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The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40 kDa exhibited specific and saturable binding properties. The dopamine antagonist [H-3]spiperone showed an average dissociation constant K-d of 0.6 +/- 0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni2+ chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively. (C) 2003 Elsevier Inc. All rights reserved.

An innovative custom-built atmospheric ionization source afforded an opportunity to perform on-line LC/MS analysis and to obtain identification of metabolites without need to rely on radioactive profiling. An HPLC with a UV detector coupled to a modified R 3010 triple quadrupole mass spectrometer was used in which nitrogen gas effectively prevented the clustering of ions with polar solvent molecules as well as acting as the nebulizing gas for the ionspray LC/MS interface. This approach was useful in elucidating the oxidative metabolism of the potent dopamine D2 agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in rat-, monkey-, and human liver microsome preparations. Microsomes from monkey showed the highest metabolic activity, with 8.3 nmol . min-1 . mg-1 protein; the human material resulted in the lowest, 1.2 nmol . min-1 . mg-1 protein. Metabolic profiling showed that the three species produced the same metabolites, but to different extents. Liver microsomes metabolized the (-) enantiomer to 2-(N-2-thienylethylamino)-5-hydroxytetralin, 2-(N-propylamino)-5-hydroxytetralin, and 2-(N-propyl-N-2-thienylethylamino)-5,6-dihydroxytetralin, a catechol metabolite. Due to the weak dopaminergic affinities of the N-dealkylated compounds and the high metabolic conversion of the catechol, no therapeutic effects of these compounds may be expected in vivo.

A method is described for the rapid production and purification of new potential dopamine agonists. Via thermal heating (refluxing in an oil bath) and microwave exposure 2-[ N -n-3-fluoropropyl- N -(4-methylphenyl)ethylamino]-5-hydroxytetralin ( 12 ), 2-[ N -n-3-fluoropropyl- N -(4-fluorophenyl)ethylamino]-5-hydroxytetralin ( 13 ), and their isotopic fluorine-18 derivatives were synthesized, respectively. The fluorine-18 label was introduced via N -fluoroalkylation with no-carrier-added (n.c.a.) 18 FCH 2 CH 2 CH 2 I. In 115 min, radiochemical yields of 11% (corrected for decay) were achieved for both compounds. The specific activity ranged from 15–75 GBq/μmol. After i.v. injection in rats, the fluorine-18 labeled compounds were evaluated for their in vivo binding to the D 2 -receptors. The radioactivity levels in the striatum, nucleus accumbens and tuberculum olfactorius were not significantly higher than in the cerebellum and frontal cortex at 15, 30 and 60 min after administration of the tetralin derivatives. Dopamine depletion with reserpine did not affect the uptake in the dopamine D 2 -receptor rich area. Remarkable high uptakes were found in the adrenal for both compounds.

The pharmacokinetics of the dopamine D2 agonist S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (1, N-0923) after intravenous, intraperitoneal, and oral administration was studied in freely moving male and female albino Wistar rats. In all cases, the dose was 1 0 mumol . kg-1. Levels of 1 in plasma were monitored up to 6 h after dosing by high-performance liquid chromatography. A biphasic disappearance pattern with a rapid distribution phase was observed in the curve of concentration in plasma versus time. Pharmacokinetic analysis based on a two-compartment model with elimination from the central compartment yielded the following parameters for male rats (mean +/-standard deviation, n = 3): elimination half-life was 108 +/- 7 min, apparent volume of distribution of the central compartment was 397 +/- 44 mL . kg-1, the plasma clearance was 32 +/- 4 mL . min-1 . kg-1, and the apparent volume of distribution at steady state was 2.29 +/- 0.25 L . kg-1. No significant difference (analysis of variance p value > 0.25) was found in the pharmacokinetic data between male and female rats. An extremely fast absorption was found after intraperitoneal administration. Maximal concentrations in plasma were often observed at the first time point 5 min after dosing. The bioavailability (mean +/- standard deviation, n = 3) was 7.9 +/- 2.7% for male rats and 6.5 +/- 2.1% for female rats. An increase of the elimination half-life of at least 40% for male rats and 300% for female rats was observed, indicating dose-dependent kinetics after intraperitoneal administration. After oral dosing, levels of 1 in plasma were extremely low and often below the quantitation limit of the assay (10 pmol . mL-1). This did not allow detailed kinetic analysis. Even in the more favorable cases, the bioavailability did not exceed 0.5%. Although the 5-0-glucuronide and 5-sulfate are the major metabolites of 1, they could not be detected in plasma, indicating efficient excretion into bile and urine.

