Therapeutic Target for Alzheimer’s Disease

Tapan Audhya, PhD. New York University, NY, USA

Health Diagnostics & Research Institute, NJ, USATaudhya@vitdiag.com

First International Congress on Updates and Advances in the field of

Clinical Nutrition and Orthomolecular Medicine2011, Valencia, Spain

Types of Alzheimer Disease (AD)

1.Familial Alzheimer Disease (FAD)

2.Early-onset Familial Alzheimer Disease (EOFAD)

3.Late-onset Alzheimer Disease (LOAD)

(sporadic Alzheimer Disease)

Cont….

FAD:a) Entirely inheritedb) Affected family members of at least two

generationc) Often starts at age 40’sd) Extremely rare 1-2% of total

Cont…

EOFAD: a) Autosomal Dominant Mutationb) Usually close to 160 mutation in chromosomes -1,

-14, -21, and 19 (APO€4)c) Virus infection in young aged) Increase in Aβ production e) Symptoms of disease appear before the age of 65 f) Approximately 10-20% of total AD are affectedg) Easily identified from muscle twitching and spasm

(myoclonus)h) Chronic Inflammation

Cont..LOAD:

a) Most common form, accounting for about 80%b) Usually show symptoms after age 65 and half of them

over 85c) May or may not be hereditaryd) Several polymorphic gene in this group has been

identified in chromosome-10 and 6e) Common symptoms are : Depression, CFS/ME, high

oxidative stress, defects in glucose and lipid metabolism, high Cortisol & glutamic acid.

f) Produce Aβ peptides and there is imbalance between Aβ production and clearance

g) Lack of B-vitamin, ω3 fatty acid & antioxidants. Low level of acetylcholine and high level of toxic chemical.

Level of Hormones in HPA-axis of AD

28 - 646 – 45β-Endorphin(pg/ml)

40 - 16014 - 90ACTH (pg/ml)

8 - 282 - 12CRF (pg/ml)

21 -333 - 16Cortisol (4 PM) (µg/dl)

30 - 5410 - 25Cortisol (8 AM)(µg/dl)

LOAD (n=88)Normal (n=67)

Test for Oxidative Stress of AD

0.09 ± 0.10.15 ± 0.05BH4/BH2

3.2 ± 0.265.3 ± 0.47SAM/SAH

9.6 ± 1.013.8 ± 1.2GSH/GSSG

1.6 ± 0.110.58 ± 0.068-OH-2dG (PL)(ng/ml)

9.8 ± 1.12.9 ± 0.32Nitrotyrosine (PL)(ng/ml)

LOADNormal

Antioxidant Enzymes in AD

18.4 ± 1.211.5 ± 1.4Super-oxide Dismutase(units/min/mg Hb)

8.2 ± 1.16.8 ± 0.85

190.6 ± 12.8165 ± 10.6Catalase(units/min/mg Hb)

AD(N=42)

Normal(N=140)Glutathione Peroxidase

(µmole/NADPH/min/mg Hb)

Test for Depression of AD

3.2 ± 0.55.4 ± 0.76Nor-epinephrine

(Platelet)(ng/109 cells)

89 ± 7.7108 ± 12.2

(M) 175 ± 15.3(F) 245 ± 25.8

Serotonin (Platelet)(ng/109 cells)

LOADNormal

Test for Methylation Block of AD

83.4 ± 7.7138 ± 11CH3-cobalamine(PL)(pmole/L)

206 ± 18550 ± 37Folic acid (RBC)(nmole/L)

6.3 ± 0.147.9 ± 0.83Folic acid (PL)(nmole/L)

LOADNormal

(B12)

Km for CH2-THF is 88.0 umole/L

{5,10,Methylene THF

5-CH3-THF dUMP

THF

MTHFR (FAD)(Reduction) (1)

Ser

dTMP

DNA synth. Repair

10 Formyl THF

5 Formyl THF

5, 10, Methenyl THF

(2)(oxidation)

Diet

Methionine

SAM

SAH

Adenosine

Meth.Synthase

HS

SerineB 6

Cystathionine

a-keto + CysteineButyrate

Succinate

Glutathione

TCACycle

HS

DNA and ProteinMethylation

Methionine is in fasting conditionHS Cysteine is after dinner (meal) conditon

MTHFR Needs FAD. It is a dimer of 77k DA; each non covalently bound to FADRequire NADH for reduction.Km for NADPH is 16 umole/L

B12

and Km for CH2-THF is 88.0 μmole/L

Folate Metabolism: Alternate Pathways

Inosine

Uric acidB6

CBS

ATP

(methyltrap) Ring

Closure

Formyl transfercoenzyme

NADPH

Glycine +P-5-P+ serine-oH-methylase

ATP + HC00H+FH4Methyl

transferase

(FH4)

MSB12

Healthy Neuron Vs. Dying Neuron in Alzheimer

Pathological Features

Schematic Diagrams of the β-amyloid Precursor Protein (APP) and its Metabolic Derivatives

Measurement of β-amyloid (Aβ) Peptide• Aβ1- 42 Found in Neurotics plaques

It is hydrophobic and prone to aggregation• Aβ1- 40 More abundant than Aβ1- 42

• Both of these peptides and their auto-antibodies are found in CSF and plasma of normal, mild cognitive impaired (MCI), AD patients and their extended families.

