Strategies for Avoiding Benzopyrone Hepatotoxicity in Lymphedema Management—The Role of Pharmacogenetics,Metabolic Enzyme Gene Identification,and Patient Selection

Abstract

Introduction:

Benzopyrones are plant-derived chemicals which have an evidenced degree of clinical efficacy in lymphedema management indicated in past trials. Unfortunately, in some of these cases idiosyncratic hepatotoxicity have been documented in a minority of patients. This review aims to tackle the problem of benzopyrone (particularly coumarin) toxicity by considering their metabolic pathways and identifying relevant alleles needed to take a targeted pharmacogenetic approach in its future use.

Methods and Results:

The nontoxic 7-hydroxylation and the toxic heterocyclic “ring-splitting” epoxidation pathways are the two main detoxification pathways in the hepatometabolism of coumarin, the former catalyzed by CYP2A6 and the latter by possibly CYP1A and CYP2E. Acetaldehyde dehydrogenase (ALDH) clears toxic aldehyde intermediates. CYP2A6 polymorphism screening methods, including genotyping, by real-time polymerase chain reaction and chromatography–mass spectroscopy functional metabolite assays; efficiency of these techniques are continually improving. ALDH polymorphisms have also been implicated, with clinically viable screening tests, rapid genotyping, and sensitive questionnaires already available for ALDH2*1/ALDH2*2. Dysfunctional polymorphisms of the above genes and others are significantly more prevalent in Eastern Asian populations, uncommon in Caucasian populations. The role of other enzymes/genes in the pathway is yet to be clarified.

Conclusion:

Although screening techniques are becoming increasingly clinically feasible, uncertainty remains on the link between the genotype, metabolic phenotype, and the exact gene products involved. These must be elucidated further before a targeted pharmacogenomic approach is fully viable. In the meantime, treatment should be avoided in those with vulnerable familial and ethnic descents if used.

Introduction

Benzopyrones are a class of drug that encompasses the coumarins and the bioflavonoid family of chemicals, including rutosides, diosmin, and quercetin. Among the most well studied of these compounds is coumarin (5,6-benzo-α-pyrone), an organic chemical which was first extracted from the Tonka bean in 1820 and has been used historically to treat a wide range of conditions, including lymphedema, chronic venous insufficiency, and asthma, as well as also showing antineoplastic properties both in itself and its derivatives.^1–3 Coumarin itself should not be confused with other dicoumarol derivatives such as warfarin; it has no anticoagulative effects. This review will focus on the applications of benzopyrones (mainly coumarin, although other benzopyrones will be considered) in lymphedematous patients.

Lymphedema is a lymphatic disease characterized by retention of fluid, accumulation of protein, and eventually fibrotic induration in the interstitial compartment as a result of attenuated lymphatic clearance. Being the “nexus” between the circulatory and immune systems, disruption of the lymphatics leads not only to edema but also predisposes to infection, inflammatory responses, and fibrotic deposition—a characteristic distinctive to edemas of this particular origin.^4,5 Etiologically this disease can occur as primarily with different ages of onset (congenital, praecox, tardum) or as secondary lymphedema as a sequela of radiation, infection (e.g., filariasis), cancer, and lymphadenectomy in the treatment/diagnosis of cancer.⁵ Despite its prominence, lymphedema continues to be an area of relative clinical inadequacy and embarrassment, with minimal emphasis placed on it in many medical programs—often leading to a lack of knowledge and inappropriate behaviors of problem minimization in treating clinicians, much to the detriment of the involved patients.⁶

Mechanism of action

The therapeutic effects of benzopyrones are hypothesized to rise through several different mechanisms—the most prominent ones, including the reduction of vascular protein leakage, increased lymphatic removal of protein, and increased proteolysis by macrophages.⁷ Reduction of vascular permeability and protein leakage result in less fluid and profibrotic protein entering the interstitium—benzopyrones are thought to mediate this effect by decreasing capillary permeability and the closure of post capillary endothelial junctions—a characteristic that explains and underlies their use in patients with chronic venous insufficiency.^7,8

Perhaps the most prominently attested mechanism of benzopyrone action is the facilitation of proteolysis, both by improved macrophage phagocytosis or increasing extracellular protease activity.⁷ It has been demonstrated that no proteolysis occurs when plasma protein is incubated with coumarin, but does proceed once macrophages are introduced into the mixture.⁹ Conversely, when the macrophages are then poisoned, proteolysis also ceases, suggesting that the macrophages are integral to the process.³ By increasing interstitial protein lysis, less osmotic vascular fluid extravasation occurs, causing less edematous exacerbation, as well as attenuation of future fibrotic development. It can therefore be seen that benzopyrones have evidenced biomechanistic effects on both the circulatory and immunological “prongs” of lymphedema pathophysiology.

