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Semen traces are considered important pieces of evidence in forensic investigations, especially those involving sexsual offenses. Recently, our research group developed a fluorescence-based technique to accurately determine the age of semen traces. However, the specific compounds resonsible for the fluoresescent behaviour of ageing semens remain unknown. As such, in this exploratory study, the aim is to identify the components associated with the fluorescent behavior of ageing semen traces. In this investigation semen stains and various biofluorophores commonly found in body fluids were left to aged for 0, 2, 4, 7, 14 and 21 days. Subsequently, thin-layer chromatography (TLC) and ultra-performance liquid chromatography (UPLC) mass spectrometry were performed to identify the biofluorophores present in semen. Several contributors to the autofluorescence could be identified in semen stain, these include tryptophan, kynurenine, kynurenic acid, and norharman. The study sheds light on the., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

D-Amino acid oxidase (DAO), a D-amino acid metabolizing enzyme, is reportedly associated with the psychiatric disease schizophrenia, suggesting a role for DAO inhibitors in its treatment. We have previously reported that DAO catalyzes the conversion of nonfluorescent 6-methylthio-D-kynurenine (MeS-D-KYN) to fluorescent 5-methylthiokynurenic acid (MeS-KYNA) in vitro. The present study aimed to determine the potential of MeS-D-KYN in evaluating DAO activity in vivo using renal microdialysis technique in rats. Male Sprague-Dawley rats were subjected to linear microdialysis probe implantation in the left kidney. Continuous perfusion of MeS-D-KYN was maintained, and DAO activity in the kidney cortex was evaluated by measuring the MeS-KYNA content in the microdialysate. The microdialysate was collected every 30 min and analyzed by high-performance liquid chromatography with fluorescence detection, monitored at 450 nm with an excitation wavelength of 364 nm. A significant production of MeS-KYNA was observed during, but not before, infusion of MeS-D-KYN, indicating that this compound is not endogenous. MeS-KYNA production was suppressed by the co-infusion of DAO inhibitor, 5-chlorobenzo[d]isoxazol-3-ol (CBIO), suggesting that MeS-D-KYN was converted to MeS-KYNA by renal DAO. Moreover, oral administration of CBIO effectively suppressed DAO activity in a dose-dependent manner. DAO converted MeS-D-KYN to MeS-KYNA in vivo, suggesting the potential of this compound in evaluating DAO activity. The use of the renal microdialysis technique developed in this study facilitates the monitoring of DAO activity in live experimental animals.

High levels of D-amino acid oxidase (DAO) are associated with neurological and psychiatric disorders, while L-amino acid oxidase (LAO) exhibits antimicrobial and antitumor properties. The enzymatic conversion of the non-fluorescent kynurenine (KYN) into the endogenous weak fluorescent kynurenic acid (KYNA) by the action of DAO has previously been reported. However, the fluorescence of KYNA can be improved by changing the substituents on the aromatic rings. In this study, we prepared different 6-phenyl-substituted KYNA derivatives and investigated their fluorescence properties. Among them, 2-MePh-KYNA showed the maximum fluorescence quantum yield of 0.881 at 340 nm excitation and 418 nm emission wavelengths. The effects of solvent properties (dielectric constant, p K a , viscosity, and proticity) on the fluorescence intensity (FLI) of the KYNA derivatives were explored. The FLI of 2-MePh-KYNA was significantly large in protic solvents. Subsequently, 2-MePh-D-KYN and 2-MePh-L-KYN were prepared with high enantiopurity (>99.25%) for the enzymatic conversion. 2-MePh-D-KYN exhibited high sensitivity (∼19 times that of a commercial DAO substrate and ∼60 times that of the previously reported MeS-D-KYN) and high selectivity, as it was not cross-reactive towards LAO, while 2-MePh-L-KYN was also converted into 2-MePh-KYNA by LAO. Furthermore, the 2-MePh-D-KYN probe successfully detected DAO in eel liver, kidney, and heparin-anticoagulated plasma in the in vitro study.

Previous studies reported significantly altered tryptophan catabolite concentrations in major depression. Thus, tryptophan catabolites were considered as potential biomarkers of depression and their modulators as potential targets for psychopharmacotherapy. However, the results were based mainly on studies with small sample sizes limiting their generalizability. Against this background, we investigated the relationship of peripheral tryptophan catabolites with depression in a population-based sample with n = 3,389 participants (with fasting status ≥ 8 h and C-reactive protein < 10 mg/L). N = 248 had clinically significant depression according to a PHQ-9 score of ≥ 10, n = 1,101 subjects had mild depressive symptoms with PHQ-9 scores between 5 and 9, and n = 2,040 had no depression. After multivariable adjustment, clinically significant depression was associated with lower kynurenine and kynurenic acid. Spearman correlation coefficients of the tryptophan catabolites with the severity of depression were very small (rho ≤ 0.080, p ≤ 0.015). None of the tryptophan catabolites could diagnostically separate depressed from not depressed persons. Concerning linear associations, kynurenine and kynurenic acid were associated only with the severity and the cognitive dimension of depression but not its somatic dimension. Tryptophan catabolites were not associated with persistence or recurrence of depression at the 5 year follow-up. The results replicated the association between kynurenine and kynurenic acid with depression. However, the associations were small raising doubts about their clinical utility. Findings underline the complexity of the relationships between depression and tryptophan catabolites. The search for subgroups of depression with a potentially higher impact of depression might be warranted., (© 2023. The Author(s).)

Kynurenic acid (KNA) and 4-hydroxyquinoline (4HQN) are photochemically active products of tryptophan catabolism that readily react with tryptophan (Trp) and tyrosine (Tyr) after optical excitation. Recently, transient absorption experiments have shown that at neutral pH Trp reacts with triplet KNA via proton-coupled electron transfer (PCET), and not via electron transfer (ET) as it was suggested before. PCET includes the stepwise transition of both electrons and protons from Trp to triplet KNA. In this work, we confirmed that PCET is the reaction mechanism by the alternative method of time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP). Further studies by TR-CIDNP revealed hydrogen transfer as the mechanism of the reaction between triplet KNA and Tyr in neutral solutions and a transition of both PCET and H-transfer mechanisms to ET under acidic conditions. 4HQN, being the chromophoric core of KNA, exhibits different spectral and photophysical properties from KNA but employs the same mechanisms for the reactions of its triplet state with Trp and Tyr at neutral and acidic pH.

