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
> 10
14
microbial cells and > 10
15
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
90
of 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
90
of 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
11/7/2013 CONFIDENTIAL 4
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
11/7/2013 CONFIDENTIAL 5
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
11/7/2013 CONFIDENTIAL 6
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
11/7/2013 CONFIDENTIAL 7
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
11/7/2013 CONFIDENTIAL 8
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 10
10
CFU BID in
yogurt format
Duration: 6 week intervention Duration: 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
11/7/2013 CONFIDENTIAL 9
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
11/7/2013 CONFIDENTIAL 10
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 10
9
CFU (100mg)
BID in capsule format BID 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 CONFIDENTIAL
11
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%
11/7/2013 CONFIDENTIAL
12
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 .
Jones et al. Eur J of Clin Nut. 2012
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
Abs 0.21 (1.16) -0.22 (0.88) 0.005
b
Rel (%) 8.91 (33.37) -4.48 (21.24) 0.006
b
apoB-100/apoA-1
Abs -0.02 (0.19) -0.09 (0.20) 0.034
b
Rel (%) -0.74 (22.82) -9.74 (17.75) 0.026
a
Fibrinogen
Table. Changes in lipid and apolipoprotein ratios and cardiovascular risk parameters
11/7/2013 CONFIDENTIAL 13
significantly reduced
CVD risk factors
including hs-CRP and
fibrinogen compared
to placebo at the 9
week endpoint.
Fibrinogen
Abs (g/l) 0.48 (0.87) -0.09 (0.96) 0.004
b
Rel (%) 17.19 (35.67) 2.95 (26.71) 0.004
b
hs-CRP (mg/l)
*
Week 0 1.63 (0.90-3.25) 2.00 (0.80-4.43)
0.005
c
Week 9 2.55 (1.50-5.40) 1.87 (0.75-5.10)
*
Geometric mean (IQR)
a
ANCOVA
b
Mann-Whitney Wilcoxon test
c
Two-factor repeated measures ANOVA
Abs, Absolute change; Rel, Relative change; LDL-C, LDL-cholesterol;
HDL-C, HDL-cholesterol; apoB-100, apolipoproteinB-100; apoA-1,
apolipoproteinA-1
Jones et al. Eur J of Clin Nut. 2012
***
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
a
t
i
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n
t
s
Placebo
L. reuteri
NCIMB 30242
22%
27%
15%
20%
25%
30%
%

o
f

p
a
t
i
e
n
t
s
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
a
t
i
e
n
t
s
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
a
t
i
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n
t
s
L. reuteri NCIMB 30242 Increases Gut BSH
Activity
L. reuteri NCIMB 30242
resulted in a significant
increase of 1.00 mol/l,
when compared to
placebo, in circulating
11/7/2013 CONFIDENTIAL
15
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 (mol/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 (mol/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
Jones et al. Eur J of Clin Nut. 2012
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
11/7/2013 CONFIDENTIAL
16
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.
0.025
Week 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.
Jones et al. Eur J of Clin Nut. 2012
2
6
10
C
h
a
n
g
e

i
n

D
B
A

(
n
m
o
l
/
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
C
h
a
n
g
e

i
n

D
B
A

(
n
m
o
l
/
l
)
Change in LDL-C (mmol/l)
11/7/2013 CONFIDENTIAL 17
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).
Jones et al. Eur J of Clin Nut. 2012
0
1
2
3
3 5
B
a
s
e
l
i
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D
B
A

(

m
o
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/
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)
Baseline LDL-C (mmol/l)
-8
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7
12
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(
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)
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)
R
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* * ** ** **
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 -carotene or dietary
intake over the intervention period
(P<0.05)
11/7/2013 CONFIDENTIAL 19
(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
11/7/2013 CONFIDENTIAL 20
Mitchell Jones MD, PhD
mitchell@micropharma.net
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 mechanism through 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 21 Patti 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
11/7/2013 CONFIDENTIAL 22
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
Crohns patients have
reduced BSH gene
abundance.
Significant depletion in
BSH gene in the
11/7/2013 CONFIDENTIAL 23
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
significantly reduced
fecal BSH activity which
is improved during
remission.
Both the conjugation of
11/7/2013 CONFIDENTIAL 24
Duboc 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 (7DH) are
the most potent ligands
LCA>CDCA>DCA>CA
Bile acid signaling in macrophages and
monocytes
Inhibition of NF-B, Il-6, Il-1 and TNF
Patients with IBD (Crohns and UC)
Alterations in commensal bacteria
Diminished concentration of bile acids
11/7/2013 CONFIDENTIAL 25
Brestoff et al. Nat Immuno. 2013.