Cardioviva Benefits

11/7/2013 CONFIDENTIAL 1

Mitchell Jones MD, PhD, MEng,

McGill University and

Micropharma Limited

The Human Body is a Community of Microbes

• Only 10% cells actually human in origin

• Remaining 90% are microorganisms

• > 1014 microbial cells and > 1015 viruses

• “Microbiota”

• Full complement of microbes in and on our bodies

• “Microbiome”

• Ecological community of microorganisms that share our body space and includes collective genomes

90of cells in the human body are

%our body space and includes collective genomes

• “Great plate count anomaly”

• In 1985 researchers demonstrated that cultivated bacteria represent small fraction of the microbes present in the environment

• Culture-independent high-throughput sequencing

• Greatly expanded repertoire of known microbes

• This has allowed for study of microbial:

• Structure and dynamics

• Relationships

• Substances produced and consumed

• Interaction with host

• Differences in health and disease

90of cells in the human body are

microorganisms

The Microbiome and Human Health

• “Forgotten organ”

• Microbiome plays major role in health

• Exposed surfaces are colonized

• skin

• respiratory system

• urogenital tract

• gastrointestinal tract

• Majority of microbes in the gut• Majority of microbes in the gut

• The known roles of gastrointestinal microbiome include:

• Metabolic• Absorbing indigestible carbohydrates, metabolizing

bile

• Synthesis• Production of vitamins K and B

• Immune• Promotion, maturation and development of innate

and cell mediated immune functions

• Maintenance of appropriate immune response to pathogens

• Maintenance of the intestinal barrier function

Microbiome Effects Our Metabolic Health“A disrupted microbiome has been associated

with a lengthening list of problems: obesity and

its opposite, malnutrition; diabetes (both type-1

and type-2); atherosclerosis and heart disease…”

- The Economist

“Scientists say a recent study suggests infants

treated with antibiotics in the first 6 months of

their life may predispose them to be overweight


their life may predispose them to be overweight

by the age of three… the earlier the exposure to

antibiotics, the more permanent the effect on

microbes in the intestine.”

- CTV News re: International Journal of Obesity

publication

“Oddly enough, the whole community starts to

resemble the microbes of someone with

metabolic syndrome – a collection of symptoms

that increase the risk of diabetes and heart

disease…”

- Discover Magazine re: Cell publication

Atherosclerosis and the Microbiome

• Oral and gut bacteria contribute to development of atherosclerosis and CVD

• Atherosclerotic plaque:

• Distinct microbiome

• “Core” signature species

• Chryseomonas (P. luteola) identified in all plaques

• Veillonella and Streptococcus in majority

• Bacterial DNA

• Correlated with leukocytes

Suggests plaque bacterial load determines • Suggests plaque bacterial load determines inflammatory status and stability

• Mechanism:

• Phagocytosis by macrophages at epithelial linings

• Reach activated endothelium of atheroma

• Leave blood stream to enter atheroma

• Transform into cholesterol-laden foam cell

• Evidence:

• Atherosclerosis-prone mice deficient in TLR2, TLR4 or MyD88 are resistant to dev. of atherosclerosis

• Gut microbiota also contributes to atherosclerotic disease through host lipid metabolism involving:

• Cholesterol catabolism and excretion

• Cholesterol absorption

• Bile metabolite associated inflammation


Microbiome, Cholesterol and Bile Acids

• Germfree animals

• Catabolize cholesterol more slowly

• Accumulate cholesterol in greater quantities,

faster rate

• Intestinal microflora responsible for accelerating

cholesterol catabolism

• Elevated levels of conjugated BA throughout • Elevated levels of conjugated BA throughout

intestine

• Significantly reduced fecal excretion

• Elevated serum cholesterol

• Mice treated with oral Abx show:

