Pharmacokinetics/Pharmacodynamics
Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of Hyzetimibe (HS-25) in Healthy Chinese Subjects
The Journal of Clinical Pharmacology 54(10) 1144–1152 © 2014, The American College of Clinical Pharmacology DOI: 10.1002/jcph.310
Zhourong Ruan, BS1, Bo jiang, MS1, Jinliang Chen, PharmD1, Xuehua Zhang, MD2, Honggang Lou, MS1, Meixiang Xiang, MD2, Qingxiang Shao, BS3, and Jian’an Wang, MD, PhD1,2
Abstract Hyzetimibe (HS-25) is a new cholesterol absorption inhibitor. We performed the first-in-human study to assess the safety, tolerability, and pharmacokinetics (including the effect of food) and pharmacodynamics (effect on blood lipid level) following single (1, 3, 5, 10, 20, and 30 mg) and multiple (5, 10, and 20 mg) ascending-dose of hyzetimibe in healthy subjects. An increase of exposure (area under the plasma concentration–time curve and maximum plasma concentration) to hyzetimibe and hyzetimibe-glucuronide (HS-25M1) was observed in an approximately dose-proportional manner. A terminal half-life of approximately 21 hours was observed with doses ranging between 5 and 30 mg. Steady state was achieved by day 8 of once-daily dosing with 1.6- and 1.2-fold accumulation for hyzetimibe and hyzetimibe-glucuronide, respectively. Food did not have any effect on hyzetimibe and hyzetimibe-glucuronide exposure. Administration of hyzetimibe once daily for 10 days reduced the levels of low-density lipoprotein cholesterol levels in healthy subjects and these recovered after discontinuation of this drug. All of the adverse events were mild or moderate in severity, and the majority of them were unrelated to hyzetimibe, with no dose-dependent trends. These findings suggest that hyzetimibe could be a potential treatment for hypercholesterolemia.
Keywords cholesterol absorption inhibitor, hyzetimibe (HS-25), first-in-human study, pharmacokinetics, pharmacodynamics
Hypercholesterolemia is a major risk factor for the development of coronary heart disease, which is a leading cause of mortality and morbidity. Elevated low-density lipoprotein cholesterol (LDL-C) levels are positively associated with the incidence of cardiovascular events.1 Evidence is mounting from clinical trials that reducing the levels of total cholesterol and LDL-C, and increasing high-density lipoprotein cholesterol (HDL-C) levels by dietary and/or pharmacological means, could lead to a reduction in the incidence of death from cardiovascular events.2,3 Therefore, recent treatment guidelines emphasize that people who have a substantial risk for coronary heart disease should meet defined target levels of LDL-C.4,5 Circulating plasma levels of cholesterol are primarily produced from 2 processes: cholesterol production from the liver and peripheral tissues, and absorption of dietary and biliary cholesterol in the gastrointestinal tract.6 Statin therapy is the mainstay for lowering of LDL-C levels, which efficiently inhibits the activity of 3-hydroxy-3methyl-glutaryl coenzyme A reductase, resulting in a reduction in cholesterol synthesis.7,8 However, many patients do not achieve the optimal targets or have partial or complete intolerance to them.5,9 Therefore, an increasing amount of attention has focused on inhibiting cholesterol absorption in the gastrointestinal tract as another treatment option.
Hyzetimibe (HS-25, 1-(4-fluorophenyl)-3(R)-[3-(4fluorophenyl)-4-hydroxybut-2(Z)-enyl]-4 (S)-(4-hydroxyphenyl)-2-azetidinone), developed by Zhejiang Hisun Pharmaceutical Co. Ltd. (Taizhou, Zhejiang, China), is a new compound belonging to the novel class of selective cholesterol absorption inhibitors, which effectively blocks intestinal absorption of cholesterol and phytosterols. In previous studies, hyzetimibe was well tolerated in rats with a maximum tolerated dose not less than 2000 mg/kg in a single dose, and 500 mg/kg in cynomolgus monkeys. No toxicity was observed with administration of 500 mg/kg once-daily for 4 weeks in
1
Center of Clinical Pharmacology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 2 Department of Cardiology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 3 Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, China Submitted for publication 22 January 2014; accepted 11 April 2014. Corresponding Author: Jian’an Wang, MD, PhD, Center of Clinical Pharmacology and Department of Cardiology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, Zhejiang, China Email: [email protected]
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Figure 1. Structural formula of hyzetimibe and its metabolites.
cynomolgus monkeys. Preclinical pharmacokinetic studies have suggested that hyzetimibe is metabolized to its active metabolite acting by different isoforms of UDP-glucuronosyltransferase in the intestine and liver (Figure 1). Hyzetimibe is rapidly and almost completely (98–99%) converted to its main active metabolite (hyzetimibe-glucuronide, HS-25M1, 1-O-[4-[trans-(2S,3R)1-(4-Fluorophenyl)-3(R)-[3-(4-fluorophenyl)-4-hydroxybut-2(Z)-enyl]-4 (S)-(4-hydroxyphenyl)-2-azetidinyl] phenyl]-b-D-glucuronic acid]) in the small intestine after dosing. Hyzetimibe can reduce cholesterol levels in high-cholesterol fed rhesus monkeys. All of these preclinical studies suggest that hyzetimibe is a potential treatment for hypercholesterolemia. In the present study, we carried out the first-in-human assessment of the safety, tolerability, pharmacokinetics, and pharmacodynamics of hyzetimibe following single and multiple ascending-dose oral administration in healthy subjects. We also evaluated the effect of a high-fat, high-calorie meal on the pharmacokinetics of hyzetimibe.
