*2.8. Statistical Analysis*

Values are represented as mean ± standard deviation (SD). A student's t-test was applied for statistical significance of differences (*p* < 0.05) and the statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA).

#### **3. Results and Discussion**

### *3.1. Pharmacokinetic Data*

The purpose of our study was to assess the effect of ticagrelor on plasma concentration-time profiles of tadalafil in rats. Drug-drug interactions may occur when CYP inducers or inhibitors are co-administered with the drug metabolized by the CYP enzyme. Several studies have demonstrated that ticagrelor is a CYP3A4 inhibitor and tadalafil is a CYP3A4 substrate [23,25]. Therefore, potential drug-drug interactions between ticagrelor and tadalafil can occur, affecting the pharmacokinetic profile, the efficacy for treatment of erectile dysfunction, and the frequency of side effects.

To identify the potential drug-drug interactions between ticagrelor and tadalafil in rats, ticagrelor was administered once a day for seven days to inhibit CYP3A in rats only for Group T. On the last day of the study, Group N and Group T received 2 mg/kg tadalafil via oral administration to assess the pharmacokinetics effect of ticagrelor on tadalafil. The plasma concentrations of ticagrelor were

sufficient to competitively inhibit CYP3A, and the effects of CYP3A inhibition on systemic exposure of tadalafil could be determined [36].

The pharmacokinetic profiles of tadalafil for Group N and Group T are shown in Figure 1. The parameters of the non-compartment analysis are listed in Table 1. The non-compartmental analysis is the model-independent method, which is based on the time course of drug concentrations. Pharmacokinetic parameters from the non-compartmental analysis are mainly used to evaluate drug exposure in oral administration of a drug. The systemic exposure of tadalafil was increased in Group T compared with Group N. Group T showed higher Cmax, AUC0–24, and AUC0-∞ than the Cmax, AUC0–24, and AUC0-∞ of Group N. These results showed significant increases in the values of AUC0–24 (1.61-fold, *p* < 0.05) and AUC0–∞ (1.66-fold, *p* < 0.05). The Cmax of Group T slightly increased 1.15-fold compared to that of Group N, but there was no significant difference (*p* = 0.3332). The ratio of ACU0–24 and AUC0–∞ between Group N and Group T exceeded the range of 0.8–1.25, where no pre-specified pharmacological effect was observed [37].

**Figure 1.** Plasma concentration-time profiles of tadalafil after oral administration to non-pretreated rats (Group N) and ticagrelor-pretreated rats (Group T). Values are represented as mean ± SD (n = 10). Dotted marks and dashed lines indicate the observed plasma concentration and the fitted pharmacokinetic profile from the one-compartment model, respectively. (**A**) Linear scale; (**B**) log scale.



a Ratio = *Value o f Group T Value o f Group N* ; maximum concentration (Cmax); area under the plasma concentration vs. time curve from 0 to 24 h (AUC0–24); area under the plasma concentration vs. time curve from 0 to infinity (AUC0–∞);time to reach maximal concentration (Tmax); half-life (T1/2); apparent total clearance (CL/F).

As a result of CYP inhibition by ticagrelor, the plasma concentration of tadalafil decreased more slowly after co-administration of ticagrelor and tadalafil than after tadalafil alone. Tmax, T1/2, and CL/F showed statistically significant differences between Groups T and Group N. In the case of Tmax, the absorption of tadalafil in Group T was delayed (Tmax of 3.22 ± 1.30 h) compared to that in Group N (Tmax of 1.45 ± 0.50 h) (*p* < 0.005). Thus, the tadalafil plasma concentration remained high until 8 h. This result indicated that the reduced metabolism of tadalafil by CYP3A inhibition caused the absorption of the drug for a longer period of time [38]. In particular, Group T showed an increased T1/2 compared to Group N (*p* < 0.005) and the CL/F value of Group T was significantly lower than that of Group N (*p* < 0.005). These results showed that the inhibition of hepatic CYP3A metabolism and the reduction of the first pass effect increased the exposure of tadalafil [39].

In general, the non-compartment analysis parameters of tadalafil showed statistically significant differences between Group N and Group T (Figure 2). In the present study, co-administration with ticagrelor increased tadalafil exposure and affected the AUC0–24, AUC0-<sup>∞</sup>, Tmax, and half-life of tadalafil. In particular, the half-life of tadalafil increased from 3.15 h in Group N to 4.47 h in Group T, resulting in the increased AUC0–24 (1.61-fold), AUC0–∞ (1.66-fold), and Tmax (2.22-fold). In addition, the result of 37% reduction of tadalafil clearance in Group T compared with Group N supported our findings. These data sugges<sup>t</sup> that ticagrelor inhibits tadalafil metabolism due to drug-drug interaction with tadalafil.

**Figure 2.** Comparisons of the non-compartment analysis parameters of tadalafil from non-pretreated rats (Group N) and ticagrelor-pretreated rats (Group T). Boxes mean 25th and 75th percentiles of data and whiskers mean 5th and 95th percentiles of data. The median and mean values are displayed as a solid line (–) and a plus mark (+) in boxes, respectively. (**A**) Cmax; (**B**) AUC0–24; (**C**) AUC0–∞; (**D**) Tmax; (**E**) T1/2; (**F**) CL/F. Maximum concentration (Cmax); area under the plasma concentration vs. time curve from 0 to 24 h (AUC0–24); area under the plasma concentration vs. time curve from 0 to infinity (AUC0–∞);time to reach maximal concentration (Tmax); half-life (T1/2); apparent total clearance (CL/F).
