Effect of an Antioxidants Mix on Cognitive Performance and Well Being: The Bacopa, Licopene, Astaxantina, Vitamin B12
Phase: NA
Conditions: Cognitive Dysfunction
Sponsor: A. Menarini Industrie Farmaceutiche Riunite S.r.l.

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Sponsor: A. Menarini Industrie Farmaceutiche Riunite S.r.l. 
 
Study Code MEIF/17/BAC-COG/001 
Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 1 of 56 
 
  
 
 
 
CLINICAL STUDY PROTOCOL 
 
 
Title: “Effect of an antioxidants mix on cognitive performance and well being: The 
Bacopa, Licopene, Astaxantina, Vitamin B12” (Acronym BLAtwelve study) 
 
STUDY CODE:   MEIF/17/BAC-COG/001 
 
 
Study type and design: randomized, double-blind, placebo-controlled, parallel-arm, 
superiority study 
 
 
Phase: not applicable (food supplement study) 
 
Protocol Version n° 0.0  
Emission Date: 09/10/2017 
 
 
 
Coordinating INVESTIGATOR:   
Prof. Giovambattista Desideri 
Institute / Address:  U.O.C. Geriatria e Lungodegenza - P.O. 
SS Filippo e Nicola di Avezzano 
Phone: +39 086 3499254; Fax: +39 086 3499244 
                                                                      e-mail giovambattista.desideri@cc.univaq.it 
 
CRO:   
 
 
 
Latis Srl 
Address: Viale Sauli 39/5 – 16121 Genova 
Phone: +39 010 562234; Fax: +39 010 540699 
e-mail info@latiscro.it  
 
SPONSOR :   
 
 
A. Menarini Industrie Farmaceutiche Riunite S.r.l.  
Address: Via Sette Santi 1-3, 50131, Firenze 
Phone: +39 055 5680459; Fax: + 39 055 56809961 
e-mail pfabrizzi@menarini.it 
 
CONFIDENTIALITY STATEMENT 
The information in this document contains trade secrets and commercial information that are privileged or confidential and may 
not be disclosed unless such disclosure is required by law. In any event, persons to whom the information is disclosed must be 
informed that the information is privileged or confidential and may not be further disclosed by them. These restrictions on 
disclosure will apply equally to all future information supplied to you which is indicated as privileged or confidential. 
 
 
 
 

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Sponsor: A. Menarini Industrie Farmaceutiche Riunite S.r.l. 
 
Study Code MEIF/17/BAC-COG/001 
Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 2 of 56 
 
  
 
 
 
T AB L E  O F  C O N T E N T S  
1. 
RESPONSIBILITIES .................................................................................................................................... 5 
2. 
PROTOCOL SYNOPSIS .............................................................................................................................. 6 
2.1 
STUDY SCHEME: ......................................................................................................................... 16 
2.2 
STUDY FLOW CHART ................................................................................................................... 17 
3. 
ABBREVIATIONS ...................................................................................................................................... 18 
4. 
BACKGROUND INFORMATION ............................................................................................................ 19 
5. 
TRIAL OBJECTIVES AND PURPOSE .................................................................................................... 20 
6. 
STUDY DESIGN .......................................................................................................................................... 21 
6.1 
RANDOMIZATION .................................................................................................................... 21 
6.2 
BLINDING / EMERGENCY UNBLINDING ............................................................................... 22 
6.3 
STUDY DURATION AND END OF STUDY DEFINITION ........................................................ 22 
7. 
SELECTION AND WITHDRAWAL OF SUBJECTS ............................................................................. 23 
7.1 
INFORMED CONSENT PROCEDURE .................................................................................... 23 
7.2 
INCLUSION CRITERIA ................................................................................................................. 24 
7.3 
EXCLUSION CRITERIA................................................................................................................ 24 
7.4 
WITHDRAWAL CRITERIA ........................................................................................................ 24 
8. 
TREATMENT OF SUBJECTS .................................................................................................................. 25 
8.1 
STUDY PRODUCTS ................................................................................................................. 25 
8.2 
HANDLING AND STORAGE .................................................................................................... 27 
8.3 
STUDY PRODUCT ACCOUNTABILITY AND RETURN .......................................................... 27 
8.4 
TREATMENT COMPLIANCE ................................................................................................... 28 
8.5 
ADMINISTRATION OF STUDY TREATMENT ......................................................................... 28 
8.6 
CONCOMITANT TREATMENTS .............................................................................................. 28 
8.7 
PROHIBITED PRIOR AND CONCOMITANT TREATMENTS ................................................. 29 
9. 
ASSESSMENT OF EFFICACY ................................................................................................................. 29 
9.1 
EFFICACY PARAMETERS ...................................................................................................... 29 
9.1.1 COGNITIVE PERFORMANCES ASSESSMENT .................................................................................... 29 
9.1.2 MOOD STATES ASSESSMENT ........................................................................................................ 30 
9.1.3 PSYCHOLOGICAL WELL-BEING ASSESSMENT ................................................................................. 30 
9.1.4 SEXUAL SATISFACTION ASSESSMENT ............................................................................................ 31 
9.1.5 BLOOD PRESSURE, METABOLIC VARIABLES AND PLASMA MARKERS OF OXIDATIVE STRESS ............ 31 
9.2 
STUDY PROCEDURES ........................................................................................................... 32 
10. ASSESSMENT OF SAFETY ........................................................................................................................ 34 
10.1. SAFETY DATA MANAGEMENT ................................................................................................ 34 
10.1.1 ADVERSE EVENT (AE) ................................................................................................................ 34 

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     CONFIDENTIAL                                                                                                              Page 3 of 56 
 
  
 
 
 
10.1.2 PRODUCT RELATIONSHIP ............................................................................................................ 34 
10.1.3 ADVERSE REACTION (AR) .......................................................................................................... 35 
10.1.4 SERIOUSNESS ............................................................................................................................ 35 
10.1.5 ADVERSE EVENT/ADVERSE REACTION INTENSITY ........................................................................ 36 
10.1.6 ADVERSE EVENT/ADVERSE REACTION EXPECTEDNESS ............................................................... 36 
10.1.7 COLLECTION, RECORDING AND REPORTING OF AES ................................................................... 36 
10.1.7.1 MANAGEMENT OF SERIOUS AES (SAES) INCLUDING LABORATORY ABNORMALITIES .................... 37 
⋅ 
REPORTING DUTIES OF THE INVESTIGATOR ................................................................................... 37 
⋅ 
REPORTING DUTIES OF THE SPONSOR ......................................................................................... 37 
10.1.7.2 MANAGEMENT OF NON-SERIOUS AES (NSAES) INCLUDING LABORATORY ABNORMALITIES ......... 38 
⋅ 
REPORTING DUTIES OF THE INVESTIGATOR ................................................................................... 38 
10.1.8 MANAGEMENT OF ANY LABORATORY ABNORMALITY ..................................................... 38 
10. STATISTICS ................................................................................................................................................ 38 
10.1 STATISTICAL METHODS (BLINDING AND RANDOMISATION) ............................................ 38 
10.2 DETERMINATION OF SAMPLE SIZE ...................................................................................... 39 
10.3 ANALYSIS POPULATIONS ...................................................................................................... 39 
10.4 ANALYSIS VARIABLES ............................................................................................................ 39 
10.5 STATISTICAL ANALYSIS ......................................................................................................... 40 
10.6 SUBGROUP ANALYSIS ........................................................................................................... 41 
10.7 PROTOCOL VIOLATIONS AND BLIND REVIEW .................................................................... 41 
10.8 STATISTICAL ANALYSIS PLAN............................................................................................... 41 
11. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ........................................................................ 42 
12. QUALITY CONTROL AND QUALITY ASSURANCE .......................................................................... 42 
12.1 DATA QUALITY CONTROL/STUDY MONITORING ......................................................................... 42 
12.2 CASE REPORT FORMS ............................................................................................................................ 43 
12.3 QUALITY ASSURANCE ............................................................................................................................ 44 
13. ETHICS ASPECTS ...................................................................................................................................... 44 
13.1 ETHICS COMMITTEE ................................................................................................................ 44 
13.2 SUBJECT’S INSURANCE .......................................................................................................... 45 
14. DATA PROTECTION LAWS COMPLIANCE ....................................................................................... 45 
15. DATA HANDLING AND RECORDS KEEPING .................................................................................... 46 
16. PUBLICATION POLICY AND RESULTS .............................................................................................. 47 
17. REFERENCES ............................................................................................................................................. 48 
18. PUBLIC PROTOCOL APPROVAL PAGE .............................................................................................. 50 
19. INVESTIGATOR’S APPROVAL PAGE .................................................................................................. 51 
 

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Study Code MEIF/17/BAC-COG/001 
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     CONFIDENTIAL                                                                                                              Page 4 of 56 
 
  
 
 
 
20. APPENDICES .............................................................................................................................................. 52 
20.1 APPENDIX  1: DECLARATION OF HELSINKI .................................................................................... 52 
 
 
 

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Study Code MEIF/17/BAC-COG/001 
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     CONFIDENTIAL                                                                                                              Page 5 of 56 
 
  
 
 
 
1. RESPONSIBILITIES 
 
Sponsor:  
  
A. Menarini Industrie Farmaceutiche Riunite S.r.l.  
 
  
Address: Via Sette Santi 1-3, 50131, Firenze 
 
Phone: +39 055 5680459  
 
Fax: +39 055 56809961                     
 
e-mail: pfabrizzi@menarini.it 
 
Clinical Trial Coordinator:                Eleonora Droghetti 
  
  
Address: Via Sette Santi 1-3, 50131, Firenze 
 
Phone: +39 055 56809665 
                   Fax: +39 055 56809961                      
 
e-mail: edroghetti@menarini.it 
 
Study Medical Expert (SME):            Paolo Fabrizzi 
 
  
Address: Via Sette Santi 1-3, Firenze 
 
Phone: +39 055 5680459                     
 
Fax: +39 055 56809961                      
 
e-mail: PFabrizzi@menarini.it 
 
Coordinating Investigator:     
Prof. Giovambattista Desideri  
Address: U.O.C. Geriatria e Lungodegenza - P.O. SS 
Filippo e Nicola di Avezzano 
 
Phone: +39 086 3499254                      
 
Fax: +39 086 3499244                     
 
e-mail: giovambattista.desideri@cc.univaq.it 
 
CRO:  
  
Latis S.r.l. 
 
  
Address: Viale Sauli 39/5 – 16121 Genova 
 
Phone: +39 010 562234                      
 
Fax: +39 010 540699                    
 
e-mail: info@latiscro.it 
 
   Statistician:  
  
Fabio Montanaro 
 
  
Address: Viale Sauli 39/5 – 16121 Genova 
 
Phone: +39 010 562234                      
 
Fax: +39 010 540699                    
 
e-mail: montanaro@latiscro.it 
 
 
 
 
 

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Study Code MEIF/17/BAC-COG/001 
Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 6 of 56 
 
  
 
 
 
 
2. PROTOCOL SYNOPSIS 
 
Title 
Acronym  
Study Code  
Effect of an antioxidants mix on cognitive performance and well-being: 
The Bacopa, Licopene, Astaxantin, Vitamin B12 
BLAtwelve study 
MEIF/17/BAC-COG/001 
Investigational Product 
Reference Therapy 
(comparator) 
Dose Regimen 
A mix of four bioactive compounds: bacopa, lycopene, astaxanthin, 
vitamin B12. 
Placebo. 
Once daily. 
Study Type and 
Design 
9-weeks double-blind, randomized, placebo-controlled, parallel-arm 
superiority study. 
Phase
Not applicable. 
Background Rationale 
Long-term oxidative stress is believed to represent one of the most 
important factor contributing to the decline of cognitive function often 
observable with aging. Oxidative stress, due to the generation of free 
radical resulting from normal metabolism, is usually maintained at low 
level 
by 
antioxidant 
system. 
However, 
in 
some 
conditions 
oxidant/antioxidant balance can be perturbed by increased generation of 
reactive oxygen species and/or decreased endogenous ability to 
counteract them [Praticò et al., 2008].  
Brain tissue is highly sensitive to oxidative stress because it has a high 
request for oxygen and has a relative weakness of antioxidant systems. 
Furthermore, brain also contains high levels of polyunsaturated fatty 
acid, making it more vulnerable to oxidative injuries [Praticò et al., 2008]. 
Altered mitochondrial function, the amyloid β peptides and the presence 
of unbound trace metal ions represent the most investigated potential 
sources of oxidative stress in the brain [Reddhy et al., 2005; Mattson 
MP, 2004]. Depending on the biomolecules attacked by reactive oxygen 
species, oxidative stress can promote peroxidation of protein, lipid and 
nucleic acids thus favouring the onset and progression of cognitive 
dysfunction during aging [Praticò D et al., 2008].   
During the last few years an increasing interest has been focused on 
antioxidants such as carotenoids, flavonoids and vitamins as potentially 
useful agents in the prevention of the onset and progression of cognitive 
dysfunction [Rao et al., 2013; de Rijk et al., 1997; Deschamps et al., 
2001; Engelhart et al., 2002]. 
In this regard, lycopene, a lipid-soluble carotenoid compound, well 
represented in tomatoes and red fruits, including watermelon, pink 
grapefruit and guava, has been proposed as a brain protective agent. In 
vitro studies indicated that lycopene protects against neuronal death 
induced by different neurotoxic compounds, including 1-methyl-4-
phenylpyridinium (MPP+), methylmercury, amyloid β, trimethyltin and 6-

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Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 7 of 56 
 
  
 
