The efficacy and safety of Fufangdanshen tablets (Radix Salviae miltiorrhizae formula tablets) for mild to moderate vascular dementia: a study protocol for a randomized controlled trial

Background Vascular dementia (VaD) is the second most common subtype of dementia after Alzheimer's disease (AD). Currently, there are no medications approved for treating patients with VaD. Fufangdanshen (FFDS) tablets (Radix Salviae miltiorrhizae formula tablets) are a traditional Chinese medicine that has been reported to improve memory. However, the existing evidence for FFDS tablets in clinical practice derives from methodologically flawed studies. To further investigate the safety, tolerability, and efficacy of FFDS tables in the treatment of mild to moderate VaD, we designed and reported the methodology for a 24-week randomized, double-blind, parallel, multicenter study. Methods/design This ongoing study is a double-blind, randomized, parallel placebo-controlled trial. A total of 240 patients with mild to moderate VaD will be enrolled. After a 2-week run-in period, the eligible patients will be randomized to receive either three FFDS or placebo tablets three times per day for 24 weeks, with a follow-up 12 weeks after the last treatment. The primary efficacy measurement will be the Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAS-cog) and the Clinician Interview-Based Impression of Change (CIBIC-plus). The secondary efficacy measurements will include the Mini Mental State Examination (MMSE) and activities of daily living (ADL). Adverse events will also be reported. Discussion This randomized trial will be the first rigorous study on the efficacy and safety of FFDS tablets for treating cognitive symptoms in patients with VaD using a rational design. Trial registration ClinicalTrials.gov: NCT01761227. Registered on 2 January 2013.


Background
Vascular dementia (VaD) is the second most common subtype of dementia after Alzheimer's disease (AD) [1], and it accounts for 11.1-15.8 % of all cases of dementia worldwide [2,3]. The EURODEM Prevalence Research Group compared the prevalence of VaD in five datasets from Europe (Finland, Italy, Sweden, and two from the United Kingdom) and found that the prevalence ranged from 0.0-1.6 % for those aged between 60 and 70 years and increased to 2.8-9.2 % for subjects aged 80-90 years. The annual incidence rate was estimated to be 3.79 per 1000 among non-demented populations. At present, the treatment of VaD focuses on primary and secondary prevention strategies because randomized clinical trials in VaD have not been able to demonstrate clinically relevant symptomatic improvement. Additionally, it has not yet been possible to establish disease-modifying effects in VaD syndrome [4]. Thus, the development of an effective treatment for VaD is important.
Fufangdanshen tablets (FFDS), a traditional herbal medicine approved by the China Food and Drug Administration (CFDA) in 2008 (Number: [2008]1919), are extracted from the Chinese herbs Salvia miltiorrhiza, Panax notoginseng, and Borneolum syntheticum and contain tanshinone, salvianolic acid, panax notoginsenosides, ginsenoside Rb1, ginsenoside Rg1, and borneol. The tablets are used to treat patients with VaD. Preclinical studies have shown that tanshinone can improve the impaired learning and memory induced by Aβ1-40 in rat models of AD [5], inhibiting AD-induced expression levels of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase II (MMP-2), reducing toxic free radicals, and suppressing oxidative injury in AD rats [6]. Salvianolic acid can inhibit glutamate release and anti-cerebral ischemic effects [7]. Borneol can improve the permeability of the blood-brain barrier [8].
Many studies have reported that FFDS tablets can improve the memory of demented mice [9] and improve the impairment of spatial discrimination and memory impairment in rat models of AD. The mechanisms may involve improvement in the brain choline acetyl transferase (ChAT) activity that is decreased in AD rats and induced by Aβ [10], lowering the toxicity of excitatory amino acids [11] and increasing the expression of vascular endothelial growth factor (VEGF) in the brains of rats during chronic cerebral ischemia [11]. Additionally, FFDS tablets could improve the learning and memory capabilities in rat models of VaD, increase the activity of superoxide dismutase, and reduce neuron apoptosis in the hippocampus [12].
A phase II clinical trial on the efficacy of FFDS tablets in VaD was carried out in five centers. The trial enrolled 231 patients; all patients were randomized to the FFDS tablets group (115 patients) or the dihydroergotoxine mesylate tablets group (116 patients). Scores on the Mini Mental State Examination (MMSE) [13] and activities of daily living significantly improved in both groups compared with baseline (P < 0.001). However, there were some limitations to this study. First, there was no placebo group. As all subjects knew that they were being treated with one of two drugs and all of the individuals who assessed the patients knew this as well, the results may have been influenced by a positive response bias. Second, the sample size was relatively small. Third, this previous study only followed up patients for 12 weeks, and this period of follow-up was too short to detect the effects of the drugs.
To further investigate the safety, tolerability, and efficacy of FFDS tablets in the treatment of mild to moderate VaD, we designed and report the methodology for a 24-week randomized, double-blind, parallel, multicenter clinical study.

