L. G. Darlington

Endogenous kynurenines as targets for drug discovery and development

The kynurenine pathway is the main pathway for tryptophan metabolism. It generates compounds that can modulate activity at glutamate receptors and possibly nicotinic receptors, in addition to some as-yet-unidentified sites. The pathway is in a unique position to regulate other aspects of the metabolism of tryptophan to neuroactive compounds, and also seems to be a key factor in the communication between the nervous and immune systems. It also has potentially important roles in the regulation of cell proliferation and tissue function in the periphery. As a result, the pathway presents a multitude of potential sites for drug discovery in neuroscience, oncology and visceral pathology.

Oxidative stress as a mechanism for quinolinic acid-induced hippocampal damage: protection by melatonin and deprenyl.

There are differences between the excitotoxic actions of quinolinic acid and N‐methyl‐D‐aspartate (NMDA) which suggest that quinolinic acid may act by mechanisms additional to the activation of NMDA receptors. The present study was designed to examine the effect of a potent antioxidant, melatonin, and the potential neuroprotectant, deprenyl, as inhibitors of quinolinic acid‐induced brain damage. Injections were made into the hippocampus of anaesthetized rats, which were allowed to recover before the brains were taken for histology and the counting of surviving neurones. Quinolinic acid (120u2003nmols) induced damage to the pyramidal cell layer, which was prevented by the co‐administration of melatonin (5u2003nmols locally plus 2×20u2003mgu2003kg−1 i.p.). This protective effect was not prevented by the melatonin receptor blocker luzindole. Neuronal damage produced by NMDA (120u2003nmols) was not prevented by melatonin. Quinolinic acid increased the formation of lipid peroxidation products from hippocampal tissue and this effect was prevented by melatonin. Deprenyl also prevented quinolinic acid‐induced damage at a dose of 50u2003nmols but not 10u2003nmols plus 2×1.0u2003mgu2003kg−1 i.p. The non‐selective monoamine oxidase inhibitor nialamide (10 and 50u2003nmols plus 2×25u2003mgu2003kg−1) did not afford protection. The results suggest that quinolinic acid‐induced neuronal damage can be prevented by a receptor‐independent action of melatonin and deprenyl, agents which can act as a potent free radical scavenger and can increase the activity of endogenous antioxidant enzymes respectively. This suggests that free radical formation contributes significantly to quinolinic acid‐induced damage in vivo.

Altered kynurenine metabolism correlates with infarct volume in stroke

Inflammation and oxidative stress are involved in brain damage following stroke, and tryptophan oxidation along the kynurenine pathway contributes to the modulation of oxidative stress partly via the glutamate receptor agonist quinolinic acid and antagonist kynurenic acid, and via redox‐active compounds such as 3‐hydroxyanthranilic acid. We have confirmed that following a stroke, patients show early elevations of plasma neopterin, S100B and peroxidation markers, the latter two correlating with infarct volume assessed from computed tomography (CT) scans, and being consistent with a rapid inflammatory response. We now report that the kynurenine pathway of tryptophan metabolism was also activated, with an increased kynurenineu2003:u2003tryptophan ratio, but with a highly significant decrease in the ratio of 3‐hydroxyanthranilic acidu2003:u2003anthranilic acid, which was strongly correlated with infarct volume. Levels of kynurenic acid were significantly raised in patients who died within 21u2003days compared with those who survived. The results suggest that increased tryptophan catabolism is initiated before or immediately after a stroke, and is related to the inflammatory response and oxidative stress, with a major change in 3‐hydroxyanthranilic acid levels. Together with previous evidence that inhibiting the kynurenine pathway reduces brain damage in animal models of stroke and cerebral inflammation, and that increased kynurenine metabolism directly promotes oxidative stress, it is proposed that oxidative tryptophan metabolism may contribute to the oxidative stress and brain damage following stroke. Some form of anti‐inflammatory intervention between the rise of S100B and the activation of microglia, including inhibition of the kynurenine pathway, may be valuable in modifying patient morbidity and mortality.

