Study by Dr Demarin on Alzheimers

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Summary: This study was designed to evaluate the effect of stabilized oral reduced nicotinam,de adenine din­ ucleotide (NADH) on cognitive functioning in patients with Alzheimer’s dsease (AD). NADHis a coenzyme that plays a key role in cellular energy production and stimulates dopamine production. In previous trials NADH has been shown to improve cognitive functioning in patients with Parkinson’s disease, depression and AD. The pre­ sent trial was a randomized, placebo-controlled, matched-pairs, double-blind, 6-month clinical study. Patients with probable AD (n = 26) were randomized to receive either stabilized oral NADH (10 mg/day) or placebo.

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Study by Dr Demarin on Alzheimers

DRUGS EXPTL. CLIN. RES. XXX(1) 27-33 (2004)


1) Department of Neurology, Sestre Milosrdnice University Hospital, Zagreb, Croatia.
2) Institute for Medical Chemistry, University of Graz, Graz, Austria.
3) Department of Neurology, Georgetown University School of Medicine, Washington, D.C., USA.
Summary: This study was designed to evaluate the effect of stabilized oral reduced nicotinam,de adenine din­ ucleotide (NADH) on cognitive functioning in patients with Alzheimer’s dsease (AD). NADHis a coenzyme that plays a key role in cellular energy production and stimulates dopamine production. In previous trials NADH has been shown to improve cognitive functioning in patients with Parkinson’s disease, depression and AD. The pre­ sent trial was a randomized, placebo-controlled, matched-pairs, double-blind, 6-month clinical study. Patients with probable AD (n = 26) were randomized to receive either stabilized oral NADH (10 mg/day) or placebo. Twelve pairs of subjects were matched for age and baseline total score on the Mattis Dementia Rating Scale (MDRS) and the Mini Mental State Examination. After 6 months of treatment, subjects treated with NADH showed no evidence of progressive cognitive deterioration and had significantly higher total scores on the MDRS com­ pared with subjects treated with placebo (p < 0.05). Analysis of MDRS subscales revealed significantly better performance by NADH subjects on measures of verbal fluency (p = 0.019), visual-constructional ability (p = 0.038) and a trend (p = 0.08) to better performance on a measure of abstract verbal reasoning. There were no differences between groups in measures of attention, memory, or in clinician ratings of dementia severity (Clinical Dementia Rating). Consistent with earlier studies, the present findings support NADH as a treatment for AD.
Introduction lar energy production by oxidative phosphorylation (1). Furthermore, NADH stimulates dopamine pro­

Reduced nicotinamide adenine dinucleotide
(NADH) is a coenzyme that plays a key role in cellu-duction {2) and regenerates tetrahydrobiopterin (3), an essential cofactor of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, and prevents nitration of this enzyme by peroxynitrite (4). In open label clinical trials, NADH has been shown to improve cognitive functioning in patients with Parkin­ son’s disease (5), depression (6) and Alzheimer’s dis-

0378-6501/2004/1 00027+6 $02.50/0 © 2004 Bioscience Ediprint Inc. 27

Demarin V et al.

