Treatment Metabolic

Plant Derived Therapeutics for the treatment Metabolic

SyndromeWilliam T. Cefalu, M.D.

Pennington Biomedical Research Center

Changing Nutrition/Health Paradigm

What food is required for me? Nutritional deficiency:

Iron - Anemia Iodine – Thyroid goiter Vitamin D - Rickets

Identifying essential nutrients required for promoting growth and sustaining life.

Vitamins Essential minerals – Calcium Essential amino acids

1920s-1980s


What does food do to me? Nutritional excess and imbalances

Calories - obesity Fiber – Colon cancer

Identifying nutrients and components that contribute to premature death

Saturated fat Cholesterol Sodium

1950s-1990s

Seven Countries Study: CHD Events areCorrelated with Saturated Fat

0 5 10 15 20% Calories from Saturated Fat

0

1

2

3

4

5

CHD

Death

s and

MI/1

00 R = 0.84

V

MC

DG

S W

BZ

UN

E

K

Keys, 1970


What does food do for me? Nutritional optimization of quality of life Identification of physiological active

components to prevent or delay premature onset of chronic disease

Phytochemicals Pre/Probiotics Fiber

1990s-Today!

Identification of Phytochemicals

Phytochemicals as Functional Components

Individual compounds in plants that have evolved in part as protective mechanisms against environmental insult

Phytochemicals with biological activity have had great utility as pharmaceuticals and pest-management agents.

Very few of these potentially active compounds have been examined thoroughly.

Functional Foods Definition

Generic description of foods, that when ingested, provide demonstrated physiological health benefits beyond simple nutritional value

Expanded Definition Similar in appearance to conventional foods;

Are consumed as part of a usual diet

How to Be a Functional Food A natural food product can be engineered to

become a functional food by Increasing specific components (Phytochemicals)

to reach a concentration more likely to express health benefits

Adding components not normally present but having a beneficial effect

Replacing a component that is excessive and harmful with one having a beneficial effect

Improving the bioavailability of components having desired health benefits

Phytochemicals and Reported Sources

Polyphenols Epicatechin Epigallocatechin Epigallocatechin

gallate

Source: Green tea; grapes, red wine Benefits: Anti-cancer; CHD protective Function: Inhibit chemical carcinogenesis and

tumor formation; inhibit cancer cell growth; antioxidant; reduces free radical/oxidative damage

Isoflavones Genistein Daidzein

Source: Soybean, flaxseed Benefits: Relieves menopausal symptoms;

prevents osteoporosis; anti-cancer; CHD protective

Function: Estrogen-like activity; inhibit growth of breast cancer cells; stimulate

Ca absorption; lower cholesterol levels

Genistein

Phytosterols -sitosterol Campesterol -sitostanol

Source: Plant oils Benefits: CHD protective Function: Inhibit cholesterol absorption

-sitosterol

Carotenoids Lycopene -carotene -cryptoxanthin Lutein

Source: Tomatoes, carrots, yams, cantaloupe, spinach, sweet potatoes; citrus

fruits Benefits: Anti-cancer; CHD protective Function: Antioxidant; free radical scavenger;

induction of cell-cell communication and growth control; inhibit tumor growth

-carotene

n3-Fatty Acids -linoleic acid Docosahexaenoic

acid Eicosapentaenoic

acid Source: Flaxseed oil, fish oil Benefits: CHD protective; anti-cancer; anti-

inflammatory Function: Lower triglycerides; inhibit platelet

aggregation; affect eicosanoid production

DHA

Flavonoids Quercetin Apigenin Luteolin Myricetin

Source: Citrus fruits; vegetables Benefits: Anti-cancer; CHD protective Function: Antioxidant; inhibit platelet aggregation;

inhibit cancer cell growth and proliferation; cytotoxic to

cancer cells

Luteolin

The Promise of Plant Therapeutics Better health through improved nutrition

can:Increase quality of lifeEnhance productivityReduce health-care costs - by preventing or delaying the onset of

chronic disease, i.e diabetes - or improving metabolic factors related to the

disease, i.e. glucose

Health claims need to be verified

with carefully controlled studies

O

Type 2 diabetes

Years from diagnosis

0 5-10 -5 10 15

Pre-diabetes

Onset Diagnosis

Insulin resistance

Post-Meal glucoseFasting glucose

“Nutraceutical (Botanical/Bioactive)Strategies”

“Pre-Diabetes”

Type 2 diabetes


0 5-10 -5 10 15

Pre-diabetes

Onset Diagnosis

Insulin secretion

Insulin “inefficiency

Ramlo-Halsted BA, Edelman SV. Prim Care. 1999;26:771-789 Nathan DM. N Engl J Med. 2002;347:1342-1349

Post-Meal glucose

Fasting glucose

Pancreas function

Progressive -Cell Failure

Natural History of Type 2 Diabetes

Rel

ativ

e R

isk

of M

I or S

trok

e

0

1

2

3

4

5

6

7

NondiabeticThroughout

2.4

>15 YrBefore Dx

10-14.9 YrBefore Dx

3.64

<10 YrBefore Dx

DiabeticThroughout

5.02

3.19

1.0

Hu FB, et al. Diabetes Care. 2002;25:1129-1134.

