Pharmacokinetics and brain distribution studies of 6-hydroxykynurenic acid and its structural modified compounds.

OBJECTIVES: 6-Hydroxykynurenic acid (6-HKA) is an organic acid component in extracts of Ginkgo biloba leaves and acts as a major contributor to neurorestorative effects, while its oral bioavailability was low. Therefore, using prodrug method to improve the bioavailability and brain content of 6-HKA is significant. METHODS: Three structural modified compounds of 6-HKA were synthesized, and ultra performance liquid chromatography-tandem mass spectrometry methods for quantification of these structural modified compounds in rat plasma and rat brain homogenate were established and comprehensively validated. The methods were effectively applied to investigate the effects of structural modification on apparent permeability coefficients in cells, the pharmacokinetics and the brain distribution in rats. KEY FINDINGS: The results illustrated that esterification can greatly improve the apparent permeability coefficient and bioavailability of 6-HKA. Comparing with direct oral administration of 6-HKA, the bioavailability of isopropyl ester was greatly improved (from 3.96 ± 1.45% to 41.8 ± 15.3%), and the contents of 6-HKA in rat brains (49.7 ± 9.2 ng/g brain) were significantly higher after oral administration. CONCLUSIONS: The bioavailability and the brain content of 6-HKA can be improved by the prodrug method. Among three structural modified compounds, isopropyl-esterified 6-HKA was the most promising treatment.

Chronic dietary supplementation with kynurenic acid, a neuroactive metabolite of tryptophan, decreased body weight without negative influence on densitometry and mandibular bone biomechanical endurance in young rats.

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan. KYNA naturally occurs in breast milk and its content increases with lactation, indicating the role of neonatal nutrition in general growth with long-term health effects. KYNA is also an antagonist of ionotropic glutamate receptors expressed in bone cells. The aim of this study was to establish the effects of chronic KYNA supplementation on bone homeostasis in young rats, using mandible as a model bone. Female and male newborn Wistar rats were divided into control and KYNA-administered groups until 60 days of age (25x101 mg/L or 25x102 mg/L in drinking water). Hemimandibles were subjected to densitometry, computed tomography analysis and mechanical testing. Rats supplemented with KYNA at both doses showed a decrease in body weight. There were no effects of KYNA administration and mandible histomorphometry. In males, a significant quadratic effect (P < 0.001) was observed in the densitometry of the hemimandible, where BMD increased in the group supplemented with 2.5x101 mg/L of KYNA. Analysis of mechanical tests data showed that when fracture forces were corrected for bone geometry and rats body weight the improvement of bone material properties was observed in male and female rats supplemented with lower dose of KYNA. This study showed that chronic supplementation with KYNA may limit weight gain in the young, without adversely affecting the development of the skeleton.

Kynurenic acid as the neglected ingredient of commercial baby formulas.

The global increase in resorting to artificial nutritional formulas replacing breastfeeding has been identified among the complex causes of the obesity epidemic in infants and children. One of the factors recently recognized to influence metabolism and weight gain is kynurenic acid (KYNA), an agonist of G protein-coupled receptor (GPR35). Therefore the aim of the study was to determine the concentration of KYNA in artificial nutritional formulas in comparison with its level in human breast milk and to evaluate developmental changes in rats exposed to KYNA enriched diet during the time of breastfeeding. KYNA levels were measured in milk samples from 25 heathy breast-feeding women during the first six months after labor and were compared with 21 time-adjusted nutritional formulas. Animal experiments were performed on male Wistar rats. KYNA was administered in drinking water. The content of KYNA in human milk increases more than 13 times during the time of breastfeeding while its level is significantly lower in artificial formulas. KYNA was detected in breast milk of rats and it was found that the supplementation of rat maternal diet with KYNA in drinking water results in its increase in maternal milk. By means of the immunoblotting technique, GPR35 was evidenced in the mucosa of the jejunum of 1-day-old rats and distinct morphological changes in the jejunum of 21-day-old rats fed by mothers exposed to water supplemented with KYNA were found. A significant reduction of body weight gain of rats postnatally exposed to KYNA supplementation without changes in total body surface and bone mineral density was observed. The rat offspring fed with breast milk with artificially enhanced KYNA content demonstrated a lower mass gain during the first 21 days of life, which indicates that KYNA may act as an anti-obesogen. Further studies are, therefore, warranted to investigate the mechanisms regulating KYNA secretion via breast milk, as well as the influence of breast milk KYNA on mass gain. In the context of lifelong obesity observed worldwide in children fed artificially, our results imply that insufficient amount of KYNA in baby formulas could be considered as one of the factors associated with increased mass gain.

