1. Villicote® Restoring The Gastrointestinal Barrier
Willem Wassenaar MD, MSc, MBA
Medical Director
Wellesley Therapeutics Inc.
© 2016

2. IBD: Topics for Discussion
Beyond Inflammation
Intestinal Barrier
Mucin Composition & Penetrability
Promoting Mucin Production

3. IBD: An Inflammatory Disease
Class
Example
Mechanism
Corticosteroids
Entocort
Reduction in the number and responsiveness of lymphocytes
Reduction in inflammatory mediators
IL-1, 2, 6 And TNF-α
5-Aminosalicylic Acid
Pentasa
Inhibits leukocyte chemotaxis, degranulation , migration and T-cell proliferation
Antimetabolites
Methotrexate
Inhibition of DNA synthesis and lymphocyte replication
TNF-α Inhibitors
Remicade
Binds to soluble and membrane associated TNF-α

4. Infliximab Treatment Results
All patients concurrently taking:
Corticosteroids with or without Azathioprine /Mercaptopurine
Treatment:
Placebo
5 mg /kg infliximab
10 mg/kg infliximab
Nielsen 2013 N Eng J Med IBD and TNFα inhibitors:
10 to 40% of Crohn’s patients do not have a clinically relevant response.
In UC as many as 50% do not have a clinically relevant response.
Rutgeerts 2005

5. From Harrison 18th edition (2012) “The normal microbiota is likely perceived inappropriately as if it were a pathogen.”
Abraham 2009

6. Intestinal Barrier Outer Loose Mucin Inner Dense Mucin Johansson 2013
From Harrison 18th edition (2012) “The normal microbiota is likely perceived inappropriately as if it were a pathogen.”
Intestinal surface barrier separating highly immunologic agents in the intestinal lumen from a highly immunoreactive submuscosal.
Johansson 2013

7. Gel Forming Mucin Mucin Domains Johansson 2013
MUC2 Mucin Molecule 2.5x106 Da
Protein core: 20% of the mucin molecule
Carbohydrate side branches
80% of the mucin molecule
N-Acetylgalactosamine linked to serine or threonine
“O” type linkage
Extensions contain 2-acetamido-2-deoxy-D-glucose, galactose fucose and sialic acid
Varying degrees of sulphation
Mucin Domains
Johansson 2013

8. Mucin Polysaccharide Profile
716a: GalNAc + GlcNAc + Sialic acid
716b: GalNAc + GalNAc + Sialic acid
Larsson 2011

9. Saccharide Structure: MW 1,372
Protein
Core
Polysaccharide Side Chain
Mucin layer could be thin: Easily penetrated by gut microbes
Mucins could be defective: Readily broken down by gut microbes
Microbes: Population that has the enzymatic capability to break down mucin
N-Acetylglucosamine
N-Acetylgalactosamine
Galactose
Sialic Acid

10. Mucin Type: Control vs Active Disease

11. Mucin Type: Control vs Active Disease

12. Measuring Mucin Integrity
Human sigmoid colon biopsy
Normal controls
UC = patients with acute inflammation
End Point
Measurement of the thickness of mucin
Penetration of beads into mucin layers
Measuring Mucin Integrity: how strong is this barrier or how functional is this barrier?
Johansson 2013

13. Penetrability of Mucin
Top of Mucin Layer
Green beads 2μm diameter; red beads 0.5 μm diameter Fluospheres Invitrogen. Left to incubate for 40 mins then the distance in relationship to the epithelium was measured
Epithelial Layer

14. Penetrability of Mucin
Top of Mucin
Epithelium

15. Thickness & Penetrability of Mucin
Outer Loose Mucin Layer
Inner Dense Mucin Layer
D) Relation between penetrable and impenetrable mucus of the 2μm green beads. C control, MO IP pat with Mayo endoscopic score 0 and impenetrable mucus, MO P Mayo endoscopic score 0 and penetrable mucus. Mayo endoscopic score of 1 to 3.
Green beads 2μm diameter; red beads 2μm diameter

16. In Summary
Compared to Controls, Patient with Ulcerative Colitis have a mucin layer that:
Has fewer complex carbohydrates
Is thinner
More easily penetrated

17. Research Question
Can N-Acetylglucosamine supplementation drive mucin production and restore intestinal health?

18. Why is N-Acetylglucosamine Critical?
Knocking out the enzyme that converts glucosamine to N-acetylglucosamine is lethal
N-Acetylglucosamine is the 2nd most important sugar in human breast milk
N-Acetylglucosamine reused in the production of human glycoproteins
N-acetylglucosamine is a precursor for:
N-acetylgalactosamine
Sialic Acid

19. Surplus N-Acetylglucosamine Drives Cellular End Product
Experimental evidence:
In Vitro
Human peritoneal mesothelial cells
In Vivo
Mouse model of intestinal permeability
Human before and after biopsies in UC and Crohn’s patients

20. Human Mesothelial Cells
Experimental Setup
Human peritoneal mesothelial cells derived from omentum at surgery
Culture media supplemented with
Glucose
2-acetamido-2-deoxy-D-glucose
Endpoint production of:
Hyaluronan
Sulphated glycosaminoglycans
Breborowicz 1998

