This study was an open-label and single-center clinical trial (China Food and Drug Administration registration: 2013L01977). The protocol was approved by the Ethics Committee of First Affiliated Hospital with Nanjing Medical University and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guideline. All participants gave written informed consent prior to the enrollment.

A total of 328 subjects were enrolled for the genetic analysis of gastric H⁺/K⁺-ATPase polymorphisms. Among this pharmacogenetic population, 51 subjects participating in the PK and PD study were sampled for genotyping of CYP2C19 and ABCB1 additionally.

Eligible subjects for PK and PD study were selected from healthy Chinese subjects aged 18–40 years, with a body mass index of 19–24 kg/m². Subjects were examined to be healthy on the basis of medical history, physical examination, laboratory examination, and 12-lead electrocardiogram. The following exclusion criteria were applied to subjects in PK and PD study: a history of clinically significant cardiovascular, hepatic, renal or gastrointestinal diseases; a history of nervous system or muscle disease; seizure or other psychiatric disorders; a history of known allergy or intolerance to any drugs; a history of tobacco, alcohol, or drug abuse; a positive outcome of Helicobacter pylori infection test; those with abnormalities in clinical laboratory parameters; reception of an experimental drug or donation of blood 3 months prior to the first dose; pregnant or nursing female.

All subjects were confined to the Phase I unit beginning with the evening before baseline intubation and kept to a regular schedule. An overnight fasting (12 h) was required before administration, while standard meals were provided 4 h post-dose.

Dexlansoprazole injection (30 mg per vial) used in the PK and PD study was manufactured by Nanjing Yoko Pharma Co., Ltd. (Nanjing, China). Subjects received an intravenous infusion of 20 or 30 mg dexlansoprazole injection in sterile saline solution (100 mL/h, 60 min). The study drug was administrated with an infusing pump (SA213, Lifepum, Beijing, China) and given through an intravenous catheter in the antecubital vein opposite to the one from which blood samples were taken.

To determine the pharmacokinetic properties of dexlansoprazole injection in human, a series of venous blood samples (5 mL) were collected in heparinized tubes at 0 (pre-dose), 10, 20, 40, 60, and 75 min, 1.5, 2, 2.5, 3, 4, 6, 9, 12, 16, 24, and 36 h post-dose. Samples were centrifuged immediately at 4°C for 10 min at 3,500 revolutions per minute, then separated and stored at −80°C for PK analysis.

A chiral liquid chromatography-mass spectrometry method was developed and validated for the simultaneous determination of (R)- and (S)-lansoprazole in human plasma, with the calibration curve of 500–3,000 ng/mL for both (R)- and (S)-lansoprazole (Sun et al., 2015). Baseline chiral separation was achieved on a Chiralpak IC column. The mass spectrometric analysis was performed using a QTrap 5500 mass spectrometer (AB Sciex, Concord, Ontario, Canada).

The PK parameter of C_max (maximum concentration) and T_max (the time to C_max) were obtained directly from the observed data. Other PK parameters including t_1/2 (elimination half-life), AUC^PK (area under the plasma concentration-time curve), V_d (volume of distribution), and CL (clearance) were calculated using non-compartmental methods with Phoenix WinNonlin version 6.4 (Pharsight Inc., CA, USA).

An ambulatory pH recorder (Orion II, MMS, USA) with a disposable pH catheter (VersaFlex, MMS, USA) was used to monitor intragastric pH. The electrode was placed in water for at least 10 min and then calibrated using standard buffer solutions (pH 1.07 and 7.01) before intubation. Both calibration and intubation processes were conducted under the guidance of MMS Virtual Instructor Program. The catheter, trans nasally placed 10 cm under the lower esophageal sphincter, was identified by a sharp decrease in pH (Wang et al., 2010, 2011). Subjects were intubated on baseline and the day of dosing at about 7:30 a.m., and the intragastric pH data were recorded continuously for 24 h from 8:00 a.m. Intragastric pH was sampled every second, and the data were stored in a MMS Database program (MMS, USA). The PD parameters including Median (pH), Mean (pH), T_pH≥3 (%), T_pH≥4 (%), T_pH≥6 (%), T_pH≥6, and AUC^pH−t were analyzed by the MMS Database program.

Median (pH) and mean (pH) were well-investigated in most studies, and the percentage time of pH ≥ 4 (or pH ≥ 4 holding time ratio) was the benchmark for predicting clinical efficacy of PPIs in treating acid-related diseases (Metz et al., 2009). Novel parameter T_pH≥6 (time to first gastric pH ≥ 6, min) and AUCτ 1-τ 2pH-t (area under pH-time curve from τ1 to τ2, pH·s) were defined to evaluate the velocity and extent of pharmacodynamics with more accuracy. All parameters were calculated and compared according to time periods of 0–24, 0–4, 4–10, 10–14, and 14–24 h post dosing.

