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Old and new results show that both catechol-O-methyltransferase (COMT) forms are found in all mouse tissues, demonstrating that COMT is a ubiquitous enzyme. Some novel findings are obvious when considering differences between old and new distribution data. In addition to the brain, membrane-bound form of COMT (MB-COMT) is found also in most peripheral mouse tissues at about equal amounts as soluble form of COMT (S-COMT), suggesting that their functions do not need to be very different. There are large differences between the species in the relative distribution of S-COMT and MB-COMT. According to the new data, it is evident that even in the animal tissues MB-COMT is not associated with the plasma membranes but with intracellular membranes, and that S-COMT resides not only in the cytoplasm but even in the nucleus., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In animals, different types of COMT inhibitors, irrespective of their brain penetration, are pro-nociceptive in several models of acute and inflammatory pain. Similarly, COMT knock-out mice are more sensitive to nociceptive stimuli, whereas in mice over-expressing a high activity COMT variant nociceptive sensitivity is decreased. COMT knock-out mice also show altered response to opioids and stress-induced analgesia. In different rat models of neuropathic pain, the action of nitecapone is opposite: it is antinociceptive and antiallodynic. Complex actions of low COMT activity may be caused by enhanced adrenergic and dopaminergic activities that play different and even contrasting roles at different parts of the nociceptive system. Also compensatory changes in other neurotransmitters may occur. Pro-nociceptive effects seem to be caused by increased activation of peripheral adrenergic β(2)- and β(3) -receptors. Other properties of COMT inhibitors, like scavenging of oxygen and nitrogen radicals, may be important in antiallodynic effects found in neuropathic pain models. Increased number of µ-opioid receptors in certain brain areas may be responsible of enhanced opioid effects associated with a low COMT activity. In human pain studies, a low COMT activity is often associated with increased pain sensitivity in experimental pain models and with increased pre- and postoperative pain in acute clinical situations. As a rule, a simultaneous occurrence of several SNPs within the haplotype, causing low COMT activity, is more often associated with pain than any single SNP alone. In experimental pain studies, all negative findings resulted from concentrating solely on SNP rs4680 (Val158Met). Virtually all studies assessing haplotypes were able to confirm an association of a low COMT and increased pain. In chronic clinical pain, the effect of COMT polymorphisms depends on the pain conditions. Hence, in neuropathic and cancer pains, COMT activity is meaningless but in some chronic musculoskeletal pain conditions and migraine or headache low COMT activity appears to increase incidence and symptoms. A low COMT activity also increases availability of opioid receptors and may enhance opioid analgesia and adverse effects at least in cancer pains., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Duplication of human chromosome 22q11.2 is associated with elevated rates of mental retardation, autism and many other behavioral phenotypes. However, because duplications cover 1.5-6 Mb, the precise manner in which segments of 22q11.2 causally affect behavior is not known in humans. We have now determined the developmental impact of over-expression of an approximately 190 kb segment of human 22q11.2, which includes the genes TXNRD2, COMT and ARVCF, on behaviors in bacterial artificial chromosome (BAC) transgenic (TG) mice. BAC TG mice and wild-type (WT) mice were tested for their cognitive capacities, affect- and stress-related behaviors and motor activity at 1 and 2 months of age. An enzymatic assay determined the impact of BAC over-expression on the activity level of COMT. BAC TG mice approached a rewarded goal faster (i.e. incentive learning), but were impaired in delayed rewarded alternation during development. In contrast, BAC TG and WT mice were indistinguishable in rewarded alternation without delays, spontaneous alternation, prepulse inhibition, social interaction, anxiety-, stress- and fear-related behaviors and motor activity. Compared with WT mice, BAC TG mice had an approximately 2-fold higher level of COMT activity in the prefrontal cortex, striatum and hippocampus. These data suggest that over-expression of this 22q11.2 segment enhances incentive learning and impairs the prolonged maintenance of working memory, but has no apparent effect on working memory per se, affect- and stress-related behaviors or motor capacity. High copy numbers of this 22q11.2 segment might contribute to a highly selective set of phenotypes in learning and cognition during development.

