Inflammopharmacology. 2021 Oct;29(5):1261-1278. doi: 10.1007/s10787-021-00871-2. Epub 2021 Sep 12.

Limitations of drug concentrations used in cell culture studies for understanding clinical responses of NSAIDs.

Inflammopharmacology

Garry G Graham, Kieran F Scott

PMID: 34510275 DOI: 10.1007/s10787-021-00871-2

Abstract

In this review, the in vitro cellular effects of six nonsteroidal anti-inflammatory drugs (NSAIDs), salicylate, ibuprofen, naproxen, indomethacin, celecoxib and diclofenac, are examined. Inhibition of prostanoid synthesis in vitro generally occurs within the therapeutic range of plasma concentrations that are observed in vivo, consistent with the major action of NSAIDs being inhibition of prostanoid production. An additional probable cellular action of NSAIDs has been discovered recently, viz. decreased oxidation of the endocannabinoids, 2-arachidonoyl glycerol and arachidonyl ethanolamide. Many effects of NSAIDs, other than decreased oxidation of arachidonic acid and endocannabinoids, have been put forward but almost all of these additional processes are observed at supratherapeutic concentrations when the concentration of albumin, the major protein that binds NSAIDs, is taken into account. However, one exception is salicylate, a very potent inhibitor of the neutrophilic enzyme, myeloperoxidase, the inhibition of which leads to reduced production of the inflammatory mediator, hypochlorous acid, and inhibition of the inflammation associated with rheumatoid arthritis.

© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.

Keywords: 2-Arachidonoyl glycerol; Albumin; Arachidonoyl ethanolamide; Celecoxib; Cyclooxygenase; Diclofenac; Enantiomer; Ibuprofen; Indomethacin; Myeloperoxidase; Naproxen; Non-steroidal anti-inflammatory drug; Protein binding; Racemate; Salicylate; Therapeutic plasma concentrations