The parameter AUCτ 1-τ 2pH-t was calculated as follows:

Where t represented the time duration measured by seconds, AUCτ 1-τ 210-t was the area between the pH line = 10 and the pH-time curve, which was generated by the MMS software.

Baseline-adjusted PD parameters were calculated as follows: for example, ΔAUC^pH−t = AUC^pH−t − AUC^{pH−tbaseline}, ΔMean (pH) = Mean (pH) − Mean (pH)^baseline, and ΔMedian (pH) = Median (pH) − Median (pH)^baseline.

Genomic DNA was extracted from leucocytes of blood samples using a commercially available kit (RelaxGene Blood DNA System, Tiangen, Beijing, China) according to the manufacturer's protocol. A total of 14 exons of ATP4A gene were sequenced using primers as Oksanen (Oksanen et al., 2006) published. Genotyping for H⁺/K⁺-ATPase (rs2733743), CYP2C19^*2 (rs4244285), CYP2C19^*3 (rs4986893), CYP2C19^*17 (rs12248560), and ABCB1 C3435T (rs1045642) variant alleles was performed by polymerase chain reaction followed by Sanger sequencing. DNA sequencing was processed using BigDye® Terminator Cycle Sequencing v3.1 kit and DNA Analyzer (3730xl, Applied Biosystems, USA), which was provided by Realgene Biotech (Nanjing, China) and Sangon Biotech (Shanghai, China). Subjects participating in PK and PD studies were genotyped into three groups according to CYP2C19 wild-type gene, ^*2 and ^*3, namely homEM (^*1/^*1), hetEM (^*1/^*2 and ^*1/^*3), and PM (^*2/^*2, ^*2/^*3, and ^*3/^*3).

Descriptive statistics was utilized to summarize demographic and biologic characteristics of subjects. Continuous variables (PK and PD parameters) were expressed as Mean ± SD, and categorical variables as frequencies and percentages. Genotype frequencies were tested for Hardy-Weinberg equilibrium. Statistical comparison of PK and PD parameters was performed by an independent sample test and one-way analysis of variance (ANOVA) with two-sided P < 0.05 considered statistically significant. Statistical analysis was performed using SPSS software version 22.0 (IBM Corp., USA).

Demographic and biologic variables are summarized in Table 1. Genotype and allele frequencies of gastric H⁺/K⁺-ATPase (rs2733743), CYP2C19^*2 (rs4244285), and ABCB1 C3435T (rs1045642) were distributed according to the Hardy-Weinberg equilibrium in the study population. No CYP2C19^*17 (rs12248560) mutant was observed in the PK and PD population. In the pharmacogenetic analysis of the gene encoding the gastric H⁺/K⁺-ATPase α-subunit, one non-synonymous variation (rs2733743) was found in most subjects with 115 heterozygotes (35.1%) and 123 homozygotes (37.5%). The recorded SNP rs2733743, a homozygous A → G transition (c.823T>C) in ATP4A, alters amino acid 265 of gastric H⁺/K⁺-ATPase α subunit from valine to alanine (Yang et al., 2012). Subjects were then classified into three types as gastric H⁺/K⁺-ATPase wildtype, heterozygote and homozygote (AA, AG, and GG, respectively) accordingly.

Mean plasma concentration-time profile of dexlansoprazole after single intravenous administration is shown in Figure 1A. The PK parameters for dexlansoprazole injection grouped by dose are summarized in Table 2. The C_max and AUC^PK exhibit dose proportionality with no statistical difference in ln (C_max/dose) and ln (AUC^PK/dose) between the 20 and 30 mg groups. The PK parameters of t_1/2, CL, and V_d were independent of dosage, and no significant gender-related difference was observed in PK parameters except that the V_d of male subjects was slightly higher than that of female subjects.

CYP2C19 PMs presented a slower elimination and higher exposure (C_max and AUC^PK) compared to CYP2C19 homEMs, while the elimination and exposure of CYP2C19 hetEMs were intermediate between that of PMs and homEMs (Figures 1B,C). The PK parameters grouped by CYP2C19 phenotype are summarized in Table 2. In the 30 mg group, CYP2C19 PMs exhibit significantly higher C_max, AUC^PK, and t_1/2 than homEMs and presented remarkably higher AUC^PK and t_1/2 than those of CYP2C19 hetEMs. CYP2C19 hetEMs shows a significantly higher C_max and AUC^PK compared to CYP2C19 homEMs. As the previous study (Kodaira et al., 2009) revealed, no statistical significant differences were observed among ABCB1 genotypes.

The 24-h intragastric pH-time curves before and after intravenous dosing are presented in Figure 2A. The rapidity of onset of increase in intragastric pH was dose-dependent with the time to first gastric pH ≥ 6 (T_pH≥6) of 76 (± 29) min for the 20 mg group and 58 (± 25) min for the 30 mg group. The mean intragastric pH, pH ≥ 4 and pH ≥ 6 holding times (T_pH≥4 (%), T_pH≥6 (%), the percentage of time that intragastric pH was above 4 or 6) for the 30 mg group was obviously higher than the 20 mg group. The baseline-adjusted intragastric pH-time curves (post-dose pH minus the baseline value) are shown in Figure 2B, and the baseline-adjusted intragastric pH was dose-dependent.