Catechol-O-methyl transferase (COMT) methylates catechols, such as L-dopa and dopamine, and COMT deficient mice show dramatic shifts in the metabolite levels of catechols. Increase in catechol metabolite levels can, in principle, lead to oxidative stress but no indices of oxidative stress have been reported in COMT-knockout (KO) mice [Forsberg MM, Juvonen RO, Helisalmi P, Leppanen J, Gogos JA, Karayiorgou M, et al. Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)-deficient mice. Naunyn Schmiedebergs Arch Pharmacol 2004;370:279-89.]. Here we perform a proteomic based analysis of the livers of COMT-KO mice in search for potential compensatory mechanisms developed to cope with the effects of disrupted catechol metabolism. We found sex specific changes in proteins connected to stress response. Our results show that alterations in protein levels contribute to the homeostatic regulation in the liver of COMT deficient mice.

Catechol-O-methyltransferase (COMT) polymorphisms modulate pain and opioid analgesia in human beings. It is not clear how the effects of COMT are mediated and only few relevant animal studies have been performed. Here, we used old male Comt gene knock-out mice as an animal model to study the effects of COMT deficiency on nociception that was assessed by the hot plate and tail flick tests. Stress-induced analgesia was achieved by forced swim. Morphine antinociception was measured after 10 mg/kg of morphine subcutaneously. Morphine tolerance was produced with subcutaneous morphine pellets and withdrawal provoked with subcutaneous naloxone. In the hot plate test, morphine-induced antinociception was significantly greater in the COMT knock-out mice, compared to the wild-type mice. This may be due to increased availability of opioid receptors as suggested by previous human studies. In the tail flick test, opioid-mediated stress-induced analgesia was absent and morphine-induced analgesia was decreased in COMT knock-out mice. In the hot plate test, stress-induced analgesia developed to all mice regardless of the COMT genotype. There were no differences between the genotypes in the baseline nociceptive thresholds, morphine tolerance and withdrawal. Our findings show, for the first time, the importance of COMT activity in stress- and morphine-induced analgesia in mice. COMT activity seems to take part in the modulation of nociception not only in the brain, as suggested earlier, but also at the spinal/peripheral level.

Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. Although POP has been considered to be a soluble cytoplasmic peptidase, significant levels of activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. Here we isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. The kinetic properties of detergent-soluble mPOP are fully comparable to those of POP; however, when attached to the membranes in its natural conformation, mPOP is significantly less active and, moreover, it migrates anomalously in SDS/PAGE. Our results are the first to show that membrane-bound and cytoplasmic POP are encoded by variants of the same gene.

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes proline-containing peptides shorter than 30-mer. It has been suggested that POP is associated with cognitive functions and inositol 1,4,5-triphosphate (IP(3)) signaling. However, little is known about the distribution and physiological role of POP in the brain. We used immunohistochemistry to determine the cellular and subcellular distribution of POP in the rat brain. POP was specifically expressed in the glutamatergic pyramidal neurons of the cerebral cortex, particularly in the primary motor and somatosensory cortices, and also in the CA1 field of hippocampus. Purkinje cells of the cerebellum were also intensively immunostained for POP. Double immunofluorescence indicated that POP was present in the gamma-aminobutyric acid (GABA)ergic and cholinergic interneurons of the thalamus and cortex but not in the nigrostriatal dopaminergic neurons. POP did not colocalize with astrocytic markers in any part of the rat brain. We used postembedding immunoelectron microscopy to determine the distribution of POP at the subcellular level. POP was mainly present in neuronal cytosol and membranes, hardly at all in neuronal plasma membrane, but more extensively in intracellular membranes such as the rough endoplasmic reticulum and Golgi apparatus. Our findings point to a role for POP--evidently modifying neuropeptide levels--in excitatory and inhibitory neurotransmission in the central nervous system via glutamatergic, GABAergic, and cholinergic neurotransmission systems. Furthermore, according to our results, POP may be involved in thalamocortical neurotransmission, memory and learning functions of the hippocampal formation, and GABAergic regulation of voluntary movements. Subcellular distribution of POP points to a role in protein processing and secretion., ((c) 2008 Wiley-Liss, Inc.)

A unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease was used to determine an effective dose to abolish circling behaviour of the continuous intrastriatal infusions of L-dopa via osmotic minipumps into the lesioned striatum. This 2-week L-dopa treatment evoked a dose-dependent decrease in the contralateral rotations induced by acute intraperitoneal L-dopa and carbidopa that was sustained at least for 10 weeks. The minimum effective dose of intrastriatal L-dopa was 3 microg/hr. Striatal [3H]-spiperone binding was significantly increased by the 6-OHDA lesion, reflecting a permanent, lesion-induced, up-regulation of dopamine D2 receptors. Furthermore, striatal dopamine and its metabolites as well as the level of tyrosine hydroxylase were significantly reduced by 6-OHDA. None of these parameters were restored by the 2-week L-dopa infusions but, unexpectedly, the rotational response did not become normalized after discontinuation of L-dopa infusions. Nigral 6-OHDA lesions suppressed ipsilateral striatal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations, and 5-HT uptake sites while tryptophan hydroxylase was not changed in the striatum. When studying the cause of the sustained circling behaviour, we found that intrastriatal L-dopa infusion dose-dependently elevated striatal tryptophan hydroxylase much above the levels of intact side and 5-HT uptake sites to the level of intact side, but the striatal 5-HT levels exhibited no significant recovery while 5-HIAA levels were partially restored. These data support the view that a long-term ipsilateral activation of the serotonergic innervation occurs after L-dopa infusions into the lesioned striata.

In the present study, we assessed the effect of single graded doses of a putative anxiolytic compound, the 5-HT(2A/C )antagonist, deramciclane fumarate (EGIS-3886), on the dopamine efflux and metabolism in nucleus accumbens and striatum and thus evaluated the dose window for deramciclane to cause adverse effects related to the brain dopaminergic system. Dual probe in vivo microdialysis in freely moving rats was used to compare the effects of graded doses of deramciclane fumarate (3, 10 and 30 mg/kg), 5-HT(2A/C )antagonist ritanserin (1 mg/kg) and a partial 5-HT(1A) agonist buspirone hydrochloride (5 mg/kg) on the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in nucleus accumbens and striatum assayed by high performance liquid chromatography with electrochemical detection. The indirect dopamine agonist, D-amphetamine sulfate (2 mg/kg), was used as a positive control. Ritanserin, buspirone and deramciclane 3 and 10 mg/kg had no significant effects on the extracellular dopamine levels in either brain area but deramciclane 30 mg/kg significantly increased accumbal dopamine as well as DOPAC and HVA in both brain areas. As expected, the positive control D-amphetamine significantly increased both striatal and accumbal dopamine levels. The effects of buspirone or the highest deramciclane dose and D-amphetamine on DOPAC and HVA levels were opposite; buspirone and deramciclane increased while D-amphetamine decreased the metabolite levels in both brain areas. The results indicate that a single high dose of deramciclane has the neuroleptic- or buspirone-like effect, particularly in mesolimbic regions. There is at least a 5-fold margin between the anxiolytic and neuroleptic doses of deramciclane in the rat.

Accumulating evidence from clinical and preclinical studies shows that catechol-O-methyltransferase (COMT) plays a significant role in dopamine metabolism in the prefrontal cortex, but not in the striatum. However, to what extent dopamine overflow in the prefrontal cortex and striatum is controlled by enzymatic degradation versus reuptake is unknown. We used COMT deficient mice to investigate the role of COMT in these two brain regions with in vivo voltammetry. A real-time analysis of evoked dopamine overflow showed that removal of dopamine was twofold slower in the prefrontal cortex of mice lacking COMT than in wild-type mice, indicating that half of the dopamine decline in this brain region results from COMT-mediated enzymatic degradation. Lack of COMT did not influence dopamine overflow/decline in the dorsal striatum. COMT-deficient mice demonstrated a small (20-25%) but consistent increase in evoked dopamine release in the prefrontal cortex, but not in the dorsal striatum. Cocaine affected equally dopaminergic neurotransmission in the prefrontal cortex in both genotypes by prolonging 3-4 times dopamine elimination from extracellular space. Paradoxically, this happened without increase of the peak levels of evoked dopamine release. The present findings represent the first demonstration of the significant contribution of COMT in modulating the dynamics of dopamine overflow in the prefrontal cortex and underscore the therapeutic potential of manipulating COMT activity to alter dopaminergic neurotransmission in the prefrontal cortex.