 
 
hydroxydopamine [Di Matteo et al., 2009; Yi et al.;2013]. Furthermore, 
animal experiments using rat models demonstrated that lycopene 
prevents brain injury caused by focal or global ischemia and reperfusion 
[Fujita et al., 2013] and alleviates cognition dysfunction induced by 
colchicine and rotenone [Kaur et al., 2011]. A population-based follow-up 
study demonstrated that males in the highest quartile of serum lycopene 
concentrations exhibited 59 and 55% lower risks of ischemic stroke 
when compared with males in the lowest quartile [Karppi et al., 2012].  
Astaxanthin is a xanthophyll carotenoid nutrient known for having potent 
antioxidant and anti-inflammatory actions thanks to molecular properties 
that precisely position it within cell membranes and circulating 
lipoproteins [Kidd, 2011; Lim SY et al. 2016]. Astaxanthin has shown a 
variety of brain benefits under experimental conditions [Kidd, 2011; Lim 
SY et al. 2016]. 
Bacopa monniera is a creeping herb extensively investigated for its 
pharmacological and therapeutic effects. Its ethanol extract contains a 
mixture of triterpenoid saponins designated as bacosides A and B 
[Chatterjee et al., 1963; Chatterjee et al., 1965]. In vitro studies using 
Bacopa Monnieri have shown that it inhibits free radical formation and 
DNA damage in a dose dependent manner [Russo et al.;2003]. 
Promising indications for use in humans include improving cognition in 
the elderly and in patients with neurodegenerative disorders [Stough C 
et al., 2013]. 
Vitamin B12, also called cobalamin, is a water-soluble vitamin that has a 
key role in the normal functioning of the brain and nervous system, and 
for the formation of red blood cells [Aisen et al.; 2008]. Serum levels in 
the subclinical low-normal range (<250 ρmol/L) are associated with 
Alzheimer's disease, vascular dementia and Parkinson's disease [Moore 
et al.; 2012] while some evidences suggest that vitamin B12 
administration might be useful in preserving brain health. 
Starting from these evidences it is conceivable that a food supplement 
containing bacopa, lycopene, astaxantina, vitamin B12 could be effective 
in improving brain health. 
Aim of the Study 
To evaluate the influence of a mix of four bioactive compounds – 
bacopa, lycopene, astaxanthin and vitamin B12 – on cognitive 
performance, mood state and well-being in subjects aged ≥ 60 years 
with no evidence of cognitive dysfunction. 
Primary Objective 
Evaluation of changes in Trail Making Test (TMT) scores from baseline 
(V2) to 8 weeks of treatment (V4), analyzed in the following hierarchical 
order: TMT-B, TMT-A and TMT B-A. 
Secondary Objectives 
1. Change of Verbal Fluency Test (VFT) score from baseline (V2) to 8 
weeks of treatment (V4). 
2. Changes of Montreal Cognitive Assessment (MoCA) score from 
baseline (V2) to 8 weeks of treatment (V4). 
3. Changes of Mini Mental State Examination (MMSE) score from 

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Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 8 of 56 
 
  
 
 
 
baseline (V2) to 8 weeks of treatment (V4). 
4. Changes of Rey Auditory Verbal Learning Test (AVLT) from baseline 
(V2) to 8 weeks of treatment (V4). 
5. Changes of psychological well-being as assessed by General Health 
Questionnaire (GHQ-12) from baseline (V2) to 8 weeks of treatment 
(V4). 
6. Changes of mood states as assessed by the Profile of Mood Stated 
(POMS) from baseline (V2) to 8 weeks of treatment (V4). 
7. Changes of sexual satisfaction as evaluated by the New Sexual 
Satisfaction Scale (NSSS) from baseline (V2) to 8 weeks of treatment 
(V4). 
8. Changes of metabolic parameters from baseline (V2) to 4 weeks of 
treatment (V3) and from baseline (V2) to 8 weeks of treatment (V4): 
     A. glucose  
     B. insulin 
     C. Homeostatic Model Assessment - Insulin Resistance (HOMA-IR)      
derived from glucose and insulin 
     D. total cholesterol  
     E. Low Density Lipoprotein (LDL) cholesterol 
     F. High Density Lipoprotein (HDL) cholesterol 
     G. triglycerides  
     H. uric acid 
9. Changes of plasma markers of oxidative stress from baseline (V2) to 
4 weeks of treatment (V3) and from baseline (V2) to 8 weeks of 
treatment (V4): 
     A. 8-iso-Prostaglandin F2alpha  
     B. Plasma malondialdehyde 
Subjects 
characteristics 
Number of Subjects 
Cognitively-intact individuals aged 60 years or more. 
No. 80 (including a possible 15% for screening failure and drop-out) 
participants with a ratio of 1:1 for the 2 treatment groups.   
Number of Centers & 
Countries  
1 center, Geriatric Division, P.O. SS Filippo e Nicola, Avezzano, Italy 
Study Duration 
(specify different study 
phases) 
First Pts In (FPI) 
Last Pts Last Visit 
(LPLV): 
4 months 
 
January 2018 
April 2018 

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Study Code MEIF/17/BAC-COG/001 
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Study Procedures  
Cognitive functions, mood state, self-perceived well being and sexual 
satisfaction will be assessed at baseline and after 8 weeks of regular 
consumption of the mix of the four antioxidant compounds. 
Furthermore, blood pressure, metabolic variables and plasma markers of 
oxidative stress will be assessed at baseline, after 4 and after 8 weeks of 
regular consumption of the antioxidant compounds. 
 
A. COGNITIVE PERFORMACES WILL BE EVALUATED BY TMT, 
VFT, MOCA, MMSE AND AVLT: 
TMT, which explores visual-conceptual and visual-motor tracking, is a 
frequently used neuropsychological test because of its sensitivity to brain 
damage. TMT is administered in two parts. Part A is a visual-scanning, 
timed task where participants are asked to connect with lines 25 circles 
numbered from 1 to 25 as quickly as possible. The test is terminated 
after 5 minutes even if not completed. In Part B participants are asked to 
connect circles containing numbers (from 1 to 13) or letters (from A to L) 
in an alternate numeric/alphabetical order. The test is terminated in 
every case after 10 minutes even if not completed. The TMT B-A score 
calculated as the difference between TMT-B and TMT-A times is 
considered a measure of cognitive flexibility relatively independent of 
manual dexterity. 
VFT is a short test of verbal functioning. Participants are given 1 min to 
produce as many unique words as possible within a semantic category 
(category fluency) or starting with a given letter (letter fluency). The 
participant's score in each task is the number of unique correct words. 
MoCA assesses a broader array of cognitive domains (e.g., 
attention/executive functioning, visuospatial abilities and language) and it 
has been demonstrated to be able to detect cognitive impairment with 
scores ranging from 0 to 30. 
MMSE is a widely used screening tool for cognitive impairment and 
covers five areas of cognitive function including orientation, attention, 
calculus, recall and language with scores ranging from 0 to 30. 
AVLT is a neuropsychological assessment designed to evaluate the 
nature and severity of memory dysfunction and to track changes in 
memory function. The examiner reads aloud a list of 15 words at the rate 
of one per second. The participant is then asked to repeat all words from 
the list that she/he can remember. This procedure is carried out a total of 
five times. After a 15-minute delay, the participant is again asked to 
recall as many words as possible from the first list. The participant is 
then requested to read a list of words and asked to indicate whether 
each word was from the first list. The score for each trial is the number of 
words correctly recollected. 
 
B. MOOD STATES WILL BE ASSESSED BY POMS: 
POMS is a widely used tool in assessing mood states that has already 

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Study Code MEIF/17/BAC-COG/001 
Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 10 of 56 
 
  
 
 
 
been validated for Italy. Higher scores reflect mood decrements, except 
for the vigor subscale, where higher scores reflect improved mood. 
Taking into consideration the two components characteristic of the 
POMS that measure negative affect and positive affect, respectively, the 
data collected with the questionnaire will be analyzed both as global 
score and by separately considering a Positive Affect Scale (PAS-
POMS, 48 items, score ranging from 0 to 192) and a Negative Affect 
Scale (NAS-POMS, 10 items, score ranging from 0 to 40). 
 
C. PSYCHOLOGICAL WELL-BEING WILL BE ASSESSED BY GHQ-
12: 
GHQ-12 is a relevant instrument for measuring psychological well-being. 
It has been extensively evaluated in terms of validity and reliability as a 
one dimensional indicator of the severity of psychological morbidity, and 
it has already been validated for Italy. 
 
D. SEXUAL SATISFACTION WILL BE EVALUATED BY THE NSSS: 
NSSS is a 20 item, multidimensional, composite measure of sexual 
satisfaction based on a five-dimension, conceptual model that 
emphasized the importance of multiple domains of sexual behavior 
including sexual sensations, sexual awareness and focus, sexual 
exchange, emotional closeness, and sexual activity. 
 
E. BLOOD PRESSURE, METABOLIC VARIABLES, AND PLASMA 
MARKERS OF OXIDATIVE STRESS WILL BE ASSESSED AT 
BASELINE, AFTER 4 AND 8 WEEKS OF REGULAR CONSUMPTION 
OF THE ANTIOXIDANT COMPOUNDS: 
- clinic systolic and diastolic blood pressure levels will be recorded in the 
morning, using a validated oscillometric device with appropriately sized 
cuffs on the non-dominant upper arm after 5 min resting in a seated 
position; the first blood pressure measurement will be discarded and the 
subsequent three consecutive blood pressure readings, taken at 3-min 
intervals, will be recorded. The average of these latter measures will be 
considered for statistical analysis. [O'Brien et al., 2005]. 
- Metabolic profile will be evaluated by the following variables: 
A. glucose (normal range 60-100 mg/dL) 
B. insulin (normal range 2.7-10.4 mcUI/mL) 
C. HOMA-IR (derived from glucose and insulin) 
D. total cholesterol (normal range 150-200 mg/dL) 
E. LDL cholesterol (normal range 40-130 mg/dL) 
F. HDL cholesterol (normal range 35-90 mg/dL) 
G. triglycerides (normal range 60-170 mg/dL) 
H. uric acid (normal range 3.5 – 7.0 mg/dL) 
- Oxidative stress will be evaluated by measuring circulating levels of 2 

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Study Code MEIF/17/BAC-COG/001 
Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 11 of 56 
 
  
 
 
 
soluble biomarkers: 
A. 8-iso-Prostaglandin F2alpha (no predefined normal range) 
B. Plasma malondialdehyde (no predefined normal range) 
 
 
The study will last approximately two months for each subject.  
A total of 4 study visits will be performed as follows: 
VISIT 1 (screening, day -7 to Visit 2):  
Subjects who provide written informed consent will undergo screening 
assessments to evaluate eligibility for the study. Subjects who meet all 
inclusion criteria and none of the exclusion criteria will be eligible for the 
study and will undergo the following evaluations: 
1) Collection of medical history and information about eventual 
concomitant medications 
2) Complete physical evaluation including a) anthropometric parameters 
(height, weight, waist circumference), b) cardiovascular system, c) 
respiratory system, d) gastrointestinal system and e) respiratory system 
3) Blood pressure measurement 
Subjects eligible for the study will meet with a dietician in order to 
evaluate current diet habits and correct any nutritional insufficiencies. 
Participants will be then instructed to maintain their usual lifestyle and 
intake of fruits and vegetables and to avoid any food supplement. 
 
VISIT 2 (randomization/baseline, 7 days after V1): 
Subjects eligible for the study will undergo the following evaluations: 
1) Collection of information about eventual concomitant medications 
2) Complete physical evaluation including a) anthropometric parameters 
(weight, waist circumference), b) cardiovascular system, c) respiratory 
system, d) gastrointestinal system and e) respiratory system 
3) Blood pressure measurement 
4) Neuropsychological evaluation (TMT, VFT, MoCA, MMSE, AVLT) 
5) Evaluation of mood state (POMS) 
6) Evaluation of quality of life (GHQ12) 
7) Evaluation of sexual satisfaction (NSSS) 
8) Blood samples to assess metabolic parameters (glucose, insulin, total 
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, uric acid) 
9) Blood samples to assess plasma markers of oxidative stress (8-iso-
Prostaglandin F2alpha, malondialdehyde) 
Subjects will be randomly assigned to two parallel groups in a 1:1 ratio 
and treated with a mix of four bioactive compounds: bacopa, lycopene, 

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Study Code MEIF/17/BAC-COG/001 
Protocol Version n° 0.0 – 09 October 2017 
  
 
 
 
     CONFIDENTIAL                                                                                                              Page 12 of 56 
 
  
 
 
 
astaxanthin and vitamin B12, (1025,00 mg/daily) or placebo (1025,00 
mg/daily, for 2 months. 
During Visit 2 one month of treatment of IMP (45 tablets) will be given to 
each subject. 
 
VISIT 3 (Week 4, treatment period, 30 days ± 3 days after V2): 
Subjects will undergo to the following evaluations: 
1) Collection of information about eventual concomitant medications 
2) Complete physical evaluation including a) anthropometric parameters 
(weight, waist circumference), b) cardiovascular system, c) respiratory 
system, d) gastrointestinal system and e) respiratory system 
3) Blood pressure measurement 
4) Blood samples to assess metabolic parameters (glucose, insulin, total 
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, uric acid) 
5) Blood samples to assess plasma markers of oxidative stress (8-iso-
Prostaglandin F2alpha, malondialdehyde) 
Eventual adverse events will be checked and evaluated. 
IMP return/compliance check of first month of therapy. 
During Visit 3 another one month of treatment of IMP (45 tablets) will be 
given to each subject. 
 
VISIT 4 (Week 8, final visit, 30 days ± 3 days after V3): 
Subjects will undergo to the following evaluations: 
1) Collection of information about eventual concomitant medications 
2) Complete physical evaluation including a) anthropometric parameters 
(weight, waist circumference), b) cardiovascular system, c) respiratory 
system, d) gastrointestinal system and e) respiratory system 
3) Blood pressure measurement 
4) Neuropsychological evaluation (TMT, VFT, MoCA, MMSE, AVLT) 
5) Evaluation of mood state (POMS) 
6) Evaluation of quality of life (GHQ12) 
7) Evaluation of sexual satisfaction (NSSS) 
8) Blood samples to assess metabolic parameters (glucose, insulin, total 
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, uric acid) 
9) Blood samples to assess plasma markers of oxidative stress (8-iso-
Prostaglandin F2alpha, malondialdehyde) 
Eventual adverse events will be checked and evaluated. 