The PD parameters are summarized in Table 3. The PD parameters after intravenous administration of 20 or 30 mg dexlansoprazole were significant increased (P < 0.05) compared to baseline. There was a significant elevation in PD parameters in the 30 mg group than the 20 mg group during initial time (0–4 h), overnight hours (14–24 h), and the whole time (0–24 h) (P < 0.05) after intravenous dosing at about 8:00 a.m. Mean and median intragastric pH for the 30 mg group was significantly higher than those of the 20 mg group during 0–4, 14–24, and 0–24 h after dosing. A similar increment was observed in delta(Δ)-values (values after dosing minus the corresponding values at baseline) of mean and median intragastric pH from 20 to 30 mg. No significant differences in mean and median pH were noticed between 20 and 30 mg during the 4–10 h interval, except that Δ-mean pH for the 30 mg group was higher than that of the 20 mg group (4.2 ± 1.2 vs. 3.3 ± 1.4, P = 0.043). Statistically significant differences in T_pH≥3 (%) and T_pH≥4 (%) were observed during time intervals of 0–24, 0–4, and 14–24 h. Pairwise comparisons of baseline-adjusted T_pH≥3 (%) and T_pH≥4 (%) were significantly greater for 30 mg compared with 20 mg during 0–24, 0–4, and 14–24 h intervals. Significant differences were observed in both T_pH≥6 (%) and its Δ-values between dosage groups during 0–24, 0–4, 4–10, and 14–24 h intervals. Statistically significant higher values of novel PD parameter AUCτ 1-τ 2pH-t (area under pH-time curve from τ1 to τ2) were obtained with the 30 mg group compared with the 20 mg group during 0–24, 0–4, and 14–24 h intervals. Corresponding Δ-values for AUC0-24 hpH-t, AUC0-4hpH-t, AUC4-10hpH-t, and AUC14-24 hpH-t were significantly higher with the 30 mg group vs. the 20 mg group. No statistically significant difference of above-mentioned PD parameters and Δ-values was observed between the 20 and 30 mg groups during 10–14 h post-dosing.

PD parameters divided by CYP2C19 phenotype in the 30 mg group are shown in Figures 3A1–A4, and mean values are summarized in Table 4. No significant differences in PD parameters were noticed between CYP2C19 homEMs, hetEMs, and PMs across all time periods, except that T_pH≥6 (%) was higher with PMs than homEMs during the 14–24 h interval. However, analysis in Δ-values for mean (pH), median (pH), T_pH≥3 (%), T_pH≥4 (%), T_pH≥6 (%), and AUCτ 1-τ 2pH-t showed statistically significant variations between homEMs and PMs during the 0–24 and 14–24 h intervals. Statistically significant difference between hetEMs and homEMs were found in Δ-T_pH≥6 (%) during the 14–24 h interval additionally.

PD parameters divided by gastric H⁺/K⁺-ATPase rs2733743 wildtypes, heterozygotes and homozygotes in the 30 mg group are shown in Figures 3B1–B4, and mean values are summarized in Table 5. No significant differences in PD parameters were noticed between gastric H⁺/K⁺-ATPase homEMs, hetEMs, and PMs across all time periods. However, analysis in Δ-values for mean (pH), median (pH), T_pH≥3 (%), T_pH≥4 (%), T_pH≥6 (%), and AUCτ 1-τ 2pH-t showed statistically significant variation between homEMs and PMs during 0–24, 0–4, and 14–24 h intervals. Statistically significant differences between hetEMs and homEMs were found in Δ-median pH, Δ-mean pH, Δ-T_pH≥6 (%), and Δ- AUC4-10 hpH-t during 0–24, 14–24, or 0–4 h intervals, respectively.

PD parameters divided by gastric H⁺/K⁺-ATPase rs2733743 wildtypes, heterozygotes, and homozygotes based on CYP2C19 EMs in the 30 mg group are shown in Figures 3C1–C4, and mean values are summarized in Table 6. No significant differences in PD parameters were noticed between gastric H⁺/K⁺-ATPase homEMs, hetEMs, and PMs across all time periods, except that T_pH≥4 (%) was higher with homEMs than hetEMs during 0–4 h interval. However, analysis in Δ-values for mean (pH), median (pH), T_pH≥3 (%), T_pH≥4 (%), T_pH≥6 (%), and AUCτ 1-τ 2pH-t showed statistically significant higher values in gastric H⁺/K⁺-ATPase homEMs than those in PMs during 0–24, 0–4, and 14–24 h intervals. Statistically significant differences between hetEMs and homEMs were found in Δ-median pH, Δ-mean pH, Δ-T_pH≥6 (%), and Δ- AUC4-10 hpH-t during 0–24, 14–24, or 0–4 h intervals, respectively.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.