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1 Journal of Neurological Sciences [Turkish] 32:(1)# 43; , Research Article Combination Treatment of Atorvastatin and Fasudil Ameliorate Cerebral Vasospasm after Subarachnoid Hemorrhage Renguo LUO, Jie FAN, Jie DUAN, Lin FENG, Xiaohong YIN Department of Neurosurgery Affiliated Hospital North Sichuan Medical College Sichuan Nanchong, China Summary Objective: Cerebral vasospasm (CV) and early brain injury remain major causes of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). The aims of this study were to investigate whether combination treatment, with atorvastatin as an inhibitor of RhoA and fasudil as an inhibitor of Rho-kinase, prevents the CV. Methods: Atovastatin, fasudil, or combination treatment were administrated. Ischemic lesion size was measured using computed tomography on the last available scan. Angiographic narrowing was semiquantitatively assessed in patients with vasospasm. The primary end point was the occurrence of cerebral infarction or symptomatic vasospasm (SV) caused by CV. The secondary end point was clinical outcome 6 months later. Results: In the trial population, cerebral infarction(20.5% vs.27.5% vs.29.3%) and SV(20.5% vs.26.8% vs. 27.5%) were significantly lower in the combination treatment group than in fasudil and atovastatin groups (both P<0.05). No differences were found between statin, fasudil, and combination groups regarding rescue therapy intensity (P>0.05), mrs (P=0.06), and GOS (P>0.05) at 6 months. In the overall population, patients with SV had poorer clinical outcomes (P=0.05). Conclusion: Combination treatment reduces the incidence, severity and the ischemic consequences of vasospasm. Although no improvement of clinical outcome was seen in the combination population, there was a tendency for a better clinical outcome. Key words: Atorvastatin; Fasudil; Cerebral Vasospasm; Subarachnoid Hemorrhage Subaraknoid Kanama Sonrası Serebral Vazospazmı Atorvastatin ve Fasudil Kombine Tedavisi Düzeltir Özet Giriş: Anevrizmal subaraknoid kanama (SAH) sonrası gelişen serebral vazospazm (CV) ve erken beyin yaralanması morbidite ve mortalitenin ana nedenleri arasındadır. Bu çalışmanın amacı bir RHoA inhibitörü olan atorvastatin ile bir Rho-kinaz inhibitörü olan fasudilin birlikte kullanılmasının CV yi önleyip önleyemeyeceğini araştırmaktır. Yöntemler: Atovastatin, fasudil, ya da kombinasyonu tedavi olarak verildi. İskemik lezyon alanı en son olası BT tetkikinde ölçüldü. Anjigrafik daralma yarıkantitatif olarak vazospazmlı hastalarda değerlendirildi. Primer son nokta nedeni CV olan serebral infarktın varlığı ya da semptomatik vasospazmın (SV) görülmesi oldu. İkincil varış noktası ise 6 ay sonraki klinik sonuç idi. Sonuçlar: Deneme örneğinde serebral infarkt (20,5% vs.27,5% vs.29,3%) ve SV(20,5% vs.26,8% vs. 27,5%) tekli fasudil ve atorvastatin tedavi gruplarına göre kombine tedavi grubunda önemli ölçüde daha düşük bulundu. (her iki P<0,05). Statin, fasudil ve kombinasyon gruplarında kurtarma tedavisi yoğunluğu (p>0,05), mrs (p=0,06) ve GOS (p>0,05) bazında 6 40

2 ay sonrasında bir farklılık bulunmadı. Tüm denek örneğinde SV hastaları en kötü klinik sonuçları gösterdi (P=0,05). Yargı: Kombinasyon tedavisi vazospazmım iskemik sonuçlarını, görülme sıklığını ve şiddetini azaltmaktadır. Klinik sonuç olarak herhangi bir iyileştirme sonucu olamamakla birlikte daha iyi bir klinik sonuç oluşturma eğilimi vardır. Anahtar Kelimeler: Atorvastatin; Fasudil; Serebral Vazospazm; Subaraknoid Kanama INTRODUCTION Aneurysmal subarachnoid hemorrhage (SAH) is a major cause of stroke, as approximately 15% stroke cases are due to ruptured intracranial aneurysms. (7) Outcome after SAH depends on several factors, including the severity of the initial event, the peri-ictal medical management, various surgical variables, and the incidence of SAH induced complications. Cerebral vasospasm (CV) is the most frequent and unpredictable complication after SAH and leads to adverse outcomes. (15) The development of vasospasm is related to a number of pathological processes, including endothelial damage, smooth muscle cell contraction, and inflammatory reactions. (31) Recent studies suggested that Rho/Rho kinase pathway is an important target in the