Cyp 3 1 Assignment Web

Abstract

Pharmacogenetics affects both pharmacokinetics and pharmacodynamics, thereby influencing an individual's response to drugs, both in terms of response and adverse reactions. Within the area of pharmacogenetics, findings of genetic variation influencing drug levels have been more prevalent, and variation in the cytochrome P450 (CYP) enzymes is one of the most common causes. Much of the work concerning sequence variations in CYPs aims at finding biomarkers of use for individualised treatment, thereby increasing the treatment response, lowering the number of side effects and decreasing the overall cost of treatment regimens. For over ten years, the Human Cytochrome P450 Allele Nomenclature (CYP-allele) website (http://www.cypalleles.ki.se/) has offered a database of genetic information on CYP variants, along with effects at the molecular as well as clinical level. Thus, this database serves as an assembly of past, current and soon-to-be published information on CYP alleles and their outcome effects. The website is used by academic researchers and companies (eg as a tool in drug development and for outlining new research projects). By providing peer-reviewed genetic information on CYP enzymes, the CYP-allele website has become increasingly popular and widely used. Recently, NADPH cytochrome P450 oxidoreductase (POR), the electron donor for CYP enzymes, was included on the website, which already contains 29 CYP genes, hence POR alleles are now also designated using the star allele (POR*) nomenclature. Although most CYPs on the CYP-allele website are involved in the metabolism of xenobiotics, polymorphic enzymes with endogenous functions are also included. Each gene on the CYP-allele website has its own webpage that lists the different alleles with their nucleotide changes, their functional consequences and links to publications in which the allele has been identified and/or characterised. Thus, the CYP-allele website offers a rapid online publication of new alleles, as well as providing an overview of peer-reviewed data.

Keywords: pharmacogenetics, adverse drug reactions, drug response, haplotypes, drug metabolism, cytochrome P450 oxidoreductase (POR)

Introduction

Only 30-60 per cent of patients respond properly to treatment with antidepressants, beta-blockers, statins and antipsychotic agents [1]. Approximately two days of prolonged hospital visits are caused by adverse drug reactions (ADRs), and, in the USA, about 100,000 deaths are estimated to be due to ADRs every year [1]. These data emphasise an important problem that can be mostly explained by variable pharmacokinetics due, to a large extent, to differences in the activity of cytochrome P450 (CYP) enzymes involved in the metabolism of the drugs. Variable metabolism, in turn, can be caused by variation in the CYP genes, as is the focus of the current review.

Cytochromes P450

There are 57 active CYP genes in the human genome, which are divided into 18 families. The first three families (CYP1-3) are generally involved in the metabolism of exogenous substances such as drugs, whereas CYP families with higher numbers are usually involved in the metabolism of endogenous substances. CYP enzymes are responsible for 75-80 per cent of all phase I-dependent metabolism and for 65-70 per cent of the clearance of clinically used drugs [2,3]. Variation in CYP genes results in phenotypes classically defined as ultrarapid, extensive, intermediate and poor metabolisers. An ultrarapid metaboliser (UM) generally carries duplicated or multi-duplicated gene copies of the same allele, whereas intermediate (IM) and poor metabolisers (PM) characteristically carry one and two defective alleles (eg gene inactivation or deletion), respectively. The term extensive metaboliser (EM) is normally used for subjects carrying two alleles giving normal activity of the CYP enzyme (also called the *1 or consensus allele). The metaboliser phenotypes are mainly used for describing drug metabolism, but genetic variation in CYPs with endogenous functions, such as in sterol, steroid, bile acid and fatty acid homeostasis, have also been well characterised, some of which give rise to disease states.

CYP3A4 accounts for about 50 per cent of all CYP-dependent drug metabolism, although individuals' capacity for CYP3A4-mediated drug metabolism is highly variable. No common genetic variants can account for this variation, despite the fact that 20 different alleles have been described. By contrast, CYP2C19 and CYP2D6 are highly polymorphic and together account for about 40 per cent of the metabolism of clinically used drugs. In addition, CYP1A2, CYP2A6 and CYP2B6 are polymorphic enzymes that significantly contribute to xenobiotic metabolism. The characterised genetic polymorphism of these enzymes provides a basis for the possibility to adjust drug dosage and choice of drug therapy according to genotype,[4-8] which includes avoidance of ADRs, since polymorphic CYPs are frequently the cause of these, either due to the formation of high levels of metabolites or because of decreased metabolism of the parent drug.