In this work, the interaction between Kynurenic acid (KYNA) and several natural and modified cyclodextrins (CDs) is carried out. Among all the CD tested, HPβ-CD showed the strongest complexation constant (K F ), with a value of 270.94 ± 29.80 M -1 . Between natural (α- and β-) CDs, the complex of KYNA with β-CD was the most efficient. The inclusion complex of KYNA with CDs showed a strong influence of pH and temperature. The K F value decreased at high pH values, when the pK a was passed. Moreover, an increase of the temperature caused a decrease in the K F values. The thermodynamic parameters of the complexation (ΔH°, ΔS° and ΔG°) were studied with negative entropy, enthalpy and spontaneity of the process at 25 °C. Moreover, the inclusion complex was also characterized using FTIR and TGA. Finally, molecular docking calculations provided different interactions and their influence in the complexation constant., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Kynurenic acid (KYNA, 4-oxoquinoline-2-carboxylic acid), an intermediate of the tryptophan metabolism, has been recognized to exert different neuroactive actions; however, the need of how it or its aminoalkylated amide derivative N -(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-oxo-1,4-dihydroquinoline-2-carboxamide (KYNA-A4) exerts any effects on ion currents in excitable cells remains largely unmet. In this study, the investigations of how KYNA and other structurally similar KYNA derivatives have any adjustments on different ionic currents in pituitary GH 3 cells and hippocampal mHippoE-14 neurons were performed by patch-clamp technique. KYNA or KYNA-A4 increased the amplitude of M-type K + current ( I K(M) ) and concomitantly enhanced the activation time course of the current. The EC 50 value required for KYNA- or KYNA-A4 -stimulated I K(M) was yielded to be 18.1 or 6.4 μM, respectively. The presence of KYNA or KYNA-A4 shifted the relationship of normalized I K(M) -conductance versus membrane potential to more depolarized potential with no change in the gating charge of the current. The voltage-dependent hysteretic area of I K(M) elicited by long-lasting triangular ramp pulse was observed in GH 3 cells and that was increased during exposure to KYNA or KYNA-A4. In cell-attached current recordings, addition of KYNA raised the open probability of M-type K + channels, along with increased mean open time of the channel. Cell exposure to KYNA or KYNA-A4 mildly inhibited delayed-rectifying K + current; however, neither erg -mediated K + current, hyperpolarization-activated cation current, nor voltage-gated Na + current in GH 3 cells was changed by KYNA or KYNA-A4. Under whole-cell, current-clamp recordings, exposure to KYNA or KYNA-A4 diminished the frequency of spontaneous action potentials; moreover, their reduction in firing frequency was attenuated by linopirdine, yet not by iberiotoxin or apamin. In hippocampal mHippoE-14 neurons, the addition of KYNA also increased the I K(M) amplitude effectively. Taken together, the actions presented herein would be one of the noticeable mechanisms through which they modulate functional activities of excitable cells occurring in vivo.

The application of kynurenic acid (KYNA) as an electron-rich aromatic system in the modified Mannich reaction has been examined. The extension possibility of the reaction was tested by using amines occurring in a number of bioactive products, such as morpholine, piperidine, or N -methylpiperazine and aldehydes of markedly different reactivities, like formaldehyde and benzaldehyde. The influence of substituents attached to position 3 on the aminoalkylation was also investigated. Thus, reactions of 3-carbamoyl-substituted precursors with tertiary amine containing side-chains were also tested to afford new KYNA derivatives with two potential cationic centers. By means of NMR spectroscopic measurements, supported by DFT calculations, the dominant tautomer form of KYNA derivatives was also determined.

Kynurenine (kyn) and kynurenic acid (kyna) are well-defined metabolites of tryptophan catabolism collectively known as "kynurenines", which exert regulatory functions in host-microbiome signaling, immune cell response, and neuronal excitability. Kynurenine containing peptides endowed with opioid receptor activity have been isolated from natural organisms; thus, in this work, novel opioid peptide analogs incorporating L-kynurenine (L-kyn) and kynurenic acid (kyna) in place of native amino acids have been designed and synthesized with the aim to investigate the biological effect of these modifications. The kyna-containing peptide ( KA1 ) binds selectively the m-opioid receptor with a Ki = 1.08 ± 0.26 (selectivity ratio m/d/k = 1:514:10000), while the L-kyn-containing peptide ( K6 ) shows a mixed binding affinity for m, d, and k-opioid receptors, with efficacy and potency (E max = 209.7 + 3.4%; LogEC 50 = -5.984 + 0.054) higher than those of the reference compound DAMGO. This novel oligopeptide exhibits a strong antinociceptive effect after i.c.v. and s.c. administrations in in vivo tests, according to good stability in human plasma (t 1/2 = 47 min)., Competing Interests: The authors declare no conflict of interest.

Kynurenic acid demonstrates antioxidant, neuroprotective and free radical scavenging properties. However, low aqueous solubility of kynurenic acid limits its therapeutic activity. In the present study, cyclodextrin nanosponges were used to improve the solubility and therapeutic activity of kynurenic acid. The formation of kynurenic acid loaded nanosponge was confirmed by different characterization techniques. The solubility of kynurenic acid was significantly increased with nanosponge (111.1 μg/ml) compared to free kynurenic acid (16.4 μg/ml) and β-cyclodextrin (28.6 μg/ml). High drug loading (19.06%) and encapsulation efficiency (95.31%) were achieved with NS. The particle size and zeta potential of kynurenic acid loaded nanosponge was around 255.8 nm and -23 mV respectively. Moreover, higher solubilization of kynurenic acid loaded nanosponge produced better antioxidant activity compared to free kynurenic acid. The kynurenic acid loaded nanosponge and blank nanosponge were found nontoxic in the cytotoxicity assay. Thus, these studies demonstrated that nanosponges can be used as a carrier for the delivery of kynurenic acid., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A sensitive, rapid and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to assay tryptophan (TRP) and its nine metabolites, including kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), 3-indolepropionic acid (IPA) and 3-indoleacetic acid (IAA) in human plasma. Tryptophan-d5 (TRP-d5) and carbamazepine (CAR) were applied to the method quantification, where TRP-d5 was the corresponding internal standard (IS) for TRP and KYN, and CAR was the corresponding IS for the other analytes. Plasma samples were processed by deproteinisation with acetonitrile, followed by separation on an Acquity UPLC HSS T3 column by using gradient elution with 0.1% (v/v) formic acid in water and acetonitrile and detection by electrospray ionisation tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) within a total run time of 5 min. The calibration ranges were 3-600 ng/mL for 3-HK, 1.5-300 ng/mL for 5-HT, 25-5000 ng/mL for KYN, 1-200 ng/mL for XA, 100-20,000 ng/mL for TRP, 5-1000 ng/mL for KYNA, 2-400 ng/mL for 3-HAA, 2.5-500 ng/mL for 5-HIAA and 10-2000 ng/mL for IAA and IPA. All intra- and inter-day analytical variations were acceptable. Matrix effect and recovery evaluation proved that matrix effect can be negligible, and sample preparation approach was effective. The newly developed method can simultaneously determine a panel of TRP metabolites and was successfully applied in the clinical study characterising TRP metabolism in healthy volunteers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Kynurenic acid (KYNA), a metabolite of tryptophan, as an excitatory amino acid receptor antagonist is an effective neuroprotective agent in case of excitotoxicity, which is the hallmark of brain ischemia and several neurodegenerative processes. Therefore, kynurenine pathway, KYNA itself, and its derivatives came into the focus of research. During the past fifteen years, our research group has developed several neuroactive KYNA derivatives, some of which proved to be neuroprotective in preclinical studies. In this study, the synthesis of these KYNA derivatives and their evaluation with divergent molecular characteristics are presented together with their most typical effects on the monosynaptic transmission in CA1 region of the hippocampus of the rat. Their effects on the basic neuronal activity (on the field excitatory postsynaptic potentials: fEPSP) were studied in in vitro hippocampal slices in 1 and 200 μM concentrations. KYNA and its derivative 4 in both 1 and 200 μM concentrations proved to be inhibitory, while derivative 8 only in 200 μM decreased the amplitudes of fEPSPs. Derivative 5 facilitated the fEPSPs in 200 μM concentration. This is the first comparative study which evaluates the structural and functional differences of formerly and newly developed KYNA analogs. Considerations on possible relations between molecular structures and their physiological effects are presented.

