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Methylene Blue Research

methylene blue

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#31 geddarkstorm

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Posted 09 March 2009 - 05:31 PM

Neurons are postmitotic, so can they even go senescent? Senescence doesn't seem to me to be a very useful thing to target. From p. 233 of Ending Aging

The use of the word "senescent" to describe these cells is, however, a bit misleading. When people hear about these cells, they often assume that cellular "senescence" is the ultimate fate of all of the cells in the body with age, and that the entry of "young" cells into this senescent state is the underlying cause of aging. [. . .] In fact, senescent cells are generally held to be extremely rare even in very aged people.


I don't quite think that is completely accurate in the sense of how we kinda use "senescence" these days. It's absolutely true in that non-replicating cells are said to be senescent, and differentiated cells for the most part do not replicate. On the other hand, cells that are losing their biological function and role, and usually experiencing mitochondrial dysfunction, is what is kinda also termed as "senescence", and that is a big part of aging, or an additional meaning to the word anyways. Right or wrong use of the word aside, that's how I use it (and consequently why frequently in the literature "senescence" is thought to cause aging; the question is what causes "senescence").

On the other hand, part of aging is the loss of the function of stem cells (possibly in part due to losing telomere length later on in life), and there are stem cells for the brain, retina, and just about every other tissue in the body. Without them, we can't regenerate and of course age rapidly to death. So, there you want to stop senescence in every term of the word.

His solution is to just target senescent cells for elimination. But if you want to slow the process down, alternatives to MB include resveratrol, carnosine, MitoQ (And maybe ubiquinol too. It did help those SAMP mice.), Alcar/RALA, and oleuropein. Most of that list also treats mitochondrial dysfunction, so I wouldn't expect MB to provide much in the way of an additional benefit to the heavier supplement stacks.


Most of those are great choices and highly useful, but they do lack one interesting thing that MB has, that is certain direct chemistry. MitoQ, ubiquinone for example, is already abundant and everywhere, so increasing its abundance is mostly going to help in cases where the loss of ubiquinone is playing a major part in the cause or symptoms displayed. All of the others, sans resveratrol, fall mostly into that same catagory, though resveratrol acts in a totally different manner to signal stress response and mitochondrial biogenesis, it still isn't going to answer mitochondrial dysfunction, most likely (it mostly only answers the Sirt1 loss part of aging, still have mitochondrial dysfunction and telomere length to contend with).

MB on the other hand, has a unique chemical role likely not directly monitored or balanced by the body (which limits the impacts of many of the other supplements). It can directly take back-flow (ROS) producing electrons from Complex I, directly drive electron flow through the electron transport chain (ECC) via cytochrome c (ubiquinone is already part of the ECC, and more of it won't do much unless it was depleted and rate limiting), and stimulates the increase of Complex IV, the oxygen redox center and one of the ECC flux limiters. Maybe there is something else that does those things and better or not, but I have yet to run into it. Let alone its in vivo effects, such as significant enhancement of the rat brain at least, which is a simple result of ECC increase. If other factors did they same, we'd expect to see that too.

There's a lot of stuff out there though, so who knows ;)

That was an acute effect, and I believe that subtle chronic effects are much harder to discover.


Maybe, but significant changes in rate are usually easy enough to see via meta analysis, at least. We've had no lack of time or lack of use to see it.

To put the photosensitizing thing to rest, there was an interesting bit of info in the intro of this paper:

"The ability of MB to act as a redox indicator, explains the
rapid reduction to stainless MBH, which is not photo-
dynamically active when given to the organism orally or
intravenously [25]. Therefore, PDT with systemically
applied MB failed in the treatment of superficial bladder
cancer [26].
"

Indeed, MB is not used systemically for PDT, as it is non reactive when given orally. Instead it is directly injected at high concentration before it's useful.

In fact, that same paper also found that MB was unsuccessful at killing cancer cells below levels that were already toxic to the cells. They used 1% MB and directly injected 0.1ml (1mg of MB, or 50mg/kg levels injected into the mouse if as a whole, but actually a lot more considering just into the tumor, size of which was not reported) into the mouse tumor (which subsequently turned deep blue) and found it inhibited tumor growth without PDT the same that 0.1% MB inhibited with PDT, and only 1% killed any cells. Also, 0.01% MB was not photoreactive even with direct injection to the tumor and did not affect tumor growth (that is a 0.5mg/kg injection into the mouse if as a whole; contrast to the 0.015mg/kg of 1mg orally to a human).

The lack of action of MB when given orally is likely because ~75% is converted to the leuco form (makes no difference for the mitochondrial effects, as seen in vivo and in vitro, but it means the effective MB+ concentration of a 100nM dose is only ~25nM), so there's no way to get the absurdly high amounts needed for photosensitization in vivo.

Edited by geddarkstorm, 09 March 2009 - 05:46 PM.


#32 AgeVivo

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Posted 09 March 2009 - 08:42 PM

Apparently there has been evidence of MB increasing life span in mice, but since we're taking about a much lower dose than that used for most medicinal purposes, it's been largely overlooked.

Were did you see the MB increases life span of mice?
except this paper where it extends the life span of mice... with cancer:

Antitumor effect of methylene blue in vivo
Methylene blue can inhibit the growth of Ehrlich ascitic tumor, L1210 leukemia and P388 leukemia in mice. The average life span of the treated animals was obviously longer than that of the controls.

it has also been shown, at lower than medicinal doses, in rats, to significantly increase learning of all types, and memory

Which paper is that?

If we were to test (MPrize @home) if MB extends mouse lifespan,
  • would you advize MB or smthg else?
  • which formulation?
    Based on the current UK clinical trial of MB against Alzheimer, MB would be effective when dissolved in the stomach, but not when absorbed through the intestines (!)
  • which concentration in water?
    Warning, unlike humans mice die very easily from diarrhea and 'because the 100-mg dose dissolved in the intestines, it was more likely to cause diarrhea, which was reported by about 30% of patients and was the most common adverse event'
  • where to buy it?
    "the drug used in his trial was "much purer" than the formulation currently available"
  • which placebo (blue food colorant) to buy?
    certainly in your rat paper. For the UK trial, 321 patients were randomized to 30 mg, 50 mg, 100 mg or placebo

    Thanks


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#33 geddarkstorm

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Posted 09 March 2009 - 09:26 PM

Apparently there has been evidence of MB increasing life span in mice, but since we're taking about a much lower dose than that used for most medicinal purposes, it's been largely overlooked.

