Inhibition Of Aromatic Amino Acid Decarboxylase Research Articles

Ophthalmate is a new regulator of motor functions via CaSR: implications for movement disorders.

Dopamine's role as the principal neurotransmitter in motor functions has long been accepted. We broaden this conventional perspective by demonstrating the involvement of non-dopaminergic mechanisms. In mouse models of Parkinson's disease, we observed that L-DOPA elicited a substantial motor response even when its conversion to dopamine was blocked by inhibiting the enzyme aromatic amino acid decarboxylase (AADC). Remarkably, the motor activity response to L-DOPA in the presence of an AADC inhibitor (NSD1015) showed a delayed onset, yet greater intensity and longer duration, peaking at 7 h, compared to when L-DOPA was administered alone. This suggests an alternative pathway or mechanism, independent of dopamine signalling, mediating the motor functions. We sought to determine the metabolites associated with the pronounced hyperactivity observed, using comprehensive metabolomics analysis. Our results revealed that the peak in motor activity induced by NSD1015/L-DOPA in Parkinson's disease mice is associated with a surge (20-fold) in brain levels of the tripeptide ophthalmic acid (also known as ophthalmate in its anionic form). Interestingly, we found that administering ophthalmate directly to the brain rescued motor deficits in Parkinson's disease mice in a dose-dependent manner. We investigated the molecular mechanisms underlying ophthalmate's action and discovered, through radioligand binding and cAMP-luminescence assays, that ophthalmate binds to and activates the calcium-sensing receptor (CaSR). Additionally, our findings demonstrated that a CaSR antagonist inhibits the motor-enhancing effects of ophthalmate, further solidifying the evidence that ophthalmate modulates motor functions through the activation of the CaSR. The discovery of ophthalmate as a novel regulator of motor function presents significant potential to transform our understanding of brain mechanisms of movement control and the therapeutic management of related disorders.

Gut bacterial aromatic amine production: aromatic amino acid decarboxylase and its effects on peripheral serotonin production

ABSTRACT Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.

Pacjent z chorobą Parkinsona i bólem neuropatycznym – opis przypadku

Parkinson’s disease is a complex neurodegenerative process involving various structures in the nervous system. Parkinson’s disease is diagnosed mainly on the basis of clinical symptoms resulting from damage to the nigrostriatal pathway, which gives the characteristic motor symptoms. Therefore, traditional symptomatic treatment consists of administering dopamine precursor levodopa with an aromatic amino acid decarboxylase inhibitor or directly stimulating the striatal dopamine receptors. The problem of dopaminergic therapy is the limited influence on the symptoms of the disease, including pain. Central pain is one type of pain that occurs in Parkinson’s disease patients and dopaminergic therapy may be of limited value in treating it. Proper diagnosis of the problem and the application of appropriate chronic pain therapy are essential for the quality of life of patients. The author presents the case of a 65-year-old man with Parkinson’s disease and chronic pain who required modification of dopaminergic treatment and implementation of central neuropathic pain therapy.

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine.

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.

Comparative examination of levodopa pharmacokinetics during simultaneous administration with lactoferrin in healthy subjects and the relationship between lipids and COMT inhibitory activity in vitro

ABSTRACT Background: Lactoferrin (bLF) is an iron-binding multifunctional protein that is abundant in milk. In mice, it inhibits catechol-O-methyltransferase (COMT) activity and increases blood levodopa levels. However, the clinical effects are unknown. Objective: The objective of this study was to determine the effect of bLF on the kinetics of levodopa in blood. Design: The effects of the concomitant administration of a combined formulation of levodopa and an aromatic amino acid decarboxylase inhibitor and bLF on the concentration of levodopa in blood and its metabolism were assessed in eight healthy subjects. In addition, we analyzed the association with clinical factors and evaluated whether clinical factors affected the COMT inhibitory activity of bLF in vitro. Results: Although not statistically significant, the peak plasma concentration (C max) of levodopa increased by 18.5%. From the results of the stratified analysis of total cholesterol, a relationship with ΔC max was predicted. Therefore, bLF was reacted with cholesterol in the presence of lecithin and sodium deoxycholate in vitro to evaluate COMT inhibitory activity, and an increase in inhibitory activity was observed. By contrast, the ester compound cholesteryl oleate had no effect. The inhibitory activity of free fatty acids, which are known to interact with bLF, was also enhanced. Conclusion: The COMT inhibitory activity of bLF is not effective in elevating blood levodopa levels. However, in humans with high lipid levels, such as cholesterol, interactions may enhance the inhibitory effect, resulting in the enhanced absorption of levodopa. Trial registration: ID, UMIN000026787, registered 30 March 2017; URL, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000030749 Trial registration: UMIN Japan identifier: UMIN000026787

The Gut Microbiome: A Therapeutically Targetable Site of Peripheral Levodopa Metabolism.