• Increased biliary BA output

• Decreased fecal bile output

• Increased serum cholesterol

• Thus, the deconjugation function of the

intestinal microbiome, results in

• Increased levels of circulating unconjugated bile

• Maintenance of normal levels of cholesterol


Microbiome Regulates Bile Metabolism

• Bile acid synthesis under negative control through bile acid nuclear receptor (FXR)

• Germ free (GF) animals have:

• Lower unconjugated and total BA

• Lower fecal excretion of bile

Reduced BA diversity• Reduced BA diversity

• BA diversity is lower in GF and antibiotic-treated tissues compared with conventional animals

• Bile acid receptors (TGR5, FXR, VDR, PXR) activation by:

• Unconjugated BA (CDCA, CA)

• Secondary BAs (DCA, LCA)

• Thus GF animals have little bile acid receptor agonist activity


Bile Acid Dysmetabolism and Heart Disease

• Cholesterol eliminated from the body by hepatic catabolism of cholesterol into bile acids

• Coronary Artery Disease (CAD) patients:• Significantly decreased bile

acid excretion levels than non-acid excretion levels than non-CAD patients

• Additional risk factor for CAD development

• Interestingly, studies of responders and non-responders to statin therapy have shown:• Metabolism of BAs by the gut

microbiome is associated with a response to statin therapy


Design of First Randomized Controlled Trial

Objective: To evaluate the cholesterol lowering efficacy and safety of a L. reuteri NCIMB 30242 yogurt formulation in mildly hypercholesterolemic adults.

Design: Randomized, double blind, placebo controlled, parallel arm, multi-center trial

Investigational Product: 1 x 1010 CFU BID in yogurt format

Duration: 6 week interventionDuration: 6 week intervention

ITT: N 114 subjects (n 58 placebo, n 56 treatment)

Primary endpoint: LDL-C at 6 week endpoint relative to placebo

Secondary endpoints: LDL-C, TC, apoB-100, non-HDL-C, HDL-C, TG, biochemistry, hematology, fecal microflora, fecal deconjugated bile

Additional endpoints: Demographic data, diet journal data, GI questionnaire data

ClinicalTrials.gov Identifier: NCT01185795


Jones et al. Br J Nutr. 2012

L. reuteri NCIMB 30242 Lowers LDL-C 8.9%

Over 6 Weeks• LDL-C: -8.92% (P=0.02)*

• TC: -4.81% (P=0.03)*

• apoB-100: -6.81% (P=0.046)*

• non-HDL-C: -6.01% (P=0.025)*

• No change in TG and HDL-C*

• Balanced diet income between


• Balanced diet income between treatment groups**

• No change in fecal bile acids*

• No adverse events

• No change in safety parameters that considered associated with treatment or clinically significant

*ANCOVA/Kruskal-Wallis

**ANOVA/Kruskal-Wallis

Jones et al. Br J Nutr. 2012

Design of Second Randomized Controlled Trial

Objective: To evaluate the cholesterol lowering efficacy and safety of a L. reuteri NCIMB 30242 capsule formulation in mildly hypercholesterolemic adults over 9 weeks.

Design: Randomized, double blind, placebo controlled, parallel arm, multi-center trial

Investigational Product: 2.9 x 109 CFU (100mg) BID in capsule formatBID in capsule format

Duration: 9 week intervention

ITT: N 127 subjects

Primary endpoint: LDL-C at 9 week endpoint relative to placebo

Secondary endpoints: LDL-C, TC, apoB100, apoA1, non-HDL-C, HDL-C, TG, hs-CRP, fibrinogen, free and esterified cholesterol, bile acid profile, plant sterols, biochemistry, hematology

Additional endpoints:

Demographic data, diet journal data, GI questionnaire data

ClinicalTrials.gov Identifier: NCT01341613

11/7/2013 CONFIDENTIAL11

Jones et al. Eur J of Clin Nut. 2012

L. reuteri NCIMB 30242 Lowers LDL-C 11.6%

Over 9 Weeks

• L. reuteri NCIMB 30242

resulted in significant

reductions in LDL-C of

11.64%, TC of 9.14%,

non-HDL-C of 11.30%


non-HDL-C of 11.30%

and ApoB-100 of

8.41% at the 9 week

endpoint compared to

placebo.