References

1. Aggarwal S, Taneja N, Lin L et al (2000) Indomethacin-induced apoptosis in oesophageal adenocarcinoma cells involves upregulation of Bax and translocation of mitochondrial cytochrome C independent of COX-2 expression. Neoplasia 2(4):346–356 - PubMed
2. Alper AB Jr, TomLin H, Sadhwani U et al (2006) Effects of the selective cyclooxygenase-2 inhibitor analgesic celecoxib on renal carbonic anhydrase enzyme activity: a randomized, controlled trial. Am J Ther 13:229–235 - PubMed
3. Arisan ED, Ergul Z, Bozdag G (2018) Diclofenac induced apoptosis via altering PI3K/Akt/MAPK signaling axis in HCT 116 more efficiently compared to SW480 colon cancer cells Mol. Biol Rep 45(6):2175–2184 - PubMed
4. Avcıkurt AS, Oğuzhan Korkut O (2018) Effect of certain non-steroidal anti-inflammatory drugs on the paraoxonase 2 (PON2) in human monocytic cell line U937. Arch Physiol Biochem 124(4):378–382 - PubMed
5. Baba M, Yuasa S, Niwa T et al (1993) Effect of human serum on the in vitro anti-HIV-1 activity of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) derivatives as related to their lipophilicity and serum protein binding. Biochem Pharmacol 45(12):2507–2512 - PubMed
6. Bao W, Luo Y, Wang D et al (2018) Sodium salicylate modulates inflammatory responses through AMP-activated protein kinase activation in LPS-stimulated THP-1 cells. J Cell Biochem 119(1):850–860 - PubMed
7. Bayoumi AE, Perez-Pertejo Y, Zidan HZ et al (2003) Cytotoxic effects of two antimolting insecticides in mammalian CHO-K1 cells. Ecotoxicol Environ Saf 55(1):19–23 - PubMed
8. Beaven MA, Bayer BM (1980) Factors influencing the uptake and disposition of indomethacin-[14C] in cell cultures. Biochem Pharmacol 29(14):2055–2061 - PubMed
9. Bensen RC, Standke SJ, Colby et al (2021) Single cell mass spectrometry quantification of anticancer drugs: proof of concept in cancer patients. ACS Pharmacol Transl Sci 2021(4):96–100 - PubMed
10. Brody TM (1956) Action of sodium salicylate and related compounds on tissue metabolism in vitro. J Pharmacol Exp Ther 117(1):39–51 - PubMed
11. Burczynski FJ, Wang GQ, Elmadhoun B et al (2001) Hepatocyte [3H]-palmitate uptake: effect of albumin surface charge modification. Can J Physiol Pharmacol 79(10):868–875 - PubMed
12. Chae HJ, Chae SW, Reed JC, Kim HR (2004) Salicylate regulates COX-2 expression through ERK and subsequent NF-kappaB activation in osteoblasts. Immunopharmacol Immunotoxicol 26(1):75–91 - PubMed
13. Chan KK, Vyas KH, Brandt KD (1987) In vitro protein binding of diclofenac sodium in plasma and synovial fluid. J Pharm Sci 76(2):105–108 - PubMed
14. Chang CY, Li JR, Wu CC et al (2018) Indomethacin induced glioma apoptosis involving ceramide signals. Exp Cell Res 365(1):66–77 - PubMed
15. Cieslik KA, Zhu Y, Shtivelband M, Wu KK (2005) Inhibition of p90 ribosomal S6 kinase-mediated CCAAT/enhancer-binding protein beta activation and cyclooxygenase-2 expression by salicylate. J Biol Chem 280(18):18411–18417 - PubMed
16. Davies NM, McLachlan AJ, Day RO, Williams KM (2000) Clinical pharmacokinetics and pharmacodynamics of celecoxib: a selective cyclo-oxygenase-2 inhibitor. Clin Pharmacokinet 38(3):225–242 - PubMed
17. de Vries BJ, van den Berg WB, Vitters E, van de Putte LB (1986) The effect of salicylate on anatomically intact articular cartilage is influenced by sulfate and serum in the culture medium. J Rheumatol 13(4):686–693 - PubMed
18. Denoon T, Sunilkumar S, Ford S (2020) Acetoacetate enhances oxidative metabolism and response to toxicants of cultured kidney cells. Toxicol Lett 280(1):48–56 - PubMed
19. Dong Z, Huang C, Brown RE, Ma WY (1997) Inhibition of activator protein 1 activity and neoplastic transformation by aspirin. J Biol Chem 272(15):9962–9970 - PubMed
