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


METHODS
We estimated the inhibitory response of reference compounds: 1400 W 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
1400 W 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.


CONCLUSIONS
The results proclaim that these cell-based assays can act as ideal tools for screening new anti-inflammatory/anti-allergic compounds.
Department of Cellular Assay, R&D Centre, Natural Remedies Pvt. Ltd, Veerasandra Industrial Area, Bangalore, India. cvc@naturalremedy.com
C V
CV
Chandrasekaran
R
R
Edwin Jothie
Preeti
P
Kapoor
Anumita
A
Gupta
Amit
A
Agarwal
D000818:Animals; D018926:Anti-Allergic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D000893:Anti-Inflammatory Agents / Q000008:administration & dosage / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D051381:Rats; D015203:Reproducibility of Results
D018926:Anti-Allergic Agents; D000893:Anti-Inflammatory Agents