• Results were significant

even at 6 weeks when

compared to placebo .


ANCOVA or Mann Whitney U; *P<0.05, **P<0.01, ***P<0.001

L. reuteri NCIMB 30242 Improves CVD Risk

• L. reuteri NCIMB

30242 resulted in

significantly improved

lipid ratios at the 6

week point and 9

week endpoint and

significantly reduced

Placebo (n 61) L. reuteri (n 66)

Mean (SD) Mean (SD) P

LDL-C/HDL-C

AbsΔ 0.21 (1.16) -0.22 (0.88) 0.005b

RelΔ (%) 8.91 (33.37) -4.48 (21.24) 0.006b

apoB-100/apoA-1

AbsΔ -0.02 (0.19) -0.09 (0.20) 0.034b

RelΔ (%) -0.74 (22.82) -9.74 (17.75) 0.026a

Fibrinogen

Table. Changes in lipid and apolipoprotein ratios and cardiovascular risk parameters



CVD risk factors

including hs-CRP and

fibrinogen compared

to placebo at the 9

week endpoint.

Fibrinogen

AbsΔ (g/l) 0.48 (0.87) -0.09 (0.96) 0.004b

RelΔ (%) 17.19 (35.67) 2.95 (26.71) 0.004b

hs-CRP (mg/l) *

Week 0 1.63 (0.90-3.25) 2.00 (0.80-4.43)0.005c

Week 9 2.55 (1.50-5.40) 1.87 (0.75-5.10)* Geometric mean (IQR)a ANCOVA b Mann-Whitney Wilcoxon testc Two-factor repeated measures ANOVA

AbsΔ, Absolute change; RelΔ, Relative change; LDL-C, LDL-cholesterol;

HDL-C, HDL-cholesterol; apoB-100, apolipoproteinB-100; apoA-1,

apolipoproteinA-1


***

L. reuteri NCIMB 30242 Improves CVD Risk Profile

hs-CRP CVD Risk Profile Fibrinogen CVD Risk Profile

**• L. reuteri NCIMB

30242 capsules

resulted in a

greater

percentage of

*

14%

19%

10%

12%

14%

16%

18%

20%

% o

f p

ati

en

ts

Placebo

L. reuteri

NCIMB 3024222%

27%

15%

20%

25%

30%

% o

f p

ati

en

ts

Placebo

L. reuteri

NCIMB 30242

Figure: % of patients lowering hs-CRP risk profile after

taking placebo or L. reuteri NCIMB 30242 over 9 weeks.

Criteria for hs-CRP levels and associated CVD risk: low

risk < 1mg/l; average risk 1-3 mg/l; high risk > 3mg/l.

Figure: % of patients lowering fibrinogen risk profile

after taking placebo or L. reuteri NCIMB 30242 over

9 weeks. Criteria for fibrinogen levels and

associated CVD risk: low risk < 2.36 g/l; average risk

2.36 - 2.77 g/l; high risk > 2.77 g/l.

percentage of

patients reducing

hs-CRP and

fibrinogen CVD

risk profile at the

9 week endpoint

when compared

to placebo.

2%

4%

5%5%

0%

2%

4%

6%

8%

10%

Risk profile

decreased by 2

risk groups

Risk profile

decreased by 1

risk group

Risk profile

decreased by

at least 1 risk

group

% o

f p

ati

en

ts

0%

2% 2%

5%

0%

5%

10%

15%

Risk profile

decreased by 2

risk groups

Risk profile

decreased by 1

risk group

Risk profile

decreased by at

least 1 risk

group

% o

f p

ati

en

ts

L. reuteri NCIMB 30242 Increases Gut BSH

Activity

• L. reuteri NCIMB 30242

resulted in a significant

increase of 1.00 µmol/l,

when compared to

placebo, in circulating


placebo, in circulating

deconjugated bile acids

at the 9 week endpoint.