Study design
This ongoing study was designed as a randomized, double-blind, parallel, placebo-controlled, multicenter trial (six centers). It involves a single-blind run-in (and washout) period using placebo only (2 weeks) and a double-blind treatment phase after randomization (24 weeks). The study is being carried out at six centers in China. It has been approved by the China Food and Drug Administration (code: SFDA [2008] I919) and also by the Institutional Review Board of Dongzhimen Hospital, Beijing University of Chinese Medicine and has been registered with Clinical-Trials.gov (ClinicalTrials.gov: NCT01761227). If there is any amendment to the protocol, approval must again be sought from the Ethics Committee. The patients and responsible caregivers will provide written informed consent. The study will be conducted according to Good Clinical Practice Guidelines and the principles of the Declaration of Helsinki. The protocol design is based on the guidelines of Consolidated Standards of Reporting Trials (CONSORT), and the study results will also be reported according to these guidelines.

Participants
This trial will enroll both outpatient and inpatient Chinese-speaking males and females aged 45-80 years old, weighing 45-90 kilograms, and meeting the diagnostic criteria of probable VaD established according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) [14], the National Institute of Neurological Disorders and Stroke, and the Association Internationale pour la Recherche et l'Enseignement en Neurosciences (NINDS-AIREN) [15].
Participants' inclusion criteria are described as follows: Patients who confirm any of the following excluding criteria conditions will not be enrolled in the study: subjects with AD and any other secondary types of dementia (e.g., post-traumatic dementia, dementia associated with Parkinson's disease, or cerebral tumor); depressive pseudodementia and other mental disorders; a history of epilepsy; patients suffering from psychotic episodes; psychomotor excitation; MRI scans not showing vascular lesions in the brain; a history of drug or alcohol abuse in the past 6 months; acute or uncontrolled chronic illnesses; history of hypersensitivity to the treatment drugs; concomitant drugs with the potential to interfere with the study outcomes (including anticonvulsant agents, psychotropic drugs, drugs with psychiatric side effects, anticoagulants, cholinomimetic agents, short-acting benzodiazepines, or any other drugs that contain Salvia miltiorrhiza, Panax notoginseng, and/ or Borneolum syntheticum); participation in another clinical study.
All subjects will undergo an MRI scan before they are randomized. Participants will be excluded if the MRI presents medial temporal lobe atrophy adjusted for age. Scans will be performed routinely at each site and will be considered adequate if the images are of sufficient technical quality to be read accurately for the purposes of determining study inclusion. After receiving this imaging information, investigators will consider the clinical history, examination, and laboratory evaluations to assign the designation of "probable VaD" or "possible VaD."

Study medication
All trial medication will be supplied by the Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Company Limited (China). The FFDS tablets are composed of Salvia miltiorrhiza (DanShen in Chinese), Panax pseudo-ginseng (SanQi in Chinese), and Borneolum syntheticum (BingPian in Chinese). Each tablet weighs 0.3 g and contains tanshinone, salvianolic acid, panax notoginsenosides, ginsenoside Rb1, ginsenoside Rg1, and borneol. The FFDS tables were produced in a single batch (batch number: 080401, Placebo batch number: 081101) in strict compliance with standards of Good Manufacturing Practice (GMP).
During the 2-week placebo washout period, all patients will receive three placebo tablets three times per day. During the double-blind, 24-week intervention, the patients will receive either three FFDS or placebo tablets (three times per day), at least 2 hours apart from taking any routine Western medication. To preserve blinding, the placebo tablets have an identical taste and appearance to the FFDS tablets.
Concomitant use of anticonvulsants, antipsychotics, cholinomimetic drugs, anticholinergic agents, anti-Parkinson drugs, cholinesterase inhibitors, memantine, nootropic drugs, nimodipine, other cognition enhancers, or any drugs containing Salvia miltiorrhiza and Panax notoginseng will be forbidden. The investigator must record the concomitant drugs, including the name of the drug, daily dose, reason for using, and date of termination.

