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What is traditional Japanese Kampo medicine
1. International Surgical Week
August 29, 2011 Yokohama, Japan
What is traditional Japanese Kampo medicine
An Overview of Basic and Clinical Challenges
Asahikawa
Toru Kono
Division of Gastroenterologic and General Surgery
Department of Surgery, Asahikawa Medical University
Tokyo
Mt. Taisetu National Park, Asahikawa
(Scenery from the window of my office)
3. Herbal medicinal products in the world
China and other countries
• Many doctors are concerned about the
quality of herbal medicinal products
• One reason is that some herbal medicinal
products are contaminated with
unexpected pharmaceutical ingredients
(e.g. antibiotics, pesticides)
• EBM is not enough
4. What is Kampo ?
• Kampo is traditional Japanese medicine
• Kampo is prescribed by Japanese medical
doctors
• Kampo is covered by the National Health
Insurance
• Kampo products from Tsumura are not
considered as CAM products in Japan
5. Kampo
• Over the last few years, FDA began
shifting its focus on Kampo because of its
exceptionally high quality and
standardized ingredients
• Kampo is primarily extract granules and
their pharmacological actions have been
elucidated at the molecular level
6. What is daikenchuto (DKT, TJ-100) ?
- TU-100 in the US -
Most frequently prescribed Kampo medicine
Approved in 1986 by the Japanese government
Improves gastrointestinal motility and
prevents postoperative adhesion and paralytic
ileus after abdominal surgery
7. Annual sales of Daikenchuto (TU-100)
sales
X 100 80 200 packages
million yen X 10000
60
40
20
0
2005 2006 2007 2008 2009 2010
8. What is daikenchuto (TU-100) ?
Composed of 3 medicinal herbs
* Japanese pepper
* Processed ginger
* Ginseng radix
and maltose powder
9. Manufacturing process of TU-100
Japanese Pepper Processed Ginger Ginseng Radix
Mix herbs spray drying via addition of heat (203ºF) extract powder
Maltose powder
Extracted powder TU-100
Mixture of extract powder and maltose powder is at a ratio of 1:8
10. TU-100 final components
Extract of 3 medicinal herbs
( Japanese pepper,
Magnesium stearate (lubricant processed ginger,
ginseng radix )
Lactose hydrate
Maltose powder
Clinical dose of TU-100 per day: 7.5 to 15g
weights in parentheses show doses per day
11. O
MeO O
MeO
How to check the quality HO
6-shogaol HO (10-shogaol)
hydroxy-α-sanshool
of TU-100 MeO
O
NH HO
8-shogaol
hydroxy MeO
O OH O
HO
O OH
α-sanshool
HO
6-gingerol 6-shogaol
MeO
HO 10-gingerol
ginsenoside Rg1
Glc
O ginsenoside Rb1
Circled ingredients are the
HO 6
Glc-Glc
HO
O
MeO
O OH
characteristic markers
hyperoside
HO O
OH HO
O-Glc 2
Glc-Glc O
HO
8-gingerol for quality control of TU-100
OH
O Gal quercitrin OH
OH O
HO O
OH
OH O
O Rha
ginsenoside
Rb1
No unexpected pharmaceutical ingredients
No toxins, pesticides, microbes and heavy metals
(impurity) O OH
MeO
α-sanshool O
NH
HO 10-gingerdion
H
γ-sanshool NH
OH O
N O
H MeO
O N
hydroxy-β-sanshool HO
10-dehydrogingerdion
β-sanshool O HO
Three-dimensional high-performance liquid chromatography of TU-100
Confidential data in TSUMURA
Kono T. et al J Gastroenterology 2011 (in press)
15. TU-100 reports of adverse effects
from 1986 to 2006
Only 46 cumulative cases during 20 years
Annual sales of > 9 million packages
All AEs are not severe and patients have
fully recovered
• liver dysfunction
• liver damage
• jaundice
• pneumonitis
• pancytopenia
• oral mucositis
17. TU-100 Main Clinical Applications and Mechanism
• Prevention and treatment of paralytic
bowel obstruction after abdominal
surgery, orally or through ileus tube
Shibata C, et al. Surgery 1999;126(5): 918-24
Nagano T, et al. Biol Pharm Bull 1999;22(10):1131-3
Jin XL, et al. Dig Dis Sci 2001;46(6):1171-6
Sato Y, et al. Biol Pharm Bull 2004;27(11): 1875-77
Several important neuronal factors are involved in the
mechanism, one of which is calcitonin gene-related peptide
(CGRP), a neuropeptide produced in the nervous tissue
by the sensory neurons.
