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

doi:10.4236/pp.2013.49093

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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

Open Access PP

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

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

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

*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.

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

664

(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. Subj ects

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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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. Ph ar ma c oki netic 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-

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,

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Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl

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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. Sa f ety Assessmen t s

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. Chem i c al s

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-(bu t-2-yn-1- yl)-1-[(4 -cyanoph enyl)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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667

(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:

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).













HtHsHc Hs100

where Ht = test release caused by a compound, Hs =

spontaneous release, and Hc = total cellular histamine

content [17].

2.2.5. Effects of Compounds on Histamine Release

from Rat Peritoneal Mast Cells and Basophils 2.2.6. Stati s tical Analysi s

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(-).

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

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(-).

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.

Mast cells (104 cells/180 μL/tube) or whole blood cells

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 6 22.5 ± 2.0 169.80 ± 4.07 62.02 ± 6.01 21.48 ± 1.30

10 mg 6 23.7 ± 3.8 175.95 ± 3.69 68.87 ± 4.22 22.27 ± 1.50

20 mg 6 26.7 ± 5.4 170.05 ± 4.90 59.77 ± 5.14 20.65 ± 1.18

40 mg 6 23.5 ± 1.5 170.12 ± 7.76 59.48 ± 4.72 20.55 ± 0.94

80 mg 6 26.2 ± 4.9 169.68 ± 6.62 62.48 ± 5.26 21.70 ± 1.45

All E3024-treated 30 24.5 ± 3.9 171.12 ± 5.77 62.52 ± 5.86 21.33 ± 1.37

All placebo-treated 18 23.9 ± 2.4 171.34 ± 6.38 63.78 ± 6.63 21.69 ± 1.67

All subjects 48 24.3 ± 3.4 171.20 ± 5.94 63.00 ± 6.12 21.47 ± 1.48

MI, body mass index. Mean ± standard deviation.

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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 Cma x and AUC0-inf obtained

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.

1000

100

0.1

06 12 18 24303642 48

5 mg

10 mg

20 mg

40 mg

80 mg

Plasma concentration (ng/mL)

(a)

Time (h)

20 40 60 80100020 4060 80100

Dose (mg) Dose (mg)

(b) (c)

300

600

900

1200

1500 5000

4000

3000

2000

1000

Y = 8.22X Y = 42.57X

max

(ng/mL)

AUC

0-inf

(ng·h/mL)

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.

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Table 2. Summary of pharmacokinetic parameters for plasma E3024 obtained by model-independent analysis.

E3024 dose

Parameter 5 mg 10 mg 20 mg 40 mg 80 mg

Cmax (ng/mL) 36.7 ± 3.2 87.6 ± 15.5 152.4 ± 37.9 323.9 ± 30.7 819.3 ± 247.5

tmax (h) 1.50 (1.00 - 2.00) 0.83 (0.67 - 2.00) 1.50 (0.67 - 3.00) 1.50 (1.00 - 2.00) 1.25 (0.33 - 2.00)

AUC0-inf (ng·h/mL) 223.0 ± 38.1 440.4 ± 71.9 812.6 ± 183.8 1684.9 ± 314.9 3571.1 ± 623.2

t1/2 (h) 5.34 ± 1.43 5.75 ± 1.68 6.08 ± 1.21 11.68 ± 5.20 9.43 ± 2.16

CL/F (L/h) 23.0 ± 4.0 23.3 ± 4.1 25.4 ± 4.3 24.4 ± 4.3 23.1 ± 4.9

Vz/F (L) 175.1 ± 47.7 186.5 ± 30.0 217.9 ± 31.0 416.8 ± 194.9 318.6 ± 108.6

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.

E3024 dose

Parameter 5 mg 10 mg 20 mg 40 mg 80 mg

Ae (mg) 3.16 ± 0.56 5.58 ± 0.93 11.01 ± 2.03 20.93 ± 3.14 47.23 ± 14.05

fe (%) 63.2 ± 11.1 55.8 ± 9.3 55.0 ± 10.1 52.3 ± 7.9 59.0 ± 17.6

CLR (mL/min) 236.6 ± 14.2 211.2 ± 6.3 227.2 ± 13.6 208.4 ± 14.7 215.7 ± 42.3

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.

ash developed in four subjects each of the 40 mg and

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100

-20

0 6 121824

5 mg

10 mg

20 mg

40 mg

80 mg

Inhibition of DPP-IV activity (%)

