Accepted Manuscript

Characterization of the effects of L-4-chlorokynurenine on nociception in rodents

Tony L. Yaksh, Ph.D, Robert Schwarcz, Ph.D, H. Ralph Snodgrass, Ph.D

PII: S1526-5900(17)30552-7
DOI: 10.1016/j.jpain.2017.03.014
Reference: YJPAI 3409

To appear in: Journal of Pain

Received Date: 6 January 2017

Revised Date: 16 March 2017
Accepted Date: 30 March 2017

Please cite this article as: Yaksh TL, Schwarcz R, Snodgrass HR, Characterization of the effects of L-4-
chlorokynurenine on nociception in rodents, Journal of Pain (2017), doi: 10.1016/j.jpain.2017.03.014.

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Characterization of the effects of L-4-chlorokynurenine on nociception in rodents

Running title: Analgesic actions of glycine B receptor antagonist

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Tony L. Yaksh, Ph.D. 1,4, Robert Schwarcz, Ph.D. 2, H. Ralph Snodgrass, Ph.D. 3

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Department of Anesthesiology, University of California, San Diego, La Jolla CA 92093, USA

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Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore,
MD 21228
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VistaGen Therapeutics, Inc., 343 Allerton Ave, South San Francisco, CA 94080

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Corresponding author: Tony L. Yaksh, Ph.D., Department of Anesthesiology, University of
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California, San Diego, La Jolla CA 92093, tyaksh@ucsd.edu; TEL; 619-543-3597; FAX: 619-
543-6070
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Disclosures.
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Dr. Yaksh was supported by VistaGen Therapeutics and Dr. Schwarcz was supported by
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VistaGen Therapeutics and NIH grants R43DA018515, U01NS04821401A1, R43NS047808

and R21MH099345. Dr. Schwarcz has an equity position in VistaGen Therapeutics. Dr.
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Snodgrass is an employee of, and has an equity position in, VistaGen Therapeutics, which has

commercial rights to L-4-chlorokynurenine (AV-101). Dr. Yaksh performed the research under
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UCSD Laboratory Service Agreement with VistaGen and has no other financial interests.
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ABSTRACT

Upon systemic administration in rats, the prodrug L-4-chlorokynurenine (4-Cl-KYN) (AV-101) is

rapidly absorbed, actively transported across the blood-brain barrier, and converted in

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astrocytes to 7-chlorokynurenic acid (7-Cl-KYNA), a potent and specific antagonist of the GlyB

co-agonist site of the NMDA receptor. We examined the effects of 4-Cl-KYN in several rat

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models of hyperalgesia and allodynia and determined the concentrations of 4-Cl-KYN and

newly produced 7-Cl-KYNA in serum, brain and spinal cord. Adult male rats were given 4-Cl-

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KYN (56, 167, 500 mg/kg), the NMDA receptor antagonist MK-801 (0.1, 0.3, 1.0 mg/kg) or

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gabapentin (33, 100, 300 mg/kg) intraperitoneally (IP), and were then examined on rotarod,
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intraplantar formalin-evoked flinching, thermal escape in the normal and carrageenan-inflamed

paw, and allodynia following sciatic nerve ligation. Our conclusions show that after systemic
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delivery, the highest two doses (167 and 500 mg/kg) of 4-Cl-KYN yielded brain concentrations

of 7-Cl-KYNA exceeding its IC50 at the GlyB site and resulted in dose-dependent anti-
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hyperalgesia in the four models of facilitated processing associated with tissue inflammation
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and nerve injury. Based on the relative dose requirements for analgesic actions and side effect

profiles from these experiments, 4-Cl-KYN is predicted to have anti-hyperalgesic efficacy and a
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therapeutic ratio equal to gabapentin and superior to MK-801.

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Perspective
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These studies demonstrate that systemic administration of the prodrug 4-Cl-KYN produces

high CNS levels of 7-Cl-KYNA, a potent and highly selective antagonist of the NMDA receptor.

Compared to other drugs tested, 4-Cl-KYN has robust anti-nociceptive effects with a better

side effect profile, highlighting its potential for treating hyperpathic pain states.

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Key Words. 7-chlorokynurenic acid, L-4-chlorokynurenine, glycine site antagonist,

neuropathic pain, inflammatory pain, NMDA receptor

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INTRODUCTION
Tissue and nerve injury leads to the development of facilitated pain states in which the afferent

traffic generated by otherwise innocuous or mildly aversive thermal and mechanical stimuli

evokes a behavioral response consistent with a more intense stimulus24,60. A variety of

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convergent preclinical studies have emphasized the importance of the role played by the spinal

NMDA/glutamate-gated channel in the development of this facilitated state9,59. Thus, NMDA

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receptor antagonists reduce electrophysiological15-17 and behavioral indices of the facilitated

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states initiated by repetitive small afferent activation (windup) and surrogate paradigms of

ongoing activation such as the intraplantar formalin-evoked flinching model12,22,43,64,

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inflammation-evoked hyperalgesia13,19,48, and nerve injury-evoked hyperalgesia7,65,68. Notably,
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NMDA receptor function is regulated by several distinct mechanisms, often including the

strychnine-insensitive glycine (GlyB) site43,56. This co-agonist site recognizes the endogenous
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amino acids glycine and D-serine as agonists and is required for channel activation38. GlyB
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antagonists have comparable effects in established models of pain, such as wind-up18 and

hyperalgesia-induced by intraplantar formalin, tissue and nerve injury5,37,39,58.