18. TU-100 Main Clinical Applications and Mechanism
• Improvement in intestinal blood flow
• Beneficial effects are due to the mobilization of
endogenous calcitonin family of peptides and their
receptor-associated factors
• Calcitonin gene-related peptides (CGRP
• Adrenomedullin (ADM
Murata P, et al. Life Sci 2002;70:2061-2070
Kono T, et al. J Surgical Res 2008;150:78-84
Kono T, et al. Surgery 2009;146(5):837-840
Kono T, et al. J Crohn’s and Colitis 2010;4:161-170
Kono T, et al. J Gastroenterology 2011 (in press)
19. CGRP and ADM: Physiological Effects
Gastrointestinal motility (CGRP)
Vasodilation (CGRP, ADM)
Secretion (CGRP)
Anti-inflammatory cytokine (CGRP, ADM)
Anti-microbe (ADM)
CGRP and ADM play pivotal roles in
understanding TU-100’s effects
20. CGRP and ADM
Different sites of production and expression
CGRP is synthesized almost exclusively in neuronal tissues
(e.g. nerve terminals)
ADM is mainly distributed in non-neuronal tissues (e.g.
epithelial cells, smooth muscle cells, etc.)
Dawn in Tokyo
21. Sensory nerve fibers positive for CGRP in
intestinal mucosa
CGRP
Mucosa (crypt)
CGRP
22. ADM production by intestinal epithelial cells
Small Intestine Large Intestine
Rat
Small Intestine Large Intestine
Human
24. TU-100 injection site
Practical experiment scenery
A tube was inserted
via the cecum into
the proximal colon
TU-100 is dissolved
Laser probe
Cecum
Distal Colon
Recording site
Plastic wrap to keep warm and to prevent colon
the tissue from becoming dehydrated.
25. Effect of TU-100 intraluminal administration on Colonic
Vascular Conductance (VC; index of blood flow)
The time-course of changes in colonic VC
0.24
TU-100
0.20
100 mg/kg
300 mg/kg
0.16
10 mg/kg
Vehicle
0.12
0.08
0.04
0.00
0 15 30 45 60 75 90
Kono T., et al., J. Surg Res 2008
26. Next, we examined the mechanism of
vasodilation using blocker drugs:
CGRP receptor blocker (CGRP8-37)
Nitric oxide synthase inhibitor (L-NAME)
Vasoactive intestinal polypeptide (VIP)
receptor blocker ([4-Cl-DPhe6, Leu17]-VIP)
Substance P (SP) receptor blocker (spantide)
28. TU-100 increases blood flow of the
small intestine as well as colon
0.25
D.W. 5 mL/kg (N=7)
TJ-100 2700 mg/kg (N=6)
TU-100 100
## ##
0.20 ** *
#
VC
0.15
0.10
0.05
0 15 30 45 60 75 90 105 120
Time (min)
Two-way ANOVA: treatment, [F(1, 99)=32.96 p<0.0001], time, [F(8, 99)=3.69,
p=0.0008]. *P<0.05, **p<0.01 versus pre-administration (0 min) (Dunnett’s test).
#, ## p<0.05, 0.01 versus D.W. group (Student’s t-test).
29. Some of you may be wondering
if TU-100 works like capsaicin............
Kyoto, Genkoan
30. TU-100 does not work like capsaicin
BCTC did not abolish TU-100 effect
BCTC: selective anti-capsaicin
200 200 BCTC (3mg/kg, i.v.) + D.W. (5 mL/kg, i.d.)
BCTC (3mg/kg, i.v.) + TJ-100 (900 mg/kg, i.d.)
160 Capsaicin (3 mg/kg, i.d.) 160
BCTC (3mg/kg, i.v.) + Capsaicin (3 mg/kg, i.d.)
120 120
VC (%)
VC (%)
80 80
40 40
0 0
-40 -40
0 15 30 45 60 75 90 0 15 30 45 60 75 90
Time (min) Time (min)
31. TU-100-induced vasodilation is observed by
in vivo dynamic microscopy
Before After
The difference in background color (yellow) is due to TU-100 infusion
Arrows are pointing to the same area TU-100
32. What is the correlation between
CGRP and ADM receptors?
