Pharmacology & Pharmacy, 2013, 4, 663-678
`Published Online December 2013 (http://www.scirp.org/journal/pp)
`http://dx.doi.org/10.4236/pp.2013.49093
`
`663
`
`Safety, Tolerability, Pharmacokinetics and
`Pharmacodynamics of E3024, a Novel and Selective
`Dipeptidyl Peptidase-IV Inhibitor, in Healthy Japanese
`Male Subjects: Rash Development in Men and Its Possible
`Mechanism*
`
`Yutaka Takeuchi1, Masayuki Namiki2, Yasumi Kitahara1, Setsuo Hasegawa3,4, Akihiro Ohnishi5,
`Nobuyuki Yasuda6, Takashi Inoue6, Richard Clark6, Kazuto Yamazaki6#
`1Clinical Development, Japan/Asia Clinical Research PCU, Eisai Co., Ltd., Tokyo, Japan; 2Clinical Pharmacology, Clinical Science,
`SOCS CFU, Eisai Co., Ltd., Tokyo, Japan; 3Sekino Clinical Pharmacology Clinic, Tokyo, Japan; 4Present Address: Pharmaspur, Inc.,
`Tokyo, Japan; 5Department of Laboratory Medicine, Daisan Hospital, Jikei University School of Medicine, Tokyo, Japan; 6Tsukuba
`Research Laboratories, Eisai Co., Ltd., Tsukuba, Japan.
`Email: #k5-yamazaki@hhc.eisai.co.jp
`
`Received October 16th, 2013; revised November 28th, 2013; accepted December 2nd, 2013
`
`Copyright © 2013 Yutaka Takeuchi et al. This is an open access article distributed under the Creative Commons Attribution License,
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`
`
`ABSTRACT
`E3024 (3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate) is a dipeptidyl
`peptidase-IV (DPP-IV) inhibitor that was expected to be an antidiabetic agent. Its safety, tolerability, pharmacokinetics
`(PK), and pharmacodynamics (PD) were investigated in a randomized, double-blind, placebo-controlled, ascending
`single-dose study in 48 healthy Japanese male subjects. Fasted subjects were orally administered E3024 (5, 10, 20, 40,
`or 80 mg) or placebo. E3024 was rapidly absorbed, with tmax values ranging between 0.33 and 3 h after dosing. The
`mean t1/2 ranged from 5.34 to 11.68 h. AUC0-inf and Cmax increased dose-proportionately. PK-PD relationship of E3024
`was evaluated by using an Imax model, indicating that plasma E3024 concentrations and inhibitory effects of plasma
`DPP-IV activity were well correlated. The IC50 value was calculated as 33.7 ng/mL, which was consistent with in vitro
`data. Thus, E3024 showed a good PK profile and inhibited DPP-IV dose-dependently. Of 30 subjects administered
`E3024, 12 (40%) experienced adverse events (AEs). Dose escalation to 160 mg was abandoned owing to undesired
`subjective/objective findings in 4 of 6 subjects receiving 40 mg and 5 of 6 subjects receiving 80 mg. The most promi-
`nent AE was rash, but there were no serious AEs or deaths. The maximum tolerated dose was considered to be 20 mg.
`We hypothesized that histamine was a cause of the rash induction, and examined blood histamine levels of normal
`Fischer rats treated with E3024. Blood histamine levels were increased significantly by E3024 at 500 mg/kg (p < 0.001),
`but not by vildagliptin or valine-pyrrolidide (DPP-IV inhibitors) at the same dose. No blood histamine increases were
`observed in genetically mast cell-deficient Ws/Ws rats treated with E3024 at 500 mg/kg. In in vitro assays, E3024 in-
`duced histamine release from normal rat peritoneal mast cells in a concentration-dependent manner, but not from baso-
`phils. The structure-activity relationship study suggested that a piperazine group N-linked to the 2-position of the
`5,6-membered fused heterocyclic rings was a key structural element for triggering histamine release.
`
`Keywords: Dipeptidyl Peptidase-IV Inhibitor; Rash; Histamine; Structure-Activity Relationship
`
`1. Introduction
`Dipeptidyl peptidase-IV (DPP-IV) inhibitors have been
`*This clinical study was sponsored by Eisai Co., Ltd. Dr. Hasegawa
`was the director of the study site. Dr. Ohnishi was a paid consultant to
`Eisai and other pharmaceutical companies.
`#Corresponding author.
