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The purpose of this study was to investigate the influence of different methods of skin preservation on the percutaneous permeation of two drugs with varied physicochemical properties (melatonin and nimesulide). Hairless rat skin was freshly excised and immediately stored at three different storage conditions: (a) 4°C in minimal essential medium eagle (MEM), (b) frozen at −22°C and (c) frozen at −22°C in 10% glycerol (as a cryoprotective agent). The permeation of melatonin and nimesulide from saturated solutions was studied using the skin stored at various cold storage conditions for 6 months. At 4°C, the flux of melatonin was similar to fresh skin for up to 7 days (P>0.05) and increased 2.4-fold at 14 days (P<0.001). The flux of melatonin was similar to fresh skin for 14 days with skin at −22°C (P>0.05) and then steadily increased from 30 days onwards and at 180 days, the flux was 5-fold greater than fresh skin (P …