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     CONFIDENTIAL                                                                                                              Page 13 of 56 
 
  
 
 
 
IMP return/compliance check. 
Inclusion Criteria 
1) Subjects aged ≥60 years. 
2) Subjects who provide written Informed Consent to the study. 
Exclusion Criteria 
1) Subjects with cognitive dysfunctions or clinically significant coexisting 
medical conditions (cardiovascular disease, cerebrovascular events, 
overt dementia defined by MMSE <27 or other neurological disorders, 
thyroid disorders, or inflammatory diseases) 
2) Subjects with a score on the Geriatric Depression Scale (GDS) >11 in 
order to avoid confounding due to the influence of concomitant 
depression on the performance on cognitive tests 
3) Current smokers 
4) Habitual users of antioxidant supplements (including vitamins C and 
E) 
5) Habitual consumers of chocolate or other cocoa products (daily 
consumption of any amount) 
6) Subjects under treatments with medications known to have 
antioxidant properties (including statins and glitazones) or to interfere 
with cognitive functions (including benzodiazepines and antidepressants)
7) Subjects with hypersensitivity to any component of the study 
medications 
8) Subjects who are participating in or having participated in another 
clinical trial within the previous three months. 
Efficacy Evaluation 
 
• Primary Efficacy Endpoint: 
changes of TMT scores from baseline (V2) to 8 weeks of treatment 
(V4). Changes will be assessed according to the following 
hierarchical order: TMT-B, TMT-A, TMT B-A. 
• Secondary Efficacy Endpoints: 
a) changes of VFT score from baseline (V2) to 8 weeks of treatment 
(V4); 
b) changes of MoCA score from baseline (V2) to 8 weeks of 
treatment (V4); 
c) changes of MMSE score from baseline (V2) to 8 weeks of 
treatment (V4); 
d) changes of AVLT score baseline from baseline (V2) to 8 weeks of 
treatment (V4); 
d) changes of GHQ-12 score baseline from baseline (V2) to 8 weeks 
of treatment (V4); 
e) changes of POMS score from baseline (V2) to 8 weeks of 
treatment (V4);  

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     CONFIDENTIAL                                                                                                              Page 14 of 56 
 
  
 
 
 
f) changes of NSSS score from baseline (V2) to 8 weeks of 
treatment (V4);  
g) changes of metabolic parameters (glucose, insulin, HOMA-IR , 
total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides 
and uric acid from baseline (V2) to 4 weeks of treatment (V3) and 
from baseline (V2) to 8 weeks of treatment (V4);  
h) changes of circulating levels of 8-iso-Prostaglandin F2alpha and 
malondialdehyde from baseline (V2) to 4 weeks of treatment (V3) 
and from baseline (V2) to 8 weeks of treatment (V4). 
• Exploratory analyses:  
a) correlation between changes of cognitive scores (TMT, VFT, 
MoCA, MMSE, AVLT) and changes of blood pressure and 
metabolic parameters including indices of oxidative stress; 
b) assessment of the effect of time in the change for the primary 
endpoint. 
Safety Evaluation 
 
All adverse events will be duly assessed for identifying any emergent 
safety finding. 
Statistical 
Assumptions 
 
 
 
• 
Sample size calculation 
The study will be based on an estimated sample size of 68 subjects, with 
a ratio of 1:1 for the 2 treatment groups, which has been calculated to be 
adequate to achieve 90% power to detect a large effect size (f=0.40) 
using an ANCOVA model with a baseline covariate and an α of 0.05 
between treatment and control (G*power version 3.1.9.2). To consider a 
possible 15% dropout rate, 80 subjects will be enrolled. The three 
primary variables (changes in TMT-B, TMT-A and TMT B-A scores) will 
be analyzed in a hierarchical order and no adjustment for sample size is 
needed. 
All randomized subjects receiving at least a treatment dose and having 
the post-randomization efficacy evaluation will be included in the ITT 
population for efficacy analysis. All randomized subjects without protocol 
deviations affecting the primary endpoint will be included in the Per-
Protocol (PP) analysis. All randomized subjects receiving at least a 
treatment dose will be included in the safety analysis. 
• 
Efficacy analyses 
The primary variables (changes of TMT-B, TMT-A and TMT B-A scores), 
will be analyzed in a hierarchical order using an ANCOVA model with the 
treatment group (main effect) and baseline as covariates. All the other 
continuous variables (changes of test scores, changes of metabolic 
parameters, changes of circulating levels of 8-iso-Prostaglandin F2alpha 
and malondialdehyde) will be analyzed by using an ANCOVA model with 
the treatment group (main effect) and baseline as covariates. As 
additional covariate for testing the homogeneity of the regression 
coefficients, the interaction term of treatment*baseline will be included in 
the model and removed if not significant. 

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     CONFIDENTIAL                                                                                                              Page 15 of 56 
 
  
 
 
 
Categorical variables (gender, eventually present cardiovascular risk 
factors or pharmacological treatment) will be tested by using a Chi-
Square test or the Fisher exact test if necessary.  
• 
Exploratory analyses  
Pearson correlation will be used to evaluate correlations between 
changes of cognitive scores (TMT, VFT, MoCA, MMSE, AVLT) and 
changes of blood pressure and metabolic parameters including indices 
of oxidative stress. Spearman nonparametric correlation will be also 
applied when one or both of the variables are not assumed to be 
normally distributed and interval (but are assumed to be ordinal).  
In order to evaluate and quantify the effect of time itself (i.e. 8 weeks 
after the treatment intake) in the change for the primary endpoint a two-
way analysis of variance (treatment*time) with time as a repeated 
measure will be also implemented. 
• 
Safety analysis 
Adverse events will be summarized and compared between the two 
treatment groups. Study product tolerability will be investigated and 
compared between treatment groups. 
 
 
 

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     CONFIDENTIAL                                                                                                              Page 16 of 56 
 
  
 
 
 
 
2.1 
Study Scheme:   
 
 
 
 
 
 
      Treatment A ------------ Follow Up ------------ Final Visit                                         
  
Screening --------------- 
 
 
 
                  Treatment B ------------- Follow Up ------------- Final Visit                                       
        V1------------------------------V2----------------------------V3---------------------------------V4 
(day -7 to Visit 2)                     (7 days after V1)                (30 days ± 3 days after V2)              (30 days ± 3 days after V3) 
 
 
 

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     CONFIDENTIAL                                                                                                              Page 17 of 56 
 
  
 
 
 
 
2.2 
Study Flow Chart 
 
 
 
NOTE:  
a. Blood pressure will be taken with the subject in the sitting position (subject seated for 5 minutes). 
Heart rate will be taken in a resting state (5 minutes). 
b. Blood samples will be taken after an overnight fasting 
c. Neuropsychological testing will be performed in a quiet room before taking the daily dose of the 
tested food supplement 
 
 
Day -7 to 1 
 
 
Day 1 
 
 
Week 4 (± 3 
days) 
Week 8 (± 3 
days) 
 
Last visit 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Screening 
Randomization
Treatment  
Treatment  
Inclusion/exclusion criteria 
 
 
 
 
Informed consent 

 
 
 
Randomization 
 

 
 
Medical history 

 
 
 
Concomitant medications 




Physical examination 




Dietitian visit 

 
 
 
Blood pressure 




MMSE, GDS 

 
 
 
Neuropsychological evaluation (TMT, VFT, 
MoCA, MMSE, AVLT) 
 

 

Evaluation of mood state (POMS) 
 

 

Evaluation of quality of life (GHQ-12) 
 

 

Evaluation of sexual satisfaction (NSSS) 
 

 

Blood samples to assess metabolic 
parameters (glucose, insulin, total 
cholesterol, LDL cholesterol, HDL 
cholesterol, triglycerides, uric acid) 
 



Blood samples to assess plasma markers 
of oxidative stress (8-iso-Prostaglandin 
F2alpha, malondialdehyde) 
 
 



IMP dispense treatment 
 


 
IMP return/compliance check 
 
 


Adverse events 
 




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     CONFIDENTIAL                                                                                                              Page 18 of 56 
 
  
 
 
 
3. ABBREVIATIONS 
AE 
Adverse Event 
AR 
Adverse Reaction 
AVLT 
Rey Auditory Verbal Learning Test 
CA 
Competent Authority 
CRA 
Clinical Research Associate 
CRF 
Case Report Forms 
CRO 
Contract Research Organisation 
DCF 
Data Clarification Form 
DNA 
DeoxyriboNucleic Acid 
GCP 
Good Clinical Practice 
GDS 
Geriatric Depression Scale 
GHQ-12 
General Health Questionnaire 12 
HOMA-IR 
Homeostasis Model Assessment of Insulin Resistance  
IEC 
Independent Ethics Committee 
ICF 
Informed Consent Form 
ICH 
International Conference of Harmonisation 
ICH-GCP 
International Conference of Harmonisation  - Good Clinical Practice 
ITT 
Intent to Treat 
LDL 
Low-density-lipoprotein 
HDL 
High-density-lipoprotein 
MedDRA 
Medical Dictionary for Regulatory Activities 
MMSE 
Mini Mental State Examination 
MoCA 
Montreal Cognitive Assessment 
NSAE 
Non-Serious Adverse Event 
NSSS 
New Sexual Satisfaction Scale 
POMS 
Profile Of Mood Stated 
PT 
Preferred Term 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAR 
Serious Adverse Reaction 
SOC 
System/Organ Class 
TMF 
Trial Master File 
TMT 
Trail Making Test 
VFT 
Verbal Fluency Test 
 
 
 

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     CONFIDENTIAL                                                                                                              Page 19 of 56 
 
  
 
 
 
4. BACKGROUND INFORMATION 
Long-term oxidative stress is believed one of the most important factor contributing to the 
decline of cognitive function often observable with aging. Oxidative stress, due to the 
generation of free radical resulting from normal metabolism, is usually maintained at low 
level by antioxidant system. However, in some conditions oxidant/antioxidant balance can 
be perturbed by increased generation of reactive oxygen species and/or decreased 
endogenous ability to counteract them [Praticò et al., 2008].  
 
Brain tissue is highly sensitive to oxidative stress because it has a high request for oxygen 
and has a relative weakness of antioxidant systems. Furthermore, brain also contains high 
levels of polyunsaturated fatty acid, making it more vulnerable to oxidative injuries [Praticò 
et al., 2008]. Altered mitochondrial function, the amyloid β peptides and the presence of 
unbound trace metal ions represent the most investigated potential sources of oxidative 
stress in the brain [Reddhy et al., 2005; Mattson MP, 2004]. Depending on the 
biomolecules attacked by reactive oxygen species, oxidative stress can promote 
peroxidation of protein, lipid and nucleic acids thus favouring the onset and progression of 
cognitive dysfunction during aging [Praticò D et al., 2008].   
 
During the last few years an increasing interest has been focused on antioxidants such as 
carotenoids, flavonoids and vitamins as potentially useful agents in the prevention of the 
onset and progression of cognitive dysfunction [Rao et al., 2013; de Rijk et al., 1997; 
Deschamps et al., 2001; Engelhart et al., 2002]. 
 
In this regard, lycopene, a lipid-soluble carotenoid compound, well represented in 
tomatoes and red fruits, including watermelon, pink grapefruit and guava, has been 
proposed as a brain protective agent. In vitro studies indicated that lycopene protects 
against neuronal death induced by different neurotoxic compounds, including 1-methyl-4-
phenylpyridinium (MPP+), methylmercury, amyloid β, trimethyltin and 6-hydroxydopamine 
[Di Matteo et al., 2009; Yi et al.;2013]. Furthermore, animal experiments using rat models 
demonstrated that lycopene prevents brain injury caused by focal or global ischemia and 
reperfusion [Fujita et al., 2013] and alleviates cognition dysfunction induced by colchicine 
and rotenone [Kaur et al., 2011]. A population-based follow-up study demonstrated that 
males in the highest quartile of serum lycopene concentrations exhibited 59 and 55% 
lower risks of ischemic stroke when compared with males in the lowest quartile [Karppi et 
al., 2012].  
 
Astaxanthin is a xanthophyll carotenoid nutrient known for having potent antioxidant and 
anti-inflammatory actions thanks to molecular properties that precisely position it within 
cell membranes and circulating lipoproteins [Kidd, 2011; Lim SY et al. 2016]. Astaxanthin 

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     CONFIDENTIAL                                                                                                              Page 20 of 56 
 
  
 
 
 
has shown a variety of brain benefits under experimental conditions [Kidd, 2011; Lim SY 
et al. 2016]. 
 
Bacopa monniera is a creeping herb extensively investigated for its pharmacological and 
therapeutic effects. Its ethanol extract contains a mixture of triterpenoid saponins 
designated as bacosides A and B [Chatterjee et al., 1963; Chatterjee et al., 1965]. In vitro 
studies using Bacopa Monnieri have shown that it inhibits free radical formation and DNA 
damage in a dose dependent manner [Russo et al.;2003]. Promising indications for use in 
humans include improving cognition in the elderly and in subjects with neurodegenerative 
disorders [Stough C et al., 2013]. 
 
Vitamin B12, also called cobalamin, is a water-soluble vitamin that has a key role in the 
normal functioning of the brain and nervous system, and for the formation of red blood 
cells [Aisen et al.; 2008]. Serum levels in the subclinical low-normal range (<250 ρmol/L) 
are associated with Alzheimer's disease, vascular dementia and Parkinson's disease 
[Moore et al.; 2012] while some evidences suggest that vitamin B12 administration might 
be useful in preserving brain health. 
 
Starting from these evidences it is conceivable that a food supplement containing bacopa, 
lycopene, astaxantina, vitamin B12 could be effective in improving brain health. 
The purpose of this study is to evaluate if the mix of these four bioactive compounds, 
orally administered for 8 consecutive weeks, can be of help on cognitive performance, 
mood state and well-being in a target population with no evidence of cognitive 
dysfunctions. 
The study will be conducted in compliance with this protocol, GCP and the applicable 
regulatory requirements. 
 
5. TRIAL OBJECTIVES AND PURPOSE 
The aim of this study is to evaluate the influence of a mix of four bioactive compounds – 
bacopa, lycopene, astaxanthin and vitamin B12 – on cognitive performance, mood state 
and well-being in subjects aged ≥ 60 years with no evidence of cognitive dysfunction. 
The primary objective of the study is to evaluate the changes in TMT scores from baseline 
(V2) to 8 weeks of treatment (V4), analyzed in the following hierarchical order: TMT-B, 
TMT-A and TMT B-A 
Secondary objectives of this study are to evaluate changes from baseline (V2) to 8 weeks 
of treatment (V4) in Verbal Fluency Test (VFT) score, Montreal Cognitive Assessment 
(MoCA) score, Mini Mental State Examination (MMSE) score, Rey Auditory Verbal 
Learning Test (AVLT), psychological well-being as assessed by General Health 

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     CONFIDENTIAL                                                                                                              Page 21 of 56 
 
  
 
 
 
Questionnaire (GHQ-12), mood states as assessed by the Profile of Mood Stated 
(POMS), sexual satisfaction as evaluated by the New Sexual Satisfaction Scale (NSSS).  
Changes of metabolic parameters from baseline (V2) to 4 weeks of treatment (V3) and 
from baseline (V2) to 8 weeks of treatment (V4) will be also evaluated as secondary 
objectives (glucose, insulin, HOMA-IR, total cholesterol, LDL cholesterol, HDL cholesterol, 
triglycerides, uric acid). 
Changes of plasma markers of oxidative stress from baseline (V2) to 4 weeks of treatment 
(V3) and from baseline (V2) to 8 weeks of treatment (V4) will be evaluated as secondary 
objectives (8-iso-Prostaglandin F2alpha, Plasma malondialdehyde). 
Finally the safety and tolerability of the study product will be assessed. 
6. STUDY DESIGN 
This study has been designed as 9 weeks double-blind, randomized, placebo-controlled, 
parallel-arm, superiority study. 
The study will be conducted in 1 Italian clinical site and will involve about 80 subjects.  
 