pathogenesis of various vascular diseases. (13,33) The G protein-coupled receptor is activated by clot-derived substances that alter the subarachnoid space after SAH and that induce the activation of RhoA followed by activation of Rho-kinase. (30) Fasudil, the Rho-kinase inhibitor, protects neurons, improves neurological function, inhibits ischemic cerebral injuries (24), and prevents and reverses CV. (37) Some study (12,20) indicate that fasudil significantly improves the clinical outcome in SAH patients by preventing and reversing CV. However, the other studies (16,38) do not support this finding. Statins, inhibitors of the 3-hydroxy-3- methylglutarylcoenzyme A (HMG-CoA) reductase, are widely used as cholesterollowering drugs. Studies have shown that high concentrations of statins enhance endothelial function (11), and protect against stroke, (10) by mechanisms involving inhibition of RhoA. (35) These so-called pleiotropic effects of statins are thought to be responsible for the observed improvement in flow-mediated vasodilation, (9) increased numbers of circulating endothelial progenitor cells, (9) and perhaps the reduction in vascular inflammation. (22) However, the full effects of the statins on cerebral vasospasm after SAH remain controversial. (14) Although neither fasudil nor statin show remarkable effects in preventing cerebral vasospasm after SAH, a combination use of these drugs might effectively suppress the Rho-kinase pathway. Therefore, in this prospective, randomized, efficacy study, atorvastatin and fasudil were used to examine whether combination therapy prevent CV and improve clinical outcomes after SAH onset. MATERIAL AND METHODS Participants A total of 128 patients were enrolled at our department between April 2010 to April All patients underwent SAH surgery and randomly assigned (1:1:1) to atorvastatin group, fasudil group, and combination group with atorvastatin and fasudil. Patients in all groups were admitted to the stroke intensive care unit. Treatment after surgery included intracranial pressure control and cisternal drainage. A computer-generated randomisation code was used to randomise patients. Inclusion criteria were radiological confirmatory evidence of an aneurysmal subarachnoid haemorrhage (by CT angiography (CTA), magnetic resonance angiography (MRA), or digital 41

3 subtraction angiography (DSA), age years, presentation less than 72h from ictus, and first surgery patients. Exclusion criteria were patient taking statin therapy at presentation, pregnancy or of child-bearing potential, no reasonable prospect of survival, known renal or hepatic impairment, patient not fully independent before bleed, strong suspicion of drug or alcohol misuse, patient taking contraindicated medication (amiodarone, amlodipine, verapamil, or potent CYP3A4 inhibitors), patient taking warfarin-type drugs, metabolic disease(such as diabetes), or suspected additional life-threatening disease. We obtained ethics approval from the Ethics Committee of our hospital. Informed written consent from were obtained from all patients or their legal representative. Procedures Patients started treatment as soon as possible within 72 h of the ictus, for a period of up to 3 weeks. Patients were randomized to the atorvastatin (LIPITOR, Pfizer, USA), fasudil (ERIL Injection, Asahi Kasei Corporation, Japan), and combination therapy group, and treatment with atorvastatin, fasudil, or combination therapy was started within 24 hours after aneurysm surgery and continued for 21 consecutive days. Fasudil 30mg was prescribed for intravenous administration three times a day. Atorvastatin was administrated 20mg a day. The drugs were stopped when patients were discharge, and continuation of trial medication thereafter was deemed unnecessary. Commencement of study drug was not dependent on aneurysm treatment. Outcomes The patients were followed daily for one month after SAH. The primary outcome was cerebral infarction or symptomatic vasospasm (SV) caused by CV. Secondary outcomes were the assessment of delayed ischaemic deficit requiring rescue therapy, modified Rankin Scale (mrs) scores, and Glasgow Outcome Scale(GOS) at 6 months. CV was defined as a newly detected, low-density area identified by CT when all other causes