Key nutrients in the management of synaptic failure a result of Alzheimer s disease

Key nutrients in the management of synaptic failure a result of Alzheimer s disease Patrick Kamphuis, PhD Global Head of Medical and Scientific Affairs Nutricia, Schiphol, The Netherlands

Disclosure statement Employee of Nutricia Research No other relevant disclosures

Alzheimer s disease: multiple pathologies, multiple approaches T Amyloid Plaques A R G Neurofibrillary tangles AD Synapse loss Synapse loss E T Neurotransmitter deficits

Saturated fatty acid Synapses are built from neuronal membranes Opportunity for a dietary approach Choline Phosphate dendritic spine Glycerol neurite Axon

Nutritional precursors enhance the synthesis rate of neuronal membranes The Kennedy pathway for biosynthesis of neuronal membrane Synapses consist principally of neuronal membranes dendritic Axon spine terminal neurite Dendritic spine Neurite Axon Kennedy & Weiss (1956) J. Bio. Chem. 222, 193-214

Lower nutrient intake due to poor food choices in early AD Lower intake from food sources of DHA vitamin B-6 vitamin B-12 Folate vitamin C vitamin E Lower intake of Fruits Nuts Seeds Fish Higher intake of Sugars & Snacks Shatenstein et al., J Am Diet Assoc, 2007 Phillips et al., Nutr Neurosci, 2012; Hart et al., Nutr Soc, 2013

Nutrition and the brain link with Alzheimer s disease (AD)? Deficiencies in essential nutrients can result in neurological symptoms Diet and nutrient intake may change with age In AD patients, poor nutrition may be related to failure to remember to eat, changes in eating preferences, and/or olfactory changes Epidemiological evidence suggests potential relationship between nutrient intake and dementia B-Vitamins Important nutrients Antioxidants vitamins C and E involved in synaptic Dietary fats membrane synthesis Dietary patterns i.e., Mediterranean diet

Relative Risk Incidence (per 100 persons year) Nutrients and risk of AD Rotterdam study Fish consumption 1 7 6 AD Dementia 5 4 3 2 1 0 <95 96-133 >133 mg/d Vitamin C intake 0 daily > weekly < weekly never Engelhart et al., JAMA 2002 Collaboration in Dementia project Barberger-Gateau et al., BMJ, 2002 Dangour, Vellas et al., J Alz Dis 2010. Systematic review on Bvits and Fatty acids

But single nutrient intervention doesn t benefit Nutrient Author Journal n3 PUFAs B-vitamins Quinn 2010 Freund-Levi 2006 Smith 2010 Aisen 2008 JAMA Arch Neurol Plos JAMA #Subjects/ Duration 402 18 months 174 6 months 271 24 months 409 18 months Outcome DHA compared with placebo did not slow the rate of cognitive and functional decline in mild-moderate AD patients. No delay the rate of cognitive decline. However, positive effects were observed in a small group of patients with very mild AD (MMSE>27) Accelerated rate of brain atrophy in mild cognitive impairment can be slowed by treatment with homocysteine-lowering B vitamins. No cognitive benefit. Post hoc (Jeneren 2015) links reduced atrophy to high DHA/EPA sub group. This regimen of high-dose B vitamin supplements does not slow cognitive decline in individuals with mild to moderate AD. McMahon 2006 N Eng J Med 276 24 months The results of this trial do not support the hypothesis that homocysteine lowering with B vitamins improves cognitive performance. Vitamin E / Antioxidants Vitamin D2 Ginkgo biloba Dysken 2014 Petersen 2005 Galasko 2012 Stein 2011 DeKosky 2008 JAMA N Eng J Med Arch Neurol J Alz Disease JAMA 304 Mean f-up 27 months 769 36 months 52 16 weeks 32 8 weeks 3069 median f-up 6.1a Among patients with mild to moderate AD, 2000 IU/d of alpha-tocopherol compared with placebo resulted in slower functional decline. Vitamin E had no benefit in patients with mild cognitive impairment. However, this treatment (vitamin E + vitamin C plus α-lipoic acid) raised the caution of faster cognitive decline We conclude that high-dose vitamin D provides no benefit for cognition or disability over low-dose vitamin D in mild-moderate AD Ginkgo biloba at 120 mg twice a day was not effective in reducing either the overall incidence rate of dementia or AD incidence in elderly individuals with normal cognition or those with MCI.

High adherence to Mediterranean diet reduces risk of cognitive impairment AD MCI MMSE Psaltopoulou et al, Ann Neurol 2013

Epidemiological evidence suggests dietary patterns can reduce AD risk Epidemiological evidence suggests certain dietary patterns can reduce AD risk: Mediterranean Diet 1,2 Based on higher intakes of fish, vegetables, fruit DASH diet 3 and monounsaturated fats As above with emphasis on reduced intake of dairy and salt MIND / Nordic / Finnish diet 4,5 As per Mediterranean diet but with less fish and fruit and more green vegetables and berries 1. Scarmeas N, et al. Mediterraneandiet and risk for Alzheimer s disease. AnnNeurol 2006;59:912 21. 2. Martinez-Lapiscina EH, et al. Mediterranean diet improves cognition: the PREDIMED-NAVARRA randomised trial. J Neurol Neurosurg Psychiatry 2013;84:1318 25 3 Tangney CC, et al. Relation of DASH- and Mediterranean-like dietary patterns on cognitive decline in older persons. Neurology 2014;83:1410 6. 4 Morris MC, et al. MIND diet associated with reduced incidence of Alzheimer s disease. Alzheimer s & Dementia 11 (2015) 1007-1014 5 Akesson A, et al. Health effects associated with foods characteristic of the Nordic diet. Food Nutr. Res57 ( 2013), 10.3402.

