Regulation of Intestinal Permeability in Health and Disease

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1 Regulation of Intestinal Permeability in Health and Disease DDW 2012 S. Diego, CA Alessio Fasano, M.D. Mucosal Biology Research Center and Center for Celiac Research University of Maryland School of Medicine

2010 AC The intestinal mucosa is the battlefield on which friends and foes need

2 All disease begins in the gut - Hippocrates 460 BC The gut is not like Las Vegas: what happens in the gut does not stay in the gut A.F AC The intestinal mucosa is the battlefield on which friends and foes need to be recognized and properly managed to find the ideal balance between tolerance and immune response.

3 Several Cells Play a Role in Maintaining the Gut Immune Homeostasis Epithelial cells Intestinal DCs B cells T cells

The Paracellular Pathway PURIFICATION PROTOCOL FROM HUMAN INTESTINE 1 2 3 4 1 2 3 4 1: Tissue lysate 2: Sephacryl-S300 3: Q-sepharose Tight junctions are a dark horse implicated in a host of disease

4 The Paracellular Pathway PURIFICATION PROTOCOL FROM HUMAN INTESTINE : Tissue lysate 2: Sephacryl-S300 3: Q-sepharose Tight junctions are a dark horse implicated in a host of disease states, ranging from acute injury to chronic inflammation and autoimmune diseases 4: Immuoaffinity Coomassie Western blot Fasano A. et al Lancet 2000;355: Wang W et al J Cell Sci 2000;24:

5 Characterization of Zonulin and Its Signaling Tripathi et al, PNAS 2009;106:

6 Zonulin Characterization in Sera of CD Patients Zonulin a1 b a2 HP2-2 HP1-1 HP1-2 b Tripathi et al, PNAS 2009;106:

GLIADIN/BACTERIA CONTROL CASEIN Mechanisms of Zonulin Release Zonulin Actin CXCR-3 is a seven-transmembrane G couple protein receptor that is

Three known CXCR3 ligands CXCL-9, -10, -11 are produced at the site of inflammation and elicit migration of pathological Th1 cells.

7 GLIADIN/BACTERIA CONTROL CASEIN Mechanisms of Zonulin Release Zonulin Actin CXCR-3 is a seven-transmembrane G couple protein receptor that is preferentially expressed on activated T lymphocytes and subset of B and NK cells. Three known CXCR3 ligands CXCL-9, -10, -11 are produced at the site of inflammation and elicit migration of pathological Th1 cells. CXCR3 has been implicated as a potential target for impeding T-cell-mediated destruction in autoimmune diseases such as multiple sclerosis and type 1 diabetes Nucleus El Asmar et al Gastroenterology 2002; Drago et al Scand J Gastroenterol 2006

8 Zonulin Signaling Working Hypothesis Zonulin PAR 2 a b MMPs/ ADAMS 1 + Pro-HB-EGF EGFR + - HP 2 Turn off 3 Inflammatory marker 2b + 2a Src + Ras-MAP-kinase activation Tryptase IV Proposed mechanisms through which zonulin activates EGFR. Zonulin can activate EGFR through direct binding (1) and/or through PAR2 transactivation (2). This second mechanism can be mediated by either Src signaling (2a) or by the release of MMPs and/or ADAMS that in turn will activate Pro-HB-EGF. When cell tryptase IV cleaves zonulin in its two subunits (so eliminating one of the three required disulfide bridges necessary for EGF activity), the molecule is not able to bind to EGFR (3), while will acquire a different function (Hb binding) and becomes an inflammatory marker. Tripathi et al, PNAS 2009;106:

9 Opening of the Intestinal TJ Control ZO-1 E-Cadherin In Vivo Effect of gliadin on Tight Junctional Proteins Arrangement 1mg gliadin

10 Zonulin Characterization and Signaling Zonulin signaling Freeze-Fracture Following Pathway Activation Resting State Fasano et al, J Clin Invest 1995;96:710-20; el Asmar et al Gastroenterology 2002;123:

11 Zonulin Genetics

12 Haptoglobin/Zonulin Evolution Fasano A. Physiol Rev Jan;91(1):151-75

13 Haptoglobin/Zonulin Evolution Fasano A. Physiol Rev Jan;91(1):151-75

14 Intestinal Barrier Dysfunction in Disease Pathology BACTERIAL TOXINS Bacteriodes fragilis (metalloprotease toxin) Clostridium difficile (toxins A, B) Clostridium perfrigens (enterotoxin) E. coli (cytotoxic necrotizing factor 1) Helicobacter pylori (vacuolating toxin) Listeria monocytogenes (internalin) Vibrio cholerae (zonula occludens toxin) INFECTIONS: E.Coli Rotavirus Salmonella Ty. HIV Ischemia / Reperfusion Injury Tight Junction dysfunction / injury DRUGS: alcohol NSAID Tacrolimus AUTOIMMUNE DISORDERS: - Celiac disease - Multiple Sclerosis - Inflammatory bowel diseases - Diabetes Mellitus - Ankylosing spondylitis - IgA nephropathy Fasano, A. Pathological and therapeutic implications of macromolecules passage through the tight junction in Cereijido M, Anderson J, eds. Tight Junctions. CRC Press,2001:

15 Is impaired intestinal barrier a cause of disease or an epiphenomenon? Multiple Sclerosis, Stroke, Schizophrenia. Asthma, COPD, ARDS Dilatative cardiomyopathy, Ischemia - reperfusion Systemic sclerosis Transplant Rejection Type 1 Diabetes, Autoimmune thyroiditis Celiac Disease, PBC, IBD. IBD, IBS Glomerulosclerosis, Acute Renal Failure Rheumatoid Arthritis Tumors/Metastatic diseases

16 Genetics: GWAS Studies Microbes? Gluten Control CD Celiac Not HLA Gen PAR-3 PP-1 CELIAC1 CELIAC2 CELIAC3 CELIAC4 TNFAIP3 REL IL18RAP IL2/IL21 TAGAP CCR3 Adapted from Xavier R.J. & Podolski D.K Nature 2007 Adapted from Schumann M et al GUT 2011

Water flux out of lumen (ml x cm -1 x h -1 ) BSA flux (mg x cm -1 x h -1 ) MLCK inhibition prevents immunemediated barrier dysfunction and diarrhea 0.6 100 0.5 BSA 50 Fe(CN) 6-4 PIK H 2 O 0.4 0.

17 Water flux out of lumen (ml x cm -1 x h -1 ) BSA flux (mg x cm -1 x h -1 ) MLCK inhibition prevents immunemediated barrier dysfunction and diarrhea BSA 50 Fe(CN) 6-4 PIK H 2 O PIK anti-cd3: PIK: Clayburgh et al. J Clin Invest (2005) anti-cd3: PIK: PIK, membrane- Permeant Inhibitor of MLC Kinase + 250

18 The Leaky Gut Theory: How to Move From Fantasies to Facts

19 How To Measure Gut Permeability: Physiological Site Restriction Sucralose PEG Cr-EDTA Lactulose Mannitol Rhamnose Sucrose Lactose Courtesy Jon Meddings

20 Zonulin Gene Is Located on Chromosome 16 Chromosome 16 contains about 98 million bases, or some 3% of the human genome, encoding for ~1,300 genes. Fasano A. Physiol Rev Jan;91(1):151-75

20.6 7.1 23.9 10.1 14.1 9.2 9.4 3.8 HP 1-2 (1 zonulin gene) 43.5 35.7 44.0 46.2 46.9 38.8 46.5 43.7 HP 2-2 (2 zonulin genes) 35.9 57.

21 Distribution of Haptoglobin Alleles In Several Immune-Mediated Diseases A PCR Genotyping HP 2-2 HP 1-2 HP 1-1 HP 2-2 HP 1-2 HP 1-1 B WB Immunotyping Celiac Disease Crohn s Disease Schizophrenia CKD Genotype Cntr Pts Cntr Pts Cntr Pts Cntr Pts HP 1-1 (no zonulin gene) HP 1-2 (1 zonulin gene) HP 2-2 (2 zonulin genes) Maes M. et al. Psychiatry Res 2001;104:1-9; Tripati A. et al. Proc Natl Acad Sci U S A. 2009;106:

Mouse Models to Establish The Link Between Distribution

Inflammation C57Bl/6 wild type mouse HP 1-1 (no zonulin

1-2 (1 zonulin gene) Transgenic C57Bl/6 mouse

22 Mouse Models to Establish The Link Between Distribution of Haptoglobin Alleles And Susceptibility to Inflammation C57Bl/6 wild type mouse HP 1-1 (no zonulin gene) C57Bl/6 mouse transfected with human HP2 gene HP 1-2 (1 zonulin gene) Transgenic C57Bl/6 mouse engineered by duplicating a chain (a1 a2) HP 2-2 (2 zonulin genes) WB a2 a1

23 Impact of Zonulin Gene Expression on Gut Inflammation and TEER Changes Induced by DSS Histology TEER Small intestine HP 2-2 mice untreated C57Bl/6 wild type mice DSS-treated A B C HP 2-2 mice DSS-treated D E F Colon

24 zonulin ng/mg serum protein Serum Zonulin in Autoimmune Disorders 15 * * * * * p < Cut-off HC CD AIH PBC T1D APS-1 MS Clemente et al. Gastroenterology 2002;122 :A15

