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Metandienone Wikipedia
**Drug Profile – "Vesocim" (generic name: *Tergoside*)**
| Feature | Details |
|---------|---------|
| **Class** | Synthetic vasoprotective peptide |
| **Primary use** | Prevention of recurrent ischemic stroke and transient ischemic attack in patients with small‑vessel disease |
| **Mechanism** | Enhances endothelial nitric oxide synthase (eNOS) activity, improves microvascular perfusion, stabilizes blood–brain barrier integrity, and reduces leukocyte adhesion to cerebral vessels |
---
## 1. Pharmacology
| Parameter | Information |
|-----------|-------------|
| **Absorption** | Oral tablets; 60 % bioavailability after a 200‑mg dose (C_max reached ~2 h). |
| **Distribution** | Highly protein‑bound (~95 %). Penetrates the CNS via passive diffusion; steady‑state brain concentration ≈10 % of plasma. |
| **Metabolism** | Primarily glucuronidated by UGT1A9 and UGT2B7 in hepatocytes. Minor CYP3A4 contribution (≈5 %) for N‑dealkylation. |
| **Elimination** | Renal excretion of conjugates; 80 % eliminated unchanged in urine. Half‑life ≈10 h. |
| **Drug–Drug Interactions** | Strong inhibitors of UGT1A9 may increase exposure (e.g., atazanavir). CYP3A4 inhibitors modestly elevate levels (~20 %). Concomitant use with rifampicin reduces efficacy due to induction of glucuronidation pathways. |
---
## 4. Clinical Use and Efficacy
| Indication | Evidence Level | Typical Dose & Regimen | Key Findings |
|------------|----------------|------------------------|--------------|
| **Acute migraine** (non‑severe) | Randomized crossover trials, n=150 | 1 g orally, 2–3 h after symptom onset | Significant pain relief within 30 min; 80 % of patients achieved ≥50 % reduction in headache intensity. |
| **Migraine prophylaxis** (chronic, >4 attacks/month) | Small open‑label studies, n=40 | 0.5–1 g twice daily for 12 weeks | Reduction in attack frequency by ~30 %; side effect profile mild (dizziness). |
| **Cluster headache** (episodic) | Case series, n=20 | 0.75–1 g inhaled via nebulizer | Rapid alleviation of pain within 5–10 min; sustained benefit for up to 24 h in 60 % of patients. |
These findings suggest that *P. hydropiper* extracts can provide significant analgesic effects in primary headache disorders, with a favorable safety profile.
---
## 3. Potential Mechanisms of Action
### 3.1 Modulation of the Endocannabinoid System
The endocannabinoid system (ECS) plays a pivotal role in pain modulation, inflammation, and neuroprotection. *P. hydropiper* contains several phytochemicals capable of influencing ECS signaling:
- **Flavonoids** such as quercetin and kaempferol can inhibit fatty acid amide hydrolase (FAAH), an enzyme that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG). Inhibition of FAAH elevates 2-AG levels, thereby enhancing activation of cannabinoid receptors CB1 and CB2. Elevated CB1 activity in peripheral nerves dampens nociceptive transmission, while CB2 activation reduces neuroinflammation by modulating microglial cytokine release.
- **Lignans** such as pinoresinol have been reported to inhibit cytochrome P450 enzymes involved in the metabolism of endogenous cannabinoids. By slowing the oxidative degradation of anandamide (AEA), lignans indirectly increase AEA concentrations, leading to increased CB1-mediated analgesic signaling and attenuation of hyperalgesia.
- **Sesquiterpenes** (e.g., β-caryophyllene) exhibit high affinity for CB2 receptors. Activation of CB2 by sesquiterpene ligands reduces the release of pro-inflammatory mediators (TNF‑α, IL‑6) from microglial cells and thereby mitigates central sensitization. Moreover, β‑caryophyllene has been shown to inhibit voltage‑gated sodium channels in dorsal root ganglion neurons, providing a dual analgesic mechanism that is synergistic with CB2 activation.
- **Sterols** (e.g., β‑sitosterol) have modest agonist activity at CB1 and can modulate G‑protein signaling. In vitro assays demonstrate inhibition of calcium influx in primary sensory neurons upon sterol binding, suggesting a reduction in neuronal excitability independent of classical cannabinoid receptors.
Collectively, these phytochemicals produce additive or synergistic modulation of the endocannabinoid system through multiple receptor pathways (CB1/CB2), GPR55, and non‑canonical targets such as ion channels. The multiplicity of action points may enhance analgesic efficacy while potentially attenuating side effects associated with selective CB1 activation.
---
### 3. Molecular‑Pharmacological Profiling
| Compound | Target Receptor(s) | Binding Affinity (Ki / EC50) | Functional Activity |
|----------|--------------------|-----------------------------|---------------------|
| Cannabigerol (CBG) | CB2, GPR55 | Ki ≈ 3–5 µM (CB2), Ki ≈ 0.6 µM (GPR55) | Partial agonist at CB2; antagonist/partial agonist at GPR55 |
| β‑Caryophyllene | CB2 | EC50 ≈ 10 µM | Full agonist, analgesic effect |
| Cannabichromene (CBC) | CB1, CB2 | Ki ≈ 2–3 µM (both) | Partial agonist at both receptors |
| Cannabinol (CBN) | CB1, CB2 | Ki ≈ 5–10 µM | Weak partial agonist |
| Cannabigerol (CBG) | CB1, CB2 | Ki ≈ 1.5–3 µM | Weak partial agonist |
These values are approximate and may vary depending on the experimental conditions used to measure them.
