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Pharmaceutical-Grade Methylene Blue Trihydrate (MB·3H₂O)

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Comprehensive Clinical Evidence Evaluation for NTRPX Systems

Document Version: 1.0
Date: January 23, 2026
Classification: Internal R&D Evaluation
Compound: Methylene Blue Trihydrate (MB·3H₂O), ≥99.5% HPLC purity
Chemical Name: Methylthioninium chloride trihydrate

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Executive Summary

RECOMMENDATION: DO NOT INCLUDE IN NTRPX SYSTEMS Pharmaceutical-grade methylene blue presents a compelling theoretical mechanism but fails to meet NTRPX standards for three critical reasons:
  1. Insufficient independent replication of cognitive enhancement effects in healthy adults
  2. Zero human RCTs demonstrating athletic/physical performance benefits
  3. Serious drug interaction profile (MAO-A inhibition) creating unacceptable liability for a consumer product
While the mechanistic rationale is scientifically elegant and the compound has legitimate medical applications, the evidence base does not support inclusion in All Systems Go (Boost, Sustain, Recover) or Sprint Systems.

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Table of Contents

  1. Compound Overview
  2. Mechanism of Action
  3. Clinical Evidence Hierarchy
  4. Safety Profile & Contraindications
  5. NTRPX Systems Fit Analysis
  6. Verdict & Rationale
  7. References

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1. Compound Overview

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Identity

PropertyValue
Chemical FormulaC₁₆H₁₈ClN₃S·3H₂O
Molecular Weight373.90 g/mol (trihydrate)
CAS Number7220-79-3 (trihydrate)
AppearanceDark green crystalline powder
SolubilityFreely soluble in water (blue solution)
pKa~0 (strongly basic)

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Regulatory Status

  • FDA Status: Grandfathered drug; approved for methemoglobinemia (Provayblue®)
  • WHO Essential Medicines List: Yes (for methemoglobinemia, malaria)
  • WADA Status: NOT prohibited
  • Supplement Status: Sold as “nootropic” supplement without FDA oversight

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Historical Context

Methylene blue was synthesized in 1876 as a textile dye, becoming the first fully synthetic drug used in medicine. Paul Ehrlich and Ramón y Cajal used it as a supravital nervous tissue stain in the 1890s. It has legitimate medical applications for methemoglobinemia, carbon monoxide poisoning, cyanide poisoning, and vasoplegic syndrome.

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2. Mechanism of Action

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2.1 Primary Mechanism: Mitochondrial Electron Cycling

Methylene blue’s purported nootropic effects derive from its unique redox chemistry in mitochondria: 
┌─────────────────────────────────────────────────────────────────┐
│               MITOCHONDRIAL ELECTRON TRANSPORT CHAIN            │
├─────────────────────────────────────────────────────────────────┤
│                                                                 │
│   NADH → Complex I → CoQ → Complex III → Cyt c → Complex IV → O₂│
│              ↓                                    ↑             │
│         [electron                            [electron          │
│          leakage]                             donation]         │
│              ↓                                    ↑             │
│            ROS ←──────── MB ──────────────────────┘             │
│                     (alternative                                │
│                      electron                                   │
│                      carrier)                                   │
│                                                                 │
│   MB accepts electrons from NADH, bypasses Complexes I-III,     │
│   donates directly to cytochrome c → increases Complex IV       │
│   activity → enhanced ATP production + reduced ROS              │
│                                                                 │
└─────────────────────────────────────────────────────────────────┘
Key biochemical effects at LOW doses (0.5-4 mg/kg):
  • Increases cytochrome c oxidase (Complex IV) activity by up to 138%
  • Increases cellular oxygen consumption by up to 70%
  • Boosts ATP production by approximately 30-40%
  • Reduces superoxide/ROS generation
  • Upregulates Nrf2/ARE antioxidant signaling

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2.2 Hormetic Dose-Response (CRITICAL)

Methylene blue exhibits a biphasic (hormetic) dose-response curve—the same property that makes it mechanistically interesting also makes it dangerous in consumer products: 
        ↑ Benefit
        │
        │           ╭───────╮
        │          ╱         ╲
        │         ╱           ╲
        │        ╱             ╲
        │       ╱               ╲
   0────┼──────╱─────────────────╲────────────────→ Dose
        │                         ╲
        │                          ╲
        │                           ╲
        ↓ Harm                       ╲____

