Pharmacokinetic study of single- and multiple-dosing with metolazone tablets in healthy Chinese population

Background Metolazone is a diuretic, saluretic and antihypertensive chemical compound from the quinazoline category that possesses medicinal features similar to those of other thiazide diuretic drugs. However, the pharmacokinetics of metolazone in the Chinese population has rarely been studied. This study aimed to examine the pharmacokinetic characteristics, safety characteristic, and tolerability of metolazone in healthy Chinese subjects after single and multiple doses taken orally as well as the effects that food and gender have on oral metolazone pharmacokinetic parameters. Methods An open-label, randomized, and single- and multiple-dosing investigation was performed in healthy Chinese subjects. The investigation included 3 study groups: the 0.5 mg, 1 mg and 2 mg dose groups were the single-dose study groups in the first stage. Eligible volunteers were randomly and orally administered a single 0.5 mg, 1 mg, or 2 mg metolazone tablet. The 0.5 mg dose group was also part of the multiple-dose study group, and the 1 mg dose group was the food-effect study group in the second stage. Human plasma samples were gathered pre-dosing and up to 48 h after dosing. The human plasma sample concentration of metolazone was quantified using a validated liquid chromatography tandem mass spectrometry method. Pharmacokinetic data were calculated by a noncompartmental analysis method using WinNonlin version 6.4. Tolerability was evaluated based on adverse events, medical examination, 12-lead ECG, and other clinical laboratory exams. Results Thirty eligible subjects (15 men and 15 women) were registered in our investigation and completed all of the study stages. The AUC and Cmax showed dose proportionality after a single dose based on the linear-regression analysis. A comparison of the pharmacokinetic data revealed that the differences between the male and female groups were not statistically significant. The tmax of metolazone was increased by approximately 100% in the fed condition. Metolazone was well tolerated at the tested dose, and no adverse effects were observed. Conclusions Single dosing with 0.5 mg, 1 mg, or 2 mg metolazone yielded linear plasma pharmacokinetic properties in healthy Chinese subjects. Multiple oral doses of metolazone did not display significantly different distributions or elimination characteristics from those observed for a single dose. Gender factors did not appear to influence the pharmacokinetic parameter variation of metolazone. The tmax of metolazone increased in the fed condition. Metolazone was well tolerated at the tested dose in this study. Trial registration This investigation is retrospectively registered at chictr.org.cn (ChiCTR-IIR-17012929, October 09 2017).

Metolazone pharmacokinetics data are obtainable from previous studies on Chinese volunteers, including male and female volunteers and those participating in multiple oral dose administrations [1][2][3][4][5]. However, there are no data on the effects that food and gender have on this drug in Chinese volunteers. Therefore, this investigation was conducted to investigate the pharmacokinetic characteristics of metolazone in the Chinese population. It was a registered study permitted by the China Food and Drug Administration (approval No. 2005 L04730) before our clinical trial.
According to a previously reported study, single doses of 0.5 mg, 1 mg and 2 mg metolazone are rapidly and completely absorbed, with the peak plasma concentrations attained approximately 1.5 h following administration. The mean elimination half-life of metolazone has been observed to be between 6 and 8 h, and the area under the curve is reported to range from 30 ng•h/ml to 160 ng•h/ml [6].
Our study is a phase I clinical study of metolazone in Chinese volunteers. Our investigation assessed the safety character, tolerability, and pharmacokinetic parameters of single (0.5 mg, 1 mg and 2 mg) and multiple (0.5 mg) oral doses of metolazone. This study is the first to evaluate the role that food and gender have on this drug in Chinese volunteers. Few studies examining metolazone have been reported in the literature, and our investigation provides pharmacokinetic information for future reference.

Inclusion and exclusion criteria
Our inclusion criteria included healthy male and female Chinese subjects who were 18 to 45 years of age. The body mass index of qualified volunteers was between 18 and 24. Other inclusion criteria comprised the physical status diagnosed by health and family history, a medical examination, 12-lead ECG values, laboratory exams (e.g., haematology, blood biochemistry, hepatic function, urinalysis, hepatitis B surface antigen, and exams for alcohol and other substance abuse) and smoking habits.
Our exclusion criteria included allergies or cardiopathic disease history, pulmonary, renal, hepatic, gastrointestinal, haematologic abnormalities, and severe or long-lasting disease. Additional exclusion criteria included participating in any other clinical study, having taken drugs within the previous 30 days, pregnant or lactating women, and women who were not taking effective precautions to avoid pregnancy.