Rekdal, VM, Bess, EN, Bisanz, JE, Turnbaugh, PJ, Balskus, EP. Discovery and inhibition of an interspecies gut bacterial pathway for levodopa metabolism. Science 2019; 364( 6445):eaau6323. https://doi.org/10.1126/science.aau6323 Much research has recently focused on the gut–brain axis and its influences on neurodegenerative processes.1 Less studied is the impact of the microbiome on treatment of Parkinson's disease (PD) and other neurodegenerative disorders.2 Levodopa (l-dopa) remains the primary symptomatic treatment for PD. Therapeutic efficacy depends on achieving sufficient central nervous system concentrations, a factor largely influenced by first-pass metabolism outside the brain.2, 3 The major extracerebral site of l-dopa metabolism is the gut. Here, l-dopa undergoes decarboxylation either via human aromatic amino acid decarboxylase (AADC) or by gut organisms.2 Peripheral metabolism to dopamine not only reduces l-dopa availability for central nervous system penetration but also produces unwanted side effects.2 Clinicians therefore coadminister peripheral AADC inhibitors, for example, carbidopa. Despite this blockade, up to 56% of administered l-dopa fails to reach the brain.4 There is significant interindividual variability in serum l-dopa levels following standard oral administration, producing variable therapeutic responses.2, 3 Gut microbiota metabolize l-dopa through a 2-step pathway (initial decarboxylation to dopamine followed by dehydroxylation to m-tyramine).2 Variations in gut microbiota therefore contribute to such therapeutic variations. In this study, Rekdal and colleagues2 characterized the organisms, genes, and enzymes responsible for microbiome-related l-dopa metabolism and explored therapeutic manipulation of this pathway. First, using genome-mining approaches, they identified enterococcus species, particularly Enterococcus faecalis as the major l-dopa decarboxylator (via the enzyme tyrosine decarboxylase [TyrDC]).2 Next, they identified Eggerthella lenta as the strain responsible for dehydroxylating dopamine to m-tyramine.2 Although this step does not influence l-dopa therapeutic efficacy, through regulating peripheral dopamine clearance, it is an important determinant of l-dopa side effects. These findings, alongside demonstrations of l-dopa metabolism in previously nonmetabolizing samples on the addition of E. faecalis, confirmed it as the dominant l-dopa decarboxylator in human microbiota.2 The authors proceeded to determine that host AADC inhibitors, for example, carbidopa, were ineffective against microbiome-related decarboxylation; activity against TyrDC was about 200 times less than toward host AADC.2 Interestingly, another compound, α-fluoromethyltyrosine, a nontoxic selective inhibitor of TyrDC but not AADC, completely inhibited l-dopa decarboxylation in gut microbiota samples.2 In a mouse model colonized with TyrDC-producing E. faecalis, α-fluoromethyltyrosine significantly increased peak l-dopa serum concentrations when compared with a vehicle control.2 This study highlights the role of the microbiome in explaining some of the variability in l-dopa responsiveness in PD. The identification of TyrDC as the predominant mediator of microbiome-associated l-dopa decarboxylation offers a potential biomarker for reduced l-dopa efficacy in certain populations. This also raises the future possibility of personalized, biomarker-driven specific TyrDC inhibition as a therapeutic addition to classic AADC inhibitors. Such therapies, through reducing peripheral l-dopa metabolism, would likely increase peak central nervous system l-dopa levels, thereby improving clinical outcomes. Potential complications of increasing synaptic dopaminergic pulsatility (motor fluctuations, increased risk of dyskinesia), however, will also need to be borne in mind during future studies of any such compounds.5 (1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the first draft, B. Review and Critique. E.M.: 1A, 1B, 1C, 3A K.P.B.: 1A, 1B, 3B We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. Informed patient consent and institutional review board approval was not necessary for this work. No specific funding was received for this work. The authors declare that there are no conflicts of interest relevant to this work. EM reports no financial disclosures.KPB holds research grants from EU Horizon 2020 and has received honoraria to speak at meetings or to attend advisory boards from Ipsen, Cavion, Allergan, Teva Lundbeck and Bial pharmaceutical companies. He also receives royalties from Oxford University Press and a stipend for MDCP editorship.

Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism.