Autumn in Kyoto, Shodaji temple
33. ADM and CGRP antagonists, both receptor
blockers abolished TU-100-induced blood flow
DKT alone (N=8)
DKT + ADM22-52 (N=7)
DKT + CGRP 8-37 (N=6)
CVC
Time (min)
34. CGRP and ADM receptors are very unusual
Calcitonin receptor-like receptor (CRLR)
is an immature receptor.
Only when RAMP (receptor activating
membrane protein) binds to CRLR does
it mature into a “real” receptor that is capable
of binding ADM and/or CGRP.
35. Association of CRLR with a RAMP dictates the specificity of ligand binding
CRLR CRLR CRLR
N N N
C C C
RAMP1 RAMP2 RAMP3
N N N N N N
C C C
C C C
CGRP receptor ADM receptor ADM/CGRP receptor
36. TU-100 up-regulates CGRP and its receptor components mRNA
TU-100 1.5
CRLR *
- + 1
CRLR 504 bp 0.5
0
DKT(-) DKT(+)
Ramp-1 230 bp
1.5
Ramp-1
1
*
CGRP 102 bp
0.5
GAPDH 249 bp 0
DKT(-) DKT(+)
1
Evaluation of RNA from the rat colon CGRP *
0.75
after TU-100 administration using RT-PCR.
0.5
0.25
0
DKT(-) DKT(+)
Kono T., et al., J. Surg Res 2008
The intensity of each band was normalized to
that of the corresponding band of GAPDH.
The data are presented as mean ± SE (n = 4).
Bars show SEM. Compared with control, *p < 0.05.
37. TU-100 up-regulates RAMP2, 3 (ADM/CGRP receptor)
CRLR RAMP2 RAMP3
N N RAMP2
C
C
N N
RAMP3
Cont TU-100 Cont TU-100 Cont TU-100
C
C
CGRP GAPDH
ADM receptor
ADM/CGRP receptor
Cont TU-100 Cont TU-100
38. ADM production by epithelial cell line of rat small intestine
IEC-6 cell
ADM (green)
Dashed line: Isotype Control Ab (rabbit IgG)
Red line: Anti-ADM Ab
IEC-6 cell (rat intestinal epithelial cell line) was stained with anti-ADM antibody, and
analyzed by flow cytometer using FACScalibur and by immunocytochemistry.
Kono, J Crohn’s Colitis 2010
39. TU-100 enhances ADM production by intestinal epithelial
cell lines in a time-dependent manner
900 µg/ml TU-100
ADM concentration ( pg/ml )
Control
Culture time ( hour )
IEC-6 cell was cultured with or without 900 µg/ml of DKT for 12, 24, 48, 72, or 96 h.
N=3. *, **: p<0.05, 0.01 vs. no TU-100 control, respectively.
Kono, J Crohn’s Colitis 2010
40. TU-100 enhances ADM production by intestinal epithelial
cell lines in a dose-dependent manner
a) IEC-6 b) IEC-18
ADM concentration ( pg/mL )
ADM concentration ( pg/mL )
TU-100 concentration (µg/ml )
TU-100 was added to IEC-6 (a) and IEC-18 (b) cells at concentrations of 90, 270, 900 or 2700
mg/ml. N=4.
*, **: p<0.05, 0.01 vs. no TU-100 control, respectively.
41. TU-100 increases ADM production from
small and large intestinal epithelial (IE) cells
Cells TU-100 concentration (µg/ml)
IE cells of small intestine
IE cells of large intestine
Intestinal epithelial (IE) cells were isolated from the small or large intestine of normal
mice and stimulated with TU-100 (90, 270, or 900 µg/ml) at 1 x106 cells/ml in 96-well
round plates for 24 h. Concentrations (pg/ml) of adrenomedullin (ADM) in culture
fluids were determined using the EIA method. N=4-6, *, **: p<0.05, 0.01 versus no
TU-100 control, respectively.