Systematic review & meta-analysis: nutrient availability in AD According to Preferred Reporting Items for Systematic Reveiws and Meta-Analysis (PRISMA) guidelines Analyses by independent statistics

3 studies 10 studies 11 studies 9 studies 6 studies 37 studies 31 studies 8 studies 20 studies 11 studies Lower blood status in AD of nutrients essential for synaptic membrane synthesis Systematic review and meta-analysis of literature % Cognitive Intact elderly * 1,2,3 * * * *** *** *** *** * 90 80 70 60 50 1:Trushina (2013) PLOS 2:Olde Rikkert (2014) JAD 3: Wang (2014) J Prot Res Meta-analyses De Wilde et al., poster AAIC 2014 Meta-analyses Lopes da Silva (2013) Alz Dement

12 studies 31 studies 12 studies 4 studies 9 studies 5 studies 5 studies 7 studies 4 studies 12 studies Lower brain status in AD of nutrients essential for synaptic membrane synthesis Brain nutrient levels in AD versus cognitively healthy elderly 110 % Cognitively Healthy elderly 90 * 1 * 1 ns * 1 * 1 * 1 * 1 1 * 2 ns 3 ns 1 80 70 60 50 De Wilde, AAIC 2016

Increased nutritional need in AD Higher nutrient need due to synaptic membrane loss? Decreased synapse number in AD Nutrient control of membrane and synapse formation Lower nutrients levels in AD? Lower intake, and compromised endogenous synthesis Lower levels in blood Lower levels in the brain

Development of a Medical Food containing Dietary precursors and cofactors 400 mcg Folate 625 mg UMP 300 mg EPA 400 mg Choline 80 mg Vit C 1200 mg DHA 3 mcg Vit B12 40 mg Vit E 60 mcg Selenium 106 mg Phospholipids 1 mg Vit B6

Development of a Medical Food containing Dietary precursors and cofactors Stimulating synapse membrane formation requires specific nutrients Uridine (UMP), Omega-3 fatty acids, Phosholipids & Choline, B-Vitamins, Antioxidants Lower Nutrient status Increased nutritional need cannot be met by the regular diet HYPOTHESIS: Souvenaid successfully addresses an unmet nutritional need in people with AD by increasing their intake of these dietary precursors and co-factors

% of patients 2008 - FIRST HUMAN TRIAL SHOWED THAT SOUVENAID IMPROVED MEMORY SCORES IN PEOPLE WITH MILD AD 50 40 30 20 10 0 Improved memory in mild and very mild AD Control worsened unchanged improved Souvenaid 50 40 30 20 10 0 worsened unchanged improved

completed & published The Souvenaid clinical trial program amci due to AD Mild AD Moderate AD S-Connect ADAS-cog n=527 (MMSE 14-24); stable on AD drugs; US Souvenir I Souvenir II Souvenir II Open Label WMS-r & ADAS-cog n=225 NL, Ger, Bel, UK, US NTB + EEG n=259 NL, Ger, Bel, Fr, It, Sp Safety + Compliance + NTB This project receives funding from NL STW This project receives funding from the NL Food & Nutrition Delta project, FND N 10003 This project receives funding from the NL Food & Nutrition Delta project, FND N 10003

Souvenir II: design and methodology Multi-country randomized, controlled trial in drug naive mild AD over 24 weeks, with a further 24 weeks open label phase Primary outcome: Memory Domain NTB (z-score) at 24 weeks RAVLT immediate, delayed, recognition and VPA immediate and delayed Baseline Characteristics Control (n = 129) Souvenaid (n = 130) n=259 Outcome parameters Souvenaid (n=130) Souvenaid Control (n=129) Souvenaid 0 12 24 48 Double blind phase Open Label Age (y) 73.2 (8.4) 74.4 (6.9) Sex: males (n[%]) 64 (49.6) 68 (52.3) Years of education on top of primary school Values are mean±sd, unless stated otherwise 6.6 (4.6) 6.5 (4.8) Total MMSE score 25.1 (2.9) 25.1 (2.8) Duration AD since diagnosis (months) (median[range]) 2.0 (0.0-88.0) 1.0 (0.0-70.0) BMI (kg/m2) 26.7 (4.2) 26.1 (4.1)

Sustained improvement in memory performance over 24 and 48 weeks Primary: memory domain z-score (p=0.023) Seconday: NTB composite z-score trend (p=0.053) 24 wk vs baseline p = 0.035 24 wk vs baseline p = 0.090 24 wk vs baseline p = 0.035 Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36

