プロフィール紹介文
A Double-blind Crossover Trial Of Methandienone Dianabol, CIBA In Moderate Dosage On Highly Trained Experienced Athletes
**Title:**
*Effect of a Novel β‑Blocker on Cardiac Output and Systemic Vascular Resistance in Patients with Heart Failure: A Randomized, Double‑Blind Study*
---
## 1️⃣ Abstract
A double‑blind, randomized, placebo‑controlled trial was conducted to evaluate the cardiovascular effects of **β‑Blocker X**, a newly developed selective β₁‑adrenergic antagonist. Eighty adults (age 35–75 yr) with NYHA class II–III heart failure and LVEF < 40 % were enrolled at 3 tertiary centers. Participants received either β‑Blocker X (25 mg daily, titrated to 100 mg over 8 weeks) or matching placebo for 12 months.
**Primary endpoint:** change in left ventricular end‑diastolic dimension (LVEDD) measured by transthoracic echocardiography at baseline and month 12.
**Secondary endpoints:** changes in LV mass, LVEF, exercise capacity (6‑minute walk test), BNP levels, and adverse events.
At 12 months, mean LVEDD decreased by 4.8 mm (p < 0.001) in the β‑Blocker X group versus a 1.2 mm increase (p = 0.07) in placebo (between‑group difference: −6.0 mm; p < 0.001). LV mass decreased by 12 g/m² (p < 0.01) and LVEF improved from 45 % to 52 % (p < 0.005). BNP fell by 35 %, and exercise capacity increased by 15 %. Adverse events were mild: dizziness in 8 % of patients, transient hypotension in 5 %. No hospitalizations or deaths occurred during the study.
The trial demonstrates that a structured, supervised aerobic program can reverse structural remodeling, enhance systolic function, and improve functional status in patients with HCM. These findings support incorporating cardiac rehabilitation into routine management for eligible individuals, particularly those with preserved exercise capacity but symptomatic diastolic dysfunction. The authors call for larger multicenter trials to confirm benefits across diverse phenotypes and to assess long‑term safety, including arrhythmic risk.
---
### 3. Comparative Table of the Two Articles
| Feature | Article 1 (Meta‑analysis) | Article 2 (RCT) |
|---------|---------------------------|-----------------|
| **Design** | Systematic review + meta‑analysis of RCTs | Single‑center, prospective, randomized controlled trial |
| **Sample Size** | 12 000+ participants across 25 trials | 300 participants total (150 per arm) |
| **Intervention(s)** | Standardized anti‑arrhythmic drugs (class I/III), compared to placebo or other drugs | Novel anti‑arrhythmic agent versus standard therapy in high‑risk atrial fibrillation |
| **Primary Outcome** | Composite of all‑cause mortality, arrhythmia recurrence, hospitalisation | 30‑day composite of major adverse cardiac events (death, MI, stroke) |
| **Secondary Outcomes** | Cardiovascular morbidity, drug toxicity, quality of life | Long‑term rhythm control, biomarker changes, neurocognitive scores |
| **Statistical Analysis** | Meta‑analysis with random effects; subgroup analysis by age, comorbidities; risk ratios (RR) and 95% CIs | Kaplan‑Meier survival curves; Cox proportional hazards models; intention‑to‑treat principle |
| **Key Findings** | Statistically significant reduction in mortality with certain antiarrhythmic agents; heterogeneity across studies | No significant difference in primary endpoint, but improved rhythm control and fewer hospitalizations in the treatment arm |
---
## 1. Background & Rationale
### 1.1 Disease Burden
- **Cardiovascular diseases (CVD)** are the leading cause of mortality worldwide (~17.9 million deaths annually).
- A significant proportion of CVD deaths are attributable to *arrhythmogenic conditions* such as atrial fibrillation, ventricular tachycardia, and sudden cardiac death.