`
`considered highly attractive for the treatment of type 2
`diabetes, as the inhibition of DPP-IV results in an in-
`crease of the endogenous active glucagon-like peptide-1
`(GLP-1) levels [1-5]. Of this class of drugs, sitagliptin
`(MK-0431) [6], vildagliptin (LAF237) [7], saxagliptin
`(BMS-477118) [8], alogliptin (SYR-322) [9], linagliptin
`
`Open Access PP
`
`AstraZeneca Exhibit 2082
`Mylan v. AstraZeneca
`IPR2015-01340
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`664
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`Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl
`Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism
`
`(BI-1356) [10] and anagliptin [11] have all been launched
`into the market for the treatment of type 2 diabetes.
`E3024
`(3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-
`dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate) (Fig-
`ure 1) with a molecular weight of 282.91 is a novel,
`highly selective and competitive DPP-IV inhibitor syn-
`thesized by Eisai Co., Ltd. [12-14]. E3024 inhibited the
`DPP-IV activity in human, mouse, rat and canine plasma
`with IC50 (concentration required for 50% of the maxi-
`mum inhibition) values of 0.14, 0.28, 0.40 and 0.36
`mol/L, respectively. In an oral glucose tolerance test
`using Zucker fa/fa rats, E3024 dose-dependently in-
`creased plasma insulin levels and reduced the area under
`the curve (AUC) of delta blood glucose at doses of 1 and
`3 mg/kg. E3024 had no effect on fasting blood glucose
`levels in normal rats at doses of 1 or 10 mg/kg. These
`non-clinical data had suggested that E3024 would be a
`novel antidiabetic agent in the treatment of postprandial
`hyperglycemia with a low risk of causing hypoglycemia.
`The objectives of the present studies were: 1) to evalu-
`ate the safety, tolerability, pharmacokinetics (PK) and
`pharmacodynamics (PD) after single oral dose of E3024
`in healthy Japanese male subjects, and 2) to examine
`possible mechanisms of rash development observed in
`this clinical trial, using normal and genetically mast cell-
`deficient rats.
`
`2. Materials and Methods
`2.1. Clinical Study
`This study was conducted at Sekino Clinical Pharmacol-
`ogy Clinic, Tokyo, Japan, in accordance with the ethical
`
`
`principles of the Declaration of Helsinki, good clinical
`practice in Japan, and International Conference on Har-
`monization guidelines. The clinical study protocol and
`informed consent documents were approved by the insti-
`tutional review board of Sekino Clinical Pharmacology
`Clinic. Informed consent was obtained from all subjects
`in writing before implementation of any study-related
`procedures.
`
`2.1.1. Study Design
`This study was performed as a randomized, double-blind,
`placebo-controlled, escalating single-dose study. Differ-
`ent groups of eight subjects each were orally adminis-
`tered single doses of E3024 (5, 10, 20, 40, or 80 mg, n =
`6) or placebo (n = 2 for 5, 10, 80 mg; n = 6 for 20, 40 mg)
`after an overnight fast of 10 h.
`E3024 was supplied by Eisai Co., Ltd. E3024 was ad-
`ministered in film-coated tablets containing 1, 10, or 40
`mg of E3024. Placebo was administered in visually
`matching tablets.
`
`2.1.2. Subjects
`Healthy Japanese male subjects between 20 and 39 years
`of age and with body mass index (BMI) of 18.5 to 25.0
`kg/m2 were eligible for participation in this study. Sub-
`jects were excluded if they had a known history of any
`significant drug or food allergy, a significant organ dys-
`function, or any clinically significant deviation from
`normal in medical history, physical examination findings,
`vital signs, electrocardiogram, or laboratory test results.
`Subjects with gastrointestinal, hepatic, renal, respiratory,
`or cardiovascular diseases; congenital metabolic disorder;
`
`
`
`Figure 1. Chemical structures of E3024, ER-319441-15, ER-319433-15, and ER-463809-15.
`
`
`
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`Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl
`Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism
`
`665
`
`lected from a cutaneous vein in the forearm into a so-
`dium-heparinized tube. Samples were centrifuged (4˚C at
`1500× g for 15 min) to obtain plasma. Urine samples
`were collected before dosing and at the following inter-
`vals: 0 to 6 h, 6 to 12 h, 12 to 24 h, 24 to 48 h, 48 to 72 h,
`and 72 to 96 h after dosing, for determination of E3024
`in human urine. Plasma and urine samples were stored at
`−20˚C until sample analysis.