Methods Ethics The study was performed at the Center of Clinical Pharmacology, the Second Affiliated Hospital of
Zhejiang University School of Medicine (Hangzhou, Zhejiang, China). The study protocols and amendments were reviewed and approved by the China Food and Drug Administration and the Human Subject Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine. The study is registered at www.chinadrugtrials.org.cn (CTR20130033). The study was performed in accordance with Good Clinical Practices and the ethical principles enunciated in the revised Declaration of Helsinki (revised version of Seoul, 2008). Subjects provided written informed consent before participating in any study-related procedures. Subjects Male and female healthy subjects (female subjects needed to be non-nursing, non-pregnant, and not of childbearing potential) between the ages of 18 and 45 years who had a body mass index (BMI) between 19 and 24 kg/m2 inclusive and body weight not less than 45 kg were eligible for the study. Subjects were considered healthy by the principal investigator based on a detailed medical history, full physical examination, clinical laboratory test, 12-lead electrocardiogram (ECG), and vital signs. Subjects were required to be nonsmokers with no history of alcohol or drug abuse. Prescription, herbal, and over-the-counter medications were prohibited for 30 days prior to and throughout the whole study. Subjects
1146 were excluded for the following reasons: a clinically significant history of medical illness; positive blood screen for HIV and hepatitis tests; any hospital admission or major surgery within 90 days prior to screening; donation or blood collection of more than 450 mL of blood, or acute loss of blood during the 90 days prior to screening; and heavy tea or coffee drinkers of more than 1 L/day. Female subjects of childbearing potential were required to have a negative pregnancy test upon study entry. All subjects were willing and able to give written informed consent, and to adhere to study restrictions. Study Design The first part of the overall study was a first-in-human phase I study using a randomized, double-blind, placebocontrolled, parallel-group, ascending-dose design. Sixty healthy adult subjects (30 men and 30 women) were enrolled in 6 dose cohorts (cohort 1, 1 mg; cohort 2, 3 mg; cohort 3, 5 mg; cohort 4, 10 mg; cohort 5, 20 mg; cohort 6, 30 mg). In cohorts 3–5, a total of 12 subjects each were randomized in a 5:1 ratio to receive a single dose of hyzetimibe (n ¼ 10) or matching placebo (n ¼ 2). In cohorts 1, 2, and 6, a total of 8 subjects each were randomized in a 3:1 ratio to receive HS-25 (n ¼ 6) or placebo (n ¼ 2). The starting dose (1 mg) was calculated according to applicable regulatory guidance,10,11 using the No Observable Adverse Effect Level (NOAEL) in the most sensitive species (500 mg/kg/day in Sprague– Dawley rats and cynomolgus monkeys) and a conservative standard cross-species safety factor of 4800. Each dose of hyzetimibe or placebo was taken orally with water (240 mL) after a minimum 10-hour fast. Regular standardized meals were provided 4 hours after drug administration. Water was allowed as desired, except for 1 hour before and 2 hours after drug administration. Subjects were admitted to the clinical center 1 day prior to drug administration for baseline procedures, and remained in the clinical center for 3 overnight stays and 3 outpatient visits until discharge. The decision to proceed to each higher dose cohort was based on a review of safety data collected for 6 days after administration, and if it was considered acceptable by the investigator and sponsor. Multiple once-daily doses of hyzetimibe (5, 10, and 20 mg) for 10 days were also studied under a randomized, double-blind, placebo-controlled, parallel-group, ascending-dose design. Three sequential cohorts of 12 healthy subjects (6 men and 6 women) each were enrolled. Within each cohort, 2 subjects were randomized to placebo and 10 to hyzetimibe. Sixteen male healthy subjects were enrolled for an open-label, randomized, two-period, cross-over study. They were randomized to receive a single oral dose of 10 mg hyzetimibe with 240 mL water after a 10-hour fast
