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Tutorials

What Happens to Drugs Inside the Body?

The wise nootropic user knows two things: his goals with nootropic use, & how to use nootropics to achieve those goals. The latter is an area not well explored by many nootropics users. Knowledge of how individual agents work guides the design of stacks. Certain drugs may work synergistically (both together produce a greater effect than either alone), additively (both agents work in combination), or antagonistically (counteract each other). Thus, it is essential to understand what exactly happens after a drug is ingested.

The science of pharmacology serves to explain how the ultimate pharmacodynamic effect is produced (the end result of a drug, e.g. improved long-term memory). This is accomplished by studying two aspects of what happens to drugs within the body: mechanisms & pharmacokinetics.

Mechanisms

The most common way in which all drugs work is by modulating a receptor. The body is full of thousands of different types of receptors. These receptors are usually small proteins embedded in the surface of cells. There are 3 types of receptors:

  • Enzyme-linked receptors include enzymes & receptors that lead to the direct activation of enzymes.
  • Ion channel-linked receptors are regulated by concentrations of positively & negatively charged ions, such as Ca2+ & Cl. Na+ channels play a central role in neurotransmission.
    • Lidocaine, a Na+ channel blocker, suppresses conduction of action potentials between neurones, resulting in nullification of pain signals, hence its use as a local anaesthetic.
Receptors exhibit selectivity for ligands like locks & keys. The degree of compatibility between a ligand & a receptor is referred to as affinity.
  • G-protein-coupled receptors (GPCRs) receive signals by binding with ligands.
    • A ligand is something that binds to a receptor & produces an effect. Some examples are hormones, neurotransmitters, & drugs. When a ligand binds a receptor, it can produce any of 4 effects:
      • A receptor agonist is a ligand which produces stimulatory effects upon binding, triggering or potentiating a biochemical pathway ultimately leading to a response in the body.
        • When histamine binds H1 receptors, it triggers the immune system in such a way that symptoms of an allergic reaction are produced.
      • A receptor antagonist is a ligand which does not exert an effect upon binding, leading to the extinguishment of a somatic response as the stimulating ligand is unable to make contact with the occupied receptor. Antagonists can be competitive (meaning they reversibly bind to the receptor) or non-competitive (irreversibly bind receptors). Non-competitive receptor antagonists are more potent.
        • Aspirin is an irreversible COX-enzyme antagonist. Its inhibitory effects on platelets are permanent– following discontinuation, normal platelet function does not return for ~10 days- the lifespan of a platelet. For this reason, aspirin should be held for at least 1 week prior to major surgeries.
      • A partial agonist is a ligand which has properties of both an agonist & an antagonist ultimately producing a mild stimulatory effect.
        • Aripiprazole (Abilify) is an antipsychotic with partial agonist activity. Aripiprazole’s mild modulation at the D2 receptor produces modest reductions in some psychotic symptoms but avoids undesirable side effects due to over-antagonism of dopamine activity caused by other antipsychotics.
      • An inverse agonist is a ligand which exerts the opposite effect upon binding to a receptor, reversing a somatic process.
        • When an antihistamine drug such as diphenhydramine (Benadryl) binds H1 receptors, it does not merely occupy the receptor, preventing histamine from binding, but actually promotes the opposite biochemical pathway, actively countering the allergic reaction.

Visualised dose-response curves for various ligand-receptor interactions.
Visualised dose-response curves for various ligand-receptor interactions.

 

Pharmacokinetics

Pharmacokinetics (PK) refers to what happens to the drug after it has been ingested in the body. It is divided into 5 major areas, denominated by the acronym ADME-Tox:

  1. Absorption is the first phase of pharmacokinetic interest. Here we consider aspects of the drug’s transit into the bloodstream.
    • The route of administration (oral, sublingual, inhalation, intravenous, etc.)
    • Foods, as well as most drugs that are taken by mouth, are absorbed in the small intestine
    • How long a drug takes to be absorbed
    • The maximum amount that is absorbed (peak concentration, CMAX) & the time it takes to achieve CMAX (TMAX)
    • The onset, which is the time it takes for drug’s effects to begin
  2. Distribution is where we examine where the drug goes in the body.
    • The bioavailability (F), which refers to the concentration of the drug at the site of action (for nootropics, this would be the brain)
    • The volume of distribution (VD) which represents the degree of absorption into fat tissue. This is important because the amount of available drug is that which is present in the blood
    • Protein binding refers to the amount of drug in the blood that is bound to proteins such as albumin where it is inactive. These proteins act as carriers of the drug, preventing it from degradation. When drugs compete for binding proteins, or chronic malnutrition leads to decreased production of plasma proteins, there is a potential for increased levels of drug in the blood
  3. Metabolism has to do with biochemical pathways of breakdown or altering the structure of the drug molecule.
    • The liver is the major site of drug metabolism
    • Cytochrome P450 is a key group of enzymes in the liver that is responsible for the degradation of many drugs. Individual enzymes include 2C9, 2D6, 3A4, & so on. When 2 drugs happen to compete for the same enzyme, there is potential for a drug interaction. Understanding CYP450 is understanding the majority of drug interactions (will be covered later!)
    • Other drugs are able to undergo alternate pathways of hepatic (via the liver) metabolism (e.g. oxidation) or non-hepatic metabolism (e.g. proteolysis)
    • Prodrugs are parent compound which are inactive when ingested but become activated when undergoing metabolism. Usually, this is through the cleavage of a portion of the molecule. Active compounds, too, may have active metabolites. Keep these in mind as the metabolites can continue to contribute to the pharmacodynamic effect of the drug.
  4. Elimination is how the parent compound of the drug & its metabolites are removed from the body. There are two major pathways of elimination: hepatic (faecal) & renal (via the kidneys; urination).
    • Dysfunction of either system could lead to accumulation of drugs that are excreted by that pathway, possibly worsening side effects
    • Some drugs can be excreted via alternate pathways if the major pathway of removal is compromised
The ideal dosage of a drug achieves levels above the MEC but does not exceed the MTC.
The ideal dosage of a drug achieves levels above the MEC but does not exceed the MTC.
  • Toxicology is the propensity of a drug to cause harm & via which mechanisms.
    • «The dose makes the poison»: every agent has the ability to cause harm given the correct amount.
    • The minimum toxic concentration (MTC) is the level at which a drug’s toxicity becomes clinically significant. This is often compared with the minimum effective concentration (MEC), where a drug’s desired effects start to work. The difference between MTC & MEC is the therapeutic range. The narrower the therapeutic range, the higher the risk of toxicity.
    • Some mechanisms by which antidotes can work include binding the toxic molecule, preventing it from triggering receptors or promoting recovery processes (e.g. antioxidants)

Pharmacokinetic
Pharmacokinetic pathways & major organs.

 

Summary

  • Mechanisms are how drugs produce their effect. Most drugs work by affecting a receptor.
  • Pharmacokinetics is the study of the changes that happen to the drug after it is introduced into the body. It is broken down into 5 major areas: absorption, distribution, metabolism, elimination, & toxicology.
  • Pharmacodynamics is the outcome effect of a drug, such as reduced depressive symptoms.
  • An understanding of mechanisms & pharmacokinetics of drugs can be useful in designing stacks which exploits the characteristics of each agent for an optimised pharmacodynamic effect.
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Health

6 Supplements to Boost Your Immune System

Immune system functioning is something that is often taken for granted. Even when one is not feeling sick, the immune system is working every hour of every day by neutralising cancerous growth, clearing wastes from the body, and fighting developing infections[1]. Certain supplements have the potential to provide a boost to your immune system function for fighting off illnesses caused by infections and for supporting general well-being.

Zinc

zincThe beneficial effects of zinc for symptoms of the common cold were first documented 30 years ago[2]. Since then, a modest body of literature has formed which supports the efficacy of zinc as a mild palliative aid for cold symptoms. Prophylactic administration of zinc (to prevent cold) has not been substantiated[3]. Further studies are needed as the current data are not fully conclusive.

Zinc modulates various components of both the innate (e.g. neutrophils, macrophages)[4] and adaptive (e.g. B- & T-lymphs)[5][6] immune systems.

Effectiveness

A 2015 Cochrane review of 16 therapeutic trials (n = 1387) found that administration within 24h of cold onset at a dose of over 75 mg per day had the following effects[7]:

  • Zinc significantly reduced the duration of cold symptoms by a mean difference of -1.03 days
  • Zinc had no effect on the severity of cold symptoms.
  • Individuals who took zinc were about half as likely to be symptomatic after 7 days of treatment, were less likely to develop a cold, less likely to be absent from school, and were less likely to be prescribed antibiotics (note: although antibiotics should not be prescribed for the common cold as it is caused by a virus, they are nevertheless frequently given due to patient insistence.).

Although the authors noted high heterogeneity within the data, these data appear to corroborate general findings from the literature. Zinc appears to be mildly effective for reducing symptoms of the common cold.

Formulations

Zinc is available in many salt forms. The most common over-the-counter formulations are zinc oxide, zinc acetate, and zinc gluconate[8]. Other formulations vary in bioavailability, with zinc monomethionine being the most absorbed. The amount of ionic zinc content seems to be correlated to therapeutic efficacy[9], however, the acetate and gluconate salts have been most studied.

The usual dosage ranges from 4.5 to 24 mg of elemental zinc taken every 1 to 2 hours during waking hours as cold symptoms persist[10]. The regimen is typically continued for 5 to 14 days or until symptoms subside.

Cautions

High doses (≥100 mg) of or prolonged exposure (≥10 years) to zinc may be unsafe[11], and has been linked to adverse sequelae such as prostate cancer. Be aware of elemental zinc content in multivitamins. Pregnant or breastfeeding women should limit their zinc intake to less than 40 mg/day.

