Amino Acid Decarboxylase Inhibitor Research Articles

Colorimetric determination of carbidopa in anti-Parkinson drugs based on 4-hydroxy-3-methoxybenzaldazine formation by reaction with vanillin

In this paper is reported the selective colorimetric detection and quantification of carbidopa, an inhibitor of aromatic amino acid decarboxylase, in the co-presence of levodopa as dopamine precursor in pharmaceutical formulations for the treatment of Parkinson’s disease. The method is based on the selective condensation reaction between the hydrazine group from carbidopa and the formyl functional group of vanillin, a natural flavoring agent, in acidified alcoholic solution. The yellow color development (λmax ~ 420 nm) due to the formation of 4-hydroxy-3-methoxybenzaldazine (HMOB) was observed for carbidopa only, whereas levodopa, lacking the hydrazine group, did not color the solution, as expected. The calibration curves for two tablet formulations of levodopa in combination with carbidopa (4:1) were superimposable with levodopa/carbidopa (4:1), as well as carbidopa alone, in standard solution, i.e., the excipients and additives did not interfere with carbidopa determination, corresponding to a mean recovery about 105%. The linear dynamic range was between 5.00 and 50.0 mg L−1 with very good reproducibility within this range (CVav% about 3–4%) and very good sensitivity, with limits of quantification of about 1 mg L−1. The colorimetric method developed here is very simple, inexpensive, and effective for drug estimation and quality control of pharmaceutical formulations.Graphical abstract

Formulation and evaluation of chitosan based polyelectrolyte complex of levodopa for nasal drug delivery

Because chitosan is biodegradable, biocompatible, non-toxic, and mucoadhesive, it is commonly used in the formulation of nasal drug delivery nanoparticles employing polyelectrolyte complexes. However, chitosan's lower solubility in aqueous and alkaline conditions limits its use in the pharmaceutical and biomedical fields. This needs the development of improved chemically altered chitosan mimics that can overcome the solubility barrier. Although Levodopa is an alternative in the treatment of Parkinson's disease, it has a low oral bioavailability and very low brain absorption due to its extensive degradation by aromatic amino acid decarboxylase in the peripheral circulation. As a result, levodopa and carbidopa, a peripheral amino acid decarboxylase inhibitor, are given together. The nose to brain medication delivery of levodopa desolate via the olfactory pathway and the trigeminal neurons has been investigated in an effort to improve brain uptake and avoid degradation of levodopa in peripheral circulation and the use of carbidopa in combination. Ionic interactions mediate charged functional groups of former polysaccharides at dissimilar pH situations (pH 5, 8, 10, and 12) and identify polyelectrolyte complexes based on chitosan and pectin during physicochemical (particle size and zeta potential) and solid-state characterizations.

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.

Droxidopa alters dopamine neuron and prefrontal cortex activity and improves attention-deficit/hyperactivity disorder-like behaviors in rats

Finding alternative treatments for attention-deficit/hyperactivity disorder (ADHD) is crucial given the safety and efficacy problems of current ADHD medications. Droxidopa, also known as L-threo-dihydroxyphenylserine (L-DOPS), is a norepinephrine prodrug that enhances brain norepinephrine and dopamine levels. In this study, we used electrophysiological tests to examine effects of L-DOPS on the prefrontal cortex (PFC) and dopamine neurons in the ventral tegmental area. We also conducted behavioral tests to assess L-DOPS’ effects on ADHD-like behaviors in rats. In chloral hydrate-anesthetized rats, PFC local field potentials oscillated between the active, depolarized UP state and the hyperpolarized DOWN state. Mimicking the effect of d-amphetamine, L-DOPS, given after the peripheral amino acid decarboxylase inhibitor, benserazide (BZ), increased the amount of time the PFC spent in the UP state, indicating an excitatory effect of L-DOPS on PFC neurons. Like d-amphetamine, L-DOPS also inhibited dopamine neurons, an effect significantly reversed by the D2-like receptor antagonist raclopride. In the behavioral tests, BZ + L-DOPS improved hyperactivity, inattention and impulsive action of the adolescent spontaneously hypertensive rat (SHR/NCrl), well-validated animal model of the combined type of ADHD. BZ + L-DOPS also reduced impulsive choice and impulsive action of Wistar rats, but did not ameliorate the inattentiveness of Wistar Kyoto rats (WKY/NCrl), proposed model of the ADHD-predominantly inattentive type. In conclusion, L-DOPS produced effects on the PFC and dopamine neurons characteristic of drugs used to treat ADHD. BZ + L-DOPS ameliorated ADHD-like behaviors in rats suggesting its potential as an alternative ADHD treatment.

