RESUMEN
The kynurenine pathway (KP) is the main path for tryptophan metabolism, and it represents a multitude of potential sites for drug discovery in neuroscience, including pain, stroke, and epilepsy. L-kynurenine (LKYN), the first active metabolite in the pathway, emerges to be a prodrug targeting glutamate receptors. The safety, tolerability, pharmacokinetics, and pharmacodynamics of LKYN in humans have not been previously investigated. In an open-label, single ascending dose study, six participants received an intravenous infusion of 50, 100, and 150 µg/kg LKYN and new six participants received an intravenous infusion of 0.3, 0.5, 1, and 5 mg/kg LKYN. To compare the pharmacological effects between species, we investigated in vivo the vascular effects of LKYN in rats. In humans, LKYN was safe and well-tolerated at all dose levels examined. After infusion, LKYN plasma concentration increased significantly over time 3.23 ± 1.12 µg/mL (after 50 µg/kg), 4.04 ± 1.1 µg/mL (after 100 µg/kg), and 5.25 ± 1.01 µg/mL (after 150 µg/kg) (p ≤ 0.001). We observed no vascular changes after infusion compared with baseline. In rats, LKYN had no effect on HR and MAP and caused no dilation of dural and pial arteries. This first-in-human study of LKYN showed that LKYN was safe and well-tolerated after intravenous infusion up to 5 mg/kg over 20 minutes. The lack of change in LKYN metabolites in plasma suggests a relatively slow metabolism of LKYN and no or little feed-back effect of LKYN on its synthesis. The therapeutic potential of LKYN in stroke and epilepsy should be explored in future studies in humans.
Asunto(s)
Circulación Cerebrovascular/efectos de los fármacos , Quinurenina/efectos adversos , Profármacos/efectos adversos , Adulto , Animales , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Epilepsia/tratamiento farmacológico , Femenino , Voluntarios Sanos , Humanos , Infusiones Intravenosas , Quinurenina/administración & dosificación , Quinurenina/farmacocinética , Masculino , Proyectos Piloto , Profármacos/administración & dosificación , Profármacos/farmacocinética , Ratas , Accidente Cerebrovascular/tratamiento farmacológico , Adulto JovenRESUMEN
Manipulation of kynurenic acid (KYNA) level through kynurenine aminotransferase-2 (KAT-2) inhibition with the aim of therapy in neuro-psychiatric diseses has been the subject of extensive recent research. Although mouse models are of particular importance, neither the basic mechanism of KYNA production and release nor the relevance of KAT-2 in the mouse brain has yet been clarified. Using acute mouse brain slice preparations, we investigated the basal and L-kynurenine (L-KYN) induced KYNA production and distribution between the extracellular and intracellular compartments. Furthermore, we evaluated the effect of specific KAT-2 inhibition with the irreversible inhibitor PF-04859989. To ascertain that the observed KYNA release is not a simple consequence of general cell degradation, we examined the structural and functional integrity of the brain tissue with biochemical, histological and electrophysiological tools. We did not find relevant change in the viability of the brain tissue after several hours incubation time. HPLC measurements proved that mouse brain slices intensively produce and liberate KYNA to the extracellular compartment, while only a small proportion retained in the tissue both in the basal and L-KYN supplemented state. Finally, specific KAT-2 inhibition significantly reduced the extracellular KYNA content. Taken together, these results provide important data about KYNA production and release, and in vitro evidence for the first time of the function of KAT-2 in the adult mouse brain. Our study extends investigations of KAT-2 manipulation to mice in a bid to fully understand the function; the final, future aim is to assign therapeutical kynurenergic manipulation strategies to humans.
Asunto(s)
Encéfalo/metabolismo , Ácido Quinurénico/metabolismo , Transaminasas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Inmunohistoquímica/métodos , Ácido Quinurénico/farmacología , Quinurenina/metabolismo , Quinurenina/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Transaminasas/antagonistas & inhibidoresRESUMEN
Introdução: A suplementação com ácido fólico (AF) é recomendada em algumas condições para evitar a deficiência de folato, como para mulheres no período periconcepcional e durante a gestação. Atualmente, existe uma preocupação quanto ao consumo excessivo de AF pela população pelo uso de suplementos com altas doses dessa vitamina. As vitaminas B6 e B2 agem como cofatores no metabolismo de um carbono, e o uso de altas doses de AF pode influenciar o metabolismo de ambas vitaminas e, consequentemente, interferir em metabolismos importantes das quais elas participam, como a via das quinureninas, e no sistema imune. Objetivo: Avaliar os efeitos da intervenção diária com uma alta dose de AF (5 mg) por 90 dias sobre marcadores do estado das vitaminas do complexo B, e as consequências sobre os metabólitos da via das quinureninas e o sistema imune em adultos saudáveis. Material e Métodos: 64 indivíduos saudáveis foram submetidos à intervenção diária com 5 mg de AF por 90 dias. Coletas de sangue foram realizadas antes (baseline) e após 45 e 90 dias de intervenção. As concentrações séricas de folato e vitamina B12 foram avaliadas por métodos microbiológicos. As concentrações séricas das vitaminas B6 (piridoxal 5'-fosfato (PLP), piridoxal (PL) e ácido 4-piridóxico (PA)), B2 (riboflavina e flavina mononucleotídeo (FMN)), B1 (tiamina e tiamina monofosfato (TMP)) e B3 (ácido nicotínico, nicotinamida e N1-metilnicotinamida), bem como de triptofano, quinurenina e metabólitos, foram avaliadas por