Combining modern science and traditional medicine in the fight against Aboriginal type 2 diabetes: The experience of the CIHR Team in Aboriginal Anti-diabetic Medicines

Combining modern science and traditional medicine in the fight against Aboriginal type 2 diabetes: The experience of the CIHR Team in Aboriginal Anti-diabetic Medicines Pierre S. Haddad PhD Department of Pharmacology Université de Montréal http://www.taam-emaad.umontreal.ca NAHO 2011 Speaker Series, Ottawa, February 23, 2011 This talk is dedicated to the memory of Elders Sam Awashish, René Coon Come, Smally Petawabano and Sally Matthews

1) Introduction
Type 2 diabetes
Traditional medicine and diabetes
2) Boreal forest plants
CIHR Team in Aboriginal Antidiabetic Medicines
Project flow diagram
Ethnobotany
Phytochemistry
Pharmacology & Toxicology
Initial screening using in vitro bioassays
In vivo validation
Mechanisms of action
Case of W7 & AD01
Clinical studies
Ethics
4) Concluding remarks
5) Acknowledgements
Presentation outline

PREVALENCE OF DIABETES WHO

Acquired Diet, physical inactivity, Increased FFA, aging, glucotoxicity Decreased Insulin sensitivity Deficiency of  cells TYPE 2 DIABETES Type 2 diabetes (dysregulated high blood sugar) Genetic IR Mutations, anti-insulin antibodies, GLUT4 mutations Compensatory hyperinsulinemia (Insulin resistance) Postprandial hyperglycermia Type 2 diabetes (symptoms)

How is blood sugar controlled? TZD Metformin Skeletal Muscle Activation of AMPK Insulin Energy depletion Activation of Akt Glucose uptake GLUT4 expression & translocation Activation of Akt Fat Inhibition of AMPK Expression of C/EBP-  & PPAR  Activation of C/EBP-  Differentiation of adipocytes Accumulation of fat Hyperglycemia ACC activity Liver Activation of AMPK Activation of Akt Glucose production SREPB-1 expression Gene expression of lipogenic enzymes Fatty acid oxidation Fatty liver Hepatic insulin sensitivity Fatty acid synthesis

According to WHO:
Obesity/T2D = worldwide epidemic
Traditional medicines and NHP = First line healthcare for 75% of the world population today
800-1200 antidiabetic NHP are identified worldwide (e.g. fenugreek, bitter gourd, nopal, ginseng)
However…
Little evidence-based data available (although 80% of tested NHP possess promising biological activities)
Problems = product quality : i.e. botanical ID , source, preparation, dosage
Our work:
Mediterranean Nigella ( Nigella sativa )
Moroccan Argan oil ( Argania spinosa )
Canadian low bush blueberry ( Vaccinium angustifolium )
Cashew seed ( Anacardium occidentale )
Boreal forest plants (Cree Traditional Medicine)

According to WHO:
Obesity/T2D = worldwide epidemic
Traditional medicines and NHP = First line healthcare for 75% of the world population today
800-1200 antidiabetic NHP are identified worldwide (e.g. fenugreek, bitter gourd, nopal, ginseng)
However…
Little evidence-based data (although 80% of tested NHP possess promising biological activities)
Problems = product quality : i.e. botanical ID , source, preparation, dosage
Our work:
Mediterranean Nigella ( Nigella sativa )
Moroccan Argan oil ( Argania spinosa )
Canadian low bush blueberry ( Vaccinium angustifolium )
Cashew seed ( Anacardium occidentale )
Boreal forest plants (Cree Traditional Medicine)
Diabetes more pronounced in aboriginal populations

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of W7 & AD01
Clinical studies
Ethics
5) Acknowledgements

Population
15,000 Eeyouch (Cree)
9 communities
4 communities involved
in research project
2 communities in the process of ratifying the Research Agreement
The People & their territory: The Eeyouch (Cree) of Eeyou Istchee Legaré et al , 2004 – Project of Diabetes Surveillance amongst the Cree of Eeyou Istchee

Two problems addressed by the research project
The near abandonment of Cree healing practices and practitioners from the 1950s or so.
2) The sudden rise of the diabetes ‘epidemic’ from the 1990s.

Problem 1: The near abandonment of Eeyou medicine
Allopathic medical care arrived gradually in the region from the 1930s. By the 1960s, all existing communities had a nursing station or clinic.
Cree Board of Health & Social Services of James Bay became responsible for all services after 1978.
No official recognition of any type of Eeyou (Cree) medicine or healing practices until very recently.