The human gut microbiota metabolizes the Parkinson’s disease medication Levodopa (L-dopa), potentially reducing drug availability and causing side effects. However, the organisms, genes, and enzymes responsible for this activity in patients and their susceptibility to inhibition by host-targeted drugs are unknown. Here, we describe an interspecies pathway for gut bacterial L-dopa metabolism. Conversion of L-dopa to dopamine by a pyridoxal phosphate-dependent tyrosine decarboxylase from Enterococcus faecalis is followed by transformation of dopamine to m-tyramine by a molybdenum-dependent dehydroxylase from Eggerthella lenta. These enzymes predict drug metabolism in complex human gut microbiotas. Although a drug that targets host aromatic amino acid decarboxylase does not prevent gut microbial L-dopa decarboxylation, we identified a compound that inhibits this activity in Parkinson’s patient microbiotas and increases L-dopa bioavailability in mice.INTRODUCTIONParkinson’s disease is a debilitating neurological condition affecting more than 1% of the global population aged 60 and above. The primary medication used to treat Parkinson’s disease is levodopa (L-dopa). To be effective, L-dopa must enter the brain and be converted to the neurotransmitter dopamine by the human enzyme aromatic amino acid decarboxylase (AADC). However, the gastro-intestinal tract is also a major site for L-dopa decarboxylation, and this metabolism is problematic because dopamine generated in the periphery cannot cross the blood-brain barrier and causes unwanted side effects. Thus, L-dopa is coadministered with drugs that block pe-ripheral metabolism, including the AADC in-hibitor carbidopa. Even with these drugs, up to 56% of L-dopa fails to reach the brain. Moreover, the efficacy and side effects of L-dopa treatment are extremely heterogeneous across Parkinson’s patients, and this variability cannot be completely explained by differences in host metabolismRATIONALEPrevious studies in humans and animal models have demonstrated that the gut microbiota can metabolize L-dopa. The major proposed pathway involves an initial decarboxylation of L-dopa to dopamine, followed by conversion of dopamine to m-tyramine by means of a distinctly microbial dehydroxylation reaction. Although these metabolic activities were shown to occur in complex gut microbiota samples, the specific organisms, gene, and enzymes responsible were unknown. The effects of host-targeted inhibitors such as carbidopa on gut microbial L-dopa metabolism were also unclear. As a first step toward understanding the gut microbiota’s effect on Parkinson’s disease therapy, we sought to elucidate the molecular basis for gut microbial L-dopa and dopamine metabolism.RESULTSHypothesizing that L-dopa decarboxylation would require a pyridoxal phosphate (PLP)–dependent enzyme, we searched gut bacterial genomes for candidates and identified a conserved tyrosine decarboxylase (TyrDC) in Enterococcus faecalis. Genetic and biochemical experiments revealed that TyrDC simultaneously decarboxylates both L-dopa and its preferred substrate, tyrosine. Next, we used enrichment culturing to isolate a dopamine dehydroxylating strain of Eggerthella lenta, a species previously implicated in drug metabolism. Transcriptomics linked this activity to a molybdenum cofactor–dependent dopamine dehydroxylase (Dadh) enzyme. Unexpectedly, the presence of this enzyme in gut bacterial genomes did not correlate with dopamine metabolism; instead, we identified a single-nucleotide polymorphism (SNP) in the dadh gene that predicts activity. The abundance of E. faecalis, tyrDC, and the individual SNPs of dadh correlated with L-dopa and dopamine metabolism in complex gut microbiotas from Parkinson’s patients, indicating that these organisms, genes, enzymes, and even nucleotides are relevant in this setting.We then tested whether the host-targeted AADC inhibitor carbidopa affected L-dopa decarboxylation by E. faecalis TyrDC. Carbidopa displayed greatly reduced potency toward bacteria and was completely ineffective in complex gut microbiotas from Parkinson’s patients, suggesting that this drug likely does not prevent microbial L-dopa metabolism in vivo. To identify a selective inhibitor of gut bacterial L-dopa decarboxylation, we leveraged our molecular understanding of gut microbial L-dopa metabolism. Given TyrDC’s preference for tyrosine, we examined tyrosine mimics and found that (S)-α-fluoromethyltyrosine (AFMT) prevented L-dopa decarboxylation by TyrDC and E. faecalis as well as complex gut microbiota samples from Parkinson’s patients. Coadministering AFMT with L-dopa and carbidopa to mice colonized with E. faecalis also increased the peak serum concentration of L-dopa. This observation is consistent with inhibition of gut microbial L-dopa metabolism in vivo.CONCLUSIONWe have characterized an interspecies pathway for gut bacterial L-dopa metabolism and demonstrated its relevance in human gut microbiotas. Variations in these microbial activities could possibly contribute to the heterogeneous responses to L-dopa observed among patients, including decreased efficacy and harmful side effects. Our findings will enable efforts to elucidate the gut microbiota’s contribution to treatment outcomes and highlight the promise of developing therapies that target both host and gut microbial drug metabolism.