Based on the hypothesis that simultaneous stimulation of dopamine-D1 and -D2 receptors could be beneficial for the treatment of Parkinson's disease7,13, we prepared 5,6,7,8-tetrahydro-6-[propyl[2-(2-thienyl)ethyl]amino]-1,2-naphthalenediol (PTAT, 4), the diol analogue of N-0437 (3), as a potential mixed D1/D2-receptor agonist. The synthesis of 4 was carried out via a new strategy. The crucial step in this strategy was the conversion of 1-diazo-4-(2,3-dimethoxyphenyl)-2-butanone (15) to the key intermediate 5,6-dimethoxy-2-tetralone (9) via a rhodium(II)-acetate-catalysed cyclisation, followed by transformation under the influence of trifluoroacetic acid. Pharmacological evaluation of the dopaminergic properties of PTAT (4) revealed that this compound displays high affinity for both D1 and D2 receptors and has the same ability as DPAT (1), a mixed D1/D2-receptor agonist, to induce locomotor activity in mice rendered acutely akinetic with reserpine.

Brain microdialysis was used to investigate whether different calcium concentrations (1.2 and 3.4 mmol/l) of the perfusion fluid influenced the effects of D2 agonists on the release of dopamine in the striatum. We used the D2 agonists (-)N-0437 and (+)PHNO. After both local and systemic administration of (-)N-0437 and (+)PHNO, differences were apparent between their effects at 1.2 mmol/l calcium and 3.4 mmol/l calcium Ringer's solution. Although the drugs induced a similar maximal decrease in the release of dopamine with both calcium concentrations, the potency of the effect was significantly greater at 1.2 mmol/l when compared to 3.4 mmol/l calcium Ringer's solution. Thus, when measuring pharmacological effects of dopaminergic agents, it seems essential to use a Ringer's solution containing the physiological calcium concentration in brain microdialysis.

The extracellular concentration of dopamine (DA) and 3,4-dihydroxyphenylacetic acid in the substantia nigra (SN) and striatum was estimated by microdialysis. The dialysate content of DA from the SN was recorded during infusion of a DA uptake blocker (nomifensine; 5-mu-mol/L) dissolved in the perfusion fluid. Perfusion of tetrodotoxin (1-mu-mol/L) produced a virtually complete disappearance of nigral and striatal DA release. Dendritic as well as terminal release of DA was inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of gamma-butyrolactone (750 mg/kg, i.p.). The systemic administration (0.3 mg/kg, i.p.) as well as infusion (1-mu-mol/L) of the D2 agonist (-)-N-0437 [2-(n-propyl-N-2-thienylethylamino)-5-hydroxytetralin] produced a significant decrease in the release of DA in both the striatum and the SN. DA levels were recorded in the striatum both with and without addition of nomifensine to the perfusion fluid. The decrease in the striatum after (-)-N-0437 was suppressed in the presence of nomifensine. Infusion (1-mu-mol/L) as well as systemic administration (40 mg/kg) of sulpiride caused a similar increase in the release of striatal DA; this increase was, in both experiments, potentiated by nomifensine coinfusion. Sulpiride administration induced a small increase in the release of nigral DA. Infusion of (-)-N-0437 or (-)-sulpiride into the nigra caused a moderate decrease and increase, respectively, of striatal DA level. It was possible to estimate the relative contribution of nigral and striatal autoreceptors to the typical changes in striatal DA release seen after peripheral administration of D2 agonists or antagonists. These results suggest that the dendritic release of DA fulfills classical release criteria.

The potent and selective D2-agonist N-0437 [2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin] undergoes considerable first-pass metabolism after oral administration due to glucuronidation of the phenolic group. In an attempt to improve its bioavailability, seven ester prodrugs of N-0437 were sythesized, i.e the acetyl-, propionyl-, isobutyryl-, pivaloyl-, 2-aminophenyl 2-methoxy-phenyl- and 2,4-dimethylphenyl-analogues. In vivo activities were assessed by measuring contralateral turning after transdermal administration of N-0437 and its prodrugs to rats with unilateral 6-OHDA lesions of the nigrostriatal pathway, From time-effect curves the area under the curve for separate time intervals was taken as a measure of dopaminergic activity during that interval. It was found that slowly hydrolyzing prodrugs, which are known to show an improved duration of action after oral administration, are devoid of activity after transdermal application. The acetyl-, the propionyl- and the isobutyryl analogues, which are prodrugs with a relatively high hydrolysis rate, were found to have interesting and promising profiles following transdermal application.