(a)

Time (h)

Placebo

100

-10

Inhibition of DPP-IV activity (%)

(b)

Plasma concentration (ng/mL)

Predicted

50 100 150 250200 300

Observed

Figure 3. (a) Time course of inhibition of plasma dipeptidyl peptidase-IV (DPP-IV) activity after single oral doses of E3024 (5

- 80 mg) or placebo in healthy adult male subjects. Each plot represents mean values and standard deviations (six subjects for

each E3024 group, 18 subjects for placebo); (b) Relationship between plasma E3024 concentration and inhibition of DPP-IV

activity. Observed: Plots of 1/5 of actual plasma E3024 concentrations and inhibition of DPP-IV activity at each time point

(plasma was diluted to 1/5 in assay for DPP-IV activity). Predicted: Imax model prediction of plasma E3024 concentrations

and inhibition of DPP-IV activity.

5 mg

10 mg

20 mg

40 mg

80 mg

Active GLP-1 (pmol/L)

Time after lunch (h)

Placebo

1 2 3 012 3

Time after dinner (h)

Figure 4. Time course of active glucagon-like peptide-1 (GLP-1; GLP-1-[7-36]amide and GLP-1-[7-37]) concentrations after

single oral doses of E3024 (5 - 80 mg) or placebo in healthy adult male subjects. Each plot represents mean values and stan-

ard deviations (six subjects for each E3024 group, 18 subjects for placebo). d

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80 mg groups. The onset of rash was 31 - 83 and 4 - 28

min after dosing in the 40 mg and 80 mg groups, respec-

tively. Additional tests were conducted for the subjects

with rash. In the four subjects receiving 40 mg, no

changes were observed in eosinophil fraction or non-

specific IgE, and DLST was also negative. The results of

additional tests of the four subjects receiving 80 mg are

shown in Table 5. No changes were observed in eosino-

phil fraction, IgE, or serotonin, but three of the four sub-

jects (subjects A, C, and D) showed a trend to increased

histamine levels immediately after the onset of rash fol-

lowing study drug administration. Substance P showed

an upward tendency in two subjects (subjects B and D).

Although all AEs occurring in this study were mild

Table 4. Summary of adverse events after single oral dose of E3024.

E3024 dose

Placebo (n = 18)5 mg (n = 6)10 mg (n = 6)20 mg (n = 6)40 mg (n = 6) 80 mg (n = 6)

Number (%) of subjects experiencing

any adverse events 0 (0.0%) 1 (16.7%) 1 (16.7%) 1 (16.7%) 4 (66.7%) 5 (83.3%)

Headache 0 0 0 0 0 1

Conjunctival hyeremia 0 0 0 0 0 4

Diarrhea 0 0 0 0 0 3

Erythema 0 0 1 0 0 0

Pruritus 0 0 0 0 1 1

Rash 0 0 0 0 4 4

Feeling hot 0 0 0 0 0 4

ALT increased 0 0 0 1 0 0

Amylase increased 0 1 0 0 0 0

Lipase increased 0 0 0 0 0 1

ALT, alamine aminotransferase.

Table 5. Individual data for additional tests in subjects having rash in the 80 mg group.

Subject Time of treatment Eosinophil (%) Non-specific IgE (U/mL)Serotonine (ng/mL)Histamine (ng/mL) Substance P (pg/mL)

Day -1 10.6 106 - 1.09

Rash onset (0.33 h after dose) 8.0 144 141 1.60

24 h after dose 7.2 157 166 0.76

96 h after dose 5.4 187 159 1.28

Not tested

Day -1 2.3 112 - 0.41 105.24

Rash onset (1 h after dose)2.1 154 198 0.42 176.09

24 h after dose 2.4 162 153 0.29 120.70

96 h after dose 1.4 195 189 0.28 100.74

Day -1 0.8 112 - 0.24 115.71

Rash onset (1.5 h after dose)0.6 154 114 0.43 116.39

24 h after dose 0.5 162 131 0.26 114.58

96 h after dose 0.7 195 149 0.19 105.35

Day -1 2.0 59 - 0.42 83.74

Rash onset (0.67 h after dose) 1.3 57 165 0.57 127.67

24 h after dose 1.4 65 159 0.92 80.83

96 h after dose 2.3 84 190 0.73 80.75

gE, immunoglobulin E.

