hGAT-1 (IC50 = 0.26 μM); rGAT-1 (IC50 = 1.2 μM); rGAT-2 (IC50 = 297 μM); hGAT-3 (IC50 = 333 μM); rGAT-3 (IC50 = 1140 μM); hBGT-3 (IC50 = 300 μM)

The mechanism of action of CI-966 hydrochloride is to specifically block GABA reuptake in neurons and glial cells[4].

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gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The synaptic action of GABA is terminated by rapid uptake into presynaptic terminals and surrounding glial cells. Molecular cloning has revealed the existence of four distinct GABA transporters termed GAT-1, GAT-2, GAT-3, and BGT-1. Pharmacological inhibition of transport provides a mechanism for increasing GABA-ergic transmission, which may be useful in the treatment of various neuropsychiatric disorders. Recently, a number of lipophilic GABA transport inhibitors have been designed and synthesized, which are capable of crossing the blood brain barrier, and which display anticonvulsive activity. We have now determined the potency of four of these compounds, SK&F 89976-A (N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid), tiagabine ((R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3- piperidencarboxylic acid), CI-966 ([1-[2-[bis 4-(trifluoromethyl)phenyl]methoxy]ethyl]-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid), and NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride), at each of the four cloned GABA transporters, and find them to be highly selective for GAT-1. These data suggest that the anticonvulsant activity of these compounds is mediated via inhibition of uptake by GAT-1 [1].

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Molecular biology has revealed the presence of four high-affinity GABA transporters in the brain, GAT-1, GAT-2, GAT-3, and BGT-1, the latter transporting both GABA and the osmolyte Betaine. We have shown that known GABA uptake inhibitors such as SK&F 89976-A, CI-966, and Tiagabine exhibit high affinity and selectivity for GAT-1 [3].

When given to PTZ-trained rats, CI-966 hydrochloride causes intermediate levels of Pentylenetetrazol (PTZ)-lever responding[4]. Gamma-aminobutyric acid activity in the CA1 pyramidal layer is enhanced in situ by CI-966 hydrochloride. Under urethane anesthesia, CI-966 hydrochloride is given systemically to Sprague-Dawley rats via intraperitoneal injection (5 mg/kg). A very varied but often considerable augmentation of the suppression of hippocampal population spikes by GABA administered by microiontophoresis in the CA1 region occurs twenty to thirty minutes after injection[5]. In a number of animal models, CI-966 hydrochloride demonstrates anticonvulsant characteristics. When fed 1.39 mg/kg, dogs absorb CI-966 hydrochloride orally with a tmax of 0.7 hours. Rats administered oral 5 mg/kg exhibit a mean tmax of 4.0 hours. After intravenous administration of identical dosages, the average elimination t1/2 for rats and dogs is 4.5 hours and 1.2 hours, respectively. In both species, CI-966 hydrochloride has 100% oral bioavailability[6].

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A new potent, blood-brain barrier permeable gamma-aminobutyric acid (GABA) uptake blocker, 1-[2-[bis[4-(trifluoromethyl)-phenyl]methoxy]ethyl]-1,2,5,6- tetrahydro-3-pyridinecarboxylic acid (CI-966) was administered systemically by i.p. injection (5 mg/kg) in Sprague-Dawley rats under urethane anaesthesia. Twenty to thirty minutes after injection there was a highly variable, but overall significant, enhancement of the inhibition of hippocampal population spikes by GABA applied by microiontophoresis in the CA1 region. Like the effect of nipecotic acid (applied locally by iontophoresis), the potentiation by CI-966 was clearest when GABA was applied in or near the stratum pyramidale where its action normally is weakest and shows the most pronounced fading. This change in GABA potency is most simply explained by a reduction in GABA uptake.[5]

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CI-966 exhibits anticonvulsant properties in various animal models. The drug acts by inhibiting synaptic uptake of gamma-aminobutyric acid (GABA). Oral absorption of CI-966 in dogs given 1.39 mg/kg is rapid with a tmax of 0.7 hr. In rats given 5 mg/kg oral, a mean tmax of 4.0 hr was observed. Following iv administration of the same respective doses, elimination t1/2 in dogs and rats averaged 1.2 and 4.5 hr. Absolute oral bioavailability of CI-966 was 100% in both species. Following oral dosing of [14C]CI-966 HCl to dogs, fecal, and urinary excretion accounted for 89% and 2.3% of the 14C dose, respectively. In bile-duct cannulated rats, biliary excretion is the major elimination pathway of radioactivity (75%). Urinary and fecal excretion accounted for 4.1 and 12%, respectively. CI-966 does not induce or inhibit mouse hepatic mixed function oxidases, as determined by hexobarbital sleeping time[6].

Cell Lines. [3]
In the present study we employed rat GAT-2(rGAT-2)6 and the human homologs of GAT-1 (hGAT-1, whichwe have recloned),20 GAT-3 (hGAT-3),9 and BGT-1 (hBGT-1). Stable cell lines for each of these clones were generated inLM(tk") cells using the calcium phosphate method and selec-tion in G-418, as described previously. Cells were grownunder standard conditions (37 °C, 5% COa) in Dulbecco’smodified Eagles’s medium.