Were did you see the MB increases life span of mice?
except this paper where it extends the life span of mice... with cancer:

Antitumor effect of methylene blue in vivo
Methylene blue can inhibit the growth of Ehrlich ascitic tumor, L1210 leukemia and P388 leukemia in mice. The average life span of the treated animals was obviously longer than that of the controls.


If you read through the thread, you'd see where the NTP toxicology study on MB was done. In that study on toxicity of MB, they saw that "Survival of dosed male and female groups exceeded that of the vehicle controls in a generally dose-related manner."

it has also been shown, at lower than medicinal doses, in rats, to significantly increase learning of all types, and memory

Which paper is that?


There are several papers: where it increases significantly discrimination learning, memory (and here), and improves brain metabolism (and memory). There are more papers than these, but I just grabbed a few representative ones.


If we were to test (MPrize @home) if MB extends mouse lifespan,

  • would you advize MB or smthg else?
  • which formulation?
    Based on the current UK clinical trial of MB against Alzheimer, MB would be effective when dissolved in the stomach, but not when absorbed through the intestines (!)
  • which concentration in water?
    Warning, unlike humans mice die very easily from diarrhea and 'because the 100-mg dose dissolved in the intestines, it was more likely to cause diarrhea, which was reported by about 30% of patients and was the most common adverse event'
  • where to buy it?
    "the drug used in his trial was "much purer" than the formulation currently available"
  • which placebo (blue food colorant) to buy?
    certainly in your rat paper. For the UK trial, 321 patients were randomized to 30 mg, 50 mg, 100 mg or placebo

    Thanks


Methylene blue is absorbed very well through the gut, at 50-75%, so oral administration is no problem what so ever. MB answers only one of the, what I believe to be, three primary pathways of aging -- that is it helps to fix mitochondria dysfunction by directly interacting with and enhancing (even driving) the electron transport chain. It also can drive glucolysis and keep flux going via chemical interactions. MB is usually given in an aqueous solution, so just MB with water is all one needs, food has no real impact that I know of either. You want to dilute MB down however, at least to 0.01%, to avoid staining.

MB can be bought in many locations, but it differs greatly per place in this country.

Any blue food colorant should do, as long as it isn't MB! haha

#34 geddarkstorm

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Posted 09 March 2009 - 10:58 PM

Oh, and about dosage, which I don't think you asked about?, but which deserves mention:

To get around 100nM in a human (generally have five liters of blood, and also dry weight), you want around 1mg of MB for a standard person. That is, about 0.015mg/kg (0.01-0.02), this is estimated to be most effective at enhancing mitochondria, oxidative defense, and other metabolic parameters in human cells. So, if you wanted to do a "life span" trial in humans, you'd want to use 0.1mg, 1mg, and 10mg.

Another interesting thing to consider is the brain studies in rats, which generally use 1mg/kg, or a human effective dose of 0.17mg/kg. Since the enhancement follows from the same effect by MB that enhances mitochondria, and the ECC in particular, the doses above may likely cover it, since 100nM sweet spot was for human cells not rats. On the other hand, the rough 10mg would give the straight equivalent dose if there isn't a species effect. Remember, that this is 10 times the "optimal" dose seen in vitro in human cells, and that 10 times over that optimal dose halved the beneficial effects of MB in general, but at least they were still there. So a test using 0.1mg, 1mg, and 10mg would allow a nice spread in a trial. (10mg is still over 20 times less than what has been used medicinally for nearly a century or more)

Note, this is still a lot lower than the amount being kicked around for anti-Alzheimer's use (60mg). That's because they are shooting for levels to directly break up tau and beta plaques. Alternatively, one may be able to fight Alzheimer's by preventing the characteristic mitochondria dysfunction that accompanies the disease, which would need the lower amounts I talked about above.

#35 krillin

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Posted 10 March 2009 - 04:17 AM

Maybe there is something else that does those things and better or not, but I have yet to run into it. Let alone its in vivo effects, such as significant enhancement of the rat brain at least, which is a simple result of ECC increase. If other factors did they same, we'd expect to see that too.

Why doesn't the alcar/RALA brain rejuvenation (PMID: 11854529) count?

Maybe, but significant changes in rate are usually easy enough to see via meta analysis, at least. We've had no lack of time or lack of use to see it.

We do have a lack of motivation: it's a grandfathered drug usually used for short periods of time. Congenital methemoglobinemia is about the only reason it'd be given chronically and "Although an autosomal recessive form of methemoglobinemia was described in 1845, it is so rare that no known incidence and prevalence has been established."

#36 gattaca

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Posted 10 March 2009 - 05:42 AM

Methylene Blue is pretty safe. Find me a clean source to buy from and I'll try it, as the board's guinea pig.

A CLEAN source. I'm not eating aquarium products or slurping down mexican holistic remedies like some kind of junkie.

#37 geddarkstorm

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Posted 11 March 2009 - 02:33 AM

Why doesn't the alcar/RALA brain rejuvenation (PMID: 11854529) count?


Where did I say they didn't count? r-Lipoic acid an essential cofactor for the pyruvate dehydrogenase complex (glycolysis), while carnitine is the molecule used to transport long chain fatty acids across the mitochondrial membrane so that they can be broken down (fatty acid oxidation). RALA and ALCAR have their vital uses for driving metabolism of sugars and fats and stimulating metabolic pathways, and since their levels decline with age, they can obviously partially rejuvenate mitochondria and cells in cases where those pathways are becoming defunct, such as during aging. That is, supplementing with them will potentially reverse dysfunction associated with their decline, and enhance function of tissues like muscle and brain, another reason they are so useful. However, neither lipoic acid nor ubiquinone (nor carnitine, though this link isn't about it) extend life span by themselves (but, they do increase health, no doubt about that!) in a mouse nor prevent age related transcriptional alterations in metabolic pathways. This is because their loss isn't a cause, but a symptom of aging. Finally, neither directly interacts with the ECC or can directly donate electrons and drive ECC function as far as I know, like MB apparently can (one reason it's a major antidote to cyanide). Therefore they aren't like MB in that sense, which was what I was talking about; I haven't yet run into other supplement factors that do that same action, though that doesn't mean there aren't any out there (which was another point I made ;)).