Clinical efficacy of benzopyrones

Benzopyrones have been trialled in the treatment of lymphedema with varying but promising results. Trials such as the one conducted by the Casley-Smiths in 1996 have demonstrated encouraging outcomes, showing significant reductions in limb volume when oral and topical coumarin is coadministered with complex physical therapy (CPT).¹⁰ Out of 628 limbs, three fourths of the limbs treated with CPT + oral and topical benzopyrones experienced a reduction in edema of 29% or higher, compared to 19% of limbs treated with CPT alone.¹⁰ Depending on the measure of edema used, oral and topical benzopyrones improved the decrease in edema over the whole year from 150% to 300% in limbs with CPT only.¹⁰

Conversely, an influential large-scale trial by Loprinzi et al. demonstrated statistically negligible effects of coumarin on arm volume in breast cancer treatment patients.¹¹ One hundred forty women with lymphedema on the ipsilateral arm of their treatment were treated with standard doses of either coumarin or placebo over 6 months, followed by 6 months of the opposite treatment. At both 6 and 12 months, no statistically significant difference was measured between the two groups; at 6 months the average changes in volume were 21 mL in placebo group and 58 mL in benzopyrone group (p = 0.8). This trial did not incorporate CPT or manual lymphatic drainage (MLD) alongside medication therapy which could explain the results; however, other sources have suggested that benzopyrones are effective alone without CPT.¹²

A meta-analysis of 50 trials from 37 authors in 8 countries by Casley-Smiths demonstrated an average annual edema reduction of 55% (p < 0.001, standard error = 7.8%, 95% CI = 40–71).¹³

Legal status

Although there are still several fine details to be clarified on a molecular level in terms of the mechanism of action, benzopyrones have consistently demonstrated some degree of clinical efficacy in lymphedema patients, which is to be discussed further below. Despite this, however, coumarins (and related compounds) have been tightly regulated by various therapeutic goods authorities around the world due to a probability of it causing hepatotoxicity, both in animal models and human cases. As such, coumarin has been banned as a food additive in the United States, limited to a certain concentration in beverages in the European Union and similarly restricted or banned in other nations.^1,14

Methods

A literature search of online databases, including MEDLINE, PubMed, and Scopus, was conducted using “benzopyrone” or “coumarin” as keyword, combined with “hepatotoxicity” or “liver injury.” Article titles were screened to include studies relevant to pharmacogenetics, metabolic pathways, detoxification mechanisms, screening approaches, and contraindications to use. Articles were excluded if they were duplicates, not in English or had no translations available, primarily explored hepatoprotectivity/antioxidant properties, or were otherwise unsuitable. Ethical consideration in the conduct and reporting of the research was not required.

Results

Hepatotoxicity and metabolism of coumarin

Despite its reasonably well established clinical efficacy, perhaps the most significant “downfall” of benzopyrones lies in their risk of idiosyncratic hepatotoxicity in certain patients. Different values are reported from different sources regarding the rate of hepatic injury and toxicity, with rates ranging from 0.3% to 6% incidence.^11,13,15

It should be emphasized that the metabolism and toxicity incidence of coumarin varies significantly between species, with rats being greatly susceptible, mice less so, and humans being more resistant than both of the above.¹⁶ In fact, in humans therapeutic doses are significantly lower than doses which typically cause hepatic damage—the LD₅₀ of coumarin in rats (which are far more susceptible than humans) is 290 mg/kg, whereas the higher boundary for therapeutic doses in human is 500 mg/daily—magnitudes lower than doses shown to cause damage in the “normal” metaboliser.¹ Exposures through food and cosmetics constitute even more negligible doses, despite the legal restrictions in place.¹

However, the incidence of toxicity in the minority cannot be ignored and must be addressed by analyzing coumarin's hepatotoxicity mechanistically. To do this, we must consider the metabolic pathways that it undertakes during detoxification (Fig. 1).

FIG. 1.

Hepatic detoxification pathways of coumarin metabolism.

First, coumarin undergoes Phase I metabolism in one of two manners: oxidation into 7-hydroxycoumarin (umbelliferone or 7-HC) or formation of coumarin-3,4-epoxide (CE). 7-HC is a nontoxic metabolite, whereas CE is a reactive intermediate, which can either be conjugated with glutathione to CE-SG or spontaneously react to form o-hydroxyphenylacetaldehyde (o-HPA), with the former being inert and the latter being a known hepatotoxic aldehyde.¹⁶ o-HPA is then converted to o-HPE or o-HPAA, both being inactive metabolites.

