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Safety and efficacy of herbal medicine for acute intracerebral hemorrhage (CRRICH): a multicentre randomised controlled trial

Objective To evaluate the safety and efficacy of removing blood stasis (RBS) herbal medicine for the treatment of acute intracerebral haemorrhage (AICH) within a 6-hour time window. Study design A randomised, multicentre, double-blind, placebo-controlled study performed in 14 hospitals in China. Participants and interventions Patients with AICH were randomly assigned to receive a placebo, the ICH-1 (Intracerebral Haemorrhage) formula (eight herbs, including the RBS herbs hirudo and tabanus) or the ICH-2 formula (six herbs without the RBS herbs hirudo and tabanus) within 6 hours of ICH onset. Outcomes The primary safety outcome was the incidence of haematoma enlargement at 24 hours and at 10 days after treatment. The secondary outcome was the incidence of poor prognosis (mortality or modified Rankin Scale score ≥5) assessed at 90 days after symptom onset. Results A total of 324 subjects were randomised between October 2013 and May 2016: 105 patients received placebo; 108 patients received the ICH-1 formula; and 111 patients received the ICH-2 formula. The incidence of haematoma enlargement at 24 hours was 7.8% in the placebo group, 12.3% in the ICH-1 group and 7.5% in the ICH-2 group; the incidence of haematoma enlargement on day 10 was 1.1% in the placebo group, 1.1% in the ICH-1 group, and 3.1% in the ICH-2 group, with no significant differences among the groups (P>0.05). The mortality rates were 3.8% in the placebo group, 2.8% in the ICH-1 group, and 0.9% in the ICH-2 group; the incidences of poor prognosis were 7.1% in the placebo group, 6.0% in the ICH-1 group and 4.8% in the ICH-2 group at 3 months, with no significant differences among the groups (p>0.05). However, the overall frequency of treatment-emergent adverse events in the ICH-1 group (12.1%) was higher among the three groups (5.8% and 2.8%, respectively, p<0.05). All three cases of serious adverse events were in the ICH-1 group. Conclusions Ultra-early administration of ICH-1 formula for AICH patients did not exert significant beneficial effects on clinical outcomes but increased the risk of bleeding, which probably resulted from the inclusion of RBS herbal medicines in ICH-1. Trialregistration number NCT01918722.

the placebo group, 1.1% in the ICH-1 group, and 3.1% in the ICH-2 group, with no significant differences between the groups (P>0.05). The mortality rates were 3.8% in the placebo group, 2.8% in the ICH-1 group, and 0.9% in the ICH-2 group,and the incidences of poor prognosis were 7.1% in the placebo group, 6.0% in the ICH-1 group, and 4.8% in the ICH-2 group at 3 months, with no significant differences between the groups (P>0.05). However, the overall frequency of adverse events in the ICH-1 group (12.1%) was higher than that in the placebo and ICH-2 groups (5.8% and 2.8%, respectively, P<0.022). The 3 cases of serious adverse events were all in the ICH-1 group. approximately 15% [1] of all acute strokes which is the third most common cause of death in most Western countries, following coronary heart disease and cancer. [2,3] HICH has been the leading cause of death in China in recent years, [4] being responsible for approximately one-third of the total number of deaths from stroke worldwide. [5] Available specific medical and surgical treatments do not improve prognosis substantially, [7,8] and intracerebral hemorrhage (ICH) still accounts for a higher proportion of stroke in Chinese people compared with that in white populations. [6] Therefore, traditional Chinese medicine (TCM) physicians in China widely use removing blood stasis (RBS) herbs for the treatment of HICH, which was proven previously to provide some benefit for HICH patients in a meta-analysis. [9] RBS herbal medicine maybe promotes hematoma absorption and improves recovery of neurological impairment [10,11] , which is already included by a guidelines for the management of spontaneous intracerebral hemorrhage [24] .However, sufficient evidence of long-term benefits and risks of superacute stage is lacking. RBS herbals, such as hirudo, that act as a thrombin inhibitor with anticoagulant pharmacological effects, may cause re-bleeding risk in clinical practice. [12] A total of 90% of hematoma growth, which plays an important role in prognosis, occurs within a 6-h time window. [13] Therefore, confirmation of the safety and efficacy of RBS herbal medicines in the treatment of early-stage HICH is urgently needed.
We performed a retrospective study that demonstrated that RBS herbal medicine for HICH patients was safe and effective. [14] However, little additional evidence of the safety and efficacy of this treatment exists.
We designed a multicenter, three-group, prospective, randomized, double-blinded and placebo-controlled clinical trial on hematoma enlargement in HICH patients treated with RBS herbal medicine within a 6-h time window from symptom onset (CRRICH) to evaluate its safety

Study Design
The CRRICH trial was a multicenter, randomized, double-blinded, placebo-controlled, parallel-group study to assess the safety and efficacy of RBS herbal medicine in HICH patients. This trial is registered at clinicaltrials.gov (NCT01918722), and the research design proposal has been published. [15] Participants Patients with a Glasgow Coma Scale [GCS] score ≥6 were enrolled if they were ≥18 years of age and had a clinical diagnosis of ICH confirmed with computed tomography within 6h of onset. All participants signed an informed consent form. Patients who suffered secondary ICH resulting from trauma, brain tumor, blood diseases, arteriovenous malformation or aneurysm were excluded. A detailed patient history was recorded. S3 Table in the online Data Supplement provides a full list of study inclusion and exclusion criteria during the screening period.