Sustained improvement in memory performance over 24 and 48 weeks Primary: memory domain z-score (p=0.023) Active - Active: p=0.038 Control - Active: p=0.029 Seconday: NTB composite z-score trend (p=0.053) 24 wk vs baseline p = 0.035 24 wk vs baseline p = 0.090 24 wk vs baseline p = 0.035 Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36 Olde Rikkert et al. J Alzheimers Dis. 2015

DHA (% total fatty acids erythrocyte membrane) Souvenaid increases nutrient blood levels - Data from Souvenir II & Open label extension 45 Control Souvenaid Souvenaid Uridine Folate pla DHA 40 Choline B12 pla EPA 35 edha B6 Vit E 30 0 Sample size (N) Double-blind phase 0 24 eepa 129 128 114 119 Open-label phase Hcy 48 87 93 Time (weeks) Selenium

Souvenaid increases nutrient blood levels - Data from Souvenir II & Open label extension Uridine Folate pla DHA Choline B12 pla EPA edha B6 Vit E eepa Hcy Selenium

Souvenaid increases EEG biomarkers for functional connectivity, derivatives of synaptic activity Level 3: network analysis In AD: disruption of optimal organization Stam 2004 Level 2: connectivity analysis In AD: decreased functional connectivity Babiloni et al. 2004 Level 1: basic signal analysis De Waal et al., PlosOne, 2014; Scheltens et al., J Alz Dis, 2012 In AD: slowing of the oscillations Snaedal et al. 2010

Souvenaid increases EEG biomarkers for functional connectivity, derivatives of synaptic activity Level 3: network analysis In AD: disruption of optimal organization p = 0.009 p = 0.053 Stam 2004 Level 2: connectivity analysis In AD: decreased functional connectivity p = 0.011 Babiloni et al. 2004 Level 1: basic signal analysis De Waal et al., PlosOne, 2014; Scheltens et al., J Alz Dis, 2012 In AD: slowing of the oscillations p = 0.019 Snaedal et al. 2010

Lower levels of multiple key nutrients in early AD (MMSE 25) compared to age-matched healthy controls P = 0.046 Baseline data from 79 AD patients Data collected at single timepoint for 93 healthy subjects matched for age, gender and country No significant differences B12 (p=0.158), Folate (p=0.309) Olde-Rikkert et al., J Alz Dis, accepted

MRS Study: Design & methodology Goal: A Magnetic Resonance Spectroscopy (MRS) study to explore the effects of Souvenaid on brain phospholipid metabolites in patients with mild Alzheimer s disease dementia (AD) Experimental set up: 4-week, randomised, controlled, double blind, parallel-group, single-centre, exploratory study in 33 drug naïve mild AD patients Rijpma et al. Neurobiol Aging 2016 39 (S1) S7-8

The Souvenaid clinical trial program amci due to AD Mild AD Moderate AD S-Connect ADAS-cog n=527 (MMSE 14-24); stable on AD drugs; US Souvenir I Souvenir II Open Label MRS study WMS-r & ADAS-cog n=225 NL, Ger, Bel, UK, US NTB + EEG n=259 NL, Ger, Bel, Fr, It, Sp Safety + Compliance + NTB 31P and 1H-MRS 30 drug-naïve, NL This project receives funding from NL STW This project receives funding from the NL Food & Nutrition Delta project, FND N 10003 This project receives funding from the NL Food & Nutrition Delta project, FND N 10003 LipiDiDiet NTB + MRI / CSF n=311(mmse 24); Ger, NL, FI, SW Funded by the EU FP7 project LipiDiDiet, Grant Agreement N 211696 FDG-PET MIND-AD FDG-PET 40 drug-naïve, NL Feasibility, tbd This project receives funding by the NL NWO-FCB This project is funding by the Joint Programming Neurodegenerative Disease Research

Favorable safety profile across clinical trial program Souvenaid is well tolerated High compliance across all the trials: >95% Trial Total trial n= Total AEs Control AEs Active AEs P-value SI 225 107 49 58 ns SII 259 239 135 104 ns S-Connect 527 903 445 458 ns SII open label 201 135 135 - LDD 311 270 138 132 ns AE s are consistent with that expected for elderly population with AD No SAE s considered to be related to the study product NNH values for Souvenaid are very high or not calculable, in accordance with its good safety and tolerability profile

