Pathway Methylene Blue Pathway, Pharmacodynamics

Pharmacodynamics
A stylized diagram showing the mechanisms that can cause methemoglobin production in erythrocytes and the control mechanisms to prevent methemoglobinemia, including methylene blue treatment which requires NADPH from the Pentose Phosphate Pathway.
Methylene Blue Pathway, Pharmacodynamics
methylene blue methylene blue riboflavin cyb5a cyb5r3 cyb5a blvrb blvrb g6pd oxidative pathway pentose phosphate pathway
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Description

Methylene Blue
Methylene blue has many uses - in vivo it is indicated for use as a therapy for drug-induced methemoglobinemia, can be used for the treatment of infections, pathologies or poisoning, and as a dye for diagnostics. It is also commonly used as a dye in vitro - for example as a component in staining of cells, tissues, DNA, parasites and bacteria [Articles:21235292, 14714878, 21316815]. Examples of clinical use include anti-malarial regimes, treatment of ifosfamide-induced neurotoxicity (though treatment has been reported ineffective), an antidote for cyanide poisoning, visualization of fallopian tubes or ruptured membranes, a marker of tumors, and even as a potential therapy for septic shock, ischemic brain injury and Alzheimer disease [Articles:14962363, 18840018, 20151259, 17278152, 9605379, 11048840, 17928358, 20731659, 14714878, 20575634, 20959759] (Moldenhauer Brooks, 1936, The Scientific Monthly, 43:585-586). As a photodynamic therapy it could be used to treat psoriasis, West Nile virus infection, AIDS-related Kaposi's sarcoma, anti-biotic resistant bacterial strains, and decontaminate blood before transfusion [Articles:19180895, 16118025, 16942436, 19193212, 22232515, 14714878]. Methylene blue has also contributed to drug development as the chemical basis of other therapeutic drugs, including the anti-malarial chloroquine, the antihistamine promethazine, and anti-psychotic chlorpromazine [Article:14714878].

Methemoglobinemia
Methemoglobinemia is an increase in the methemoglobin (MetHb) content of red blood cells (RBCs) [Articles:22024786, 7073040]. Methemoglobin (MetHb) is formed when heme iron atoms within hemoglobin are oxidized, and can no longer bind oxygen or carbon dioxide [Articles:22024786, 8416301, 7073040, 15862084, 10533013]. Normal levels of MetHb in circulating red blood cells (RBCs) are around 1% - methemoglobinemia occurs when these levels increase, and can be due to inherited factors (hereditary) or induced by exogenous oxidizing agents such as therapeutic drugs (acquired) [Articles:22024786, 7073040]. Cyanosis occurs at around 15% MetHb, and tissue hypoxia can occur as levels rise further - MetHb levels of 70% or above can be fatal [Article:7073040].

Pathway Description
The Methylene Blue Pathway focuses on the mechanisms involved in oxidation of hemoglobin to MetHb and protection from this process or reduction of MetHb back to hemoglobin. Hemoglobin in RBCs has interchangeable structural forms that enable the uptake of oxygen and release of carbon dioxide in the lungs and the release of oxygen and uptake of carbon dioxide in the tissues (reviewed extensively in [Article:9435331]). Deoxyhemoglobin is the tense conformation - as oxygen binds to one heme group, the structure relaxes and affinity for oxygen increases, enabling oxygen molecules to rapidly bind the remaining three heme groups (oxyhemoglobin) [Articles:9435331, 8416301]. MetHb is formed through the oxidation of one or more heme iron atoms within deoxyhemoglobin from a ferrous to the ferric state (Fe2+ to Fe3+), by compounds such as hydrogen peroxide (H2O2) and superoxide (O2-) [Articles:22024786, 8416301, 7073040, 15862084, 10533013].

To prevent the formation of MetHb, RBCs have several different mechanisms that work by either reducing ROS in the cell to prevent MetHb formation (including mechanisms described in the Oxidative Stress Regulatory Pathway), or reverse the ferric iron back to a ferrous state by reduction [Articles:7073040, 15862084, 22024786]. The main RBC enzyme that reduces MetHb in vivo is the soluble form of cytochrome b5 reductase (CYB5R3, also known as NADH-dependent MetHb reductase or diaphorase-1), utilizing the electron donor NADH to reduce cytochrome b5 (CYB5A) which can then in turn reduce MetHb [Articles:22024786, 8416301, 7073040, 18318771, 10533013]. More than forty genetic variants within the CYB5R3 gene have been associated with recessive congenital methemoglobinemia [Article:18318771]. Extracellular NADH in the presence of lactose dehydrogenase can enhance the rate of MetHb reduction in human erythrocytes [Article:15833275].