Kono, J Crohn’s Colitis 2010
42. Which of the active ingredients in TU-100?
Kyoto Darumaji
43. Ingredient Analysis of TU-100
• TU-100 is composed of only 4 components:
Japanese pepper 2.2%
processed ginger 5.6%
ginseng radix 3.3%
maltose powder 88.9%
44. Japanese pepper and processed ginger are
key components for ADM release
ADM release ( ng/ml )
Cont. 80 800 10 100 10 100 10 100 ( µg/mL )
Maltose Processed Ginseng Japanese
Ginger radix pepper
Components of TU-100
Epithelial cell line of rat small intestine, IEC-6, was plated in 96-well flat plates at 1 x104 cells/well. The next
day, culture fluids were replaced with fresh medium containing test sample. Cells were allowed to incubate
an additional 24 h, and the supernatants were harvested. Concentrations (pg/ml) of adrenomedullin (ADM)
in culture fluids were determined using the EIA method. Maltose syrup and extracts of three medicinal herbs
(processed ginger, ginseng radix, and Japanese pepper) were evaluated at concentrations described in
figure. N=3. **: p<0.01 versus control (Dunnett test)
Kono, J Crohn’s Colitis 2010
46. Mechanism of TU-100-induced hyperemia
6-shogaol, hydroxy- -sanshool
Lumen
How do
they
Microvascular
stimulate?
vessel
Neuronal tissue
(Sensory nerve) Non-Neuronal tissue
(Epithelial cells)
ADM
Nerve endings CGRP
vasodilatation Improvement in
Microvascular Vessel microcirculation
47. Role of TU-100 as
TRP Channel Activator
Activation of transient receptor potential A1 expressed
in intestinal epithelial cell increases intestinal blood flow
via release of adrenomedullin
Inhibition of postoperative adhesion formation by Daikenchuto, a releaser
of endogenous adrenomedullin in intestinal tract
Florida
48. What is a Transient Receptor Potential (TRP)
channel?
TRP channel is a 6-transmembrane Ca2+ channel
49. Natural ligands like mint, cinnamon and chili pepper
stimulate specific TRP channels
AITC (mustard oil)
Menthol (mint) Capsaicin (‘hot’ chili peppers)
CNA (cinnamon)
Do the crude drugs (or constituents) in Kampo stimulate also?
TRPA1 channel
TRPM8 channel TRPV1 channel
C
N
TRP channel
Figure modified from David D. McKemy. Molecular Pain. 2005;1:16.
50. Hypothesis
TU-100 and TRP channels
Pharmacological effects of Kampo may
be defined by TRP channel activity
keywest
51. Do the TU-100 active ingredients,
hydroxy- -sanshool and 6-shogaol,
activate
TRP channels?
Florida
52. Hydroxy- -sanshool, 6-shogaol are TRPA1, TRPV1 agonists
Molecular biological profiles of hydroxy- -sanshool
Molecular biological profiles of 6-shogaol
54. Intestinal epithelial cells express TRPA1 and ADM
but not TRPV1
IEC-6 DRG
Fig.6. intestinal epithelial cell line IEC-6 TRPA1
expressed mRNA of TRPA1 and ADM
RT-PCR analysis was performed for TRPA1, TRPV1, TRPV1
ADM and β-Actin in IE-6 cell, and dorsal root ganglion
(DRG) isolated from normal rats using KOD PCR kit ADM
(Toyobo). The PCR products of 30-cycle amplification
of the mRNAs were resolved on a 2% agarose gel. β-Actin
DDW2011 poster presentation
55. TRPA1 expression in intestinal epithelial cells
The nucleus is dyed blue by DAPI
56. Hypothesis
TU-100
(6-shogaol, hydroxy- -sanshool
Stimulation
TRPA1
in intestinal epithelial cells
Enhanced ADM release from
intestinal epithelial cells
57. TRPA1 agonist only stimulates ADM production in IEC-6 cell
ADM release ( pg/mL )
TRPV1, CB1,CB2 TRPA1 TRPV1 TRPV1-V3
KCNK3
-
(Cont.) anandamide allyl capsaicin 2-aminoethoxy
Isothiocyanate diphenyl borate
(mustard oil)
Various TRP agonists were dissolved in DMSO and added at concentrations of 0.3, 3, 30 mol/L to
cultivation of IEC-6 cell (rat intestinal epithelial cell line). DMSO concentration was 0.3% in all wells. Next
day, ADM concentrations in culture fluid were measured by the EIA method. Data are shown as average/SE
of 3 well. ** : P<0.01 significant at the Dunnett determination
58. Treatment of TRPA1 siRNA diminishes
ADM enhancing activity of TU-100
300 2000
TU-100 TRPA1 agonist
allyl isothiocyanate (AITC)
Percentage to no stimulus control
Cinnamaldehyde (CNA)
1500
ADM production
200 1000
500
100
0
NC A1 NC A1
No stimulus No stimulus
IEC-6 cell was incubated for 3 days in presence of 0.3 μmol/L siRNA specific to TRPA1. Culture fluids were replaced with
fresh medium containing the respective siRNA and stimulus (900, 2700 μg/ml DKT, 30 μmol/L AITC or 100 μmol/L CNA).
