The α2C-adrenoceptor antagonist, ORM-10921, exerts antidepressant-like effects in the Flinders Sensitive Line rat.

Depression involves deficits in monoaminergic neurotransmission. Differential roles for α2A, B and C subtypes of the α2-adrenoceptor (AR) are evident, with selective α2C-AR antagonists purported to have antidepressant and procognitive properties. However, this has not been demonstrated in a genetic animal model of depression. The role of the α2C-AR in modulating two key depression-related behaviours in the Flinders Sensitive Line (FSL) rat was studied using a dose-response analysis following subcutaneous administration with the selective α2C-AR antagonist ORM-10921 (0.03; 0.3 mg/kg), the nonselective α2-AR antagonist idazoxan (3 mg/kg), or vehicle once daily for 14 days. Behaviour in the novel object recognition test, forced swim test (FST) and locomotor activity test was assessed. To ratify the validity of the FSL model, the reference tricyclic antidepressant imipramine (15 mg/kg, intraperitoneally) was used as a comparator drug in the FST. FSL rats demonstrated significantly increased immobility and recognition memory deficits versus Flinders Resistant Line controls, with imipramine significantly reversing said immobility. Similarly, ORM-10921 at both doses but not idazoxan significantly reversed immobility in the FST as well as attenuated cognitive deficits in FSL animals. We conclude that selective α2C-AR antagonism has potential as a novel therapeutic strategy in the treatment of depression and cognitive dysfunction.

Test-retest reliability of (11)C-ORM-13070 in PET imaging of α2C-adrenoceptors in vivo in the human brain.

PURPOSE: α2C-Adrenoceptors share inhibitory presynaptic functions with the more abundant α2A-adrenoceptor subtype, but they also have widespread postsynaptic modulatory functions in the brain. Research on the noradrenergic system of the human brain has been hampered by the lack of suitable PET tracers targeted to the α2-adrenoceptor subtypes. METHODS: PET imaging with the specific α2C-adrenoceptor antagonist tracer [(11)C]ORM-13070 was performed twice in six healthy male subjects to investigate the test-retest reliability of tracer binding. RESULTS: The bound/free ratio of tracer uptake relative to nonspecific uptake into the cerebellum during the time interval of 5 - 30 min was most prominent in the dorsal striatum: 0.77 in the putamen and 0.58 in the caudate nucleus. Absolute test-retest variability in bound/free ratios of tracer ranged from 4.3 % in the putamen to 29 % in the hippocampus. Variability was also <10 % in the caudate nucleus and thalamus. Intraclass correlation coefficients (ICC) ranged from 0.50 in the hippocampus to 0.89 in the thalamus (ICC >0.70 was also reached in the caudate nucleus, putamen, lateral frontal cortex and parietal cortex). The pattern of [(11)C]ORM-13070 binding, as determined by PET, was in good agreement with receptor density results previously derived from post-mortem autoradiography. PET data analysis results obtained with a compartmental model fit, the simplified reference tissue model and a graphical reference tissue analysis method were convergent with the tissue ratio method. CONCLUSION: The results of this study support the use of [(11)C]ORM-13070 PET in the quantitative assessment of α2C-adrenoceptors in the human brain in vivo. Reliable assessment of specific tracer binding in the dorsal striatum is possible with the help of reference tissue ratios.

Validation of [(11) C]ORM-13070 as a PET tracer for alpha2c -adrenoceptors in the human brain.

This study explored the use of the α2C -adrenoceptor PET tracer [(11) C]ORM-13070 to monitor α2C -AR occupancy in the human brain. The subtype-nonselective α2 -AR antagonist atipamezole was administered to eight healthy volunteer subjects to determine its efficacy and potency (Emax and EC50 ) at inhibiting tracer uptake. We also explored whether the tracer could reveal changes in the synaptic concentrations of endogenous noradrenaline in the brain, in response to several pharmacological and sensory challenge conditions. We assessed occupancy from the bound-to-free ratio measured during 5-30 min post injection. Based on extrapolation of one-site binding, the maximal extent of inhibition of striatal [(11) C]ORM-13070 uptake (Emax ) achievable by atipamezole was 78% (95% CI 69-87%) in the caudate nucleus and 65% (53-77%) in the putamen. The EC50 estimates of atipamezole (1.6 and 2.5 ng/ml, respectively) were in agreement with the drug's affinity to α2C -ARs. These findings represent clear support for the use of [(11) C]ORM-13070 for monitoring drug occupancy of α2C -ARs in the living human brain. Three of the employed noradrenaline challenges were associated with small, approximately 10-16% average reductions in tracer uptake in the dorsal striatum (atomoxetine, ketamine, and the cold pressor test; P < 0.05 for all), but insulin-induced hypoglycemia did not affect tracer uptake. The tracer is suitable for studying central nervous system receptor occupancy by α2C -AR ligands in human subjects. [(11) C]ORM-13070 also holds potential as a tool for in vivo monitoring of synaptic concentrations of noradrenaline, but this remains to be further evaluated in future studies.