The Human Cytochrome P450 (CYP) Allele Nomenclature website

Introduction

Important work in the CYP area focused on the identification and characterisation of polymorphic human CYP genes, which, in turn, created the need for a unified nomenclature system. Thus, in 1999, a nomenclature committee was formed with the aim of creating a platform for present and future allele nomenclature. Thus, the Human Cytochrome P450 Allele Nomenclature (CYP-allele) website (http://www.cypalleles.ki.se/) was launched with the purpose of managing allele designations, facilitating rapid online publication and providing a summary of alleles and their associated effects. The nomenclature system chosen for the CYP-allele website was based on recognised nomenclature guidelines [9-13].

Currently, the website covers the nomenclature for polymorphic alleles of 29 CYP enzymes and NADPH cytochrome P450 oxidoreductase (POR)[14] (see Table ​1). The CYP2B6, CYP2C9, CYP2C19 and CYP2D6 genes are particularly polymorphic, all with a high number of functionally different alleles. Each of the genes at the CYP-allele website has its own webpage that lists the various alleles with their nucleotide changes, molecular and functional consequences in vitro as well as in vivo, and also publications identifying or characterising the alleles. In addition, links to the National Center for Biotechnology Information (NCBI) single nucleotide polymorphism database (dbSNP) and papers with allele frequencies are presented. The number of visits is relatively constant over time, about 36,000 per year, and the website is highly cited in publications in the field of pharmacogenomics.

Table 1

Polymorphic genes covered on the CYP-allele website

Inclusion criteria for CYP alleles

The designation of an allele (such as CYP2B6*4) ideally requires determination of all sequence variations in the gene, although sequencing the intronic regions is generally not necessary. On the website, a gene is considered as the sequence from 5 kilobases upstream from the transcription start site to 500 base pairs downstream of the last exon. If a regulatory element has been characterised at a more distant part of the gene, however, it too is considered to belong to the gene. All known sequence variations within an allele are described on the CYP-allele website, although new allele numbers are currently only designated for alleles that contain at least one functional variation causing consequences such as amino acid substitutions, translation terminations, splice defects, differential transcription rates etc. Nevertheless, the allele is required to be well characterised regarding linkage or lack of linkage of the consequential SNP with other nucleotide variations, including those in exons, intron-exon junctions and flanking regions. Inferring haplotypes by program analyses is generally avoided, although such alleles have occasionally been included on the CYP-allele website. When the characterised sequence variant is found in different constellations with non-causative (eg silent) ones, the different combinations are defined as sub-alleles and receive letters in addition to the number (eg CYP2B6*4A, CYP2B6*4B). When several effective polymorphisms are present on the same allele, however, the allelic number given is based on the polymorphism that causes the most severe consequence, such as a splice defect (eg CYP2C19*2A), so alleles that additionally contain sequence variants with less severe effects will share the same allele number, together with an additional letter (eg CYP2C19*2B). Combinations of variants that are also present alone and that are considered similarly effective (eg different amino acid substitutions) are given unique allele numbers (eg CYP2B6*6). Notably, the earliest described alleles on the website do not follow the nomenclature system, but the allelic designations have remained, for practical reasons.

Submission of new alleles

When new alleles are identified, relevant information is sent to the Webmaster. Inclusion criteria (http://www.cypalleles.ki.se/criteria.htm) involve complete characterisation of the gene sequence, covering exons and exon-intron junctions at the minimum, investigation of linkage with other sequence variants and potential in vitro or in vivo findings. It is advised that the authors of a manuscript that describes a novel allele contact the Webmaster before submission, in order to review the data and assign a new allele name to be used in the manuscript. Usage of star allele designations that have not been approved by the nomenclature committee is strongly discouraged, because of the apparent risk of confusion and of using the same allele name for different variants. All information sent to the Webmaster is kept strictly confidential until publication of the manuscript or until the authors request it to be released. Thus, there are likely to be allele names designated by the CYP-allele website that have not yet been published, further emphasising the importance of refraining from using unauthorised allele names.

The Webmaster (and in rare cases also the Editorial and/or Advisory Board) reviews the submission to evaluate whether there are enough data to support a new allele designation. Only peer-reviewed data are thus published on the CYP-allele website. Papers describing additional characterisation of a known allele--with respect to, for example, in vitro or in vivo activity--are also peer reviewed and can be linked to the respective allele on the webpage. Suggestions of papers that should be included with respect to further characterisation of alleles are appreciated.