We do have a lack of motivation: it's a grandfathered drug usually used for short periods of time. Congenital methemoglobinemia is about the only reason it'd be given chronically and "Although an autosomal recessive form of methemoglobinemia was described in 1845, it is so rare that no known incidence and prevalence has been established."


There are three different types of it. Two are recessive (type I "benign" and type II "severe", both are different genetic causes) while the third is a dominate mutant, also known as Hemoglobin M (which methylene blue cannot treat, unlike MB's use to treat the first two). Around 33 different mutations have been identified that lead to the first two types of methemoglobinemia, found in diverse populations from Chinese to African American. Since it follows Mendelian genetics, it is rare but not unheard of. Most likely a major limit in calculating a population rate is how often it might go undiagnosed, in particular for the type I (especially since it can be misdiagnosed as hypertension).

None the less, there's a reason why there's an NTP toxicology study on MB (2008 too, MB is not a grandfather drug, it's actively being explored and analyzed for a variety of purposes -- especially if it'll be used in Alzheimer's, you can bet it's under a lot of scrutiny!), which looked at it chronically in rats and mice, and at very high doses. I think we have quite a good idea what chronic effects of it are in mammals therein (we'll certainly find out even more from the Alzheimer's studies, too). Remember, that MB works amazingly well as an antioxidant, in vivo, at moderate concentrations, but all antioxidants I know of can become pro-oxidant at very high levels. Even so, it's highly unlikely you can chronically ingest enough to cause photosensitization or oxidative stress (need even more than the 24mg/kg used in malaria treatment, which affects the parasite specifically due to its unique reductases; that's a huge MB dose; the LD50 for MB is somewhere between 200mg/kg and 500mg/kg as calculated from rats and mice respectively), as you'll hit its other, nastier, more potent and acute toxic effects at the high doses needed to cross that threshold, like MB induced methemogloginemia (at over 15mg/kg dosage, and you'll possibly even start to turn a pretty hue of blue by that point).

If you want to keep discussing potential or not chronic affects, I should point out the chronic effects of a good number of the supplements taken around here, especially at the doses they are taken, are unknown. On top of that, the 0.015mg/kg I'm talking about for MB is so much lower than we see used anywhere else, including toxicology studies, that I doubt one has to worry: the benefits are potentially huge, and the risks can easily be mitigated by a variety of other useful supplements and antioxidants, especially vitamin C and E, even resveratrol.

Methylene Blue is pretty safe. Find me a clean source to buy from and I'll try it, as the board's guinea pig.

A CLEAN source. I'm not eating aquarium products or slurping down mexican holistic remedies like some kind of junkie.


Lol, I love the way you put that. Though I have you beat, been using it since August.

If you are concerned about purity, try the science grade - most such chemicals are much purer than you'll ever get in the consumer world, but it may cost you a pretty penny. www.fishersci.com

Remember, methylene blue has only two CAS numbers, 61-73-4 for the aqueous solution and 7220-79-3 for the powder. Anything else may or may not be methylene blue, and "New Methylene Blue" is NOT the same chemical at all, and quite likely toxic.

Edited by geddarkstorm, 11 March 2009 - 03:01 AM.


#38 s123

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Posted 11 March 2009 - 09:34 AM

After reading the paper about methylene blue that started this discussion, I decided to study methylene blue as an MFURI project. Although, it’s not yet clear if I will find a lab where I can do this. I would use yeast (Saccharomyces cerevisiae) to study the effects of methylene blue on lifespan and ATP production and compare it to some other interventions including calorie restriction.
Experimental design (as proposed on this moment):

- Yeast cells + MB stored in the dark
- Yeast cells + MB exposed to light resembling human exposure to sunlight
- Yeast cells on CR
- Yeast cells + MB and on CR
- Yeast cell control group

What I hope to answer the following questions:

- Is there a difference between the lifespan of yeast + MB stored in the dark versus exposed to light? If there’s no difference then I will do the next experiments without controlling the light exposure.
- Is there a difference in lifespan and ATP production between cells on MB and control cells?
- Does CR lengthens the lifespan of yeast (probably yes because it has already been proven) and what's the difference in lifespan of cells on CR versus cells on MB?
- Is there a difference between the ATP production of cells on CR versus control cells.
- Is MB and CR together more effective in lengthening the lifespan than either of the two alone.

Another downside of methylene blue is that it inhibits the absorption of thiamine in yeast cells [1] but that will probably not be a problem in the concentrations that we talk about. They saw an inhibition of 12,5% of thiamine uptake by yeast cells after incubation with 1µM of MB.
[1] Inhibition of thiamine transport in baker's yeast by methylene blue. Experientia 36 (1980).

Edited by s123, 11 March 2009 - 09:36 AM.


#39 geddarkstorm

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Posted 11 March 2009 - 04:46 PM

After reading the paper about methylene blue that started this discussion, I decided to study methylene blue as an MFURI project. Although, it’s not yet clear if I will find a lab where I can do this. I would use yeast (Saccharomyces cerevisiae) to study the effects of methylene blue on lifespan and ATP production and compare it to some other interventions including calorie restriction.
Experimental design (as proposed on this moment):

- Yeast cells + MB stored in the dark
- Yeast cells + MB exposed to light resembling human exposure to sunlight
- Yeast cells on CR
- Yeast cells + MB and on CR
- Yeast cell control group

What I hope to answer the following questions:

- Is there a difference between the lifespan of yeast + MB stored in the dark versus exposed to light? If there’s no difference then I will do the next experiments without controlling the light exposure.
- Is there a difference in lifespan and ATP production between cells on MB and control cells?
- Does CR lengthens the lifespan of yeast (probably yes because it has already been proven) and what's the difference in lifespan of cells on CR versus cells on MB?
- Is there a difference between the ATP production of cells on CR versus control cells.
- Is MB and CR together more effective in lengthening the lifespan than either of the two alone.

