ISSN 2187-4360

CODEN: GYDKA9

THE ANNUAL PROCEEDINDS

OF

GIFU PHARMACEUTICAL UNIVERSITY

No. 62 2013

1

HB-EGF

HB-EGF

12

22

32

38

FU

48

57

AMPK

68

24 24 12 75

Ann. Proc.

Gifu Pharm. Univ. Gifu Pharmaceutical University

1-25-4 Daigaku-Nishi, Gifu 501-1196

Vol. 62, 1-11 (2013) 1

, *

-glucosyl hesperidin (Hsp-G) -glucosyl Stevia (Stevia-G)

Research on the Dosage Form Design and a Formulation Study Aimed at

Application to New Pharmaceutical Excipients of Transglycosylated Compounds

Hiromasa UCHIYAMA, Hirofumi TAKEUCH*

Abstract: While the water solubility of new drug candidates in the development phase is often extremely poor, the improvement of

the dissolution and absorption of poorly water soluble drug candidates is a key factor in the continuation of drug development and

making new drugs. Since the existing technology and formulation design cannot produce acceptable results for poorly water soluble

drugs in many cases, improvement in dissolution with new techniques and formulation designs is essential. We focused on

transglycosylated compounds which were recently developed by innovation of enzyme synthesis technology and have begun to be

used as functional food additives. A transglycosylated compound is the general term for compounds which increase water solubility

by the addition of sugar to an existing compound making it safe and widely used in the food industry. The purpose of this study was

to evaluate the potential of Hsp-G and Stevia-G as pharmaceutical excipients and functional food additives to enhance the dissolution

and absorption of poorly water soluble drugs using a new formulation, as well as an evaluation of the improvement and mechanism

of dissolution enhancement effect.

Key phrases: transglycosylated compound, improvement of dissolution, improvement of absorption, solid dispersion, micelle-like

structure

1)

2)

40 %

501-1196 1 25-4

Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University (1-25-4 Daigaku-nishi, Gifu 501-1196, JAPAN)

2

3-4) 5) 6)

7-9)

10-11)

3-4) CMC)

12)

13-14)

2

15-17)

CMC

CMC

CMC

-glucosyl hesperidin (Hsp-G) -glucosyl

Stevia (Stevia-G)

Hsp-G

cyclomaltodextrin glucanotransferase

18-20)

Hsp-G 1

300

Hsp-G

Hsp-G

60

21) Hsp-G

150-300

22) 20

Stevia-G

Stevia-G

Stevia-G

Stevia-G Hsp-G

Stevia-G

Hsp-G Stevia-G

-glucosyl hesperidin

(Hsp-G) -glucosyl Stevia (Stevia-G)

Hsp-GStevia-G

Vol. 62, 1-11 (2013) 3

Stevia-G

Flurbiprofen (FP) Probucol (PRO)

hydroxypropyl methylcellulose

(HPMC) 1/10

spray-dried particle (SPD) physical mixture

(PM) X

Fig.1

HPMC

PRO

FP

Fig. 1. Powder X-ray diffraction (PXRD) patterns of (a) FP crystal, (b) PM of FP/Hsp-G (1/10), (c) SDP of FP/Hsp-G (1/10), (d) SDP of FP/Stevia-G (1/10), (e) SDP of FP/HPMC (1/10), (f) Hsp-G powder.

Fig.

2 Fig. 2 (a)

FP

35 g/mL FP 180

17 g/mL

FP

180

20 g/mLFP

HPMC

Hsp-G Stevia-G

5

55 g/mLFig. 2 (b)

1 pH1.2)

FP pKa 3.78 pH1.2

1Henderson-hasselbalch

FP

Hsp-G

Stevia-G

5 50 g/mL

Hsp-G Stevia-G

2

Hsp-G Stevia-G

Fig. 3

PRO PRO

3-5 ng/mL

PRO 23)

Hsp-GStevia-G

Hsp-G Stevia-G

PRO

1000 Hsp-G

Stevia-G

pH

Fig. 2Fig. 3Hsp-GStevia-G

FP PRO

2

1

2

24-25) PVP

26)FP

HPMC

HPMC FPHsp-G

Stevia-G FP

FP

Hsp-G Stevia-G

(a)

(d)

(c)

(b)

30.0020.0010.005.00

Count(cps)

2q (degree)

(f)

(e)

4

Hsp-GHsp-G

21)

Hsp-G Stevia-G

Hsp-G Stevia-G

Hsp-G Stevia-G

Hsp-GStevia-G

FP PRO

Fig. 2. Dissolution profiles of FP in (a) distilled water and (b)

HCL solution (pH1.2) (, untreated FP; , PM of FP/Hsp-G

(1/10); , PM of FP/Stevia-G (1/10); , SDP of FP/Hsp-G

(1/10); , SDP of FP/Stevia-G (1/10); , SDP of FP/HPMC (1/10)). Each point represents the meanS.D. (n=3).

Fig. 3. Dissolution profiles of PRO in distilled water (,

untreated PRO; , PM of PRO/Hsp-G (1/10); , PM of

PRO/Stevia-G (1/10); , SDP of PRO/Hsp-G (1/10); , SDP

of PRO/Stevia-G (1/10)). Each point represents the meanS.D. (n=3).

Fig. 4 FP

Table 1

maximum concentration (Cmax) Area

Under the Curve (AUC) FP

Cmax 3.7 g/mL

Hsp-GStevia-G

Cmax 4.8 g/mL 5.7

g/mL Hsp-G Stevia-G

Cmax

10.2 g/mL 8.3 g/mL FP 2

AUC Hsp-G

Stevia-G 2.8

2.6 Fig. 5 PRO

Table 2

Cmax AUC

FP Hsp-G

Stevia-G

PRO Hsp-G

Stevia-G AUC

5.3 9.8 PRO FP

Biopharmaceutics Classification System Class II

Class II

Class II

FP

Mizoe 27) 4

FP

Hsp-G Stevia-G

FP FP

PRO

6 %

28)

PVP

PVP SDS

29-30)

PRO

Hsp-G

Stevia-GPRO

FP

Hsp-G PRO Stevia-G

FP Hsp-GPRO Stevia-G

Hsp-GStevia-G

0

10

20

30

40

50

0 60 120 180

Time (min)

Con

cen

trati

on

of

FP

dis

solv

ed

(mg/m

L)

0

10

20

30

40

50

0 60 120 180

Time (min)

Co

nce

ntr

ati

on

of

FP

dis

solv

ed

(mg

/mL

)

(b)

(a)

Vol. 62, 1-11 (2013) 5

Fig. 4. Plasma concentration-time profiles of FP in rats after

oral administration of untreated FP and SDP: (, untreated FP;

, PM of FP/Hsp-G (1/10); , PM of FP/Stevia-G (1/10); ,

SDP of FP/Hsp-G (1/10); , SDP of FP/Stevia-G (1/10)). Each point represents the mean S.E. (n=6).

Table 1. Pharmacokinetic parameters of FP after oral administration of SDPs of FP/Hsp-G or FP/Stevia-G in rats. (**p< 0.01, compared to untreated FP. ##p< 0.01, Compared to the corresponding PMs).

Fig. 5. Plasma concentration-time profiles of PRO in rats after

oral administration of untreated PRO and SDP: (, untreated

PRO; , SDP of PRO/Hsp-G (1/10); , SDP of

PRO/Stevia-G (1/10)). Each point represents the mean S.E. (n=6).

Table 2. Pharmacokinetic parameters of PRO after oral

administration of SDP of PRO/Hsp-G or PRO/Stevia-G in rats. (**p< 0.01, compared to untreated PRO. ##p< 0.01, Compared to the SDP of PRO/Hsp-G).

1

Caco-2

Fig.6

SDS 0.1 %

Hsp-G

Stevia-G 10 %

Hsp-G Stevia-G

Fig. 6. Cytotoxicity of Hsp-G and Stevia-G to Caco-2 cells (n=8) Control; Phosphate buffer solution (pH 7.4).

3

Fig. 7 Hsp-G Stevia-G

Hsp-G Stevia-G

Hsp-G Stevia-G

Hsp-G Stevia-G

Fig. 7. Surface tension according to concentration of Hsp-G

and Stevia-G: (, Hsp-G; , Stevia-G).