Conclusions

The CYP-allele website is widely used and well acknowledged within the scientific community. It serves the purpose of a unified and easily accessible nomenclature system for CYP enzymes, as well as for the CYP electron donor POR. The purpose of the CYP-allele website is to facilitate rapid online publication as well as providing a summary of the characteristics of specific alleles. The database has proven to be a useful resource in the area of pharmacogenomics.

References

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Abstract

It is well recognized that herbal supplements or herbal medicines are now commonly used. As many patients taking prescription medications are concomitantly using herbal supplements, there is considerable risk for adverse herbal drug interactions. Such interactions can enhance the risk for an individual patient, especially with regard to drugs with a narrow therapeutic index such as warfarin, cyclosporine A and digoxin. Herbal drug interactions can alter pharmacokinetic or/and pharmacodynamic properties of administered drugs. The most common pharmacokinetic interactions usually involve either the inhibition or induction of the metabolism of drugs catalyzed by the important enzymes, cytochrome P450 (CYP). The aim of the present article is to provide an updated review of clinically relevant metabolic CYP-mediated drug interactions between selected herbal supplements and prescription drugs. The commonly used herbal supplements selected include Echinacea, Ginkgo biloba, garlic, St. John's wort, goldenseal, and milk thistle. To date, several significant herbal drug interactions have their origins in the alteration of CYP enzyme activity by various phytochemicals. Numerous herbal drug interactions have been reported. Although the significance of many interactions is uncertain but several interactions, especially those with St. John’s wort, may have critical clinical consequences. St. John’s wort is a source of hyperforin, an active ingredient that has a strong affinity for the pregnane xenobiotic receptor (PXR). As a PXR ligand, hyperforin promotes expression of CYP3A4 enzymes in the small intestine and liver. This in turn causes induction of CYP3A4 and can reduce the oral bioavailability of many drugs making them less effective. The available evidence indicates that, at commonly recommended doses, other selected herbs including Echinacea, Ginkgo biloba, garlic, goldenseal and milk thistle do not act as potent or moderate inhibitors or inducers of CYP enzymes. A good knowledge of the mechanisms of herbal drug interactions is necessary for assessing and minimizing clinical risks. These processes help prediction of interactions between herbal supplements and prescription drugs. Healthcare professionals should remain vigilant for potential interactions between herbal supplements/medicines and prescription drugs, especially for drugs with a narrow therapeutic index are used.

Keywords: herbal drug interactions, CYP, dietary supplements, herbal medicines, botanical supplements, drug interactions

Introduction

Products containing biologically active phytochemicals are often defined as “herbal” or “botanical” supplements. They are derived or extracted from a broad range of plant sources. Herbs and herbal supplements are used by people for purpose of treating medical or psychiatric disorders. Herbal supplements are now commonly used in both Eastern and Western countries. Herbal medicines or traditional medicines have a long history of use in the Eastern countries, such as China and India, and continue to be used today (Philp, 2004[142]). The use of herbal supplements in the United States has grown at a rapid rate with a 380 % increase between 1990 and 1997 (Brevoort, 1998[22]; Eisenberg et al., 1998[38]). The Dietary Supplement Health and Education Act (DSHEA) of 1994 permits the direct marketing of herbal products and supplements without prior proof of safety or efficacy. Herbal supplements can be purchased in several forms. The most common formulation of herbal supplements are encapsulated extracts. Herbal extracts, because they are crude products, usually contain many natural phytochemicals. They are also accessible as tinctures and beverages (Markowitz et al., 2008[112]). They can be ingredients in nutrition and sport drinks; powders, and “energy” bars. Patients may also consume food products containing certain phytochemical ingredients to treat common diseases. For instance, cranberry juice is widely used for urinary tract infections and garlic for hyperlipidemia (Markowitz et al., 2008[112]).