`Analysis was performed by Bioanalysis Section,
`Clinical Research Center at Eisai Co., Ltd. (Tokyo, Ja-
`pan). For quantitative determination of E3024, plasma
`and urine samples were analyzed by a validated liquid
`chromatographic-tandem mass spectrometry (LC/MS/MS)
`method. This method was based on solid-phase extrac-
`tion using Empore extraction disk plates (3M, St. Paul,
`MN) in a 96-well format, with 0.02 mL (plasma) or
`0.005 mL (urine) eluent samples injected into the LC/
`MS/MS.
`Pharmacokinetic parameters were calculated from
`plasma and urine concentrations of E3024 by model-in-
`dependent analysis using WinNonlin Professional ver-
`sion 4.1 (Pharsight Corp., Mountain View, CA). The
`dose-proportionality of maximum observed concentra-
`tion (Cmax) and area under the plasma concentration-time
`curve from 0 to infinity (AUC0-inf) obtained from model-
`independent analysis was assessed both visually and us-
`ing a power model (Y = αXβ; X, dose; Y, Cmax or
`AUC0-inf). Dose proportionality was assessed based on
`whether 95% confidence intervals (CIs) of β lay within
`the range from 0.7 to 1.3 [15].
`
`2.1.5. Pharmacodynamic Assessments
`For DPP-IV activity assay, blood samples were collected
`before dosing and at 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 12,
`and 24 h after dosing. For active GLP-1 and glucagon,
`blood collection was performed before the meals provided
`at 4.5 and 10.5 h after dosing (lunch and dinner, respec-
`tively); and at 0.33, 0.67, 1, 1.5, 2, and 3 h after the
`meals provided at 4.5 and 10.5 h after dosing. Blood (2
`mL) was withdrawn into tubes containing ethylenedia-
`minetetraacetic acid (EDTA) alone (plasma DPP-IV ac-
`tivity assay and plasma active GLP-1) or EDTA plus
`aprotinin (plasma glucagon). For serum insulin, C-pep-
`tide and glucose, blood (3 mL) was withdrawn into se-
`rum separator tubes. For active GLP-1 samples, 50 μL of
`DPP-IV inhibitor solution (Linco Research, Inc., St.
`Charles, MO) was added to each tube within 30 sec after
`collection, and the tubes were gently mixed and placed
`on ice water immediately. After centrifugation, plasma
`and serum samples were stored at −20˚C or below until
`assayed.
`Pharmacodynamic parameters were measured at Mi-
`tsubishi Kagaku Bio-clinical Laboratories, Inc. (now,
`
` a
`
` positive test result for hepatitis B surface antigen, hepa-
`titis C antibody or human immunodeficiency virus; or
`alcohol or drug abuse (or a positive urine drug test result
`at screening) were excluded from participation. Subjects
`were excluded if they had a known history of any gas-
`trointestinal surgery that could impact upon absorption of
`the study drug. Subjects were also excluded if they had
`experienced a weight change >10% from screening to
`baseline. Furthermore, any subject was excluded who
`had received blood within three months or donated blood
`(400 mL within three months or 200 mL within 30 days
`of study start), or ingested any investigational medication
`within four months before study start. Subjects were pro-
`hibited from any prescription drugs and over-the-counter
`(OTC) acid controllers within 30 days prior to and during
`the study, and other OTC medications within seven days
`prior to and during the study.
`
`2.1.3. Procedures
`Screening procedures, including medical history taking,
`physical examination, 12-lead electrocardiography (ECG),
`clinical laboratory evaluations, vital signs measurement,
`and urine drug screening, were performed from 30 days
`before study drug administration along with the assess-
`ment of inclusion/exclusion criteria. Eligible subjects
`were admitted to the study site on the day prior to dosing
`for base line evaluations. Subjects were required to ab-
`stain from food and beverages, except water, for at least
`10 h prior to check-in. After the check-in evaluation was
`completed, subjects were provided with an appropriate
`meal(s); thereafter, they were required to fast (abstain
`from food and fluids, except water) overnight for at least
`10 h prior to drug administration on the following day.
`Subjects took the study drug with 200 mL of water in a
`fasted state. Water was allowed ad libitum, except from 2
`h before dosing to 1 h after dosing. Subjects were re-
`quired to abstain from food up to 4.5 h after dosing.