Overall, the main pathway in humans is the 7-hydroxylation pathway, which is likely to account for our relative resistance to liver damage compared to other species. However, the heterocyclic ring-splitting pathway, which forms CE and produces the toxic acetaldehyde o-HPA, is still thought to proceed to some degree and is hypothesized to be the predominating pathway operating in patients with hepatotoxicity.^16,17

The cytochrome p450 CYP2A6 enzyme is thought to catalyze the 7-hydroxylation route, while CYP1A and CYP2E are suggested to catalyze the ring-splitting route.¹⁸ Aldehyde dehydrogenases (ALDHs) are responsible for the oxidation of the aldehyde o-HPA to the carboxylic acid o-HPAA, while glutathione-S-transferases have the role of conjugating CE to CE-SG.¹⁶ Coumarin has also been observed to undergo 3-hydroxylation to 3-HC through CYP3A4 catalysis, although there is disagreement as to whether this progresses to 3,4 epoxidation and subsequent o-HPA formation, or if the two form distinct pathways.¹⁹

Deficiencies in these enzymes can lead to abnormalities in detoxification, for example, if the 7-hydroxylation route is defective (e.g., CYP2A6 is dysfunctional), then metabolites will build up and shunt down the alternative toxic pathway, producing CE and then toxic o-HPA, leading to potential injury. Similarly, if there is deficient activity of ALDH then o-HPA will be cleared at a lower rate, leading to buildup of toxic products. Differences in glutathionation have also been considered, but are shown to not affect incidence of toxicity between species.¹⁶ A variety of genetic polymorphisms can lead to these enzyme deficits, as described below in the Pharmacogenetics section.

It is important to note that the hepatotoxicity of other benzopyrones and flavonoids has not been as well studied, despite these compounds showing similar efficacy in many trials. The literature search has yielded no records of hepatotoxicity of micronized purified flavonoid fractions (MPFFs) or other flavonoids in humans or other species.

Pharmacogenetics

In a time where genetic sequencing and testing is continually becoming exponentially cheaper and clinically viable, a genetic approach to pharmacology may very well be a potential solution to avoiding adverse effects and toxicity in many drugs, coumarins not excluded. By identifying the exact genetic cause and allelic variance responsible for metabolic traits that lead to coumarin intolerance, it may be practically feasible to avoid administering these medications to the intolerant and/or individualizing doses to patients, thereby significantly decreasing or negating the risk of hepatotoxicity altogether.^17,20

The role of CYP2A6

A current hypothesis is that polymorphisms in CYP2A6, being the gene/enzyme responsible for detoxifying coumarin to a benign 7-HC, mainly accounts for the toxicity in human cases.^17,21 This is a cytochrome P450 enzyme which is involved in the metabolic detoxification of several substances, including coumarin, Aflatoxin B1, certain carcinogens, nitrosamine, several antineoplastics, and nicotine, the latter of which has been a topic of research interest in association with CYP2A6 for its relevance in smoking addiction and withdrawal.^21–23

There are over 10 variant alleles of CYP2A6 which code for dysfunctional enzymes, potentially leading to an accumulation of coumarin and the shuttling of metabolites down the “ring-splitting” hepatotoxic pathway in affected carriers.¹⁷ This is exemplified by one subject who was found to be homozygous for the CYP2A6*2 allele showing no urinary 7-HC 8 hours after coumarin administration but 50% o-HPAA excretion, suggesting a complete reliance on the epoxidation pathway, while his immediate family members (CYP2A6*2 heterozygotes) demonstrated near normal urinary 7-HC excretion with minimal o-HPAA metabolites.²⁴

There have been noted to be significant genomic variance between ethnic populations with different incidences of mutations. For example, CYP2A6*2 present in 2% of the Caucasian population, while CYP2A6*4 (which results in complete dysfunction if homozygous) is present in 15%–20% of the Asian population.²⁵ A study on a Chinese population (n = 120) measuring urinary 7-HC after coumarin dosage found that 13.3% of participants were poor metabolizers with low CYP2A6 activity, with no sex-related difference in incidence.²⁶ Similar trials on Iranian (n = 151), Turkish (n = 100), and Finnish (n = 110) populations found that 10.6%, 4%, and <1% were poor metabolizers, respectively (the “slower” metabolizers in the Finnish study were fast enough to be considered normal by the measures in the other studies).^27–29 These results suggest a gradual “gradient” of coumarin metabolism speed (possibly consistent with genetic frequencies) across the Eurasian continent, lowest in Eastern Asia, medium in the middle east, and highest in Europe.