The perception that herbal supplements and medicines, legally categorized as over-the-counter dietary supplements, may reduce or enhance the effects of prescription drugs has earned our attention slowly (Cott, 2008[31]). Herbal supplements usually have many active phytochemical constituents. Therefore, the possibility of interactions is increased when compared with the likelihood of interactions between two prescription drugs. Presently, most herbal drug interactions are documented as case series or individual case reports (Sood et al., 2008[162]). The common misconception is that herbal medicines or supplements are prepared from natural plants so therefore are safe to be taken concomitantly with prescription drugs. However, this does not guarantee that they are safe, free from adverse effects or toxicity, and devoid of any drug-drug interaction potential (Hermann and von Richter, 2012[78]). The overwhelming majority of herbal use involves “self-medication” and these herbal products and supplements are commonly taken concomitantly with conventional medications with little or no information available on potential herbal drug interactions. Patients may feel embarrassed that they are self-medicating with complementary therapies and others may not consider that herbs are medicines in the conventional sense. A study in the United States revealed that only one-third of patients told their physician they were taking herbal remedies or supplements (Kennedy et al., 2008[94]). In fact, there have been increasing reports on herbal drug interactions. Drug interactions can be caused by some natural products which are not be consumed as medicines. Grapefruit and grapefruit juice is a common example. The first major concern of herbal drug interactions was recognised in 1989 when grapefruit juice was reported to increase the blood concentrations of felodipine, an anti-hypertensive calcium channel antagonist (Bailey et al., 1998[11]). Later in 1996, a similar grapefruit drug interaction was found with terfenadine, a non-sedative antihistamine (Bailey et al., 1998[11]; Benton et al., 1996[13]). Successive studies implied that these effects of grapefruit were caused by inhibition of intestinal CYP3A4 and a drug transporter protein, P-glycoprotein (P-gp) by furanocoumarins (e.g. 6’, 7’-dihydroxybergamottin, bergamottin and bergapten), naturally-occurring ingredients in grapefruit (Bailey et al., 2000[9]; Goosen et al., 2004[56]; Ho et al., 2001[81]; Lin et al., 2012[103]; Paine et al., 2005[136]). Serious adverse drug reactions arising from grapefruit and terfenadine interaction was one of the main reasons for withdrawal of terfenadine from the market in 1998. These adverse drug reactions were due to inhibition of CYP3A4 by grapefruit active ingredients led to marked increase in blood terfenadine concentrations. This subsequently caused prolonged QT intervals and following cardiac arrhythmias. Grapefruits have also been shown to increase the plasma concentration and bioavailability of several drugs that are substrates for cytochrome P450 (CYP) 3A4 enzyme (Bailey et al., 2007[10]; Greenblatt et al., 2003[59]). This means that grapefruit interacts with most clinically used drugs as more than 50 % of prescription drugs are metabolized by CYP3A4 (Guengerich, 1999[61]; Nebert and Russell, 2002[132]). This made substantial concern regarding food or herbal drug interactions, and has also stimulated numerous research to fulfill the knowledge in this particular area. Cruciferous vegetables is an additional example, it has been involved in the interactions with a number of CYP1A2-substrate drugs (Ioannides, 1999[84]; Zhou et al., 2004[203]). An understanding of these herbal drug interactions has great benefit for drug therapy as it helps us to predict herbal drug interactions.

Herbal supplements and cancer chemoprevention

For many years it has been recognized that the use of herbs as medicines plays an important role in nearly every culture, including Asia, Africa, Europe and the Americas. Recent surveys suggest that one in three Americans use dietary supplements daily. It is interesting that the rate of herbal usage is much higher in cancer patients (Pierce et al., 2002[143]; Richardson et al., 2000[152]; Rock, 2003[155]; Wargovich et al., 2001[180]), in some cases, up to 50 % of patients treated in cancer centers. Many of these supplements are herbal in nature (Wargovich et al., 2001[180]). Herbal supplements are generally taken for two main reasons. The first reason is that they are used to alleviate symptoms of illness. For example, the widespread use of St. John’s wort for relief of depression, the use of Echinacea for relief of cold symptoms, and use of Ginkgo biloba for improvement in cognition (Wargovich, 2001[179]). The second reason is that herbal supplements are used specifically with the hopes of preventing disease or reducing the risk for certain diseases. Examples of this include green tea, grape seed extract and other flavonoid-rich botanicals which are taken because of their natural antioxidants (Mandlekar et al., 2006[111]). The use of garlic and its supplement preparation is another example, which has been demonstrated in animals, to prevent cancer (Wargovich, 1997[179]; Wargovich et al., 2001[180]).