As a caution, zinc may interfere with the absorption of certain drugs[12], such as quinolone and tetracycline antibiotics, as well as other mineral supplements, such as calcium or iron. Notify your doctor and pharmacist if you are taking zinc. Zinc should be taken 2 hours before or 6 hours after quinolone & tetracycline antibiotics.

Side effects are relatively mild and limited. Bad taste and nausea are frequently reported[13].

There is a concern that doses higher than 40 mg per day may interfere with copper absorption, leading to anaemia[14]. One study has tested the effects of long-term coadminstration of zinc & copper; through 6 years of supplementation no significant adverse effects were reported[15].




Garlic

garlicGarlic is a culinary herb that has been largely investigated for its benefits on cardiovascular health, which stem from modest reductions in blood pressure and cholesterol levels[16]. In addition to these helpful effects, garlic may also have an immunostimulating effect.

The active constituent in garlic is a compound called allicin, which has been shown to promote the activity of various components of the immune system, evidenced by enhanced phagocytosis, lymphocyte proliferation, and inhibition of immunosuppressive processes, among other mechanisms. Allicin has also demonstrated intrinsic antimicrobial activity demonstrated in vitro.

Formulations & Dosage

Garlic can be consumed in a variety of forms. In general, the recommended allicin intake is 2 to 5 mg per day[17]. The following dosages approximate this amount. Be advised that various formulations may vary in allicin content and pharmacologic properties [18]. Further information about various formulations and intake can be found here.

  • 2 to 5 g of fresh garlic (cut, not crushed & uncooked[19])
  • 0.4 to 1.2 g of dried powder
  • 2 to 5 mg of oil
  • 300 to 1000 mg of extract

Warnings

Increased garlic intake should be avoided in individuals with a bleeding disorder (e.g. haemophilia or other coagulopathy) or planned surgery within 2 weeks[20]. Because allicin has been shown to inhibit platelet aggregation and TxA2 synthesis, individuals consuming large amounts of garlic are at an increased bleeding risk. Exercise caution if you are taking «blood thinners» such as warfarin or aspirin and consult your doctor and pharmacist before use.

Individuals with diabetes, GI infection, and inflammatory bowel disease should also exercise caution[21].

Allicin has been observed to substantially inhibit the CYP2E1 isozyme, reducing its activity by up to 39%[22]. Consequently, elevations in drugs metabolised by 2E1 may be expected. These include paracetamol and alcohol.

As an inducer of the CYP3A4 isozyme[23], an increased consumption of garlic may stimulate the metabolism of certain drugs including birth control.

Adverse Effects

Gastrointestinal upset is the most common complaint with supplemental garlic consumption[24]. This entails heartburn, intestinal gas and bloating, nausea and vomiting, and diarrhoea. A tolerance may develop to these side effects over time. Certain extracts may be associated with less gastrointestinal upset.




Reishi Mushroom

ganoderma lucidum

The Reishi mushroom is a fungal remedy which has been used in traditional Chinese medicine. Its major studied properties include antioxidant, antineoplastic, and immunomodulatory activity[25].

Its pharmacologic effects derive from many bioactive molecules, which include various polysaccharides and triterpenes, among others. Polysaccharides seem to be involved in modulating the immune system[26], which entails stimulating the proliferation and differentiation of T-lymphocytes & NK cells, for example, when the immune system is weakened, and attenuating TNFα and some interleukin activity when the immune system is over-functioning (e.g. auto-immune disease).

Warnings

This supplement should be avoided in individuals with an autoimmune disease (e.g. multiple sclerosis) or immunosuppression therapy (e.g. for transplant recipients)[27]. Ganoderma‘s immunomodulating effects may trigger disease flares or organ rejection.

Use within 2 weeks of surgery is also not recommended as with garlic extracts[28]. Some constituents in Ganoderma inhibit platelet aggregation, producing a clinically significant effect at doses over 3 grams per day. Thus, individuals taking Ganoderma may be at a higher risk of bleeding.

Ganoderma may interact with certain medications for diabetes and blood pressure by contributing to their effects[29]. In individuals receiving antidiabetics such as insulin or glipizide, supplementing Ganoderma may increase the risk of hypoglycaemia, or dangerously low blood sugar which may lead to a seizure. Individuals taking medications for blood pressure, such as lisinopril, may be at a higher risk of hypotension, or very low blood pressure which may lead to shock.

Adverse Effects

The most frequently reported side effects with Ganoderma supplementation are bleeding[30], rash, dizziness, and headache.




Lion’s Mane Mushroom

lion's maneHericium is another medicinal fungus used in traditional Chinese therapeutics as well as in cuisine. It is named for its characteristic spines. H. erinaceus has been observed to produce various beneficial effects when consumed, ranging from enhancement of metabolic processes (such as fat metabolism)[31] to neurogenesis[32], to name a few.

Hericium erinaceus preparations contain molecules which may stimulate or suppress elements of the immune system[33]. Polysaccharides have been observed to promote macrophage activity while some other components of Lion’s mane preparations have been shown to inhibit chemotaxis[34].

Lion’s mane is available as powdered mushroom or the more potent powdered extract (in different strengths, e.g. 10:1 extract with 30% polysaccharide content). The typical dosage is as follows: 1 g of the purified extract by mouth three times a day with food[35]. As with all of these supplements, a lower starting dosage with a scheduled titration may be considered.

Adverse Effects

Hericium may cause itching. If this occurs without signs and symptoms of an allergic reaction such as hives, it is most likely due to nerve sensitivity in the setting of elevated Nerve Growth Factor (NGF)[36]. The long-term safety of Hericium is uncharacterised.




Vitamin C

vitamin c

This water-soluble vitamin has been traditionally recommended as an immune system booster specifically with regard to the common cold[37]. However, the data are highly conflictive as to whether supplementing vitamin C actually helps[38].

Vitamin C is purported to support the immune system through its function as an antioxidant, protecting host cells from oxidative stress caused by pathogens.

Stronger evidence supports the use of vitamin C to marginally reduce the duration of cold symptoms by approximately 1 to 1.5 days. A 2013 Cochrane meta-analysis found in 24 trials that studied the general population, supplementing vitamin C did not significantly reduce the risk of developing a cold (RR 0.97, 95% CI 0.94–1.00)[39]. The same meta-analysis found that regular vitamin C supplementation could modestly reduce the duration and the severity of cold symptoms. Adults experienced an 8% reduction (3–12%) and children a 14% reduction (7-21%).

Formulations & Dosage

While vitamin C is better absorbed at lower doses, it appears that higher doses are required to achieve benefit. A dosage of 1–3 g by mouth daily is recommended starting at the onset of symptoms & continued up to 3-5 days or as long as symptoms persist[40].

Labdoor has published rankings for the best vitamin C formulations.

Warnings

Individuals with G6PD deficiency should exercise caution with higher doses of vitamin C, as there is a risk of precipitating haemolytic anaemia[41].

Vitamin C is generally well-tolerated[42]. At high doses, however, adverse effects may present themselves. These include gastrointestinal upset marked by nausea, vomiting and diarrhoea and flushing of the skin.




Colostrum

colostrumColostrum is a rich pre-milk fluid derived from cows during the first 2–4 days after birth. This substance contains immune factors, amino acids, minerals, and other nutrients which promote the growth of the offspring[43]. Nutrients from colostrum support general metabolic functioning while other compounds may have intrinsic antimicrobial activity. Colostrum in particular provides a substantial amount of secretory immunoglobulin A (IgA), which is an antibody involved in the humoral immune response[44].

It is debatable whether adults would benefit from consumption of colostrum as much as newborns[45]. Newborns have a relatively undeveloped gastrointestinal system which does not degrade food intake to the extent that a fully developed gastrointestinal system would. An adult’s stomach would more likely break down many of the helpful constituents present in colostrum. Colostrum has been used to prevent some gastrointestinal infections in children, such as infectious diarrhoea & gastroenteritis. It may also be effective in preventing these infections in immunocompromised patients, such as individuals with AIDS. However, the efficacy of colostrum as an immune system booster in the healthy adult population has not been substantiated.

Warnings

Because colostrum is a dairy product, it is not recommended for individuals with lactose intolerance or dairy allergy.

Heat-denatured bovine immunoglobulin present in colostrum may contribute to atherosclerotic processes[46]. Patients with high cholesterol should discuss with their doctor or pharmacist before use.

Colostrum is generally well-tolerated. The most common side effects experienced with colostrum are gastrointestinal in nature, such as nausea, vomiting, diarrhea, intestinal gas, and bloating.




Conclusion

There are many different ways to augment your immune system, reducing your risk of becoming sick & attenuating symptoms of being sick. Before starting any substance, research how it works, how to take it (e.g. when to start, how much, when to stop, etc.), and any warnings (e.g. colostrum should not be tried in individuals with a dairy allergy). Stay safe!