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7-Chlorokynurenic acid (7-Cl-KYNA) is a potent and specific GlyB antagonist28 and has been
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shown to have potent antinociceptive actions after neuraxial delivery8,18,32,48,54. Interestingly,

though effects after systemic delivery have been reported36,69, 7-Cl-KYNA has poor central
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bioavailability3,27,47. Its bioprecursor L-4-chlorokynurenine (4-Cl-KYN), in contrast, has no

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activity at the GlyB site on its own52, but is actively transported into the brain by the large

neutral amino acid transporter after systemic administration27. In the brain, kynurenine

aminotransferases in astrocytes convert 4-Cl-KYN to 7-Cl-KYNA, which is then readily

released and can act on neuronal GlyB sites30,52,61. 4-CI-KYN can therefore serve as a prodrug

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to therapeutically address facilitated pain states in which central NMDA receptor activation is

believed to play a contributory role23,60,63.

This study systematically examined the analgesic and behavioral profile of systemically

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delivered 4-Cl-KYN and two standard compounds used in the field (MK-801 and gabapentin)

on the rotarod, the carrageenan-evoked thermal hyperalgesia, the formalin test, and the Chung

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model of neuropathy. Dose-response curves were generated and compared with the

behavioral profiles. We further assessed the central levels of 4-Cl-KYN and 7-Cl-KYNA in

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serum, brain and spinal cord.

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MATERIALS AND METHODS

Animals

Adult male (225-250 g) Holtzman (Rotarod, Carrageenan model, Formalin model) and

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Sprague-Dawley (Chung model) (Harlan Industries, Indianapolis, IN) rats were employed.

Animals were maintained in an AAALAC-approved vivarium and individually housed with

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absorbent bedding material with ad libitum access to rat chow and water, and maintained on a

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12/12 day-night cycle. All animal studies were carried out per protocols approved by the

Institutional Animal Care and Use Committee of the University of California, San Diego and

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were in compliance with the USDA Animal Welfare Act (USDA, Title 9 Code of Federal
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Regulations Part 3, Federal Register 15 February 1991).

Drugs
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Drugs employed included enantiomerically pure L-4-Cl-KYN (AV-101, VistaGen Therapeutics,

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Inc.), MK-801 hydrogen maleate (Sigma-Aldrich, St. Louis, MO), and gabapentin (Sigma-
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Aldrich). Unless otherwise stated, all agents were prepared from stock solutions in 0.9% NaCl.

To prepare a stock solution of 4-Cl-KYN (50 mg/mL), the powder was dissolved in a small
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volume of 1 M NaOH and titrated to pH 7.4 with 0.1 M phosphate-buffered saline (PBS).

Several doses of 4-Cl-KYN (19, 56, 167 and 500 mg/kg), MK-801 (0.1, 0.3, 1 and 3 mg/kg) and
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gabapentin (3, 33, 100 and 300 mg/kg) were tested. Test compounds or 0.9% NaCl were
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administered intraperitoneally (IP) at 0.1 ml/100 g body weight.

Behavioral assessment

General observations. General behavioral assessments were made during each 4 hr interval of

post-injection observation and specifically included: i) spontaneous vocalization, ii) biting and

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chewing of body surface and hind paws, iii) loss of hind limb placing and stepping reflex (hind

paw lifting and planting of paw evoked by dragging of the dorsum of the paw over the edge of

a table), iv) loss of hind limb weight bearing (evoked by lifting the animal by the thorax and

forcing bipedal weight bearing by the hind limbs), and v) loss of righting reflex (tested by

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placing the animal supine). Assessments were noted as “present” or “absent”. Adverse

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assessments were represented by noting the presence of vocalization and biting/chewing and

the absence of placing, stepping and a righting response.

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Motor performance. Motor function was assessed using a rotating drum device (Economex,

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Columbus Instruments, Columbus, OH), and animals were tested one at a time. Prior to
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initiation of the study, rats went through a training period of two days. On the day of the

experiment, animals were tested for baseline performance and again 15, 30, 60, 120 and 240
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min after drug injection. Baseline time range for each rat was a minimum of 60 sec, with a

cutoff of 180 sec. Before the trial/test run, rats were placed inside a plexiglass housing for 15
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to 20 min to acclimate. The rotarod timer/wheel was started at a 4 RPM setting. The rats were
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then placed on the wheel, with the tail facing outside of the compartment, and allowed to run

for a few seconds. The acceleration button for 1 RPM/sec was then activated, and the time
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when the animal fell from the rotarod was recorded (“response”). In between trial runs, rats

were returned to the plexiglass houses. Data are presented as the mean and standard error of
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the raw scores (seconds on rotarod up to 180 sec) plotted vs. time after drug delivery (effect-
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time curves). For statistical analysis, a drug effect index (DEI) was calculated for each animal.