Prolyl oligopeptidase (POP) is a serine endoprotease that hydrolyses peptides shorter than 30-mer. POP may have a role in inositol 1,4,5-triphosphate (IP(3)) signaling and in the actions of antidepressants, and POP inhibitors have exhibited antiamnesic and neuroprotective properties. However, little is known about the distribution of POP protein in the brain. We used immunohistochemistry to localize POP enzyme in the human whole hemisphere and in the rat whole brain. In humans, the highest POP densities were observed in caudate nucleus and putamen, hippocampus and cortex. In the rat, the highest POP densities were found in substantia nigra, hippocampus, cerebellum and caudate putamen. In general, the distribution of POP in human and rat brains was very similar and resembled that of IP(3) receptors. Our findings are support for a role of POP in movement regulation, cognition and possibly in IP(3) signaling. The expression of POP in processing nuclei further supports its function beyond neuropeptide metabolism.

Background: In the CNS, the heterotrimeric G protein Galphai2 is a minor Galpha subunit with restricted localization in the ventricular regions including the ependymal cilia. The localization of Galphai2 is conserved in cilia of different tissues, suggesting a particular role in ciliary function. Although studies with Galphai2-knockout mice have provided information on the role of this Galpha subunit in peripheral tissues, its role in the CNS is largely unknown. We used intracerebroventricular (icv) antisense administration to clarify the physiological role of Galphai2 in the ventricular system., Results: High resolution MRI studies revealed that continuous icv-infusion of Galphai2-specific antisense oligonucleotide caused unilateral ventricular dilatation that was restricted to the antisense-receiving ventricle. Microscopic analysis demonstrated ependymal cell damage and loss of ependymal cilia. Attenuation of Galphai2 in ependymal cells was confirmed by immunohistochemistry. Ciliary beat frequency measurements on cultured ependymal cells indicated that antisense administration resulted in ciliary stasis., Conclusion: Our results establish that Galphai2 has an essential regulatory role in ciliary function and CSF homeostasis.

A series of dicarboxylic acid azacycle l-prolyl-pyrrolidine amides was synthesized, and their inhibitory activity against prolyl oligopeptidase (POP) from porcine brain was tested. Three different azacycles were tested at the position beyond P3 and six different dicarboxylic acids at the P3 position. l-Prolyl-pyrrolidine and l-prolyl-2(S)-cyanopyrrolidine were used at the P2-P1 positions. The IC(50) values ranged from 0.39 to 19000 nM. The most potent inhibitor was the 3,3-dimethylglutaric acid azepane l-prolyl-2(S)-cyanopyrrolidine amide. Molecular docking (GOLD) was used to analyze binding interactions between different POP inhibitors of this type and the POP enzyme. The data set consisted of the novel inhibitors, inhibitors published previously by our group, and well-known reference compounds. The alignments were further analyzed using comparative molecular similarity indices analysis. The binding of the inhibitors was consistent at the P1-P3 positions. Beyond the P3 position, two different binding modes were found, one that favors lipophilic structures and one that favors nonhydrophobic structures.

Dopamine supersensitivity occurs in schizophrenia and other psychoses, and after hippocampal lesions, antipsychotics, ethanol, amphetamine, phencyclidine, gene knockouts of Dbh (dopamine beta-hydroxylase), Drd4 receptors, Gprk6 (G protein-coupled receptor kinase 6), Comt (catechol-O-methyltransferase), or Th-/-, DbhTh/+ (tyrosine hydroxylase), and in rats born by Cesarean-section. The functional state of D2, or the high-affinity state for dopamine (D2High), was measured in these supersensitive animal brain striata. Increased levels and higher proportions (40-900%) for D2High were found in all these tissues. If many types of brain impairment cause dopamine behavioral supersensitivity and a common increase in D2High states, it suggests that there are many pathways to psychosis, any one of which can be disrupted.