Exposure to Acute and Chronic Fluoxetine has Differential Effects on Sociability and Activity of Serotonergic Neurons in the Dorsal Raphe Nucleus of Juvenile Male BALB/c Mice

Although the neurobiological mechanisms underlying autism spectrum disorder (ASD) are still unknown, dysregulation of serotonergic systems has been implicated in the etiology of ASD, and serotonergic antidepressant drugs are often prescribed to treat some symptoms of ASD. The BALB/c strain of mice express a dysregulated serotonergic system and a phenotype that is relevant to ASD. In this study, juvenile male BALB/c mice were exposed to the selective serotonin reuptake inhibitor fluoxetine either chronically (18 mg/kg/day in drinking water, post-natal day (PND) 28–39) or acutely (18 mg/kg, i.p.; PND40), or to vehicle control conditions (0.9% sterile saline, i.p.; PND40), prior to being exposed to the three-chambered sociability test (SAT; PND40). One cohort of mice then received an injection of the aromatic amino acid decarboxylase inhibitor, NSD-1015, and one hour later brain tissue was collected for quantification of 5-hydroxytryptophan accumulation in the dorsal raphe nucleus (DR) as a measure of TPH2 activity. For the second cohort, brain tissue was collected ninety minutes after the onset of the social phase of the SAT and prepared for immunohistochemical staining for c-Fos and TPH2 to measure the activation of serotonergic neurons within subregions of the DR. Acute fluoxetine decreased social behavior, while chronic fluoxetine increased social behavior compared with vehicle-treated controls. Furthermore, acute and chronic fluoxetine treatments were without effect on TPH2 activity but differentially affected populations of serotonergic neurons in the DR. These data are consistent with the hypothesis that serotonergic systems are implicated in social behavior that is relevant for ASD.

Pharmacokinetics of Rytary®, An Extended-Release Capsule Formulation of Carbidopa-Levodopa.

Parkinson’s disease (PD) is a chronic progressive neurological disorder characterized by resting tremor, rigidity, bradykinesia, gait disturbance, and postural instability. Levodopa, the precursor to dopamine, coadministered with carbidopa or benserazide, aromatic amino acid decarboxylase inhibitors, is the most effective and widely used therapeutic agent in the treatment of PD. With continued levodopa treatment, a majority of patients develop motor complications such as dyskinesia and motor ‘on-off’ fluctuations, which are, in part, related to the fluctuations in plasma concentrations of levodopa. A new extended-release (ER) carbidopa–levodopa capsule product (also referred to as IPX066) was developed and approved in the US as Rytary® and in the EU as Numient®. The capsule formulation is designed to provide an initial rapid absorption of levodopa comparable to immediate-release (IR) carbidopa–levodopa, and to subsequently provide stable levodopa concentrations with reduced peak-to-trough excursions in plasma concentrations in order to reduce motor fluctuations associated with pulsatile stimulation of dopamine receptors and to minimize dyskinesia. Phase III studies of this ER carbidopa–levodopa capsule formulation in patients with PD have shown a significant reduction in ‘off’ time compared with IR carbidopa–levodopa and carbidopa–levodopa–entacapone. We present a review of the clinical pharmacokinetics and pharmacodynamics of this ER product of carbidopa–levodopa in healthy subjects and in patients with PD.Electronic supplementary materialThe online version of this article (doi:10.1007/s40262-017-0511-y) contains supplementary material, which is available to authorized users.

Spotlight on opicapone as an adjunct to levodopa in Parkinson's disease: design, development and potential place in therapy.

Parkinson’s disease (PD) is a progressive, chronic, neurodegenerative disease characterized by rigidity, tremor, bradykinesia and postural instability secondary to dopaminergic deficit in the nigrostriatal system. Currently, disease-modifying therapies are not available, and levodopa (LD) treatment remains the gold standard for controlling motor and nonmotor symptoms of the disease. LD is extensively and rapidly metabolized by peripheral enzymes, namely, aromatic amino acid decarboxylase and catechol-O-methyltransferase (COMT). To increase the bioavailability of LD, COMT inhibitors are frequently used in clinical settings. Opicapone is a novel COMT inhibitor that has been recently approved by the European Medicines Agency as an adjunctive therapy to combinations of LD and aromatic amino acid decarboxylase inhibitor in adult PD patients with end-of-dose motor fluctuations. We aimed to review the biochemical properties of opicapone, summarize its preclinical and clinical trials and discuss its future potential role in the treatment of PD.

11C]5-HTP and microPET are Not Suitable for Pharmacodynamic Studies in the Rodent Brain

The PET tracer [(11)C]5-hydroxytryptophan ([(11)C]5-HTP), which is converted to [(11)C]5-hydroxytryptamine ([(11)C]5-HT) by aromatic amino acid decarboxylase (AADC), is thought to measure 5-HT synthesis rates. But can we measure these synthesis rates by kinetic modeling of [(11)C]5-HTP in rat? Male rats were scanned with [(11)C]5-HTP (60 minutes) after different treatments. Scans included arterial blood sampling and metabolite analysis. 5-HT synthesis rates were calculated by a two-tissue compartment model (2TCM) with irreversible tracer trapping or Patlak analysis. Carbidopa (inhibitor peripheral AADC) dose-dependently increased [(11)C]5-HTP brain uptake, but did not influence 2TCM parameters. Therefore, 10 mg/kg carbidopa was applied in all subsequent study groups. These groups included treatment with NSD 1015 (general AADC inhibitor) or p-chlorophenylalanine (PCPA, inhibitor of tryptophan hydroxylase, TPH). In addition, the effect of a low-tryptophan (Trp) diet was investigated. NSD 1015 or Trp depletion did not affect any model parameters, but PCPA reduced [(11)C]5-HTP uptake, and the k3. This was unexpected as NSD 1015 directly inhibits the enzyme converting [(11)C]5-HTP to [(11)C]5-HT, suggesting that trapping of radioactivity does not distinguish between parent tracer and its metabolites. As different results have been acquired in monkeys and humans, [(11)C]5-HTP-PET may be suitable for measuring 5-HT synthesis in primates, but not in rodents.

