Modafinil Suppresses Hypertrophic Scar Formation by Inhibiting Adenosine Deaminase and Activating Adenosine Signaling

Abstract

Modafinil (MF) is a clinically approved wake-promoting agent with emerging anti-inflammatory and anti-fibrotic effects, although its upstream molecular target has remained undefined. Here, we identify adenosine deaminase (ADA) as a previously unrecognized target mediating the therapeutic actions of MF. Its S- and R-isomers (MF-S and MF-R) robustly increased intracellular cAMP levels in fibroblasts with efficacy comparable to NECA, despite minimal direct binding to adenosine receptors, and suppressed K_Ca3.1 channel activity via a PKA-dependent mechanism. MF-S markedly upregulated CD39 and CD73, leading to increased adenosine availability. Pharmacological inhibition of CD73 with AB680 abolished MF-S-induced increases in cAMP and Epac levels and reversed suppression of TGFβ-induced collagen expression. Consistently, MF-S attenuated canonical profibrotic signaling by inhibiting TGFβ-induced Smad4 upregulation. In vivo, MF-S significantly reduced hypertrophic scarring in a rabbit ear model, with efficacy comparable to Contratubex. Mechanistically, MF-S directly inhibited purified ADA at subnanomolar concentrations and suppressed cellular ADA activity in fibroblast and immune cells. Collectively, these findings establish ADA inhibition as a key upstream mechanism by which MF enhances adenosine-cAMP signaling to suppress inflammation and fibrosis, highlighting MF and its isomers as promising therapeutic candidates for inflammatory and fibrotic diseases.

Keywords

Modafinil / Adenosine deaminase / Adenosine / cAMP signaling / Anti-fibrotic effect / Hypertrophic scar

Supplementary Materials

The following supporting information can be found at: https://www.sciepublish.com/article/pii/842, Figure S1: MF-S inhibited Smad4 upregulation by TGFβ in human lung fibroblasts. Smad4 protein levels were measured by Western blot. Blots shown are representative of 9 experiments performed with 9 different cultures. Data are presented as the mean ± SEM values. ** p < 0.01.

Statement of the Use of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this manuscript, the authors used ChatGPT (version 4.0) solely for grammar checking. After using this tool, the authors reviewed and edited the content as needed and assume full responsibility for the content of the published article.

Author Contributions

Conceptualization, S.C., K.-C.K. and S.-H.S.; methodology, S.C., J.-A.K. and S.-H.S.; formal analysis, S.C., J.-A.K. and S.-H.S.; writing—original draft preparation, S.C. and K.-C.K.; writing—review and editing, S.C. and S.-H.S.; supervision, S.C. and S.-H.S.; funding acquisition, S.C., K.-C.K. and S.-H.S. All authors have read and agreed to the published version of the manuscript.

Ethics Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Seoul National University Bundang Hospital, Gyeonggi-do, Republic of Korea (BA-2108-326-008-02, issued at 27 August 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2022R1A2C1092484 and NRF-2022R1A2C1007823), Republic of Korea and intramural research promotion grants from Ewha Womans University, School of Medicine.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

[1] Gerrard P, Malcolm R. Mechanisms of modafinil: A review of current research. Neuropsychiatr. Dis. Treat. 2007, 3, 349-364. DOI:10.2147/ndt.s12160183

[2] Jung J, Youm J, Kang J, Kim AY, Suh JK, Kang HY. Assessing Condition-Specific Adverse Event Profiles of Modafinil for Labelled and Off-Label Uses: A Systematic Review and Meta-Analysis. Basic Clin. Pharmacol. Toxicol. 2026, 138, e70147. DOI:10.1111/bcpt.70147

[3] Ghazanfar S, Farooq M, Qazi SU, Chaurasia B, Kaunzner U. The use of modafinil for the treatment of fatigue in multiple sclerosis: A systematic review and meta-analysis of controlled clinical trials. Brain Behav. 2024, 14, e3623. DOI:10.1002/brb3.3623

[4] Sousa A, Dinis-Oliveira RJ. Pharmacokinetic and pharmacodynamic of the cognitive enhancer modafinil: Relevant clinical and forensic aspects. Subst. Abus. 2020, 41, 155-173. DOI:10.1080/08897077.2019.1700584

