Juha-Matti Savola

Fipamezole (JP-1730) is a potent alpha2 adrenergic receptor antagonist that reduces levodopa-induced dyskinesia in the MPTP-lesioned primate model of Parkinson's disease.

Previous studies in the MPTP‐lesioned primate model of Parkinsons disease have demonstrated that α2 adrenergic receptor antagonists such as idazoxan, rauwolscine, and yohimbine can alleviate L‐dopa–induced dyskinesia and, in the case of idazoxan, enhance the duration of anti‐parkinsonian action of L‐dopa. Here we describe a novel α2 antagonist, fipamezole (JP‐1730), which has high affinity at human α2A (Ki, 9.2 nM), α2B (17 nM), and α2C (55 nM) receptors. In functional assays, the potent antagonist properties of JP‐1730 were demonstrated by its ability to reduce adrenaline‐induced 35S‐GTPγS binding with KB values of 8.4 nM, 16 nM, 4.7 nM at human α2A, α2B, and α2C receptors, respectively. Assessment of the ability of JP‐1730 to bind to a range of 30 other binding sites showed that JP‐1730 also had moderate affinity at histamine H1 and H3 receptors and the serotonin (5‐HT) transporter (IC50 100 nM to 1 μM). In the MPTP‐lesioned marmoset, JP‐1730 (10 mg/kg) significantly reduced L‐dopa–induced dyskinesia without compromising the anti‐parkinsonian action of L‐dopa. The duration of action of the combination of L‐dopa and JP‐1730 (10 mg/kg) was 66% greater than that of L‐dopa alone. These data suggest that JP‐1730 is a potent α2 adrenergic receptor antagonist with potential as an anti‐dyskinetic agent in the treatment of Parkinsons disease.

Central α2-adrenoceptors are highly stereoselective for dexmedetomidine, the dextro enantiomer of medetomidine

To determine whether different alpha 2-adrenoceptor-mediated functions have different stereochemical requirements in the central nervous system, we studied the pharmacological activities of the purified optical enantiomers of medetomidine (dl-4-[1(2,3-dimethylphenyl)ethyl]-1H-imidazole), a specific and selective agonist of alpha 2-adrenoceptors. We found that dexmedetomidine (the dextro enantiomer) had the pharmacological activity of medetomidine. Dexmedetomidine had hypotensive and bradycardic actions in anaesthetized rats as well as sedative (decreased spontaneous locomotor activity and prolonged sleep induced by hexobarbital in rats), analgesic (attenuated a writhing response induced by acetic acid in mice) and midriatic actions in rats. The potency of dexmedetomidine was slightly greater than that of medetomidine. 1-Medetomidine was generally without pharmacological activity, but it showed some sedative and analgesic properties at high doses. Although the findings obtained with l-medetomidine might indicate some deviation from strict homogeneity, these experiments demonstrate that the different alpha 2-adrenoceptor-mediated functions have similar stereochemical requirements in the central nervous system.

α2-Adrenoceptor regulation of adenylyl cyclase in CHO cells: Dependence on receptor density, receptor subtype and current activity of adenylyl cyclase

Chinese hamster ovary (CHO) cells stably transfected to express different densities of the human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes, were used to characterize the regulation of adenylyl cyclase activity by alpha2-adrenoceptor agonists. In isolated cell membranes, activation of alpha2A- and alpha2C-adrenoceptors did not affect basal enzyme activity, but activation of alpha2B-adrenoceptors stimulated adenylyl cyclase activity. The extent of stimulation was dependent on the receptor density and was insensitive to pertussis toxin treatment. In the presence of 10 microM forskolin all three receptor subtypes mediated inhibition of adenylyl cyclase activity in a pertussis toxin-sensitive manner. In experiments performed with intact cells the same pattern could be seen: the basal production of cAMP was not affected when alpha2C-adrenoceptors were activated, but activated alpha2B-adrenoceptors mediated stimulation of cAMP production. In the presence of forskolin, both receptor subtypes mediated inhibition of cAMP production. Our results suggest that alpha2B-adrenoceptors are coupled to both Gi and Gs proteins. The signal transduction pathway to which the receptor is coupled is not dependent on receptor density, but its effect on adenylyl cyclase regulation is dependent on the current activity of adenylyl cyclase. The results also suggest that the alpha2A- and alpha2C-subtypes are preferentially coupled to Gi and transduce only inhibition of adenylyl cyclase activity in transfected CHO cells. At low densities of alpha2C-adrenoceptors, clonidine was a partial agonist, but in clones expressing high levels of alpha2C-adrenoceptors, clonidine acted as a full agonist by inhibiting cAMP accumulation with the same efficacy as (-)-noradrenaline. This demonstrates that receptor reserve can mask partial agonist activity.