`Subjects received a standardized meal at 4.5 (lunch) and
`10.5 (dinner) h after dosing to assess the pharmacody-
`namic effects of E3024 on GLP-1, insulin, C-peptide,
`glucagon, and glucose. The total energy of each meal
`was 800 kcal, with a nutrient breakdown of 25% fat, 15%
`protein, and 60% carbohydrate. Subsequent meals were
`provided as per the regular meal schedule at the site.
`Subjects were to maintain an upright (seated or standing)
`position for at least 4.5 h following administration of the
`study drug.
`
`2.1.4. Pharmacokinetic Assessments
`Blood samples were collected at 0 (pre-dose), 0.33, 0.67,
`1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, and 96 h after ad-
`ministration of the study drug for determination of E3024
`in human plasma. Blood samples (3 mL each) were col-
`
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`Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl
`Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism
`
`Mitsubishi Chemical Medience Corp., Tokyo, Japan).
`DPP-IV enzyme activity was determined via incubation
`of 20-μL EDTA-treated human plasma (5-fold dilution in
`assay) with the substrate, glycyl-L-proline 7-amido-4-
`methyl-coumarin hydrobromide (H-Gly-Pro-AMC·HBr;
`0.08 mmol/L in assay) at room temperature for 10 min by
`measurement of the release of 7-amino-4-methyl-cou-
`marin with a multifunctional microplate reader (excita-
`tion 360 nm; emission 465 nm). Enzyme activity (1
`mU/mL) was defined as the amount of enzyme required
`to degrade 1 nmole of substrate per min in 1 mL of reac-
`tion solution (mU/mL = nmol/mL·min). The range of
`reliable quantitation was 0.05 to 20.0 mU/mL. Active
`GLP-1 (GLP-1-[7-36]amide and GLP-1-[7-37]) was as-
`sayed with an enzyme-linked immunosorbent assay
`(ELISA) kit (Linco Research, Inc.). The lower limit of
`reliable quantitation was estimated to be 5.00 pmol/L. If
`concentrations could be calculated from measured fluo-
`rescence intensity, the values were used in the analysis
`even if less than 5.00 pmol/L. If concentrations could not
`be calculated, the measured values were defined as zero.
`Insulin, C-peptide, glucagon, and glucose concentrations
`were measured by standard methods in the laboratory, i.e.
`immunoradiometric assay for insulin, radioimmunoassay
`for glucagon and C-peptide, and enzymatic assay for
`glucose.
`For pharmacodynamic parameters, the values meas-
`ured and changes from baseline at each time point were
`summarized using descriptive statistics by dose. Percent
`inhibition of plasma DPP-IV activity for each subject
`was plotted against plasma E3024 concentration, and an
`Imax model (effect = Imax·C/(IC50 + C); where C is plasma
`E3024 concentration) was used to determine the IC50
`values.
`
`2.1.6. Safety Assessments
`The following data were collected during the study to
`assess safety: physical examination findings, vital signs
`(blood pressure, pulse rate, respiratory rate, and body
`temperature), body weight, 12-lead ECGs, and clinical
`laboratory parameters (hematology, biochemistry and
`urinalysis). In the case of a clinically significant abnor-
`mal value, the evaluation was to be repeated until the
`value was within an acceptable or normal range. AEs
`were to be followed to resolution.
`From subjects who had rash in the 40-mg group, blood
`samples were collected for measurement of non-specific
`immunoglobulin E (IgE) at 24 and 96 h, and drug-in-
`duced lymphocyte stimulation test (DLST) at 96 h after
`dosing. In the same way, from subjects who had rash in
`the 80-mg group, blood samples were collected for
`measurement of IgE, serotonin, histamine, and substance
`P at onset of rash (the nearest pharmacokinetic time
`
`point), 24 and 96 h after dosing. Blood samples, which
`were collected for clinical laboratory tests the day before
`dosing, were also used to obtain baseline data for IgE,
`histamine and substance P. These additional assays were
`performed at the study site for IgE, and at Mitsubishi
`Kagaku Bio-clinical Laboratories, Inc. for DLST, sero-
`tonin, histamine and substance P.
`The numbers of subjects with AEs were tabulated. For
`clinical laboratory parameters (except urinalysis), vital
`signs, body weight, and 12-lead ECG parameters, the
`values measured and changes from baseline at each time
`point were summarized using descriptive statistics by
`dose. For urinalysis, cross tables were prepared.
