2-Hydroxyisocaproic acid: A novel in vitro protease activity modulator with potential benefits for arthritis treatment

Main Article Content

Tuomo Karila, MD, PhD, Orthopaedic Surgeona Taina Tervahartiala, DDS, Docent, PhD Beniamin Cohen, LLM Timo Sorsa, DDS, Professor, PhD, Dipl Perio

Abstract

Osteoarthritis affects approximately half a billion people worldwide and creates a significant economic burden, accounting for up to 2.5% of the national gross domestic product. Despite extensive research, a disease-modifying osteoarthritis drug remains unavailable. 2-hydroxyisocaproic acid is a physiological substance and the 2-hydroxy analogue of the essential amino acid leucine. This study investigates the potential of 2-hydroxyisocaproic acid to down-regulate key proteases involved in articular cartilage degradation. Specifically, we explore the use of 2-hydroxyisocaproic acid to inhibit the activity of matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5, aiming to reduce the degradation of type II collagen and aggrecan, respectively, in osteoarthritis. Our findings demonstrate that 2-hydroxyisocaproic acid modulates and reduces the activity of both matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Notably, 2-hydroxyisocaproic acid’s reversible inhibition of these enzymes does not involve covalent bonding, positioning it as an enzyme modulator or down-regulator rather than a direct inhibitor.

Article Details

How to Cite
KARILA, Tuomo et al. 2-Hydroxyisocaproic acid: A novel in vitro protease activity modulator with potential benefits for arthritis treatment. Medical Research Archives, [S.l.], v. 12, n. 7, july 2024. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/5468>. Date accessed: 05 aug. 2024. doi: https://doi.org/10.18103/mra.v12i7.5468.
Section
Research Articles

References

1. Huang Z, Ding C, Li T, Yu SP. Current status and future prospects for disease modification in osteoarthritis. Rheumatology (Oxford). May 1 2018;57(suppl_4):iv108-iv123. doi:10.1093/rheumatology/kex496

2. Hunter DJ, Bierma-Zeinstra S. Osteoarthritis. Lancet. Apr 27 2019;393(10182):1745-1759. doi:10.1016/S0140-6736(19)30417-9

3. Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum. Jun 2001;44(6):1237-47.

4. Yao Q, Wu X, Tao C, et al. Osteoarthritis: pathogenic signaling pathways and therapeutic targets. Signal Transduct Target Ther. Feb 3 2023; 8(1):56. doi:10.1038/s41392-023-01330-w

5. Quicke JG, Conaghan PG, Corp N, Peat G. Osteoarthritis year in review 2021: epidemiology & therapy. Osteoarthritis Cartilage. Feb 2022;30(2): 196-206. doi:10.1016/j.joca.2021.10.003

6. Lo J, Chan L, Flynn S. A Systematic Review of the Incidence, Prevalence, Costs, and Activity and Work Limitations of Amputation, Osteoarthritis, Rheumatoid Arthritis, Back Pain, Multiple Sclerosis, Spinal Cord Injury, Stroke, and Traumatic Brain Injury in the United States: A 2019 Update. Arch Phys Med Rehabil. Jan 2021;102(1):115-131. doi:10.1016/j.apmr.2020.04.001

7. Hiligsmann M, Cooper C, Arden N, et al. Health economics in the field of osteoarthritis: an expert's consensus paper from the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO). Semin Arthritis Rheum. Dec 2013;43(3):303-13. doi:10.1016/j.semarthrit.2013.07.003

8. Honorati MC, Bovara M, Cattini L, Piacentini A, Facchini A. Contribution of interleukin 17 to human cartilage degradation and synovial inflammation in osteoarthritis. Osteoarthritis Cartilage. Oct 2002; 10(10):799-807. doi:10.1053/joca.2002.0829

9. Anderson DD, Chubinskaya S, Guilak F, et al. Post-traumatic osteoarthritis: improved understanding and opportunities for early intervention. J Orthop Res. Jun 2011;29(6):802-9. doi:10.1002/jor.21359

10. Huskisson EC, Dieppe PA, Tucker AK, Cannell LB. Another look at osteoarthritis. Annals of the rheumatic diseases. Oct 1979;38(5):423-8. doi:10.1136/ard.38.5.423

11. Dingle JT. Articular Damage in Arthritis and Its Control. Annals of Internal Medicine. 1978;88 (6):821-826. doi:10.7326/0003-4819-88-6-821