        └──┬──┘    └────┬────┘     └────┬────┘
         Low      Therapeutic        High
       (<0.5)     (0.5-4 mg/kg)    (>7 mg/kg)
Dose RangeEffectMechanism
0.5-4 mg/kgBeneficialElectron donation, enhanced ETC efficiency
4-7 mg/kgDiminishing returnsApproaching saturation
>7 mg/kgHarmfulElectron theft from ETC, pro-oxidant, toxicity
>10 mg/kgDangerousParadoxical methemoglobinemia, hemolysis
Implication for NTRPX: The narrow therapeutic window leaves minimal margin for error. Individual variation in absorption, metabolism, and body weight means a “one-size-fits-all” dose is inherently problematic.

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2.3 Secondary Mechanisms

PathwayEffectRelevance
MAO-A InhibitionIncreases serotonin, NE, DACREATES DRUG INTERACTION RISK
Nitric Oxide SynthaseInhibits NOS at high dosesMay reduce blood flow at high doses
Guanylate CyclaseInhibits cGMP productionAntidepressant mechanism
Tau AggregationInhibits tau protein aggregationAD research focus
Amyloid-βMay reduce Aβ accumulationAD research focus

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3. Clinical Evidence Hierarchy

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3.1 Summary Evidence Table

Evidence TypeCountQualityRelevance to NTRPX
Meta-analyses in healthy adults0N/A❌ None exist
Independent RCTs (healthy cognition)0N/A❌ All from single lab
RCTs from Gonzalez-Lima lab2-3Moderate⚠️ No independent replication
RCTs in disease states (AD, PTSD)4-5Mixed⚠️ Disease, not optimization
Exercise/performance RCTs0N/A❌ Zero studies
Preclinical (animal/in vitro)50+Good⚠️ Mechanism established

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3.2 Key Human Studies

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Study 1: Rodriguez et al. 2016 (THE Primary Evidence)

Publication: Radiology, 281(2):516-26 [PMC5084971]
Design: Double-blind, randomized, placebo-controlled
Population: n=26 healthy adults (ages 22-62)
Intervention: Single dose 280mg oral USP methylene blue (~4 mg/kg)
Outcomes:
  • Memory retrieval improved by 7% (P=0.01)
  • Increased fMRI activity in bilateral insular cortex during sustained attention
  • Increased fMRI activity during short-term memory tasks
Strengths:
  • Rigorous design (double-blind, placebo-controlled)
  • fMRI biomarker confirmation
  • Used USP-grade pharmaceutical MB
Limitations:
  • Very small sample (n=26)
  • Single research group (no independent replication)
  • Acute study only (1 hour post-dose)
  • No long-term or chronic dosing data
NTRPX Assessment: This is the ONLY high-quality RCT showing cognitive enhancement in healthy humans. A single small study from one lab is insufficient for NTRPX standards.

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Study 2: Functional Connectivity Study (Rodriguez et al. 2017)

Publication: Neuroimage [PMC5018244]
Design: Double-blind, randomized, placebo-controlled
Population: n=28 healthy adults
Intervention: Single dose 280mg oral methylene blue
Outcomes:
  • Enhanced resting-state functional connectivity
  • Modulated task-related network deactivation
  • NO significant cognitive performance improvement (only neural correlates)
NTRPX Assessment: Supports mechanism but did NOT show behavioral/cognitive benefit.

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Study 3: Fear Extinction (Telch et al. 2014)

Design: RCT
Population: n=42 subjects with claustrophobia
Intervention: 260mg/day methylene blue
Outcomes:
  • Improved fear extinction at 1-month follow-up
  • Improved contextual memory
NTRPX Assessment: Disease state (phobia), not cognitive optimization in healthy individuals.

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Study 4: PTSD (Zoellner et al. 2017)

Design: RCT
Population: n=26 patients with PTSD
Intervention: 260mg daily for 3 months
Outcomes: Moderate cognitive-enhancing effect vs. waitlist control NTRPX Assessment: Disease state, waitlist control (not true placebo), small sample.