The rotarod DEI is the area under the effect-time curve (sec*min: 0-4 hrs), normalized by

dividing by the rotarod response (in sec), observed before drug delivery at time 0. The smaller

the DEI, the greater the negative impact of a given drug dose on rotarod performance. The DEI

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was plotted for each drug dose and vehicle group, and plotted vs. dose. A best fit regression

line was calculated for each drug.

Inflammatory hyperalgesia. Thermal escape latency for each hind paw was assessed in rats

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prepared with a unilateral hind paw inflammation initiated by the intraplantar injection of

carrageenan (2%/100 µL). To assess hindpaw thermal escape latencies, a modified

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Hargreaves box was used. This system focuses a light beam onto the plantar surface of the

paw through a glass surface upon which the rat stands. In the absence of stimulation, surface

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temperature was maintained at 30˚C. Withdrawal of the paw to the stimulus was defined as

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the response. Lack of response within 20 sec was cause to terminate the test and to assign a
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score of “20”. Drugs were delivered 30 min prior to the carrageenan injection. Data were

presented as the mean and standard error of the mean (SEM) of the raw scores (response
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latency in sec), plotted vs. time after drug delivery (effect-time curves). For statistical analysis,

a DEI was calculated for each animal. For the carrageenan model, the calculated DEI was the
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area under the normalized effect-time curve (0-4 hrs) where the normalized effect was
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calculated as follows: Post-drug response latency – Pre-response latency/Pre-response

latency time x 100. The smaller the thermal escape DEI the less the hyperalgesic state. DEIs
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were plotted vs. dose for each drug dose and vehicle group. A best fit regression line was

calculated for each drug.

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Formalin-evoked flinching. In the formalin model, a small metal band (0.5 g) was loosely

placed around the rat’s right hind paw 30 min prior to dosing. The animal was placed in a

cylindrical Plexiglas chamber for adaptation (minimum of 30 min). Fifty µl of 5% formalin was

then injected into the dorsal surface of the right hind paw. Subsequently, the rat was placed

into the chamber of the automated formalin apparatus where movement of the formalin-

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injected paw was monitored, and the number of paw flinches was tallied every minute over the

next 60 min64. Upon completion of the test, the animal was removed from the chamber and

euthanized. The flinches per minute were plotted vs. time after the formalin injection. For

statistical analysis, the data for each animal were expressed as a DEI [mean and SEM of the

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cumulative flinching for each rat for phase 1 (1-9 min) and phase 2 (10-60 min)]. The DEI for

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each drug treatment and vehicle group was plotted vs. dose, and a best fit regression line was

calculated for each drug.

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Chung model of neuropathy: For creating the neuropathic preparation, the nerve ligation

originally described by Kim and Chung29 was performed to induce an allodynic state. Briefly,

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the left L-5 and L-6 spinal nerves were isolated adjacent to the vertebral column under

isoflurane anesthesia (1.0-1.2% in air) and ligated with a 6-0 silk suture distal to the dorsal root
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ganglion. At recovery, each animal received a single subcutaneous (SC) dose of an anti-

inflammatory agent (Carprofen; 5 mg/kg) and lactated Ringer’s solution (2 mL/100 g, s.c.).
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Rats were allowed a minimum 7-day postoperative recovery period. They were then examined
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for baseline tactile threshold values (measured as force, in grams, at 50% threshold). They

were then dosed with one of three concentrations of either the test article, positive controls
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(MK-801, gabapentin), or vehicle (0.9% NaCl). All drugs and doses were given in similar

volumes and tested at several time points after dosing. To assess tactile thresholds, rats were
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placed in a clear plastic, wire mesh-bottomed cage, divided into individual compartments. Rats
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were allowed to accommodate, and baseline thresholds were assessed prior to drug

treatment. To determine the 50% mechanical threshold for paw withdrawal, von Frey hairs

were applied to the plantar mid-hind paw, avoiding the tori (footpads). The eight von Frey hairs

used were designated by [log (10 * force required to bend hair, mg)] and a range from 0.4-15.1

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g (#'s 3.61--5.18). Each hair was pressed perpendicularly against the paw with sufficient force

to cause slight bending, and held for approximately 6-8 sec. A positive response was noted if

the paw was sharply withdrawn. Flinching immediately upon removal of the hair was also

considered a positive response. Absence of a response ("-") was cause to present the next

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consecutive stronger stimulus; a positive response ("+") was cause to present the next weaker

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stimulus. Stimuli were presented successively until either six data points were collected, or the

maximum or minimum stimulus was reached. If a minimum stimulus was reached and positive

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responses still occurred, the threshold was assigned an arbitrary minimum value of 0.25 g; if a

maximum stimulus was presented and no response occurred, a maximum threshold value of

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15 g was assigned. If a change in response occurred, either "-" to "+' or "+" to "-", causing a
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change in the direction of stimulus presentation from descending to ascending or vice versa,

four additional data points were collected after the change. The resulting pattern of responses
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was tabulated, and the 50% response threshold was computed using the formula:
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log (threshold, mg x 10) = Xf + k_; where:

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Xf = value of the last von Frey hair applied;

k = correction factor based on pattern of responses (from calibration table)
_ = mean distance in log units between stimuli.
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The 15.1-g hair was selected as the upper limit for testing6. Data were presented as the mean
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and SEM of the raw scores (response threshold in grams) plotted vs. time. For statistical

analysis, the data were expressed in comparison to control as a Drug Effect Index (DEI): Post-

drug response threshold – Pre-response threshold / Pre-response threshold time x 100 was

calculated. The DEI, i.e. the area under the time-effect curve over the observation interval (0-4

hrs), was calculated for each animal.