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Transport Assay. [3]
GABA transport was measured asdescribed previously,6 with the following modifications. Cells grown in 24-well plates (well diameter 18 mm) were washedthree times with HEPES-buffered saline (HBS, in mM: NaCl,150; HEPEs, 20; CaCla, 1; glucose, 10; KC1, 5; MgCla, 1; pH7.4) and allowed to equilibrate on a 37 °C slide warmer. After10 min the medium was removed, and unlabeled drugs in HBSwere added (450 /¿L/well). Transport was initiated by adding50 pL per well of a concentrated solution of [3H]GABAin HBS(final concentration = 50 nM). Nonspecific uptake was definedin parallel wells with 1 unlabeled GABA and was subtractedfrom total uptake (no competitor) to yield specific uptake; alldata represent specific uptake. Plates were incubated at 37°C for 10 min and then washed rapidly three times with ice-cold HBS, using a 24-position plate washer. Cells were solubilized with 0.05%sodium deoxycholate/0.1 N NaOH (0.25 mL/well), an aliquotneutralized with 1 N HC1, and radioactivity was determinedby scintillation counting. Protein was quantified in an aliquotof the solubilized cells using a BIO-RAD protein assay kit,according to the manufacturer’s directions.Lipophilic inhibitors were dissolved in DMSO. The finalconcentration of DMSO in the transport assay was <2%, andcontrol experiments demonstrated that this concentration hadno significant effect on transport.

Animal/Disease Models: Eight male SD (Sprague-Dawley) rats[4]
Doses: 0.3-30 mg/kg
Route of Administration: Injection IP in a volume of 1 mL/kg
Experimental Results: Dose dependent decreases in rates of responding occurred following CI-966 administration.

In dogs, 1.39 mg/kg of CI-966 was rapidly absorbed after oral administration, with a time to peak concentration (tmax) of 0.7 hours. In rats, 5 mg/kg of CI-966 resulted in a mean tmax of 4.0 hours. After intravenous administration of the same dose, the mean elimination half-life (t1/2) in dogs and rats was 1.2 hours and 4.5 hours, respectively. The absolute oral bioavailability of CI-966 was 100% in both animals. In dogs, fecal and urinary excretion of [14C]CI-966 HCl accounted for 89% and 2.3% of the 14C dose, respectively. In cannulated rats, bile excretion was the main route of radioactive material clearance (75%). Urinary and fecal excretion accounted for 4.1% and 12%, respectively. CI-966 did not induce or inhibit mixed-function oxidases in mouse liver according to hexobarbital sleep time assays. [6]

198692 Rats Oral LD50 894 mg/kg Sensory organs and special senses: tearing; eyes; behavior: coma; gastrointestinal tract: hypermotility, diarrhea Drug development research, 28(65), 1993
198692 Mice Oral LD50 703 mg/kg Behavior: seizures or effects on epilepsy threshold; gastrointestinal tract: hypermotility, diarrhea; kidneys, ureters and bladder: other changes Drug development research, 28(65), 1993

[1]. Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1. Eur J Pharmacol. 1994 Oct 14;269(2):219-24.

[2]. GABA potentiation: a logical pharmacological approach for the treatment of acute ischaemic stroke. Neuropharmacology. 2000 Jul 10;39(9):1483-94.

[3]. Design, synthesis and evaluation of substituted triarylnipecotic acid derivatives as GABA uptake inhibitors: identification of a ligand with moderate affinity and selectivity for the cloned human GABA transporter GAT-3. J Med Chem (IF: 7.

[4]. Discriminative stimulus effects of presynaptic GABA agonists in pentobarbital-trained rats. Pharmacol Biochem Behav.1994 Jan;47(1):5-11.

[5]. Systemic CI-966, a new gamma-aminobutyric acid uptake blocker, enhances gamma-aminobutyric acid action in CA1 pyramidal layer in situ. Can J Physiol Pharmacol. 1990 Sep;68(9):1194-9.

[6]. Pharmacokinetics, mass balance, and induction potential of a novel GABA uptake inhibitor, CI-966 HCl, in laboratory animals. Pharm Res. 1993 Oct;10(10):1442-5.