`
`2.2. Non-Clinical in Vivo and in Vitro Studies
`2.2.1. Chemicals
`E3024, vildagliptin, valine-pyrrolidide (a DPP-IV in-
`hibitor [16]), ER-319441-15 (trifluoroacetate salt form of
`ER-319441 (2-(3-amino-piperidin-1-yl)-3-but-2-ynyl-5-
`methyl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one)),
`ER-319433-15 (trifluoroacetate salt form of ER-319433
`(2-{[7-(but-2-yn-1-yl)-1-[(4-cyanophenyl)methyl]-6-oxo-
`8-(piperazin-1-yl)-6,7-dihydro-1H-purin-2-yl]methyl}
`benzamide)), and ER-463809-15 (trifluoroacetate salt form
`of ER-463809 (2-({8-(3-aminopiperidin-1-yl)-7-(but-2-
`yn-1-yl)-1-[(4-cyanophenyl)methyl]-6-oxo-6,7-dihydro-
`1H-purin-2-yl}methyl)benzamide)) were synthesized in
`our laboratories. Chemical structures of ER-319441-15,
`ER-319433-15 and ER-463809-15 are indicated in Fig-
`ure 1. A23187 (a calcium ionophore) and dimethyl sul-
`foxide (DMSO) were purchased from Sigma-Aldrich (St.
`Louis, MO). Methylcellulose (MC) was obtained from
`Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
`
`2.2.2. Animals
`Five-week-old normal male Fischer (F344/Jcl) rats were
`purchased from CLEA Japan, Inc. (Tokyo, Japan). Five-
`week-old male Slc:WsRC-Ws/Ws (Ws/Ws) and Slc:
`WsRC-+/+ (+/+; wild-type homozygous) rats were ob-
`tained from Japan SLC, Inc. (Hamamatsu, Japan). The
`rats were provided with a commercial diet (MF; Oriental
`Yeast, Tokyo, Japan) and water ad libitum, and were
`kept under conventional conditions of controlled tem-
`perature, humidity and lighting (22  2˚C, 55  5% and a
`12-hr light/dark cycle with lights on at 07:00 a.m.). All
`procedures were conducted according to the Eisai Ani-
`mal Care Committee’s guideline.
`
`2.2.3. Determination of Plasma Compound
`Concentrations in Rats
`Compounds were suspended in 0.5% MC, and adminis-
`tered to Fischer rats aged eight weeks orally (10 mL/kg).
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`Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl
`Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism
`
`667
`
`After 30 min later, blood samples were taken from the
`tail vein (~200 μL). Plasma was obtained after centrifu-
`gation. The concentrations of compounds were deter-
`mined by the LC/MS/MS method.
`
`2.2.4. Effects of Compounds on Blood Histamine
`Levels
`Compounds or vehicle (0.5% MC, 10 mL/kg) were orally
`administered to seven-week-old Fischer rats. Blood (20
`μL) was taken from the tail vein at 0, 0.5 or 1 h after ad-
`ministration, and was mixed with saline containing 50
`mg/mL EDTA (20 μL). Blood histamine levels were de-
`termined using a Histamine ELISA kit (Immunotech;
`Medical & Biological Laboratories Co., Ltd., Nagoya,
`Japan).
`
`2.2.5. Effects of Compounds on Histamine Release
`from Rat Peritoneal Mast Cells and Basophils
`Peritoneal exudate cells (PECs) were used for studies on
`histamine release from rat mast cells [17]. Seven-week-
`old Fischer rats were sacrificed by exsanguination from
`the carotid artery under deep diethyl ether anesthesia.
`PECs were recovered by washing the peritoneal cavity
`with the injection of 10 mL of Ca2+-free Dulbecco’s
`phosphate-buffered saline (D-PBS(-)) containing 5 units/
`mL of heparin and 0.1% bovine serum albumin, followed
`by gentle massage for 90 sec. The peritoneal fluids were
`pooled and spun down at 1200 rpm for 5 min at 4˚C.
`Cells were washed in D-PBS(-) three times, and resus-
`pended in D-PBS(-).
`We used whole blood cells to investigate histamine
`release from basophils according to the method of Kowal
`et al. [18]. Heparinized whole blood was obtained from
`the posterior vena cava of seven-week-old Fischer rats
`under deep diethyl ether anesthesia, and whole blood was
`diluted to 1/25 with D-PBS(-).