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

Mammalian Taste Bud Cells Utilize Extragemmal 5-Hydroxy-L-Tryptophan to Biosynthesize the Neurotransmitter Serotonin.

Serotonin or 5-hydroxytryptamine (5-HT) is an important neurotransmitter that is found in mammalian taste buds and can regulate the output of intragemmal signaling networks onto afferent nerve fibers. However, it is unclear how 5-HT is produced, synthesized locally inside taste buds or absorbed from outside sources. In this study, we attempt to address this question by delineating the process of possible 5-HT biosynthesis within taste buds. First, we verified that the rate-limiting enzyme tryptophan hydroxylase (TPH2) responsible for converting L-tryptophan into the intermediate 5-hydroxy-L-tryptophan (5-HTP) is expressed in a subset of type II taste bud cells (TBCs) whereas the enzyme aromatic L-aromatic amino acid decarboxylase (AADC) capable of converting 5-HTP into 5-HT is found in type III TBCs. And abolishment of TPH2 did not affect the production of intragemmal 5-HT or alter TBCs; the mutant mice did not show any changes in behavioral responses to all five primary taste qualities: sweet, umami, bitter, salty, and sour. Then we identified that 5-HTP as well as AADC are abundant in type III TBCs; and application of an AADC inhibitor significantly blocked the production of 5-HT in taste buds. In contrast, administration of an inhibitor on serotonin-reuptake transporters had minimal impact on the 5-HT amount in taste buds, indicating that exogenous 5-HT is not a major source for the intragemmal transmitter. Taken together, our data indicate that intragemmal serotonin is not biosynthesized de novo from tryptophan; instead, it is produced by AADC-mediated conversion of 5-HTP absorbed from the plasma and/or nerve fibers into 5-HT. Thus, our results suggest that the overall bodily 5-HTP level in the plasma and nervous system can regulate taste buds’ physiological function, and provide an important molecular mechanism connecting these peripheral taste organs with the circulatory and nervous systems.

Levodopa in Parkinson's Disease: Current Status and Future Developments.

BackgroundEver since the pioneering reports in the 60s, L-3,4-Dioxyphenylalanine (levodopa) has represented the gold standard for the treatment of Parkinson’s Disease (PD). However, long-term levodopa (LD) treatment is frequently associated with fluctuations in motor response with serious impact on patient quality of life. The pharmacokinetic and pharmacodynamic properties of LD are pivotal to such motor fluctuations: discontinuous drug delivery, short half-life, poor bioavailability, and narrow therapeutic window are all crucial for such fluctuations. During the last 60 years, several attempts have been made to improve LD treatment and avoid long-term complications.MethodsResearch and trials to improve the LD pharmacokinetic since 1960s are reviewed, summarizing the progressive improvements of LD treatment.ResultsInhibitors of peripheral amino acid decarboxylase (AADC) have been introduced to achieve proper LD concentration in the central nervous system reducing systemic adverse events. Inhibitors of catechol-O-methyltransferase (COMT) increased LD half-life and bioavailability. Efforts are still being made to achieve a continuous dopaminergic stimulation, with the combination of oral LD with an AADC inhibitor and a COMT inhibitor, or the intra-duodenal water-based LD/ carbidopa gel. Further approaches to enhance LD efficacy are focused on new non-oral administration routes, including nasal, intra-duodenal, intrapulmonary (CVT-301) and subcutaneous (ND0612), as well as on novel ER formulations, including IPX066, which recently concluded phase III trial.ConclusionNew LD formulations, oral compounds as well as routes have been tested in the last years, with two main targets: achieve continuous dopaminergic stimulation and find an instant deliver route for LD.