Non-Diabetic Diabetes

Cardiovascular Risk in Pre-diabetes

Pre-Diabetes

Type 2 diabetes


0 5-10 -5 10 15

Pre-diabetes

Onset Diagnosis

Insulin resistance

Ramlo-Halsted BA, Edelman SV. Prim Care. 1999;26:771-789 Nathan DM. N Engl J Med. 2002;347:1342-1349

Post-Meal glucose

Fasting glucose

Natural History of Type 2 Diabetes

“Nutraceutical (Botanical/Bioactive)Strategies”

Phytochemicals and Reported Sources

Blueberries as a Therapy for Type 2 Diabetes

• Literature Review– Blueberries are a folk remedy in Canada for

treatment of diabetes (Martineau LC et al., Phytomedicine, 2006)

– Blueberries have been found to:• Reduce blood glucose concentrations in rats and

humans (Abidov M et al, 2006 ; DeFuria J et al, 2009)

• Increase glucose uptake in muscle and fat cells (Tri Vuong et al, 2006)

• Protect against obesity in rats (DeFuria J et al, 2009)

PBRC Blueberry Research Study • Primary Objective

– To evaluate the effect of blueberry bioactives on improving a pathophysiologic parameter contributing to the development of type 2 diabetes in humans

• Hypothesis– Increased consumption of blueberry

bioactives will result in an increase in whole body insulin action, i.e. insulin sensitivity, in pre-diabetic individuals

Study Design

Baseline

Blueberry Group

Placebo (Control)

Screening

Insulin Sensitivity Test

1 2 3 4 5 6 7 8 9

End

End

Visits (Weeks)

*Randomized*Started consuming smoothies

Hyperinsulinemic Euglycemic Clamp

“Gold Standard” for objectively assessing whole body insulin action

Nutritional Value (per 16oz) Bioactives PlaceboEnergy, kcal 239 234Carbohydrate, g 48.5 48.6 Fiber, g 4.2 4.3Protein, g 11.9 11.1Fat, g 0.08 0.08 Saturated Fat, g 0.05 0.05

Each subject consumed two 16oz smoothies per day

16 oz Smoothie (Bioactives) =

Nutritional Value of Intervention

Placebo Smoothie

Stull AJ et al. J Nutr. 2010 Oct;140(10):1764-8.

The Ability of Insulin to Work in All ParticipantsFrom Beginning to End of Study

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

Subject Numbers

% ∆

Ins

ulin

Sen

sitiv

ity

Bioactives Group

Placebo

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Placebo (Control)Group

Bioactives Group

Individual Response in Insulin Sensitivity

Technical Variability

Blueberry Bioactives Improved Insulin Sensitivity

Blueberry Placebo 0

5

10

15

20

25 22.2

4.9

%∆

Insu

lin S

ensit

ivity

∆ insulin Sensitivity = ability of insulin to work from week 0 to week 6

4-Fold

Botanicals showing Promise to Favorably modulate Carbohydrate

Metabolism

• Bitter Melon ( Momordica charantia)

• Hoodia (Hoodia gordonii) • Artemisia dracunculas and related sp.

0 6 120.0

0.5

1.0

1.5

2.0LFD HFD BM

****

#

Insu

lin (n

g/m

l)

Weeks

0 6 120

50

100

150

200

250 LFD HFD BM

Glu

cose

(mg/

dl)

** #

Weeks

A

B

C

Figure 3

0 20 40 60 80 100 120100150200250300350400 HFD

BM

Glu

cose

(mg/

dl)

Time (min)

IPGTT***

******

***#

#

#

D

0 20 40 60 80 100 1200

50

100

150

200HFDBM

Glu

cose

(mg/

dl)

Time (min)

IPITT

# #

***

****** ***

***E

F

LFD HFD BM0

300060009000

1200015000

Plasma leptin concentration in mice(p

mol

/ml)

Groups

****** ##

0 6 120.00.20.40.60.81.01.2 LFD HFD BM

HO

MA

-IR

*

***

###***

Weeks

Bitter Melon and Glucose tolerance

IRS-2

IRS-1

-actin

Group LFD HFD BM

IR

GLUT4

PI 3K

Akt1

Akt2

Skeletal muscleIRS-1 p (Tyr612)

Akt1 p (ser473)

Insulin - + - + - +Group LFD HFD BM

IRS-1

Akt1

Akt2

Akt2 p (ser474)

Mouse # 1 2 3 4 8 9 7 11 13 15 16 18Insulin - + - + - + - + - + - +Group LFD HFD BM

0.0

0.5

1.0

1.5

2.0

2.5

LFD HFD BM

Basal

Insulin

Fold

of

LFD

at

basa

l

**

**

*

###

Bitter Melon and Glucose tolerance

Figure 4.