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and non-serious, the incidence of AEs tended to increase

when 40 mg or more of E3024 was administered, and

their manifestation including rash also suggested toler-

ability-related problems. Consequently, it was concluded

that for single dose administration of E3024, the maxi-

mum permissible dose that would not induce tolerability

problems was 20 mg.

3.5. Effects of E3024 on Blood Histamine Levels

of Normal Fischer Rats

We speculated that histamine was involved in rash de-

velopment in men treated with E3024. Therefore, we

treated Fischer rats with E3024 to examine if this in-

creased blood histamine levels. Blood histamine levels

were about 0.5 μmol/L in pre-treatment of E3024 in this

rat strain. Thirty min after oral administration of E3024,

significant increases in blood histamine levels were ob-

served in the 500 (p < 0.001) and 750 mg/kg groups (p <

0.001) (Figure 5(a)). Next, we examined if the well-

known DPP-IV inhibitors, vildagliptin and valine-pyr-

rolidide, caused increases in blood histamine levels. Oral

administration of vildagliptin at 500 mg/kg induced no

elevation of blood histamine levels 0.5 h after treatment

(Figure 5(b)). Similarly, neither did valine-pyrrolidide

increase blood histamine levels (data not shown). Plasma

concentrations of E3024, vildagliptin and valine-pyr-

rolidide were 52, 54 and 221 mol/L, respectively. Re-

garding E3024, in rat blood the concentration was about

18-fold higher than the Cmax for the 80-mg group of the

E3024 clinical trial which was 819 ng/mL corresponding

to 2.89 mol/L.

3.6. Effects of E3024 on Blood Histamine Levels

of Mast Cell-Deficient (Ws/Ws) Rats and

Wild-Type Homozygous (+/+) Rats

We investigated whether E3024 increased blood hista-

mine levels in genetically mast cell-deficient rats, com-

paring their wild-type homozygous rats. In +/+ rats,

E3024 treatment increased blood histamine concentra-

tions in a dose-dependent manner (Figure 6(a)). A sig-

nificant increase in blood histamine was found at 1 h in

500 mg/kg treatment (p < 0.001). On the other hand, no

effects were detected in Ws/Ws rats treated with 500

mg/kg E3024 (Figures 6(a) and (b)), although treatment

with the same dose caused increases in blood histamine

to >12 mol/L in +/+ rats.

3.7. In Vitro Histamine Release from Rat

Peritoneal Mast Cells and Basophils, and

Structure-Activity Relationship (SAR) Study

To examine which cells, mast cells or basophiles, were

involved in E3024-induced histamine release, we col-

lected these cells from normal Fischer rats and subjected

them to in vitro assays. At first, we studied the effects of

vildagliptin and our DPP-IV inhibitors (E3024, ER-

319441-15, ER-319433-15 and ER-463809-15) on his-

tamine release from rat mast cells. Concerning the

chemical structures, the piperazin-1-yl group of E3024

and ER-319433-15 was replaced with a 3-amino-

0.0

Blood histamine (

mol/L)

(a)

Time after administration (h)

0.5 0.0

0.5

1.0

2.0

3.0

1.5

2.5

(b)

Blood histamine (μmol/L)

Veh ic le

E3024 (250 mg/kg)

E3024 (500 mg/kg)

E3024 (750 mg/kg)

Veh i cle

Vildagliptin

E3024

Figure 5. (a) Changes in blood histamine levels in Fischer rats treated with vehicle (0.5% methylcellulose, 10 mL/kg) or

E3024 (250, 500 or 750 mg/kg) (n = 8). Blood histamine was determined before and 0.5 h after administration. The data were

analyzed by two-way repeated measures analysis of variance followed by Bonferroni’s test as a post hoc test. ***, p < 0.001; (b)

Blood histamine levels 0.5 h after vehicle, vildagliptin (500 mg/kg) or E3024 (500 mg/kg) in Fischer rats (n = 8). *, p < 0.05.

Values are expressed as the mean ± standard error of the mean.