Lower vitamin, mineral and omega/3 fatty acid nutrient folate status in folate AD: Meta-analyses n mean sd n mean sd n mean sd n mean sd n mean sd n mean study control control control AD AD AD study control control control study AD AD AD control control control AD AD Folate Agarwal '10 127 15.7 30.1 32 15.0 14.7 Agarwal '10 Anello '04 127 181 15.7 15.7 30.1 5.9 Agarwal 32 15.0 '10 180 14.3 14.7 127 5.7 15.7 30.1 32 15.0 Anello '04 Asita De Silva '05 181 21 15.7 19.7 5.9 9.7 Anello 180 '04 14.3 23 15.9 5.7 8.4 181 15.7 5.9 180 14.3 Asita De Silva '05 Cascalheira '09 21 36 19.7 20.4 9.7 1.7 Asita 23 De 15.9 Silva '05 8.4 19 18.8 5.3 21 19.7 9.7 23 15.9 Cascalheira '09 Clarke '98 36 108 20.4 22.9 1.7 10.0 Cascalheira 19 18.8 '09 5.3 36 164 17.6 10.7 20.4 1.7 19 18.8 Clarke '98 Dominguez '05 108 19 22.9 29.6 10.0 9.0 Clarke 164 '98 17.6 29 17.9 10.7 108 7.2 22.9 10.0 164 17.6 Dominguez '05 Faux '11 19 760 29.6 30.3 9.0 12.7 Dominguez 29 17.9 '05 7.2 19 205 29.4 14.5 29.6 9.0 29 17.9 Faux '11 Galimberti '08 760 23 30.3 19.8 12.7 6.2 Faux 205 '11 29.4 29 8.6 14.5 760 2.8 30.3 12.7 205 29.4 Galimberti '08 Galluci '04 23 42 19.8 14.1 6.2 11.1 Galimberti 29 8.6 '08 137 11.6 2.8 6.1 23 19.8 6.2 29 8.6 Galluci '04 Hogervorst '02 42 62 14.1 24.9 11.1 11.3 Galluci 137 11.6 '04 66 15.9 6.1 42 11.3 14.1 11.1 137 11.6 Hogervorst '02 Irizarry '05 62 88 24.9 35.2 11.3 32.9 Hogervorst 66 15.9'02 145 29.9 11.3 62 21.3 24.9 11.3 66 15.9 Irizarry '05 Joosten '97 88 49 35.2 8.6 32.9 3.2 Irizarry 145 29.9 '05 52 7.9 21.3 88 4.2 35.2 32.9 145 29.9 Joosten '97 Karimi '09 49 49 8.6 15.9 3.2 8.6 Joosten 52 7.9 '97 51 14.5 4.2 6.6 49 8.6 3.2 52 7.9 Karimi '09 Koseoglu '07 Koseoglu '07 Lelhuber '00 49 40 40 19 15.9 28.1 28.1 14.3 8.6 3.4 3.4 9.3 Karimi 51 '09 14.5 51 21.4 Koseoglu 51 21.4 '07 19 10.0 6.6 4.4 4.4 3.4 49 40 15.9 28.1 8.6 3.4 51 51 14.5 21.4 Lelhuber '00 Li '04 19 30 14.3 37.2 9.3 21.2 Lelhuber 19 10.0 '00 30 29.2 3.4 19 12.7 14.3 9.3 19 10.0 Li '04 Linnebank '10 30 60 37.2 14.1 21.2 7.7 Li 30'04 29.2 60 15.6 12.7 30 7.0 37.2 21.2 30 29.2 Linnebank '10 Lovati '07 60 76 14.1 15.6 7.7 7.9 Linnebank 60 15.6'10 108 8.2 7.0 5.3 60 14.1 7.7 60 15.6 Lovati '07 Malaguarnera '04 76 30 15.6 13.6 7.9 3.2 Lovati 108 '07 8.2 30 10.6 5.3 3.2 76 15.6 7.9 108 8.2 Malaguarnera '04 Mizrahi '04 30 155 13.6 4.8 3.2 2.6 Malaguarnera 30 10.6 '04 3.2 75 4.3 3.2 30 13.6 3.2 30 10.6 Mizrahi '04 Morillas-Ruiz '10 155 48 4.8 28.8 2.6 7.7 Mizrahi 75 4.3 '04 52 21.8 3.2 8.7 155 4.8 2.6 75 4.3 Morillas-Ruiz '10 Parnetti '92 48 26 28.8 14.1 7.7 1.1 Morillas-Ruiz 52 21.8 '10 8.7 52 9.5 1.1 48 28.8 7.7 52 21.8 Parnetti '92 Postiglione '01 26 74 14.1 8.5 1.1 3.2 Parnetti 52 9.5 '92 74 5.7 1.1 2.1 26 14.1 1.1 52 9.5 Postiglione '01 Quadri '05 74 79 8.5 16.8 3.2 5.5 Postiglione 74 5.7 '01 111 13.1 2.1 5.9 74 8.5 3.2 74 5.7 Quadri '05 Ravaglia '04 79 29 16.8 16.6 5.5 7.3 Quadri 111 13.1 '05 51 11.1 5.9 4.3 79 16.8 5.5 111 13.1 Ravaglia '04 Regland '92 29 32 16.6 20.0 7.3 18.0 Ravaglia 51 11.1 '04 53 16.7 4.3 29 15.5 16.6 7.3 51 11.1 Regland '92 Religa '03 32 100 20.0 17.1 18.0 12.2 Regland 53 16.7 '92 99 19.3 15.5 32 7.7 20.0 18.0 53 16.7 Religa '03 Selley '02 Selley '02 Serot '01 100 25 25 28 17.1 25.1 25.1 13.2 12.2 4.7 4.7 4.8 Religa 99 '03 19.3 27 14.7 Selley 27 '02 14.7 30 12.1 7.7 4.3 4.3 4.9 100 25 17.1 25.1 12.2 4.7 99 27 19.3 14.7 Serot '01 Villa '09 28 18 13.2 19.0 4.8 4.1 Serot 30 '01 12.1 20 16.8 4.9 4.7 28 13.2 4.8 30 12.1 Villa '09 18 19.0 4.1 Villa 20 '09 16.8 Unadjusted Overall (REML, I-squared=88%, P<0.001) 4.7 18 19.0 4.1 20 16.8 Unadjusted Overall (REML, I-squared=88%, P<0.001) Unadjusted Overall (REML, I-squared=88%, P<0.001) Age adjusted Overall (REML meta-regression, P<0.001) Age adjusted Overall (REML meta-regression, P<0.001) Age adjusted Overall (REML meta-regression, P<0.001) vitamin B12vitamin B12 mean weight sd n mean mean sd n mean weight mean sd