Although a body of evidence points to the importance of CYP2A6, there currently still remains uncertainty on the effect of the genotypes on the metabolic phenotype and exactly which gene products are involved. A study by Burian et al. dosed 231 patients with 90 g/day coumarin and found that 16 patients had CYP2A6*2 polymorphisms, with 9 patients showing hepatic injury with elevated liver enzymes, and only 1 of the 9 patients having the polymorphism, the other 8 were homozygous wild types.³⁰ The conclusion drawn from this was that CYP2A6 polymorphisms did not affect toxicity.³⁰

This unexpected result could possibly be explained by mutations/abnormalities in other enzymes in the pathway (e.g., ALDH, CYP3A4, CYP2E, CYP1A, and so on) or other predisposing factors present such as medication interactions (as described further in the article in Further factors influencing hepatotoxicity and patient selection). Vassallo et al. postulate that it is in fact the affinity for oxidative clearance of o-HPA by ALDH which is the crucial factor in determining the risk of coumarin toxicity rather than CYP2A6 dysfunction, based on the observed reliance of o-HPAA formation on NAD⁺ presence and its inhibition by disulfiram in rat, mouse, and human cytosolic samples.¹⁶

The role of ALDH and other enzymes in the pathway

ALDH is a mitochondrial dehydrogenase enzyme responsible for catalyzing the oxidative detoxification of acetaldehyde to acetate, consuming and reducing NAD⁺ in the process. It has been studied for its roles in alcohol intolerance and carcinogenesis.³¹ Up to 19 ALDH isoforms have been documented, the most relevant in this instance being the ALDH1 and ALDH2 isoforms, latter of which has well established associations with alcohol intolerance (i.e., the alcohol “flush”), bladder, liver, esophageal cancers, and other malignancies if dysfunctional.^32,33 These pathologies are all mechanistically due to acetaldehyde accumulation, the same mechanism proposed to be responsible for coumarin hepatotoxicity in its ineffective removal of o-HPA.

ALDH2 is encoded by the gene of the same name (rs671), located at chromosome 12q24.³⁴ It is the most comprehensively studied ALDH gene, having several polymorphisms (ALDH2*1, ALDH2*2) due to single-nucleotide Glu504Lys substitution which produces defective products.³⁵ The ALDH2*2 polymorphism results in a completely dysfunctional enzyme if homozygous and, therefore, produces the most severe symptoms during alcohol consumption, whereas homozygous ALDH2*1 patients demonstrate a less severe phenotype, with some residual enzyme function.³⁶ ALDH2*2 is the most common polymorphism, present in 8% of the world's population—560 million people.³⁷

Population studies have demonstrated that certain ethnic groups possess higher rates of ALDH dysfunction, similar to CYP2A6 polymorphisms. A trial by Yoshida et al. on autopsied livers (n = 10) showed that 50% of Japanese and liver samples of other oriental individuals had a normal ALDH1 but indeed an atypical dysfunctional ALDH2, whereas nearly all Caucasian liver samples possessed both functional isoforms.³⁸ A larger study also on autopsied livers of Japanese individuals (n = 40) demonstrated abnormal ALDH phenotypes in 52% of specimens which showed up to 90% inhibition by disulfiram, compared to the normal 20%–30%.³⁹

Although this association between ALDH dysfunction and disordered coumarin metabolism has been suggested, further trials need to investigate the relationship between ALDH gene polymorphisms and indicators of the predominating coumarin metabolic pathway (e.g., urinary 7-HC and o-HPAA) to confirm or refute this hypothesis.

The enzymes CYP2E, CYP1A, and CYP3A4 are also supposedly relevant in coumarin metabolism and epoxidation; however, their role in toxicity is still poorly understood.

Interestingly, due to the wide range of alleles implicated by various studies and the multifactorial nature of the toxicity, it can be hypothesized that individuals prone to toxicity are not isolated point-mutation, single-enzyme deficients, but all-round poor hepatic metabolizers who are intolerant to multiple medications and substances—etiologically genetic in origin, present more prominently in certain ethnic populations over others (i.e., East Asians more than Caucasians). The consideration of this “general principle” during prescription of these agents may well serve to avoid cases adverse to hepatic events, although admittedly crude and “shotgun approach” in nature.