Amphetamine decreases α2C-adrenoceptor binding of [11C]ORM-13070: a PET study in the primate brain.

BACKGROUND: The neurotransmitter norepinephrine has been implicated in psychiatric and neurodegenerative disorders. Examination of synaptic norepinephrine concentrations in the living brain may be possible with positron emission tomography (PET), but has been hampered by the lack of suitable radioligands. METHODS: We explored the use of the novel α2C-adrenoceptor antagonist PET tracer [(11)C]ORM-13070 for measurement of amphetamine-induced changes in synaptic norepinephrine. The effect of amphetamine on [(11)C]ORM-13070 binding was evaluated ex vivo in rat brain sections and in vivo with PET imaging in monkeys. RESULTS: Microdialysis experiments confirmed amphetamine-induced elevations in rat striatal norepinephrine and dopamine concentrations. Regional [(11)C]ORM-13070 receptor binding was high in the striatum and low in the cerebellum. After injection of [(11)C]ORM-13070 in rats, mean striatal specific binding ratios, determined using cerebellum as a reference region, were 1.4±0.3 after vehicle pretreatment and 1.2±0.2 after amphetamine administration (0.3mg/kg, subcutaneous). Injection of [(11)C]ORM-13070 in non-human primates resulted in mean striatal binding potential (BP ND) estimates of 0.65±0.12 at baseline. Intravenous administration of amphetamine (0.5 and 1.0mg/kg, i.v.) reduced BP ND values by 31-50%. Amphetamine (0.3mg/kg, subcutaneous) increased extracellular norepinephrine (by 400%) and dopamine (by 270%) in rat striata. CONCLUSIONS: Together, these results indicate that [(11)C]ORM-13070 may be a useful tool for evaluation of synaptic norepinephrine concentrations in vivo. Future studies are required to further understand a potential contribution of dopamine to the amphetamine-induced effect.

¹¹C-ORM-13070, a novel PET ligand for brain α2C-adrenoceptors: radiometabolism, plasma pharmacokinetics, whole-body distribution and radiation dosimetry in healthy men.

PURPOSE: (11)C-labelled 1-[(S)-1-(2,3-dihydrobenzo[1,2]dioxin-2-yl)methyl]-4-(3-methoxy-methylpyridin-2-yl)-piperazine ((11)C-ORM-13070) is a novel PET tracer for imaging of α2C-adrenoceptors in the human brain. Brain α2C-adrenoceptors may be therapeutic targets in several neuropsychiatric disorders, including depression, schizophrenia and Alzheimer's disease. To validate the use of (11)C-ORM-13070 in humans, we investigated its radiometabolism, pharmacokinetics, whole-body distribution and radiation dose. METHODS: Radiometabolism was studied in a test-retest setting in six healthy men. After intravenous injection of (11)C-ORM-13070, blood samples were drawn over 60 min. Plasma samples were analysed by radio-HPLC for intact tracer and its radioactive metabolites. Metabolite-corrected plasma time-activity curves were used for calculation of pharmacokinetics. In a separate group of 12 healthy men, the whole-body distribution of (11)C-ORM-13070 and radiation exposure were investigated by dynamic PET/CT imaging without blood sampling. RESULTS: Two radioactive metabolites of (11)C-ORM-13070 were detected in human arterial plasma. The proportion of unchanged (11)C-ORM-13070 decreased from 81 ± 4 % of total radioactivity at 4 min after tracer injection to 23 ± 4 % at 60 min. At least one of the radioactive metabolites penetrated into red blood cells, while the parent tracer remained in plasma. The apparent elimination rate constant and corresponding half-life of unchanged (11)C-ORM-13070 in arterial plasma were 0.0117 ± 0.0056 min(-1) and 73.6 ± 35.8 min, respectively. The organs with the highest absorbed doses were the liver (12 µSv/MBq), gallbladder wall (12 µSv/MBq) and pancreas (9.1 µSv/MBq). The mean effective dose was 3.9 µSv/MBq, with a range of 3.6 - 4.2 µSv/MBq. CONCLUSION: (11)C-ORM-13070 was rapidly metabolized in human subjects after intravenous injection. The effective radiation dose of (11)C-ORM-13070 was in the same range as that of other (11)C-labelled brain receptor tracers. An injection of 500 MBq of (11)C-ORM-13070 would expose a subject to 2.0 mSv of radiation. This supports the use of (11)C-ORM-13070 in repeated PET scans, for example, in receptor occupancy trials with novel drug candidates.