Subjects will be randomly allocated to one of the following groups: 
- 
Group I: mix of the four bioactive compounds (bacopa, lycopene, astaxanthin and 
vitamin B12), once a day for 8 weeks per os; 
- 
Group II: placebo, once a day for 8 weeks per os. 
 
 
 
The study will be double blind. Neither the study staff at clinical sites (Investigators, 
nurses, pharmacist) nor the subject will be aware of the treatment assigned.  
 
Each participant will attend 4 visits over a total period of about 9 weeks. 
 
6.1 RANDOMIZATION 
Each subject for whom written consent is obtained will be assigned a five-digit screening 
code, consisting of the site number (e.g. 01) and a progressive number within the site: for 
example the first subject screened will be assigned the code 01-001, the second one 01-
002 etc. 
All screened subjects will receive the code irrespective of whether or not they will be 
randomized. If a subject discontinues from the study at any time, the code will not be re-
used. 
All subjects who will sign the informed consent and receive the screening code will be 
entered into a Subject’s Register, containing the name and surname of the subjects and 
the date they have signed the consent form. 
The randomization list will be generated by Latis S.r.l., using the PROC PLAN of SAS 9.4 
for Windows (SAS Institute Inc., Cary, NC, USA). Once eligibility of a subject is 
established (see Inclusion/Exclusion Criteria) the study treatment will be assigned through 

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     CONFIDENTIAL                                                                                                              Page 22 of 56 
 
  
 
 
 
envelopes randomization system: the site will be provided with sealed envelopes, 
numbered in progressive number starting from R-001, containing the treatment kit to be 
assigned to the subject. The Investigator will open the first available envelope in 
progressive order. Inside the envelope there will be the kit number of the treatment to be 
assigned to that subject. Opened envelopes will be signed and dated by the Investigator 
and stored in the Investigator’s File.  
The Investigator will keep record of all enrolled subjects in the Subject’s Enrolment Log: 
the subject screening number, the date of consent, the treatment assigned to the subject, 
if applicable, or the reason for not being randomized will be recorded. 
 
6.2 BLINDING / EMERGENCY UNBLINDING 
The study is designed as a double blind study and neither the subject nor the clinical site 
personnel (Investigator, sub-Investigator, study nurse, psychologists, pharmacist) will 
know which treatment is being administered. The identity of the treatments can’t be 
revealed except in an emergency under the discretion of the Investigator.  
The Principal Investigator will receive a study treatment identification key in the form of 
sealed envelopes containing the kit number and the corresponding treatment. 
The envelope can be opened only in case of an emergency presenting the need to 
disclose the identification of the study treatment assigned to the subject, for the purpose of 
establishing the appropriate therapy. Once the code is broken for a subject, this subject 
shall be withdrawn from the study, with the completion of the final study evaluation, 
indicating the specific reason of the subject withdrawal. 
The Study Monitor must be notified immediately by the Investigator of any emergency 
unblinding; the date and time, along with the reason for the unblinding, will be noted. 
Treatment codes will not be freely available to the Investigator or personnel monitoring the 
study until after the study completion and database lock. 
 
6.3 STUDY DURATION AND END OF STUDY DEFINITION 
 
Each participant will attend 4 visits over a total period of about 9 weeks: an initial 
screening phase of 7 to 1 days and then a treatment phase of 8 weeks.    
 
Duration of the treatment: 8 weeks in a double blind scheme (food supplement versus 
placebo). 
 
The enrolment period is foreseen to be of 2 months and the overall expected study 
duration is about 4-5 months. 
Unless premature interruption occurs, the end of the study will be the closure visit at 
clinical site. 
 
 

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     CONFIDENTIAL                                                                                                              Page 23 of 56 
 
  
 
 
 
7. SELECTION AND WITHDRAWAL OF SUBJECTS 
Subjects aged ≥60 years with no evidence of cognitive dysfunction will be enrolled at the 
Geriatric Division of the P.O. SS Filippo e Nicola, Avezzano, Italy. 
No. 80 (including a possible 15% for screening failure and drop-out) subjects will be 
involved in this study. 
 
7.1 INFORMED CONSENT PROCEDURE 
Prior to the subject’s enrolment into the study and before performing any study-related 
procedures, the Investigator - or its authorised delegate - shall obtain the subject’s written, 
dated and signed informed consent to participate into the study and to the confidential 
disclosure, processing and transferring necessary documentation of the subject’s health 
and personal data to the CRO, the Sponsor and its Affiliates, the competent Health 
Authorities and any other institutions (even if located outside the European Economic 
Area), as legally required and in accordance with the applicable privacy laws. 
Institution and Investigator undertake to duly inform subjects about personal data 
processing and the relevant applicable privacy rights before their participation into the 
study. 
 
Prior to be submitted to the subject, the Informed Consent form will be approved in the 
corresponding local language and in accordance with local laws and regulations by the 
IEC. 
Subjects will be given information and fully comprehensive explanation in easily 
understandable terms of the study procedures, regarding the benefits, restrictions, 
discomforts, and risks in taking part in the study, the properties of the study product, the 
method of assignment to treatments, and any medically accepted and readily available 
treatment other than the study product. 
Subjects will also be informed about the measures taken to ensure their confidentiality 
according to the pertinent legislation. The Investigator will provide the subject with an 
emergency telephone number. 
After being duly informed and interviewed by the Investigator, the subject freely has to 
date and sign the ICF before being enrolled into the study and before undergoing any 
study procedure. The Investigator must store the original of the signed ICF in the 
Investigator’s File, and the subject will be provided with a copy of it. 
If a protocol amendment would affect the terms of the ICF, it will be revised to reflect the 
protocol change and submitted to IEC for approval.  
The Investigator will ensure that this new consent form is signed by all subjects 
subsequently entered in the study and those currently in the study, if affected by the 
amendment. 
 

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7.2 
INCLUSION CRITERIA  
For inclusion in the study, all of the following inclusion criteria must be fulfilled: 
 
1) Subjects aged ≥60 years. 
2) Subjects who provide written Informed Consent to the study. 
 
7.3 
EXCLUSION CRITERIA  
Subjects will not be considered eligible for this study if they fulfill any of the following 
exclusion criteria: 
 
1) Subjects with cognitive dysfunctions or clinically significant coexisting medical 
conditions (cardiovascular disease, cerebrovascular events, overt dementia defined 
by MMSE <27 or other neurological disorders, thyroid disorders, or inflammatory 
diseases) 
2) Subjects with a score on the Geriatric Depression Scale (GDS) >11 in order to 
avoid confounding due to the influence of concomitant depression on the 
performance on cognitive tests 
3) Current smokers 
4) Habitual users of antioxidant supplements (including vitamins C and E) 
5) Habitual consumers of chocolate or other cocoa products (daily consumption of any 
amount) 
6) Subjects under treatments with medications known to have antioxidant properties 
(including statins and glitazones) or to interfere with cognitive functions (including 
benzodiazepines and antidepressants) 
7) Subjects with hypersensitivity to any component of the study medications.  
8) Subjects who are participating in or having participated in another clinical trial within 
the previous three months. 
 
7.4 
WITHDRAWAL CRITERIA 
The subject may withdraw from the study at any time without explanation, without losing 
the right to future medical care. The participation of the subject may, at any moment, be 
terminated by the Investigator, if considered appropriate. 
Subjects who discontinue from the study early will complete an early termination visit (Visit 
4). 
Study treatment must be terminated during the study for any of the following reasons: 
• Request of the subject (consent withdrawal); 
• An AE occurs that, in the opinion of the Investigator, makes unsafe for the subject 
to continue in the study; 
• Investigator deems it to be in the best interest of the subject to discontinue; 

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     CONFIDENTIAL                                                                                                              Page 25 of 56 
 
  
 
 
 
• Failure to comply adequately with the dosing, evaluations, or other requirements of 
the study 
The Investigator must immediately notify the C.R.O (see C.R.O. officer contacts on page 
37) by telephone or fax when a subject has been discontinued/withdrawn due to an AE. 
The reason for the withdrawal must be well documented in the CRF. 
Withdrawn subjects will not be replaced as already foreseen in the sample size estimation. 
Any deviation from the protocol (to be classified as major or minor) will be accepted only in 
case of emergency and/or after a written agreement with the Sponsor. 
 
8. TREATMENT OF SUBJECTS 
Subjects will be randomly allocated to one of the following groups: 
 
- 
Group I: mix of the four bioactive compounds (bacopa, lycopene, astaxanthin and 
vitamin B12) for 8 weeks; 
- 
Group II: placebo for 8 weeks. 
 
8.1 STUDY PRODUCTS 
The product under investigation is a new food supplement composed by four bioactive 
ingredients: bacopa, lycopene, astaxanthin and vitamin B12. The product and its placebo 
will be administered to subjects in oral tablets to be taken once a day for 8 weeks. 
 
Study product 1:  
Name: food supplement (a mix of bacopa, lycopene, astaxanthin and vitamin B12) 
Formulation: tablets 
Route of administration: oral 
 
Table 1: food supplement tablet composition 
Average amounts of characterizing ingredients
Quantity
(mg/dose) 
% NRVs
Astaxanthin 
160.000 
15.610 
Lycopene 
100.000 
9.756 
Bacopa (Bacopa monnieri (L.) Pennel) aerial part e.s. tit. 50% 
bacosides 
80.000 
7.805 
Cyanocobalamin (vitamin B12) 
6.000 
0.585 
Microcrystalline Cellulose 
619.434 
60.433 
Coating agent: hydroxypropylmethylcellulose 
15.819 
1.543 
Antiagglomerating agents: crosslinked sodium 
carboxymethylcellulose 
15.000 
1.463 

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     CONFIDENTIAL                                                                                                              Page 26 of 56 
 
  
 
 
 
Antiagglomerating agents: polyethylene glycol 
8.500 
0.829 
Antiagglomerating agents: magnesium vegetable stearate 
8.000 
0.780 
Antiagglomerating agents: silicon dioxide 
6.000 
0.585 
Dye: titanium dioxide 
3.651 
0.356 
Antiagglomerating agent: vegetable stearic acid 
2.434 
0.237 
Dye: oxide of iron (red) 
0.150 
0.015 
Dry: Patent blue V 
0.013 
0.001 
                                                                Total
1025.000
100.000
 
 
Study product 2:  
Name: placebo 
Formulation: tablets 
Route of administration: oral 
 
Table 2: placebo tablet composition 
Average amounts of characterizing ingredients
Quantity 
(mg/dose) 
%  NRVs
Microcrystalline Cellulose 
994.434 
97.018 
Coating agent: hydroxypropylmethylcellulose 
15.819 
1.543 
Antiagglomerating agents: magnesium vegetable stearate 
5.000 
0.488 
Dye: titanium dioxide 
3.651 
0.356 
Antiagglomerating agents: silicon dioxide 
3.000 
0.293 
Antiagglomerating agent: vegetable stearic acid 
2.434 
0.237 
Antiagglomerating agents: polyethylene glycol 
0.500 
0.049 
Dye: oxide of iron (red) 
0.150 
0.015 
Dry: Patent blue V 
0.013 
0.001 
                                                                                                     
Total 
1025.000
100.000
 
Tablets of the food supplement and placebo will be manufactured and packaged (primary 
and secondary packaging) by Fine Foods & Pharmaceuticals N.T.M. S.p.A. Zingonia-
Verdellino (BG), Italy. Study products will be then labeled, supplied to the clinical site, 
returned from the clinical site and destroyed by Euromed Clinical Supply Services 
(EUROMED), Cantù (CO) - Italy 
No difference will be detectable in the presentation of the food supplement and placebo 
tablets. 

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Study Code MEIF/17/BAC-COG/001 
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     CONFIDENTIAL                                                                                                              Page 27 of 56 
 
  
 
 
 
Tablets will be packaged in blisters (primary packaging) and in boxes (secondary 
packaging). 
Each box will contain 3 blisters with 15 tablets each. The subject’s kit will be composed by 
two boxes, named 1 and 2, in order to cover the 4 + 4 weeks study treatment periods. 
Subjects will receive at each visit enough tablets for daily treatment (one tablet/day) until 
the next visit. The number of tablets provided will be in large excess (45 tablets), with 
respect to the hypothetical need (28-31 tablets for 4 weeks ± 3 days at each phase).  
Blisters and boxes will be labelled according to Annex 13 requirements. Boxes will have a 
tear-off portion of label to be attached on the appropriate section of the CRF.  
 
Each treatment will be defined by a three-digit number and by the numbers 1 and 2 to 
indicate the first and the second box to be used (i.e. treatment 015-1 and 015-2, 034-1 
and 034-2). Treatment numbers will be randomized. 
 
8.2 HANDLING AND STORAGE 
The study product shall be carefully stored at the study site, in a safe area and separately 
from other drugs/products. It shall not be exposed to direct sunlight or heat. 
The Investigator’s staff shall maintain a record of the study product delivery to the study 
site and inventory at the study location. 
After study conclusion, all unused study product shall be returned and destroyed by 
EUROMED after written Sponsor approval. 
 