Genetic screening strategies and practicalities

In theory, if carriers of the defective alleles are identified through screening, the drug can be withheld or dosing can be adjusted to match the patient so as to avoid hepatotoxicity. One must consider the costs and practicalities of such a process, however, when factoring in the potential efficacy of the drug in reducing limb volume, the wider implications for the legal status of the substance, and the fact that the rapidly developing technologies will lead to progressive decreases in costs of these tests over time; the potential benefits may indeed trump the costs.²⁰

Screening for ALDH2 polymorphisms is simple and unequivocally clinically viable, with a rapid simultaneous genotyping test involving high-resolution melting assays available that can yield results in 2 hours for ∼$0.50 per test, as well as an effective two-question screening tool which demonstrated 90% sensitivity and 88% specificity in a male Japanese population.^37,40,41

In contrast, CYP2A6 genotyping methods are still developing, proving a challenge in the high sequence homology between CYP2A6 and its neighboring CYP2A genes.²³ Fogli et al. demonstrate a practical approach to this in assessing the nicotine profiles of a group of smokers (n = 66) by simultaneously analyzing nicotine metabolite ratios and patient genotype from blood samples.⁴² Liquid chromatography–mass spectrometry was used to assay metabolite ratio, and CYP2A6 genotype was determined with real-time polymerase chain reaction (PCR).⁴² Wassenar et al. further describe the use of an SYBR green allele amplification method in real time PCR, which offers improvement in time and cost efficiency compared to traditional end point PCR methods by bypassing the need for gel electrophoresis in visualization.²³ Another recently studied method utilizes a gas chromatography–mass spectrometry “cocktail approach” allowing for the genotyping of multiple cytochrome P450s at once.⁴³

Overall, despite the emergence of increasingly clinically practical and efficient modes of pharmacogenetic screening, the genetic correlation must be solidified further before a fully realized pharmacogenomic approach is fully viable for benzopyrone candidates.

Further factors influencing hepatotoxicity and patient selection

A variety of other factors besides genetics may play a role in the predisposition toward benzopyrone toxicity, which is important especially when considering prescription of this treatment to prospective candidates. One factor is the use of medications which as inducers and/or inhibitors of relevant enzymes can shift the balance toward hepatotoxicity, as well as previous or existing hepatic pathology. The role of alcohol and smoking is also of note but not fully understood, both being known to be metabolized by the same enzymes in the coumarin pathway (ALDH and CYP2A6, respectively), therefore acting as metabolic competitors.

Inducers and inhibitors

Tamoxifen, an antiestrogen taken in breast cancer (therefore commonly used concomitantly with coumarin), has also been implicated in potentiating coumarin toxicity by inducing CYP3A4 independent of CYP2A6,¹⁷ the former of which has been implicated in toxicity, being induced by aflatoxin B1—an agent whose toxicity has been shown to be augmented by coumarin on human liver S9 samples, possibly as a result of CYP3A4 activation.⁴⁴

Nicotine is also metabolized by CYP2A6 and has been studied extensively for this reason. It has been identified as a competitive inhibitor of coumarin breakdown, with smokers showing varying evidence of impaired coumarin metabolism; one study (n = 37) demonstrated significantly lower concentrations of 7-HC in the smoking group compared to nonsmokers (p < 0.001),⁴⁵ whereas another study demonstrated no such significant difference (n = 194).⁴⁶ Further studies are needed to clarify whether this is a clinically relevant effect. Identification of further inducers and inhibitors of enzymes in this pathway are still progressing.

Liver and alcohol history

A past history or currently active liver disease also constitutes a significant risk factor in coumarin toxicity; both Hepatitis A and alcoholic liver cirrhosis have been implicated in CYP2A6 impairment and poor 7-HC excretion,^21,47 a characteristic consistent with the tendency of these stimuli to produce generalized liver injury and dysfunction.

Given the evidence on the importance of ALDH in detoxification of o-HPA, it follows that disulfiram, an inhibitor of ALDH that is commonly used to treat chronic alcoholism, is likely to drive toxicity in patients as it has done in laboratory trials.^16,39 By extension, medications which exert a “disulfiram-like effect” of ALDH inhibition can be inferred to have a similar effect. These include a variety of antimicrobials, including metronidazole, chloramphenicol, and furazolidone, among a number of others, although recent studies suggest that metronidazole does not pose a clinically significant effect.⁴⁸

The above effects are important when considering the treatment of patients with chronic alcoholism and active microbial infections, for which they may be on medications which form a dangerous combination with coumarin. Interestingly, the concurrent use of these medications have not been taken into account by influential past trials where cases of coumarin hepatotoxicity were demonstrated and evaluated, potentially acting as confounders.¹¹ Future studies should identify, quantify, and confirm the extent of these risk factors.

Overall, if all predisposing factors are correctly identified and mitigated, there is potential to devise a comprehensive contraindication list and therapeutic monitoring regime for these agents to facilitate their safe usage and exclude at-risk candidates.