Sample size calculations
Because there are few studies on the treatment of VaD, the sample size was calculated based on data from a study of AD treatment using the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAScog) [17]. The previous study reported that patients who received donepezil (5 mg/day) showed a mean improvement of 0.67 ± 0.51; the patients who received the placebo showed a worse score of 1.82 points. Using a one-sided test with a significance level of 0.05 and power of 90 %, the minimum number was 88 per group to detect a difference of 1.5 points on the ADAS-cog. To achieve an adequate number to detect the safety of the FFDS tablets, the number of the patients was increased to 100 per group. Considering a greater rate of discontinuation of 20 %, the sample size was increased to 120 per group to ensure adequate patients to complete the study.
The randomization is stratified according to center using SAS statistical software (version 9.13) (SAS Institute Inc., Cary, NC, USA). Balanced randomization generated by SAS statistical software is carried out in three steps (in blocks of four) by a statistician with no access to information on the patients or physicians. Patients will be randomized in a ratio of 1:1 to receive the FFDS or placebo tablets. The randomized code is generated in the randomization process and sealed in an envelope. Statisticians assign the medication group according to the randomization code. Afterwards, the sealed randomization codes and each available medication number are sent out to each center. Blinding will be broken only if a patient's trial medication requires specific emergency treatment. Once the blinding is broken, the patient will be managed as off-trial.

Procedures
Two methods are being used to recruit participants with VaD. The first source of subjects is from memory clinics of six public hospitals of different centers, and the second source is those who respond to advertisements published in local newspapers. Figure 1 shows the schedule of enrollment.
The neuropsychological assessments, laboratory determinations, measurements of vital signs (including temperature, blood pressure, and electrocardiogram), and neurological tests (including an examination of cranial nerves, motor coordination, muscle tonus, and power) will be evaluated before patients are enrolled. The diagnosis of probable VaD will be based on the neurological test and confirmed by the criteria of the NIND-AIREN and DSM-IV, the HIS, and the Hamilton Depression Rating Scale (HAMD) [18]. For differential diagnosis, the Activities of Daily Living (ADL) [19] and a scan of the brain are also necessary.
All patients will receive an assessment at baseline (0 week), 12 weeks, and 24 weeks, and an additional assessment will be performed 12 weeks after completing the assigned double-blind medication. The study timeline and endpoints are shown in Table 1.
Any adverse events will be recorded during the duration of the trial. Patients will receive four clinical assessments.
Patients, caregivers, the study investigator, any other personnel involved in the study, and the investigating staff of the Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Company Limited will remain blinded until all patients complete the study and all data are collected.

Efficacy measurements
The primary efficacy measurements are the ADAS-cog [20] and the Clinician Interview-Based Impression of Change (CIBIC-plus) [21]. The ADAS-cog contains 12 items dealing with word recall, naming objects and fingers, commands, constructions and ideational praxis, orientation, word recognition, spoken language ability and comprehension of spoken language, word-finding difficulty, and attention. The scores of the ADAS-cog range from 0 to 75, and a higher score indicates higher impairment.
The CIBIC-plus is used to assess the global clinical status of the demented patient relative to baseline based on information from a semi-structured interview with the patient and the caregiver, and was designed specifically to evaluate the severity of cognitive dysfunctions characteristic of AD patients [21]. The score of the CIBIC-plus ranges from 1 to 7; a score of 1-3 indicates improvement, 4 means no change, and 5-7 indicates decline.
The secondary efficacy measurements include the MMSE and ADL [19]. The MMSE is used to assess the extent of cognitive dysfunction. The ADL mainly will be used to measure the basic activities and the instrumental activities of daily living. The ADL scale can be divided into the Physical Self-Maintenance Scale (PSMS) and the Instrumental Activities of Daily Living (IADL). The PSMS relates to physical activities, such as toileting, mobility, dressing, and bathing; the IADL contains eight items, such as shopping, cooking, doing laundry, handling finances, using the telephone, mode of transportation, responsibility for own medication, and housekeeping.

