Effect of urethral wall injection of replication-defective herpes simplex virus-mediated gene transfer of kynurenine aminotransferase on urethral pressure in spinal cord-injured rats.

AIMS: We determined whether or not replication-defective herpes simplex virus vector-mediated kynurenine aminotransferase II (HSVrd-KAT II) suppressed the tonic activity of the urethral sphincter in spinal cord-injured (SCI) rats. METHODS: Thirty-six adult female Sprague-Dawley rats were used to produce a spinal cord injury model. One week after spinalization, HSVrd-KAT II was injected into the urethral wall of rats and another two groups of SCI rats were treated with saline and HSVrd as controls. Three weeks after viral injection, the urethral pressure profile (UPP), continuous cystometry, and gene expression in the L6-S1 spinal cords were evaluated in all three groups. RESULTS: In the HSVrd-KAT II group, the maximum urethral closure pressure (Pclo.max) and maximum voiding pressure were significantly decreased (23.6-24.9% and 31.6-30.9%, respectively), in addition to an increase in voiding efficiency(48.8-76%), compared with the sham and HSVrd groups. The KAT II protein and mRNA levels were significantly increased in HSV-KAT II group compared with the HSVrd group. CONCLUSION: KAT II gene therapy effectively reduced the urethral pressure, improving detrusor-sphincter dyssynergia (DSD), and detrusor overactivity (DO), probably by blocking the N-methyl-D-aspartate receptor (NMDAr) in the L6-S1 spinal cord. Neurourol. Urodynam. 36:1046-1051, 2017. © 2016 Wiley Periodicals, Inc.

The modulatory effect of anandamide on nitroglycerin-induced sensitization in the trigeminal system of the rat.

BACKGROUND: One of the human and animal models of migraine is the systemic administration of the nitric oxide donor (NO) nitroglycerin (NTG). NO can provoke migraine-like attacks in migraineurs and initiates a self-amplifying process in the trigeminal system, probably leading to central sensitization. Recent studies suggest that the endocannabinoid system is involved in nociceptive signal processing and cannabinoid receptor (CB) agonists are able to attenuate nociception in animal models of pain. AIM: The purpose of the present study was to investigate the modulatory effects of a CB agonist anandamide (AEA) on the NTG-induced expression of transient receptor potential vanilloid type 1 (TRPV1), neuronal nitric oxide synthase (nNOS), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and kynurenine aminotransferase-II (KAT-II) in the upper cervical spinal cord (C1-C2) of the rat, where most of the trigeminal nociceptive afferents convey. METHODS: A half hour before and one hour after NTG (10 mg/kg) or placebo injection, adult male Sprague-Dawley rats (n = 44) were treated with AEA (2 × 5 mg/kg). Four hours after placebo/NTG injection, the animals were perfused and the cervical spinal cords were removed for immunohistochemistry and Western blotting. RESULTS AND CONCLUSION: Our results show that NTG is able to increase TRPV1, nNOS, NF-κB and COX-2 and decrease KAT-II expression in the C1-C2 segments. On the other hand, we have found that AEA modulates the NTG-induced changes, thus it influences the activation and central sensitization process in the trigeminal system, probably via CBs.

Reduction of brain kynurenic acid improves cognitive function.

The elevation of kynurenic acid (KYNA) observed in schizophrenic patients may contribute to core symptoms arising from glutamate hypofunction, including cognitive impairments. Although increased KYNA levels reduce excitatory neurotransmission, KYNA has been proposed to act as an endogenous antagonist at the glycine site of the glutamate NMDA receptor (NMDAR) and as a negative allosteric modulator at the α7 nicotinic acetylcholine receptor. Levels of KYNA are elevated in CSF and the postmortem brain of schizophrenia patients, and these elevated levels of KYNA could contribute to NMDAR hypofunction and the cognitive deficits and negative symptoms associated with this disease. However, the impact of endogenously produced KYNA on brain function and behavior is less well understood due to a paucity of pharmacological tools. To address this issue, we identified PF-04859989, a brain-penetrable inhibitor of kynurenine aminotransferase II (KAT II), the enzyme responsible for most brain KYNA synthesis. In rats, systemic administration of PF-04859989 dose-dependently reduced brain KYNA to as little as 28% of basal levels, and prevented amphetamine- and ketamine-induced disruption of auditory gating and improved performance in a sustained attention task. It also prevented ketamine-induced disruption of performance in a working memory task and a spatial memory task in rodents and nonhuman primates, respectively. Together, these findings support the hypotheses that endogenous KYNA impacts cognitive function and that inhibition of KAT II, and consequent lowering of endogenous brain KYNA levels, improves cognitive performance under conditions considered relevant for schizophrenia.

