pharmaceuticals Article Kynurenic Acid Accelerates Healing of Corneal Epithelium In Vitro and In Vivo Anna Matysik-Wo´zniak 1,* , Waldemar A. Turski 2 , Monika Turska 3,4 , Roman Paduch 1,5 , Mirosław Ła ´ncut 6, Paweł Piwowarczyk 7 , Mirosław Czuczwar 7 and Robert Rejdak 1 1 Department of General and Pediatric Ophthalmology, Medical University of Lublin, 20-079 Lublin, Poland; [email protected] (R.P.); [email protected] (R.R.) 2 Department of Experimental and Clinical Pharmacology, Medical University of Lublin, 20-090 Lublin, Poland; [email protected] 3 Department of Pharmacology, Faculty of Health Sciences, Medical University of Lublin, 20-093 Lublin, Poland; [email protected] 4 Postgraduate School of Molecular Medicine, Medical University of Warsaw, 02-091 Warsaw, Poland 5 Department of Virology and Immunology, Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University, 20-033 Lublin, Poland 6 Center for Experimental Medicine, Medical University of Lublin, 20-090 Lublin, Poland; [email protected] 7 2nd Department of Anesthesiology and Intensive Care, Medical University of Lublin, 20-081 Lublin, Poland; [email protected] (P.P.); [email protected] (M.C.) * Correspondence: [email protected]; Tel.: +48-81532-8601 Abstract: Kynurenic acid (KYNA) is an endogenous compound with a multidirectional effect. It Citation: Matysik-Wo´zniak,A.; possesses antiapoptotic, anti-inflammatory, and antioxidative properties that may be beneficial in the Turski, W.A.; Turska, M.; Paduch, R.; treatment of corneal injuries. Moreover, KYNA has been used successfully to improve the healing Ła´ncut,M.; Piwowarczyk, P.; outcome of skin wounds. The aim of the present study is to evaluate the effects of KYNA on corneal Czuczwar, M.; Rejdak, R. Kynurenic and conjunctival cells in vitro and the re-epithelization of corneal erosion in rabbits in vivo. Normal Acid Accelerates Healing of Corneal human corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) lines were Epithelium In Vitro and In Vivo. used. Cellular metabolism, cell viability, transwell migration, and the secretion of IL-1β, IL-6, and Pharmaceuticals 2021, 14, 753. IL-10 were determined. In rabbits, after corneal de-epithelization, eye drops containing 0.002% and https://doi.org/10.3390/ph14080753 1% KYNA were applied five times a day until full recovery. KYNA decreased metabolism but did not Academic Editors: affect the proliferation of the corneal epithelium. It decreased both the metabolism and proliferation of Ioannis Tsinopoulos, Ioanna Mylona conjunctival epithelium. KYNA enhanced the migration of corneal but not conjunctival epithelial cells. and Lampros Lamprogiannis KYNA reduced the secretion of IL-1β and IL-6 from the corneal epithelium, leaving IL-10 secretion unaffected. The release of all studied cytokines from the conjunctival epithelium exposed to KYNA was Received: 4 July 2021 unchanged. KYNA at higher concentration accelerated the healing of the corneal epithelium. These Accepted: 28 July 2021 favorable properties of KYNA suggest that KYNA containing topical pharmaceutical products can be Published: 30 July 2021 used in the treatment of ocular surface diseases. Publisher’s Note: MDPI stays neutral Keywords: kynurenic acid; corneal epithelium; conjunctival epithelium; accelerated healing with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction One of the most important challenges in modern ophthalmic pharmacology is how to accelerate the healing of corneal epithelial defects and prevent the migration of con- Copyright: © 2021 by the authors. junctival epithelium onto the cornea. In addition, the reduction of pain and prevention Licensee MDPI, Basel, Switzerland. of scarring of the conjunctiva after surgery (especially anti-glaucoma surgery) or trauma This article is an open access article will be welcomed. So far, many attempts have been considered in topical therapy of oc- distributed under the terms and ular surface including blood derivatives [1], saliva [2], conditioned media from human conditions of the Creative Commons amniotic epithelial cells [3], the growth hormone [4], and erythropoietin [5], albeit without Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ satisfactory outcomes. 