8.3 STUDY PRODUCT ACCOUNTABILITY AND RETURN 
The Investigator is responsible of ensuring accountability of the study product, including 
reconciliation of the study products and maintenance of records. 
Upon receipt of the study products, the Investigator (or designee) will check the contents 
and acknowledge receipt by signing (or initialing) and dating the documentation provided 
by the C.R.O and returning it to the C.R.O. A copy will be retained in the Investigator File. 
The dispensing of the study product will be carefully recorded on the appropriate 
accountability forms provided by the C.R.O and an accurate accounting will be available 
for verification by the Study Monitor at each monitoring visit. 
Study product accountability records will include: 
• Confirmation of study product receipt at the clinical site. 
• The inventory at the site of study product provided by the C.R.O.  
• The intake of doses by each subject. 
• The return to EUROMED or alternative disposition of unused study product. 
The Investigator should maintain records that adequately document: 
• That the subjects were provided the doses specified by the clinical study 
protocol/amendment(s), and 

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     CONFIDENTIAL                                                                                                              Page 28 of 56 
 
  
 
 
 
• That all study products provided by EUROMED was fully reconciled. 
To the purpose of accountability, the subject will be instructed to always return the unused 
containers at each visit. 
Unused study product must not be discarded or used for any purpose other than the 
present study. Study product that has been dispensed to a subject must not be re-
dispensed to a different subject. 
The Study Monitor will periodically collect the study product accountability forms and will 
check all returns (both unused and used containers) before arranging for their return to 
EUROMED.  
 
8.4 TREATMENT COMPLIANCE  
The subject compliance for study treatment period is calculated by the following formula: % 
compliance = number of tablets actually taken x 100 / expected number of tablets which 
should have been taken.  
The global subject compliance will be calculated as the percentage of the number of tablets 
actually taken by the subject over the number of tablets expected to be taken.  
The number of tablets actually taken will be calculated as the difference between the number 
of tablets handled out to the subject and the number of unused tablets returned or declared 
lost by the subject.  
The expected number of tablets taken will be calculated using the difference (in days) 
between Visit 3 and Visit 2 and between Visit 4 and Visit 3. A subject that has taken at least 
80% and no more than 120% of the required product intake since the last visit will be 
considered compliant. 
 
8.5 ADMINISTRATION OF STUDY TREATMENT 
Subjects will be randomly allocated to one of the following groups: 
- 
Group I: mix of the four bioactive compounds (bacopa, lycopene, astaxanthin and 
vitamin B12), 1025,00 mg/daily); 
- 
Group II: placebo, 1025,00 mg/daily. 
Both treatments, the food supplement and placebo, shall be taken once a day for 8 
consecutive weeks: one tablet a day, with a glass of water, possibly at the same time of 
the day and away from meals. 
 
8.6 CONCOMITANT TREATMENTS 
Any medications (other than those excluded by the clinical study protocol) that are 
considered necessary for the subjects’ welfare and will not interfere with the study product 
may be given at the Investigator’s discretion. 
The Investigator will record all concomitant medications taken by the subject at study 
inclusion and during the study, in the appropriate section of the CRF. 

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     CONFIDENTIAL                                                                                                              Page 29 of 56 
 
  
 
 
 
Any additional concomitant therapy that becomes necessary during the study and any 
change to concomitant treatments must be recorded in the corresponding section of the 
CRF, noting the name, dose, duration and indication of each product. 
 
8.7 PROHIBITED PRIOR AND CONCOMITANT TREATMENTS 
At screening and during the study the following concomitant treatments are not allowed: 
• Antioxidant supplements (including vitamins C and E) 
• Medications known to have antioxidant properties (including statins and glitazones) 
• Medications known to interfere with cognitive functions (including benzodiazepines 
and antidepressants). 
Habitual use of chocolate or other cocoa product should be avoided during the study. 
If the treatment with one or more of the above mentioned products becomes necessary 
during the study, then the subject will be withdrawn from the study.  
 
9. ASSESSMENT OF EFFICACY 
9.1 EFFICACY PARAMETERS 
Cognitive functions, mood state, self-perceived well being and sexual satisfaction will be 
assessed at baseline and after 8 weeks of regular consumption of the mix of the four 
antioxidant compounds or placebo. 
  
Blood pressure, metabolic variables and plasma markers of oxidative stress will be 
assessed at baseline, after 4 and after 8 weeks of regular consumption of the antioxidant 
compounds or placebo. 
 
9.1.1 Cognitive Performances Assessment 
Cognitive performances will be evaluated by TMT, VFT, MoCA, MMSE and AVLT. 
 
-Trail Making Test (TMT) is a frequently used neuropsychological test because of its 
sensitivity to brain damage. It explores visual-conceptual and visual-motor tracking. TMT 
is administered in two parts. Part A is a visual-scanning, timed task where participants are 
asked to connect with lines 25 circles numbered from 1 to 25 as quickly as possible. The 
test is terminated after 5 minutes even if not completed. In Part B participants are asked to 
connect circles containing numbers (from 1 to 13) or letters (from A to L) in an alternate 
numeric/alphabetical order. The test is terminated in every case after 10 minutes even if 
not completed. The TMT B-A score calculated as the difference between TMT-B and 
TMT-A times is considered a measure of cognitive flexibility relatively independent of 
manual dexterity. 
 

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     CONFIDENTIAL                                                                                                              Page 30 of 56 
 
  
 
 
 
-Verbal Fluency Test (VFT) is a short test of verbal functioning. Participants are given 1 
min to produce as many unique words as possible within a semantic category (category 
fluency) or starting with a given letter (letter fluency). The participant's score in each task 
is the number of unique correct words. 
 
-Montreal Cognitive Assessment (MoCA) evaluates a broader array of cognitive 
domains (e.g., attention/executive functioning, visuospatial abilities and language) and it 
has been demonstrated to be able to detect cognitive impairment with scores ranging from 
0 to 30. 
 
-Mini Mental State Examination (MMSE) is a widely used screening tool for cognitive 
impairment and covers five areas of cognitive function including orientation, attention, 
calculus, recall and language with scores ranging from 0 to 30. 
 
-Rey Auditory Verbal Learning Test (AVLT) is a neuropsychological assessment 
designed to evaluate the nature and severity of memory dysfunction and to track changes 
in memory function. The examiner reads aloud a list of 15 words at the rate of one per 
second. The participant is then asked to repeat all words from the list that she/he can 
remember. This procedure is carried out a total of five times. After a 15-minute delay, the 
participant is again asked to recall as many words as possible from the first list. The 
participant is then requested to read a list of words and asked to indicate whether each 
word was from the first list. The score for each trial is the number of words correctly 
recollected. 
 
9.1.2 Mood States Assessment 
Mood states will be assessed by POMS. 
 
-Profile of Mood Stated (POMS) is a widely used tool in assessing mood states that has 
already been validated for Italy. Higher scores reflect mood decrements, except for the 
vigor subscale, where higher scores reflect improved mood. Taking into consideration the 
two components characteristic of the POMS that measure negative affect and positive 
affect, respectively, the data collected with the questionnaire will be analyzed both as 
global score and by separately considering a Positive Affect Scale (PAS-POMS, 48 items, 
score ranging from 0 to 192) and a Negative Affect Scale (NAS-POMS, 10 items, score 
ranging from 0 to 40). 
 
9.1.3 Psychological Well-Being Assessment 
Psychological well-being will be assessed by GHQ-12. 
 

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     CONFIDENTIAL                                                                                                              Page 31 of 56 
 
  
 
 
 
-General Health Questionnaire (GHQ-12) is a relevant instrument for measuring 
psychological well-being. It has been extensively evaluated in terms of validity and 
reliability as a one dimensional indicator of the severity of psychological morbidity, and it 
has already been validated for Italy. 
 
9.1.4 Sexual Satisfaction Assessment 
Sexual satisfaction will be evaluated by the NSSS. 
 
-New Sexual Satisfaction Scale (NSSS) is a 20 item, multidimensional, composite 
measure of sexual satisfaction based on a five-dimension, conceptual model that 
emphasized the importance of multiple domains of sexual behavior including sexual 
sensations, sexual awareness and focus, sexual exchange, emotional closeness, and 
sexual activity. 
 
9.1.5 Blood Pressure, Metabolic Variables and Plasma Markers Of Oxidative 
Stress 
 
-Clinic systolic and diastolic blood pressure levels will be recorded in the morning, 
using a validated oscillometric device with appropriately sized cuffs on the non-dominant 
upper arm after 5 min resting in a seated position; the first blood pressure measurement 
will be discarded and the subsequent three consecutive blood pressure readings, taken at 
3-min intervals, will be recorded. The average of these latter measures will be considered 
for statistical analysis. [O'Brien et al., 2005]. 
 
-Metabolic profile will be evaluated by the following variables: 
1. glucose (normal range 60-100 mg/dL) 
2. insulin (normal range 2.7-10.4 mcUI/mL) 
3. HOMA-IR (derived from glucose and insulin) 
4. total cholesterol (normal range 150-200 mg/dL) 
5. LDL cholesterol (normal range 40-130 mg/dL) 
6. HDL cholesterol (normal range 35-90 mg/dL) 
7. triglycerides (normal range 60-170 mg/dL) 
8. uric acid (normal range 3.5 – 7.0 mg/dL) 
 
-Oxidative stress will be evaluated by measuring circulating levels of 2 soluble 
biomarkers: 
A. 8-iso-Prostaglandin F2alpha (no predefined normal range) 
B. Plasma malondialdehyde (no predefined normal range) 
 

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     CONFIDENTIAL                                                                                                              Page 32 of 56 
 
  
 
 
 
9.2 STUDY PROCEDURES 
The study will last approximately 9 weeks for each subject.  
A total of 4 study visits will be performed as follows. 
 
VISIT 1 (screening, day -7 to Visit 2):  
Subjects who provide written informed consent will undergo screening assessments to 
evaluate eligibility for the study. Subjects who meet all inclusion criteria and none of the 
exclusion criteria will be eligible for the study and will undergo to the following evaluations: 
1) Collection of medical history and information about eventual concomitant medications 
2) Complete physical evaluation including  
a) anthropometric parameters (height, weight, waist circumference) 
b) cardiovascular system  
c) respiratory system  
d) gastrointestinal system  
e) respiratory system 
3) Blood pressure measurement 
4) MMSE 
5) GDS. 
 
Subjects eligible for the study will meet with a dietician in order in order to evaluate current 
diet habits and correct any nutritional insufficiencies. Participants will be then instructed to 
maintain their usual lifestyle and intake of fruits and vegetables and to avoid any food 
supplement. 
 
VISIT 2 (randomization, 7 days after V1): 
Subjects eligible for the study will undergo to the following evaluations: 
1) Collection of information about eventual concomitant medications 
2) Complete physical evaluation including 
a) anthropometric parameters (weight, waist circumference)  
b) cardiovascular system  
c) respiratory system  
d) gastrointestinal system  
e) respiratory system 
3) Blood pressure measurement 
4) Neuropsychological evaluation (TMT, VFT, MoCA, MMSE, AVLT) 
5) Evaluation of mood state (POMS) 
6) Evaluation of quality of life (GHQ-12) 
7) Evaluation of sexual satisfaction (NSSS) 
8) Blood samples collection to assess metabolic parameters (glucose, insulin, total 
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, uric acid) 

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     CONFIDENTIAL                                                                                                              Page 33 of 56 
 
  
 
 
 
9) Blood samples collection to assess plasma markers of oxidative stress (8-iso-
Prostaglandin F2alpha, malondialdehyde). 
10) Adverse events check and evaluation 
 
Subjects will be randomly assigned to two parallel groups in a 1:1 ratio and treated with a 
mix of the four bioactive compounds (bacopa, lycopene, astaxanthin and vitamin B12, –
(1025,00 mg/daily) or placebo (1025,00 mg/daily).  Subjects will intake one tablet/day for 
about 8 weeks. 
 
During Visit 2 one box of study treatment (containing 45 tablets) will be given to each 
subject. 
 
VISIT 3 (treatment period, 30 days ± 3 days after V2): 
Subjects will undergo to the following evaluations: 
1) Collection of information about eventual concomitant medications 
2) Complete physical evaluation including  
a) anthropometric parameters (weight, waist circumference),  
b) cardiovascular system 
c) respiratory system 
d) gastrointestinal system  
e) respiratory system 
3) Blood pressure measurement 
4) Blood samples collection to assess metabolic parameters (glucose, insulin, total 
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, uric acid) 
5) Blood samples collection to assess plasma markers of oxidative stress (8-iso-
Prostaglandin F2alpha, malondialdehyde) 
6) Adverse events check and evaluation 
7) Study product return/compliance check of first month of therapy. 
 
During Visit 3 another one month of treatment of study product (45 tablets) will be given to 
each subject. 
 
VISIT 4 (final visit, 30 days ± 3 days after V3): 
Subjects will undergo to the following evaluations: 
1) Collection of information about eventual concomitant medications 
2) Complete physical evaluation including  
a) anthropometric parameters (weight, waist circumference)  
b) cardiovascular system  
c) respiratory system  
d) gastrointestinal system  

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     CONFIDENTIAL                                                                                                              Page 34 of 56 
 
  
 
 
 
e) respiratory system 
3) Blood pressure measurement 
4) Neuropsychological evaluation (TMT, VFT, MoCA, MMSE, AVLT) 
5) Evaluation of mood state (POMS) 
6) Evaluation of quality of life (GHQ-12) 
7) Evaluation of sexual satisfaction (NSSS) 
8) Blood samples collection to assess metabolic parameters (glucose, insulin, total 
cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, uric acid) 
9) Blood samples collection to assess plasma markers of oxidative stress (8-iso-
Prostaglandin F2alpha, malondialdehyde) 
8) Adverse events check and evaluation 
7) Study product return/compliance check of second month of therapy. 
  
 
10. ASSESSMENT OF SAFETY 
All adverse events will be duly assessed for identifying any emergent safety finding. 
 
10.1. SAFETY DATA MANAGEMENT 
10.1.1 Adverse Event (AE) 
Any untoward medical occurrence in a subject or clinical trial subject administered a 
medicinal product and which does not necessarily have a causal relationship with this 
treatment. 
An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product. 
 
10.1.2 Product Relationship 
The relationship between an AE and study products will be judged according to the 
following categories: 
1. Certain: The AE occurs in a plausible time relation to the administration of the 
product and cannot be explained by a concurrent disease or other drugs or 
chemicals. The response to withdrawal of the product (dechallenge) should be 
clinically 
plausible. 
The 
event 
must 
be 
definitive 
pharmacologically 
or 
phenomenologically, using a satisfactory rechallenge procedure if necessary. 
2. Probable: The AE occurs in a reasonable time relation to the administration of the 
product, it is unlikely to be attributed to a concurrent disease or other drugs or 
chemicals and it follows a clinically reasonable response on withdrawal 
(dechallenge). 
Rechallenge 
information 
(AE 
reappearance 
after 
product 
reintroduction) is not required to fulfil this definition. 