Randomization and Blinding
Patients were randomized to receive placebo, ICH-1, or ICH-2 in

Primary and Secondary Outcomes
The hematoma enlargement rate, which is a radiographic outcome, was the primary outcome in the study. Hematoma enlargement was operationally defined as an increase in hematoma volume of >33% or 12.5 ml as measured on image analysis on the 24-h CT compared with the baseline CT scan. Hematoma volume was measured using the ABC/2 Coniglobus formula [16] at 24 h after onset. The same measurement was performed on days 10-14 as a primary outcome measure. The following secondary end points were assessed: change in NIHSS score at 3 months; mortality at 3 months; and poor prognosis rate, which was defined as the proportion of patients who died or were severely disabled based on an mRS score ≥5 at 3 months. [17] Safety data focused on treatment-emergent AEs (TEAEs). TEAEs were defined as AEs that first occurred or worsened (increased in severity) after the first dose of study drug. The

Statistical Analyses
All reported efficacy analyses were predefined and based on full were assigned a Barthel index score of 0 and mRS score of 6, and the last recorded NIHSS score was carried forward. Hematoma enlargement rate at 24 h (primary end point), the rate of poor prognosis and the incidence of total adverse events between the three groups were analyzed using the χ 2 test. The hematoma enlargement rate on days 10-14 (primary end point) and mortality between the three groups were compared using Fisher's exact test. Mean differences in NIHSS score between groups were analyzed using one-way ANOVA. Demographic characteristics and serious AEs were summarized using descriptive statistics.
Power and sample size were determined using relative hematoma volume at 24 h as the primary outcome variable. A total of 285 participants were required at the final follow-up to provide 90% power with a two-sided alpha level at 0.05 to test the primary effectiveness, which was calculated using the PASS (Power Analysis and Sample Size)  were needed for primary outcome randomization assuming a dropout rate of 20%, with 120 patients per treatment arm. The statistical package STATA (version 13.1) was used for all statistical analyses.

Patient and Public Involvement statement
While we put forward the research question and outcome measures, the patients' priorities, experience, and preferences were fully considered by a survey. Patients were involved in the recruitment to and conduct of the study.The results would be disseminated to study participants by posting a letter. The CRRICH trial was a randomised controlled trials, patients themselves had fully assessed the burden of the intervention.
Patient advisers should also be thanked in the contributorship statement/acknowledgements.

Funding statement and Ethical statement
The   ICH, one patient for surgery within 24 h and one patient withdrew consent). Therefore, only the 319 participants who underwent randomization and received study medication were included in the FAS and safety analysis set based on ITT. Twenty-three patients in the FAS did not completely adhere to the protocol and dropped out during the treatment period. A total of 296 patients were included in the PPS (see Figure 1 for patient flow diagram). The mean dropout rate was <8%. Table 1 presents the demographics and baseline characteristics of the FAS, which were well balanced with no significant between-group differences. The mean age was 62.5±12.7 years (mean±SD, n=319); 65.5% of the patients were male, and 98.7% were ethnic Han. The median GCS score was 15 (range, 3 to 15), and the mean NIHSS score was 8 (range, 0 to 47). The deep gray matter was involved in 93.4% of cases, and the lobar regions were involved in 6.6% of cases. The baseline characteristics of the three treatment groups were similar. The mean volume of the ICH at baseline for all patients was 11.0 ml (range, 0.3 to 86.7 ml), which was similar in the three groups (

Primary Outcome (Radiographic Outcomes)
A total of 319 baseline CT scans and 315 CT scans at 24 h were available for analysis. The volume enlargement rates of ICH were 7.8% in the placebo group, 12.3% in the group ICH-1 and 7.5% in the group ICH-2. There was no significant difference between the three groups in enlargement rates of ICH on days 10-14 day were 1.1% in the placebo group, 3.1% in the group ICH-2 and 1.1% in the group ICH-1. There was also no significant difference between the three groups in the enlargement rate of the hematoma volume on days 10-14 (P=0.625) ( Table 2).

Secondary Outcomes (Clinical Outcomes)
Mortality at 3 months was approximately 2.6% in the three groups (Table 2, Figure 2). Poor prognosis (i.e., the proportion of patients who died or were severely disabled, mRS ≥5) did not differ significantly between the three groups ( Table 2). The distributions of outcomes on the modified Rankin scale ( Figure 3) were similar between the three groups.
The differences in the NIHSS scores at 3 months between the three groups were not significant ( Table 2).

