The Institutional Review Board of Seoul National University Hospital and the Korean Ministry of Food and Drug Safety approved the study protocol and informed consent form. This study was conducted in accordance with the Korean Good Clinical Practice guidelines and the principles of the Declaration of Helsinki. The study was registered in the public clinical trial registry on September 29, 2020 (ClinicalTrials.gov identifier: NCT04568772). Eligible subjects were recruited through a recruitment notice at the Seoul National University Hospital Clinical Trials Center, including the website (https://ctcr.snuh.org/). Prior to any study-related procedures, written informed consent was obtained from all individual subjects.

2.1 Study Population

Eligible subjects were healthy CYP2C19 extensive metabolizers carrying the CYP2C19 *1/*1 diplotype who were 19–50 years old with a body mass index (BMI) of 19.0–30.0 kg/m2. Subjects who had evidence or a history of gastrointestinal disorders likely to influence drug absorption and/or who ingested drug metabolizing inducers (e.g., barbiturates) or inhibitors (e.g., clarithromycin) within 4 weeks prior to the first administration and/or other medications within 2 weeks prior to the first administration were excluded from the study. The exclusion criteria also included aspartate aminotransferase or alanine aminotransferase values 1.5 times greater than the upper normal limit or Modification of Diet in Renal Disease (MDRD) glomerular filtration ratio (GFR) values < 80 mL/min. The enrolled subjects were prohibited from intake of any medication without the prior permission of the investigators and the consumption of grapefruit products, caffeine, or alcohol throughout the study. The sample size was determined empirically based on the exploratory and descriptive characteristics of the study without calculating study power for any statistical hypothesis.

2.2 Study Design

This study had a two-part, randomized, open-label, two-sequence, three-period crossover design (Fig. 1). Each sequence consisted of the following three treatments (i.e., one control treatment and two co-administration treatments): a single oral administration of atovaquone/proguanil 250/100 mg alone [control], 6-day pretreatment with once-daily oral doses of tegoprazan 50 mg, followed by a single oral administration of atovaquone/proguanil 250/100 mg concomitant with tegoprazan 50 mg [co-administration], and 6-day pretreatment with once-daily oral doses of esomeprazole 40 mg, followed by a single oral administration of atovaquone/proguanil 250/100 mg concomitant with esomeprazole 40 mg (Part 1) or 6-day pretreatment with once-daily oral doses of vonoprazan 20 mg, followed by a single oral administration of atovaquone/proguanil 250/100 mg concomitant with vonoprazan 20 mg (Part 2) [co-administration]. In accordance with the guideline for clinical drug interaction studies, the dose of each study drug was set to the therapeutic dose for a substrate, proguanil, and to the approved maximum dose for perpetrators, tegoprazan, vonoprazan, and esomeprazole, respectively [17]. The following drug products were used: atovaquone/proguanil 250/100 mg (Malarone® Tab., GlaxoSmithKline Inc., Seoul, Republic of Korea), tegoprazan 50 mg (K-CAB® Tab. 50 mg, HK inno.N Corp., Seoul, Republic of Korea), esomeprazole 40 mg (Nexium® Tab. 40 mg, AstraZeneca Pharmaceutical Co., Ltd., Seoul, Republic of Korea), or vonoprazan 20 mg (Takecab® Tab. 20 mg, Takeda Pharmaceutical Co., Ltd., Tokyo, Japan).

Fig. 1

The enrolled subjects were randomly assigned to one of two sequences in each part and received their respective treatment with 150 mL of water after at least 10 h of overnight fasting. The respective treatment was separated by a 7-day washout period, which was set based on the turnover half-life (t½) of CYP2C19 and the t½ of the study drugs [14, 16, 18,19,20].

For PK analyses of proguanil and cycloguanil, blood samples were obtained at pre-dose and 1, 2, 3, 4, 6, 8, 10, 24, and 48 h after atovaquone/proguanil dosing, and urine samples were collected at pre-dose and 0–8, 8–24, and 24–48 h after atovaquone/proguanil dosing. At each blood sampling point, approximately 8 mL of blood was collected in a sodium heparin tube and then centrifuged at 4 °C and 3000 rpm for 10 min, and the supernatant was separated in Eppendorf tubes® and stored at −70 °C until analysis. At each urine collection interval, the collected urine was gently mixed, and separated and stored in the same manner as the blood samples.

2.3 CYP2C19 Genotyping

To identify CYP2C19 extensive metabolizers, DNA was extracted from whole blood using a Maxwell® CSC Blood DNA Kit and Maxwell® CSC Instrument (Promega, Madison, WI, USA), and TaqMan allelic discrimination assays were performed in a real-time polymerase chain reaction (PCR) System (Applied Biosystems, Foster City, CA, USA). A 10-µL PCR mixture was prepared with 5 µL of 2× TaqMan Universal Master Mix II, 0.5 µL of 20× Drug Metabolism Genotyping Assay Mix, 3.5 µL of DNase-free water, and 1 µL of DNA. Genotyping for the CYP2C19*2 allele (rs4244285, assay ID: C__25986767_70) and CYP2C19*3 allele (rs4986893, assay ID: C__27861809_10) was performed using validated TaqMan Genotyping Assays. The PCRs were carried out in the order of initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 seconds and annealing/extension at 60 °C for 1 minute. The allelic discrimination results were determined using 7500 Real-Time PCR System software version 2.0.6 (Applied Biosystems, Foster City, CA, USA). Based on the genotyping results, only CYP2C19 extensive metabolizers carrying the CYP2C19 *1/*1 diplotype were included in the study.