### 1.2 Existing Evidence
- Numerous observational studies suggest that specific antiarrhythmic medications reduce morbidity and mortality.
- However, the magnitude of benefit remains uncertain due to heterogeneity in study designs, populations, and endpoints.
---
## 2. Objectives
### Primary Objective
- **To determine whether treatment with antiarrhythmic medication reduces all-cause mortality compared to placebo or standard care in patients with arrhythmogenic cardiac conditions.**
### Secondary Objectives
1. Evaluate the impact on *cardiovascular-specific* outcomes (e.g., heart failure hospitalizations, arrhythmia recurrence).
2. Assess safety and tolerability profiles.
3. Examine subgroup effects by age, sex, comorbidities, and baseline arrhythmia severity.
---
## 3. Study Design
- **Type:** Multicenter, randomized, double-blind, placebo-controlled trial.
- **Duration:** Minimum follow-up of 2 years (median 24 months).
- **Population:**
- Inclusion Criteria:
- Adults ≥18 years with documented arrhythmia (e.g., atrial fibrillation, ventricular tachycardia) confirmed by ECG/monitoring.
- Stable baseline medication regimen for at least 4 weeks.
- Exclusion Criteria:
- Severe uncontrolled heart failure (NYHA III-IV), active ischemic disease, or other major comorbidities limiting life expectancy <2 years.
- **Intervention:**
- Experimental drug targeting arrhythmia pathways (e.g., sodium channel blocker).
- Standard of care as per guidelines for all participants.
- **Control:**
- Placebo + standard of care.
- **Outcomes:**
- Primary efficacy endpoint: Composite of hospitalization for heart failure or cardiovascular death within 12 months.
- Secondary endpoints: Arrhythmia burden (via implantable devices), quality-of-life scores, all-cause mortality.
- Safety endpoints: Incidence of proarrhythmic events, syncope, adverse drug reactions.
- **Sample Size Calculation:**
- Assuming a control event rate of 15% for the composite endpoint over one year, expecting a relative risk reduction to 10% with the intervention (RRR ≈ 33%). With α=0.05 (two-sided) and power 80%, using a log-rank test approximation yields approximately 1,200 participants per arm (total N≈2,400). This accounts for 5% loss-to-follow-up.
- **Randomization & Blinding:**
- Centralized computer-generated random sequence with permuted blocks stratified by site. Double-blind design: matching placebo tablets identical in appearance.
- **Follow-Up Schedule:**
- Baseline visit (enrollment), then visits at 3, 6, 12, 18, and 24 months to assess outcomes, adherence, adverse events, and collect blood samples for biomarker analysis.
---
### (b) Critical Commentary on the Protocol
> **Dr. Maria Santos (Ethics Reviewer):**
> "While I appreciate the rigorous design, there are several ethical concerns. First, the exclusion of participants with comorbidities may limit generalizability and potentially deny individuals who could benefit from the intervention access to participation. Second, the 24-month follow-up requires sustained participant commitment; we must ensure robust retention strategies and consider incentives that do not coerce."
> **Dr. Luis Almeida (Statistical Consultant):**
> "From a statistical standpoint, stratifying by sex is sound, but we should also account for baseline disease severity as a covariate in the analysis to reduce residual confounding. Additionally, we need to predefine subgroup analyses and adjust for multiple comparisons."
> **Dr. Marta Silva (Ethics Officer):**
> "The informed consent process must clearly explain potential risks and benefits, especially since this is a randomized trial with no standard-of-care control arm. We should also establish an independent data monitoring committee to oversee safety."
---
### 4. "What If" Scenarios
#### Scenario A: Inclusion of Patients Without Diabetes
- **Impact on Trial Design**: Removing the diabetes requirement would broaden eligibility, potentially increasing enrollment speed but also introducing heterogeneity in metabolic status.
- **Statistical Power**: The sample size calculation must account for increased variability; larger cohorts may be needed to maintain power.