Efficient gene-targeting in rat embryonic stem cells by CRISPR/Cas and generation of human kynurenine aminotransferase II (KAT II) knock-in rat.

The relative proportion of kynurenine aminotransferase (KAT) I-IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, we had to give up the production because of no recombinant ESCs. Accordingly, we developed a method to improve the efficiency of homologous recombination (HR) in ESCs by the combination with the CRISPR/Cas system. Co-electroporation of Cas9 plasmid, single guide RNA plasmid and hKAT II KI vector increased the number of drug-resistant colonies and greatly enhanced the HR efficiency from 0 to 36 %. All the clones which we obtained showed the same sequence as designed. These recombinant clones resulted in chimeras that transmitted the hKAT II KI allele to their offspring. hKAT II KI rats showed no reduction of KATs mRNA expression and the amount of kynurenic acid was similar between the hKAT II KI rats and the wild type in their brains. These results indicate that the methodology presented in this report can overcome the problem encountered in conventional gene-targeting that prevented production of humanized rats.

Kynurenine pathway dynamics in patients with schizophrenia spectrum disorders across the disease trajectory.

The aim of this study was to evaluate how schizophrenia spectrum disorders (SSD) and applied long-term (5.1 years) antipsychotic (AP) treatment affect the serum levels of tryptophan (Trp) metabolites. A total of 112 adults (54 first-episode psychosis [FEP] patients and 58 control subjects [CSs]) participated in the study. The investigated changes in the metabolite levels appeared against a background of persistent increase in BMI and waist circumference among the patients. Regarding the kynurenine (KYN) pathway, the strongest changes were seen in AP-naïve FEP patients. Trp, KYN, kynurenic acid (KYNA), and anthranilic acid (ANT) levels were significantly reduced in blood samples from patients in the early stage of the disease. Furthermore, 3-OH-kynurenine (3-HK) and quinolinic acid (QUIN) levels were somewhat lower in these patients. Most of these changes in the KYN pathway became weaker with AP treatment. The levels of serotonin and its metabolite 5-HIAA tended to be higher at 5.1 years in patients showing the relation of elevated serotonin turnover to increased BMI and waist circumference. The similar trend was evident for the ratio between xanthurenic acid (XA) and KYNA with strong link to the elevated BMI. Altogether, the present study supports the role of Trp-metabolites in the development of obesity and metabolic syndrome in SSD patients.

Alterations in rat prefrontal cortex kynurenic acid levels are involved in the enduring cognitive dysfunctions induced by tetrahydrocannabinol exposure during the adolescence.

Introduction: Cannabis abuse during adolescence is a risk factor for cognitive impairments in psychiatric disorders later in life. To date, the possible causal relationship between cannabinoids, kynurenic acid (KYNA; i.e., a neuroactive metabolite of tryptophan degradation) and cognition has not been investigated in adolescence. Early exposure to delta 9-tetrahydrocannabinol (THC; i.e., the main psychotropic component of cannabis) causes enduring cognitive deficits, which critically involve impaired glutamatergic function in the prefrontal cortex (PFC). In addition, prenatal cannabis exposure results in enduring increases in PFC KYNA levels. Based on these findings, the effects of chronic THC exposure in rats, during another critical period of neurodevelopment particularly sensitive to perturbation by exogenous stimuli, such as adolescence, have been investigated. Methods: Male Wistar rats were chronically treated with vehicle or ascending intraperitoneal (i.p.) doses of THC starting on postnatal day (PND) 35 until PND 45. In adulthood (PND 75), cognitive assessment (Y-maze) and extracellular KYNA/glutamate levels were measured in the PFC by in vivo microdialysis, before and after a challenge with KYN (5 mg/kg i.p., the biological precursor of KYNA). By using the selective, brain-penetrable KAT II inhibitor PF-04859989, we then examined whether blockade of KYNA neosynthesis prevents the cognitive impairment. Results: Compared to vehicle-treated controls, extracellular basal KYNA levels were higher in the PFC of adult rats chronically exposed to THC in adolescence (p < 0.01). No changes were observed in extracellular glutamate levels. Following a challenge with KYN, extracellular KYNA levels similarly increased in both groups (i.e., vehicle- and THC-treated; p < 0.001 and p < 0.01, respectively). Chronic adolescent THC exposure negatively affected short-term memory (reduced spontaneous alternation), in adult animals (p < 0.001), while PF-04859989 (30 mg/kg i.p.) restored the cognitive impairment (p < 0.05). Discussion: We propose that the observed alterations in PFC KYNA signaling might be involved in the cognitive dysfunction induced by the exposure to THC during the adolescence. In the translational realm, these experiments raise the prospect of prevention of KYNA neosynthesis as a possible novel approach to counteract some of the detrimental long-term effects of adolescence cannabis use.