Figure 2
Kaplan-Meier Survival Curves. Group placebo with 4 cases dead, respectively at day 4,5,8,9; Group ICH 1 with 3 cases dead, respectively at day 4,5,9; Group ICH 2 with 1 case dead at day 3.A possible small benefit of treatment with ICH 1,ICH 2 was evident from 10 to 90 days after treatment. evaluates global disability and handicap. Scores range from 0 (no symptoms or disability) to 6 (death). There were no significant differences between the three groups.    (Table 2). These data suggest that RBS treatment for ICH patients within 6 h of symptom onset is a safety concern. However, no data were available to suggest a mechanism for this effect.

Adverse
This result contrasts with the clinical benefit demonstrated in a previous meta-analysis [9] , which included 9 randomized-controlled bleeding or intracerebral re-bleeding) counteracted the benefit to some extent.
The primary outcome of the rate of hematoma enlargement, which is an independent prognostic determinant of mortality and poor prognosis, [17] was also used to assess safety. The hematoma enlargement rates at 24 h in the three groups in the CRRICH trial (placebo, ICH-2, and ICH-1) were 7.8%, 7.5%, and 12.3%, respectively, which were not significantly different ( Table 2). The mean hematoma enlargement rate was only 9.2%, which was far lower than the mean level of 18-30%. [27] The main reasons of lower hematoma expansion incidence may include the following: (1) the investigators' inclination toward surgery in HICH patients with large hemorrhage volumes within 24 h of onset, which resulted in the exclusion of patients susceptible to re-bleed in the study; and (2) patients with terrible and unstable conditions who were prone to hematoma expansion were excluded by investigators privately for fear that deterioration risk would rise during study.
Hematoma growth is an independent determinant of death and disability. [18] Therefore, hematoma expansion may be an attractive therapeutic target. [19,20] However, no proven specific therapies or  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   21 treatments exist to prevent hematoma expansion or improve outcomes after ICH. Sufficient evidence-based medical research to prove that mini-traumatic surgeries and the present available medicines provide ideal treatment is lacking. [7,8] Herbal medicine in China has been used to treat ICH for at least 2,000 years, and RBS herbal medicines are listed in the 2010 Chinese Pharmacopeia. [21] Dr. Si reported the earliest available literature on HICH with RBS herbal medicine therapy in 1981. [22] Another RBS medicine, Naoxuekang capsule, was approved as routine treatment for HICH patients by the Chinese state food and drug administration [21] .

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Introduction
Acute intracerebral hemorrhage accounts for approximately 15% [1] of all acute strokes; stroke is the third most common cause of death in most Western countries, following coronary heart disease and cancer. [2,3] AICH has been the leading cause of death in China in recent years [4] and is responsible for approximately one-third of the total number of deaths from stroke worldwide. [5]  Available specific medical and surgical treatments do not improve prognosis substantially, [6,7] and AICH still accounts for a higher proportion of stroke in Chinese people than in white populations. [8] Therefore, traditional Chinese medicine (TCM) physicians in China widely use removing blood stasis (RBS) herbs for the treatment of AICH.
Meta-analysis showed that RBS herbal therapy for AICH could improve the neurological function deficit, reduce the volume of hematoma and perihematomal edema, and lower the mortality rate and dependency. [9] It is possible that RBS herbal medicine promotes hematoma absorption and improves the recovery of neurological impairment [10,11] ; RBS herbal medicine has already been included by guidelines for the management of spontaneous ICH. However, sufficient evidence of long-term benefits and risks of superacute stage is lacking. RBS herbals, such as Hirudo nipponica Whitman, which acts as a thrombin inhibitor with anticoagulant pharmacological effects, may cause rebleeding risk in clinical practice. [12] A total of 90% of hematoma growth, which plays an important role in prognosis, occurs within a 6-h time window. [13] Therefore, a confirmation of the safety and efficacy of RBS herbal medicines in the treatment of early-stage AICH is urgently needed.
We performed a retrospective study that demonstrated that RBS herbal medicine for AICH patients was safe and effective. [14] However, little additional evidence of the safety and efficacy of this treatment

Study Design
The CRRICH trial was a multicenter, randomized, double-blinded, placebo-controlled, parallel-group study to assess the safety and efficacy of RBS herbal medicine in AICH patients. This trial is registered at clinicaltrials.gov (NCT01918722), and the research design proposal has been published. [15] Participants

Randomization and Blinding
Patients were randomized to receive placebo, ICH-1, or ICH-2 in    were reported during each visit. Participants were randomly assigned to one of three treatment groups at a ratio of 1:1:1 during double-blinded treatment.

Primary and Secondary Outcomes
The incidence of hematoma enlargement, which was defined as the percentage of participants experienced hematoma enlargement, was the primary outcome of the study. Hematoma enlargement was operationally defined as an increase in hematoma volume of >33% or 12.5 ml, as measured on image analysis on the 24-h CT compared with the baseline CT scan. Hematoma volume was measured using the ABC/2 Coniglobus formula [16] at 24 h after onset. The same measurement was performed on days 10-14 as a primary outcome measure. The following secondary end points were assessed: change in NIHSS score at 3 months; mortality at 3 months; and poor prognosis rate, which was defined as the proportion of patients who died or were severely disabled based on an mRS score ≥5 at 3 months. [17] Safety data focused on treatment-emergent AEs (TEAEs).

