71.05; H, 7.05; Cl, 9.53; N, 3.77; O, 8.60

85375-15-1

85375-15-1 ( free base);85375-85-5 (HCl);

92409

Light yellow to khaki solid powder

531.4ºC at 760 mmHg

275.2ºC

5.044

1

3

6

25

427

0

Cl.O=C(C1CCCN(CC/C=C(\C2C=CC=CC=2)/C2C=CC=CC=2)C1)O

TXQKSMSLZVKQBI-UHFFFAOYSA-N

InChI=1S/C22H25NO2/c24-22(25)20-13-7-15-23(17-20)16-8-14-21(18-9-3-1-4-10-18)19-11-5-2-6-12-19/h1-6,9-12,14,20H,7-8,13,15-17H2,(H,24,25)

1-(4,4-diphenylbut-3-enyl)piperidine-3-carboxylic acid

SKF89976A HCl; 85375-85-5; Skf 89,976A; SKF89,976A; 1-(4,4-diphenylbut-3-en-1-yl)piperidine-3-carboxylic acid; N-(4,4-Diphenyl-3-butenyl)nipecotic acid; N-(4,4-Diphenyl-3-butenyl)homo-beta-proline; SKF-89976; CHEMBL38686; SKF 89976-A; SKF-89976-A; SKF89976A hydrochloride

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 : ~100 mg/mL (~268.89 mM)
H2O : ~20 mg/mL (~53.78 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.72 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 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.72 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 25 mg/mL (67.22 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)