References   [ + ]

1. Owen JA, Punt J, Stranford SA. Immunology. 7th ed. New York: W.H. Freeman & Company; c2013. Chapter 1, Overview of the Immune System.
2. Eby GA, Davis DR, Halcomb WW. Reduction in duration of common colds by zinc gluconate lozenges in a double-blind study. Antimicrobial Agents & Chemotherapy 1984;25:20-4.
3, 10, 11, 14. Zinc. Natural Medicines®. Therapeutic Research Center, Somerville, Massachusetts, USA. [updated 17 Jun 2015].
4. Sheikh A, Shamsuzzaman S, Ahmad SM, Nasrin D, Nahar S, Alam MM, Al Tarique A, Begum YA, Qadri SS, Chowdhury MI, Saha A, Larson CP, & Qadri F. Zinc influences innate immune responses in children with enterotoxigenic Escherichia coli-induced diarrhea. J Nutr 2010;140(5):1049-1056.
5. Licastro F, Chiricolo M, Mocchegiani E, et al. Oral zinc supplementation in Down’s syndrome subjects decreased infections & normalized some humoral & cellular immune parameters. J Intellect Disabil Res 1994;38:149-62.
6. Stabile A, Pesaresi MA, Stabile, AM, Pastore M, Sopo SM, Ricci R, Celestini E, & Segni G. Immunodeficiency & plasma zinc levels in children with Down’s syndrome: a long-term follow-up of oral zinc supplementation. Clin Immunol.Immunopathol. 1991;58(2):207-216.
7. Singh M, Das RR. Zinc for the common cold. Cochrane Database of Systematic Reviews 2013, Issue 6. Art. No.: CD001364. DOI: 10.1002/14651858.CD001364.pub4.
8. DiSilvestro RA. Handbook of Minerals as Nutritional Supplements. CRC Press: Boca Raton, Florida; 2004.
9. Eby GA. Zinc lozenges as cure for the common cold– a review and hypothesis. Med Hypotheses. 2010 Mar;74(3):482-92.
12, 13. Micromedex® 1.0 (Healthcare Series), (electronic version). Truven Health Analytics, Greenwood Village, Colorado, USA. Available at: http://www.micromedexsolutions.com/
15. Age-Related Eye Disease Study Research Group. The effect of five-year zinc supplementation on serum zinc, serum cholesterol and hematocrit in persons randomly assigned to treatment group in the age-related eye disease study: AREDS Report No. 7. J Nutr. 2002 Apr;132(4):697-702.
16, 20, 21, 24. Garlic. Natural Medicines®. Therapeutic Research Center, Somerville, Massachusetts, USA. [updated 14 Feb 2015].
17. World Health Organisation. Monographs on Selected Medicinal Plants, Volume I. Geneva: WHO; 1999.
18. Kasuga S, Uda N, Kyo E, Ushijima M, Morihara N, & Itakura Y. Pharmacologic activities of aged garlic extract in comparison with other garlic preparations. J Nutr 2001;131(3s):1080S-1084S.
19. Song K & Milner JA. The influence of heating on the anticancer properties of garlic. J Nutr 2001;131(3s):1054S-1057S.
22. Gurley BJ, Gardner SF, Hubbard MA, et al. Cytochrome P450 phenotypic ratios for predicting herb-drug interactions in humans. Clin Pharmacol Ther 2002;72:276-87.
23. Piscitelli SC, Burstein AH, Welden N, et al. The effect of garlic supplements on the pharmacokinetics of saquinavir. Clin Infect Dis 2002;34:234-8.
25, 27, 28. Reishi Mushroom. Natural Medicines®. Therapeutic Research Center, Somerville, Massachusetts, USA. [updated 16 Feb 2015].
26. Xu Z, Chen X, Zhong Z, Chen L, & Wang Y. Ganoderma lucidum polysaccharides: immunomodulation & potential anti-tumor activities. Am.J.Chin Med. 2011;39(1):15-27.
29. Gao Y, Lan J, Dai X, Ye J, & Zhou S. A Phase I/II Study of Ling Zhi Mushroom.
30. Tao J & Feng KY. Experimental & clinical studies on inhibitory effect of ganoderma lucidum on platelet aggregation. J Tongji Med Univ 1990;10:240-3.
31. Hiwatashi K, Kosaka Y, Suzuki N, Hata K, Mukaiyama T, Sakamoto K, et al. Yamabushitake mushroom (Hericium erinaceus) improved lipid metabolism in mice fed a high-fat diet. Biosci Biotechnol Biochem. 2010;74(7):1447-51.
32. Wong KH, Naidu M, David RP, Bakar R, Sabaratnam V. Neuroregenerative potential of lion’s mane mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (higher Basidiomycetes), in the treatment of peripheral nerve injury (review). Int J Med Mushrooms. 2012;14(5):427-46.
33. Kim YO, Lee SW, Oh CH, Rhee YH. Hericium erinaceus suppresses LPS-induced pro-inflammation gene activation in RAW264.7 macrophages. Immunopharmacol Immunotoxicol. 2012 Jun;34(3):504-12.
34. Abdulla MA, Fard AA, Sabaratnam V, Wong KH, Kuppusamy UR, Abdullah N, et al. Potential activity of aqueous extract of culinary-medicinal Lion’s Mane mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (Aphyllophoromycetideae) in accelerating wound healing in rats. Int J Med Mushrooms. 2011;13(1):33-9.
35. Mori K, Inatomi S, Ouchi K, Azumi Y, Tuchida T. Improving effects of the mushroom Yamabushitake (Hericium erinaceus) on mild cognitive impairment: a double-blind placebo-controlled clinical trial. Phytother Res. 2009 Mar;23(3):367-72.
36. Tanaka A, Matsuda H. Expression of nerve growth factor in itchy skins of atopic NC/NgaTnd mice. J Vet Med Sci. 2005 Sep;67(9):915-9.
37, 40, 41, 42. Vitamin C. Natural Medicines®. Therapeutic Research Center, Somerville, Massachusetts, USA. [updated 17 Mar 2015].
38, 39. Hemilä H, Chalker E. Vitamin C for preventing & treating the common cold. Cochrane Database of Systematic Reviews 2013, Issue 1. Art. No.: CD000980. DOI: 10.1002/14651858.CD000980.pub4.
43. Thapa, BR. Health factors in colostrum. Indian J Pediatr 2005;72(7):579-581.
44. He F, Tuomola E, Arvilommi H, Salminen S. Modulation of human humoral immune response through orally administered bovine colostrum. FEMS Immunol Med Microbiol. 2001 Aug;31(2):93-6.
45. Bovine Colostrum. Natural Medicines®. Therapeutic Research Center, Somerville, Massachusetts, USA. [updated 14 Feb 2015].
46. Annand, JC. Denatured bovine immunoglobulin pathogenic in atherosclerosis. Atherosclerosis 1986;59(3):347-351.
Categories
Tutorials

How to Understand Clinical Research, Part II: Quality of Evidence

The ability to critically understand & judge the data from a study is crucial in making decisions on whether a new drug is safe & effective. As we will see with studies regarding nootropics, the answer is not always clear. Understanding concepts of validity, bias, & limitation can help in the evaluation of any study.

Internal validity

Internal validity is analogous to the inner workings of a clock
Internal validity is analogous to the inner workings of a clock– a study with strong internal validity will produce results that truly reflect what the investigators sought to explore, like how a clock with well-adjusted inner workings will accurately display the time.

Internal validity is the strength of the study’s purported causal or associative relationship.

Higher level studies, such as randomised controlled trials & meta-analyses, seek to demonstrate a causal relationship (e.g. drug A causes improved cognitive function). Lower level studies, such as cohort studies & case-control studies provide evidence that demonstrates an association between a cause & effect (not as strong of an assumption: drug A is associated with improved cognitive function). The tighter the study’s internal validity, the more reliance we can have that drug A does indeed cause or is associated with improved cognitive function, rather than any other conclusion (i.e. has no effect on, worsens cognitive function).

Bias

Bias comprises confounding factors which may compromise a study’s internal validity.

For example, consider a study with 2 treatment groups testing the effect of a new drug on cognitive function. The group that receives the drug is generally more educated, while the group that receives placebo is generally less educated.

How much faith would you have if the investigators concluded that the drug significantly improved cognitive function?

This is an example of sample selection bias. We will introduce more forms of bias later that could impair internal validity & thus the ability to truly believe that a study’s results are relevant to the study question.

External validity

External validity is the generalisability of a study’s findings to populations beyond the study sample.

External validity should only be assessed after a study is found to be internally valid. If a study is not found to be internally valid, then its findings could not be said to truly answer the study question; & thus there would be no reason to evaluate whether its results should be generalised to others.While internal validity is susceptible to bias, external validity is counterbalanced by limitation. These are characteristics of the study sample which add to & restrict the population to whom the results may be generalised.

Always consider the type of people who were enrolled in the study
Always consider the type of people who were enrolled in the study & whether what worked (or didn’t work) for them would work for you.

Consider the previous example of a study, but with both groups comprising generally older subjects otherwise comparable at baseline. Absent other confounding variables, the study could be said to be internally valid: if the investigators reported a significant improvement in cognitive function, then this result would be probably accurate. However, whether we could assume that this drug would work for younger individuals would be up for question as it has not been tested in this population. This limitation of generalisability applies to other demographic information such as race, sex, & even geographic location, & may include comorbidity (the presence of other health conditions), diet, & other factors depending on how the data is to be used. Including more diverse individuals within a study may decrease limitations & increase external validity, but possibly at the expense of internal validity (without randomisation). Conversely, creating a more uniform sample could increase internal validity but introduce more limitations.

Other forms of bias

Other forms of bias that could impair internal validity include:

  • Sample selection bias is when the treatment groups are not equal at baseline due to demographic differences.
  • Intervention selection bias is present if different forms of the experimental variable are used. This is a risk when the study protocol is ambiguous; for this reason study protocols are usually very detailed so as to prevent deviation. Consider the previous study with 2 treatment groups. Assume the groups are balanced at baseline. However, among the individuals in the group receiving the study drug, two different manufacturers of the drug are used. This could potentially produce variation in the results, providing an imperfect picture of how well the drug actually works. To limit this type of bias, it would be more prudent to select one manufacturer or have two treatment groups (one for each manufacturer).
  • Measurement bias is when there exist variations in how outcomes are measured. If the study drug group took an easier cognitive test than the placebo group, then the results would show that they performed better, when in fact the comparison was not equal.
  • Outcome bias occurs if the selected endpoints do not correspond to the desired outcome of interest. For instance, if the researchers claiming to measure cognitive performance instead administered a personality test.
  • Attrition bias is when more subjects from one group leave the study than in the other. Although the two groups may have been equal at baseline, attrition may result in unequal groups later on in the study which can result in confounding due to imbalanced characteristics (e.g. if all the young subjects left from one group) or simply due to number (sample size too small to detect a difference).