Ethics
The protocol, informed consent, and related documents of this investigation were reviewed by the Independent Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology (Wuhan, China). The entire study process was managed in compliance with the Declaration of Helsinki regarding medical ethics and the guiding principles of the International Conference on Harmonization Guidelines for Good Clinical Practice. All volunteers were required to provide informed consent before being screened for enrolment.

Study design and treatments
Administration of the research drug and human sample collection were completed in the Phase I Study laboratory of the Tongji Hospital clinical trial center, which was under direct medical management by the Huazhong University of Science and Technology. The studied metolazone tablets (the structures are shown in Fig. 1, batch No. 20140114) were provided by Libang Pharmaceuticals Co., Ltd. (Xian, China).
The study enrolled 30 (15 male and 15 female) healthy Chinese subjects. Based on a computer-generated table of random numbers, 30 volunteers were assigned in a 1:1:1 proportion to randomly take a single 0.5 mg, 1 mg, or 2 mg metolazone oral tablet.
This study was designed as an open-label, randomized, single-centre, single-and multiple-dose pharmacokinetic study that included 3 study groups. Groups 1-3 were single-dose study groups in the first stage; qualified volunteers were randomly administered a single 0.5 mg, 1 mg, or 2 mg metolazone tablet. The 0.5 mg dose group was the multiple-dose study group in the second stage, and the 1 mg dose group was the food effects study group in the second stage. All volunteers arrived at the Phase I Study laboratory 1 day ahead of the drug administration. They were instructed to avoid eating overnight (10 h); then, they were provided standard food after taking a single oral dose (either a 0.5 mg, 1 mg, or 2 mg metolazone tablet) on the dose day in every single oral dose study period. The metolazone tablets were taken with 250 mL of water. More drinking water was allowed 2 h after the dose. Then, the volunteers were provided a unified food (924 kcal; 18% protein, 49% carbohydrate, and 33% fat) 4 and 10 h after the single oral dose administration. Human plasma samples (3 mL) were collected through the indwelling catheter, which was placed in a forearm vein before and at 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 36 and 48 h after dosing.
In our multiple oral dose administration stage, the volunteers administered a 0.5 mg metolazone tablet once each day for 6 days. Human plasma samples (3 mL) were taken before dosing on the third, fourth, fifth, sixth, seventh, eighth, and ninth days. On the last day of the multiple oral dose, human plasma samples (3 mL) were gathered before and at 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 36 and 48 h after dosing, and this phase was followed by a single oral dose.
The food effect study was conducted on the 1 mg dose group. There was a single oral dose 2-period, 2sequence crossover study to assess the influence of food on the pharmacokinetic parameters of metolazone tablets in 10 Chinese volunteers. In each administration period, a 1 mg metolazone tablet was orally administered to volunteers under the following fasting or food (high-fat breakfast) situations: (1) after an allnight (at least 10 h) empty stomach or (2) after an all-night, 10-h empty stomach followed by a high-fat caloric meal. The washout period between the fast and fed states was 7 days. The high-fat breakfast was 924 cal, with fat comprising 59% of the total caloric content.
The subjects were prohibited from using other drugs, including non-prescription drugs, for 14 days before and during the investigation process. They were prohibited from drinking alcohol, smoking, severe physical activity and caffeine-containing drinks throughout the investigation period.
The biological samples were transferred into plastic tubes, heparin sodium was added to the tubes as an anticoagulant before blood sample collection, and the tubes were instantaneously centrifuged at 3500 rpm. The centrifugation time and temperature were 10 min and 4°C, respectively. The supernatant liquid was drawn into a polypropylene tube and then stored at −70°C until drug determination.

Metolazone assays
Human plasma concentrations of metolazone were determined via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method established and validated previous to our clinical trial [7][8][9][10][11]. The determination of the metolazone concentration in the human plasma samples was completed at the Research Center for Drug Metabolism of Tongji University. Chromatography was conducted with an Agilent 1200 HPLC connected to a 6410 Triple Quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray source operating in positive ionization mode and a Thermo AQUASIL C18 column (50 mm × 2.1 mm; 5 μm), and the column temperature was sustained at 30°C. The mobile phase consisted of methanol with 0.1% formic acid in water (10:90, v/v) and was set at a flow rate of 0.5 mL/ min. Human plasma samples were kept frozen at −70°C and protected from light. Plasma samples were spiked with an internal standard and then extracted by partitioning with ethyl acetate. The determination was conducted by triple quadrupole mass spectrometry to monitor the precursorto-product ion pairs m/z 366.1-259.0 and m/z 306.1-236.1 for metolazone and the internal standard, respectively. The mean accuracy ranged from 91.6% to 106.1%, and the precision of the analysis ranged from 5.7% to 19.0%. The study was analysed for metolazone concentrations by means of an LC-MS/MS method with a concentration range from 0.0198 to 79.0400 ng/mL.