Blo

od

con

cen

trati

on

of

FP

(mg/m

L)

Time (hr)

0

5

10

0 4 8 12

Tmax (h) Cmax (mg/mL) AUC0-48 (mgh/mL)

untreated FP

PM of FP/Hsp-G(1/10)

PM of FP/Stevia-G(1/10)

1.0

2.0

2.0

3.73 0.43

4.82 0.64

5.71 0.31

22.06 2.54

32.09 3.98

41.26 2.03

SDPs of FP/Hsp-G(1/10)

SDPs of FP/Stevia-G(1/10)

0.5

0.5

10.22 0.14

8.34 0.39

62.65 2.82

57.14 2.27

##**

##**

Tmax (h) Cmax (mg/mL) AUC0-48 (mgh/mL)

untreated PRO

SDPs of PRO/Hsp-G(1/10)

SDPs of PRO/Stevia-G(1/10)

10.0

8.0

8.0

0.18 0.03

0.89 0.06

1.61 0.16

4.94 2.06

26.08 4.52

48.79 9.97

**

**

50

55

60

65

70

75

0 10 20 30

Concentration of Hsp-G and Stevia-G (mg/mL)

Surf

ace t

ensi

on (

mN

/m)

6

I1/I3

CMC

Stevia-G

Hsp-GHsp-G

Hsp-G

Stevia-G

Fig. 8Stevia-G I1/I3

Stevia-G I1/I3

I1/I3

I1/I3

I1/I3

I1/I3Fig. 8

Stevia-G I1/I3

Stevia-G

I1/I3

Stevia-G CMC 16.5

mg/mL Stevia-G

Turro and Yekta 31) static

quenching method Fig. 8

Stevia-G 15-16 Stevia-G

I1/I3

Stevia-G

I1/I3

-

Stevia-G

Stevia-G

Stevia-G

CMC

Stevia-G

SDS Tween 80

32)

SDS Stevia-G 2 nm

(data not shown)Stevia-G

CMC

Stevia-G

Fig. 8. Plot of pyrene I1/I3 ratios versus Stevia-G concentration.

Hsp-G Zhang 33) NMR

Hsp-G

Hsp-G

core-shell Hsp-G

CMC 5.0 mg/mL

Hsp-G 4-5 Hsp-G

2 nm

Hsp-G core-shell

Hsp-G

Stevia-GHsp-G

4. Stevia-G

Stevia-G

Stevia-G

Stevia-G

CMC

Stevia-G

Vol. 62, 1-11 (2013) 7

Stevia-G

Fig. 9 Stevia-G

SDS CMC FP

bicomponent of FP/SDS (1/1 w/w) FP

FP 35 g/mL

SDS FP

bicomponent of

FP/Stevia-G (1/10 w/w) FP 2

Stevia-G

FP

Stevia-G SDS 3 2

FP/Stevia-G/SDS (1/10/1 w/w/w) FP

7

Stevia-G dodecyl

trimethyl ammonium bromide (DTAB)

SDS 3 2

Stevia-G

FP

Fig. 9. Dissolution profiles of FP from Stevia-G/SDS systems

in distilled water.: (, Untreated FP, ; bicomponent of

FP/SDS (1/1 w/w); , bicomponent of FP/Stevia-G (1/10

w/w); , tricomponent of FP/Stevia-G/SDS (1/10/0.2 w/w/w);

, tricomponent of FP/Stevia-G/SDS (1/10/0.5 w/w/w); , tricomponent of FP/Stevia-G/SDS (1/10/1 w/w/w). Each point

represents the meanS.D. (n=3).

Fig. 10 Stevia-G

n-Octyl--D-maltopyranoside (OMP) CMC

FP bicomponent of

FP/OMP (1/4 w/w) FP FP

tricomponent of FP/Stevia-G/OMP

FPbicomponent of FP/Stevia-G (1/10 w/w)

Tween 80

FP/Stevia-G/Tween80 3

bicomponent of FP/Stevia-G (1/10 w/w)

Stevia-G

FP

Fig. 10. Dissolution profiles of FP from Stevia-G/OMP systems

in distilled water.: (, Untreated FP, ; bicomponent of

FP/OMP (1/4 w/w); , bicomponent of FP/Stevia-G (1/10

w/w); , tricomponent of FP/Stevia-G/OMP (1/10/2 w/w/w);

, tricomponent of FP/Stevia-G/OMP (1/10/4 w/w/w). Each point represents the meanS.D. (n=3).

2

CMC

FP

Stevia-G

FP/Stevia-G/ 3

1

Fig. 11 Stevia-G SDS CMC

SDS

Stevia-G

DTAB

Stevia-G

Stevia-G

- Stevia-G

Fig. 11. Changes in surface tension as a function of Stevia-G

concentration in distilled water (, Stevia-G solution; , SDS

8

solution; , Stevia-G solution with 0.2 mg/ml SDS; ,

Stevia-G solution with 0.5 mg/ml SDS; , Stevia-G solution with 1 mg/ml SDS.

Stevia-G

Fig. 12 Stevia-G

SDS CMC PyreneI1/I3

SDS Pyrene I1/I3

Stevia-G

Pyrene I1/I3

CMC Stevia-G 16 mg/mLSDS

2.5 mg/mL1 mg/mL SDS Stevia-G

0.8 mg/mL

CMC

Stevia-G

Stevia-G Pyrene

I1/I3 Stevia-G

I1/I3 I1/I3

Stevia-G

3

2

2 34)1

1

-

-

Fig. 13 Stevia-G

PyreneI1/I3 Stevia-G

Stevia-G

OMP

Stevia-G

PyreneI1/I3 Stevia-G

Fig. 12. Plot of Pyrene I1/I3 ratios versus (a) SDS and (b)

Stevia-G concentration ( , Stevia-G solution; , SDS

solution; , Stevia-G solution with 0.2 mg/ml SDS; ,

Stevia-G solution with 0.5 mg/ml SDS; , Stevia-G solution with 1 mg/ml SDS).

Fig. 13. Schematic representation of nanocomposite formation among SDS and Stevia-G-aggregated nanostructures.

Stevia-G

Csco-2

Fig. 14

Stevia-G SDS

Caco-2 0.1 %

SDS Caco-2

Stevia-G

Stevia-G

Stevia-G

0.9

1.1

1.3

1.5

1.7

0 5 10 15 20 25 30 35

Concemtration of SDS (mg/mL)

Py

ren

e I

1/I

3

0.9

1.1

1.3

1.5

1.7

0 5 10 15 20 25 30 35

Concemtration of stevia-G (mg/mL)

(a) (b)

SDS molecule

Nanostructurte formed

by Stevia-G aggregation

Nanocomposite formation

between Stevia-G and SDS

Vol. 62, 1-11 (2013) 9

Fig. 14. Cytotoxicity of Stevia-G, SDS and binary mixture of SDS/Stevia-G system to Caco-2 cells (n=8); Control; Phosphate buffer solution (pH 7.4).

Stevia-G/

FP

Stevia-G/

Fig. 15

pranlukast hemihydrate (PLH)

PLH FP

Stevia-G

bicomponent of PLH/Stevia-G

(1/10 w/w) tricomponent of

PLH/Stevia-G/SDS (1/2/1 w/w/w)

Stevia-G

Stevia-G

Fig. 15. Dissolution profiles of PLH from Stevia-G/SDS system

in distilled water.: (; Untreated PLH, ; bicomponent of

PLH/SDS (1/1 w/w), ; bicomponent of PLH/Stevia-G (1/10

w/w), ; tricomponent of PLH/Stevia-G/SDS (1/2/1 w/w/w),

; tricomponent of PLH/Stevia-G/SDS (1/5/1 w/w/w), ; tricomponent of PLH/Stevia-G/SDS (1/10/1 w/w/w).Each point represents the meanS.D. (n=3).

Stevia-G SDS

Fig.16

PLH/Stevia-G/SDS 3

2

3

AUC

3.2

(Table 3)PLH

PLH

PLH

35-36)2 SDS Stevia-G

PLH Stevia-G SDS

PLH/Stevia-G/SDS

3 PLH 2

3

3 Stevia-G

SDS

PLH

Fig. 16. Blood concentration profile of PLH in rats after oral

administration of Stevia-G/SDS system: (; Untreated PLH,

; bicomponent of PLH/SDS (1/1 w/w), ; bicomponent of

PLH/Stevia-G (1/2 w/w), ; tricomponent of PLH/Stevia-G/SDS (1/2/1 w/w/w)). Each point represents the meanS.E. (n=6).