Problem 2: Diabetes in northern Quebec Cree community Years Modified from Cree Board of Health and Social Services of James Bay - CBHSSJB (2005) Crude prevalence of T2D in Eeyou Istchee area 21.6% 7.6% 25.5% 7.0% 17.7% 8.1% Kuzmina E, Lejeune P, Dannenbaum D, Torrie J. 2010. CREE Diabetes Information System CDIS): 2009 Annual Update. C hisasibi, Québec: Cree Board of Health and Social Services of James Bay 2009

Explore Boreal forest plants traditionally used by the Cree to target symptoms related to diabetes
Goal: identify anti-diabetic plants which can be used by the community to help treat diabetes
How? - Collaborative Multidisciplinary Approach
Cree Traditional Knowledge
Ethnobotany
Phytochemistry
Pharmacology
Nutrition
Endocrinology
CREE RESEARCH PROJECT

Cree Nations Traditional Knowledge Medicinal plants CIHR Team in Aboriginal Antidiabetic Medicines (2003-2006) 2006-2011 Nutrition (Johns) Ethnobotany (Cuerrier) Phytochemistry (Arnason) Clinical/ Observational Trials (Chiasson, Yale) Education/ Integration (CBHSSJB) Pharmacology (Haddad, Prentki, Musallam, Bennett, Sirois) Toxicology (Foster)

Ethnobotanical study (4 communities) Collection of 17 species (potentially antidiabetic) Preparation of the extracts In vitro assays In vivo validation Isolation and identification of active principles Clinical studies Mechanisms of action Standardized plant extracts Quality controlled traditional preparations standardization Bioguided fractionation Toxicology All the species The most active species The active species PROJECT FLOW DIAGRAM

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of W7 & AD01
Clinical studies
Ethics
5) Acknowledgements

Population
15 000 Cree
9 communities
A total of 109 Cree Elders interviewed (4 communities; 2003/2004/2008)
Interviews based on 15 symptoms associated with T2D.
Species cited were ranked according to:
Frequency of citation
Number of symptoms
Quality of symptoms
Ethnobotany (Cuerrier Lab: Montreal Botanical Garden) James Kawapit Legaré et al, 2004 – Project of Diabetes Surveillance amongst the Cree of Eeyou Istchee

Ethnobotany results in Mistissini Leduc et al., J. Ethnopharmacol. 2006; 105(1-2):55-63

Larix laracenia Tamarack Watnagan (Inner bark) Picea mariana Black spruce Inaahtkw (Cones) Alnus incana Speckled alder Atushpi (Inner bark) Rhododendron groenlandicum Labrador Tea Kachichepukw (Leaves) Pinus banksiana Jack pine Ushchishk (Cones) Abies balsamea Balsam fir Inaasht (Inner bark) Sarracenia purpurea Pitcher plant Ayigadash (Whole plant) Sorbus decora Mountain ash Muskuannanatuk (Inner bark) Leduc et al., J. Ethnopharmacol. 2006; 105(1-2):55-63 Top 8 Mistissini plants screened

Other Botanical Studies
IMPACT ASSESSMENT STUDIES:
Evaluate the impact of different collecting intensities (e.g. 0, 20%, 50%, 80%) on a given medicinal plant species.
Evaluate the impact of collecting at various stages in plant growth.
Inform sustainable management of the resource.
GEOGRAPHICAL GRADIENTS:
Evaluate the variation in phytochemical content and biological activity along North-South (latitudinal) anD inland-coastal gradients for a given medicinal plant species
Discussions with Elders on impact of environmental changes

Phytochemistry (Arnason Lab: University of Ottawa)
Preparation of plant extracts
Classic ethanolic extract of ground material prepared for screening assays
Hot water extracts adjusted to better reproduce traditional preparations
Characterization of plant extracts
Many challenges: No methods No reference standards Phytochemical markers often unknown No active principles identified
Isolation and identification of active compounds
Metabolomics approach for known compounds (mostly phenolics)
Classical IR, NMR and MS for new compounds