The in vivo investigation of kynurenic acid (KYNA) and its analogs is one of the recent exciting topics in pharmacology. In the current study we assessed the biological effects of these molecules on bdelloid rotifers ( Philodina acuticornis and Adineta vaga ) by monitoring changes in their survival and phenotypical characteristics. In addition to longitudinal (slowly changing) markers (survival, number of rotifers alive and body size index), some dynamic (quickly responding) ones (cellular reduction capacity and mastax contraction frequency) were measured as well. KYNA and its analogs increased longevity, reproduction and growth, whereas reduction capacity and energy-dependent muscular activity decreased conversely. We found that spermidine, a calorie restriction mimetic, exerted similar changes in the applied micro-invertebrates. This characterized systemic profile evoked by the above-mentioned compounds was named beneficial physiologic attenuation. In reference experiments, using a stimulator (cyclic adenosine monophosphate) and a toxin (sodium azide), all parameters changed in the same direction (positively or negatively, respectively), as expected. The currently described adaptive phenomenon in bdelloid rotifers may provide holistic perspectives in translational research.

A sensitive and specific bioanalytical method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 6-hydroxykynurenic acid (6-HKA) in a small quantity of rat plasma has been developed and comprehensively validated. Tolbutamide (Tol) was used as the internal standard (IS). An aliquot of 50 μL rat plasma sample was deproteinized by 150 μL methanol, and then 100 μL of its supernatant was mixed with 100 μL water after centrifugation. Chromatographic separation was performed on a ZORBAX Eclipse Plus C 18 Rapid Resolution HD column (2.1 mm × 100 mm, 1.8 μm) with a gradient mobile phase consisting of water containing 2 mM ammonium formate and methanol at a flow rate of 0.2 mL/min over a total run time of 5.0 min. The mass spectrometer was operated under multiple reactions monitoring (MRM) mode with the transitions m/z 206.1 → 160.0 for 6-HKA and m/z 269.0 → 170.0 for Tol. The linear range was 2.5-250 ng/mL with the square regression coefficient (r 2 ) of 0.997. The lower limit of quantification (LLOQ) was 2.5 ng/mL (Relative Error, RE +1.6% and RSD 4.8%, n = 5). The intra- and inter-day precision and accuracy was <13.3%. The mean recovery of 6-HKA in rat plasma was between the range of 99.3-103.4%. This method was successfully applied to study the pharmacokinetics of 6-HKA in rats after oral administration at a single dose of 20.0 mg/kg and after tail intravenous injection at a single dose of 2.0 mg/kg. Pharmacokinetic parameters bioavailability, C max, oral , T max, oral , AUC 0-24h, oral , AUC 0-∞, oral , AUC 0-24h, iv and AUC 0-∞, iv were 3.96%, 152.0 ± 85.5 ng/mL, 0.4 ± 0.1 h, 340.0 ± 136.3 ng/mL ∗ h, 369.5 ± 135.0 ng/mL ∗ h, 906.6 ± 324.1 ng/mL*h and 932.9 ± 336.5 ng/mL ∗ h, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fractionation of the bioactive extract of a culture of the marine derived actinomycete Streptomyces cyaneofuscatus M-157 led to the isolation of the known 3-hydroxyquinaldic acid ( 4 ), its amide ( 5 ) and three new derivatives ( 1 ⁻ 3 ) containing different amino acid residues. The structures of the new molecules ( 1 ⁻ 3 ), including their absolute configuration, were determined by the analysis of their ESI-TOF MS and one-dimensional (1D) and two-dimensional (2D) NMR spectra and advanced Marfey's analysis of their hydrolyzation products. Compound 3 spontaneously dimerized in solution to give the disulfide derivative 6 . Unfortunately, none of the new compounds isolated confirmed the antimicrobial activity found in the bacterial extract, perhaps indicating that such antibacterial activity might be due to presence in the extract at the trace level of larger bioactive 3-hydroxyquinaldic acid derivatives from which compounds 1 ⁻ 3 are biosynthetic precursors. Cytotoxicity tests confirmed the moderate and weak IC 50 values of 15.6 and 51.5 µM for compounds 5 and 1 , respectively.

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite kynurenic acid (KYNA), an antagonist at both N-methyl-d-aspartate (NMDA) and α7 nicotinic acetylcholine (α7nACh) receptors, has been implicated in cognitive processes in health and disease. As KYNA levels are elevated in the brains of patients with schizophrenia, a malfunction at the glutamatergic and cholinergic receptors is believed to be causally related to cognitive dysfunction, a core domain of the psychopathology of the illness. KYNA may play a pathophysiologically significant role in individuals with schizophrenia. It is possible to elevate endogenous KYNA in the rodent brain by treating animals with the direct bioprecursor kynurenine, and preclinical studies in rats have demonstrated that acute elevations in KYNA may impact their learning and memory processes. The current protocol describes this experimental approach in detail and combines a) a biochemical analysis of blood kynurenine levels and brain KYNA formation (using high-performance liquid chromatography), b) behavioral testing to probe the hippocampal-dependent contextual memory (passive avoidance paradigm), and c) an assessment of sleep-wake behavior [telemetric recordings combining electroencephalogram (EEG) and electromyogram (EMG) signals] in rats. Taken together, a relationship between elevated KYNA, sleep, and cognition is studied, and this protocol describes in detail an experimental approach to understanding function outcomes of kynurenine elevation and KYNA formation in vivo in rats. Results obtained through variations of this protocol will test the hypothesis that the KP and KYNA serve pivotal roles in modulating sleep and cognition in health and disease states.