Statistical Analyses
All

Patient and Public Involvement Statement
While we put forward the research question and outcome measures, the patients' priorities, experience, and preferences were fully considered by a survey. Patients were involved in the recruitment to and conduct of the study. The results would be disseminated to study participants by posting a letter. The CRRICH trial was a randomized controlled trial, and the patients themselves had fully assessed the burden of the intervention.
Patient advisors should also be thanked in the contributorship statement/acknowledgments.

Funding and Ethical Statements
The

Study Population
Patient recruitment began in October 2013, and the study ended in Twenty-three patients in the FAS did not completely adhere to the protocol and dropped out during the treatment period. A total of 296 patients were included in the PPS (see Figure 1 for patient flow diagram).
The mean dropout rate was <8%. Fourteen percent of patients were treated within 3 h after symptom onset.
Two patients were treated outside of the 6-h time window. The timing of the treatment was similar in the three groups (Table 1).  * The values are expressed as counts and % within group, and plus-minus values are the means±SD. Percentages may not total 100 because of rounding. GCS, Glasgow Coma Scale (Scores range from 15 (normal) to 3 (deep coma)); NIHSS, National Institutes of Health Stroke Scale (Scores range from 0 (normal) to 42 (coma with quadriplegia)); BP, blood pressure.

Primary Outcome (Radiographic Outcomes)
At 24 h, a total of 319 baseline CT scans and 315 CT scans were available for analysis. The incidence of hematoma enlargement were 7.8% in the placebo group, 12.3% in the ICH-1 group and 7.5% in the ICH-2 group. There was no significant difference in the incidence of hematoma enlargement at 24 h among the three groups (P=0.409). The incidence of hematoma enlargement on days 10-14 were 1.1% in the placebo group, 3.1% in the ICH-2 group and 1.1% in the ICH-1 group.
There was also no significant difference among the three groups in the incidence of hematoma enlargement on days 10-14 (P=0.625) ( Table 2).

Secondary Outcomes (Clinical Outcomes)
Mortality at 3 months was approximately 2.6% in the three groups (Table 2). Poor prognosis (i.e., the proportion of patients who died or were severely disabled, mRS ≥5) did not differ significantly among the three groups. It is the least patients of poor prognosis occurred in the ICH-2 treatment, but no statistical difference compared with placebo  Table 2). The distributions of outcomes on the modified Rankin Scale (Figure 2) were similar among the three groups. The differences in the NIHSS scores at 3 months among the three groups were not significant (Table 2).

Figure2
Clinical Outcome at 90 Days According to the Modified Rankin Scale. The modified Rankin Scale evaluates global disability and handicap. Scores range from 0 (no symptoms or disability) to 6 (death). There were no significant differences among the three groups.

Adverse Events
There were altogether 35 AEs reported by investigators, of which 22 AEs may have been TEAEs. All the TEAEs occurred during the double-blinded treatment period (within 2 weeks). TEAEs occurred in 13 (12.1%) ICH-1-treated patients, 3 (2.8%) ICH-2-treated patients and 6 (5.8%) placebo-treated patients. The patterns of TEAEs were similar in the 3 groups, but the overall frequencies of TEAEs among the three groups were significantly different (P=0.022), as shown in Table 2. And the paired comparisons showed that the test for difference in the overall TEAEs rate between group ICH-1 and group ICH-2 yields a p-value of 0.029. Table 3 shows the TEAEs that occurred during the study. The most frequent event was diarrhea, and its incidence was similar in the 3 treatment groups . Treatment was discontinued because of TEsAEs in 3    The values are expressed as n/N (%) within group. All adverse events are listed in order of frequency from the overall study period (90 days); adverse events were collected from investigator reports. The principal investigator at each site determined whether an adverse event or serious adverse event was related to the intervention. TEAEs, treatment-emergent adverse events. *indicates TEAEs.

Discussion
RBS administration within 6 h after the symptom onset of ICH did not significantly reduce hematoma growth and failed to improve survival or functional outcome at 90 days. Conversely, compared to the ICH-2, the incidence of TEAEs showed an increasing trend in the ICH-1 group (P=  Table   2). These data suggest that RBS treatment for ICH patients within 6 h of symptom onset is a safety concern. However, no data were available to suggest a mechanism for this effect.
As can be seen from the table 2, ICH-2 treatment was least vulnerable to poor prognosis and TEAEs. Therefore, ICH-2 formula would have the potential to be an effective herbal drug for ICH patients of superacute stage.

