# Abstract

 

## Background Plasmalogens (Pls) reportedly decreased in postmortem brain and in the blood of patients with Alzheimer's disease (AD). Recently we showed that intraperitoneal administration of Pls improved cognitive function in experimental animals. In the present trial, we tested the efficacy of oral administration of scallop-derived purified Pls with respect to cognitive function and blood Pls changes in patients with mild AD and mild cognitive impairment (MCI). 

## Methods The study was a multicenter, randomized, double-blind, placebo-controlled trial of 24 weeks. Participants were 328 patients aged 60 to 85 years who had 20 to 27 points in Mini Mental State Examination-Japanese (MMSE-J) score and five or less points in Geriatric Depression Scale-Short Version-Japanese (GDS-S-J). They were randomized to receive either 1 mg/day of Pls purified from scallop or placebo. The patients and study physicians were masked to the assignment. The primary outcome was MMSE-J. The secondary outcomes included Wechsler Memory Scale-Revised (WMS-R), GDS-S-J and concentration of phosphatidyl ethanolamine plasmalogens (PlsPE) in erythrocyte membrane and plasma. This trial is registered with the University Hospital Medical Information Network, number UMIN000014945. 

## Findings Of 328 patients enrolled, 276 patients completed the trial (140 in the treatment group and 136 in the placebo group). In an intention-to-treat analysis including both mild AD (20 ≤ MMSE-J ≤ 23) and MCI (24 ≤ MMSE-J ≤ 27), no significant difference was shown between the treatment and placebo groups in the primary and secondary outcomes, with no severe adverse events in either group. In mild AD patients, WMS-R improved significantly in the treatment group, and the between group difference was nearly significant (P = 0.067). In a subgroup analysis of mild AD patients, WMS-R significantly improved among females and those aged below 77 years in the treatment group, and the between-group differences were statistically significant in females (P = 0.017) and in those aged below 77 years (P = 0.029). Patients with mild AD showed a significantly greater decrease in plasma PlsPE in the placebo group than in the treatment group. 

## Interpretation Oral administration of scallop-derived purified Pls may improve cognitive functions of mild AD. 

## Funding The Japanese Plasmalogen Society. # Results 

## Study Participants A total of 328 patients were enrolled at 25 hospitals or clinics in Kyushu, Kanto, and Kansai regions in Japan from November 15, 2014 to October 8, 2015. They were randomly assigned to either of the two treatment groups (166 to Pls group and 162 to placebo group). Of the 328 enrolled patients, 285 continued to participate in the study for 12 weeks, and 276 completed the 24 week study period. Those who completed the study included 11 patients (4.0%) who failed to use 80% or more of the provided test substance. [Fig. 1](#) shows the trial profile. No significant difference was noted between the groups with respect to age, MMSE-J, GDS-S-J, WMS-R (0 min), WMS-R (30 min), erythrocyte PlsPE, and plasma PlsPE, at baseline, although the male-to-female ratio was significantly lower in the placebo group ([Table 1](#)). 

## Clinical Efficacy 

### Primary Analysis In an intention-to-treat analysis, there was no significant difference between the treatment and placebo groups in the primary and secondary outcomes of cognitive function. The MMSE-J score showed a nearly significant improvement in the treatment group while no such improvement was observed in the placebo group, resulting no statistically significant between-group difference. The WMS-R (0 min) and WMS-R (30 min) score each showed a significant improvement in both groups, but there was no statistically significant between-group difference. Erythrocyte PlsPE increased and plasma PlsPE decreased to different degrees after treatment in both Pls and placebo groups. There was no statistically significant between-group difference at any points of time, while the decrease in plasma PlsPE seemed greater in the placebo group ([Table 2](#)). 

### Subgroup Analysis We examined the change in cognitive function in mild AD patients and MCI patients separately. No notable change was seen in MMSE-J in either group. WMS-R (0 min) and WMS-R (30 min) improved statistically significantly in the treatment group, and the improvement seemed greater in the Pls treatment group with respect to the 0 min value (P = 0.067) and the 30-min value (P = 0.078) ([Table 3](#)). 

