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In the 1980s, Orion Pharma, then a mid-ranking Nordic area pharmaceutical company, established a drug development programme on the inhibition of catechol O-methyltransferase (COMT). This enzyme, which plays an important role in the inactivation of catecholamine neurotransmitters and drugs with a catechol structure, thus came under consideration as a target in the innovative translational and clinical programme we describe in this historical review. The starting point was the conjecture that a peripherally acting COMT inhibitor might improve entry of levodopa into the brain. This had potentially significant implications for the medical treatment of Parkinson's disease (PD). The rationale was that more efficient delivery of levodopa to the brain might allow the high therapeutic doses of levodopa to be reduced and the dose interval to be extended. Elucidation of structure–activity relations paved the way for the discovery and development of entacapone, a 5-nitrocatechol that was a potent and highly specific inhibitor of COMT. Experience in phase III clinical trials established that entacapone, used as an adjunct to regular or controlled-release levodopa preparations (also including a peripherally acting dopa-decarboxylase inhibitor), increased ON-time and reduced OFF-time and improved clinical condition in patients with PD experiencing wearing-off, often with a reduced daily levodopa dose. Several of these studies also identified that entacapone improved patients' quality of life and was cost-effective. Subsequently, entacapone has been amalgamated into a triple-combination preparation (Stalevo®) with levodopa and carbidopa to create a flexible and convenient drug therapy for patients with PD who have end-of-dose motor fluctuations not stabilised on levodopa/dopa-decarboxylase inhibitor treatment. This review offers a historical perspective on a successful programme of drug development by researchers who played central roles in the progress from exploratory hypothesis to registered pharmaceutical product. [ABSTRACT FROM AUTHOR]

L-dopa is still the gold standard in the symptomatic treatment of Parkinson's disease (PD), and thus, it is the most commonly used drug in the non-clinical assessment of new drug therapies to PD, including those intended to improve the effect of L-dopa. In unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD, the results from L-dopa-induced rotation tests are often unpredictable. While repeated administration of L-dopa improves the rotation, the exact mechanisms underlying the extensive variability in rotation responses between rats and testing times are unclear. In the present study, we aimed to assess whether the route of administration (oral or intraperitoneal) or the form of L-dopa (base or methyl ester) is associated with the extensive variation in rotation responses to L-dopa in 6-OHDA rats. We also wanted to examine the dependence between L-dopa (base or methyl ester)-induced rotational behaviour and the extent of dopamine and dopa decarboxylase enzyme loss in the lesioned striatum. It was found that variation in plasma levels of L-dopa as well as the administration route explains a part of the variability in rotation. There were small but significant differences in striatal dopamine depletion (indicative of degree of lesion) between the groups, which may partially account for the various patterns in L-dopa-induced rotational behaviour. While apomorphine-induced rotation test is a useful tool for primary screening of the success of 6-OHDA lesion, it is not useful at predicting the rotational performance of 6-OHDA rats to L-dopa. The exact mechanisms and causes of the variability in the rotation responses to L-dopa in 6-OHDA rats still remain to be clarified. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Rationale. We have earlier found that 1) COMT inhibitors did not enhance amphetamine-induced dopamine efflux into striatal extracellular, that 2) they did not increase dopamine levels in striatal tissue and that 3) they did not potentiate amphetamine-induced turning behavior of hemiparkinsonian rats. Further, when COMT knockout mice were challenged with l-dopa or a dopamine transporter (DAT) inhibitor, an accumulation of dopamine occurred and the neurochemical and locomotor effects of l-dopa and GBR 12909 were modified accordingly. Objective. Since DAT inhibitors and amphetamine apparently have different mechanisms of action, we were interested to see how COMT knockout mice would react to d-amphetamine treatment. Methods. We measured the effects of d-amphetamine on locomotor activity and on the levels of catecholamines and their metabolites in striatal microdialysis fluid and in striatal, hypothalamic and cortical brain regions of COMT gene disrupted mice. Striatal dopamine receptor binding was also determined. Results. After d-amphetamine administration, the DOPAC content in homozygous mice was 3-fold in the striatum, 17- to 18-fold in the cortex and 7- to 8-fold in the hypothalamus higher than in wild-type control mice, and there were no indications of genotype×sex interactions. However, the lack of COMT did not potentiate d-amphetamine-induced dopamine levels in brain tissue or in striatal extracellular fluid. d-Amphetamine-induced (10 mg/kg) hyperlocomotion was less suppressed in male COMT knockout mice than in their wild-type counterparts. Striatal dopamine D[sub 1] and D[sub 2] receptor levels in male mice were not altered by COMT gene disruption. Conclusions. Changes in COMT activity modulates dopamine metabolism but the behavioral effects of d-amphetamine in male mice only to a small extent, and this action does not seem to depend on the actual extracellular dopamine concentration. Nor is it mediated through compensatory changes in dopamine D[sub 1] and D[sub 2] receptor levels. In dopaminergic neurons, the contribution of intracellular COMT remains secondary in conditions when dopamine is released by d-amphetamine. [ABSTRACT FROM AUTHOR]