The mammalian kynurenine aminotransferase (KAT) enzymes are a family of related isoforms that are pyridoxal 5'-phosphate-dependent, responsible for the irreversible transamination of kynurenine to kynurenic acid. Kynurenic acid is implicated in human diseases such as schizophrenia where it is found in elevated levels and consequently KAT-II, as the isoform predominantly responsible for kynurenic acid production in the brain, has been targeted for the development of specific inhibitors. One class of compounds that have also shown inhibitory activity towards the KAT enzymes are estrogens and their sulfate esters. Estradiol disulfate in particular is very strongly inhibitory and it appears that the 17-sulfate makes a significant contribution to its potency. The work here demonstrates that the effect of this moiety can be mirrored in existing KAT-II inhibitors, from the development of two novel inhibitors, JN-01 and JN-02. Both inhibitors were based on NS-1502 (IC50: 315 μM), but the deliberate placement of a sulfonamide group significantly improved the potency of JN-01 (IC50: 73.8 μM) and JN-02 (IC50: 112.8 μM) in comparison to the parent compound. This 3-4 fold increase in potency shows the potential of these moieties to be accommodated in the KAT-II active site and the effect they can have on improving inhibitors, and the environments in the KAT-II have been suitably modelled using docking calculations.

Photoinduced generation of radicals in the eye lens may play an important role in the modification of proteins leading to their coloration, aggregation, and insolubilization. The radicals can be formed via the reactions of photoexcited endogenous chromophores of the human lens with lens proteins, in particular with tryptophan residues. In the present work we studied the reactions induced by UV-A (315-400nm) light between kynurenic acid (KNA), an effective photosensitizer present in the human lens, and N-acetyl-L-tryptophan (NTrpH) under aerobic and anaerobic conditions. Our results show that the reaction mechanism strongly depends on the presence of oxygen in solution. Under aerobic conditions, the generation of singlet oxygen is the major channel of the effective NTrpH oxidation. In argon-bubbled solutions, the quenching of triplet KNA by NTrpH results in the formation of KNA • - and NTrp • radicals. Under laser pulse irradiation, when the radical concentration is high, the main pathway of the radical decay is the back electron transfer with the restoration of initial reagents. Other reactions include (i) the radical combination yielding NTrp dimers and (ii) the oxygen atom transfer from KNA • - to NTrp • with the formation of oxidized NTrp species and deoxygenated KNA products. In continuous-wave photolysis, even trace amounts of molecular oxygen are sufficient to oxidize the majority of KNA • - radicals with the rate constant of (2.0 ± 0.2) × 10 9 M -1 s -1 , leading to the restoration of KNA and the formation of superoxide radical O 2 • - . The latter reacts with NTrp • via either the radical combination to form oxidized NTrp (minor pathway), or the electron transfer to restore NTrpH in the ground state (major pathway). As the result, the quantum yields of the starting compound decomposition under continuous-wave anaerobic photolysis are rather low: 1.6% for NTrpH and 0.02% for KNA. The photolysis of KNA with alpha-crystallin yields the same deoxygenated KNA products as the photolysis of KNA with NTrpH, indicating the similarity of the photolysis mechanisms. Thus, inside the eye lens KNA can sensitize both protein photooxidation and protein covalent cross-linking with the minor self-degradation. This may play an important role in the lens protein modifications during the normal aging and cataract development., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dermal fibrosis, characterized by excessive extracellular matrix (ECM), is a pathological condition with limited effective therapeutic modalities. Lack of an antiscarring dressing further impedes the preventive measures for this condition. Here, we develop a new antiscarring dressing and investigate its potential as a slow-releasing vehicle for kynurenic acid (KynA), an antifibrotic agent. KynA was incorporated into polymethyl methacrylate (PMMA) nanofibers, containing increasing concentration of polyethylene glycol (PEG). Fibre morphology, water absorption capacity, surface hydrophilicity, in vitro drug release profile, and in vivo antifibrotic effects were investigated. Increasing concentrations of PEG (1-20%) significantly increased surface hydrophilicity, water absorption capacity, and drug release. Based on the obtained release profiles, PMMA + 10% PEG was the preferred formulation for sustained KynA release up to 120 hours. In vitro studies confirmed the preservation of KynA antifibrotic properties during electrospinning, indicated by fibroblasts proliferation suppression and ECM expression modulation. In vivo application of KynA-incorporated films significantly inhibited collagen (23.89 ± 4.79 vs. 6.99 ± 0.41, collagen-I/β-actin mRNA expression, control vs. treated) and fibronectin expression (7.18 ± 1.09 vs. 2.31 ± 0.05, fibronectin/β-actin mRNA expression, control vs. treated) and enhanced the production of an ECM-degrading enzyme (2.03 ± 0.88 vs. 11.88 ± 1.16 MMP-1/β-actin mRNA expression, control vs. treated). The fabricated KynA-incorporated films can be exploited as antifibrotic wound dressings. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2334-2344, 2016., (© 2016 Wiley Periodicals, Inc.)

This work clearly demonstrates an evaluation process that is easily performed and is simply based on the fitting of temperature-dependent surface plasmon resonance (SPR) sensorgrams to provide detailed thermodynamic characterization of biologically relevant interactions. The reversible binding of kynurenic acid (KYNA) on human glutamate receptor (GluR1) polypeptide (GluR1270-300)-modified gold surface has been studied at various temperatures under physiological conditions by two-dimensional SPR experiments. The registered sensorgrams were fitted by using different kinetic models without application of any commercial software. Assuming that the association of GluR1270-300-KYNA complex is first order in both reactants, the association (ka) and dissociation (kd) constants as well as the equilibrium constants (KA) and the Gibbs free-energy change (ΔG°) were given at 10, 20, 30, and 40 °C. Moreover, the enthalpy (ΔH° = -27.91 kJ mol(-1)), entropy (ΔS° = -60.33 J mol(-1) K(-1)), and heat capacity changes (ΔCp = -1.28 kJ mol(-1) K(-1)) of the model receptor-ligand system were also calculated using a spreadsheet program. Negative values of ΔG° and ΔH° indicate the exothermic formation of a stable GluR1270-300-KYNA complex, because the |ΔH| > |TΔS| relation suggests an enthalpy-driven binding process. The negative ΔH° and ΔS° values strongly support the formation of a salt bridge between KYNA and the positively charged residues of the polypeptide (Arg, Lys) at pH 7.4, confirmed by molecular docking calculations as well.