• Beside brain, the peptides are present in various human tissues, such as, platelets, liver, skeletal muscles, kidney, lungs, skin, intestine and sc. tissue in ratio 90/10

• A transporter dependent movement is involved in transporting Aβ from CSF to plasma via interstitial fluid (ISF). Two such transporters are LRP-1* and RAGE*. Insulin helps the clearance of Aβ peptide from the brain.

LPR-1 – Low density lipoprotein receptor-related protein-1RAGE – Receptor for advanced glycation end products

Effect of Dissociation of Plasma Amyloid Protein

0

0.2

0.4

0.6

0.8

1

1.2

Control (N=35) AD (N= 23)

ControlAD

Effect of Dissociation of Plasma Amyloid Protein

Control (N = 35) AD (N = 25)

Before

After Before

After

Level of Plasma Aβ(1- 40) and Aβ(1- 42) of Geriatric Population with LOAD

20.6 – 30.8538 - 726

26.1 – 47.6240 – 407

30.3 – 51.6218 – 245

17.8 – 40.0187 – 212

14.2 – 21.6159 – 178

5.6 – 10.168 - 138

Pg/mlPg/ml

15.9 – 19.0177.6 – 200.23786 – 90

16.2 – 18.3167.2 – 180.64681 – 85

19.7 – 30.7153.4 – 177.94976 – 80

11.6 – 21.5130.2 – 166.66271 – 75

9.6 – 15.9110.6 – 135.62566 - 70

2.2 – 6.750.2 – 100.73160 - 65

Aβ(1- 42)Aβ(1- 40)NAge

Level of CSF Aβ(1- 40) and Aβ(1- 42) in Populationwith Different ADAS-cog Score

(ng/ml)(ng/ml)

Aβ(1- 40)

14.615.34237-52Severe

6.413.64530-35Moderate

18.114.24716 -28Mild

7.420.63910 -20Questionable

3.216.4410 -5Normal

Aβ(1- 42)NADAS-cogScore

Level of Plasma Aβ(1- 40) and Aβ(1- 42) &ADAS-cog Score

157.4 – 198.8643 – 128437-52Severe

187.6 – 251.3503 – 72130-35Moderate

142.7 – 192.3309 – 516 11-28Mild

54.6 – 81.4230-26810-20Questionable

8.2 – 15.7142-1590-5Normal

Aβ(1- 42)pg/ml

Aβ(1- 40)pg/ml

ADAS-cog Score

Level of Aβ Peptides in the Tissue of Frontal Cortex in AD Patient

1913 ± 1341521 ± 32911Late Onset AD(ADAS-cog > 28

2748 ± 1311931 ± 40610

(pmole/g tissue)(pmole/g tissue)Early Onset FAD(ADAS-cog > 28)

A β (1- 42)A β (1- 40)NDementia Type

Dissociated Antibody Conc. Vs. Age

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control (N=35)60 70 80

AGE ( CONTROL)

N =25

N=21

N=18

Dissociated Antibody Conc. Vs. Age

0

0.2

0.4

0.6

0.8

1

1.2

AD (N=47)

60

70

80

90

AD

N=21

N=27

N=27N=15

4343

107107

5353

4444

6969NN

Cognitive Cognitive

Vit. K Vit. K

Cognitive Cognitive NMDA NMDA receptor receptor antagonist, antagonist, glutamate glutamate

Cognitive Cognitive

DepressionDepression

4.24.29090Vit Vit –– EE(natural)(natural)

25 Iu25 Iu/kg /kg BWBW

43.043.05656Huperzine Huperzine -- AA(200 (200 µµgg/day)/day)

30.130.19090PSPS1.0g /day1.0g /day

39.039.0168168PCPC1.4g/day1.4g/day

26.126.19090ArginineArginine1.5 g/day1.5 g/day

%%RespondersResponders

TreatmentTreatmentPeriod (days)Period (days)AgentsAgents

9696

6464

129129

9494

7171

6262NN

Energy Energy Nor epi Nor epi DopamineDopamine

Memory Memory Bl. Viscosity Bl. Viscosity

CognitiveCognitiveDepression Depression Blood Blood viscosityviscosity

%%RespondersResponders

TreatmentTreatmentPeriod (days)Period (days)AgentsAgents

46.046.06060CDPCDP--cholinecholine(1gm/day)(1gm/day)

20.020.06060VinpocetineVinpocetine(25 mg/day)(25 mg/day)

67.467.49090ωω33 Fatty acidFatty acid(2 g/day)(2 g/day)