Another downside of methylene blue is that it inhibits the absorption of thiamine in yeast cells [1] but that will probably not be a problem in the concentrations that we talk about. They saw an inhibition of 12,5% of thiamine uptake by yeast cells after incubation with 1µM of MB.
[1] Inhibition of thiamine transport in baker's yeast by methylene blue. Experientia 36 (1980).


That would be awesome. The experimental design looks solid, and even publishable depending on how deep you want to do the analysis of effects (I haven't yet seen any other papers that look at it like this ;)). Concentrations of MB are the only other consideration, and you might want a spread. Especially since microbes are considerably more sensitive to it and PDT than our cells, and the majority of MB in vivo is in the non-light reactive leuco form. This may or may not include yeast (35.2uM MB growth inhibited ~93% of the fungus C. albicans in response to PDT), but it's a consideration when looking at trying to determine a "human effective" dose for these organisms. Maybe four different doses, 1nM, 10nM, 100nM, and 1uM would be useful, but I don't know how many resources you have to devote to such a project. Also, the leuco form may make for another nice control for the light batch, reducing MB into leucoMB and raising a batch of the yeast in light on that would strengthen data on potential phototoxic effects from MB+. Perhaps leucoMB has other properties too unexplored you could see. But again, doing all that would make it quite a bigger project, and any experimentation by you is appreciated (I would do it myself with Pichia pastoris and insect SF9 cells, but I would get in trouble for using lab resources that way -- too afield from the cellular sensors we are working on).

Coincidentally, I've been in contact with a researcher in a lab that works on MB in human cell culture, specifically on its effects on respiration. I don't think they have done any experiments like your well thought out one, but I'll ask.

Edited by geddarkstorm, 11 March 2009 - 04:59 PM.


#40 maxwatt

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Posted 11 March 2009 - 06:02 PM

Methylene Blue increases the NAD/NADH ratio, which is indicative of possible life-extending properties.

I have been looking for things that increase the NAD+/NADH ratio, as this seems to be an artifact in common with CR, exercise, CR mimetis such as resveratrol, and SIRT1, and NAMPT and AMPK. One supplement with its own thread that purports to raise this ration is Benegene. This is an old study below, but I think the results are valid interesting though what they measured was a change in NADPH/NADH. It also indicates a positive effect on glucose metabolism, another effect shared by putative CR mimetics.

I've been looking for things to raise the NAD/NADH ratio, and there are not that many available. (Caffeine and L-serine, anyone?)

BTW, the MB solution sold as an aquarium fungicide is probably safe. It's sterile, tropical fish are exquisitely sensitive to toxic substances, and the concentrations we are considering would dilute it considerably.

Effect of Methylene Blue on pyridine nucleotides and insulin secretion of rat pancreatic islets
Journal Diabetologia
Publisher Springer Berlin / Heidelberg
ISSN 0012-186X (Print) 1432-0428 (Online)
Issue Volume 17, Number 1 / July, 1979

Pages 41-44

H. P. T. Ammon1, 2 and E. J. Verspohl1, 2

(1) Abteilung für Pharmakologie der Universität Erlangen-Nürnberg, FRG
(2) Pharmazeutisches Institut der Universität Tübingen, FRG
Received: 12 September 1978 Revised: 5 February 1979

Summary Methylene Blue, which is known to oxidise NADPH in red blood cells, was used to assess a possible role of NADPH in the glucose-stimulated secretion of insulin. When islets from rats were incubated with 3 mg/ml of glucose, Methylene Blue (0.5, 1.0, 2.0 or 5.0 g/ml) significantly decreased the concentration of NADPH, increased that of NADP+ and decreased the NADPH/NADP+ ratio in a dose-dependent manner. This effect was associated with inhibition of the glucose-induced insulin release. No significant change of NADH, NAD+ and ATP could be observed. [??? I find this puzzling, possibly due to insulin release in medium.] It is suggested that the secretory response of the pancreatic islet to glucose stimulation depends on the NADPH/NADP+ ratio.


FULL TEXT

Edited by maxwatt, 11 March 2009 - 10:17 PM.


#41 geddarkstorm

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Posted 12 March 2009 - 11:36 PM

There's also this paper talking about MB mediated partial hypoxia protection of cells. They used 40uM of MB, so that's far too high for in vivo I think, but they concluded that:

"These results indicate that partial protection
against cell injury by methylene blue during
hypoxic exposure is due to oxidation of NADH to
NAD+, resulting in an increase in the NAD+/NADH
ratio.
"

The cool thing is that MBH (leucoMB) can then go and donate its proton/electron to cytochrome c, which directly uses it to drive a proton through Complex IV and generate more ATP. So it recovers otherwise lost energy. Now, MB isn't going to be nearly as effective at generating ATP as NADH, since MB's donated electron will only drive proton pumping by one complex instead of three like NADH. But, a high NADH ratio means there's a lot of lost energy not being processed, and a loss of metabolic flux that requires NAD+; so MB appears to be able to scavenge back up the electrons/protons and put them to work again and keep metabolism flowing. This is even cooler with the proposed ability of MB to recapture back-flow electrons from Complex I, which otherwise are lost and produce super oxide. MBs ratio to cytochrome c is apparently critical for carrying this aspect out, and requires the very low amounts of MB we see in the original paper at the start of this thread.

On an aside, I would like to whole heartily thank Krillin for the challenges and the insightful questions he's posed. I hope neither him or anyone else thought we were "fighting', as an informed opposing voice is absolutely critical, I feel, for evaluating, discussing, and getting to the truth of matters -- especially ones as mightily complex as these. I appreciate the views he's brought, and I hope everyone following this thread have gained a greater understanding of these matters, I certainly have.

Any thoughts on this source? It costs $30.50 for 25g in a 1% aqueous solution.

It's made by

Acros Organics N.V.
One Reagent Lane
Fair Lawn, NJ 0741

and has CAS# 61-73-4.