20. Duffield-Lillico AJ, Boyle JO, Zhou XK (2009) Levels of prostaglandin E metabolite and leukotriene E(4) are increased in the urine of smokers: evidence that celecoxib shunts arachidonic acid into the 5-lipoxygenase pathway. Cancer Prev Res 2(4):322–329 - PubMed
21. Duggan KC, Hermanson DJ, Musee J et al (2011) (R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2. Nat Chem Biol 7(11):803–809 - PubMed
22. Edelman MJ, Wang X, Hodgson L et al (2017) Phase III randomized, placebo-controlled, double-blind trial of celecoxib in addition to standard chemotherapy for advanced non-small-cell lung cancer with cyclooxygenase-2 overexpression: CALGB 30801 (alliance). J Clin Oncol 35(19):2184–2192 - PubMed
23. Emoto A, Ushigome F, Koyabu N et al (2002) H - PubMed
24. Evans AM, Nation RL, Sansom LN et al (1991) Effect of racemic ibuprofen dose on the magnitude and duration of platelet cyclo-oxygenase inhibition: relationship between inhibition of thromboxane production and the plasma unbound concentration of S(+)-ibuprofen. Br J Clin Pharmacol 31(2):131–138 - PubMed
25. Evans AM, Nation RL, Sansom LN, Bochner F, Somogyi AA (1989) Stereoselective plasma protein binding of ibuprofen enantiomers. Eur J Clin Pharmacol 36(3):283–290 - PubMed
26. Ferreira SH, Moncada S, Vane JR (1971) Aspirin and indomethacin abolish prostaglandin release from the spleen. Nat New Biol 231:237–239 - PubMed
27. Foreman JE, Sorg JM, McGinnis KS (2009) Regulation of peroxisome proliferator-activated receptor-beta/delta by the APC/beta-CATENIN pathway and nonsteroidal antiinflammatory drugs. Mol Carcinog 48(10):942–952 - PubMed
28. Fujii Y, Suhara Y, Sukikara Y (2019) Elucidation of the interaction between flavan-3-ols and bovine serum albumin and its effect on their in-vitro cytotoxicity. Molecules 24:3667 - PubMed
29. Furst DE, Tozer TN, Melmon KL (1979) Salicylate clearance, the resultant of protein binding and metabolism. Clin Pharmacol Ther 26(3):380–389 - PubMed
30. Geary TG, Akood MA, Jensen JB (1983) Characteristics of chloroquine binding to glass and plastic. Am J Trop Med Hyg 32(1):19–23 - PubMed
31. Geisslinger G, Schaible HG (1996) New insights into the site and mode of antinociceptive action of flurbiprofen enantiomers. J Clin Pharmacol 36(6):513–520 - PubMed
32. Geisslinger G, Menzel-Sollowek S, Beck WS, Brune K (1993) R-flurbiprofen isomeric ballast or active entity of the racemic compound? Variability in response to anti-rheumatic drugs. Agents Actions 44(Suppl):31–36 - PubMed
33. Gierse JK, Hauser SD, Creely DP et al (1995) Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase. Biochem J 305(2):479–484 - PubMed
34. Gillette JR (1965) Reversible binding as a complication in relating the in vitro effect of drugs to their in in vivo activity. Drugs Enzymes 4:9–22 - PubMed
35. Günsberg M, Bochner F, Graham G, Imhoff D, Parsons G, Cham B (1984) Disposition of and clinical response to salicylates in patients with rheumatoid disease. Clin Pharmacol Ther 35(5):585–593 - PubMed
36. Hallbrink M, Oehlke J, Papsdorf G, Bienert M (2004) Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration. Biochim Biophys Acta 1667(2):222–228 - PubMed
37. Hamberg M (1972) Inhibition of prostaglandin synthesis in man. Biochem Biophys Res Commun 49(3):720–726 - PubMed
38. Hawley SA, Fullerton MD, Ross FA et al (2012) The ancient drug salicylate directly activates AMP-activated protein kinase. Science 336(6083):918–922 - PubMed
39. Higgs GA, Salmon JA, Henderson B, Vane JR (1987) Pharmacokinetics of aspirin and salicylate in relation to inhibition of arachidonate cyclooxygenase and antiinflammatory activity. Proc Natl Acad Sci USA 84(5):1417–1420 - PubMed
40. Housby JN, Cahill CM, Chu B et al (1999) Non-steroidal anti-inflammatory drugs inhibit the expression of cytokines and induce hsp70 in human monocytes. Cytokine 11(2):347–358 - PubMed