Evaluation of the effects of a specific α2-adrenoceptor antagonist, atipamezole, on α1- and α2-adrenoceptor subtype binding, brain neurochemistry and behaviour in comparison with yohimbine

In the present study we evaluated the α1- and α2-adrenoceptor subtype binding, central α2-adrenoceptor antagonist potency, as well as effects on brain neurochemistry and behavioural pharmacology of two α2-adrenoceptor antagonists, atipamezole and yohimbine. Atipamezole had higher selectivity for α2- vs. α1-adrenoceptors than yohimbine regardless of the subtypes studied. Both compounds had comparable affinity for the α2A-, α2C- and α2B-adrenoceptors, but yohimbine had significantly lower affinity for the α2D-subtype. This may account for the fact that significantly higher doses of yohimbine than atipamezole were needed for reversal of α2-agonist (medetomidine) -induced effects in rats (mydriasis) and mice (sedation and hypothermia). The effect on central monoaminergic activity was estimated by measuring the concentrations of transmitters and their main metabolites in whole brain homogenate. At equally effective α2-antagonising doses in the rat mydriasis model, both drugs stimulated central noradrenaline turnover (as reflected by increase in metabolite levels) to the same extent. Atipamezole increased dopaminergic activity only slightly, whereas yohimbine elevated central dopamine but decreased central 5-hydroxytryptamine turnover rates. In behavioural tests, atipamezole (0.1–10u2007mg/kg) did not affect motor activity but stimulated food rewarded operant (FR-10) responding (0.03–3u2007mg/kg) whereas yohimbine both stimulated (1u2007mg/kg) and decreased (≥ 3u2007mg/kg) behaviour in a narrow dose range in these tests. In the staircase test, both antagonists increased neophobia, but in the two compartment test only yohimbine (≥ 3u2007mg/kg) decreased exploratory behaviour. The dissimilar effects of the antagonists on neurochemistry and behaviour are thought to be caused by non α2-adrenoceptor properties of yohimbine. In conclusion, the α2-antagonist atipamezole blocked all α2-adrenoceptor subtypes at low doses, stimulated central noradrenergic activity and had only slight effects on behaviour under familiar conditions, but increased neophobia. The low affinity for the α2D-adrenoceptor combined with its unspecific effects complicates the use of yohimbine as pharmacological tool to study α2-adrenoceptor physiology and pharmacology.

Two human α2-adrenoceptor subtypes α2A-C10 and α2B-C2 expressed in Sf9 cells couple to transduction pathway resulting in opposite effects on cAMP production

Two human alpha-2 adrenoceptor subtypes alpha-2A-C10 and alpha-2B-C2 expressed in Sf9 cells couple to transduction pathways resulting in opposite effects on cAMP production

Functional analysis of the human α2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.

Characterization of [3H]atipamezole as a radioligand for α2-adrenoceptors

Atipamezole (MPV-1248, 4-(2-ethyl-2,3-dihydro-1H-inden-2-yl)-1H-imidazole), a potent alpha 2-adrenoceptor antagonist, was tritiated to high specific activity. We then compared [3H]atipamezole and [3H]rauwolscine as radioligands for alpha 2-adrenoceptors in rat cerebral cortex, neonatal rat lung, and human platelets. (-)-Noradrenaline and phentolamine were used to define specific alpha 2-adrenergic binding. Unlabelled atipamezole was used in a similar manner to define saturable, high-affinity non-adrenergic binding. [3H]Atipamezole binding to human platelets (Kd 1.3 nM) and rat brain membranes (Kd 0.5 nM) equilibrated rapidly and was displaced in the expected manner by alpha 2-adrenergic ligands. In contrast, [3H]atipamezole binding in neonatal rat lung membranes was only effectively inhibited by unlabelled atipamezole, and by high concentrations of idazoxan. The total density of binding sites for [3H]atipamezole was clearly in excess of the density of alpha 2-adrenoceptors in this tissue, as defined by [3H]rauwolscine binding. We conclude that [3H]atipamezole binds with high affinity to alpha 2-adrenoceptors in human platelets and rat cerebral cortex, and that the compound can be used to investigate alpha 2-adrenoceptor properties and drug actions in these tissues. In neonatal rat lung, [3H]atipamezole identified an additional population of binding sites, distinct from both classical alpha 2-adrenoceptors and idazoxan-defined imidazoline receptors. The pharmacological identity of these binding sites remains to be elucidated. This non-adrenergic component in the binding characteristics of [3H]atipamezole complicates its use as a general alpha 2-adrenoceptor radioligand.