[5] Minzenberg MJ, Carter CS. Modafinil: A review of neurochemical actions and effects on cognition. Neuropsychopharmacology 2008, 33, 1477-1502. DOI:10.1038/sj.npp.1301534

[6] Lindsay SE, Gudelsky GA, Heaton PC. Use of modafinil for the treatment of attention deficit/hyperactivity disorder. Ann. Pharmacother. 2006, 40, 1829-1833. DOI:10.1345/aph.1H024

[7] Zamanian MY, Karimvandi MN, Nikbakhtzadeh M, Zahedi E, Bokov DO, Kujawska M, et al. Effects of Modafinil (Provigil) on Memory and Learning in Experimental and Clinical Studies: From Molecular Mechanisms to Behaviour Molecular Mechanisms and Behavioural Effects. Curr. Mol. Pharmacol. 2023, 16, 507-516. DOI:10.2174/1874467215666220901122824

[8] Battleday RM, Brem AK. Modafinil for cognitive neuroenhancement in healthy non-sleep-deprived subjects: A systematic review. Eur. Neuropsychopharmacol. 2015, 25, 1865-1881. DOI:10.1016/j.euroneuro.2015.07.028

[9] Amini MJ, Seighali N, Arabazadeh Bahri R, Ala M, Mohammad Jafari R, Dehpour AR. Repurposing of modafinil as an anti-inflammatory drug: A systematic review of experimental studies. Naunyn Schmiedeberg’s Arch. Pharmacol. 2025, 398, 15599-15609. DOI:10.1007/s00210-025-03964-9

[10] Li H, Kim JA, Jo SE, Lee H, Kim KC, Choi S, et al. Modafinil exerts anti-inflammatory and anti-fibrotic effects by upregulating adenosine A_2A and A_2B receptors. Purinergic Signal. 2024, 20, 371-384. DOI:10.1007/s11302-023-09973-8

[11] Choi S, Kim JA, Li H, Jo SE, Lee H, Kim TH, et al. Anti-inflammatory and anti-fibrotic effects of modafinil in nonalcoholic liver disease. Biomed. Pharmacother. 2021, 144, 112372. DOI:10.1016/j.biopha.2021.112372

[12] Zager A. Modulating the immune response with the wake-promoting drug modafinil: A potential therapeutic approach for inflammatory disorders. Brain Behav. Immun. 2020, 88, 878-886. DOI:10.1016/j.bbi.2020.04.038

[13] Kamel M, Ahmed SM, Abdelzaher W. The potential protective effect of modafinil in intestinal ischemic reperfusion-induced in rats. Int. Immunopharmacol. 2020, 88, 106983. DOI:10.1016/j.intimp.2020.106983

[14] Yousefi-Manesh H, Rashidian A, Hemmati S, Shirooie S, Sadeghi MA, Zarei N, et al. Therapeutic effects of modafinil in ischemic stroke; possible role of NF-kappaB downregulation. Immunopharmacol. Immunotoxicol. 2019, 41, 558-564. DOI:10.1080/08923973.2019.1669045

[15] Abbasi Y, Shabani R, Mousavizadeh K, Soleimani M, Mehdizadeh M. Neuroprotective effect of ethanol and Modafinil on focal cerebral ischemia in rats. Metab. Brain Dis. 2019, 34, 805-819. DOI:10.1007/s11011-018-0378-0

[16] Yousefi-Manesh H, Shirooie S, Hemati S, Shokrian-Zeini M, Zarei N, Raoufi M, et al. Protective effects of modafinil administration on testicular torsion/detorsion damage in rats. Exp. Mol. Pathol. 2019, 111, 104305. DOI:10.1016/j.yexmp.2019.104305

[17] Dejban P, Rahimi N, Takzare N, Dehpour AR. Biochemical and histopathological evidence for the beneficial effects of modafinil on the rat model of inflammatory bowel disease: Involvement of nitric oxide pathway. Pharmacol. Rep. 2020, 72, 135-146. DOI:10.1007/s43440-019-00054-5

[18] Brandão WN, Andersen ML, Palermo-Neto J, Peron JP, Zager A. Therapeutic treatment with Modafinil decreases the severity of experimental autoimmune encephalomyelitis in mice. Int. Immunopharmacol. 2019, 75, 105809. DOI:10.1016/j.intimp.2019.105809