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The smaller the nerve injury DEI, the less the allodynic state. The DEIs were plotted vs. dose

for each drug dose and vehicle group, and a best fit regression line was calculated for each

drug.

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Drug levels

Animals. Rats were injected IP with one of three 4-Cl-KYN doses (19, 167 or 500 mg/kg). At

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0.5, 1.5 or 4 hrs post-injection, rats were deeply anesthetized under isoflurane until the righting

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reflex was lost. Blood samples (tail clip), brain and spinal cord (hydraulic extrusion) tissue

samples were collected and stored at –70°C prior to analysis.

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Drug Analysis. For 4-Cl-KYN measurement, tissue was thawed and sonicated in distilled water
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(1:10, w/v). A 50 µl aliquot of the homogenate was further diluted with water (1:1, v/v) and

acidified with 25 µl of 6% perchloric acid (PCA). After centrifugation (5 min, 12,000 x g), an
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aliquot of the supernatant was diluted (1:1, v/v) with high pressure liquid chromatography
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(HPLC) mobile phase, i.e. 0.1 M ammonium acetate and 18% acetonitrile (pH 4.65). To
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measure the blood content of 4-Cl-KYN, red cells were removed by centrifugation, and a 20 µl

aliquot of the supernatant plasma was diluted with water (1:5, v/v) and deproteinated with 25 µl
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of 6% PCA. After centrifugation, an aliquot of the supernatant was further diluted (1:100, v/v) in

the HPLC mobile phase described above. 4-Cl-KYN was subsequently measured in
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appropriate aliquots of the tissue and plasma extract by HPLC, using a 5 µM reverse phase
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C18 column (Adsorbosil, Alltech, Deerfield, IL, USA) and a flow rate of 1.0 ml/min. 4-Cl-KYN

was detected by UV absorption at 365 nm (160 Absorbance Detector, Beckman, Fullerton, CA,

USA). The retention time of 4-Cl-KYN was approximately 5 min.

For measurement of 7-Cl-KYNA, a 50 µl aliquot of the original tissue homogenate was diluted

(1:1, v/v) with HPLC mobile phase, i.e. 50 mM sodium acetate buffer containing 0.25 M zinc

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acetate and 10% acetonitrile (pH 6.2). To assess levels of 7-Cl-KYNA in blood, an aliquot of

the deproteinated plasma sample was diluted (1:100, v/v) in the same HPLC mobile phase. 7-

Cl-KYNA was then determined in appropriate aliquots of the tissue and plasma extract by

HPLC analysis. Samples were applied to a 3 µm reverse phase HR-80 C18 column (ESA,

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Chelmsford, MA, USA). After elution at a flow rate of 1.0 ml/min, 7-Cl-KYNA was detected

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fluorimetrically using an excitation wavelength of 344 nm and an emission wavelength of 398

nm (S200 fluorescence detector, Perkin-Elmer, Beaconsfield, UK). 7-Cl-KYNA eluted with a

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retention time of 5-6 min.

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Statistical analysis AN
Data graphics and statistical analysis were performed using GraphPad Prism 6, v6.0c

(GraphPad Software, Inc., La Jolla, CA) mounted on a MacBook, Air, OS X v10.6.8). The
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primary analysis was the DEI vs. dose curve for each drug on each of the 4 measurements

(rotarod, carrageenan thermal escape for inflamed and non-inflamed paws, formalin flinching
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(phase 1 and phase 2) and tactile thresholds after nerve injury. For each end point, the mean
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and standard deviation (SD) of the saline DEI was determined. Best fit regression lines were

plotted, and the statistical significance of the slope was determined. If the slope of the dose-
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response curve for that drug in that treatment was statistically significant (p<0.05), the
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estimated drug dose with 95% confidence interval required to produce an effect equal to a
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reduction of 3 SDs less than the saline DEI was calculated as a measure of a significant drug

effect. The use of three SDs was considered to represent a robust criterion for defining drug

action across treatments, as 99.73% of randomly distributed values lie within this range, even

for non-normally distributed variables44.

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RESULTS

Tissue levels of 4-Cl-KYN and its active metabolite 7-Cl-KYNA

A total of 27 rats (n=3 per group per time point) were injected IP with 4-Cl-KYN (19, 167 or 500

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mg/kg), and animals were euthanized at various time points (30, 90 or 240 min). For the first

1.5 hrs, significant levels (0.15-1.3 µM in the brain and spinal cord, and up to 100 µM in the

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serum) of the active metabolite (7-Cl-KYNA), i.e. concentrations close to or above the IC50

(0.56 µM) of 7-Cl-KYNA at the GlyB site28, were recovered from animals receiving 167 mg/kg

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and 500 mg/kg 4-Cl-KYN (Fig. 1). The ratio of 7-Cl-KYNA to 4-Cl-KYN was quite similar across

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doses and times, but was skewed toward the formation of 7-Cl-KYNA in spinal cord and serum
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(each ~1:10 on average) compared to the brain (~1:50 on average).

