Presence Of L-DOPA Research Articles

Pharmacodynamic, pharmacokinetic and rat brain receptor occupancy profile of NLX-112, a highly selective 5-HT1A receptor biased agonist.

NLX-112 (i.e., F13640, befiradol) exhibits nanomolar affinity, exceptional selectivity and full agonist efficacy at serotonin 5-HT1A receptors. NLX-112 shows efficacy in rat, marmoset and macaque models of L-DOPA induced dyskinesia (LID) in Parkinson's disease and has shown clinical efficacy in a Phase 2a proof-of-concept study for this indication. Here we investigated, in rats, its pharmacodynamic, pharmacokinetic (PK) and brain 5-HT1A receptor occupancy profiles, and its PK properties in the absence and presence of L-DOPA. Total and free NLX-112 exposure in plasma, CSF and striatal ECF was dose-proportional over the range tested (0.04, 0.16 and 0.63mg/kg i.p.). NLX-112 exposure increased rapidly (Tmax 0.25-0.5h) and exhibited approximately threefold longer half-life in brain than in plasma (1.1 and 3.6h, respectively). At a pharmacologically relevant dose of 0.16 mg/kg i.p., previously shown to elicit anti-LID activity in parkinsonian rats, brain concentration of NLX-112 was 51-63 ng/g from 0.15 to 1h. In microPET imaging experiments, NLX-112 showed dose-dependent reduction of 18F-F13640 (i.e., 18F-NLX-112) brain 5-HT1A receptor labeling in cingulate cortex and striatum, regions associated with motor control and mood, with almost complete inhibition of labeling at the dose of 0.63 mg/kg i.p.. Co-administration of L-DOPA (6mg/kg s.c., a dose used to elicit LID in parkinsonian rats) together with NLX-112 (0.16mg/kg i.p.) did not modify PK parameters in rat plasma and brain of either NLX-112 or L-DOPA. Here, we demonstrate that NLX-112's profile is compatible with 'druggable' parameters for CNS indications, and the results provide measures of brain concentrations and 5-HT1A receptor binding parameters relevant to the anti-dyskinetic activity of the compound.

Smart-Phone-Assisted Optical Biosensors Based on Silk-Fibroin-Decorated Reduced Graphene Oxide Quantum Dots for Fluorescent Turn-On Recognition of l-Dopa

Levodopa (l-dopa) is a gold standard neurological drug for symptomatic medication of Parkinson’s disease. The long-term use of l-dopa therapy frequently leads to l-dopa-induced dyskinesia and several other non-motor flocculants for fluctuating plasma concentration. To alleviate the adverse effect of dopamine replacement therapy, sensitive detection of l-dopa is still challenging. Therefore, this study presents a simple strategy to develop an inexpensive, easy-to-use, smartphone-based fluorescence turn-on sensory system based on the aggregation-induced emission enhancement phenomenon for instant low-level detection of l-dopa in biological samples. The sensor was fabricated using silk-fibroin-decorated reduced graphene oxide quantum dots, which exhibit a progressive increase of fluorescence intensity in the presence of l-dopa. By measuring the turn-on efficiency of the sensor in elevated concentrations of l-dopa, the limit of detection (LOD) was evaluated as 76.18 nM over a linear range of 0–35 μM. We have further designed a smartphone-based electronic device involving a 365 nm light emitting diode attached below the rare camera for capturing the change of fluorescent color of the solution during sensing. From these images, red, green, and blue values are analyzed using a mobile phone application, and the corresponding LOD was found to be 0.29 μM in a similar linear range of concentration. This simple, cost-effective, and rapid screening gadget is very demanding for on-spot detection of the analytes in remote areas where sophisticated instrumentation is not usually available.