A second enzyme, flavin reductase (NADPH) (also known as BLVRB, biliverdin reductase B, NADPH-MetHb reductase, NADPH-MetHb-diaphorase) undertakes around 5% of this activity in RBCs under normal conditions, requiring NADPH and an electron acceptor cofactor [Articles:22024786, 7073040, 10533013, 8687377]. Riboflavin, FAD and FMN can all act as electron acceptors to then reduce MetHb [Articles:869945, 7400118, 698125].

Pharmacodynamics of methylene blue
Another such cofactor for BLVRB that greatly accelerates the reduction of MetHb is methylene blue [Articles:22024786, 7073040, 10533013]. Methylene blue is reduced to leukomethylene blue by BLVRB, accepting electrons from NADPH [Article:7073040]. Leukomethylene blue acts as an electron donor to reduce MetHb to hemoglobin, converting back to methylene blue in a cyclical redox reaction [Articles:7073040, 10533013]. Conversely, because methylene blue is an oxidizing agent, at high concentrations it can cause methemoglobinemia by oxidizing hemoglobin [Articles:7073040, 10533013]. The efficacy of methylene blue treatment can be affected by numerous factors, for example an aniline intermediate may block RBC uptake of methylene blue [Article:12845393].

Pharmacogenetics
Almost 330 million people are estimated to have a deficiency in the G6PD enzyme, with the highest prevalence found in Africa, the Middle East and Asia [Article:19233695]. G6PD deficient individuals are more susceptible to RBC oxidative stress induced by exogenous agents such as therapeutic drugs because NADPH production cannot meet the demand required by regulatory mechanisms (see also the Pentose Phosphate Pathway and Oxidative Stress Pathway) [Articles:7949118, 4154443, 2633878]. The variants within the G6PD gene that have been identified (currently >180) are categorized into WHO classes (I-V) depending on the extent of enzyme deficiency and clinical manifestations they confer (see the PharmGKB G6PD VIP summary) [Articles:22237549, 22293322, 6075369, 5316621, 2633878].

Methylene blue is an effective treatment for reducing MetHb, however it is associated with adverse reactions in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals (Table 1). Drug labels for methylene blue contraindicate or advise precaution for use in G6PD deficient individuals due to a risk of hemolytic anemia and/ or methemoglobinemia. Early in vitro studies demonstrate that the rate of MetHb reduction in G6PD deficient RBCs incubated with methylene blue and glucose is severely reduced compared to normal RBCs [Article:14056871]. However, this can be increased by incubating G6PD deficient and 'normal' RBCs together - possibly due to the diffusion of leukomethylene blue into G6PD deficient RBCs [Article:14056871].

Due to dependency on NADPH, methylene blue treatment is often ineffective at ameliorating methemoglobinemia in G6PD deficient patients [Articles:5091568, 9590495, 17444323, 15587250, 18561168, 11418378], and may exacerbate the condition and/or induce hemolysis in individuals with G6PD deficiency (see Table 1) [Articles:16493607, 15842651, 5091568, 11048840, 15787927, 22015451, 17444323, 11418378, 15587250, 11824767] [Article:22024786]. In fact, failure of methylene blue to reduce methemoglobin was developed as a test for G6PD deficiency [Articles:13804322, 13872968].

