**1. Introduction**

Erectile dysfunction (ED), the most prevalent complaint in males, is the persistent inability to maintain an erection [1]. Various factors, such as age and presence of cardiovascular diseases, influence the incidence of ED [2,3]. Particularly, vascular diseases, such as coronary artery disease (CAD), are related to a high prevalence of ED [4–6]. It has been reported that 42% of patients between the ages of 40 and 60 years are affected by this condition [7–9]. In addition, a high rate of ED prevalence (~75%) has been investigated in CAD patients [10,11]. The most commonly prescribed oral drugs for ED are the 5-phosphodiesterase (PDE5) inhibitors, and the drugs for CAD are the platelet aggregation inhibitor [12,13]. Therefore, PDE5 may be co-administered to CAD patients already receiving platelet aggregation inhibitors.

Tadalafil (Cialis®), the approved PDE5 inhibitor, is used in the treatment of ED and is one of the most frequently prescribed PDE5 inhibitors [14]. In addition, tadalafil exhibits a longer clinical efficacy (up to 36 h) than sildenafil or vardenafil [15,16]. Because of the long duration of action, a once-a-day dose regimen improves the life quality of patients. Tadalafil is classified as a cytochrome P450 (CYP) 3A4 substrate and is mainly metabolized by CYP3A4 to catechol, which is extensively bound to form methyl catechol glucuronide, a major circulating metabolite of tadalafil through methylation [17].

Ticagrelor (Brilinta ®), the platelet aggregation inhibitor, belongs to P2Y12 receptor antagonists and is used for the treatment of CAD [18,19]. It provides the superior and more sustained inhibition of platelet aggregation than clopidogrel, another P2Y12 receptor antagonist [20]. Recent studies have reported that ticagrelor acts as an inhibitor of CYP3A4 [21,22]. When ticagrelor was co-administered with atorvastatin, ticagrelor acted as a CYP inhibitor, increasing the maximal plasma concentration of atorvastatin and the area under the plasma concentration-time curve from 0 to infinity by 23% and 36%, respectively [23]. Therefore, drug-drug interaction by co-administration of tadalafil and ticagrelor can inhibit the metabolism of CYP3A4 substrates, such as tadalafil. Despite the possibility of co-administration, the pharmacokinetic interactions between ticagrelor and tadalafil are still uncertain.

Because ticagrelor is an inhibitor of CYP3A4 that accounts for about 15–30% of the total CYP enzyme in humans, the co-administration potentially a ffects e fficacy and safety by altering tadalafil exposure [24]. In healthy male volunteers, the plasma concentrations of tadalafil have been shown to increase with CYP3A4 inhibitors, such as ritonavir [25]. Although tadalafil is well tolerated, patients have experienced side e ffects, such as headaches, stomachaches, back pain, muscle aches, nasal congestion, redness, limb pain, dizziness, or blurred vision due to the high exposure of tadalafil [26,27]. In other studies, the side e ffects were increased when doses of tadalafil were higher, indicating that side e ffects were somewhat related to blood concentration of tadalafil [28,29]. Thus, the combination of tadalafil with ticagrelor also warranted investigation and a drug-drug interaction study should be conducted. However, pharmacokinetic interactions between tadalafil and ticagrelor have not been reported in vivo models.

The objective of our study was to evaluate the e ffect of ticagrelor on the plasma concentration-time profiles of tadalafil in rats. This study was performed with a parallel design consisting of a non-pretreated group and a ticagrelor-pretreated group. The ticagrelor-pretreated group received oral administration of ticagrelor for seven days of the pretreatment period to inhibit CYP3A. Tadalafil was then orally administered on the seventh day. The non-compartment analysis was performed to analyze the pharmacokinetic profile and the one-compartment model was successfully applied to compare the pharmacokinetics between the two groups. This study assumed that potential drug-drug interactions between ticagrelor and tadalafil could have a clinical impact on the patients.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Tadalafil, ticagrelor, and paclitaxel (internal standard (IS)) were kindly obtained from Korea United Pharma Inc. (Seoul, Korea). Dimethyl sulfoxide, formic acid, and polyethylene glycol 400 (PEG 400) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were purchased from J.T. Baker (Phillipsburg, NJ, USA). Analytical grade reagents were used throughout this study. Overall, distilled water was used.

#### *2.2. LC-MS*/*MS Analysis of Tadalafil*

The concentrations of tadalafil were analyzed by a liquid chromatography tandem-mass spectrometry (LC-MS/MS) system equipped with Agilent 1290 series and Agilent 6495 Triple Quad LC/MS (Agilent Technologies, Santa Clara, CA, USA). A YMC-Triart C18 column (50 × 2.0 mm, 1.9 μm; YMC Inc., Wilmington, NC, USA) was used. The mobile phase was a mixture of 0.2% formic acid in acetonitrile and 0.2% formic acid in distilled water (50:50, *v*/*v*), and the flow rate was 0.4 mL/min. The temperature of the column and autosampler were set as 30 ◦C and 4 ◦C, respectively. The positive ion mode using Agilent jet stream electrospray ionization (AJS-ESI) was applied to record the scan mass spectra. The ion transitions of tadalafil and IS were set as 390.4 →268.1 m/z and 876.4 →308.1 m/z, respectively, and detected with a multiple reaction monitoring (MRM) mode. The collision energies for tadalafil and IS were 10 V and 30 V, respectively. The cell accelerator voltage was 5 V and the dwell time was set as 200 ms. The source parameters were set as follows: Gas temperature 200 ◦C, gas flow 14 L/min, nebulizer 20 psi, sheath gas heater 250 ◦C, sheath gas flow 11 L/min, capillary 3000 V, and nozzle voltage 1500 V.

In this analysis, the most abundant ion transition of tadalafil (390.4 →268.1 m/z) was selected to determine the lowest limit of quantification (LLOQ), and the LLOQ of tadalafil was 3 ng/mL. The range of calibration curve of tadalafil was set to 3–6670 ng/mL. The curve was written with a weighted linear regression (1/x) and showed excellent linearity with R<sup>2</sup> > 0.997. The method has shown accurate and reproducible results within acceptable tolerances (less than 20% coe fficient of variation (CV) at LLOQ and less than 15% CV at all other concentrations) [30]. The acquired LC-MS/MS data were processed with Agilent analysis software (Agilent MassHunter Quantitative Software Version B.07.00, Agilent Technologies, Santa Clara, CA, USA).