With respect to cancer chemoprevention, herbal supplements can act through several mechanisms to provide a protective effect. Induction of phase I and phase II metabolic enzymes by herbal products is one of the common mechanisms (Wargovich et al., 2001[180]). Garlic as well as many organosulfur compounds derived from garlic have been demonstrated to possess strong chemopreventive activity against experimentally induced cancers of the skin, esophagus, stomach, colon, liver, lung and mammary gland (Cerella et al., 2011[26]; Chandra-Kuntal et al., 2013[29]; Wargovich, 1997[179]; Wargovich et al., 2001[180]). One of the active constituents in garlic, diallyl sulfide, is a potent inhibitor of the phase I enzyme CYP2E1 (Brady et al., 1988[19]). CYP2E1 is involved in the metabolic activation of several environmental and dietary carcinogens, e.g., nitrosamines (Yang et al., 2001[193]; Zhou et al., 2003[202]). In addition, diallyl sulfide significantly increases a number of phase II enzymes, including glutathione S-trans-ferase, UDP-glucuronosyltransferase, and quinone reductase. These phase II enzymes are responsible for the detoxification of many carcinogens (Wargovich, 1997[179]; Wargovich et al., 2001[180]).

Cytochrome P450 (CYP)

CYP enzymes are responsible for detoxification of a wide range of xenobiotics including drugs, environmental pollutants, and cancer-causing agents (i.e., carcinogens). They are also involved in the biosynthesis of cholesterol and other important lipids such as prostacyclins and thromboxane A2 which are implicated in the causation of many cardiovascular diseases (Guengerich, 1999[61]; Nebert and Russell, 2002[132]). In fact, the role of CYP enzymes in protecting the body against foreign chemicals is as crucial as that of antibodies in dealing with invading organisms. Most chemical carcinogens require metabolic activation by CYP, to their genotoxic intermediates (Guengerich and Shimada, 1991[62]; Badal et al., 2012[7]). In some instances, these activated metabolites are subjected to detoxification by conjugation reactions. Thus, activities or levels of CYP enzymes may be one of the important host factors which determine whether exposure to the carcinogen results in cancer or not (Maliakal et al., 2011[110]; Nebert, 1991[131]). The CYP1A subfamily (CYP1A1 and CYP1A2) plays a vital role in the metabolism of two important classes of environmental carcinogens: polycyclic aromatic hydrocarbons and arylamines. Many clinically important drugs are metabolized by this specific isoenzyme such as caffeine, theophylline, verapamil and clozapine. CYP2D6 is responsible for the metabolism of more than 30 clinically important drugs, such as metoprolol and several other β-blockers, antiarrhythmics, antidepressants, neuroleptics and morphine-related drugs (Gonzalez, 1992[54]). More than 50 % of all prescription drugs are metabolized by CYP3A4 (Guengerich, 1999[61]; Nebert and Russell, 2002[132]). The activity of CYP can be influenced by many factors; including genetic composition of the host, certain medications, and exposure to certain dietary and environmental chemicals (Weisburger and Chung, 2002[185]). It has been demonstrated that vegetables, particularly cruciferous ones (e.g., cabbage, brussel sprouts and cauliflower), induce CYP enzymes; namely CYP1A1 and CYP1A2 (Murray, 2006[128]; Yoshida et al., 2004[196]; Zhou et al., 2004[203]). Cigarette smoking is known to induce CYP1A1 and CYP1A2; the high levels of these CYP enzymes have been linked to an increased risk of lung and colon cancer (Guengerich and Shimada, 1991[62]; Smith et al., 2001[161]; Zhou et al., 2010[206]).

CYP3A4 is the most important human CYP isozyme as it is involved in the metabolism of most clinically used drugs (Guengerich, 1999[61]; Nelson et al., 1996[133]; Nebert and Russell, 2002[132]). Human CYP3A4 is expressed in the prostate, breast, gut, colon and small intestine. However, its expression is most abundant in the liver which accounts for 30 % of the total CYP protein content (Guengerich, 1999[61]; Lown et al., 1997[107]; Shimada et al., 1994[159]; Watkins et al., 1987[182]). CYP3A4 plays an important role in the oxidation of both testosterone and estrogen. Activity of CYP3A4 can be inhibited or induced by drugs, herbs, pesticides, and carcinogens. Individual variation in CYP3A4 levels may play a role in breast and prostate carcinogenesis through modulation of sex hormone metabolite levels. Alternatively, CYP3A4 can be involved in bioactivation of exogenous carcinogens. It has been shown that CYP3A4 is involved in activation of many environmental carcinogens. These include polycyclic aromatic hydrocarbons, heterocyclic amines, aflatoxin B1, and nitrosamines (Guengerich and Shimada, 1991[62]; Patten et al., 1997[138]; Shimada et al., 1994[159]). Furthermore, ingestion of polycyclic aromatic hydrocarbons and heterocyclic amines, and their metabolism mediated by CYP3A4 has been shown to result in the formation of carcinogen DNA adducts in mammary tissues (Lightfoot et al., 2000[102]).