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or 30 minutes after consumption of a standard high-fat, high-calorie breakfast. This alternate treatment was administrated after a 5-day washout period. Meal composition was based on a standard high-fat, highcalorie (~940 calories) breakfast, which consisted of 2 fried eggs, 85 g sausage, 96 g toasted bread, and 250 mL whole milk. Meals were consumed within 30 minutes. The study drugs (tablets of HS-25 and placebo, both identical in appearance) were manufactured by Zhejiang Hisun Pharmaceutical Co. Ltd. according to Good Manufacturing Practice guidelines. Safety Assessment All available data from subjects who received hyzetimibe or placebo were included in the summaries of safety data. For all studies, safety assessments were performed during the whole study period. Safety assessments included monitoring of adverse events (AEs), physical examinations, vital signs, clinical laboratory tests, and 12-lead ECG. Data on AEs were obtained from information volunteered by subjects or the investigators’ review of their vital signs, ECG, and laboratory test results. Sample Collection In the single ascending-dose and food effect studies, blood samples (3.0 mL) for determination of plasma hyzetimibe and hyzetimibe-glucuronide concentrations were collected by an indwelling catheter or direct venipuncture. Blood samples were collected within 30 minutes prior to drug administration (t ¼ 0), and 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 18, 24, 36, 48, 72, 96, and 120 hours after administration. In the multiple ascending-dose study, 3-mL blood samples were collected prior to dosing (0 hour), and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 18, and 24 hours after dosing on days 1 and 10. Blood samples were also collected prior to dosing (0 hour) on days 8 and 9; and at 36, 48, 72, 96, and 120 hours after dosing on day 10. The blood samples were centrifuged (3000 rpm) within 30 minutes of collection using a refrigerated centrifuge to separate the plasma. The plasma samples were frozen after collection and maintained at 70°C until analysis. Urine samples for measurement were collected in cohort 4 (10 mg) of the single ascending-dose study. Urine samples were collected within 30 minutes prior to dosing (t ¼ 0) and over the following intervals: 0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, and 96–120 hours post-dose. The volume of urine sample collected over each interval was measured, recorded, and mixed with an appropriate amount of acetic acid to reach a final concentration (0.1%, v/v). One 5-mL aliquot from each collection interval was then separated for analysis. The urine samples were frozen and maintained at 70°C.
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Analysis Pharmacokinetic Analysis. Plasma concentrations of hyzetimibe and hyzetimibe-glucuronide were determined using a liquid chromatography/tandem mass spectrometry analysis method with calibration ranging from 0.1 to 100 and 1 to 1000 ng/mL (correlation coefficient R2 0.99), with a lower limit of quantitation at 0.1 and 1 ng/mL, respectively. This method was validated based on 2001 FDA bioanalytical method validation guidance and 2009 EMA validation of bioanalytical methods. Hyzetimibe and hyzetimibe-glucuronide were extracted from plasma by protein precipitation using methanol. Stable isotopelabeled hyzetimibe and hyzetimibe-glucuronide were used as internal standards. Chromatographic separation of analytes was performed using a ZORBAX-SB-C18 column (1.8 mm, 2.1 50 mm2, Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of methanol and water (5/95, v/v) at a flow rate of 0.3 mL/min with gradient elution. All of the analytes were monitored by an API 5500 tandem mass spectrometer on negative ionization mode with Analyst(r) 1.6 software (Applied Biosystems, Foster City, CA, USA). The intra-run precision of quality control (QC) samples was 13.44% for hyzetimibe and 12.15% for hyzetimibe-glucuronide, which is within acceptance criteria (coefficient of variation, CV 15%). Urine concentrations of hyzetimibe and hyzetimibe-glucuronide were determined using the same method as that for plasma samples, with the calibration ranging from 0.1 to 50 and 1 to 5000 ng/mL (correlation coefficient R2 0.99), respectively. The intra-run precision was 7.53% for hyzetimibe and 9.19% for hyzetimibe-glucuronide. The stability of hyzetimibe, hyzetimibe-glucuronide and their internal standards was acceptable under a variety of conditions for samples (ie, 4 hours at room temperature, 3 cycles of freeze–thaw, and 94 days at 70°C). Pharmacokinetic parameters were calculated using a standard non-compartmental method with WinNolin software (Version 6.3, Pharsight Corporation, Mountain View, CA, USA). The maximum plasma concentration (Cmax) and the time to maximum concentration (tmax) were obtained from observed data of individual drug plasma concentration–time data. The terminal elimination rate constant (lz) was determined by least-squares regression analysis of the log-linear portion of the terminal phase. The area under the plasma concentration time curve from time zero to infinity (AUC0–1) was calculated as AUC0–t þ Cz/lz, where Cz is the last measurable plasma concentration at time t. The terminal half-life (t1/2) was calculated as 0.693/lz. Urine excretion pharmacokinetic parameters, such as the elimination rate constant (lz), the maximum excretion rate (Max-Rate), the time to maximum excretion rate (tMax-Rate), the amount of drug excretion in urine, and the percentage of urine excretion (%), were calculated.
