Theo B.A. Mulder

In vivo dopamine receptor binding studies with a non-radioactively labeled agonist, dipropyl-5,6-ADTN.

Evidence is presented that a low dose of peripherally administered N,N-dipropylamino-5,6-dihydroxytetralin (DiPr-5,6-ADTN) specifically labels dopamine (DA) receptors in rat brain. Concentrations of this potent DA receptor agonist were determined by a highly selective method using reversed phase liquid chromatography and amperometric detection. The binding characteristics satisfy all criteria regarding saturability, stereospecificity, regional distribution and relation with pharmacological effects that are associated with DA receptor interactions. A rough estimation of the density of binding sites in the striatum resulted in values of 60-70 pmol/g. Lesioning the nigrostriatal pathway does not significantly alter the amount of ligand bound, nor do pretreatments with serotonergic, alpha-adrenergic or beta-adrenergic antagonists. DiPr-5,6-ADTN has thus been shown to be a useful ligand for labeling central DA receptors and a powerful tool in the study of DA-ergic mechanisms.

THE SIGNIFICANCE OF COMT ACTIVITY IN CONTROLLING DOPAMINE AGONIST LEVELS IN BRAIN AND SERUM - STUDIES WITH A PRODRUG AND A METABOLITE OF 6,7-ADTN

After i.p. administration of the dibenzoylester of 2-amino-6,7-dihydroxytetralin (DB-6,7-ADTN) a metabolite was found in rat brain and serum, which was identified as 2-amino-6-hydroxy-7-methoxytetralin (7-O-MeADTN). By means of HPLC coupled with amperometric detection, time-concentration curves of 7-O-MeADTN in rat brain and serum were determined after 100 mumol/kg DB-6,7-ADTN. These showed a rapid formation and homogeneous distribution of high peak levels (4 nmol/g) of 7-O-MeATN. Brain and serum concentrations of 6,7-ADTN after 100 mumol/kg DB-6,7-ADTN, determined during catechol-O-methyltransferase (COMT) inhibition by tropolone, were 5--7 times higher than those during normal COMT activity, thus equalling 5,6-ADTN concentrations after 100 mumol/kg DB-5,6-ADTN. The greater susceptibility of 6,7-ADTN to metabolic degradation by COMT was confirmed by preliminary results of in vitro studies, which showed that two methoxy derivatives are formed from 6,7-ADTN and one, in very small amounts, from 5,6-ADTN. 7-O-MeADTN had no dopaminergic activity of its own, as after i.p. injection of 100 mumol/kg it was devoid of behavioural and biochemical effects typical for DA agonists. A homogeneous distribution and high peak concentrations (20 nmol/g after 15 min) were found in rat brain after this dose of the metabolite. The results indicate that a substantial amount of 6,7-ADTN, in contrast to 5,6-ADTN, is metabolized by COMT and that differences between brain concentrations of both isomers are almost exclusively due to differences in susceptibility for COMT. This has implications for the design of new DA agonists.

Kinetic and pharmacological profiles of the in vitro binding of the potent dopamine agonist [3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes

The in vitro binding of the new tritiated dopaminergic ligand [3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes is described. Binding assays were performed in 50 mM Tris-HCl pH 7.5 containing 1 mM EDTA and 0.01% ascorbic acid at 25 degrees C for 30 min. Specific binding at 0.5 nM [3H]DP-5,6-ADTN and 5 mg/ml membrane suspension was very high (87-93%) and was found to be linearly related with homogenate concentration over the range of 0.5-10 mg/ml (r = 0.9968). (+)Butaclamol 1 microM was used to define specific binding. Specific binding disappeared completely within 20 min when the tissue was incubated at 55 degrees C. Association and dissociation curves (obtained after addition of 1 microM (-)-DP-5,6-ADTN) were converted to linear logarithmic plots and the kinetic constants were computed (k1 = 0.054 min-1 and k-1 = 0.0188 min-1) as were the t1/2 for association (3.52 min) and dissociation (20.8 min). This resulted in an apparent KD of 0.34 nM. Increasing concentrations of [3H]DP-5,6-ADTN (0.05-3.0 nM) were found to saturate the receptor site. Linear transformation of the saturation curves and presentation of the curves as Eadie-Hofstee plots showed that only one set of receptors was labeled (nHill was 0.995 +/- 0.07) with a high affinity constant KD of 0.57 +/- 0.10 nM and a maximum number of binding sites Bmax of 18.6 +/- 2.4 pmol/g tissue. Various compounds were tested for their potency to displace the specific binding of [3H]DP-5,6-ADTN to striatal membranes (2.5 mg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Reversed-phase liquid chromatography with amperometric detection of lipophilic dopamine analogues and determination of brain and serum concentrations after sample clean-up on small sephadex G-10 columns.

