DL-3-n-butylphthalide improves cerebral hypoperfusion in patients with large cerebral artherosclerotic stenosis. A single-center, randomized, double-blind, placebo-controlled study. CURRENT

Background: DL-3-n-butylphthalide (NBP) was demonstrated to increase the cerebral blood flow (CBF) in the animal models, but there are no clinic studies to verify this. We aimed to explore the effect of NBP on improving cerebral hypoperfusion caused by cerebral large-vessel stenosis. Methods: In this single-center, randomized, double-blind, placebo-controlled study, 120 patients with severe carotid atherosclerotic stenosis and cerebral hypoperfusion in the ipsilateral middle cerebral artery (MCA) were included and randomly assigned into the NBP or placebo group as 1:1 radio. Patients in NBP and placebo group received 200mg and 20mg of NBP capsules three times daily for four weeks respectively. Single photon emission computed tomography (SPECT) were used to assess regional CBF (rCBF) in four regions of interest (ROIs) corresponding to MCA before and 12 weeks after the treatment. The rC BF change for every ROI and the CBF change in whole MCA territory for every patient after therapy were classified into amelioration, stabilization and deterioration respectively. Results: 48 NBP patients (6 with bilateral stenosis) and 46 placebo patients (8 with bilateral stenosis) completed the trial. Both groups had 54 stenotic carotid arteries and 216 ROIs for rCBF change analysis. After therapy, the rCBF in ROIs increased in NBP group (83.5%±11.4% vs. 85.8%±12.5%, p=0.000), but had no change in placebo group (86.9%±11.6% vs. 87.8%±11.7%, p=0.331). There was higher percentages of ROIs with rCBF amelioration and stabilization in NBP group than in placebo group (93.1% vs. 79.2%, p=0.000). Ordinal regression analysis showed that NBP independently made more patients to have whole CBF amelioration in ipsilateral MCA than placebo (Wald-χ2=5.247, OR=3.31, p=0.022). Conclusions: NBP

controlled study, 120 patients with severe carotid atherosclerotic stenosis and cerebral hypoperfusion in the ipsilateral middle cerebral artery (MCA) were included and randomly assigned into the NBP or placebo group as 1:1 radio. Patients in NBP and placebo group received 200mg and 20mg of NBP capsules three times daily for four weeks respectively.
Single photon emission computed tomography (SPECT) were used to assess regional CBF (rCBF) in four regions of interest (ROIs) corresponding to MCA before and 12 weeks after the treatment. The rC BF change for every ROI and the CBF change in whole MCA territory for every patient after therapy were classified into amelioration, stabilization and deterioration respectively. Results: 48 NBP patients (6 with bilateral stenosis) and 46 placebo patients (8 with bilateral stenosis) completed the trial. Both groups had 54 stenotic carotid arteries and 216 ROIs for rCBF change analysis. After therapy, the rCBF in ROIs increased in NBP group (83.5%±11.4% vs. 85.8%±12.5%, p=0.000), but had no change in placebo group (86.9%±11.6% vs. 87.8%±11.7%, p=0.331). There was higher percentages of ROIs with rCBF amelioration and stabilization in NBP group than in placebo group (93.1% vs. 79.2%, p=0.000). Ordinal regression analysis showed that NBP independently made more patients to have whole CBF amelioration in ipsilateral MCA than placebo (Wald-χ2=5.247, OR=3.31, p=0.022). Conclusions: NBP may improve the cerebral hypoperfusion in the patients with carotid artery atherosclerotic stenosis. Trial registration: Chinese Clinical Trial Registry, ChiCTR1900028005, registered December 8th 2019-Retrospectively registered, http://www.chictr.org.cn/edit.aspx?pid=45490&htm=4. 4 Background Hypoperfusion caused by large intracranial and extracranial atherosclerotic stenosis can impair clearance of distal emboli, which is an alternative important mechanism of ischemic stroke, in addition to thromboembolism [1][2]. Approximal 20% 30% of ischemic strokes in the United States were attributed to cerebral large-vessel stenosis [3]. The study of China National Stroke Registry Large showed that large arterial atherosclerotic stenosis accounted for 45% ischemic strokes in China [4]. In addition, many studies showed that chronic cerebral hypoperfusion was associated with a higher prevalence of cognitive impairment [5]. Therefore, improving cerebral hypoperfusion may prevent ischemic stroke and cognitive impairment. Although carotid endarterectomy (CEA) and carotid artery stenting (CAS) can normalize cerebral hemodynamics by increasing the lumen diameter and providing a better cerebral irrigation, they are not suitable to all the patients with cerebral large-vessel stenosis [6]. The augmentation of collateral vessels may also enhance the cerebral blood flow in these patients. Some pharmacologic approaches such as volume expansion, hemodilution, vasodilatation, and induced hypertension, have been suggested to increase collateral perfusion, but unfortunately, the trials of these drugs failed to demonstrate an overall clinical benefit among the participants [7]. Consequently, the American Heart Association's 2017 guidelines advised against the common use of these medicines to enhance collateral circulation [8].
Dl-3-butylphthalide (NBP) is a synthetic chiral compound based on l-3-butylphthalide, which is originally isolated from seeds of Apium graveolens [9]. The systematic review showed that the combination of NBP and standard anti-ischemic stroke agents was superior to standard drugs alone in the therapy of patients with acute ischemic stroke, based on both the Barthel index and National Institutes of Health Stroke Scale, as a neuroprotective drug [10]. The numerous animal studies also showed that NBP decreased the ischemic brain area and improved the neurological deficits. NBP could improve the cerebral microcirculation and cerebral blood flow (CBF) by vasodilatation and angiogenesis in these animal studies [11][12][13][14]. However, there are still no clinic studies to verify that NBP can improve decreased CBF in the human being.
This clinical trial was carried out to explore the effect of NBP capsules on improving cerebral hypoperfusion in the ipsilateral middle cerebral artery (MCA) territory caused by the severe carotid atherosclerotic stenosis. This study may provide an evidence-based clinical strategy to prevent ischemic stroke and vascular cognitive impairment from cerebral large-vessel stenosis and hypoperfusion.