It has been hypothesized that alcoholic beverages, particularly red wine, offer extra cardiovascular protection due to their rich content of antioxidant flavonoid compounds. Many flavonoids, such as quercetin, resveratrol and naringenin, have been shown to alter the activity of CYP enzymes (Chan and Delucchi, 2000[27]; Niestroy et al., 2012[134]). This effect could also be associated with a protective property against cancer. One target of the chemopreventive effect of naturally occurring flavonoids, such as grape constituents, could be inhibition of xenobiotic metabolizing phase I enzymes, i.e., CYP. Alternatively, it could be the induction of phase II conjugation enzymes, such as UDP-glucuronosyltransferase and glutathione S-transferase, that are responsible for the detoxification of carcinogens. Among the main human CYPs, CYP1A1 and CYP1A2 isoforms are involved in the activation of many procarcinogens; including polycylic aromatic hydrocarbons, polycyclic amines and aflatoxin B1 (Gonzalez and Gelboin, 1994[55]). Resveratrol, a phytoalexin present in grapes, has been demonstrated to be an aryl hydrocarbon receptor antagonist which inhibits the induction of the CYP1A1 enzyme (Chan and Delucchi, 2000[27]). CYP2E1 is responsible for the activation of volatile organic solvents and precarcinogenic N-nitrosamines (Gonzalez and Gelboin, 1994[55]; Yang et al., 2001[193]; Zhou et al., 2003[202]). Human CYP3A4 activates aflatoxin B1 and the biotransformation of many clinically used drugs (Gonzalez, 1992[54]). Moreover, it has been shown that resveratrol inactivated CYP3A4 in a time- and NADPH-dependent manner (Chan and Delucchi, 2000[27]; Chan et al., 1998[28]). The data suggest that resveratrol is an effective mechanism-based inactivator of CYP3A4.

From the above information, it clearly shows that developed countries recognize and value the importance of research on complementary alternative medicines, including herbal medicines. This is the case especially in the USA and UK where there are established research funding agencies, such as the Office of Dietary Supplements (ODS) under the National Institutes of Health (NIH) and the “Prince of Wales Foundation for Integrated Health”, respectively (The Office of Dietary Supplements website, Ernst et al., 2006[39]). These emphasize the relevance and clinical importance of pursuing research on herbal medicines.

Mechanisms of herbal drug interactions: general considerations

Like drug-drug interactions, herbal drug interactions result from the same principles. These principles are based on the same pharmacokinetic (i.e. changes of plasma drug concentration) and pharmacodynamic (i.e. drugs interacting at receptors) interactions. The currently recognized pharmacokinetic interactions between clinical drugs and herbs or herbal dietary supplements indicate that a number of herbs, most remarkably St. John’s wort, can alter the plasma concentration of different conventional medicines metabolized by CYP, and/or are transported by P-gp (Izzo, 2012[85]). P-gp is presented in the intestine, liver and kidney; it performs an important role in the absorption, distribution, or excretion of drugs. P-gp appears to limit the cellular transport from intestinal lumen into epithelial cells and also enhances the excretion of drugs via hepatocytes and renal tubules into the adjacent luminal space.

