Doshisha University

有毒ガスでありながら生体内シグナルガスとしても注目される
一酸化炭素の生体内除去およびデリバリーツール
内因性の一酸化炭素の産生 について

一酸化炭素
CO

heme metabolism

有毒ガス，火災の死亡原因，排気ガス，タバコ，練炭自殺…etc.

metabolic products
O O

heme oxygenase
NN

NH

FeIII
NN

生理活性ガス，ヘムタンパク質へのシグナル，抗炎症作用，臓器保存効果…etc.

HN+ Fe２+CO
HOOC

HOOC

COOH

COOH

biliverdinheme(Fe3+ hemin)

有毒ガス ＝ 生体内で作用する＝ 微量は生物に必要NO

N

HN

細胞内シグナル
ガスとして機能CO-ヘモグロビンとして循環

Knockout/knockdown methods
are available.

H2S
COO O

N

HO-1

N

NH+ Fe2+ + COFeIII
NN

HOOC

HN

N

HN

HOOC

COOH

COOH

Knockout/knockdown cannot be available for CO.
COの生理 機能には不明な点が多く，注目ターゲット

一酸化炭素の捕捉物 質hemoCDについて
一酸化炭素の捕捉物質hemoCDについて
OCH3O

H3CO
O

O

H3CO

OCH3H3CO

OCH3OCH3O
OCH3H3COO

OCH3H3COO

H3COO

H3COO

O

OCH3H3COO

SSNO

OCH3H3CO

SO3–

OCH3
H3COO
O
OCH3OCH3N

N+O
O

H3CO

S 33 S

OCH3H3CO動物体内のCOを尿中へと強制的に追い出す

SO3-O

OCH3OHO

OCH3 H3CO O
OCH3O

H3CO

O

O

O

O
OCH3OCH3H3CO

O

O
HO
H3COO

H3CO

OCH3–O S
3NFeIIN

SO3–NNN
FeII
N
N

-O S
3SO3-OCH3O

SO
OCH3SO3–OCH3

Py3CD

-O S
3CO

Fe(II)

Fe(II)N

N
S

oxy-hemoCD

hemoCDFeIITPPSO2SSA CO-removal agent
in the living organisms

CO-hemoCD (in urine)
Angew. Chem. Int. Ed. 2010, 49, 1312–1315.

An Hb/M b functional model

Fe(II)NS

hemoCDS

P50 (O2) / Torr

P50 (CO) / Torr

Hb (T-state)

22-S

O2

COHb (R-state)

0.22

0.0013Fe(II)

Fe(II)

N

hemoCD

100.000015

NS

oxy-hemoCDSS

CO-hemoCDAdministration ofhemoCD

K. Kano, H. Kitagishi et al Inorg. Chem. 2006,45, 4448–4460.

HemoCD is a useful CO receptor in aqueous media.

Endogenous CO-depleted state

Pseudo-knockdown
of CO in mice

内在性COを欠乏した疑ノックダウンマウス

北岸 宏亮 Hiroaki KITAGISHI
同志社大学リエゾンオフィス

同志社大学理工学部 機能分子・生命化学科

https://kikou.doshisha.ac.jp/

T E L：0774-65-6223
E-mail：jt-liais@mail.doshisha.ac.jp

Doshisha University

有毒ガスでありながら生体内シグナルガスとしても注目される
一酸化炭素の生体内除去およびデリバリーツール
一酸化炭素の捕捉物質hemoCDについて
血中COヘモグロビンの定量
動物体内のCOを尿中へと強制的に追い出す
O2

COFe(II)

Fe(II)NSSoxy-hemoCDSNSThe CO-Hb content (%) in the blood
was measured by gas chromatography.

S

oxy-hemoCD(1 mM, 0.15 mL)

CO-hemoCD (in urine)
Angew. Chem. Int. Ed. 2010, 49, 1312–1315.