Benzopyrones other than coumarin

It should also be noted that several trials have demonstrated at least some degree of clinical efficacy of other benzopyrones such as diosmin, hesperidin, and several rutins (by similar mechanisms as coumarin), while no reports of hepatotoxicity were reported in any of these compounds.^12,49,50 The metabolism and hepatotoxicity of these compounds should be also evaluated further to solidify their use in clinical settings.

Conclusion

Benzopyrones are low-cost, accessible plant-derived agents which have demonstrated a promising degree of clinical efficacy in both past studies and clinical practice, especially when used in conjunction with other therapies such as CPT or MLD. However, the tendency for certain members of this family to cause hepatotoxicity (although in a statistically minor but occasionally severe manner) has posed a barrier to its commonplace use, as well as to the trust of many relevant clinicians, leading to its “fall from favor” in the lymphedema community. Yet with ever-progressing technological advancements at our fingertips, as well as a newfound knowledge of past failures and oversights, it is perhaps time to once again consider strategies for their reimplementation in modern practice.

One important strategy is the development of evidence-based clinical guidelines around contraindications, drug interactions, and therapeutic monitoring regimes to avoid putting vulnerable populations at risk. At the current moment, we recommend caution if not complete avoidance of benzopyrone usage in patients regularly taking agents implicated in driving toxicity (including tamoxifen, disulfiram, its mimics, and possibly nicotine and alcohol), patients with a significant history of liver disease/hepatitis/hepatotoxicity/alcoholism and patients of a “hepatically vulnerable” familial or ethnic descent (i.e., Eastern Asians). Urinary 7-HC and o-HPAA concentrations are potentially effective measures of coumarin metabolism and predictors of hepatotoxicity; standard Liver Function Tests are likely to be of little utility in monitoring given the reaction's idiosyncratic nature.

A pharmacogenetic approach is another such strategy; however, many specifics regarding the genotypic correlation to metabolic phenotypes in both populations and individuals remain to be clarified before fully reliable clinical pharmacogenetic practices can be applied to benzopyrones in lymphedema patients. That being said, pharmacogenetic technologies have developed substantially in the last 10 years since the previous review of the same nature,¹⁷ to the point where practically viable options are actually being applied effectively in clinical settings, such as in the case of ALDH screening.

Although there has been little progress in the way of applying pharmacogenetics to benzopyrone use clinically in these last 10 years since there has been almost complete avoidance of its use, the prerequisite technologies and knowledge base are certainly continuing to advance. It follows that successful implementation of these methods in the future truly depends on the initiative of interested parties in undertaking the experimentation needed to solidify an evidence base, whether it be by primary or secondary intention.

As it stands, there is much to be elucidated, as well as various economic and practical barriers, to the return of benzopyrones. However, taking into account the fact that many of these therapeutic yet potentially dangerous agents are cheap and easily accessible online for lymphedema patients to purchase (largely unregulated by food and drug authorities) and self-administer unmonitored, is it not the responsibility of the professional and scientific community to comprehend, develop, and pave the way for their safe usage in the future?

Footnotes

Acknowledgments

The authors thank Professor Sandro Michelini for the enlightening academic discourse and advisorship, as well as other members of the International Lymphatic Framework () and Australasian Lymphology Association who provided valuable commentary and feedback.

Author Disclosure Statement

No competing financial interests exist.

References

Lake

. Coumarin metabolism, toxicity and carcinogenicity: Relevance for human risk assessment. Food Chem Toxicol, 1999; 37:423–453.

Marshall

, MJL, Edmonds

, Williams

, Butler

, Ryles

, Weiss

, Urban

, Bueschen

, Markiewicz

, et al. An updated review of the clinical development of coumarin (1,2-benzopyrone) and 7-hydroxycoumarin. J Cancer Res Clin Oncol, 1994; 120 Suppl:S39–S42.

Musa

, Cooperwood

, Khan

, Omar

. A review of coumarin derivatives in pharmacotherapy of breast cancer. Curr Med Chem, 2008; 15:2664–2679.

Rockson

. The lymphatics and the inflammatory response: Lessons learned from human lymphedema. Lymphat Res Biol, 2013; 11:117–120.

Rockson

. The unique biology of lymphatic edema. Lymphat Res Biol, 2009; 7:97–100.

Granger

, Skeff

, Chaite

, Rockson

. Lymphatic biology and disease: Is it being taught? Who is listening?. Lymphat Res Biol, 2004; 2:86–95.

Casley-Smith

, Casley-Smith

. High Protein Oedemas and the Benzo-Pyrones. JB Lippincott Company; 1986; pp. 269–276.

Nicolaides

. Edema in chronic venous insufficiency and the effect of modern pharmacotherapy. Angiology, 2000; 51:1–2.