Pharmacodynamic Analysis. Blood samples for pharmacodynamics analysis were collected in the multiple ascending-dose study at screening visits as follows: day 1, day 10 (prior to last dosing) and day 15 (end-of-study visit). Changes in LDL-C levels from day 1 to day 10 were assessed. Statistical Analysis. To assess dose proportionality following single doses of hyzetimibe, the pharmacokinetic parameters AUC0–t and Cmax of 6 doses were lntransformed using the Power model: ln(Y) ¼ a þ bln(Dose), where Y is AUC0–t or Cmax. A linear relationship between pharmacokinetic parameters and dose was considered if 90% confidence intervals (CIs) of the slope b were within the predefined range 1þ
lnðuLÞ lnðuHÞ ;1 þ lnðRÞ lnðRÞ
where uL ¼ 0.8, uH ¼ 1.25, and R is the ratio of the highest dose and the lowest dose. To assess the effect of food on the pharmacokinetics of hyzetimibe, analysis of variance was performed for the logarithms of Cmax and AUC0–t. No effect of food was concluded if 90% CIs for ratios of geometric means for Cmax and AUC0–t, with and without food, were contained entirely within the equivalence interval of 80–125%. Statistical analyses were performed using SAS software (Version 9.1; SAS Institute, Inc., Cary, NC, USA).
Results Subjects A total of 60 healthy subjects (30 men and 30 women) were enrolled in the single ascending-dose study. Their age was 22.4 2.6 years, height was 166.3 7.6 cm, weight was 57.8 7.5 kg, and BMI was 21.0 1.5 kg/m2. Fifty-nine (98.3%) subjects were randomized to receive hyzetimibe (n ¼ 47) or placebo (n ¼ 12), and 58 (96.7%) subjects’ blood samples were collected for the pharmacokinetics study. One subject withdrew consent prior to dosing and another subject did not complete the blood collection process. Both of these subjects were excluded from evaluation of pharmacokinetics. The multiple ascending-dose study included a total of 36 healthy subjects (18 men and 18 women). Their age was 22.6 2.3 years, height was 166.5 7.6 cm, weight was 58.3 6.1 kg, and BMI was 21.0 1.3 kg/m2. All of them completed the whole study. The 16 male healthy subjects enrolled in the food effect study were aged 23.6 3.7 years, height was 171.6 4.4 cm, weight was 64.7 4.2 kg, and BMI was 22.0 1.4 kg/m2. All of them completed the whole study.
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Pharmacokinetics Single Ascending-Dose Study. The mean hyzetimibe and hyzetimibe-glucuronide plasma concentration versus time curves for subjects in each cohort are shown in Figures S1–S6. Following a single dose, hyzetimibe and hyzetimibe-glucuronide plasma concentrations showed multiple peaks, while hyzetimibe-glucuronide plasma concentrations appeared to have a multiphasic elimination profile with an initial relatively rapid decline, followed by a more gradual terminal phase. The pharmacokinetic parameters are shown in Table 1. Mean terminal t1/2 values ranged from 16 to 31 hours (hyzetimibe) or from 17 to 24 hours (hyzetimibe-glucuronide), and appeared to be independent of the dose (dose range, 5–30 mg). We found that tmax of hyzetimibe and hyzetimibe-glucuronide was similar, the median tmax of which occurred between 1.00 and 1.75 hours and between 0.75 and 2.00 hours, respectively, after administration. The Power model was used to assess dose proportionality following single doses of hyzetimibe. For hyzetimibe, the 90% CIs of the slope b of AUC0–t and Cmax were 0.784, 1.036 and 0.732, 1.023, respectively. For hyzetimibe-glucuronide, the 90% CIs of the slope b of AUC0–t and Cmax were 0.808, 1.040 and 0.750, 1.028, respectively. All of the 90% CIs of the slope b were not within the predefined range (0.934, 1.066) in which a dose-linearity relationship could be considered. The direction of the deviation was lower than expected from fitting the AUC0–t and Cmax (Figure 2). These results suggested that the
main pharmacokinetic parameters of hyzetimibe and hyzetimibe-glucuronide were positively correlated with single doses ranging from 1 to 30 mg. However, linearity was not present because of individual differences among the subjects. Urine samples for the pharmacokinetic profile after single administration (10 mg) were collected in corresponding intervals. All of the subjects who had a dose of 10 mg hyzetimibe were included in assessment of urine pharmacokinetics. The mean t1/2 was 23.93 hours for hyzetimibe and 19.11 hours for hyzetimibe-glucuronide, and these were similar to those of plasma concentrations. The accumulative urine excretion–time curve and excretion rate–time curve are shown in Figure S7. Urine pharmacokinetic parameters are shown in Table S1. Approximately 20% of the administrated hyzetimibe dose (10 mg) was excreted in the glucuronide-conjugated form by urine and a small amount (0.11% of the dose) of unconjugated form was found in urine. Multiple Ascending-Dose Study. The mean plasma concentration versus time profiles of hyzetimibe and hyzetimibe-glucuronide after administration of escalating multiple once-daily doses to subjects are shown in Figure 3A and B. The steady-state (day 10) pharmacokinetic parameters of hyzetimibe and hyzetimibe-glucuronide are shown in Table 2. The differences among trough concentrations on days 8–10 were
Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of Hyzetimibe (HS-25) in Healthy Chinese Subjects
The Journal of Clinical Pharmacology 54(10) 1144–1152 © 2014, The American College of Clinical Pharmacology DOI: 10.1002/jcph.310
