*3.1. Study Design and Demographic Analysis*

The bioequivalence data (from reference formulation) collected from 24 healthy Korean males were used in this PK study for tiropramide. For the PK modelling, a total of 288 tiropramide plasma concentrations were available. There was complete information on height, age, and body weight for the 24 participants. Additionally, we successfully collected information on the total proteins, albumin, creatinine, AST, ALT, ALP, and the creatinine clearance levels of each participant, according to the method described above (Section 2.2). The related demographic information about the participants are shown in Table 1.


**Table 1.** Demographic information of the studied subjects (*n* = 24).

\* Body surface area (BSA) was determined on the basis of the Monsteller equation as follows: p (height (cm) × weight (kg)/3600); \*\* body mass index (BMI) was calculated as follows: body weight (kg)/height<sup>2</sup> (m<sup>2</sup> ); \*\*\* creatinine clearance was determined on the basis of the Cockcroft–Gault equation as follows: [(140-age) × body weight (kg)]/[serum creatinine (mg/dL) × 72].

#### *3.2. Genetic Analysis*

Genotyping was performed on all 24 individuals who participated in this study. The analyzed genotypes were *ABCB1* (1236C>T, 2677G>T/A, and 3435C>T), *CYP2D6* (\*1 and \*10), *OCT2* (808G>T), and *PEPT1* (1287G>C) genes. The results are presented in Table 2. The *ABCB1* 1236C>T genotyping revealed that 6 (25.00%) subjects had the mutant type (TT), 13 (54.17%) subjects had the heterozygous type (CT), and 5 (20.83%) subjects had the homozygous wild type (CC). *ABCB1* 2677G>T/A genotyping revealed that eight (33.33%) subjects had the mutant type (TT or AT or TA or AA), nine (37.50%) subjects had the heterozygous type (GT or GA), and seven (29.17%) subjects had the homozygous wild type (GG). *ABCB1* 3435C>T genotyping revealed that 3 (12.50%) subjects had the mutant type (TT), 12 (50.00%) subjects had the heterozygous type (CT), and 9 (37.50%) subjects had the homozygous wild type (CC). *CYP2D6* genotyping revealed that 6 (25.00%) subjects had the homozygous \*10 allele (\*10/\*10), 14 (58.33%) subjects had the heterozygous \*10 allele (\*1/\*10), and 4 (16.67%) subjects had the homozygous \*1 allele (\*1/\*1). *CYP2D6*\*2, \*4, \*5, \*14A/B, \*36, and \*47 alleles were not detected in any of the subjects of this study. We classified the *CYP2D6* genotypes into three groups to investigate the impact of *CYP2D6* genotypes on the PKs of tiropramide as follows: extensive metabolizers (EMs) (\*1/\*1), heterozygous intermediate metabolizers (IMs) (\*1/\*10), and homozygous IMs (\*10/\*10), on the basis of the reports of the difference in *CYP2D6* enzyme activity according to *CYP2D6* genotypes [9,17]. *OCT2* 808G>T genotyping revealed that 8 (33.33%) subjects had the heterozygous type (GT) and 16 (66.67%) subjects had the homozygous type (GG). *PEPT1* 1287G>C genotyping revealed that 3 (12.50%) subjects had the mutant type (CC), 2 (8.33%) subjects had the heterozygous type (GC), and 19 (79.17%) subjects had the homozygous wild type (GG).


**Table 2.** Genetic information of the studied subjects (*n* = 24).

#### *3.3. Determination of Plasma Tiropramide Concentrations*

After the oral administration of a 100 mg dose, plasma concentrations of tiropramide were determined by a column-switching semi-micro HPLC method (as mentioned in Section 2.4). This method analyzed tiropramide at a run time of 25 min per sample, enabling PK studies on tiropramide. The linearity of calibration curve for tiropramide was excellent (*r* <sup>2</sup> = 0.99) in human plasma, ranging from 2 to 500 ng/mL. Moreover, the calculation formula of the calibration curve was as follows: *y* = 384.51*x* + 320.45 (*p* < 0.01), where *y* is the peak area of tiropramide and *x* is the concentration (ng/mL) of the tiropramide. In addition, the lower limit of quantitation (LLOQ) for tiropramide was as low as 2 ng/mL, and was sufficient for PK studies after the oral administration of tiropramide tablet to humans. This assay has been validated for specificity, accuracy, precision, and sensitivity in order to be applied to accurate PK studies. There were no significant interferences derived from system or endogenous substances peaks, and we confirmed the identical tiropramide peak spectrum with the diode array detector. Intra-batch (*n* = 5) accuracies for tiropramide ranged from 100.70% to 113.50% with precision (coefficient of variation, CV) of < 13.57%. Inter-batch (*n* = 5) accuracies for tiropramide ranged from 98.00% to 111.42% with precision (CV) of < 9.23%.

#### *3.4. Pharmacokinetic (NCA) Analysis*

The observed plasma concentration–time profiles of tiropramide in the 24 subjects after oral administration of the 100 mg dose are presented in Figure 1.

In most individuals, the concentration of tiropramide in blood was measured up to 12 h after dosing. The PK parameters of tiropramide according to the genotypes (*ABCB1* 1236C>T, *ABCB1* 2677G>T/A, *ABCB1* 3435C>T, *CYP2D6*, *OCT2* 808G>T, and *PEPT1* 1287G>C) calculated by NCA are presented in Table 3. According to the NCA results for each genotype, the diversity of PK parameters in each group was not significant (*p* > 0.05). Although the higher mean AUC0-t, AUC0-∞, and Cmax values but lower mean CL/F values were calculated in the mutant type group (TT) than in the wild homozygous type groups (CC) in the *ABCB1* 1236C>T gene, this was not statistically significant considering the high SD. In the *CYP2D6* gene, higher mean AUC0-t, AUC0-∞, and Cmax values were calculated for IMs (\*1/\*10 and \*10/\*10) than EMs (\*1/\*1), but this was also not statistically significant considering the high SD. In addition, although the higher mean Cmax, AUC0-t, and AUC0-<sup>∞</sup> values and lower mean CL/F value were calculated in wild homozygous type groups (GG) than in mutant type group (CC) in the *PEPT1* 1287G>C gene, this was not statistically significant considering the high SD. The AUC0-t, AUC0-∞, Cmax, half-life, and Tmax values calculated by the NCA analysis of tiropramide were 254.31 ± 197.38 h· ng/mL, 280.34 ± 199.96 h· ng/mL, 69.07 ± 59.74 ng/mL, 3.41 ± 1.99 h, and 1.74 ± 0.63 h, respectively.

**Figure 1.** Log-transformed plasma concentration–time profiles of tiropramide in 24 subjects (**A**) and the mean curves (**B**). The vertical bars represent standard deviation of the mean.


