## 2 Results
### Viral Non-Structural Protein
#### 2.2.2 3C Protease  
As described previously, 3C protease cleaves the viral polyprotein at eight sites, including those to release 3 AB and 3A proteases [61]. Due to the role 3C protease plays in EV processing, it makes the protease an essential target against EV. Indeed, 3C-protease inhibitors tend to be more effective than envioxime and pleconaril, which were more effective than vapendavir and pirodavir [64]. The 3C protease is a protease found in picornaviruses and performs several functions. In coxsackieoviruses, 3C protease activates apoptosis via several caspases (caspase-3, caspase-8, and caspase-9) [65]. Moreover, in rhinoviruses, 3C protease targets OCT-1 transcription factor for proteolytic cleavage [65; 66]. These aspects make this a target to be favored. Promising molecules have been evaluated in clinical trials since discovering the role of 3C protease inhibitors. * Rupintrivir  
The historic 3C inhibitor of enterovirus is rupintrivir (Agouron Pharmaceuticals, Inc., San Diego, CA, USA), first synthesized in 1999 [67]. Three years later, a randomized, double-blind, placebo-controlled phase 1 clinical trial was published to evaluate the pharmacokinetic parameter of the RV inhibitor [53]. The drug was delivered to 36 male patients, ranging from 18 to 50 years, as a nasal spray solution containing 4 or 8 mg, one or six times per day, for 7 days. Rupintrivir was measured with low concentration (<0.52 ng/mL in plasma and <2% in nasal wash) at the end of treatment. Few metabolite concentrations were also observed. However, rupintrivir was identified as safe and well tolerated over time, with substantial drug detection for 9 h after administration. In 2003, a randomized, double-blind, placebo-controlled phase 2 clinical trial was published to evaluate the efficacy of rupintrivir against RV-A39 and RV-A21 infections [54]. The virus was administered to patients as nasal drops. A dose was given 6 h prior to the viral challenge for the prophylaxis study and 24 h after for the treatment. A total of 8 mg of the drug was delivered two or five times a day for 7 days to 202 patients (83 males and 119 females), ranging in age from 18 to 60 years, as a nasal spray solution. The active cohort had lower mean total daily symptoms than the placebo cohort (\(p\) = 0.014) and a higher incidence reduction on viral culture (\(p\) = 0.04-2 doses per day group/\(p\) = 0.03-5 doses per day group). It also has a decrease in viral titers (\(p\) < 0.05) but no significant difference in cold cases. The mean cumulative nasal discharge was lower for drug-treated patients (\(p\) = 0.05-2 doses per day group/\(p\) = 0.12-5 doses per day group). Finally, no significant local nasal adverse effects were declared. * V-7404  
In 2005, a phase 1 clinical trial was published to evaluate the pharmacokinetic parameters of anti-rhinoviral V-7404 (Pfizer Global Research and Development, San Diego, CA, USA), a rupintrivir derivative, administered in one single dose orally for fasted or fed patients [55; 68]. The oral route has more risk of adverse effects than the nasal route but can access other RV replication sites for diseases localized elsewhere than in the respiratory tract. A total of 14 male patients, ranging from 18 to 50 years, received 200, 500 (fasted or fed), 1000, or 2000 mg of drug. The study concluded that for 500 mg doses, the free maximum serum concentration (C\({}_{\text{max}}\)) was superior to the compound's half maximal effective concentration (EC50), inhibiting 80% of RV serotypes. In 2018, a randomized, double-blind, placebo-controlled phase 1 clinical trial was conducted to evaluate the V-7404 safety and pharmacokinetic parameters against EV infections [56]. For this study, 77 patients (33 males and 44 females) aged 18 to 45 years were recruited to receive an oral solution. Different weights (200, 500, 1000, and 2000 mg) were tested in one single dose of one or two doses per day for 2 weeks. V-7404 was well tolerated, even with multiple doses, and with acceptable safety.