Structure directing agent assisted solvothermal synthesis of octahedral WO3·nH2O large microcrystals: Study of electrochemical and optical properties

In the present study, the growth of tungsten trioxide crystals by solvothermal method was investigated in the presence of polyethylene glycol (PEG) and L-3,4-dihydroxyphenylalanine (L-dopa) as the structure directing agents in acetonitrile solution. The change of crystal faces consisting of stacked layers to very smooth facets is attributed to the change of the crystal growth mechanism from a three-step mechanism in the absence of PEG and L-dopa to a two-step mechanism in their presence. The rate of formation and growth of crystals in the presence of 5 w% of PEG increases significantly compared to those grown in the absence of PEG. The crystals solvothermally grown in the presence of L-dopa were of the octahedral WO3·0.5H2O with fully smooth faces, and those grown in the presence of PEG were the octahedral and rectangular microcrystals of WO3·H2O The transparency and electro-optical properties of the prepared crystal layers are improved in the presence of PEG. The dynamic potential electropolymerization of aniline and the formation of a thin layer of polyaniline on tungsten trioxide crystals were performed and the properties of the obtained core–shell hybrid film were studied. It was shown that covering the crystals with a very thin layer of polyaniline helps to stabilize their electrochemical and optical properties.

Improved natural melanin production by Aspergillus nidulans after optimization of factors involved in the pigment biosynthesis pathway

BackgroundMelanin is a natural pigment that can be applied in different fields such as medicine, environment, pharmaceutical, and nanotechnology. Studies carried out previously showed that the melanin produced by the mel1 mutant from Aspergillus nidulans exhibits antioxidant, anti-inflammatory, and antimicrobial activities, without any cytotoxic or mutagenic effect. These results taken together suggest the potential application of melanin from A. nidulans in the pharmaceutical industry. In this context, this study aimed to evaluate the effect of factors L-tyrosine, glucose, glutamic acid, l-DOPA, and copper on melanin production by the mel1 mutant and to establish the optimal concentration of these factors to maximize melanin production.ResultsThe results showed that l-DOPA, glucose, and copper sulfate significantly affected melanin production, where l-DOPA was the only factor that exerted a positive effect on melanin yield. Besides, the tyrosinase activity was higher in the presence of l-DOPA, considered a substrate required for enzyme activation, this would explain the increased production of melanin in this condition. After establishing the optimal concentrations of the analyzed factors, the melanin synthesis was increased by 640% compared to the previous studies.ConclusionsThis study contributed to elucidating the mechanisms involved in melanin synthesis in A. nidulans as well as to determining the optimal composition of the culture medium for greater melanin production that will make it possible to scale the process for a future biotechnological application.

Serotonin, melatonin and their precursors and metabolites and vitamin D3 derivatives in honey.

We are commenting recent discoveries on the presence of L-DOPA, dopamine, 5-hydroxytryptophan, tryptamine, serotonin, N-acetylserotonin, melatonin, 2-hydroxymelatonin, AFMK and AMK in honey. Serotonin and melatonin, products of the tryptophan metabolism, are widely produced in nature, serving as hormones, neurotransmitters, biological regulators, neurotransmitters and antioxidants, in a context dependent fashion. Dopamine and tryptamine are important neurotransmitters across different species. Honey is used as one of the most popular healthy food substances. Detection of above molecules in honey accompanied by detection of vitamin D3 and its hydroxyderivatives, is consistent with their detection in insects and plants. Their presence in honey enhances spectrum of its beneficial effects for human health and implicates that these molecules must play important role in social insects physiology, bees development and colony functions.

Monophenolase assay using excitation-emission matrix fluorescence and ELMAN neural network assisted by whale optimization algorithm

Tyrosinase plays a vital role for melanogenesis and inherently involves both monophenolase activity and diphenolase activity. Monophenolase catalyzes hydroxylation of tyrosine to l-DOPA (L-3,4-dihydroxyphenylalanine). Real-time monophenolase assay method is of outstanding interest for both scientific research and industrial application. A combined strategy of three-dimensional excitation-emission matrix (EEM) fluorescence spectra and artificial neural network was developed to determine monophenolase activity. A quantitation system for tyrosine in presence of l-DOPA was designed based on ELMAN neural network. Principal component analysis (PCA) was conducted to reduce the dimensionality of fluorescence spectra. Four principal components was used as input variables. Whale optimization algorithm (WOA) was implemented to optimize the initial weights and threshold network. Real-time concentration of tyrosine in monophenolase reaction was monitored to calculate the initial velocity for tyrosine consumption. The exclusive monophenolase activity without interference from diphenolase reaction was determined. Limit of detection (LOD) for monophenolase assay is 0.0113 U mL−1. Using the proposed method, enzyme kinetics for monophenolase was investigate. Km was calculated as 14.16 μM. Inhibitor for monophenolase was screened by using model molecule kojic acid with IC50 of 3.49 μM. The assay method exhibited a promising prospect to characterize the kinetics and inhibitor of monophenolase.