12. Sokolove J, Lepus CM. Role of inflammation in the pathogenesis of osteoarthritis: latest findings and interpretations. Ther Adv Musculoskelet Dis. Apr 2013;5(2):77-94. doi:10.1177/1759720X12467868

13. Lawrence RC, Felson DT, Helmick CG, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum. Jan 2008;58(1):26-35.

14. Barter M, Bui C, Young D. Epigenetic mechanisms in cartilage and osteoarthritis: DNA methylation, histone modifications and microRNAs. Osteoarthritis and cartilage. May 2012;20(5):339-349. doi:10.1016/j.joca.2011.12.012

15. Loughlin J. The genetic epidemiology of human primary osteoarthritis: current status. Expert Rev Mol Med. May 24 2005;7(9):1-12. doi:10.1017/ S1462399405009257

16. Glyn-Jones S, Palmer A, Agricola R, et al. Osteoarthritis. The Lancet. Jul 25 2015;386(9991): 376-387. doi:10.1016/S0140-6736(14)60802-3

17. Bajaj S, Shoemaker T, Hakimiyan AA, et al. Protective effect of P188 in the model of acute trauma to human ankle cartilage: the mechanism of action. Journal of orthopaedic trauma. Sep 2010; 24(9):571. doi:10.1097/BOT.0b013e3181ec4712

18. Goldring MB, Marcu KB. Cartilage homeostasis in health and rheumatic diseases. Arthritis Res Ther. 2009;11(3):224. doi:10.1186/ar2592

19. Nagase H, Kashiwagi M. Aggrecanases and cartilage matrix degradation. Arthritis Res Ther. 2003;5(2):94.

20. Karsdal MA, Madsen SH, Christiansen C, Henriksen K, Fosang AJ, Sondergaard BC. Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity. Arthritis Res Ther. 2008;10(3):R63. doi:10.1186/ar2434

21. Troeberg L, Nagase H. Proteases involved in cartilage matrix degradation in osteoarthritis. Biochim Biophys Acta. Jan 2012;1824(1):133-45. doi:10.1016/j.bbapap.2011.06.020

22. Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis. Front Biosci. Jan 1 2006;11(1):529-543. doi:10.2741/1817

23. Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol. Jan 2011;7(1):33-42. doi:10.1038/nrrheum.2010.196

24. Wojdasiewicz P, Poniatowski LA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators Inflamm. 2014;2014:561459. doi:10.1155/2014/561459

25. Wang M, Sampson ER, Jin H, et al. MMP13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther. Jan 8 2013;15(1):R5. doi:10.1186/ar4133

26. Murphy G, Nagase H. Progress in matrix metalloproteinase research. Molecular aspects of medicine. 2008;29(5):290-308.

27. Vilen S-T, Salo T, Sorsa T, Nyberg P. Fluctuating roles of matrix metalloproteinase-9 in oral squamous cell carcinoma. The Scientific World Journal. 2013;2013

28. Lauhio A, Farkkila E, Pietilainen KH, et al. Association of MMP-8 with obesity, smoking and insulin resistance. Eur J Clin Invest. Sep 2016;46(9):757-65. doi:10.1111/eci.12649

29. Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res. 2002;4(3):157-64. doi:10.1186/ar401

30. Shiomi T, Lemaitre V, D'Armiento J, Okada Y. Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseases. Pathol Int. Jul 2010;60( 7):477-96. doi:10.1111/j.1440-1827.2010.02547.x

31. Roach HI, Yamada N, Cheung KS, et al. Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions. Arthritis Rheum. Oct 2005;52(10):3110-24. doi:10.1002/art.21300

32. Mehana E-SE, Khafaga AF, El-Blehi SS. The role of matrix metalloproteinases in osteoarthritis pathogenesis: An updated review. Life sciences. Oct 1 2019;234:116786. doi:10.1016/j.lfs.2019.116786

33. Sandy JD, Flannery CR, Neame PJ, Lohmander LS. The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. J Clin Invest. May 1992; 89(5):1512-6. doi:10.1172/JCI115742

34. Lohmander LS, Neame PJ, Sandy JD. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum. Sep 1993;36(9):1214-22. doi:10.1002/art.1780360906

35. Hoffer LJ, Taveroff A, Robitaille L, Mamer OA, Reimer ML. Alpha-keto and alpha-hydroxy branched-chain acid interrelationships in normal humans. J Nutr. Sep 1993;123(9):1513-21. doi:10.1093/jn/123.9.1513

36. Wishart DS, Guo A, Oler E, et al. HMDB 5.0: the Human Metabolome Database for 2022. Nucleic Acids Res. Jan 7 2022;50(D1):D622-D631. doi:10.1093/nar/gkab1062

37. Broadhurst MJ, Brown PA, Johnson WH, Lawton G, inventors; Hoffmann-La Roche Inc, Nutley, N.J., assignee. Amino acid derivatives. patent 5,304,549. patent application 823,212. 1994.