This result contrasts with the clinical benefit demonstrated in a
previous meta-analysis [9] , which included 9 randomized controlled clinical trials with 798 individuals, demonstrating that RBS therapy for acute ICH reduced brain hematoma and cerebral edema volumes, improved the neural function and reduced the mortality and disability rates with fewer TEAEs. The present trial revealed significant heterogeneity compared with the studies included in the meta-analysis, possibly because of differences in the intervention time window. This research is the first prospective, multicenter, randomized, double-blind, prior studies that examined these effects within a 24-h or later intervention time window. Earlier RBS administration increased the risk of bleeding events (e.g., gastrointestinal bleeding or intracerebral rebleeding), which counteracted the benefit to some extent.
The primary outcome of the incidence of hematoma enlargement, which is an independent prognostic determinant of mortality and poor prognosis, [18] was also used to assess safety. The incidence of hematoma enlargement at 24 h in the three groups in the CRRICH trial (placebo, ICH-2, and ICH-1) were 7.8%, 7.5%, and 12.3%, respectively, and they were not significantly different ( Table 2). The mean incidence of hematoma enlargement was only 9.2%, which was far lower than the mean level of 18-30%. [18] The main reasons for lower hematoma expansion incidence may include the following: (1)  Hematoma growth is an independent determinant of death and disability. [19] Therefore, hematoma expansion may be an attractive therapeutic target. [20,21] However, no proven specific therapies or treatments exist to prevent hematoma expansion or improve outcomes after ICH. Sufficient evidence-based medical research to prove that mini-traumatic surgeries and the present available medicines provide ideal treatment is lacking. [6,7] Herbal medicine in China has been used to treat ICH for at least 2,000 years, and RBS herbal medicines are listed in the 2010 Chinese Pharmacopeia. [22] Dr. Si reported the earliest available literature on AICH with RBS herbal medicine therapy in 1981. [23] Another RBS medicine, Naoxuekang capsule, was approved as routine treatment for AICH patients by the Chinese State Food and Drug Administration [22] (Naoxuekang capsule, register No. Z10960009). RBS herbal medicines have been studied in AICH patients in more than 200 clinical studies over the past 30 years, and they have gradually come to be considered a regular therapy. [24] AICH management guideline in China cited this treatment. [25] However, safety evidence in the early stage of AICH is lacking. Some studies demonstrated that 30% of ICH patients suffered continued bleeding within 6 h of onset [26] . Some research has provided low-level evidence of the safety of RBS herbal medicine for superacute cerebral hemorrhage, but whether RBS herbal medicine administration in    1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  removing blood stasis (RBS) herbs hirudo and tabanus) or the ICH-2 formula (6 herbs without the RBS herbs hirudo and tabanus) within 6 h of ICH onset. Outcomes: The primary safety outcome was the incidence of hematoma enlargement at 24 h and at 10 days after treatment. The secondary outcome was the incidence of poor prognosis (mortality or modified Rankin Scale (mRS) score ≥5) assessed at 90 days after symptom onset.
Results: A total of 324 subjects were randomized between October 2013 and May 2016: 105 patients received placebo; 108 patients received the ICH-1 formula; and 111 patients received the ICH-2 formula. The incidence of hematoma enlargement at 24 h was 7.8% in the placebo group, 12.3% in the ICH-1 group, and 7.5% in the ICH-2 group; the incidence of hematoma enlargement on day 10 was 1.1% in the placebo group, 1.1% in the ICH-1 group, and 3.1% in the ICH-2 group, with no significant differences among the groups (P>0.05). The mortality rates were 3.8% in the placebo group, 2.8% in the ICH-1 group, and 0.9% in the ICH-2 group; the incidences of poor prognosis were 7.1% in the placebo group, 6.0% in the ICH-1 group, and 4.8% in the ICH-2 group at 3 months, with no significant differences among the groups (P>0.05). However, the overall frequency of treatment-emergent adverse events(TEAEs) in the ICH-1 group (12.1%) was higher among the three groups (5.8% and 2.8%, respectively, P<0.05). All 3 cases of serious adverse events were in the ICH-1 group.
Conclusions: Ultra-early administration of ICH-1formula for AICH patients did not exert significant beneficial effects on clinical outcomes but increased the risk of bleeding, which probably resulted from the inclusion of RBS herbal medicines in ICH-1.