StephenB


Considering ~1mg per day of MB is what we've been talking about (~0.015mg/kg) -- which actually is about 450 nM with 75% absorption, but I've estimated four times higher to give room for absorption/elimination kinetics and dry weight; still others might want to drop the MB intake down to ~0.25mg or ~0.0038mg/kg, which would be about 110nM straight up, though that's quite less likely to get to or sustain for any length 100nM in cells I think -- then that 25g bottle will last you 25,000 days, or about 51 years if we account for leap years....

Yeah, that's a pretty cheap deal. They have the most comprehensive MSDS sheet I've ever seen, and seem ok. They say "certified" so hopefully that means they include a certificate of analysis, or you might be able to call them up for one.

Edited by geddarkstorm, 13 March 2009 - 12:09 AM.


#42 s123

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Posted 13 March 2009 - 07:21 AM

MB acts as a photosensitisers when illuminated with UV-light. Most humans spend 90% or so of their time in buildings or cars. UV-light is unable to penetrate glass. So, you would only experience the photosensitization effect when you are exposed to direct sun and so, I would say that this won’t be a problem for most humans.

#43 tintinet

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Posted 13 March 2009 - 11:19 PM

"Phenothiazinium dyes such as MB+ or TB+ absorb strongly at approximately 600–670 nm..." -That's red visible light.

Most glass absorbs the short wavelength UV (UV-C 190 - 280 nm and UV-B
280 - 320 nm), but transmits the longer UV-A (320 - 400 nm) radiation.

#44 AgeVivo

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Posted 15 March 2009 - 01:22 PM

Thank you Aydenpride, geddarkstorm and s123 for orienting me towards a MB-mouse-survival analysis.

A. NTP survival data
Here is actually the full report of the NTP toxicology test: http://ntp.niehs.nih...0_web_FINAL.pdf (free access)
I extracted the main results concerning lifespan:
Posted Image
Posted Image
Posted Image
Posted Image

Rapid conclusion:

in the paper (2) they link to the study above and say that there is weak evidence of life span extending effects in mice at 2,5 mg/kg.
(2)
Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways.
Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN.
FASEB J. 2008 Mar;22(3):703-12. Epub 2007 Oct 10.

In fact in mice the data suggest a clear trend towards life-extension, especially around 12.5 and 25 mg/kg/day and perhaps higher.
In rats the results are quite equivocal: in males there is no effect except a quite positive effet at 25 mg/kg/day, in females MB seems to be dangerous. One would need to carefully read the report to distinguish accidental death (!) and cancer deaths. It would also perhaps suggest why NB seems beneficial for mice but not rats

Parenthesis: I don't understand why the NTP stops experiments at the age of 2!! I gives a very poor statistics concerning long-term safety, in case of poorly dangerous products. 50 animals per group would have been a lot if the experiment had last longer (say 5 months), or if they had started the experiment with >= 5-month-old animals. At a time when everyone is concerned about safety in the long run, it would great if someone here/some lobby could tell the NTP to decay or extend their experiments by a few months at least when the tested product shouldn't be particularly dangerous.

Question: is that NTP study the main data we have so far concerning MB mouse survival?

Idea/MFURI?: it would be nice to go over such NTP studies to list the compounds that seem to extend lifespans. On pubmed by searching "National Toxicology Program"[Corporate Author] i see that there are 274 such studies, most of them probably concern products that we can easily find, like MB.
One can look at them at http://ntp.niehs.nih...tml?col=010stat
Example: MB: http://ntp.niehs.nih...BE2C5310A792BB3


B. Dosage

You want to dilute MB down however, at least to 0.01%, to avoid staining.
(...) about dosage, which I don't think you asked about?
(...) about 0.015mg/kg (0.01-0.02), this is estimated to be most effective at enhancing mitochondria, oxidative defense, and other metabolic parameters in human cells. (...) this is still a lot lower than the amount being kicked around for anti-Alzheimer's use (60mg).

In fact in MPrize @home we would not do daily gavage, but put MB in water (and change the water every week or other week, or more frequently in MB is modified with time in water???). Mice will drink a certain quantity of water per week so the dillution of MB in water is what makes the right dosage.

Every administration route has its trouble. A trouble with gavage is that the dosage is all in one shot and therefore it can lead at the same time to more serious adverse effects and no effect most of the time (difference between having 3 beers a day and 3 beers at lunch every day). So based on the NTP data my first guess would be a dillution in the drinking water corresponding to say 20 mg/kg/day? (hey, I might be wrong ;-). I'm not sure how that relates to your numbers, in particular you seem to suggest that the human equivalent would typically be 1 or 2 mg/kg/day?

Thanks

Edited by AgeVivo, 15 March 2009 - 01:23 PM.


#45 geddarkstorm

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Posted 16 March 2009 - 11:23 PM

The doses in mice and rats are very high, much higher than we want, in my opinion (and that's just my opinion based on the human cell in vitro work). The mice did see an upward trend with even huger doses, but I think that might mean there's a biphasic property experienced in mice - they still had adverse toxic events, even if they lived longer. Lower doses might have seen a similar life span increasing "hump", maybe even better. Regrettably, they didn't use very very small doses of MB and see how that affected life span. We only know that in human cells, in vitro, that 100nM is the optimal for extending life and health (don't forget "and health" as extending life while sacrificing health is a not a good thing I feel personally). Therefore, this minuscule amount is what I believe is best.

Hm, if you really want to do a life span study on mice with that low a dose.. perhaps you could split into two groups for each dosage level, one which takes it all at once (how ever that is done in mice), and one which just has it at the water to drink how ever much? Calculating the proper amount in their water will depend on how much water they drink daily, verses the amount of blood an average mouse has, as we want to see if 100nM in the body works across the whole organism the same in mice as human cells in vitro. This is completely different than 100nM in their water, completely so -- that would give far too little in their bodies.

I think, and this is really just a guess, that the NTP study meant a total effect per day amount of MB, based on how much they drink or eat (however it was administered), not simply what was in their water.