The herbicide paraquat alters growth and melanin production on the Cryptococcus neoformans/Cryptococcus gattii species complex

Paraquat (1,10-dimethyl-4,4-bipyridinium dichloride; PQ) is a free-radical producing herbicide that affects cell membranes and can upset the environmental balance of microorganisms present in soil, such as Cryptococcus spp. This study aimed to evaluate the in vitro activity of PQ against Cryptococcus spp. in planktonic and biofilm forms, as well as the protective effect of antioxidant agents against the antifungal effect of PQ and the kinetics of melanin production in response to PQ. Susceptibility to PQ was evaluated by microdilution. Cryptococcus sp. strains exposed to PQ were grown in media with ascorbic acid (AA) and glutathione (GSH). Melanin production was assessed in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) + PQ. The minimum inhibitory concentration of PQ against Cryptococcus spp. ranged from 8 to 256 µg/mL. Furthermore, PQ reduced biofilm formation. AA and GSH restored the fungal growth of Cryptococcus spp. exposed to PQ. In addition, l-DOPA + PQ delayed melanin production by 24 and 48 h for C. deuterogattii and C. neoformans sensu lato, respectively, suggesting that PQ induces a fitness trade-off in melanin production. Taken together, our data suggest that the antifungal effect of PQ against Cryptococcus spp. possibly exerts selective pressures interfering with biofilm formation and melanin production by these yeasts.

Reduced graphene quantum dot based versatile platform for l-dopa sensing: Fluorescence turn-on, filter paper, and air-stable flexible electronic devices

L-dopa is a chiral drug that has been extensively used for the treatment of Parkinson’s disease. The concentration of L-dopa plays a vital role in the treatment, and this work emphasizes on designing a versatile platform for the quantitative and selective detection of L-dopa using reduced graphene quantum dot (rGQD). The platform is efficient in three different sensing methods, viz. fluorescence turn-on, filter paper-based sensing, and electronic measurements. The rGQD undergoes a dramatic fluorescence turn-on in the presence of L-dopa in aqueous media and artificial urine with a detection limit as low as 1.307 µM and 1.217 µM respectively. Experimental evidence revealed that the fluorescence enhancement is attributed to aggregation-induced emission mechanism. The system is also applicable for visual detection of L-dopa using filter paper strips treated with rGQD and develops bright fluorescence under ultra-violet irradiation. Apart from this, electrical sensors for L-dopa were also developed by preparing rGQD treated poly-vinyl alcohol films, and the increase in the current conduction with varying concentrations of L-dopa was observed. The films produce 2.5 order higher current conduction at 300 μM of L-dopa with a detection limit of about 13.136 µM. At the final stage, we have designed flexible electronic devices using these materials that develop 1.3 order higher current for L-dopa. The devices exhibit excellent air stability under varied relative humidity conditions as well as high bending stability for potential practical applications.

Anti-melanogenesis and anti-tyrosinase properties of aryl-substituted acetamides of phenoxy methyl triazole conjugated with thiosemicarbazide: Design, synthesis and biological evaluations

A series of aryl phenoxy methyl triazole conjugated with thiosemicarbazides were designed, synthesized, and evaluated for their tyrosinase inhibitory activities in the presence of l-dopa and l-tyrosine as substrates. All the compounds showed tyrosinase inhibition in the sub-micromolar concentration. Among the derivatives, compound 9j bearing benzyl displayed exceptionally high potency against tyrosinase with IC50 value of 0.11 μM and 0.17 μM in the presence of l-tyrosine and l-dopa as substrates which is significantly lower than that of kojic acid as the positive control with an IC50 value of 9.28 μM for l-tyrosine and 9.30 μM for l-dopa. According to Lineweaver–Burk plot, 9j demonstrated an uncompetitive type of inhibition in the kinetic assay. Also, in vitro antioxidant activities determined by DPPH assay recorded an IC50 value of 68.43 μM for 9i. The melanin content of 9j was determined on B16F10 melanoma human cells which demonstrated a significant reduction of the melanin content. Moreover, the binding energies corresponding to the same ligand as well as computer-aided drug-likeness and pharmacokinetic studies were also carried out. Compound 9j also possessed metal chelation potential correlated to its high anti-TYR activity.

