Homocysteine lowering clinical trial on Alzheimer’s disease

This is a 26-week, double-blind, placebo-controlled, randomized clinical trial conducted in En Chu Kong Hospital, Taiwan from July 2003 to March 2006. The inclusion criteria were history of cognitive decline that was gradual in onset and progressive over a period of more than 6 months; clinical diagnosis of mild to moderate AD based on criteria set forth by the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) ¹⁰⁴; a score of 10-26 on the Mini-Mental State Examination (MMSE) ^{105, 106}; and a screening score of 1-2 on Clinical Dementia Rating Scale (CDR) ¹⁰⁷. Exclusion criteria were the following: history of epilepsy; clinically significant hepatic, renal, pulmonary, metabolic or endocrine disturbances, or significant cardiovascular disease that would impede the subject's ability to complete the trial; vascular dementia or evidence of cerebrovascular disease; low serum levels of folic acid (<5 ng/ml) and vitamin B12 (<180 pg/ml); use of any vitamin supplements and all the agents for the treatment of dementia (approved, experimental, or over the counter) except for

acetylcholine esterase inhibitors (AChEIs); cognitive impairment due to acute cerebral

trauma, hypoxic cerebral damage, infection, primary or metastatic cerebral neoplasm, significant endocrine or metabolic disease, or mental retardation. Computer tomography (CT) or Magnetic radioimaging (MRI) was performed on all study participants to rule out structural brain lesion (i.e. stroke, tumor, chronic subdural hematoma, etc.)

For ethical considerations, all of the 89 enrolled participants were also prescribed with AChEIs, the FDA approved treatment for AD patients. Except for one taking rivastigmin (® Exelone), all of the other patients were concurrently taking donepezil (® Aricept) in addition to our study regimens. All of the patients were then randomly allocated to receive the add-on therapy of either multi-vitamin or placebo according to a 1:1 ratio for six months. All of the study personnel and participants were blinded to treatment assignments and randomization was performed using a computer-generated randomization list. Efficacy measurements for the double-blinded phase were performed at baseline and week 26.

The study was conducted in accordance with Declaration of Helsinki and was approved by the hospital’s Institution Review Board. Written informed consent was given by each patient or patient’s representative or by the caregiver participating in this study.

3.4.2 Study regimens

The study regimens used mecobalamin preparation (trade name Methycobal) capsule (Eisai Co.) containing vitamin B12 0.5 mg with an active methyl base, and another over-the-counter vitamin named Pramet®FA (Abbott Laboratories Services Corp.) containing folic acid 1 mg, pyridoxine HCl 5 mg, iron ferrous 60 mg, nicotinamide 10 mg, calcium carbonate 250 mg, riboflavin 2 mg, thiamine mononitrate 3 mg, calcium pantothenate 1 mg, ascorbic acid 100 mcg, iodine 100 mcg, copper 150 mcg, vitamin B12 3 mcg, vitamin A 4000 I.U., and vitamin D3 400 I.U. In Taiwan, Pramet®FA tablet

was originally used as a kind of nutritional supplement for pregnant women. The placebo of Pramet®FA and Methycobal were made by the Genovate company (the manufacturer for Abbott Corp. in Taiwan) and Eisai Pharmaceutics Company respectively and were identical in size and color to vitamin supplements.

3.4.3 Assessment and outcome measures

Patients were assessed at the beginning with detailed history taking, blood tests,

neuro-imaging studies, and neuropsychological tests. Screening cognitive tests included the scale of CDR and MMSE. Baseline blood tests included standard fasting venous blood sample for blood chemistry (liver and renal function profile, fasting sugar), complete blood count (CBC), thyroid function (T3, TSH), syphilis blood test (VDRL, TPHA), and serum levels of B12, folic acid, and homocysteine.