• This suggests bile salt

hydrolysis in the GI

lumen and supports the

MOA.Placebo (n 61) L. reuteri (n 66)

Mean (SD) Mean (SD) P*

Conjugated Bile Acids (µmol/l)

Week 0 2.33 (3.05) 2.43 (2.85)0.738

Week 9 2.22 (2.08) 2.49 (3.20)

Deconjugated Bile Acids (µmol/l)

Week 0 1.81 (2.79) 1.82 (2.48)0.025

Week 9 1.62 (2.02) 2.64 (3.64)

* Two-factor repeated measures ANOVA

Table. Conjugated and deconjugated bile acid concentrations at baseline and

endpoint


Reduced Sterol Absorption Responsible for

Cholesterol Reduction• L. reuteri NCIMB 30242

capsules resulted in

significant reductions in

campesterol of 41.5%,

sitosterol of 34.2%,

stigmasterol of 40.7% and

Placebo (n 61) L. reuteri (n 66)

Mean (SD) Mean (SD) P*

Campesterol (ng/mL)

Week 0 2840.8 (1471.0) 3064.2 (1860.9)0.025

Week 9 3438.3 (2041.2) 2805.0 (1649.8)

Table. Plant sterols concentrations at baseline and endpoint


stigmasterol of 40.7% and

total plant sterols of

38.9%, when compared

to placebo, at the 9 week

endpoint.

• This suggests reduced

absorption of cholesterol

as PS are surrogate

markers for cholesterol

absorption.

0.025Week 9 3438.3 (2041.2) 2805.0 (1649.8)

Sitosterol (ng/mL)

Week 0 1368.9 (682.1) 1520.1 (891.1)0.031

Week 9 1636.8 (945.3) 1428.9 (868.5)

Stigmasterol (ng/mL)

Week 0 68.3 (51.3) 68.4 (49.4)0.042

Week 9 81.2 (78.7) 62.0 (54.2)

Total PS (ng/mL)

Week 0 4278.0 (2156.1) 4652.7 (2756.9)0.027

Week 9 5156.4 (3008.7) 4295.9 (2524.6)* Two-factor repeated measures ANOVA on log transformed values

PS, Plant sterols.


2

6

10

Ch

an

ge

in

DB

A (

nm

ol/

l)

Placebo

L. reuteri

NCIMB 30242

Bile Deconjugation Correlated to Reductions

in LDL-C• A significant association

was observed in subjects

taking L. reuteri NCIMB

30242 (Spearman

correlation, r=-0.369,

P=0.003) while no

association was

observed in subjects

-10

-6

-2-2 -1 0 1 2

Ch

an

ge

in

DB

A (

nm

ol/

l)

Change in LDL-C (mmol/l)


Figure: Individual changes in plasma deconjugated bile acids (DBA) and

serum LDL-cholesterol (LDL-C) over the intervention period.

observed in subjects

taking Placebo

(Spearman correlation,

r=0.086, P=0.516).

• It was also noted that

the regression

coefficients of the

Placebo and L. reuteri

NCIMB 30242 groups

were significantly

different (P=0.012).


0

1

2

3

3 5

Ba

seli

ne

DB

A (

µm

ol/

l)

Baseline LDL-C (mmol/l)

-8

-3

2

7

12

Re

lati

ve

ch

an

ge

ov

er

inte

rve

nti

on

pe

rio

d (

%)

Placebo

Cardioviva

L. reuteri NCIMB 30242 Reduces Cholesterol

Esters Assoc. with CVD• L. reuteri NCIMB 30242

capsules reduced total

cholesteryl esters by

6.3% (P=0.015)

compared to placebo.