Heterocyclic Cathinones as Inhibitors of Kynurenine Aminotransferase II-Design, Synthesis, and Evaluation.

Kynurenic acid is a neuroprotective metabolite of tryptophan formed by kynurenine aminotransferase (KAT) catalyzed transformation of kynurenine. However, its high brain levels are associated with cognitive deficit and with the pathophysiology of schizophrenia. Although several classes of KAT inhibitors have been published, the search for new inhibitor chemotypes is crucial for the process of finding suitable clinical candidates. Therefore, we used pharmacophore modeling and molecular docking, which predicted derivatives of heterocyclic amino ketones as new potential irreversible inhibitors of kynurenine aminotransferase II. Thiazole and triazole-based amino ketones were synthesized within a SAR study and their inhibitory activities were evaluated in vitro. The observed activities confirmed our computational model and, moreover, the best compounds showed sub-micromolar inhibitory activity with 2-alaninoyl-5-(4-fluorophenyl)thiazole having IC50 = 0.097 µM.

Galantamine-Memantine combination in the treatment of Alzheimer's disease and beyond.

Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly population worldwide. Despite the major unmet clinical need, no new medications for the treatment of AD have been approved since 2003. Galantamine is an acetylcholinesterase inhibitor that is also a positive allosteric modulator at the α4ß2 and α7nACh receptors. Memantine is an N-methyl-d-aspartate receptor modulator/agonist. Both galantamine and memantine are FDA-approved medications for the treatment of AD. The objective of this review is to highlight the potential of the galantamine-memantine combination to conduct randomized controlled trials (RCTs) in AD. Several studies have shown the combination to be effective. Neurodegenerative diseases involve multiple pathologies; therefore, combination treatment appears to be a rational approach. Although underutilized, the galantamine-memantine combination is the standard of care in the treatment of AD. Positive RCTs with the combination with concurrent improvement in symptoms and biomarkers may lead to FDA approval, which may lead to greater utilization of this combination in clinical practice.

The kynurenine pathway in schizophrenia and bipolar disorder.

The kynurenine pathway of tryptophan degradation generates several neuroactive compounds. Of those, kynurenic acid is an N-methyl-d-aspartate (NMDA) and alpha7 nicotinic receptor antagonist. The kynurenic acid hypothesis of schizophrenia is built upon the fact that kynurenic acid blocks glutamate receptors and is elevated in schizophrenia. Kynurenic acid tightly controls glutamatergic and dopaminergic neurotransmission and elevated brain levels appear related to psychotic symptoms and cognitive impairments. Contributing to enhanced production of kynurenic acid, the expression and enzyme activity of kynurenine 3-monooxygenase (KMO) are reduced in schizophrenia and in bipolar patients with a history of psychosis. The kynurenine pathway is also critically regulated by cytokines, and, indeed, the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 are elevated in schizophrenia and bipolar disorder and stimulate the production of kynurenic acid. One physiological mechanism controlling the activity of the kynurenine pathway originates from the protein sorting nexin 7 (SNX7). This glial signaling pathway initiates a caspase-8-driven activation of IL-1ß that induces tryptophan-2,3-dioxygenase 2 (TDO2), an enzyme in the kynurenine pathway. A recent study shows that a genetic variation resulting in decreased expression of SNX7 is linked to increased central levels of kynurenic acid and ultimately to psychosis and cognitive dysfunction in bipolar disorder. Experimental studies highlight the detrimental effects of increased synthesis of kynurenic acid during sensitive periods of early brain development. Furthermore, experimental studies strongly support inhibition of kynurenine aminotransferase (KAT) II as a novel target and a valuable pharmacological strategy in the treatment of psychosis and for improving cognitive performance relevant for schizophrenia. This article is part of the Special Issue entitled 'The Kynurenine Pathway in Health and Disease'.

Oral administration of a specific kynurenic acid synthesis (KAT II) inhibitor attenuates evoked glutamate release in rat prefrontal cortex.