of formation of low density areas, for instance, low-density areas around the hematoma, were excluded. (27) We defined SV as a documented arterial vasospasm consistent with new neurologic deterioration, either transient or permanent, occurring on days 4 to 30 after SAH onset. (27) CT was performed whenever clinical deterioration was noted. The presence of arterial vasospasms was diagnosed by mean arterial velocities >120 cm/s in the middle or anterior cerebral arteries or >90 cm/s in the basilar arteries measured using transcranial Doppler ultrasonography. (21) Patients with these findings typically underwent CT angiography to confirm the presence of vasospasms. (28) We confined reporting of adverse events in this study to serious adverse events and those that we deemed to be related to trial medication. Statistical analysis Statistical analyses were performed using Statistical Analysis Software versions 8.2 and 9.1 (SAS Institute Inc., Cary, North Carolina, USA). Characteristics at baseline were compared using the Wilcoxon rank sum test. An X 2 test was used to compare class variables. The inhibitory effects of statin and fasudil on cerebral infarction and SV were evaluated by multivariate logistic regression analysis. The significance level was set at P < RESULTS Clinical and Demographic Data The clinical and demographic data of the 120 remaining patients are shown in Table 1. There were no significant differences in age, sex, aneurysm location, Fisher classification of CT findings, mean time from onset of SAH to treatment, and treatment modality between the three groups(all P>0.05). Severe SAH (WFNS grades IV and V) was present in 9 patients in the combination group, 8 in atorvastatin group and 9 in fasudil group, and the 42

4 WFNS grades did not differ significantly between the 3 groups( P>0.05). Cerebral infarction and SV The frequency of cerebral infarction and SV was evaluated in all patients. The incidence of cerebral infarction caused by CV occurred in 8/39 patients (20.5%) in combination group, 11/40 (27.5%) in fasudil group, and 12/41 (29.3%) in atorvastatin group, which is significantly lower in the combination treatment group than in the other two groups (20.5% vs. 27.5% vs. 29.3%, P< 0.05). Fewer patients had cerebral infarction in the combination group than in the atorvastatin or fasudil group (10.3 vs. 19.5% vs.17.5%, P<0.05). Multivariate analysis revealed an adjusted odds ratio of 0.37 (95% confidence interval , P=0.33) for cerebral infarction by combination treatment. The frequency of SV in all 120 patients also was significantly lower in the combination group than in atorvastatin or fasudil group (20.5% vs. 26.8% vs. 27.5%, P=0.048; Table 2). Multivariate analysis revealed an adjusted odds ratio of 0.46 (95% confidence interval , P=0.044) for SV inhibition by combination treatment. Clinical Outcomes Use of extended hypervolaemic therapy differed between 3 groups (P= 0.03) (Table 3), although the duration of extended hypervolaemic therapy was the same (8 days in all 3 groups). The need for hypertensive inotropic, angioplasty, and steroids support showed no differences between 3 groups (all P>0.53). Clinical outcomes were assessed using the mrs. The percentages of patients with a favorable outcome (mrs=0-2) did not differ significantly between the three groups (63.4% vs. 67.5% vs. 69.2%, P= 0.33) on 6 months after SAH onset (Table 3). The effect of the occurrence of SV on the clinical outcome also was examined. Fewer patients with SV had a favorable outcome compared with those without SV on 6 months (60% vs. 70%, P = 0.05) after the onset of SAH (Table 4). The occurrence of SV did not affect the outcomes in patients in the combination group on 6 months after SAH onset, but patients in the atorvastatin and fasudil group with SV had significantly poorer outcomes on 6 months (Table 4). Adverse events were observed in 6/39(15.4%) patients in the combination group, in 5/40(12.5%) in fasudil group and in 6/42(14.3%) in atorvastatin group, with no significant difference between the three groups (P>0.05). Table 1 Basic clinical and demographic characteristic of patients Atorvastatin Fasudil group Combination group group Age, years 51±6.2 50±5.9 52±6.3 Male sex n(%) 23(56.1) 22(55.0) 24(61.5) WFNS SAH grade n(%) I 18(43.9) 17(42.5) 16(41.0) II 8(19.5) 8(20.0) 7(17.9) III 7(17.1) 6(15.0) 7(17.9) IV 3(7.3) 5(12.5) 4(10.2) V 5(12.2) 4(10.0) 5(12.8) Fisher grade n (%) 1 5(12.2) 4(10.0) 6(15.4) 2 8(19.5) 8(20.0) 7(17.9) 3 12(29.3) 13(32.5) 11(28.2) 4 16(39.0) 15(37.5) 15(38.5) Location of aneurysm n (%) 43