Study design
This was a single-center, randomized, double-blind, placebo-controlled trial that enrolled

Patients
The patients, who had both severe atherosclerotic stenosis in the carotid artery and cerebral hypoperfusion in the ipsilateral MCA territory, were invited to participate in this study. Eligibility criteria included the following: (1) 30-80 years of age; (2) ≥70% stenosis or occlusion in the internal carotid artery (ICA) and/or the first sphenoidal segment (M1) of MCA; (3) cerebral hypoperfusion in the ipsilateral MCA territory; (4) no transient ischemic attacks (TIA) or ischemic strokes within 2 weeks; (5) no ≥50% stenosis or occlusion in the subclavian artery, vertebral artery, basilar artery and posterior cerebral artery (6) no or ≤15mm infarction in the territory of MCA and cerebellum on CT or MRI. We excluded the following subjects:(1) cerebral stenosis caused by other diseases than atherosclerosis, such as autoimmune disorder, vasculitis etc; (2) willing to carry out CAS or CEA; (3) contraindications for assessing cerebral stenosis and perfusion; (4) other cerebral diseases (such as infection, degeneration, demyelination etc.) which influence cerebral perfusion; (5) pregnant or lactating women; (6) severe cardiac, pulmonary, hepatic or renal diseases etc. and life expectancy ≤6 months; (7) psychotic; (8) allergy to NBP.

Randomization and drug treatment
Patients were randomly assigned in a 1:1 ratio to therapy and placebo groups. Both groups received three times daily oral NBP 200mg or 20mg (ineffective dose) for 4 weeks respectively. Patients also received optimized medical therapy (OMT) against cerebral artery atherosclerotic stenosis, including antiplatelet aggregation, lipid-lowering, antihypertension, hypoglycemia, etc. However, any vascular dilation drug was contraindicated for the patients.
An independent statistician from Chinese Beijing Medical University created the randomization list by the compute. Every kit of drug was labeled with sequential numbers corresponding to the randomization list. The successive patients were distributed with the kits from lowest number to highest number. All kits of drugs had identical appearance and similar smell. The patients and investigators were blinded to the drug allocation.

Clinical assessment and Follow-up
Initial assessments of the patients before the therapy included demographic characters, the atherosclerotic risk factors (hypertension, diabetes mellitus, hyperlipidemia, coronary heart disease and smoking), cerebral stenotic location and degree, integrity of the circle of Willis (CoW), and CBF. The medication compliance of NBP and its adverse reactions were followed up by telephone after 4 weeks. CBF was assessed again after 12 weeks. The cerebral artery stenosis and integrity of CoW were assessed by magnetic resonance angiography (MRA), computed tomography angiography (CTA), or digital subtraction angiography (DSA). The extracranial artery stenotic degree was calculated according to North American Symptomatic Carotid Endarterectomy Trial Collaborators (NASCET) and the intracranial artery stenotic degree was calculated according to Warfarin-Aspirin Symptomatic Intracranial Disease (WASID). CBF was assessed by single photon emission computed tomography (SPECT).