LC-MS/MS. A proteína C-reativa ultrassensível (PCRus) foi determinada por imunoturbidimetria, e as concentrações séricas de interleucina (IL)-6, IL-8, IL-10, interferon gama (IFN-γ) e fator de necrose tumoral alfa (TNF-α) foram avaliadas por ensaio multiplex. A expressão de RNAm de DHFR (dihidrofolato redutase), MTHFR (metilenotetrahidrofolato redutase), IL8, TNFA e IFNG em leucócitos mononucleares (PBMC) foram avaliadas por PCR em tempo real. O número de células T regulatórias (Treg) (CD3+, CD4+, CD25high, FoxP3+, CD127-) foi avaliado após incubação dos PBMC com PMA e ionomicina ou veículo por 18h, por imunofenotipagem. Resultados: Houve um grande aumento das concentrações de folato sérico após 45 e 90 dias de intervenção com AF. Não houve diferença nas concentrações de vitamina B12 antes e após a intervenção. As concentrações séricas de PLP foram semelhantes antes e após a intervenção, entretanto, um aumento de PL sérico foi observado após 45 e 90 dias, e de PA após 45 dias, quando comparado ao baseline. Riboflavina e FMN foram maiores após 45 e 90 dias em relação ao baseline. A tiamina sérica foi menor após 45 dias, e as concentrações de TMP foram maiores após 90 dias quando comparados aos períodos anteriores. Não houve diferença nas concentrações de vitamina B3 antes e após a intervenção. Dentre os metabólitos da via das quinureninas, apenas o ácido antranílico apresentou aumento após 45 e 90 dias, enquanto o ácido picolínico diminuiu após 90 dias. PCRus, IL-6, IL-8, IL-10, IFN-γ e TNF-α séricos foram semelhantes no baseline e após a intervenção. Um aumento da expressão de RNAm de DHFR e TNFA foi observado após, respectivamente, 90 dias e 45 e 90 dias de intervenção. Após 90 dias de intervenção com AF, foi observada diminuição do número de células Treg após estímulo com PMA e ionomicina. Conclusão: O uso diário de 5 mg de AF foi associado a alterações nas concentrações séricas de marcadores do estado de vitaminas do complexo B e da via das quinureninas, bem como a diminuição do número de células Treg
Introduction: Folic acid (FA) supplementation is recommended in some conditions to avoid folate deficiency, as women during periconceptional period and pregnancy. Currently, there is a concern about the excessive consumption of FA by population by using supplements with high doses of this vitamin. Vitamins B6 and B2 are cofactors of enzymes of one carbon metabolism and, consequently, may disturb key metabolism in which they participate, as kynurenine pathway, and the immune system. Aim: To assess the effects of a daily intervention with high dose of FA (5 mg) for 90 days on biomarkers of complex B vitamins status and its outcomes in kynurenine pathway metabolites and immune system in healthy adults. Material and Methods: 64 healthy individuals were submitted to a daily intervention with 5 mg of FA for 90 days. Blood samples were collected before (baseline) and after 45 and 90 days of intervention. Serum folate and vitamin B12 were assessed by microbiological assays. Serum vitamin B6 (pyridoxal 5'-phosphate (PLP), pyridoxal (PL) and 4-pyridoxic acid (PA)), vitamin B2 (riboflavin and flavin mononucleotide (FMN)), vitamin B1 (thiamin and thiamin monophosphate)) and vitamin B3 (nicotinic acid, nicotinamide and N1-methylnicotinamide), as well as tryptophan, kynurenine and metabolites, were assessed by LC-MS/MS. C-reactive protein (hs-CPR) was assessed by immunoturbidimetry, and serum interleukin (IL)-6, IL-8, IL-10, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) were assessed by multiplex assay. Mononuclear leukocytes mRNA expression of DHFR (dihydrofolate reductase), MTHFR (methylenetetrahydrofolate reductase), IL8, TNFA and IFNG were assessed by real time PCR. Regulatory T Cell (Treg) number (CD3+, CD4+, CD25high, FoxP3+, CD127-) was determined after mononuclear leukocytes incubation with PMA and ionomycin or vehicle for 18h, by immunophenotyping. Results: A great increase on serum folate was observed after 45 and 90 days of FA intervention. No differences in serum vitamin B12 were observed before and after intervention. Serum PLP was similar before and after intervention, however, an increase in serum PL was observed after 45 and 90 days, and in PA after 45 days, when compared to baseline. Riboflavin and FMN were increased after 45 and 90 days than in baseline. Serum thiamine was decreased after 45 days than in baseline. Serum TMP was increased after 90 days when compared with previous timepoints. No differences in vitamin B3 were observed after and before FA intervention. Among kynurenine pathway metabolites, anthranilic acid was increased after 45 and 90 days, while picolinic acid was decreased after 90 days. hs-CPR, serum IL-6, IL-8, IL-10, IFN-γ and TNF-α were similar at baseline and after intervention. An increase on mRNA expression of DHFR and TNFA was observed after, respectively, 90 days and 45 and 90 days of intervention. After 90 days of FA intervention, it was observed a decrease on Treg cell number after PMA and ionomycin stimulation. Conclusion: Daily use of 5 mg of FA was associated with changes in serum markers of B-complex vitamins status and kynurenine pathway, as well as decreased number of Treg cells
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Riboflavina/farmacocinética , Vitamina B 6/farmacocinética , Ácido Fólico/administración & dosificación , Ácido Fólico/análisis , Tiamina/farmacocinética , Linfocitos T Reguladores/clasificación , Niacinamida/farmacocinética , Quinurenina/farmacocinéticaRESUMEN