Tryptophan metabolism and oxidative stress in patients with chronic brain injury

The kynurenine pathway generates the excitotoxic N‐methyl‐d‐aspartate receptor agonist, quinolinic acid and the glutamate antagonist, kynurenic acid, as well as free‐radical generators. We investigated the status of the pathway following severe brain injury sustained at least 1u2003year previously in 15 patients compared with controls. At baseline, patients with brain injury showed increased levels of neopterin, erythrocyte sedimentation rate, C‐reactive protein and peroxidation products in the blood compared with controls, indicating persistent inflammation and oxidative stress. At baseline and following tryptophan depletion, more tryptophan was converted to kynurenine in patients than controls, but less kynurenine was converted into the neuroprotectant, kynurenic acid. This suggests that neuroprotection by kynurenic acid may be inadequate in brain‐damaged patients even many years after injury. On tryptophan loading, patients metabolized more kynurenine into kynurenic acid than controls, a process which may be neuroprotective. In addition, lower levels of 3‐hydroxykynurenine and 3‐hydroxyanthranilic acid in patients after tryptophan loading should be protective since these compounds generate free radicals. The results suggest that for brain‐damaged patients, increased activation of the kynurenine pathway, oxidative stress and raised levels of inflammation continue many years after the original insult, possibly contributing to the continuing cerebral dysfunction in these patients.

Levels of Purine, Kynurenine and Lipid Peroxidation Products in Patients with Inflammatory Bowel Disease

The factors affecting gut activity in inflammatory bowel disease are unclear, but purines and kynurenines may be involved in the regulation of neuronal activity and therefore gut motility and secretion. We have measured the serum levels of these compounds in patients and in sex- and age-matched controls. Purines and kynurenines were analysed using HPLC. The levels of tryptophan and its metabolites 3-hydroxykynurenine, 3-hydroxyanthranilic acid and xanthurenic acid were unchanged in all patients. However, the levels of kynurenine and kynurenic acid were significantly elevated in patients with inflammatory bowel disease when compared to control subjects. There were no significant differences between patients and controls for any of the purines analysed or for neopterin. In the inflammatory bowel disease patients serum lipid peroxidation products were significantly elevated when compared to control subjects, suggesting the presence of increased oxidative stress consistent with inflammatory activity. The elevated level of kynurenic acid may represent either a compensatory response to elevated activation of enteric neurones, or a primary abnormality, which induces a compensatory increase in gut activity, but may indicate a role for kynurenine modulation of glutamate receptors in the symptoms of inflammatory bowel disease.

Blood 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and melatonin levels in patients with either Huntington's disease or chronic brain injury

Following a study of oxidative tryptophan metabolism to kynurenines, we have now analysed the blood of patients with either Huntingtons disease or traumatic brain injury for levels of 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA) and melatonin. There were no differences in the baseline levels of these compounds between patients and healthy controls. Tryptophan depletion did not reduce 5‐HT levels in either the controls or in the patients with Huntingtons disease, but it increased 5‐HT levels in patients with brain injury and lowered 5‐HIAA in the control and Huntingtons disease groups. An oral tryptophan load did not modify 5‐HT levels in the patients but increased 5‐HT in control subjects. The tryptophan load restored 5‐HIAA to baseline levels in controls and patients with brain injury, but not in those with Huntingtons disease, in whom 5‐HIAA remained significantly depressed. Melatonin levels increased on tryptophan loading in all subjects, with levels in patients with brain injury increasing significantly more than in controls. Baseline levels of neopterin and lipid peroxidation products were higher in patients than in controls. It is concluded that both groups of patients exhibit abnormalities in tryptophan metabolism, which may be related to increased inflammatory status and oxidative stress. Interactions between the kynurenine, 5‐HT and melatonin pathways should be considered when interpreting changes of tryptophan metabolism.

Changes in synaptic transmission and protein expression in the brains of adult offspring after prenatal inhibition of the kynurenine pathway