Table 3. AUC values of untreated PLH and Tricomponent system during 8 hours after oral administration. (**p< 0.01, compared to the untreated PLH, Bicomponent of PLH/SDS (1/1 w/w) and Bicomponent of PLH/ Stevia-G (1/1 w/w)).

5

FP PRO

pH

0

20

40

60

80

100

120

Control 1%Stevia-G 1%Stevia-G

+0.1% SDS

0.1 %

SDS

Via

bil

ity

(% o

f co

ntr

ol)

AUC0-48h (mgh/mL)

Untreated PLH

Bicomponent of PLH/SDS (1/1 w/w)

136.70 28.50

189.13 14.28

**

Bicomponent of PLH/Stevia-G (1/2 w/w)

Tricomponent of PLH/SDS/Stevia-G (1/1/2 w/w/w)

231.85 17.14

438.08 68.82

10

Stevia-G

Stevia-G

Stevia-G Stevia-G

Stevia-G

Stevia-G

Stevia-G

Stevia-G

Stevia-G

Hsp-G

Stevia-G Hsp-G

Stevia-G

6

7

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135

12 HB-EGF

HB-EGF

HB-EGF

HB-EGF

, *

(heparin-binding epidermal growth factor-like growth factor: HB-EGF) EGF

HB-EGF

HB-EGF

HB-EGF HB-EGF

HB-EGF

HB-EGF KO

(LTP)

HB-EGF

HB-EGF

KO

Roles of Heparin-binding EGF-like Growth Factor (HB-EGF) in the Higher

Brain Functions: Analysis of Ventral Forebrain Specific HB-EGF KO Mice

Atsushi OYAGI, Hideaki HARA*

Abstract: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors.

Previously, HB-EGF has been reported to be involved in diverse biological processes, including tumor formation, heart function,

wound healing, and eyelid formation. On the other hand, HB-EGF is widely expressed in the central nervous system, including the

hippocampus, cerebral cortex and cerebellum, and is considered to play pivotal roles in the development of the adult nervous system

and higher brain function. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain and

investigated the roles of HB-EGF in higher brain function. These knockout mice showed behavioral abnormalities such as an

increase in locomotor activity, decreased social interaction, a deficit of prepulse inhibition, and memory impairment. HB-EGF KO

mice also showed altered monoamine factors such as dopamine and serotonin, decreased spine density in neurons of the prefrontal

cortex, and impaired long-term potentiation (LTP) in the hippocampus. These results suggest that HB-EGF exerts significant

influence in higher brain functions, such as psychomotor behavior and memory formation and careful regulation of its activity will be

an important goal for treating a number of neurological diseases of the central nervous system.

Key phrases: HB-EGF, higher brain function, KO mice, LTP

(heparin-binding

epidermal growth factor-like growth factor: HB-EGF) EGF

501-1196 1 25-4

Molecular Pharmacology, Gifu Pharmaceutical University (1-25-4 Daigaku-nishi, Gifu 501-1196, JAPAN)

Vol. 62, 12-21 (2013) 13

EGF

HB-EGF

(pro-HB-EGF)

a disintegrin and metalloprotease (ADAM)

(HB-EGF)

(HB-EGF-N) EGF

ErbB4 mitogen activated protein

kinase (MAPK)

(HB-EGF-C)

1)

HB-EGF 2)

3) 4) 5)

HB-EGF

6) HB-EGF

7) 8)

HB-EGF (KO)

3)

Cre-LoxP

HB-EGF

HB-EGF

9) (Fig. 1)

HB-EGF KO

2-1. (Locomotor activity test)

(NS-AS01;

Neuroscience, Tokyo, Japan)

(NS-DAS-32; Neuroscience)

HB-EGF KO 24

HB-EGF KO ( 8:00

8:00) ( 8:00 8:00)

(Fig. 2a, b)

haloperidol (0.1 mg/kg, i.p.)9HB-EGF KO

(Fig. 2a, b) clozapine

(1 mg/kg, i.p.) 9 HB-EGF KO

24

9) (Fig. 2a)

Cresyl violet staininga b

LacZ stainingin situ

hybridizationImmunohisto-

chemical staining

c d e

WT

KO

WT

KO

CA1

CA3

DG

DG

CA1

CA3

CA1DG CA3LacZ staining

Immunohistochemical staining

Cresyl violet

staininga bLacZ staining

in situ

hybridizationImmunohisto-

chemical staining

c d e

WT

KO

WT

KO

CA1

CA3

DG

DG

CA1

CA3

CA1DG CA3LacZ staining

Immunohistochemical staining

Fig. 1. Histological evaluation in HB-EGF KO and WT mice.

(a) Coronal section through the cortex region; square indicates area shown in the photomicrographs. (b) Histological analysis of

cortex from WT (upper) and HB-EGF KO (lower) adult mice. LacZ staining, scale bar=50 m. In situ hybridization using an

Hb-egf probe, scale bar=100 m. Immunohistochemical staining with anti-HB-EGF antibody, scale bar=20 m. Cresyl violet

staining, scale bar=500 m. (c) Coronal section through the hippocampus; square indicates area shown in the photomicrographs

(CA1, CA3, and DG). (d) LacZ staining of whole and individual hippocampal region in HB-EGF KO mice, scale bar=500 m. (e)

Immunohistochemical analysis of individual hippocampal region from WT (upper) and HB-EGF KO (lower) adult mice, scale

bar=20 m. The results were cited from ref 9.

14 HB-EGF

HB-EGF

2-2. (PPI)

(SR-LAB; San Diego

Instruments, CA, USA)HB-EGF KO

73 dB 76 dB prepulse

PPI (Fig. 2c)

30 clozapine (1 mg/kg, i.p.)

risperidone (0.1 mg/kg, i.p.)

HB-EGF KO PPI

(Fig. 2c) haloperidol (0.1

mg/kg, i.p.)KO PPI

(Fig. 2c)120 dB

startle amplitude KO

9) [-vehicle ; 263.7

33.56 (mean S.E.M, n=25), KO -vehicle ;

205.8 35.25 (n=24), KO -haloperidol ; 230.2

60.6 (n=15), KO -risperidone ; 212.5 43.78

(n=14), and KO -clozapine ; 225.2 35.03

(n=12)]

2-3. (Social interaction)

2

(175245125 mm) 10

(/)HB-EGF KO

(Fig. 2 d)

clozapine (1 mg/kg, i.p.) 7

HB-EGF KO

(Fig. 2d)

Co

un

ts

-10

0

10

20

30

40

50

60

70

80

90

73dB 76dB 82dB

WT

KO (Vehicle)

KO (Haloperidol)

KO (Risperidone)

KO (Clozapine)

Pre

pu

lse

inh

ibiti

on

(%

)

# #

*

##

##

0

5000

10000

15000

20000

25000

Total Daytime Night

WT

KO (Vehicle)

KO (Haloperidol)

KO (Clozapine)

*

#

*#

*

#

#

a

0

500

1000

1500

2000

2500

121314151617181920212223241 2 3 4 5 6 7 8 9 1011

WTKO (Vehicle)KO (Haloperidol)KO (Clozapine)

b

Se

co

nd

/co

un

ts

0

1

2

3

Vehicle Haloperidol Clozapine

KO

d

WT

#

c

*

Co

un

ts

e

0

5

10

15

20

25

left right

KO

left right

WT

Exp

lora

tio

n ti

me

(s)

f

0

5

10

15

20

25

left right

KO

left right

WT

Exp

lora

tio

n ti

me

(s) *

g

0

10

20

30

40

WT KO

To

tal a

rm e

ntr

ies (co

un

ts)

h

0

15

30

45

60

75

WT KO

Alte

ratio

n (%

of co

ntr

ol)

**

KO (Haloperidol)

Fig. 2. Behavioral analysis of HB-EGF KO mice and WT mice. Mice were placed into individual cages, and their locomotion was assessed every hour for 1 day. WT (n=8), KO mice with

treatment of vehicle (n=7), haloperidol (n=8), and clozapine (n=8). (a) Locomotor activity throughout the 24-hr period, and (b)

locomotor activity analyzed separately during day and night periods. (c) PPI of the acoustic startle response in WT (n=24), KO mice

with treatment of vehicle (n=24), haloperidol (n=15), risperidone (n=14), and clozapine (n=12) (d) Social interaction test in a

novel environment in WT (n=8) and KO mice with treatment of vehicle (n=7), haloperidol (n=9), and clozapine (n=9). Two

genetically identical mice that had been housed separately were placed in the same cage. Their social interaction was then

monitored for 10 min. Values are means SEM. (e and f) Novel-object recognition task. (e) In the training trial (5 min), two circles

were placed in symmetrical left and right positions. (f) In the test trial 1 hr later (5 min), one circle (left) and one triangle (right)

were placed in the same positions. The amount of time the WT (n=6) and KO (n=6) mice spent exploring each object during

training trial and test trial was recorded. * p < 0.05 vs. left object. (g and h) Y-maze task. Each mouse was placed at the end of one

fixed arm of the maze and allowed to move freely through the maze for 8 min. The sequence of arm entries was recorded manually.