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0.0

Blood histamine (μmol/L)

Time after administration (h)

0.5

+/+; Vehicle

+/+; E3024 (250 mg/kg)

+/+; E3024 (500 mg/kg)

1.0

+/+; E3024 (250 mg/kg)

Ws/Ws; Vehicle

Ws/Ws; E3024 (500 mg/kg)

(a)

(b)

0.00.5 1.0

Time after administration (h)

0.00

0.05

0.10

0.15

Blood histamine (µmol/L)

Figure 6. (a) Changes in blood histamine levels in genetically mast cell-deficient (Ws/Ws) and wild-type normal (+/+) rats. We

treated +/+ rats with vehicle (0.5% methylcellulose, 10 mL/kg) or E3024 (250 or 500 mg/kg), and Ws/Ws rats with vehicle or

E3024 (500 mg/kg) (n = 7). The data were analyzed by two-way repeated measures analysis of variance followed by Bon-

ferroni’s test as a post hoc test. ***, p < 0.001; (b) Changes in blood histamine levels in Ws/Ws rats are extracted from (a). Val-

ues are expressed as the mean ± standard error of the mean.

piperidin-1-yl group to give ER-319441-15 and ER-

463809-15, respectively (Figure 1). E3024 and ER-

319433-15 treatment triggered a significant histamine

release in a concentration-dependent fashion (Figure

7(a)). On the other hand, neither ER-319441-15 nor

ER-463809-15 caused histamine release like vildagliptin.

In the case of whole blood, no histamine release was

found until 300 mol/L of E3024, but it was detected in

the treatment of A23187 at 1 mol/L (Figure 7(b)). To

confirm if this in vitro observation was reproduced in in

vivo, we administered ER-319441-15 orally to Fischer

rats. Although blood histamine was significantly in-

creased 0.5 h after E3024 treatment at 500 mg/kg (p <

0.01) again, ER-319441-15 caused no increases in blood

histamine at 500 mg/kg (Figure 7(c)).

4. Discussion

Pharmacokinetic profiles following administration of

single oral doses of E3024 were assessed in healthy adult

male volunteers. E3024 was absorbed immediately after

administration and eliminated at a mean t1/2 of 5.34 to

11.68 h. The CL/F was almost constant regardless of

dose level, while Vz/F levels showed an upward tendency

in high-dose (40 mg and 80 mg) groups. Inappropriate

evaluation of the terminal elimination phase in low-dose

groups was considered to have influenced the above re-

sults. Urinary profiles of E3024 revealed that E3024 was

excreted in the urine at a mean cumulative excretion rate

of 52.3% to 63.2%. Therefore, it was estimated that at

least 52.3% to 63.2% of the orally administered dose

would be absorbed. In addition, the CLR was greater than

the glomerular filtration rate of approximately 125

mL/min in healthy adults (70 kg) [19], demonstrating

active secretion of E3024 from glomeruli into the urine.

To assess the pharmacodynamic profile of E3024 on

single oral administration to healthy male adult volun-

teers, DPP-IV activity was measured up to 24 h after

dosing, and active GLP-1, insulin, C-peptide, glucagon

and glucose levels were measured up to 3 h after lunch

and dinner, which started at 4.5 and 10.5 h after dosing,

respectively. E3024 inhibited plasma DPP-IV activity

dose-dependently. A good correlation was found between

plasma E3024 concentration and inhibition of DPP-IV

activity, and the IC50 value was calculated at 33.7 ng/mL

from the Imax model. It was reported that when E3024

was added to human plasma, the IC50 value was 0.14

mol/L [12], which was equivalent to 39.61 ng/mL. The

IC50 value from this clinical study was similar to that

previously obtained from the in vitro study. Thus, the

pharmacological effect could be explained by the plasma

drug concentration.

GLP-1 is an incretin that is released from L-cells in the

intestine postprandially as active GLP-1, and then is rap-

idly degraded to inactive GLP-1 in the body by DPP-IV,

a type of serine proteases. Therefore, it was expected that

endogenous active GLP-1 would be increased by inhibi-

tion of DPP-IV [1-5]. In the placebo group in this study,

the level of active GLP-1 increased immediately after

food intake, reached a maximum concentration 20 min

after eating, and then decreased. In the E3024 groups

administered with doses of 20 mg or more, although

there were large variations in measured values, the in-

crease in active GLP-1 after eating was larger than that in

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0.0

0.5

1.0

2.0

3.0

1.5

2.5

(b)

Blood histamine (μmol/L)

Vehicle

ER-319441-15

E3024

Vehicle

Vildagliptin

E3024

(c)

(a)

Histamine release (% of total)