Application of the PET ligand [11C]ORM-13070 to examine receptor occupancy by the α2C-adrenoceptor antagonist ORM-12741: translational validation of target engagement in rat and human brain.

BACKGROUND: Availability of the α2C-adrenoceptor (α2C-AR) positron emission tomography (PET) tracer, [11C]ORM-13070, and the α2C-AR antagonist ORM-12741 allows probing of the roles of this G-protein coupled receptor subtype in brain function, both in healthy humans and in patients with various brain disorders. This translational study employed [11C]ORM-13070 autoradiography and PET to determine α2C-AR occupancy by ORM-12741 in rat and human brain, respectively. RESULTS: ORM-12741 has high affinity (Ki: 0.08 nM) and potent antagonist activity (Kb: 0.04 nM) as well as selectivity (Ki estimates for the human α2A-AR and α2B-AR were 8.3 nM and 0.8 nM, respectively) for the human α2C-AR subtype. [11C]ORM-13070 had highest uptake in the basal ganglia of rat and human brain. Pretreatment with ORM-12741 inhibited [11C]ORM-13070 binding in rat striatum in a time- and dose-dependent manner at 10 and 50 µg/kg (s.c.) with an EC50 estimate of 1.42 ng/mL in rat plasma, corresponding to protein-free drug concentration of 0.23 nM. In the living human brain, time- and dose-related α2C-AR occupancy was detected with EC50 estimates of 24 ng/mL and 31 ng/mL for the caudate nucleus and putamen, respectively, corresponding to protein-free concentrations in plasma of 0.07 nM and 0.1 nM. Modelling-based maximum α2C-AR occupancy estimates were 63% and 52% in the caudate nucleus and the putamen, respectively. CONCLUSIONS: ORM-12741 is a selective α2C-AR antagonist which penetrates the rat and human brain to occupy α2C-ARs in a manner consistent with its receptor pharmacology. Trial registration number and date of registration: ClinicalTrial.cov NCT00829907. Registered 11 December 2008. https://clinicaltrials.gov/ .

Disease-modifying effect of atipamezole in a model of post-traumatic epilepsy.

Treatment of TBI remains a major unmet medical need, with 2.5 million new cases of traumatic brain injury (TBI) each year in Europe and 1.5 million in the USA. This single-center proof-of-concept preclinical study tested the hypothesis that pharmacologic neurostimulation with proconvulsants, either atipamezole, a selective α2-adrenoceptor antagonist, or the cannabinoid receptor 1 antagonist SR141716A, as monotherapy would improve functional recovery after TBI. A total of 404 adult Sprague-Dawley male rats were randomized into two groups: sham-injured or lateral fluid-percussion-induced TBI. The rats were treated with atipamezole (started at 30min or 7 d after TBI) or SR141716A (2min or 30min post-TBI) for up to 9 wk. Total follow-up time was 14 wk after treatment initiation. Outcome measures included motor (composite neuroscore, beam-walking) and cognitive performance (Morris water-maze), seizure susceptibility, spontaneous seizures, and cortical and hippocampal pathology. All injured rats exhibited similar impairment in the neuroscore and beam-walking tests at 2 d post-TBI. Atipamezole treatment initiated at either 30min or 7 d post-TBI and continued for 9 wk via subcutaneous osmotic minipumps improved performance in both the neuroscore and beam-walking tests, but not in the Morris water-maze spatial learning and memory test. Atipamezole treatment initiated at 7 d post-TBI also reduced seizure susceptibility in the pentylenetetrazol test 14 wk after treatment initiation, although it did not prevent the development of epilepsy. SR141716A administered as a single dose at 2min post-TBI or initiated at 30min post-TBI and continued for 9 wk had no recovery-enhancing or antiepileptogenic effects. Mechanistic studies to assess the α2-adrenoceptor subtype specificity of the disease-modifying effects of atipametzole revealed that genetic ablation of α2A-noradrenergic receptor function in Adra2A mice carrying an N79P point mutation had antiepileptogenic effects after TBI. On the other hand, blockade of α2C-adrenoceptors using the receptor subtype-specific antagonist ORM-12741 had no favorable effects on the post-TBI outcome. Finally, to assess whether regulation of the post-injury inflammatory response by atipametzole in glial cells contributed to a favorable outcome, we investigated the effect of atipamezole on spontaneous and/or lipopolysaccharide-stimulated astroglial or microglial cytokine release in vitro. We observed no effect. Our data demonstrate that a 9-wk administration of α2A-noradrenergic antagonist, atipamezole, is recovery-enhancing after TBI.

