Clinical Safety and Efficacy Evaluation of the Blood-cooling and Stasis-removing Formula as an Adjunct to Atorvastatin in the Treatment of Hemorrhagic Moyamoya Disease.

1. Research Design This clinical trial was a single-center, interventional, randomized, single-blind, placebo-controlled study. Patients diagnosed with hemorrhagic moyamoya disease at the outpatient and emergency departments of Jiangsu Provincial People's Hospital, who met the inclusion criteria, were selected as subjects. Subjects were kept blinded to the intervention they received (experimental group/control group); informed consent was provided to ensure treatment implementation and safety. The investigational drug was uniformly numbered by the center's pharmacy department and allocated according to the randomization table. Randomization and allocation concealment were achieved through the center's randomization system. Referring to previous studies, a total of 60 subjects were enrolled. The enrolled subjects were coded from 1 to 60 and randomly ranked by computer. The first 30 subjects were selected as the experimental group, receiving a blood-cooling and stasis-removing formula, supplemented with atorvastatin (Pfizer, 10mg, National Drug Approval Number H20051407, one tablet once daily). The remaining 30 subjects served as the control group, receiving a placebo, supplemented with atorvastatin (Pfizer, 10mg, one tablet once daily).
2. Study population 2.1 Sample size calculation Based on the MR3T plain scan + contrast examination of moyamoya disease, based on four parameters: TTP, MTT, rCBF and rCBV, the infarct-type moyamoya disease from poor to good is: II2, II1, I2, I1, which are recorded as 1, 2, 3, 4 points, which are rank variables. The improvement of perfusion score after intervention is greater than or equal to 1 point. It is estimated that the improvement rate of the control group (atorvastatin + placebo) is 50%[9], and the improvement rate of the experimental group (atorvastatin + blood-cooling and stasis-removing formula) is 85%[10]. With α=0.05 (two-sided) and the test power of 80%, the sample size is estimated by comparing the two independent sample rates. Each group needs to enroll about 27 cases. Considering a 10% dropout rate, each group is planned to enroll 30 cases, for a total of 60 subjects.

The calculation is as follows:

Significance level: α = 0.05 (two-tailed); Statistical power: 1-β = 0.80; Improvement rate in the control group: p₁ = 0.50 Improvement rate in the experimental group: p₂ = 0.85; Difference: Δ = p₂ - p₁ = 0.35

The common formula for comparing two rates (1:1 allocation) is as follows:

Substituting: Zα/2≈1.96Z_{α/2} ≈ 1.96Zα/2≈1.96 (α=0.05 two-sided) Z1-β≈0.84 (efficiency 80%) n≈26.9⇒Take 27 cases/groups Adjust by 10% dropout rate: n'=1-0.1058≈30⇒Take 30 cases/group; A total of 60 cases were included in the experimental group and the control group.

2.2 Selection Criteria

1. Age: 18-60 years old;

2. Meets the Western medical diagnostic criteria for Moyamoya disease. Refer to the recommendations in the "Chinese Expert Consensus on the Diagnosis and Treatment of Moyamoya Disease and Moyamoya Syndrome (2024 Edition)":

   The diagnosis was confirmed as Moyamoya disease (8B22.B) and stroke (A07.01.01).

   • DSA findings include: stenosis or occlusion of the distal ICA and/or the initial segments of the ACA and/or MCA; abnormal vascular network at the base of the skull in the arterial phase; ② MRI and MRA findings: stenosis or occlusion of the terminal segment of the ICA and/or the initial segment of the ACA and/or MCA; abnormal vascular network in the basal ganglia (more than 2 obvious flow voids in the basal ganglia on one scan plane, suggesting the presence of an abnormal vascular network); ③ Excluding other subtypes of Moyamoya disease that have been diagnosed as Moyamoya disease;
3. Traditional Chinese Medicine diagnostic criteria for Moyamoya disease: It meets the diagnostic criteria for "stroke" with phlegm and blood stasis obstructing the collaterals, and has a dark tongue, white or yellow greasy coating, wiry or slippery or thin and hesitant pulse, etc., and has 2 or more main symptoms or 1 main symptom + 2 secondary symptoms to be diagnosed.
4. The basic treatment has remained stable for nearly one month, and no additional medication is needed;