When cognitive tests are administered to a group of subjects, two possible biases could confound the results:

  • Statistical regression to the mean is a type of bias wherein on the second administration of the same test to the same subjects, the worst performers from the first administration tend to perform better & the best performers tend to perform worse on the second exam. They regress to the mean.
  • The testing effect is when subjects who take the same test become familiar with the style & better at taking the test. Although they might perform better on subsequent applications of the same cognitive test, it may not be because the study drug resulted in cognitive improvement.
  • Other forms of biases which are less commonly implicated but could still undermine a study’s findings include maturation bias & history bias.

Summary

  • Validity, bias, & limitations are key aspects of study designs to consider when researching & evaluating clinical data. The strength of evidence is best with high internal & external validity, & low risk of bias.
  • Internal validity is the strength of a study design to determine a causal or associative relationship. Studies with highly controlled experimental methodology (which we will discuss in depth later) exhibit tight internal validity minimizing the effect of biases.
  • External validity is the extent to which findings from internally valid studies may be generalized to populations beyond the study sample. Being aware of limitations to external validity guides the extrapolation of study data.

If you are interested in reading more about validity & bias, & how to apply them when reading an article, I highly recommend the Cochrane Foundation’s tool for bias risk assessment. It has since been widely used in meta-analyses when deciding whether to include articles.

Categories
Tutorials

How to Understand Clinical Research, Part IV: Sample Data

In previous articles, we have learned how to access & read journal articles, how to interpret validity as a measure of the strength of evidence, and the various types of studies. With this foundation, we should have a basic idea of what to look for in a study. The next step would be developing our skills of interpreting the actual data from the article, which can be expressed as charts, tables, graphs, or text. The next two articles will be focused on the two major types of results in studies: sample data & endpoint data.

Sample Data

people-312122_1280

Sample data are information about the subjects who participated in the study. Although not what most people think of when they think of study results, sample data can profoundly influence one’s interpretation of endpoint data.

The first thing to consider in sample data is the sample size (n), or how many people participated in the study. Ideally, studies should enrol enough individuals to detect a treatment effect. For example, drug A may slightly improve cognitive function, but perhaps only in 15% of people. If only 50 patients are enrolled in the study, & 25 receive the study drug while the other half receives placebo, then only 3.75 individuals will likely experience the slight improvement in cognitive function. This small change is unlikely to be detected, especially when tested against a placebo effect.

How many subjects are needed, then, in order to truly detect a treatment effect? There exist statistical tests which guide investigators in all aspects of study design, including calculating the minimum sample size needed to detect a treatment effect. This process usually involves:

  1. estimating the magnitude of the treatment effect based upon previous studies,
  2. estimating the number of subjects needed to truly see that treatment effect, &
  3. minimizing the effects of chance & randomness

The first two parts should seem fairly intuitive after the previous example. The third requirement is a new concept, & is based on the following components (This is an area of biostatistics that gets a little complicated with over-thinking, but can be understood with enough time. Comment if you have questions!):

  • The hypothesis (H1) is what is predicted to happen in the experiment. It should be explicitly defined, such as: drug A shortens rapid recall time in comparison to placebo.
  • The null hypothesis (H0) is the opposite of the hypothesis- it is what is predicted to happen if there is no treatment difference: drug A does not affect rapid recall time in comparison to placebo.

Next we have the concepts of true positives/negatives & false positives/negatives. This is usually discussed in terms of the null hypothesis, which can sometimes be confusing if you overthink it.

Screen Shot 2015-09-21 at 18.39.04

The leftmost column represents the real result. If drug A really shortens rapid recall time, then in the best case scenario, the researchers will detect this difference & claim a treatment difference. This means rejecting the null hypothesis & accepting the hypothesis. If they fail to detect this difference (maybe because they did not enrol enough participants) then we would see the false negative scenario.

On the other hand, if drug A really is ineffective, then we would either see the true negative scenario or a false positive scenario. Both the false positive & false negative scenarios emerge out of chance & confounding (e.g. some subjects on the placebo have a good day while taking the recall test, or some start taking phenylpiracetam without telling the investigators, or some subjects in the treatment group take the recall test after a night of heavy drinking; these are all examples of biases that could compromise internal validity).

  • A false positive is called a type I error. The chance of a type I error happening is denoted as α (alpha).
  • A false negative is called a type II error. The chance of a type II error happening is denoted as β (beta).
  • When β is subtracted from 1 (1 – β), this difference is called the power of a study. The power is the chance of finding a true treatment effect (true positive). To bring the discussion back to prior to this talk on hypotheses, true & false results, & errors, power is a key component in determining what sample size we need to detect a treatment effect. The standard power in a study is 80% (1 – β = 0.80). At 80% power, we have an 80% chance of correctly rejecting the H0 & claiming that there is a treatment difference when, in fact, there is one. We achieve sufficient power by enrolling enough subjects, as power & sample size are proportionally related (⊕ n → ⊕ 1 – β ).

*Although power higher than 80% is possible, usually it is not done because:

  • Initial increases in sample size lead to higher increases in power than further increases, as shown in the following graph.
  • Enrolling many subjects requires a lot of funding.
  • α & β are inversely proportional- that means that as you increase your power (1 – β) & β decreases, then α will increase- meaning there is a higher risk of a false positive. At 80% power, there is a 20% chance of a false negative & a 5% chance of a false positive. 80% power is considered a balance between the type I error rate & the type II error rate.
Initial increases in sample size lead to more substantial increases in power than later increases. Select a sample size where power ≥ 0.8 for the best chance of detecting the treatment effect- here it is a little less than 120 patients. Enrolling fewer patients would lead to a higher risk of a false positive.

So, we have established that power is a key driver of how many patients to enroll. We need to enroll enough patients so that the treatment effect does emerge & we can reliably detect it, with minimal concern for confounding & chance.

A few other odds & ends to consider in sample data:

  • Ideally, all treatment groups will be equal or similar in size
  • All treatment groups should remain similar throughout the study- if more subjects drop out of one group than another, that could lead to attrition bias. If a disproportionate amount of patients drops out from the study drug group, that should be a warning sign– investigate why they dropped out (side effects?).
  • Baseline characteristics are traits of the subjects in the study at the beginning before the study drug or placebo has been administered. These data, as previously mentioned, are typically presented in a standard table 1. It is important to skim this table for (1) significant differences between the groups & (2) generalisability/external validity- whether the results could be applied to you.
modafinil vs placebo
Discussion of power in a small RCT comparing modafinil vs. placebo. [1]

Summary

  • Sample data describe the characteristics of participants in a study.
  • Sample size (n) is the amount of subjects that are enrolled in a study. It is far from an arbitrary number– the determination of how many people are needed in a study is predicated upon the anticipated magnitude of the treatment effect, the number of subjects needed to have that effect emerge, & minimising «statistical noise» by reducing the risk of false positives (α) & false negatives (β).
  • Power (1 – β) is the chance of a true positive. Researchers typically prefer 80% power as it balances the chance of a false negative with the chance of a false positive (both error rates cannot be controlled simultaneously). Power is achieved by increasing the number of patients in a study. For those interested, power & its determinants can be mathematically expressed as: ⊕ Δ , σ, ⊕ n → ⊕ δ → ⊕ ( 1 – β ) where Δ is the true difference between groups, σ is the standard deviation, n is the sample size, & δ is the non-centrality parameter. An equation form would be δ = ( Δ ÷ σ ) √ ( n ÷ 2 )
  • Other key sample data to consider are the baseline, interim, & final sample sizes between the groups as well as the baseline characteristics.

References   [ + ]

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Tutorials

How to Understand Clinical Research, Part III: Types of Studies

In clinical research, there exist different types of studies which serve particular purposes. These studies are distinguished based on their experimental design (how the study is conducted) & the kind of data they produce– from the way a study is designed, we can draw certain expectations about the grade of evidence it produces.

Experimental designs can be described in several ways. A basic division of study designs can be made on how test subjects are enrolled, which significantly determines the study’s strength in describing a relationship between a cause (an experimental variable such as a drug to be tested) & an effect (an outcome such as cognitive performance). This particular way of classifying studies results in two main families of studies: observational studies & assignment studies.

Observational Studies

Observational studies are conducted in order to determine associations between certain prior exposures (e.g. a drug) & outcomes of interest (e.g. death).

Here, participants are selected based on exposure or outcome, depending on the type of observational study. These are more prevalent in research on nootropics as randomised controlled trials (RCTs) are generally conducted with larger samples requiring more funding. Small observational studies build up a body of evidence which provide the grounds for an RCT, a process called «hypothesis-generating» (as opposed to hypothesis-testing). Cohort studies & case-control studies are two major types of observational studies, both of which involve following a group of patients over a period of time.

Subjects in cohort studies are selected based on their having received a particular exposure, then they are followed prospectively (forward in time) until a certain outcome of interest (e.g. death) occurs. RCTs are also prospective.

Depiction of observational studies. Prospective cohort studies work from the perspective of the left viewer, while retrospective case-control studies work from the perspective of the right viewer.

In a case-control study, subjects are selected based on their exhibiting a certain outcome & tracing their history back (retrospectively) to find out whether they have had a certain exposure (e.g. used a particular drug). Retrospective case-control studies are especially useful when studying rare diseases.