Behavioral outcome measures

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General behavioral observations. In this series of studies, MK-801 (0.1-3 mg/kg), gabapentin
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(3-300 mg/kg) or 4-Cl-KYN (19-500 mg/kg) did not result in vocalization, biting or scratching, or
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loss of placing and stepping, or the loss of the righting reflex end points during the 4 hr interval

after drug delivery (data not shown). Though not systematically examined or quantified, mice
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receiving MK-801, but not gabapentin or 4-Cl-KYN, showed progressive increases in

spontaneous activity.
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Motor function: Figure 2 illustrates the time course of changes in rotarod performance as a
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function of time, and the dose-response curves for the calculated DEI after treatment with

saline, or multiple doses of 4-Cl-KYN, MK-801 and gabapentin. MK-801 and gabapentin dose-

dependently suppressed rotarod performance (Table 1). The calculated DEI ED3SD doses for

MK-801 and gabapentin were 0.3 and 902 mg/kg, respectively. As shown in the time course

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data, 4-Cl-KYN showed some decline in motor function at the highest dose (500 mg/kg), but

the slope of the drug response curve did not reach statistical significance.

Formalin model

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Intraplantar injection of formalin resulted in a significant incidence of flinching during phase 1

(0-9 min) and phase 2 (10-60 min). The time course of changes in flinching as a function of

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time after formalin and the dose response curve for the calculated DEI of the inflamed paw

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after treatment with saline or several doses of 4-Cl-KYN, MK-801 or gabapentin are shown in

Figure 3. For 4-Cl-KYN, the slopes of the DEI response regression line for both phase 1 and

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phase 2 showed statistical significance (p<0.05). The calculated ED3SD doses for 4-Cl-KYN in
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phase 1 and phase 2 were 119 and 257 mg/kg, respectively (Table 1). For MK-801, the slope

of the DEI response regression line was not significant for either of the two phases. Although
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no dose of MK-801 exceeded the criteria of 3SD for phase 1 or phase 2, significant

spontaneous activity was judged to interfere with formalin flinching assessments after
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treatment. The slope of the DEI response regression line for gabapentin was not significant in
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phase 1, but showed statistical significance in phase 2 (p<0.05). The calculated ED3SD dose for

gabapentin in phase 2 was 74 mg/kg (Table 1).

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Carrageenan model
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Thermal escape: The baseline thermal escape latencies prior to intraplantar carrageenan were
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10.3 ± 0.5 sec. Following carrageenan, there was a time-dependent fall in thermal escape

latency in the injected paw, such that saline control latencies were 4.3 ± 0.7 sec at 2 hrs (Fig.

4). No change was noted in the latency of the contralateral paw (data not shown). Figure 4

illustrates changes in escape latency performance as a function of time and dose-response

curves for the calculated DEI of the inflamed paw after treatment with saline or several doses

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of 4-Cl-KYN, MK-801 or gabapentin. The slope of the DEI dose-response regression line was

statistically significant for the three compounds (Table 1). The thermal escape DEI ED for 4-
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Cl-KYN, MK-801 and gabapentin was 320, 0.8 and 160 mg/kg, respectively. None of the three

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compounds had any effect on escape latency of the uninjured paw at any dose (data not

shown).

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Inflammation: Baseline paw thickness was 4.9 ± 0.2 mm (mean ± SEM). Intraplantar injection

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of carrageenan resulted in a significant increase in the thickness of the inflamed paw. Thus,

paw thickness in vehicle treated animals was 10.6 ± 0.3 mm. None of the three agents tested

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(4-Cl-KYN, MK-801, gabapentin) had any effect on paw thickness at the highest doses used
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(data not shown).

Adverse events: 4-Cl-KYN had no untoward effect on motor function or other behaviors tested
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at any dose used. MK-801 had no effect on behavior at 0.1 mg/kg, whereas 0.3 mg/kg and 1
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mg/kg caused increased locomotor activity, as expected (data not shown). Gabapentin caused

little behavioral deficits, except for one of 8 rats, which showed sedation at 90, 120 and 180
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min after receiving 100 mg/kg of the drug.

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Chung neuropathy

In normal animals, tactile thresholds were routinely 15 g or greater. After nerve ligation for 7
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days, von Frey testing revealed a tactile threshold of 2.3 ± 0.2 g, indicating tactile allodynia.
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No change was noted in the threshold of the contralateral paw (data not shown). Figure 5

illustrates changes in thermal escape latency as a function of time and dose-response curves

for the calculated DEI of the inflamed paw after treatment with saline, or several doses of 4-Cl-

KYN, MK-801 or gabapentin. Saline had no effect, but the slopes of the DEI response

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regression line for 4-Cl-KYN, MK-801 and gabapentin were statistically significant (p<0.05).

