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What is traditional Japanese Kampo medicine

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  • 1. International Surgical Week August 29, 2011 Yokohama, JapanWhat is traditional Japanese Kampo medicineAn Overview of Basic and Clinical Challenges Asahikawa Toru Kono Division of Gastroenterologic and General Surgery Department of Surgery, Asahikawa Medical UniversityTokyo Mt. Taisetu National Park, Asahikawa (Scenery from the window of my office)
  • 2. Herbal medicinal products in the world Autumn in Kyoto, Kinkakuji temple
  • 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 medicineApproved 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 packagesmillion 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-100Japanese Pepper Processed Ginger Ginseng RadixMix 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 HOThree-dimensional high-performance liquid chromatography of TU-100 Confidential data in TSUMURA Kono T. et al J Gastroenterology 2011 (in press)
  • 12. Absorption of TU-100 Tokyo Kanazawa castle, Japan
  • 13. Pharmacokinetics of TU-100 after single oral administration to healthy Japanese volunteers (A) Japanese pepper (B) Processed ginger (C) Ginseng radix 1000 10 1 6S 2.5g GRB1 2.5g HAS 2.5g 6S 5g GRB1 5g 100 HAS 5g GRB1 10g 6S 10g HAS 10g 1 10S 2.5g GRG1 2.5gPlasma consentration(ng/mL) Plasma consentration(ng/mL) Plasma consentration(ng/mL) 10S 5g GRG1 5g 10 HBS 2.5g 0.1 10S 10g GRG1 10g HBS 5g HBS 10g 1 0.1 0.1 0.01 0.01 0.01 0.001 0.001 0.001 0 4 8 12 16 20 24 28 32 36 40 0 0.5 1 1.5 2 2.5 3 3.5 4 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hr) Time (hr) Time (hr) -●- HAS -○- HSB -■- 6S -□- 10S -▲- GRB1 HAS: hydroxy α-sanshool 1400 1.4 4 HSB: hydroxy β-sanshool AUC0-last ( ng・hr/mL) AUC0-last ( ng・hr/mL) 3.5 AUC0-last ( ng・hr/mL) 1200 1.2 3 6S: 6-shogaol 1000 1 2.5 10S: 10-shogaol 800 0.8 2 GRB1: ginsenoside RB1 600 0.6 1.5 400 0.4 1 200 0.2 0.5 0 0 0 0 5 10 0 5 10 0 5 10 Dose ( g ) Dose ( g ) Dose ( g ) Munekage M. et al, Drug Metab Dispos. 2011( in press)
  • 14. TU-100Adverse Effects
  • 15. TU-100 reports of adverse effects from 1986 to 2006Only 46 cumulative cases during 20 yearsAnnual sales of > 9 million packagesAll AEs are not severe and patients have fully recovered • liver dysfunction • liver damage • jaundice • pneumonitis • pancytopenia • oral mucositis
  • 16. TU-100Main Clinical ApplicationsandMechanism
  • 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 ADMDifferent sites of production and expressionCGRP 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
  • 23. We investigated whether TU-100 hasany effect on intestinal blood flow viaCGRP and/or ADM
  • 24. TU-100 injection site Practical experiment scenery A tube was inserted via the cecum into the proximal colonTU-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)
  • 27. Effects of antagonists on TU-100-induced blood flow increase 0.28 0.24 0.20 0.16 Vehicle+TU-100 L-NAME+TU-100 0.12 CGRP1ra+TU-100 0.08 0.04 0.00 0 15 30 45 60 75 90 0.36 0.32 Vehicle+TU-100 0.28 VIPra+TU-100 0.24 SPra+TU-100 0.20 0.16 0.12 0.08 0.04 0.00 0 15 30 45 60 75 90
  • 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 wonderingif 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 120VC (%) 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 AfterThe 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 receptorblockers 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 GAPDHADM receptorADM/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) cellsCells TU-100 concentration (µg/ml)IE cells of small intestineIE cells of large intestineIntestinal epithelial (IE) cells were isolated from the small or large intestine of normalmice and stimulated with TU-100 (90, 270, or 900 µg/ml) at 1 x106 cells/ml in 96-wellround plates for 24 h. Concentrations (pg/ml) of adrenomedullin (ADM) in culturefluids were determined using the EIA method. N=4-6, *, **: p<0.05, 0.01 versus noTU-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-100Epithelial cell line of rat small intestine, IEC-6, was plated in 96-well flat plates at 1 x104 cells/well. The nextday, culture fluids were replaced with fresh medium containing test sample. Cells were allowed to incubatean 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 infigure. N=3. **: p<0.01 versus control (Dunnett test) Kono, J Crohn’s Colitis 2010
  • 45. Random test for active ingredients on ADM production Test sample Concentration Growth activity ADM concentration ( µmol/L ) ( OD 465-630 nm ) ( pg/ml ) Up or DownControl (0.3% DMSO) 1.419 ± 0.045 85.3 ± 4.9Processed ginger 6-gingerol 3 1.429 ± 0.016 99.7 ± 12.4 30 1.400 ± 0.025 93.0 ± 6.5 6-shogaol 3 1.504 ± 0.019 100.0 ± 9.3 30 1.363 ± 0.023 246.3 ± 5.0 ** UpGinseng radix ginsenoside Rb1 3 1.399 ± 0.014 98.0 ± 2.6 30 1.366 ± 0.047 90.7 ± 9.2 ginsenoside Rg1 3 1.393 ± 0.067 89.0 ± 5.1 30 1.375 ± 0.040 95.7 ± 3.8 ginsenoside Rd 3 1.379 ± 0.045 88.3 ± 5.4 30 1.386 ± 0.026 103.3 ± 12.3 protopanaxadiol 3 1.366 ± 0.032 87.7 ± 6.4 30 0.086 ± 0.001 * * 9.3 ± 4.7 ** DownJapanese pepper xanthoxylin 3 1.429 ± 0.030 89.3 ± 7.4 30 1.395 ± 0.012 99.0 ± 12.1 (-)-sinigrin 3 1.451 ± 0.007 76.7 ± 2.4 30 1.399 ± 0.004 80.7 ± 0.9 hydroxy-α-sanshool 3 1.449 ± 0.009 110.3 ± 12.5 30 1.557 ± 0.013 * 120.3 ± 8.8 * Up 100 1.493 ± 0.043 162.0 ± 1.7 * * UpMaltose powder maltose 30 1.484 ± 0.019 106.7 ± 11.0 300 1.467 ± 0.031 106.7 ± 11.7Growth activity was evaluated by XTT method. Medium alone: 0.085±0.015 in XTT method, < 10 pg/ml in ADM EIA method.*, **: p<0.05, 0.01 vs. Control, respectively. Kono, J Crohn’s Colitis 2010