During early brain development, N-methyl-d-aspartate (NMDA) receptors are involved in cell migration, neuritogenesis, axon guidance and synapse formation, but the mechanisms which regulate NMDA receptor density and function remain unclear. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at NMDA receptors and we have previously shown that inhibition of the pathway using the kynurenine-3-monoxygenase inhibitor Ro61-8048 in late gestation produces rapid changes in protein expression in the embryos and effects on synaptic transmission lasting until postnatal day 21 (P21). The present study sought to determine whether any of these effects are maintained into adulthood. After prenatal injections of Ro61-8048 the litter was allowed to develop to P60 when some offspring were euthanized and the brains removed for examination. Analysis of protein expression by Western blotting revealed significantly reduced expression of the GluN2A subunit (32%) and the morphogenetic protein sonic hedgehog (31%), with a 29% increase in the expression of doublecortin, a protein associated with neurogenesis. No changes were seen in mRNA abundance using quantitative real-time polymerase chain reaction. Neuronal excitability was normal in the CA1 region of hippocampal slices but paired-pulse stimulation revealed less inhibition at short interpulse intervals. The amount of long-term potentiation was decreased by 49% in treated pups and recovery after low-frequency stimulation was delayed. The results not only strengthen the view that basal, constitutive kynurenine metabolism is involved in normal brain development, but also show that changes induced prenatally can affect the brains of adult offspring and those changes are quite different from those seen previously at weaning (P21). Those changes may be mediated by altered expression of NMDAR subunits and sonic hedgehog.

Possible mediation of quinolinic acid-induced hippocampal damage by reactive oxygen species.

Summary. Several differences exist between quinolinic acid and N-methyl-D-aspartate (NMDA) in the potency and pharmacology of their neurotoxic actions in the brain, suggesting that quinolinic acid may act by mechanisms additional to the activation of NMDA receptors, possibly involving lipid peroxidation. In the present review, studies are considered which have attempted to determine whether free radicals might contribute to the neuronal damage induced by quinolinic acid. Following Injections into the hippocampus of anaesthetised rats, quinolinic acid induced damage is prevented by melatonin, by an action not blocked by the melatonin receptor blocker luzindole. Deprenyl, but not the non-selective monoamine oxidase inhibitor nialamide, also prevent quinolinic acid-induced damage. In vitro, seversl groups have shown that quinolinic acid can induce lipid peroxidation of brain tissue The results suggest that free radical formation contributes significantly to quinolinic acid-induced damage in vivo.

Kynurenine and neopterin levels in patients with rheumatoid arthritis and osteoporosis during drug treatment.

The kynurenine pathway from tryptophan generates compounds which can act on glutamate receptors in peripheral tissues or modulate free radical activity. We have measured the concentrations of several of these compounds in the plasma of patients with rheumatoid arthritis (RA) and osteoporosis (OP) before treatment with drugs and then at monthly intervals for 6 months during treatment. Kynurenine analysis was performed by HPLC. Compared with healthy controls, RA patients showed significantly decreased baseline levels of tryptophan, 3-hydroxykynurenine and 3-hydroxyanthranilic acid and increased levels of kynurenine and xanthurenic acid, while kynurenic acid concentrations were normal. Different results were recorded from patients with OP with only a significant reduction in tryptophan and 3-hydroxyanthranilic acid when compared with healthy controls. During 6 months of treating the RA patients with prednisolone or methotrexate, and the OP patients with raloxifene or etidronate and calcium there were significant therapeutic responses and a significant trend towards a reduction in levels of neopterin in RA patients receiving methotrexate but no changes in the profiles of tryptophan metabolites. The results are consistent with the induction of indoleamine-2,3-dioxygenase (IDO) in both RA and OP but with far greater activation of the pathway in the much more inflammatory condition, i.e. RA. It is concluded that there are changes in the kynurenine pathway, which may modify the activation of tissue glutamate receptors, in RA and OP, but that these are not affected by the drug treatments studied.

The role of kynurenines in the production of neuronal death, and the neuroprotective effect of purines

The kynurenine metabolic pathway from tryptophan accounts for a large proportion of the metabolism of this amino acid in the brain. Although a major route for the generation of the essential co-factor nicotinamide adenine dinucleotide (NAD), two components of the pathway have marked effects on neurons. Quinolinic acid is an agonist at N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors, while kynurenic acid is an antagonist and, thus, a potential neuroprotectant. The levels of quinolinic acid are known to increase substantially following cerebral insults or infection, and has been most clearly implicated in the AIDS-dementia complex. The actions of quinolinic acid and NMDA show subtle differences, however, which suggest other factors contributing to cell damage. In this article we review the evidence that free radicals may be involved in the neurotoxic effects of quinolinic acid and consider the possibility that quinolinic acid might be involved in Alzheimers disease. Finally, adenosine receptor ligands can modulate neuronal damage, supporting the view that they may represent suitable targets for the development of novel neuroprotectant drugs for the treatment of Alzheimers and other neurodegenerative disorders.