Zhourong Ruan, BS1, Bo jiang, MS1, Jinliang Chen, PharmD1, Xuehua Zhang, MD2, Honggang Lou, MS1, Meixiang Xiang, MD2, Qingxiang Shao, BS3, and Jian’an Wang, MD, PhD1,2
Abstract Hyzetimibe (HS-25) is a new cholesterol absorption inhibitor. We performed the first-in-human study to assess the safety, tolerability, and pharmacokinetics (including the effect of food) and pharmacodynamics (effect on blood lipid level) following single (1, 3, 5, 10, 20, and 30 mg) and multiple (5, 10, and 20 mg) ascending-dose of hyzetimibe in healthy subjects. An increase of exposure (area under the plasma concentration–time curve and maximum plasma concentration) to hyzetimibe and hyzetimibe-glucuronide (HS-25M1) was observed in an approximately dose-proportional manner. A terminal half-life of approximately 21 hours was observed with doses ranging between 5 and 30 mg. Steady state was achieved by day 8 of once-daily dosing with 1.6- and 1.2-fold accumulation for hyzetimibe and hyzetimibe-glucuronide, respectively. Food did not have any effect on hyzetimibe and hyzetimibe-glucuronide exposure. Administration of hyzetimibe once daily for 10 days reduced the levels of low-density lipoprotein cholesterol levels in healthy subjects and these recovered after discontinuation of this drug. All of the adverse events were mild or moderate in severity, and the majority of them were unrelated to hyzetimibe, with no dose-dependent trends. These findings suggest that hyzetimibe could be a potential treatment for hypercholesterolemia.
Keywords cholesterol absorption inhibitor, hyzetimibe (HS-25), first-in-human study, pharmacokinetics, pharmacodynamics
Hypercholesterolemia is a major risk factor for the development of coronary heart disease, which is a leading cause of mortality and morbidity. Elevated low-density lipoprotein cholesterol (LDL-C) levels are positively associated with the incidence of cardiovascular events.1 Evidence is mounting from clinical trials that reducing the levels of total cholesterol and LDL-C, and increasing high-density lipoprotein cholesterol (HDL-C) levels by dietary and/or pharmacological means, could lead to a reduction in the incidence of death from cardiovascular events.2,3 Therefore, recent treatment guidelines emphasize that people who have a substantial risk for coronary heart disease should meet defined target levels of LDL-C.4,5 Circulating plasma levels of cholesterol are primarily produced from 2 processes: cholesterol production from the liver and peripheral tissues, and absorption of dietary and biliary cholesterol in the gastrointestinal tract.6 Statin therapy is the mainstay for lowering of LDL-C levels, which efficiently inhibits the activity of 3-hydroxy-3methyl-glutaryl coenzyme A reductase, resulting in a reduction in cholesterol synthesis.7,8 However, many patients do not achieve the optimal targets or have partial or complete intolerance to them.5,9 Therefore, an increasing amount of attention has focused on inhibiting cholesterol absorption in the gastrointestinal tract as another treatment option.
Hyzetimibe (HS-25, 1-(4-fluorophenyl)-3(R)-[3-(4fluorophenyl)-4-hydroxybut-2(Z)-enyl]-4 (S)-(4-hydroxyphenyl)-2-azetidinone), developed by Zhejiang Hisun Pharmaceutical Co. Ltd. (Taizhou, Zhejiang, China), is a new compound belonging to the novel class of selective cholesterol absorption inhibitors, which effectively blocks intestinal absorption of cholesterol and phytosterols. In previous studies, hyzetimibe was well tolerated in rats with a maximum tolerated dose not less than 2000 mg/kg in a single dose, and 500 mg/kg in cynomolgus monkeys. No toxicity was observed with administration of 500 mg/kg once-daily for 4 weeks in
1
Center of Clinical Pharmacology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 2 Department of Cardiology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 3 Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, China Submitted for publication 22 January 2014; accepted 11 April 2014. Corresponding Author: Jian’an Wang, MD, PhD, Center of Clinical Pharmacology and Department of Cardiology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, Zhejiang, China Email: [email protected]
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Figure 1. Structural formula of hyzetimibe and its metabolites.
cynomolgus monkeys. Preclinical pharmacokinetic studies have suggested that hyzetimibe is metabolized to its active metabolite acting by different isoforms of UDP-glucuronosyltransferase in the intestine and liver (Figure 1). Hyzetimibe is rapidly and almost completely (98–99%) converted to its main active metabolite (hyzetimibe-glucuronide, HS-25M1, 1-O-[4-[trans-(2S,3R)1-(4-Fluorophenyl)-3(R)-[3-(4-fluorophenyl)-4-hydroxybut-2(Z)-enyl]-4 (S)-(4-hydroxyphenyl)-2-azetidinyl] phenyl]-b-D-glucuronic acid]) in the small intestine after dosing. Hyzetimibe can reduce cholesterol levels in high-cholesterol fed rhesus monkeys. All of these preclinical studies suggest that hyzetimibe is a potential treatment for hypercholesterolemia. In the present study, we carried out the first-in-human assessment of the safety, tolerability, pharmacokinetics, and pharmacodynamics of hyzetimibe following single and multiple ascending-dose oral administration in healthy subjects. We also evaluated the effect of a high-fat, high-calorie meal on the pharmacokinetics of hyzetimibe.