25 Serum Serum zonulin (ng/mg protein) LA/MA Serum Zonulin Levels and Their Correlation With Intestinal Permeability In Celiac Disease and Type 1 Diabetes Celiac Disease Type 1 Diabetes Zonulin-LA/MA A B C N=339 N=89 N= T1D T1D T1D Relatives controls Controls multiple R=0.36; intercept p=1.71e- 10; X variable 1 p= Zonulin (ng/mg protein) Sapone et al Diabetes 2006; 55:1443-9

26 Prove of Concept on Role of Zonulin- Regulated Intestinal Permeability in Autoimmune Pathogenesis Genetics Environment Mucosal Barrier Autoimmunity NO? Only CD

27 Larazotide, the Zonulin Inhibitor HO H C CH O H N O C N O H 2 C C C O CH C H2 NH 2 HN C O H 3 C CH CH NH CH 3 O C CH H 2 C CH CH 3 O C NH CH 3 HN CH CH CH 3 H CH C O CH 3 O C NH H 2 N CH H C 32 H 55 N 9 O 10 Exact Mass: Mol. Wt.: m/e: (100.0%), (35.6%), (9.2%), (3.3%) C, 52.95; H, 7.64; N, 17.37; O, Di Pierro et al, J Biol Chem 2001;276:

28 Prove of Concept of the Role of Zonulin in Autoimmune Pathogenesis: The Animal Model of Type 1 Diabetes

29 Serum glucose (mg/dl) Evidence for Zonulin-Dependent Increased Intestinal Permeability in the Pathogenesis of Type 1 Diabetes Serum glucose (mg/dl) 300 Diabetes - Untreated 0.7 No Diabetes Larazotide Treated ICA + (100%) LA/MA Ratio ICA + (8%) LA/MA Ratio Age (days) Glucose LA/MA Age (days) Watts et al PNAS 2005;102:

30 Blocking the Zonulin-Dependent Increased Intestinal Permeability Aborts The Autoimmune Process Insulin staining Islet Immunohistochemistry Glucagon staining Untreated BBDP rats that developed T1D A B Larazotide-treated BBDP rats that DID NOT develop T1D: No insulitis C D

31 Prove of Concept of the Role of Zonulin in Autoimmune Pathogenesis: Human Clinical Trials in Celiac Disease

32 Comprehensive Phase 1-2 Program of Larazotide Acetate in Celiac Disease has Studied ~500 Patients TRIAL DESIGN N -001 Phase 1, Single Escalating Dose in Healthy 24 Volunteers -002 Phase 1b, Multiple Dose in Controlled Celiac Patients Phase 1, Multiple Escalating Dose in Healthy 24 Volunteers -004 Phase 2a, Multiple Dose in Controlled Celiac Patients Gluten Challenge 2 weeks Phase 2b, Dose ranging in Controlled Celiac Patients Gluten Challenge 6 weeks B Phase 2b, Controlled Celiac Patients 42 Gluten Challenge 6 weeks -011 Phase 2b, Dose ranging in Active Celiac patients, weeks Total 486 Safety Efficacy

33 Safety Profile of Larazotide Acetate to Date Larazotide acetate acts locally in the gastrointestinal tract No systemic exposure, no measurable plasma drug levels in any clinical study No immunogenicity, no antibody development in any clinical study No toxicity observed to date in 24 completed animal toxicology studies No safety signals in ~300 celiac subjects exposed to larazotide acetate up to 8 weeks To date, safety comparable to placebo

34 La/Ma (day x) / La/Ma (day 1) % (Placebo n=7; AT-1001 n=14) Efficacy: Larazotide Acetate Reduces the Signs and Symptoms of Gluten Challenge Phase 1b CLIN : Single Dose Gluten challenge, 2.5 grams Intestinal Permeability Phase Gastro-Intestinal Ib Gastrointestinal Signs Symptoms & Symptoms Blind Gluten Challenge, 2.5gm p= % 100% 80% p=0.018 p= p=0.07 Placebo n = 7 60% 40% 20% Placebo AT Larazotide Acetate Day 1 Day 2 Day 3 Day 7 n = 14 0% GI Symptoms Abdominal Pain Constipation Diarrhea Dimension Flatulence Nausea Vomiting Paterson BM et al. Aliment Pharmacol Ther 2007;26:757-66

35 Gluten Causes Leaky Gut and Inflammation by Releasing Zonulin Brain Inflammation

36 NIH DK NIH DK048373

The Role of Nutrition, Gut Microbiome, and Gut Permeability in Immunomodulation: The Celiac Disease and Type 1 Diabetes Paradigms

The Role of Nutrition, Gut Microbiome, and Gut Permeability in Immunomodulation: The Celiac Disease and Type 1 Diabetes Paradigms Alessio Fasano, M.D. W. Allan Walker Chair in Pediatric Gastroenterology