ease (AD) (7). The ability of stabilized oral NADH to be absorbed and to cross the blood brain barrier was recently demonstrated by Rex et al. (8), who showed a 20% increase in the NADH level in rat cortex follow­ ing oral administration of NADH. Previously, following 1-week intravenous infusion of NADH to Parkinson’s disease patients, Kuhn et al. (9) reported increased levels and bioavailability of plasma levodopa (AUC 25% increase and Cmax 40% increase). Although cholinergic dysfunction, a primary manifestation of AO, is the main target of current pharmacologic treat­ ment of AD, there is considerable evidence of dys­ function of the dopaminergic neurotransmitter sys­ tem in AD (10). As the dopaminergic system is impor­ tant not only for motor performance but also for cog­ nitive functions, attempts to restore deficits of the dopaminergic system are and should be further con­ sidered as a therapeutic approach for AD (11, 12).
Based on these findings, NADH may be a suit­ able candidate for further investigation in the treat­ ment of AD based on its ability to facilitate dopamine synthesis.
As a prerequisite for a study with human sub­ jects, Birkmayer and Nadlinger (13) investigated the safety and potential toxicity of the stabilized, orally absorbable form of reduced NADH in rats in order to demonstrate the safety of this drug in a chronic study. There were no deaths associated with the study drug and no treatment-relate<i clinical signs. No differ­ ences were observed between the control and the treated groups in terms of hematology or clinical chemistry parameters. There was no apparent treat­ ment-related effect on urine analysis parameters or on either absolute or relative organ weight. Further­ more, no macroscopic evidence of specific target organ toxicity associated with the test drug was ob­served. The results of this study and the fact that the daily dose of 5 mg per day administered to rats in this stucjy corresponds to a dose of 175 mg per day in a 70-kg human indicate that stabilized oral NADH 5 mg
28 tablets can be generally regarded as safe.

A series of studies have examined the use of NADH in AD. The first study was an open-label 8- to 12-week trial with oral stabilized NADH (ENADA®, Prof. Birkmayer Gesundheitsprodukte GmbH, A-1090 Wien, Schwarzspanierstr. 15, Austria; 1O mg per day) in 17 patients with AD. The results showed improve­
ment (8.35X points ± 2.45; p < 0.001) on the Mini
Mental State Examination (MMSE) and on the Global Deterioration Scale (1.82 points ± 0.39, p < 0.001) (7). The initial double-blind, placebo-controlled, par­ allel group study was a pilot project designed to eval­ uate the effect of NADH on cognitive functions par­ ticularly sensitive to changes in AD severity (i.e., ver­
bal fluency and verbal memory). Primary outcome measures were the Hopkins Verbal Learning Test (14) and Verbal Fluency Test (15). Seventeen subjects (NADH, n = 9; placebo, n = 8) completed the study. Randomization groups differed at baseline on mea­ sures of overall verbal memory, dementia rating and attention. After 6 months of treatment, NADH sub­ jects improved and placebo subjects declined on measures of verbal memory (p < 0.04). Similarly, improved verbal fluency was found for NADH sub­ jects and decreased fluency was found for placebo subjects at 6 months (p < 0.04). The baseline differ­ ences in cognitive functioning indicated the need for better subject matching in subsequent small popula­ tion trials (16).
The next study was conducted with 22 AD pa­ tients (NADH, n = 12; placebo, n = 10) with the same
cognitive outcome measures. Groups were matched for baseline on the MMSE and age. The results de­ monstrated a trend for better verbal fluency (p = 0.056) and for better overall performance on the Mattis Dementia Rating Scale (MDRS) (particularly the flu­ ency and memory measures). However, the results showed significant baseline differences between placebo subjects and NADH subjects on the MORS, confounding interpretation of the results (16).

NADH treatment of Alzheimer’s disease
The present study was designed to rigorously match subjects a priori on the MDRS allowing for bet­ ter analysis of the impact of NADH on cognitive func­ tioning in AD.

Patients and methods

Patients were recruited from the outpatient clinic of the Neurology Department, University Hospital, Zagreb, Croatia and were enrolled in the study if they met the following criteria: diagnosis of probable AD according to criteria of the National Institute of Neuro­ logical and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association, age between 50 and 80 years, MMSE score of between 13 and 25, MDRS between 70 and 125, naive to treatment with NADH and no use of donepezil or tacrine within 6 months of enrollment in the study. Patients were not required to discontinue other medications. Seventy-five patients were screened and 25 met all the inclusion criteria. Informed consent was provided by caregivers and by patients capable of providing consent before entering into the study. The study was approved by the Croatian Ministry of Health.
In this double-blind, placebo-controlled, matched­
pairs study, each patient made eight visits w the clin­ ic over 6 months to examine the effects of stabilized
oral NADH on cognitive functioning and to monitor for adverse effects and compliance. The primary out­ come variable, chosen a priori, was the MDRS. After a 2-week drug compliance trial (passed by all pa­ tients), participants underwent a neurological exami­ nation, routine blood and urine laboratory tests and baseline cognitive testing. Random allocation to the two groups was computer-generated offsite; the key was stored at Birkmayer Institute (Vienna, Austria) until the end of the study. Patients were randomly as­ signed to receive NADH 5 mg (ENADA®, The Enada Team), 2 tablets QD (n = 13} or matching placebo tablets (n = 13). Patients were monitored by neurological examinations at the eight study visits, and both pa­ tients and caregivers were questioned about adverse effects.