1 Yield is expressed as (mass of recovered extract / mass dry plant material) x 100% 2 Total phenolics expressed as quercetin equivalents (μg) / mg extract Spoor et al., Can. J. Physiol. Pharmacol. 84:847-858, 2006 Phytochemistry (Arnason Lab: University of Ottawa) Total phenolics and identified marker compounds of each plant extract Plants % Yield 1 Total Phenolics (μg/mg) 2 Identified Phenolic Marker Compounds A. balsamea 15.3 % 97.6 p-coumaric acid, gallocatechin A. incana 26.1 % 305.9 catechins L. laricina 23.8 % 208.0 taxifolin, hydroxystilbenes P. mariana 21.0 % 163.7 p-coumaric acid, hydroxystilbenes P. banksiana 9.0 % 318.0 taxifolin, catechin, procyanidins R. groenlandicum 31.0 % 188.5 chlorogenic acid, catechins, procyanidins, quercetin glycosides S. purpurea 25.2 % 85.4 taxifolin, flavonol glycosides (quercetin, kaempferol, myricitin) S. decora 8.9 % 59.6 quercetin and quercetin glycosides

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of W7 & AD01
Clinical studies
Ethics
5) Acknowledgements

In vitro Pharmacology – in vitro (Haddad & Prentki Labs: University of Montreal) Glucose absorption Glucose production Glucose uptake Glitazone-like activity Insulin Secretion Primary anti-diabetic activity (glycemia-lowering activities) Glucose uptake Adipokines secretion Pancreas Liver Skeletal Muscle Fat Intestines FRACTIONATION

Mechanisms of action Skeletal Muscle Activation of AMPK Insulin Energy depletion Activation of Akt Glucose uptake GLUT4 expression & translocation Activation of Akt Fat Inhibition of AMPK Expression of C/EBP-  & PPAR  Activation of C/EBP-  Differentiation of adipocytes Accumulation of fat Hyperglycemia ACC activity Liver Activation of AMPK Activation of Akt Glucose production SREPB-1 expression Gene expression of lipogenic enzymes Fatty acid oxidation Fatty liver Hepatic insulin sensitivity Fatty acid synthesis

In vitro Pharmacology-Toxicology (Johns, Foster, Arnason & Bennett: McGill, University of Ottawa) CYP450 Pro/anti- Inflammation Neuroprotection against low & high glucose P-glycoprotein transport Toxicology (potential Herb-Drug interaction) Secondary anti-diabetic activity (protection against diabetic complications) Cardiomyocyte electrotoxicity Anti-oxidant activity Anti-glycation activity Heart Liver Brain Intestines Blood Cell-free assays

Pharmacology – in vivo (Haddad Lab: University of Montreal)

Novel ethnobotanical approach established & validated
Out of the 17 plants:
None : stimulate insulin secretion
10 : potentiate glucose transport to some degree (short or long term; muscle or fat)
8 : enhance adipocyte differentiation (a few confirmed PPARg agonists)
10 : cytoprotective in pre-neuronal cells; 8 in endothelial cells
9 : potent anti-oxidants
7 : strongly inhibit intestinal glucose transport
6 : anti-inflammatory activity; 8 show pro-inflammatory activity
12 : prevent AGE formation
most exhibit a low to moderate inhibition of human CYPs
6 plants confirmed anti-hyperglycemic in vivo ; 1 plant confirmed anti-obesity in vivo
Several novel phenolic active compounds isolated by bioassay-guided fractionation and identified by NMR and MS
Mechanisms of action: similar to Metformin (uncoupling and/or inhibition of mitochondrial respiration; AMPK activation)
SUMMARY OF DATA TO DATE

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of AD01
Clinical studies
Ethics
5) Acknowledgements

How is blood sugar controlled? Skeletal Muscle Activation of AMPK Insulin Energy depletion Activation of Akt Glucose uptake GLUT4 expression & translocation Activation of Akt Fat Inhibition of AMPK Expression of C/EBP-  & PPAR  Activation of C/EBP-  Differentiation of adipocytes Accumulation of fat Hyperglycemia ACC activity Liver Activation of AMPK Activation of Akt Glucose production SREPB-1 expression Gene expression of lipogenic enzymes Fatty acid oxidation Fatty liver Hepatic insulin sensitivity Fatty acid synthesis

9 -10 of the 17 Cree anti-diabetic plants decrease hepatic glucose output by different mechanisms 9 plants significantly decrease G-6Pase Nachar, Haddad et al., unpublished % activation of GS 10 plants significantly activate GS % inhibition of G-6Pase Hepatic glucose production Expression of G-6Pase & PEPCK Activity of G-6Pase Hepatic glucose storage AMPK Akt Activity of GSK-3  Activity of GS Hepatic glucose output