Cognitive deficits represent core symptoms in schizophrenia (SZ) and predict patient outcome; however, they remain poorly treated by current antipsychotic drugs. Elevated levels of the endogenous alpha7 nicotinic receptor negative allosteric modulator and NMDA receptor antagonist, kynurenic acid (KYNA), are commonly seen in post-mortem tissue and cerebrospinal fluid of patients with SZ. When acutely or chronically elevated in rodents, KYNA produces cognitive deficits similar to those seen in the disease, making down-regulation of KYNA, via inhibition of kynurenine aminotransferase II (KAT II), a potential treatment strategy. We determined, in adult Wistar rats, if the orally available KAT II inhibitor BFF816 a) prevents KYNA elevations in prefrontal cortex (PFC) after a systemic kynurenine injection and b) reverses the kynurenine-induced attenuation of evoked prefrontal glutamate release caused by stimulation of the nucleus accumbens shell (NAcSh). Systemic injection of kynurenine (25 or 100 mg/kg, i.p.) increased KYNA levels in PFC (532% and 1104% of baseline, respectively). NMDA infusions (0.15 µg/0.5 µL) into NAcSh raised prefrontal glutamate levels more than 30-fold above baseline. The two doses of kynurenine reduced evoked glutamate release in PFC (by 43% and 94%, respectively, compared to NMDA alone). Co-administration of BFF816 (30 or 100 mg/kg, p.o.) with kynurenine (25 mg/kg, i.p.) attenuated the neosynthesis of KYNA and dose-dependently restored NMDA-stimulated glutamate release in the PFC (16% and 69%, respectively). The ability to prevent KYNA neosynthesis and to normalize evoked glutamate release in PFC justifies further development of KAT II inhibitors for the treatment of cognitive deficits in SZ.

A systemically-available kynurenine aminotransferase II (KAT II) inhibitor restores nicotine-evoked glutamatergic activity in the cortex of rats.

Kynurenic acid (KYNA) is a tryptophan metabolite that acts in the brain as an endogenous antagonist at multiple receptors, including glutamate and α7 nicotinic acetylcholine receptors. Increased levels of KYNA have been demonstrated in the brain of patients with a range of neurocognitive disorders, including schizophrenia, and are hypothesized to contribute to cognitive symptoms. Reducing KYNA levels by administering inhibitors of enzymes of the kynurenine pathway, particularly kynurenine aminotransferase II (KAT II), has been proposed as a treatment for such cognitive impairments. Here we report that administration of a systemically available KAT II inhibitor, PF-04859989, restores glutamate release events ("transients") evoked by pressure ejections of nicotine into the prefrontal cortex of rats exhibiting elevated KYNA levels. Nicotine-evoked glutamatergic transients can be reliably evoked and recorded after repeated pressure ejections of nicotine over 4-5 h. Systemic administration of l-kynurenine (100 mg/kg; i.p.) significantly increased frontal cortical KYNA levels and greatly attenuated the amplitude of nicotine-evoked glutamatergic transients. Systemic administration of PF-04859989 30 min prior to administration of l-kynurenine, but not when administered 30 min after l-kynurenine, restored glutamatergic transients recorded up to 75 min after the administration of the KAT II inhibitor. Furthermore, the KAT II inhibitor significantly reversed l-kynurenine-induced elevations of brain KYNA levels. The KAT II inhibitor did not affect nicotine-evoked glutamatergic transients in rats not pre-treated with l-kynurenine. Because PF-04859989 restores evoked glutamate signaling it therefore is a promising therapeutic compound for benefiting the cognitive symptoms of schizophrenia and other disorders associated with elevated brain KYNA levels.

Discovery of Brain-Penetrant, Irreversible Kynurenine Aminotransferase II Inhibitors for Schizophrenia.

Kynurenine aminotransferase (KAT) II has been identified as a potential new target for the treatment of cognitive impairment associated with schizophrenia and other psychiatric disorders. Following a high-throughput screen, cyclic hydroxamic acid PF-04859989 was identified as a potent and selective inhibitor of human and rat KAT II. An X-ray crystal structure and (13)C NMR studies of PF-04859989 bound to KAT II have demonstrated that this compound forms a covalent adduct with the enzyme cofactor, pyridoxal phosphate (PLP), in the active site. In vivo pharmacokinetic and efficacy studies in rat show that PF-04859989 is a brain-penetrant, irreversible inhibitor and is capable of reducing brain kynurenic acid by 50% at a dose of 10 mg/kg (sc). Preliminary structure-activity relationship investigations have been completed and have identified the positions on this scaffold best suited to modification for further optimization of this novel series of KAT II inhibitors.