5. Can understand and agree to comply with the testing requirements and limitations.

   2.3 Exclusion Criteria

   1. Individuals with coexisting cognitive impairment or severe mental illness (ICD-10: F20-F48, F60-F69), such as Alzheimer's disease, other neurodegenerative diseases, Parkinson's disease, multiple sclerosis, demyelinating/hereditary central nervous system diseases, neurodevelopmental abnormalities, uncontrollable epilepsy, and other neurological diseases that researchers determine can significantly affect motor or cognitive function;
   2. Pregnant women and breastfeeding patients;
   3. Individuals with severe dysfunction of organs such as the heart, liver, kidneys, and lungs; individuals with severe congenital malformations (including congenital encephalocele, congenital hydrocephalus, congenital spina bifida, Argyll syndrome, etc., and congenital heart diseases with obvious circulatory and respiratory symptoms such as ventricular septal defect, tetralogy of Fallot, etc.); individuals with hematologic disorders; individuals with severe infections, tumors, etc.; individuals with positive HIV/syphilis/hepatitis B/C serological tests;
   4. Currently participating in other drug clinical trials;
   5. History of intolerance/allergy to any component of the investigational drug;
   6. Hemorrhagic stroke within 12 months prior to enrollment; or a history of infarct-type moyamoya disease or other types of moyamoya disease;
   7. Use of medications prohibited under this protocol within 30 days prior to enrollment; presence of alcohol or drug dependence;
   8. Carotid/cerebrovascular surgery (including interventional treatment) may be required during the trial.

   2.4 Subject Withdrawal Criteria

   1. Unable to or unwilling to comply with the requirements of the plan;
   2. The subject/legal representative withdrew informed consent;
   3. Concomitant medications/treatments prohibited under this protocol must be used;
   4. A serious adverse event occurs, or although the SAE (Serious Adverse Event) criteria are not met, the investigator believes that continuing the study would jeopardize the health/well-being;
   5. Neck and brain surgery (including interventional treatment) is required;
   6. Poor compliance or failure to follow the visit/procedure;
   7. Other circumstances where the researcher believes, based on reasonable medical judgment, that withdrawal should be initiated.
   8. Clinically significant protocol deviations/violations that the investigator or sponsor decides to exclude.

   From the perspective of participant safety, for example, if a participant experiences a situation where they are no longer suitable to continue the study, including: worsening of their condition, serious adverse events, poor compliance, etc.

   3. Research intervention measures 3.1 Study intervention measures Subjects enrolled in the experimental group were treated with a blood-cooling and stasis-removing formula (200ml per dose, twice a day, one dose each time, decocted and taken orally, a formula agreed upon by Nanjing Municipal Hospital of Traditional Chinese Medicine), supplemented with atorvastatin (produced by Pfizer, USA, 10mg, National Drug Approval Number H20051407, one tablet once a day). The medication was started after the diagnosis of hemorrhagic moyamoya disease, and the course of treatment was 12 weeks (3 months).

   Subjects in the control group received a placebo (placebo preparation: 10% of the original drug formula mixed with rice and edible flavoring to create a control placebo with similar color, taste, and properties) as supplemental treatment, along with atorvastatin (Pfizer Inc., 10mg, National Drug Approval Number H20051407, one tablet once a day), which was started after diagnosis of hemorrhagic moyamoya disease and continued for 12 weeks.

   If a study drug is involved, specify its name, dosage form, specifications, source, manufacturer, batch number, expiration date, and storage conditions. If the control drug is a placebo, it should meet the requirements for placebo preparation. Explain the basis and rationale for selecting the control drug. Include the dosage, route of administration, method of administration, frequency of administration, treatment duration, and follow-up period.

   Note: The blood-cooling and stasis-removing formula (200ml per dose, twice a day, one dose each time, decocted and taken orally, an in-hospital formula of Nanjing Municipal Hospital of Traditional Chinese Medicine) was provided by the collaborating research unit, Nanjing Municipal Hospital of Traditional Chinese Medicine. It was accepted and confirmed by the chief physician of the Department of Traditional Chinese Medicine of our hospital who participated in the research (see the list of participants) before being distributed to the subjects in the experimental group.

   3.2 Combined medication If a participant has a chronic disease requiring long-term medication, such as hypertension, diabetes, or rheumatoid arthritis, they may continue to take any antihypertensive drugs (e.g., ACEIs, ARBs, calcium channel blockers, diuretics, beta-blockers), hypoglycemic drugs (biguanides, sulfonylureas, alpha-glucosidase inhibitors, DPP-4 inhibitors, GLP-1 receptor agonists, insulin), or rheumatoid immunotherapy drugs that they were already taking before the study. No additional medications may be added after enrollment.