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3. Possible: The AE occurs with a reasonable time relation to the administration of 
the product, but it could also be explained by a concurrent disease or other drugs 
or chemicals. Information on product withdrawal (dechallenge) may be lacking or 
unclear. 
4. Unassessable: The relationship cannot be judged, because of the information is 
insufficient or contradictory and cannot be supplemented or verified. 
5. Unlikely: A causal relationship cannot be definitively ruled out, but 
– other drugs, chemicals, or underlying disease provide plausible explanations 
and/or 
– the temporal relation to the administration of the product makes a causal 
relation improbable. 
6. Not Related: Any of the following are present: 
– existence of a clear alternative explanation, and/or 
– unreasonable temporal relationship between product and event, and/or 
– non-plausibility. 
 
10.1.3 Adverse Reaction (AR) 
An AR is any untoward and unintended response to an investigational product related to 
any dose administered. 
The definition implies a reasonable possibility of a causal relationship between the event 
and the investigational product. This means that there are facts (evidence) or arguments 
to suggest a causal relationship. 
An AR is considered any AE for which the relationship is considered as: 
1. Certain 
2. Probable 
3. Possible 
4. Unassessable 
An AE is not considered as AR when the relationship is judged as: 
5. Unlikely 
6. Not related 
 
10.1.4 Seriousness 
An AE/AR is considered Serious when: 
• results in death; 
• is life-threatening; 
Note: Life-threatening is considered any AE in which the subject was at risk of death at 
the time of the event; it does not refer to an event which hypothetically might have caused 
death if it was more severe. 
• requires insubject hospitalisation or prolongation of existing hospitalisation; 

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• results in persistent or significant disability/incapacity; 
• is a congenital anomaly/birth defect; 
• is another medically important condition that may jeopardise the subject or may 
require intervention to prevent one of the outcomes listed above. Any suspected 
transmission of an infectious agent via a medicinal product is considered serious 
and should be assessed under the category of medically important events in the 
absence of other seriousness criteria. 
An AE/AR is considered Non-serious when it does not fulfill the conditions for the 
definition of Serious AE/AR. 
 
10.1.5 Adverse Event/Adverse Reaction Intensity 
The intensity level of a Serious or a Non-serious AE or AR is attributed according to the 
following definitions: 
• Mild: does not interfere with routine activities; in case of laboratory tests, when there 
is a mild abnormality. 
• Moderate: interferes with the routine activities; in case of laboratory tests, when 
there is a moderate abnormality. 
• Severe: makes it impossible to perform routine activities; in case of laboratory tests, 
when there is a significant abnormality. 
 
10.1.6 Adverse Event/Adverse Reaction Expectedness 
An AE/AR is considered Unexpected when the nature, severity, or outcome of the AE/AR 
is not consistent with the information known about the product. 
 
10.1.7 Collection, Recording and Reporting of AEs  
At each visit the Investigator will collect and assess any occurred subjective or objective 
AE occurred to each subject after his/her signature of the informed consent. 
The Investigator should manage as AE any laboratory test abnormality (newly occurring 
after the study product administration or worsening of previously known abnormalities) 
considered as clinically relevant: i.e. values significantly above or under normal range or 
which require an intervention or diagnostic tests, or may result in the study product 
discontinuation. 
Any AE communicated by the subject or by the subject’s relatives or delegates through 
phone calls, letters or emails will also be collected and assessed. 
The Investigator shall record on the respective CRF AE recording pages/AE form any 
recognised AE, both serious and non-serious, whether or not thought to be product-
related, observed in or reported by the subject (or relatives/delegates), specifying the 
judgement on the causal relationship with the study treatment. 

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Any available information and diagnostic measure (laboratory and instrumental tests, 
procedures, etc.) shall be recorded in and/or attached to the concerned CRF 
pages/sections. 
The Investigator must report all the collected information on any Serious AE (whether or 
not thought to be related to the investigational product), providing the concerned SAE 
reporting form by email or fax, after the first knowledge of the occurrence of the case, to: 
 
CRO Officer: Laura Michellini 
 
Email: michellini@latiscro.it 
 
 
Fax: + 39 010 540699 
 
Tel: +39 010 562234 
 
Mobile: +39 347 4785898 
 
When relevant, also the CRF pages concerning medical history, concomitant medication, 
and laboratory tests will be placed at Sponsor disposal by email or fax. 
 
10.1.7.1 Management of Serious AEs (SAEs) including laboratory abnormalities 
⋅ 
Reporting Duties of the Investigator 
The Investigator must report all the collected information on any SAE (whether or not 
thought to be related to the investigational product), as above specified, no later than 24 
hours after the first knowledge of the occurrence of the case. 
Any further information and supporting documentation that become available (copies of 
laboratory reports, tests, procedures, autopsy evidence of the cause of death, etc.) shall 
be provided no later than 24 hours after the knowledge, by the Investigator to the CRO by 
email or fax, to be forwarded to the Sponsor. 
The Investigator must also comply with the local applicable obligation(s) on the reporting 
of ARs to the local concerned Regulatory Authority/Ethics Committee. 
⋅ 
Reporting Duties of the Sponsor 
The Sponsor shall ensure that all relevant information about any SAE (whether or not 
thought to be related to the investigational product), is expeditiously reported to the 
competent Authority and Ethics Committee as required, with these deadlines after the first 
knowledge, intended as the day when the CRO receives the notification of the SAE: 
• Fatal and life threatening unexpected cases, no later than 7 days; 
• Other unexpected serious cases, no later than 15 days. 
The Sponsor shall ensure that all relevant information and supporting documentation that 
subsequently becomes available, is also expeditiously reported as follow-up information 
according to the above mentioned deadlines. 
Since the study is blinded, the subject’s code will be broken before the expedited 
reporting to the Competent Authority and the Ethics Committee. 

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Furthermore the following safety issues will be subjected to expedited management for the 
identification of possible necessary actions: 
• SAEs associated with the study procedures 
• Potential clinically significant findings emerging from non-clinical studies 
• An anticipated end or suspension for safety reasons of another study with the same 
study product. 
When appropriate and applicable the Sponsor will arrange the adequate information also 
to the Investigators. 
 
10.1.7.2 
Management 
of 
Non-serious 
AEs 
(NSAEs) 
including 
laboratory 
abnormalities 
⋅ 
Reporting Duties of the Investigator 
The Investigator must record all the collected information on any NSAE (whether or not 
thought to be related to the investigational product) on the appropriate section of the CRF. 
10.1.8 MANAGEMENT OF ANY LABORATORY ABNORMALITY 
Any laboratory test abnormality which is considered by the Investigator as AE is to be 
managed as above detailed (see 10.1.7). 
 
 
10. STATISTICS 
 
10.1 STATISTICAL METHODS (BLINDING AND RANDOMISATION) 
Once eligibility will be established, subjects will be randomized to receive either a mix of 
the four bioactive compounds or placebo in a 1:1 ratio and a double-blind manner. 
Descriptive statistics of all relevant variables will be performed. Continuous variables will 
be summarized by the number of subjects (N), mean, standard deviation, median, 
minimum, maximum. Where appropriate, 95% confidence intervals for the target variables 
will be estimated. Categorical variables will be summarized by the number (N) and the 
proportion of subjects (%).  
The significance level of statistical tests will be set at 0.05. Details of statistical analysis 
are provided in the following paragraphs. Further details will be given in a Statistical 
Analysis Plan that will be issued before database lock. 
No adjustment for multiplicity is needed. Missing values will not be replaced. No interim 
analysis is planned. 
The statistical analysis will be performed using SAS 9.4 for Windows (SAS Institute Inc., 
Cary, NC, USA). 
 

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Sponsor: A. Menarini Industrie Farmaceutiche Riunite S.r.l. 
 
Study Code MEIF/17/BAC-COG/001 
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10.2 DETERMINATION OF SAMPLE SIZE  
The primary objective of this study will be assessed using three primary variables 
(changes in TMT-B, TMT-A and TMT B-A) in a hierarchical order. According to the “Points 
to consider on multiplicity issues in clinical trials”, issued by the Committee for Proprietary 
Medicinal Products (CPMP - 2002), no sample size adjustment for multiplicity is needed. 
The study will be based on an estimated sample size of 68 subjects, with a ratio of 1:1 for 
the 2 treatment groups, which has been calculated to be adequate to achieve 90% power 
to detect a large effect size (f=0.40) using an ANCOVA model with a baseline covariate 
and an α of 0.05 between treatment and control (G*power version 3.1.9.2). To consider a 
possible 15% dropout rate, 80 subjects will be enrolled.  
 
10.3 ANALYSIS POPULATIONS 
The study will be analyzed using an Intent-to-Treat (ITT) approach. All randomized 
subjects receiving at least a treatment dose and having the post-randomization efficacy 
evaluation will be included in the ITT population for efficacy analysis.  
All subjects without protocol deviations affecting the primary endpoint will be included in 
the Per-Protocol (PP) analysis. Protocol deviations affecting the protocol can include 
deviations to the treatment dose or assessment schedule, violations of some 
inclusion/exclusion criteria, etc. Detailed list of these deviations will be created and 
included in the SAP. 
All randomized subjects receiving at least a treatment dose will be included in the safety 
analysis. 
 
10.4 ANALYSIS VARIABLES 
10.4.1 Primary Efficacy Endpoint 
The primary efficacy endpoints are the changes of TMT scores from baseline (V2) to 8 
weeks of treatment (V4), analyzed in the following hierarchical order: TMT-B, TMT-A and 
TMT B-A. 
 
10.4.2 Secondary Efficacy Endpoints 
Secondary efficacy endpoints of the study are:  
a) changes of VFT score from baseline (V2) to 8 weeks of treatment (V4); 
b) changes of MoCA score from baseline (V2) to 8 weeks of treatment (V4); 
c) changes of MMSE score from baseline (V2) to 8 weeks of treatment (V4); 
d) changes of AVLT score baseline from baseline (V2) to 8 weeks of treatment (V4); 
e) changes of GHQ-12 score baseline from baseline (V2) to 8 weeks of treatment (V4); 
f) 
changes of POMS score from baseline (V2) to 8 weeks of treatment (V4);  
g) changes of NSSS score from baseline (V2) to 8 weeks of treatment (V4);  

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h) changes of metabolic parameters (glucose, insulin, HOMA-IR, total cholesterol, LDL 
cholesterol, HDL cholesterol, triglycerides and uric acid from baseline (V2) to 4 weeks 
of treatment (V3) and from baseline (V2) to 8 weeks of treatment (V4);  
i) 
changes of circulating levels of 8-iso-Prostaglandin F2alpha and malondialdehyde 
from baseline (V2) to 4 weeks of treatment (V3) and from baseline (V2) to 8 weeks of 
treatment (V4). 
  
10.4.3 Exploratory Endpoints 
Exploratory endpoints of this study are: 
 
a) correlation between changes of cognitive scores (TMT, VFT, MoCA, MMSE, AVLT) 
and changes of blood pressure and metabolic parameters including indices of 
oxidative stress; 
b) quantify the effect of time in the change for the primary endpoint. 
 
10.5 STATISTICAL ANALYSIS  
Efficacy analysis 
The primary variables (changes of TMT-B, TMT-A and TMTB-A scores) will be analyzed 
using an ANCOVA model with the treatment group (main effect) and baseline as 
covariates. A fixed sequence multiple test will be performed, with the following hierarchical 
order: 1) changes of TMT B, 2) changes of TMT A and 3) changes of TMT B-A scores. 
All the other continuous variables (changes of test scores, changes of metabolic 
parameters, changes of circulating levels of 8-iso-Prostaglandin F2alpha and 
malondialdehyde) will be analyzed using an ANCOVA model with the treatment group 
(main effect) and baseline as covariates.  As additional covariate for testing the 
homogeneity of the regression coefficients the interaction term of treatment*baseline will 
be included in the model and removed if not significant. 
Categorical variables (gender, eventually present cardiovascular risk factors or 
pharmacological treatment) will be tested by using a Chi-Square test or the Fisher exact 
test if necessary.  
 
Explorative analysis  
o Pearson correlation will be used to evaluate correlations between changes of cognitive 
scores (TMT scores, VFT, MoCA, MMSE, AVLT) and changes of blood pressure and 
metabolic parameters including indices of oxidative stress. Spearman nonparametric 
correlation will be also applied when one or both of the variables are not assumed to be 
normally distributed and interval (but are assumed to be ordinal).  
o in order to evaluate and quantify the effect of time itself (i.e. 8 weeks after the treatment 
intake) in the change for the primary endpoint a two-way analysis of variance 
(treatment*time) with time as a repeated measure will be also implemented. 

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Safety analysis 
All adverse events will be duly assessed for identifying any emergent safety finding. 
Adverse events will be coded using the last updated version of the Medical Dictionary for 
Regulatory Activities (MedDRA) dictionary to give a preferred term (PT) and a 
system/organ class term (SOC) for each event.  
The number of AEs, study product-related AE, serious AE, severe AE will be summarized 
by treatment arm. The number of subjects who experienced at least one AE and the 
number of subjects withdrawn due to AE will also be summarized. 
AEs occurred after the first treatment intake will be considered Treatment Emergent 
Adverse Events (TEAEs), selected and analyzed separately. 
For each SOC and preferred term, summaries will be made with respect to the proportion 
of subjects having at least one occurrence of that event during the trial and the total 
number of events. The incidence of AEs in each treatment arm will be presented overall, 
by SOC and preferred term, and additional grouping by severity and relationship to the 
trial treatment. The comparisons will be analyzed using chi-square test. 
 
10.6 SUBGROUP ANALYSIS 
No subgroup analysis is planned. 
 
10.7 PROTOCOL VIOLATIONS AND BLIND REVIEW 
Protocol deviations will be registered during the course of the study. Protocol deviations 
can be identified in various ways: during onsite monitoring, during remote data checking, 
during or after data entry, thanks to data checks on CRFs or automated data checks 
during or after data entry. The impact of each deviation and the opportunity to drop 
subjects following a deviation will be discussed. 
Blind data review will be performed prior to database lock, to assess data completeness 
and protocol deviations. 
 
10.8 STATISTICAL ANALYSIS PLAN 
The reference document for the Statistical Analysis Plan is the ICH Topic E9 Statistical 
Principles for Clinical Studies: Note for Guidance on Statistical Principles in Clinical 
Studies. 
Further details of the statistical analyses will be provided in the Statistical Analysis Plan 
(SAP), which will be finalized prior to breaking the blind. 
 