Pharmacokinetic parameters and statistical analysis
The pharmacokinetic data were summarized using a noncompartmental analysis method calculated using WinNonlin version 6.4 (Pharsight Corporation, Mountain View, CA, USA). C max and t max were calculated from the human plasma concentration time characters for metolazone. The AUC 0-t was obtained based on the linear trapezoidal method. The summation of AUC 0-t and Ct/λ was applied to calculate the value of AUC 0-t. The value of Ct was the last measured concentration of human plasma.
To obtain the slope as the value of λ, the logtransformed concentration-time curve was used to conduct linear regression. The ratio of 0.693/λ was applied as the t 1/2 value. SAS version 9.2 (SAS Institute, Inc., Cary, NC, USA) was used for all statistical analyses in this study. Independent-sample t tests or nonparametric tests were applied to measure statistically significant differences between the pharmacokinetic data. A linearregression model was adopted to measure the dose proportionality.
The dose proportionality of metolazone over the dose range from 0.5 to 2 mg was estimated through matching an appropriate model. It was assumed that a linear relationship existed between the natural log transformed pharmacokinetic parameter (AUClast, AUCinf, and Cmax) and the natural log transformed dose in the model. The proportionality constant and its corresponding 90% confidence interval (CI) were contrasted with the modified acceptance range. For all analytical results, P < 0.05 was considered statistically significant.

Tolerability assessment
The tolerability assessments consisted of recording and monitoring all adverse events. The volunteers were arranged physical examinations continuously in the Phase I Study laboratory throughout the study. All adverse events were observed and recorded by trained study nurses or researchers. The vital signs were examined throughout the study as follows: pre-dosing and 0.5, 1, 2, 4 and 24 h post-dosing on each dosing day. The medical examinations were assessed based on the screening, predosing, and 24-h post-dosing on every dose day. All abnormal data of the volunteers were documented on the case-report form throughout the study. The laboratory tests, including the 12-lead ECG, haematology, blood biochemistry, hepatic function and urinalysis, were termly examined throughout the study. All laboratory examinations were conducted at the relevant laboratory at Tongji Hospital, Huazhong University of Science and Technology.

Safety analysis
The safety evaluation of this study was examined at screening and baseline and throughout the study using medical examinations, most important signs (systolic and diastolic blood pressure, pulse rate, respiratory rate, and temperature), 12-lead ECGs values, laboratory examinations (serum chemical analysis, haematologic examining, including coagulation parameters, and urinalysis) and adverse event judgement.