0 10 30 100 10 30 100 300 103010013 101310

Ve hi c le

E3024

A23187

ER-319441-15

ER-463809-15

ER-319433-15

Histamine release (% of total)

10 100 300 30 1

Concentration (μmol/L)

Figure 7. In vitro histamine release from peritoneal mast cells (a) and whole blood (b) of Fischer rats by several dipeptidyl

peptidase-IV inhibitors. (a) Peritoneal exudate cells were treated with vehicle (dimethyl sulfoxide; DMSO), vildagliptin (10,

30 or 100 µmol/L), E3024 (10, 30, 100 or 300 µmol/L), ER-319441-15 (10, 30 or 100 µmol/L), ER-319433-15 (1, 3 or 10 µmol/L)

or ER-463809-15 (1, 3 or 10 µmol/L) (n = 3). A broken line indicates vehicle’s value; (b) Whole blood was treated with vehicle

(DMSO), E3024 (10, 30, 100 or 300 µmol/L) or A23187 (1 µmol/L) (n = 3); (c) Blood histamine levels 0.5 h after vehicle (0.5%

methylcellulose, 10 mL/kg), E3024 (500 mg/kg) or ER-319441-15 (500 mg/kg) in Fischer rats (n = 8). The data were analyzed

by one-way analysis of variance followed by Dunnett’s test as a post hoc test. **, p < 0.01; ***, p < 0.001. Values are expressed

as the mean ± standard error of the mean.

the placebo group, suggesting that E3024 could enhance

the increase of active GLP-1 after eating. If the study

drug was administered just before eating, it is expected

that a greater enhancement of active GLP-1 would have

been obtained, and there may have also been obvious

changes in insulin, C-peptide, glucagon or glucose. Al-

though this study was originally designed to examine the

postprandial change in active GLP-1 levels when E3024

was administered just prior to food intake in a later por-

tion of the study, we could not conduct this portion be-

cause the study was discontinued before reaching this

point.

In this study, E3024 was administered to healthy

adults, but the drug had been administered to patients

with type 2 diabetes, and a greater enhancing effect may

have been obtained. Active GLP-1 can enhance glucose-de-

pendent insulin secretion from islet



cells as well as

have an inhibitory action on glucagon secretion [1-5].

However, E3024 showed no dose-dependent effects on

the postprandial (after-lunch or -dinner) insulin, C-pep-

tide, glucagon, or glucose change. Because healthy adults,

the target subjects in this study, had normal insulin secre-

tion and did not show postprandial hyperglycemia, it

might be difficult to detect the changes in these parame-

ters in these subjects. On the other hand, if the study drug

is administered to patients with type 2 diabetes, the

changes in these endpoints might be more evident.

Subjective symptoms and objective findings after sin-

gle oral doses of 5, 10, 20, 40, and 80 mg of E3024 in 48

subjects were as follows: one event (redness) in one sub-

ject in the 10 mg group; five events (rash, pruritus) in

four subjects in the 40 mg group; 18 events (rash, feeling

hot, conjunctival hyperemia, headache, diarrhea, pruritus)

in five subjects in the 80 mg group. Although all these

events were mild, a greater variety of symptoms and a

larger number of the events were observed in the 80 mg

group compared with the 40 mg group. These events

suggested that drug administration at greater than 80 mg

might be capable of eliciting more numerous and more

severe events. Thus, administration of 160 and 320 mg of

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E3024 was not conducted.

In eight subjects reporting rash, no changes in eosino-

phil fraction or non-specific IgE, the parameters for type

1 (immediate) allergy, were observed. DLST was per-

formed on four subjects to examine for the possibility of

drug hypersensitivity due to delayed allergy, also with

negative results. In three subjects from the 80 mg group,

an increasing tendency in histamine level was observed

immediately after the onset of rash following study-drug

administration. In view of the extremely early onset of

rash, the dose-dependency in the rash development pro-

file, and the increase in blood histamine levels, histamine

release due to the direct action of E3024 on mast cells

was considered most likely to have caused the rash. Be-

cause there has been no report of such a frequent devel-

opment of rash associated with other DPP-IV inhibitors,

it is considered unlikely that the rash following E3024

administration had been induced by DPP-IV inhibition. It

has been suggested that assessment of the selectivity of

DPP-IV inhibition over dipeptidyl peptidase-8 (DPP-8)