2.4 Determination of Plasma and Urine Proguanil and Cycloguanil Concentrations

The plasma and urine concentrations of proguanil and cycloguanil were determined using validated high-performance liquid chromatography (HPLC; Agilent 1260/1290 Infinity system, Agilent Technologies) coupled with tandem mass spectrometry (MS/MS; API4000, AB Sciex for plasma samples and Agilent 6460, Agilent Technologies for urine samples). For plasma specimens, 50 μL of plasma was mixed with 10 μL of internal standard solution (0.2 μg/L proguanil-d4 in 50% acetonitrile and 0.1 μg/L cycloguanil-d4 in 50% acetonitrile, respectively), followed by 1 mL of 100% acetonitrile. Mixed solutions were centrifuged at 4 °C and 14,000 rpm for 5 min. The supernatant was dried at 40 °C and dissolved with 100 μL of 30% acetonitrile, followed by centrifugation at 4 °C and 14,000 rpm for 5 min. Then, 5 μL of the supernatant was subjected to HPLC-MS/MS analysis. For urine specimens, 50 μL of urine was mixed with 10 μL of internal standard solution (proguanil-d4 12.5 μg/L in 50% acetonitrile and cycloguanil-d4 10 μg/L in 50% acetonitrile, respectively), followed by 930 μL of 100% acetonitrile. Mixed solutions were centrifuged at 4 °C and 14,000 rpm for 5 min. The supernatant was dried at 40 °C and dissolved with 500 μL of 0.1% formic acid in 20% acetonitrile, followed by centrifugation at 4 °C and 14,000 rpm for 5 min. Then, 5 μL of the supernatant was subjected to HPLC-MS/MS analysis.

The lower limits of quantification for proguanil and cycloguanil were 1 μg/L and 0.5 μg/L, respectively, in plasma and 200 μg/L and 100 μg/L, respectively, in urine. The accuracies for proguanil and cycloguanil were 92.87–100.19% and 96.10–98.68%, respectively, in plasma samples and 96.65–99.75% and 99.28–100.95%, respectively, in urine samples. The precision coefficients of variation for proguanil and cycloguanil were ≤ 5.65% and ≤ 6.49%, respectively, in plasma samples and ≤ 2.32% and ≤ 2.69%, respectively, in urine samples.

2.5 Pharmacokinetic Analysis

The PK parameters of proguanil and cycloguanil were derived using a non-compartmental method in Phoenix® WinNonlin® version 8.2 (Certara, St. Louis, MO, USA). The area under the concentration–time curve (AUC) from 0 to last measurable time point (AUClast) was calculated using the linear-up and log-down trapezoidal rule and extrapolated to infinity (AUCinf) using the terminal elimination rate constant (λz). Cmax and the time to reach Cmax (Tmax) were determined directly from the observed plasma concentration–time profiles. The elimination t½ was calculated as ln(2) divided by λz, and the apparent clearance (CL/F) was calculated as the dose divided by AUCinf. The fraction excreted unchanged in the urine (fe) was calculated as the cumulative amount of proguanil in urine up to 48 h post-dose divided by the dose. The metabolic ratio was calculated as the AUCinf ratio of cycloguanil to proguanil, and the apparent formation clearance (CLF/F) was calculated as the cumulative amount of cycloguanil in urine up to 48 h post-dose divided by AUClast of proguanil.

2.6 Safety Evaluation

Safety was evaluated based on adverse event (AE) monitoring, clinical laboratory tests, vital signs, physical examination, and 12-lead electrocardiogram (ECG) throughout the study. Each finding from the safety evaluation was assessed regarding its clinical significance and relationship with the treatment by the investigators.

2.7 Statistical Analysis

Statistical analyses were performed using SAS® version 9.4 (SAS Institute Inc., Cary, NC, USA). The demographic characteristics were compared between parts and sequences in each part using Wilcoxon's rank sum test. For control and tegoprazan co-administration treatments, the PK data from Part 1 and Part 2 were pooled. The PK parameters of proguanil and cycloguanil were summarized by treatment and compared between administered alone and co-administered with tegoprazan, vonoprazan, or esomeprazole. Analysis of variance was performed for log-transformed AUC and Cmax, and the effects on the PK of proguanil and cycloguanil were compared by estimating the geometric mean ratios of each co-administration to control and the corresponding confidence intervals. Additionally, CYP2C19 metabolism-related PK parameters, the metabolic ratio and CLF/F of cycloguanil were evaluated using Dunnett’s t test.