Assignment Studies

Assignment studies enroll subjects to either test or control groups.

Assignment studies are subdivided based on (1) whether the allocation of subjects into test groups is randomised & (2) whether a control group is present.

Randomised controlled trials (RCTs) are generally considered to be the gold standard of clinical evidence for their strong internal validity, & are used to demonstrate causal relationships between experimental variables & outcomes. Randomly assigning patients to either treatment or control groups theoretically establishes equal groups, as any differences in age, race, comorbidity, or other features are equally distributed (eliminates sample selection bias). Prospective follow-up & the presence of a control group allows for comparison of the experimental variable (e.g. a new drug) against a standard treatment (to demonstrate a better treatment effect) or placebo (to demonstrate a treatment effect).

Mohamed AD, Lewis CR. Modafinil increases the latency of response in the Hayling Sentence Completion Test in healthy volunteers: a randomised controlled trial. PLoS One. 2014 Nov 12;9(11):e110639.
Standard CONSORT diagram depicting enrolment, allocation, follow-up, & analysis of subjects from an RCT comparing modafinil vs. placebo.

Synthetic Studies

Systematic reviews & meta-analyses critically evaluate the literature by consolidating the results of several studies focused on the same topic.

Synthetic studies are more recent study designs that have been developed out of a need to draw from the existing evidence on a topic. Prior to the rise of systematic reviews & meta-analyses, studies were selectively cited which led to bias (e.g. selecting only the studies which supported the use of a drug & either intentionally or unwittingly omitting the others which found significant side effects). Nowadays, both are considered the highest forms of clinical evidence, producing strong inferences of treatment effects. Synthetic studies are part of the trend of comparative efficacy analyses (CEAs): gathering data on several major drugs used for the same purpose & determining which are superior. Collecting findings from multiple RCTs & observational studies can produce a more complete picture of a drug’s safety & efficacy- in other words, considering the ‘big picture’. However, before drawing conclusions, one must be cognisant of differences between the studies that have been gathered (e.g. study protocol, different doses used, different sample characteristics).

Systematic reviews present findings from a pre-defined, reproducible search of the literature- that is, the authors exhaustively describe the methods they used to search databases & how they selected which studies to include in their systematic review, usually with a pre-defined criteria set. The importance of reproducibility is to reduce bias from selective inclusion of studies– this is a weakness of narrative reviews, in which the author performs a search & simply chooses which studies to include.

Meta-analyses are systematic reviews where the gathered data is then combined, producing an estimate of the true treatment effect from the pooled data. This is commonly expressed in what’s called a Forest plot, which shows the individual trials included in the systematic review as well as a diamond representing the estimate of the true efficacy or safety measure of the drug (how to read & interpret different tables & graphs will be covered later!).

An example Forest plot, with included trials listed on the left & their findings on the right. The findings are also plotted, with the diamond representing the composite of the studies’ results.

Summary

As we have seen, the design of a clinical trial can provide a quick way to judge its findings.

  • Observational studies produce evidence of associations by following patients over a period of time. Patients are selected based on a specific previous exposure in the case of prospective cohort studies or for a certain outcome in the case of retrospective case-control studies.
  • Assignment studies produce evidence of causal relationships by assigning patients to multiple groups including a comparator arm. The most prominent example of an assignment study is the randomised control trial, which has become the standard for clinical data.
  • Synthetic evaluations of the literature, such as systematic reviews & meta-analyses, draw on existing studies to better approximate treatment effects of drugs of interest.
  • Other experimental designs which are less commonly relevant to the area of nootropics include cross-sectional observational studies & non-randomised controlled trials.
Categories
Cognitive Health Memantine Nootropics Recovery

Can Nootropics Help With Drug Abuse and Addiction?

In recent years, evidence has compiled suggesting a common pathologic mechanism underlying addictive behaviours of several substances. Dysregulation of glutamatergic neurotransmission within the prefrontal cortex (PFC) and nucleus accumbens (NA) appears to predispose to a higher tendency towards drug-seeking behaviour.

Thus far, this mechanism has been associated with the addiction potential of cocaine, heroin, nicotine, cannabis, & alcohol, with possible implications for other substances and even non-drug-related compulsive habits such as pathological gambling. Discovery of this shared pathology has led to the investigation of the potential application of existing agents, such as Memantine and n-acetylcysteine.

Could nootropics targeting elements in this key glutamatergic circuit reduce symptoms and complications of substance use disorders?

Glutamate Spillover

«Glutamate spillover» refers to the pathologic cascade in brain chemistry that occurs with chronic abuse of certain substances that results in reinforcement of the behaviour[1].

McClure EA, Gipson CD, Malcolm RJ, Kalivas PW, Gray KM. Potential role of N-acetylcysteine in the management of substance use disorders. CNS Drugs. 2014 02;28(2):95-106.

Prolonged exposure to substances of abuse leads to several maladaptive changes in the glutamatergic PFC-NA pathway, specifically:

  • Downregulation of glial glutamate transporter-1 (GLT1) expression in the nucleus accumbens. By removing glutamate from the extrasynaptic space, GLT1 prevents inappropriate excitatory stimulation due to an accumulation of the excitatory neurotransmitter.
  • Decreased ability of presynaptic metabotropic glutamate receptor 2 (mGluR2) to inhibit glutamate release. In normal physiology, mGluR2 autoreceptors manage a feedback loop where increased extracellular glutamate levels trigger a reduction in the presynaptic release of glutamate. This auto-regulatory mechanism also serves to prevent an extracellular accumulation of glutamate.

When glutamate spillover within the non-synaptic extracellular space does occur as a result of the combination of these processes, the following sequelae are may manifest:

  • Stimulation of postsynaptic mGluR5, AMPA and NMDA receptors.
  • Upregulation of AMPA and NMDA receptors (increased synaptic plasticity).
  • Stimulation of extrasynaptic glutamate receptors may also occur.

Increased excitatory tone due to these two processes culminates in impaired inhibition with regard to drug-seeking behaviour as well as increased risk of relapse. Furthermore, persistently elevated glutamatergic tone may lead to neurotoxicity secondary to excessive Ca2+ ion influx. This pathology has also been associated in neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s disease.

n-acetylcysteine

n-acetylcysteine (NAC) is a cysteine precursor that has a long history of use for indications ranging from bronchopulmonary disorders to paracetamol overdose. It produces many beneficial effects through a variety of mechanisms ranging from supporting antioxidant processes to suppressing over-reactive immune responses to inhibiting apoptosis. NAC’s glutamatergic modulation, however, is of key interest in managing substance use disorders[2].

Brown RM, Kupchik YM, Kalivas PW. The story of glutamate in drug addiction & of n-acetylcysteine as a potential pharmacotherapy. JAMA Psychiatry. 2013 09;70(9):895-7.

NAC is converted to L-cysteine in vivo, which enhances the activity of the cysteine/glutamate exchange transporter positioned near the pre-synaptic terminal. This increases the concentration of extracellular glutamate, resulting in increased tonic activation of pre-synaptic mGluR2 autoreceptors. This causes a subsequent decrease in glutamate release. NAC also increases expression of GLT1 and the cysteine/glutamate exchange transporter, promoting the removal of glutamate from the extrasynaptic space and ‹putting it back› in the pre-synaptic area. These effects in concert have been shown to mitigate the complications from glutamate spillover, & have been tested in several small trials with promising results.

  • When administered in patients with a history of cocaine addiction, NAC was shown to decrease self-reported cocaine use within the 28 days of treatment (mean 8.1 days out of 28 days before treatment & 1.1 days during treatment, p = 0.001)[3], desire to use cocaine (F = 5.07; df = 1,13; p = 0.05), & response to cocaine cues (F = 4.79, df = 1,13, p = 0.05)[4]. A magnetic resonance spectroscopy study confirmed elevated glutamate levels in the dorsal anterior cingulate cortex of cocaine users when compared against non-users (t(7) = 3.08, p = 0.02), and also showed a reduction 1 hour after a single 2.4 g dose of NAC[5].
  • With regard to cannabis, 2.4 g/day NAC decreased craving in one 4-week open label study of 24 patients[6]; in a double-blind placebo-controlled trial, subjects given 2.4 g/day NAC in addition to counselling were 2.4 times more likely to test negative on urinalysis (95%CI 1.1 to 5.2) but there was no difference in number of reported days of cannabis use[7].

The dosage for managing consequences of substance use disorders in trials ranged from 1.2 to 2.4 g by mouth daily. Benefits on neurochemistry may occur with single doses although significant alterations in behaviour may take days to weeks. The pharmacodynamic effect also depends upon the history of substance use and individual predisposition to addictive behaviour.

NAC is significantly protein-bound (80%). It is metabolised in the liver via non-CYP450 pathways. NAC and its metabolites are primarily eliminated in the urine, with a half-life of 5.6 hours in adults[8].

NAC is generally well-tolerated. Nausea, vomiting, rash, and fever have been reported.

Memantine

Memantine (Namenda®) is an uncompetitive NMDA receptor antagonist most commonly used in the management of moderate-to-severe Alzheimer’s disease. In addition to its glutamatergic modulation, memantine also acts as an agonist at the D2 and nicotinic acetylcholinergic receptors (nAChR). Memantine binds and inhibits NMDA receptors with low-to-moderate affinity, most effectively in states of excess glutamatergic activity (such as in substance use disorder). By blocking NMDA receptors, memantine decreases glutamatergic tone[9].

Clapp P, Bhave SV, Hoffman PL. How adaptation of the brain to alcohol leads to dependence: a pharmacological perspective. Alcohol Res Health. 2008;31(4):310-39.
Neurochemical effects of alcohol intoxication in various contexts.