ADM concentrations in the 24 h culture fluids were measured by the EIA method. N=3 to 4, NC: Negative control siRNA,
A1: TRPA1 siRNA,
59. Hypothesis: Mechanism of TU-100`s effect in promoting colonic blood flow
TU-100
(Hydroxy- -sanshool, 6-shogaol)
Increased
Intestinal epithelial cells concentration
in blood
TRPA1 (absorption)
ADM up-regulation
ADM release in blood
Vasodilatation
60. How does TU-100
behave inside the
Epithelial body ?
cell
adrenomedullin
Improvement of
Microvascular
Circulation
Hydroxy sanshool
Shogaol
Gingerol
62. The number of Crohn's disease patients has steadily
increased over the past 30 years in Japan
Incidence
Japan
30,000
Patients/100,000 people/Year
Over 500,000 Crohn’s patients in USA
63. Distribution of Crohn’s disease patients in Japan
Asahikawa
(my home)
Low High incidence
Tokyo
Emergency helicopter transport of
IBD patient from rural area to Asahikawa
Ministry of Health, Labour and Welfare, 2000
64. Overnight workshop at the hot springs hosted annually for
patients of IBD Friendship Society of Asahikawa
Once a year doctors and patients get together to enjoy hot spring
65. CGRP and ADM in Crohn’s disease
CGRP in the intestine of Crohn’s disease
patients is decreased compared with that of
normal intestine.
Decrease of CGRP contributes to microvascular
dysfunction in Crohn’s disease patients.
66. Submucosal neuronal network, including CGRP, in the
intestine of Crohn’s disease patients was decreased
compared with normal intestine
Normal ileum Crohn’s disease ileum
Neuronal marker, PGP9.5 (red) and DAPI (blue
67. Selective loss of neuropeptide CGRP, but not ADM,
in Crohn’s disease model and human
Kono T. et al J Gastroenterology 2011 (in press)
It has been reported that blood flow is decreased by more than
50% in the terminal ileum and colon of Crohn’s disease patients
Gastroenterology. 1977;72:388-96. Gut. 1986;27:542-9.
68. Measurement of colonic blood flow in TNBS-treated rat
0.14
normal colon
0.12
TNBS treated colon
0.10
0.08
CV
0.06
0.04
0.02
0.00
0 15 30 45 60 75 90
min
The involved segment of the ischemic colon (VC value: ≤ 0.06)
in TNBS-induced colitis rats was identified by measurement of colonic blood flow,
while that of control rats was indicated as 0.10±0.01.
Kono T. et al J Gastroenterology 2011 (in press)
69. CGRP and ADM in Crohn’s disease (CD)
Several investigators have reported therapeutic
effects of CGRP in experimental CD models.
ADM has been reported to have therapeutic
effects in experimental CD models as well.
Can exogenous CGRP and ADM be candidate
therapeutic agents for CD ?
Answer is NO!
70. Exogenous CGRP and ADM cannot be
therapeutic agents for Crohn’s disease
First, metabolic clearance is too fast
Second, administration of both peptides in
excess may induce decrease in peripheral
vascular resistance
Third, it is difficult to control delivery of both
peptides to diseased intestine
Therefore, it is desirable to develop an agent that
increases endogenous CGRP and ADM such as TU-100
71. TU-100 and Crohn’s disease
Our hypothesis is that TU-100 acts as an
endogenous CGRP and ADM enhancer and
exerts therapeutic effects on CD via endogenous
CGRP and ADM
• Improves intestinal blood flow
• Suppresses anti-inflammatory cytokine production
• Anti-microbial
Specifically, ADM can be targeted instead of decreased-CGRP
in CD
72. TU-100 improves colonic blood flow
in ischemic colon
0.20 TU-100, 900 mg/kg (N=7) TU-100
Distilled Water (N=5) +ischemic
Distilled Water (Normal; N=6) †† colon
††
†† ** ††
0.15 ** **
†† † **
* *
Normal
VC
0.10
0.05 ischemic
colon
0.00
0 15 30 45 60 75 90
Time (min)
The basal colonic vascular conductance (VC) of the ischemic colon indicated 0.06 or less which was lower than that of the
normal colon (0.10 ± 0.01). Changes of the VC induced by test sample are expressed as the mean ± S.E.M. (a) DKT (900
mg/kg) and vehicle (distilled water) were evaluated. Factorial two-way ANOVA analysis revealed significant effects of group
[F(4, 144)=21.17 p<0.0001] and time [F(5, 144)=3.95, p=0.0022]. *p<0.05, **p<0.01 versus pre-administration (0 min)
(Dunnett’s test). †, ††: p<0.05, 0.01 versus the vehicle control, respectively (Student’s t-test).