SPECT procedures and Region of interest (ROI) selection
SPECT brain scans were carried out in a dark and quiet environment according to our previous study [6]. 925 MBq of ethylcysteinate dimer ( 99m TC-ECD, China institute of atomic energy, China) was injected into the antecubital vein. The images were taken by the SPECT scanner (Infinia Hawkeye; General Electric Company, USA) after one hour. The images were taken per 6° during rotation of 360° and at a rate of 35s per frame. The data were acquired on a 128×128 matrix.
ROI selection and data analysis were also according to previous studies [6,15].
NeuroMatch Software package (GE Medical Systems; Segami Corporation, Columbia, MD, USA) was used for the ROI selection. Four slices of cerebral hemisphere were selected on orbitomeatal line (OML) +55mm, +58mm, +61mm, and +64mm.Six pairs of sphenoid mirrored ROIs over cortical gray matter were drawn at each slice of cerebral hemisphere.
Four middle ROIs corresponding to the MCA territory were selected, and the mean value of four slices in each ROI was used for data analysis. However, mirrored ROIs were also drawn in one slice of cerebellar hemisphere (OML+15mm) for normalization of cerebral hemodynamic parameters.

Definition of CBF and cerebral hypoperfusion
The ratio of radioactive counting obtained from the ROI in the cerebral hemisphere to that from the ROI in the ipsilateral cerebellar hemisphere was calculated automatically by computer for the cerebral hemodynamic normalization, which was defined as CBF. The CBF in each ROI was called as regional CBF (rCBF). The cerebral hypoperfusion was defined as rCBF stenosis ≤90% and/or rCBF m irror rCBF stenosis ≥90% in any ROI corresponding to the ipsilateral MCA territory [15,16].

Classifications of CBF change after treatment
As for a ROI in the ipsilateral MCA of carotid artery stenosis, its rCBF change was classified according to the difference between pre-treatment and post-treatment rCBF.
The rCBF amelioration was defined as rCBF after rCBF before ≥10%, and the rCBF deterioration was defined as rCBF after rCBF before ≤-10%, and the rCBF stabilization was defined as -10% rCBF after rCBF before 10% [15].
As for a patient with carotid artery stenosis, his whole CBF change in the ipsilateral MCA territory was classified according to the number of ROIs with rCBF deterioration or amelioration after treatment (4 ROIs in the unilateral stenosis and 8 ROIs in the bilateral stenosis). First, the whole CBF deterioration was defined when a patient had ≥1, ≥2, ≥3 or ≥4 ROIs with rCBF deterioration in the ipsilateral MCA territory. Correspondingly, the whole CBF amelioration was defined when a patient had ≥1, ≥2, ≥3 or ≥4 ROIs with rCBF amelioration in the ipsilateral MCA territory. At last, the whole CBF stabilization was defined in the other patients than CBF deterioration and amelioration.

Statistical analysis
Continuous variables with normal distribution were presented as mean and standard deviation (SD), and continuous variables with non-normal distribution as median and interquartile range. Categorical variables were reported as the percentage of patients in the subgroup. In univariate analysis, the Student t test or the Mann-Whitney U test was used for two class comparisons of continuous variable, and one-way ANOVA or Kruskal Wallis H test for multiple-class comparisons of continuous variables. χ 2 test was used for the comparisons of categorical variables. Multivariable ordinal regression models were performed with the whole CBF change after treatment as dependent variable, and NBP as independent variable. The statistical significance was defined as p≤0.05. All the resultant data were analyzed by the software of SPSS 16.0 (IBM, Amon, New York, USA).

Results
A total of 120 patients with cerebral hypoperfusion in the ipsilateral MCA of carotid artery stenosis, participated in this study and underwent randomization. Among them, 60 were assigned to NBP group and 60 were assigned to placebo group. As shown in Figure 1, 48 patients among NBP group and 46 patients among placebo group completed treatment and follow-up. Therefore, 94 patients were included in the efficacy analysis and 26 patients (21.7%) were lost to follow up in this study. Baseline demographics and clinical characteristics were well-balanced between NBP group and placebo group, which were listed in Table 1.

rCBF outcomes for ROI analysis
Six patients had bilateral carotid artery stenosis and cerebral hypoperfusion in 48 NBP patients, and eight patients had bilateral carotid artery stenosis and cerebral hypoperfusion in 46 placebo patients. Therefore, both NBP and placebo groups had a total of 54 stenotic carotid arteries with cerebral hypoperfusion in ipsilateral MCA respectively.