Fate and distribution of kynurenic acid administered as beverage.

BACKGROUND: Kynurenic acid (KYNA) is a biologically active metabolite of tryptophan exerting action on several receptors located in the brain and periphery. KYNA can be synthesized endogenously or supplied in the diet. It was documented that KYNA is present in various types of food. However, its presence in beverages was not yet investigated. Here, we measured content of KYNA in tea and coffee as well as analyzed distribution and fate of intragastrically administered labelled KYNA in mice. METHODS: 16 and 13 studied samples of tea and coffee, respectively were of commercial origin. Tea and coffee infusions were prepared according to the producers' guidelines. KYNA content in beverages was measured by means of HPLC detection. Adult male mice were used for analysis of fate of intragastrically administered labelled KYNA and collected samples were analyzed using liquid scintillation counter. RESULTS: KYNA was identified in all studied beverages. Amounts of KYNA found in various types of beverages differed significantly. The highest content of KYNA in tea and coffee was 8.7 µg/100 ml and 0.63 µg/100 ml, respectively. It was found that KYNA administered intragastrically as a liquid is absorbed from the digestive system and readily excreted in urine. The atypical kinetics of KYNA distribution were found in intestinal content of cecum, where it appeared later and persisted longer than in other tissues. CONCLUSIONS: Our data show that tea and coffee intake may contribute to KYNA content in the human organism. The distribution pattern of KYNA delivered as a liquid suggests that it either directly affects digestive system's functioning and intestinal microbiome composition, or participates in the whole body pool of KYNA.

Topical dura mater application of CFA induces enhanced expression of c-fos and glutamate in rat trigeminal nucleus caudalis: attenuated by KYNA derivate (SZR72).

BACKGROUND: Migraine is a debilitating neurological disorder where trigeminovascular activation plays a key role. We have previously reported that local application of Complete Freund's Adjuvant (CFA) onto the dura mater caused activation in rat trigeminal ganglion (TG) which was abolished by a systemic administration of kynurenic acid (KYNA) derivate (SZR72). Here, we hypothesize that this activation may extend to the trigeminal complex in the brainstem and is attenuated by treatment with SZR72. METHODS: Activation in the trigeminal nucleus caudalis (TNC) and the trigeminal tract (Sp5) was achieved by application of CFA onto the dural parietal surface. SZR72 was given intraperitoneally (i.p.), one dose prior CFA deposition and repeatedly daily for 7 days. Immunohistochemical studies were performed for mapping glutamate, c-fos, PACAP, substance P, IL-6, IL-1ß and TNFα in the TNC/Sp5 and other regions of the brainstem and at the C1-C2 regions of the spinal cord. RESULTS: We found that CFA increased c-fos and glutamate immunoreactivity in TNC and C1-C2 neurons. This effect was mitigated by SZR72. PACAP positive fibers were detected in the fasciculus cuneatus and gracilis. Substance P, TNFα, IL-6 and IL-1ß immunopositivity were detected in fibers of Sp5 and neither of these molecules showed any change in immunoreactivity following CFA administration. CONCLUSION: This is the first study demonstrating that dural application of CFA increases the expression of c-fos and glutamate in TNC neurons. Treatment with the KYNA analogue prevented this expression.

Perifornical hypothalamic pathway to the adrenal gland: Role for glutamatergic transmission in the glucose counter-regulatory response.

Adrenaline is an important counter-regulatory hormone that helps restore glucose homeostasis during hypoglycaemia. However, the neurocircuitry that connects the brain glucose sensors and the adrenal sympathetic outflow to the chromaffin cells is poorly understood. We used electrical microstimulation of the perifornical hypothalamus (PeH) and the rostral ventrolateral medulla (RVLM) combined with adrenal sympathetic nerve activity (ASNA) recording to examine the relationship between the RVLM, the PeH and ASNA. In urethane-anaesthetised male Sprague-Dawley rats, intermittent single pulse electrical stimulation of the rostroventrolateral medulla (RVLM) elicited an evoked ASNA response that consisted of early (60±3ms) and late peaks (135±4ms) of preganglionic and postganglionic activity. In contrast, RVLM stimulation evoked responses in lumbar sympathetic nerve activity that were almost entirely postganglionic. PeH stimulation also produced an evoked excitatory response consisting of both preganglionic and postganglionic excitatory peaks in ASNA. Both peaks in ASNA following RVLM stimulation were reduced by intrathecal kynurenic acid (KYN) injection. In addition, the ASNA response to systemic neuroglucoprivation induced by 2-deoxy-d-glucose was abolished by bilateral microinjection of KYN into the RVLM. This suggests that a glutamatergic pathway from the perifornical hypothalamus (PeH) relays in the RVLM to activate the adrenal SPN and so modulate ASNA. The main findings of this study are that (i) adrenal premotor neurons in the RVLM may be, at least in part, glutamatergic and (ii) that the input to these neurons that is activated during neuroglucoprivation is also glutamatergic.