[19] Bibani RH, Tench CR, George J, Manouchehrinia A, Palace J, Constantinescu CS. Reduced EDSS progression in multiple sclerosis patients treated with modafinil for three years or more compared to matched untreated subjects. Mult. Scler. Relat. Disord. 2012, 1, 131-135. DOI:10.1016/j.msard.2012.03.004

[20] Han J, Chen D, Liu D, Zhu Y. Modafinil attenuates inflammation via inhibiting Akt/NF-kappaB pathway in apoE-deficient mouse model of atherosclerosis. Inflammopharmacology 2018, 26, 385-393. DOI:10.1007/s10787-017-0387-3

[21] Pal China S, Pal S, Chattopadhyay S, Porwal K, Mittal M, Sanyal S, et al. The wakefulness promoting drug Modafinil causes adenosine receptor-mediated upregulation of receptor activator of nuclear factor kappaB ligand in osteoblasts: Negative impact of the drug on peak bone accrual in rats. Toxicol. Appl. Pharmacol. 2018, 348, 22-31. DOI:10.1016/j.taap.2018.04.006

[22] Choi S, Kim MY, Joo KY, Park S, Kim JA, Jung JC, et al. Modafinil inhibits K(Ca)3.1 currents and muscle contraction via a cAMP-dependent mechanism. Pharmacol. Res. 2012, 66, 51-59. DOI:10.1016/j.phrs.2012.02.009

[23] Tavares LP, Negreiros-Lima GL, Lima KM, PMR ES, Pinho V, Teixeira MM, et al. Blame the signaling: Role of cAMP for the resolution of inflammation. Pharmacol. Res. 2020, 159, 105030. DOI:10.1016/j.phrs.2020.105030

[24] Raker VK, Becker C, Steinbrink K. The cAMP Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases. Front. Immunol. 2016, 7, 123. DOI:10.3389/fimmu.2016.00123

[25] Insel PA, Murray F, Yokoyama U, Romano S, Yun H, Brown L, et al. cAMP and Epac in the regulation of tissue fibrosis. Br. J. Pharmacol. 2012, 166, 447-456. DOI:10.1111/j.1476-5381.2012.01847.x

[26] Campo GM, Avenoso A, D’Ascola A, Prestipino V, Scuruchi M, Nastasi G, et al. Protein kinase a mediated anti-inflammatory effects exerted by adenosine treatment in mouse chondrocytes stimulated with IL-1beta. Biofactors 2012, 38, 429-439. DOI:10.1002/biof.1040

[27] Hewer RC, Sala-Newby GB, Wu YJ, Newby AC, Bond M. PKA and Epac synergistically inhibit smooth muscle cell proliferation. J. Mol. Cell Cardiol. 2011, 50, 87-98. DOI:10.1016/j.yjmcc.2010.10.010

[28] Roach KM, Bradding P. Ca²⁺ signalling in fibroblasts and the therapeutic potential of K_Ca3.1 channel blockers in fibrotic diseases. Br. J. Pharmacol. 2020, 177, 1003-1024. DOI:10.1111/bph.14939

[29] Organ L, Bacci B, Koumoundouros E, Kimpton WG, Samuel CS, Nowell CJ, et al. Inhibition of the K(Ca)3.1 Channel Alleviates Established Pulmonary Fibrosis in a Large Animal Model. Am. J. Respir. Cell Mol. Biol. 2017, 56, 539-550. DOI:10.1165/rcmb.2016-0092OC

[30] Lee WR, Kim KH, An HJ, Kim JY, Lee SJ, Han SM, et al. Apamin inhibits hepatic fibrosis through suppression of transforming growth factor beta1-induced hepatocyte epithelial-mesenchymal transition. Biochem. Biophys. Res. Commun. 2014, 450, 195-201. DOI:10.1016/j.bbrc.2014.05.089

[31] Prisinzano T, Podobinski J, Tidgewell K, Luo M, Swenson D. Synthesis and determination of the absolute configuration of the enantiomers of modafinil. Tetrahedron Asymmetry 2004, 15, 1053-1058. DOI:10.1016/j.tetasy.2004.01.039