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of AD01
Clinical studies
Ethics
5) Acknowledgements

MODE OF ACTION OF BOREAL PLANTS ? Altered Mitochondrial Respiration

Mechanism of action of Boreal plants: Insulin-dependent (p-Akt) and -independent (p-AMPK  and p-ACC) activities Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

Mechanism of action of Boreal plants: Insulin-dependent (p-Akt) and -independent (p-AMPK  , p-ACC) activities Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

Effect of Abies balsamifera on mitochondrial respiration Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

Effect of plant extracts on mitochondrial respiration: Uncoupling Inhibiting Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

L6-wt myotubes L6-myc myoblasts Glut-4 Translocation Assay Eid, Sweeney, Haddad et al., unpublished Insulin Plant Z Insulin Plant Z α-Glut4 α-myc (myc)

WORKING HYPOTHESIS FOR BOREAL PLANTS : Altered Mitochondrial Respiration ??

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of W7 & AD01
Clinical studies
5) Acknowledgements

Screening for “ glitazone-like ” activity (Adipogenesis) Harbilas et al. Can. J. Physiol. Pharmacol. 2009 Boreal Plants from Cree Pharmacopeia W7 AD01

Body Weight Food Intake *AD02 250 mg/kg and W7 125 and 250 mg/kg significantly prevent body weight gain compared to the control DIO group between day 20 and 60 of treatment Effects of AD02 & W7 on prevention of DIO AD02 125 mg/kg AD02 250 mg/kg W7 125 mg/kg W7 250 mg/kg AD02 125 mg/kg AD02 250 mg/kg W7 125 mg/kg W7 250 mg/kg Harbilas, Haddad et al., unpublished * Food intake significantly lower in animals treated with W7 250 mg/kg compared to DIO control group between day 30 and 60 of treatment

Body Weight Food Intake W7 W7 Harbilas, Haddad et al., unpublished Effects of W7 on DIO treatment W7 W7

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of W7 & AD01
Clinical studies
Ethics
5) Acknowledgements

Neuroprotection against low/ high glucose In vitro Anti-Inflammatory properties Inhibitor of ATP synthase 3 compounds Insulin secretion (at high glucose) AD01 Ouchfoun, Haddad et al., unpublished Harbilas et al., CJPP 2009 Martineau, Spoor et al., JEP 2010 Nistor et al., JEP 2010 Sugar absorption (10’) FRACTIONATION Sugar production Fat production Pancreas Liver Skeletal Muscle Fat Brain Intestines Blood Sugar uptake (1h) Sugar uptake (18h) Sugar uptake (1 & 18h)

Fractionation guided by stimulation of adipogenesis Fractionation : J-A Guerrero (Arnason) Bioassay : M Ouchfoun (Haddad) Leaves of AD01 Compound 1 Compound 2 Compound 3 Ouchfoun, Haddad et al., unpublished

Fractionation guided by stimulation of adipogenesis Compound 1 Compound 2 Compound 3

AD01 in DIO model

Weight (~6%) In vivo Glucose (~9 %) AD01 ( Ethanolic Extract) Fat hormons (~10%) Brains (Analysed by Bennett) Ouchfoun, Haddad et al., unpublished Whole Body Pancreas Blood Weight (~14%) Abdominal (~14%) Brown (~7%) Lipid content (~ 42 %) Insulin (~65%) Liver Fat Skeletal Muscle Brain

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of AD01
Clinical studies
Ethics
5) Acknowledgements

Pilot # 1 Original clinical Trial proposed Pilot # 2

« Putting Traditional Medicine First » Current all-inclusive observational studies 1. Inclusion criteria Pre-diabetic or Type 2 diabetes with duration  5 years since diagnosis Already treated with herbal remedies for diabetes or wishes to start de-novo treatment with herbal remedies Male or female 18 years or older HbA1C between <10% 2. Exclusion criteria Type 1 diabetes Type 2 diabetes patients requiring insulin therapy Type 2 diabetes >5 years duration? Renal disease with overt proteinuria (ACR > 30 mg/mmol) and/or eGFR < 60 ml/m Liver disease with ALT 3 times above upper limit of normal Unstable coronary artery disease (CAD) or uncontrolled hypertension Uncontrolled hypothyroidism Alcohol or drug abuse Pregnancy or lactation Any major illnesses Any medication that can impair glucose metabolism (i.e. glucocorticoids) Unable to sign voluntary consent 1. Efficacy HbA1C and Fructosamine Fasting plasma glucose 75 gram 2 hr post OGTT (pre-diabetic only) Fasting insulin level Capillary blood glucose profile Blood biochemistry/ Lipid/ Inflammatory profile Blood pressure Morphometric measurements 2. Safety Urea, creatinine, eGFR, and electrolytes Liver function tests (ALT, AST, GGT, bilirubin, and alkaline phosphatase, INR) Complete blood count ECG Urine analysis, ACR

