• 의약후보물질 최적화를 위한 독성평가

• 화학물질의 체내 대사체 구조 규명 (1 상 및 2 상 대사체 )

• 화학물질의 체내 대사경로 연구

• 장내 미생물에 의한 화학물질 대사 연구

• 약물 상호작용 연구

연구분야

• 간독성 및 면역독성 평가기술 (in vitro 및 in vivo)

• 대사체 구조 결정 (LC-MS)

• 약물 상호작용 평가 기술• 간세포 분리 및 일차배양 기술• CYP 효소활성 측정기술

보유기술

연구분야 소개

The elimination of xenobiotics depends on their conversion to water-soluble metabolites by a process known as biotransfor-mation, which is catalyzed by en-zymes in the liver and other tis-sues.

Phase and Phase ReactionsⅠ Ⅱ

ABSORPTION METABOLISM ELIMINATION

  Phase Ⅰ Phase Ⅱ  

  Conjugate  

Drug metabolite with modi-fied activity Conjugate

UrineFeces

Inactive drug metabolite Conjugate  

     

  Lipophilic Hydrophilic

m in0 5 1 0 1 5 2 0 2 5 3 0 3 5

m A U

0

2

4

6

8

1 0

1 2

1 4

UV Chromatograms of Rutaecarpine and its Metabolites

m in0 5 1 0 1 5 2 0 2 5 3 0 3 5

m A U

0

2

4

6

8

1 0

1 2

1 4 without NGS

with NGS

Rutaecarpine

Rutaecarpine

Metabolites

2 6 0 .0

2 7 1 .0

0 .0

0 .5

1 .0

1 .5

2 .0

4x 1 0

In ten s .

1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5 2 5 0 2 7 5 3 0 0 m /z

2 7 3 .0

2 4 3 .0

MS2 Spectra of Parent: Rutaecarpine

NH2

O+

NH

CH+

NH

m/z 169 EE+

NH

O+

m/z 120 EE+

NH

N

O

+

m/z 145 EE+

NH

CH+

m/z 142 EE+

N

NH

N

O

++H

Rutaecarpine [M+H]+ 288

[M+H]+288120.0

142.0145.0

185.0168.9

NH

NH

NH+

m/z 185 OE+

NH

N

N

O

NH

N

N

O

HO 10

NH

N

N

OOH

NH

N

N

O

HO

11

NH

N

N

O

OH3

NH

N

N

OOH9

Rutaecarpine

M2

M1

M4

M5

M3

CYP3A4

CYP3A4/1A2/2C9CYP3A4

CYP1A2/2C9/3A4CYP3A4/1A2/2C9

Proposed Metabolic Pathway of Rutae-carpine in Human Liver Microsomes

Only in vitro

β-Glucuronida-tion and Sulfa-tion

β-Glucuronida-tion and Sulfa-tion

β-Glucuronida-tion and Sulfa-tion

0 200 500 1000

Dose-response

1-Bromopropane (mg/kg)

IU/m

l

10

100

1000

10000

Time-course

10

100

1000

10000

100000

0 6 12 24 48

Time (hr)

IU/m

l

****

**

**

Acute effects of 1-BP on serum activity of ALT and histopathology of liver tissue

X 100 magnification

Vehicle

200 mg/kg

500 mg/kg

1000 mg/kg

6 hr

24 hr

48 hr

12hr

SRBCs (5×108)

Results: AFCs/106 spleen cellsor AFCs/spleen (×103)

Splenocytes, SRBC, Complement (guinea pig) and 0.5% agar containing 0.05% DEAE-dextran

Petri dish (100×15 mm)

0 h3 h later37oC

Cover glass(24×40 mm)

Splenocytes counting(Coulter counter)

Stereomaster plaque viewer, ×20

AFCs

Sensitization, ip Single splenocyte

After 30 min

Spleen

4 days later

1-BP treated BALB/c mice, po

T-dependent antibody response to SRBC

CID Spectrum of Glutathione

++H+

Glutathione [M+H]+ 308

[M+H]+[M+H-75]+

[M+H-146]+

[M+H-129]+

HOOC

NH2HN

O

NH

COOH

O

SH

a b c

a

bc

100 150 200 250 300 350 400m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