Opportunistic use of catecholamine neurotransmitters as siderophores to access iron by Pseudomonas aeruginosa.

Iron is an essential nutrient for bacterial growth and the cause of a fierce battle between the pathogen and host during infection. Bacteria have developed several strategies to access iron from the host, the most common being the production of siderophores, small iron-chelating molecules secreted into the bacterial environment. The opportunist pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a wide panoply of xenosiderophores, siderophores produced by other microorganisms. Here, we demonstrate that catecholamine neurotransmitters (dopamine, l-DOPA, epinephrine and norepinephrine) are able to chelate iron and efficiently bring iron into P. aeruginosa cells via TonB-dependent transporters (TBDTs). Bacterial growth assays under strong iron-restricted conditions and with numerous mutants showed that the TBDTs involved are PiuA and PirA. PiuA exhibited more pronounced specificity for dopamine uptake than for norepinephrine, epinephrine and l-DOPA, whereas PirA specificity appeared to be higher for l-DOPA and norepinephrine. Proteomic and qRT-PCR approaches showed pirA transcription and expression to be induced in the presence of all four catecholamines. Finally, the oxidative properties of catecholamines enable them to reduce iron, and we observed ferrous iron uptake via the FeoABC system in the presence of l-DOPA.

Opportunistic Use by <i>Pseudomonas</i> Aeruginosa of Catecholamine Neurotransmitters as Siderophore to Access Iron

Iron is an essential nutrient for bacteria growth and microorganisms have developped several strategies to access iron, the most common being the production of siderophores, iron-chelating molecules secreted into the bacterial environment. Here, we demonstrate that catecholamine neurotransmitters (dopamine, L-DOPA, epinephrine, and norepinephrine) are able to chelate iron and bring iron into Pseudomonas aeruginosa cells via an energy-dependent transport mechanism that relies on TonB-dependent transporters (TBDTs). Using bacterial growth assay under strong iron-restricted conditions, we show that the TBDTs involved are PiuA and PirA. PiuA exhibited more pronounced specificity for dopamine uptake, whereas PirA specificity appeared to be higher for L-DOPA and norepinephrine. Proteomic and qRT-PCR approaches showed pirA transcription and expression to be induced in the presence of all four catecholamines. Finally, the oxidative properties of catecholamines enable them to reduce iron, and we observed ferrous iron uptake via the FeoABC system in the presence of L-DOPA.

Inhibition of Enzymatic Browning by Onion (Allium cepa L.): Investigation on Inhibitory Mechanism and Identification of Active Compounds

Presence of browning or black-spot in fresh foods can adversely affect consumer acceptance. Onion has been reported to exert inhibitory activity against browning reaction. This research aimed to uncover the mechanism and identify active compounds in onion responsible for PPO inhibitors based on metabolomic approach. Onion was fractioned using different solvents, i.e n-hexane; chloroform; ethyl acetate; water, respectively. As a result, ethyl acetate fraction (EAF) of the onion demonstrated the strongest inhibition to PPO in comparison with other fractions, i.e. n-hexane, chloroform, and water. The reversible inhibitory activity of PPO by EAF occurred with presence of L-DOPA as substrate through competitive inhibition and Cu chelation in the active side of the PPO. Based on 1H-NMR (X) score plot and PPO inhibition (Y) using OPLS, NMR signals revealed that active compounds accounting for inhibition of PPO included quercetin, kaempferol, cyanidin 3.4’-di-O-β-glucopyranoside, quercetin 4-O-β-D-glucopyranoside, cyanidin 7-O-(3”-O-glucosyl-6”-O-malonyl-β-glucopyrano-side)-4’-O-β-glucopyranoside, cyanidin 3-(6”-O-malonyl) laminaribioside’.

