A Randomized, Double Blind, Vehicle Controlled, Parallel, Phase Ii Study to Evaluate Ecacy and Safety of Bac in Patients With Alzheimer’s Disease or Vascular Dementia

Background: Dementia causes a long-term and gradual cognitive decline and there is no treatment to slow or stop the progression of dementia. Methods: Middle cerebral artery occlusion in rats was employed to investigate the effects of BAC in neural inammation. A phase II study was conducted in Alzheimer’s disease or vascular dementia patients. BAC or matched vehicle was applied topically on the subject’s external nasal skin, scalp, and neck, twice daily, 30 grams per day for 12 weeks. Cognitive evaluation questionnaires including ADAS-Cog, ADCS-ADL, MMSE, NPI, CIBIS, and CIBIC-plus scores were performed to evaluate the ecacy. A responder analysis was conducted with stratication of different sub-groups of receiving/not receiving dementia medication or donepezil hydrochloride and type of dementia. Results: The responder analysis showed that the naïve and not using donepezil hydrochloride sub-groups in the BAC-treated group had a higher responder rate compared to using donepezil hydrochloride and any dementia medication sub-groups. The BAC group showed a higher percentage of responders than the vehicle group in the mixed dementia population. Conclusion: BAC was safe for human use. BAC improved the responder rate in naïve dementia patients regardless of dementia type and in patients not receiving donepezil hydrochloride. The bigger sample size is required to conrm the ecacy of BAC. we investigated the inammatory molecule expression in the brain tissues of the middle cerebral artery occlusion (MCAO) following BAC treatment. we are presenting here a randomized, double-blind, vehicle-controlled, parallel, phase II study was designed to evaluate the ecacy and safety of BAC in patients with AD, VaD, or mixed dementia. The to evaluate the ecacy of in or

heavy metal-induced AD mice and the VaD-resembled rats when treated with BAC (unpublished data). In the present study, we investigated the in ammatory molecule expression in the brain tissues of the middle cerebral artery occlusion (MCAO) rats following BAC treatment. Besides, we are presenting here a randomized, double-blind, vehicle-controlled, parallel, phase II study was designed to evaluate the e cacy and safety of BAC in patients with AD, VaD, or mixed dementia. The primary objective of the clinical study was to evaluate the e cacy of BAC in patients with AD or VaD. The secondary objective of this study was to evaluate the safety of BAC patients with AD or VaD.

Materials And Methods
Middle cerebral artery occlusion (MCAO) rats as an animal model of dementia A total of 18 male Wistar rats (8 weeks old) weighing 250-330 grams were purchased from the BioLASCO Taiwan Co., Ltd. Rats were randomly divided into three groups (N = 6 in each group): sham group, middle cerebral artery occlusion (MCAO) group, and MCAO + BAC group: treated with BAC for 7 days after MCAO. The procedure of MCAO from previously reported studies was followed and described as below [18,19]. Rats were anesthetized by 3% iso urane in oxygen and maintained anesthetized using 2% iso urane in oxygen. A ventral midline incision was made to identify the left common carotid artery (LCCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA and the proximal LCCA were tied with a 6 − 0 silk suture, followed by blocking ICA blood ow with an arterial clip. The blood ow blocking procedure was conducted by using proper embolization wire (MCAO sutures, per weight of the rat) to insert from the distal end of LCCA and enter the brain through ICA. After inserted an 18 mm of nylon suture, the resistance that was felt indicated the blockage of the middle cerebral artery and caused cerebral ischemia. After 45 minutes of ischemia, the embolization wire was removed to allow blood reperfusion. The incision was closed and animals were transferred to the cages. The rats in the MCAO + BAC group were applied with 2 grams of BAC to the whole area of head and neck following by the gentle massage for 30 seconds, and then BAC was applied to the nose for each rat. Topical BAC treatment was repeated for 1 week and the brain tissues were collected for further analysis.
All the experimental procedures were performed according to the speci cations of the Guideline for Animal Experiments of Animal Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.