BACKGROUND AND AIMS: Neuropathic pain is a significant medical problem needing more effective treatments with fewer side effects. Overactive glutamatergic transmission via N-methyl-d-aspartate receptors (NMDARs) are known to play a role in central sensitization and neuropathic pain. Although ketamine, a NMDAR channel-blocking antagonist, is often used for neuropathic pain, its side-effect profile and abusive potential has prompted the search for a safer effective oral analgesic. A novel oral prodrug, AV-101 (l-4 chlorokynurenine), which, in the brain, is converted into one of the most potent and selective GlyB site antagonists of the NMDAR, has been demonstrated to be active in animal models of neuropathic pain. The two Phase 1 studies reported herein were designed to assess the safety and pharmacokinetics of AV-101, over a wide dose range, after daily dosing for 14-days. As secondary endpoints, AV-101 was evaluated in the capsaicin-induced pain model. METHODS: The Phase 1A study was a single-site, randomized, double-blind, placebo-controlled, single oral ascending dose (30-1800mg) study involving 36 normal healthy volunteers. The Phase 1B study was a single-site randomized, double-blind, placebo-controlled, study of multiple ascending doses (360, 1080, and 1440mg/day) of AV-101 involving 50 normal healthy volunteers, to whom AV-101 or placebo were administered orally daily for 14 consecutive days. Subjects underwent PK blood analyses, laboratory assessments, physical examination, 12-lead ECG, ophthalmological examination, and various neurocognitive assessments. The effect of AV-101 was evaluated in the intradermally capsaicin-induced pain model (ClinicalTrials.gov Identifier: NCT01483846). RESULTS: Two Phase 1, with an aggregate of 86 subjects, demonstrated that up to 14 days of oral AV-101, up to 1440mg per day, was safe and very well tolerated with AEs quantitively and qualitatively like those observed with placebo. Mean half-life values of AV-101 were consistent across doses, ranging with an average of 1.73h, with the highest Cmax (64.4µg/mL) and AUC0-t (196µgh/mL) values for AV-101 occurring in the 1440-mg dose group. In the capsaicin induce-pain model, there was no significant change in the area under the pain time curve (AUPC) for the spontaneous pain assessment between the treatment and the placebo groups on Day 1 or 14 (the primary endpoint). In contrast, there were consistent reductions at 60-180min on Day 1 after dosing for allodynia, mechanical hyperalgesia, heat hyperalgesia, and spontaneous pain, and on Day 14 after dosing for heat hyperalgesia. CONCLUSIONS: Although, AV-101 did not reach statistical significance in reducing pain, there were consistent reductions, for allodynia pain and mechanical and heat hyperalgesia. Given the excellent safety profile and PK characteristics demonstrated by this study, future clinical trials of AV-101 in neuropathic pain are justified. IMPLICATIONS: This article presents the safety and PK of AV-101, a novel oral prodrug producing a potent and selective GlyB site antagonist of the NMDA receptor. These data indicate that AV-101 has excellent safety and PK characteristics providing support for advancing AV-101 into Phase 2 studies in neuropathic pain, and even provides data suggesting that AV-101 may have a role in treating depression.
Asunto(s)
Voluntarios Sanos , Hiperalgesia , Ácido Quinurénico/análogos & derivados , Quinurenina/análogos & derivados , Neuralgia/tratamiento farmacológico , Fármacos Neuroprotectores/farmacocinética , Adulto , Estudios Cruzados , Femenino , Humanos , Hiperalgesia/tratamiento farmacológico , Ácido Quinurénico/administración & dosificación , Ácido Quinurénico/sangre , Ácido Quinurénico/farmacocinética , Quinurenina/administración & dosificación , Quinurenina/sangre , Quinurenina/farmacocinética , Masculino , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/sangre , Dimensión del DolorRESUMEN
The kynurenine pathway of tryptophan metabolism has an important role in mediating the behavioral effects of inflammation, which has implications in understanding neuropsychiatric comorbidity and for the development of novel therapies. Inhibition of the rate-limiting enzyme, indoleamine 2,3-dioxygenase (IDO), prevents the development of many of these inflammation-induced preclinical behaviors. However, dysregulation in the balance of downstream metabolism, where neuroactive kynurenines are generated, is hypothesized to be a functionally important pathogenic feature of inflammation-induced depression. Here we utilized two novel transgenic mouse strains to directly test the hypothesis that neurotoxic kynurenine metabolism causes depressive-like behavior following peripheral immune activation. Wild-type (WT) or kynurenine 3-monooxygenase (KMO)-deficient (KMO-/-) mice were administered either lipopolysaccharide (LPS, 0.5 mg kg-1) or saline intraperitoneally. Depressive-like behavior was measured across multiple domains 24 h after immune challenge. LPS precipitated a robust depressive-like phenotype, but KMO-/- mice were specifically protected from LPS-induced immobility in the tail suspension test (TST) and reduced spontaneous alternations in the Y-maze. Direct administration of 3-hydroxykynurenine, the metabolic product of KMO, caused a dose-dependent increase in depressive-like behaviors. Mice with targeted deletion of 3-hydroxyanthranilic acid dioxygenase (HAAO), the enzyme that generates quinolinic acid, were similarly challenged with LPS. Similar to KMO-/- mice, LPS failed to increase immobility during the TST. Whereas kynurenine metabolism was generally increased in behaviorally salient brain regions, a distinct shift toward KMO-dependent kynurenine metabolism occurred in the dorsal hippocampus in response to LPS. Together, these results demonstrate that KMO is a pivotal mediator of hippocampal-dependent depressive-like behaviors induced by peripheral LPS challenge.