38. van den Berg WB. Osteoarthritis year 2010 in review: pathomechanisms. Osteoarthritis Cartilage. Apr 2011;19(4):338-41. doi:10.1016/j.joca.2011.01.022

39. Rowan AD, Litherland GJ, Hui W, Milner JM. Metalloproteases as potential therapeutic targets in arthritis treatment. Expert Opin Ther Targets. Jan 2008;12(1):1-18. doi:10.1517/14728222.12.1.1

40. Westermarck HW, Hietala P, Jaarma M, Sorsa T, Vaara M, inventors; Oy Extracta Ltd, assignee. Antimicrobial and anti-inflammatory compounds. EPO patent EP0871438B1. patent application EP0871438A1. 1996.

41. Nieminen MT, Hernandez M, Novak-Frazer L, et al. DL-2-hydroxyisocaproic acid attenuates inflammatory responses in a murine Candida albicans biofilm model. Clin Vaccine Immunol. Sep 2014;21(9):1240-1245. doi:10.1128/CVI.00339-14

42. Tervahartiala T, Ingman T, Sorsa T, Ding Y, Kangaspunta P, Konttinen YT. Proteolytic enzymes as indicators of periodontal health in gingival crevicular fluid of patients with Sjogren's syndrome. Eur J Oral Sci. Feb 1995;103(1):11-6. doi:10.1111 /j.1600-0722.1995.tb00004.x

43. Hanemaaijer R, Sier CF, Visser H, et al. MMP-9 activity in urine from patients with various tumors, as measured by a novel MMP activity assay using modified urokinase as a substrate. Ann N Y Acad Sci. Jun 30 1999;878:141-9. doi:10.1111/j.1 749-6632.1999.tb07680.x

44. Hanemaaijer R, Visser H, Konttinen YT, Koolwijk P, Verheijen JH. A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sjogren's syndrome contain increased latent and active gelatinase-B levels. Matrix Biol. Dec 1998;17(8-9):657-65. doi:10.1016/s0945-053x(98)90116-0

45. Peterson JT. The importance of estimating the therapeutic index in the development of matrix metalloproteinase inhibitors. Cardiovasc Res. Feb 15 2006;69(3):677-87. doi:10.1016/j.cardiores.2005.11.032

46. Karila T, Tervahartiala T, Cohen B, Sorsa T. The collagenases: are they tractable targets for preventing cartilage destruction in osteoarthritis? Expert Opin Ther Targets. Feb 2 2022:1-13. doi:10.1080/14728222.2022.2035362

47. Matter H, Schudok M, Schwab W, et al. Tetrahydroisoquinoline-3-carboxylate based matrix-metalloproteinase inhibitors: design, synthesis and structure-activity relationship. Bioorg Med Chem. Nov 2002;10(11):3529-44. doi:10.1016/s0968-0896(02)00215-8

48. Nakai R, Salisbury CM, Rosen H, Cravatt BF. Ranking the selectivity of PubChem screening hits by activity-based protein profiling: MMP13 as a case study. Bioorg Med Chem. Feb 1 2009;17(3): 1101-8. doi:10.1016/j.bmc.2008.03.018

49. Adhikari N, Halder AK, Mallick S, Saha A, Saha KD, Jha T. Robust design of some selective matrix metalloproteinase-2 inhibitors over matrix metalloproteinase-9 through in silico/fragment-based lead identification and de novo lead modification: Syntheses and biological assays. Bioorg Med Chem. Sep 15 2016;24(18):4291-4309. doi:10.1016/j.bmc.2016.07.023

50. Johnson LL, Dyer R, Hupe DJ. Matrix metalloproteinases. Curr Opin Chem Biol. Aug 1998;2(4):466-71. doi:10.1016/s1367-5931(98)80122-1

51. Settle S, Vickery L, Nemirovskiy O, et al. Cartilage degradation biomarkers predict efficacy of a novel, highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: confirmation by multivariate analysis that modulation of type II collagen and aggrecan degradation peptides parallels pathologic changes. Arthritis Rheum. Oct 2010;62(10):3006-15. doi:10.1002/art.27596