Structure-activity relationship of quinoline derivatives as potent and selective alpha(2C)-adrenoceptor antagonists.

Starting from two acridine compounds identified in a high-throughput screening campaign (1 and 2, Table 1), a series of 4-aminoquinolines was synthesized and tested for their properties on the human alpha(2)-adrenoceptor subtypes (alpha(2A), alpha(2B), and alpha(2C)). A number of compounds with good antagonist potencies against the alpha(2C)-adrenoceptor and excellent subtype selectivities over the other two subtypes were discovered. For example, (R)-{4-[4-(3,4-dimethylpiperazin-1-yl)phenylamino]quinolin-3-yl}methanol 6j had an antagonist potency of 8.5 nM against, and a subtype selectivity of more than 200-fold for, the alpha(2C)-adrenoceptor. Investigation of the structure-activity relationship identified a number of structural features, the most critical of which was an absolute need for a substituent in the 3-position of the quinoline ring. The 3-position on the piperazine ring was also found to play an appreciable role, as substitutions in that position exerted a significant and stereospecific beneficial effect on the alpha(2C)-adrenoceptor affinity and potency. Replacing the piperazine ring proved difficult, with 1,4-diazepanes representing the only viable alternative.

The α2C-adrenoceptor antagonist, ORM-10921, has antipsychotic-like effects in social isolation reared rats and bolsters the response to haloperidol.

Early studies suggest that selective α2C-adrenoceptor (AR)-antagonism has anti-psychotic-like and pro-cognitive properties. However, this has not been demonstrated in an animal model of schizophrenia with a neurodevelopmental construct. The beneficial effects of clozapine in refractory schizophrenia and associated cognitive deficits have, among others, been associated with its α2C-AR modulating activity. Altered brain-derived neurotrophic factor (BDNF) has been linked to schizophrenia and cognitive deficits. We investigated whether the α2C-AR antagonist, ORM-10921, could modulate sensorimotor gating and cognitive deficits, as well as alter striatal BDNF levels in the social isolation reared (SIR) model of schizophrenia, comparing its effects to clozapine and the typical antipsychotic, haloperidol, the latter being devoid of α2C-AR-activity. Moreover, the ability of ORM-10921 to augment the effects of haloperidol on the above parameters was also investigated. Animals received subcutaneous injection of either ORM-10921 (0.01mg/kg), clozapine (5mg/kg), haloperidol (0.2mg/kg), haloperidol (0.2mg/kg)+ORM-10921 (0.01mg/kg) or vehicle once daily for 14days, followed by assessment of novel object recognition (NOR), prepulse inhibition (PPI) of startle response and striatal BDNF levels. SIR significantly attenuated NOR memory as well as PPI, and reduced striatal BDNF levels vs. social controls. Clozapine, ORM-10921 and haloperidol+ORM-10921, but not haloperidol alone, significantly improved SIR-associated deficits in PPI and NOR, with ORM-10921 also significantly improving PPI deficits vs. haloperidol-treated SIR animals. Haloperidol+ORM-10921 significantly reversed reduced striatal BDNF levels in SIR rats. α2C-AR-antagonism improves deficits in cognition and sensorimotor gating in a neurodevelopmental animal model of schizophrenia and bolsters the effects of a typical antipsychotic, supporting a therapeutic role for α2C-AR-antagonism in schizophrenia.

alpha(2A)-Adrenoceptors regulate d-amphetamine-induced hyperactivity and behavioural sensitization in mice.