The calculated Chung ED3SD doses for 4-Cl-KYN, MK-801 and gabapentin were 180, 0.3 and

40 mg/kg, respectively. No drug-related behavioral deficits were noted for 4-Cl-KYN. As in the

carrageenan studies, 0.1 mg/kg MK-801 had no effect on behavior, whereas behavioral

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deficits, including increased locomotor activity, were observed at 0.3 mg/kg and 1 mg/kg of the

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agent.

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DISCUSSION
The present studies, performed in rats, were designed to determine the pharmacokinetic

parameters of 4-Cl-KYN, to study its behavioral and side effect profile in multiple pain models,

and to compare this compound with MK-801, a well-studied NMDA receptor antagonist11, and

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gabapentin, an agent considered to act by altering facilitated states through an interaction with

the α2∂ subunit of voltage-gated calcium channels14. As the effects of 4-Cl-KYN are mediated

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at least in part by its transport into the CNS and subsequent conversion to an active metabolite

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(7-Cl-KYNA)3,27, we examined the presence of both compounds in serum, brain and spinal

cord, and detected significant concentrations of 4-Cl-KYN and proportional amounts of 7-Cl-

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KYNA as soon as 30 min after administration of the prodrug. Notably, the concentrations of 7-
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Cl-KYNA, in brain and spinal cord were in most cases above the IC50 of the compound at the

GlyB site28. In addition to the mechanistic and therapeutic implications discussed below, the
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presence of substantial concentrations of 7-Cl-KYNA in the serum bodes well for using
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peripheral measurements of the compound as a biomarker in future clinical trials.

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Analgesic profile

Our results demonstrate that 4-Cl-KYN, MK-801 or gabapentin, given IP, had no effect on
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thermal nociception in the un-inflamed paw but produced a dose-dependent block of thermal

hyperalgesia in the inflamed paw after carrageenan. In addition, these agents reduced
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phase 2 responses of formalin-induced pain, and reversed the tactile allodynia in the Chung
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neuropathic pain model. Generally comparable antihyperalgesic results were observed with

MK-801 and gabapentin, which were employed as active test controls. The one exception was

that, in contrast to MK-801 and gabapentin, 4-Cl-KYN did reduce the phase 1 response after

formalin (Fig. 3).

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The carrageenan model induces potent inflammatory effects characterized by erythema and

volume increases in the injected paw. This inflammatory response was not altered by any of

the agents examined in the present study, consistent with their presumed lack of anti-

inflammatory properties. These results are consistent with a body of literature suggesting that

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antagonists of the GlyB site of the NMDA receptor are analgesically active in models of

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mechanically induced hyperalgesia33, formalin-evoked phase 2 flinching and the allodynia

otherwise observed after nerve injury37,45,46. The profile of analgesic activity observed here for

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4-Cl-KYN supports the conclusion that this agent has potent anti-hyperalgesic actions in

models of facilitated processing produced by peripheral tissue inflammation and nerve injury.

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Importantly, these effects, though requiring large doses of drug, were dose-dependent and
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unencumbered by evident side effects over the range of doses examined. Thus, based on

relative analgesic dose requirements and side effect profile, systemic 4-Cl-KYN appears to
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have efficacy and a therapeutic ratio equal to gabapentin and greater than MK-801.
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Adverse effects upon motor function

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In all nociceptive assay models, except for the formalin test, the rank order of potency was

MK-801 > gabapentin > 4-Cl-KYN. However, MK-801 produced dose-dependent changes on
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the rotarod over the range of doses examined, with the effects being greater than 2SD from
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control at doses of 1 mg/kg and higher. This activity precludes interpretation of the drug’s
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analgesic effects in the formalin test. In contrast, 4-Cl-KYN, even at the highest doses used,

did not approach the 2SD criteria used to demarcate a significant effect on rotarod

performance. This is in line with previous work, which showed that agents targeting the GlyB

site are distinguished by a lack of the side effects seen with the use of channel blockers of the

NMDA receptor (e.g. MK-801 and ketamine)9.

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Mechanisms of action

The prodrug, 4-Cl-KYN, is essentially inactive at the GlyB site of the NMDA receptor (IC50:

~150 µM)52. Orally administered 4-Cl-KYN is rapidly absorbed through the gut (Wallace, et al.

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Randomized, Double-Blind, Placebo Controlled, Dose-Escalation Study: Investigation of the

Safety, Pharmacokinetics, and Antihyperalgesic Activity of L-4-chlorokynurenine in Healthy

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Volunteers, 2017, manuscript submitted) and then actively transported into the CNS via the

large neutral amino acid transporter27. Subsequently, 4-Cl-KYN accumulates in astrocytes

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where kynurenine aminotransferases (KATs) catalyze its irreversible conversion to 7-Cl-

KYNA30,40,41,61. 7-Cl-KYNA61.

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KAT inhibition therefore
AN blocks the formation of Of

pharmacodynamic relevance in this context, KAT activity is up-regulated in response to various

pathological processes such as seizures and neurodegenerative events21,34,62. KAT is likely

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similarly enhanced in association with neuropathic and inflammatory pain, where spinal

astrocytes and other glial cells are known to change morphology and increase in number, and
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thus play a critical role in the development and maintenance of allodynia and hyperalgesia4,42.
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In all these cases, increased KAT activity results in greater synthesis of 7-Cl-KYNA from 4-Cl-

KYN preferentially at the site(s) where pathological changes take place34,62. This locally
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restricted, enhanced production of 7-Cl-KYNA serves to down-regulate NMDA receptor

function and thereby attenuates nerve and tissue injury.