Western blot analysis
To prepare protein extraction, the frozen brains were homogenized in T-PER™ reagent (Tissue Protein Extraction Reagent, #78510, Thermo Fisher Scienti c/Invitrogen, Carlsbad, CA) supplemented with a cocktail of protease inhibitors (Complete™ Protease Inhibitor Cocktail, Roche/Sigma Chemical Co., St. Louis, MO), followed by centrifugation at 10000 × g for 10 minutes to harvest the supernatant. 50 µg total protein for each sample was loaded in 12% SDS gel. After electrophoresis, the protein was transferred to polyvinylidene di uoride (PVDF) membranes. After blocking for 1 hour with blocking buffer (5% of BSA in TBST), the membranes were probed with primary antibodies overnight at 4 °C. The following antibodies were used: IL-1β (1:500; #500-P80; Peprotech, New Jersey, USA) and GAPDH (1:50000; #6004-1-lg; Proteintech, Chicago, USA). After washing, the membranes were incubated with horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit secondary antibody (Jackson ImmunoResearch, PA, USA) at room temperature for 1 hour. The blots were developed using chemiluminescence, captured by CCD chemiluminescence imaging system (ChemiDoc XRS Imaging System, Bio-Rad, Hercules, CA) and the intensity of each band was quanti ed using NIH Image J software.

Clinical study design and population
For this clinical study, it was a randomized, double-blind, vehicle-controlled, and parallel phase II trial to evaluate the e cacy and safety of BAC in patients with AD, VaD, or mixed dementia. The sample size of treatment and control groups was 45 versus 15 patients (3:1). The study participants were recruited from 10 study centers across the United States from January 30, 2017 to November 01, 2018.
The main inclusion and exclusion criteria were described below and may be referred to NCT02886494 at ClinicalTrials.gov for details. At each study site, eligible patients were strati ed to either AD (including mixed dementia) or non-Alzheimer's disease.
The main inclusion criteria were (1) with a diagnosis of one of the following diseases: i. VaD according to the NINDS-AIREN International Workshop criteria, ii. AD according to the NIAAA criteria, or iii. mixed dementia (possible AD with the cerebrovascular disease) according to the NIAAA criteria; (2) with mild-to-moderate dementia (score of the MMSE de ned as between 10 to 24 and score of ADAS-Cog as at least 12). The main exclusion criteria were (1) with large vessel thrombosis (thrombotic stroke occurring in large arteries); (2) with radiological evidence of other brain disorders (subdural hematoma, post-traumatic/post-surgery); (3) with dementia caused by other brain diseases except for AD and VaD.

Outcomes
The primary endpoint of this study was the change in Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score at Week 12 compared to baseline, which measures the severity of the most prominent symptoms of AD including disturbances in memory, language, praxis, and attention.
Secondary endpoints included changes in ADAS-cog score at other visits; CIBIC-plus score at all visits, which is the Clinician's Interview-Based Impression of Severity/Clinician's Interview-Based Impression of Change Plus Caregiver Input (CIBIS/CIBIC-Plus), a widely used in anti-dementia drug trials for global assessment in the outcome measurement [17]; change in Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) Inventory compared to baseline, which measures the competence of patients with AD in their basic and instrumental activities of daily living [20]; change in Mini-Mental State Examination (MMSE) at all visits compared to baseline, which evaluates cognitive functions among elderly on orientation, attention, memory, language, and visual-spatial skills [21]; and change in Neuropsychiatric Instrument (NPI) score compared to baseline, which assesses neuropsychiatric symptoms and psychopathology of patients with AD and other neurodegenerative disorders.
The "responder" analyses of the change in ADAS-Cog and MMSE score from the baseline to Week 4, 8, and 12 were performed. The "responder" analyses consisted of two steps: (1) The ADAS-Cog/MMSE score at Week 4, 8, or 12 was deducted by the ADAS-Cog/MMSE score at the baseline; (2) based on the score difference, patients were then dichotomized into "responder" or "non-responder". If a patient made progress or remained unchanged in ADAS-Cog/MMSE score, he/she was considered as a "responder", whereas a patient who deteriorated in ADAS-Cog/MMSE score was considered as a "non-responder". The patients were further strati ed into 12 subgroups of receiving/not receiving any dementia medication (DM), receiving/not receiving any donepezil hydrochloride (DH), and type of dementia (all patients, AD patients, and mixed dementia patients).
The safety measurements included the incidence of adverse events and changes in physical examinations and laboratory monitoring parameters.