32.032.03030Ginko biloba (761)Ginko biloba (761)160 mg/day160 mg/day

36.036.0365365Folic acidFolic acid600 600 µµg/dayg/day

44.044.090 90 Methyl CobalamineMethyl Cobalamine1 mg/day S.C1 mg/day S.C

Ginkgo Biloba Extract(EGb – 761)

Flavonoids (24%)

┼

Terpenoid (6%)

┼

Isorhamnetin glycosideKaempferolQuercetin

Ginkgolides A, B, C

Depression Depression Cognitive Cognitive

%%ResponderResponder

ss

TreatmentTreatmentPeriod (days)Period (days)NNAgentsAgents

16.316.31811817676VitaminVitamin--AA600 Iu/kg600 Iu/kg

16.416.440404747HBOTHBOT1.3/40/24%1.3/40/24%

63.463.4365365132132LithiumLithium150 mg/day150 mg/day

16.816.83653654747LL--carnitinecarnitine1.0 gm/day1.0 gm/day

18.418.48484121121NN--acetyl Cysteineacetyl Cysteine1.5 g/day1.5 g/day

Structure of (A) Curcumin (B) demethoxycurcumin and(C) bisdemethoxycurcumin

Alzheimer Disease Assessment Scale-Cognitive Subscale (ADAS-cog) 11-item

Memory and new learning 0 – 35Word recall 0 – 10

(mean number of words not recalled)Orientation 0 - 8(one point for each incorrect response)Word Recognition 0 – 12(mean number of incorrect responses)Remembering test Instructions 0 – 5Language 0 - 25

Commands 0 - 5 Spoken language ability 0 – 5Naming objects/fingers 0 – 5 Word-finding difficulty 0 – 5 Comprehension 0 – 5

Praxis 0 - 10Constructional praxis 0 – 5Ideational praxis 0 – 5

Total 0 - 70Increasing scores indicate worsening cognitive function

Score Range

Change on ADAS-cog Sub-items at Wk 26

0.012*0.4210.032*

0.013*0.1060.060

< 0.001*0.037*0.0950.023*0.014*

pvalue†

Placebo (N = 159)

Mean ± SDCurcuminoids

N= 290)

142142142

141137143134142135141139N

259258259

256249259245258249258255N

–0.2 ± 1.00.0 ± 1.0

–0.3 ± 1.0

–0.1 ± 1.2–0.2 ± 3.6–0.1 ± 1.7–0.1 ± 1.3–0.1 ± 0.9–0.1 ± 1.1

0.0 ± 1.30.1 ± 1.3

Change atWk 26

Item 11: comprehensionItem 10: word finding difficultyItem 9: spoken language ability

Item 8: remembering test instructions

Item 7: word recognitionItem 6: orientationItem 5: ideational praxisItem 4: naming objects/fingersItem 3: constructional praxisItem 2: commandsItem 1: word recallADAS-cog sub-item

0.2 ± 1.00.1 ± 1.00.1 ± 1.2

0.2 ± 1.30.5 ± 3.30.4 ± 1.80.4 ± 1.50.1 ± 0.80.2 ± 1.00.6 ± 1.21.0 ± 1.3

Change atWk 26

*p < 0.05† p value based on van Elteren test

Treatment Difference at Wk 26 on ADAS-cog by States

Illinois (N = 19)S. Dakota (N = 21)Wisconsin (N = 41)New Jersey (N = 63)New York (N = 59)Texas (N = 63)California (N = 48)Arizona (N = 118)Florida (N = 18)N. Dakota (N = 17)Montana (N = 19)Iowa (N = 45)

–10 0 10 20

LS mean difference (95% CI).

–20

–39.67

–29.92

46.92

Favor CurcuminoidsFavor placebo

Placebo Controlled Supplement StudyPlacebo Controlled Supplement Study

# of Volunteers = 476 (309 M + 167 F)# of Volunteers = 476 (309 M + 167 F)

6 Month 6 Month

208(134M + 74 F)

RespondersResponders

1. Howthorne Effect 2. Placebo Effect & Drift

6 Month

238(154M + 84 F)

6 Month6 Month

Responders

6 Month

Responders

6 Month6 Month

Responders

6 Month

238(155M + 83 F)

6 Month

Responders

6 Month

Responders

36 (18%)(31M + 5F)

6 Months

Responders

172 (76%)(93M + 79 F)

226(143M + 83 F)

6 Months

Curcuminoids – 2 gm/day + Vitamin D3 2000 iu/day

Possible Bioactivity of Curcuminoids

• D3 interacts & Curcuminoids to stimulate Aβ peptide clearance via macrophage (Type I- II)

• Curcuminoids enhanced memory in Aβamyloid infused rat model of AD

• It inhibits acetylcholine esterase and stimulate creatine kinase in vitro and invivo in rat model.

• Curcuminoids and vit. E mixture control inflammation of brain by modulating Aβinduced toxicity.

Acetylcholinesterase (mU/ml) Acetylcholine (nM)

Assay of Ach & Ach-esterase