5 30.8Anterior 12(29.3) 11(27.5) 12(30.8) communicating Posterior 9(22.0) 8(20.0) 8(20.5) communicating Internal carotid 4(9.8) 4(10.0) 4(10.3) Middle cerebral 8(19.5) 9(22.5) 7(17.9) Posterior circulation 4(9.8) 4(10.0) 5(12.8) Other 3(7.3) 4(10.0) 3(7.7) Mean time from onset of 2.1± ± ±0.4 SAH to treatment, days Treatment modality n(%) Clipping 13(31.7) 12(30.0) 11(28.2) Coiling 23(56.1) 24(60.0) 24(61.5) Clipping+ coiling 3(7.3) 2(5.0) 3(7.7) Other 2(4.9) 2(5.0) 1(2.6) Table 2 Incidence of Cerebral infarction and Symptomatic vasospasm Atovastatin Fasudil Combination P value Odds ratio P value group(n=41) group(n=40) group(n=39) (X 2 test) (95%CI) Cerebral infarction n (%) 8/41(19.5) 7/40(17.5) 4/39(10.3) ( ) Symptomatic vasospasm n (%) 11/41(26.8) 11/40(27.5) 8/39(20.5) ( ) Atorvastatin group(n=41) Table 3 Clinical outcome measures Fasudil group (n=40) Combination group(n=39) P value Rescue therapy n (%) Extended 10(24.4) 9(22.5) 5(12.8) 0.03 hypervolaemic therapy Inotropic support 8 (19.5) 9 (22.5) 8 (12.3) 0.55 Angioplasty 3 (7.3) 4(10.0) 3 (7.7) 0.46 Steroids 7 (17.1) 8 (20.0) 7 (17.9) 0.67 mrs at 6 months n (%) 26 (63.4) 27 (67.5) 28 (71.2) 3-4 n (%) 11 (26.8) 10 (25.9) 8 (20.5) 5-6 n (%) 5 (12.2) 5 (12.5) 3 (7.8) GOS at 6 months n (%) 8 (19.5) 9 (22.5) 6 (15.4) 4 n (%) 4 (9.8) 4 (10.0) 3 (7.7) 5 n (%) 29 (70.1) 29 (72.5) 30 (76.9) 44

6 Table 4 Favorable Clinical outcomes in patients with or without SV on six months after the onset of SAH With SV Without SV P value Total patients 18/30 (60.0) 63/90 (70.0) 0.05 Atorvastatin group 6/11 (54.5) 20/30 (66.7) 0.04 Fasudil group 6/11 (54.5) 21/29 (72.4) 0.02 Combination group 6/8 (75.0) 22/31 (71.0) 0.35 Note: Scores of 0-2 on the mrs were considered to be favorable outcomes. DISCUSSION In our study, combination treatment significantly reduced vasospasm incidence, vasospasm severity, and vasospasminduced brain ischemia as assessed by CT. The results of this randomized study confirm that combination treatment of atorvastatin and fasudil significantly inhibits CV and SV after SAH onset. The present study also shows that combination treatment affords benefit tendency in clinical outcomes after acute aneurysmal SAH, comparing to atorvastatin or fasudil treatment. The Rho/Rho-kinase pathway has been considered as playing an important role in sustained contraction during cerebral vasospasm after aneurysmal SAH. (26) After SAH, it is thought that the Rho/Rho-kinase pathway is activated (5) by many cytokines. Rho-kinase activates MLC kinase resulting (39) in sustained contraction of smooth muscle cells. Inhibition of the Rho/Rho-kinase pathway is therefore considered to represent potential therapy preventing cerebral vasospasm. Fasudil is a selective inhibitor of Rho-kinase, mediating an increase in the myocardial NO content. (36) In addition, fasudil is indicated to have pleiotropic effects for cerebral vasospasm, such as protection from endothelial cell damage and antiinflammatory effects. (17) Several clinical studies have shown that fasudil suppresses cerebral vasospasm and the associated cerebral ischemic symptoms; however, it has also been demonstrated as failing to show sufficient effect in terms of preventing cerebral vasospasm as single use. (29) On the other hand, statins were isolated about 40 years ago as a molecules could inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, reducing serum cholesterol concentrations. (24) Statins that decreased serum cholesterol and reduced heart disease and stroke were identified. (1) In addition to the reduction of cholesterol synthesis, decreased synthesis of farnesylpyrophosphate and geranylgeranylpyrophosphate might also occur. These compounds bind to proteins such as heterotrimeric G proteins (Ras, Rho, and others) in a process called protein prenylation. (2,3) RhoA was shown to be inhibited by statin. (23) Thus, statins might affect many cellular signal transduction pathways. In