Optimization of cell-based assays to quantify the anti-inflammatory/allergic potential of test substances in 96-well format

• Published in: Inflammopharmacology Vol. 19; no. 3; pp. 169 - 181
• Main Authors: Chandrasekaran, C. V., Edwin Jothie, R., Kapoor, Preeti, Gupta, Anumita, Agarwal, Amit
• Format: Journal Article
• Language: English
• Published: Basel SP Birkhäuser Verlag Basel 01.06.2011
• Subjects: Allergology; Animals; Anti-Allergic Agents - administration & dosage; Anti-Allergic Agents - pharmacology; Anti-Inflammatory Agents - administration & dosage; Anti-Inflammatory Agents - pharmacology; Biomedical and Life Sciences; Biomedicine; Cell Line; Cell Line, Tumor; Data Interpretation, Statistical; Dermatology; Drug Evaluation, Preclinical - methods; Gastroenterology; Humans; Immunology; Inhibitory Concentration 50; Mice; Pharmacology/Toxicology; Rats; Reproducibility of Results; Research Article; Rheumatology; Dexamethasone; CV; Captopril; Z′ factor; 1400W dihydrochloride; Ketotifen fumarate; Celecoxib; Acetylsalicylic acid; Inflammatory mediators
• ISSN: 0925-4692; 1568-5608
• DOI: 10.1007/s10787-010-0065-1

Objective There is an insistent need for robust, reliable, and optimized assays for screening novel drugs targeting the inflammatory/allergic markers. The present study describes about the optimization of eight cell-based assays utilizing mammalian cell lines in 96-well format for quantifying anti-inflammatory/allergic drug candidates. Materials and methods We estimated the inhibitory response of reference compounds: 1400W dihydrochloride on LPS-induced NO release, celecoxib on LPS-induced PGE 2 production and dexamethasone on LPS-induced pro-inflammatory cytokines IL-1 beta, IL-6, and TNF-alpha production by J774A.1 murine macrophages. Response of acetylsalicylic acid and celecoxib was studied on A23187-induced TXB 2 production; captopril on A23187-stimulated LTB 4 production by HL-60 cells. Effect of ketotifen fumarate was evaluated on A23187-elicited histamine release by RBL-2H3 cells. Each experiment was repeated twice to assess the reproducibility and suitability of the assays by determining appropriate statistical tools viz. %CV, S/B and Z′ factor. Results 1400W dihydrochloride was capable of inhibiting LPS-induced NO levels (IC 50  = 10.7 μM). Dexamethasone attenuated LPS-induced IL-1 beta (IC 50  = 70 nM), IL-6 (IC 50  = 58 nM) and TNF-alpha (IC 50  = 44 nM) release, whereas celecoxib, a specific COX-2 inhibitor showed marked reduction in LPS-induced PGE 2 (IC 50  = 23 nM) production. Captopril (IC 50  = 48 μM) and ketotifen fumarate (IC 50  = 36.4 μM) demonstrated potent inhibitory effect against A23187-stimulated LTB 4 and histamine levels, respectively. Both acetylsalicylic acid (IC 50  = 5.5 μM) and celecoxib (IC 50  = 7.9 nM) exhibited concentration-dependent decrease in TXB 2 production. Results for all the cell assays from two experiments showed a Z′ factor varying from 0.30 to 0.99; the S/B ratio ranged from 2.39 to 24.92; %CV ranged between 1.52 and 20.14. Conclusion The results proclaim that these cell-based assays can act as ideal tools for screening new anti-inflammatory/anti-allergic compounds.