Human urinary kallidinogenase or edaravone combined with butylphthalide in the treatment of acute ischemic stroke

Abstract Aim The effectiveness of neuroprotective agents is still unclear. Here we analyzed the clinical outcomes of acute ischemic stroke (AIS) patients treated with human urinary kallidinogenase (HUK) or edaravone (Eda) combined with butylphthalide (NBP). Methods From January 2016 to December 2017, a total of 165 AIS patients were enrolled in this open‐label, randomized controlled clinical study. Patients were randomly allocated into HUK group and Eda group in a ratio of 2:1. All the patients received basic treatments and NBP (200 mg p.o. qid) while HUK group received 0.15 PNA unit of HUK injection (ivgtt. qd) and Eda group received 30 mg Eda (ivgtt. bid) for 14 consecutive days. Independency rate [12‐month modified Rankin Scale (mRS) score ≤ 1] and related factors were compared between the two groups. Results Twelve‐month mRS score of the HUK group (1, IQR 0~1) was significantly lower compared with Eda group (2, IQR 1~3, p < .0001). The HUK treatment achieved an independency rate of 79.1% while the Eda treatment only had 45.3% (p < .0001). Further binary logistic regression showed that recurrent stroke (RR: 0.1, 95% CI: 0.0~0.1, p = .038) and HUK treatment (RR: 4.2, 95% CI: 1.1~16.5, p = .041) could significantly affect patients' 12‐month outcomes. Conclusion Human urinary kallidinogenase combined with NBP can enhance AIS patients' long‐term independency rate, and the effectiveness of HUK combined therapy is better than Eda.

mechanical thrombectomy, neuroprotective agents do play an important and irreplaceable role. Hence in 2018 Chinese AIS diagnosis and treatment guidelines, individualized application of butylphthalide (NBP) and human urinary kallidinogenase (HUK) therapy can be taken into consideration according to the results of some randomized controlled trials (level IIb) (Chinese Society of Neurology, 2018).

NBP was approved for ischemic stroke by the State Food and
Drug Administration in China in 2002 for the potential of inhibiting platelet aggregation (Peng, Zeng, Feng, & Wang, 2004), reducing ischemia-induced oxidative damage , improving microcirculation (Liu et al., 2007), attenuating mitochondrial dysfunction (Chen et al., 2018), and reconstructing the impaired neuronal network (Zhao, Liu, Li, Zhang, & Wang, 2019). In a Chinese randomized, double-blind trial of 573 ischemic stroke patients, NBP treatment (both intravenous and oral) was safe and could improve outcomes at the third month after stroke (Cui et al., 2013).
The kallikrein-kinin system (KKS) is a complex endogenous enzyme system taking part in the regulation of heart disease, kidney disease, inflammation, cancer, and many other diseases Emanuelia & Madeddu, 2003). Recently, it is also proved to protect against ischemic stroke (Chen et al., 2010;Zhang, Tao, Liu, & Wang, 2012). HUK can selectively dilate arterioles in the ischemic area (Nagano, Suzuki, Hayashi, & Asano, 1992), enhancing angiogenesis and neurogenesis (Stone et al., 2009), increasing regional cerebral blood flow, inhibiting apoptosis and inflammation (Xia et al., 2006), promoting glial cell migration (Lu et al., 2008), and improving neurological deficits after AIS (Ling et al., 2008). Meanwhile, edaravone (Eda) was first approved by the Japanese Ministry of Health in 2001 for the treatment of ischemic stroke. It scavenges free radicals (Higashi, Jitsuiki, Chayama, & Yoshizumi, 2006), inhibits lipid peroxidation and oxidative damage to brain cells, endothelial cells, and nerve cells (Yoshida et al., 2006), and reduces the effects of cerebral ischemia and edema (Nakamura et al., 2008), thus decreasing the tissue damage caused by acute cerebral infarction.
Since HUK ($71.5 per day) and Eda ($53.6 per day) are expensive in China, usually patients can only choose one drug in combination with NBP ($7.5 per day). Up till now, there is still no convincing evidence so we conducted this study to compare the effectiveness of HUK and Eda combined with NBP in the treatment of AIS, aiming to help healthcare givers and patients make cost-effective decisions.

| Patients
Patients aged 18-85 years who were diagnosed with AIS were en- confirmed by head CT or MRI; and (c) with the first onset and onset time less than 48 hr. Exclusion criteria included: (a) bleeding disorder or bleeding trends in latest one month; (b) incomplete hepatic and renal function; (c) medical history of peptic ulcer, hemorrhagic stroke, brain tumor, and brain trauma; (d) patients who were eligible to IV thrombolytics or mechanical thrombectomy; (e) patients whose pertinent data could not be evaluated at the time of stroke onset; or (f) incomplete follow-up or noncompliance with the study treatment.
The stroke subtypes were defined according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification system (Bamford, Sandercock, Dennis, Warlow, & Burn, 1991). The degree of carotid artery stenosis (CAS) was performed by radiologists who had more than 10 years of vascular ultrasonography experience using Philips IU 22 (Philips Healthcare), and severe stenosis was defined as 70%~99% according to the North American Symptomatic Carotid Endarterectomy Trial (NASCET) (Ferguson et al., 1999).
This study was reviewed and approved by the Ethics Committee at the Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine. Written informed consent was obtained from each participant before enrollment.

| Treatments
In this open-label study, the 165 study subjects were randomly allocated in a 2:1 ratio into two treatment groups with random allocation sequence determined by a computer-generated randomization chart. (Shijiazhuang Pharma Group) orally four times a day and basic treatment including antithrombosis, blood pressure and blood glucose control, statins, and dehydrating agents according to disease condition.