Time-course of kynurenic acid concentration in mouse serum following the administration of a novel kynurenic acid analog.

The changes in concentration of kynurenic acid (KYNA) in different biological samples are of great interest in the pathomechanism and medication of several disorders, and especially those affecting the nervous system. Besides the recent pharmaceutical advances targeting the kynurenine pathway, there is a constant need for further drug development through the synthesis of novel analogs. Reliable analytical methods should be set up to monitor the metabolism and effects of these analogs in both preclinical experiments and human studies. Following a sample preparation procedure based on protein precipitation, new high-performance liquid chromatographic methods with fluorescence and mass spectrometric detection were developed for the determination of KYNA and a novel KYNA analog (N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride; KYNA amide) in mouse serum samples. The analytical parameters obtained in the validation procedure suggest that the developed method with mass spectrometric detection is simple, fast, accurate and suitable for the measurement of KYNA and its analogs. The results reveal the good in vivo stability of the novel KYNA amide.

Nucleus raphe pallidus participates in midbrain-medullary cardiovascular sympathoinhibition during electroacupuncture.

We have shown that electroacupuncture (EA) inhibits sympathoexcitatory rostral ventrolateral medulla (rVLM) neurons and reflex responses following activation of a long-loop pathway in the arcuate nucleus and ventrolateral periaqueductal gray (vlPAG). Additionally, EA at P 5-6 acupoints (overlying the median nerve) activates serotonin-containing neurons in the nucleus raphé pallidus (NRP), which, in turn, inhibit rVLM neurons. Although direct projections from the vlPAG to the rVLM exist, it is uncertain whether an indirect pathway through the NRP serves an important role in vlPAG-rVLM cardiovascular modulation. Therefore, the splanchnic nerve (SN) was stimulated to induce cardiovascular sympathoexcitatory reflexes, and EA was applied at P 5-6 acupoints in α-chloralose-anesthetized cats. A single-barreled recording electrode was inserted into the NRP or rVLM. Microinjection of DL-homocysteic acid (DLH) into the vlPAG increased the NRP neuronal response to SN stimulation (5 ± 1 to 12 ± 2 spikes/30 stim). Likewise, EA at P 5-6 for 30 min increased the NRP response to SN stimulation (3 ± 1 to 10 ± 2 spikes/30 stim), an effect that could be blocked by microinjection of kynurenic acid (KYN) into the caudal vlPAG. Furthermore, the reflex increase in blood pressure induced by application of bradykinin to the gallbladder and the rVLM cardiovascular presympathetic neuronal response to SN stimulation was inhibited by injection of DLH into the vlPAG, a response that was reversed by injection of KYN into the NRP. These results indicate that EA activates the vlPAG, which excites the NRP to, in turn, inhibit rVLM presympathetic neurons and reflex cardiovascular sympathoexcitatory responses.

NMDA receptors mediate feeding elicited by neuropeptide Y in the lateral and perifornical hypothalamus.

Neuropeptide Y (NPY) and N-methyl-d-aspartate (NMDA) receptors in the lateral (LH) and perifornical hypothalamus (PFH) are believed to be involved in the stimulation of feeding behavior. To investigate the possibility that neurons with these receptors interact to stimulate eating, the NMDA receptor antagonists d-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) or 7-chlorokynurenic acid (7-CK) were injected into the LH or PFH of satiated rats 5 min prior to NPY in the same site and subsequent food intake was measured 1, 2, and 4 h postinjection. The injection of NPY (78 pmol/0.3 microl aCSF) in the PFH produced an average food intake of 9.7 g in 4 h, compared to the intake of 1.3 g after the artificial cerebrospinal fluid (aCSF) vehicle. D-AP5 (1, 10, or 20 nmol/0.3 microl aCSF) pretreatment suppressed NPY-induced eating, with the 20 nmol dose of D-AP5 producing up to an 80% suppression of elicited food intake down to 1.9 g in 4 h. Similar effects were produced with the LH as the injection site. Illustrating the specificity of the NMDA receptor antagonist's suppression of NPY-elicited feeding, D-AP5 suppressed NMDA-elicited feeding but did not affect the eating response induced by kainic acid. Consistent with the effects of D-AP5, the NMDA receptor antagonist 7-CK (40 nmol/0.3 microl dimethyl sulfoxide, DMSO) suppressed feeding elicited by NPY in the LH by 78%. Collectively, the findings suggest that the feeding elicited by NPY is dependent upon the activation of the NMDA receptors in the LH and PFH.