  • 46. Mechanism of TU-100-induced hyperemia 6-shogaol, hydroxy- -sanshool Lumen How do they Microvascular stimulate? vesselNeuronal 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 ActivatorActivation of transient receptor potential A1 expressedin intestinal epithelial cell increases intestinal blood flowvia release of adrenomedullinInhibition of postoperative adhesion formation by Daikenchuto, a releaserof 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 channelFigure modified from David D. McKemy. Molecular Pain. 2005;1:16.
  • 50. Hypothesis TU-100 and TRP channelsPharmacological 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 agonistsMolecular biological profiles of hydroxy- -sanshoolMolecular biological profiles of 6-shogaol
  • 53. Do intestinal epithelial cells express TRPA1 and TRPV1 channels? 2010 DDW New Orleans, Hilton
  • 54. Intestinal epithelial cells express TRPA1 and ADMbut 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 theEpithelial body ? cell adrenomedullin Improvement of Microvascular Circulation Hydroxy sanshool Shogaol Gingerol
  • 61. TU-100 and Crohn’s disease Milano
  • 62. The number of Crohns 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 AsahikawaOnce 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 diseasepatients is decreased compared with that ofnormal intestine. Decrease of CGRP contributes to microvasculardysfunction 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 therapeuticeffects of CGRP in experimental CD models. ADM has been reported to have therapeuticeffects in experimental CD models as well.Can exogenous CGRP and ADM be candidatetherapeutic 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 intestineTherefore, it is desirable to develop an agent thatincreases endogenous CGRP and ADM such as TU-100
  • 71. TU-100 and Crohn’s diseaseOur hypothesis is that TU-100 acts as anendogenous CGRP and ADM enhancer andexerts therapeutic effects on CD via endogenousCGRP and ADM • Improves intestinal blood flow • Suppresses anti-inflammatory cytokine production • Anti-microbialSpecifically, ADM can be targeted instead of decreased-CGRPin 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 thenormal colon (0.10 ± 0.01). Changes of the VC induced by test sample are expressed as the mean ± S.E.M. (a) DKT (900mg/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 AfterHow 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 0.00In 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)
  • 75. TU-100-induced endogenous ADM can improve intestinal blood flowAnti-ADM antibody abolished TU-100’s effect 0.20 TU-100 + Rabbit IgG (N=6) TU-100 + Anti-ADM IgG (N=7) 0.15 † †† † ** * ** † † † * * VC 0.10 * 0.05 0.00 0 15 30 45 60 75 90 105 120 Time after administration of TU-100 (min)
  • 76. TU-100 directly induced blood flow increase in Crohn’s disease ( colon artificial anus) Thermographs before aftercolon artificial anus a b c skin temp 33 32 31 30 29 28 27 26 DKT sprinkled on the artificial anus 25
  • 77. Crohn’s disease and intestinal blood flowSlovenia
  • 78. Blood flow is a very important factorin pathogenesis of Crohn’s disease Ileal ulcers tend to occur along themesenteric 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 sideMesenteric side stenosis stenosis
  • 80. Who can answer the prepotency ofthe Crohn’s disease?Hypothesis:Primary pathological abnormalityin Crohn’s disease is in themesenteric 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 marginlong 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 responseIntestinal inflammation induced by intrarectal administration of hapten reagentTNBS in ethanol solution. Simultaneous administration of TNBS and ethanol isrequired to induce TNBS colitis, because ethanol disrupts the epithelial layer andexposes 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
  • 84. Hematoxylin and Eosin Massons Trichrome ControlTNBS-induced colitis
  • 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 modelConcentrations 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 responsesConcentrations 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 SplenocytesConcentration 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-TNFHydroxy- -sanshool Anti-IFN- OO vasodilatationHO 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 workwas performedin collaborationwithDr KanekoDr OmiyaDr KosekiDr SuzukiDr HiraDr WatanabeDr EbisawaDr ChisatoDr Chiba Polar bear is the main attraction Thank you for your at Asahiyama Zoo in Asahikawa, attention the most popular zoo in Japan