In vivo study blinding and bias

The in vivo studies were conducted as randomized, blinded, and controlled animal trials. Animals were allocated to different treatment groups at random using envelopes containing specifics codes, in order to minimize bias and control variation. The investigator administering the treatments and conducting the preclinical testing throughout the study was blinded to the treatment allocation. A major limitation to the blinding, which we had expected, was that diclofenac 1% gel (Voltaren®) had to be applied three times a day in accordance with the manufacturer's instructions, whereas the other treatments were applied once daily. The main purpose of the study was to evaluate the effect of loperamide liposomal gel in an animal model of chronic inflammatory pain following once daily dosing. This is to compare whether our once daily formulation would be more effective than standard topical NSAIDs, which must be applied three or four times daily for efficacy. The effect of NSAIDs on pain and inflammation is already well-established in the literature and clinic, and we have used it as a positive control in our previous studies (Hua and Cabot, 2013; Iwaszkiewicz and Hua, 2014). In addition, the no treatment and naive control groups also affected the blinding, as they did not receive any treatments throughout the study. Despite these limitations, the most important controls that required blinding in this study were the empty liposomal gel and free loperamide gel groups, in order to assess the effect of the individual components of the loperamide liposomal gel formulation. The loperamide liposomal gel, empty liposomal gel, and free loperamide gel were identical in dosing regimen and appearance. Results of the preclinical testing were also supported by histological analysis to further reduce any bias, and this was performed by investigators blinded to the treatment allocation and hypothesis of the study. The blinded investigators conducting the histological analysis were different to the investigator conducting the preclinical testing.