Enhanced dopamine D2 autoreceptor function in the adult prefrontal cortex contributes to dopamine hypoactivity following adolescent social stress.

Adult psychiatric disorders characterized by cognitive deficits reliant on prefrontal cortex (PFC) dopamine are promoted by teenage bullying. Similarly, male Sprague-Dawley rats exposed to social defeat in mid-adolescence (P35-39) show impaired working memory in adulthood (P56-70), along with decreased medial PFC (mPFC) dopamine activity that results in part from increased dopamine transporter-mediated clearance. Here, we determined if dopamine synthesis and D2 autoreceptor-mediated inhibition of dopamine release in the adult mPFC are also enhanced by adolescent defeat to contribute to later dopamine hypofunction. Control and previously defeated rats did not differ in either DOPA accumulation following amino acid decarboxylase inhibition (NSD-1015 100mg/kg ip.) or total/phosphorylated tyrosine hydroxylase protein expression, suggesting dopamine synthesis in the adult mPFC is not altered by adolescent defeat. However, exposure to adolescent defeat caused greater decreases in extracellular dopamine release (measured using in vivo chronoamperometry) in the adult mPFC upon local infusion of the D2 receptor agonist quinpirole (3 nM), implying greater D2 autoreceptor function. Equally enhanced D2 autoreceptor-mediated inhibition of dopamine release is seen in the adolescent (P40 or P49) mPFC, which declines in control rats by adulthood. However, this developmental decrease in autoreceptor function is absent following adolescent defeat, suggesting retention of an adolescent-like phenotype into adulthood. Current and previous findings indicate adolescent defeat decreases extracellular dopamine availability in the adult mPFC via both enhanced inhibition of dopamine release and increased dopamine clearance, which may be viable targets for improving treatment of cognitive deficits seen in neuropsychiatric disorders promoted by adolescent stress.

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.

Novel formulations and modes of delivery of levodopa.

Ever since its early clinical use in the 1960s, levodopa has remained the gold standard of symptomatic efficacy in the drug treatment of Parkinson's disease (PD). Motor response fluctuations and drug-induced dyskinesias seriously compromise the unparalleled symptomatic efficacy of l-dopa during long-term treatment. Discontinuous drug delivery resulting from the short half-life of l-dopa and erratic gastrointestinal absorption plays a major role in the pathophysiology of these motor complications. Several approaches to improve the pharmacokinetics and ways of administration of l-dopa are in different stages of clinical development and include novel formulations as well as nonoral routes of drug delivery. IPX066 is a novel extended-release l-dopa capsule that has successfully completed phase III clinical trials while the l-dopa prodrug XP21279 and a gastric retention formulation ("accordion pill") are in earlier phases of clinical development. Novel enzyme inhibitors enhancing l-dopa efficacy and half-life are also still being developed, including a novel catechol-O-methyltransferase inhibitor with once-daily pharmacokinetics, and there are studies testing the effects of increasing the dose of amino acid decarboxylase inhibitors given concomitantly with l-dopa. Intrajejunal infusion of a gel formulation of l-dopa/carbidopa is in clinical use in Europe, and its efficacy to smooth out motor fluctuations has recently been shown in a randomized, controlled trial. Subcutaneous and intrapulmonal delivery routes of l-dopa have reached phase III of clinical development. After more than 50 years of clinical use, l-dopa not only remains the gold standard of symptomatic efficacy, but it also remains a drug in active clinical development.