Upregulation of NMDA receptors has been observed with chronic alcohol consumption. Abrupt discontinuation of alcohol removes GABAergic suppression, resulting in the characteristic acute sequelae of alcohol withdrawal (symptoms of excitotoxicity): seizures, hallucinations, tachycardia, and shock. By inhibiting these receptors, memantine may theoretically attenuate symptoms of alcohol withdrawal.

  • In one RCT of 18 moderate alcohol drinkers (10-30 drinks/week), 30 mg/day memantine significantly decreased alcohol craving before alcohol consumption in comparison to 15 mg/day and placebo[10]. Another placebo-controlled RCT with 10-40 mg/day showed no difference[11].
  • A subsequent study of 38 patients utilising 20-40 mg/day memantine showed dose-dependent reductions in cue-induced craving[12].
  • In another RCT of 127 male patients undergoing alcohol withdrawal, administration of 10 mg memantine three times a day decreased apparent withdrawal symptom severity, dysphoria, and need for diazepam[13].
  • Administration of 60 mg significantly alleviated subjectively-rated symptoms of naloxone-induced opioid withdrawal in 8 heroin-dependent patients[14].
  • In a study of 67 heroin-dependent subjects, 10-30 mg/day memantine significantly reduced heroin craving, depression, and state & trait anxiety compared to placebo after 3 weeks of use. A separate treatment arm using amitriptyline 75 mg/day achieved similar results but with a higher incidence of side effects and a higher dropout rate[15].
  • Clinical data on application in cocaine[16],[17] and nicotine abuse[18] is less promising.

The dosage for mitigating substance use disorders in trials ranged from 5 to 60 mg, with 30 mg by mouth once daily showing the best effects for alcohol abuse and 30 to 60 mg by mouth once daily shown to be most effective in limited trials for opioid dependence. Safety is best characterised at doses up to 30 mg, as this dosage is used in Alzheimer’s disease. Memantine is typically initiated at 5 mg daily then titrated by 5 mg per week up to the goal dose (30 to 60 mg depending upon the indication).

Memantine undergoes favourable non-hepatic metabolism; its metabolites are minimally active. Individuals with a history of kidney disease should consult a doctor or pharmacist before use, as memantine undergoes significant renal elimination (74% is excreted in the urine). The half-life of memantine ranges from 60-80 hours.

The most common side effects noted at therapeutic doses higher than 7 mg/day are dizziness, headache, confusion, anxiety; increased blood pressure; cough; & constipation[19].

Summary

  • Disrupted regulation of glutamatergic pathways in the prefrontal cortex-nucleus accumbent pathway has been implicated as an underlying pathology among several substance use disorders, including cocaine, alcohol, and opioid dependence.
  • Therapies such as n-acetylcysteine (NAC) and memantine have demonstrated efficacy in attenuating the symptoms of some of these disorders in small trials.

References   [ + ]

1. McClure EA, Gipson CD, Malcolm RJ, Kalivas PW, Gray KM. Potential role of n-acetylcysteine in the management of substance use disorders. CNS Drugs. 2014 02;28(2):95-106.
2. Brown RM, Kupchik YM, Kalivas PW. The story of glutamate in drug addiction & of n-acetylcysteine as a potential pharmacotherapy. JAMA Psychiatry. 2013 09;70(9):895-7.
3. Mardikian PN, LaRowe SD, Hedden S, Kalivas PW, Malcolm RJ. An open-label trial of n-acetylcysteine for the treatment of cocaine dependence: a pilot study. Prog Neuropsychopharmacol Biol Psychiatry. 2007;31:389-94.
4. LaRowe SD, Myrick H, Hedden S, Mardikian P, Saladin M, McRae A, et al. Is cocaine desire reduced by n-acetylcysteine? Am J Psychiatry. 2007;164:1115-7.
5. Schmaal L, Veltman DJ, Nederveen A,van den Brink W, Goudriaan AE. n-acetylcysteine normalizes glutamate levels in cocaine- dependent patients: a randomized crossover magnetic resonance spectroscopy study. Neuropsychopharmacology. 2012;37:2143-52.
6. Gray KM, Watson NL, Carpenter MJ, LaRowe SD. n-acetylcysteine (NAC) in young marijuana users: an open-label pilot study. Am J Addict. 2010;19:187-9.
7. Gray KM, Carpenter MJ, Baker NL, DeSantis SM, Kryway E, Hartwell KJ, et al. A double-blind randomized controlled trial of n-acetylcysteine in cannabis-dependent adolescents. Am J Psychiatry. 2012;169:805-12.
8. Medscape® 5.1.2, (electronic version). Reuters Health Information, New York, New York.
9. Zdanys K, Tampi RR. A systematic review of off-label uses of memantine for psychiatric disorders. Prog Neuro-Psychopharmacol Biol Psychiatry. 2008 8/1;32(6):1362-74.
10. Bisaga A, Evans SM. Acute effects of memantine in combination with alcohol in moderate drinkers. Psychopharmacology 2004;172:16–24.
11. Evans SM, Levin FR, Brooks DJ, Garawi F. A pilot double-blind treatment trial of memantine for alcohol dependence. Alcoholism: Clin Exp Res 2007;31(5):775–82.
12. Krupitsky EM, Neznanova O, Masalov D, Burakov AM, Didenko T, Romanova T, et al. Effect of memantine on cue-induced alcohol craving in recovering alcohol-dependent patients. Am J Psychiatry 2007a;164(3):519–23.
13. Krupitsky EM, Rudenko AA, Burakov AM, Slavina TY, Grinenko AA, Pittman B, et al. Antiglutamatergic strategies for ethanol detoxification: comparison with placebo & diazepam. Alcoholism: Clin Exp Res 2007b;31(4):604–11.
14. Bisaga A, Comer SD, Ward AS, Popik P, Kleber HD, Fischman MW. The NMDA antagonist memantine attenuates the expression of opioid physical dependence in humans. Psychopharmacology 2001(157):1–10.
15. Krupitsky EM, Masalov DV, Burakov AM, Didenko TY, Romanova TN, Bespalov AY, et al. A pilot study of memantine effects on protracted withdrawal (syndrome of anhedonia) in heroin addicts. Addict Disord Treat 2002;1(4):143–6.
16. Collins ED, Vosburg SK, Ward AS, Haney M, Foltin RW. Memantine increases cardiovascular but not behavioral effects of cocaine in methadone-maintained humans. Pharmacol Biochem Behav 2006;83(1):47–55.
17. Collins ED, Ward AS, McDowell DM, Foltin RW, Fischman MW. The effects of memantine on the subjective, reinforcing, & cardiovascular effects of cocaine in humans. Behav Pharmacol 1998;9(7):587–98.
18. Thuerauf N, Lunkenheimer J, Lunkenheimer B, Sperling W, Bleich S, Schlabeck M, et al. Memantine fails to facilitate partial cigarette deprivation in smokers—no role of memantine in the treatment of nicotine dependency? J Neural Transm 2007;114:351–7.
19. Micromedex® 1.0 (Healthcare Series), (electronic version). Truven Health Analytics, Greenwood Village, Colorado, U.S.A. Available at: http://www.micromedexsolutions.com/
Categories
Tutorials

How to Understand Clinical Research, Part I: Accessing and Reading Research

researchA major part of the intrigue surrounding nootropics has to do with the fact that many of these compounds have not been largely studied. In effect, we are gambling with our neurochemistry in order to gain some benefit in our mental functions. But we are not without resources which can improve our chances of using nootropics safely & effectively. While the body of evidence behind nootropic agents is not large, it is growing, & will likely continue to grow with increasing rapidity as public interest in nootropics increases. Drawing upon this background of research can help us understand how nootropic agents work, in whom they work, & what the risks are. To this end, I will be launching a multi-part tutorial on how to understand & interpret clinical trials, designed for both novices & more advanced users. Topics we’ll cover include:

  • Validity & bias
  • Types of studies & hierarchy of evidence
  • Reading results (graphs & tables)
  • Methodology
  • Basic biostatistics

How to access clinical research

Pubmed search
A Pubmed search with limitations for clinical trials, free full text, & publication within the last 5 years.

First of all, clinical research is generally accessible by online databases through universities or hospitals. If you have access to some of these databases through your institution, such as Medline or Ebscohost, I highly recommend familiarizing yourself with them. For those without institutional database access, Pubmed offers a large open-access catalog of many journal articles. Google Scholar is another option for finding studies, but does not offer advanced search functions.

Pubmed screenshot 2
The abstract is shown, as well as full-text links on the right side.

Here are some other tips when searching for journal articles:

  • I would generally recommend limiting one’s search to full text articles, as abstracts do not often reveal the full story of a study.
  • Searching by MeSH terms (analogous to tags for topics) usually provides more relevant results than a basic keyword search. This involves searching for a MeSH term, selecting it, then running the search.
  • More recent results (preferably within the last 5 years) are preferable, as scientific research can move fast. Depending on the topic area, however, you might find yourself stretching your search to include up to 10 years.
  • Be aware of the country of origin of the article, as standards for publication may vary.
  • Authors who write many articles on the same topic may be biased &/or highly knowledgeable.
Pubmed screenshot 3
Select the desired MeSH term, add it to your search on the right side, then run your search.

Structure of a journal article

So you have located a journal article of interest. Fortunately, every journal article generally follows a similar structure. This organisation is designed to present the details of the study in an intuitive order.