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7-Cl-KYNA is a potent and selective antagonist of the GlyB site of the NMDA receptor (IC50 =

0.56 µM), and its affinity is approximately 20 times that of the endogenous tryptophan

metabolite kynurenic acid28. Furthermore, in distinction to kynurenic acid, 7-Cl-KYNA does not

have affinity for the α7 nicotinic acetylcholine receptor26, nor does it exhibit pharmacologically-

relevant binding to 50 other ion channels, receptors, and transporters67. In view of this high

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selectivity and the fact that the tissue levels of 7-Cl-KYNA in brain and spinal cord reach high

nanomolar to low micromolar concentrations after systemic prodrug administration, it is very

likely that the antinociceptive effects described here, as well as the previously reported

neuroprotective35,51,70, anti-epileptic53,71 and antidepressant29,30,47 effects of 4-Cl-KYN/7-Cl-

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KYNA, are mediated by NMDA receptor inhibition.

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Aside from the modulation of NMDA receptor function by inhibition of the GlyB site, 4-Cl-KYN

may provide an additional therapeutic benefit due to its potential to down-regulate the

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production of quinolinic acid (QUIN). This is due to the fact that 4-Cl-KYN is also metabolized

to 4-Cl-3-hydroxyanthranilic acid21, a highly potent, selective inhibitor of 3-hydroxyanthranilic

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acid oxygenase (IC50: ~6 nM), the immediate biosynthetic enzyme of the excitotoxic NMDA

receptor agonist quinolinic acid (QUIN)51,55. Elevated QUIN levels may be causally involved in
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various neurodegenerative processes and diseases57-62 and are believed to be especially

significant when the immune system – and consequently the neosynthesis of QUIN – is
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activated2,10,20,31,50,53. The transformation of systemically applied 4-Cl-KYN to 4-Cl-3-

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hydroxyanthranilic acid may therefore play an especially relevant role in the antinociceptive

effects seen in pain models that are associated with an inflammation-related accumulation of
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QUIN in the spinal cord25,66.

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Clinical implications
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In general, our data in the preclinical models used here are consistent with studies suggesting

that drugs targeting the GlyB site of the NMDA receptor are likely to be effective and well

tolerated in humans1,35,49,57 However, a randomized, double-blind study in humans failed to

show efficacy of one such antagonist (GV196771A) in treating neuropathic pain induced by

diabetic neuropathy, postherpetic neuralgia, complex regional pain syndrome, or peripheral

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nerve injury.57 This lack of efficacy may be due, in part, to insufficient brain penetrance of the

drug, suggesting that a GlyB/NMDA receptor antagonist with greater neuraxial bioavailability

may be more effective in treating facilitated pain states. Indeed, though the results were not

statistically significant, 4-Cl-KYN (AV-101) was recently shown in a small clinical study to

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alleviate allodynia pain, mechanical hyperalgesia, and heat hyperalgesia (Wallace, et al.

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Randomized, Double-Blind, Placebo Controlled, Dose-Escalation Study: Investigation of the

Safety, Pharmacokinetics, and Antihyperalgesic Activity of L-4-chlorokynurenine in Healthy

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Volunteers, 2017, manuscript submitted).

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In conclusion, the present set of experiments suggests that 4-Cl-KYN, applied systemically,
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can exert significant beneficial effects in several models of facilitated pain processing at doses

which have no impact on motor function. Furthermore, together with its excellent safety profile
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in animals and humans (Wallace, et al. Randomized, Double-Blind, Placebo Controlled, Dose-

Escalation Study: Investigation of the Safety, Pharmacokinetics, and Antihyperalgesic Activity

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of L-4-chlorokynurenine in Healthy Volunteers, 2017, manuscript submitted), the data

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presented here justify clinical studies of 4-Cl-KYN as a potential treatment for neuropathic

pain.
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Highlights

• The GlyB site regulates activation of the NMDA ionophore, which is involved in pain
mechanisms.

• 4-Cl-KYN is actively transported into the brain, but does not bind to the NMDAR GlyB

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sites.

• In astrocytes, 4-Cl-KYN is converted to 7-Cl-KYNA, a potent and selective GlyB NMDAR

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antagonist.

• In the present studies, IP administration of 4-Cl-KYN resulted in concentrations of 7-Cl-

KYNA in brain and spinal cord exceeding the IC50 for the GlyB site.

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• IP administration of 4-Cl-KYN reduces tissue/nerve injury pain with minimal adverse
effects.

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Acknowledgements: We thank Mr. Damon McCumber for his excellent work in performing

the in vivo studies, and Ms. Song-Chu Lee for the determination of 4-Cl-KYN and 7-Cl-KYNA.
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62. Wu HQ, Rassoulpour A, Goodman JH, Scharfman HE, Bertram EH, Schwarcz R.
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cord injury. J Neurotrauma. 23:866-881, 2006.