weight difference (95% CI) (%) AD n mean sd n mean sd study control control difference control (95% CI) AD AD (%) AD difference (95% CI) (%) Vitamin B12 study control control control AD AD AD -0.71 (-8.03, 6.61) 1.8 14.7 Anello '04 181 283 211 180 278 221-0.71 (-8.03, 6.61) Anello 1.8-0.71 '04 181 283 (-8.03, 6.61) 211 1.8 180 278 221 Asita De Silva '05 21 365-1.40 (-2.60, 103-0.20) 23 357 4.1 99 5.7-1.40 (-2.60, -0.20) Asita De 4.1Silva '05 21 365-1.40 (-2.60, 103-0.20) 23 357 4.1 99 Basun '94 99 305-3.85 (-9.24, 128 1.54) 22 246 2.4 103 8.4 Basun '94 99 305 128 22 246 103 Basun '94 117 290-3.85 (-9.24, 137 1.54) 26 294 2.4-3.85 169 (-9.24, 1.54) 2.4-1.59 (-4.03, 0.85) 3.7 5.3 Basun '94 117 290 137 26 294 169 Basun '94 112 249-1.59 (-4.03, 117 0.85) 28 259 3.7 105-1.59 (-4.03, 0.85) 3.7-5.30 (-7.80, -2.80) Basun 3.7 '94 112 249 117 28 259 105 10.7 Basun '94 58 239-5.30 (-7.80, 116-2.80) 15 319 3.7 143-5.30 (-7.80, -2.80) 3.7-11.70 (-16.51, -6.89) Basun 2.6 '94 58 239 116 15 319 143 7.2 Basun '94 6 198 41 3 279 91-11.70 (-16.51, -6.89) Basun 2.6 '94 6 198-11.70 41 (-16.51, -6.89) 3 279 2.6 91 Cascalheira '09 36 345-0.94 (-3.12, 26 1.24) 19 198 3.8 94 14.5-0.94 (-3.12, 1.24) Cascalheira 3.8 '09 36 345-0.94 (-3.12, 26 1.24) 19 198 3.8 94 Clarke '98 108 253-11.19 (-13.91, 100-8.47) 164 236 3.6 112 2.8 Clarke '98 108 253 100 164 236 112 Dominguez '05 19 713-11.19 (-13.91, 256-8.47) 29 468 3.6 218-11.19 (-13.91, -8.47) 3.6-2.50 (-6.01, 1.01) 3.2 6.1 Dominguez '05 19 713 256 29 468 218 Faux '11 760 300-2.50 (-6.01, 132 1.01) 205 299 3.2 139-2.50 (-6.01, 1.01) 3.2-9.06 (-12.99, -5.13) Faux '11 3.0 760 300 132 205 299 139 11.3 Galluci '04 42 354-9.06 (-12.99, 297-5.13) 137 277 3.0 169-9.06 Galluci '04 42 354 (-12.99, -5.13) 297 3.0 137 277-5.30 (-13.00, 2.40) 1.7 169 Glaso '04 18 435 263 20 350 264 21.3-5.30 (-13.00, 2.40) Glaso '04 1.7 18 435-5.30 (-13.00, 263 2.40) 20 350 1.7 264 Hogervorst '02 62 346-0.68 (-2.14, 116 0.78) 66 261 4.0 108 4.2 Hogervorst '02 62 346 116 66 261 108 Irizarry '05`"' 88 390-0.68 (-2.14, 179 0.78) 145 429 4.0-0.68 220 (-2.14, 0.78) 4.0-1.36 (-4.37, 1.65) 3.4 6.6 Irizarry '05`"' 88 390 179 145 429 220 Joosten '97 49 210-1.36 (-4.37, 91 1.65) 52 210 3.4 150-1.36 (-4.37, 1.65) 3.4-6.68 (-8.28, -5.08) Joosten 4.0 '97 49 210 91 52 210 150 4.4 Karimi '09 49 269 211 51 288 182-6.68 (-8.28, -5.08) 4.0-6.68 (-8.28, -5.08) 4.0-4.30 (-8.75, 0.15) Karimi 2.8 '09 49 269 211 51 288 182 Koseoglu '07 40 288 15 51 207 15 3.4-4.30 (-8.75, 0.15) Koseoglu 2.8-4.30 '07 40 288 (-8.75, 0.15) 15 2.8 51 207 15 Kristensen '93 20 256-8.00 (-16.84, 103 0.84) 26 179 1.4 92 12.7-8.00 (-16.84, 0.84) Kristensen 1.4 '93 20 256-8.00 (-16.84, 103 0.84) 26 179 1.4 92 Lelhuber '00 19 283 1.57 (-1.08, 139 4.22) 19 260 3.6 95 7.0 Lelhuber '00 19 283 139 19 260 95 Li '04 30 314 1.57 (-1.08, 184 4.22) 30 217 3.6 1.57 134 (-1.08, 4.22) 3.6-7.36 (-9.41, -5.31) 3.8 5.3 Li '04 30 314 184 30 217 134 Linnebank '10 60 248-7.36 (-9.41, 103-5.31) 60 272 3.8 192-7.36 (-9.41, -5.31) 3.8-3.00 (-4.60, -1.40) Linnebank 4.0 '10 60 248 103 60 272 192 Malaguarnera '04 30 437 58 30 370 77 3.2-3.00 (-4.60, -1.40) 4.0-3.00 (-4.60, -1.40) 4.0-0.50 (-1.33, 0.33) Malaguarnera 4.2 '04 30 437 58 30 370 77 Mizrahi '04 155 351 175 75 323 136 3.2-0.50 (-1.33, 0.33) Mizrahi 4.2 '04 155 351-0.50 (-1.33, 175 0.33) 75 323 4.2 136 Morillas-Ruiz '10 48 441-6.99 (-10.21, 217-3.77) 52 454 3.3 300 8.7-6.99 (-10.21, -3.77) Morillas-Ruiz 3.3 '10 48 441-6.99 217 52 454 300 Nagga '03 101 307 153 47 274 80 (-10.21, -3.77) 3.3-4.59 (-5.11, -4.07) 4.2 1.1 Nagga '03 101 307 153 47 274 80 Nilsson '04 51 257-4.59 (-5.11, 129-4.07) 159 254 4.2 97-4.59 (-5.11, -4.07) 4.2-2.80 (-3.67, -1.93) 4.2 Nilsson '04 51 257 129 159 254 97 2.1 O'Neill '90 9 290 93 9 313 82-2.80 (-3.67, -1.93) 4.2-2.80 (-3.67, -1.93) 4.2-3.70 (-5.34, -2.06) O'Neill 4.0 '90 9 290 93 9 313 82 Parnetti '92 26 260 117 52 262 