41. Ikegaki N, Hicks SL, Regan PL, Jacobs (2014) S(+)-ibuprofen destabilizes MYC/MYCN and AKT, increases p53 expression, and induces unfolded protein response and favorable phenotype in neuroblastoma cell lines. Int Oncol 44(1):35–43 - PubMed
42. Janssen A, Maier TJ, Schiffmann S et al (2006) Evidence of COX-2 independent induction of apoptosis and cell cycle block in human colon carcinoma cells after S- or R-ibuprofen treatment. Eur J Pharmacol 540(1–3):24–33 - PubMed
43. Jaradat MS, Wongsud B, Phornchirasilp S et al (2001) Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H2 synthases by ibuprofen, naproxen and indomethacin. Biochem Pharmacol 62(12):1587–1595 - PubMed
44. Johnson AJ, Hsu AL, Lin HP et al (2002) The cyclo-oxygenase-2 inhibitor celecoxib perturbs intracellular calcium by inhibiting endoplasmic reticulum Ca2+-ATPases: a plausible link with its anti-tumour effect and cardiovascular risks. Biochem J 366(3):831–837 - PubMed
45. Kawamori T, Rao CV, Seibert K, Reddy BS (1998) Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res 58(3):409–412 - PubMed
46. Kettle AJ, Winterbourn CC (1991) Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs. Biochem Pharmacol 41(10):1485–1492 - PubMed
47. Knudsen JF, Carlsson U, Hammarstrom P et al (2004) The cyclooxygenase-2 inhibitor celecoxib is a potent inhibitor of human carbonic anhydrase II. Inflammation 28(5):285–290 - PubMed
48. Kokoska ER, Smith GS, Deshpande Y et al (1998) Indomethacin increases susceptibility to injury in human gastric cells independent of PG synthesis inhibition. Am J Physiol G275(4):G620-628 - PubMed
49. Lee EJD, Williams K, Day R, Graham G, Champion D (1985) Stereoselective disposition of ibuprofen enantiomers in man. Br J Clin Pharmacol 19:669–674 - PubMed
50. Lejal N, Tarus B, Bouguyon E et al (2013) Structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza a virus. Antimicrob Agent Chemother 57(5):2231–2242 - PubMed
51. Lin JH, Cocchetto DM, Duggan DE (1987) Protein binding as a primary determinant of the clinical pharmacokinetic properties of non-steroidal anti-inflammatory drugs. Clin Pharmacokinet 12(6):402–432 - PubMed
52. Lucas S (2016) Indomethacin. Headache 56(2):436–446 - PubMed
53. Lucena G, Reyes-Botella C, García-Martínez O et al (2016) Effect of NSAIDs on the aminopeptidase activity of cultured human osteoblasts. Mol Cell Endocrinol 426:146–154 - PubMed
54. Madunic J, Horvat L, Majstorovic I et al (2017) Sodium salicylate inhibits urokinase. Activity in MDA MB-231 breast cancer cells. Clin Breast Cancer 17(8):629–637 - PubMed
55. Manzano-Moreno FJ, Costela-Ruiz VJ, Melguizo-Rodríguez L et al (2018) Inhibition of VEGF gene expression in osteoblast cells by different NSAIDs. Arch Oral Biol 92:75–78 - PubMed
56. Mitchell JA, Akarasereenont P, Thiemermann C et al (1993) Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc Natl Acad Sci USA 90(24):11693–11697 - PubMed
57. Mitchell JA, Saunders M, Barnes PJ et al (1997) Sodium salicylate inhibits cyclo-oxygenase-2 activity independently of transcription factor (nuclear factor κB) Role of arachidonic acid. Mol Pharmacol 51:907–912 - PubMed
58. Morgan AGM, Babu D, Michail K, Siraki AG (2017) An evaluation of myeloperoxidase-mediated bio-activation of NSAIDs in promyelocytic leukaemia (HL-60) cells for potential cytotoxic selectivity. Toxicol Lett 280(1):48–56 - PubMed
59. Mutter R, Lu B, Carbone DP et al (2009) A phase II study of celecoxib in combination with paclitaxel, carboplatin, and radiotherapy for patients with inoperable stage IIIA/B non-small cell lung cancer. Clin Cancer Res 15(6):2158–2165 - PubMed
60. Novakova I, Subileau EA, Toegel S et al (2014) Transport rankings of non-steroidal antiinflammatory drugs across blood-brain barrier in vitro models. PLoS One 9(1):e86806 - PubMed