Spontaneous retinal activity is tonic and does not drive tectal activity during activity-dependent refinement in regeneration.

During development, waves of activity periodically spread across retina to produce correlated activity that is thought to drive activity-dependent ordering in optic fibers. We asked whether similar waves of activity are produced in the retina of adult goldfish during activity-dependent refinement by regenerating optic fibers. Dual-electrode recordings of spontaneous activity were made at different distances across retina but revealed no evidence of retinal waves in normal retina or during regeneration. Retinal activity was tonic and lacked the episodic bursting associated with waves. Cross-correlation analysis showed that the correlated activity that was normally restricted to near neighbors (typically seen across 100-200 microm and absent at >500 microm) was not altered during regeneration. The only change associated with regeneration was a twofold reduction in ganglion cell firing rates. Because spontaneous retinal activity is known to be sufficient to generate refinement during regeneration in goldfish, we examined its effect on tectal activity. In normal fish, acutely eliminating retinal activity with TTX rapidly reduced tectal unit activity by >90%. Surprisingly, during refinement at 4-6 weeks, eliminating retinal activity had no detectable effect on tectal activity. Similar results were obtained in recordings from torus longitudinalis. After refinement at 3 months, tectal activity was again highly dependent on ongoing retinal activity. We conclude that spontaneous retinal activity drives tectal cells in normal fish and after regeneration but not during activity-dependent refinement. The implications of these results for the role of presynaptic activity in refinement are considered.

NMDA receptor involvement in neuroplastic changes induced by neonatal capsaicin treatment in trigeminal nociceptive neurons.

NMDA receptor involvement in neuroplastic changes induced by neonatal capsaicin treatment in trigeminal nociceptive neurons. J. Neurophysiol. 78: 2799-2803, 1997. This study examines whether 1) the neonatal loss of C-fiber afferents results in neuroplastic changes in the mechanoreceptive field (RF) properties and spontaneous activity of nociceptive neurons in trigeminal subnucleus caudalis (medullary dorsal horn) of adult rats, and that 2) N-methyl--aspartic acid (NMDA) receptor mechanisms are involved in these neuroplastic changes. Compared with vehicle-treated (i.e., control, CON) rats, capsaicin-treated (CAP) rats showed a marked increase in neuronal spontaneous activity and RF size per se, but these neuroplastic changes could be significantly reduced by MK-801 (1 mg/kg, iv), a noncompetitive NMDA receptor antagonist; RF size and spontaneous activity remained unchanged in CON rats after MK-801 administration and in CAP rats after vehicle (saline, iv). Administration of 7-chlorokynurenic acid intrathecally (5 microgram/10 microliter), an antagonist of strychnine-insensitive glycine bindin sites on the NMDA receptor, also significantly reduced neuronal RF size and spontaneous activity in CAP rats, but not in CON rats. These data provide evidence that C-fiber afferents play a role in shaping the properties of nociceptive neurons and that the neuroplastic changes involve NMDA receptor mechanisms.

Role of rostral ventrolateral medulla in centrally mediated pressor responses.

The region of the rostral ventrolateral medulla (RVLM) plays an important role in central nervous system regulation of cardiovascular function. The initial purpose of these studies was to determine whether synaptic activation of excitatory amino acid (EAA) receptors in the RVLM might mediate central pressor responses. Blockade of EAA receptors in the RVLM with kynurenic acid abolished pressor responses evoked by stimulation of sciatic nerve afferents but had no effect on increases in arterial pressure produced by stimulation of hypothalamic sites. To determine whether synaptic transmission in the RVLM, independent of EAA receptor activation, was a prerequisite for the production of hypothalamic pressor responses, axonal conduction and/or synaptic transmission were pharmacologically interrupted in the RVLM. Blockade of synaptic transmission with muscimol or kainic acid attenuated, but did not eliminate, hypothalamic pressor responses. Concurrent blockade of synaptic and axonal transmission in the RVLM with lidocaine produced the greatest reduction of hypothalamic pressor responses. Collectively, these results suggest that central pressor responses are not uniformly mediated by synaptic activation of neurons within the RVLM. Instead, a combination of synaptic transmission and axonal conduction within and possibly outside the region of the RVLM may be required for the production of many centrally mediated pressor responses.