Asunto(s)
Depresión/inducido químicamente , Depresión/inmunología , Hipocampo/efectos de los fármacos , Hipocampo/inmunología , Inflamación/inmunología , Quinurenina 3-Monooxigenasa/deficiencia , Quinurenina/farmacocinética , Quinurenina/toxicidad , Animales , Femenino , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Oxidorreductasas/deficienciaRESUMEN
Sub-chronic tryptophan depletion (SCTD) is proposed as an animal model for depression. Aims were to test the hypothesis and optimise the time of SCTD-induced depression-related behaviour and associated biochemical changes. Sprague Dawley rats were treated with a low tryptophan (TRP) containing diet for 0, 7 or 14 days. Peripheral and central neurochemical markers were measured. SCTD-induced depression-related behaviour was assessed by the forced swim test (FST). Model sensitivity to antidepressants was tested by concomitant treatment with paroxetine. SCTD-induced significant reductions in weight gain and measures of peripheral and central TRP. Corticosterone, aldosterone and kynurenine (K), increased whilst kynurenic acid (KA), an NMDA antagonist decreased. 5-HT(2) receptor binding Bmax was enhanced but was reversed by paroxetine. Corticosterone and aldosterone were significantly negatively-correlated to weight gain. SCTD increased floating time and reduced swimming time in the FST but were reversed by paroxetine. Aldosterone was increased at 7 and 14 days, whereas other changes maximised at 14 days. Aldosterone may be an early marker or causal link for depression development. Increased corticosterone and brain tissue 5-HT-receptor density may be correlates of depressive behaviour. Consequential increases in NMDA signalling through increased K/KA ratios suggest the model may be useful for testing novel antidepressants.
Asunto(s)
Depresión/etiología , Triptófano/deficiencia , Aldosterona/sangre , Análisis de Varianza , Animales , Peso Corporal/fisiología , Encéfalo/metabolismo , Proteínas de Unión al Calcio/sangre , Catecolaminas/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Corticosterona/sangre , Proteínas de Unión al ADN/sangre , Depresión/patología , Dieta/efectos adversos , Modelos Animales de Enfermedad , Ácido Quinurénico/metabolismo , Quinurenina/metabolismo , Quinurenina/farmacocinética , Masculino , Proteínas del Tejido Nervioso/sangre , Nucleobindinas , Paroxetina/uso terapéutico , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéutico , Natación/psicología , Factores de Tiempo , Tritio/farmacocinéticaRESUMEN
PURPOSE: Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens. METHODS: The t-Boc-protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC. RESULTS: At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in beta-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys. CONCLUSIONS: These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.