Introduction
Acute intracerebral hemorrhage(AICH) accounts for approximately 15% [1] of all acute strokes; stroke is the third most common cause of death in most Western countries, following coronary heart disease and cancer. [2,3] AICH has been the leading cause of death in China in recent years [4] and is responsible for approximately one-third of the total number of deaths from stroke worldwide. [5] Available specific medical and surgical treatments do not improve prognosis substantially, [6,7] and AICH still accounts for a higher proportion of stroke in Chinese people than in white populations. [8] Therefore, traditional Chinese medicine (TCM) physicians in China widely use removing blood stasis (RBS) herbs for the treatment of AICH. Meta-analysis showed that RBS herbal therapy for AICH could improve the neurological function deficit, reduce the volume of hematoma and perihematomal edema, and lower the mortality rate and dependency. [9] It is possible that RBS herbal medicine promotes hematoma absorption and improves the recovery of neurological impairment [10,11] ; RBS herbal medicine has already been included by guidelines for the management of spontaneous ICH. However, sufficient evidence of long-term benefits and risks of superacute stage is lacking. RBS herbals, such as Hirudo nipponica Whitman, which acts as a thrombin inhibitor with anticoagulant pharmacological effects, may cause rebleeding risk in clinical practice. [12] A total of 90% of hematoma growth, which plays an important role in prognosis, occurs within a 6-h time window. [13] Therefore, a confirmation of the safety and efficacy of RBS herbal medicines in the treatment of early-stage AICH is urgently needed.
We performed a retrospective study that demonstrated that RBS herbal medicine for AICH patients was safe and effective. [14] However, little additional evidence of the safety and efficacy of this treatment exists. We designed a multicenter, three-group, prospective, randomized, double-blinded and placebo-controlled clinical trial on hematoma enlargement in AICH patients treated with RBS herbal medicine within a 6-h time window from symptom onset (CRRICH) to evaluate its safety and efficacy in AICH patients and to provide a high level of evidence for clinical practice.

Study Design
The CRRICH trial was a multicenter, randomized, double-blinded, placebo-controlled, parallel-group study to assess the safety and efficacy of RBS herbal medicine in AICH patients. This trial is registered at clinicaltrials.gov (NCT01918722), and the research design proposal has been published. [15] Participants Patients with a Glasgow Coma Scale [GCS] score ≥6 were enrolled if they were ≥18 years of age and had a clinical diagnosis of ICH confirmed with computed tomography within 6 h of onset. All participants signed an informed consent form. If the participants did not have the capacity to sign the informed consent for serious condition or illiteracy, the researcher will explain the informed consent to the patients or their authorized immediate family. Weighing the pros and cons of both sides, their immediate family will decide whether to sign the informed consent on behalf of the patient. Patients who suffered secondary ICH resulting from trauma, brain tumor, blood diseases, arteriovenous malformation or aneurysm were excluded. A detailed patient history was recorded. S1 Table in the online-only Data Supplement provides a full list of study inclusion and exclusion criteria during the screening period.

Randomization and Blinding
Patients were randomized to receive placebo, ICH-1, or ICH-2 in a 1:1:1 ratio with a stratification and block size of 6 using the PROC PLAN process in SAS software version 9.13. Stratified block and randomization were concealed using sequentially numbered opaque envelopes. Persons involved in the study, including the investigators, patients and data analysts, were blinded to treatment assignment during double-blinded treatment until the end of follow-up. Only the data administrators were permitted access to unblinded data.  Table in the online-only Data Supplement). The study consisted of a screening and randomization period (≤6 h poststroke), a 10-day double-blinded treatment period and a 3-month follow-up period (S1 Figure in the online-only Data Supplement). Participants were randomly assigned to one of three treatment groups at a ratio of 1:1:1 during the double-blinded treatment (shown in the S3 and S4 Table in the online-only Data Supplement): (1) The ICH-1 group received the ICH-1 formula, which consisted of 8 herbs including RBS medicines; (2) The ICH-2 group received the ICH-2 formula, which consisted of 6 herbal medicines without RBS herbs(Hirudo nipponica Whitman and Tabanus bivittatus Matsumura) in the same doses as those in the ICH-1 group, with the remaining dose filled with a placebo; and (3) The placebo group received a placebo that included dextrin and farina. Each unit of TCM medicine or placebo was dissolved in 200 ml of boiling water. Each patient received 200 ml orally or through a nasogastric tube two times daily for 10 days (S3 Table in the online-only Data Supplement).
Patients were followed up for 3 months after the 10-day double- blinded treatment period. Patients were hospitalized in stroke units and received standard stroke care in accordance with local guidelines, which included general supportive care, treatment of acute complications, and rehabilitation. Astringents and other RBS drugs were prohibited. Visits were scheduled at baseline, 24 h poststroke, and at the end of the treatment period, with a single visit scheduled 3 months poststroke (S1 Figure in the online-only Data Supplement). Adverse events (AEs), treatment compliance, and concomitant medication use were reported during each visit. Participants were randomly assigned to one of three treatment groups at a ratio of 1:1:1 during double-blinded treatment.