Kono T. et al J Gastroenterology 2011 (in press)
73. Effect of TU-100 on colonic blood flow
in ischemic colon
Before After
How about TU-100’s effect on hyperemic colon?
Does TU-100 exert an effect or NOT?
74. TU-100 increases VC in ischemic lesion (CV < 0.6) only
and never in hyperemic lesion (active) (CV > 0.2)
0.40 0.40
Vascular Conductance 0.35 0.35 大建中湯 100 mg/kg (N=7)
Vascular Conductance
大建中湯 100 mg/kg (N=7)
0.30 0.30
0.25 0.25
0.20 ischemic 0.20
0.15 0.15 hyperemic
0.10 0.10
0.05 0.05
0.00 0.00
In hyperemic segments, endogenous ADM and CGRP
0 15 30 45 60 75 90 0 15 30 45 60 75 90
Time (min) Time (min)
have been used up so there is nothing left
200 200
大建中湯 100 mg/kg (N=7)
for TU-100 to act on
Vascular Conductance (%)
Vascular Conductance (%)
160 160 大建中湯 100 mg/kg (N=7)
120 120
80 80
40 40
0 0
-40 -40
0 15 30 45 60 75 90 0 15 30 45 60 75 90
Time (min) Time (min)
Kono T. et al J Gastroenterology 2011 (in press)
76. TU-100 directly induced blood flow increase
in Crohn’s disease ( colon artificial anus)
Thermographs
before after
colon artificial anus
a b c skin temp
33
32
31
30
29
28
27
26
DKT sprinkled on the artificial anus
25
78. Blood flow is a very important factor
in pathogenesis of Crohn’s disease
Ileal ulcers tend to occur along the
mesenteric margin of the bowel wall in CD
and experimental models of CD
J Clin Pathol. 1997;50:1013-7.
Aliment Pharmacol Ther. 1999;13:531-5.
Aliment Pharmacol Ther. 2000;14:241-5.
79. Crohn’s disease ileum
Mesenteric side
Mesenteric side
Mesenteric side
stenosis
stenosis
80. Who can answer the prepotency of
the Crohn’s disease?
Hypothesis:
Primary pathological abnormality
in Crohn’s disease is in the
mesenteric blood supply
Lancet. 1989;2:1057-62.
81. Schematic diagram of human small intestine in Crohn’s disease
Normal Remission Mucosal barrier Active
flora
vessel
bacteria
inflammation
* granuloma ulcer
Mesenteric Mesenteric
margin margin
long artery
short artery
CGRP glanulomatous
Blood flow vasculitis
Lancet. 1989;2:1057-62.
No connection between the submucosal plexuses derived from short artery and
* long artery The association might well be explained in terms of granulomatous vasculitis
affecting small end-arteries that specifically supply the mesenteric margin
82. Is TU-100 an effective treatment for
Crohn’s disease?
Adelaide, Australia
83. Crohn’s disease model
• 2,4,6-TRINITROBENZE SULFONIC ACID (TNBS)-
INDUCED COLITIS:Th1-mediated response
Intestinal inflammation induced by intrarectal administration of hapten reagent
TNBS in ethanol solution. Simultaneous administration of TNBS and ethanol is
required to induce TNBS colitis, because ethanol disrupts the epithelial layer and
exposes the underlying Lamina propria to bacterial components.
Intestinal inflammation induced by intrarectal administration of TNBS
has many of the characteristic features of CD in humans
85. Experimental Schedule
Assessment of colonic damage was performed
on day 3 by macroscopic/microscopic observations
and cytokine contents.