Methods Ethics The study was performed at the Center of Clinical Pharmacology, the Second Affiliated Hospital of
Zhejiang University School of Medicine (Hangzhou, Zhejiang, China). The study protocols and amendments were reviewed and approved by the China Food and Drug Administration and the Human Subject Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine. The study is registered at www.chinadrugtrials.org.cn (CTR20130033). The study was performed in accordance with Good Clinical Practices and the ethical principles enunciated in the revised Declaration of Helsinki (revised version of Seoul, 2008). Subjects provided written informed consent before participating in any study-related procedures. Subjects Male and female healthy subjects (female subjects needed to be non-nursing, non-pregnant, and not of childbearing potential) between the ages of 18 and 45 years who had a body mass index (BMI) between 19 and 24 kg/m2 inclusive and body weight not less than 45 kg were eligible for the study. Subjects were considered healthy by the principal investigator based on a detailed medical history, full physical examination, clinical laboratory test, 12-lead electrocardiogram (ECG), and vital signs. Subjects were required to be nonsmokers with no history of alcohol or drug abuse. Prescription, herbal, and over-the-counter medications were prohibited for 30 days prior to and throughout the whole study. Subjects
1146 were excluded for the following reasons: a clinically significant history of medical illness; positive blood screen for HIV and hepatitis tests; any hospital admission or major surgery within 90 days prior to screening; donation or blood collection of more than 450 mL of blood, or acute loss of blood during the 90 days prior to screening; and heavy tea or coffee drinkers of more than 1 L/day. Female subjects of childbearing potential were required to have a negative pregnancy test upon study entry. All subjects were willing and able to give written informed consent, and to adhere to study restrictions. Study Design The first part of the overall study was a first-in-human phase I study using a randomized, double-blind, placebocontrolled, parallel-group, ascending-dose design. Sixty healthy adult subjects (30 men and 30 women) were enrolled in 6 dose cohorts (cohort 1, 1 mg; cohort 2, 3 mg; cohort 3, 5 mg; cohort 4, 10 mg; cohort 5, 20 mg; cohort 6, 30 mg). In cohorts 3–5, a total of 12 subjects each were randomized in a 5:1 ratio to receive a single dose of hyzetimibe (n ¼ 10) or matching placebo (n ¼ 2). In cohorts 1, 2, and 6, a total of 8 subjects each were randomized in a 3:1 ratio to receive HS-25 (n ¼ 6) or placebo (n ¼ 2). The starting dose (1 mg) was calculated according to applicable regulatory guidance,10,11 using the No Observable Adverse Effect Level (NOAEL) in the most sensitive species (500 mg/kg/day in Sprague– Dawley rats and cynomolgus monkeys) and a conservative standard cross-species safety factor of 4800. Each dose of hyzetimibe or placebo was taken orally with water (240 mL) after a minimum 10-hour fast. Regular standardized meals were provided 4 hours after drug administration. Water was allowed as desired, except for 1 hour before and 2 hours after drug administration. Subjects were admitted to the clinical center 1 day prior to drug administration for baseline procedures, and remained in the clinical center for 3 overnight stays and 3 outpatient visits until discharge. The decision to proceed to each higher dose cohort was based on a review of safety data collected for 6 days after administration, and if it was considered acceptable by the investigator and sponsor. Multiple once-daily doses of hyzetimibe (5, 10, and 20 mg) for 10 days were also studied under a randomized, double-blind, placebo-controlled, parallel-group, ascending-dose design. Three sequential cohorts of 12 healthy subjects (6 men and 6 women) each were enrolled. Within each cohort, 2 subjects were randomized to placebo and 10 to hyzetimibe. Sixteen male healthy subjects were enrolled for an open-label, randomized, two-period, cross-over study. They were randomized to receive a single oral dose of 10 mg hyzetimibe with 240 mL water after a 10-hour fast