Asunto(s)
Envejecimiento/metabolismo , Cristalinas/metabolismo , Glutatión/metabolismo , Quinurenina/farmacocinética , Cristalino/metabolismo , Animales , Unión Competitiva/fisiología , Bovinos , Cisteína/síntesis química , Cisteína/farmacocinética , Glutatión/química , Histidina/síntesis química , Histidina/farmacocinética , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cetonas/metabolismo , Quinurenina/síntesis química , Cristalino/efectos de la radiación , Procesamiento Proteico-Postraduccional/fisiología , Rayos UltravioletaRESUMEN
Kynurenine (KYN), a tryptophan metabolite, is a precursor of kynurenic acid, which is an antagonist of N-methyl-d-aspartate receptor. In this study, an enantiomeric separation of d,l-KYN derivatized with the benzofurazan fluorescence reagent 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) (DBD-d,l-KYN) was first investigated by using a high-performance liquid chromatography (HPLC) with several chiral columns. As a consequence, DBD-d,l-KYN was enantiomerically separated on a cellulose-type chiral column (CHIRALCEL OJ-RH) with a mobile phase of H(2)O/CH(3)CN/MeOH (40/50/10) containing 0.1% acetic acid. Under this condition, the separation factor and resolution were 1.48 and 1.28, respectively. Next, a column-switching HPLC consisting of both octadecylsilica and chiral columns was developed and used to determine both d- and l-KYN enantiomers in 10 microL of rat plasma following the intraperitoneal administration of d,l-KYN to rats (10 mg kg(-1)). The result revealed that the concentration of l-KYN was higher than that of d-KYN, suggesting that d-KYN was eliminated faster than l-KYN.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Quinurenina/química , Quinurenina/farmacocinética , Animales , Análisis Químico de la Sangre/métodos , Celulosa/química , Técnicas de Química Analítica/métodos , Indicadores y Reactivos/farmacología , Infusiones Parenterales/métodos , Quinurenina/administración & dosificación , Masculino , Oxazoles/química , Ratas , Ratas Sprague-Dawley , Dióxido de Silicio/química , Espectrometría de Fluorescencia/métodos , Estereoisomerismo , Sulfonamidas/químicaRESUMEN
The amino acid tryptophan is a precursor for the neurotransmitter serotonin as well as for kynurenic and quinolinic acids. These latter molecules are antagonists and agonists, respectively, of the excitatory amino acid glutamate and arise through the kynurenine pathway of tryptophan metabolism. Significant differences exist in the sites and physiological control of serotonin versus kynurenine. While serotonin is formed within serotonin neurons (in the brain and intestine) and neuroendocrine cells of the intestine, kynurenine is formed by liver cells (as a precursor to nicotinic acid) and in macrophages, activated by inflammatory cytokines. Our studies are based on the hypothesis that inhibition of kynurenine metabolism (at the kynurenine hydroxylase [KH] step) allows the amino acid to be converted to kynurenic acid, a neuroprotective antagonist of excitatory amino acid receptors. Inhibition of KH also prevents formation of the neurotoxic species 3-hydroxykynurenine and quinolinic acid. To accomplish this end, inhibitors were identified and are described.
Asunto(s)
Encéfalo/metabolismo , Quinurenina/farmacocinética , Animales , Encéfalo/efectos de los fármacos , Isótopos de Carbono/farmacocinética , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Concentración 50 Inhibidora , Quinurenina/antagonistas & inhibidores , Luz , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ácido Quinolínico/farmacocinética , Factores de Tiempo , Tritio/farmacocinéticaRESUMEN
L-Kynurenine and quinolinic acid are neuroactive L-tryptophan-kynurenine pathway metabolites of potential importance in pathogenesis and treatment of neurologic disease. To identify precursors of these metabolites in brain, [(2)H(3) ]-L-kynurenine was infused subcutaneously by osmotic pump into three groups of gerbils: controls, CNS-localized immune-activated, and systemically immune-activated. The specific activity of L-kynurenine and quinolinate in blood, brain and systemic tissues at equilibrium was then quantified by mass spectrometry and the results applied to a model of metabolism to differentiate the relative contributions of various metabolic precursors. In control gerbils, 22% of L-kynurenine in brain was derived via local synthesis from L-tryptophan/formylkynurenine versus 78% from L-kynurenine from blood. Quinolinate in brain was derived from several sources, including: local tissue L-tryptophan/formylkynurenine (10%), blood L-kynurenine (35%), blood 3-hydroxykynurenine/3-hydroxyanthranilate (7%), and blood quinolinate (48%). After systemic immune-activation, however, L-kynurenine in brain was derived exclusively from blood, whereas quinolinate in brain was derived from three sources: blood L-kynurenine (52%), blood 3-hydroxykynurenine or 3-hydroxyanthranilate (8%), and blood quinolinate (40%). During CNS-localized immune activation, > 98% of both L-kynurenine and quinolinate were derived via local synthesis in brain. Thus, immune activation and its site determine the sources from which L-kynurenine and quinolinate are synthesized in brain. Successful therapeutic modulation of their concentrations must take into account the metabolic and compartment sources.
Asunto(s)
Encéfalo/metabolismo , Encefalitis/metabolismo , Inflamación/metabolismo , Quinurenina/metabolismo , Ácido Quinolínico/metabolismo , Animales , Compartimentos de Líquidos Corporales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Femenino , Gerbillinae , Inflamación/inducido químicamente , Inyecciones Subcutáneas , Riñón/metabolismo , Quinurenina/administración & dosificación , Quinurenina/farmacocinética , Lipopolisacáridos , Hígado/metabolismo , Pulmón/metabolismo , Espectrometría de Masas , Modelos Biológicos , Unión Proteica/fisiología , Albúmina Sérica/metabolismo , Distribución Tisular , Tritio , Triptófano/metabolismoRESUMEN
This study describes the functional characterization of a specific mosquito transaminase responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). The enzyme was purified from Aedes aegypti larvae by ammonium sulfate fractionation, heat treatment, and various chromatographic techniques, plus non-denaturing electrophoresis. The purified transaminase has a relative molecular mass of 42,500 by SDS-PAGE. N-terminal and internal sequencing of the purified protein and its tryptic fragments resolved a partial N-terminal sequence of 19 amino acid residues and 3 partial internal peptide sequences with 7, 10, and 7 amino acid residues. Using degenerate primers based on the partial internal sequences for PCR amplification and cDNA library screening, a full-length cDNA clone with a 1,167-bp open reading frame was isolated. Its deduced amino acid sequence consists of 389 amino acid residues with a predicted molecular mass of 43,239 and shares 45-46% sequence identity with mammalian alanine glyoxylate transaminases. Northern analysis shows the active transcription of the enzyme in larvae and developing eggs. Substrate specificity analysis of this mosquito transaminase demonstrates that the enzyme is active with 3-HK, kynurenine, or alanine substrates. The enzyme has greater affinity and catalytic efficiency for 3-HK than for kynurenine and alanine. The biochemical characteristics of the enzyme in conjunction with the profiles of 3-HK transaminase activity and XA accumulation during mosquito development clearly point out its physiological function in the 3-HK to XA pathway. Our data suggest that the mosquito transaminase was evolved in a manner precisely reflecting the physiological requirement of detoxifying 3-HK produced in the tryptophan oxidation pathway in the mosquito.