Kono T, et al. J Crohn’s and Colitis 2010
86. TU-100 decreases mucosal cytokines
TNF and IFN in CD model
Concentrations of cytokines in protein extracts of the colonic mucosa were determined by ELISA.
N = 6. *, **: p<0.05, 0.01 versus colitis control, respectively.
Kono T, et al. J Crohn’s and Colitis 2010
87. TU-100 treatment protects against development of
TNBS-induced mucosal damage in colon
Representative photographs
Vehicle TU-100
(water) (900mg/kg)
Macroscopically visible damage was evaluated 3 days after TNBS instillation.
Mucosal damage was scored on a 0-8 scale.
TU-100 was given orally at 900 mg/kg. Clinical severity was monitored by mucosal damage score,
necrotic area of colonic mucosa. N=9 (Naive), 13 (colitis groups). *, **: p<0.05, 0.01 versus TNBS/Water
(colitis vehicle control), respectively.
Kono T, et al. J Crohn’s and Colitis 2010
88. TU-100 decreases
systemic inflammatory responses
Concentrations of cytokines in protein extracts of the colonic mucosa were determined by ELISA.
N = 6. (b) Serum amyloid A (SAA) concentration in plasma was determined by ELISA. n = 11.
SAA of naive and vehicle (50% EtOH) control mice were less than 0.001 mg/ml, respectively.
*, **: p<0.05, 0.01 versus colitis control, respectively.
Kono T, et al. J Crohn’s and Colitis 2010
89. Crohn’s disease model
• Oxazolone:Th2-polarized type colitis
6
Significant at P = 0.03
Score ( Max: 11 )
4
2
0
Water Water TJ-100
50%EtOH ir OXN ir ( Colitis )
90. ADM inhibits production of cytokines including
IFN and TNF by rat immune cells
γ α
Splenocytes
Concentration
Peritoneal macrophages
ADM addition ( μmol/L )
Splenocytes (upper) and peritoneal macrophages (lower) were isolated from normal SD rats, and cultured in presence of 1 µg/ml
LPS. ADM was added at final concentrations of 0.01, 0.1, or 1 µmol/L. N=3. *, **: p<0.05, 0.01 vs. LPS control, respectively.
91. ADM does not work like infliximab: it inhibits TNF production
infliximab binds TNF infliximab releases TNF
from the target cell membrane
infliximab
infliximab
infliximab kills TNF cell ADM inhibits TNF production
infliximab
Membrane binding TNF Membrane binding TNF
ADM
92. Antimicrobial effect of ADM
PAMP>ADM=Human b-defensin-2>human neutrophil peptide-1
Adrenomudullin (ADM) Proadrenomudullin N-terminal 20 peptide
(PAMP)
brush border membrane
Intestinal
TRPA1 Epithelial cell
O
N
OH Anti-TNF
Hydroxy- -sanshool
Anti-IFN-
O
O
vasodilatation
HO
6-Shogaol Microvascular Vessel
ADM
ADM, PAMP release
release
Enteric flora can be controlled by TU-100 via ADM
93. Conclusions
• TU-100 is an endogenous CGRP and
ADM enhancer via TRPA1 channel
• TU-100 improves intestinal blood flow
but is unlikely to be a direct vasodilator
• ADM inhibits TNF and IFN
production from immune cells
• ADM suppresses systemic
inflammatory responses (CRP)
• ADM exerts influence on enteric flora
• TU-100 ameliorates both Th-1 and Th-2
polarized type animal CD models
94. Strategy
• TU-100 may be a new type of medication for
Crohn’s disease
• Unlike infliximab, TU-100 is not for severe
conditions
• TU-100 is mainly effective in moderate or
mild or conditions
• TU-100 may help prolong remission periods
after infliximab-induced remission
• TU-100 may help decrease the frequency
and dosage of antibody treatment
Kono T, et al. Surgery 2009;146(5):837-840
95. This work
was performed
in collaboration
with
Dr Kaneko
Dr Omiya
Dr Koseki
Dr Suzuki
Dr Hira
Dr Watanabe
Dr Ebisawa
Dr Chisato
Dr Chiba
Polar bear is the main attraction Thank you for your
at Asahiyama Zoo in Asahikawa, attention
the most popular zoo in Japan