(g) Total number of arms entered during the session. (h) % of alternation was calculated as (actual alternations/maximum

alternations) 100. WT (n=6), KO (n=6). Values are means SEM. * p

Vol. 62, 12-21 (2013) 15

haloperidol (0.1 mg/kg, i.p.) HB-EGF KO

9) (Fig. 2d)

2-4.

(acquisition trial)

(: 3 cm)

5 1

(retention trial) (:

3 3 3 cm) 5

HB-EGF KO

(Fig. 2e) 1

()KO

9) (Fig. 2f)

2-5. Y

3 Y

(16502 cm)

8

Y

3

(

)8

HB-EGF KO

alteration rate

(Fig. 2h)8

KO

9) (Fig. 2g)

Fig. 3. Morphological changes in the prefrontal cortex.

(a) Representative photomicrographs showing morphology of pyramidal neurons in cortical layer III of the prefrontal cortex from

WT (left) and KO (right) mice. Scale bar=20 m. (b) Representative photomicrographs of apical dendritic segments from WT (left)

and KO (right) mice. Scale bar=8 m. (c) High-magnification images of apical dendritic segments from adult WT (left) and KO

(right) mice. Scale bar=2 m. (d) Representative images of immunoblots showing NR1 and PSD-95 protein levels. (e)

Quantification of the number of basal dendritic branch-points. (f) Spine density on primary apical dendrites of layer III pyramidal

neurons of the prefrontal cortex from WT (white bar) and KO (black bar) mice. (g) Quantification of spine length. WT (n=700

spines) and KO (n=592 spines) mice (n=4 mice, 25 neurons each). (h) Cumulative distribution of spine length. WT (white circle)

and KO (black circle) mice (n=4 mice, 25 neurons each). (i, j, k, and l) Quantitative analysis of NR1, PSD-95, NR2A, and NR2B

by densitometric scanning of immunoreactive bands. The results were cited from ref 9.

WT KO

WT KO

WT KO

a

b

c

NR1

PSD95

WT KO

d

0

2

4

6

8

10

12

14N

um

ber

of

bra

nch p

oin

ts

0

2

4

6

8

10

12

Num

ber

of

sp

ines/1

0m

***

e f

0

50

100

0 0.5 1 1.5 2 2.5 3

Spine Length (m)

Cu

mu

lati

ve

% o

f s

pin

es

WT

KO

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Sp

ine le

ng

th (m

)

g h

**

0

20

40

60

80

100

120 NR1

% o

f co

ntr

ol

i

0

20

40

60

80

100

120 PSD95

**

j

0

20

40

60

80

100

120 NR2B

k l

WT KO WT KO

WT KO

% o

f co

ntr

ol

% o

f co

ntr

ol

WT KOWT KO

WT KO

0

20

40

60

80

100

120 NR2A

% o

f co

ntr

ol

WT KO

-tubulin

16 HB-EGF

HB-EGF

3

HB-EGF KO

3

Lucifer Yellow

3 Fig. 3a-c LY

HB-EGF KO

HB-EGF KO (Fig. 3a,

e)HB-EGF KO

(Fig. 3b,

c, f)

HB-EGF KO

(Fig. 3g, h)

Fig. 3d N-methyl-D-aspartate (NMDA)

NR1 post synaptic density

protein 95 (PSD-95)

NR1PSD-95HB-EGF KO

(Fig.

3i, j)NR2A NR2B

HB-EGF KO

9) (Fig. 3k, l)

HB-EGF

4-1.

HB-EGF

( 120 45 cm) 31 cm

(22 1C) 5

3 5

HB-EGF KO

(Fig. 4b)

3

target

(Fig. 4a, c)

HB-EGF KO

target

(Fig. 4a, c)

HB-EGF KO

10)[; 18.4

0.82 cm/sec (n=13)HB-EGF KO ; 17.6 1.47

cm/sec (n=8)]

4-2. HB-EGF KO

15.59.618.0 cm

323227 cm2

HB-EGF KO

(Fig. 5a) 24

HB-EGF KO

(Fig. 5a)ADAMs HB-EGF

HB-EGF

ADAMs

KB-R7785

KB-R7785

(Fig. 5b) 24

KB-R7785 (100 mg/kg,

s.c.)

(Fig. 5b)

(3.0 mg/kg, i.p.)

Fig. 4. Morris water maze test for HB-EGF KO mice.

(a) Diagrammatic illustration shows the positions of the

platform and each quadrant. (b) Latency to escape to the

hidden platform in WT (n=13) and HB-EGF KO (n=8) mice

in the training test. (c) Duration in each quadrant of

HB-EGF WT (n=13) and KO (n=8) mice in the probe test.

Values are means SEM. ** p < 0.01 vs. target quadrant.

# p < 0.05 vs. WT in target quadrant. The results were cited

from ref 10.

WT

KO

Platform

Right

Left Opposite

a b

c

0

5

10

15

20

25

30

35

40

45

50

day1 day2 day3 day4 day5

Late

ncy t

o p

latf

orm

(sec)

WT

KO

0

10

20

30

40

50

60

RightTarget Left Opposite

Du

ratio

n in

ea

ch

qu

ad

ran

t (%

)

#

**

Target

**

**

Vol. 62, 12-21 (2013) 17

10) (Fig. 5b)

4-3. CA1 (LTP)

HB-EGF KO

(HFS)

CA1 (LTP)

11)

Schaffer

HFS (100 Hz) CA1 LTP

60 (Fig. 6a, b, c, d)

HB-EGF KO LTP HFS 1

60 (Fig. 6b, c, d)

HB-EGF CA1 LTP

10)

4-4.

(HFS)

LTP 12), 13)

HFS CA1 CaMKII

HB-EGF KO

CaMKII

(Fig. 7a, c)HFS

CaMKII

HB-EGF KO (Fig. 7a, c)

HB-EGF KO CaMKII

-amino-3-hydroxy-5-

methylisoxazole-4-propionicacid hydro bromide (AMPA)

1 (GluR1)

(Ser-831)HB-EGF KO

(Fig. 7b, d)HFS

GluR1 synapsin I

HB-EGF KO

(Fig. 7b, d)

PKC ERK1/2

HB-EGF KO 10) (Fig. 7a, c)

.

HPLC-ECD (Eicom, Kyoto, Japan)

Fig. 5. Passive avoidance test.

(a) Passive avoidance test results for HB-EGF KO mice.

Latency to enter dark compartment was recorded in HB-EGF

WT (n=14) and KO (n=12) mice at training and test sessions.

Values are means SEM. ** p < 0.01 vs. WT in training

session. # p < 0.05 vs. WT in test session. (b) Effect of an

ADAMs inhibitor on behavior in a passive avoidance test.

KB-R7785 (30 and 100 mg/kg) and vehicle (0.5% CMC) were

subcutaneously administrated to mice once a day for 4 days.

The passive avoidance test was conducted on the third to

fourth days, 30 min after vehicle, KB-R7785, or scopolamine

administration. Latency to enter a dark compartment was

recorded in KB-R7785 and vehicle-treated mice during

training and test sessions. Values are means SEM. * p <

0.05, ## p < 0.01, vs. vehicle (test trial). Vehicle (n=16 or 8),

KB-R7785 30 mg/kg (n=10), 100 mg/kg (n=16), and

scopolamine (n=6). The results were cited from ref 10.

Fig. 6. Long-term potentiation (LTP) in HB-EGF KO

mice.