Statistical analysis
Changes in the e cacy scores including ADAS-cog score, ADCS-ADL score, MMSE score, and NPI score were performed by ANCOVA with the treatment group as a factor and with baseline score as a covariate. The CIBIS/CIBIC-plus score at all post-treatment visits was presented by the transition table. The Fisher's exact test was performed to test the "responder" differences in ADAS-Cog and MMSE scores between treatment groups. The safety outcomes determined by the incidence of adverse events were reported by treatment groups and by physiological systems. All treatment group comparisons were conducted with a signi cance level of 0.05, using 2-tailed tests. All the statistical analyses were performed by Statistical Analysis Software (SAS) 9.4 (SAS institute September 2017).

BAC treatment reduced the expression of pro-IL-1β in MCAO rats
The middle cerebral artery occlusion (MCAO) ischemic stroke model was considered to be a reliable model to study dementia. We evaluated the anti-in ammation effects of BAC in MCAO rats with BAC topical treatment by 7 days after cerebral ischemia reperfusion. The cytokine interleukin-1β (IL-1β) has been implicated in the contribution to ischemic brain damage [22]. Western blot showed that pro-IL-1β (precursor of IL-1β) signi cantly increased in the MCAO group compared to the sham group (Fig. 1). This con rmed a successful induction of ischemic stroke in the rats. Treatment with BAC signi cantly decreased the pro-IL-1β expression in the brain of MCAO + BAC rats ( Fig. 1). This result indicated that BAC treatment may reduce brain in ammation in MCAO rats.

Demographics, dementia history, and drug exposure
To further explore the e cacy and safety of BAC in patients with AD, VaD or mixed dementia, a clinical trial was conducted in the US. Details of subject disposition are shown in Fig. 2. In total 119 patients were screened, and 80 eligible study patients were enrolled in the study, with sixty patients being randomized to BAC treated group and twenty patients to vehicle-treated group. A total of 21 patients did not complete the study, and among them, 15 patients voluntarily withdrew consent.
Generally, the patients in the BAC and vehicle groups showed similar baseline characteristics, including age, gender, body weight, height, body mass index (BMI), and race. The mean duration of dementia history was 3.64 years. A total of 55 (68.8%), 17 (21.3%), and 8 (10.0%) patients were with Alzheimer's disease (AD), mixed dementia, and vascular dementia (VaD), respectively. There was no particular group difference regarding dementia types and duration. The demographic characteristics, dementia history, and the extent of drug exposure are summarized in Table 1. The duration of exposure to BAC versus vehicle was 76.7 days versus 73.9 days. No statistically signi cant difference in the extent of drug exposure was observed between treatment groups. The details of drug exposure are also summarized in Table 1. The primary e cacy outcome was the change in the ADAS-Cog score at Week 12 compared to baseline. After 12 weeks of BAC treatment, patients showed improvement in ADAS-Cog score by 0.7, which was not signi cantly different from patients treated with vehicle ( Table 2). In contrast, patients treated with vehicle appeared to show score reduction as early as Week 4, but the scores uctuated in the remaining period of the study. The results of secondary e cacy endpoints were summarized in Table 2. As for CIBIS/CIBIC-plus analysis, the distribution of CIBIS/CIBIC-plus score was similar in the BAC and vehicle-treated groups throughout the study period (Table 3). In contrast, the patients who use dementia medication and patients who use donepezil receiving BAC treatment gradual decline in responders in time, and those receiving vehicle treatment showed uctuation in the percentage of responders.