150 5.9-3.70 (-5.34, -2.06) 4.0-3.70 Parnetti '92 26 260 (-5.34, -2.06) 117 4.0 52 262-5.47 (-8.36, -2.58) 3.5 150 Postiglione '01 74 780 211 74 491 144 4.3-5.47 (-8.36, -2.58) Postiglione 3.5 '01 74 780-5.47 (-8.36, 211-2.58) 74 491 3.5 144 Quadri '05 79 275-3.30 (-10.80, 96 4.20) 111 272 1.7 108 15.5 Quadri '05 79 275 96 111 272 108 Ravaglia '00 13 327-3.30 (-10.80, 69 4.20) 34 282 1.7-3.30 24 (-10.80, 4.20) 1.7 2.15 (-0.68, 4.98) 3.5 7.7 Ravaglia '00 13 327 69 34 282 24 Ravaglia '04 29 308 2.15 (-0.68, 127 4.98) 51 292 3.5 96 2.15 (-0.68, 4.98) 3.5-10.35 (-12.79, -7.91) Ravaglia 3.7 '04 29 308 127 51 292 96 4.3 Regland '92 32 261 110 53 251 93-10.35 (-12.79, -7.91) 3.7-10.35 (-12.79, -7.91) 3.7-1.04 (-3.54, 1.46) Regland 3.7'92 32 261 110 53 251 93 Religa '03 100 305 178 99 234 103 4.9-1.04 (-3.54, 1.46) Religa 3.7-1.04 '03 100 305 (-3.54, 1.46) 178 3.7 99 234 103 Selley '02 25 322-2.26 (-5.05, 68 0.53) 27 254 3.5 77 4.7-2.26 (-5.05, 0.53) Selley 3.5 '02 25 322-2.26 (-5.05, 68 0.53) 27 254 3.5 77 Villa '09 18 369-3.88 (-5.13, 199-2.64) 20 334 126 Villa '09 18 369 199 20 334 126 Unadjusted Overall (REML, I-squared=87%, -3.88 (-5.13, -2.64) -3.88 P=0.001) (-5.13, -2.64) -4.33 (-5.83, -2.84) Unadjusted Overall (REML, I-squared=87%, P=0.001) Age adjusted Overall (REML meta-regression, -4.33 (-5.83, -2.84) P<0.001) -4.33 (-5.83, -2.84) Age adjusted Overall (REML meta-regression, P<0.001) weight mean difference (95% CI) (%) -5.00 (-49.58, 39.58) 3.0-8.10 (-67.86, 51.66) 2.7-59.00 (-108.88, -9.12) 2.9 4.00 (-65.54, 73.54) 10.00 (-34.52, 54.52) 2.5 3.0 80.00 (1.72, 158.28) 2.4 81.00 (-27.07, 189.07) 1.8-146.87 (-190.18, -103.56) 3.0-17.00 (-42.49, 8.49) 3.3-245.35 (-385.21, -105.49) 1.4-1.40 (-22.61, 19.81) mean difference (95% CI) -5.00 (-49.58, 39.58) -8.10 (-67.86, 51.66) -59.00 (-108.88, -9.12) 4.00 (-65.54, 73.54) 10.00 (-34.52, 54.52) 80.00 (1.72, 158.28) 81.00 (-27.07, 189.07) -146.87 (-190.18, -103.56) -17.00 (-42.49, 8.49) -245.35 (-385.21, -105.49) -1.40 (-22.61, 19.81) -76.62 (-170.85, 17.61) -85.00 (-252.77, 82.77) -85.00 (-123.89, -46.11) 39.00 (-12.78, 90.78) 0.00 (-48.08, 48.08) 19.19 (-58.11, 96.49) -80.52 (-86.84, -74.20) -77.00 (-134.24, -19.76) -22.81 (-98.58, 52.96) -96.30 (-177.82, -14.78) 24.00 (-31.13, 79.13) -67.20 (-101.74, -32.66) -27.60 (-68.94, 13.74) 13.01 (-89.05, 115.07) -33.00 (-70.60, 4.60) -3.00 (-41.48, 35.48) 22.88 (-58.08, 103.84) 1.93 (-58.64, 62.50) -289.00 (-347.20, -230.80) -3.00 (-32.19, 26.19) -45.00 (-83.50, -6.50) -16.00 (-69.20, 37.20) -10.42 (-56.02, 35.18) -71.43 (-111.80, -31.06) -68.80 (-108.19, -29.41) -35.80 (-142.91, 71.31) -35.83 (-57.30, -14.36) -47.88 (-70.75, -25.01) -76.62 (-170.85, 17.61) 2.1-85.00 (-252.77, 82.77) 1.1 Similar -85.00 (-123.89, -46.11) results 3.1 for 39.00 (-12.78, 90.78) 2.9 0.00 (-48.08, 48.08) 2.9 19.19 (-58.11, 96.49) 2.4 plasma levels of -80.52 (-86.84, -74.20) 3.4-77.00 (-134.24, -19.76) 2.8-22.81 (-98.58, 52.96) 2.4 vitamins -96.30 (-177.82, -14.78) 2.3C and E, 24.00 (-31.13, 79.13) 2.8-67.20 (-101.74, -32.66) 3.2-27.60 (-68.94, 13.74) & DHA 3.1 13.01 (-89.05, 115.07) 3.3 1.9-33.00 (-70.60, 4.60) 3.1-3.00 (-41.48, 35.48) 3.1 22.88 (-58.08, 103.84) 2.3 1.93 (-58.64, 62.50) 2.7-289.00 (-347.20, -230.80) 2.8-3.00 (-32.19, 26.19) 3.2-45.00 (-83.50, -6.50) 3.1-16.00 (-69.20, 37.20) 2.8-10.42 (-56.02, 35.18) 3.0-71.43 (-111.80, -31.06) 3.1-68.80 (-108.19, -29.41) 3.1-35.80 (-142.91, 71.31) 1.9-35.83 (-57.30, -14.36) -47.88 (-70.75, -25.01) -20-15 -10-5 0 5 10-400 -300-200 -100 0 100 200-20 -15-10 -5 0-20 5-15 10-10 -5 0 5 10-400 -300-200 -100 0 100 200 AD lower AD higher AD lower AD higher AD lower AD higher AD lower AD higher AD lower AD higher 5.64 (3.26-8.02); p < 0.001 Unadjusted overall mean difference (95% CI) 35.83 (14.36-57.30); p = 0.001 4.33 (2.84-5.83); p <0.0.001 Age-adjusted overall mean difference (95% CI) 47.88 (25.01-70.75); p < 0.001

