Animal Studies
The goal of the studies described herein are to develop first-in-class agents for the chemoprevention of colorectal cancer in humans. Rodents are widely regarded as essential models for early pharmacology and toxicology studies, and mice were chosen owing to extensive published data demonstrating CRC chemoprevention by conventional PDE5i. For all animal studies, six-week-old C57BL6 mice were purchased from Jackson Laboratories (Bar Harbor, ME), and allowed to acclimate to the Augusta University animal facility for at least 2 weeks prior to experimentation. Mice were housed in shoebox cages with 5 mice of the same sex per cage with water and irradiated rodent chow (Teklad 8904)ad libitum . Cages were maintained in conventional specific pathogen free environment at 24°C, and a 12 h light/dark cycle. On completion of experiments or at any signs of morbidity, mice were euthanized by controlled CO2 delivery for humane asphyxiation followed by decapitation. The experimental design included equal numbers of randomly chosen male and female mice for the proof-of-principle pharmacokinetic studies, but only male mice were used for the surgical procedure and for the colon proliferation studies since our previous studies have found no difference in the response to cGMP elevation on colon homeostasis or colon cancer prevention. Statistical analysis was only undertaken for studies where each group size was at least n = 5. All studies carried out with animals were approved by the Augusta University Institutional Animal Care and Use Committee. Descriptions of animal studies are compliant with the ARRIVE guidelines (Percie du Sert et al. , 2020) and recommendations made by the British Journal of Pharmacology (Lilley et al. , 2020).