experimental studies, statin was shown to ameliorate cerebral vasospasm, improve cerebral autoregulation, and reduce vasospasmrelated injury, by upregulating enos. (18) But, effects of statins on cerebral vasospasm and neurological outcome after SAH were still controversial. (5,8) Therefore, we speculated that a combination treatment of statin, for RhoA and enos, and fasudil, for Rho-kinase, might suppress the Rho/Rho-kinase pathway more intensely than the single use of statin or fasudil alone. In animal study, results indicate that combination treatment could extensively prevent cerebral vasospasm due to the synergic effect of combining pitavastatin and fasudil on the Rho/Rho-kinase pathway and on enos. (19) The decreased activity of Rho-kinase induced by fasudil 45

7 may play a role as a feedback to reduce RhoA activity. (32) On the other hand, the combination group showed a statistically significant improvement in clinical outcome after acute aneurysmal SAH, suggesting that a combination of statin and fasudil could achieve a synergic effect. Its reduction in activity by statin may be increased by the depression of RhoA activity, and its reduction in activity by fasudil may due to the direct effect of fasudil on Rho-kinase. Study showed a reduction of enos expression in the endothelial cells after SAH. The enos expression was enhanced in the combination group and the statin group, but its expression was not enhanced in the fasudil group. (38) Therefore, despite the incomplete effects of statin or fasudil alone in the prevention of cerebral vasospasm, a combination treatment can achieve adequate effects in this clinical study. In our study, the combination treatment can greatly induce remarkable amelioration of cerebral vasospasm, which may suggest the Rho/Rho-kinase pathway was one of the main pathways inducing sustained contraction of smooth muscle cells during cerebral vasospasm. Although the combination treatment reduced the development of SV and cerebral infarction in this study, we also found that clinical outcomes in patients in the combination and control groups were not different. Study suggests that cerebral infarction after SAH contributes to poor outcome. (4) A placebo group was not included due to the ethical implications of withdrawing antispasmodic therapy in patients with SAH in this study. The incidences of cerebral vasospasm in fasudil and statin groups are low (17.5% and 19.5%, respectively), comparing with the incidences of 38%-44% in the control groups in previous studies. (27,34) All three groups were administrated of antispasmodic drugs to prevent further worsening of outcomes when SV occurred in each group. Therefore, the clinical outcomes in patients in the fasudil or statin group in this study were so favorable that no significant differences were detected between the three groups. We only found a benefit tendency in clinical outcomes after combination treatment (mrs comparison, p=0.06) We also found that patients without SV had more favorable outcomes compared with patients with SV, and clinical outcomes in the combination treatment group were affected by the development of SV (Table 4). Furthermore, among patients with SV, those in the combination group had more favorable outcomes than those in the fasudil or statin group, which may suggest that combination treatment inhibits the development of SV after SAH and also prevents worsening of SV through cerebral vasospasm inhibition and multiple pleiotropic effects. In conclusion, the results of this study show that combination treatment inhibits the development of SV and CV after SAH onset. The may be due to inhibition of the Rho-kinase pathway, enhancement of enos expression, neuroprotective and multiple pleiotropic effects. We also showed that combination treatment can be safely used to prevent vasospasm after the onset of SAH with no significant adverse effects. Further well-designed controlled trials involving a larger number of patients are required to confirm the effect of atorvastatin and fasudil combination treatment on the clinical outcomes of patients with SAH. Correspondence to: Xiaohong Yin Received by: 19 June 2014 Revised by: 05 January 2015 Accepted: 05 January

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