Effect of VSL#3 on Bone Mineral Density in Postmenopausal Women

Osteoporosis has a devastating impact on quality of life of postmenopausal women, and is a
significant cause of disability and morbidity. Many drugs are approved for the prevention and
treatment of osteoporosis, but are associated with high costs and side effects. Some data
from animal studies suggests that supplementation with probiotics can safely treat and
prevent osteoporosis. The probiotic VSL#3 is commercially available, is safe for human
consumption, and has been widely used in human clinical trials, and has known
health-promoting effects in both children and adults.

The double-blind, randomized, placebo-controlled trial of VSL#3 will be conducted in a
population of ambulatory, otherwise healthy, postmenopausal women for 12 months. Control and
VSL#3-treated postmenopausal women will be matched by age (± 3 years). Study visits will
include all or some of the following procedures: a medical exam, urine collection, height and
weight measurement, a blood draw to assess bone biomarkers, a DEXA (dual energy X-ray
absorptiometry) scan to measure bone density, and health questionnaires.

The primary endpoint is the change in bone mineral density (BMD) at the L1-4 lumbar spine
over one year of study. Changes in BMD at the femoral neck and total hip area will be
secondary endpoints. All BMD data will also be used as a tool for future studies power
calculation and design. Additional endpoints will include changes in bone turnover markers
and inflammatory/osteoclastogenic cytokines. Measurements of indices of bone turnover and
cytokine levels will provide much needed mechanistic information.The data will allow to
establish whether VSL#3 prevents bone loss and/or increases bone mass by regulating bone
resorption, formation, or both.

Inclusion Criteria:

1. Willing and able to give written informed consent for participation in the study,

2. Age range 50-65 years,

3. Menopausal status (defined by >1 yr since last menstrual period or FSH level in the
postmenopausal range),

4. Ambulatory,

5. Body mass Index (BMI) must be ≥ 18 and ≤ 32 kg/m2 at screening,

6. Bone mineral density (BMD), expressed as T-scores, must be > - 2.5 in the lumbar spine
(L1-L4), the femoral neck, and the total hip, as measured by dual energy X-ray
absorptiometry (DXA),

7. Commitment not to use any products that may influence the study outcome (see below),

8. Ability to understand and comply with the requirements of the study.

Exclusion Criteria:

1. Premenopausal status,

2. History of >1 previous atraumatic bone fractures after age 50;

3. Presence of established osteoporosis (T-score ≤ - 2.5, in the lumbar spine, femoral
neck or total hip as measured by screening DXA),

4. History of immunological or bone-related disorders including: HIV infection, Type I
diabetes mellitus, bone marrow or organ transplantation; Inflammatory bowel disease
(ulcerative colitis, Crohn's disease); multiple myeloma; osteomalacia; osteosarcoma;
Paget's disease; rheumatoid arthritis; systemic lupus erythematous; parathyroid
disorders,

5. Uncontrolled type II diabetes mellitus (HgbA1c ≥ 7% within the last 12 months),

6. History of bariatric surgery or other forms of malabsorption (including documented
celiac disease, or chronic diarrhea),

7. Alcohol abuse,

8. Clinically significant chronic kidney disease (stage ≥ 2, with total serum creatinine
level > 2.5 mg/dL and calculated glomerular filtration rate (GFR) < 60 mL/min by the
Modification of Diet in Renal Disease (MDRD equation),

9. Clinically significant cardiovascular disease (myocardial infarction, cerebral
vascular accident or acute congestive heart failure within the previous 12 months,

10. Any malignancies, other than localized skin squamous cell carcinoma, diagnosed within
the previous 5 years, or any history of metastatic cancer,

11. History of use of oral supplement products containing probiotic bacteria (more than
once per week) within four weeks prior to baseline,

12. Current use (within the past 8 weeks) of any medication with known influences on the
immune or skeletal system (e.g. immune modulation therapy, systemic glucocorticoids,
systemic steroid hormones,

13. Use of oral or injectable bisphosphonates for more than 1 year within the last 5
years,

14. Current or past use (within 1 year) of Denosumab, Teriparatide, Raloxifene, hormone
replacement therapy (HRT), calcitonin, or any other anti-resorptive agent other than
bisphosphonates used for the prevention and treatment of osteoporosis,

15. Use of antibiotics during the previous two months or frequent user of antibiotics (>2
courses during the previous 12 months) for any cause,

16. Smoking or use of nicotine-containing products during the last six months,

17. Known hypersensitivity to any of the ingredients in the VSL#3 or the placebo study
drug,

18. serum or plasma 25-hydroxyvitamin D [25(OH)D] concentration < 12 ng/mL,

19. uncontrolled thyroid disease (abnormal blood TSH level within the last 12 months
and/or changing dose of thyroid replacement therapy within the last 12 months).