6-[18F]Fluoro-l-DOPA Uptake in the Rat Pancreas is Dependent on the Tracer Metabolism

6-[(18)F]fluoro-L-3,4-dihydroxyphenyl alanine ([(18)F]FDOPA) positron emission tomography (PET) is a diagnostic tool which can detect malignancies of the pancreas. We aimed to study whether the manipulation of the [(18)F]FDOPA metabolic pathway would change the (18)F-behavior to provide a biochemical foundation for PET imaging of rat pancreas with [(18)F]FDOPA. Inhibitors of aromatic amino acid decarboxylase, catechol-O-methyltransferase, monoamine oxidases A and B, or their combinations on [(18)F]FDOPA uptake, metabolism, and the regional distribution in the rat pancreas was evaluated using in vivo PET/computed tomography imaging, chromatographic metabolite analyses, and autoradiography. Enzyme inhibition generally increased the uptake of [(18)F]FDOPA derived (18)F-radioactivity in rat pancreas. Dependent on which enzymatic pathway is blocked (or a combination of pathways), different radiolabeled metabolites in pancreas are responsible for this increase in uptake. Altering the metabolism of [(18)F]FDOPA by using various enzymatic inhibitors increased the radioactivity uptake and changed the radiometabolic profile in the pancreas allowing better discrimination between pancreas and surrounding tissues of rat. However, these manipulations did not separate islets from the exocrine pancreas. Elucidating the metabolic behavior of [(18)F]FDOPA provides a biochemical foundation of PET imaging of the rat pancreas.

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.

Formulation and characterization of intranasal mucoadhesive nanoparticulates and thermo-reversible gel of levodopa for brain delivery

Levodopa is the drug of choice in the treatment of Parkinson’s disease but it exhibits low oral bioavailability (30%) and very low brain uptake due to its extensive metabolism by aromatic amino acid decarboxylase in the peripheral circulation. Hence, levodopa is co-administered with carbidopa, a peripheral amino acid decarboxylase inhibitor. In an attempt to improve brain uptake and to avoid degradation of levodopa in peripheral circulation and the use of carbidopa in combination, nose to brain drug delivery of levodopa alone via the olfactory route and the trigeminal nerves has been investigated. Chitosan nanoparticles loaded with levodopa (CNL) were prepared and were incorporated in a thermo-reversible gel prepared using Pluronic PF127 (CNLPgel). The preparation of CNL and CNLPgel was optimized for formulation parameters such as chitosan:TPP ratio, drug load Pluronic concentration to obtain desired particle size of CNL, gelling temperature, gelling time and mucoadhesive strength of CNLPgel. Rheological studies indicated a change in the rheological behavior of plain pluronic gel from Newtonian system at 30 °C to pseudoplastic behavior at 35 °C on incorporation of CNL. In vitro release studies from CNL obeyed Higuchi kinetic model, whereas the drug release from CNLPgel followed the Hixson–Crowell model. In vivo studies indicated a maximum recovery of the drug in brain following intranasal administration of CNL suspension in saline closely followed by the drug dispersed in plain pluronic gel.

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.

Role of Endothelial AADC in Cardiac Synthesis of Serotonin and Nitrates Accumulation

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation.

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.

Recent advances in pharmacological therapy of Parkinson’s disease: Levodopa and carbidopa protective effects against DNA oxidative damage

Parkinson’s disease is one of the most common progressive neurodegenerative disorder. It is characterized by the depletion of dopamine in the dopaminergic neurons of the striatum of the brain. Pharmacological treatment involves the administration of a dopamine precursor, levodo- pa (L-Dopa), which crosses the blood-brain barrier and replaces the loss of dopamine in the brain. One of the main drawbacks of the ad- ministration of L-Dopa is its short half-life, due to the presence of enzymes, such as the amino acid decarboxylase (AADC), able to rapidly me- tabolize L-Dopa. For this reason the intake of L-Dopa takes always place together with an AADC inhibitor such as carbidopa. The assumption of carbidopa increases L-Dopa half-life, but several patients need to increase the dosage of the pharmacological therapy during the progression of the disease. Another area of dispute is represented by the possibility that L-Dopa can exert a toxic effect on the cells, both in peripheral and in central nervous system, increasing the production of ROS following its conversion to dopamine. Past studies reported toxic effects of L-Dopa in vitro and show conflicting data in in vivo experiments. More recent studies have however shown that L-dopa may exert a protective and antioxidant effect on dopaminergic cells, and its combination with carbidopa in pharmacological treatment amplifies antioxidant capability.