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or 30 minutes after consumption of a standard high-fat, high-calorie breakfast. This alternate treatment was administrated after a 5-day washout period. Meal composition was based on a standard high-fat, highcalorie (~940 calories) breakfast, which consisted of 2 fried eggs, 85 g sausage, 96 g toasted bread, and 250 mL whole milk. Meals were consumed within 30 minutes. The study drugs (tablets of HS-25 and placebo, both identical in appearance) were manufactured by Zhejiang Hisun Pharmaceutical Co. Ltd. according to Good Manufacturing Practice guidelines. Safety Assessment All available data from subjects who received hyzetimibe or placebo were included in the summaries of safety data. For all studies, safety assessments were performed during the whole study period. Safety assessments included monitoring of adverse events (AEs), physical examinations, vital signs, clinical laboratory tests, and 12-lead ECG. Data on AEs were obtained from information volunteered by subjects or the investigators’ review of their vital signs, ECG, and laboratory test results. Sample Collection In the single ascending-dose and food effect studies, blood samples (3.0 mL) for determination of plasma hyzetimibe and hyzetimibe-glucuronide concentrations were collected by an indwelling catheter or direct venipuncture. Blood samples were collected within 30 minutes prior to drug administration (t ¼ 0), and 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 18, 24, 36, 48, 72, 96, and 120 hours after administration. In the multiple ascending-dose study, 3-mL blood samples were collected prior to dosing (0 hour), and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 18, and 24 hours after dosing on days 1 and 10. Blood samples were also collected prior to dosing (0 hour) on days 8 and 9; and at 36, 48, 72, 96, and 120 hours after dosing on day 10. The blood samples were centrifuged (3000 rpm) within 30 minutes of collection using a refrigerated centrifuge to separate the plasma. The plasma samples were frozen after collection and maintained at 70°C until analysis. Urine samples for measurement were collected in cohort 4 (10 mg) of the single ascending-dose study. Urine samples were collected within 30 minutes prior to dosing (t ¼ 0) and over the following intervals: 0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, and 96–120 hours post-dose. The volume of urine sample collected over each interval was measured, recorded, and mixed with an appropriate amount of acetic acid to reach a final concentration (0.1%, v/v). One 5-mL aliquot from each collection interval was then separated for analysis. The urine samples were frozen and maintained at 70°C.
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Analysis Pharmacokinetic Analysis. Plasma concentrations of hyzetimibe and hyzetimibe-glucuronide were determined using a liquid chromatography/tandem mass spectrometry analysis method with calibration ranging from 0.1 to 100 and 1 to 1000 ng/mL (correlation coefficient R2 0.99), with a lower limit of quantitation at 0.1 and 1 ng/mL, respectively. This method was validated based on 2001 FDA bioanalytical method validation guidance and 2009 EMA validation of bioanalytical methods. Hyzetimibe and hyzetimibe-glucuronide were extracted from plasma by protein precipitation using methanol. Stable isotopelabeled hyzetimibe and hyzetimibe-glucuronide were used as internal standards. Chromatographic separation of analytes was performed using a ZORBAX-SB-C18 column (1.8 mm, 2.1 50 mm2, Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of methanol and water (5/95, v/v) at a flow rate of 0.3 mL/min with gradient elution. All of the analytes were monitored by an API 5500 tandem mass spectrometer on negative ionization mode with Analyst(r) 1.6 software (Applied Biosystems, Foster City, CA, USA). The intra-run precision of quality control (QC) samples was 13.44% for hyzetimibe and 12.15% for hyzetimibe-glucuronide, which is within acceptance criteria (coefficient of variation, CV 15%). Urine concentrations of hyzetimibe and hyzetimibe-glucuronide were determined using the same method as that for plasma samples, with the calibration ranging from 0.1 to 50 and 1 to 5000 ng/mL (correlation coefficient R2 0.99), respectively. The intra-run precision was 7.53% for hyzetimibe and 9.19% for hyzetimibe-glucuronide. The stability of hyzetimibe, hyzetimibe-glucuronide and their internal standards was acceptable under a variety of conditions for samples (ie, 4 hours at room temperature, 3 cycles of freeze–thaw, and 94 days at 70°C). Pharmacokinetic parameters were calculated using a standard non-compartmental method with WinNolin software (Version 6.3, Pharsight Corporation, Mountain View, CA, USA). The maximum plasma concentration (Cmax) and the time to maximum concentration (tmax) were obtained from observed data of individual drug plasma concentration–time data. The terminal elimination rate constant (lz) was determined by least-squares regression analysis of the log-linear portion of the terminal phase. The area under the plasma concentration time curve from time zero to infinity (AUC0–1) was calculated as AUC0–t þ Cz/lz, where Cz is the last measurable plasma concentration at time t. The terminal half-life (t1/2) was calculated as 0.693/lz. Urine excretion pharmacokinetic parameters, such as the elimination rate constant (lz), the maximum excretion rate (Max-Rate), the time to maximum excretion rate (tMax-Rate), the amount of drug excretion in urine, and the percentage of urine excretion (%), were calculated.
