GBR 12935

GBR 12935 is a potent and selective inhibitor of dopamine uptake with IC50 value of 3.7nM [1, 2].

GBR 12935 is found to have multiple binding sites in rat and human brains. One of these is the classic DA uptake site of the dopamine transporter. The [3H] GBR 12935 binding reveals specific and selective labeling of the DA transport complex. This binding site is found to be identical or similar to the binding sites for substrates of DA uptake. GBR 12935 inhibits DA uptake with IC50 value of 3.7nM in vitro. In dopamine transporter transfected COS-7 cells, GBR 12935 shows a Kd value of 1.08nM [1,2 and 3].

Another binding site of GBR 12935 is a piperazine acceptor site and is found to be the CYP2D isoform. This site is present not only in brain but also in the liver. Among the ten forms of PY450, GBR 12935 binds to CYP2D6 with highest activity. The Kd value is 42.2nM. The binding of GBR 12935 to the piperazine acceptor site can be inhibited by inhibitors of CYP2D6 but not dopamine [3].

References:
[1] Andersen P H. Biochemical and pharmacological characterization of [3H] GBR 12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex. Journal of neurochemistry, 1987, 48(6): 1887-1896.
[2] Matecka D, Rothman R B, Radesca L, et al. Development of novel, potent, and selective dopamine reuptake inhibitors through alteration of the piperazine ring of 1-[2-(diphenylmethoxy) ethyl]-and 1-[2-[bis (4-fluorophenyl) methoxy] ethyl]-4-(3-phenylpropyl) piperazines (GBR 12935 and GBR 12909). Journal of medicinal chemistry, 1996, 39(24): 4704-4716.
[3] Hiroi T, Imaoka S, Chow T, et al. Specific binding of 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenyl propyl) piperazine (GBR-12935), an inhibitor of the dopamine transporter, to human CYP2D6. Biochemical pharmacology, 1997, 53(12): 1937-1939.

Environmental enrichment enhances sensitization to GBR 12935-induced activity and decreases dopamine transporter function in the medial prefrontal cortex.[Pubmed:14684252]

Behav Brain Res. 2004 Jan 5;148(1-2):107-17.

Rats raised in an enriched condition (EC) during development display increased hyperactivity to the effect of acute amphetamine compared to rats raised in an impoverished condition (IC). The present study determined whether environmental enrichment differentially alters the effects of GBR 12935 administration, a selective dopamine transporter (DAT) inhibitor. Acutely, EC rats showed a greater, dose-dependent GBR 12935-induced increase in activity compared to IC rats; however, basal activity for EC rats was lower than for IC rats. After repeated GBR 12935, only EC rats exhibited behavioral sensitization. Kinetic analysis of DAT function in medial prefrontal cortex (mPFC) revealed that the maximal velocity of [3H]dopamine ([3H]DA) uptake in EC rats was less than in IC rats (4.9 +/- 0.6 and 7.7 +/- 0.6 pmol/min/mg, respectively), but not in striatum or nucleus accumbens. Furthermore, GBR 12935-induced inhibition of DAT function, [3H]GBR 12935 binding density and DA content in mPFC, striatum and nucleus accumbens were not different between EC and IC rats. However, dihydroxyphenylacetic acid content in mPFC was lower in EC than IC rats, whereas no differences were found in striatum and nucleus accumbens. These results suggest that EC-induced changes in activity may be due to decreased DAT function and decreased DA metabolism in the mPFC.

Effect of 1,2,3,4,-tetrahydroisoquinoline administration under conditions of CYP2D inhibition on dopamine metabolism, level of tyrosine hydroxylase protein and the binding of [3H]GBR 12,935 to dopamine transporter in the rat nigrostriatal, dopaminergic system.[Pubmed:15120584]

Brain Res. 2004 May 29;1009(1-2):67-81.