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Study 5: Alzheimer’s Disease Phase II (Wischik et al. 2015)

Design: RCT, 24 weeks
Population: n=321 mild-moderate AD patients
Intervention: 69, 138, or 228 mg/day methylthioninium chloride
Outcomes:
  • Patients with moderate AD: ADAS-cog improved 5.42 points vs. placebo
  • Patients with mild AD: NO significant cognitive benefit
  • Reduced decline in cerebral blood flow
NTRPX Assessment: Disease state (AD). Notably, mild AD showed no benefit—suggesting MB may only help impaired, not optimal, cognition.

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Study 6: Postoperative Cognitive Dysfunction (Deng et al. 2021)

Design: Open-label RCT
Population: Elderly patients undergoing major non-cardiac surgery
Intervention: 2 mg/kg IV methylene blue
Outcomes: Reduced incidence of postoperative delirium and cognitive dysfunction NTRPX Assessment: Disease state (surgery-induced cognitive impairment), IV administration.

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3.3 Exercise & Physical Performance Evidence

STATUS: ZERO HUMAN RCTs
ClaimEvidence LevelSource
”Boosts ATP production”Preclinical/mechanisticCell culture studies
”Improves endurance”TheoreticalExtrapolated from mitochondrial mechanism
”Reduces muscle fatigue”TheoreticalNo human performance studies
”Enhances oxygen utilization”PreclinicalRat brain studies
”Speeds recovery”TheoreticalNo human studies
Critical Finding: Every performance claim in the supplement industry is based on extrapolation from: (1) mitochondrial mechanisms established in cell culture, or (2) cognitive studies that measured fMRI, not physical performance. No study has measured VO2max, time to exhaustion, power output, or any athletic metric.

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4. Safety Profile & Contraindications

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4.1 CRITICAL: MAO-A Inhibition & Serotonin Syndrome

This is the disqualifying safety concern for NTRPX integration. Methylene blue is a potent reversible MAO-A inhibitor at nanomolar concentrations (Ki for MAO-A inhibition is in the nanomolar range). This creates severe interactions with:
Drug ClassExamplesInteraction Severity
SSRIsfluoxetine, sertraline, escitalopram⛔ LIFE-THREATENING
SNRIsvenlafaxine, duloxetine⛔ LIFE-THREATENING
MAOIsphenelzine, selegiline⛔ LIFE-THREATENING
TCAsclomipramine, amitriptyline⛔ SEVERE
Opioidstramadol, meperidine, fentanyl⛔ SEVERE
Otherdextromethorphan, buspirone, St. John’s Wort⚠️ MODERATE-SEVERE
FDA Black Box Warning (2011): The FDA issued a safety communication warning of serious CNS reactions when methylene blue is given to patients on serotonergic medications. Serotonin Syndrome Symptoms:
  • Mental status changes (agitation, confusion, hallucinations)
  • Autonomic instability (tachycardia, hyperthermia, diaphoresis)
  • Neuromuscular symptoms (tremor, rigidity, myoclonus)
  • Can progress to seizures, coma, death
Market Reality: Approximately 13% of US adults take antidepressants. Including MB in a consumer supplement creates unacceptable risk of serotonin syndrome in a significant portion of potential users.

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4.2 Absolute Contraindication: G6PD Deficiency

Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects approximately 400 million people worldwide and ~2-3% of the US population. Mechanism of Harm:
  • MB requires NADPH to function
  • G6PD deficiency → insufficient NADPH production
  • MB administration → NADPH depletion → hemolytic anemia
  • Can cause severe, life-threatening hemolysis
Affected Populations (Higher Prevalence):
  • African descent (~10-14%)
  • Mediterranean descent (~4-12%)
  • Middle Eastern descent (~5-10%)
  • Southeast Asian descent (~variable)
NTRPX Implication: Including MB would require either: (1) mandatory G6PD testing before purchase, or (2) accepting risk of causing hemolytic anemia in ~2-10% of users depending on demographics.

