Bifantis ® ( Bifidobacterium infantis 35624)

1. Bifantis®(Bifidobacterium infantis 35624) Clinical Data In Irritable Bowel Syndrome (IBS)

2. Irritable Bowel Syndrome • Functional GI condition characterized by abdominal pain associated with a change in bowel habits (diarrhea, constipation or alternating between the two) • 2nd only to common cold in causes of workplace absenteeism • Accounts for approximately 2.4 to 3.5 million physician visits annually—average of 4 to 6 visits per patient per year • Total annual costs (direct and indirect) of IBS in the United States have been estimated to be approximately $30 billion, excluding prescription and over-the-counter drug costs

3. Diagnosis of IBS • No diagnostic test or associated pathology— diagnosis made primarily by exclusion • Current standard for diagnosis is the Rome III criteria: Recurrent abdominal pain or discomfort, at least 3 days/month in the last 3 months associated with two or more of the following: 1. Improved with defecation • Onset associated with a change in frequency of stool • Onset associated with a change in form (appearance) of stool Criterion fulfilled for the last 3 months with symptom onset at least 6 months prior to diagnosis

4. Establishment of the Microflora • Digestive environment (the microflora) is established early in life (as an infant) • Primarily 5 species: bifidobacteria, bacteriodes, eubacterium, fusobacterium and peptostreptococcus • In a healthy state, primary functions of microflora are: • a “natural defense system” • providing nutrients and metabolic processes necessary for proper diet and nutrition • Changes occur due to diet, infection, stress, antibiotic use, travel, etc. • Altered flora has been documented in the literature associated with IBS • In particular, decreased levels of bifidobacteria have been found in IBS subjects

5. Defining Probiotics • Probiotics are “living microorganisms which, upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition” • Use of probiotics can be traced to the Ancient Roman Historian Plinio (76 AD) who advocated the use of fermented milk for the treatment of GI infections • Modern probiotics were first described by Metchnikoff in 1907: “ingested bacteria, in the form of yogurt and other fermented foods, could beneficially affect the normal gut flora”

6. Desirable Selection Criteria for Probiotics Should: • be of human origin • be nonpathogenic • be resistant to processing • be resistant to gastric acidity and bile toxicity • adhere to gut epithelial tissue • colonize the GI tract • produce antimicrobial substances • modulate immune response • influence metabolic activities • be documented and assessed independently Lee & Salminen 1995

7. Probiotics for IBS • All probiotics are currently marketed in the U.S. as dietary supplements • A few probiotics have been demonstrated to have benefits in digestive disorders: • Traveler’s diarrhea, antibiotic-induced diarrhea • Several probiotic products make claims of benefits in IBS; however, they are not backed by solid evidence: • Scarcity of well-controlled clinical trials published • Poor-quality control results and formulation in inability to sustain live bacteria in product • Use of strains that are not purified

8. Bifidobacterium infantis 35624 • Only probiotic species known to be isolated from a healthy human colon • Demonstrates ability to adhere to mucosal tissue • Complete genome has been sequenced • No regions that code for pathogenicity were found • Formulated into a capsule that is shelf-stable at room temperature • Industry-leading quality control methods being used in production to ensure viability and purity of finished product

9. Bifidobacterium infantis AH 35624

10. Initial Clinical Trial in IBS • Double-blind, placebo-controlled, parallel 14-week study in 77 male and female IBS suffererers: • 2-week run-in, 8 weeks of treatment with 4 weeks of follow-up • Milk-based formulation at dosage of 1 x 1010 CFU per day • Compared B. infantis 35624 to L. salivarius 43331 and placebo • Results indicated improvement in abdominal pain, bloating and bowel movement difficulty (composite score) with B. infantis–but not the Lactobacillus strain. • Also found a marked immunologic difference between IBS patients and controls. O’Mahony et al. Gastroenterology 2005 (128):541-551.

11. Figure 1. Comparison of the effects of placebo, L. salivarius UCC43331 and B. infantis 35624 on a composite score of IBS symptoms.