0.3

CO-H b / %Administration ofhemoCD

blood sampling

Fe(II)A CO-removal agent
in the living organisms

NS

O2

Endogenous CO-depleted state

0.2%

CO-ヘモグロビンの量がほとんどゼロになった

0.2%

0.2

*
0.1

0.03%

**

すぐに定常値へと回復した。すなわちCOの産生 が亢進された？

0Pseudo-knockdownof CO in micecontrol

30 min90 min

180 minTime after the administration

内在性COを欠乏した疑ノックダウンマウス****

9HO-1induction

6HemoCDは微量COの検出試薬としても使える

The expression of HO-1 strongly
increased in the hemoCD-treated mice.
****

oxy-hemoCD
in PBS

3**

removal of medium

1) cell count2) sonication3) ﬁltrationcollection of the cells

O20inin
PBS oCD 0 minmm
38090m1eh
FbOxy-hemoCD

1 x 106 cells
adhered on the dishFe(II)cell suspensionSS

oxy$hemoCD
NFeNNH HNHO-1NNHN

spectrumc (b + Na2S2O4)

Absorbance

OONRemoval of
3+Fe2+CO
CO$hemoCDendogenous CO.

spectrumb (a + CO)

spectruma (ﬁltrate)

HOOC

COOH

ﬁltrate in cuvatte

NCO/ pmol
M/ pmolper
per 106 cells
cells
COH
mo
x
1/bactin

12

J. Am. Chem. Soc. 2016, 138, 5417-5425.

HOOC

COOH

350

hemin

450
550
Wavelength / nm

300

**

200

1000

HepG2

HeLa

RAW264cancer cells

650

NIH3T3CHO

SH-SY5Y

normal cells

induc&on
細胞内にある内在性COの定量に成功！

CO恒常性維持のためのフィードバックシステムの発見！

J. Am. Chem. Soc. 2017, 139, 5984.

J. Am. Chem. Soc. 2016, 138, 5417-5425.

Measurement
forbiologicalCO
Measurement for biological CO

内在性COの欠乏は，体内時計のリズムを乱す

１) ガスセンサー/ガスクロマトグラフィー法

BMAL1 CLOCKor NPAS2
Per,Cry

ヒトの場合：呼気のCOを測定し，血中CO量を推定する
血液/組織：COの結合したヘムを酸化し，遊離COの気相分析を行う

２) 赤外レーザー分光法
細胞内COを感度良くリアルタイム計測可能だが，特殊装置が必要

degradation
1 dayPER CRY

Circadian time(Y. Morimoto, et al., Am. J. Physiol. 2001, 280, H483)

PerCry３) ケミカルプローブの利用
E-box

CO選択的な蛍光プローブ (BODIPY-Pd錯体) 定量性には乏しい

clock genes(C. J. Chang, et al., J. Am. Chem. Soc. 2012, 134, 15668)

HemoCD アッセイNEW!

Hbなどの生体内のあらゆるヘムタンパク質からCOを奪い，吸光度測定により簡単かつ正確にCOを定量可能

SO3–

外来性COの臓器分布計測
新生児黄疸の早期診断法への応用etc.

N II NFeN
N–O S
3

SO3–N
S–O S
3

Sci. Rep. 2018, 8, 11996 (12 pages).

北岸 宏亮 Hiroaki KITAGISHI
同志社大学リエゾンオフィス

同志社大学理工学部 機能分子・生命化学科

https://kikou.doshisha.ac.jp/

T E L：0774-65-6223
E-mail：jt-liais@mail.doshisha.ac.jp

Doshisha University

有毒ガスでありながら生体内シグナルガスとしても注目される
一酸化炭素の生体内除去およびデリバリーツール
一酸化炭素徐放試薬CORMについて

CO RM -4 0 1

Cellular uptake of CORM-401
CORM-401 (50 µM in D-MEM/10% or 1% FBS)
血清の成分はCORMの細胞導入を阻害
Incubation for 2 h
Silva, T. S., et al., J. Am. Chem. Soc. 2011, 133, 1192-1195.

wash by PBS
5 µM oxy-hemoCD1CO-hemoCD1 / cell (x 10-16 mol)R. Motterlini

JCI Insight, 3, e123485 (2018).