179.0

162.0233.0

308.0290.1

Fragmentation Mechanism and Proposed Structures of GSH

HOOC

NH2HN

O

O

SH

NH

COOH

O

SH

H2NNH

COOH

O

SH

HOOC

NH2HN

O

NH

COOH

O

SH

++H+

++H+

HN OHOOC

[M+H]+

308 [M+H-75]+ 233.0

[M+H-146]+ 162.0

[M+H-129]+ 179.0

ab

ca b c

Loss of neutral glycine

Loss of amino glutamateLoss of glutamate

Metabolism of 1-BP

Br

1-Bromo-propane

CYPα-hydroxyla-tion Br

OH

3-Bromo-2-propanol

HOOC

NH2 HN

O

NH

COOH

O

SOH

GST

S-(2’-Hydroxyl-1’-propyl) glutathione

H3CHN

O

COOH

S

OH

N-acetyl-S-(2-hydrox-ypropyl)cysteine

Br O

GST

H3CHN

O

COOH

S

N-acetyl-S-propyl cysteine

Propene (Volatile metabolite) NADPH- depen-dence

CYPGST

Br OH

3-Bromopropionic acid

3-Bromo-1-propanol

H3CHN

O

COOH

S

COOH

COOHBr

H3CHN

O

COOH

S

OH

N-acetyl-S-(3-hydrox-ypropyl)cysteine

GST

N-acetyl-S-(2-carboxyethyl) cysteine

GST

ALDH

Propionaldehyde

O O

OH

Propionic acid (Ether extract) NADPH- de-pendence

HOOC

NH2 HN

ONH

COOH

O

S

S-propyl glutathione, m/z 350

ODehydrohalogena-tion (Enzyme-medi-ated)

OH

OH

1,2-Propane-diol (Ether ex-tract) NADPH- dependence

C3 oxida-

tion

CYP

Excretion in

urine

Excretion in urine

Barnsley et al., 1966, Jones and Walsh, 1979 Tachizawa et al., 1982

Effects of 1-BP on content of GSH and formation of S-propyl GSH in liver and spleen: Dose-re-sponse

Hepatic GSHn

mo

le/m

g p

rote

in

0

200

400

600

800

Splenic GSH

1-Bromopropane (mg/kg)

nm

ole

/mg

pro

tein

0

50

100

150

**

**

**

**

Hepatic propyl GSH

nm

ole

/mg

pro

tein

0

150

300

450

600

Splenic propyl GSH

1-Bromopropane (mg/kg)

nm

ole

/mg

pro

tein

0

3

6

9

12

0 200 500 10000 200 500 1000

clean

ing agen

t

chemical intermediates

S-propyl GSH (1-BP)

S-3-79/81bromopropyl GSH (1,3-DBP)

S-2-79/81bromopropenyl GSH (2,3-DBPE)

Expo-sure

Glutathione

spra

y fo

rm

extraction solvents

chemical synthesesstabilizer the wool

Dependent mechanism

Suppressed the antibody response to SRBCsSuppressed the splenic in-tracellular IL-2 production

Spleen

Increased the serum ALT ac-tivity and tis-sue damage

Depletion of GSHIncreased oxidative stress

Liver

Br

1

2

3

Br

1

2

3

Br Br

1

2

3Br

Br

1

2

3

Br

Br

Br

1

2

3

Br

Br

1

2

3

HOOC

HN

NH

COOH

NH2 O

OS

HOOC

HN

NH

COOH

NH2 O

OS

HOOC

HN

NH

COOH

NH2 O

OS

OHO

HOOC

HN

NH

COOH

NH2 O

OS

Br

HOOC

HN

NH

COOH

NH2 O

OS

Br

S-Isopropyl GSH (2-BP)

S-2-hydroxypropyl(oxopropyl) GSH& their mercapturic acid (1,2-DBP)

Overall ranking of toxicity:1,2-DBP, 1,3-DBP > 1-BP >> 2-BP

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