and dipeptidyl peptidase-9 (DPP-9) is important for ob-

taining an optimal safety profile of DPP-IV inhibitors in

the treatment of type 2 diabetes [20]. The Food and Drug

Administration (FDA) requested conduct of skin lesion

assessments in monkeys for all DPP-IV inhibitors, based

on findings of necrotizing skin lesions due to some

DPP-IV inhibitors. The FDA considers the skin lesion a

result of off-target inhibition of DPP-8 or DPP-9. Be-

cause E3024 was shown to be a highly selective DPP-IV

inhibitor which did not inhibit DPP-8 or DPP-9 activity

[12], we speculate that inhibition of DPP-8 or DPP-9 is

not the cause of the rashes observed with E3024.

We inferred that rash in the clinical trial was related to

histamine release which was caused by E3024 directly.

Then, we examined if E3024 increased histamine levels

in rats. The reasons why we chose rats were: 1) we had

pharmacokinetic and pharmacodynamics data of E3024

in rats, and 2) there is a mutant rat strain in which mast

cells are deficient. We found that E3024 increased blood

histamine levels in normal rats. On the other hand, valine-

pyrrolidide and vildagliptin had no effects on blood his-

tamine levels, even in the presence of sufficient plasma

concentrations. However, there is a large difference in

the concentrations of E3024 causing rash in men and

increasing blood histamine in the rat. This may be due to

different sensitivity between the species: men may be

more sensitive to E3024 than rats.

Ws/Ws rats are deficient in both mucosal-type and

connective tissue-type mast cells [21]. The defected gene

of Ws is c-kit, receptor-type tyrosine kinase [22]. c-kit is

a receptor of stem cell factor essential for migration, dif-

ferentiation, and proliferation of cells, such as hemato-

poietic stem cells, neural crest-derived melanocytes.

Thus, Ws/Ws mutant rats manifest depigmentation, ane-

mia, and mast cell deficiency [21]. Histamine is pro-

duced in mast cells, basophils, and entero-chromaffin-

like (ECL) cells. Basophils of Ws/Ws rats are not differ-

ent from wild-type rats in number, and produce histamine

[23]. In addition, histamine is synthesized and stored in

ECL cells of Ws/Ws rats [24]. In our study, E3024 in-

creased blood histamine in wild-type homozygous (+/+)

rats, while no response was observed in Ws/Ws rats. In

view of the difference in histamine-producing cells be-

tween Ws/Ws and wild-type rats, we concluded that

E3024 acted specifically on mast cells to release hista-

mine. The cell-specific effect was also confirmed by the

observation that histamine was released from normal rat

mast cells, but not from basophils in vitro.

From the in vitro study, we obtained the following

SAR: comparing between E3024 and ER-319441-15, the

presence of a piperazin-1-yl group on position 2 of the

imidazo[4,5-d]pyridazine may be a causal structure for

induction of histamine release in rats. Similarly, com-

parison of ER-319433-15 and ER-463809-15 revealed

that the piperazin-1-yl group on position 8 of purine is

important in determining whether histamine release oc-

curs. These results showed that a key structure causing

histamine release is piperazine linked to a 5,6-membered

fused heterocyclic core, namely, either a pyrimidine ring

fused to an imidazole ring, or a pyridazine ring fused to

an imidazole ring. More interestingly, this piperazine-

associated histamine release can be avoided by substitu-

tion with 3-amino-piperidine, while maintaining DPP-IV

inhibitory activity (data not shown).

Unfortunately, we cannot demonstrate whether or not

the 3-amino-piperidin-1-yl compounds do not induce

rash clinically. However, among marketed DPP-IV in-

hibitors, linagliptin (8-[(3R)-3-amino-piperidin-1-yl]-7-

(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl)

methyl]-3,7-dihydro-1H-purine-2,6-dione) [10] has a

structure very similar to our compounds and contains a

3-amino-piperidin-1-yl group, not a piperazin-1-yl group,

on position 8 of the purine (Figure 8). High incidence of

rash as observed in the Phase I trial of E3024 has not

been reported in clinical trials of linagliptin: the first-

in-man study was performed as a randomized, doubled-

blind, placebo-controlled Phase I trial in which 63

healthy, male, Caucasian volunteers received the treat-

ment (47 received linagliptin; 16 received placebo) [25].