Table 1: Cases of adverse reactions reported after methylene blue treatment in G6PD deficient individuals

Study details Reported trigger of methemoglobinemia Consequence of subsequent methylene blue treatment G6PD deficiency? Test carried out? Reference
Case study, male adult (Filipino) with metastatic renal cell carcinoma Triapine Developed jaundice, hemoglobinuria, hemolysis G6PD deficient, enzyme activity assay [Articles:16493607, 15842651]
Case study, male adult (Mexican-American) Cleaning liquid containing aniline and toluene No change in MetHb levels, hemolysis on day 2 which may have been triggered by aniline, toluene, methylene blue (or the subsequent vitamin c* treatment) G6PD deficiency A- variant, quantitative and qualitative enzyme activity assays [Article:5091568]
Report of three premature neonates who were exposed to methylene blue prenatally (two males, one female) Not applicable Development of severe hemolysis resulting in hyperbilirubinemia, all required exchange transfusions G6PD deficiency was confirmed in two of the three cases, enzyme activity assay [Article:11048840]
Case study, 26-month old male Nail removal fluid (containing nitroethane) MetHb levels were only transiently reduced, and rose, but were resolved by RBC exchange. G6PD deficient, enzyme activity, and 53% HbA, 41% HbS sickle trait. [Article:9590495]
Case study, 23-year old woman (India) Aniline, was also given vitamin c* No improvement. Developed hemolysis and required transfusion. G6PD deficient, methemoglobin reduction method [Article:17444323]
A 3 month year old who had undergone cardiac surgery Could not define, but the authors discuss it may be due to the effects of cardiopulmonary bypass on enzyme activity, or glyceryl trinitrate treatment, or other cardiac or respiratory causes. Low dose of methylene blue was given over 10 minutes, and MetHb levels reduced. Developed jaundice and mild hematuria attributed to hemolysis. Prior knowledge of a partial G6PD deficiency [Article:15787927]
A case study (n=1), 40-year old male Fungicide containing copper-8-hydroxyquinolate. He was initially treated with vitamin c*. Methemoglobinemia and hemolysis developed. Methylene blue was administered, along with continued treatment with vitamin c*. Treatment was not effective, and hemolysis became more severe. Underlying G6PD deficiency, as well as inhibition of G6PD activity by copper. Activity assay. [Article:15587250]
A case study, adult, in a trial of 80 patients (patients with known G6PD deficiency were not enrolled) Rasburicase MetHb levels decreased but hemolysis worsened Previously unknown G6PD deficiency, test not reported. [Article:22015451]
A case study, 12-year old male (Laotian) Rasburicase Treatment was not effective G6PD deficient, enzyme activity test. [Article:18561168]
A case study, a male patient (Jordanian) with chronic renal failure Metoclopramide (impaired renal function and cytochrome b5 reductase deficiency also contributed) Also given vitamin c*. Treatment was not effective, and his condition worsened, and likely developed hemolysis. The patient died. CYB5R3 deficient, quantitative assay, and G6PD deficient, enzyme activity assay. [Article:11418378]
A case study, 25-year old male Aniline Symptoms diminished and MetHb levels reduced, however hemolytic anemia developed several days later. G6PD deficiency, assay. [Article:11824767]
A case study, 23-year old female undergoing a laparoscopy Methylene blue (administered through the cervix to visualize the fallopian tubes) Cyanosis was treated with vitamin c* rather than methylene blue G6PD deficiency, enzyme levels [Article:9605379]
n=409 children were genotyped, n=88 were G6PD deficient. Dose-finding study - treated with combination of chloroquine and different doses of methylene blue. Burkino Faso Not applicable In one child, hemoglobin dropped below 5g/dl on day 5, and in 7 children, hemoglobin value dropped >3g/dl In the case <5g/dl = G6PD deficient hemizygous male. 3 out of 7 children with >3g/dl drop in hemoglobin were G6PD deficient. Genotyping method or variant not described [Article:17026773]
Pooled analysis of four randomized control trials (n=1005 children) (includes PMID: 16179085, 17026773 above, 18286187 and unpublished data). A 21-month old girl and a 28-month old boy, both with malaria Not applicable n=844 children were treated with methylene blue combined with other anti-malarial drugs. In these two patients, hemoglobin levels fell to equal to or less than 5g/dl (indicating severe anemia) Female was heterozygous for G6PD A-, the male was hemizygous (likely that described above in PMID: 17026773]). Specifics of genotyping were not provided [Article:23135803]

*N.B. - it should be noted that vitamin c (ascorbic acid) at high concentrations has also been associated with inducing hemolysis in G6PD deficient individuals [Articles:1138591, 1976956], though is considered safe at therapeutic doses in patients who do not have WHO Class I G6PD variants [Articles:7949118, 20701405].