   In particular, drugs/health products containing the following ingredients are prohibited: succinic acid and its salts (such as reamberin, remaxol, cytoflavin, etc.), vitamin B6 and its derivatives, antioxidant/anti-hypoxia drugs (containing 3-hydroxypyridine derivatives), various nootropic drugs (such as citicoline, α-glycerophosphorylcholine, piracetam, pyridoxine derivatives, GABA derivatives/analytes, ginkgo, neuropeptides/peptide preparations, cerebral circulation improvers such as vinorelbine/cinnarizine, plant-derived adaptogens, etc.).

   4. Follow-up 4.1 Follow-up time and frequency This study included two in-hospital follow-up visits for Moyamoya disease using MR3T (plain scan + contrast-enhanced MRI), both conducted at our hospital. The first visit was one week before the intervention, and the second was the day after the end of the treatment course (3 months). Patients were accompanied by physicians from the research team, and appointments were scheduled in advance via telephone. Both follow-up visits were completed at the hospital by the same research team following a standardized procedure.

   In addition, this study also included a three-year telephone follow-up, with clinicians from the research team calling patients every two weeks to understand medication adherence and the occurrence of adverse events.

   4.2 Follow-up Content Clinical examination and testing: Inquire about the patient's medical history and medication use in detail, measure vital signs, and instruct the patient to have a complete blood count and blood biochemistry (electrolytes, liver function) every 2 weeks to 1 month, and record whether any adverse clinical events occur.

   Scale assessment: At each follow-up visit, the clinical symptom improvement, FMA, MBI, MoCA and other pre-set functional scales need to be scored separately. The same set of assessment tools and assessors should be used and the assessments should be conducted at the same time period as much as possible to reduce measurement bias.

   Imaging examinations: A standardized MRI examination protocol was adopted, namely Moyamoya disease MR3T (plain scan + contrast), which was performed once one week before intervention and once after the clinical course of treatment. The same type of MRI machine and fixed scanning parameters were used. Experienced radiologists reviewed the images and recorded the preset imaging parameters (TTP, MTT, rCBF, rCBV) without knowing the patient group.

   All adverse events (AEs)/several adverse events (SAEs) should be completed item by item according to the pre-designed Safety Follow-up Record Form, including the time of occurrence, severity, treatment measures, correlation with the investigational drug, and outcome, and should be summarized and analyzed regularly. In the event of a serious adverse event (SAE), the investigator should immediately provide appropriate clinical treatment and report to the project leader and ethics committee within 24 hours in accordance with GCP and ethical requirements.

   4.3 Handling of Loss to Follow-up and Withdrawal If a subject discontinues medication or withdraws from the trial midway, the investigator should arrange for them to complete the clinical and imaging examinations at the endpoint visit to obtain key outcome data; if they are unable to complete the visit, the reason for loss to follow-up should be recorded and included in the follow-up statistical analysis.

   5. Evaluation indicators/study endpoints 5.1 Primary therapeutic indicators:

   Imaging improvement indicators:

   Cerebral perfusion parameters were examined using 3T MRI (plain scan + contrast) for Moyamoya disease, and cerebral artery parameters, including mean transit time (MTT), cerebral blood flow (CBF), time to peak flow (TTP), and cerebral blood volume (CBV), were recorded.

   CBF: In normal adults, gray matter is approximately 50-80 ml/100g/min, and white matter is approximately 20-30 ml/100g/min; CBV: Normal gray matter is about 4-5 ml/100g, and white matter is about 2-3 ml/100g; MTT (Mean Transit Time): Normally about 3-5 seconds; a time exceeding 6 seconds may indicate abnormal perfusion.

   TTP: Normally, a difference of ≤2 seconds between the TTP and the contralateral healthy area is considered normal.

   Note: Commonly used cerebral perfusion staging:

   Stage I-1: TTP prolonged, MTT, rCBF, and rCBV normal; Stage I-2: TTP and MTT are prolonged, rCBF is normal, and rCBV is normal or slightly elevated; Phase II-1: TTP and MTT are prolonged and rCBF is decreased, while rCBV is basically normal or slightly decreased; Phase II2: TTP and MTT are prolonged, while rCBF and rCBV decrease. Effective: Infarct-type moyamoya disease is graded from worst to best as follows: II-2, II-1, I-2, I-1. The scores are determined by the chief physician of the radiology department of our hospital based on relevant imaging data, and are assigned as 1, 2, 3, and 4 points respectively, representing a rank variable. An improvement of 1 point or more in the perfusion score after intervention is considered an improvement.