Asunto(s)
Aedes/enzimología , Alanina Transaminasa/genética , Alanina Transaminasa/metabolismo , Quinurenina/análogos & derivados , Quinurenina/farmacocinética , Transaminasas/genética , Transaminasas/metabolismo , Alanina Transaminasa/química , Alanina Transaminasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Inactivación Metabólica , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transaminasas/química , Transaminasas/aislamiento & purificaciónRESUMEN
Kynurenine 3-mono-oxygenase, one of the key enzymes of the "kynurenine pathway", catalyses the formation of 3-hydroxykynurenine and may direct the neo-synthesis of quinolinic and kynurenic acids. While 3-hydroxykynurenine and quinolinic acid have neurotoxic properties, kynurenic acid antagonizes excitotoxic neuronal death. Here we report that the expression and activity of kynurenine 3-mono-oxygenase significantly increased in the spinal cord of rats with experimental allergic encephalopathy, an experimental model of multiple sclerosis. As a consequence of this increase, the spinal cord content of 3-hydroxykynurenine and quinolinic acid reached neurotoxic levels. We also report that systemic administration of Ro 61-8048, a selective kynurenine 3-mono-oxygenase inhibitor, reduced the increase of both 3-hydroxykynurenine and quinolinic acid, and caused accumulation of kynurenic acid. In the brain and spinal cord of the controls, kynurenine 3-mono-oxygenase immunoreactivity was located in granules (probably mitochondria) present in the cytoplasm of both neurons and astroglial cells. In the spinal cord of rats with experimental allergic encephalopathy, however, cells with a very intense kynurenine 3-mono-oxygenase immunoreactivity, also able to express class II major histocompatibility complex and inducible nitric oxide synthase, were found in perivascular, subependymal and subpial locations. These cells (most probably macrophages) were responsible for the large increase in 3-hydroxykynurenine and quinolinic acid found in the spinal cords of affected animals. The results show that cells of the immune system are responsible for the increased formation of 3-hydroxykynurenine and quinolinic acid, two neurotoxic metabolites that accumulate in the central nervous system of rats with experimental allergic encephalomyelitis. They also demonstrate that selective kynurenine 3-mono-oxygenase inhibitors reduce the neo-synthesis of these toxins.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Quinurenina/farmacocinética , Oxigenasas de Función Mixta/metabolismo , Médula Espinal/enzimología , Animales , Astrocitos/enzimología , Encéfalo/enzimología , Gránulos Citoplasmáticos/enzimología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Inmunohistoquímica , Ácido Quinurénico/metabolismo , Quinurenina/análogos & derivados , Quinurenina 3-Monooxigenasa , Esclerosis Múltiple , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Wistar , Sulfonamidas/farmacología , Tiazoles/farmacologíaRESUMEN
L-3-Hydroxykynurenine (L-3-HK) and quinolinate (QUIN) are two metabolites of the kynurenine pathway, the major route of tryptophan degradation in mammals. L-3-HK is a known generator of highly reactive free radicals, whereas QUIN is an endogenous excitotoxin acting specifically at N-methyl-D-aspartate (NMDA) receptors. This study was designed to examine possible synergistic interactions between L-3-HK and QUIN in the rat brain in vivo. Intrastriatal coinjection of 5 nmol L-3-HK and 15 nmol QUIN, i.e. doses which caused no or minimal neurodegeneration on their own, resulted in substantial neuronal loss, determined both behaviourally (apomorphine-induced rotations) and histologically (quantitative assessment of lesion size). The excitotoxic nature of the lesion was verified by tyrosine hydroxylase immunohistochemistry, showing the survival of dopaminergic striatal afferents. There was also a relative sparing of large striatal neurons, and neurodegeneration was prevented both by NMDA receptor blockade (using CGP 40116) and free radical scavenging [using N-tert-butyl-alpha-(2-sulphophenyl)-nitrone, S-PBN]. The pro-excitotoxic features of L-3-HK were especially pronounced at low QUIN doses and were not observed when QUIN was substituted by NMDA. Notably, the effect of L-3-HK was not due to its intracerebral conversion to QUIN and was duplicated by equimolar D,L-3-HK. These data indicate that an elevation of L-3-HK levels constitutes a significant hazard in situations of excitotoxic injury. Pharmacological interventions aimed at decreasing L-3-HK formation may therefore be particularly useful for the treatment of neurological diseases which are associated with an abnormally enhanced flux through the kynurenine pathway.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Quinurenina/análogos & derivados , N-Metilaspartato/toxicidad , Ácido Quinolínico/toxicidad , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/farmacología , Vías Aferentes/efectos de los fármacos , Vías Aferentes/fisiología , Animales , Bencenosulfonatos/farmacología , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Sinergismo Farmacológico , Complejo IV de Transporte de Electrones/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Depuradores de Radicales Libres/farmacología , Quinurenina/farmacocinética , Quinurenina/farmacología , Masculino , Actividad Motora/efectos de los fármacos , Ácido Quinolínico/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
In order to complete pharmacokinetic studies on the central vs. peripheral origin of several tryptophan metabolites, we infused gerbils with labelled kynurenine (2H4 or 15N2). Osmotic minipumps charged with kynurenine solutions were surgically implanted subcutaneously in adult female gerbils (50-60 g). After a variable number of hours, the gerbils were sacrificed and organs taken for determination of labelled/unlabelled kynurenine ratios using mass spectrometric assay of a pentafluorobenzyl derivative as described previously. Surprisingly high ratios of 2H to 1H-kynurenine were measured in the kidney (0.25-0.40) and urine (4.0-8.0), although the ratio of deuterium labelled to endogenous kynurenine remained below detection limits (< 0.05) in serum and other tissues. Infusion of greater quantities of 2H4-kynurenine confirmed these observations in gerbils in which ratios of 2H4-to-1H kynurenine were measurable in serum and tissues. Synthesis and infusion of 15N2-kynurenine demonstrated that these effects were not due to deuterium isotope substitution. The data demonstrate a non-equilibrium between infused and endogenous kynurenine, which is related to differential rates of protein binding and the rapid clearance of free, infused kynurenine by kidney.