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4.3 Other Safety Considerations

ConcernDetailsRisk Level
Blue urine/stoolExpected, harmlessLow (cosmetic)
GI upsetNausea, abdominal discomfortLow-Moderate
PregnancyCategory X teratogen (intra-amniotic data)⛔ Contraindicated
NursingUnknown excretion in breast milk⚠️ Avoid
Renal impairmentReduced clearance⚠️ Dose adjustment needed
High-dose toxicityMethemoglobinemia, cardiac effectsAt doses >7 mg/kg
PhotosensitivityMB is a photosensitizerLow at oral doses

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5. NTRPX Systems Fit Analysis

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5.1 All Systems Go: Boost

CriterionMethylene BlueNTRPX StandardPass/Fail
Acute cognitive enhancement7% memory improvement (1 RCT, n=26)Multiple independent RCTs❌ FAIL
Energy/alertnessTheoretical (mitochondrial)Proven acute effect❌ FAIL
Safety for daily useMAO-A inhibition, drug interactionsBroad population safety❌ FAIL
Synergy with existing stackUnknown; potential conflictsDemonstrated compatibility❌ FAIL
Verdict for Boost: DOES NOT FIT

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5.2 All Systems Go: Sustain

CriterionMethylene BlueNTRPX StandardPass/Fail
Chronic cognitive support1 study (PTSD, not healthy)Long-term RCTs in target population❌ FAIL
NeuroprotectionStrong preclinical, limited humanHuman validation required⚠️ INSUFFICIENT
Safe for long-term useUnknown; no chronic studies in healthy adultsLong-term safety data❌ FAIL
Consistent dosingHormetic curve requires precisionForgiving dose-response❌ FAIL
Verdict for Sustain: DOES NOT FIT

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5.3 All Systems Go: Recover

CriterionMethylene BlueNTRPX StandardPass/Fail
Recovery enhancementZero human studiesRCTs showing recovery benefits❌ FAIL
Anti-inflammatoryPreclinical onlyHuman inflammation data❌ FAIL
Sleep qualityNo dataSleep architecture studies❌ FAIL
Muscle recoveryNo dataRecovery biomarker studies❌ FAIL
Verdict for Recover: DOES NOT FIT

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5.4 Sprint Systems

CriterionMethylene BlueNTRPX StandardPass/Fail
Acute performanceZero RCTsDemonstrated acute ergogenic effect❌ FAIL
Exercise capacityZero RCTsVO2max/TTE/power studies❌ FAIL
EnduranceTheoretical onlyHuman endurance trials❌ FAIL
WADA complianceNot prohibitedClean status✅ PASS
Safety in exerciseUnknownSafe under exertion❌ UNKNOWN
Verdict for Sprint: DOES NOT FIT

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6. Verdict & Rationale

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6.1 Final Recommendation

┌─────────────────────────────────────────────────────────────────┐
│                                                                 │
│   RECOMMENDATION: DO NOT INCLUDE IN NTRPX SYSTEMS               │
│                                                                 │
│   Confidence: HIGH                                              │
│                                                                 │
│   Applies to: All Systems Go (Boost, Sustain, Recover)          │
│               Sprint Systems                                     │
│                                                                 │
└─────────────────────────────────────────────────────────────────┘

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6.2 Evidence Gap Analysis

What would be needed to reconsider:
GapCurrent StateRequired Evidence
Independent replication0 studies outside Gonzalez-Lima lab≥2 independent RCTs showing cognitive enhancement
Sample sizen=26 (largest healthy adult study)n≥100 adequately powered study
Chronic dosingNo long-term data in healthy adults≥12-week chronic administration study
Exercise performanceZero studies≥1 RCT with performance endpoints
Drug interaction mitigationMAO-A inhibition is intrinsicCannot be mitigated without changing the molecule
G6PD risk~2% population contraindicatedCannot be mitigated in consumer product

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6.3 Comparison to NTRPX Standards

NTRPX PrincipleMethylene Blue Status
”Evidence over theory”❌ Strong theory, weak human evidence
”Proven over promising”❌ Promising mechanism, unproven in target use
”No compromises on safety”❌ MAO-A inhibition, G6PD contraindication
”Independent replication required”❌ Single research group
”Target population validation”❌ Mostly disease states, not optimization
”Broad population applicability”❌ Excludes SSRI users (~13%), G6PD deficient (~2%)

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6.4 Why the Biohacker Enthusiasm is Misleading

Methylene blue has gained significant popularity in biohacker communities based on:
  1. Mechanistic elegance - The mitochondrial electron cycling mechanism is scientifically compelling
  2. Historical pedigree - 140+ years of medical use
  3. Subjective reports - User testimonials (not controlled data)
  4. Influencer promotion - High-profile endorsements without evidence review
  5. Longevity association - Connection to mitochondrial health/aging research
The problem: None of this constitutes evidence of efficacy for cognitive or physical performance enhancement in healthy adults. The biohacker community has conflated “interesting mechanism” with “proven benefit.”