1) Introduction
Type 2 diabetes
Ethnobotany
Phytochemistry
In vivo validation
Case of AD01
Clinical studies
Ethics
5) Acknowledgements

Weak legal protection of Indigenous Knowledge in Canada
Ratified Convention on Biodiversity in 1992
However, no legislation to protect IK
Intellectual property laws not well adapted
Funding agencies’ policies exist since 2008
Thus, Best Approach =
Agreements to govern transfer and use of IK

Specific concerns of Elders, communities, Grand Council of the Cree and Cree Board of Health concerning this project
Safety issues in using traditional medicines for diabetes care
Use of Eeyou knowledge without consent
Ownership of intellectual property
Question of partnership between Elders and Researchers
Misuse of medicines by others (health concern)

History of Agreement
Project started before the agreement was signed (2003)
Took 5 years of negotiations for Interim Agreement (2008)
One other year for Final Agreement (2009)
Led to better agreement
All parties acted as if agreement was in force
Copy of the Research Agreement is available on our website:
http://www.taam-emaad.umontreal.ca/about%20us/agreement.html
(Lay language summary available in French, English and Cree (Inland &Coastal)
Paper copies also available upon request

Eeyou anti-diabetic plants Research Agreement
Parties:
4 Cree (Eeyou) communities, (with 2 additional)
Cree Board of Health & Social Services of James Bay
3 Universities (Montréal, McGill, Ottawa)
1 university hospital (CHUM)

Important principles of agreement
Confidentiality and Eeyou control over TK
Review of publications
Collaborative research
Joint ownership of intellectual property
Benefit-sharing

Our in vitro bioassays are good tools to screen for anti-obesity and anti-diabetic NHP stemming from traditional medicines
CIHR-TAAM: not a drug discovery program
Ethics in Aboriginal Health Research
Respect for Cree culture and knowledge
Transparency about the project with Cree communities
Sharing ideas and knowledge (Central role of Elders)
Steering committee = Cree + Academics
Protection of Aboriginal Traditional Knowledge and related Intellectual Property
Knowledge translation :Traditional medicine  scientific language
ID of active compounds is for quality control and standardization of preparations
Use evidence-based approach to favor inclusion of traditional botanical preparations into Cree diabetes care
consistency, clinical efficacy, safety, herb-drug interaction, active ingredients and site of action
Concluding Remarks

ACKNOWLEDGEMENTS & TEAM CIHR-TAAM Annual retreat at Mistissini (2010) Haddad lab

MEGWETCH!!

AD01 strongly stimulated adipogenesis AD01

AD01 activates Insulin pathway in the skeletal muscle in vivo pAkt pAMPK pACC GLUT4 AMPK Akt GLUT4 expression & translocation Glucose uptake Ouchfoun, Haddad et al., unpublished

AD01 activates Insulin pathway and AMPK pathway in the liver in vivo pAkt pAMPK p-IKK  SREBP-1 Sugar production Akt Ouchfoun, Haddad et al., unpublished Hepatic insulin sensitivity Fatty acid oxidation AMPK Expression of SREBP-1 Fatty liver

AD01 tends to stimulate the adipogenic program in the abdominal fat in vivo p = 0.07 PPAR  C/EBP  C/EBP  Ouchfoun, Haddad et al., unpublished Adipogenesis Expression of C/EBP  Fat accumulation Expression of PPAR  Insulin sensitivity

Insulin actions and insulin resistance

Combining modern science and traditional medicine in the fight against Aboriginal type 2 diabetes: The experience of the CIHR Team in Aboriginal Anti-diabetic Medicines

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Combining modern science and traditional medicine in the fight against Aboriginal type 2 diabetes: The experience of the CIHR Team in Aboriginal Anti-diabetic Medicines Presentation Transcript

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