Stimulants, such as d-amphetamine, enhance the release of dopamine in the central nervous system (CNS) and induce locomotor activation in mice. When amphetamine is administered repeatedly, the locomotor activation is progressively increased. This behavioural sensitization may be associated with the development of drug craving, addiction and dependence. Also noradrenergic mechanisms participate in the mediation of the effects of psychostimulants. In this study we show that mice lacking the alpha(2)-adrenoceptor subtype A (alpha(2A)-AR knock-out (KO) on C57Bl/6J background) are supersensitive to the acute locomotor effects of d-amphetamine (5 mg/kg) in a novel environment compared to wild-type (WT) control mice. When both genotypes were treated repeatedly with d-amphetamine (2 mg/kg) they developed locomotor hyperactivation (sensitization), but its amplitude was lower in alpha(2A)-AR KO mice. Development of hyperactivation was reduced in both genotypes by pretreatment with the selective alpha(2)-adrenoceptor antagonist, atipamezole (1 mg/kg). Acute atipamezole also attenuated the expression of d-amphetamine-induced behavioural sensitization especially in WT mice. Interestingly, alpha(2A)-AR KO mice failed to exhibit persistent sensitization after 2 weeks of abstinence from repeated d-amphetamine. Rewarding properties of d-amphetamine, measured by conditioned place preference, were similar in both genotypes. These findings indicate that d-amphetamine-induced acute and sensitized locomotor effects are controlled by alpha(2)-adrenoceptors. Drugs antagonizing the alpha(2A)-adrenoceptor subtype may provide a novel approach for reducing drug sensitization and motor complications caused by dopaminergic agents.

Application of cross-species PET imaging to assess neurotransmitter release in brain.

RATIONALE: This review attempts to summarize the current status in relation to the use of positron emission tomography (PET) imaging in the assessment of synaptic concentrations of endogenous mediators in the living brain. OBJECTIVES: Although PET radioligands are now available for more than 40 CNS targets, at the initiation of the Innovative Medicines Initiative (IMI) "Novel Methods leading to New Medications in Depression and Schizophrenia" (NEWMEDS) in 2009, PET radioligands sensitive to an endogenous neurotransmitter were only validated for dopamine. NEWMEDS work-package 5, "Cross-species and neurochemical imaging (PET) methods for drug discovery", commenced with a focus on developing methods enabling assessment of changes in extracellular concentrations of serotonin and noradrenaline in the brain. RESULTS: Sharing the workload across institutions, we utilized in vitro techniques with cells and tissues, in vivo receptor binding and microdialysis techniques in rodents, and in vivo PET imaging in non-human primates and humans. Here, we discuss these efforts and review other recently published reports on the use of radioligands to assess changes in endogenous levels of dopamine, serotonin, noradrenaline, γ-aminobutyric acid, glutamate, acetylcholine, and opioid peptides. The emphasis is on assessment of the availability of appropriate translational tools (PET radioligands, pharmacological challenge agents) and on studies in non-human primates and human subjects, as well as current challenges and future directions. CONCLUSIONS: PET imaging directed at investigating changes in endogenous neurochemicals, including the work done in NEWMEDS, have highlighted an opportunity to further extend the capability and application of this technology in drug development.

Levosimendan alone and in combination with valsartan prevents stroke in Dahl salt-sensitive rats.

The effects of levosimendan on cerebrovascular lesions and mortality were investigated in models of primary and secondary stroke. We aimed to determine whether the effects of levosimendan are comparable to and/or cumulative with those of valsartan, and to investigate whether levosimendan-induced vasodilation has a role in its effects on stroke. In a primary stroke Dahl/Rapp rat model, mortality rates were 70% and 5% for vehicle and levosimendan, respectively. Both stroke incidence (85% vs. 10%, P<0.001) and stroke-associated behavioral deficits (7-point neuroscore: 4.59 vs. 5.96, P<0.001) were worse for vehicle compared to levosimendan. In a secondary stroke model in which levosimendan treatment was started after cerebrovascular incidences were already detected, mean survival times were 15 days with vehicle, 20 days with levosimendan (P=0.025, vs. vehicle), 22 days with valsartan (P=0.001, vs. vehicle), and 31 days with levosimendan plus valsartan (P<0.001, vs. vehicle). The respective survivals were 0%, 16%, 20% and 59%, and the respective incidences of severe lesions were 50%, 67%, 50% and 11%. In this rat model, levosimendan increased blood volume of the cerebral vessels, with significant effects in the microvessels of the cortex (∆R=3.5±0.15 vs. 2.7±0.17ml for vehicle; P=0.001) and hemisphere (∆R=3.2±0.23 vs. 2.6±0.14ml for vehicle; P=0.018). Overall, levosimendan significantly reduced stroke-induced mortality and morbidity, both alone and with valsartan, with apparent cumulative effects, an activity in which the vasodilatory effects of levosimendan have a role.