61. Oliveira IM, Borges A, Borges F, Manuel Simoes M (2019) Repurposing ibuprofen to control Staphylococcus biofilms. Eur J Med Chem 166:197e205 - PubMed
62. Pantziarka P, Sukhatme V, Bouche G et al (2016) Repurposing drugs in oncology (ReDO)—diclofenac as an anti-cancer agent. Ecancermedicalscience 10:610 - PubMed
63. Parepally JM, Mandula H, Smith QR (2006) Brain uptake of nonsteroidal anti-inflammatory drugs: ibuprofen, flurbiprofen, and indomethacin. Pharm Res 23(5):873–881 - PubMed
64. Patrignani P, Panara MR, Greco A et al (1994) Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases. J Pharmacol Exp Ther 271(3):1705–1712 - PubMed
65. Paulson SK, Kaprak TA, Gresk CJ et al (1999) Plasma protein binding of celecoxib in mice, rat, rabbit, dog and human. Biopharm Drug Dispos 20(6):293–299 - PubMed
66. Rainsford KD (2015) Ibuprofen, discovery, development and therapeutics. John Wiley & Sons Ltd, Chichester - PubMed
67. Rosenkranz B, Fischer C, Meese CO, Frolich JC (1986) Effects of salicylic and acetylsalicylic acid alone and in combination on platelet aggregation and prostanoid synthesis in man. Br J Clin Pharmacol 21(3):309–317 - PubMed
68. Rowland M, Riegelman S, Harris PA, Sholkoff SD (1972) Absorption kinetics of aspirin in man following oral administration of an aqueous solution. J Pharm Sci 61(3):379–385 - PubMed
69. Rudkowski R, Graham GG, Champion GD, Ziegler JB (1990) The activation of gold complexes by cyanide produced by polymorphonuclear leukocytes. I. The effects of aurocyanide on the oxidative burst of polymorphonuclear leukocytes. Biochem Pharmacol 39(11):1687–1695 - PubMed
70. Seyberth HW, Sweetman BJ, Frolich JC, Oates JA (1976) Quantification of the major urinary metabolite of the E prostaglandins by mass spectrometry: evaluation of the method’s application to clinical studies. Prostaglandins 11(2):381–397 - PubMed
71. Shaw RJ, Cantley LC (2012) Ancient sensor for ancient drug. Science 336:813–814 - PubMed
72. Song D, Hsu LF, Au JLS (1996) Binding of taxol to plastic and glass containers and protein under in vitro conditions. J Pharm Sci 85(1):29–31 - PubMed
73. Stamp LK, Khalilova I, Tarr JM, Senthilmohan R, Turner R, Haigh RC, Winyard PG, Kettle AJ (2012) Myeloperoxidase and oxidative stress in rheumatoid arthritis. Rheumatology 51:1796–1803 - PubMed
74. Stevenson MA, Zhao MJ, Asea A et al (1999) Salicylic acid and aspirin inhibit the activity of RSK2 kinase and repress RSK2-dependent transcription of cyclic AMP response element binding protein- and NF-kappa B-responsive genes. J Immunol 163(10):5608–5616 - PubMed
75. Stichtenoth DO, Tsikas D, Gutzki FM, Frölich JC (1996) Effects of ketoprofen and ibuprofen on platelet aggregation and prostanoid formation in man. Eur J Clin Pharmacol 51:231–234 - PubMed
76. Stichtenoth DO, Marhauer V, Tsikas D et al (2005) Effects of specific COX-2-inhibition on renin release and renal and systemic prostanoid synthesis in healthy volunteers. Kidney Int 68(5):2197–2220 - PubMed
77. Syggelos SA, Giannopoulou E, Gouvousis PA et al (2007) In vitro effects of non-steroidal anti-inflammatory drugs on cytokine, prostanoid and matrix metalloproteinase production by interface membranes from loose hip or knee endoprostheses. Osteoarthr Cartil 15(5):531–542 - PubMed
78. Szebeni J, Weinstein JN (1991) Dipyridamole binding to proteins in human plasma and tissue culture media. J Lab Clin Med 117(6):485–492 - PubMed
79. Tognon G, Frapolli R, Zaffaroni M et al (2004) Fetal bovine serum, but not human serum, inhibits the in vitro cytotoxicity of ET-743 (Yondelis, trabectedin), an example of potential problems for extrapolation of active drug concentrations from in vitro studies. Cancer Chemother Pharmacol 53(1):89–90 - PubMed