Neuropsychological testing was repeated at 10 weeks and 6 months. using the following measures: Hopkins Verbal Learning Test (14), Verbal Fluency Test (15), CogScreen® Matching to Sample Test (17) and Clinical Dementia Rating Scale (18).
MDRS (19} testing was performed at baseline and after 6 months. The primary outcome measure was total score on the MDRS.
We hypothesized that NADH would reduce dete­ rioration and possibly improve cognitive functioning in AD. The analytic approach employed to test this hypothesis was Student’s t-test for paired samples (SPSS-PC Version 10.1) comparing mean change from baseline to 6 months.

Twenty-six patients were matched a priori into 13 study pairs. One patient was withdrawn from the study because of psychiatric hospitalization prior to initiating treatment. The matched subject of this pair was allowed to participate in the study but data for this subject is not included in the analyses (Fig. 1). All analyses were conducted with the 12 remaining pairs. The age range of the 24 patients included in the analyses was from 52 to 79 years; the median age was 68.5 years. The MMSE scores at basline ranged fwrn 13 to 25 with a median of 19.2. The MDRS scores at baseline ranged from 74 to 125 with a median of 107. The randomized groups were not statistically different with respect to age, gender, months since diagnosis, or dementia scores on the
MDRS and MMSE at baseline. 29

Demarin V et al.
IAssessed for eligibility (n = 75) j

Randomized (n = 26)


Analyzed (n = 12) Excluded from analysis (n = 1)2

Fig. 1 Study participant flow. ‘Psychiatric hospitalization prior to treatment initiation. 2No matched reduced nicotinamide adenine dinucleotide (NADH) subject.
Table I shows the findings for the two groups at baseline and 6 months. Change from baseline scores (6 months from baseline) revealed a mean in­ crease in MDRS total score for NADH subjects of
1.25 (± 1.94) and a mean decrease in MDRS total score for placebo subjects of -7.92 (± 5.05) (Fig. 2). The difference between NADH and placebo groups

in the primary outcome measure was significant (T = 1.85, df = 11, p < 0.05). Analysis of MDRS sub­ scales revealed that the group differences in the MDRS total score were primarily due to the initiation/ presecvation scale (T = 2.35, df = 11;p = 0.019), the
construction scale (T = 1.97, df = 11, p = 0.038) and
the conceptualization scale (T = 1.51, df = 11; p =
Table I Changes in the Mattis Dementia Rating Scale (total and subscales) after 6 months of treatment with 10 mg oral reduced nicotinamide adenine dinuc/eotide (NAOHJ

Changes in score
Group 1 Group 2
p-value stabilized oral NADH Control
MDRTOT28 0.046 -1.25 7.92
MDRIP28 0.019 -0.17 3.17
MDRCCP28 0.80 -0.67 2.25
• MDRCON28 0.038 -0.08 0.75
MDRTOT28 = Mattis Dementia Rating Scale (MDRS) total score; MDRIP28 = MDRS lnitiation/Perseveration subscale; MDRCCP28 = MDRS
30 conceptualization subscale; MDRCON28 = MDRS construction subscale.