Piller

. Variation in acid and neutral protease activity in rats with thermal oedema together with the influence of various benzopyrones. Arzn-Forsh (Drug Res), 1977; 23:1069–1073.

10.

Casley-Smith

, Casley-Smith

. Treatment of lymphedema by complex physical therapy with and without oral and topical benzopyrones: What should therapists and patients expect. Lymphology, 1996; 29:76–82.

11.

Loprinzi

, Kugler

, Sloan

, et al. Lack of effect of coumarin in women with lymphedema after treatment for breast cancer. N Engl J Med, 1999; 340:346–350.

12.

Piller

, Morgan

, Casley-Smith

. A double-blind, cross-over trial of O-(beta-hydroxyethyl)-rutosides (benzo-pyrones) in the treatment of lymphoedema of the arms and legs. Br J Plast Surg, 1988; 41:20–27.

13.

Casley-Smith

. Benzo-pyrones in the treatment of lymphoedema. Int Angiol, 1999; 18:31–41.

14.

Wang

, Avula

, Zhao

, Smillie

, Nanayakkara

NPD

, Khan

. Characterization and distribution of coumarin, cinnamaldehyde and related compounds in Cinnamomum spp. by UPLC-UV/MS combined with PCA. Planta Med, 2010; 76:31.

15.

Therapeutic Goods Administration. Australian adverse drug reactions. Canberra, AU: Adverse Drug Reaction Advisory Committee, 1995; 14:1.

16.

Vassallo

, Hicks

, Daston

, Lehman-McKeeman

. Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity. Toxicol Sci, 2004; 80:249–257.

17.

Farinola

, Piller

. CYP2A6 polymorphisms: Is there a role for pharmacogenomics in preventing coumarin-induced hepatotoxicity in lymphedema patients?. Pharmacogenomics, 2007; 8:151–158.

18.

Born

, Caudill

, Fliter

, Purdon

. Identification of the cytochromes P450 that catalyze coumarin 3,4-epoxidation and 3-hydroxylation. Drug Metab Dispos, 2002; 30:483–487.

19.

Lewis

DFV

, Ito

, Lake

. Metabolism of coumarin by human P450s: A molecular modelling study. Toxicol in Vitro, 2006; 20:256–264.

20.

Farinola

, Piller

. Pharmacogenomics: Its role in re-establishing coumarin as treatment for lymphedema. Lymphat Res Biol, 2005; 3:81–86.

21.

Pelkonen

, Rautio

, Raunio

, Pasanen

. CYP2A6: A human coumarin 7-hydroxylase. Toxicology, 2000; 144:139–147.

22.

Kubota

, Nakajima-Taniguchi

, Fukuda

, et al. CYP2A6 polymorphisms are associated with nicotine dependence and influence withdrawal symptoms in smoking cessation. Pharmacogenomics J, 2006; 6:115–119.

23.

Wassenaar

, Zhou

, Tyndale

. CYP2A6 genotyping methods and strategies using real-time and end point PCR platforms. Pharmacogenomics, 2015; 17:147–162.

24.

Hadidi

, Zahlsen

, Idle

, Cholerton

. A single amino acid substitution (Leu160His) in cytochrome P450 CYP2A6 causes switching from 7-hydroxylation to 3-hydroxylation of coumarin. Food Chem Toxicol, 1997; 35:903–907.

25.

Mizutani

. PM frequencies of major CYPs in Asians and Caucasians. Drug Metab Rev, 2003; 35:99–106.

26.

, Huang

S-L

, Zhu

R-H

, Han

X-M

, Zhou

H-H

. Phenotypic polymorphism of CYP2A6 activity in a Chinese population. Eur J Clin Pharmacol, 2002; 58:333–337.

27.

Hassanzadeh Khayyat

, Vahdati Mashhadian

, Eghbal

, Jalali

. Inter-individual variability of coumarin 7-hydroxylation (CYP2A6 activity) in an Iranian population. Iran J Basic Med Sci, 2013; 16:610–614.

28.

Iscan

, Rostami

, Güray

, Pelkonen

, Rautio

. Interindividual variability of coumarin 7-hydroxylation in a Turkish population. Eur J Clin Pharmacol, 1994; 47:315–318.

29.

Rautio

, Kraul

, Kojo

, Salmela

, Pelkonen

. Interindividual variability of coumarin 7-hydroxylation in healthy volunteers. Pharmacogenomics, 1992; 2:227–233.

30.

Burian

, Freudenstein

, Tegtmeier

, Naser-Hijazi

, Henneicke-von Zepelin

, Legrum

. Single copy of variant CYP2A6 alleles does not confer susceptibility to liver dysfunction in patients treated with coumarin. Int J Clin Pharmacol Ther, 2003; 41:141–147.