### Subgroup Analysis by Gender and Age Furthermore, we analyzed the cognitive function in patients with mild AD patients by gender and age ([Table 4](#)). Mild AD patients were divided into two groups based on the median value, 77 years or younger and 78 years or older. In the patients aged 77 years or younger, WMS-R (30 min) increased statistically significantly in the treatment group, showing a significant between-group difference (P = 0.029). In female patients with mild AD, WMS-R (30 min) improved significantly in the treatment group, with a significant between-group difference (P = 0.017). There was no notable between-group difference in the analysis of MCI patients by gender and age. 

### Changes in Blood Plasmalogens The changes in blood PlsPE levels among mild AD patients were examined ([Table 5](#)). Plasma PlsPE decreased in the placebo group, showing a significant between-group difference (P = 0.016). There was a statistically significant elevation of erythrocyte PlsPE at week 16 in the treatment group and at week 24 in placebo. However, the changes were not significantly different between the two groups. 

## Clinical Safety [Table 6](#) summarizes adverse events. There was no notable difference in the occurrence of adverse events between the two groups. <figure> Fig. 1 <p><img src="" /></p> <figcaption>Trial profile.</figcaption> </figure> ::::table-wrap

::: caption
Baseline characteristics.
:::

Table 1

Variable Pls group (n = 145) Placebo group (n = 140) P value[a](#)

______________________________________________________________________

Male, n (%) 65 (44.8) 45 (32.1) 0.03
MCI, n (%) 62 (42.8) 59 (42.1) 0.92
Age in year 76.4 (6.0) 76.5 (5.6) 0.94
MMSE-J 24.0 (2.4) 24.2 (2.2) 0.38
GDS-S-J 2.32 (1.54) 2.36 (1.55) 0.83
WMS-R (0 min) 4.21 (3.71) 4.34 (4.03)[b](#) 0.78
WMS-R (30 min) 2.50 (3.73) 2.77 (4.07)[b](#) 0.56
Erythrocyte PlsPE 8.03 (1.05) 8.11 (0.96) 0.49
Plasma PlsPE 3.65 (1.31) 3.93 (1.28) 0.08

Values are mean (SD) unless otherwise specified.

MMSE-J = Mini Mental State Examination-Japanese, GDS-S-J = Geriatric
Depression Scale-Short Version-Japanese, WMS-R = Wechsler Memory
Scale-Revised, min = minute, PlsPE = phosphatidyl ethanolamine
plasmalogens.

Chi-square test for proportion and unpaired *t*-test for mean.

Number of the patients was 139.:::: ::::table-wrap
::: caption
Mean difference from the baseline.
:::

Table 2

Variable Week Pls group Placebo group P value

______________________________________________________________________

n Mean (95% CI) n Mean (95% CI)\
MMSE-J 12 145 0.43 (0.04; 0.80) 140 0.33 (− 0.08; 0.73) 0.73
24 140 0.40 (− 0.01; 0.81) 136 0.32 (− 0.14; 0.77) 0.79\
28 81 0.69 (0.12; 1.26) 79 0.44 (− 0.14; 1.03) 0.54\
GDS-S-J 12 145 0.37 (0.05; 0.68) 140 0.39 (0.06; 0.73) 0.91
24 140 0.20 (− 0.12; 0.52) 136 0.18 (− 0.16; 0.51) 0.92\
28 81 0.17 (− 0.32; 0.66) 80 0.18 (− 0.34; 0.69) 1.00\
WMS-R (0 min) 12 145 0.66 (0.21; 1.20) 139 0.86 (0.36; 1.36) 0.55
24 140 1.41 (0.91; 1.92) 135 1.39 (0.87; 1.92) 0.95\
28 80 2.30 (1.48; 3.12) 78 2.46 (1.67; 3.25) 0.78\
WMS-R (30 min) 12 145 0.48 (0.11; 0.83) 139 0.35 (− 0.05; 0.75) 0.65
24 140 1.09 (0.59; 1.59) 135 1.10 (0.61; 1.59) 0.99\
28 80 2.09 (1.19; 2.98) 78 1.92 (1.19; 2.66) 0.78\
Erythrocyte PlsPE 8 145 0.13 (− 0.03; 0.30) 139 0.08 (− 0.09; 0.24) 0.64
16 140 0.24 (0.10; 0.37) 138 0.22 (0.06; 0.37) 0.86\
24 139 0.26 (0.10; 0.41) 135 0.35 (0.19; 0.52) 0.40\
28 139 0.27 (0.12; 0.42) 133 0.26 (0.09; 0.43) 0.95\
Plasma PlsPE 8 116 − 0.48 (− 0.70; − 0.27) 116 − 0.77 (− 0.98; − 0.56) 0.06
16 122 − 0.59 (− 0.80; − 0.39) 118 − 0.72 (− 0.93; − 0.50) 0.42\
24 140 − 0.24 (− 0.42; − 0.06) 136 − 0.38 (− 0.60; − 0.17) 0.31\
28 138 − 0.59 (− 0.77; − 0.40) 131 − 0.63 (− 0.81; − 0.44) 0.77