52. Cai H, Agrawal AK, Putt DA, et al. Assessment of the renal toxicity of novel anti-inflammatory compounds using cynomolgus monkey and human kidney cells. Toxicology. Apr 5 2009;258(1):56-63. doi:10.1016/j.tox.2009.01.006

53. Mortimore GE, Pösö AR, Kadowaki M, Wert JJ, Jr. Multiphasic control of hepatic protein degradation by regulatory amino acids. General features and hormonal modulation. J Biol Chem. Dec 5 1987;262(34):16322-7.
doi:https://doi.org/10.1016/S0021-9258(18)49257-5

54. Stockel-Maschek A, Stiebitz B, Koelsch R, Neubert K. Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1: Synthesis and kinetic characterization as aminopeptidase P inhibitors. Bioorg Med Chem. Aug 15 2005;13(16): 4806-20. doi:10.1016/j.bmc.2005.05.040

55. Walser M, inventor; Composition for promotion of protein synthesis and suppression of urea formation in the body utilizing alpha-hydroxy-acid analogs of amoni acids. US patent 1 444 621. patent application 32122/73. 30. April 1973 1973.

56. Walser M, inventor; The Johns Hopkins University, Baltimore, Md., assignee. Therapeutic compositions comprising alpha-hydroxy analogs of essential amino acids and their administration to humans for promotion of protein synthesis and suppression of urea formation. U.S. patent 4,100,160. patent application 669,588. Mar. 23, 1976 1978.

57. Boebel KP, Baker DH. Comparative utilization of the alpha-keto and D- and L-alpha-hydroxy analogs of leucine, isoleucine and valine by chicks and rats. J Nutr. Oct 1982;112(10):1929-39. doi:10.1093/jn/112.10.1929

58. Tischler ME, Desautels M, Goldberg AL. Does leucine, leucyl-tRNA, or some metabolite of leucine regulate protein synthesis and degradation in skeletal and cardiac muscle? J Biol Chem. Feb 25 1982;257(4):1613-21.

59. Lindgren S, Jonsson U, Sandberg G, Granelli K, inventors; Kabivitrum AB, assignee. Energy substrate containing hydroxycarboxylic acid and a glycerol ester. patent EP 367734 A1. patent application 89850290.1. 06.09.1989 1990.

60. Lindgren S, Sandberg G, Enekull U, Werner T, inventors; Kabivitrum Ab, assignee. Energy substrate containing hydroxycarboxylic acid. patent EP 363337 A1. patent application 89850292.7. 1990.

61. Hietala P, Karila TAM, Seppälä TA, Tähtivuori K, inventors; Nutrient supplement and use of the same. Finland patent application PCT/FI2005/050365. 2005.

62. Westermarck HW, Hietala P, Jaarma M, Sorsa T, Vaara M, inventors; Exracta Oy, assignee. Use of alpha-hydroxy acids in the manufacture of a medicament for the treatment of inflammation. patent application PCT/FI96/00363. 1997.

63. Westermarck HW, Apajalahti J, Hietala P, Jaarma M, inventors; Oy Extracta Ltd, assignee. Use of hydroxy acid or a product containing the same in animal feed. patent US 6,203,835 B1. patent application 08/981,108. 2001.

64. Lang CH, Pruznak A, Navaratnarajah M, et al. Chronic alpha-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy. Am J Physiol Endocrinol Metab. Aug 1 2013;305(3):E416-28. doi:10.1152/ajpendo.00618.2012

65. Mamer OA, Laschic NS, Scriver CR. Stable isotope dilution assay for branched chain alpha-hydroxy-and alpha-ketoacids: serum concentrations for normal children. Biomed Environ Mass Spectrom. Oct 1986;13(10):553-8. doi:10.1002/bms.1200131007

66. Lancaster G, Mamer OA, Scriver CR. Branched-chain alpha-keto acids isolated as oxime derivatives: relationship to the corresponding hydroxy acids and amino acids in maple syrup urine disease. Metabolism. Mar 1974;23(3):257-65. doi:10.1016/0026-0495(74)90064-x

67. Jakobs C, Sweetman L, Nyhan WL. Hydroxy acid metabolites of branched-chain amino acids in amniotic fluid. Clin Chim Acta. Jul 16 1984;140 (2):157-66. doi:10.1016/0009-8981(84)90340-1

