Phase 1 study to access safety, tolerability, pharmacokinetics, and pharmacodynamics of kynurenine in healthy volunteers

Abstract 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.

loss of Trp 3 ; tryptophan-2,3-dioxygenase (TDO) in the liver, as well as indoleamine-2,3-dioxygenase 1 (IDO1) and 2 (IDO2) extrahepatic. 4 The principal branch for tryptophan metabolism generates Lkynurenine (LKYN), quinolinic acid (QUIN), and nicotinamide, whereas, the side branches generate kynurenic acid (KYNA) and xanthurenic acid ( Figure 1). 5 The KP gained a considerable scientific interest by discovering that QUIN could excite neurons in the central nervous system (CNS) by acting as an agonist at the N-methyl-D-aspartate (NMDA) receptor, 6 while KYNA is the only endogenous NMDA receptor antagonist. 7 This observation raised the possibility that the KP could be involved in various CNS phenomena, including synaptic plasticity, neurodegeneration, 8 epilepsy, 9 and schizophrenia. 10 Since then, the KP has been implicated in various conditions including cognitive ability, 11,12 migraine, 13 inflammation-associated depressive symptoms, 14 heart failure, 15 stroke, 16 and invasive cancer cells. 17 LKYN is the source for all the other kynurenine metabolites, and it is readily transported across the blood-brain barrier (BBB) by a neutral amino acid carrier. 18 KYNA emerges to be a promising therapeutic drug for several neurological disorders, 1 but its use as a neuroprotective agent is rather restricted due to limited ability to cross the BBB. 18 Interestingly, the generation of KYNA endogenously in brain slices by perfusion with LKYN has a greater inhibitory action than a similar concentration applied exogenously. 9 Furthermore, peripheral treatment with LKYN dose-dependently increases the concentration of KYNA in the brain, offering an opportunity for the treatment of stroke and neurodegenerative disorders. 16,[19][20][21][22][23] However, the safety, tolerability, and physiological effect of LKYN in vivo in human is yet to be elucidated. In the present study, we investigated the pharmacovigilance, pharmacokinetic and pharmacological effects of intravenous LKYN infusion in healthy volunteers. We added a series of in vivo studies in rats for a translational comparison of the vascular pharmacology of LKYN.

| Animal
Experiments using the closed cranial window model were performed on seven male Sprague-Dawley rats (295-340 g; Taconic, Denmark) under approval number 2014-15-0201-00256 from the Danish Animal Experiments Inspectorate. All rats were group housed in Tecniplast 1354G Eurostandard type IV polycarbonate cages (L*W*H: 60*38*20 cm; Brogaarden, Denmark) using a 12-hour light/ dark cycle with lights on at 06.00 am. Individual opaque red polycarbonate shelters (20*11.5*16 and 15*9*9 cm, respectively), together with an aspen biting stick (10*2*2 cm; Tapvei, Estonia) and piece of hemp rope suspended from the cage lid, were provided in each homecage for retreat and enrichment purposes. Bedding consisted of Enviro-Dri nesting material (Brogaarden, Denmark). Standard rat chow (Altromin) and tap water were available ad libitum in the animals' homecage environment. Humidity ranged from 45-65%. All in vivo experiments were performed between 8 am and 4 pm. One unit represents one animal.