Asunto(s)
Quinurenina/metabolismo , Quinurenina/farmacocinética , Animales , Encéfalo/metabolismo , Deuterio , Femenino , Cromatografía de Gases y Espectrometría de Masas , Gerbillinae , Infusiones Intravenosas , Riñón/metabolismo , Quinurenina/administración & dosificación , Isótopos de Nitrógeno , Factores de Tiempo , Distribución TisularRESUMEN
We examined the effects of kynurenine metabolites administered into the right cerebroventricle (1 micromol) on the electrocorticogram (ECoG) of rats to establish the role of kynurenines on brain function. Kynurenine, anthranilic acid, quinaldic acid, xanthurenic acid or 8-hydroxyquinaldic acid showed no effect on ECoG throughout the recording period of 4 hours. 3-Hydroxykynurenine had a transient suppressive effect on the ECoG, while kynurenic acid caused a slight suppression of ECoG activity. 3-Hydroxyanthranilic acid (3-OH-An), a metabolite of 3-hydroxykynurenine, induced spike discharges with a long latency (60-230 min). 3-OH-An is thought to be metabolized to o-aminophenol, 3-methoxyanthranilic acid, quinolinic acid, 2-ketoadipic acid and picolinic acid. Among 3-OH-An metabolites, only o-aminophenol induced spike discharges several minutes after administration, lasting for 60 min. On the other hand, quinolinic acid suppressed ECoG, though 3-methoxyanthranilic acid, 2-ketoadipic acid and picolinic acid had no effects on ECoG. These electrocorticographic findings suggest that 3-OH-An may induce spike discharges after it is metabolized in the brain to o-aminophenol.
Asunto(s)
Electroencefalografía/efectos de los fármacos , Quinurenina/farmacología , Quinurenina/farmacocinética , Ácido 3-Hidroxiantranílico/farmacología , Animales , Biotransformación , Depuradores de Radicales Libres/farmacología , Inyecciones Intraventriculares , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
3-Hydroxykynurenine (3-HK) is a potential endogenous neurotoxin whose increased levels have been described in several neurodegenerative disorders. Here, we characterized in vitro neurotoxicity of 3-HK. Of the tested kynurenine pathway metabolites, only 3-HK, and to a lesser extent 3-hydroxyanthranilic acid, were toxic to primary cultured striatal neurons. 3-HK toxicity was inhibited by various antioxidants, indicating that the generation of reactive oxygen species is essential to the toxicity. 3-HK-induced neuronal cell death showed several features of apoptosis, as determined by the blockade by macromolecule synthesis inhibitors, and by the observation of cell body shrinkage with nuclear chromatin condensation and fragmentation. In addition, 3-HK toxicity was dependent on its cellular uptake via transporters for large neutral amino acids, because uptake inhibition blocked the toxicity. Cortical and striatal neurons were much more vulnerable to 3-HK toxicity than cerebellar neurons, which may be attributable to the differences in transporter activities of these neurons. These results indicate that 3-HK, depending on transporter-mediated cellular uptake and on intracellular generation of oxidative stress, induces neuronal cell death with brain region selectivity and with apoptotic features, which may be relevant to pathology of neurodegenerative disorders.