80. Tran POT, Gleason CE, Robertson RP (2002) Inhibition of interleukin-1β-induced COX-2 and EP3 gene expression by sodium salicylate enhances pancreatic islet β-cell function. Diabetes 51:1772–1778 - PubMed
81. Tuynman JB, Vermeulen L, Boon EM et al (2008) Cyclooxygenase-2 Inhibition Inhibits c-met kinase activity and wnt activity in colon cancer. Cancer Res 68(4):1213–1220 - PubMed
82. van Wijngaarden J, van Beek E, van Rossum G et al (2007) Celecoxib enhances doxorubicin-induced cytotoxicity in MDA-MB231 cells by NF-kappaB-mediated increase of intracellular doxorubicin accumulation. Eur J Cancer 43(2):433–442 - PubMed
83. Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs. Nat New Biol 231:232–235 - PubMed
84. Villanueva M, Heckenberger R, Strobach M, Palmer M, Schror K (1993) Equipotent inhibition by R(-)-, S(+)- and racemic ibuprofen of human polymorphonuclear cell function in vitro. Br J Clin Pharmacol 35:235–242 - PubMed
85. Vital-Reyes VS, Rodrıguez-Burford C, Oelschlager DK et al (2006) Cell density influences the effect of celecoxib in two carcinoma cell lines. Biotech Histochem 81(1):51–54 - PubMed
86. Wagner S, Lang S, Popp T et al (2019) Evaluation of selective and non-selective cyclooxygenase inhibitors on sulfur mustard-induced pro-inflammatory cytokine formation in normal human epidermal keratinocytes. Toxicol Lett 312:109–117 - PubMed
87. Wang J-L, Lin K-L, Chou C-T et al (2012) Effect of celecoxib on Ca - PubMed
88. Wang C, Wang F, Lin F et al (2019) Naproxen attenuates osteoarthritis progression through inhibiting the expression of prostaglandin-endoperoxide synthase. J Cell Physiol 234(8):12771–12785 - PubMed
89. Warner TD, Giuliano F, Vojnovic I et al (1999) Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci USA 96(13):7563–7568 - PubMed
90. Warner TD, Vojnovic I, Bishop-Bailey D et al (2006) Influence of plasma protein on the potencies of inhibitors of cyclooxygenase-1 and -2. FASEB J 20(3):542–544 - PubMed
91. Waskewich C, Blumenthal RD, Li H et al (2002) Celecoxib exhibits the greatest potency amongst cyclooxygenase (COX) inhibitors for growth inhibition of COX-2-negative hematopoietic and epithelial cell lines. Cancer Res 62(7):2029–2033 - PubMed
92. Weber A, Casini A, Heine A et al (2004) Unexpected nanomolar inhibition of carbonic anhydrase by COX-2-selective celecoxib: new pharmacological opportunities due to related binding site recognition. J Med Chem 40:550–557 - PubMed
93. Whitehouse MW (1964) Biochemical properties of anti-inflammatory drugs–III. Uncoupling of oxidative phosphorylation in a connective tissue (cartilage) and liver mitochondria by salicylate analogues: relationship of structure to activity. Biochem Pharmacol 13:319–336 - PubMed
94. Williams CS, Watson AJ, Sheng H et al (2000) Celecoxib prevents tumor growth in vivo without toxicity to normal gut: lack of correlation between in vitro and in vivo models. Cancer Res 60(21):6045–6051 - PubMed
95. Willis JV, Kendall MJ, Jack DB (1981) The influence of food on the absorption of diclofenac after single and multiple oral doses. Eur J Clin Pharmacol 19:33–37 - PubMed
96. Wu GS, Zou SQ, Liu ZR et al (2003) Celecoxib inhibits proliferation and induces apoptosis via prostaglandin E2 pathway in human cholangiocarcinoma cell lines. World J Gastroenterol 9(6):1302–1306 - PubMed
97. Xu XM, Sansores-Garcia L, Chen XM (1999) Suppression of inducible cyclooxygenase 2 gene transcription by aspirin and sodium salicylate. Proc Natl Acad Sci USA 96:5292–5297 - PubMed
98. Ye CG, Wu WKK, Yeung JHK et al (2011) Indomethacin and SC236 enhance the cytotoxicity of doxorubicin in human hepatocellular carcinoma cells via inhibiting P-glycoprotein and MRP1 expression. Cancer Lett 304:90–96 - PubMed

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