| Closed cranial window model
The rats were anaesthetized by an intraperitoneal injection of pentobarbital (65 mg/kg). A rectal thermometer connected to a heating pad was used to maintain the body temperature at 37.5°C. The trachea was cannulated and connected to a ventilator (SAR-830/P Ventilator, CWE Inc.). The femoral vein and artery were cannulated on both sides using BTPE-10, Polyethylene tubing, 0.011 × 0.024 in (0.28 × 0.60 mm) and BTPU-040, Polyurethane tubing, 0.025 × 0.040 in (0.63 × 1.02 mm), and secured with suture. The arteries were used for continuous blood F I G U R E 1 A summary of tryptophan (Trp) metabolism. The kynurenine pathway represents 99% of Trp metabolism. The principal branch (red arrows) for tryptophan metabolism generates L-kynurenine (LKYN), quinolinic acid (QUIN), and nicotinamide pressure measurements and sampling of arterial blood for blood gas analysis. The veins were used for a continuous infusion of anesthesia (pentobarbital 50 mg/ml; 0.15-0.23 ml/hour). A free-floating carotid catheter for drug infusion was placed in the right carotid artery using tissue glue as previously described. 24 The rat was placed in a stereotaxic frame, and the right parietal bone was exposed and thinned to transparency using a dental drill. To avoid overheating of the bone and underlying tissues while drilling, the surface of the bone was repeatedly washed with cold saline. The cranial window was then covered with mineral oil to prevent it from drying, and dural and pial arteries were viewed with an intravital microscope consisting of a Kappa CF8/5 digital camera (Kappa optronics GmbH, Gleichen, Germany) connected to a Leica Model MZ 16 microscope with a 0.5X 10445929 Video Objective (Leica Microsystems, Brønshøj, Denmark). The diameter of the arteries was monitored with a video dimension analyzer (V94, Living Systems Instrumentation Inc., Burlington, VT, USA) and recorded and analyzed together with the mean arterial blood pressure with Perisoft (Version 2.5.5; Perimed AB, Järfälla, Sweden). Before initiation of the experiments, the tracheal cannula was connected to a ventilator, a blood gas analysis was made, and the ventilator was adjusted if needed. Another blood gas analysis was made halfway through the experiment.

| Data treatment and statistical analysis
The effectiveness of the test substances is based on measurements of three parameters: changes in the diameter of dural and pial arteries and changes in mean arterial blood pressure.
The artery diameter was measured in arbitrary units and mean arterial blood pressure in mmHg. Dilation of the arteries and changes in mean arterial blood pressure were calculated as percentage change from the baseline, which is defined as the average of the 60 seconds preceding administration of test substance. Vessel diameter was measured at the peak response occurring 1 to 2 minutes after drug administration. The group size was estimated as a function of the desired effect size (approximately 50% change versus corresponding vehicle treatment) where we assumed a significance level of 5% and a power of 90%. 25 Statistical analyses were made using GraphPad Prism 8 with the mixed effects model for datasets with missing values (a missing value in the second CGRP administration group) were used to analyze the overall effects of treatments followed by Bonferroni's multiple comparisons test. Groups were considered significantly F I G U R E 2 A. Design of the animal experiments. Intracarotid (ic) infusion of saline, CGRP, and vehicle (LKYN) were performed on seven male Sprague-Dawley rats. There was 10 min between each dose of LKYN. A closed cranial window model was used to measure vascular changes in dural and pial arteries. B. Design of human study. Twelve healthy volunteers; six to the first experiment study and six to the second experiment study. In the first experiment study, the participants received a continuous intravenous infusion of 50, 100, and 150 µg/kg LKYN in the mentioned order over 20 min on three days separated by at least 1 week. In the second experiment study, the participants received a continuous intravenous infusion of 0.3, 0.5, 1, and 5 mg/kg LKYN in the mentioned order over 20 min on four days separated by at least 1 week. Blood and urine samples were collected only during the first experiment study different when the p-value was less than 0.05. All values are given as mean ± SEM.

| Human
We recruited a total of twelve healthy volunteers, six to the first experiment and six to the second experiment. None of the participants had previously participated in similar provocation studies. All participants were recruited through the Danish test subject Web site (www.forso gsper son.dk). All participants gave written informed consent before inclusion. The female patients were required to have sufficient contraception (contraceptive pill or intrauterine device/system (IUD/IUS)). Exclusion criteria were any type of primary headache (except episodic tension-type headache no more than one day per month), previous serious somatic or psychiatric diseases, or intake of daily medication including prophylactic migraine treatment, except oral contraceptives. A full medical examination and ECG were performed on the day of recruitment.

| Data availability
The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Experiment 1
In an open-label design, the participants received a continuous intra-