68. Ehling S, Reddy TM. Direct analysis of leucine and its metabolites β-hydroxy-β-methylbutyric acid, α-ketoisocaproic acid, and α-hydroxyisocaproic acid in human breast milk by liquid chromatography–mass spectrometry. Journal of agricultural and food chemistry. Sep 2 2015;63(34):7567-7573. doi:10.1021/acs.jafc.5b02563

69. Ward ME, Politzer IR, Laseter JL, Alam SQ. Gas chromatographic mass spectrometric evaluation of free organic acids in human saliva. Biomed Mass Spectrom. Apr 1976;3(2):77-80. doi:10.1002/bms.1200030207

70. Ehling S, Reddy TM. Investigation of the presence of beta-hydroxy-beta-methylbutyric acid and alpha-hydroxyisocaproic acid in bovine whole milk and fermented dairy products by a validated liquid chromatography-mass spectrometry method. J Agric Food Chem. Feb 19 2014;62(7):1506-11. doi:10.1021/jf500026s

71. Yamamoto A. STUDIES ON FLAVORS OF SAKE. 2. SEPARATION AND IDENTIFICATION OF HYDROXY CARBONIC ACID. NIPPON NOGEI KAGAKU KAISHI. 1961;35(7):619.

72. Van Wyk CJ, Kepner RE, Webb AD. Some Volatile Components of Vitis Vinifera Variety White Riesling. 2. Organic Acids Extracted from Wine. Journal of Food Science. 2006;32(6):664-668. doi:10.1111/j.1365-2621.1967.tb00859.x

73. Begemann WJ, Harkes PD, inventors; Process for enhancing a fresh cheese flavour in foods U.S. patent US3853996. 1974b.

74. Smit BA, Engels WJ, Wouters JT, Smit G. Diversity of L-leucine catabolism in various microorganisms involved in dairy fermentations, and identification of the rate-controlling step in the formation of the potent flavour component 3-methylbutanal. Appl Microbiol Biotechnol. Apr 2004;64(3):396-402. doi:10.1007/s00253-003-1447-8

75. Tressl R, Kossa T, Renner R, Koeppler H. Gas chromatographic-mass spectrometric investigation of volatile compounds of hops, wort, and beer and their formation. I. Organic acids in wort and beer. Monatsschrift fuer Brauerei. 1975; 28(5):109-118. doi:10.1007/BF01274254

76. Broadbent JR, Gummalla S, Hughes JE, Johnson ME, Rankin SA, Drake MA. Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese. Appl Environ Microbiol. Aug 2004;70(8):4814-20.

77. Axel C, Brosnan B, Zannini E, Furey A, Coffey A, Arendt EK. Antifungal sourdough lactic acid bacteria as biopreservation tool in quinoa and rice bread. Int J Food Microbiol. Dec 19 2016;239 :86-94. doi:10.1016/j.ijfoodmicro.2016.05.006

78. Loh LX, Ng DHJ, Toh M, Lu Y, Liu SQ. Targeted and Nontargeted Metabolomics of Amino Acids and Bioactive Metabolites in Probiotic-Fermented Unhopped Beers Using Liquid Chromatography High-Resolution Mass Spectrometry. J Agric Food Chem. Nov 24 2021;69 (46):14024-14036. doi:10.1021/acs.jafc.1c03992

79. Mei S, He Z, Zhang J. Identification and analysis of major flavor compounds in radish taproots by widely targeted metabolomics. Front Nutr. 2022;9:889407. doi:10.3389/fnut.2022.889407

80. Friedrich M, Murer H, Sterchi E, Berger EG. Transport of L-leucine hydroxy analogue and L-lactate in human small intestinal brush border membrane vesicles. Eur J Clin Invest. Feb 1992;22 (2):73-8. doi:10.1111/j.1365-2362.1992.tb01939.x

81. Mann GE, Pearson JD, Sheriff CJ, Toothill VJ. Expression of amino acid transport systems in cultured human umbilical vein endothelial cells. J Physiol. Mar 1989;410:325-39. doi:10.1113/jphysiol.1989.sp017535

82. Abumrad NN, Wise KL, Williams PE, Abumrad NA, Lacy WW. Disposal of alpha-ketoisocaproate: roles of liver, gut, and kidneys. Am J Physiol. Aug 1982;243(2):E123-31. doi:10.1152/ajpendo.1982.243.2.E123