In the present series of studies, attention was focussed particularly on dopaminergic D2 receptor compounds, with emphasis on the enantiomers of the potent and selective dopamine D2 receptor agonist N-0437. Drugs that display activity at D2 receptors are of great interest as potentially new therapeutic agents for the treatments of Parkinson’s disease and schizophrenia. With these therapeutic uses in mind, the pharmacological profiles of the enantiomers of N-0437 were considered. The pathophysiology of Parkinson’s disease, and to a lesser extent of schizophrenia, is thought to be related to disturbances in the functioning of the basal ganglia. Therefore, in order to understand the neuropathology of these diseases, it is essential to gain further insight into the normal functioning of the basal ganglia. Hence, experiments in this thesis were also directed to further clarification of neuronal interactions in the basal ganglia. In summary, the aims were (1) to characterize the pharmacological properties of the enantiomers of the D2 agonist N-0437; (2) to assess their potential as therapeutic agents in the treatment of Parkinson’s disease and schizophrenia; (3) to study dopamine-GABA interactions in the basal ganglia.

The absorption of the dopamine D2 agonist S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxy-tetralin hydrochloride (1; N-0923) was studied in fasted and non-fasted male Albino Wistar rats after intragastric administration of 10.0 mumol . kg-1. Blood samples up to 120 min were obtained from the portal vein and the levels of 1 were monitored with a sensitive HPLC method.The maximal fraction of the drug reaching the liver invariable was less than 1% of the dose. Maximal plasma levels of 30 pmol . ml-1 were found within 12 min after dosing in fasted animals. Plasma concentrations of 1 were measurable up to 60 min, after which they were below the quantitation limit of the assay (10 pmol . ml-1). This did not allow detailed kinetic analysis.Analysis of -the portal blood samples obtained after an oral dosing of 10.0 mumol . kg-1 1 spiked with 0.37 MBq tritium labelled drug showed that the amount of unchanged drug which reaches the liver invariably was comparable with the concentrations found in the study without radioactivity.In vitro incubation of 1 with gastric juice and gut contents showed no degradation. Therefore, it can be concluded that 1 undergoes an extensive metabolism in the gastrointestinal mucosa.

The determination of the enantiomeric impurity, i.e, the percentage of (+) N-0437 (= N-0924) in several batches of (-) N-0437 (= N-0923) by chiral HPLC is described. Enantiomeric impurities were calculated based on the peak areas of the two baseline separated enantiomers in the chromatogram. The enantiomeric impurities found in different batches ranged from 0.02% to 0.11%. Calibration curves of the two isomers of N-0437 (Fig. 1,) were made twice to study the reproducibility and linearity of the method. The absorbance ratio, N-0923/N-0924, was found to be 1.02 with a relative standard deviation (RSD) of 9% over the whole concentration range used for the calibration curves.

An innovative custom-built atmospheric ionization source afforded an opportunity to perform on-line LC/MS analysis and to obtain identification of metabolites without need to rely on radioactive profiling. An HPLC with a UV detector coupled to a modified R 3010 triple quadrupole mass spectrometer was used in which nitrogen gas effectively prevented the clustering of ions with polar solvent molecules as well as acting as the nebulizing gas for the ionspray LC/MS interface. This approach was useful in elucidating the oxidative metabolism of the potent dopamine D2 agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in rat-, monkey-, and human liver microsome preparations. Microsomes from monkey showed the highest metabolic activity, with 8.3 nmol . min-1 . mg-1 protein; the human material resulted in the lowest, 1.2 nmol . min-1 . mg-1 protein. Metabolic profiling showed that the three species produced the same metabolites, but to different extents. Liver microsomes metabolized the (-) enantiomer to 2-(N-2-thienylethylamino)-5-hydroxytetralin, 2-(N-propylamino)-5-hydroxytetralin, and 2-(N-propyl-N-2-thienylethylamino)-5,6-dihydroxytetralin, a catechol metabolite. Due to the weak dopaminergic affinities of the N-dealkylated compounds and the high metabolic conversion of the catechol, no therapeutic effects of these compounds may be expected in vivo.