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http://dx.doi.org/10.1503/jpn.120228

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FIGURE LEGENDS

Figure 1. Pharmacokinetics of 4-Cl-KYN and 7-Cl-KYNA. Time-dependent concentrations

of 4-Cl-KYN and 7-Cl-KYNA in brain (A), spinal cord (B) and serum (C) at three time points

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after the IP delivery of one of three doses of 4-Cl-KYN (19, 167, and 500 mg/kg). Each time

point and dose presents the mean (nM) and SD of 3 rats. The respective brain (D), spinal cord

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(E) and serum (F) ratios of the active metabolite (7-Cl-KYNA) to 4-Cl-KYN are presented. Note

that all Y-axes are presented on a log scale.

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Figure 2. Rotarod performance. Time-effect curves plotting time on rotarod (mean ± SEM)

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per 180 sec test epochs as a function of time after: A) 4-Cl-KYN, B) MK-801 or C) gabapentin.
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D) Dose response curves plot the Drug Effect Index (AUC) vs. dose for IP saline, 4-Cl-KYN,

MK-801 or gabapentin. The dashed line indicates the Drug Effect Index (AUC) for saline - 3SD.
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Statistical analysis is presented in Table 1.

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Figure 3. Formalin evoked flinching. Time-effect curves plotting flinches/min (mean ± SEM)
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after intraplantar formalin in animals pretreated with: A) 4-Cl-KYN, B) MK-801 or C)

gabapentin. Dose response curves plot the cumulative flinching for D) Phase 1, and E)
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Phase 2 vs. dose for IP saline, 4-Cl-KYN, MK-801 or gabapentin. The dashed line indicates

the Drug Effect Index (AUC) + 2SD. Statistical significance of slope of dose response curve for
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phase 1: 4-Cl-KYN p=0.0002; MK-801: p=0.7095; gabapentin: p=0.4704; and for phase 2: 4-
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Cl-KYN: p=0.0011; MK-801: p=0.0146; gabapentin: p=0.3271;

Figure 4. Carrageenan evoked thermal hyperalgesia. Time-effect curves plotting response

latency in sec of the inflamed paw (mean ± SEM) as a function of time after: A) 4-Cl-KYN, B)

MK-801 or C) gabapentin. Carrageenan was delivered into the ipsilateral paw at 30 min (heavy

dashed line) prior to the delivery of drug at time 0 (light dashed line). D) Dose-response curves

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plot the Drug Effect Index (AUC) vs. dose for IP saline, 4-Cl-KYN, MK-801 or gabapentin. The

dashed line indicates the Drug Effect Index (AUC) for saline -3SD. Statistical analysis is

presented in Table 1.

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Figure 5. Nerve injury tactile allodynia. A unilateral nerve ligation was performed in rats at -

7 days. The Figure illustrates the time-effect curves plotting tactile stimulus in grams (mean ±

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SEM) required to evoke a withdrawal as a function of time after: A) 4-Cl-KYN, B) MK-801 or C)

gabapentin. D) Dose-response curves plotting the Drug Effect Index (AUC) vs. dose for IP

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saline, 4-Cl-KYN, MK-801 or gabapentin. The dashed line indicates the Drug Effect Index

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(AUC) - 3SD. Statistical analysis is presented in Table 1.
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TABLE LEGEND

Table 1 Title: Drug Effect Index Dose Response Curve Slope and calculated ED2SD
for 4-Cl-KYN, MK-801 and gabapentin on Rotarod, Carrageenan
thermal escape, Chung injury tactile allodynia and formalin evoked
flinching.

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Table 1 Legend:
# Number is the Drug Effect Index, which is 3 SD less than the saline response.
ED 3SD: intersection of the dose response curve for each drug

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* = Slope statistically different from saline.

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Yaksh, et al.

Table 1 Drug Effect Index Dose Response Curve Slope and calculated ED2SD
for 4-Cl-KYN, MK-801 and gabapentin on Rotarod, Carrageenan
thermal escape, Chung injury tactile allodynia and formalin evoked
flinching.

Saline Drug Dose ED 3SD (mg/kg)

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Model Effect Index Response Calculated for
(-3SD)# Slope Statistically Significant
Slopes

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Rotarod 263
4-Cl-KYN -189 -
MK-801 -139* 0.3 ( 0.003-1)

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gabapentin -313* 902 (343-5861)

Carrageenan 290
4-Cl-KYN -670* 320(166-4149)

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MK-801 -904* 0.8 (0.4-5)
gabapentin -1030* 160(97-400)
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Chung -473
4-Cl-KYN -1070* 184(108-340)
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MK-801 -1689* 0.3(0.25-0.44)

gabapentin -1404* 40(6-72)
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Formalin Phase 1 243

4-Cl-KYN -211* 119 (64 – 181)
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MK-801 -50 -
gabapentin -17 -
Formalin Phase 2 1179
4-Cl-KYN -829* 257 (157-612)
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MK-801 -800* 1.5(0.7-23)

gabapentin -602* 74(13-168)
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# Number is the Drug Effect Index, which is 3 SD less than the saline response.
ED 3SD: intersection of the dose response curve for each drug
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* = Slope statistically different from saline.

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