  • The abstract is a short summary of study’s methods & results.
  • The background section reviews the current state of understanding in the topic area of interest. The investigators conducting the study also explain what they are trying to show.
  • In the methods portion, key details of how the study works are defined:
    • Endpoints or outcomes are what is being measured, such as performance on a cognitive test
    • Experimental variables are what is being tested, such as the study drug & the control against which it is compared (placebo or standard treatment)
    • The type of individuals the investigators wanted to analyse in their study as test subjects
    • The allocation or assignment of enrolled individuals to either treatment groups (who receive the study drug) or control groups is typically visualized in a flowchart
    • Statistical tests used to analyse the data
  • The results section is where authors list their findings only (without interpretation). These include:
    • Baseline characteristics– a description of the final sample. Most often, this is summarized in table labelled as Table 1. When reading this section, think about the age, race, geography, & health status of the sample, & whether they are similar to you.
    • Outcomes– how did people who took the drug do in comparison to those who took the control? These data will be presented in tables, charts, graphs, & text.
  • Considered to be the most important section, the discussion area is where investigators interpret the results- what they mean, whether they are significant, where there could be error, the weaknesses of their study, & areas for further research. What is stated in this section can sometimes be highly contentious.

Some tips for reading an article:

  • The background section is not usually necessary unless if the topic area is new to the reader- if you are extensively researching a drug by reading multiple articles, you will find that many of their background sections are similar. However, if you don’t understand what’s covered in the background section, bring yourself up to speed with other resources such as Wikipedia.
  • Some prefer to read the abstract first to obtain a rapid summary of the study, then the discussion second for a detailed look at how the authors felt about the findings.
  • Always compare the raw numbers from the results section against the authors’ interpretation in the discussion section. Never take what the authors state at face value. Do the numbers actually show what they claim is happening?
  • Yes, the word «data» is plural.
  • I personally prefer to print out the pdf article & write comments on the hard copy as I read.
  • It’s not uncommon to read the same article several times. These subjects are quite advanced, & many details are important.
  • Check the articles cited in the bibliography for other studies that might be related to your topic.
Table 1.
A (very short) table 1. More meticulous studies will list more baseline characteristics of test subjects.

Summary

In review, using clinical data can provide a powerful edge when making decisions about nootropics. The informed nootropic user is better able to discern which nootropics are safe & effective.

  • Clinical research is accessible within databases which are offered through institutions. The general public can access some research through resources such as Pubmed or Google Scholar.
  • All journal articles follow the same general format consisting of an abstract, background, methods, results, & discussion sections. Knowing where to find what information you need within an article can make reading articles faster.
Categories
Life Extension

Epitalon, Part II: Mechanism of Action

Peptide bioregulators, like Epitalon, Cortexin or Thymalin work via pleiotropic mechanisms, providing support to diverse areas of day-to-day cellular and tissue-related functions. The culmination of these small supportive mechanisms is believed to produce the purported anti-ageing effect of peptide bioregulators and, in particular, Epitalon. In this article, a few central mechanisms contributing to this preserving effect will be highlighted.

Physiology

To begin, peptides are small chains of amino acids (the basic units of organic matter) linked by amide bonds, & can be thought of as «small proteins». Proteins are larger macromolecules comprised of many more amino acids linked together. Despite their size, however, peptides play many important roles in the normal functioning of the human body. Some examples include insulin, which is responsible for controlling levels of glucose within the blood, and substance P, a peptide playing a major role in the perception of pain.

Telomere Elongation

With each cellular replication, the telomeres (in green) are shortened. The maximum number of times that DNA can be replicated is called the Hayflick Limit. Once this number is reached, the cell undergoes apoptosis or programmed cell death. Telomerase is an enzyme which restores telomere length.

Epitalon is a synthetic peptide which were originally developed based upon the action of epithalamin, a hormone produced by the pineal gland. This hormone was found to stimulate the production of telomerase, an enzyme which plays a role in maintaining telomere length. Telomeres are non-coding terminal regions of DNA strands which preserve the integrity of the strand. With each revision, telomeres are shortened until the DNA strand cannot be further replicated. This process is highly implicated in the ageing process. Elongating telomeres theoretically extend the lifespan of a copy of DNA and allows it to replicate more times than usual. This was the theory behind the development of Epitalon®, a synthetic version of epithalamin which also stimulates the production of telomerase. Indeed, this theory has been confirmed in vitro in human cell cultures[1].

Neutralisation of Harmful Free Radicals

The role of antioxidants in mitigating damage from free radicals.

Cytotoxicity secondary to free radical damage has been implicated in the ageing process[2]. Administration of epithalon has also been shown to exert an antioxidant effect[3]. The presence of toxic compounds within the body can lead to the formation of reactive oxygen species (ROS) which can damage DNA, leading to cellular death &/or mutations leading to the formation of cancerous cells.

Inhibition of Cancer Formation & Growth

Maintaining the integrity of the genetic information stored in DNA is one of the mechanisms in which peptide bioregulators work to prevent cancer.

The anti-carcinogenic effect of Epitalon has also been explored in several animal studies. Epitalon has shown beneficial effects in animal models of breast[4] & colorectal cancer[5] without significant rates of adverse effects. Purported mechanisms include inhibition of carcinogenic receptor expression (Human Epithelial growth factor Receptor 2, also known as HER2, which is over-expressed in breast cancer) & retardation of metastasis[6],[7].

Attenuation of Inflammation

fire

Inflammation is a normal immune response that can become dysregulated and potentiated in a broad spectrum of disorders from rheumatoid arthritis to ulcerative colitis and even has been implicated in psychiatric disorders. The inflammatory process is dependent upon intercellular communication mediated by biomolecules such as cytokines, C-reactive protein, and other acute phase reactants[8]. Epithalamin has been observed to play a role in the regulation of these molecules[9] & thus attenuate the inflammatory response[10].

Endocrine Regulation

Hormones released by endocrine glands play signalling roles that coordinate different organs of the body.

Epitalon has been shown to help regulate endocrine activity in the body. Hormones are responsible for many key signalling circuits between cells which on a larger scale comprise the functions of large organs. For example, melatonin is a hormone which regulates the circadian rhythm, an internal biological clock. Endogenous melatonin production has been observed to decrease with ageing. A 2007 study of Epitalon administration in elderly patients found that the compound helped to restore pineal gland function & increased release of melatonin[11], which is purported to be the mechanism behind the restoration of sleep. Other studies have found that Epitalon exerts regulatory effects on gonadotropic hormones (FSG, LG, prolactin), which are involved in sexual & reproductive functions[12].

Summary

These are but a few of the many mechanisms by which Epitalon support normal functioning of the human body, and ultimately produce their anti-ageing effect. Telomerase activation, neutralisation of free radicals, oncostasis, modulation of inflammatory mediators, and endocrine regulation are all ways in which peptide bioregulators can help to ultimately prolong life. Peptide bioregulators as a class are still relatively uncharacterised, and most of the available clinical data are from Russian studies. As interest in this class of drugs grows, more mechanisms of action may be discovered, and the true treatment effects of Epitalon and peptide bioregulators may begin to be better understood.

References   [ + ]

1. Khavinson VK, Bondarev IE, Butyugov AA. Epithalon peptide induces telomerase activity & telomere elongation in human somatic cells. Bull Exp Biol Med. 2003 Jun;135(6):590-2.
2. Hekimi S, Lapointe J, Wen Y. Taking a “good” look at free radicals in the aging process. Trends In Cell Biology. 2011;21(10) 569-76.
3. Anisimov VN, Arutjunyan AV, Khavinson VK. Effects of pineal peptide preparation Epithalamin on free-radical processes in humans and animals. Neuro Endocrinol Lett. 2001;22(1):9-18.
4. Anisimov VN, Khavinson VK, Provinciali M, Alimova IN, Baturin DA, Popovich IG, et al. Inhibitory effect of the peptide epitalon on the development of spontaneous mammary tumours in HER-2/neu transgenic mice. Int J Cancer. 2002 Sep 1;101(1):7-10.
5. Anisimov VN, Khavinson VK, Popovich IG, Zabezhinski MA. Inhibitory effect of peptide Epitalon on colon carcinogenesis induced by 1,2-dimethylhydrazine in rats. Cancer Lett. 2002 Sep 8;183(1):1-8.
6. Kossoy G, Zandbank J, Tendler E, Anisimov V, Khavinson V, Popovich I, et al. Epitalon and colon carcinogenesis in rats: proliferative activity & apoptosis in colon tumors & mucosa. Int J Mol Med. 2003 Oct;12(4):473-7.
7. Kossoy G, Anisimov VN, Ben-Hur H, Kossoy N, Zusman I. Effect of the synthetic pineal peptide epitalon on spontaneous carcinogenesis in female C3H/He mice. In Vivo. 2006 Mar-Apr;20(2):253-7.
8. Owen JA, Punt J, Stranford SA. Immunology. 7th ed. New York: W.H. Freeman & Company; (c) 2013.
9. Labunets IF, Butenko GM, Korkushko OV, Shatilo VB. Effect of epithalamin on the rhythm of immune and endocrine systems functioning in patients with chronic coronary disease. Bull Exp Biol Med. 2007 Apr;143(4):472-5.
10. Khavinson VKh. Peptide regulation of aging. Proceedings of the 17th World Congress of the International Association of Gerontology; Jul 1–6 2001; Vancouver, Canada. Gerontology: 2001; 47(1).
11. Korkushko OV, Lapin BA, Goncharova ND, Khavinson VK, Shatilo VB, Vengerin AA, et al. Normalizing effect of the pineal gland peptides on the daily melatonin rhythm in old monkeys & elderly people. Adv Gerontol. 2007;20(1):74-85.
12. Slepushkin VD, Mordovin VF, Zoloev GK, Iakovleva RA, Khavinson VK. Effect of the epiphysial preparation epithalamin on the gonadotropic function of the hypophysis. Probl Endokrinol (Mosk). 1983 Nov-Dec;29(6):51-4.
Categories
Methylene Blue Nootropics Reviews

Methylene Blue as a Nootropic? (Review)

Since its initial synthesis in 1886, the phenothiazine derivative methylene blue (MB) has been established as a highly versatile chemical agent with a diverse span of uses, ranging from treating malaria to dying textiles[1]. Within the past few years, preclinical research has suggested a possible neuroprotective benefit from MB administration. MB is believed to promote neuronal cell health by supporting mitochondrial function. Animal studies have yielded promising results in neurocognitive tests[2][3]. Here is what you need to know if you’re interested in using methylene blue.