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6.5 Appropriate Use Cases (Outside NTRPX)

Methylene blue does have legitimate applications:
Use CaseEvidence LevelAppropriate?
Methemoglobinemia (medical)FDA-approved✅ Yes
Vasoplegic syndrome (medical)Clinical evidence✅ Yes
Research toolN/A✅ Yes
Individual biohacker experimentationN/APersonal risk acceptance
Consumer supplement productInsufficient❌ No

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7. References

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Primary Clinical Studies

  1. Rodriguez P, Zhou W, Barrett DW, et al. Multimodal Randomized Functional MR Imaging of the Effects of Methylene Blue in the Human Brain. Radiology. 2016;281(2):516-526. [PMC5084971]
  2. Rodriguez P, Singh AP, Malloy KE, et al. Methylene blue modulates functional connectivity in the human brain. Neuroimage Clin. 2017;15:252-257. [PMC5018244]
  3. Telch MJ, Bruchey AK, Rosenfield D, et al. Effects of post-session administration of methylene blue on fear extinction and contextual memory in adults with claustrophobia. Am J Psychiatry. 2014;171(10):1091-1098.
  4. Zoellner LA, Telch M, Foa EB, et al. Enhancing Extinction Learning in Posttraumatic Stress Disorder With Brief Daily Imaginal Exposure and Methylene Blue. J Clin Psychiatry. 2017;78(7):e782-e789.
  5. Wischik CM, Staff RT, Wischik DJ, et al. Tau aggregation inhibitor therapy: an exploratory phase 2 study in mild or moderate Alzheimer’s disease. J Alzheimers Dis. 2015;44(2):705-720.
  6. Deng Y, Wang R, Li S, et al. Methylene blue reduces incidence of early postoperative cognitive disorders in elderly patients undergoing major non-cardiac surgery. J Clin Anesth. 2021;68:110108.

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Mechanism & Review Articles

  1. Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue. Prog Neurobiol. 2012;96(1):32-45. [PMC3265679]
  2. Tucker D, Lu Y, Zhang Q. From Mitochondrial Function to Neuroprotection—An Emerging Role for Methylene Blue. Mol Neurobiol. 2018;55(6):5137-5153. [PMC5826781]
  3. Gonzalez-Lima F, Auchter A. Protection against neurodegeneration with low-dose methylene blue and near-infrared light. Front Cell Neurosci. 2015;9:179. [PMC4428125]
  4. Bruchey AK, Gonzalez-Lima F. Behavioral, Physiological and Biochemical Hormetic Responses to the Autoxidizable Dye Methylene Blue. Am J Pharmacol Toxicol. 2008;3(1):72-79. [PMC2867617]

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Safety & Drug Interactions

  1. Gillman PK. Methylene blue implicated in potentially fatal serotonin toxicity. Anaesthesia. 2006;61(10):1013-1014.
  2. Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity: inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. Br J Pharmacol. 2007;152(6):946-951. [PMC2078225]
  3. FDA Drug Safety Communication: Updated information about the drug interaction between methylene blue and serotonergic psychiatric medications. 2011.
  4. Methylene Blue - StatPearls. NCBI Bookshelf. [NBK557593]
  5. Blue cures blue but be cautious. J Lab Physicians. 2011;3(2):127-129. [PMC3249703]

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Document Control

VersionDateAuthorChanges
1.02026-01-23NTRPX R&DInitial comprehensive evaluation
This document represents NTRPX’s internal evaluation based on available clinical evidence as of the document date. The recommendation to exclude methylene blue from NTRPX Systems is based on the compound’s failure to meet NTRPX’s evidence standards, not a judgment on its potential for future development or individual biohacker use.