Phase I/II Randomized Clinical Study of Cycles of a New Formulated FMD in Prefrail Elderly.

A phase I/II randomized clinical study will be performed to assess feasibility, safety, and efficacy (improvement of the response to vaccination, but also functional status improvement or maintenance) of two cycles of FMD (1 week) administered 30 days apart, preceding flu vaccination in pre-frail elderly subjects. The study will enrol 40 pre-frail subjects aged 65-80 years. Subjects will be excluded if they have known immunosuppressive disorders or medications or have not received influenza vaccination in the previous two years. Subjects will be randomized 1:1 to Arm A (FMD) or Arm B (regular diet). Subjects in Arm A will receive the 5 day FMD diet once a month for 2 months (2 cycles completed in 40 days: 5-30-5).

This is based on the regimen adopted in our ongoing study with younger subjects but involves one less cycle to minimize weight loss, and other potential side effects in elderly subjects. The primary endpoint will be diet safety: the side effects of the diet will be assessed by weight change, headaches, blood pressure, and by closely monitoring body composition, i.e. lean body mass, by bio-impedance measurements, handgrip strength and DEXA scans. The secondary endpoints will include: i) the compliance with the FMD; ii) the improvement of frailty parameters and prevention of disability in patients 65-80 years of age; iii) the improvement in immune parameters after 2 cycles of the diet and will be based on measures of improved T cell response, reduced inflammation, improved lymphoid/myeloid ratios and HSC/pre-HSC number, improved B cell numbers and CSR; iv) the efficacy of the influenza vaccine assayed by higher antibody titer levels. In addition, we will also document the number of flu episodes over the course of the trial. The trial will be carried out over a period of 3 years and all subjects will be vaccinated in the fall of each year with the current preparation of the inactivated virus influenza vaccine (Fluzone® high dose, Sanofi Pasteur Inc.) at the San Martino Hospital. During the flu season each participant will receive weekly reminders to call in and report any influenza like symptoms. These will include respiratory symptoms (cough, sore throat, shortness of breath and nasal stuffiness) and systemic symptoms (headache, malaise, fatigue, fever or feverishness and muscle aches). Flu-like illnesses will be documented based on the report of two respiratory symptoms or one respiratory and one systemic symptom when influenza is known to be circulating in the local community. A laboratory diagnosis of influenza illness will be confirmed from the results of nasopharyngeal swabs for virus culture, and/or a pre- to post-illness 4-fold or greater rise in antibody titer. Blood samples will be collected at baseline, after 2 diet cycles prior to vaccination and at 4 weeks after vaccination. The study period for this trial will be 2-5 day diet cycles (5-30-5) followed by a single flu vaccination per year. Each diet cycle will consist of 5 days of FMD followed by 30 days of the subjects' usual diet plus a plant-based daily protein and fat supplement (20 grams of proteins + 30 grams of olive oil and 30 grams (total) of walnuts, hazelnuts and almonds). This daily supplement serves at minimizing weight loss caused by the 5 day FMD. At the first visit, subjects will undergo a physical evaluation to assess health and frailty status, and medical history will be noted. Blood samples will be collected for baseline measurements.

Subjects in the FMD arm will be given a 2 cycle supply of the diet with instructions. Before receiving the second FMD cycle, patients will undergo a second physical examination at the geriatrics Clinic of the University of Genoa which will include the assessment of body composition and the administration of the second FMD cycle will only be allowed in the absence of a decrease in lean body mass (vs. baseline) as detected by BIA, handgrip strenght and by DEXA. Two weeks after completion of second diet cycle, subjects will return for physical evaluation, blood collection and vaccination. The third and final visit will be 4 weeks following vaccination, the subjects will undergo a physical evaluation and assessment of body composition and blood samples will be collected. At this visit, subjects will also be instructed to call the research coordinator to report malaise, discomfort, influenza or respiratory symptoms. Further follow-up will be over the phone to monitor onset of influenza. Nasopharyngeal swabs for virus culture and confirmation of influenza will be collected if possible within 72 hours of a subject reporting symptoms of influenza. DIET: Subjects randomized to the restricted diet arm (A) will be provided with all food to be consumed during each of the three cycles. The exact components of the diet will depend on results obtained in Task

1 but the diet is anticipated to be approximately 30% calorie restricted and 50% protein restricted but supplemented with 50% of the RDA in vitamins and minerals and also supplemented with both nonessential and essential amino acids identified in animal studies to be effective. Amino acid supplementation levels will depend on published clinical studies demonstrating safety and will not exceed the recommended daily levels. Subjects in the control arm will receive dietary advice per the standard practice of the treating physician. This will include consultation with a registered dietitian. A major focus will be placed on ensuring that subjects do not lose weight (unless obese at baseline) nor lose lean body mass. They will also be asked to maintain a diary of the food consumed and approximate amounts.

VACCINATION: Subjects will be vaccinated with the standard dose of the Fluzone® high dose vaccine from Sanofi Pasteur Inc. for each year. This vaccine has four times the normal antigen contained in the regular flu vaccine and is specially designed for individuals 60 and older.