Assessment of dopamine (DA) synthesis rate in selected parts of the rat brain with central noradrenergic lesion after administration of 5-HT3 receptor ligands

The study objective was to determine the effect of central noradrenergic system lesions performed in the early extrafetal life period on dopamine synthesis in the rat brain. The content of L-dihydroxyphenylalanine (L-DOPA) was assessed in the frontal lobe, thalamus, hypothalamus and brain stem of rats by high-pressure chromatography with electrochemical detection (HPLC/ED) after administration of 5-HT3 receptor ligands. Adult male Wistar rats which underwent central noradrenergic lesions by DSP-4 administration (50 mg/kg m.c. i.p.) on day 1 and 3 of life received i.p. injections of the aromatic amino acid decarboxylase inhibitor (NSD-1050) in a dose of 100 mg/kg b.w. Next, 30 min after NSD-1050 injection, the animals were decapitated by guillotine. Selected brain structures were dissected and L-DOPA content was determined by HPLC/ED. A statistically significant reduction was found in DA synthesis in the group of animals with DSP-4 lesions induced by PBG (1-phenylbiguanide, 7.5 mg/kg b.w. i.p.) and ondansetron (1.0 mg/kg b.w. i.p.). Morphine and PBG had no major effect on DA synthesis in the cerebral cortex of both control animals and in rats with noradrenergic lesions. The assessment of the effect of DSP-4 lesions on L-DOPA content in the brain stem after administration of morphine (7.5 mg/kg b.w. s.c.), PBG (7.5 mg/kg b.w. i.p.) or ondansetron (1.0 mg/kg b.w. i.p.) separately or jointly showed a statistically significant increase in the synthesis of DA in animals with DSP-4 lesions, as compared to the control group exposed to 0.9% NaCl and morphine. The analysis of the effect of DSP-4 lesions on L-DOPA content in the thalamus and hypothalamus revealed no statistically significant differences between the control groups of rats and those with DSP-4 lesions. As shown by this model, permanent noradrenergic lesions in animals in the early extra-fetal period result in increased reactivity of the central dopamine system.

Abstract 2680: Improved imaging of orthotopic BxPC3 pancreatic adenocarcinoma xenograft using animal PET/CT.

Abstract Treatment of pancreatic adenocarcinoma is difficult because the location of the lesion is often unknown. 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA) is used for imaging of neuroendocrine tumours, which take up and decarboxylate amine precursors. [18F]FDOPA use in diagnostic pancreatic imaging has increased, since the exocrine pancreas synthesises dopamine. We studied the uptake of [18F]FDOPA in healthy and diseased pancreas. Mice bearing orthotopic BxPC3 pancreatic adenocarcinoma and sham-operated controls were pre-treated with enzyme inhibitors of aromatic amino acid decarboxylase (AADC), catechol-O-methyl transferase (COMT), monoamine oxidase A (MAO-A) or combinations of these inhibitors. Mice were injected with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) or [18F]FDOPA and scanned via positron emission tomography/computed tomography. The absolute [18F]FDOPA uptake was determined from selected tissues using a well counter. The intratumoural biodistribution pattern of [18F]FDOPA was recorded autoradiographically. The main FDOPA metabolites present in pancreatic samples were determined with high-performance liquid chromatography. [18F]FDG uptake was high in pancreatic tumours, while [18F]FDOPA uptake was highest in healthy pancreas and significantly lower in tumour-bearing pancreas, independent of pre-treatment. [18F]FDOPA uptake in pancreas was lowest with vehicle pre-treatment and highest with pre-treatment with the inhibitor of AADC. When mice received COMT+MAO-A inhibitors, uptake was high in healthy pancreas but low in tumour-bearing pancreas. Unequal uptake may be due to blockade of metabolism and increased availability of [18F]fluorodopamine due to the employed enzyme inhibitors. Combined use of [18F]FDG and [18F]FDOPA is suitable for imaging pancreatic tumours. Pre-treatment with COMT+MAO-A inhibitors improved the differentiation of pancreas from surrounding tissue and healthy pancreas from tumour. Citation Format: Johanna M. Tuomela, Jani Seppänen, Laura Haavisto, Sarita Fosrback, Tero Vahlberg, Olof Solin, Merja Haaparanta. Improved imaging of orthotopic BxPC3 pancreatic adenocarcinoma xenograft using animal PET/CT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2680. doi:10.1158/1538-7445.AM2013-2680