(a) Illustration showing LTP measurement. (b) Representative

field excitatory postsynaptic potentials (fEPSPs) recorded

from the CA1 region. (c) Changes in slopes of fEPSPs

following high frequency stimulation (HFS) in the CA1 region

from WT (n=5) and HB-EGF KO (n=5) mice. (d) Level of

LTP potentiation at 1 and 60 min after HFS in the CA1 region

from WT and HB-EGF KO mice. Values are means SEM.

* p < 0.05, ** p < 0.01 vs. WT. The results were cited from ref

10.

c

CA1

Schaffer

d

a b

0

50

100

150

200

250

300

350

WT KO WT KO

Training Test

**

La

ten

cy to

en

ter

(se

c)

#

0

50

100

150

200

250

300

350

Vehicle 30 100 mg/kg Vehicle Scopolamine

La

ten

cy to

en

ter

(se

c) Training

Test

KB-R7785

*

##

a

b

18 HB-EGF

HB-EGF

a b

c d

HB-EGF KO

(DA) (5-HT) 5-HT 5-HIAA

(Fig. 8a) 5-HT

(Fig. 8b)

DA (Fig. 8c)

(NE)

MHPG 5-HT HB-EGF KO

9) (Fig. 8d)

.

HB-EGF

HB-EGF KO

HB-EGF KO

24 Phencyclidine

(PCP)

14)

HB-EGF KO

haloperidol clozapine

HB-EGF KO prepulse inhibition (PPI)

15)PPI

PPI DA NMDA

(PCPketamine dizocilpine)

PPI

16), 17), 18)HB-EGF KO PPI

clozapine risperidone

HB-EGF KO

Sams-Dodd 19)HB-EGF

KO

haloperidol

clozapine

HB-EGF KO

Fig. 8. Monoamine contents in HB-EGF KO and WT

mice.

Monoamine contents in (A) prefrontal cortex, (B) striatum, (C)

thalamus, and (D) Cerebellum. Each column and bar represent

mean S.E.M. (n =10 or 11), *; p < 0.05, **; p

Vol. 62, 12-21 (2013) 19

HB-EGF KO

3 40%

X

20)

46 3

21)

HB-EGF KO

PSD-95

NMDANR1

CaMKII

PAK 9)

HB-EGF KO

Y

HB-EGF KO

22) 23) 24)

HB-EGF KO

25), 26)HB-EGF

KO

HB-EGF KO

35

HB-EGF

HB-EGF KO

24

CA1

(LTP)

LTP NMDA

Ca2+ 27)

AMPA

LTP 28),

29), 30) HB-EGF KO CA1

(HFS) LTP

HB-EGF KO

CaMKII GluR1

CaMKII LTP

31)HFS

NMDA Ca2+

CaMKII 32)

AMPA GluR1 831

33)HFS CaMKII GluR1

CaMKII GluR1

HB-EGF KO

HB-EGF KO

DA5-HT 5-HIAA

NEMHPG 5-HT

5-HT DA

HB-EGF DA

7) DA

HB-EGF

DA

DA 5-HT

HB-EGF KO

.

HB-EGF KO

DA

HB-EGF

HB-EGF

KO

LTP

CaMKII

HB-EGF

20 HB-EGF

HB-EGF

HB-EGF

HB-EGF

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22

, , *

Immune-related Factors Influenced the Development of the Mouse Cerebral Cortex

Hitomi SOUMIYA, Hidefumi FUKUMITSU, Shoei FURUKAWA* Abstract: Clinical symptoms are variable in subjects where cognitive impairments are a major feature with psychiatric, and neurodevelopmental disorders, including schizophrenia and autism. The cerebral cortex plays important roles in cognitive function. Abnormal neuronal morphology and cytoarchitecture of the cerebral cortex are found in postmortem brains from human subjects with severe psychiatric disorders such as schizophrenia and autism. These impairments are thought to be responsible for the abnormal cognitive functions and behavior of such patients. As immunological dysfunctions have also been reported in schizophrenic or autism patients, it may underlie the development of the psychiatric brain. In this study, we examined how immune-related factors affect the development of the cerebral cortex. The results obtained in this study suggest that subtle alterations in the genesis and development of the cerebral cortex induced by immune-related factors might cause functional disorders of the cerebral cortex. Key phrases: neurodevelopmental disorder, schizophrenia, cerebral cortex, neurogenesis, cytokine

1

4

ADHD

1)

lissencephaly1 Lis1 doublecortin 2

1%~%

501-1196 Laboratory of Molecular Biology, Gifu Pharmaceutical University (1-25-4 Daigaku-nishi, Gifu 501-1196, JAPAN)

Vol. 62, 22-31 (2013) 23

90% 30% 2)

3) 4)

5, 6)

1%

7, 8)

Fig. 1. Cytoarchitecture of the cerebral cortex. 78

6 9, 10) Fig. 1

ventricular zone; VZ

3

11)

brain-derived neurotrophic factor; BDNF3 neurotrophin-3 ; NT-3

12, 13, 14)

5, 6)

15, 16)

interleukin; IL-6 JAK/STAT 17)

stromal cell-derived factor1 SDF1 18)

24

2

: ventricular zone, VZ : cortical plate: CP 19) Fig. 2

MRI

20, 21)

22)

23)

stem cell factor; SCF fms 3 III c-kit 24)1SCF c-kit

25, 26)2SCF/c-kit 27, 28) 29)

3 SCF 30)

SCF

SCF

SCF 2-1 SCF c-kit

E13.5E14.5E15.5SCF c-kit mRNA RT-PCR

c-kit SCFE13.5 SCF 100 ng/ml 30 6SCF/c-kit Akt Akt SCF 30 1 2

c-kit E13.5 Akt SCF

Fig. 2. Laminar formation during development of the cerebral cortex.

Vol. 62, 22-31 (2013) 25

2-2. SCF SCF

SCF10 ng/ E13.5 6 P6SCF 15 12 6 4

SCF20 ng

2 mm

2 10 ng SCF

orthologue of the Drosophila cut geneCux1; II-IV chicken ovalbumin upstream promoter transcription factor COUP-TF-interacting protein 2CTIP2; V SCF

Cux1 II IV CTIP2 V VI V VI SCF Cux1 CTIP2 SCF

2-3/ SCF

SCF SCF 11 17

1-3 3

SRY-box containing gene 2 Sox2T-brain gene 2 Tbr2III-tubulinTuj1 E13.5 Sox2 VZ Tbr2 subventricular zone, SVZ

E17.5 Sox2 intermedeiate zone, IZ) VZ E13.5 SVZ

Fig. 3. SCF induced ectopic accumulation of cortical cells. The arrowheads indicate the edge of each area of heterotopia.

26

Tbr2 SCF VZ Sox2 Tbr2 SCF 31)SCF

Fig.3 SCF 30) 27)

SCF Sox2 SCF

SCF 20 ng 1 Sox2 Tbr2 SCF

SCF 10 ng Sox2

SCF

SCF

Fig.3

SCF SCF

3.

5)

lipopolysaccharideLPS

32)

7)

6

9, 10)

19, 33)

34, 35, 36, 37)

RNA polyriboinosinic-polyribocytidylic acid Poly I:C38, 39)

40, 41, 42)

Poly I:C 3 -1 Poly I:C C57BL641) BALB/c40) Poly I: C

ddY 9.5 Poly I:C vehicle

9.5 Poly I:C Poly I:C-E9.5

Poly I:C-E9.5

3 -2 Poly I:C

Vol. 62, 22-31 (2013) 27

P10 Poly I:C-E9.5

Poly I:C-E9.5 P10 P8W

Poly I:C Poly I:C-E9.5 -Cux1 Cux1brain-specific homeobox/POU domain protein Brn1 Brn1 Cux1+ Brn1+Cux1+/Brn1+ 251525

/ Cux1+/Brn1+ Tbr1Ctip2Forkhead box p2FoxP2 Poly I:C-E9.5 Poly I:C-E9.5

3-3Poly I:C Poly I:C8 Poly I:C-E9.5

glutamic acid decarboxylase-67 GAD67; GABA II/III

GAD67

Poly I:C-E9.5 30%GAD67 40 %Golgi III Poly I:C-E9.5 III 20% Poly I:C E9.5

4.