AbstractParkinson's disease is a major neurological disorder that primarily affects the nigral dopaminergic cells. Nigral histamine innervation is altered in human postmortem Parkinson's disease brains. However, it is not known if the altered innervation is a consequence of dopamine deficiency. The aim of the present study was to investigate possible changes in the H3 receptor system in a well-characterized model of Parkinson's disease – the 6-hydroxydopamine (6-OHDA) lesioned rats. Histamine immunohistochemistry showed a minor increase of the fibre density index but we did not find any robust increase of histaminergic innervation in the ipsilateral substantia nigra on the lesioned side. In situ hybridization showed equal histidine decarboxylase mRNA expression on both sides in the posterior hypothalamus. H3 receptors were labelled with N-alpha-[3H]-methyl histamine dihydrochloride ([3H] NAMH). Upregulation of binding to H3 receptors was found in the substantia nigra and ventral aspects of striatum on the ipsilateral side. An increase of GTP-γ-[35S] binding after H3 agonist activation was found in the striatum and substantia nigra on the lesioned side. In situ hybridization of H3 receptor mRNA demonstrated region-specific mRNA expression and an increase of H3 receptor mRNA in ipsilateral striatum. Thus, the histaminergic system is involved in the pathological process after 6-OHDA lesion of the rat brain at least through H3 receptor. On the later stages of the neurotoxic damage, less H3 receptors became functionally active. Increased H3 receptor mRNA expression and binding may, for example, modulate GABAergic neuronal activity in dopamine-depleted striatum. [ABSTRACT FROM AUTHOR]

Several neurotrophic factors (NTF) are shown to be neuroprotective and neurorestorative in pre-clinical animal models for Parkinson’s disease (PD), particularly in models where striatal dopamine neuron innervation partially exists. The results of clinical trials on late-stage patients have been modest. Subthalamic deep brain stimulation (STN DBS) is a proven treatment for a selected group of advanced PD patients. The cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic protein, but its effects in animal models of late-stage PD have remained under-researched. The interactions of NTF and STN DBS treatments have not been studied before. We found that a nigral CDNF protein alone had only a marginal effect on the behavioral deficits in a late-stage hemiparkinsonian rat model (6-OHDA MFB). However, CDNF improved the effect of acute STN DBS on front limb use asymmetry at 2 and 3 weeks after CDNF injection. STN lesion—modeling chronic stimulation—had an additive effect in reducing front limb use in the cylinder test and apomorphine-induced rotation. The combination of CDNF and acute STN DBS had a favorable effect on striatal tyrosine hydroxylase. This study presents a novel additive beneficial effect of NTF and STN DBS, which might be explained by the interaction of DBS-induced endogenous NTFs and exogenously injected CDNF. SNpc can be reached via similar trajectories used in clinical STN DBS, and this interaction is an important area for future studies. [ABSTRACT FROM AUTHOR]

Abstract: The misfolding and aggregation of α-synuclein (aSyn) eventually lead to an accumulation of toxic forms that disturb normal neuronal function and result in cell death. aSyn rich inclusions are seen in Parkinson's disease, dementia with Lewy bodies and other synucleinopathies. Prolyl oligopeptidase (PREP) can accelerate the aggregation process of aSyn and the inhibition of PREP leads to a decreased amount of aggregated aSyn in cell models and in aSyn transgenic mice. In this study, we investigated the effect of 5- and 28-day PREP inhibitor (KYP-2047) treatments on a mouse strain carrying a point mutation in the aSyn coding gene. Following PREP inhibition, we found a decrease in high molecular-weight oligomeric aSyn and a concomitant increase in the amount of the autophagosome marker, LC3BII, suggesting enhanced macroautophagy (autophagy) and aSyn clearance by KYP-2047. Moreover, 28-day treatment with KYP-2047 caused significant increases in striatal dopamine levels. In cell culture, overexpression of PREP reduced the autophagy. Furthermore, the inhibition of PREP normalized the changes on autophagy markers (LC3BII and p62) caused by an autophagy inhibition or aSyn overexpression, and induced the expression of beclin 1, a positive regulator of autophagy. Taken together, our results suggest that PREP inhibition accelerates the clearance of protein aggregates via increased autophagy and thus normalizes the cell functions in vivo and in vitro. Therefore, PREP inhibition may have future potential in the treatment of synucleinopathies. [Copyright &y& Elsevier]

