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)

In neurons and glial cells, CI-966 specifically inhibits GABA reuptake to produce its effects [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 rats trained in pentylenetetrazol (PTZ) administration, CI-966 causes moderate levels of PTZ lever responses [4]. Gamma-aminobutyric acid's in situ actions in the CA1 pyramidal layer are amplified by CI-966. Sprague-Dawley rats were given systemic administration of CI-966 via intraperitoneal injection (5 mg/kg) while under urethane anesthesia. Micro-iontophoretic GABA had a typically significant, but highly variable, inhibitory effect on the hippocampal group peak in the CA1 area 20 to 30 minutes after injection [5]. Different animal models demonstrate the anticonvulsant effects of CI-966. When given orally to dogs, 1.39 mg/kg CI-966 was absorbed quickly, peaking 0.7 hours later. Rats receiving an oral dose of 5 mg/kg showed a mean tmax of 4.0 hours. In dogs and rats, elimination t1/2 averaged 1.2 and 4.5 hours following intravenous administration of the same dosage. In both species, CI-966 has a 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: 8 male SD (SD (Sprague-Dawley)) rats [4]
Doses: 0.3-30 mg/kg
Route of Administration: intraperitoneal (ip) injection in a volume of 1 mL/kg
Experimental Results: Dose-dependent decrease in response rate after 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.

1-[2-[bis[4-(trifluoromethyl)phenyl]methoxy]ethyl]-3,6-dihydro-2H-pyridine-5-carboxylic acid is a diarylmethane. 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 major primary event leading to cell death after acute hypoxic-ischemic stroke, GABAergic drugs may have neuroprotective effects. This review explores the evidence for acute inhibition of GABAergic function after ischemic injury and reviews data indicating that increasing brain GABA concentration has neuroprotective effects, as well as the neuroprotective effects of some (but not all) GABAergic drugs. 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 exert neuroprotective effects when administered to stroke animal models after ischemic injury. In contrast, benzodiazepines, especially barbiturates, are potent GABA(A) receptor enhancers, but their efficacy as neuroprotective agents is less than satisfactory. The diversity of GABA(A) receptor subtypes and the in vivo efficacy of certain GABA(A) receptor ligands in animal models of stroke suggest that GABA receptor 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 biology has 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 transport both GABA and the osmotic regulator betaine. We have demonstrated that known GABA uptake inhibitors, such as SK&F 89976-A, CI-966, and Tiagabine, have high affinity and selectivity for GAT-1. This paper describes the design and synthesis of a series of novel triarylnipoic acid derivatives and evaluates their activity as GABA uptake inhibitors. The lead compound for this series of compounds is 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, (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic acid, 4(S) was identified as a novel ligand selective for GAT-3. 4(S) has an IC50 of 5 μM on GAT-3, 21 μM on GAT-2, greater than 200 μM on GAT-1, and 140 μM on BGT-1. This compound will become an important tool for evaluating the role of GAT-3 in neurological function. [3]

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In an experiment training rats to distinguish between 5 mg/kg pentobarbital (PB) and saline, the discriminative stimulation effects of indirect-acting GABAergic drugs were compared with those of pentobarbital (PB) and midazolam. The experiment used a double-bar fixed ratio 32 food fortification program. Both PB and midazolam were dose-dependent substitutes for the training dose of PB, and only reduced the response rate at doses higher than the complete substitute dose. Valproic acid (an antiepileptic drug and GABA transaminase inhibitor) was also a substitute for PB, but only at doses that inhibited the response rate. Vigabatrin (an irreversible GABA transaminase inhibitor) was not a substitute for PB, but it did reduce the response rate in a dose-dependent manner. 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

473.143

C, 58.35; H, 4.47; F, 24.08; N, 2.96; O, 10.14

110283-79-9

CI-966 hydrochloride;110283-66-4

198693

Typically exists as solid at room temperature

1.34g/cm3

514.1ºC at 760mmHg

264.7ºC

2.16E-11mmHg at 25°C

1.517

5.484

1

10

7

33

643

0

FC(F)(F)C1C=CC(C(C2C=CC(C(F)(F)F)=CC=2)OCCN2CCC=C(C(O)=O)C2)=CC=1

CMHQDSBIBSKHFP-UHFFFAOYSA-N

InChI=1S/C23H21F6NO3/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)

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

CI966; CI 966; 1-(2-(Bis(4-(trifluoromethyl)phenyl)methoxy)ethyl)-1,2,5,6-tetrahydropyridine-3-carboxylic acid; CI966; DTXSID90149194; 1-(2-(Bis(4-(trifluoromethyl)phenyl)methox)ethyl)-1,2,5,6-tetrahydro-3-pyrdinecarboxylic acid; DTXSID30149193; CI-966

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.)