Enzyme inhibition of dopamine metabolism alters 6-[18F]FDOPA uptake in orthotopic pancreatic adenocarcinoma

BackgroundAn unknown location hampers removal of pancreatic tumours. We studied the effects of enzyme inhibitors on the uptake of 6-[18F]fluoro-l-3,4-dihydroxyphenylalanine ([18F]FDOPA) in the pancreas, aiming at improved imaging of pancreatic adenocarcinoma.MethodsMice bearing orthotopic BxPC3 pancreatic adenocarcinoma were injected with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) and scanned with positron emission tomography/computed tomography (PET/CT). For [18F]FDOPA studies, tumour-bearing mice and sham-operated controls were pretreated with enzyme inhibitors of aromatic amino acid decarboxylase (AADC), catechol-O-methyl transferase (COMT), monoamine oxidase A (MAO-A) or a combination of COMT and MAO-A. Mice were injected with [18F]FDOPA and scanned with PET/CT. The absolute [18F]FDOPA uptake was determined from selected tissues using a gamma counter. The intratumoural biodistribution of [18F]FDOPA was recorded by autoradiography. The main [18F]FDOPA metabolites present in the pancreata were determined with radio-high-performance liquid chromatography.Results[18F]FDG uptake was high in pancreatic tumours, while [18F]FDOPA uptake was highest in the healthy pancreas and significantly lower in tumours. [18F]FDOPA uptake in the pancreas was lowest with vehicle pretreatment and highest with pretreatment with the inhibitor of AADC. When mice received COMT + MAO-A inhibitors, the uptake was high in the healthy pancreas but low in the tumour-bearing pancreas.ConclusionsCombined use of [18F]FDG and [18F]FDOPA is suitable for imaging pancreatic tumours. Unequal pancreatic uptake after the employed enzyme inhibitors is due to the blockade of metabolism and therefore increased availability of [18F]FDOPA metabolites, in which uptake differs from that of [18F]FDOPA. Pretreatment with COMT + MAO-A inhibitors improved the differentiation of pancreas from the surrounding tissue and healthy pancreas from tumour. Similar advantage was not achieved using AADC enzyme inhibitor, carbidopa.

L-dopa-induced dopamine synthesis and oxidative stress in serotonergic cells

l-dopa is a precursor for dopamine synthesis and a mainstay treatment for Parkinson's disease. However, l-dopa therapy is not without side effects that may be attributed to non-dopaminergic mechanisms. Synthesized dopamine can be neurotoxic through its enzymatic degradation by monoamine oxidase (MAO) to form the reactive byproduct, hydrogen peroxide and hydroxyl radicals or through auto-oxidation to form highly reactive quinones that can bind proteins and render them non-functional. Since l-dopa could be decarboxylated by aromatic amino acid decarboxylase (AADC) present within both dopamine and serotonin neurons, it was hypothesized that serotonin neurons convert l-dopa into dopamine to generate excessive reactive oxygen species and quinoproteins that ultimately lead to serotonin neuron death. To examine the effects of l-dopa on serotonin neurons, the RN46A-B14 cell line was used. These immortalized serotonergic cell cultures were terminally differentiated and then incubated with varying concentrations of l-dopa. Results show that RN46A-B14 cells contain AADC and can synthesize dopamine after incubation with l-dopa. Furthermore, l-dopa dose-dependently increased intracellular reactive oxygen species (ROS) and cell death. Dopamine, ROS production and cell death were attenuated by co-incubation with the AADC inhibitor, NSD-1015. The MAO inhibitor, pargyline, also attenuated cell death and ROS after l-dopa treatment. Lastly, quinoprotein formation was enhanced significantly by incubation with l-dopa. Taken together, these data illustrate that serotonergic cells can produce dopamine and that the accumulation of dopamine after l-dopa and its subsequent degradation can lead to ROS production and death of RN46A-B14 serotonergic cells.

Benserazide dosing regimen affects the response to L-3,4-dihydroxyphenylalanine in the 6-hydroxydopamine-lesioned rat