Abstract: Amantadine is commonly given to alleviate L-DOPA-induced dyskinesia of Parkinson’s disease (PD) patients. Animal and human evidence showed that amantadine may also exert neuroprotection in several neurological disorders. Additionally, it is generally believed that this neuroprotection results from the ability of amantadine to inhibit glutamatergic NMDA receptor. However, several lines of evidence questioned the neuroprotective capacity of NMDA receptor antagonists in animal models of PD. Thus the cellular and molecular mechanism of neuroprotection of amantadine remains unclear. Using primary cultures with different composition of neurons, microglia, and astroglia we investigated the direct role of these glial cell types in the neuroprotective effect of amantadine. First, amantadine protected rat midbrain cultures from either MPP+ or lipopolysaccharide (LPS), two toxins commonly used as PD models. Second, our studies revealed that amantadine reduced both LPS- and MPP+-induced toxicity of dopamine neurons through 1) the inhibition of the release of microglial pro-inflammatory factors, 2) an increase in expression of neurotrophic factors such as GDNF from astroglia. Lastly, differently from the general view on amantadine’s action, we provided evidence suggesting that NMDA receptor inhibition was not crucial for the neuroprotective effect of amantadine. In conclusion, we report that amantadine protected dopamine neurons in two PD models through a novel dual mechanism, namely reducing the release of pro-inflammatory factors from activated microglia and increasing the expression of GNDF in astroglia. [Copyright &y& Elsevier]

Abstract: Ret is the canonical signaling receptor for glial cell line-derived neurotrophic factor (GDNF), which has been shown to have neuroprotective effects when administered prior to neurotoxic challenge. A missense Meth918Thr mutation causes the constitutive activation of Ret, resulting in multiple endocrine neoplasia type 2 B (MEN2B). To clarify the role of Ret signaling in neuroprotection, we studied the effects of the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) on the dopaminergic system of mice carrying the MEN2B mutation. We found that MEN2B mice were significantly more resistant to nigral tyrosine hydroxylase (TH)-positive cell loss induced by unilateral striatal 6-OHDA than Wt mice. However, 6-OHDA caused profound dopamine (DA) depletion in the striatum of both MEN2B and Wt mice. Systemic MPTP caused similar DA depletion and a decrease in TH-immunostaining in the striatum of MEN2B and Wt mice. Neither neurotoxin induced a compensatory increase in striatal metabolite/DA ratios in the MEN2B mice, possibly contributing to an increased amphetamine-induced turning behavior observed in behavioral assessments of these mice. Thus, our data suggest that activated Ret protects DA cell bodies in the substantia nigra pars compacta, but does not protect DA axons in the striatum. [Copyright &y& Elsevier]

Abstract: Numerous reports have described the effects of quercetin in models of neurodegenerative diseases, or cancer, resulting in a very complex and sometimes paradoxical picture. Understanding how quercetin causes either protection or cell death in the same model is both tempting and essential. We used the 6-OHDA-induced toxicity model in SH-SY5Y cells, applying graded concentrations of quercetin for a variable time period and following cell viability with MTT-assay and LDH-release as well as caspase-3-like activity. We detected a time-dependent action of quercetin and distinguished an early protective effect from a late toxic one. In addition, we revealed a narrower therapeutic dose-range of quercetin than previously reported in the literature, demonstrating that the toxic effects of quercetin occurred at a concentration only 2-fold higher than the one that produced the greatest protection. We also demonstrated, to our knowledge for the first time, that quercetin itself directly inhibits caspase-3-like activity in a dose-dependent manner. Finally, single doses of quercetin failed to protect against 6-OHDA toxicity in a unilateral rat model of Parkinson''s disease. In conclusion, our data may offer an explanation for the dualistic effect of quercetin reported in the literature. In fact, in most studies suggesting quercetin protection against oxidative stressors, the experimental setting failed to include prolonged exposure, and therefore the toxic effects may have been missed. This study supports previous in vivo studies that cast doubt on the efficacy of quercetin against neurodegenerative diseases due to its delayed toxicity. [Copyright &y& Elsevier]