#46 AgeVivo

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Posted 17 March 2009 - 08:22 AM

The doses in mice and rats are very high (...) they still had adverse toxic events, even if they lived longer. Lower doses might have seen a similar life span increasing "hump", maybe even better.(...) in human cells, in vitro, that 100nM is the optimal for extending life

Uh... for in vivo work i trust in vivo doses more than in vitro doses: it is frequent that the in vitro mechanisms that are studied are not the right ones that matter in vivo. To be convinced I need to read the thread again in search of small doses that have effects in vivo.

optimal for extending life and health (don't forget "and health"


For us, of course. I agree that the optimal dose for lifespan may not be the optimal dose for comfort, altough they are likely to be very close (NB: life-dose isn't necessarily greater than comfort-dose; it depends of the relative discomfort of various dying causes).
For mice, the main precise measurement is lifespan and i'd like to avoid useless results such as "this MB dose seemed to slightly extend life (far from significant) but mice seemed quite healthier". I also would like to avoid "during the first months this MB dose started to extend life much and mice looked much better but after a few month MB-mice where obviously ill and started dying very fast".

two groups for each dosage level, one which takes it all at once (how ever that is done in mice)

no:
i) we are looking for meaningful (useful) results and for a first MPrize @home trials i guess we will not be enough people to allow us to test several doses. ii) 'all at once' requieres complex administration

Calculating the proper amount in their water will depend on how much water they drink daily (...) the NTP study meant a total effect per day amount of MB(...) not simply what was in their water

yes that's what i meant but you put it better. The NTP did gavage: a known quantity is poor directly in the stomach (mice don't

The doses in mice and rats are very high (...) they still had adverse toxic events, even if they lived longer. Lower doses might have seen a similar life span increasing "hump", maybe even better.(...) in human cells, in vitro, that 100nM is the optimal for extending life

Uh... for in vivo work i trust in vivo doses more than in vitro doses: it is frequent that the in vitro mechanisms that are studied are not the right ones that matter in vivo. To be convinced I need to read the thread again in search of small doses that have effects in vivo.

optimal for extending life and health (don't forget "and health"


For us, of course. I agree that the optimal dose for lifespan may not be the optimal dose for comfort, altough they are likely to be very close (NB: life-dose isn't necessarily greater than comfort-dose; it depends of the relative discomfort of various dying causes).
For mice, the main precise measurement is lifespan and i'd like to avoid useless results such as "this MB dose seemed to slightly extend life (far from significant) but mice seemed quite healthier". I also would like to avoid "during the first months this MB dose started to extend life much and mice looked much better but after a few month MB-mice where obviously ill and started dying very fast".

two groups for each dosage level, one which takes it all at once (how ever that is done in mice)

no:
i) we are looking for meaningful (useful) results and for a first MPrize @home trials i guess we will not be enough people to allow us to test several doses. ii) 'all at once' requieres complex administration protocols that

Calculating the proper amount in their water will depend on how much water they drink daily (...) the NTP study meant a total effect per day amount of MB(...) not simply what was in their water

yes that's what i meant but you put it better. The NTP did gavage: a known quantity is poor directly in the stomach (mice don't

#47 AgeVivo

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Posted 01 May 2009 - 11:26 AM

We are probably soon starting MPrize @ home with methylene blue.
MB can also be bought on eBay; btw a very creative guy tells you have to make a home-made bread-based rember-like trick to turn your pee blue ;) . The MB dose isn't decided yet.
Posted Image
We'll probably need a control food colorant that also turns the pee blue, such as Blue Brilliant FCF (E133) or Indigotine (E132), or another blue colorant such as Prussian Blue; i'm not sure it makes the pee blue too. There are medicines such as indometacin that would do the trick but if they increase or reduce lifespan... If anyone might participate or is aware of MB concentrations/control/color, don't hesistate to post or to send at email at longevity#agevivo.com (#=@). Thanks

Edited by AgeVivo, 01 May 2009 - 11:30 AM.


#48 s123

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Posted 05 May 2009 - 04:41 AM

Does anyone have the data from the clinical trials with Rember?

#49 abelard lindsay

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Posted 06 May 2009 - 01:16 PM

Just bumping this to see if anyone has been using the Kordon product, and if so, what were your results?

#50 Lufega

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Posted 07 May 2009 - 01:46 AM

How much of the Kordon product would be enough for life extension purposes? 1-2 drops per day?

#51 AgeVivo

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Posted 07 May 2009 - 05:02 PM

Can we use leucomethylene blue rather than methylene blue for the MPrize @ home?

1) Does it avoid the need for a blue control?
For MPrize @ home I was told to use leucomethylene blue rather than methylene blue, because it has no color rather than the blue color.
Except for pee perhaps?... if no one knows i'll check to see if the blue pee is visible in my cage, at 100nM in the drinking water :)

2) Does it have the same effects?
The proposed mechanism* of life extension with MB looks very much like the one proposed with oxaloacetic acid (benagene): the mitochondrial function is improved because there is a cycling between reduced and oxidized NADP that requires a cycling between methylene blue and leucomethylene blue (like a cycling between oxoloacetic acid and malic acid). In this thread it was suggested that administering malic acid would therefore probably not increase the mitochondrial function like oxoloacetic acid... can we say the same for leucomethylene blue??

*: Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways (free full text)
Atamna, H., Nguyen, A., Schultz, C., Boyle, K., Newberry, J., Kato, H., Ames, B. N.

Methylene blue (MB) has been used clinically for about a century to treat numerous ailments. We show that MB and other diaminophenothiazines extend the life span of human IMR90 fibroblasts in tissue culture by >20 population doubling (PDLs). MB delays senescence at nM levels in IMR90 by enhancing mitochondrial function. (...) Flavin-dependent enzymes are known to use NAD(P)H to reduce MB to leucomethylene blue (MBH2), whereas cytochrome c reoxidizes MBH2 to MB. (...) We propose that the cellular senescence delay caused by MB is due to cycling between MB and MBH2 in mitochondria



#52 AgeVivo

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Posted 12 May 2009 - 02:09 PM

I've just looked at my 2 mice after 10 days of absence; their hair looks nicer and they seem more active than usual. It happens that i had left them with approx 200nM MB (MB for fish... that contains other things as well) in the drinking water. Of course it is subjective, but I was far from expecting my mice to look different than usual (and especially after only ten days)

For MPrize @ home (...) i'll check to see if the blue pee is visible in my cage, at 100nM in the drinking water

While the drinking water is blue/green, I don't see blue pee (my cage is blue, perhaps it would be different with a white cage).