Alpha2A-adrenoceptors are important modulators of the effects of D-amphetamine on startle reactivity and brain monoamines.

Amphetamines are commonly used to treat attention-deficit hyperactivity disorder, but are also widely abused. They are employed in schizophrenia-related animal models as they disrupt the prepulse inhibition (PPI) of the acoustic startle response. The behavioral effects of amphetamines have mainly been attributed to changes in dopamine transmission, but they also involve increases in the synaptic concentrations of norepinephrine (NE). alpha2-Adrenoceptors (alpha2-ARs) regulate the excitability and transmitter release of brain monoaminergic neurons mainly as inhibitory presynaptic auto- and heteroreceptors. Modulation of acoustic startle and its PPI by the alpha2A-AR subtype was investigated with mice lacking the alpha2A-AR (alpha2A-KO) and their wild-type (WT) controls, without drugs and after administration of the alpha2-AR agonist dexmedetomidine or the antagonist atipamezole. The interaction of D-amphetamine (D-amph) and the alpha2-AR-noradrenergic neuronal system in modulating startle reactivity and in regulating brain monoamine metabolism was assessed as the behavioral and neurochemical responses to D-amph alone, or to the combination of D-amph and dexmedetomidine or atipamezole. alpha2A-KO mice were supersensitive to both neurochemical and behavioral effects of D-amph. Brain NE stores of alpha2A-KO mice were depleted by D-amph, revealing the alpha2A-AR as essential in modulating the actions of D-amph. Also, increased startle responses and more pronounced disruption of PPI were noted in D-amph-treated alpha2A-KO mice. alpha2A-AR also appeared to be responsible for the startle-modulating effects of alpha2-AR drugs, since the startle attenuation after the alpha2-AR agonist dexmedetomidine was absent in alpha2A-KO mice, and the alpha2-AR antagonist atipamezole had opposite effects on the startle reflex in alpha2A-KO and WT mice.

Alpha2-adrenergic drug effects on brain monoamines, locomotion, and body temperature are largely abolished in mice lacking the alpha2A-adrenoceptor subtype.

alpha(2)-ARs regulate brain monoaminergic function by inhibiting neuronal firing and release of monoamine neurotransmitters, noradrenaline (NA), serotonin (5-HT) and dopamine (DA). Both alpha(2A)- and alpha(2C)-AR inhibit monoamine release in vitro in brain slices, but the in vivo roles of individual alpha(2)-AR subtypes in modulating monoamine metabolism have not been characterised. Metabolism of brain monoamine neurotransmitters, locomotor activity and body temperature were investigated in mice with targeted inactivation of the gene encoding alpha(2A)-AR (alpha(2A)-knockout, alpha(2A)-KO) and wild-type (WT) mice after treatment with the alpha(2)-AR agonist dexmedetomidine and the antagonist atipamezole. Dexmedetomidine caused profound hypothermia (up to 14.7 degrees C mean reduction in rectal temperature) and locomotor inhibition in WT mice, and inhibited the turnover of NA, 5-HT and DA, but increased NA turnover in alpha(2A)-KO mice. alpha(2)-AR agonist-induced hypothermia and locomotor inhibition were attenuated, but not totally abolished, in alpha(2A)-KO mice. These results suggest that alpha(2A)-ARs are principally responsible for the alpha(2)-AR mediated inhibition of brain monoamine metabolism, but other alpha(2)-ARs, possibly alpha(2C)-ARs, are also involved, especially in the striatum. However, secondary effects of the physiological alterations caused by drug administration, especially hypothermia, may have contributed to the observed neurochemical changes in WT mice.

The alpha2-adrenoceptor antagonist atipamezole reduces the development and expression of d-amphetamine-induced behavioural sensitization.