MMSE-J = Mini Mental State Examination-Japanese, GDS-S-J = Geriatric
Depression Scale-Short Version-Japanese, WMS-R = Wechsler Memory
Scale-Revised, min = minute, PlsPE = phosphatidyl ethanolamine
plasmalogens.:::: ::::table-wrap
::: caption
Mean difference from the baseline, in patients with MMSE-J score 20 to
23\.
:::

Table 3

Variable Week Pls group Placebo group P value

______________________________________________________________________

n Mean (95% CI) n Mean (95% CI)\
MMSE-J 12 55 0.31 (− 0.44; 1.06) 49 0.35 (− 0.43; 1.12) 0.94
24 50 0.06 (− 0.76; 0.88) 48 0.19 (− 0.59; 0.96) 0.82\
28 25 − 0.12 (− 1.48; 1.24) 25 0.36 (− 0.78; 1.50) 0.58\
GDS-S-J 12 55 0.33 (− 0.28; 0.94) 49 0.67 (0.02; 1.32) 0.44
24 50 0.20 (− 0.45; 0.85) 48 0.25 (− 0.28; 0.78) 0.91\
28 25 0.72 (− 0.66; 2.10) 25 0.18 (− 0.71; 1.03) 0.48\
WMS-R (0 min) 12 55 0.53 (− 0.13; 1.19) 48 0.73 (0.12; 1.34) 0.66
24 50 1.24 (0.49; 1.99) 47 0.30 (− 0.39; 0.99) 0.067\
28 24 1.29 (0.04; 2.55) 24 1.08 (0.46; 1.70) 0.76\
WMS-R (30 min) 12 55 0.47 (− 0.02; 0.96) 48 0.13 (− 0.25; 0.50) 0.27
24 50 0.78 (0.04; 1.52) 47 0.02 (− 0.37; 0.41) 0.078\
28 24 0.96 (− 0.33; 2.24) 24 0.29 (− 0.26; 0.84) 0.33

MMSE-J = Mini Mental State Examination-Japanese, GDS-S-J = Geriatric
Depression Scale-Short Version-Japanese, WMS-R = Wechsler Memory
Scale-Revised, min = minute.:::: ::::table-wrap
::: caption
Mean difference from the baseline, by gender and age, in patients with
MMSE-J score 20 to 23.
:::

Table 4

Gender/age Week Pls group Placebo group P value

______________________________________________________________________

n Mean (95% CI) n Mean (95% CI)\
Female WMS-R (0 min) 12 27 1.04 (0.02; 2.05) 30 0.80 (0.04; 1.56) 0.70
WMS-R (30 min) 12 27 0.93 (0.10; 1.75) 30 0.17 (− 0.37; 0.70) 0.11\
WMS-R (0 min) 24 25 1.52 (0.35; 2.69) 29 0.48 (− 0.47; 1.43) 0.16\
WMS-R (30 min) 24 25 1.08 (0.06; 2.10) 29 − 0.21 (− 0.68; 0.26) 0.02\
Male WMS-R (0 min) 12 28 0.04 (− 0.84; 0.92) 18 0.61 (− 0.53; 1.76) 0.41
WMS-R (30 min) 12 28 0.04 (− 0.51; 0.58) 18 0.06 (− 0.47; 0.58) 0.96\
WMS-R (0 min) 24 25 0.96 (− 0.06; 1.98) 18 0.00 (− 1.05; 1.05) 0.19\
WMS-R (30 min) 24 25 0.48 (− 0.66; 1.62) 18 0.39 (− 0.32; 1.10) 0.90\
77 or younger WMS-R (0 min) 12 20 1.30 (− 0.14; 2.74) 21 1.19 (0.37; 2.01) 0.89
WMS-R (30 min) 12 20 1.15 (0.05; 2.25) 21 0.10 (− 0.22; 0.41) 0.06\
WMS-R (0 min) 24 19 2.21 (0.61; 3.81) 21 0.48 (− 0.52; 1.47) 0.06\
WMS-R (30 min) 24 19 1.84 (0.10; 3.58) 21 0.00 (− 0.41; 0.41) 0.03\
78 or older WMS-R (0 min) 12 35 0.09 (− 0.57; 0.74) 27 0.37 (− 0.54; 1.28) 0.60
WMS-R (30 min) 12 35 0.09 (− 0.37; 0.54) 27 0.15 (− 0.50; 0.79) 0.87\
WMS-R (0 min) 24 31 0.65 (− 0.07; 1.36) 26 0.15 (− 0.86; 1.17) 0.41\
WMS-R (30 min) 24 31 0.13 (− 0.42; 0.68) 26 0.04 (− 0.61; 0.69) 0.83