Cryptococcus neoformans – New Science for Discovering Melanin Modifiers

Aim: The present study was taken up to establish the effect of niacinamide on phenoloxidase lead melanogenesis and to prove the reliability of C. neoformans based screening methodology.
 Methods: The organism was grown in the Minimal media in presence and absence of L- DOPA and Niacinamide and checked for its pigment producing ability at different time intervals.
 Results: Niacinamide did not affect the pigmentation in Cryptococcus neoformans in the absence or presence of L-Dopa.
 Conclusion: Cryptococcus neoformans as a biological tool for studying the mechanism of action of various melanin promoters/ inhibitors. The present study highlights the importance and usefulness of Cryptococcus neoformans based screening invention as it is cost effective rapid and ‘living cell model’.

Malassezia pachydermatis from animals: Planktonic and biofilm antifungal susceptibility and its virulence arsenal

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to>64 μg/mL for azole derivatives, 1 to >16 μg/mL for amphotericin B and 0.03 to 0.25 μg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.

Effects of the Serotonin 5-HT1A Receptor Biased Agonists, F13714 and F15599, on Striatal Neurotransmitter Levels Following L-DOPA Administration in Hemi-Parkinsonian Rats.

Peak-dose dyskinesia is associated with the dramatic increase in striatal dopamine levels that follows L-DOPA administration. The 'false neurotransmitter' hypothesis postulates that the latter is likely due to an aberrant processing of L-DOPA by serotonergic neurons. In keeping with this hypothesis, two highly selective 'biased agonists' of 5-HT1A receptors-namely F13714 and F15599 (NLX-101)-were recently shown to exhibit exceptionally potent anti-dyskinetic activity without impairing L-DOPA therapeutic properties despite their differential targeting of 5-HT1A receptor sub-populations. In this study, we investigated whether these two compounds dampened peak L-DOPA-induced dopamine microdialysate levels in the striatum of hemi-parkinsonian rats. Acute administration of either F13714 (0.04 and 0.16mg/kg i.p.) or F15599 (0.16 and 0.64mg/kg, i.p.) blunted L-DOPA (2mg/kg)-induced increases in dopamine microdialysate levels in the denervated striatum (following unilateral injection of 6-OHDA into the medial forebrain bundle). No significant changes were observed on the intact side of the brain. Concurrently, both drugs profoundly reduced striatal serotonin levels on both sides of the brain. In addition, F13714 and F15599, in the presence of L-DOPA, produced a dose-dependent increase in glutamate levels, but this effect was restricted to later time points. These finding support the interpretation that F13714 and F15599 mediate their anti-dyskinetic effects by blunting of the peak in dopamine levels via activation of somatodendritic serotonin 5-HT1A receptors and the consequent inhibition of serotonergic neurons. This study adds to the growing body of evidence supporting the development of a potent 5-HT1A receptor agonist for treatment of peak-dose dyskinesia.

Post-translational incorporation of 3,4-dihydroxyphenylalanine into the C terminus of α-tubulin in living cells.

The C-terminal tyrosine (Tyr) of the α-tubulin chain is subjected to post-translational removal and readdition in a process termed the "detyrosination/tyrosination cycle". We showed in previous studies using soluble rat brain extracts that l-3,4-dihydroxyphenylalanine (l-Dopa) is incorporated into the same site as Tyr. We now demonstrate that l-Dopa incorporation into tubulin also occurs in living cells. We detected such incorporation by determining the "tyrosination state" of tubulin before and after incubation of cells in the presence of l-Dopa. The presence of a tubulin isospecies following l-Dopa incubation that was not recognized by antibodies specific to Tyr- and deTyr-tubulin was presumed to reflect formation of Dopa-tubulin. l-Dopa was identified by HPLC as the C-terminal compound bound to α-tubulin. l-Dopa incorporation into tubulin was observed in Neuro 2A cells and several other cell lines, and was not due to denovo protein biosynthesis. Dopa-tubulin had microtubule-forming capability similar to that of Tyr- and deTyr-tubulin. l-Dopa incorporation into tubulin did not notably alter cell viability, morphology, or proliferation rate. CAD cells (a neuron-like cell line derived from mouse brain) are easily cultured under differentiating and nondifferentiating conditions, and can be treated with l-Dopa. Treatment of CAD cells with l-Dopa and consequent increase in l-Dopa-tubulin resulted in reduction of microtubule dynamics in neurite-like processes.