83. Sumi K, Sakuda M, Munakata K, Nakamura K, Ashida K. alpha-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFalpha/ IFNgamma Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube. Nutrients. Jul 13 2021;13(7)doi:10.3390/nu13072391

84. Schauder P, Matthaei D, Henning HV, Scheler F, Langenbeck U. Blood levels of branched-chain amino acids and alpha-ketoacids in uremic patients given keto analogues of essential amino acids. Am J Clin Nutr. Jul 1980;33(7):1660-6. doi:10.1093/ajcn/33.7.1660

85. Matthews DE, Harkin R, Battezzati A, Brillon DJ. Splanchnic bed utilization of enteral alpha-ketoisocaproate in humans. Metabolism. Dec 1999;48(12):1555-63. doi:10.1016/s0026-0495(99)90245-7

86. Selis D, Pande Y, Smoczer C, et al. Cytotoxicity and Genotoxicity of a New Intracanal Medicament, 2-hydroxyisocaproic Acid-An In Vitro Study. J Endod. May 2019;45(5):578-583. doi:10.1016/j.joen.2019.01.012

87. Smoczer C, Park YK, Herrington JB, et al. A Potential Intracanal Medicament, 2-Hydroxyisocaproic Acid (HICA): Cytotoxicity, Genotoxicity, and Its Effect on SCAP Differentiation. Dentistry Journal. 2023;11(12):270. doi:10.3390/dj11120270

88. Malfait A, Tortorella M. The “elusive DMOAD”: Aggrecanase inhibition from laboratory to clinic. Clin Exp Rheumatol. 2019;37(Suppl 120):S130-34.

89. Guilak F, Nims RJ, Dicks A, Wu C-L, Meulenbelt I. Osteoarthritis as a disease of the cartilage pericellular matrix. Matrix Biology. 2018;71:40-50.

90. Losina E, Thornhill TS, Rome BN, Wright J, Katz JN. The dramatic increase in total knee replacement utilization rates in the United States cannot be fully explained by growth in population size and the obesity epidemic. J Bone Joint Surg Am. Feb 1 2012;94(3):201-7. doi:10.2106/JBJS.J.01958

91. Little CB, Hunter DJ. Post-traumatic osteoarthritis: from mouse models to clinical trials. Nat Rev Rheumatol. Aug 2013;9(8):485-97. doi:10.1038/nrrheum.2013.72

92. Cinque ME, Dornan GJ, Chahla J, Moatshe G, LaPrade RF. High Rates of Osteoarthritis Develop After Anterior Cruciate Ligament Surgery: An Analysis of 4108 Patients. Am J Sports Med. Jul 2018;46(8):2011-2019. doi:10.1177/0363546517730072

93. Oiestad BE, Holm I, Aune AK, et al. Knee function and prevalence of knee osteoarthritis after anterior cruciate ligament reconstruction: a prospective study with 10 to 15 years of follow-up. Am J Sports Med. Nov 2010;38(11):2201-10. doi:10.1177/0363546510373876

94. Oiestad BE, Holm I, Engebretsen L, Risberg MA. The association between radiographic knee osteoarthritis and knee symptoms, function and quality of life 10-15 years after anterior cruciate ligament reconstruction. Br J Sports Med. Jun 2011;45(7):583-8. doi:10.1136/bjsm.2010.073130

95. Barenius B, Ponzer S, Shalabi A, Bujak R, Norlen L, Eriksson K. Increased risk of osteoarthritis after anterior cruciate ligament reconstruction: a 14-year follow-up study of a randomized controlled trial. Am J Sports Med. May 2014;42(5):1049-57. doi:10.1177/0363546514526139

96. Bluteau G, Gouttenoire J, Conrozier T, et al. Differential gene expression analysis in a rabbit model of osteoarthritis induced by anterior cruciate ligament (ACL) section. Biorheology. 2002;39(1-2):247-58.

97. Malemud CJ, Islam N, Haqqi TM. Pathophysiological mechanisms in osteoarthritis lead to novel therapeutic strategies. Cells Tissues Organs. 2003;174(1-2):34-48. doi:10.1159/000070573

98. Glasson SS, Askew R, Sheppard B, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 2005;434(7033):644.

99. Heim-Riether A, Taylor SJ, Liang S, et al. Improving potency and selectivity of a new class of non-Zn-chelating MMP-13 inhibitors. Bioorg Med Chem Lett. Sep 15 2009;19(18):5321-4. doi:10.1016/j.bmcl.2009.07.151