 

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11. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
The Investigator agrees to allow Sponsor/CRO auditors/monitors to have direct access to 
his/her study records for review, being understood that the auditors/monitors are bound by 
professional secrecy, and as such will not disclose any personal identity or personal 
medical information. 
The Sponsor and the CRO must ensure that the Investigator keeps secret from third 
parties any confidential information disclosed or provided by the Sponsor and regarding 
the Sponsor and its study-related products. The Investigator agrees to use such 
information only to accomplish the present study tasks and not to use it for any other 
purposes without the prior written consent by the Sponsor. A confidentiality agreement will 
be signed between the Sponsor and the main Investigator. 
Study documents provided by the Sponsor will be kept appropriately to ensure their 
confidentiality. 
 
 
12. QUALITY CONTROL AND QUALITY ASSURANCE 
 
12.1 DATA QUALITY CONTROL/STUDY MONITORING 
The study will be monitored by CRO’s adequately qualified and trained clinical Monitor. 
Before the start of the study, the Clinical Monitor responsible for the study site has the 
task to assess the adequacy of the study site and the staff involved. After start, the study 
will be monitored on a regular basis throughout the study period to ensure the proper 
conduct of the clinical study.  
The purposes of clinical study monitoring are to verify that the rights and well-being of the 
study subjects are protected, that the reported study data are accurate, complete and 
verifiable against the source documents, and that the study is conducted in accordance 
with the current protocol, Good Clinical Practice guideline and applicable regulatory 
requirements. 
During the monitoring visits, Monitors will verify the following including but not limited to: 
subject’s informed consent, subject’s eligibility, safety data and reporting, subject’s 
questionnaires, quality of source documents and CRF data against subject’s medical 
records.  
If inconsistencies are identified, the corresponding corrections to the CRF data will have to 
be made by the Investigator or designated person. Monitors will also check treatment 
compliance, accrual, study product handling, including dispensing procedures and 
accountability logs, delegation of responsibilities within the Investigator’s team, relevant 
communications with family doctors, if any, ancillary equipment and facilities, including 
refrigerators and freezers, local labs, etc.  

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The Investigator and other site staff involved in the study must allocate enough time to the 
Monitor at these visits. 
 
12.2 CASE REPORT FORMS 
Data collected during the study will be recorded in the Case Report Form (CRF), which is 
confidential. Data reported on the CRF have to be consistent with the source documents. 
The Investigator must ensure the accuracy, the completeness and the consistency of the 
data entered in the CRF. 
On the CRF, subjects will be identified by the subject number, assigned at the Screening 
Visit. The subject number will be a number composed by the site code (01) and a 
progressive number (001, 002, 003…etc). 
During each subject’s study visit, the Investigator or designee will collect and report study 
data in the relevant subject’s chart, documenting all significant observations. 
Any contact with the subject via telephone or other means that provides significant clinical 
information must be documented in the source data and will be promptly entered in the 
CRF. 
A paper CRF will be used for recording subject’s study data.  
The CRFs must be completed for all subjects who have been included in the study. The 
Investigator will review all CRFs and sign and date them for each subject, verifying that the 
information is complete, true and correct. 
All CRF entries are to be done in black ballpoint ink. Changes and/or additions to data 
entered on original CRFs must be made in the following manner: the original entry will be 
lined out with a single line through the error (neither erasures nor correction fluid may be 
used) so as to leave it legible. The correction will be entered, initialed, and dated by the 
person making the correction. The Investigator or study personnel authorized by the 
Investigator may enter corrections on the original CRFs. 
All fields on the CRF must be completed as applicable. 
Subjects will be provided with paper questionnaires. Such documents will be filled by the 
subject during the study, to record data concerning cognitive performance, mood states, 
psychological well-being and sexual satisfaction.  
It is responsibility of the Investigators to instruct the study participants on how to fill in 
questionnaires in a clear way and preferably in black ball-point pen. The questionnaires 
are anonymous, each subject is identified through the subject screening number. At the 
end of the study a copy of all questionnaires will be stored in the Investigator’s File. It is 
responsibility of the Investigators to correctly enter the data collected on the 
questionnaires in the relevant sections on the e-CRF. Questionnaires will be considered 
source data. 
 

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After the CRFs are approved and signed by the Investigator, the monitor will collect and 
forward them to the Data Management Department where they will be processed. Data 
Clarification Forms generated by Data Management after data entry will be sent to the 
study site for resolution. The Investigator is responsible for the review and approval of all 
query resolutions. 
The data collected by CRFs will be entered in a dedicated database under the 
responsibility of Latis. The access to the database will be controlled by user-specific 
account and password combinations and audit trailed. Checks to assist during the data 
entry and to assess the appropriateness and consistence of data will be developed. 
 
12.3 QUALITY ASSURANCE 
Upon request by the Sponsor, on-site study audits may be conducted in order to ensure 
the study is in compliance with GCP, applicable regulatory requirements, and the study 
protocol. The auditing activities may also be conducted after study completion. 
In addition, Regulatory Authorities may wish to conduct on-site inspections (during the 
study or after its completion). If a Regulatory Authority notifies the Investigator of an 
inspection or visits the site unannounced for purposes of conducting an inspection, the 
Investigator must inform the Sponsor and CRO immediately. The Investigator will make all 
efforts to facilitate the conduct of the audits and inspections giving access to all necessary 
facilities, data and documents. 
Any result or information arising from the inspection will be immediately communicated by 
the Investigator to the Sponsor. The Investigator will take all appropriate measures 
required by the Sponsor to implement corrective actions for all problems found during 
audits or inspections. 
 
 
13. ETHICS ASPECTS 
 
This study will be carried out in compliance with the study protocol, the recommendations 
on biomedical research on human subjects of the Declaration of Helsinki, International 
Conference of Harmonisation – Good Clinical Practice (ICH-GCP) Guidelines, EU-
Directive 2001/20 and national requirements of the participating country. 
All clinical work conducted under this protocol is subject to GCP rules. This includes 
audits/inspections by the Sponsor, and/or by national/international Health Authority 
representatives at any time. All Investigators must agree to the inspection of the study site, 
facilities, and of study related records by the Health Authority representatives and/or by the 
Sponsor, and/or its delegates, which must be performed in accordance with national laws 
concerning personal data protection. 
 
13.1 ETHICS COMMITTEE 
Before starting the study in a study site, study protocol and relevant documentation 

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(Subject information leaflet, the informed consent Form and the study protocol) must be 
submitted to and approved by the Independent Ethics Committee (IEC) of the participating 
centre. The study will be then notified to the Competent Authority (CA). 
In addition, all local national legal requirements for the conduct of a clinical study have to 
be followed prior to the start of the study. Any amendment to the protocol, before 
implementation, will be submitted to the IEC for approval, after prior discussion between 
the Sponsor and the Co-ordinating Investigator. 
The CA and IEC will be informed about any changes in the study protocol, the end of the 
study, or the premature study termination as appropriate and within the requested time 
period. 
 
13.2 SUBJECT’S INSURANCE 
For subjects participating in the study, Sponsor has stipulated an insurance policy in 
accordance with local regulatory requirements. 
Details on the insurance company, the insurance number and conditions will be made 
available to subjects in the ICF and/or provided as a separate document, in accordance 
with national requirements. 
A copy of the insurance certificate will be provided to each Investigator and will be filed in 
the Investigator’s File at the sites and in the study’s Trial Master File (TMF). 
The Investigator must notify to Sponsor immediately upon notice of any claims or lawsuits. 
 
14. DATA PROTECTION LAWS COMPLIANCE 
By signing the study protocol, the Institution and the Investigator (including their appointed 
staff) acknowledge that the performance of the study will imply processing of personal 
data. Personal data processing is regulated by the Sponsor national legislation as well as 
local laws (Italy). The Sponsor specifies that strict compliance with the applicable data 
protection laws by any parties and relevant employees who take part in the study will be 
an essential condition for the appointment of and the collaboration with research 
institutions, investigators and the CRO. 
The parties shall acknowledge that according to the applicable privacy laws, Sponsor and 
Institution/s will act as independent data controllers while CRO and Investigator/s will act 
as data processors.  
Given the sensitive nature of data processed in the frame of the Study, the parties 
undertake to take adequate safety measures (physical, logical, organizational, technical, 
etc) to warrant that data will always be processed safely and in compliance with the Italian 
D.Lgs.196/03. 
Investigator and Institution (including their personnel) shall comply with the applicable 
privacy laws on the protection of personal data.  
Investigator must assure the subject’s anonymity will be maintained. The Investigator will 
keep a separate log of the subject’s study numbers, names, addresses and telephone 

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numbers. The Investigator will maintain this for the longest period of time allowed from 
own institution and in any case, till further communication from Sponsor. 
 
15. DATA HANDLING AND RECORDS KEEPING 
The Investigator should keep all study-related documents, as specified in ICH/GCP 
Section 8 “Essential Documents for the Conduct of a Clinical Trial” and all study 
documents as specified by the applicable regulatory requirement(s), in the Investigator's 
Trial Center File. 
The Investigator will take measures to prevent accidental or premature destruction of 
these documents. 
Essential documents must be retained for minimum 7 years.  
If the responsible Investigator retires, relocates, or for other reasons withdraws from the 
responsibility of keeping the study records, custody must be transferred to a person who 
will accept the responsibility.  
The Sponsor must be notified in writing of the name and address of the new custodian.  
If it becomes necessary for the Sponsor and/or a Regulatory Authority to review any 
documentation related to this study, the Investigator must permit access to such 
documentation. 
Any difficulty in storing original documents should be discussed with the Sponsor 
personnel prior to initiation of the study. 
The protocol must be read thoroughly by everybody whom the information therein 
concerns and the instructions must be exactly followed. 
Changes in the study protocol will require a protocol amendment. Such amendments will 
be agreed upon and approved in writing by all signatories of the protocol. If amendments 
are substantial, i.e. are likely to have an impact on the safety of the subjects, or to change 
the interpretation of the scientific documents in support of the conduct of the study, or if 
they are otherwise significant, the IEC has to approve these amendments before 
implementation. 
Changes which have no significant impact on medical or scientific validity of the study will 
be agreed upon and approved in writing by all signatories of the protocol and the IEC will 
be notified of this protocol amendment. 
Any substantial amendments of the protocol will be integrated in an updated study 
protocol. The Principal Investigator will ensure full compliance with the updated study 
protocol.  
 
 

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16. PUBLICATION POLICY AND RESULTS 
By signing the study protocol, the Investigator (and his/her appointed staff) affirms that any 
information and all the study documents provided by the Sponsor will be maintained strictly 
confidential. 
None of this material may be disclosed to any party not directly involved in the study 
without written permission from Sponsor. 
The Investigator will supply the Sponsor with all the data/results from the study.  
All information concerning the study, the study product as well as data and results of the 
study are confidential and property of the Sponsor. The Sponsor will prepare the final 
report, including the statistical and clinical evaluations. The Investigator’s agreement and 
signature will be obtained and a copy will be provided to the Investigator. 
Sponsor reserves the exclusive right to publish and present data and results of the present 
study at scientific meetings, or to submit these clinical study data to national and 
international Regulatory Authorities. The Investigator may not use the results of this study 
for publication or presentation without written authorization from Sponsor. 
 
 
 
 

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17. REFERENCES 
 
Agca CA, Tuzcu M, Gencoglu H, Akdemir F, Ali S, Sahin K, Kucuk O. Lycopene 
counteracts the hepatic response to 7,12-dimethylbenz[a]anthracene by altering the 
expression of Bax, Bcl-2, caspases, and oxidative stress biomarkers. Pharm Biol. 2012 
Dec;50(12):1513-8. doi: 10.3109/13880209.2012.688057. Epub 2012 Sep 14. 
 
Aisen PS, Schneider LS, Sano M, Diaz-Arrastia R, van Dyck CH, Weiner MF, Bottiglieri T, 
Jin S, Stokes KT, Thomas RG, Thal LJ; Alzheimer Disease Cooperative Study.High-dose 
B vitamin supplementation and cognitive decline in Alzheimer disease: a randomized 
controlled trial. JAMA. 2008 Oct 15;300(15):1774-83. doi: 10.1001/jama.300.15.1774. 
 
Chatterjee N, Rastogi RP, Dhar ML (1963) Chemical examination of Bacopa munniera 
Wettst.: Part I – Isolation of chemical constituents. Indian J Chem, : 212–215. 
 
Committee for Proprietary Medicinal Products (CPMP). Points to consider on multiplicity 
issues in clinical trials. EMEA 2002. 
  
De Rijk MC1, Breteler MM, den Breeijen JH, Launer LJ, Grobbee DE, van der Meche FG, 
Hofman A “Dietary antioxidants and Parkinson disease. The Rotterdam Study. Arch 
Neurol. 1997 Jun;54(6):762-5. 
 
Deschamps V, Barberger-Gateau P, Peuchant E, Orgogozo JM. Nutritional factors in 
cerebral aging and dementia: epidemiological arguments for a role of oxidative stress. 
Neuroepidemiology. 2001 Feb;20(1):7-15. 
 
Di Matteo V, Pierucci M, Di Giovanni G, Dragani LK, Murzilli S, Poggi A, Esposito E. Intake 
of tomatoenriched diet protects from 6-hydroxydopamine-induced degeneration of rat 
nigral dopaminergic neurons J Neural Transm Suppl. 2009;(73):333-41. 
 
Engelhart M, Geerlings MI, Ruitenberg A, Van Swieten JC, Hofman A, Witteman JC, 
Breteler MM., Diet and risk of dementia: Does fat matter?: The Rotterdam Study. 
Neurology. 2002 Dec 24;59(12):1915-21. 
 
Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea FM Astaxanthin: a review of its 
chemistry and applications. Crit Rev Food Sci Nutr. 2006;46(2):185-96. 
 
Higuera-Ciapara I1, Felix-Valenzuela L, Goycoolea FM. Crit Rev Food Sci Nutr. 
2006;46(2):185-96 Astaxanthin: a review of its chemistry and applications. 
 
Jenner P, Olanow CW “Oxidative stress and the pathogenesis of Parkinson's disease 
Neurology” 1996 Dec;47(6 Suppl 3):S161-70.Karppi J, Laukkanen JA, Sivenius J, 
Ronkainen 
K, 
Kurl 
S. 
Neurology. 
2012 
Oct 
9;79(15):1540-7. 
doi: 

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10.1212/WNL.0b013e31826e26a6 Serum lycopene decreases the risk of stroke in men: a 
population-based follow-up study. 
 