The effect of catechol-O-methyltransferase (COMT) deficiency on methamphetamine-induced hydroxyl radical production in the brain was assessed by the salicylate trapping method. Methamphetamine-induced hyperthermia was also studied. Furthermore, the effect of COMT deficiency on the activities of glutathione S-transferase, quinone reductase and liver mono-oxygenases was assessed with and without l-dopa challenge. Finally, two alternative pathways of l-dopa metabolism were evaluated. Methamphetamine increased 2,3-dihydroxybenzoic acid levels only slightly (n.s.) at the lowest dose level (2.5 mg/kg x 4 i.p.). This was accompanied by a simultaneous increase in salicylate levels so that the 2,3-dihydroxybenzoic acid/salicylate ratio decreased correspondingly. Most importantly, no COMT genotype-dependent changes were observed. However, hyperthermia was induced even at the lowest methamphetamine dose, the COMT-deficient mice being most sensitive. COMT deficiency did not significantly change the activities of liver glutathione S-transferase, quinone reductase or 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylation. In COMT-deficient female mice, l-dopa (30-80 mg/kg b.i.d. for 2 days) did not induce any significant changes in liver or brain glutathione S-transferase and quinone reductase activity or liver 7-ethoxyresorufin O-deethylation activity. The levels of l-dopa conjugates in urine were also negligible in COMT-deficient mice. Skin tyrosinase activity was increased in 7- to 8-day-old hairless COMT-deficient pups. The present results suggest that despite the increased hyperthermic response, COMT deficiency does not increase methamphetamine-induced hydroxyl radical production or change significantly the activity of certain enzymes involved in defense against reactive oxygen species. In conclusion, we found no evidence of increased oxidative stress in the liver or brain of adult mice lacking COMT activity.

In order to assess the importance of the chronic increase in precursor availability on central histaminergic mechanisms in rats, nine male Wistar rats received L-histidine orally at a dose of 1000 mg/kg, twice daily (07.00 h and 19.00 h) for 1 week; 9 rats were used as controls. Brain tissue histamine and tele-methylhistamine levels, as well as plasma histamine concentration were assayed. Binding properties and regional distribution of the autoregulatory histamine H3 receptors in brain were studied with [3H]-R-alpha-methylhistamine receptor binding and autoradiography. In L-histidine loaded rats, tissue histamine levels in cortex, hypothalamus, and rest of the brain were significantly increased by 40%-70%. Histamine concentrations in cerebellum and plasma, and tele-methylhistamine concentrations in cortex and hypothalamus did not change. The binding properties of H3 receptors in cortex were not altered. However, there were changes in the regional distribution of [3H]-R-alpha-methylhistamine binding sites, suggestive of a region-selective up-/down-regulation of histamine H3 receptors or their receptor sub-types. These results imply that following repeated L-histidine administration in the rat (1) there is enhanced synthesis of brain histamine not reflected in its functional release; (2) the excess of histamine is sequestered and stored rather than being metabolized; (3) histamine H(3) receptor binding properties are not altered, whereas receptor density is changed in selected regions. In conclusion, these results demonstrate that the neuronal mechanisms controlling histamine synthesis, storage, and release are adaptable and allow the sequestration of the excess of histamine in order to prevent excessively high neuronal activity.