| Outcome measurements
The primary outcome in this study was 12-month independency rates of HUK therapy compared with Eda therapy using the modified Rankin Scale (mRS). Twelve-month mRS score ≤ 1 was defined as independency (Gocmen, Arsava, Oguz, & Topcuoglu, 2018). Secondary outcomes were (a) nosocomial infection rates; (b) 12-month recurrent stroke rates; and (c) factors related to the independency rate.
Twelve-month mRS scores were obtained by telephone follow-up.

| Statistical analysis
Statistical analysis was performed using the software IBM SPSS Statistics v.19 (SPSS Inc.). Baseline characteristics of the study patients were analyzed using chi-square or Fisher's exact test as appropriate for categorical variables and Student's t test or Mann-Whitney test for continuous variables. Categorical variables were reported as number or percentage; continuous variables fitting the normal distribution were expressed as mean ± standard deviation (SD), whereas median (1st to 3rd quartile, interquartile ratio) was used for nonfitting variables. In addition, binary logistic regression analysis was performed to assess factors affecting 12-month independency rate with feed-forward model. A p-value <.05 was considered statistically significant.

| Baseline characteristics of all the patients
The baseline clinical and laboratory characteristics of the study patients are summarized in Table 1. Overall, there were no statistically significant differences in patient clinical characteristics between the two study groups except that more patients had smoking history (43.6% vs. 21.8%, p = .004) and transient ischemic attack (TIA) history (25.5% vs. 0, p < .0001) in Eda group. Patients' TOAST subtypes were not quite the same in our study: 20.9% of the HUK group was small-artery occlusion (SAO) while nearly a half (43.8%) of patients in the Eda group was cardioembolism (CE, p < .0001). Moreover, the National Institute of Health stroke scale (NIHSS) scores before treatment were 2 (IQR 0-11) and 3 (IQR 2-7) for the HUK and Eda groups, respectively (p = .035), and the two groups differed significantly in low-density lipoprotein (LDL, 2.5 ± 0.9 vs. 3.1 ± 0.9, p < .0001) and homocysteine (Hcy, 14.6 ± 9.0 vs. 20.5 ± 13.5, p = .0001) concentration.
Seven patients (6.4%) in the HUK group and 5 (9.1%) in the Eda group accepted anticoagulant therapy (p = .537) based on their disease severity and eligibility.

| Secondary outcomes
No significant difference was found in nosocomial infection rates and 12-month recurrent stroke rates between the two groups (Table 2).

| D ISCUSS I ON
Butylphthalide has been widely used in Chinese AIS patients since its approval in 2002, and after that, several Chinese multicenter, open-label clinical studies demonstrated its effectiveness and safety (Cui & Li, 2006;Cui et al., 2005Cui et al., , 2008. NBP has a variety of biological activities including suppressing platelet aggregation and thrombosis (Peng et al., 2004), improving cerebral microcirculation (Liu et al., 2007;Yan, Feng, & Zhang, 1998) and reducing ischemia-induced oxidative damage ), all of which were associated with brain injury caused by ischemic stroke. However, the mechanism of NBP for the treatment of AIS is still unrevealed. One possible way was up-regulation of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1 alpha expression, which was associated with angiogenesis (Cao, Deji, Li, He, & Zhou, 2009;Liao et al., 2009 neuronal cells due to mitochondrial dysfunction (Siesjö et al., 1999).
Eda was shown to capture and reduce excessive ROS, thus preventing brain damage (Watanabe, Tanaka, Watanabe, Takamatsu, & Tobe, 2004). In 2003, Eda Acute Infarction Group reported that 30 mg Eda-treated patients had significantly lower mRS, both short-term and long-term (Edaravone Acute Infarction Study Group, 2003). Another retrospective study in 70 patients with lacunar infarction investigated patients who received 30 mg Eda twice daily for 14 days and found a significant odds ratio effect in patients that received Eda treatment (Mishina et al., 2005).
Like Eda, HUK was reported to suppress oxidative stress through tissue KKS (Chao & Chao, 2005). And like NBP, HUK could induce the expression of VEGF, leading to new blood vessels formation by transfer of kinins (Emanueli & Madeddu, 2004). Furthermore, kinin and kallidin, which were transferred from kininogen hydrolysis by HUK, could combine with bradykinin B1 receptor produced under induction of ischemic brain tissue to release NO and relax vascular smooth muscle (Lagneux, Adam, & Lamontagne, 2003;Li et al., 2008;Sangsree, Brovkovych, Minshall, & Skidgel, 2003 Pharmaceutical companies never give up exploring novel neuroprotective agents. For example, compound 10b, a hybrid of Eda and a ring-opening derivative of NBP, has recently been reported to significantly reduce the infarct volume and neurological deficits in rats, and its neuroprotective effects have been more pronounced compared with NBP, Eda, or NBP + Eda (Hua et al., 2015). Since the reason why neuroprotective agents were not recommended by 2018 AHA/ASA guidelines was that no neuroprotective agent met the quality of evidence, we should conduct more large-scale clinical trials to confirm the safety and efficacy of neuroprotective agents instead of stop using them.

| CON CLUS ION
Human urinary kallidinogenase combined with NBP can enhance AIS patients' long-term independency rate, and the effectiveness of HUK combined therapy is better than Eda. HUK combined with NBP may be a more cost-effective treatment for AIS patients.

This study was supported by Hangzhou Health Science and
Technology Plan the mechanism of human urinary kallikrein on cerebral infarction based on nitric oxide synthase system (2018A10).

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
All original data will be available when contact the correspondence author by email.