• Cholesteryl ester

saturated fatty acids by

8.8% (P=0.002) -13

Total cholesteryl

esters

Total saturated

cholesteryl esters

Palmitic acid

(16:0)

Stearic acid (18:0) Total saturated

cholesteryl esters

+ Oleic acid (18:1)

Re

lati

ve

ch

an

ge

ov

er

inte

rve

nti

on

* ***** **

• Over 80% of circulating cholesterol is bound as a CE

• Epidemiologic studies show that CE are most closely associated

with CVD (Gertler et al.; Circulation)

• Lower levels of cholesterol esters have been associated with

reduced CVD risk.

• In patients with hypercholesterolemia, CEs are highly saturated

• Saturated + oleic CEs are found in atherosclerotic plaques

8.8% (P=0.002)

compared to placebo.

• Of the saturated fatty

acids, palmitic acid and

stearic acid were

reduced by 8.8%

(P=0.001) and 16.8%

(P=0.003) respectively

as compared to placebo.

• Cholesteryl ester

saturated fatty acids +

oleic acid were reduced

by 5.2% (P=0.039) as

compared to placebo.Jones et al. Heart and Stroke Sci Sessions. 2012

L. reuteri NCIMB 30242 increases circulating

25-hydroxyvitamin D

• No significant differences between

capsule groups in serum vitamin A,

vitamin E, or β-carotene or dietary

intake over the intervention period

(P<0.05)


(P<0.05)

• L. reuteri NCIMB 30242 increased

serum 25-hydroxyvitamin D by 14.9

nmol/L, or 25.5%, over the

intervention period, which was a

significant mean change relative to

placebo of 17.1 nmol/L, or 22.4%,

respectively (P<0.003)

Jones et al. J Clin Endocrinol Metab. 2013


Mitchell Jones MD, PhD

[email protected]

T: 514-987-4151

Serum bile acids contribute to improved

glucose and lipid metabolism

• Improved lipid and glucose

metabolism may be

mediated by bile acids

through TGR5 mechanismthrough TGR5 mechanism

• Total BA increased following

gastric bypass surgery

• Total BA inversely correlated

with 2h post meal glucose

and fasting triglycerides and

positively correlated with

adiponectin and peak GLP-1

11/7/2013 CONFIDENTIAL 21Patti et al. Obesity 2009

Dysbiosis is a pathophysiologic cause of IBD

• IBD is caused by an

abnormal immune

response to the gut

bacteria in people

who are genetically


who are genetically

predisposed.

• Reduced bile acid

hydrolysis (BSH)

activity a significant

factor in the onset

and progression of

IBD as immune

response

Duboc et al. Gut. 2012.

bsh gene reduced in Microbiome of IBD

patients

• Crohn’s patients have

reduced BSH gene

abundance.

• Significant depletion in

BSH gene in the


Ogilvie et al. Gut. 2012.

BSH gene in the

Firmicutes phylogenic

division containing

Lactobacilli.

• UC patients may have

increased BSH activity

in the Actinobacteria

division

BSH activity reduced in IBD and improved in

remission

• IBD patients have


fecal BSH activity which

is improved during

remission.

• Both the conjugation of

11/7/2013 CONFIDENTIAL 24Duboc et al. Gut. 2012.

• Both the conjugation of

bile acids and the

presence of appropriate

quantities of secondary

bile acids appear to play

a role.

• FXR and TGR5 are

sensors for the products

of appropriate bile acid

metabolism.

Bile acid Regulation of Immune cells

• Bile acids are the primary ligands for bile acid receptors:

• FXR (nuclear)

• TGR5 (cell surface)

• FXR and TGR5 are highly expressed

• GI and liver tissues

• Immune monocytes and macrophages• Immune monocytes and macrophages

• Metabolic products of deconjugation (BSH) and dehydroxylation (7αDH) are the most potent ligands

• LCA>CDCA>DCA>CA

• Bile acid signaling in macrophages and monocytes

• Inhibition of NF-κB, Il-6, Il-1β and TNFα

• Patients with IBD (Crohn’s and UC)

• Alterations in commensal bacteria

• Diminished concentration of bile acids


Brestoff et al. Nat Immuno. 2013.

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