21. Hyaluronan Synthesis
2500
2000
1500
1000
500 xx xx xx xx
Hyaluronan ng/mg protein x xx
Synthesis of hyaluronan by human peritoneal mesothelial cells in culture. Results are the mean from 7 experiments on different primary cell lines x: P<0.05 vs. control; xx P<0.01 vs. control.
24 hours in culture.
10 mM
20 mM
40 mM
80 mM
Synthesis of hyaluronan by human peritoneal mesothelial cells
x: P<0.05 vs. control; xx P<0.01 vs. control.
Control
2-Acetamido-2-deoxy-D-glucose
Glucose

22. Sulphated Glycosaminoglycans Synthesisxx
2800
2100
1400
700 x x x
Sulphated GAGs ng/mg protein
Synthesis of glycosaminoglycans by human peritoneal mesothelial cells in culture. Results are the mean from 8 experiments on different primary cell lines x: P<0.05 vs. control; xx P<0.01 vs. control.
24 hrs
72 hrs
120 hrs
Synthesis of sulphated glycosaminoglycans by human peritoneal mesothelial cells .
40mM of monosaccharide added.
Control
2-Acetamido-2-deoxy-D-glucose
Glucose

23. In Vivo Evidence

24. Prevention of Intestinal Injury
In Vivo Mouse Model
Indomethacin by gavage increases intestinal injury and permeability
Procedure
Indomethacin 8mg/kg by gavage X 3 days (0 to 2)
N-Acetylglucosamine added to drinking water at 0, 0.2, 2 and 20 mg/kg
Pre-treated for 5 days (-5 to 0)
Treatment for 12 days
Endpoint: Measure of intestinal inflammation
Myeloperoxidase activity assay (inflammatory cell infiltrates)
Result
0.2 mg/kg same as placebo
20 mg/kg very significant reduction in myeloperoxidase (injury)
Myeloperoxidase is most abundantly expressed in neutrophil granulocytes .[3] It is a lysosomal protein stored in azurophilic granules of the neutrophil. MPO has a heme pigment, which causes its green color in secretions rich in neutrophils, such as pus and some forms of mucus.
MPO produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion (Cl-) (or the equivalent from a non-chlorine halide) during the neutrophil's respiratory burst. It requires heme as a cofactor. Furthermore, it oxidizes tyrosine to tyrosyl radical using hydrogen peroxide as an oxidizing agent.[5]
Hypochlorous acid and tyrosyl radical are cytotoxic, so they are used by the neutrophil to kill bacteria and other pathogens.
One unit is the amount of MPO that can produce 1.0 nmole of taurine chloramine (hypochlorous acid) at pH 6.5 and 25C during 30 minutes in the presence of 100 mM chloride and 100 mM of hydrogen peroxide.
0.2 mg/kg; 2 mg.kg; 20 mg/kg
Klompus 2007

25. Impact on NSAID Intestinal Injury
One unit is the amount of MPO that can produce 1.0 nmole of taurine chloramine (hypochlorous acid) at pH 6.5 and 25C during 30 minutes in the presence of 100 mM chloride and 100 mM of hydrogen peroxide.
Klompus 2007

26. Human N-Acetylglucosamine Trials
Clinical Conditions
Crohn’s disease
Ulcerative colitis
End Points
Clinical
Biopsy evidence of an increase in tissue glycosaminoglycans
Salvatore 2000

27. N-Acetylglucosamine in Crohn’s Disease
Oral administration 3 to 6 grams/day
N= 10
Clinical improvement
9/10 showed reduction in symptoms
Onset of benefit
1 to 3 months
Of those showing improvement most experienced improvement in 1 month

28. N-Acetylglucosamine in Ulcerative Colitis
Single agent treatment
Enema administration
Ulcerative colitis N= 6
5 clear improvement over 1 to 3 months
1 partial improvement

29. Biopsy Results Pretreatment biopsies
Repeat biopsies in 6 to 8 weeks after the initiation of N-Acetylglucosamine
End point
increase in tissue glycosaminoglycans

30. Increase In Glycosaminoglycan Content
Percent differences
Epithelium pre = 79.7 post % change +80%
Matrix pre 98.6 post % change +74%
Units represent an increase in optical density indicating more glycosaminoglycans.
Measurement of glycosaminoglycans tissue content based on optical density
Specific for epithelial:Heparan sulphate; Dermatan sulphate; Chondroitin
Pretreatment: ±9.9
Posttreatment: ± P< 0.05

31. Adverse Events
“No adverse side-effects of treatment were noted in any patient, apart from mild stinging on rectal insertion in one.”
Salvatore 2000

32. In Summary An intact mucin layer is essential for proper gut function
N-Acetylglucosamine provides the building blocks for intestinal mucin and glycosaminoglycan production when cellular production and dietary sources may be insufficient

33. For More Information Sales and Marketing contact
Laurie Middleton-Crump
For Medical / Technical contact
Willem Wassenaar MD
Wellesley Therapeutics Inc.
Toronto, Canada
Phone ; Fax
Villicote is a registered trade mark used under license