Safety assessment
Studies have reported that the adverse events of FFDS tablets were as follows [22]: a total of 11 patients suffered adverse events. Five patients suffered allergic reactions, such as rash or allergic asthma; two patients suffered hypokalemia, with symptoms like bloating and fatigue; one patient also showed significant sinus bradycardia, one patient showed tuberculosis hemoptysis, one showed thrombocytopenia, and one patient suffered hematuria with back pain and fever. Safety will be assessed at baseline (week 0), midstudy (week 12), and at the end of treatment (week 24). The safety assessment will include the following: (1) physical examination of vital signs, including breathing, heart rate, and blood pressure; (2) electrocardiography; (3) laboratory testing, including urine and blood; and (4) any adverse events that may occur, including the types of adverse events, time of occurrence, duration, treatment measures, and evaluation of the correlation between the tested drugs and the adverse event (positive, probable, possible, or not correlated); the severity of the adverse event (mild, moderate, and severe) must be evaluated. A mild adverse event is described as one that could induce mild symptoms, but the symptoms could be well tolerated, and there is no necessity to receive treatment or discontinue the study drugs. A moderate adverse event is defined as symptoms that could not be well tolerated and could affect the subject's ADLs; a moderate adverse event means that the tested drug must be stopped. Severe adverse events include any event that is fatal, considered to be lifethreatening, or requires hospitalization.

Compliance strategy
To maximize subjects' compliance, we will try to prevent dropouts by providing ongoing support to patients. A direct telephone line set up for this clinical trial will enable the study team to communicate personally with the patients. If a patient is lost to follow-up, we will call to ask the reason and attempt to schedule a meeting at the patient's convenience.

Statistical analysis
Statistical analyses will be conducted in three populations. The intent-to-treat (ITT) population will consist of all of the randomized population who take at least one dose of medication and at least one primary efficacy evaluation on treatment. The fully evaluated (FE) population will include all randomized patients who have received at least 80 % of the assigned 24 weeks of double-blind medication with a complete record of efficacy variables, with no major protocol violations. The safety set (SS) population will include all of the randomized population who receive at least one dose of the study medication, with at least one safety record post baseline.
The efficacy analysis will be conducted with the ITT and FE populations. The ADAS-cog and CIBIC-plus will be analyzed using the last observation carried forward (LOCF) method for the replacement of missing observations. The ADAS-cog and MMSE will be assessed by analysis of covariance (ANCOVA) with baseline scores as covariates and the treatment group and center as factors. The Cochran-Mantel-Haenszel test will be used for CIBIC-plus scores. The occurrence of adverse events will be analyzed using frequency calculations and descriptive statistics.

Discussion
Currently, there is no medication approved for treating patients with VaD. This ongoing randomized, placebo-controlled, 24-week clinical trial will be the first rigorous testing of FFDS tablets for the treatment of patients with VaD. Success in this clinical trial will provide evidence for the use of FFDS tablets in the treatment of VaD.
A series of neuropathological studies have shown that the diagnostic accuracy of the NINDS-AIREN criteria is 90 %, and the sensitivity is 0.20-0.58 [23,24]; the HIS also showed high specificity (87-95 %) and a relatively lower sensitivity for the diagnosis of VaD (43-50 %) [24,25]. Thus, in the present ongoing study, the diagnosis of VaD is based on the criteria of the NINDS-AIREN and the HIS. Additionally, the present study uses structural neuroimaging to differentiate VaD from AD to enroll proper VaD subjects.
The United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have issued guidelines on the criteria of efficacy with symptomatic improvement clinical trials [26][27][28]. Both of these guidelines emphasize three domains as efficacy endpoints: cognition endpoint, ADLs (functional endpoint), and overall clinical response as reflected by global assessment (global endpoint). Based on these guidelines, in the present study we are using each of these three endpoints as efficacy endpoints. In conclusion, the results of the present study are expected to provide evidence for the efficacy and safety of FFDS tablets for the treatment of patients with VaD.

Trial status
The trial has been approved and registered. At the time of manuscript submission, the study has been actively enrolling subjects and, as of this writing, has a total of 212 subjects. The study is ongoing.  Method used to implement the random allocation sequence (e.g., numbered containers or central telephone), clarifying whether the sequence was concealed until interventions were assigned 7 Implementation 10 Who generated the allocation sequence, who enrolled participants, and who assigned participants to their groups 7 Blinding (masking) 11 Whether or not participants, those administering the interventions, and those assessing the outcomes were blinded to group assignment. If done, how the success of blinding was evaluated 7 Statistical methods 12 Statistical methods used to compare groups for primary outcome(s); methods for additional analyses, such as subgroup analyses and adjusted analyses