Kynurenine aminotransferase (KAT) is a pyridoxal-5'-phosphate (PLP) dependent enzyme that catalyses kynurenine (KYN) to kynurenic acid (KYNA), a neuroactive product in the tryptophan metabolic pathway. Evidence suggests that abnormal levels of KYNA are involved in many neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease and schizophrenia. Reducing KYNA production through inhibiting kynurenine aminotransferase 2 (KAT2) would be a promising approach to understanding and treating the related neurological and mental disorders. In this study we used an optimized codon sequence to overexpress histidine-tagged human KAT2 (hKAT2) using an Escherichia coli expression system. After a single step of Ni-NTA based purification the purified protein (>95%) was confirmed to be active by an HPLC based activity assay and was crystallized using the hanging-drop vapour diffusion method. The crystal system represents a novel space group, and a complete X-ray diffraction data set was collected to 1.83 Å resolution, and higher resolution data than for any reported native human KAT2 structure. The optimised method of protein production provides a fast and reliable technique to generate large quantities of active human KAT2 suitable for future small-molecule lead compound screening and structural design work., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Core-shell nanoparticles (CSNPs) were developed to get over therapeutic amount of kynurenic acid (KYNA) across the blood-brain barrier (BBB). Bovine serum albumin (BSA) was used as core for encapsulation of KYNA and the BSA/KYNA composite was finally encapsulated by poly(allylamine) hydrochloride (PAH) polymer as shell. In the interest of the optimization of the synthesis the BSA and KYNA interaction was studied by two-dimensional surface plasmon resonance (SPR) technique as well. The average size of d~100 nm was proven by dynamic light scattering (DLS) and transmission electron microscopy (TEM), while the structure of the composites was characterized by fluorescence (FL) and circular dichroism (CD) spectroscopy. The in vitro release properties of KYNA were investigated by a vertical diffusion cell at 25.0 °C and 37.5 °C and the kinetic of the release were discussed. The penetration capacity of the NPs into the central nervous system (CNS) was tested by an in vitro BBB model. The results demonstrated that the encapsulated KYNA had significantly higher permeability compared to free KYNA molecules. In the neurobiological serial of in vivo experiments the effects of peripherally administered KYNA with CSNPs were studied in comparison with untreated KYNA. These results clearly proved that KYNA in the CSNPs, administrated peripherally is suitable to cross the BBB and to induce electrophysiological effects within the CNS. As the neuroprotective properties of KYNA nowadays are proven, the importance of the results is obvious., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The reactions of photoexcited kynurenic acid (KNA) with bovine α-crystallins under anaerobic conditions proceed via the electron transfer from tryptophan (Trp) and tyrosine (Tyr) residues to the triplet KNA molecules. The subsequent radical reactions lead to the protein aggregation and insolubilization. The absorption of the photolyzed proteins at 335 nm as well as their total fluorescence significantly increases, while the tryptophan-related fluorescence decreases. It has been established that the alterations of the protein optical properties are related to the modifications of Trp residues. Intrinsic lens antioxidants ascorbate (Asc) and glutathione (GSH) that are present in the human lens at the millimolar level effectively block the formation of the observed light-induced protein modifications. The protective effect of Asc was attributed to its ability to quench highly reactive triplet states, while the role of GSH, most likely, corresponds to the reduction of photochemically formed radicals into a diamagnetic state. The results obtained disclose the possible mechanism of UVA-induced modifications of the lens crystallins, leading to the formation of cataract, and the role of major lens antioxidants Asc and GSH in the protection of the lens proteins.

Elution profiles of kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) were examined by high-performance liquid chromatography (HPLC) using a triazole-bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 O-rich mobile phases, respectively. The present HPLC method using a triazole-bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d-kynurenine (d-KYN) by d-amino acid oxidase (DAO) using Cl-KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d-KYN with DAO. Production of KYNA from d-KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia., (Copyright © 2015 John Wiley & Sons, Ltd.)

Free-radical scavenging by tryptophan and eight of its metabolites through electron transfer was investigated in aqueous solution at physiological pH, using density functional theory and the Marcus theory. A test set of 30 free radicals was employed. Thermochemical and kinetic data on the corresponding reactions are provided here for the first time. Two different pathways were found to be the most important: sequential proton loss electron transfer (SPLET) and sequential double proton loss electron transfer (SdPLET). Based on kinetic analyses, it is predicted that the tryptophan metabolites kynurenic acid and xanthurenic acid are the best free-radical scavengers among the tested compounds; they were estimated to be at least 24 and 12 times more efficient than Trolox for scavenging (•)OOH. These findings are in line with previous reports suggesting that the antioxidant activity that has been attributed to tryptophan is actually due to its metabolites, and they demonstrate the particular importance of phenolic metabolites to such activity. Graphical Abstract Kynurenic acid (KNA) and xanthurenic acid (XNA) are the major contributors to the free-radical scavenging activity of tryptophan.

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood-brain barrier and converted via the kynurenine pathway to N-formyl-L-kynurenine, which is further degraded to L-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC-MS/MS method to quantify L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-L-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098-100 ng/ml for 3HK, 9.8-20,000 ng/ml for KYN, 0.49-1000 ng/ml for KYNA and AA. The method was linear (r>0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Kynurenic acid (KYNA) was found to be an antagonist of iontropic glutamate receptors and alpha7 nicotinic acetylcholine receptors. Furthermore, it was documented that KYNA is an agonist of G-protein coupled GPR35 receptors which are mainly present in the gastrointestinal tract. It was also found that KYNA is present in the gastrointestinal tract and that its concentration gradually increases along it. The origin of KYNA in the gastrointestinal tract is not known. Both might be synthesized from tryptophan in it or absorbed from food and other dietary products. Therefore, the aim of the study was to investigate the concentration of KYNA in animal feed. The results indicate that the highest concentration of KYNA was found in animal feeds intended for livestock. The lower amount of KYNA was detected in animal feeds for fish. Interestingly, the lowest amount of KYNA was found in dog and cat feeds. Furthermore, an analysis of KYNA content in animal food ingredients was conducted. The concentration of KYNA found in one of the ingredients – rapeseed meal – was several times higher in comparison to animal feeds studied. The content of KYNA in the remaining feed ingredients tested was significantly lower. This is the first report on the concentration of KYNA in animal feeds. There is a need for further detailed analysis leading to establishing a set of guidelines for animal feeding., (Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.)

Though migraine and neurodegenerative disorders have a high socioeconomic impact, their therapeutic management has not been fully addressed. Their pathomechanisms are not completely understood, but glutamateinduced excitotoxicity, mitochondrial disturbances and oxidative stress all seem to play crucial roles. The overactivation of glutamate receptors contributes to the hyperexcitability observed in migraine and also to the neurodegenerative process. The kynurenine pathway of the tryptophan metabolism produces the only known endogenous Nmethyl- D-aspartate receptor antagonist, kynurenic acid, which has been proven in different preclinical studies to exert a neuroprotective effect. Influencing the kynurenine pathway might be beneficial in migraine and neurodegenerative diseases, and in the normalization of glutamatergic neurotransmission and the prevention of excitotoxic neuronal damage. The synthesis of kynurenic acid analogues may offer a valuable tool for drug development.