Cortical stimulation relieves parkinsonian pathological activity invitro.

Previously, we have shown that chemical excitatory drives such as N-methyl-d-aspartate (NMDA) are capable of activating the striatal microcircuit exhibiting neuronal ensembles that alternate their activity producing temporal sequences. One aim of this work was to demonstrate whether similar activity could be evoked by delivering cortical stimulation. Dynamic calcium imaging allowed us to follow the activity of dozens of neurons with single-cell resolution in mus musculus brain slices. A train of electrical stimuli in the cortex evoked network activity similar to the one induced by bath application of NMDA. Previously, we have also shown that the dopamine-depleted striatal microcircuit increases its spontaneous activity generating dominant recurrent ensembles that interrupt the temporal sequences found in control microcircuits. This activity correlates with parkinsonian pathological activity. Several cortical stimulation protocols such as transcranial magnetic stimulation reduce motor signs of Parkinsonism. Here, we show that cortical stimulation invitro temporarily eliminates the pathological activity from the dopamine-depleted striatal microcircuit by turning off some neurons that sustain this activity and recruiting new ones that allow transitions between network states, similar to the control circuit. When cortical stimulation is given in the presence of L-DOPA, parkinsonian activity is eliminated during the whole recording period. The present experimental evidence suggests that cortical stimulation such as that generated by transcranial magnetic stimulation, or otherwise, may allow reduce L-DOPA dosage.

Interaction of catecholamine precursor l-Dopa with lysozyme: A biophysical insight

The current study comprises of an inclusive biophysical study, enlightening the binding of L-3, 4-dihydroxyphenylalanine (l-Dopa) with human lysozyme (HL) and hen egg white lysozyme (HEWL). Spectroscopic and molecular docking tools have been utilized to study the interaction of l-Dopa with both HL and HEWL. Spectrofluorimetric measurements exhibited that l-Dopa quenched the HL and HEWL intrinsic fluorescence. A binding constant (Kb) of ∼104M−1 for both HL and HEWL was obtained, asserting a significant binding. Negative value of ΔG affirmed that the reaction between proteins and l-Dopa was spontaneous. Far-UV CD spectra revealed a boost to the proteins helical content in the presence of l-Dopa. Furthermore, DLS measurements displayed the decrease in hydrodynamic radii (Rh) of HL and HEWL in the presence of l-Dopa. Molecular docking studies established that l-Dopa formed complexes with both the proteins through hydrogen bonding and hydrophobic interaction. The present study characterizing the l-Dopa interaction with lysozyme could be noteworthy in realizing both pharmaco-dynamics and/or −kinetics of drugs used in various diseases.

Molecularly imprinted cyclodextrin nanosponges for the controlled delivery of L-DOPA: perspectives for the treatment of Parkinson’s disease

ABSTRACTBackground: L-DOPA is an amino acid precursor to the neurotransmitter dopamine that is extensively used as a prodrug for the treatment of Parkinson’s disease. However, L-DOPA is an unstable compound: when exposed to light or added to aqueous solutions, it may degrade, compromising its therapeutic properties.Methods: In this work, a new type of drug-loaded cyclodextrin-based nanosponge, obtained using molecular imprinting, is described for the prolonged and controlled release of L-DOPA. The molecularly imprinted nanosponges (MIP-NSs) were synthesized by cross-linking β-cyclodextrin with 1,1ʹ-carbonyldiimidazole in DMF in the presence of L-DOPA as a template molecule. TGA, DSC and FTIR analyses were performed to characterize the interactions between L-DOPA and the two nanosponge structures. Quantitative NMR spectroscopy was used to determine the amount and the affinity of L-DOPA entrapped in the nanosponges. The in vitro L-DOPA release kinetics from the NSs were quantitatively determined by HPLC analysis.Results: The MIP-NSs show a slower and more prolonged release profile than the non-imprinted nanosponges. No degradation of the L-DOPA hosted in the MIP-NSs was observed after long-term storage at room temperature.Conclusions: The MIP-NSs are a promising alternative for the storage and controlled delivery of L-DOPA.

Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine.

Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.