COの継続投与は，体重増加を効果的に抑制する＝ダイエットピル？

1 x 106 cell / mL

Removal ofmedium

Incubation 2 hmeasurement by
UV-vis spectrum

R ESEA R CH A R THepG2
I CLE cells

水溶 性，3CO/molecule放出

D-10 1 mL
CORMs 50 µM

disrupt cells bysonication
O8HO

7

2.3 x 10-16 mol/cell

65

2.1 x 10-16 mol/cell

S COS MnI COCOCO
CORM-401

血清を減らすことで、細胞へのCO送達量が2.1倍増加した

43

細胞導入率
10% FBS : 0.4%
1% FBS : 0.8%

210CH3N

without CORM

CORM+
10%FBS

CORM+
1%FBS

投与量に対して非常に少ない

Figure 2. Quantification of endogenous CO in cells after the additionof 50 µM CORM-401 by hemoCD. Cells were incubated in D-MEM/10% or 1% FBS.

Collection of cells
Cell countSonication
Filtration

PBS 1 mL
Oxy-hemoCD

Removal ofmedium

Measured by UV-vis

CORM401-E
 大量合成できるCORM401から，簡便で後処理がほとんFigure 1. CORM-401 reduces body weight gain and improves insulin resistance in high-fat diet–induced obesity. Mice received a standard diet (SD) or
high-fat diet (HFD) for 14 weeks. CORM-401 (30 mg/kg) was given by oral gavage starting either at the beginning of (HFD + CORM-T) or after 6 weeksof
どなくメチルエステル化に成功した(HFD + CORM-P) HFD. Carbonmonoxy hemoglobin (COHb) was measured after oral gavage with CORM-401 (A). Body weight (BW ) was measured everyweek (B) and BW gain was calculated as the percentage of the basal weight (C). A glucose tolerance test (GTT) was performed at week 13 (D), and data
represent the area under the curve (GTT AUC) (E). An insulin tolerance test (ITT) was performed at week 14 (F), and data represent the area under the curve(ITT AUC) (G). Fasting total cholesterol (H), fasting triglycerides (I), fasting glucose (J), fasting insulin (K), calculated HOMA-IR (L), and fasting leptin (M)were measured after 14 weeks in all groups examined. Values represent the mean ±SEM. n = 4–6 mice per group (A); n = 8–10 mice per group (B–M). *P <0.05 vs. control group (SD), # P < 0.05 vs. HFD group, values not designated with symbols are not statistically di f erent, Student’s t test or 1-way or 2-way
ANOVA with Fisher multiple comparison test.

 従来のCORM401よりも5倍以上の高い胞内取り込み効率を示した

 COの研究をするための研究用試薬としての需要は高い

to basal levels by CORM -401 in the H FD + CORM -P group but not in H FD + CORM -T group. I n contrast,
mRNA expression of PPar-α, Pgc1-α, and Cpt1 was decreased by H FD but remained unchanged after CORM 401 treatment (data not shown). Signiﬁ cant remodeling in other fat pads, particularly in brown adipose tissue(BAT), was evident in the reduction of weight and lipid content in both groups administered with CORM 401 (Supplemental Figure 3, A–E). Similarly to that in eW AT, mRNA expression of the metabolic genes
PPar-γ, Vegf, adiponectin, Pgc1-α, and Ucp1 was decreased in BAT of H FD -fed obese mice and fully or partiallyrestored in the H FD + CORM -P group (Supplemental Figure 3G ). Conversely, gene expression of metabolic
markers in subcutaneous adipose tissue was not affected by CORM -401 (Supplemental Figure 3F). D espite
the fact that H FD did not induce a high-grade inﬂammation in our model, CORM -401 still modulated local
inﬂammation in obese mice. First, the increased macrophage inﬁltration induced by H FD was virtually abolished by CORM -401 administration (Figure 2, F and G ), although the induction of Ccl2 was unchanged by
the compound (Figure 2H ). Second, CORM -401 decreased Hmox-1 expression induced by H FD (Figure 2I )北岸 宏亮 Hiroaki KITAGISHI
同志社大学リエゾンオフィス
insight.jci.org

https://doi.org/10.1172/jci.insight.123485

北岸宏亮，高山実花子，R. Motterlini, 特許出願中

同志社大学理工学部 機能分子・生命化学科

3
https://kikou.doshisha.ac.jp/

T E L：0774-65-6223
E-mail：jt-liais@mail.doshisha.ac.jp