The interaction between kynurenic acid (KYNA) and two peptide fragments (ca. 30 residues) of Human Glutamate Receptor 201-300 (GluR1) using surface plasmon resonance (SPR) spectroscopy was investigated. Because of the medical interest in the neuroscience, GluR1 is one of the important subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). AMPARs are ionotoropic glutamate receptors, which are mediating fast synaptic transmission and are crucial for plasticity in the brain. On the other hand, KYNA has been suggested to have neuroprotective activity and it has been considered for apply in therapy in certain neurobiological disorders. In this article the adsorption of the GluR1201-230 and GluR1231-259 peptides were studied on gold biosensor chip. The peptides were chemically bonded onto the gold surface via thiol group of L-cysteine resulted in the formation of peptide monolayer on the SPR chip surface. Because the GluR1231-259 peptide does not contain L-cysteine the Val256 was replaced by Cys256. The cross sectional area and the surface orientation of the studied peptides were determined by SPR and theoretical calculations (LOMETS) as well. The binding capability of KYNA on the peptide monolayer was studied in the concentration range of 0.1-5.0 mM using 150 mM NaCl ionic strength at pH 7.4 (±0.02) in phosphate buffer solutions. In order to determine the binding enthalpy the experiments were carried out between +10°C and +40°C. The heat of adsorption was calculated by using adsorption isotherms at different surface loading of KYNA on the SPR chip., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the present contribution, we have explored ground and excited state spectroscopic properties of an antiexcitotoxic and anticonvulsant drug, Kynurenic acid (KA), through steady-state absorption, emission and time-resolved emission spectroscopy and quantum chemical calculations. The main focus of this article is to illustrate the effects of various parameters such as the nature of the solvents and pH of the medium on the spectral properties of KA which confirms the presence of different neutral and ionic species in the ground and excited states. The molecule KA exists mainly as keto- or anionic form in the ground state, whereas the main contribution of its emission is arising from the keto tautomer in the excited state. Quantum chemical calculations by Density Functional Theory (DFT) method has been effectively employed to correlate the experimental findings. The ground and excited state properties of KA ascertained by means of experimental and theoretical method reveal that it resembles well with other two compounds, 4-hydroxyquinoline and xanthurenic acid formed by the decomposition of UV filters., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aggregation behavior and critical aggregation concentration (CAC) values of daptomycin in aqueous solutions were evaluated under the external factors of pH, temperature, daptomycin concentration, and calcium ions concentration by using the complementary characterization techniques, fluorescence, dynamic and static light scattering, and nuclear magnetic resonance (NMR) spectroscopy. On the basis of the intrinsic fluorescence resonance energy transfer of daptomycin, the CAC values were identified by an upward inflection of the fluorescence emission from Kyn-13 at 460 nm. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM at pH 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Kynurenine aminotransferase (KAT) is a homodimeric pyridoxal protein that mediates the catalytic conversion of kynurenine (KYN) to kynurenic acid (KYA), an endogenous N-methyl-d-aspartate (NMDA) receptor antagonist. KAT is involved in the biosynthesis of glutamic and aspartic acid, functions as a neurotransmitter for the NMDA receptor in mammals, and is regulated by allosteric mechanisms. Its importance in various diseases such as schizophrenia makes KAT a highly attractive drug target. Here, we present the crystal structure of the Pyrococcus horikoshii KAT (PhKAT) in complex with pyridoxamine phosphates (PMP), KYN, and KYA. Surprisingly, the PMP was bound to the LYS-269 of phKAT by forming a covalent hydrazine bond. This crystal structure clearly shows that an amino group of KYN was transaminated to PLP, which forms a Schiff's base with the LYS-269 of the KYN. Thus, our structure confirms that the PMPs represent an intermediate state during the KAT reaction. Thus, PhKAT catalyzes the sequential conversion of KYN to KYA via the formation of an intermediate 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD), which is spontaneously converted to KYA in the absence of an amino group acceptor. Furthermore, we identified the two entry and exit sites of the PhKAT homodimer for KYN and KYA, respectively. The structural data on PhKAT presented in this manuscript contributes to further the understanding of transaminase enzyme reaction mechanisms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The photophysics and photochemistry of kynurenic acid (KNA) and kynurenine yellow (KNY) in neutral aqueous solutions were investigated using time-resolved optical spectroscopy. Both molecules have similar quinoline-like structures, the only difference being the absence of conjugation in the nitrogen containing cycle in KNY. The main channel of S(1) excited state decay in the case of partially-unconjugated KNY is the solvent assisted S(1) → S(0) radiationless transition via intermolecular hydrogen bonds (Φ(IC) = 0.96), whereas, in the case of fully-conjugated KNA, it is intersystem crossing to the triplet state (Φ(T) = 0.82). The major intermediate products of the singlet excited KNY deactivation are the triplet state (Φ(T) = 0.022) and, most probably, the enol form (Φ(enol) = 0.012), which decay with the formation of 2,3-dihydro-4-hydroxyquinoline and 4-hydroxyquinoline, respectively. The results obtained show that KNA and KNY, which are products of the decomposition of the UV filter kynurenine, are significantly more photoactive and less photostable than the parent molecule.

Introduction: The use of herbal medicines is common among people living in rural areas and increasingly popular in urbanized countries. Kynurenic acid (KYNA) is a metabolite of kynurenine possessing anti-inflammatory, anti-oxidative and pain reliving properties. Previous data indicated that the content of KYNA in the synovial fluid of patients with rheumatoid arthritis is lower than in patients with osteoarthritis. Rheumatoid arthritis is a chronic, systemic inflammatory disorder affecting about 1% of the world's population., Aim: The aim of the presented study was to investigate the content of KYNA in 11 herbal preparations used in rheumatic diseases., Materials and Methods: The following herbs were studied: bean pericarp, birch leaf, dandelion root, elder flower, horsetail herb, nettle leaf, peppermint leaf and willow bark. An anti-rheumatic mixture of the herbs Reumatefix and Reumaflos tea were also investigated. The herbs were prepared according to producers' directions. In addition, the herbal supplement Devil's Claw containing root of Harpagophytum was used. KYNA content was measured using the high-performance liquid chromatography method, and KYNA was detected fluorometrically., Results: KYNA was found in all studied herbal preparations. The highest content of KYNA was found in peppermint, nettle, birch leaf and the horsetail herb. The lowest content of KYNA was found in willow bark, dandelion root and in the extract from the root of Harpagophytum., Conclusion: These findings indicate that the use of herbal preparations containing a high level of KYNA can be considered as a supplementary measure in rheumatoid arthritis therapy, as well as in rheumatic diseases prevention.

