# Abstract

Herein contains the synopsis of my research on MB as a rarely used, highly experimental modifier of mitochondrial metabolism. MB can act as an alternative electron acceptor/donor enhancing the efficiency of energy production. MB tends to accumulate in areas of high metabolic need (e.g. brain tissue). [Phase 3 clinical trials] are under way using Rember© for treatment of Alzheimer’s Disease showing great promise. MB’s action is dose-dependent with many interactions that vary by concentration.

The purification and formulation of MB is paramount to ensure safety and efficacy. Impurities including heavy metals and similarly structured dyes are commonly found in commercial pharmaceutical formulations. The pharmacokinetics of MB depends highly on the structure of the dye and the absorption dynamics. MB can be bought OTC as dry powder or solution in lab, reagent, and sometimes pharmaceutical grade. Purification in a home lab is possible.

## Claims

• Methylene blue can be made safe and non-toxic when properly prepared and administered orally.
• Methylene blue can function as an alternative electron accepter/donor and bypass certain complexes of the ETC.
• Methylene blue has dose dependent effects and proper formulation is essential.

## Author’s Conclusions

MB’s interactions with MAO are the limiting factor in my personal use. The benefits (if real at all) are not sufficient to deprive myself of aged cheddar cheese. I will likely pick up this project again when the completed clinical trial has been published.

## Plain Language Summary

MB may increase performance in many aspects of life including memory, attention, general physical performance, and possibly help prevent certain diseases like cognitive decline. MB has been used for more than a century and has a well known safety profile. Caution is advised for attempting to supplement with MB at home, however. Many over the counter forms may be contaminated with impurities that cause adverse health effects. The effects are radically different at varying doses.

## Abstract

Herein contains the synopsis of my research on MB as a rarely used, experimental modifier of mitochondrial metabolism. MB can act as an alternative electron acceptor/donor enhancing the efficiency of energy production. MB tends to accumulate in areas of high metabolic need (e.g. brain tissue) due to MB’s aromatic and redox nature. [Phase 3 clinical trials] are under way using Rember© for treatment of Alzheimer’s Disease showing great promise. MB’s action is dose-dependent with many interactions that vary by concentration.

The purification and formulation of MB are paramount to ensure safety and efficacy. Impurities including heavy metals and similarly structured dyes are commonly found in commercial pharmaceutical formulations. The pharmacokinetics of MB depends highly on the structure of the dye and the absorption dynamics. MB can be bought OTC as dry powder or solution in lab, reagent, and sometimes illegal pharmaceutical grade and purified at home using easily obtainable chemicals and equipment.

## Physical Characteristics

### Moities

Acetyl-, Leuco-, Hydrates-

## Pharmacokinetics

MB’s absorption is affected by pH, oxidation status, MB has a substantially alterted pharmacokinetics from the more traditional zenobiotics due to the Mitochondrial Membrane Potential (MMP).

### Interactions

Given as the in-vitro EC50:

• Acetylcholinesterase inhibition (1 $$\mu$$M)
• Nitric Oxide Synthase inhibition (5 $$\mu$$M)
• Oxidation of Cysteine Residues (2-30 $$\mu$$M)
• $$\beta$$-Amyloid Aggregation inhibition (2.3-12.4 $$\mu$$M)
• Monoamine Oxidase B inhibition (5.5 $$\mu$$M)
• Glutamatergic inhibition (5-50 $$\mu$$M)
• Noradrenaline Uptake inhibition (50 $$\mu$$M)
• Guanylate Cyclase inhibition (60 $$\mu$$M)
• Monoamine Oxidase A inhibition (0.16 $$\mu$$M)
• Enhanced Mitochondrial $$\beta$$ Oxidation (0.3 $$\mu$$M)

Apparently when MOAIs are mixed with tyramine containing foods, a hypertensive crisis can occur in certain sensitive individuals… Namely, me.

## Methylene Blue Preparation

### Purification

Aiming for >98% purity. Minimizing heavy metal contamination and similarly structured dyes. Rubber gloves are your friend since MB dyes just about anything and at extremely low concentrations. Each method of purification can be applied in any order or combination. As with any purification, more is better but at the cost of greater product loss. Most contamination will be in the form of inorganic metal salts and like structured dyes. Soluble impurities can be made soluble by the addition of a precipitant. Recrystallization allows separation of precipitated product from dissolved impurities. Organic extraction removes similar dyes. I am currently purifying a Methylene Blue product bought online labeled as 93% dye content. Procedures will be administered in this order:

1. Dissolve MB in excess water
3. Filter MB solution and discard filtrete
4. Organic Extraction
6. Heating to vaporize dissolved organic solvent
7. Hot Salt Recrystallization
8. (optional) Cool Acid Recrystallization for recovery

#### Hot Salt Recrystallization

1. 20g crude MB dissolved in 1700 ml H2O at 65$$^{\circ}$$C
3. Cool mixture to 22$$^{\circ}$$C over 3.5 hours
4. Vacuum filter resulting crystals and dry at 75$$^{\circ}$$C for 16 hours
• optional washing with chilled, saturated salt solution

#### Cool Acid Recrystallization

1. 20g crude MB dissolved in 1700 ml H2O at 65$$^{\circ}$$C
2. Cool to 22$$^{\circ}$$C
3. Adjust pH to 1 using aqueous HCl
4. Vacuum filter suspension and dry at 75$$^{\circ}$$C over 16 hours
• optional washing with chilled, HCl solution