Peripheral aromatic amino acid decarboxylase (AADC) inhibitors, such as benserazide, are routinely used to potentiate the effects of L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson's disease (PD) and in experimental models of PD. However, there is little information available on the optimal dose or the timing of administration relative to L-DOPA treatment. We now assess the effect of dose, timing, and supplemental administration of benserazide on the rotational response induced by L-DOPA in unilateral 6-hydroxydopamine-lesioned rats. L-DOPA (12.5 mg/kg, p.o.) concomitant with benserazide (3.125-15 mg/kg, p.o.) produced a dose-dependent increase in contraversive rotation compared with the effects of L-DOPA alone. The optimal L-DOPA response was achieved with 10 mg/kg of benserazide and this dose was used in subsequent experiments. When L-DOPA treatment was delayed for 1, 2, or 3 h after benserazide, the rotational response declined suggesting loss of AADC inhibition. Unexpectedly, there was also a progressive decline in response when benserazide and L-DOPA were given together but at increasingly later time points of 08.00, 09.00, 10.00, and 11.00 h. To assess supplemental administration of benserazide, an additional dose was given 2 h after the initial benserazide/L-DOPA treatment. This produced a further increase in the number of contralateral rotations indicating that the effect of benserazide declines while plasma levels of L-DOPA are maintained. Therefore, optimization of the dose and timing of benserazide administration is essential to achieve a consistent L-DOPA response in 6-hydroxydopamine-lesioned rats. These findings may have implications for the way in which peripheral AADC inhibitors are used in the treatment of PD.

Mirtazapine Relieves Limb Paralysis of Unknown Etiology: A Three-Year Case Follow-Up

Back to table of contents Previous article Next article LettersFull AccessMirtazapine Relieves Limb Paralysis of Unknown Etiology: A Three-Year Case Follow-UpChien-Han Lai, M.D., M.Sc.Chien-Han LaiSearch for more papers by this author, M.D., M.Sc.Published Online:1 Oct 2011https://doi.org/10.1176/jnp.23.4.jnpe18AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: “Mrs. M” is a housewife who has had bilateral limb paralysis (LP) of her legs for the last 3.3 years; she had no significant brain or lumbar spine findings in MRI of brain or lumbar-spine, biochemistry examination, and substance or toxicity screenings by many departments of internal and surgical medicine. She was transferred to our clinic because there were no pathologic results of medical examinations. No significant psychosocial stressors, depression (Hamilton Rating Scale for Depression [Ham-D]), or anxiety symptoms (Hamilton Rating Scale for Anxiety (Ham-A) were noted, except bilateral LP of legs, with decreased muscle power (MP: score 2), mild anorexia, low back pain (visual analog scale (VAS) score: 5), and insomnia. No other neurologic signs were found except bilateral LP of legs. Mirtazapine (30 mg/day) was used for the above complaints with unknown etiology, and Mrs. M had relief of LP with less pain feelings (VAS: 3), increased mp, better sleep, and appetite within 2 weeks. LP improved progressively in the following 1 month, with MP returning to normal (score: 5), pain relief (VAS: 1), and ability to walk without assistance. She continued mirtazapine use during the last 3 years; however, she complained that LP and pain would relapse with cessation of mirtazapine (MP: 3 and VAS: 4 if she discontinued mirtazapine use for longer than 1 month). She was still under continuous therapy with mirtazapine (30 mg/day) with good control of LP and pain up to the present (MP: 5; VAS: 1). There has been no significant psychopathology of depression (Ham-D: 0–1) or anxiety (Ham-A: 0–2) during these 3 years.DiscussionMirtazapine could enhance firing rates of dopaminergic neurons and adrenergic neurons in frontocortical and corticolimbic areas, such as frontal cortex, striatum, nucleus accumbens, and hippocampus.1 Dopaminergic-enhancing agents, such as L-dopa, has been reported to facilitate motor recovery in hemiplegic stroke patients.2 The precursor of norepinephrine or aromatic amino acid decarboxylase inhibitor could elevate norepinephrine level in cerebral cortex and hippocampus of sensorimotor cortex-ablated rats. The increase in norepinephrine level was correlated with motor-function recovery.3 Serotonin could give a significant number of stroke patients better outcomes of motor function after fluoxetine treatment, above and beyond the depression-treating effects.4Dept. of Psychiatry Buddhist Tzu-Chi General Hospital Taipei Branch Taipei, Taiwan Institute of Brain Science National Yang Ming University Taipei City, Taiwane-mail: [email protected]com.tw.Support: Buddhist Tzu-Chi General Hospital, Taipei Branch hospital project TCRD-TPE-97-02.1. Millan MJ , Gobert A , Rivet JM , et al.: Mirtazapine enhances frontocortical dopaminergic and corticolimbic adrenergic, but not serotonergic, transmission by blockade of alpha2-adrenergic and serotonin2C receptors: a comparison with citalopram. Eur J Neurosci 2000; 12:1079–1095Crossref, Medline, Google Scholar2. Scheidtmann K , Fries W , Muller F , et al.: Effect of levodopa in combination with physiotherapy on functional motor recovery after stroke: a prospective, randomised, double-blind study. Lancet 2001; 358:787–790Crossref, Medline, Google Scholar3. Kikuchi K , Nishino K , Ohyu H : Increasing CNS norepinephrine levels by the precursor L-dopa facilitates beam-walking recovery after sensorimotor cortex ablation in rats. Brain Res 2000; 860:130–135Crossref, Medline, Google Scholar4. Dam M , Tonin P , De Boni A , et al.: Effects of fluoxetine and maprotiline on functional recovery in poststroke hemiplegic patients undergoing rehabilitation therapy. Stroke 1996; 27:1211–1214Crossref, Medline, Google Scholar FiguresReferencesCited byDetailsCited ByNone Volume 23Issue 4 Fall 2011Pages E18-E18 Metrics Support: Buddhist Tzu-Chi General Hospital, Taipei Branch hospital project TCRD-TPE-97-02.PDF download History Published online 1 October 2011 Published in print 1 October 2011