Experiment 2
In an open-label design, the participants received a continuous intra- Headache characteristics, including intensity and accompanying symptoms, vital signs, and adverse events were recorded before and then every 10 minutes until 120 minutes after the beginning of infusion. Headache intensity was recorded on numerical rating scale (NRS) from 0 to 10. The participants were discharged from the hospital after finishing the measurements and asked to complete a headache diary every hour until 24 hours after start of infusion. The diary included headache characteristics and accompanying symptoms, any rescue medication, adverse events, and premonitory symptoms (unusual fatigue, yawning, neck stiffness, mood swings). The quantification of the analytes was based on a multiple reaction monitoring (MRM) coupled to stable isotope dilution. The limit of quantification (LOQ) and coefficient of variation of kynurenine, kynurenic acid and tryptophan were 0.5, 0.1, and 0.1 ng/mL and 5.4%, 2.2%, and 3.4%, respectively. The recovery of kynurenine, kynurenic acid, and tryptophan was 87%, 95%, and 90%, respectively. Linear rage of quantification for kynurenine, kynurenic acid, and tryptophan was 0.05-6500, 50-10000, and 5-15000 ng/mL, respectively. All data were acquired, analyzed, and processed using the Thermo Scientific Xcalibur software (version 4.1, Thermo Scientific, Bremen, Germany).

| Data analysis and statistics
All values are presented as mean values ±SD, except headache scores which are presented as median values. Baseline was defined as T 0 before the start of infusion of each dose. We calculated AUC according to the trapezium rule 31 to obtain a summary.
Difference in AUC for headache intensity scores, HR and mean arterial pressure (MAP), V MCA (0-2 hours), STA diameter and RA diameter between the seven doses was tested using the Friedman

test. All analyses were performed with SPSS Statistics version 19
for Windows, and P-value <0.05 was considered as the level of significance.

| Pial arteries
Intra carotid artery infusion of CGRP caused no increase in pial artery diameter. The effect after the first CGRP infusion was −2.2 ± 4.8% (n = 6) as compared to saline that induced a response of −5.3 ± 3.6% (n = 6) ( Figure 3B). There was no difference between the two administrations of CGRP (p = 0.69; n = 5-6) with the second CGRP response amounting to 4.7 ± 1.1% (n = 5). As found in dural arteries, there was no dilation after infusion of LKYN in cumulative doses (p > 0.05). On the contrary, a significant (p < 0.01) constriction of −6.5 ± 1.8% (n = 6) was observed at 2 mg/kg LKYN when compared to the vehicle response of 1.1 ± 2.5% (n = 6). We believe this is a false-positive outcome as the change in diameter was very small and all other doses of LKYN were highly nonsignificant (p > 0.999) when compared to the vehicle response ( Figure 3B). with responses between 1.8 ± 1.6% at 2 mg/kg and −8.0 ± 6.7% mg/ kg at 5 µg/kg ( Figure 3C).

| Humans
Six healthy volunteers (4 women and 2 men) completed the first experiment ( Figure 2B

| Pharmacokinetic effects
LKYN plasma concentration increased significantly over time after

| Hemodynamic variables
We found no change in HR, mean arterial blood pressure (MAP), V MCA , and STA and RA diameter after LKYN infusion compared with baseline (p > 0.05) (Figure 4).

| DISCUSS ION
This is the first human study with intravenous infusion of LKYN, and the major findings were that systemic LKYN administration was safe, tolerable, and had no physiological effects in either species. To translate animal dose to human equivalent dose (HED), we used the following formula 35 : HED = animaldosex animalkm humankm . Km factor for monkeys is 12, whereas km factor for a 70 kg human is 40. Thus, the dose to be used in humans is 5-10 mg/kg. Since this is the first human study to investigate the physiological effect of intravenous In the present study, no change was found in KYNA plasma concentration. This observation suggests the following: 1 slow metabolism of LKYN, and thus, blood samples for upcoming studies must be The lack of vascular effect in our study is in line with these findings.
Whether higher dose of LKYN might cause vascular changes cannot be ruled out.
Kynurenine metabolites are reduced in chronic migraine patients and cluster headache patients compared with healthy controls. 43,44 Given that L-kynurenine may exert vasodilating effects similar to nitric oxide by increasing cyclic guanosine monophosphate and initiating downstream cascades (known to trigger migraine attacks) 40,[45][46][47] , it has been suggested that the KP may represent a potential therapeutic target in migraine. 13 In the present study, LKYN infusion caused no headache. Although the first LKYN dose 50 µg/kg provoked mild headache, the following doses did not induce headache, and therefore, placebo effect might explain the provoked headache. Collectively, the present data suggest that LKYN is unlikely involved in initiation of migraine. the therapeutic potential of LKYN in these conditions should be explored in future studies in humans.

ACK N OWLED G M ENTS
The authors thank all participating patients and laboratory techni-