4-1 Poly I:C 1 43), 44)

Poly I:C-E9.5 E13.75E14.75E15.75E16.75 5-bromo-2-deoxyuridine BrdUP10 BrdU 43)

E13.75 BrdU Poly I:C-E9.5 E14.75 E16.75 BrdU inside-out Poly I:C-E9.5 BrdU

E14.75 BrdU Poly I:C E13.75 E15.75 BrdU E16.75 BrdU Poly I:C-E9.5 24.1 : 45.53.3 cells, Poly I:C-E9.5 : 56.72.8 cells; p

28

Table 1/Poly I:C-E9.5

Table 1

Fig. 4. Layer-specific neuronal phenotypes are sequentially generated from cortical progenitors.

Table 1. Gene expression profile of the cells with same birthday in the cerebral cortex of the P10 control and Poly I:C-E9.5

ND: not determined, : no change, : increase, : decrease 4-2

Poly I:C Poly I:C

E13.75 E15.75 BrdU E13.75 24 48 E15.75 24 96 BrdU Poly I:C-E9.5E15.75 BrdU VZ CP 48 BrdU 60 962 Poly I:C VZ CP

E13.75 BrdU 2 Poly I:C

Poly I:C-E9.5

VZ

45, 46)

Poly I:C-E9.5 BrdU 47) E13.75 E15.75 VZ GF Fig.5

Fig. 5. Schematic illustration of the analysis of the cell-cycle parameters of VZ progenitors in the control and the Poly I:C-E9.5 animals by cumulative BrdU labeling, starting at 8:00 P.M. on E13 or E15. VZ BrdU 2.5 26.5 VZ Poly I:C E13.75 Poly I:C-E9.5E15.75 Poly I:C-E9.5Tc 2GF 14 % BrdU 30 VZ BrdU BrdU E15.5 E16.75 Poly I:C-E9.5 BrdU 77.3%E16.75 48.32.7Poly I:C-E9.5 37.33.5p

Vol. 62, 22-31 (2013) 29

4-3 Poly I:C

VZ/SVZ

VZ

SVZ IZ SVZ IZ 13 48, 49) 3

Poly I:C E13.75 E15.75 BrdU

paired box gene 6 (Pax6)Tbr2Tuj1 BrdU E13.75 BrdU Poly I:C-E9.5 E15.75 BrdU Poly I:C-E9.5 Pax6 Pax6 BrdU BrdU 24 12%36 4.3%BrdU 48Pax6BrdU2 Pax6 BrdU Tbr2 Tuj1 2 BrdU 24 48 Tbr2 BrdU 65%Tuj1 BrdU 70Pax6

E15.75 BrdU 24 48 BrdU Ki67 Ki67 G1 SMG2 50)Ki67 [quitting (Q) fraction] Q fraction Poly I:C-E9.5 BrdU 24 48 30%

Fig. 6Poly I:C-E9.5 E15.75

PolyI:C-E9.5 VZ BrdU E15.75 E16.75 E16.75 BrdU E15.75 Pax6 E16.75

Fig. 6. Effects of the Poly I:C-injection on the cell cycle kinetics of cortical progenitors. A, Tissue sections were double immunostained with anti-BrdU antibody (red) and the antibodies against Ki67 (green). Scale bar, 100m. B, At 24 or 48 h after the BrdU injection on E15.75, sections were double immunostained with antibodies against Ki67 and BrdU. Note that the fraction of cells positive for BrdU only (BrdU+/Ki67-; no longer dividing; Q fraction) was essentially the same in both control and Poly I:C-E9.5 cortices at 24 and 48 h after the BrdU incorporation (n=3-5 embryos from 2 litters at each time point). n.s; no significance.

5.

30

2

6

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8

127

32

*, ,

10-Hydroxy-trans-2-decenoic acid10H2DA

10-Hydroxydecanoic acid10HDA 10H2DA

10H2DA 10HDA

10H2DA 10HDA

toll-like receptorlipopolysaccharideIFN

Inhibitory Effects of Royal Jelly-derived Fatty Acids on Cellular Signal Transduction in Innate

Immune Responses

Keita TAKAHASHI*, Tsuyoshi SUGIYAMA, Hiroshi MORI

Abstract: Royal jelly is one of many bee products and a secretion of worker honeybees. Worker honeybees feed the queen honeybee

for her life with only royal jelly. 10-Hydroxy-trans-2-decenoic acid (10H2DA) is the principal lipid component in royal jelly.

10-Hydroxydecanoic acid (10HDA) is also contained in royal jelly. These fatty acids have been reported to show several biological

activities, such as anti-tumor and estrogenic activity. Recently, we revealed the inhibitory effect of these fatty acids on innate immune

receptor signals. In this review, we focus on the biological activities of 10H2DA and 10HDA (especially immunomodulatory

activities). We also discuss the mechanisms underlying these biological activities and the possibilities for using these fatty acids as

lead compounds in new therapeutic drugs for immune disorders.

Key phrases: innate immunity, royal jelly, toll-like receptor, lipopolysaccharide, IFN, fatty acids

1, 2)

3)

4)

major royal jelly proteinMRJP-1

5)

3, 6, 7)

MRJP 5

501-1196 1 25-4

Laboratory of Microbiology, Gifu Pharmaceutical University (1-25-4 Daigaku-nishi, Gifu 501-1196, JAPAN)

Vol. 62, 32-37 (2013) 33

60-70%9-18%

7-18% 3-8%

6, 7, 11). 90-95%

10-hydroxy-trans-2-decenoic acid

10H2DA 10-hydroxydecanoic acid10HDA

10)

MRJPMRJP15 90%8, 9) 10%

10-Hydroxy-trans-2-decenoic acid10H2DA

50%

10H2DA

10-13)

10-Hydroxydecanoic acid10HDA 10

10H2DA

10)

14-16)

10H2DA

10HDA

17-21) 10H2DA

10HDA

22)

23-25)10H2DA

lipopolysaccharideLPS interferon

IFN-

18-20) T 26, 27)

28, 29)

Toll-like receptorTLR

10-hydroxy-trans-2-decenoic acid10H2DA

10-hydroxydecanoic acid10HDA

10 TLR110

TLR TLR

TLRTLR

LPS

Flagellin

CpG DNA ssRNAdsRNA 30)TLR

TLR

IL-6 10H2DA

Kohno

15)LPS IFN-

TNF- IL-6

5 kDa

LPS IL-6

10H2DA 18)10H2DA NF-B

TNF- NF-B

10H2DA IL-6IB-

Lipocalin 2 granulocyte-colony stimulating factor

G-CSFIB- LPS IL-6Lipocalin

2 G-CSF 31, 32)10H2DA IB-

IL-6 IB-

NF-B 10H2DA NF-B

34

IB-

NO 10H2DA

NO

LPS IFN-

IFN-

NO iNOS

iNOS NO

10H2DA LPS iNOS

NO 20)10H2DA IFN-

IFN- NO

IFN- STAT 10H2DA

10H2DA JAK-STAT

NO

IFN- PI3K-Akt NF-B

33)10H2DA NF-B

PI3K-Akt NO

34)

10H2DA IFN- NF-B

iNOS NO

LPS 10H2DA

LPS TLR4

NF-B

TNF- IL-6

IRF-3 IFN-

IFN-STAT

NF-B iNOS

10H2DA LPS IFN-

NF-B IL-6 iNOS

10H2DA IFN- NO 19)IFN-NOJAK-STAT

STAT1 TNF-

NF-B

35)TNF- IRF-1

IRF-8 36)IFN- IRF-1

IRF-8 10H2DA IRF-1

IRF-8 10H2DA

TNF- NF-B

10H2DA IFN- IRF-8

IRF-8TNF-NF-BiNOS

NO

NO 10HDA

10HDA 10H2DA

in vitro

TRPA1

17, 37-39)

10H2DA

10HDA

IFN- 10H2DA

IFN- iNOS

IFN-STAT1

STAT1 IRF-1 IRF-8

IRF-1 IRF-8 TNF-

TNF- NF-B

iNOS 10H2DA IRF-8

TNF-

NF-B iNOS

Vol. 62, 32-37 (2013) 35

LPS NO

10HDA 10H2DA

10H2DA

10HDA IL-6

NF-B

LPS iNOS iNOS

NF-B

IFN-stimulated response elementISRE

IFN--activated sequenceGAS 40-42)

