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| 243P London, UK Pharmacology 2016 |
Power analysis of murine mBSA delayed type hypersensitivity: therapeutic and prophylactic dexamethasone as positive control.
Introduction
The 3Rs are legislated in the EU Animal Welfare Directive 2010/63/EU and the Animal (Scientific Procedures) Act, 1986. We are investigating the statistical power of inflammation models (Bashford & Seed, 2016). Murine methylated bovine serum albumin (mBSA) type-IV hypersensitivity is a classical integrative model of immunity, comprising both acute (immune complex) and chronic (cell mediated) phases, used in immune suppressant research. Dexamethasone (DEX) is a potent anti-inflammatory steroid used to calibrate maximum effect. Drug action on the sensitisation and challenge steps can be determined through prophylactic dosing or immediately prior to challenge. Detailed time courses are demanding however.
Method
Two archive time courses of 5 groups were chosen. Female c57/bl6 mice (n=10) were immunised under anaesthesia (100μL Freund’s complete adjuvant/1mg mBSA 1:1 emulsion, tail base s.c.) and subplantar challenge (1mg mBSA/100uL) at 14 days. 3.0mg/kg DEX and 3 drug groups were administered p.o. prophylactically (days 0-14, PDEX) or therapeutically (-24hours, TDEX). Increase in paw swelling over contralateral control was measured blind by plethysmography (Ugo Basil) six times, from 0 to 48 hours (mean±sem, 2-way ANOVA (Prism-v7). Mean DEX difference was calculated (Vehicle-DEX). Power for single time points was assessed using PASS 14 (2015, NCSS, USA) for Dunnett;s test, and Minimum Detectable Difference (MDD) at σ=0.05. PPL and PIL (ASPA, 1986), Queen Mary College University of London.
Results:
Actual
Calculated
| Groupshr | Control | DEX difference | 2-Way ANOVA | Power | MDD α=0.05 | N, DEX α=0.05 | N, Drug (50%Dex) |
| PDEX 6 | 0.056±0.05 | -0.036(64.3%) | P<0.001 | 0.8099 | 0.04 | 12 | 53 |
| 24 | 0.111±0.013 | -0.049(44.1%) | P<0.001 | 0.664 | 0.1 | 40 | 143 |
| 48 | 0.117±0.011 | -0.063(53.9%) | P<0.001 | 0.875 | 0.09 | 18 | 310 |
| TDEX 6 | 0.047±0.007 | -0.011(75.6%) | P<0.01 | 0.7289 | 0.05 | 12 | 40 |
| 24 | 0.087±0.012 | -0.076(12.6%) | NS | 0.102 | |||
| 48 | 0.081±0.008 | -0.051(37.0%) | P<0.05 | 0.615 | 0.06 | 15 | 55 |
Table 1: Difference in DTH paw inflammation with PDEX and TDEX treatment, and the extrapolated values for power, MDD, n to equate the DEX response, and 50% DEX response for individual times.
Conclusions
DEX does not maximally inhibit DTH inflammation with either protocol. The DEX difference is lower and betters the MDD only at 6hrs. Restricting to a single time increases ‘n’ to 12 to reach p<0.05, and is higher for 24 and 48 hours. For a drug to reach significance at half the maximal effect of DEX, the ethical cost is too high.
References
E Bashford-Snell, M Seed. pA2-Online 13(3):410(P).