12. 300 p=0.001 p=0.001 Pre Pre treatment treatment Post Post treatment treatment 250 200 150 100 50 0 Healthy B. infantis 35624 L. salvarius 4331 Placebo Volunteers Figure 5. O’ Mahony et al. Gastroenterology 2005 (128)541- 551. Figure 5. Comparison of PBMC IL-10/IL-12 ratios at baseline and following therapy with placebo, L. salivarius UCC43331 and B. infantis 35624 with that of a normal control period. 300 300 p=0.001 p=0.001 Pre Pre treatment treatment Post Post treatment treatment 250 250 200 200 ratio ratio 12 12 - - 150 150 10:IL 10:IL - - IL IL 100 100 50 50 0 0 Healthy Healthy B. infantis 35624 B. infantis 35624 L. salvarius 4331 L. salvarius 4331 Placebo Placebo Volunteers Volunteers ’ ’

13. Second Clinical Trial in IBS • Double-blind, placebo-controlled, parallel 8-week study in 362 female IBS suffererers: • 2-week run-in, 4 weeks of treatment with 2 weeks of follow-up • Capsule formulation at 3 dose levels of B. infantis 35624: • 1 x 1010 CFU per day • 1 x 108 CFU per day • 1 x 106 CFU per day • Compared B. infantis 35624 to placebo • Results indicated improvement for all the cardinal symptoms of IBS: pain, bloating and bowel movement difficulty with B. infantis 35624. • Also found benefits for normalization of bowel movement frequency across all IBS subtypes.

14. Figure 2. Comparison of effects of placebo and Bifidobacterium infantis 35624 on abdominal pain/discomfort. Whorwell et al. Am J Gastroenterol 2006;101:1581-1590.

15. Figure 4. Comparison of effects of placebo and Bifidobacterium infantis 35624 on IBS composite score. Whorwell et al. Am J Gastroenterol 2006;101:1581-1590.

16. Figure 5. Comparison of effects of placebo and Bifidobacterium infantis 35624 on Subjects’ Global Assessment (SGA) of IBS symptoms. Positive response rates recorded at wk 4 at the end of therapy–“yes” or “no” response: “Please consider how you felt in the past week in regard to your IBS, in particular your general well-being, and symptoms of abdominal discomfort or pain, bloating or distension and altered bowel habit. Compared to the way you felt before beginning the medication, have you had adequate relief of your IBS symptoms?” Whorwell et al. Am J Gastroenterol 2006;101:1581-1590.

17. Distribution by IBS Subtype Quigley et al. Presentation at ACG, 2005. Honolulu, Hawaii.

18. Average Bowel Movements per Day Average = 1-2 BM/day Baseline Percentile Subject Distribution (n=182) Normalization Analysis • For analysis, “normalization” defined as movement toward 1-2 BM/day (25th to 75th percentile) • 2-week baseline data (actual number of daily BMs) used to determine distribution across study: Quigley et al. Presentation at ACG, 2005. Honolulu, Hawaii.

19. Response Rate–Normalization Effect Percent of subjects outside 25th-75th percentile at baseline (1-2.29 BM/day) that moved to “normal range” at Week 4–Treatment difference of 23% P=0.05 Bifantis 1 x 108 Placebo Note: There was no significant change among subjects who began study in 25th to 75th percentile for either Bifido or Placebo. Quigley et al. Presentation at ACG, 2005. Honolulu, Hawaii.

20. Tolerability of B. infantis 35624 In the second study of 362 total subjects: • 17 subjects withdrew due to adverse events (AE’s) • 9 from the placebo group • 8 from the three treatment groups combined • The majority were occasioned by worsening of IBS symptoms. • The overall incidence of all AE’s was similar in the four groups % with IBS-like symptoms • 48% placebo 29% • 37% 1 x 106 37% • 52% 1 x 108 28% • 43% 1 x 1010 24% • The incidence of severe AE’s adjudged as treatment related was highest in the placebo group at 9%; rates for the three treatment groups were 0%, 1% and 2%, respectively Quigley et al. Gastroenterology 2006;130 (S2):A493.

21. Conclusions • Two well-controlled, properly powered studies have demonstrated effectiveness of B. infantis 35624 in the management of IBS. • Benefits of B. infantis 35624 are evident regardless of IBS subtype • B. infantis 35624 results in a “normalization” effect. • Increases frequency of BMs in subjects with less than one BM daily (constipation) • Decreases frequency of BMs in subjects with more than 2.5 BMs daily (diarrhea)