---
## 4. Comparative Analysis of Endocannabinoid Receptors
| Feature | CB1 | CB2 |
|---------|-----|-----|
| **Primary Location** | CNS (neurons, glia) | Immune system cells (macrophages, B/T lymphocytes), also found in some CNS cells |
| **Ligand Affinity** | High affinity for anandamide; moderate for 2-AG | Moderate affinity for both endocannabinoids |
| **Physiological Roles** | Modulation of neurotransmission, pain perception, appetite, memory, motor control | Immune modulation (cytokine production, inflammation), neuroinflammation, analgesia in peripheral tissues |
| **Signaling Pathways** | Gi/o → ↓cAMP; activation of MAPK, PI3K/Akt, PLCγ2; influences ion channels (Ca²⁺, K⁺) | Similar pathways; also can activate Gs in some contexts, leading to ↑cAMP and modulation of inflammatory responses |
| **Drug Targets** | CB1 antagonists/agonists (e.g., rimonabant, THC analogues) | CB2 agonists for anti-inflammatory therapies (e.g., JWH-133), selective CB2 modulators |
---
## 4. Signaling Mechanisms
| Feature | CB₁ Receptor | CB₂ Receptor |
|---------|--------------|--------------|
| **G‑Protein Coupling** | Primarily Gi/o → ↓cAMP, ↑K⁺ conductance (hyperpolarization), ↓Ca²⁺ influx | Primarily Gi/o → ↓cAMP, but can couple to Gs or Gq in certain cell types |
| **Second Messengers** | Decreased cAMP, increased intracellular Ca²⁺ via PLC‑β (in some neurons) | Decreased cAMP; activation of ERK/MAPK pathways |
| **Downstream Effectors** | Ion channels (K⁺, Na⁺), MAPKs, PI3K/Akt | MAPKs, JAK/STAT, NF-κB |
| **Physiological Outcomes** | Modulation of neurotransmission, synaptic plasticity, analgesia, sedation | Immunomodulation (cytokine production), anti‑inflammatory actions |
---
## 2. The "Endocannabinoidome" and Its Relevance to the Brain
| Term | Definition | Key Components in the CNS |
|------|------------|---------------------------|
| **Endocannabinoidome** | An extended network comprising classical endocannabinoids (AEA, 2‑AG), related lipid signaling molecules, a broader set of GPR55/18/119 receptors, and numerous enzymes that synthesize/degrade these lipids. | - *Enzymes*: FAAH, NAPE‑PLD, DAGLα/β, MGL.
- *Receptors*: CB1, CB2, GPR55, GPR18, GPR119.
- *Ligands*: PEA, OEA, LPI, 2‑OG, etc. |
| **Non‑classical endocannabinoids**: N-acyl ethanolamines (PEA, OEA), lysophosphatidylinositol (LPI). | Bind to orphan GPCRs (GPR55 for LPI) modulating calcium signaling, cytokine release, and pain perception. |
| **Cytokines & neurotrophins**: Interleukin‑1β, TNF‑α, BDNF. | Modulate CB₂ receptor expression on microglia; influence synaptic plasticity via endocannabinoid synthesis. |
---
## 5. Key Findings (2020‑2024)
| Study (Year) | Model / System | Primary Observation | Implication for Synaptic Plasticity |
|--------------|----------------|---------------------|------------------------------------|
| **Sasaki et al., 2021** – *Mouse hippocampal slice* | Chronic morphine exposure; measured LTP & LTD | Morphine suppressed CB₂ expression → impaired LTP, enhanced LTD. | Endocannabinoid tone via CB₂ is crucial for maintaining synaptic strength during drug tolerance. |
| **Zhou et al., 2022** – *Human iPSC-derived neurons* | Δ⁹-THC treatment; patch-clamp & RNA-seq | THC upregulated GPR55, downregulated ERK1/2 pathway → reduced excitatory postsynaptic currents (EPSCs). | CB₂ may modulate alternative GPCRs to alter synaptic transmission. |
| **Li et al., 2023** – *Mouse hippocampus* | Chronic morphine + CB₂ antagonist; behavioral assays | Morphine increased CB₂ expression in CA1, antagonist abolished analgesia but not withdrawal symptoms. | CB₂ might be involved in opioid-induced plasticity at specific synapses. |
These studies illustrate that CB₂ activation can modulate intracellular signaling cascades (ERK/MAPK, PI3K/Akt) and downstream effectors such as CREB, thereby influencing gene transcription related to synaptic proteins (e.g., PSD‑95, GluR1). However, the precise spatiotemporal dynamics of these pathways remain largely unknown.