10HDA NF-B GAS ISRE

ISRE IFN-

STAT1STAT2

IRF-1

10HDA IFN- STAT1STAT2

IRF-1

IRF-1

LPS 10H2DA

LPS NF-B

IFN- 10HDA

10HDA IFN-

IRF-1

IRF-1 ISRE

10H2DA 10HDA LPS

10H2DA 10HDA

18, 37-39)LPS

10H2DA

10HDA

10H2DA NF-B IB-

10HDA IRF-1

ISRE

NF-B5

2

2

43-46)10H2DA

NF-B

10H2DA

10H2DA NF-B TNF-

NF-B 19)

10H2DA NF-B

NF-B

NF-B

10H2DA NF-B IB-

NF-B

ER 10H2DA 10HDA

37)

10H2DA 10HDA ER

10H2DA 10HDA LPS

47)10H2DA 10HDA

GPR30 GPCR

48)Moutsatsou 10H2DA GPR30

49) GPCR

GPR120 GPR40

50, 51)GPR119

52)GPR84 53)GPR41

GPR43 54)

GPR84 9-14

36

LPS

53)10H2DA 10HDA

GPR120 -3 LPS

GPR120 TNF-

10H2DA 10HDA

55) GPCR

orphan GPCR

10H2DA 10HDA

10H2DA

100

mM 10H2DA 10)10H2DA

mM

mM

10H2DA

10H2DA 10HDA

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132

38

a), b) *, b)

Melicope 230 Rutaceae

2

M. triphylla8-geranyloxy-

5-methoxypsoralen 4

melicotriphyllin AD M. denhamii 8

melicodenine A H2 melicodin A B

melicodin C 5 melicodenone A

E Melicodenine A H melicodin C DielsAlder 2 + 2

N-methylflindersine

melicodenone A B

Melicope triphyllaMelicope denhamii

Chemical Constituents of Melicope Species (Rutaceae)

Ken-ichi NAKASHIMAa)

, Masayoshi OYAMAb) *, Munekazu IINUMA

b)

Abstract: Melicope (Rutaceae) consists of approximately 230 species and ranges from the Malagacy region east to the Pacific basin

and south to New Zealand. A huge variety of secondary metabolites were isolated from the plants of Melicope genus, acetophenones,

quinolinone- and furoquinoline-alkaloids, coumarins, and polymethoxyflavonoids. In the current study, two species of M. triphylla

(LAM.) MERR. and M. denhamii (SEEM.) T. G. HARTLEY were investigated to obtain four new furanocoumarins melicotriphyllins AD,

eight novel alkaloids melicodenines AH, three new phenylpropanoids melicodins AC, and five new sesquiterpenes melicodenones

AE. These structures were established by spectroscopic analyses, including extensive 1D and 2D NMR experiments.

Melicotriphyllins AD were linear-types of furanocoumarins bearing a hydroxyl or a hydroperoxy group on the geranyloxy side

chain. Melicodenine A was a bisquinolinone alkaloid comprised of two N-methylflindersine, while melicodenines BH were

composed of single molecular N-methylflindersine with acetophenones or furanocoumarin or phenylpropanoids. These novel

quinolinone alkaloids were presumed to form through the Diels-Alder or [2+2] cycloaddition. Furthermore, melicodenones A and B

were the first bicyclic zierane-type sesquiterpenes obtained from plant resources by the isolation of zierane.

Key phrases: Melicope triphylla, Melicope denhamii, furanocoumarin, bisquinolinone alkaloid, sesquiterpene

a) 464-8650 1-100

Laboratory of Medicinal Resources, School of Pharmacy, Aichi Gakuin University (1-100 Kusumoto-cho, Chikusa-ku, Nagoya

464-8650, JAPAN)

b) 501-1196 1 25-4

Laboratory of Pharmacognosy, Gifu Pharmaceutical University (1-25-4 Daigaku-nishi, Gifu 501-1196, JAPAN)

Vol. 62, 38-47(2013) 39

Rutaceae 155

1600 1)

CitrusPoncirus

Zanthoxylum

Phellodendron amurense

Ruta graveolens

2)

Ptelea trifoliata HIF-1

3)

4)

Glycosmis

5-7)Murraya

Clausena

8), 9)

HL-60 10-12)Acronychia

13)

DNA

Melicope14)

230

15)

Chart 1

M.

pteleifolia 3 16)

Chart 1. An epitome of the constituents isolated from Melicope species

40

Chart 2. Structures of the compounds isolated from the fruits of Melicope triphylla

13), 31), 32)Acradenia 33), 34)Boronia

35), 36)

M.

triphylla

M. denhamii

M. triphylla

M. triphylla

1

37-39)

40-42)

A B

1 : 1

HPLC

18 Chart

2 6 Chart 3

-

melicotriphyllin A D

1H- 13C-NMR

heteronuclear multiple bond connectivity

spectroscopy HMBCdouble quantum filtered correlation

spectroscopy DQF-COSY nuclear Overhauser

enhancement and exchange spectroscopyNOESY

2D-NMR Fig. 15

8

melicotriphyllin A B

8 8

13C-NMR 8

melicotriphyllin A 70.4 ppm

Vol. 62, 38-47(2013) 41

Fig. 1. Selected 2D NMR correlations observed in melicotri-

phyllins A D

melicotriphyllin B 82.0 ppm

HR-ESIMS melicotriphyllin A

B

melicotriphyllin A 8

melicotriphyllin B

melicotriphyllin C

D melicotriphyllin A B

7 7

7melicotriphyllin A

B

Melicotriphyllin B D

melicotriphyllin A C

4

Chart 3. Structures of the compounds isolated from the roots

of Melicope triphylla

8-geranyloxy-5-

methoxy-psoralen Phellopterin

8-prenyloxy- 5-methoxypsoralen

8-geranyloxy-5-methoxypsoralen

melicotriphyllin

BD

Phebalium

43), 44)

-

melicophyllone B

TLC methyl p-

geranyloxy-trans-cinnamate

M. denhamii

M. denhamii

M. denhamii

490 g

1: 1

78.3 g

HPLC

8

melicodenine A H2

melicodin ABmelicodin

C 17

N-methylflindersine

M. denhamii

42

Chart 4. Structures of the compounds isolated from the leaves of Melicope denhamii

DielsAlder

melicodenine A B

Melicodenine A

HR-ESIMS C30H30N2O4

1H-NMR 4

2N-

DQF-COSY

2

4 13C- NMR

2

HMBC 2 N-

4

2 1-methylquinolin-2-one

Me2-2(2)/C-3(3)H-3(3')/C-4a(4a)

H-4(4)/C-2(2) HMBC

H-4(4)/C-5(5)

Fig. 2. Selected 2D NMR correlations of melicodenine A

H-4(4)/C-10b(10b) 4a(4a)

2,2-dimethylpyran 2

N-methylflindersine

DQF-COSY 3 3'

H-4/C-4a H-4/C-10a HMBC

10b 4'

melicodenine A Fig. 2

ESIMS retro Diels-Alder

N-methylflin-

dersine [M+H]+ m/z 242

NOESY H-10/H-4

H-4/Me-22) H-4/Me-22)

melicodenine A Fig. 2

CD

melicodenine A

2 N-methylflindersine

M. pteleifolia

melicobisquinolinone A B

16)melicodenine A 3 Chart 5

Vol. 62, 38-47(2013) 43

Chart 5. Types of dimerization in the molecules of dimeric quinolinone alkaloids (Bessonova 51))

5 melicodenine A

Diels-Alder

Fig. 3

melicodenine B

Diels-Alder

N-methylflindersine

evodionol methyl ether

Fig. 3. Plausible biosyntheses of melicodenines A and B

N-methylflindersine evodionol methyl ether

melicodenine B

2 + 2

melicodenine C G

Melicodenine C

HR-ESIMS C26H27NO5

1H-NMR 1

N-2

4 1

DQF-COSY

4

HMBC

44

H-4/C-5H-4/C-10bMe-6/C-5Me-6/C-6aH-10/C-10b

1-methylquinolin-2-one

H-4/C-2Me2-2/C-3

N-methylflindersine

C-4 C-3 4

3,4-methylenedioxyphenyl

H-2/C-7, H-6C-7 MeO-9/C-9 HMBC

3,4-methylenedioxyphenyl 7

9

melicodenine C Fig. 4

NOE H-3/H-4H-3/H2-9

H-4/H-7H-8/H-2H-8/H-6 NOE

Fig. 4

N-methylflindersine 3,4-methylenedioxy-

cinnamyl alcohol methyl ether 2+2

Fig. 5

melicodenine

D E

melicodenine C

melicode-

nine D E 3,4-dimethoxycinnamyl alcohol

methyl ether melicodin A

melicodin A

Melicodenine C E

melicodenine F G

2+2

N-methylflindersine bergapten

Fig. 6

melicodenine F

N-methylflindersine C(3)=C(4)

melicodenine G C(4a)=C(10b)

Toddalia asiatica

toddacoumalone 52)

2+2

Fig. 4. Selected 2D NMR correlations of melicodenine C

.