Is methylene blue safe in G6PD deficient individuals?
A recent evidence-based review concluded that methylene blue treatment should be avoided in patients with G6PD deficiency due to a risk of hemolysis [Article:20701405], backed by other reviews [Articles:20350285, 7949118, 19769422], the Italian G6PD Deficiency Association, and drug labels. Alternative treatments for methemoglobinemia include vitamin c, an electron donor that can reduce ROS and therefore inhibit the production of MetHb (Oxidative Stress Regulatory Pathway) [Articles:12569111, 22024786, 5091568, 9605379]. However, methylene blue is a more potent and rapid reducer of MetHb than other options, such as riboflavin, [Article:10809266], and vitamin c treatment is not always effective [Articles:17444323, 15587250, 11418378], and several NADPH-dependent mechanisms are required for the recycling of oxidized vitamin c [Articles:10657232, 9667500, 9405334].

Several studies show no association between risk of methylene blue-induced hemolysis and G6PD deficiency. No cases of severe hemolysis were observed in 24 children with G6PD deficiency in a study to treat uncomplicated malaria infection with methylene blue and chloroquine, a drug combination also safe in 74 healthy G6PD deficient men [Articles:16179085, 15655011]. Both studies were carried out in an area of West Africa where G6PD class III variants are common [Articles:16179085, 15655011, 16225660].

It has therefore been debated whether methylene blue is safe to use in G6PD deficient individuals or not. The answer may depend on the type of deficiency, as WHO class III G6PD variants are considered to confer less severe deficiency, compared to Class I or II [Articles:22149420, 6075369, 5316621, 2633878, 16179085, 15655011]. However, one argument against using methylene blue in this patient population is that testing for G6PD deficiency is still based on enzyme activity in the majority of cases around the world, rather than genotyping or characterization of the underlying variant, and so methylene blue should be administered with extreme caution to those with known G6PD deficiency [Articles:22149420, 19233695]. Of the case studies identified in extensive literature review here (Table 1), only two publications describe characterization of the G6PD electrophoretic variant (A-) and genotyping of specific the underlying genetic variants is not always reported (see G6PD A- haplotypes) [Articles:5091568, 23135803]. Another argument is that despite previous perceptions, G6PD A- should not clinically be regarded as 'mild' as patients are still at risk of life-threatening acute hemolytic anemia when challenged with a potent agent [Article:22993389], and that the distinction between WHO Class II and III G6PD variants is no longer clinically useful (Luzzatto & Poggi, 2009, Glucose-6-Phosphate Dehydrogenase Deficiency chapter, in Nathan and Oski's Hematology of Infancy and Childhood, 7th Edition). Illustrating this, a recent combined analysis of four randomized controlled trials of methylene blue treatment observed significant but small reductions in hemoglobin levels in hemi/homozygous children with class III G6PD A- compared to wildtype or heterozygotes (Table 1) [Article:23135803]. This effect was described as of limited clinical consequence (with only two children of 844 displaying severe anemia, one heterozygous, one hemizygous), though monitoring of adverse hematological events in G6PD patients was advised [Article:23135803].

As it is possible that leukomethylene blue diffuses into G6PD deficient RBCs [Article:14056871], heterozygous females may be less at risk of methylene blue-induced hemolysis, though to our knowledge this mechanism has yet to be examined.

Other genetic factors may contribute to risk - for example, individuals with CYB5R3, CAT or GSH synthetase deficiency may be at a higher risk of drug-induced methemoglobinemia and hemolytic anemia [Articles:11418378, 31928, 6765904, 1999334, 6620333, 5686480, 11167850]. Newborns are particularly susceptible to methemoglobinemia due to low CYB5R3 levels, reduced CAT and GPX1 activity [Articles:8416301, 3780953, 7073040]. Other factors including baseline hemoglobin, correlating with parasitaemia levels, may also affect risk [Article:23135803]. Similar to G6PD, hexokinase is able to enhance the rate of the PPP under conditions of oxidative stress (e.g treatment with methylene blue), as demonstrated by a lack of enhanced PPP rate in cells when hexokinase is inhibited [Article:3415698], and patients with hexokinase deficiency exhibit nonspherocytic hemolytic anemia [Article:10474511]. BLVRB deficiency was described in an individual who had sufficient G6PD activity but an abnormal methylene blue screen test [Article:4383300]. Due to the role of BLVRB in the pharmacodynamics of methylene blue, methylene treatment may be unsuccessful in BLVRB deficient patients, as reflected in EU drug labeling which contraindicates the use of the drug in these individuals [Articles:7582412, 17122537].