PS: the "MB for fish" i bought is supposed to be be used at 440 nM in case of quarantine, or simply from time to time. Different system, same order of concentration...

Edited by AgeVivo, 12 May 2009 - 03:05 PM.


#53 acash

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Posted 16 May 2009 - 04:08 AM

I have been looking for things that increase the NAD+/NADH ratio, as this seems to be an artifact in common with CR, exercise, CR mimetis such as resveratrol, and SIRT1, and NAMPT and AMPK. One supplement with its own thread that purports to raise this ration is Benegene.



Hope I am not too far off topic here, and apologize for the lateness of my response, but wanted to share a graph with you from page 661 of "Haslam JM, Krebs HA. The permeability of mitochondria to oxaloacetate and malate. Biochem J 1968; 107: 659-67", available at http://www.pubmedcen...mp;blobtype=pdf


Attached File  NAD__to_NADH_Ratio.gif   38.08KB   38 downloads

As can be seen by the graph, 20 uM concentrations of oxaloacetic acid added to the cytosol creates a 900% increase in the NAD+/NADH ratio within 1 minute. Having the NAD+/NADH ratio increase is good, but having it increase inside the mitochondria is much better. Increasing NAD+ levels inside the mitochondria increase survival (as per Yang H, Yang T, Baur JA, et al. Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival. Cell 2007; 130: 1095-107) and also protect mitochondrial DNA (as per Yamamoto HA, Mohanan PV. Effect of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA damage and seizures induced by kainic acid in mice. Toxicol Lett 2003; 143: 115-22.).

Regards.

#54 zawy

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Posted 16 May 2009 - 04:09 PM

To correct some comments made in this thread: sunlight at noon during the summer for most reasonable latitudes, including when it is straight above and at sea level, is 100 mW/cm^2. 137 mW/cm^2 is what it is at the top of the atmosphere. In the MB "activation range" of 600 to 670 nm, sunlight is 10% of it's total, 10 mW/cm^2. The 660 nm devices I made for a UK researcher to activate an MB-like compound in cancer patients was 25 mW/cm^2. So yes, they could have just stayed out in the sun 2.5 times as long to get the same dose, except that the UK doesn't ever get any sun.

#55 zawy

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Posted 17 May 2009 - 03:33 PM

One of the rat memory studies used 1 mg/kg in 200 g rats. Another rat memory abstract link in this thread (see below) used 4 mg/kg, supposedly the top of the upside-down U curve for benefit of a substance (hormesis) since they were aware of 1-4 mg/kg work.

The conversion to human doses was originally based on a theory about surface area to weight ratios which gave a power factor of P=2/3 since surface area is a square (2) and volume is a cube (3). This was found to be wrong and caloric intake/body weight measurements determined P=3/4 was the most accurate factor. But the 2/3 factor is still used by most because EPA and OSHA chose to stick with 2/3 because it gives a larger margin of safety for toxicology when converting from small animals to humans. When looking for the most accurate conversion you should use 3/4. But if research would just report intake values as mg dose per calorie intake, no conversion is needed. The equation is (human dose/kg) = (animal dose/kg)*(animal weight/human weight)^(1-P)

So, 1 mg/kg by injection for the 200 g rats used results in 16 mg by injection per 70 kg person. 4 times more for the 4 mg/kg report (also by injection). Someone said absorption of MB is 50-75%, so maybe up to 128 mg per 70 kg is optimum, assuming MB metabolizes and gets to the human brain with a similar effectiveness as in rats. This is a little uncomfortably close to the 600 mg someone reported that an adverse effect had been seen. I want to learn Spanish better before I go to Peru in a few months so I might try as high as 50 mg. I base this partly on someone here reporting seeing substantial although subjective benefit from 17 mg. The papers say "50–100 mg/kg doses decreased running wheel behavior" and "Large MB doses (50 to 100 mg/kg), however, impair memory because they lead to methemoglobin formation and impaired oxygen consumption" I consider this a seriously adverse event. 50 mg/kg is about 800 mg in a 70 kg person. I definitely want to stay 10 times below that.

Although the tests appear to show only short-term memory, I'm sure this will go a long way to helping to solidify longer-term memories. I believe repeated short-term memory encounters grow the long term memories. It's interesting that it appears to work for positive reinforcement, negative reinforcement, and unlearning past patterns when not reinforced. This last seems to have shown the largest result and like it might be useful for post-traumatic disorder and obsessive-compulsive behavior. The rats repeatedly heard a tone and then were shocked which would cause them to freeze the next time they heard the tone. The rats on MB quickly, and by a very large difference, learned to not freeze when tones were not followed by shock.

The theory is that MB is working because it increases the ATP energy of the neurons (a.k.a. increased cytochrome oxidase activity). It seems like i remember that having more glucose available also improved memory in students which led to the advice of eating breakfast before class.

These are the full texts of previous links provided on MB and rat memory:
http://www.pubmedcen...bmedid=15466319
http://www.pubmedcen...bmedid=17428524

"4 mg/kg intraperitoneal injection of MB given daily for 5 d resulted in a cumulative 38% increase in overall brain cytochrome oxidase activity measured 24 h after the last injection. MB progressively accumulates in the brain, reaching a concentration >10 times greater in the brain than in the blood 1 h after the administration."

"The impaired ability to extinguish fear to a traumatic memory in congenitally helpless rats supports the validity of this strain as an animal model for vulnerability to post-traumatic stress disorder, and this study further suggests that methylene blue may facilitate fear extinction as an adjunct to exposure therapy."

"Brain cytochrome oxidase activity in the MB-treated group was approximately 70% higher than in saline-treated rats. " (1 hour post injection?)

"Thus, the extensive depletion of extracellular glucose during training in aged rats may be associated with age-related memory impairments, an effect that might be related to - or may exacerbate - the effects on learning and memory of an absence of the increases in blood glucose levels to training as seen in young rats. Together, these findings suggest that age-related changes in both peripheral and central glucose physiology contribute to age-related impairments in memory." Which makes me wonder about possible negative effects of CR that produce lower blood sugar, and the seemingly lethargic CR people i once saw in an interview.