31.

Jelski

, Szmitkowski

. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the cancer diseases. Clin Chim Acta, 2008; 395:1–5.

32.

Agarwal

. Goedde HW. Pharmacogenetics of alcohol metabolism and alcoholism. Pharmacogenomics, 1992; 2:48–62.

33.

Masaoka

, Ito

, Soga

, Hosono

, Oze

, Watanabe

, Tanaka

, Yokomizo

, Hayashi

, Eto

, Matsuo

. Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms exacerbate bladder cancer risk associated with alcohol drinking: Gene-environment interaction. Carcinogenesis, 2016; 37:583–588.

34.

, Zhao

, Sun

, Luo

, Xiao

. ALDH2 gene polymorphism in different types of cancers and its clinical significance. Life Sci, 2016; 147:59–66.

35.

Zhao

, Wang

. Glu504Lys single nucleotide polymorphism of aldehyde dehydrogenase 2 gene and the risk of human diseases. BioMed Res Int, 2015; 2015:174050.

36.

Wall

, Luczak

, Hiller-Sturmhöfel

. Biology, genetics, and environment: Underlying factors influencing alcohol metabolism. Alcohol Res, 2016; 38:59–68.

37.

Gross

, Zambelli

, Small

, Ferreira

JCB

, Chen

C-H

, Mochly-Rosen

. A personalized medicine approach for Asian Americans with the aldehyde dehydrogenase 2*2 variant. Annu Rev Pharmacol Toxicol, 2015; 55:107–127.

38.

Yoshida

, Wang

, Davé V. Determination of genotypes of human aldehyde dehydrogenase ALDH2 locus. Am J Hum Genet, 1983; 35:1107–1116.

39.

Harada

, Misawa

, Agarwal

, Goedde

. Liver alcohol dehydrogenase and aldehyde dehydrogenase in the Japanese: Isozyme variation and its possible role in alcohol intoxication. Am J Hum Genet, 1980; 32:8–15.

40.

Yuan

X-W

, Zhou

W-J

, Shang

, Li

, Liu

Y-H

, Xu

X-M

. Rapid simultaneous genotyping of polymorphisms in ADH1B and ALDH2 using high resolution melting assay. Clin Chem Lab Med, 2013; 51:e75.

41.

Yokoyama

, Yokoyama

, Kato

, et al. Alcohol flushing, alcohol and aldehyde dehydrogenase genotypes, and risk for esophageal squamous cell carcinoma in Japanese men. Cancer Epidemiol Biomarkers Prev, 2003; 12:1227.

42.

Fogli

, Saba

, Del Re

, et al. Application of a pharmacokinetic/pharmacogenetic approach to assess the nicotine metabolic profile of smokers in the real-life setting. J Pharm Biomed Anal, 2016; 131:208–213.

43.

, Lee

, Kim

, Jung

. Development of GC-MS based cytochrome P450 assay for the investigation of multi-herb interaction. Anal Biochem, 2017; 519:71–83.

44.

Goeger

, Hsie

, Anderson

. Co-mutagenicity of coumarin (1,2-benzopyrone) with aflatoxin B1 and human liver S9 in mammalian cells. Food Chem Toxicol, 1999; 37:581–589.

45.

Poland

, Pechnick

, Cloak

, Wan

Y-JY

, Nuccio

, Lin

K-M

. Effect of cigarette smoking on coumarin metabolism in humans. Nicotine Tob Res, 2000; 2:351–354.

46.

Mahavorasirikul

, Tassaneeyakul

, Satarug

, Reungweerayut

, Na-Bangchang

. CYP2A6 genotypes and coumarin-oxidation phenotypes in a Thai population and their relationship to tobacco smoking. Eur J Clin Pharmacol, 2009; 65:377–384.

47.

Pasanen

, Rannala

, Tooming

, Sotaniemi

, Pelkonen

, Rautio

. Hepatitis A impairs the function of human hepatic CYP2A6 in vivo. Toxicology, 1997; 123:177–184.

48.

Karamanakos

, Pappas

, Boumba

, et al. Pharmaceutical agents known to produce disulfiram-like reaction: Effects on hepatic ethanol metabolism and brain monoamines. Int J Toxicol, 2007; 26:423–432.

49.

Cluzan

, Alliot

, Ghabboun

, Pascot

. Treatment of secondary lymphedema of the upper limb with CYCLO 3 FORT. Lymphology, 1996; 29:29–35.

50.

Pecking

, Fevrier

, Wargon

, Pillion

. Efficacy of Daflon 500 mg in the treatment of lymphedema (secondary to conventional therapy of breast cancer). Angiology, 1997; 48:93–98.