The transdermal penetration of the dopamine D2 agonist, N-0923, was studied in freely moving male albino Wistar rats, after cutaneous dosing of 2 μmol cm −2 in a carbomer gel. A silicone patch with an effective area of 0.65 or 1.3 cm 2 was filled with 100 or 200 mg gel, respectively. The patch was attached to the rat skin using histoacrylic glue. The drug was tested as N-0923 · HCl. The enhancing effect of azone was studied by adding 5% of the penetration enhancer to the gel. A second enhancement study was performed after pretreatment of shaved rat skin with a solution containing 5% azone at various time points, prior to administrations of drug. The pretreatment solution contained ethanol-propyleneglycol-water-azone (60 : 20 : 15 : 5 v/v). The dermal penetration of N-0923 · HCl from the carbomer gel resulted in plasma levels which were relatively low and often at or just above the limit of quantitation of the assay (10 pmol ml −1 ). Adding 5% azone to the gel resulted in undetectable plasma levels, indicating the drug not being released from the carrier. Pretreatment of the rat skin with a solution containing 5% azone resulted in a 3-fold increase in the flux vs controls. Plasma levels were detectable up to 10 h after application. All pretreated animals showed stereotypy, indicating that adequate amounts of the drug reached the brain.

The absorption of the dopamine D2 agonist S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxy-tetralin hydrochloride (1; N-0923) was studied in fasted and non-fasted male Albino Wistar rats after intragastric administration of 10.0 mumol . kg-1. Blood samples up to 120 min were obtained from the portal vein and the levels of 1 were monitored with a sensitive HPLC method. The maximal fraction of the drug reaching the liver invariable was less than 1% of the dose. Maximal plasma levels of 30 pmol . ml-1 were found within 12 min after dosing in fasted animals. Plasma concentrations of 1 were measurable up to 60 min, after which they were below the quantitation limit of the assay (10 pmol . ml-1). This did not allow detailed kinetic analysis. Analysis of -the portal blood samples obtained after an oral dosing of 10.0 mumol . kg-1 1 spiked with 0.37 MBq tritium labelled drug showed that the amount of unchanged drug which reaches the liver invariably was comparable with the concentrations found in the study without radioactivity. In vitro incubation of 1 with gastric juice and gut contents showed no degradation. Therefore, it can be concluded that 1 undergoes an extensive metabolism in the gastrointestinal mucosa.

The extracellular concentration of dopamine (DA) and 3,4-dihydroxyphenylacetic acid in the substantia nigra (SN) and striatum was estimated by microdialysis. The dialysate content of DA from the SN was recorded during infusion of a DA uptake blocker (nomifensine; 5-mu-mol/L) dissolved in the perfusion fluid. Perfusion of tetrodotoxin (1-mu-mol/L) produced a virtually complete disappearance of nigral and striatal DA release. Dendritic as well as terminal release of DA was inhibited for several hours when the nerve impulse flow in dopaminergic neurons was blocked by systemic administration of gamma-butyrolactone (750 mg/kg, i.p.). The systemic administration (0.3 mg/kg, i.p.) as well as infusion (1-mu-mol/L) of the D2 agonist (-)-N-0437 [2-(n-propyl-N-2-thienylethylamino)-5-hydroxytetralin] produced a significant decrease in the release of DA in both the striatum and the SN. DA levels were recorded in the striatum both with and without addition of nomifensine to the perfusion fluid. The decrease in the striatum after (-)-N-0437 was suppressed in the presence of nomifensine. Infusion (1-mu-mol/L) as well as systemic administration (40 mg/kg) of sulpiride caused a similar increase in the release of striatal DA; this increase was, in both experiments, potentiated by nomifensine coinfusion. Sulpiride administration induced a small increase in the release of nigral DA. Infusion of (-)-N-0437 or (-)-sulpiride into the nigra caused a moderate decrease and increase, respectively, of striatal DA level. It was possible to estimate the relative contribution of nigral and striatal autoreceptors to the typical changes in striatal DA release seen after peripheral administration of D2 agonists or antagonists. These results suggest that the dendritic release of DA fulfills classical release criteria.