`Mast cells (104 cells/180 μL/tube) or whole blood cells
`
`
`
`(180 μL of the diluted whole blood/tube) were incubated
`with a test compound or vehicle (DMSO) for 30 min at
`37˚C. A23187 was used as a control compound to release
`histamine. Addition of 0.1% Triton X-100 to the tubes
`was performed to obtain total histamine content of cells
`(Hc). Blank tubes containing only cells and buffers were
`used for non-specific spontaneous release (Hs) during the
`reaction. Histamine levels were determined using an
`ELISA kit. The percentage of histamine release induced
`by a compound was calculated according to the follow-
`ing formula:
`
`
`
`
` 
`
`Ht Hs Hc Hs
`
`
`where Ht = test release caused by a compound, Hs =
`spontaneous release, and Hc = total cellular histamine
`content [17].
`
`
`
`100
`
`
`
`2.2.6. Statistical Analysis
`Data are expressed as the mean ± standard error of the
`mean (S.E.M.). A probability (p) value < 0.05 (two-sided)
`was considered statistically significant. In the comparison
`of data, we performed two-way repeated measures analy-
`sis of variance followed by Bonferroni’s test as a post
`hoc test, or one-way analysis of variance followed by
`Dunnett’s test as a post hoc test, using GraphPad Prism
`Version 6 (GraphPad Software, Inc., San Diego, CA).
`
`3. Results
`3.1. Subject Demographics
`A total of 48 healthy Japanese male subjects were en-
`rolled. Subject demographics were similar across dose
`groups (Table 1). The mean age of enrolled subjects was
`24.3 ± 3.4 years (mean ± standard deviation); range, 20 -
`35 years), with an average BMI of 21.47 ± 1.48 kg/m2
`(range, 19.2 - 24.2 kg/m2). All subjects completed the
`study.
`
`Table 1. Demographic characteristics of the study population.
`
`E3024 dose
`
`No. of subjects
`
`Age (years)
`
`Height (cm)
`
`Body weight (kg)
`
`BMI (kg/m2)
`
`5 mg
`
`10 mg
`
`20 mg
`
`40 mg
`
`80 mg
`
`All E3024-treated
`
`All placebo-treated
`
`6
`
`6
`
`6
`
`6
`
`6
`
`30
`
`18
`
`All subjects
`48
`B MI, body mass index. Mean ± standard deviation.
`
`22.5 ± 2.0
`
`23.7 ± 3.8
`
`26.7 ± 5.4
`
`23.5 ± 1.5
`
`26.2 ± 4.9
`
`24.5 ± 3.9
`
`23.9 ± 2.4
`
`24.3 ± 3.4
`
`169.80 ± 4.07
`
`175.95 ± 3.69
`
`170.05 ± 4.90
`
`170.12 ± 7.76
`
`169.68 ± 6.62
`
`171.12 ± 5.77
`
`171.34 ± 6.38
`
`171.20 ± 5.94
`
`62.02 ± 6.01
`
`68.87 ± 4.22
`
`59.77 ± 5.14
`
`59.48 ± 4.72
`
`62.48 ± 5.26
`
`62.52 ± 5.86
`
`63.78 ± 6.63
`
`63.00 ± 6.12
`
`21.48 ± 1.30
`
`22.27 ± 1.50
`
`20.65 ± 1.18
`
`20.55 ± 0.94
`
`21.70 ± 1.45
`
`21.33 ± 1.37
`
`21.69 ± 1.67
`
`21.47 ± 1.48
`
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`Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl
`Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism
`
`from model-independent analysis was assessed both
`visually and using a power model (Y = αXβ). Plots of
`individual Cmax and AUC0-inf values against dose are pre-
`sented in Figures 2(b) and (c), respectively. Point esti-
`mates of β in Cmax and AUC0-inf were 1.077 and 0.993,
`respectively, indicating that both values were approxi-
`mately 1. The 95% CIs of β for Cmax and AUC0-inf were
`1.000 - 1.153 and 0.925 - 1.061, respectively, showing
`that both Cmax and AUC0-inf following a single oral dose
`of E3024 (5 - 80 mg) increased dose-proportionately.
`Urinary pharmacokinetic parameters of E3024 are
`provided in Table 3. Mean cumulative excretion rate
`(fraction of drug excreted unchanged in urine; fe)
`reached a plateau within 96 h after dosing and ranged
`between 52.3% and 63.2%. Mean cumulative excretion
`rate and renal clearance (CLR) remained nearly constant
`over the studied dose range.