How does Methylene Blue work?

Mitochondria are organelles within cells that play the key role of energy production. Cellular energy is stored in the form of adenosine triphosphate (ATP), one of the most important molecules in the cell. As the name suggests, ATP contains three linked phosphate groups. Removal of each group releases a large amount of energy, which is expended in supporting cellular function. Subsequent removal of ATP produces ADP (adenosine diphosphate) & AMP (adenosine monophosphate). ATP is produced within mitochondria as a final product of respiration, a series of biochemical reactions that extract energy from glucose. These biochemical reactions require oxygen & electron carriers (e.g. NADH).

Poteet E, Winters A, Yan L, Shufelt K, Green KN, Simpkins JW, et al. Neuroprotective actions of methylene blue & its derivatives. PLoS One. 2012;7(10):e48279-.
Methylene blue acts as an artificial electron carrier, promoting mitochondrial respiration. The net outcome is more energy available as ATP for cellular processes.[4]
Methylene blue supports mitochondrial respiration by functioning as an additional electron carrier[5]. MB receives electrons from NADH through mitochondrial complex I, itself being reduced to leuco-MB (MBH2). Leuco-MB then donates the electrons to cytochrome C, upon which it is recycled back to MB. These reactions serve to create a high proton (H+) concentration in the space between the inner & outer mitochondrial membranes. This leads to the passage of H+ down the concentration gradient, through mitochondrial complex V. In doing so, ADP & a phosphate (Pi) are joined to form ATP. Leuco-MB can also act as a free radical scavenger, neutralising superoxides by accepting electrons & itself becoming oxidized back to MB[6]. In this way, leuco-MB acts to prevent direct oxidative damage caused by free radicals.

Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue. Prog Neurobiol. 2012 Jan;96(1):32-45.
Methylene blue preferentially accumulates in more active neurones (A), & potentiates mitochondrial & synaptic activity (B). These processes result in increased & improved neurotransmission (C), which is thought to be the mechanism behind the neurocognitive benefits associated with MB.[7]
MB has been observed to preferentially localise in neurones that are more active[3]. Stimulated mitochondria in these neurones modulate genomic expression of proteins that further potentiate mitochondrial respiration via nuclear respiratory transcription factor (NRF-1), resulting in increased expression of cytochrome oxidase (COX), nitric oxide synthase (NOS-1), NMDA receptors, & AMPA receptors. Strengthened synaptic connections as a result of these processes result in improved memory.

The pharmacologic mechanism behind the neuroprotective activity of methylene blue is unique in that it does not involve a receptor-ligand interaction, as do most drugs. In addition, MB also exhibits an atypical dose-response curve– one that has been described as hormetic[8]. Hormesis is a phenomenon where lower doses produce optimum responses while higher doses or exposures may actually produce the opposite effect. Hormesis is an intriguing pattern that may explain the dose-responses associated with exercise & oxidative stress, where the right amount of exercise-induced oxidative stress induces a cascade of favourable physiologic adaptations that can mitigate more severe stressors[9].

Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue. Prog Neurobiol. 2012 Jan;96(1):32-45.
An example hormetic dose-response curve. Note the inverse response caused with higher doses.[10]
As previously mentioned, metabolism of MB involves reduction to leukomethylene blue (MBH2). MB is primarily eliminated in the urine (75%)[11].

What is it like?

I have tested BlueBrainBoost’s formulation with the following results. I have noticed increased energy and decreased fatigue, with an onset of up to 1 hour and duration of 2-4 hours. The only adverse effects were discolouration of the mouth and urine. I used a dosage of 10 mg (20 drops) in the morning sublingually before brushing my teeth. During this time, I was also taking 30 mg/day coluracetam, 500 mg/day ashwagandha, & 600 mg/day NAC. I suspect that coadministration of CoQ10 and creatine may have a synergistic effect based on the pharmacologic site and mechanism of action.

Is it safe?

methylene blue nootropicMethylene blue is associated with a very favourable safety profile. It is generally well-tolerated at doses lower than 2 mg/kg.[12] The most noticeable side effect of MB is blue discolouration of the oral cavity and blue or blue-green discoloration of the urine. These effects are reversible and not harmful. [13] Staining of the teeth can be removed with repeated tooth-brushing, and discolouration of the urine ceases after the drug is fully removed from the system. Other reported adverse effects include a mild headache and dizziness[14].

MB does exhibit some serotonergic activity. This is due to its inhibition of enzymatic degradation of serotonin by MAO-A. Intravenous doses higher than 5 mg/kg have led to the development of serotonin syndrome. This risk is increased in individuals already taking other serotonergic agents (e.g. tianeptine, St. John’s Wort, common antidepressants, dextromethorphan, tramadol). For these reasons, individuals at risk should avoid coadministration of MB with serotonergic agents by at least 2 weeks (or more depending upon the agent), start at low doses, & increase carefully to an effective dose.

Neurotoxicity has been associated with some preparations of MB as a result of chemical impurities. The presence of heavy metals used in the synthesis of MB can have adverse effects on neurones. Thus, only pharmaceutical grade formulations are recommended for human consumption – not lab grade, & not aquarium grade. These formulations may not meet USP standards and may contain up to 11% contaminants.

How should I take it?

Because MB’s role as a neuroprotective agent in humans is still being studied, there is as yet no recommended dosage. The animal doses used in preclinical trials roughly converts to a human equivalent dose of 0.16–0.64 mg/kg administered sublingually. Sublingual administration may produce higher bioavailability than oral administration, but causes more staining of the mouth. I calculated my dose like so:

  1. 0.16 to 0.64 mg/kg × 54.43 kg = 8.71 mg to 34.84 mg per dose
  2. 10 mg/1 mL = 8.71 mg/x mL → 0.87 mL × 20 gtt/1 mL = 17 gtt to 3.5 mL per dose SL (gtt = drops)

It should be noted that due to the hormetic dose-response curve, the response to MB may decrease with higher doses. MB is typically formulated as a 10 mg/mL solution, where 1 drop = 0.05 mL = 0.5 mg. The bottle should be shaken well before administration.

Summary

Overall, I would recommend methylene blue to individuals looking for an inexpensive extra boost in energy. I have not tested MB long enough to notice changes in cognition or memory, but the pre-clinical studies & pharmacologic literature seem to support this benefit.

  • Methylene blue supports mitochondrial respiration & strengthens synaptic connections, which may lead to decreased fatigue and enhanced cognition & recall. MB exhibits a hormetic dose-response curve.
  • The safety profile has been well-characterised, and MB has generally been shown to be well-tolerated. I believe the most important warnings are those concerning serotonin syndrome and chemical impurities.
  • The best-estimated dosage is only an approximation from animal studies. MB is not yet recommended for human consumption for the purpose of improving cognition and memory.
  • Only pharmaceutical grade formulations of MB should be used.
Methylene Blue
7.5
Focus
8
Mood
7.5
Memory
8
Stimulation
5.5
Relaxation
6.5
Safety
Reviewer 8.3
Summary
Methylene Blue improves mood, memory and energy levels, as well as mitochondrial function (and may also delay aging). I think it is a powerful tool to have in your arsenal, and the BBB solution is cheap and convenient, therefore I highly recommend it to anyone.

References   [ + ]

1. Ginimuge PR, Jyothi SD. Methylene blue: revisited. J Anaesthesiol Clin Pharmacol.
2. Callaway NL, Riha PD, Bruchey AK, Munshi Z, Gonzalez-Lima F. Methylene blue improves brain oxidative metabolism & memory retention in rats. Pharmacol. Biochem. Behav. 2004; 77:175–181.
3, 7. Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement & neuroprotection of methylene blue. Prog Neurobiol. 2012 Jan;96(1):32-45.
4. Poteet E, Winters A, Yan L, Shufelt K, Green KN, Simpkins JW, et al. Neuroprotective actions of methylene blue & its derivatives. PLoS One. 2012;7(10):e48279-.
5. Gonzalez-Lima F, Barksdale BR, Rojas JC. Mitochondrial respiration as a target for neuroprotection & cognitive enhancement. Biochem Pharmacol. 2014 Apr 15;88(4):584-93.
6. Miclescu A, Basu S, Wiklund L. Methylene blue added to a hypertonic-hyperoncotic solution increases short-term survival in experimental cardiac arrest. Crit. Care Med. 2006; 34:2806–2813.
8. Bruchey AK, Gonzalez-Lima F. Behavioral, physiological, and biochemical hormetic responses to the auto-oxidizable dye methylene blue. Am. J. Pharm. & Toxicol. 2008; 3:72–79.
9. Ji LL, Gomez-Cabrera MC, Vina J. Role of free radicals & antioxidant signaling in skeletal muscle health and pathology. Infect Disord Drug Targets. 2009;9(4):428–444.
10. Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue. Prog Neurobiol. 2012 Jan;96(1):32-45.
11. Medscape® 5.1.2, (electronic version). Reuters Health Information, New York, New York.
12, 14. Ginimuge PR, Jyothi SD. Methylene blue: revisited. J Anaesthesiol Clin Pharmacol. 2010 Oct;26(4):517-20.
13. Gillett MJ, Burnett JR. Medications and green urine. Intern Med J. 2006 01;36(1):64-6.