A

B

C14:2 C14:1 C14-OH C16 C18-OH0

5

10

15

20

25

LFD HFD BM

Acy

l ca

rniti

ne

(pm

ol/m

g pr

otei

n)

Long chain acyl carnitine in muscle tissues

*** #####***

###

C3 C4 C4-DC C6 C8:1 C12-OH0

5

10

15

20

25 LFD HFD BM

Acy

l ca

rniti

ne (pm

ol/m

g pr

otei

n)

Short/medium chain acyl carnitine in muscle tissues

#

***

###

####

***

*

*

Bitter Melon Modulates Intracellular lipids

Hoodia gordonii and Asclepias incarnata (Swamp milkweed) Belong to the

Same Plant Family

cellhealthmakeover.com abnativeplants.com

12-hydroxy (lineolon)

12-acetyl12-nicotinoyl

12-tigloyl

12-benzoyl

12-cinnamoyl

In collaboration with Analyticon

Natural Diversity of PregnanesIsolated from A. incarnata Extract

ikemagenin

An approach to the study of Phytochemicals and Human Health

Phytochemicals and Carbohydrate Metabolism

Alcoholic Extract of Artemisia dracunculus L.Russian Tarragon (PMI 5011)

Ribnicky DM, et al. Am J Physiol Endocrinol Metab. 2007

• Agriculture SOPs must be developed for each crop (Germination; Plant nutrition Pest management)

Sourcing and Standardization of Phytochemicals

Artemisia dracunculus in Hydroponics at ARC Greenhouses


• Optimization of activity and composition


Phytochemicals and Carbohydrate Metabolism

Flowering Artemisia dracunculus

LC-MS Comparison of PMI-5011 Extracts Made from Plants at Different Stages of Development

Flowering Stage

Growth/ Non-flowering Stage



• Identification of the active components for standardization (Bioactivity guided Fractionization)


5011

ADEX

ADEX

ADEX

Bioactivity GuidedFractionization

Identification and Isolation of Phytochemicals

BMI 29.5

0 4 6 8 12 hours Type 2 DM subjects:

BMI 31.1

BMI 36.8

PTP-1B

-actin

PTP-1B

-actin

PTP-1B

-actin

PTP1B Levels and Artermisa sp (5011) Human Skeletal Muscle

Time Course Studies: 5011

IRS-1

IRS-2

PTP 1B

IR

PI 3

-Actin

Fraction7 Contr 2 3 4 5 6 7 8 9 10 11 5011D

Akt-p

Insulin Signaling Parameters

Human Skeletal Muscle Culture5011 Fractions

Phosphatase

Wang ZQ et al. Metabolism. 2008 Jul;57(7 Suppl 1):S58-64.Ribnicky DM et al. Am J Clin Nutr. 2008;87(2):472S-5S.

Isolated Compounds ALR2 PTP-1B PEPCK

4,5-di-O-caffeoylquinic acid * ◊ ○ A - -

Davidigenin * ◊ † ‡ A - -

A - A

2′,4′-dihydroxy- 4- methoxydihydrochalcone * ◊ ○ † ‡ §

A A A

2,4-dihydroxy- 4- methoxydihydrochalcone * ◊ ○ † ‡

- A -

- A -

6-demethoxycapillarisin * ◊ ○

Sakuranetin ▪ ◊ ○

* - confirmed with NMR

◊ - new compound to A . dracunculus

○ - activity reported for the first timeA - active† - dihydrochalcone‡ - new compound to genus Artemisia§ - first report as a constituent of plants

▪ - flavonoidsALR2- Aldose reductase

PTP-1B - Protein tyrosine phosphatase - 1B

PEPCK - Phosphoenolpyruvate carboxykinase

Bioactives Isolated from Artemisia dracunculus and PMI-5011 by Activity-Guided Fractionation

*2′, 4′–dihydroxy-4-methoxydihydrochalcone

0

50

100

150

200

250

Labrasol 50 mg/kgchalcone

150 mg/kgchalcone

300 mg/kgchalcone

metformin 300mg/kg

bloo

d gl

ucos

e (m

g/dl

)

