Danoprevir, a potent, selective inhibitor of HCV NS3/4A protease, has a short half-life in humans. Therefore, the feasibility of a controlled release (CR) formulation to allow less frequent dosing was investigated using experimental approaches and physiological modeling to examine whether danoprevir is absorbed in the colon. Danoprevir absorption was studied in portal-vein-cannulated monkeys and in monkeys surgically modified to make intraduodenal, intrajejunal, intracolonic and oral administration possible. In portal-vein-cannulated monkeys, absorption was apparent up to 24 h after administration. The observed relative bioavailability from intracolonic delivery in the monkey was approximately 30% relative to oral administration, consistent with the model prediction of 40%. Human relative bioavailability for a tablet delivered to the colon compared with an immediate release (IR) formulation was predicted to be 4-28%. Preclinical data and modeling suggested that CR development would be challenging for this Biopharmaceutics Classification System Class IV compound. Therefore, a confirmative study in healthy volunteers was conducted to investigate the relative bioavailability of danoprevir in various regions of the gastrointestinal tract. In a randomized, open-label, crossover study, subjects received 100 mg danoprevir IR soft gel capsule, 100 mg danoprevir solution delivered to the distal small bowel and colon via an Enterion™ capsule (a remotely activated capsule for regional drug delivery) and 100 mg danoprevir powder to the colon via an Enterion™ capsule. The relative bioavailability of danoprevir (compared with IR) delivered to the colon was 6.5% for a solution and 0.6% for a powder formulation, indicating that a CR formulation is not feasible.