No increased chromosomal damage in l-DOPA-treated patients with Parkinson’s disease: a pilot study

Parkinson's disease (PD) is a neurodegenerative movement disorder affecting about 2% of the human population in old age. L-3,4-Dihydroxyphenylalanine (L-DOPA) in combination with a peripheral aromatic amino acid decarboxylase inhibition has been the most frequently prescribed drug for alleviating symptoms of PD, but a potential contribution of L-DOPA therapy to further neurodegeneration via oxidative stress is still debated. We report that the specific oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) level in peripheral blood lymphocyte DNA was elevated to 8.1 +/- 1.7 8-oxodG/10(6)dG in 17 chronically L-DOPA-treated PD patients, compared to 4.6 +/- 1.2 8-oxodG/10(6)dG in 12 controls. However, the total antioxidative capacity of plasma was increased to 1113 +/- 237 microM in the PD patients compared to 941 +/- 254 microM in controls. The frequency of micronuclei, a subgroup of chromosomal aberrations, in peripheral blood lymphocytes was not elevated compared to healthy age-matched individuals. In vitro, in a cell-free assay, dopamine and its precursor L-DOPA exhibited antioxidative capacity. On the other hand, the 8-oxodG concentration in cultured PC 12 cells was enhanced after dopamine treatment. This elevation may be below a threshold for manifestation as chromosomal damage.

Carbidopa/levodopa/entacapone: the evidence for its place in the treatment of Parkinson’s disease

Parkinson's disease (PD) is a common neurodegenerative disease. In the 1960s, it was shown that the degeneration of dopamine producing neurons in the substantia nigra (SN) caused the motor features of PD. Dopamine replacement with levodopa, a dopamine precursor, resulted in remarkable benefit. Yet, the intermittent administration of levodopa is a major cause of motor complications, such as "wearing-off" of levodopa's benefit and involuntary movements, known as dyskinesia. Therefore, agents that prolong levodopa's half-life were employed, such as carbidopa, an aromatic amino acid decarboxylase (AADC) inhibitor, and entacapone, a catechol-O-methyltransferase (COMT) inhibitor. The combination product carbidopa/levodopa/entacapone (CLE) was approved in 2003 for the treatment of PD patients. To assess the evidence for the place of CLE in the treatment of PD. CLE has a good efficacy, safety and tolerability profile, similar to that of entacapone taken separately with carbidopa/levodopa (CL). Compared to CL alone, it prolongs levodopa's benefit, and improves the quality of life but not the motor performance in PD patients with nondebilitating "wearing-off" or dyskinesia. However, it increases the dyskinesia rate in early PD patients, and has adverse events in advanced patients with significant motor complications. There is insufficient evidence regarding cost-effectiveness. CLE is an attractive alternative for patients with nondisabling "wearing-off" or dyskinesia taking CL with or without entacapone. It cannot be recommended for early PD patients, as it can induce more dyskinesia than CL alone, or in any patients who seem to have more adverse events.

Lack of differential serotonin biosynthesis capacity in genetically selected low and high aggressive mice

Reduced brain serotonin (5-HT) activity has been linked to impulsive and violent forms of aggression for decades. Despite a vast accumulation of data pertinent to the above observation, information about the possible mechanisms underlying such a decreased 5-HT functioning is virtually absent. Amongst many, reduced 5-HT biosynthetic capacity is a likely possibility in violent individuals and/or in high-aggressive animals. In order to examine this hypothesis, the current study principally aimed at the determination and comparison of the 5-HT biosynthetic capacity in three different strains of high- and low-aggressive mice obtained by artificial genetic selection. While low Tryptophan Hydroxylase (TPH) activity can be expected to lead to low 5-HT levels and pathological aggression, high TPH activity can be expected to increase 5-HT levels and normal territorial aggression. The above hypothesis was assessed by estimating the in-vivo synthesis rate and synthesis rate constant of 5-HT biochemically by measuring the accumulation of 5-hydroxytryptophan (5-HTP) following treatment with the central aromatic amino-acid decarboxylase inhibitor 3-hydroxybenzylhydrazine (NSD-1015). Surprisingly, we found no differences in the 5-HT biosynthetic capacity between the high- and low-aggressive selection lines in their prefrontal cortices and raphe nuclei, two main brain regions closely involved in aggression control. Thus, the underlying inherent genetic differences in aggressiveness observed in these artificially selected mouse strains are not due to constitutive functional differences in their TPH activity in these brain regions.