Disease context may also play a role - patients with methemoglobinemia are already displaying clinical signs of oxidative stress within their RBCs, and therefore may be more at risk of hemolysis. As an oxidizing agent, the dosage of methylene blue administered should also be considered [Articles:7073040, 10533013]. Other factors, for example baseline hemoglobin in patients with malaria infection that correlate with parasitemia levels, may also affect risk [Article:23135803].

Does methylene blue cause hemolysis in G6PD deficient individuals, or is it the agent that initiated methemoglobinemia?
Several of the same agents listed that can trigger acquired methemoglobinemia are also known to cause hemolytic anemia in G6PD deficient individuals, for example primaquine, acetanilid and toluidine [Articles:7073040, 7949118]. It is therefore difficult to pinpoint whether methylene blue is the cause of hemolysis in G6PD deficient individuals being treated for acquired-methemoglobinemia, rather than the precursor agent, though methylene blue is listed both as an agent that can cause hemolytic anemia in G6PD deficient individuals and an agent that can cause acquired methemoglobinemia [Articles:7073040, 7949118]. In one study from our literature review, the development of methemoglobinemia in a G6PD deficient patient was associated with methylene blue (Table 1) [Article:9605379].

Are G6PD deficient individuals also more susceptible to methemoglobinemia risk?
G6PD deficient individuals may be more susceptible to acquired-methemoglobinemia, though this direct association is unclear in the published literature. The development of methemoglobinemia has been linked to G6PD deficiency [Article:8785565]. Rasburicase (contraindicated in G6PD deficient patients) has been associated with both methemoglobinemia and hemolytic anemia in several reported cases of patients with G6PD deficiency [Articles:22015451, 18561168, 22190578, 16204390, 22573495, 17387701, 12942574]. Deficiency in G6PD may contribute to exacerbation of acquired-methemoglobinemia in several ways via a decrease in available NADPH in RBCs, as described above, though the NADH-dependent CYB5R3 pathway dominates reduction of MetHb [Articles:7073040, 22024786].

Authors: Ellen M. McDonagh, José M. Bautista, Ilan Youngster, Russ B. Altman, Teri E. Klein.
Citation:
McDonagh Ellen M, Bautista José M, Youngster Ilan, Altman Russ B, Klein Teri E. "PharmGKB summary: methylene blue pathway" Pharmacogenetics and genomics (2013).
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History:
Therapeutic Categories:
  • Cardiovascular and hematology agents

Entities in the Pathway

Genes (4)

Drugs/Drug Classes (2)

Relationships in the Pathway

Arrow FromArrow ToControllersPMID
CYB5A CYB5A CYB5R3 10533013, 18318771, 22024786, 7073040, 8416301
Fe2+ Hemoglobin Fe3+ Methemoglobin Hydrogen Peroxide, methylene blue, reactive oxygen species 10533013, 15862084, 22024786, 7073040, 8416301
Fe3+ Methemoglobin Fe2+ Hemoglobin FAD, FMN, Leukomethylene Blue, riboflavin, BLVRB, CYB5A 10533013, 10809266, 22024786, 698125, 7400118, 869945
Hydrogen Peroxide Hydrogen Peroxide Oxidative Stress Regulatory Pathway (Erythrocyte)
Leukomethylene Blue methylene blue
methylene blue Leukomethylene Blue BLVRB 10533013, 12845393, 18318771, 22024786, 7073040, 869945
NADH NAD CYB5R3 10533013, 18318771, 22024786, 7073040, 8416301
NADPH NADP BLVRB 10533013, 12845393, 18318771, 22024786, 7073040, 869945
NADPH NADPH G6PD 16204390, 18177777, 18226191, 20350285, 21376665
Oxidative Stress Regulatory Pathway (Erythrocyte)
reactive oxygen species reactive oxygen species Oxidative Stress Regulatory Pathway (Erythrocyte) 7073040

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