The possible effect of atipamezole, a potent and specific alpha(2)-adrenoceptor antagonist, on the development and expression of d-amphetamine-induced behavioural sensitization was evaluated in mice. Male (C57Bl/6J) mice were given daily doses of d-amphetamine (2 mg/kg). In addition, groups of mice received injections of atipamezole (0.3 or 1 mg/kg) 20 min before d-amphetamine or vehicle administration. Idazoxan (1 mg/kg) was used in some experiments to extend the results to other alpha(2)-adrenoceptor antagonists. Challenge doses of d-amphetamine were administered to the mice on days 7-9 to evaluate the effects of alpha(2)-adrenoceptor antagonists on the d-amphetamine sensitization, evidenced by increased locomotor activation. Mice treated repeatedly with d-amphetamine developed strong locomotor sensitization that was reduced by pretreatment with alpha(2)-adrenoceptor antagonists. Acute atipamezole at both doses attenuated the expression of d-amphetamine-induced sensitization. Atipamezole at 1 mg/kg alone had no effect on locomotor activity, but the lower dose (0.3 mg/kg) increased locomotor activity after repeated administration. These results indicate that alpha(2)-adrenoceptor antagonists modulate the actions of d-amphetamine in a manner not explicable by their enhancing actions on noradrenaline and dopamine release, and may thus provide a novel approach to the treatment of motor complications caused by dopaminergic agents, such as dyskinesias, and perhaps also drug dependence.

Comparison of deramciclane to benzodiazepine agonists in behavioural activity of mice and in alcohol drinking of alcohol-preferring rats.

Interactions between alcohol and traditional benzodiazepine anxiolytics hamper the treatment of alcoholism-related anxiety disorders. Serotonin 5-HT(2) receptor antagonists, such as deramciclane, are anxiolytic, and considering their pharmacological profile, they might benefit alcoholics with comorbid anxiety. We studied the effects of acute deramciclane (1, 3 and 10 mg/kg i.p.) on alcohol drinking of alcohol-preferring AA rats drinking 10% (vol/vol) ethanol solution in a 4-h limited-access paradigm. Thereafter, a 5-day repeated-treatment experiment was carried out, under corresponding test design, with deramciclane (3 mg/kg i.p.) as a test drug and midazolam (1 mg/kg i.p.) as a benzodiazepine reference compound. Deramciclane had no effect on alcohol consumption in either acute or repeated dosing study. Midazolam increased ethanol drinking, as expected, when administered on successive days. A modified functional observational battery (FOB) procedure was applied to study neurological, behavioural and autonomic effects induced by deramciclane (1-30 mg/kg po) and diazepam (1-30 mg/kg po) in mice at 30 min, 2 h and 4 h after dosing. Deramciclane had a mild dopamine D(2) receptor antagonism-like effect at the highest dose. The effects of diazepam were predictable, myorelaxation-induced motor impairment and anxiolysis-related hyperlocomotion in a novel environment being the characteristic features at the two highest doses. Deramciclane appears to be a safe and well-tolerated drug and we suggest that it might be useful in the treatment of anxiety in alcoholics.

A PET Tracer for Brain α2C Adrenoceptors, (11)C-ORM-13070: Radiosynthesis and Preclinical Evaluation in Rats and Knockout Mice.

UNLABELLED: We report the development of a PET tracer for α2C adrenoceptor imaging and its preliminary preclinical evaluation. α2C adrenoceptors in the human brain may be involved in various neuropsychiatric disorders, such as depression, schizophrenia, and neurodegenerative diseases. PET tracers are needed for imaging of this receptor system in vivo. METHODS: High-specific-activity (11)C-ORM-13070 (1-[(S)-1-(2,3-dihydrobenzo[1,4]dioxin-2-yl)methyl]-4-(3-(11)C-methoxymethylpyridin-2-yl)-piperazine) was synthesized by (11)C-methylation of O-desmethyl-ORM-13070 with (11)C-methyl triflate, which was prepared from cyclotron-produced (11)C-methane via (11)C-methyl iodide. Rats and mice were investigated in vivo with PET and ex vivo with autoradiography. The specificity of (11)C-ORM-13070 binding to α2 adrenoceptors was demonstrated in rats pretreated with atipamezole, an α2 adrenoceptor antagonist. The α2C adrenoceptor selectivity of the tracer was determined by comparing tracer binding in wild-type and α2A- and α2AC adrenoceptor knockout (KO) mice. (11)C-ORM-13070 and its radioactive metabolites in rat plasma and brain tissue were analyzed with radio-high-performance liquid chromatography and mass spectroscopy. Human radiation dose estimates were extrapolated from rat biodistribution data. RESULTS: The radiochemical yield, calculated from initial cyclotron-produced (11)C-methane, was 9.6% ± 2.7% (decay-corrected to end of bombardment). The specific activity of the product was 640 ± 390 GBq/µmol (decay-corrected to end of synthesis). The radiochemical purity exceeded 99% in all syntheses. The highest levels of tracer binding were observed in the striatum and olfactory tubercle of rats and control and α2A KO mice-that is, in the brain regions known to contain the highest densities of α2C adrenoceptors. In rats pretreated with atipamezole and in α2AC KO mice, (11)C tracer binding in the striatum and olfactory tubercle was low, similar to that of the frontal cortex and thalamus, regions with low densities of α2C adrenoceptors. Two radioactive metabolites were found in rat plasma, but only one of them was found in the brain; their identity was not revealed. The estimated effective radiation dose was comparable with the average exposure level in PET studies with (11)C tracers. CONCLUSION: An efficient method for the radiosynthesis of (11)C-ORM-13070 was developed. (11)C-ORM-13070 emerged as a potential novel radiotracer for in vivo imaging of brain α2C adrenoceptors.