The liquid chromatographic determination of N-alkylated analogues of dopamine is described. The retention of these compounds, ranging from dopamine to N,N-dibutyl-dopamine, was studied on four bonded-phase columns, of which Nucleosil 5 C18 was chosen for routine use. The compounds were detected by a rotating disc amperometric detector. Samples of rat brain and serum were taken through a clean-up step on small Sephadex G-10 columns from which the dopamine analogues eluted in the same fraction as dopamine. The overall recovery was 70--90% from brain tissue and 60--70% from serum or plasma. The limit of detection for the catechol-containing compounds in tissue was 40--100 pg, for O-methylated ones 100--200 pg. The method is applied to the determination of dopamine analogues in rat brain after peripheral administration.

In vivo dopamine receptor agonist binding in rat brain: Relation with pharmacological effects

The relation between in vivo rat striatal dopamine (DA) receptor binding of N,N-dipropyl-2-amino-5,6-dihydroxytetralin (DiPr-5,6-ADTN) and the dopaminergic effects of this compound was studied. The results indicate that maximal stereotyped biting and gnawing was associated with maximal saturable binding. At doses which maximally decreased striatal acidic DA metabolite concentrations a 25% or less occupation of binding sites was found. It is suggested that total in vivo binding is associated with stereotypy but not with the biochemical effects.

Amperometric detection of low concentrations of dopamine receptor agonists after liquid chromatographic on-column sample enrichment: Effect of O-methylation on brain concentrations of dipropyl-5, 6-ADTN and dipropyl-6, 7-ADTN

A sensitive and selective method of determination of N,N-dipropylamino-5, 6-dihydroxytetralin and its 6,7-dihydroxy isomer in rat plasma and brain tissue is described. The method is based on isolation of these dopamine receptor agonists from the biological samples on small Sephadex G-10 columns, followed by reversed phase high performance liquid chromatography with amperometric detection. Large volume (2 ml) injections result in on-column sample enrichment without a subsequent loss in column efficiency. Using this method the very low detection limit of 4 pmol/g was achieved in brain tissue. It was found that the lower in vivo potency as a centrally acting dopamine agonist of the 6,7-compared with the 5,6-isomer is almost entirely due to the lower brain concentrations achieved after peripheral administration of equimolar doses. This difference is mainly caused by different rates of O-methylation by catechol-O-methyltransferase, which follows from the fact that inhibition of this enzyme by tropolone leads to almost equal brain concentrations of the two isomers.

NEUROPHARMACOLOGICAL PROFILE OF A NEW SERIES OF DOPAMINE AGONISTS - N-N-PROPYL-HEXAHYDRONAPHTHOXAZINES

It has been demonstrated that within the series of hydroxylated (7-OH, 9-OH) and non-hydroxylated (N-0498) hexahydronaphthoxazines the 9-OH (N-0500) analogue is a very potent centrally acting DA receptor agonist. In in vitro [3H]DP-5,6-ADTN binding experiments, reflecting D-2 dopaminergic activity, N-0500 was equipotent with apomorphine and RU-29717, whereas both the 7-OH (N-0499) and N-0498 were much less effective. In in vivo tests related to DA receptor stimulation N-0500 was found to be the most active compound. In the gamma-butyrolactone model, a test for DA autoreceptor activation, N-0500 was 10 times as potent as apomorphine, but 3 times less active than RU-29717. The locomotor activity of mice was inhibited more strongly by N-0500 than by N-0499. Striatal concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid were rapidly reduced by N-0500 both after intraperitoneal and oral administration, indicating that this compound is well absorbed from the gastrointestinal tract and passes the blood-brain barrier to activate DA autoreceptors. In models for postsynaptic DA receptor stimulation (induction of stereotypy in rats, reversal of reserpine-induced immobility of mice) N-0500 was found to be as effective as RU-29717 in inducing stereotyped behaviors in rats, but was much less effective than RU-29717 in restoring the mobility of reserpinized mice, suggesting a selectivity for D-2 DA receptors by N-0500 in contrast to the mixed D-1/D-2 receptor activity of RU-29717. In in vitro binding experiments for evaluating the affinity towards other receptor types, N-0500 exhibited only a weak affinity towards 5-HT1 and alpha 2 binding sites and possessed a very weak affinity for 5-HT2 and alpha 1 receptor sites. It was concluded from these in vitro binding experiments that N-0500, has not only a very high affinity for D-2 DA receptors, but is more selective than RU-29717 and much more selective than the ergot bromocriptine. On the basis of its very potent in vivo central D-2 dopamine receptor activities and its in vitro selectivity, N-0500, being the most potent compound within the series, is a much more specifically acting drug than many of the dopaminergic ergolines and might therefore be a good candidate for the treatment of Parkinsons disease.

Characterization of the in vitro Binding of [3H] DP-5,6-ADTN to Rat Striatal Membranes

N,N-dipropyl-5,6-dihydroxyaminotetralin (DP-5,6-ADTN) is a potent DA-agonist both in vivo and in vitro (Feenstra et al., 1980).