Current concepts of Parkinson's disease (PD) postulate that interaction between neurotoxins and specific genetic background may play an important role in pathogenesis of PD. Therefore, the effect of multiple administration of 1,2,3,4-tetrahydroisoquinoline (TIQ) under conditions of CYP2D blockade on the expression of key markers of PD was studied in the rat striatum (STR) and substantia nigra (SN). TIQ administered alone (50 mg/kg i.p. twice daily for 14 days) markedly decreased the level of tyrosine hydroxylase protein (TH) in the STR; however, this effect was not accompanied by reduction of dopamine (DA) concentration and [(3)H]GBR 12,935 binding to dopamine transporter (DAT). Administration of CYP2D inhibitor, quinine, jointly with TIQ lowered the levels of TH and DA in that structure, but slightly increased DAT binding. In the SN, treatment with TIQ alone did not change TH level although it enhanced DA content and decreased [(3)H]GBR 12,935 binding to DAT in the substantia nigra pars compacta (SNc). Neither the TH level nor DA concentration was affected by the combined treatment, although DAT binding was still reduced in the SN. TIQ did not change the total DA catabolism in the STR, but caused its inhibition in the SN. It strongly depressed the levels of intraneuronal DA metabolite DOPAC and enhanced that of extraneuronal 3-MT in either structure. TIQ more weakly affected the levels of both DA metabolites in the presence of quinine. Our results suggest that endogenous TIQ may act rather as neuromodulator but not as parkinsonism-inducing neurotoxin in the rat brain.

Identification of dopamine transporter in bovine pineal gland using [3H]GBR 12935.[Pubmed:12823609]

J Pineal Res. 2003 Aug;35(1):16-23.

The mammalian pineal gland contains several neurotransmitters and receptors for amino acids, biogenic amines, and peptides. Some of these, such as D1 and D2 dopamine receptors, have been previously identified and characterized in the bovine pineal gland by our group. As a matter of fact, the density of D1 dopamine receptors in the pineal gland is higher than that of corpus striatum, suggesting that this organ must possess a high affinity dopamine transporter, which has been identified in this study by using [3H]GBR 12935 as a radiological ligand and nomifensine to determine non-specific binding. The association rate of [3H]GBR 12935 binding to the pineal membrane was examined as a function of time. The binding reached equilibrium within 45 min of incubation at 25 degrees C. The specific binding was reversible and saturable. The dissociation time course of the specific [3H]GBR 12935 binding from the bovine pineal membrane was also studied. A half-life (t1/2) of 14-min was obtained. The saturation analysis of the [3H]GBR 12935 binding revealed a dissociation equilibrium constant (Kd) of 6.0 +/- 0.9 nm and a receptor density (Bmax) of 6.9 +/- 0.3 pmol/mg protein, which were comparable with those values obtained from bovine striatum and frontal cortex. In competitive experiments, the concentrations of drugs required to inhibit 50% of the binding (IC50) were in descending order GBR 12909 > GBR 12935 > trans-flupenthixol > nomifensine > cis-flupenthixol > amitriptyline > imipramine > desipramine > dopamine > fluoxetine > fuvoxamine > d-amphetamine. However, nisoxetine, SCH 23390, norepinephrine, and serotonin were unable to displace [3H]GBR binding. These results show that drugs capable of blocking dopamine transporters were effective in displacing [3H]GBR binding; whereas specific norepinephrine and serotonin transporter inhibitors were less effective or ineffective. In addition, the dopamine transporter is ion-dependent as sodium increased [3H]GBR binding in a concentration related manner. These results indicate that a high affinity dopamine transporter exists in the bovine pineal, which may exhibit circadian periodicity, and whose physiological functions need to be delineated and characterized in future investigations.

Expansion of structure-activity studies of piperidine analogues of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935) compounds by altering substitutions in the N-benzyl moiety and behavioral pharmacology of selected molecules.[Pubmed:11806716]

J Med Chem. 2002 Jan 31;45(3):654-62.

A series of substituted N-benzyl analogues of the dopamine transporter (DAT) specific compound, 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine were synthesized and biologically characterized. Different 4'-alkyl, 4'-alkenyl, and 4'-alkynyl substituents were introduced in the phenyl ring of the benzyl moiety along with the replacement of the same phenyl ring by the isomeric alpha- and beta-naphthyl groups. Different polar substitutions at the 3'- and 4'-position were also introduced. Novel compounds were tested for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in the brain by competing for [(3)H]WIN 35 428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [(3)H]dopamine. Binding results demonstrated that alkenyl and alkynyl substitutions at the 4'-position produced potent compounds in which compound 6 with a vinyl substitution was the most potent. In vivo evaluation of three selected compounds indicated that despite their high potency at the DAT, these compounds stimulated locomotor activity (LMA) less than cocaine when tested across similar dose ranges. In a drug discrimination study procedure, none of these three compounds generalized from cocaine in mice trained to discriminate 10 mg/kg cocaine from vehicle. In a 4 h time course LMA experiment, one of our previous lead piperidine derivatives (1a) showed considerable prolonged action. Thus, in this report, we describe a structure-activity relationship study of novel piperidine analogues assessed by both in vitro transporter assays and in vivo behavioral activity measurements.