Once-daily oral doses of linagliptin were 2.5, 5, 25, 50,

100, 200, 400 and 600 mg. No rash was observed in this

trial. There was a report of a randomized, double-blind,

placebo-controlled Phase I trial enrolling eight healthy

Japanese male subjects (six received linagliptin and two

received placebo per group). Linagliptin was adminis-

tered as single escalating doses of 1, 2.5, 5 and 10 mg, or

Open Access PP

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

676

Linagliptin

Figure 8. Chemical structure of linagliptin.

multiple escalating doses of 2.5, 5 and 10 mg once daily

for 12 days [26]. One subject in the 5 mg group showed

an increase in histamine concentration. No clinical signs

or symptoms attributed to this event were observed, and

then they considered unrelated to linagliptin. In addition,

no rash was reported in 2523 patients receiving lina-

gliptin 5 mg once daily in eight randomed, double-blind,

placebo-controlled Phase III trials lasting 12 to 24 weeks

[27]. These facts may support that our speculation that

rash could be avoided by the combination of 5- and

6-membered fused heterocyclic rings and 3-amino-

piperidine.

In conclusion, we found that E3024 was a compound

with favorable pharmacokinetic profiles that inhibited

plasma DPP-IV activity dose-dependently with a good

correlation between its plasma concentrations and inhibi-

tion of DPP-IV activity. Because AEs including rash

occurred frequently when 40 mg or more of E3024 was

administered, the maximum permissible single dose of

E3024 that would not induce tolerability problems was

considered to be 20 mg. Since superior safety is required

for antidiabetic therapy, we decided not to continue fur-

ther development of E3024. The results of in vivo and in

vitro pre-clinical examinations using rats indicated that

histamine release from mast cells by E3024 was involved

in rash development in men.

5. Acknowledgements

The authors wish to express our thanks to the volunteers

who participated in this first-in-man study, and staff at

Sekino Clinical Pharmacology Clinic. The authors also

thank the Eisai study team for E3024 clinical study: Ta-

kashi Sato and Hideo Takahira for the measurement of

plasma and urine concentrations of E3024; Hiroshi

Kuroiwa for clinical data management; Hideaki Miyagi-

shi for statistical analysis; and Hiroshi Arakawa and

Shohei Nishimoto for study monitoring. The authors

highly appreciated Professor Tetsuo Shiohara, Depart-

ment of Dermatology, Kyorin University School of

Medicine, Tokyo, Japan, for his medical advice on inter-

pretation of rash developing in this study. In addition, we

are grateful to Tadashi Nagakura, Katsuhiro Moriya,

Yoshihisa Sano, Osamu Takenaka, and Junichi Naga-

kawa of Eisai Co., Ltd. for their invaluable advice and

support of pre-clinical studies.

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Abbreviations

Ae, amount of unchanged drug excreted in urine; AE,

adverse event; ALT, alanine aminotransferase; AUC,

area under the curve; AUC0-inf, area under the plasma

concentration-time curve from 0 to infinity; BMI, body

mass index; CI, confidence interval; CL, clearance; CL/F,

apparent clearance; CLR, renal clearance; Cmax, maxi-

mum observed concentration; DLST, drug-induced lym-

phocyte stimulation test; DMSO, dimethyl sulfoxide;

D-PBS, Dulbecco’s phosphate-buffered saline; DPP-IV,

dipeptidyl peptidase-IV; DPP-8, dipeptidyl peptidase-8;

DPP-9, dipeptidyl peptidase-9; ECG, electrocardiography;

ECL, entero-chromaffin-like; EDTA, ethylenediamine-

tetraacetic acid; ELISA, enzyme-linked immunosorbent

assay; F, bioavailability; FDA, Food and Drug Admini-

stration; fe, fraction of drug excreted unchanged in urine;

GLP-1, glucagon-like peptide-1; IC50, concentration re-

quired for 50% of the maximum inhibition; IgE, immu-

noglobulin E; LC/MS/MS, liquid chromatographic-tandem

mass spectrometry; MC, methycellulose; OTC, over-the-

counter; PD, pharmacodynamics; PEC, peritoneal exu-

date cell; PK, pharmacokinetics; SAR, structure-activity

relationship; t1/2, terminal half-life; tmax, time to Cmax; Vz,

volume of distribution during the terminal phase; Vz/F,

apparent volume of distribution during the terminal

phase