WMS-R = Wechsler Memory Scale-Revised, min = minute.:::: ::::table-wrap
::: caption
Mean difference from the baseline, in patients with MMSE-J score 20 to
23\.
:::

Table 5

Variable Week Pls group Placebo group P value

______________________________________________________________________

n Mean (95% CI) n Mean (95% CI)\
Erythrocyte PlsPE 8 55 0.16 (− 0.13; 0.46) 50 0.28 (− 0.03; 0.48) 0.74
16 53 0.33 (0.11; 0.57) 48 0.19 (− 0.07; 0.44) 0.39\
24 49 0.24 (− 0.03; 0.52) 48 0.40 (0.11; 0.69) 0.42\
28 49 0.22 (− 0.02; 0.46) 46 0.26 (− 0.02; 0.54) 0.85\
Plasma PlsPE 8 41 − 0.21 (− 0.50; 0.08) 39 − 0.64 (− 1.01; − 0.26) 0.05
16 43 − 0.39 (− 0.68; − 0.11) 40 − 0.86 (− 1.29; − 0.43) 0.07\
24 50 0.16 (− 0.07; 0.40) 48 − 0.34 (− 0.69; 0.00) 0.02\
28 49 − 0.35 (− 0.63; − 0.08) 46 − 0.85 (− 1.13; − 0.57) 0.01

PlsPE = phosphatidyl ethanolamine plasmalogens.:::: ::::table-wrap
::: caption
List of reported adverse events.
:::

Table 6

```
                                   Pls group (n = 169)   Placebo group (n = 167)
```

______________________________________________________________________

Neoplasms\
Esophageal cancer 0 1
Ovarian cancer 0 1
Bladder cancer 1 0
Nervous system\
Parkinsonism 1 0
Transient ischaemic attack 0 1
Eye\
Ophthalmecchymosis 0 1
Circulatory system\
Hypertension 2 1
Ischaemic heart disease 1 0
Premature ventricular contraction 0 2
Stroke 1 0
Subarachnoid hemorrhage 1 0
Respiratory system\
Cold 2 3
Aspiration pneumonia 0 1
Digestive system\
Hepatic hemangioma 1 0
Stomatitis 1 1
Gastric ulcer 1 1
Intestinal obstruction 1 0
Acute hepatic disorder 1 0
Cholecystolithiasis 1 0
Skin and subcutaneous tissue\
Herpes zoster 0 1
Oral herpes 0 2
Prurigo 0 1
Rash 1 1
Musculoskeletal system\
Gouty attack 1 0
Genitourinary system\
Renal failure 1 0
Symptoms and signs\
Diarrhoea 3 1
Akathisia 1 0
Hand numbness 0 1
Ear pain 1 0
Sore throat 1 0
Constipation 2 0
Knee pain 2 0
Shoulder pain 0 1
Muscle pain 1 0
Cough 1 1
Vomiting 0 1
Dizziness 0 3
Stomach pain 1 1
Oedema 1 3
Injury\
Contusion 1 1
Fracture 4 4
Traumatic subarachnoid hemorrhage 1 0
Meniscus injury 1 0::::


Extracted trial result:
Outcome: Plasma PlsPE (continuous)
Intervention: 1 mg/day of Pls purified from scallop
Comparator: placebo
Intervention group size: 138
Intervention mean: -0.59
Intervention standard deviation: 1.11
Comparator group size: 131
Comparator mean: -0.63
Comparator standard deviation: 1.08
Notes: Data is from after 28 weeks (final timepoint). Back calculated the SD values using CI values.