Abstract: In the present study, we examined the ability of a flavonoid quercetin to prevent 6-hydroxydopamine (6-OHDA)-induced oxygen radical formation and cytotoxicity in vitro and neurotoxicity in vivo. Quercetin (10–100 μM) had an acute significant antioxidant effect against the 6-OHDA-induced (30 μM) oxygen radical formation in catecholaminergic SH-SY5Y neuroblastoma cells. Moreover, in these cells, quercetin at 10–50 μM had a significant protective effect against 6-OHDA though at 100 μM it was itself harmful to the cells. The possible effect of quercetin in preventing neurotoxicity in unilateral medial forebrain bundle (full nigral lesion) or striatal (partial lesion) 6-OHDA rat lesion models of Parkinson''s disease was studied in three treatment schedules: a 7-day pre- or post-treatment or their combination. Rotational responses to apomorphine (0.1 mg/kg, subcutaneously) and d-amphetamine (2.5 mg/kg, intraperitoneally) were assessed at weeks 1 and 2 post-lesion. Quercetin had no consistent neuroprotective effect in either model at 50–200 mg/kg once a day or 100 mg/kg twice a day. Furthermore, no protection was observed in tyrosine hydroxylase positive nigral cell numbers, striatal fiber density or in striatal levels of dopamine. These in vitro and in vivo results cast doubt on the theory that quercetin exerts reliable neuroprotective effects against 6-OHDA-induced toxicity. In vitro, quercetin seems to be protective at low doses but damaging at high doses. [Copyright &y& Elsevier]

Abstract: We have generated a transgenic mouse line overexpressing mutated human A30P α-synuclein under the control of the prion-related protein promoter. Immunohistology revealed mutated human A30P α-synuclein protein in numerous brain areas, but no gross morphological changes, Lewy bodies, or loss of dopaminergic cell bodies. The transgenic mice displayed decreased locomotion, impaired motor coordination, and balance. In vivo voltammetry showed that A30P mice responded to longer stimulation of the ascending dopaminergic pathways with less dopamine release in striatum and had a slower rate of dopamine decline after repeated stimulations or after α-methyl-p-tyrosine-HCl treatment. However, dopamine re-uptake or transporter levels were similar in transgenic and control mice. Our data provide evidence that overexpression of mutated human A30P α-synuclein in mice leads to a reduced size of the dopamine storage pool. This is in agreement with the previously postulated involvement of α-synuclein in the turnover of transmitter vesicles and may explain the observed motor deficits in A30P mice. [Copyright &y& Elsevier]

Abstract: The mechanism of action of the neurotoxin 6-hydroxydopamine (6-OHDA) is thought to involve the generation of free radicals and subsequent apoptotic processes. We have demonstrated in vitro that the neuroimmunophilin, FK506 (10–100 nM), dose dependently and significantly restored the ROS production to the control level, increased the Bcl-2 protein level, partly inhibited the cytochrome C release from mitochondria and reduced the caspase-3 activation in SH-SY5Y cells. On the other hand, there was no significant restoration of the ATP level by FK506 and the toxin activated proteins, p53 and Bax, were not normalized by FK506. In support of these latter results, daily administration of FK506 for 7 days to rats (0.5, 1 and 3 mg/kg i.p.) did not significantly prevent the apomorphine-induced contralateral circling, measured 2 weeks after unilateral nigral lesioning. Moreover, FK506 pretreatment did not significantly lower the toxin elevated lipid peroxidation levels, indicating that oxidative stress was present even after the FK506 treatment in the lesioned striatum. Taken together, our results with FK506 are inconsistent. We confirm the antioxidant nature of FK506, that is, it blocks ROS production in SH-SY5Y cells. However, there were no significant protective effects in any apoptotic analyses in SH-SY5Y cells and in animal studies, a 7-day FK506 pre-treatment was not able to reverse the toxic effect of 6-OHDA in a rat model of Parkinson''s disease. [Copyright &y& Elsevier]

6-Hydroxydopamine (6-OHDA) is widely used to produce an animal model of Parkinson’s disease by selectively destroying the catecholaminergic nerve system of the substantia nigra. In our previous studies we noted that dopaminergic neuroblastoma cells (SH-SY5Y) die mostly via apoptosis after exposure to 6-OHDA (≤100μM) but African green monkey fibroblast (CV1-P) cells do not succumb, although in both cell lines there were increased intracellular p53 levels. This study was designed to further investigate the mechanisms underlying the p53 elevation. To test how 6-OHDA penetrates into fibroblast cells and affects p53 levels, we investigated the presence of the dopamine transporter (DAT) in CV1-P cells. We showed by western hybridization that CV1-P cells contain the DAT. The apparent entry of 6-OHDA into fibroblasts was decreased by the DAT inhibitor, 1-(2-bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenyl-propyl)piperazine (GBR 12909). Pre-treatment with GBR 12909 decreased the elevation of intracellular ROS to the control level and thus prevented the increase of p53 levels in 6-OHDA-treated CV1-P cells. Moreover, an increase of Bcl-2, an antiapoptotic protein, was detected after 6-OHDA treatment, supporting our previous results where no increase in caspase-3 activity was detected. We suggest that Bcl-2 may block the activation of the caspase cascade and protect CV1-P cells from apoptosis. [Copyright &y& Elsevier]