Whole CBF outcomes in ipsilateral MCA for patient analysis
No matter what was defined as whole CBF outcomes (deterioration, amelioration and stablization) in the ipsilateral MCA territory, the NBP group always had higher percentages of patients with whole CBF amelioration and stabilization than placebo group (Table 2).
When the whole CBF amelioration/deterioration was defined as the numbers of ROIs with rCBF amelioration/deterioration in the patients ≥1, the difference of whole CBF outcomes between two groups was not significant. On the contrary, when the whole CBF amelioration/deterioration was defined as the numbers of ROIs with rCBF amelioration/deterioration in the patients ≥2, ≥3 or ≥4, the differences of whole CBF outcomes between two groups became significant (Table 2). However, when the numbers of ROIs were defined as ≥3 and ≥4, the patients who had whole CBF amelioration/deterioration were too few to make the regression analysis. Finally, the number of ROIs ≥2 was defined as whole CBF amelioration/deterioration to analyze the regression. The ordinal regression analysis showed that after adjusted for demographics, atherosclerotic risk factors, cerebral stenotic characteristics and integrity of CoW, NBP was still an independent factor to improve and stabilize impaired CBF caused by carotid artery stenosis (Table 3). Figure 3 and Figure 4 showed the classical cases whose rCBF in the ipsilateral MCA increased in NBP group and decreased in placebo group.

Discussion
Our randomized, double-blind, placebo-controlled trial showed that four weeks of orally NBP therapy improved the rCBF in the ipsilateral MCA of carotid artery stenosis. There were higher percentages of ROIs with rCBF amelioration and stablization after treatment in NBP group than in placebo group. Compared with placebo group, NBP group had more than two times of patients who had the whole CBF amelioration in the ipsilateral MCA. Therefore, NBP was recommended to treat the acute ischemic stroke by Chinese Guidelines for the Management of Stroke since 2010 [17]. A multicentre, randomized, double-blind, placebo-controlled trial recently showed that a 6-month NBP treatment was also effective for improving cognitive and global functioning in the patients with subcortical vascular cognitive impairment no dementia (VCIND) [18]. Another retrospective case-control study showed that postoperative administration of 7-day NBP injection was able to alleviate perioperative neurological deficits after revascularization surgery for the patients with moyamoya disease [19]. NBP might play the positive effects on these diseases through the mechanism of improving the cerebral hypoperfusion [9,10], since it had been demonstrated that the ischemic stroke, vascular cognitive impairment and perioperative neurological deficits of moyamoya disease after revascularization surgery were partially attributed to acute, chronic or fluctuating cerebral hypoperfusion [1,5,19]. An animal study had showed that NBP increased cerebral microvessels and perfusion, and decreased the volume of the cerebral infarct in the rats of cold-induced ischemic stroke [14]. NBP also accelerated CBF in the chronic cerebral hypoperfusion (CCH) rats after permanent bilateral common carotid artery occlusion, and prevented CCH-induced white matter damage and cognitive impairment [11,13]. In addition, NBP was found to improve rCBF in the caudate nucleus for the rats subjected to subarachnoid hemorrhage [20]. By contrast, high-quality clinical trial was lack to demonstrate that NBP increased CBF and improved cerebral hypoperfusion.
Our study firstly demonstrated that NBP could improve the cerebral hypoperfusion in the patients with carotid artery atherosclerotic stenosis. However, some important points must be noted to explain and apply this result. First, all the patients in this study had decreased CBF before the treatment because of severe carotid artery stenosis and poor collateral circulation, which suggested that the hemodynamic impairement existed distal to cerebral artery stentosis. In addition, our study excluded the patients who had acute ischemic cerebral vascular diseases before the trial, which suggested that the patients included in this study had chronic cerebral hypoperfusion. Therefore, this trial provided the new therapy method for the patients with CCH resulted from the carotid artery stenosis [1,5,19]. Second, NBP was used with the combination of OMT against atherosclerotic ischemic stroke in this study, because these patients had the large intracranial or extracranial stenosis. Therefore, the effect of NBP on improving CCH in the patients with the cerebral artery stenosis must base on the anti-arthrosclerosis OMT, but not NBP alone [21]. Third, NBP was orally taken for 4 weeks in our study, but the CBF examination after treatment was carried out 12 weeks after the trial beginning in consideration of the radioactivity of the SPECT test. Therefore, this positive result suggested that NBP had a persistent effect on improving the cerebral hypoperfusion in the patients with the cerebral artery stenosis.
The collateral circulation of the brain may be defined as the artery-to-artery anastomotic pathways that are capable, when needed, of supplying nutrient perfusion to a brain region whose primary source of blood flow has been reduced or compromised by disease [22]. In humans, it is generally recognized that there are three pathways of cerebral collaterals. The brain's primary collateral pathway consists of the CoW the anastomotic array of arteries at the base of the brain that connect the anterior and posterior portions of the cerebral circulation. The brain's secondary collateral pathways can be accessed through the ophthalmic artery and leptomeningeal vessels when collateral flow through the CoW is inadequate. The additional collateral pathway also includes the neovascularization [22,23]. One clinic study showed that NBP injection increased much more brain's primary and secondary collaterals than the control injection in the patients of acute ischemic stroke [24]. The animal studies demonstrated that NBP promoted the new angiogenesis in the rats of acute ischemic stroke or CCH [11,12,25]. Therefore, these results supported the opinion that NBP could improve the cerebral hypoperfusion in the patients with cerebral artery stenosis by increasing the brain's collateral circulation. The mechanisms that NBP increases the brain's collateral circulation may be as follows. First, nitric oxide (NO) released from vascular endothelium was thought to participate in arteriogenesis and vasodilation and increase the cerebral collateral. NBP significantly increased the activity of NOS and production of extracellular NO in bovine aortic endothelial cells and bovine cerebral endothelial cells, which might improve the cerebral microcirculation and restore the supply of oxygen and nutrients to ischemic hemisphere [9,22]. Second, NBP also promote the angiogenesis or revascularization by up-regulating the expressions of various angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) [12,[25][26][27], or by regulating some signaling axes or pathways [28,29].
Since NBP smelled plant odor, the placebo medicine was designed as a low and ineffective dose (10 mg NBP per pill) rather than a blank one, in order to make sure of an effective double-blind in this trial. However, there were also some limitations in this study. First, this study was a single-center trial, and had a relatively small sample size and a little high lost follow-up rate. Therefore, a multiple-centers and large sample size of trial needs to verify the results in this study. Second, some patients in this study completed the CBF examination more than 12 weeks after treatment, but there was not a significant difference in the follow-up time between NBP group and placebo group. Therefore, the results in this study may not be affected by this follow-up prolongation.