4.0/). Pharmaceuticals 2021, 14, 753. https://doi.org/10.3390/ph14080753 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 20212021,, 1414,, 753x FOR PEER REVIEW 22 of of 1617 In the the search search for for an an adequate adequate candidate, candidate, we we focused focused on endogenous on endogenous compounds compounds pre- presentsent in tears. in tears. In Inour our previous previous studies, studies, tryptophan tryptophan (TRP) (TRP) and and its its immediate metabolite kynurenine were investigated [[6,7].6,7]. Kynurenic acid (KYNA) is another TRP metabolitemetabolite formed along the kynurenine pathway. KYNA is anan organic,organic, water-solublewater-soluble compoundcompound that was discovered in in dog dog urine urine in in 185 18533 by by Justus Justus von von Liebig Liebig [8]. [8]. KYNA KYNA (C (C1010HH7NO7NO3; 4-3; hydroxyquinoline-2-carboxylic4-hydroxyquinoline-2-carboxylic acid) acid) has has a mole a molecularcular weight weight of 189.17 of 189.17 and solubility and solubility >28.4 >28.4ug/mL ug/mL (https://pubchem.ncbi.nlm.nih.gov, (https://pubchem.ncbi.nlm.nih.gov accessed, accessed on 22 July on 222021). July Its 2021). chemical Its chemical struc- turestructure is shown is shown in Scheme in Scheme 1. 1. Scheme 1. Chemical structure of kynurenic acid. KYNA is is widely widely distributed distributed in in nature. nature. It Itis ispresent present in inbody body fluids fluids as blood, as blood, cerebro- cere- spinalbrospinal fluid, fluid, saliva, saliva, and and tissues tissues including including eye eye structures structures in in mammals. mammals. We We found found that it occurs naturally in tears at a concentration of 0.02 μµMM[ [9].9]. It also occurs in in plants, plants, vegeta- vegeta- bles, and herbs as a component of everyday dietdiet [[10].10]. KYNA is considered as a safe andand non-toxic substance that cannot cross the blood–brain barrier. Endogenous KYNA KYNA is is a aproduct product of ofnormal normal TRP TRP metabolism. metabolism. More More than 90% than of 90% avail- of ableavailable TRP is TRP catabolized is catabolized along alongthe kynurenine the kynurenine pathway pathway (KP) [11]. (KP) TRP [11]. is TRPconverted is converted by the firstby the and first rate-limiting and rate-limiting enzymes, enzymes, tryptophan tryptophan 2,3-dioxygenase 2,3-dioxygenase constitutively constitutively present presentin liver andin liver cytokine-activated and cytokine-activated indoleamine-2,3-dioxyg indoleamine-2,3-dioxygenaseenase (IDO) broadly (IDO) broadlydistributed distributed in body in body tissues to N-formyl kynurenine, which is metabolized to kynurenine (KYN) by tissues to N-formyl kynurenine, which is metabolized to kynurenine (KYN) by kynurenine formamidase. KYN is mainly converted to 3-hydroxykynurenine and further kynurenine formamidase. KYN is mainly converted to 3-hydroxykynurenine and further downstream metabolites. KYNA synthesis is catalyzed by kynurenine aminotransferases downstream metabolites. KYNA synthesis is catalyzed by kynurenine aminotransferases (KATs) I-IV. Previously, we demonstrated the presence of KAT I, II, and III in the healthy (KATs) I-IV. Previously, we demonstrated the presence of KAT I, II, and III in the healthy human limbal conjunctiva and the cornea. Our findings support the hypothesis that human limbal conjunctiva and the cornea. Our findings support the hypothesis that TRP TRP can be metabolized to KYNA in corneal epithelium, endothelium, and stroma [12]. can be metabolized to KYNA in corneal epithelium, endothelium, and stroma [12]. KYNA KYNA displays many biological activities. KYNA exhibits anti-inflammatory, antioxidative, displays many biological activities. KYNA exhibits anti-inflammatory, antioxidative, an- analgesic, and antimicrobial properties [13–16]. It is the only known naturally occurring algesic, and antimicrobial properties [13–16]. It is the only known naturally occurring an- antagonist of endogenous glutamate receptors in the central nervous system. KYNA in tagonist of endogenous glutamate receptors in the central nervous system. KYNA in low low micromolar concentrations antagonizes the glycine site of the N-methyl-D-aspartate micromolar concentrations antagonizes the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex and acts as a potent neuroprotectant. As a result of this activity, (NMDA)it may influence receptor important complex and neurophysiologic acts as a potent and neuroprotectant. neuropathologic As a processes result of includingthis activ- Alzheimer’sity, it may influence disease, important Huntington’s neurophysiol and Parkinson’sogic and diseases, neuropathologic multiple sclerosis,processes epilepsy, includ- ingbrain Alzheimer’s ischemia, depression, disease, Huntington’s and schizophrenia. and Parkinson’s Glutamate diseases, in high concentrationmultiple sclerosis, is known epi- lepsy,for its neurotoxicitybrain ischemia, in depression, the brain and and retina schizophrenia. [17,18]. In aGlutamate similar manner, in high it concentration may play a role is knownin neurotrophic for its neurotoxicity diseases of in cornea the brain and and the retina remaining [17,18]. ocular In a similar surface