ADAS-Cog responder analysis for all, AD, and mixed dementia patients
A responder analysis was also carried out for ADAS-Cog scores, and the results are shown in Table 6. Naïve patients and patients who have no donepezil use receiving BAC treatment showed more responders throughout the study, compared to other subgroups, and the percentage of responders was found to be maintained at approximately 70% in all study visits. Similar results were observed when patients were further strati ed into patients with AD and patients with mixed dementia. The responder changes from baseline to week 4, 8, or 12 in ADAS-Cog score by sub-groups was illustrated in Fig. 4. In general, in both BAC and vehicle groups, patients who are naïve to dementia medication and patients who do not use donepezil have more responders throughout the study, and those receiving BAC treatment showed an upward slope in the percentage of responders along with the quad-weekly visits. However, for those patients receiving the vehicle, the percentage of responders showed a reduction with a downward trend over time. In addition, patients who are taking dementia medication and patients who are taking donepezil receiving vehicle treatment, uctuation in responders was observed along with the visits. Table 6 The responder analysis of ADAS-Cog score for all, AD and mixed dementia patients BAC and 2 (10.0%) vehicle-treated patients who experienced at least 1 AE related to the administration of study medication. The most frequent AEs were of "skin and subcutaneous tissue disorders" and was experienced by 5 (8.3%) BAC and 1 (5.0%) vehicle-treated patients. The treatment-related AEs are summarized in Table 7. It is widely accepted that brain in ammation has a great impact on the development of dementia. Particularly, IL-1 expression has been found to increase in the AD patients and is associated with β amyloid progression [22]. In the MCAO induced ischemic stroke model, the in ammatory markers were analyzed to understand the anti-in ammatory effect of BAC. Our results suggested that BAC can reduce brain in ammation through the route of topical application. Since IL-1β was the only in ammatory marker detected in the present study, further investigation of other in ammatory markers could be conducted.
In the e cacy assessment, we nd no deteriorating effects in cognition, ADL, or behavior in those being treated with BAC. Furthermore, we did nd e cacy in speci c populations, including patients who are naïve to dementia medications, and patients who are not using donepezil. We suspect that the use of concomitant dementia medications may have confounded the results, and careful patient selection would be essential in further studies.
Donepezil works by altering either the level of acetylcholine or the function of the nicotinic receptor, and the effect of missing a dose will have a rapid effect on the patient's cognition. As new proteins are being constantly synthesized, the level of acetylcholine may uctuate greatly when patients are not taking these drugs. However, BAC was found to reduce in ammation and maybe not easily affected by a single miss dose of BAC. This can make BAC an attractive therapeutic as it can help patients maintain sustained cognition.
We also discovered that in the mixed dementia population, patients treated with BAC had a greater percentage of responders than those treated with vehicle. Even though with small sample size, BAC may be a bene cial new option for these patients. As there are no medications approved for mixed dementia, these patients are typically prescribed AD medications in an off-label manner. BAC has shown safety and appears as a potential option for these patients.

Limitations
The present study has some intrinsic limitations to the early stage of clinical development. The sample size of the study was small so it was challenging to nd a signi cant treatment effect on clinical endpoints. Besides, the current study includes data with a short period, meaning that it may be not enough to examine the association between BAC and disease progression. The longer study period will be valuable in future studies.

Conclusions
In the MCAO induced ischemic model, the topical application of BAC showed an anti-in ammation response. This BAC clinical trial shows that topical BAC application has no deteriorating effects in cognitive performance and may be bene cial to naïve dementia patients as evaluated in the responder analysis. BAC has been found to have a good safety pro le in patients with AD or VaD. The study was conducted in accordance with regional regulations and the Declaration of Helsinki. Written informed consent was received from all participants, and ethical approval for data acquisition was provided by Chesapeake IRB (Protocol approval with modi cation (approval date: 10 October, 2016), approval notice (MOD00206802), continuing review approval (CR00076215) and subject material approval (MOD00203871)). Informed consent was obtained from all study participants and their caregivers before their enrollment.

Consent for publication
Not applicable.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Competing interests
The authors declare that they have no con ict of interests.