The study patients were enrolled and received baseline cognitive measures, including Cognitive Abilities Screening Instrument (CASI, Chinese version, C-2.0, Liu)

108-110, Alzheimer’s Disease Assessment Scale (ADAS-Cog/11, Chinese version, Liu)

111-113, the Simplified Barthel Activities of Daily Living Index (ADL), and the Instrumental Activities of Daily Living Scale (IADL). The patients were then

interviewed by the same neurologist, who was blinded to the patients’ assigned study medication, at the outpatient department at weeks 2, 6, 10, 14, 18, 22, and 26 for physical and neurological examination. Any subjective or objective findings, as well as any favorite effects or adverse events (AEs) after treatment were recorded. At week 26, serum levels of B12, folic acid, and Hcy were re-measured. Cognitive performance and daily living function were assessed by the scores of MMSE, CASI, ADAS-Cog/11, ADL, and IADL.

3.4.4 Outcome measures

The primary efficacy outcomes were the change from baseline to week 26 in

ADAS-Cog ¹¹³ for the cognition evaluation, which includes an assessment of 11 items on the cognitive subscale of the ADAS (spoken language, comprehension, recall of test instruction, word-finding difficulty, following commands, naming, constructions, ideational praxis, orientation, word recall, word recognition). Cognitive assessments were carried out at screening baseline and at week 26 by the same independent rater who was blinded to the patients’ assigned study medication and other study information, such as adverse events.

Secondary efficacy outcomes included assessment of cognition changes from baseline to week 26 on activities by using the score of the CASI and MMSE. The CASI complete form (CASI-C) provides quantitative assessment (scoring from 0 to 100) of attention, concentration, orientation, short-term memory, long-term memory, language abilities, visual construction (copying two intersecting pentagons), list-generating fluency, abstraction, and judgment. These instruments were based on information collected independently from the subject and caregiver. Another secondary outcome measurement was an evaluation of the change from baseline to week 26 of activities of daily living function. The measurements included the ADL and the IADL. The ADL indices were measured using a 10-item for evaluating daily function (toilet, grooming, feeding, physical ambulation, dressing, bathing), while the IADL scale (based on the report of M. P. Lawton and E. M. Brody) ¹¹⁴ used an 8-item evaluation of the ability to use the telephone, shopping, food preparation, housekeeping, laundry, mode of

transportation, responsibility for own medications, ability to handle finances. Secondary outcomes also included the change of plasma Hcy, serum vitamin B12, and folic acid levels.

3.4.5 Safety evaluations

Safety and tolerability were evaluated by comparing treatment groups with respect to

physical examination findings, changes in vital signs, laboratory test abnormalities, concomitant medication use, and compliance with study medication, as well as the monitoring of adverse events (AEs) throughout the study. An AE was defined as any undesirable effect experienced by a patient during the trial, whether or not related to treatment. A serious adverse event (SAE) was defined as any AE that was life threatening or resulted in death, hospitalization, prolongation of hospitalization, or significant disability.

3.4.6 Data analysis

To compare the effects of multi-vitamin therapy and placebo on the cognitive function assessed using the ADAS-cog scale between the time of enrollment and week 26, according to an intention-to-treat strategy, the initial assumption was that there were 2 points of differences in the ADAS-cog scale between these 2 groups, with 4 of common standard deviation. We estimated the sample size required for a two-tailed significance level of 0.05 and a power of 0.90. On this basis, 43 patients were needed in each group.

Assuming the drop-out rate was 20%, the ideal number of total enrolled participants should be up to 108.

We first compared characteristic variables between the placebo and treatment groups, including baseline plasma levels of homocysteine, vitamin B12, and folic acid, as well as cognitive screening tests using the chi-square test for categorical data and the Student’s t test for continuous data. The changes in plasma concentrations of vitamin B12, folic acid, and homocysteine were then compared, from baseline to 6 months, using non-parametric Wilcoxon two sample tests because the values of the variables were not normally distributed.

Paired t–test was used to evaluate the efficacy of the intervention on cognition (MMSE, CASI, ADAS- Cog) and daily living function (IADL and ADL) by comparing

the changes from baseline to 6 months between the placebo and treatment groups. All were based on an intention-to-treat analysis. A multiple linear model was performed to assess the association between the change of homocysteine and change of cognition.

The ANCOVA test was used to compare the efficacy of intervention adjusting the effect of baseline cognition status, age, and sex variables. Linear regression was used to assess the association between the score change and the Hcy change after adjustment for baseline Hcy values, age, and sex. The number of adverse events between the placebo and treatment groups was also compared by using the chi-square test and Fish exact test if the expected number less than 5.