Summary and conclusions Multiple pathologies associated with AD: synapse loss early hallmark and a valid target for intervention Synapses are composed of neuronal membranes, phospholipids: possibility for nutritional intervention Patients with AD have lower blood and brain levels of key nutrients with an increased need due to accelerated synapse loss Studies of single nutrient supplementation show no benefit Dietary patterns suggests combination of nutrients may be beneficial Several studies have demonstrated that Souvenaid increases levels of key nutrients in plasma Recent evidence from MRS suggests enhanced phospholipid formation

NL-ENIGMA study: Effects of Souvenaid on brain glucose metabolism in early Alzheimer s disease Goal: Exploring the effect of Souvenaid vs. control after 24 weeks on brain glucose metabolism as assessed by 18F-FDG-PET imaging (absolute and relative to a reference region) FDG-PET: 18 F-FDG-PET assesses glucose metabolism in the brain, which is an index of synaptic function. Both are known to be correlated and impacted in AD Population: Subjects with MCI or mild dementia due to AD, stratified for MMSE Design: Timelines: Last patient out expected in 2017 Supported by grant from NWO (Dutch government)

Decreased brain phospholipids in AD indicates disrupted membrane integrity 4. Conclusions Phospholipids provide an optimal membrane environment for protein interactions, trafficking and function. There is increasing evidence that phospholipid changes occur during pathogenic processes in Alzheimer s disease...