Primary and Secondary Outcomes
The incidence of hematoma enlargement, which was defined as the percentage of participants who experienced hematoma enlargement, was the primary outcome of the study. Hematoma enlargement was operationally defined as an increase in hematoma volume of >33% or 12.5 ml, as measured on image analysis on the 24-h CT compared with the baseline CT scan. Hematoma volume was measured using the ABC/2 Coniglobus formula [16] at 24 h after onset. The same measurement was performed on days 10-14 as a primary outcome measure. NIHSS(National Institutes of Health Stroke Scale) scores range from 0 (normal) to 42 (coma with quadriplegia); and mRS(modified Rankin Scale) scores range from 0 (no symptoms or disability) to 6 (death). Both of them can be used to evaluate the clinical outcomes.The following secondary end points were assessed: change in NIHSS score at 3 months; mortality at 3 months; and poor prognosis rate, which was defined as the proportion of patients who died or were severely disabled based on an mRS score ≥5 at 3 months. [17] Safety data focused on treatment-emergent AEs (TEAEs). TEAEs were defined as AEs that first occurred or worsened (increased in severity) after the first dose of study drug. The patient spontaneously reported TEAEs in response to an open-ended question from the contact person at each visit. A worsened patient condition, prolonged patient hospitalization, and life-threatening TEAEs in patients were reported as serious adverse events (TEsAEs). Abnormal laboratory values that constituted a serious AE or led to the discontinuation of experimental drugs were also reported as TEsAEs. S5 Table in the online-only Data Supplement provides a full list of all primary, secondary and safety end points.

Statistical Analyses
All reported efficacy analyses were predefined and based on the full analysis set (FAS) (patients who were randomized and received any study medication) according to the intent-to-treat (ITT) principle. Supportive analyses were performed in a predefined per-protocol population set (PPS), which included FAS patients who did not have one or more key protocol deviations, including violations of inclusion and exclusion criteria. The last observation for surviving patients with missing outcome data was carried forward. Patients who had died by the 90-day follow-up were assigned a Barthel index score of 0 and an mRS score of 6, and the last recorded NIHSS score was carried forward. The incidence of hematoma enlargement at 24 h (primary end point), the rate of poor prognosis and the incidence of total TEAEs among the three groups were analyzed using the χ 2 test. The incidence of hematoma enlargement on days 10-14 (primary end point) and mortality among the three groups were compared using Fisher's exact test. The mean differences in NIHSS score among groups were analyzed using one-way ANOVA. Demographic characteristics and serious TEAEs were summarized using descriptive statistics.
Power and sample size were determined using relative hematoma volume at 24 h as the primary outcome variable. A total of 285 participants were required at the final follow-up to provide 90% power with a two-sided alpha level at 0.05 to test the primary efficacy, which was calculated using the PASS (Power Analysis and Sample Size) software program (licensed by NCSS, LLC). A total of 360 participants were needed for primary outcome randomization assuming a dropout rate of 20%, with 120 patients per treatment arm. The statistical package STATA (version 13.1) was used for all statistical analyses.

Patient and Public Involvement Statement
While we put forward the research question and outcome measures, the patients' priorities, experience, and preferences were fully considered by a survey. Patients were involved in the recruitment to and conduct of the study. The results would be disseminated to study participants by posting a letter. The CRRICH trial was a randomized controlled trial, and the patients themselves had fully assessed the burden of the intervention. Patient advisors should also be thanked in the contributorship statement/acknowledgments.

Funding and Ethical Statements
The State Administration of Traditional Chinese Medicine of the P.R.C. (SATCM) awarded Guangdong Provincial Hospital of TCM (GPHTCM) SATCM Grant No: ZDJX2012074 and provided input during the study period. This report was an independent study supported by SATCM and performed by the GPHTCM in a SATCM-GPHTCM partnership. The viewpoints stated in this manuscript are those of the authors and may not represent the opinions of SATCM, GPHTH or the Public Health Bureau. The SATCM and GPHTCM approved the decision to submit this paper for publication. Dr. Jianwen Guo had full access to all study data and had final responsibility for the decision to submit for publication. Each respective institutional review board or ethics committee approved the study protocol, which followed the established Good Clinical Practice guidelines. All patients provided written informed consent to participate in the study. An independent contract research organization, S&R (Department of Science and Research of Guangdong Provincial Hospital of Chinese medicine), managed the administration, coordination, and monitoring of the study, including researcher training, study inspection, data management and statistical analyses, with oversight by the State Administration of Traditional Chinese Medicine of the P.R.C.

Study Population
Patient recruitment began in October 2013, and the study ended in May 2016. A total of 5589 individuals with ICH were screened ( Figure  1). The most frequent reasons for exclusion in descending order were time window >6 h, refusal to participate, secondary ICH (i.e., not AICH) and other reasons. A total of 319 of the 324 randomized patients (105 placebo, 108 ICH-1, 111 ICH-2) received the assigned treatment following the elimination of five patients (one patient for missing the time window, one patient for kidney failure, one patient for secondary ICH, one patient for surgery within 24 h and one patient withdrew consent). Therefore, only the 319 participants who underwent randomization and received study medication were included in the FAS and safety analysis set based on ITT. Twenty-three patients in the FAS did not completely adhere to the protocol and dropped out during the treatment period. A total of 296 patients were included in the PPS (see Figure 1 for patient flow diagram). The mean dropout rate was <8%. Table 1 presents the demographics and baseline characteristics of the FAS, which were well balanced with no significant between-group differences. The mean age was 62.5±12.7 years (mean±SD, n=319); 65.5% of the patients were male, and 98.7% were ethnic Han. The median GCS score was 15 (range, 3 to 15), and the mean NIHSS score was 8 (range, 0 to 47). The deep gray matter was involved in 93.4% of cases, and the lobar regions were involved in 6.6% of cases. The baseline characteristics of the three treatment groups were similar. The mean volume of the ICH at baseline for all patients was 11.0 ml (range, 0.3 to 86.7 ml), which was similar in the three groups ( Table 1). The mean time from onset of symptoms to admission was 3.36±1.49 h, and the mean time from onset of symptoms to treatment was 4.10±1.38 h. Fourteen percent of patients were treated within 3 h after symptom onset. Two patients were treated outside of the 6-h time window. The timing of the treatment was similar in the three groups (Table 1).