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18. Public Protocol Approval Page 
Study Title: “Effect of an antioxidants mix on cognitive performance and well being: 
The Bacopa, Licopene, Astaxantina, Vitamin B12” – BLAtwelve study 
Code: MEIF/17/BAC-COG/001 
EUDRA-CT number: not applicable 
 
The signers confirm that they have read and approved the protocol 
 
Study Medical Expert: (name in capital letters)  ______________________________________ 
 
Signature & Date: __________________________________________   _____  /  _____  /  __________ 
 
 
 
 
 
 
 
 
 
                
 
Coordinating Investigator: (if applicable) (name in capital letters)  _________________________ 
 
Signature & Date: __________________________________________   _____  /  _____  /  __________ 
 
Statistician: (name in capital letters)  ________________________________________________ 
 
Signature & Date: __________________________________________   _____  /  _____  /  __________ 
 
 
 
 
 
 
 
 
 
 
 
 
Project Leader CRO (if applicable) (name in capital letters)  ________________________________ 
 
Signature & Date: _________________________________________   _____  /  _____  /  __________ 
 
 
 
 
 
 
 
 
 
 
 
 
Corporate Medical Director/International Scientific CU Manager: _________________________ 
 
Signature & Date: _________________________________________   _____  /  _____  /  __________ 
 
 
 
 

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19. Investigator’s approval Page (original to be kept in the Trial Center File) 
 
I understand that all information concerning the product Bacopa, Licopene, Astaxantina, Vitamin B12 supplied 
by A. Menarini Industrie Farmaceutiche Riunite S.r.l., Sponsor of the study, in connection with this study 
protocol are confidential information. This information include: Protocol, Investigator’s Brochure, Case Report 
Form, Other documents. 
 
I understand that any change in this study protocol must be approved in writing by the Sponsor, the Co-
ordinating Investigator and the Ethics Committee before implementation, except where necessary to eliminate 
apparent immediate hazard to subjects. 
 
I confirm that I will conduct the study according to this protocol (except when mutually agreed to in writing with 
the Sponsor), the Good Clinical Practice (GCP), the Declaration of Helsinki and laws and regulations in the 
Country where the study is to be conducted. 
 
I confirm that I will record and report all adverse events occurring during the study, according to this protocol. 
I confirm that I am informed about the need of data records retention, according to current regulations and that 
no data can be destroyed without the written consent of the Sponsor. 
I confirm that I will transfer adequate ownership of my responsibilities for the trial and will inform the Sponsor, in 
case I retire from my PI role. 
I confirm that in case the Trial Center File is stolen or anyhow damaged, I will promptly inform the Sponsor and 
declare it to the Competent Authorities. 
 
 
Principal Investigator: ____________________________________________________ 
 
Signature & Date: _________________________________________________________    
 
 
 
 
 
 
 
 
 
 
                         
 
 
 
 
 
 
 
 
 

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20. APPENDICES 
20.1 
Appendix  1: Declaration of Helsinki 
 
 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI 
Ethical Principles for Medical Research Involving Human Subjects 
 
Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964 
and amended by the: 
29th WMA General Assembly, Tokyo, Japan, October 1975 
35th WMA General Assembly, Venice, Italy, October 1983 
41st WMA General Assembly, Hong Kong, September 1989 
48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996 
52nd WMA General Assembly, Edinburgh, Scotland, October 2000  
53rd WMA General Assembly, Washington DC, USA, October 2002 (Note of Clarification added) 
55th WMA General Assembly, Tokyo, Japan, October 2004 (Note of Clarification added) 
59th WMA General Assembly, Seoul, Republic of Korea, October 2008 
64th WMA General Assembly, Fortaleza, Brazil, October 2013 
 
Preamble 
1.         The World Medical Association (WMA) has developed the Declaration of Helsinki as a statement of ethical principles 
for medical research involving human subjects, including research on identifiable human material and data. 
            The Declaration is intended to be read as a whole and each of its constituent paragraphs should be applied with 
consideration of all other relevant paragraphs. 
2.         Consistent with the mandate of the WMA, the Declaration is addressed primarily to physicians. The WMA 
encourages others who are involved in medical research involving human subjects to adopt these principles.  
General Principles 
3.         The Declaration of Geneva of the WMA binds the physician with the words, “The health of my patient will be my first 
consideration,” and the International Code of Medical Ethics declares that, “A physician shall act in the patient's best 
interest when providing medical care.” 
4.         It is the duty of the physician to promote and safeguard the health, well-being and rights of patients, including those 
who are involved in medical research. The physician's knowledge and conscience are dedicated to the fulfilment of this 
duty. 
5.         Medical progress is based on research that ultimately must include studies involving human subjects. 
6.         The primary purpose of medical research involving human subjects is to understand the causes, development and 
effects of diseases and improve preventive, diagnostic and therapeutic interventions (methods, procedures and treatments). 
Even the best proven interventions must be evaluated continually through research for their safety, effectiveness, efficiency, 
accessibility and quality. 
7.         Medical research is subject to ethical standards that promote and ensure respect for all human subjects and protect 
their health and rights. 

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8.         While the primary purpose of medical research is to generate new knowledge, this goal can never take precedence 
over the rights and interests of individual research subjects. 
9.         It is the duty of physicians who are involved in medical research to protect the life, health, dignity, integrity, right to 
self-determination, privacy, and confidentiality of personal information of research subjects. The responsibility for the 
protection of research subjects must always rest with the physician or other health care professionals and never with the 
research subjects, even though they have given consent. 
10.       Physicians must consider the ethical, legal and regulatory norms and standards for research involving human 
subjects in their own countries as well as applicable international norms and standards. No national or international ethical, 
legal or regulatory requirement should reduce or eliminate any of the protections for research subjects set forth in this 
Declaration. 
11.       Medical research should be conducted in a manner that minimises possible harm to the environment. 
12.       Medical research involving human subjects must be conducted only by individuals with the appropriate ethics and 
scientific education, training and qualifications. Research on patients or healthy volunteers requires the supervision of a 
competent and appropriately qualified physician or other health care professional. 
13.       Groups that are underrepresented in medical research should be provided appropriate access to participation in 
research. 
14.       Physicians who combine medical research with medical care should involve their patients in research only to the 
extent that this is justified by its potential preventive, diagnostic or therapeutic value and if the physician has good reason to 
believe that participation in the research study will not adversely affect the health of the patients who serve as research 
subjects. 
15.       Appropriate compensation and treatment for subjects who are harmed as a result of participating in research must 
be ensured. 
Risks, Burdens and Benefits  
16.       In medical practice and in medical research, most interventions involve risks and burdens. 
           Medical research involving human subjects may only be conducted if the importance of the objective outweighs the 
risks and burdens to the research subjects. 
17.       All medical research involving human subjects must be preceded by careful assessment of predictable risks and 
burdens to the individuals and groups involved in the research in comparison with foreseeable benefits to them and to other 
individuals or groups affected by the condition under investigation. 
           Measures to minimise the risks must be implemented. The risks must be continuously monitored, assessed and 
documented by the researcher.  
18.       Physicians may not be involved in a research study involving human subjects unless they are confident that the 
risks have been adequately assessed and can be satisfactorily managed. 
           When the risks are found to outweigh the potential benefits or when there is conclusive proof of definitive outcomes, 
physicians must assess whether to continue, modify or immediately stop the study.            
Vulnerable Groups and Individuals 
19.       Some groups and individuals are particularly vulnerable and may have an increased likelihood of being wronged or 
of incurring additional harm. 

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           All vulnerable groups and individuals should receive specifically considered protection. 
20.       Medical research with a vulnerable group is only justified if the research is responsive to the health needs or 
priorities of this group and the research cannot be carried out in a non-vulnerable group. In addition, this group should stand 
to benefit from the knowledge, practices or interventions that result from the research. 
Scientific Requirements and Research Protocols  
21.       Medical research involving human subjects must conform to generally accepted scientific principles, be based on a 
thorough knowledge of the scientific literature, other relevant sources of information, and adequate laboratory and, as 
appropriate, animal experimentation. The welfare of animals used for research must be respected. 
22.       The design and performance of each research study involving human subjects must be clearly described and 
justified in a research protocol. 
           The protocol should contain a statement of the ethical considerations involved and should indicate how the principles 
in this Declaration have been addressed. The protocol should include information regarding funding, sponsors, institutional 
affiliations, potential conflicts of interest, incentives for subjects and information regarding provisions for treating and/or 
compensating subjects who are harmed as a consequence of participation in the research study. 
           In clinical trials, the protocol must also describe appropriate arrangements for post-trial provisions. 
Research Ethics Committees 
23.       The research protocol must be submitted for consideration, comment, guidance and approval to the concerned 
research ethics committee before the study begins. This committee must be transparent in its functioning, must be 
independent of the researcher, the sponsor and any other undue influence and must be duly qualified. It must take into 
consideration the laws and regulations of the country or countries in which the research is to be performed as well as 
applicable international norms and standards but these must not be allowed to reduce or eliminate any of the protections for 
research subjects set forth in this Declaration.  
           The committee must have the right to monitor ongoing studies. The researcher must provide monitoring information 
to the committee, especially information about any serious adverse events. No amendment to the protocol may be made 
without consideration and approval by the committee. After the end of the study, the researchers must submit a final report 
to the committee containing a summary of the study’s findings and conclusions.  
Privacy and Confidentiality  
24.       Every precaution must be taken to protect the privacy of research subjects and the confidentiality of their personal 
information. 
Informed Consent  
25.       Participation by individuals capable of giving informed consent as subjects in medical research must be voluntary. 
Although it may be appropriate to consult family members or community leaders, no individual capable of giving informed 
consent may be enrolled in a research study unless he or she freely agrees. 
26.       In medical research involving human subjects capable of giving informed consent, each potential subject must be 
adequately informed of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliations of 
the researcher, the anticipated benefits and potential risks of the study and the discomfort it may entail, post-study 
provisions and any other relevant aspects of the study. The potential subject must be informed of the right to refuse to 
participate in the study or to withdraw consent to participate at any time without reprisal. Special attention should be given 
to the specific information needs of individual potential subjects as well as to the methods used to deliver the information. 

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           After ensuring that the potential subject has understood the information, the physician or another appropriately 
qualified individual must then seek the potential subject’s freely-given informed consent, preferably in writing. If the consent 
cannot be expressed in writing, the non-written consent must be formally documented and witnessed.       
           All medical research subjects should be given the option of being informed about the general outcome and results of 
the study. 
27.       When seeking informed consent for participation in a research study the physician must be particularly cautious if 
the potential subject is in a dependent relationship with the physician or may consent under duress. In such situations the 
informed consent must be sought by an appropriately qualified individual who is completely independent of this relationship. 
28.       For a potential research subject who is incapable of giving informed consent, the physician must seek informed 
consent from the legally authorised representative. These individuals must not be included in a research study that has no 
likelihood of benefit for them unless it is intended to promote the health of the group represented by the potential subject, 
the research cannot instead be performed with persons capable of providing informed consent, and the research entails 
only minimal risk and minimal burden. 
29.       When a potential research subject who is deemed incapable of giving informed consent is able to give assent to 
decisions about participation in research, the physician must seek that assent in addition to the consent of the legally 
authorised representative. The potential subject’s dissent should be respected. 
30.       Research involving subjects who are physically or mentally incapable of giving consent, for example, unconscious 
patients, may be done only if the physical or mental condition that prevents giving informed consent is a necessary 
characteristic of the research  group. In such circumstances the physician must seek informed consent from the legally 
authorised representative. If no such representative is available and if the research cannot be delayed, the study may 
proceed without informed consent provided that the specific reasons for involving subjects with a condition that renders 
them unable to give informed consent have been stated in the research protocol and the study has been approved by a 
research ethics committee. Consent to remain in the research must be obtained as soon as possible from the subject or a 
legally authorised representative. 
31.       The physician must fully inform the patient which aspects of their care are related to the research. The refusal of a 
patient to participate in a study or the patient’s decision to withdraw from the study must never adversely affect the patient-
physician relationship. 
32.       For medical research using identifiable human material or data, such as research on material or data contained in 
biobanks or similar repositories, physicians must seek informed consent for its collection, storage and/or reuse. There may 
be exceptional situations where consent would be impossible or impracticable to obtain for such research. In such situations 
the research may be done only after consideration and approval of a research ethics committee. 
Use of Placebo 
33.       The benefits, risks, burdens and effectiveness of a new intervention must be tested against those of the best proven 
intervention(s), except in the following circumstances: 
           Where no proven intervention exists, the use of placebo, or no intervention, is acceptable; or 
           Where for compelling and scientifically sound methodological reasons the use of any intervention less effective than 
the best proven one, the use of placebo, or no intervention is necessary to determine the efficacy or safety of an 
intervention and the patients who receive any intervention less effective than the best proven one, placebo, or no 
intervention will not be subject to additional risks of serious or irreversible harm as a result of not receiving the best proven 
intervention.  
           Extreme care must be taken to avoid abuse of this option. 
Post-Trial Provisions 

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Sponsor: A. Menarini Industrie Farmaceutiche Riunite S.r.l. 
 
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34.       In advance of a clinical trial, sponsors, researchers and host country governments should make provisions for post-
trial access for all participants who still need an intervention identified as beneficial in the trial. This information must also be 
disclosed to participants during the informed consent process. 
Research Registration and Publication and Dissemination of Results 
35.       Every research study involving human subjects must be registered in a publicly accessible database before 
recruitment of the first subject. 
36.       Researchers, authors, sponsors, editors and publishers all have ethical obligations with regard to the publication 
and dissemination of the results of research. Researchers have a duty to make publicly available the results of their 
research on human subjects and are accountable for the completeness and accuracy of their reports. All parties should 
adhere to accepted guidelines for ethical reporting. Negative and inconclusive as well as positive results must be published 
or otherwise made publicly available. Sources of funding, institutional affiliations and conflicts of interest must be declared in 
the publication. Reports of research not in accordance with the principles of this Declaration should not be accepted for 
publication. 
Unproven Interventions in Clinical Practice 
37.       In the treatment of an individual patient, where proven interventions do not exist or other known interventions have 
been ineffective, the physician, after seeking expert advice, with informed consent from the patient or a legally authorised 
representative, may use an unproven intervention if in the physician's judgement it offers hope of saving life, re-establishing 
health or alleviating suffering. This intervention should subsequently be made the object of research, designed to evaluate 
its safety and efficacy. In all cases, new information must be recorded and, where appropriate, made publicly available.