Pharmacodynamic Analysis. Blood samples for pharmacodynamics analysis were collected in the multiple ascending-dose study at screening visits as follows: day 1, day 10 (prior to last dosing) and day 15 (end-of-study visit). Changes in LDL-C levels from day 1 to day 10 were assessed. Statistical Analysis. To assess dose proportionality following single doses of hyzetimibe, the pharmacokinetic parameters AUC0–t and Cmax of 6 doses were lntransformed using the Power model: ln(Y) ¼ a þ bln(Dose), where Y is AUC0–t or Cmax. A linear relationship between pharmacokinetic parameters and dose was considered if 90% confidence intervals (CIs) of the slope b were within the predefined range 1þ
lnðuLÞ lnðuHÞ ;1 þ lnðRÞ lnðRÞ
where uL ¼ 0.8, uH ¼ 1.25, and R is the ratio of the highest dose and the lowest dose. To assess the effect of food on the pharmacokinetics of hyzetimibe, analysis of variance was performed for the logarithms of Cmax and AUC0–t. No effect of food was concluded if 90% CIs for ratios of geometric means for Cmax and AUC0–t, with and without food, were contained entirely within the equivalence interval of 80–125%. Statistical analyses were performed using SAS software (Version 9.1; SAS Institute, Inc., Cary, NC, USA).
Results Subjects A total of 60 healthy subjects (30 men and 30 women) were enrolled in the single ascending-dose study. Their age was 22.4 2.6 years, height was 166.3 7.6 cm, weight was 57.8 7.5 kg, and BMI was 21.0 1.5 kg/m2. Fifty-nine (98.3%) subjects were randomized to receive hyzetimibe (n ¼ 47) or placebo (n ¼ 12), and 58 (96.7%) subjects’ blood samples were collected for the pharmacokinetics study. One subject withdrew consent prior to dosing and another subject did not complete the blood collection process. Both of these subjects were excluded from evaluation of pharmacokinetics. The multiple ascending-dose study included a total of 36 healthy subjects (18 men and 18 women). Their age was 22.6 2.3 years, height was 166.5 7.6 cm, weight was 58.3 6.1 kg, and BMI was 21.0 1.3 kg/m2. All of them completed the whole study. The 16 male healthy subjects enrolled in the food effect study were aged 23.6 3.7 years, height was 171.6 4.4 cm, weight was 64.7 4.2 kg, and BMI was 22.0 1.4 kg/m2. All of them completed the whole study.
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Pharmacokinetics Single Ascending-Dose Study. The mean hyzetimibe and hyzetimibe-glucuronide plasma concentration versus time curves for subjects in each cohort are shown in Figures S1–S6. Following a single dose, hyzetimibe and hyzetimibe-glucuronide plasma concentrations showed multiple peaks, while hyzetimibe-glucuronide plasma concentrations appeared to have a multiphasic elimination profile with an initial relatively rapid decline, followed by a more gradual terminal phase. The pharmacokinetic parameters are shown in Table 1. Mean terminal t1/2 values ranged from 16 to 31 hours (hyzetimibe) or from 17 to 24 hours (hyzetimibe-glucuronide), and appeared to be independent of the dose (dose range, 5–30 mg). We found that tmax of hyzetimibe and hyzetimibe-glucuronide was similar, the median tmax of which occurred between 1.00 and 1.75 hours and between 0.75 and 2.00 hours, respectively, after administration. The Power model was used to assess dose proportionality following single doses of hyzetimibe. For hyzetimibe, the 90% CIs of the slope b of AUC0–t and Cmax were 0.784, 1.036 and 0.732, 1.023, respectively. For hyzetimibe-glucuronide, the 90% CIs of the slope b of AUC0–t and Cmax were 0.808, 1.040 and 0.750, 1.028, respectively. All of the 90% CIs of the slope b were not within the predefined range (0.934, 1.066) in which a dose-linearity relationship could be considered. The direction of the deviation was lower than expected from fitting the AUC0–t and Cmax (Figure 2). These results suggested that the
main pharmacokinetic parameters of hyzetimibe and hyzetimibe-glucuronide were positively correlated with single doses ranging from 1 to 30 mg. However, linearity was not present because of individual differences among the subjects. Urine samples for the pharmacokinetic profile after single administration (10 mg) were collected in corresponding intervals. All of the subjects who had a dose of 10 mg hyzetimibe were included in assessment of urine pharmacokinetics. The mean t1/2 was 23.93 hours for hyzetimibe and 19.11 hours for hyzetimibe-glucuronide, and these were similar to those of plasma concentrations. The accumulative urine excretion–time curve and excretion rate–time curve are shown in Figure S7. Urine pharmacokinetic parameters are shown in Table S1. Approximately 20% of the administrated hyzetimibe dose (10 mg) was excreted in the glucuronide-conjugated form by urine and a small amount (0.11% of the dose) of unconjugated form was found in urine. Multiple Ascending-Dose Study. The mean plasma concentration versus time profiles of hyzetimibe and hyzetimibe-glucuronide after administration of escalating multiple once-daily doses to subjects are shown in Figure 3A and B. The steady-state (day 10) pharmacokinetic parameters of hyzetimibe and hyzetimibe-glucuronide are shown in Table 2. The differences among trough concentrations on days 8–10 were