0 hr

6 hr

**

***

All treatments provided with 66% Labrasol

The Pure Active DMC-2* from Artermisia is Validated in vivo with Comparable Activity to Metformin



• Identification of the active components for standardization (Bioactivity Guided Fractionization)

• Standardized for Active Components




• Identification of the active components for standardization (Fingerprinting)

• Standardized for Active Components

• Stability Assessed


-20ºC freezer

22ºC open container

22ºC desiccator

37ºC open oven

Stability of the Extract

PMI-5011 is stable under various storage conditions

Stability was investigated over a period of 7 months by a validated HPLC method

HPLC-Chromatograms of PMI-5011, stored under different conditions


Assuring Supply of materials for research

• in vitro studies• Toxicology• in vivo preclinical • clinical

Animals Received

5011

Placebo

Intervention

Animals: KK-Ay

Intervention: 5011 Total Extract vs control conditionsEndpoint: Insulin and Glucose levels, Muscle signaling

Baseline

• Insulin Stimulation/Muscle Insulin Signaling• Serial Insulin/Glucose

Baseline

Study Design: In vivo Mechanism

Ribnicky DM, et al. Phytomedicine. 2006 Sep;13(8):550-7.

Body Weight/Food Intake

Plasma insulin

0 2 4 6 80

5

10

15

20

Control

5011

Weeks of Study

Insu

lin (p

g/m

l)

Mean + SEM, * P<0.05, ** P<0.01.

*** *

Plasma Insulin

Akt-p

Akt-1

Akt-2

AS160

Mice # 1 2 3 9 10 115011 - - - + + +

Glut4

-Actin

Control 5011

Insulin Signaling ParametersSkeletal Muscle

Bench to Bedside

Cells tested with Botanical

Human Testing in Several Phases

Animal Testing to MonitorSafety and Effectiveness

Screening

Screening

5011

Placebo

Intervention

Subjects: Obese, Insulin Resistant, n = 28Intervention: 5011 Total Extract vs PlaceboEndpoint: Insulin Sensitivity (Clamp Procedure)

• Hyperinsulinemic Euglycemic Clamps• Body Composition

Baseline

Baseline

Clinical Trial Study Design

456789

101112

Pre Post Pre Post

5011

4

6

8

10

5011Placebo

Insu

lin S

ensi

tivity

(mg/

min

/FFM

)

Placebo

BaselineEnd of Study

*

* P < .05

Clinical ResponseInsulin Sensitivity

Analysis of Compounds from PMI-5011 in

SIM for active compounds in PMI-5011

Standards 6-demethoxycapillarisin & davidigenin

Standards sakuranetin &2′, 4–dihydroxy-4′-methoxydihydrochalcone

SIM plasma analysis

SIM = Selected Ion Monitoring

285257

271285

Phytochemical CharacterizationIn Plasma by LC-MS-SIM

Time Course of Appearance

0

1

2

3

4

5

6

7

0 45 120 180 240

Minutes after Ingestion

Abu

ndan

ce (X

107 )

Plasma Appearance of Phytochemical

Mean Levels over 4 hours

0

1

2

3

4

5

6

7

5011 Placebo

Ion 257: davidigenin Ion 271 sakuranetin Ion 285: chalcone

Undetectable

Abu

ndan

ce (x

107 )

Plasma Abundance5011 Metabolites

Potential Nutraceutical (Phytochemical) Markets

1. Joint health(n3-fatty acids, glucosamine, chondroitin sulfate)

2. Gastrointestinal health(ginger, pepermint, fennel, prebiotics, probiotics)

3. Blood lipids(n3-fatty acids, oat bran, phytosterols)

4. Bone density and skeletal health(phytoestrogens, Ca, Zn)

5. Hormone replacement(isoflavones, arginine, yohimbe)

6. Body fat/Weight(herbal phen-fen, Cr, garcinia cambogia)

7. Optimal vision(lutein, zeaxanthin)

8. Stress and insomnia(St. John’s Wort, tryptophan)

9. Breast and prostate health(fruits, vegetables, saw palmetto)

10. Carbohydrate Metabolism(Chalcones, Cinnamon polyphenols)

Potential Nutraceutical (Phytochemical) Markets

The most studied Phytochemical:Wine

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 <0.1 0.1 - 0.3 0.3 - 1 1.0 - 2.0 >2.0

Drinks per Day

RelativeRisk

CVD

Non-CVD

Over 40 prospective studies have documented an inverse relationship between alcohol intake and heart disease

prevention

We Can’t Put Our Heads in the Sand any Longer !!!

Regarding Plant Therapeutics and Human Health…..