"Although earlier evidence suggested that glucose does not enhance cognitive function in healthy young adults, more recent findings suggest that glucose is effective in this population, provided the tests are sufficiently difficult. In college students, glucose consumption significantly enhanced memory of material in a paragraph. Glucose also appeared to enhance attentional processes in these students. Neither face and word recognition nor working memory was influenced by treatment with glucose. The neurobiological mechanisms by which glucose acts are under current investigation. Initial evidence suggests that glucose or a metabolite may activate release of the neurotransmitter acetylcholine in rats when they are engaged in learning."

Imagine people (and animals) of old eating a good meal, feeling good from the nutrients (positive reinforcement), sitting around letting it digest and thinking about where the food came from, and at the same time being exposed to more glucose in the brain to enhance the memory about how the food came about.

This thread belongs in nootropics section.
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#56 maxwatt

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Posted 17 May 2009 - 06:13 PM

One of the rat memory studies used 1 mg/kg in 200 g rats. Another rat memory abstract link in this thread (see below) used 4 mg/kg, supposedly the top of the upside-down U curve for benefit of a substance (hormesis) since they were aware of 1-4 mg/kg work.
.....(snip)

This thread belongs in nootropics section.

Yes and no. The effective dosages used in the memory-enhancement studies appear to be much higher than the amounts used to favorably increase the NAD/NADH ratio, which would be consistent with improved health and longevity. So higher doses (perhaps) belong with nooropics, whilst doses measured in micrograms are more appropriate for longevity.

#57 zawy

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Posted 17 May 2009 - 06:18 PM

"MB treats congenital and poison-induced methemoglobinemia; prevents the side effects of chemotherapy, and treats septic shock. The dose of MB usually used in clinical settings is between 1 and 2 mg/kg/day." So 50 mg to 150 mg would be what I'd consider as a safe max for one-time use. I'll do a memory experiment using blocks.exe ( http://www.4shared.c...ame_BLOCKS.html ) and also see how many random digits I can remember. I'll try 10 mg the first day and 20 mg the second day. If the second day doesn't show improvement over first day, then I'll wait a week for it to clear out and try 5 mg. If neither 1st or 2nd day show improvement over control, I'll forget trying to use it for temporary memory enhancement.

#58 rwac

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Posted 13 June 2009 - 11:39 PM

Can someone who has access send me this paper.

Pharmacokinetics and organ distribution of intravenous and oral methylene blue.
http://www.ncbi.nlm....pubmed/10952480

It seems like different dosages of MB cause different effects due to varying concentrations in the body.

Pharmacokinetics and organ distribution of intravenous and oral methylene blue.
Peter C, Hongwan D, Küpfer A, Lauterburg BH. Department of Clinical Pharmacology, University of Bern, Switzerland.

OBJECTIVE: To determine the pharmacokinetics and organ distribution of i.v. and oral methylene blue, which is used to prevent ifosfamide-induced encephalopathy in oncology.


METHODS: The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after i.v. and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and i.v. application.


RESULTS: The time course of methylene blue in whole blood after i.v. administration showed a multiphasic time course with an estimated terminal half-life of 5.25 h. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/ml vs 137 nmol/min/ml). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the pharmacokinetics. The urinary excretion of methylene blue and its leucoform was only moderately higher after i.v. administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall and liver but lower concentrations in whole blood and brain than i.v. methylene blue.


CONCLUSIONS: Differences in organ distribution of methylene blue are mainly responsible for the different pharmacokinetics after oral and i.v. administration. If methylene blue acts in the liver, where ifosfamide is primarily activated to reactive and potentially toxic metabolites, oral and i.v. methylene blue are likely to be equally effective. However, if the site of action is the central nervous system, i.v. methylene blue which results in much higher concentrations in brain seems preferable.



#59 tlm884

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Posted 14 June 2009 - 01:06 AM

Seeing that methylene blue is a MAOi I can't help but wonder if its a possible replacement for deprenyl. If it were a replacement for deprenyl it would be hitting two birds with one stone. Alzhemier prevention and antiaging, etc etc

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#60 tlm884

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Posted 14 June 2009 - 01:13 AM

Can someone who has access send me this paper.

Pharmacokinetics and organ distribution of intravenous and oral methylene blue.
http://www.ncbi.nlm....pubmed/10952480

It seems like different dosages of MB cause different effects due to varying concentrations in the body.

Pharmacokinetics and organ distribution of intravenous and oral methylene blue.
Peter C, Hongwan D, Küpfer A, Lauterburg BH. Department of Clinical Pharmacology, University of Bern, Switzerland.

OBJECTIVE: To determine the pharmacokinetics and organ distribution of i.v. and oral methylene blue, which is used to prevent ifosfamide-induced encephalopathy in oncology.


METHODS: The concentration of methylene blue in whole blood was measured using high-performance liquid chromatography in seven volunteers after i.v. and oral administration of 100 mg methylene blue with and without mesna. The distribution of methylene blue in different tissues was measured in rats after intraduodenal and i.v. application.


RESULTS: The time course of methylene blue in whole blood after i.v. administration showed a multiphasic time course with an estimated terminal half-life of 5.25 h. Following oral administration, the area under the concentration-time curve was much lower (9 nmol/min/ml vs 137 nmol/min/ml). Co-administration of mesna, which could influence distribution by ion-pairing, did not alter the pharmacokinetics. The urinary excretion of methylene blue and its leucoform was only moderately higher after i.v. administration (18% vs 28% dose). Intraduodenal administration to rats resulted in higher concentrations in intestinal wall and liver but lower concentrations in whole blood and brain than i.v. methylene blue.


CONCLUSIONS: Differences in organ distribution of methylene blue are mainly responsible for the different pharmacokinetics after oral and i.v. administration. If methylene blue acts in the liver, where ifosfamide is primarily activated to reactive and potentially toxic metabolites, oral and i.v. methylene blue are likely to be equally effective. However, if the site of action is the central nervous system, i.v. methylene blue which results in much higher concentrations in brain seems preferable.





I downloaded the article and put it on my school fileserver https://homepage.usa...884/imminst.pdf





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