`
`
`
`Fig. 2
`
`5 mg
`10 mg
`20 mg
`40 mg
`80 mg
`
`
`3.2. Pharmacokinetic Profiles
`Pharmacokinetic profiles of E3024 were assessed in 30
`healthy adult male subjects each receiving a single oral
`dose of E3024 (5, 10, 20, 40, or 80 mg) under fasted
`conditions. Mean pharmacokinetic profiles after the sin-
`gle dose are presented in Figure 2(a), and E3024 sin-
`gle-dose pharmacokinetic parameters are provided in
`Table 2. E3024 was rapidly absorbed after dosing, with a
`median tmax of 0.83 - 1.50 h; thereafter, concentrations
`declined with a mean t1/2 of 5.34 - 11.68 h. After admini-
`stration of 5 to 80 mg E3024, mean Cmax increased from
`37 to 819 ng/mL, and mean AUC0-inf increased from 223
`to 3571 ng⋅h/mL. Mean CL/F ranged between 23.0 and
`25.4 L/h, remaining nearly constant over the studied dose
`range. Mean Vz/F showed slightly higher values at doses
`of 40 and 80 mg.
`Dose-proportionality for Cmax and AUC0-inf obtained
`
`
`
`(a)
`
`1000
`
`100
`
`10
`
`1
`
`0.1
`
`0
`
`Plasma concentration (ng/mL)
`
`A
`
`
`
`6
`
`12
`
`18
`
`24
`Time (h)
`
`30
`
`36
`
`42
`
`48
`
`(c)
`
`Y = 42.57X
`
`C
`
`5000
`
`4000
`
`3000
`
`2000
`
`1000
`
`AUC0-inf (ng·h/mL)
`
`(b)
`
`Y = 8.22X
`
`1500
`
`1200
`
`900
`
`600
`
`300
`
`B
`
`
`Cmax (ng/mL)
`
`0
`0
`
`20
`
`80
`
`100
`
`0
`
`0
`
`20
`
`60
`40
`60
`40
`Dose (mg)
`Dose (mg)
`
`Figure 2. (a) Mean plasma E3024 concentrations (semilogarithmic plotting) after single oral doses of E3024 (5 - 80 mg) in
`healthy adult male subjects under fasted conditions. Each plotted point represents mean value and standard deviation (six
`subjects per group). Relationship between dose and Cmax (b) or AUC0-inf (c) after single oral doses of E3024 (5 - 80 mg) in
`healthy adult male subjects under fasted conditions. Each point represents an individual value. Solid lines are the results of
`application of a linear regression model from a method of least squares.
`
`80
`
`100
`
`Open Access PP
`
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`

`
`Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl
`Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism
`
`669
`
`Table 2. Summary of pharmacokinetic parameters for plasma E3024 obtained by model-independent analysis.
`
`Parameter
`
`E3024 dose
`80 mg
`40 mg
`20 mg
`10 mg
`5 mg
`819.3 ± 247.5
`323.9 ± 30.7
`152.4 ± 37.9
`87.6 ± 15.5
`36.7 ± 3.2
`Cmax (ng/mL)
`1.25 (0.33 - 2.00)
`1.50 (1.00 - 2.00)
`1.50 (0.67 - 3.00)
`0.83 (0.67 - 2.00)
`1.50 (1.00 - 2.00)
`tmax (h)
`3571.1 ± 623.2
`1684.9 ± 314.9
`812.6 ± 183.8
`440.4 ± 71.9
`223.0 ± 38.1
`AUC0-inf (ng·h/mL)
`t1/2 (h)
`9.43 ± 2.16
`11.68 ± 5.20
`6.08 ± 1.21
`5.75 ± 1.68
`5.34 ± 1.43
`23.1 ± 4.9
`24.4 ± 4.3
`25.4 ± 4.3
`23.3 ± 4.1
`23.0 ± 4.0
`CL/F (L/h)
`Vz/F (L)
`318.6 ± 108.6
`416.8 ± 194.9
`217.9 ± 31.0
`186.5 ± 30.0
`175.1 ± 47.7
`Cmax, maximum observed concentration; tmax, time to Cmax; AUC0-inf, area under the plasma concentration-time curve from 0 to infinity; t1/2, terminal half-life;
`CL, clearance; F, bioavailability; CL/F, apparent clearance; Vz, volume of distribution during the terminal phase; Vz/F, apparent volume of distribution during
`the terminal phase. Mean ± standard deviation, except tmax. tmax, median (minimum - maximum). Six subjects per group.
`
`
`Table 3. Summary of pharmacokinetic parameters for urine E3024 obtained by model-independent analysis.