Souvenaid increases levels of the biomarker phospholipids Baseline and 24-week plasma samples from the Souvenir II study Drug-naïve patients with very mild AD Polar lipid profile 5 / 7 measured phospholipids reported by Mapstone significantly increased by Souvenaid By providing nutrients which normally rate-limit phospholipid synthesis Souvenaid can: modify a biomarker profile reflecting disturbed phospholipid metabolism be useful in asymptomatic subjects with plasma lipid biomarker profiles predictive for conversion to AD * P<0.001; Souvenaid vs. Control using ANCOVA Hartmann et al. JAD, 2014

Mapstone et al. identified a biomarker panel of 10 plasma lipids that can predict conversion from cognitive healthy to MCI/AD within 2 3 years with >90% accuracy Changes may reflect the breakdown of neuronal membranes Highly publicized findings set of 10 plasma lipids, including 8 phospholipids levels are lower in converters and MCI/AD subjects

Phase Lag Index (PLI) in delta band Normalised path length in beta band Peak frequency (Hz) Normalised clustering coefficient in beta band Network parameters suggest preserved synaptic formation, function and network 9,20 9,00 8,80 8,60 8,40 1. Peak Frequency p=0.019 Control Active 3. Brain Network Organization* 1,035 1,030 1,025 1,020 1,015 p=0.009 Control Active 8,20 0,15 0,14 0 12 Time (weeks) 24 2. Phase Lag Index p=0.011 Control Active 1,010 0,935 0,930 0 12 Time (weeks) 24 p=0.053 0,925 0,13 0,920 0,12 0 12 Time (weeks) 24 ITT, MMRM, 2 df contrast, data are mean ±SE 0,915 0 12 Time (weeks) 24 Scheltens et al. J Alzheimers Dis. 2012;31:225-236. de Waal et al. Plos One 2014;9:1.

Electrical activity at the synapse EEG: Biomarker for functional connectivity 1. Basic Quantitative EEG analysis - Relative power and Peak Frequency In AD disturbed signal strength 1 2. Phase Lag Index (PLI) as Functional Connectivity measure PLI In AD loss of PLI 1 3. Clustering & Path Length are measures of Network Organization In AD disrupted organization 2 Healthy AD 1 Stam CJ et al. Brain. 2009;132:213-224. 2 Stam CJ, van Straaten ECW. Clin Neurophysiol. 2012 doi:10.1016/j.clinph.2012.01.011.

Epidemiological evidence suggests dietary patterns can reduce AD risk Adherence to a Mediterranean diet 1,2 - higher intake of fish, vegetables, fruit and monounsaturated fats, can delay cognitive decline Evidence from these and other studies of dietary patterns suggest that nutrient combinations are more effective in reducing AD risk and improving cognitive outcome than single nutrient intervention 1 Scarmeas N, et al. Ann Neurol 2006;59:912 21. 2 Martinez-Lapiscina EH, et al. J Neurol Neurosurg Psychiatry 2013;84:1318 25.

Lower nutrient status in AD preceding classic protein energy malnutrition Mi et al, Nutrition, 2013, Adapted from Jack et al. Lancet Neurology, 2010

Mean change from baseline in NTB Memory domain z-score 2011- SOUVENIR II: SOUVENAID IMPROVES MEMORY IN PATIENTS WITH MILD AD 0,25 Significant* (p=0.023) improved memory Control 0,2 Active 0,15 0,1 0,05 0 0 12 Time (weeks) 24 Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36.

Mean change from baseline in NTB Memory domain z-score 2011- SOUVENIR II: SOUVENAID IMPROVES MEMORY IN PATIENTS WITH MILD AD Significant* (p=0.023) improved memory 0,25 Control 0,2 Active 0,15 0,1 0,05 0 0 12 Time (weeks) 24 Scheltens et al. J Alzheimers Dis. 2012 31(1):225-36.

2011 Confirmation of improved memory performance in Mild AD (Souvenir II primary) Significant effect on NTB memory domain during 24 weeks (whole period trajectory; p=0.023) Trend on NTB composite score during 24 weeks (whole period trajectory; p=0.053) Statistical analysis re-run by Rush Alzheimer s Disease Center ITT, Mixed Model for Repeated Measures (trajectory, mean ± SE) Scheltens et al, J Alzheimers Dis. 2012

2012- Souvenir II open label study Change between 24 an 48 weeks (paired t-tes p=0.025 active-activ p=0.008 control-acti