- **Clinical Relevance**: Findings could generalize to all patients with COVID‑19 and cardiovascular comorbidities, but confounding by baseline glycemic control would need careful adjustment.
#### Scenario B: Alternative Primary Endpoint (Composite Cardiovascular Outcome)
- **Impact on Trial Design**: A composite endpoint (e.g., myocardial infarction, stroke, arrhythmia) may capture more events but could dilute the specific effect of a drug on inflammation‑related pathways.
- **Statistical Power**: More events increase power; however, heterogeneity in event definitions may complicate interpretation.
- **Clinical Relevance**: Clinicians might value broader cardiovascular risk reduction over singular inflammatory markers.
---
## 3. Comparative Evaluation of Drug Classes
| Drug Class | Mechanism (Pharmacodynamics) | Typical Use in COVID‑19 | Evidence Strength |
|------------|------------------------------|------------------------|-------------------|
| **Corticosteroids** (e.g., dexamethasone) | Potent anti‑inflammatory via glucocorticoid receptor; suppress cytokine production, stabilize membranes. | Standard of care for hospitalized patients requiring oxygen or ventilation. | High – RCTs and meta‑analyses demonstrate mortality benefit. |
| **IL‑6 Receptor Antagonists** (tocilizumab) | Block IL‑6 signaling via anti‑IL‑6R antibodies; reduce downstream JAK/STAT activation. | Used in severe cytokine storm; evidence mixed but some benefit in specific subgroups. | Moderate – Some RCTs show reduced progression to mechanical ventilation. |
| **JAK Inhibitors** (baricitinib) | Inhibit intracellular JAK1/2, dampening multiple cytokine pathways (IFN‑γ, IL‑6, IL‑12). | Shown to reduce viral replication and inflammation when combined with antivirals. | Moderate – RCTs indicate improved recovery times. |
| **Anti‑IL‑17A** (secukinumab) | Neutralizes IL‑17A; reduces neutrophil recruitment & cytokine amplification. | Preclinical data suggest attenuation of hyperinflammatory lung injury. | Low – No clinical trials yet in COVID‑19. |
---
## 3. Suggested Clinical Trial Design
| Element | Recommendation |
|---------|----------------|
| **Population** | Adults (≥18 y) with confirmed SARS‑CoV‑2 infection, presenting within 7 days of symptom onset, requiring supplemental oxygen but not invasive ventilation. Exclude those on chronic immunosuppression or active infections. |
| **Intervention** | Secukinumab 300 mg IV once at baseline (dose adapted from rheumatology). |
| **Comparator** | Placebo IV matched in volume and appearance. |
| **Co‑interventions** | All patients receive standard of care: dexamethasone, anticoagulation, remdesivir if indicated. No other biologics allowed. |
| **Primary Endpoint** | Time to clinical improvement defined as a ≥2‑point reduction on the WHO Ordinal Scale for Clinical Improvement or discharge from hospital within 28 days. |
| **Secondary Endpoints** | 1) All‑cause mortality at day 60; 2) Duration of mechanical ventilation; 3) Incidence of serious adverse events (sepsis, secondary infections); 4) Viral clearance by RT‑PCR at days 7 and 14; 5) Levels of IL‑6, CRP, ferritin over time. |
| **Sample Size** | Assuming a hazard ratio of 1.4 for improvement, with α=0.05 (two‑sided) and power 80%, approximately 280 participants (140 per arm) are needed; inflated to 320 to account for dropout and loss to follow‑up. |
| **Statistical Analysis Plan** | Primary analysis: Cox proportional hazards model comparing time to improvement between groups, adjusting for baseline covariates (age, sex, comorbidities). Secondary endpoints: mixed‑effects models for repeated measures of biomarkers; logistic regression for mortality at 28 days; Kaplan–Meier survival curves with log‑rank test. Sensitivity analyses will include per‑protocol population and multiple imputation for missing data. All tests two‑tailed with α=0.05. |
---
### 4. Risk Management
| **Potential Risk** | **Mitigation Strategy** |
|--------------------|-------------------------|
| Cytokine release from exosomes may worsen inflammation | Use low‑dose pilot phase; monitor cytokines (IL‑6, TNF‑α) hourly; predefine stopping rules for cytokine surge. |
| Viral entry via exosomal membrane or receptor interaction | Confirm absence of ACE2 and TMPRSS2 in final product; test for spike protein binding to exosomes in vitro. |
| Contamination with host cell proteins/viral particles | Implement stringent purification, endotoxin testing, and virus filtration steps; use size‑exclusion chromatography. |
| Immunogenicity from bovine miRNA or surface proteins | Use humanized miRNA cocktail; minimize bovine protein content through extensive washing. |
| Scale‑up failure (aggregation, loss of activity) | Pilot scale batches with monitoring of particle size, zeta potential, and antiviral potency before large‑scale production. |
---
## 4. Suggested Experimental Workflow for a New Bovine‑Derived Product
1. **Design & Production**
- Clone miRNA expression cassette into a suitable bovine cell line (e.g., MDBK).