   Ineffective: No change in perfusion score after intervention is considered ineffective.

   5.2 Secondary efficacy endpoints:

   Clinical symptom improvement indicators:

   Clinical efficacy. Excellent: Symptoms disappeared without residual neurological dysfunction; Good: Symptoms improved significantly, with or without residual neurological dysfunction; Average: Symptoms were partially relieved, with or without residual neurological dysfunction; Poor: Symptoms did not improve or even worsened; Significant relief rate = (Excellent + Good) / Total number of cases × 100%, Relief rate = (Excellent + Good + Average) / Total number of cases × 100%; Motor function, activities of daily living (ADL), and cognitive function were assessed. The Fugl-Meyer Assessment (FMA) was used to evaluate motor function before and after treatment. The Modified Barthel Index (MBI) was used to assess ADL, and the Montreal Cognitive Assessment (MoCA) was used to assess cognitive function. The FMA score is out of 100, with 0 points for inability to complete any motor activity, 1 point for partial completion, and 2 points for normal completion. The MBI score is also out of 100; a score greater than 60 indicates basic self-care ability. A score of 40-60 indicates mild disability requiring assistance. A score of 20-40 indicates severe disability requiring dependence. A score less than 20 indicates complete disability requiring complete dependence. The MoCA score is out of 30, with a normal value of ≥26. Lower scores indicate poorer motor function, ADL, and cognitive function.

   Valid: The score increased compared to before; Invalid: The score remained unchanged or decreased compared to before.

   5.3 Long-term clinical endpoint evaluation indicators: In addition to assessing short-term radiological (three months) and functional scale improvements, this study also included a survival analysis of long-term clinical outcomes, specifically calculating the incidence of any of the following events from the date of randomization to the first occurrence of the event: 1. New-onset cerebral hemorrhage; 2. Recurrent/new-onset cerebral infarction; 3. All-cause mortality.

   5.4 Safety Indicators Data is primarily collected through regular follow-ups. Assessment Items, Judgment Criteria, Assessment Timeframe, Reporting Deadline Adverse events (AEs): Any adverse changes following administration (including symptoms/signs/abnormal laboratory results), regardless of cause. Inquire at every visit from the first administration to the end of the trial; mandatory evaluation at early termination visits. Routine: Report as planned; if upgraded to an SAE, follow the SAE protocol.

   Serious adverse events (SAEs) such as death, life-threatening events, hospitalization or prolonged hospitalization, disability or deformity, events requiring medical intervention to prevent the above consequences, or medically significant events must be assessed immediately upon becoming known and reported to the sponsor within 24 hours; ethical and regulatory compliance are required.

   6. Visit arrangements and data collection during the research period Before intervention, patients' relevant clinical symptoms and functional cognitive scores were recorded (Fugl-Meyer Assessment of Motor Function (FMA) for motor function, Modified Barthel Index (MBI) for activities of daily living (ADL) for self-care ability, and Montreal Cognitive Assessment (MoCA) for cognitive function). Patients newly diagnosed with hemorrhagic moyamoya disease based on relevant symptoms (dizziness, headache, cognitive decline) and imaging examinations (CTA, DSA), and meeting the inclusion criteria, were immediately given the designed drug intervention regimen for 12 weeks. Close follow-up was conducted at 1, 3, 6, 12, and 24 months after the end of the treatment course. Follow-up included: 1. Imaging examinations (cranial CTA, DSA) to assess the improvement of perfusion function. 2. Observation of the improvement of relevant clinical symptoms, calculation of the remission rate, and simultaneous assessment of motor function using the Fugl-Meyer Assessment of Motor Function (FMA), ADL for activities of daily living (ADL), and cognitive function using the Montreal Cognitive Assessment (MoCA), calculating the corresponding scores and evaluating whether there was corresponding improvement. 3. Inquire about relevant adverse reactions (gastrointestinal reactions, dizziness, headache, abnormal liver function), and compile statistics on adverse prognostic events, such as cerebral infarction, cerebral hemorrhage, transient ischemic attack, death, etc.