Studies have shown that enhancing the function of the major inhibitory neurotransmitter GABA can reduce glutamatergic activity in the brain. Since increased glutamatergic activity is a primary event leading to cell death after acute hypoxic-ischemic stroke, GABA mimics may have neuroprotective effects. This review explores the evidence for acute inhibition of GABAergic function following ischemic injury and reviews data suggesting that increasing brain GABA concentration has neuroprotective effects, as well as the neuroprotective effects of some (but not all) GABA mimics. The GABA uptake inhibitor CI-966, the GABA(A) receptor agonist muscarinic acid, and the GABA(A) receptor mimic chlorothiazide have all been shown to have neuroprotective effects in stroke animal models following ischemic injury. In contrast, benzodiazepines, especially barbiturates, while potent GABA(A) receptor enhancers, have shown less effective neuroprotective effects. The diversity of GABA(A) receptor subtypes and the in vivo efficacy of certain GABA(A) receptor ligands in animal stroke models suggest that GABA mimicry is an underestimated approach in stroke treatment. [2] Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. Molecular biological studies have revealed the presence of four high-affinity GABA transporters in the brain: GAT-1, GAT-2, GAT-3, and BGT-1, of which BGT-1 can simultaneously transport GABA and the osmotic regulator betaine. We have demonstrated that known GABA uptake inhibitors, such as SK&F 89976-A, CI-966, and tiagabin, have high affinity and selectivity for GAT-1. This article describes the design and synthesis of a series of novel triarylpiperidic acid derivatives and evaluates their activity as GABA uptake inhibitors. The lead compound for this series of compounds was the non-selective GABA uptake inhibitor EGYT-3886, [(-)-2-phenyl-2-[(dimethylamino)ethoxy]-(1R)-1,7,7-trimethylbicyclo[2.2.1]heptane]. From this series of compounds, we identified (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic acid, and compound 4(S) is a novel ligand selective for GAT-3. The IC50 of compound 4(S) was 5 μM for GAT-3, 21 μM for GAT-2, >200 μM for GAT-1, and 140 μM for BGT-1. This compound will become an important tool for evaluating the role of GAT-3 in neural function. [3]

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The discriminative stimuli effects of indirect-acting GABAergic drugs, pentobarbital (PB), and midazolam were compared in rats trained to distinguish between 5 mg/kg pentobarbital (PB) and saline. A two-bar fixed-ratio 32 food fortification program was used. Both PB and midazolam dose-dependently replaced the training dose of PB, only decreasing the response rate at doses higher than the complete replacement dose. Valproic acid (an antiepileptic drug and GABA transaminase inhibitor) also replaced PB, but only at doses that inhibited the response rate. Vegabatrin, an irreversible GABA transaminase inhibitor, failed to replace phenobarbital, but did produce a dose-dependent decrease in the response rate. GABA uptake inhibitors, such as 1-[2-[bis[4-(trifluoromethyl)phenyl]-methoxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (CI-966) and (R(-)-N-[4,4-bis(3-methylthiophene-2-yl)but-3-enyl]nipoise (thigaben), produce PB lever responses of less than 40%. Aminooxyacetic acid (AOAA), a non-selective presynaptic GABA agonist, exhibits PB lever responses as high as 43%. These results indicate that while the discriminative stimulatory effects of the indirect GABAA agonists PB and midazolam are similar, they differ from those of presynaptic GABAergic drugs. Discriminative stimulatory characteristics also differ between GABA transaminase inhibitors and uptake inhibitors. This suggests that not all presynaptic GABA agonists possess similar behavioral characteristics. These results contribute to a deeper understanding of GABA enhancement. The similarities and differences in behavioral effects of drugs that can transmit neurotransmitters. [4]

C23H22CLF6NO3

509.87

509.119

C, 54.18; H, 4.35; Cl, 6.95; F, 22.36; N, 2.75; O, 9.41

110283-66-4

CI-966;110283-79-9

198692

White to off-white solid powder

514.1ºC at 760mmHg

264.7ºC

2.16E-11mmHg at 25°C

6.286

2

10

7

34

643

0

C1CN(CC(=C1)C(=O)O)CCOC(C2=CC=C(C=C2)C(F)(F)F)C3=CC=C(C=C3)C(F)(F)F.Cl

NUQWSOWKRTZJTO-UHFFFAOYSA-N

InChI=1S/C23H21F6NO3.ClH/c24-22(25,26)18-7-3-15(4-8-18)20(16-5-9-19(10-6-16)23(27,28)29)33-13-12-30-11-1-2-17(14-30)21(31)32;/h2-10,20H,1,11-14H2,(H,31,32);1H

1-[2-[bis[4-(trifluoromethyl)phenyl]methoxy]ethyl]-3,6-dihydro-2H-pyridine-5-carboxylic acid;hydrochloride

1-(2-(bis(4-(trifluoromethyl)phenyl)methoxy)ethyl)-1,2,5,6-tetrahydropyridine-3-carboxylic acid hydrochloride; 1-[2-[BIS[4-(TRIFLUOROMETHYL)PHENYL]METHOXY]ETHYL]-1,2,5,6-TETRAHYDROPYRIDINE-3-CARBOXYLIC ACID HYDROCHLORIDE; 1-{2-[Bis(4-(trifluoromethyl)phenyl)methoxy]ethyl}-1,2,5,6-tetrahydropyridine-3-carboxylic acid hydrochloride; 692-461-5; 110283-66-4; CI 966 hydrochloride; 3-Pyridinecarboxylic acid, 1,2,5,6-tetrahydro-1-(2-(bis(4-(trifluoromethyl)phenyl)methoxy)ethyl)-, hydrochloride; 3-Pyridinecarboxylicacid, 1-[2-[bis[4-(trifluoromethyl)phenyl]methoxy]ethyl]-1,2,5,6-tetrahydro-,hydrochloride (1:1);

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: 50 mg/mL (98.06 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.90 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.90 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.90 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.

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