#### Organic Extraction

1. Add 20% by volume immiscible organic solvent (DCM, Naptha, toluene) to MB solution
2. Mix extensively for >10 minutes
3. Allow layers to separate
4. Remove organic solvent layer by draining or siphoning
5. Repeat 1-4 times

#### Metal Precipitation

1. 4.5g crude MB dissolved in 125ml at 65$$^{\circ}$$C
2. Cool solution to room temperature
3. Prepare a separate solution of sodium carbonate (163mg Na2CO3 in 10ml H2O)
4. Add sodium carbonate solution to MB
5. Stir for >10 minutes
6. Vaccum filter and collect filtrate for further processing

#### Final Procedure

1. Dissolve 5g crude MB in 230 ml H20
2. Heat solution to 65$$^{\circ}$$C for >30 min while stirring
3. Cool solution to 10$$^{\circ}$$C
4. Prepare solution of 163mg sodium carbonate in 10ml H2O
5. Add Na2CO3 solution to MB solution at once
6. Stir continuously for 15 min
7. Vacuum filter and discard solid products
8. Add 50ml organic solvent (naphtha) to MB solution
9. Stir vigorously for 10 min
10. Siphon or separate aqueous layer from mixture ensuring no organic layer is taken
11. Discard or recycle organic layer
12. Repeat steps 8-11 twice more or as needed to yield colorless organic layer
13. Adjust resulting solution to pH five using dilute HCl
14. Heat solution to 65$$^{\circ}$$C for >10 min
15. Add 118mg sodium chloride per milliliter solution
16. Vacuum filter solution to yield purified crystals of MB
17. Dry crystals at 75$$^{\circ}$$C over 16 hours to achieve final product
18. Optional: repeat all or none of the purification procedures for greater purity or recovering product

### Formulation

Methylene Blue can be consumed as a solution through direct ingestion or formed into capsules. I began with the former for easy formulation and testing. This method leaves something to be desired for long term ease of use and definitely for taste. Depending on the oxidation state of MB, the taste can be somewhat off-putting and possibly cause some throat irritation. The reduced form is almost tasteless. The best option to mitigate these issues is to manufacture pills or capsules. Capsules are somewhat easier and completely mask the taste with little effort. Care must be taken to ensure MB is delivered in the stomach and in the reduced form as much as possible. Degradation of MB can occur in the high pH of the lower gut resulting in uneven and unpredictable effects. Again, quick-dissolving gelatin capsules are ideal delivery vehicles here. In addition, since only small amounts of MB are needed per dose, an inert filler should be added. Optional inclusion of a thixotropic suspending agent can increase optimal absorption by increasing the time for stomach passage. The addition of ascorbic acid will reduce MB in the low pH of the stomach to its more effective leuco form.

Further research is necessary to determine what effect the absorption and pharmacological effects of the alternate forms of MB would have on the results of previous trials and research. It may very well be the in vitro studies showing almost an order of magnitude increase in mitochondrial respiration with MB exposure are not translated well to humans simply because of the pharmacokinetic differences. Some research indicates that only leuco-MB crosses the BBB. Still, other evidence points to equal forms of color and colorless forms of MB in serum. Most likely this is caused by the balancing effects of other antioxidant compounds and electron donors in the blood stream. In any case, it can’t hurt to include an innocuous reducing compound in the formula.

#### Dosing

Clinical trials with Rember™ indicate good performance for patients with cognitive decline in the 30-100mg three time daily formulation. It is worth noting that the 100mg dose suffered from absorption issues and subsequently lost some of its efficacy. Many subjective reports from the community (/r/nootropics, longecity) indicate best performance at three orders of magnitude lower doses. I will summarily dismiss these reports as a combination of poor methodology, placebo, and the limitation of adverse hematological effects. The intended benefits are dose dependent so the formulation must reflect the goal of the user.

#### Liquid Formulation

Maximum concentration of MB in water is approximately 43mg/ml (4.3%). The addition of ascorbic acid further reduces solubility. It is therefore best to aim for 1-2% concentration. If an accurate scale is available, daily preparation of solutions are possible. The scale that I am using at the moment has an accuracy of only 0.01g so I usually prepare large batch which can be portioned out as needed. As an example formula for a month’s supply:

• Dissolve 5.4g MB in 270 ml H20
• Stir for >10min
• Consume 3ml three times daily

Solution is easily stored in 10ml syringes. Consumption can be done by directly squirting the solution to the back of the tongue. Although, this method results in a very blue tongue. An alternative is to dispense the solution into a larger amount of water to consume over several minutes. Of course, you could dispense an entire day’s worth of MB into a water bottle and sip throughout the day resulting in very even serum concentrations. I’m usually to busy/distracted to remember to do this method and end up with half drunk bottles at bedtime.

With those issues in mind I have chosen easily available products to create this formula. Microcrystalline cellulose will function as the filler and excipient. I was able to obtain a sample of Aerosil 200, a commercial colloidal silicon dioxide product intended as a thixotropic suspending agent. The ideal dose as indicated by the clinical trials of Rember will be between thirty and ninety milligrams.

## Unsorted

• cationic thiazine dye
• low redox potential - efficient cycling
• tyramine induced hypertensive crisis
• genotoxic effects with concurrent light exposure
• light exposure with MB alters function during in vitro experiments (photosensitized)
• major metabolites, azure B & A, and their effects on MAOs

[ETC]: Electron Transport Chain [MMP]: Mitochondrial Membrane Potential