The effect of cytosine arabinoside (Ara-C) on cell viability has been studied in African green monkey kidney fibroblasts (CV1-P). It has been shown previously that Ara-C- induced cell death in neurons is mediated by apoptosis. We investigated whether Ara-C can induce apoptosis also in CV1-P cells, and if the apoptosis is p53-associated. For comparison, human neuroblastoma cells (SH-SY5Y) were studied as a model of human neuronal cells. SYTO13/propidium iodide staining revealed condensed and fragmented nuclei in both cell lines. Ara-C treatment for 48 h induced approximately 24% apoptosis in CV1-P cells whereas approximately 55% of SH-SY5Y cells were apoptotic. Ara-C increased the level of p53 in both CV1-P and SH-SY5Y cells compared to control. The maximum level of p53 in SH-SY5Y cells was reached at 12 h and this then rapidly faded whereas CV1-P cells p53 levels remained elevated after reaching their maximum. Caspase-3 activity was 5-fold higher in human neuroblastoma cells than in monkey fibroblasts, this reflected the decreased cell viability. Our results prove that Ara-C- induced apoptosis in CV1-P cells is associated with an increase of p53 and activation of caspase-3. Ara-C-induced toxicity in CV1-P cells is modest compared to that seen in neuronal cells.

New dicarboxylic acid bis(L-prolyl-pyrrolidine) amides were synthesized, and their inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. As compared with earlier described prolyl oligopeptidase inhibitors, these new compounds have in common an L-prolyl-pyrrolidine moiety, but the typical lipophilic acyl end group is replaced by another L-prolyl-pyrrolidine moiety connected symmetrically with a short dicarboxylic acid linker. These compounds are a new type of peptidomimetic prolyl oligopeptidase inhibitor.

Background: The intrarenal natriuretic hormone dopamine (DA) is metabolised by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). Inhibition of COMT, as opposed to MAO, results in a potent natriuretic response in the rat. The present study in anaesthetized homozygous and heterozygous COMT gene deleted mice attempted to further elucidate the importance of COMT in renal DA and sodium handling. After acute intravenous isotonic sodium loading, renal function was followed., Results: COMT activity in heterozygous mice was about half of that in wild type mice and was zero in the homozygous mice. MAO activity did not differ between the genotypes. Urinary sodium excretion increased 10-fold after sodium loading in wild type mice. In heterozygous and homozygous mice, the natriuretic effects of sodium loading were only 29 % and 39 %, respectively, of that in wild type mice. Arterial pressure and glomerular filtration rate did not differ between genotypes. Baseline norepinephrine and DA excretions in urine were elevated in the homozygous, but not in heterozygous, COMT gene deleted mice. Urinary DA excretion increased after isotonic sodium loading in the wild type mice but not in the COMT gene deleted mice., Conclusions: Mice with reduced or absent COMT activity have altered metabolism of catecholamines and are unable to increase renal DA activity and produce normal natriuresis in response to acute sodium loading. The results support the hypothesis that COMT has an important role in the DA-mediated regulation of renal sodium excretion.

The aim of this in vivo study was to assess the effect of improved oral bioavailability of entacapone on its actual pharmacodynamic response, COMT inhibition in erythrocytes. Rats were administered entacapone orally as a suspension, as a plain solution, an entacapone/HP-beta-CD solution, two N-alkyl-carbamate ester prodrugs and intravenously as a solution. Also the relationship between pharmacodynamic and pharmacokinetic responses of entacapone was investigated. The administration of entacapone as a solution (plain solution pH 7.4; F=34.8% or entacapone/HP-beta-CD solution pH 3.0; F = 18.5%) resulted in significantly higher degree of COMT inhibition in erythrocytes than could be achieved by administering entacapone as a suspension (pH 3.0; F=8.9%). The inhibitory Emax model did not reveal any significant differences in EC50 estimates of entacapone suspension, entacapone/HP-beta-CD solution or entacapone solution. The overall pharmacodynamic response of entacapone (AUE; area under effect-time curve) was dependent on the pharmacokinetic response (AUC; area under concentration-time curve) irrespective of the entacapone formulation and dosage form. However, this dependency did not extend to formulations producing very high peak concentrations of entacapone in plasma; high plasma concentrations reached transiently after administration of entacapone solution had only a minor effect on the overall pharmacodynamic response (AUE). The inhibitory Emax model revealed that a plateau of COMT inhibition near to Emax is attained by plasma concentrations under 2000 ng/ml, irrespective of the formulation. This supports the results concerning the dependence of AUE on AUC.