The α2 adrenergic antagonist fipamezole improves quality of levodopa action in Parkinsonian primates

Reduction in the antiparkinsonian benefit of levodopa is a major complication of long‐term levodopa treatment in advanced Parkinsons disease (PD). Such loss of benefit arises because of reduced duration of action and appearance of disabling dyskinesia. We assess the potential of the α2 adrenergic antagonist fipamezole to reduce motor complications in parkinsonian macaques. MPTP‐lesioned macaques were treated acutely with fipamezole (10 mg/kg) alone and in combination with two doses of levodopa. Fipamezole extended both duration and quality of antiparkinsonian action of levodopa. Duration of antiparkinsonian action, on time, was increased by up to 75% while “good‐quality” on time, i.e., that not associated with disabling dyskinesia, was increased by up to 98%. Combination of fipamezole with the lower dose of levodopa provided antiparkinsonian benefit at least equivalent to that provided by the higher dose levodopa alone. However, with the combination, antiparkinsonian benefit was of much better quality. The proportion of on time without disabling dyskinesia (79%) was significantly greater than that with high dose levodopa alone (45%). Increased duration and quality of levodopa action may represent therapeutically valuable actions of α2 adrenergic antagonists.

Anti-obesity effect of MPV-1743 A III, a novel imidazoline derivative, in genetic obesity

MPV-1743 A III ((+/-)-4-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) is a novel imidazoline derivative. In this study, it was shown to bind with high affinity to alpha2-adrenoceptor subtypes alpha2A (IC50) = 0.66 +/- 0.06 nM), alpha2B (IC50) = 3.8 +/- 0.53 nM), alpha2C (IC50) = 3.1 +/- 0.61 nM) in the recombinant S115 cells and to alpha2D (IC50 = 0.94 +/- 0.10 nM) in the rat submandibular gland. MPV-1743 A III also showed remarkably high affinity to alpha1-adrenoceptors (IC50 = 150 +/- 12 nM) in the rat cerebral cortex and to imidazoline I2b-binding sites (IC50) = 150 +/- 5.0 nM) in the rat liver. The functional alpha2-adrenoceptor antagonistic effect of MPV-1743 A III was demonstrated by studying the ability of orally administered MPV-1743 A III to reverse and prevent the alpha2-adrenoceptor agonist detomidine-induced mydriasis in rat. The anti-obesity effect of MPV-1743 A III was investigated in genetically obese (fa/fa) Zucker rats in two different phases of obesity. Chronic treatment with MPV-1743 A III (0.3 3 mg/kg per day p.o. for 3 weeks) dose dependently decreased weight gain in early-phase obesity. In fully established obesity, GDP binding to mitochondria and expression of uncoupling protein mRNA were increased in brown adipose tissue by MPV-1743 A III indicating an activation of non-shivering thermogenesis. The present study shows that MPV- 1743 A III has a modest anti-obesity effect in the genetic rodent model of obesity. The relative importance of alpha2- and alpha1-adrenoceptors and imidazoline I2b-binding sites in mediating the effects of MPV-1743 A III needs further evaluation.

α2-adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner

Transfected Chinese hamster ovary cells expressing human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes were used to monitor alpha2-adrenoceptor-stimulated GTP hydrolysis. Incubation with 100 microM (-)-adrenaline resulted in stimulation of pertussis toxin-sensitive GTPase by 380% after activation of the alpha2A-subtype, by 320% after activation of the alpha2B-subtype and by 110% after activation of the alpha2C-subtype. The agonists dexmedetomidine, UK14,304 (5-bromo-6-[2-imidazoline-2-ylamino]quinoxaline) and oxymetazoline showed subtype-dependent efficacy. Dexmedetomidine was a full agonist at the alpha2B-subtype and a partial agonist at the alpha2A- and the alpha2C-subtypes. UK14,304 was a full agonist at the alpha2A-subtype and a partial agonist at the other two. Oxymetazoline showed strong partial agonism at the alpha2B-subtype (63% of adrenaline), but did not significantly activate the alpha2A- and the alpha2C-subtypes. These results agreed with cAMP accumulation experiments carried out with cell lines endogenously expressing the alpha2A-subtype (human erythroleukemia, HEL) or the alpha2B-subtype (neuroblastoma-glioma, NG108-15). The GTPase assay may thus provide a valuable tool for the identification of subtype-selective alpha2-adrenoceptor agonists.