Conclusion
Our trial showed that on the basis of OMT NBP improved the cerebral hypoperfusion in the patients with carotid artery atherosclerotic stenosis. This study provides a new therapy strategy to prevent ischemic stroke and vascular cognitive impairment due to cerebral large-vessel stenosis and hypoperfusion. The therapy and placebo medications of DL-3-n-butylphthalide were donated by the Shijiazhuang Pharmaceutical Group Co that had no other role in this study.

Author Contributions:
DC has made substantial contributions to design and has been involved in drafting of the manuscript. JS has made substantial contributions to the design and given final approval of the version to be submitted. YY has made substantial contributions to design and revised the manuscript. FY has made substantial contributions to statistical analysis. KW has contributed greatly in data collection and management. FZ has contributed to the patients' follow-up. WL has revised the manuscript. BL has contributed to SPECT technique. All authors read and approved the final manuscript.

Funding
This work was funded by Ministry of Sciences and Technology of Air Force Medical Center, PLA (No. 2014ZX09104002-002). The funder provided support for the study to be carried out and published, but not any other role during the execution of the study, analyses, interpretation of the data, or decision to submit results.

Statement of Ethics
Written informed consent was obtained from all study participants or their legal representatives. The protocol for this trial was approved by the Institutional Review Boards in the Air Force Medical Center, PLA, China.

Consent for publication
Not applicable.

Competing interests
All the authors have no conflict of interest.

Other statement
My study and manuscript reporting adhered to CONSORT (Consolidated Standards of Reporting Trials) guidelines.      Comparison of rCBF outcomes in every ROI between NBP group and placebo group. There were higher percentages of ROIs with rCBF amelioration and stablization in NBP group, but higher percentages of ROIs with rCBF deterioration in placebo group (p=0.000). CBF, cerebral blood flow; rCBF, regional CBF; ROI, regions of interest; NBP, Dl-3-butylphthalide.

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