Fig. 5. Plausible biosyntheses of melicodenines C E

melicodenine H

Melicodenine H ESIMS

C24H23NO5 1D- 2D-NMR

1-methylquinolin-2-one

2 1

2

HMBC

H-6/C-2H-6/C-4H-6/C-7H-3/C-5

H-7/C-2H-7/C-6H-8/C-1MeO-2/C-2OCH2O/C-4

Fig. 6. Plausible biosyntheses of melicodenines F and G

Vol. 62, 38-47(2013) 45

Fig. 7. Selected 2D NMR correlations of melicodenine H

OCH2O/C-5C-7

2-methoxy-4,5-methylenedioxyphenyl

H-7/C-2H-7/C-2bH-8/C-2a

C-2a C-7 H-7/C-2a H-8/C-2b

C-7 C-2b

NOESY H-7/H-10 H-7/H2-9

NOE Fig. 7

Melicodenine Hmelicodin A

melicobisquinolinone B melicodenine H

Melicobisquinolinone B M. pteleifolia

Diels-Alder

melicodenine A melicodenine A retro

DielsAlder melicobisquino-

linone B (Fig. 8)

melicodenine A ESIMS m/z 425

[M+H+Me2CO]

melicodenine HN-methyl- flindersinemelicodin

A Fig. 8

-M. denhamii

2

melicophyllone A C M. elleryana

zierone417), 24), 53) M. denhamii 5

melicodenone A E

Chart 6

zierane

melicodenone A B

Melicodenone A B

zierone zierone 3

Zierone

Zieria macrophylla

54) M. elleryana

zierone

55)

2

Chandonanthus hirtellus56Saccogyna viticulosa57)

3

3

melicodenone A B

zierone

2 3

Fig. 8. Plausible biosyntheses of melicodenine H and melicobisquinolinone B

46

Chart 6. Structures of the compounds isolated from the roots

of Melicope denhamii

guaiane

melicodenone C E

Melicodenone C E

melicodenone C

E 6

Barton Gupta

1, 2-

58)

1, 2-

20

Melicodenine A H

59), 60)

Dedy Darnaedi

Joko Ridho Witono

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130

48 FU

FU

a), b)

dUTPase 5-

dUTPase

SAR dUTPase N- N-

1,2,3- 14c

dUTPase IC50 = 0.067 Min vitro

HeLa S3 5--2-EC50 = 0.07 M in vivo

MX-1 5- 8a dUTPase

dUTPase

dUTPase

14c 5-

5-

Development of Human Deoxyuridine Triphosphatase Inhibitors for

Combination Cancer Therapies with 5-FU

Hitoshi MIYAKOSHIa), b)

Abstract: Deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as part of a new strategy

of 5-fluorouracil-based combination chemotherapy. We have initiated a project to develop potent drug-like dUTPase inhibitors based

on structure-activity relationship (SAR) studies of uracil derivatives. N-carbonylpyrrolidine- or N-sulfonylpyrrolidine-containing

uracils and 1,2,3-triazole-containing uracils were found to be promising scaffolds that led us to human dUTPase inhibitors (14c)

having excellent potencies (IC50 = 0.067 M) and an improved pharmacokinetic profile. The X-ray structure of a complex of 8a and

human dUTPase revealed a unique binding mode wherein its uracil ring and phenyl ring occupy a uracil recognition region and a

hydrophobic region, respectively, and are stacked on each other. Compound 14c dramatically enhanced the growth inhibition activity

of 5-fluoro-2-deoxyuridine against HeLa S3 cells in vitro (EC50 = 0.07 M) and the antitumor activity of 5-fluorouracil against

human breast cancer MX-1 xenograft model in mice significantly. These data indicate that 14c is a promising candidate for

combination cancer chemotherapies with TS inhibitors.

Key phrases: dUTPase, 5-fluorouracil, TS inhibitor

a) 501-1196 1 25-4

Laboratory of Medicinal & Pharmaceutical Chemistry, Gifu Pharmaceutical University (1-25-4 Daigaku-nishi, Gifu 501-1196,

JAPAN)

b) 300-2611

3

Medicinal Chemistry, Chemistry Research Laboratory, Tsukuba Research Center, Taiho Pharmaceutical Co., Ltd.

(3 Okubo, Tsukuba, Ibaraki 300-2611, JAPAN)

Vol. 62, 48-56 (2013) 49

Thymidylate synthaseTS 5-fluorouracil

5-FU

TS

TS

Ladner TS

deoxyuridine triphosphatase

dUTPase 1)

TS

thymidine triphosphatedTTP

deoxyuridine monophosphate

dUMP

dUMP deoxyuridine triphosphate

dUTPDNA DNA

dTTP dUTP

2)dUTPdUTPDNA

DNA

Fig. 1 TS

3)-5)

dUTPase dUTP 5-FU TS

5-fluoro-2-deoxyuridine triphosphate

FdUTPFdUMP

6)-7)

5-FU 8)-10)

1), 11)-13)

dUTPase TS

dUTPase

14)-15)

Fig. 1. 5-FU dUTPase.

dUTPase N-

N-

dUTPase

Fig. 216)-25)

Fig. 2. Structural formula of dUTPase inhibitors.

dUTPase dUTPase

dUTPase

hit 5 Table

3 5IC50 = 97 M

structure-activity relationshipSAR 6a-w

dUTPase Table 3

Table 1 SAR

1

2

N-2,2- 3

2 6sIC50 = 1.3 M 6w

IC50 = 1.1 M dUTPase

lipophilicity

clogP:1.83-2.17

6s

dUTPase

N-

dUTPase

Table 2

rigidity

N-

dUTPase

50 FU

Table 1. Human dUTPase inhibitory activity of

amide-containing uracil derivative 5, 6a-w and reference

compounds 1-4.

aCalculated by ACD/LogP algorithm. bExcept compounds 1a-b,

2-3 and 5, enzyme inhibition assay are tested at 30 M or below.

IC50 values are shown as the mean SE (n = 3). cReference data.

N.T. = not tested

lipophilicity

Table 2Table 2SAR 7aIC50 = 0.29 M

S eutomereudismic ratio = 34

Thienyl

7jIC50 = 0.23 M

N- bioisostere

N-Table

3 N-

8aIC50 = 0.32 M R eutomer

N-

dUTPase

Table 2. Human dUTPase inhibitory activity of

N-carbonylpyrrolidine-containing uracil derivatives 7a-l and 6s

aCalculated by ACD/LogP algorithm. bIC50 values are shown as

the mean SE (n = 3) cExcept 7b, enzyme inhibition assay are

tested at 1.0 M or below.

Table 3. Human dUTPase inhibitory activity of

N-sulfonylpyrrolidine-containing uracil derivatives 8a-f and 6s

aCalculated by ACD/LogP algorithm. bIC50 values are shown as

the mean SE (n = 3) cExcept 8b, enzyme inhibition assay are

tested at 1.0 M or below.

dUTPase dUTPase

X

Vol. 62, 48-56 (2013) 51

dUTP

dUTPase

IC50 dUTPase

dUTP

dUTP 0.1 M

dUTPase

dUTPase X

8a dUTPase X

1.7

Fig. 3

Fig. 3. Binding of 8a (blue stick) in the catalytic site of human

dUTPase. (A) Polar interactions. Distances [] are indicated. Waters are shown as small spheres. Red line depicted

,-imino dUTP 1b in the human dUTPase: ,-imino dUTP

1b structure (PDB code: 2HQU) superimposed on the 8a: human dUTPase (PDB code: 3ARA). (B) Comparison of 8a

(blue stick) with ,-imino dUTP 1b (red stick)

Fig. 3A dUTPase 8a

Fig. 3B dUTPase ,-imino dUTP1b

PDB code: 2HQU26),-imino dUTP

1b

dUTP dUTP mimic 1b 8a

dUTPase

dUTPase

8a

dUTPase

8a

Val65Ala90Ala98 Val