Stacking interaction is known to play an important role in protein folding, enzyme-substrate and ligand-receptor complex formation. It has been shown to make a contribution into the aromatic antagonists binding with glutamate ionotropic receptors (iGluRs), in particular, the complex of NMDA receptor NR1 subunit with the kynurenic acid (KYNA) derivatives. The specificity of KYNA binding to the glutamate receptors subtypes might partially result from the differences in stacking interaction. We have calculated the optimal geometry and binding energy of KYNA dimers with the four types of aromatic amino acid residues in Rattus and Drosophila ionotropic iGluR subunits. All ab initio quantum chemical calculations were performed taking into account electron correlations at MP2 and MP4 perturbation theory levels. We have also investigated the potential energy surfaces (PES) of stacking and hydrogen bonds (HBs) within the receptor binding site and calculated the free energy of the ligand-receptor complex formation. The energy of stacking interaction depends both on the size of aromatic moieties and the electrostatic effects. The distribution of charges was shown to determine the geometry of polar aromatic ring dimers. Presumably, stacking interaction is important at the first stage of ligand binding when HBs are weak. The freedom of ligand movements and rotation within receptor site provides the precise tuning of the HBs pattern, while the incorrect stacking binding prohibits the ligand-receptor complex formation.

Objective: To study the chemical constituents from the seeds of Notopterygium franchetii., Method: Ethanol extracts of seeds N. franchetii were separated and purified by such methods as normal and reversed phase column chromatographies and thin-layer chromatography and structurally elucidated by MS and NMR evidences., Result: Twenty nine compounds were separated, they were isoimperatorin (1), [3-sitosterol (2), phellopterin (3), bergapten (4), N-tetra, hexa, octacosanoylanthranilic acid (5-7), daucosterol (8), oxypeucedanin hydrate (9), umbelliferone (10), demethylfuropinnarin (11), (2S, 3S, 4R, 8E)-2-[(2'R)- 2'-hydroxydoco, trico, tetraco, entaco, hexaco sanosylamino] -octadecene-1, 3, 4-triol (12-16), (-)-oxypeucedanin (17), diosmetin (18), bergaptol-O-beta-D-glucopyranoside (19), nodakenin (20), 1'-O-beta-D-glucopyranosyl-(2R, 3S)-3-hydroxynodakenetin (21), uracil (22), decuroside V (23), 8-O-beta-D-glucopyranosyl-5-hydroxypsoralen (24), 8-O-beta-D-glucopyranosyl-5-methoxylpsoralen (25), diosmin (26), alaschanioside C (27), kynurenic acid (28) and mannitol (29)., Conclusion: All of these compounds were separated from the seeds of N. franchetii for the first time. Of them, 18, 22, 26 and 29 were firstly obtained from genus Notopterygium.

Pharmacological and histological studies of ten new amides of kynurenic acid revealed that N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride has effective neuroprotective properties. Namely, this molecule is: (1) proved to be an effective inhibitor of excitatory synaptic transmission in the CA1 region of the hippocampus both in in vitro and ex vivo studies, (2) in four vessel occlusion model of transient global forebrain ischaemia, measuring the rate of hippocampal CA1 pyramidal cell loss and preservation of long-term potentiation at Schaffer collateral-CA1 synapses, the neuroprotective potential was represented. N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride administration significantly diminished hippocampal CA1 cell loss and preserved LTP expression.

The main purpose of this study was to facilitate the delivery of kynurenic acid (KYNA) across the blood-brain barrier (BBB) by applying micelles as nanoscale containers. Non-ionic amphiphilic molecules were used for preparation of spherical micelles for delivery of kynurenic acid in aqueous solution in physiological condition. It was established that Triton X 100 and Lutensol AP 20 non-ionic surfactants are able to produce stable nanocontainers for delivery of kynurenic acid molecules. The incorporation of KYNA molecules was investigated by dynamic light scattering and the size of micelles were calculated between 5 and 10 nm in 150 mM NaCl and pH 7.5-7.6 solutions. Encapsulated kynurenic acid showed a significantly higher blood-brain barrier permeability compared with non-encapsulated kynurenic acid. The in vivo experiments showed that the encapsulated kynurenic acid is able to display effects within the central nervous system, even after its peripheral administration.

Our study demonstrated that the development of seizures during the electrically induced kindling of seizures is associated with significant changes in the concentration of kynurenic acid (KYNA) and its precursor, tryptophan (TRP). The primary finding of our study was an increase in KYNA levels and the KYNA/TRP ratio (a theoretical index of activity of the kynurenine pathway) in the amygdala and hippocampus of kindled animals. We also found decreases in the concentration of tryptophan in the hippocampus and prefrontal cortex. Changes in the concentration of KYNA and TRP in the amygdala were accompanied by a significant decrease in γ-Aminobutryic Acid (GABA) levels and an increase in the glutamate/GABA ratio. Moreover, we found a significant negative correlation between the local concentrations of KYNA and glutamate in the amygdala of kindled rats. However, there were no changes in the local concentrations of the following amino acids: glutamate, aspartate, glutamine, glycine, taurine and alanine. In conclusion, these new results suggest a modulatory influence of KYNA on the process of epileptogenesis, characterized by a negative relationship between the KYNA and glutamate systems in the amygdala.

Altered function of kynurenine pathway has emerged recently as one of the factors contributing to the pathogenesis of depression. Neuroprotective kynurenic acid (KYNA) and neurotoxic 3-hydroxykynurenine (3-HK) are two immediate metabolites of L: -kynurenine. Here, we aimed to assess the hypothesis that antidepressant drugs that may change brain KYNA/3-HK ratio. In primary astroglial cultures, fluoxetine, citalopram, amitriptyline and imipramine (1-10 μM) increased de novo production of KYNA and diminished 3-HK synthesis (24 and 48, but not 2 h). RT-PCR studies revealed that Kat1, Kat2 and kynurenine-3-monooxygenase (Kmo) gene expressions were not altered after 2 h. At 24 h, the expression of Kat1 and Kat2 genes was enhanced by all studied drugs, whereas Kmo expression was diminished by citalopram, fluoxetine and amitriptyline, but not imipramine. After 48 h, the expression of Kat1 and Kat2 was further up-regulated, and Kmo expression was down-regulated by all antidepressants. The ratio KYNA/3-HK was increased by fluoxetine, citalopram, amitriptyline and imipramine in a time-dependent manner-the effect was not observed after 2 h, modest after 24 h and robust after 48 h incubation time. Our findings indicate that the action of antidepressants may involve re-establishing of the beneficial ratio between KYNA and 3-HK. Shift in the kynurenine pathway, observed after prolonged exposure to antidepressant drugs, may partly explain their delayed therapeutic effectiveness.