`
`Parameter
`
`E3024 dose
`80 mg
`40 mg
`20 mg
`10 mg
`5 mg
`47.23 ± 14.05
`20.93 ± 3.14
`11.01 ± 2.03
`5.58 ± 0.93
`3.16 ± 0.56
`Ae (mg)
`59.0 ± 17.6
`52.3 ± 7.9
`55.0 ± 10.1
`55.8 ± 9.3
`63.2 ± 11.1
`fe (%)
`215.7 ± 42.3
`208.4 ± 14.7
`227.2 ± 13.6
`211.2 ± 6.3
`236.6 ± 14.2
`CLR (mL/min)
`Ae, amount of unchanged drug excreted in urine; fe, fraction of drug excreted unchanged in urine; CLR, renal clearance. Mean ± standard deviation. Six sub-
`jects per group.
`
`3.3. Pharmacodynamic Profiles
`The DPP-IV inhibitory activity of E3024 was measured
`up to 24 h after administration of the study drug to assess
`the pharmacodynamic profile. Figure 3(a) shows the
`time course of plasma DPP-IV activity inhibition after a
`single oral dose (5 - 80 mg) of E3024 or placebo. The
`inhibition of DPP-IV activity in the E3024 groups in-
`creased immediately after drug administration, reached
`peak levels 1 to 2 h after administration, then decreased
`to the pre-dose levels at 24 h after administration. On the
`other hand, DPP-IV inhibitory activity was not observed
`in the placebo group. The DPP-IV inhibitory activity of
`E3024 increased dose-dependently. The relationships
`between plasma concentration of E3024 and inhibition of
`DPP-IV activity after single oral administration of E3024
`(5 - 80 mg) were analyzed using the Imax model. As
`shown in Figure 3(b), the relationship between plasma
`concentration of E3024 and inhibition of DPP-IV activity
`was well-adapted to the Imax model, with an IC50 value of
`33.7 ng/mL.
`Following administration of the study drug, measure-
`ment of active GLP-1, insulin, C-peptide, glucagon, and
`glucose concentrations was performed within 3 h after
`lunch and dinner, which were started at 4.5 and 10.5 h
`after dosing, respectively. The time course of active
`GLP-1 concentrations after single oral doses of E3024 (5
`- 80 mg) or placebo is shown in Figure 4. The levels of
`active GLP-1 increased immediately after food intake
`and reached maximum concentrations 20 min after food
`intake, then decreased. In the E3024 groups receiving
`
`doses of 20 mg or more, although there were large varia-
`tions in measured values, the increase in active GLP-1
`after food intake was larger than that in the placebo
`group. In addition, the increase in active GLP-1 tended to
`be larger after lunch compared with after dinner. An in-
`crease in insulin and C-peptide, and a decrease in gluca-
`gon and glucose were observed after food intake in all
`groups, but a dose-dependent change was not observed
`(data not shown).
`
`3.4. Safety and Tolerability
`No deaths or serious AEs were reported following single
`oral doses of 5 to 80 mg E3024. AEs observed in this
`study are listed in Table 4. Twenty-three events of sub-
`jective symptoms or objective findings (erythema, rash,
`pruritus, diarrhea, feeling hot, conjunctival hyperemia,
`and headache) occurred in ten subjects and three events
`of abnormal changes in laboratory values (blood amylase
`increased, alanine aminotransferase (ALT) increased, and
`lipase increased) in three subjects. All of these AEs oc-
`curred in E3024 groups. No abnormal changes were ob-
`served in 12-lead ECG parameters, vital signs, or body
`weight. The number of AEs in each of the 5 mg, 10 mg,
`and 20 mg E3024 groups was one event in one (16.7%)
`of six subjects, while that in the 40 mg group was five
`events in four (66.7%) of six subjects and that in the 80
`mg group was 18 events in five of six subjects (83.3%).
`Thus, the incidence of AEs increased when 40 mg or
`more of E3024 was administered.
`R ash developed in four subjects each of the 40 mg and
`
`Open Access PP
`
`Page 7 of 16
`
`

`
`Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl
`Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism
`
`670
`
`
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`(a)
`
`-20
`0
`
`Placebo
`5 mg
`10 mg
`20 mg
`40 mg
`80 mg
`
`
`
`6
`
`12
`Time (h)
`
`18
`
`24
`
`(b)
`
`Observed
`Predicted
`
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`-10
`0
`
`Inhib