NADH treatment of Alzheimer’s disease

0 104
c: 102

Mattis Dementia Rating Scale (MDRS) total score; n = 24 (p < 0.05)

in total recall on the Hopkins Verbal Learning Test (1 item fewer) versus placebo subjects (2.4 fewer items), slightly improved verbal recognition memory (0.33 items) versus deterioration in placebo subjects (1.25 fewer correct recognitions}, less deterioration in ver­ bal associative fluency (1.83 items) versus placebo


Baseline 6 months Session

subjects (2.75 items), less deterioration of categori­
cal fluency (0.55 items) versus placebo subjects (1.09) items and a slight improvement (or no change) in the Clinical Dementia Rating Scale (0.04 points lower) versus placebo subjects (0.09 points higher, indicating very slightly deterioration). On the MDRS,

Fig. 2 Mattis Dementia Rating Scale before and after 6 months’ treatment with reduced nicotinamide adenine dinucleotide (NADH).

0.080} (Table I). The results show an 8-point deterio­ ration for the placebo group and a 1.25 improvement for stabilized oral NADH at 6 months (relative to baseline) on the total score for the MDRS. The im­ provement was most evident in measures of verbal fluency (naming items that could be found in super­ markets) and on a measure of visual construction skill (i.e., reproducing geometric designs). The im­ provement in the conceptualization scale ap­ proached significance. Importantly, improvement was observed not only in the mean effect of NADH but also in each member of the pairs. In 8 oi.,t of 12 pairs matched on the MDRS there was positive effect in favor of NADH (p < 0.05) (Fig. 3).
There was a significant difference between the pairs at baseline in the Clinical Dementia Rating Scale. However, the groups did not differ in initial MDRS scores or in memory testing.
Analysis of the secondary outcome measures showed no significant difference between the treat­ ment groups. However, placebo subjects demon­ strated a trend for greater decline in verbal memory
than NADH subjects. After 6 months of treatment sta­
bilized oral NADH patients showed less deterioration

nonsignificant findings were slight improvement on the memory scale for stabilized oral NADH (0.42 points, slight deterioration in placebo patients (0.08 points) and less deterioration on the attention scale (0.08 points lower) versus placebo subjects (1.67 points lower).

Medication compliance/adverse events. Overall medication compliance was 100%, based on pills re­ turned and caregiver reporting. There were no adverse reactions related to the use of the study drug observed by caregivers or reported to examiners. Furthermore, the investigators observed no abnor­ malities in blood chemistries, complete blood counts, or electrocardiogram findings.

Pair2 PaJr3 Pair 4 Palr5 Palr6 Pair7 PalrB Pair 9
Pair 10
Pair 11 Pair12

-60 -50 -40 -30 -20 -10 0 10 20
Fig. 3 Mattis Dementia Rating Scale 6-month change from baseline. 31

Demarin V. et al.


The findings of this double-blind, placebo-con­ trolled study are encouraging and consistent with those of earlier open label and double-blind studies in showing a beneficial effect of NADH on cognitive functioning in AD (7, 16).
From the previous studies we learned that careful matching of patients in cognitive variables prior to randomization is essential for valid comparison of the treatment groups.
On the measure of the validated and generally accepted measure of dementia, the MDRS, AD pa­ tients receiving stabilized orally absorbable NADH showed significantly better performance (compared with baseline) after 6 months of double-blind treat­ ment than patients receiving placebo. The MDRS subscales that contributed to the total score differ­ ence were measures of verbal fluency, construction­ al ability and conceptual abllity. At this point, it is dif­ ficult to directly compare the present findings with those reported in AD clinical trials using other thera­ pies and outcome measures (20). However, no clini­ cal studies using NADH have reported adverse or side effects during the 6-month treatment period (21). In contrast to the placebo group, no statistically significant decline was observed on any of the cogni­ tive testing batteries in patients receiving NADH. However, statistically significant improvements in cer­
tain cognitive functions were observed. These findings indicate that NADH could not only help to stabilize the disease but could also improve certain cognitive func­ tions in AD patients. The results of this study confirm and extend previous findings on the beneficial effect of NADH on cognitive functioning in AD patients.

Address for correspondence: V. Demarin, Sestre Milosrdnice University Hospital, 10 000 Zagreb, Croatia.
Tel/Fax: +38513768282


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