- Transduce cells with lentivirus containing the construct; select stable integrants.
2. **Purification of Exosomes**
- Collect conditioned media, centrifuge at 300 × g → 2 000 × g to remove debris.
- Filter (0.22 µm).
- Ultracentrifuge at 100 000 × g for 70 min; wash pellet with PBS; repeat spin.
3. **Characterization**
- Nanoparticle tracking analysis for size distribution and concentration.
- Western blot for CD63, TSG101, and the specific miRNA (qPCR).
- Electron microscopy to confirm morphology.
4. **Functional Assay**
- Treat cultured cells with exosomes; measure uptake of labeled miRNA by qRT‑PCR.
- Assess downstream effect (e.g., target gene repression).
5. **Optimization**
- Test alternative isolation methods (ultrafiltration, size‑exclusion chromatography).
- Compare yield and purity across methods.
---
## 4. Summary Checklist
| Step | Action | Key Tips |
|------|--------|----------|
|1|Define biological question|Choose exosomes or microvesicles?|
|2|Select isolation method|Ultracentrifugation (gold standard) vs precipitation, chromatography|
|3|Collect conditioned media|Minimize cell death; use low‑FBS medium|
|4|Sequential centrifugation|300 ×g → 2000 ×g → 10 000 ×g (optional) → 100 000 ×g|
|5|Resuspend pellet in PBS or buffer|Keep volume consistent for quantification|
|6|Characterize vesicles|Nanoparticle tracking, electron microscopy, Western blot for CD9/CD63/CD81|
|7|Store appropriately|-80 °C; avoid freeze–thaw cycles|
---
## 4. Example Protocol (10 mL culture)
| Step | Details |
|------|---------|
| **Cell preparation** | Grow 3 × 10⁶ cells in 10 mL serum‑free medium for 24 h. |
| **Centrifugation** | 300 g, 10 min (remove cells).
2000 g, 20 min (removing debris).
100 000 g, 70 min (ultracentrifuge; 4 °C). |
| **Resuspension** | Wash pellet with 1× PBS; spin again at 100 000 g for 30 min.
Resuspend in 200 µL of sterile PBS or buffer of choice. |
---
### Practical Tips
* Use a clean, RNase‑free system if you plan to keep the RNA intact (e.g., for qRT‑PCR).
* Always pre‑cool tubes and centrifuge at 4 °C; higher temperatures can degrade RNA.
* Store isolated material at –80 °C in small aliquots to avoid repeated freeze–thaw cycles.
---
**Bottom line:** The protocol you described (high‑speed spins, short times) is typical for extracting total nucleic acids from fungal mycelium while preserving RNA integrity. It is well suited for downstream applications such as RT‑qPCR or RNA‑seq of *T. reesei* cultures.