Pharmacokinetic parameters
Thirty volunteers (15 men and 15 women) were registered in our investigation, with a mean age (CV) of 21 (9.5%) years (range = 18-25 years), weight of 58.0 (10.2%) kg (range = 50.0-75.0 kg), and height of 167.6 (4.6%) cm (range = 155.0-181.0 cm). All of the participants completed the research. Twenty subjects' pharmacokinetics parameters were summarized for the 1 mg and 2 mg single oral dose study, and 10 volunteers' pharmacokinetic parameters were summarized for the 0.5 mg single oral dose and multiple oral dose study.
The mean human plasma metolazone concentrationtime curves are illustrated in Figs. 2, 3, 4 and 5. The mean plasma concentrations for the single 0.5 mg oral metolazone tablet are illustrated in Fig. 2. The mean plasma concentrations for the multiple 0.5 mg oral metolazone tablet are illustrated in Fig. 3. The mean plasma concentrations for the single 1 mg oral metolazone tablet under fasted and fed conditions are illustrated in Fig. 4. The mean plasma concentrations for the single 2 mg oral metolazone tablet are illustrated in Fig. 5. The important pharmacokinetic (PK) parameters for metolazone after the single oral dose taken of the 0.5 mg, 1 mg, and 2 mg metolazone tablets and multiple oral dose taken of 0.5 mg metolazone tablets in healthy Chinese subjects are presented in Table 1.
All pharmacokinetic parameters are shown using the resultant mean (SD) values. The mean (SD) pharmacokinetic data are listed in Table 1. The value of AUC and C max displayed dose proportionality after a single dose, according to the linear-regression analysis. We performed a linear regression analysis of C max , AUC 0-t and AUC 0-∞ . The results suggest that a single oral dose of 0.5 mg, 1 mg and 2 mg metolazone appeared to linear plasma pharmacokinetic parameters. The C max for the 0.5 mg to 2 mg dose increased from 8.22 to 28.88 ng/ mL, respectively, and the corresponding AUC 0-t (from 49.68 to 184.84 ng/mL/h) and AUC 0-∞ (from 50.51 to 186.58 ng/mL/h) pharmacokinetic values displayed significantly linear trends (Figs. 2, 3, 4, and 5 and Table 1).
Metolazone was rapidly absorbed at the low 0.5 and 1 mg doses, although the absorption slowed at the higher 2 mg dose. The t max was 1.63 and 1.43 h at 0.5  Table 1).
The mean half-life at the different doses was 7.3 h; there was no obvious difference among the 3 dose groups. The t 1/2 values were unchanged, even after increasing the dose (7.47 h at 0.5 mg and 7.42 h at 2 mg, respectively) (Figs. 2 and 5, Tables 2 and 4).
The mean human plasma concentration-time characters after a multiple oral dose are illustrated in Fig. 3. The mean (SD) pharmacokinetic parameters are listed in Table 1.
Through repeated administrations of metolazone, the mean human plasma concentrations reached the pre-dosing levels on Days 3 and 9 of the multiple oral dose, indicating that a steady state human plasma metolazone concentration was reached close to Day 6 during the multiple oral dose. The C min and C avg of metolazone were 0.3962 ng/mL and 1.9595 ng/mL, respectively ( Fig. 3 and Tables 1 and 5).
In contrast to the single oral dose, the elimination half-life after the multiple oral dose was slightly advanced and decreased to 7.61 h ( Table 1). The mean apparent distribution volume was 122,125 L, and the mean human plasma clearance was 11,267 L/h after the orally administered multiple dose; these two parameters were slightly changed compared with those after the single oral dose (P > 0.05) ( Table 1). Neither the C max nor AUC 0-t increased after the multiple dose administration. The accumulation index value of metolazone, which was calculated from AUC singledose/AUC multiple-dose, was 0.98 (0.28), suggesting no metolazone accumulation. Tables 2, 3, 4 and 5) shows the main pharmacokinetic data between the male and female volunteers and does not identify a major change between the groups, indicating that gender does not influence the pharmacokinetic characteristics of metolazone.

Effect of food
When metolazone was administered with a high-fat food, t max was prolonged by 100% in contrast to the fasting condition. The total exposure (AUC 0-t and AUC 0-∞ ) and C max were not influenced by administration under food conditions. The pharmacokinetic parameters for metolazone (1 mg tablet) administration under fasting or fed conditions are shown in Fig. 4. The statistical analysis is presented in Table 6.

Effect of gender
The pharmacokinetic parameters for metolazone were contrasted between males and females. The metolazone pharmacokinetic parameters were not obviously different in men and women. The pharmacokinetic data for men and women are shown in Tables 2, 3, 4 and 5.

Tolerability
In our study, all of the volunteers well tolerated not only the single oral doses of 0.5 mg, 1 mg, or 2 mg metolazone tablets but also the multiple oral doses of 0.5 mg metolazone tablets once each day for 6 days. Four volunteers exhibited stuffy and running nose symptoms that were identified as the common cold by researchers. The observed symptoms were considered slight, and one subject's cold was diagnosed as Table 1 Primary pharmacokinetic (PK) parameters of metolazone after single-dose administration of 0.5 mg, 1 mg, and 2 mg metolazone tablets and multiple-dose administration of 0.5 mg metolazone tablets in healthy Chinese volunteers   These experimental results support the good specificity and selectivity of this quantification method.
Sensitivity, linearity, precision, accuracy, and recovery The lower limit of quantitation (LLOQ) for the analysis was 0.0198 ng/mL for metolazone, with acceptable accuracy and precision. The linearity tendency was obtained for seven different concentrations of metolazone (0.0198, 0.0395, 0.1976, 0.3952, 3.9520, 9.8800, 35.5680 and 79.0400 ng/mL). The peak area response was linear over the entire concentration range of this study. To acquire the calibration data, every experiment

Stability studies
The results of the short-and long-term stock solution stability were assessed and displayed no obvious deviations from the normal values when kept at 4°C. The stability was measured by selecting the quality controls to three freeze-thaw cycles and comparing them with newly prepared quality control samples. The mean human plasma concentration of metolazone in the quality control samples did not appear to change during the period under the specified storage conditions. The long-term stability of metolazone in the LQC and HQC samples after 113 days of storage at −20 and −70°C were also investigated, and the RSD values ranged from 1.0% to 14.2%.

Discussion
We studied the pharmacokinetic characteristics of metolazone in healthy Chinese subjects. We created