Primary Outcome (Radiographic Outcomes)
At 24 h, a total of 319 baseline CT scans and 315 CT scans were available for analysis. The incidence of hematoma enlargement were 7.8% in the placebo group, 12.3% in the ICH-1 group and 7.5% in the ICH-2 group. There was no significant difference in the incidence of hematoma enlargement at 24 h among the three groups (P=0.409). The incidence of hematoma enlargement on days 10-14 was 1.1% in the placebo group, 3.1% in the ICH-2 group and 1.1% in the ICH-1 group. There was also no significant difference among the three groups in the incidence of hematoma enlargement on days 10-14 (P=0.625) ( Table 2).

Secondary Outcomes (Clinical Outcomes)
Mortality at 3 months was approximately 2.6% in the three groups ( Table 2). Poor prognosis (i.e., the proportion of patients who died or were severely disabled, mRS ≥5) did not differ significantly among the three groups. It is the least patients of poor prognosis occurred in the ICH-2 treatment, but no statistical difference compared with placebo treatment ( Table 2). The distributions of outcomes on the modified Rankin Scale (Figure 2) were similar among the three groups. The differences in the NIHSS scores at 3 months among the three groups were not significant (Table 2).

Figure2
Clinical Outcome at 90 Days According to the Modified Rankin Scale. The modified Rankin Scale evaluates global disability and handicap. Scores range from 0 (no symptoms or disability) to 6 (death). There were no significant differences among the three groups.

Adverse Events
There were altogether 35 AEs reported by investigators, of which 22 AEs may have been TEAEs. All the TEAEs occurred during the doubleblinded treatment period (within 2 weeks). TEAEs occurred in 13 (12.1%) ICH-1-treated patients, 3 (2.8%) ICH-2-treated patients and 6 (5.8%) placebo-treated patients. The patterns of TEAEs were similar in the 3 groups, but the overall frequencies of TEAEs among the three groups were significantly different (P=0.022), as shown in Table 2. And the paired comparisons showed that the test for difference in the overall  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y TEAEs rate between group ICH-1 and group ICH-2 yields a p-value of 0.029. Table 3 shows the TEAEs that occurred during the study. The most frequent event was diarrhea, and its incidence was similar in the 3 treatment groups . Treatment was discontinued because of TEsAEs in 3 (1.9%) ICH-1-treated patients. The 3 cases with TEsAEs (2 cases of gastrointestinal bleeding, 1 case of cerebral hernia) reported by investigators occurred in ICH-1-treated patients. There was no consistent pattern to this difference, and all serious TEAEs occurred within the 10day drug treatment period. There were no statistically significant differences in the incidence or type of serious TEAEs leading to death among groups (data not shown). There was no relationship between TEAEs, serious TEAEs, or mortality and poor outcome (data not shown).  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   11   Table 3 Treatment-emergent adverse events   Placebo  ICH-2  ICH- The values are expressed as n/N (%) within group. All adverse events are listed in order of frequency from the overall study period (90 days); adverse events were collected from investigator reports. The principal investigator at each site determined whether an adverse event or serious adverse event was related to the intervention. TEAEs, treatment-emergent adverse events. *indicates TEAEs.  (Table 2). These data suggest that RBS treatment for ICH patients within 6 h of symptom onset is a safety concern. However, no data were available to suggest a mechanism for this effect. The possible rebreeding mechanism is that RBS medicine, such as Hirudo nipponica Whitman, which has anticoagulant pharmacological effects, could increase bleeding risk according to the previous literature. [12] As can be seen from the table 2, ICH-2 treatment  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  previous meta-analysis [9] , which included 9 randomized controlled clinical trials with 798 individuals, demonstrating that RBS therapy for acute ICH reduced brain hematoma and cerebral edema volumes, improved the neural function and reduced the mortality and disability rates with fewer TEAEs. The present trial revealed significant heterogeneity compared with the studies included in the meta-analysis, possibly because of differences in the intervention time window. This research is the first prospective, multicenter, randomized, double-blind, placebo-controlled study to assess the efficacy and safety of ICH-1 in AICH patients within a 6-h time window from symptom onset, unlike prior studies that examined these effects within a 24-h or later intervention time window. Earlier RBS administration increased the risk of bleeding events (e.g., gastrointestinal bleeding or intracerebral rebleeding), which counteracted the benefit to some extent.