[32] Hulme EC, Trevethick MA. Ligand binding assays at equilibrium: Validation and interpretation. Br. J. Pharmacol. 2010, 161, 1219-1237. DOI:10.1111/j.1476-5381.2009.00604.x

[33] Effendi WI, Nagano T, Kobayashi K, Nishimura Y. Focusing on Adenosine Receptors as a Potential Targeted Therapy in Human Diseases. Cells 2020, 9, 785. DOI:10.3390/cells9030785

[34] Liao H, Hyman MC, Baek AE, Fukase K, Pinsky DJ. cAMP/CREB-mediated transcriptional regulation of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) expression. J. Biol. Chem. 2010, 285, 14791-14805. DOI:10.1074/jbc.M110.116905

[35] Narravula S, Lennon PF, Mueller BU, Colgan SP. Regulation of endothelial CD 73 by adenosine: Paracrine pathway for enhanced endothelial barrier function. J. Immunol. 2000, 165, 5262-5268. DOI:10.4049/jimmunol.165.9.5262

[36] Pastor-Anglada M, Perez-Torras S. Who Is Who in Adenosine Transport. Front. Pharmacol. 2018, 9, 627. DOI:10.3389/fphar.2018.00627

[37] Xu F, Liu C, Zhou D, Zhang L. TGF-beta/SMAD Pathway and Its Regulation in Hepatic Fibrosis. J. Histochem. Cytochem. 2016, 64, 157-167. DOI:10.1369/0022155415627681

[38] Amanlou M, Saboury AA, Bazl R, Ganjali MR, Sheibani S. Adenosine deaminase activity modulation by some street drug: Molecular docking simulation and experimental investigation. DARU J. Pharm. Sci. 2014, 22, 42. DOI:10.1186/2008-2231-22-42

[39] Alunni S, Orru M, Ottavi L. A study on the inhibition of adenosine deaminase. J. Enzyme Inhib. Med. Chem. 2008, 23, 182-189. DOI:10.1080/14756360701475233

[40] Hasko G, Cronstein B. Regulation of inflammation by adenosine. Front. Immunol. 2013, 4, 85. DOI:10.3389/fimmu.2013.00085

[41] Hu HH, Chen DQ, Wang YN, Feng YL, Cao G, Vaziri ND, et al. New insights into TGF-beta/Smad signaling in tissue fibrosis. Chem. Biol. Interact. 2018, 292, 76-83. DOI:10.1016/j.cbi.2018.07.008

[42] Schiller M, Verrecchia F, Mauviel A. Cyclic adenosine 3′5′-monophosphate-elevating agents inhibit transforming growth factor-beta-induced SMAD3/4-dependent transcription via a protein kinase A-dependent mechanism. Oncogene 2003, 22, 8881-8890. DOI:10.1038/sj.onc.1206871

[43] Stratton R, Shiwen X, Martini G, Holmes A, Leask A, Haberberger T, et al. Iloprost suppresses connective tissue growth factor production in fibroblasts and in the skin of scleroderma patients. J. Clin. Investig. 2001, 108, 241-250. DOI:10.1172/JCI12020

[44] Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, et al. Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: Down-regulation by cAMP. FASEB J. 1999, 13, 1774-1786. DOI:10.1096/fasebj.13.13.1774

[45] Salinthone S, Yadav V, Schillace RV, Bourdette DN, Carr DW. Lipoic acid attenuates inflammation via cAMP and protein kinase A signaling. PLoS ONE 2010, 5, e13058. DOI:10.1371/journal.pone.0013058

[46] Ohya S, Kito H. Ca(2+)-Activated K(+) Channel K(Ca)3.1 as a Therapeutic Target for Immune Disorders. Biol. Pharm. Bull. 2018, 41, 1158-1163. DOI:10.1248/bpb.b18-00078

[47] Sim JH, Kim KS, Park H, Kim KJ, Lin H, Kim TJ, et al. Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8(+) T Cells. Front. Immunol. 2017, 8, 859. DOI:10.3389/fimmu.2017.00859

[48] Nguyen HM, Grossinger EM, Horiuchi M, Davis KW, Jin LW, Maezawa I, et al. Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia. Glia 2017, 65, 106-121. DOI:10.1002/glia.23078