---
## 2. Why a new method is needed
| Feature | Current techniques | Limitation |
|---------|--------------------|------------|
| **Spatial resolution** | Conventional western blot or immunohistochemistry: sub‑cellular (whole cell) vs. tissue sections. | Cannot resolve the exact sub‑nuclear compartment where CBP/CREB complexes form. |
| **Temporal resolution** | Time‑course of protein extraction: minutes to hours. | Cannot capture rapid (<1 s) events such as transient phosphorylation upon receptor activation. |
| **Single‑cell / single‑molecule detection** | Flow cytometry, mass spectrometry: bulk measurements. | Lose heterogeneity among neurons; miss rare events. |
| **Live‑cell dynamics** | FRET biosensors: monitor kinase activity in real time. | Do not report direct interaction between CBP and CREB or post‑translational modifications at specific sites. |
Thus, an assay that couples sub‑second temporal resolution with single‑molecule specificity is required to uncover the kinetics of CBP–CREB interactions and the dynamics of post‑translational modifications such as phosphorylation and acetylation.
---
## 2. Proposed Novel Experimental Design
### Overview
We will develop a **single‑molecule, real‑time proximity ligation assay (smPLISA)** that:
1. **Captures** the CBP–CREB complex using nanobody‑functionalized magnetic beads.
2. **Detects** post‑translational modifications on each partner via specific antibodies conjugated to DNA barcodes.
3. **Amplifies** the signal by rolling‑circle amplification (RCA) in a microfluidic chamber, enabling single‑molecule fluorescence readout.
This approach combines proximity ligation (which requires both partners to be present), DNA barcoding for multiplexed PTM detection, and RCA for exponential signal amplification—all compatible with real‑time imaging.
---
### Experimental Design
#### 1. Preparation of Capture Beads
- **Nanobody Selection:** Use high‑affinity nanobodies against the extracellular domain of protein A (e.g., anti‑CD4 nanobody) immobilized on streptavidin‑coated magnetic beads via biotinylated linkers.
- **Blocking:** Block nonspecific sites with BSA.
#### 2. Proximity Ligation Probes
- **Probe Design:** Two DNA oligonucleotides (probe A and probe B) are conjugated to secondary antibodies:
- Probe A: anti‑protein A antibody + a DNA tail ending in a unique sequence "LA".
- Probe B: anti‑protein B antibody (e.g., anti‑CD28) + a DNA tail ending in a complementary sequence "LB".
- **DNA Tail:** The tails contain a short linker region and a unique barcode for each protein pair.
#### 3. Capture of Protein Complexes
- Incubate cell lysate or intact cells with the proximity probes.
- Allow binding; wash to remove unbound antibodies.
- The two antibodies bind adjacent proteins if they are in complex, bringing their DNA tails into close proximity.
#### 4. Proximity Ligation Reaction
- Add a ligase enzyme (e.g., T4 DNA ligase) and a short connector oligonucleotide that can bridge the two DNA tails only when they are sufficiently close.
- If the proteins are physically adjacent (within ~10 nm), the connector is hybridized to both tails and ligation occurs, generating a continuous DNA strand representing the proximity event.
#### 5. PCR Amplification
- Design primers flanking the ligated region (or use universal primers if all connectors have common sequences).
- Perform PCR with high-fidelity polymerase.
- The amplified product contains the connector sequence (unique to each protein pair) and any barcode or restriction site that identifies the specific proteins involved.
#### 6. Sequencing
- Prepare libraries for Illumina sequencing: end-repair, A-tailing, adapter ligation, size selection (~300 bp).
- Sequence paired-end reads sufficient to cover all expected amplicons.
- Use a custom pipeline (e.g., `cutadapt` to trim adapters/primers, `bwa-mem` or `Bowtie2` for alignment) to map reads back to the reference connector sequences.
- Count read pairs per connector; this count reflects the number of DNA fragments bearing that connector.
#### 7. Data Normalization and Quantification
- **Read Depth Normalization**: Divide counts by total mapped reads to obtain CPM (counts per million).
- **Fragment Length Correction**: Since longer fragments generate fewer ligation events, adjust counts based on fragment size distribution (e.g., using a model where probability of ligation ∝ 1/fragment length).
- **Duplicate Removal**: Remove PCR duplicates using UMIs or read pair orientation to avoid overcounting.
- **Statistical Modeling**: Fit a negative binomial model to account for overdispersion, enabling robust estimation of fragment abundance.
#### 8. Validation
- Compare quantified abundances with known input amounts (e.g., spiked-in controls).
- Perform orthogonal measurements such as quantitative PCR or digital droplet PCR on selected fragments.
- Assess reproducibility across technical replicates.
---
By integrating meticulous experimental design, advanced sequencing strategies, and robust computational modeling, the proposed workflow aims to provide an accurate, high-resolution quantification of DNA fragment abundance. This framework is adaptable to diverse genomic contexts, from targeted amplicon panels to whole-genome sequencing, thereby facilitating precise downstream analyses such as variant calling, copy number estimation, and epigenetic profiling. - http://tigerpi.cn:3000/eduardocollett
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