Pharmacological characterisation of a structurally novel α2C-adrenoceptor antagonist ORM-10921 and its effects in neuropsychiatric models.

The α2-adrenoceptors (ARs) are important modulators of a wide array of physiological responses. As only a few selective compounds for the three α2-AR subtypes (α2A , α2B and α2C ) have been available, the pharmacological profile of a new α2C-selective AR antagonist ORM-10921 is reported. Standard in vitro receptor assays and antagonism of α2, and α1-AR agonist-evoked responses in vivo were used to demonstrate the α2C-AR selectivity for ORM-10921 which was tested in established behavioural models related to schizophrenia and cognitive dysfunction with an emphasis on pharmacologically induced hypoglutamatergic state by phencyclidine or MK-801. The Kb values of in vitro α2C-AR antagonism for ORM-10921 varied between 0.078-1.2 nM depending on the applied method. The selectivity ratios compared to α2A-AR subtype and other relevant receptors were 10-100 times in vitro. The in vivo experiments supported its potent α2C-antagonism combined with only a weak α2A-antagonism. In the pharmacodynamic microdialysis study, ORM-10921 was found to increase extracellular dopamine levels in prefrontal cortex in the baseline conditions. In the behavioural tests, ORM-10921 displayed potent antidepressant and antipsychotic-like effects in the forced swimming test and prepulse-inhibition models analogously with the previously reported results with structurally different α2C-selective AR antagonist JP-1302. Our new results also indicate that ORM-10921 alleviated the NMDA-antagonist-induced impairments in social behaviour and watermaze navigation. This study extends and further validates the concept that α2C -AR is a potential therapeutic target in CNS disorders such as schizophrenia or Alzheimer's disease and suggests the potential of α2C-antagonism to treat such disorders.

Autoradiographic characterization of alpha(2C)-adrenoceptors in the human striatum.

Indirect experimental evidence suggests that drugs acting on the alpha(2C)-adrenoceptor could be useful in the treatment of neuropsychiatric disorders such as depression and schizophrenia. In rodent brain, the highest levels of alpha(2C)-adrenoceptors are found in the striatum, with lower levels in cerebral cortex and hippocampus. In human brain, because of the poor subtype-selectivity of the available alpha(2)-adrenoceptor ligands, the localization of alpha(2C)-adrenoceptors has remained unknown. Recently, a selective alpha(2C)-adrenoceptor antagonist, JP-1302, was characterized, and to assess the presence of alpha(2C)-adrenoceptors in human brain, we performed competition binding in vitro receptor autoradiography with JP-1302 and the alpha(2)-adrenoceptor subtype nonselective antagonist [ethyl-(3)H]RS79948-197 on rat and human postmortem brain sections. In striatum of both species, JP-1302 vs. [ethyl-(3)H]RS79948-197 competition binding was biphasic, identifying high- and low-affinity binding sites, whereas in cortex and cerebellum, only low-affinity binding sites were detected. The results indicate that a significant portion of the alpha(2)-adrenoceptors in striatum is of the alpha(2C) subtype, whereas non-alpha(2C)-adreocneptors predominate in cortex and cerebellum. Because the alpha(2C)-adrenoceptor subtype distribution pattern appears to be conserved between rodents and humans, results obtained from studies on the role of the alpha(2C)-adrenoceptor in rodent models of neuropsychiatric disorders may be relevant also for human diseases.