Asunto(s)
Apoptosis/fisiología , Quinurenina/análogos & derivados , Neuronas/efectos de los fármacos , Neuronas/fisiología , Estrés Oxidativo/fisiología , Sistemas de Transporte de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/metabolismo , Quinurenina/metabolismo , Quinurenina/farmacocinética , Quinurenina/farmacología , Neurotoxinas/farmacología , Ratas , Ratas WistarRESUMEN
Kynurenic acid (KA) is an excitatory amino acid receptor antagonist that is believed to play an important role in a host of diseases of the neuropsychiatric and central nervous system. A method for the determination of KA in microdialysate samples using capillary electrophoresis (CE) separation with laser-induced fluorescence (LIF) detection is described. CE is advantageous for the analysis of microdialysis samples due to its short analysis times and small sample volume requirements. Three complexation approaches were evaluated in an attempt to achieve the best limit of detection. The best approach was found to be pre-column complexation with inclusion of Zn(II) in the background electrolyte. After optimization of the zinc acetate concentration and pH, a limit of detection of 1 nM KA was achieved. However, when KA was present in the dialysate, the limit of detection increased 50-fold. Even though the endogenous levels of KA in rat brain are below this limit of detection, this methodology could be used to monitor the increase of KA levels in rat brain following dosing with its precursors, tryptophan and kynurenine.
Asunto(s)
Electroforesis Capilar/métodos , Ácido Quinurénico/análisis , Quinurenina/farmacocinética , Triptófano/farmacocinética , Zinc/química , Animales , Concentración de Iones de Hidrógeno , Inyecciones Intraperitoneales , Ácido Quinurénico/química , Ácido Quinurénico/metabolismo , Quinurenina/administración & dosificación , Quinurenina/metabolismo , Concentración Osmolar , Ratas , Espectrometría de Fluorescencia , Factores de Tiempo , Triptófano/administración & dosificación , Triptófano/metabolismoRESUMEN
An electrochemically active compound was detected in the larvae of Aedes aegypti mosquitoes and progressive accumulation of this compound was observed during larval development. The compound was purified from mosquito larvae using various chromatographic techniques and spectral analysis of the purified compound resulted in its identification as xanthurenic acid. Production of xanthurenic acid results from the transamination of 3-hydroxykynuorenine, and analysis of the biochemical pathway in xanthurenic acid production revealed the presence of a particular transaminase that has a much higher specific activity to 3-hydroxykynurenine than to kynurenine in the mosquito larvae. Concentration of xanthurenic acid is closely related to the level of this transaminase activity. Results suggest that this particular transaminase plays an important role in regulating the level of 3-hydroxykynurenine in the mosquito, A. aegypti during larval development.
Asunto(s)
Aedes/crecimiento & desarrollo , Transaminasas/metabolismo , Triptófano/metabolismo , Xanturenatos/farmacocinética , Aedes/enzimología , Animales , Quinurenina/análogos & derivados , Quinurenina/aislamiento & purificación , Quinurenina/metabolismo , Quinurenina/farmacocinética , Larva/enzimología , Larva/crecimiento & desarrollo , Xanturenatos/aislamiento & purificaciónRESUMEN
7-Chlorokynurenic acid (7-Cl-KYNA) and 5,7-dichlorokynurenic acid (5,7-Cl2-KYNA) are of therapeutic interest as potent glycine/N-methyl-D-aspartate NMDA) receptor antagonists, but are excluded from brain by the blood-brain barrier. We examined whether these compounds could be delivered to brain through their respective precursors, L-4-chlorokynurenine (4-Cl-KYN) and L-4,6-dichlorokynurenine (4,6-Cl2-KYN), which are amino acids. 4-Cl-KYN was shown to be rapidly shuttled into the brain by the large neutral amino acid transporter of the blood-brain barrier (K(m) = 105 +/- 14 microM, Vmax = 16.9 +/- 2.3 nmol min-1 g-1) and to be converted intracerebrally to 7-Cl-KYNA. 4,6-Cl2-KYN also expressed affinity for the transporter, but four-fold less than that of 4-Cl-KYN. In summary, the results show that because of their facilitated uptake 4-Cl-KYN and 4,6-Cl2KYN might be useful prodrugs for brain delivery of glycine-NMDA receptor antagonists.
Asunto(s)
Encéfalo/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Ácido Quinurénico/análogos & derivados , Quinurenina/análogos & derivados , Aminoácidos/metabolismo , Animales , Biotransformación , Ácido Quinurénico/metabolismo , Quinurenina/metabolismo , Quinurenina/farmacocinética , Masculino , Perfusión , Profármacos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Fluorescent granules that are stained with Nile blue sulfate are present in larval Malpighian tubules in the wild type strain of Drosophila melanogaster, Oregon-R. These granules emit a weak blue fluorescence and most of them are about 2 microns or more in diameter. The ommochrome precursor 3-hydroxy-kynurenine (3-HK) is actively transferred into the tubules of Oregon-R. Changes in the fluorescent granules in the Malpighian tubules on administration of ommochrome precursors were investigated in eye color mutants of Drosophila. The fluorescent granules in the tubules of the nonautonomous mutants v;bw and cn bw emit a strong blue fluorescence and most of them are about 1 micron or less in diameter. When v;bw and cn bw larvae were cultured on medium supplemented with kynurenine or 3-HK, respectively, the fluorescence intensity of their granules decreased, and their size increased. These additions resulted in almost equal accumulation of 3-HK to that in Oregon-R. On the other hand, no 3-HK accumulated in the tubules of larvae of the autonomous mutants bw;st, ltd bw and w, which lack the fluorescent granules. These findings indicate that the fluorescent granules are an important intracellular site for uptake or storage of ommochrome precursors in larval Malpighian tubules of Drosophila.