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted at two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Kynurenic acid (KYNA), an endogenous antagonist of N-methyl-D-aspartate and α(7) nicotinic acetylcholine receptors, is one of the L-tryptophan (Trp) metabolites. To compare the level of KYNA produced in the striatum of rats after independent administration of L-Trp and D-Trp, rats were intraperitoneally administered L-Trp and/or D-Trp (100 mg/kg), and a microdialysis (MD) probe was implanted in the striatum. The KYNA level in the MD samples was determined using the column-switching high-performance liquid chromatography system. KYNA levels in the MD samples increased by approximately twofold in rats that were administered D-Trp or L-Trp; this result suggests that just as L-Trp, D-Trp was also metabolized to KYNA in the striatum. Additionally, 30 min before the administration of D-Trp, rats were administered 3-methyl pyrazole-5-carboxylic acid (MPC) (50 mg/kg), which is a specific inhibitor of D-amino acid oxidase (DAAO). Pretreatment with MPC suppressed striatal KYNA production; this result suggests that DAAO, encoded by one of the susceptible genes for schizophrenia, may contribute to the production of KYNA from D-Trp in the striatum of rats., (Copyright © 2011 Wiley Periodicals, Inc.)

An aqueous solution of enantiomerically pure tryptophan (Trp), namely, D-Trp or L-Trp (100 mg/kg), was administered intraperitoneally to male Sprague-Dawley rats. The time-course profiles of the rat plasma concentrations of D-kynurenine (KYN), L-KYN, and kynurenic acid (KYNA), which are metabolites of D- or L-Trp, were investigated using high-performance liquid chromatography (HPLC) systems that were reported in our previous study. The plasma D-KYN concentration increased after the administration of D-Trp, but this increase was not observed after the administration of L-Trp. The plasma L-KYN concentration increased after the administration of L-Trp, but no significant change was observed after the administration of D-Trp. The plasma KYNA concentration drastically increased in the case of rats administered D-Trp and those administered L-Trp. Additionally, an inhibitor of D-amino acid oxidase (DAAO), 5-methylpyrazole-3-carboxylic acid (MPC) (50 mg/kg), was administrated to the rats 30 min before the administration of D-Trp. The preadministration of MPC remarkably increased the D-KYN concentration and suppressed the production of KYNA. These results suggest that DAAO may contribute to the metabolism of D-KYN to KYNA in vivo., (© 2010 Wiley-Liss, Inc.)

The synthesis and the pharmacological activity of a series of 1-aroyl derivatives of kynurenic acid methyl ester (4-oxo-quinolin-2-carboxy methyl (KYNA) esters), structurally related to NSAID indomethacin are described. The derivatives were screened in vivo for anti-inflammatory and analgesic activities. Most of the compounds exhibited good anti-inflammatory and analgesic activities. An automatic docking of the synthesized compounds was performed using X-ray structures of COX-1 and COX-2. Docking results are in good accordance with the experimental biological data.

Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations.

Kynurenic acid is an endogenous product of the tryptophan metabolism, and as a broad-spectrum antagonist of excitatory amino acid receptors may serve as a protective agent in neurological disorders. The use of kynurenic acid as a neuroprotective agent is rather limited, however, because it has only restricted ability to cross the blood-brain barrier. Accordingly, new kynurenic acid analogues which can readily cross the blood-brain barrier and exert their complex anti-excitotoxic activity are greatly needed. Such a novel analogue, 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride, has been developed and tested. In an in vitro electrophysiological study, in which its properties were compared with those of kynurenic acid, the new analogue behaved quite similarly to kynurenic acid: in the micromolar range, its administration led to a decrease in the amplitudes of the field excitatory postsynaptic potentials in the CA1 region of the hippocampus, while in nanomolar concentrations it did not give rise to inhibition, but, in fact, facilitated the field excitatory postsynaptic potentials. Moreover, the new analogue demonstrated similar protective action against PTZ-induced facilitation to that observed after kynurenic acid administration. The findings strongly suggest that the neuroactive effects of the new analogue are comparable with those of kynurenic acid, but, in contrast with kynurenic acid, it readily crosses the blood-brain barrier. The new analogue may therefore be considered a promising candidate for clinical studies.

Kynurenine aminotransferase (KAT) is a key enzyme of the kynurenine pathway along the route of tryptophan catabolism. It catalyzes the irreversible transamination reaction of L-kynurenine (L-Kyn) to kynurenic acid (KYNA), an important neuroactive metabolite that plays a role in protecting neurons from excitatory neurotransmission. Although four isoforms (KAT-I to -IV) of this enzyme have been hitherto identified, KAT-II is the enzymatic isoform that mainly accounts for the synthesis of cerebral KYNA. In this study, the transamination mechanism of L-Kyn catalyzed by KAT-II is theoretically determined by performing combined quantum mechanical and molecular mechanical (QM/MM) simulations. The results are instrumental to explore the catalytic properties of the enzyme and to provide theoretical details on the mechanism of the intramolecular condensation of the ketoacid intermediate, leading to the final product KYNA. Ultimately, they will also be of value in the future design of new KAT-II selective inhibitors.

Recent studies have demonstrated that kynurenic acid (KYNA), a compound produced endogenously by the interferon-gamma-induced degradation of tryptophan by indoleamine 2,3-dioxygenase, activates the previously orphaned G protein-coupled receptor, GPR35. This receptor is expressed in immune tissues, although its potential function in immunomodulation remains to be explored. We determined that GPR35 was most highly expressed on human peripheral monocytes. In an in vitro vascular flow model, KYNA triggered the firm arrest of monocytes to both fibronectin and ICAM-1, via beta(1) integrin- and beta(2) integrin-mediated mechanisms, respectively. Incubation of monocytes with pertussis toxin prior to use in flow experiments significantly reduced the KYNA-induced monocyte adhesion, suggesting that adhesion is triggered by a G(i)-mediated process. Furthermore, KYNA-triggered adhesion of monocytic cells was reduced by short hairpin RNA-mediated silencing of GPR35. Although GPR35 is expressed at slightly lower levels on neutrophils, KYNA induced firm adhesion of these cells to an ICAM-1-expressing monolayer as well. KYNA also elicited neutrophil shedding of surface L-selectin, another indicator of leukocyte activation. Taken together, these data suggest that KYNA could be an important early mediator of leukocyte recruitment.

The complete (1)H, (13)C and (15)N NMR spectral assignments of two new alkaloids isolated from chestnut honey and structurally related to kynurenic acid have been made using 1-D and 2-D NMR techniques, including COSY, HMQC and HMBC experiments. The new compounds have been identified as 3-(2'-pyrrolidinyl)-kynurenic acid and its gamma-lactam derivative., (Copyright (c) 2009 John Wiley & Sons, Ltd.)