Many probe drugs have been used to assess potential effects of herbs on the activity of a specific CYP enzyme. These probe drugs include midazolam, alprazolam, nifedipine (CYP3A4); debrisoquine, dextromethorphan (CYP2D6); chlorzoxazone (CYP2E1); tolbutamide, diclofenac and flurbiprofen (CYP2C9); caffeine, tizanidine (CYP1A2); and omeprazole (CYP2C19). Fexofenadine, digoxin and talinolol have been extensively used in pharmacokinetic studies as P-gp substrates (Izzo, 2012[85]). Polymorphisms in the genes for CYP enzymes and P-gp may influence the interactions mediated through these pathways (Lei et al., 2009[100]). Pharmacodynamic-based interactions between herbs and drugs have been less investigated. However, the outcomes of pharmacodynamic interactions may be either additive or synergetic (i.e. the herbal medicines potentiate the pharmacological/toxicological action of conventional drugs) or antagonistic (i.e. the herbal medicines reduce the efficacy of the drugs). Warfarin and herbal interactions are a classical example of pharmacodynamic interactions. Theoretically, augmented anticoagulant effects could be likely when warfarin is administered concomitantly with coumarin-containing herbs (some plant coumarins exert anticoagulant effects) or with anti-platelet herbs (Izzo, 2012[85]). On the other hand, vitamin K-containing herbs can antagonize the effect of warfarin (the action of warfarin is due to its ability to antagonize the cofactor function of vitamin K).

A number of recently published review articles have comprehensively written and exclusively drawn attention to the mechanisms of herbal drug interactions (Colalto, 2010[30]; Izzo, 2012[85]; Tomlinson et al., 2008[168]; Zhou et al., 2004[203], 2010[206]; Zhang et al., 2009[201]). They have provided evidence of herbs that can modify CYP or P-glycoprotein.

CYP mediated herbal drug interactions: evidence in humans

Evidence is emerging that particular herbs and herbal medicines may contain specific phytochemicals which can modulate the activity of drug metabolizing enzymes CYP and drug transporting protein, P-glycoprotein (P-gp). These modulations may lead to potential interaction between herbal medicines and prescription drugs. This review will focus on important herbal drug interactions having clinical relevance and discuss the mechanism of drug interactions which is mediated by CYP enzymes. Some interactions mainly involved with function of P-gp are omitted, but may be mentioned. Studies in animals are generally excluded because of the marked species, which makes extrapolation of such results to humans difficult (European Medicines Agency, 2013[43]; Hermann and von Richter, 2012[78]). Herbal drug interactions caused by pharmacodynamic mechanisms including partly cover toxicities, while not less important, are outside the scope of this review article. Nevertheless, these have been reviewed elsewhere (Izzo and Ernst, 2009[87]; Johne and Roots, 2005[92]).

Echinacea

Echinacea is a genus of herbaceous flowering plants (purple coneflowers), and belongs to Asteraceae family. It comprises of nine species, which are native and endemic to eastern and central North America. Marketed preparations of Echinacea arise from underground as well as aerial parts of three different species (E. purpurea, E. angustifolia, E. pallida), whereby the majority (i.e., about 80 %) of the products are based on E. purpurea. Due to its immunostimulant properties, echinacea is commonly used for the prevention and treatment of acute virus infections of the upper respiratory tract, such as the common cold and influenza (Capasso et al., 2008[25]; Ernst et al., 2006[39], 2008[40]). Recommended daily doses of echinacea products vary broadly depending on the formulation and content of the product. Usually daily doses of echinacea for adults range between 900 to 1000 mg, to be taken in 3 to 4 divided doses.

Echinacea is considered to be one of the safest herbal medicines, with few reported adverse effects and very few reports of in vivo drug interactions. Echinacea seems to cause no serious risk for drug interactions in humans (Izzo, 2012[85]). No verifiable case reports of herbal drug interactions with any echinacea product have been published to date. The effect of echinacea (E. purpurea root) on specific CYP activities in humans was evaluated by using a single dose administration of the CYP probe drugs in 12 healthy subjects (Gorski et al., 2004[57]). These CYP probes included caffeine (CYP1A2), tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (hepatic and intestinal CYP3A4). The probe drugs were administered before and after a short course of Echinacea [E. purpurea root extract 400 mg, 4 times daily (= 1600 mg/day) for 8 days], and respective plasma concentration-time profiles of the probe drugs were determined. In this study, it was found that echinacea dosing significantly decreased the oral clearance of the CYP1A2 probe caffeine by 27 % (Gorski et al., 2004[57]), indicating inhibition of in vivo CYP1A2 catalytic activity. Theophylline is a widely prescribed CYP1A2 drug that has a narrow therapeutic window. Therefore modest changes of 20 % in clearance of theophylline are considered to be clinically significant (Gorski et al., 2004[57]). There was considerable inter-individual variability in this interaction, with 2 individuals (out of 12 subjects) presenting a greater than 50 % reduction in caffeine oral clearance (Gorski et al., 2004[57]). The co-administration of echinacea and theophylline may give rise to an increased incidence of toxicity developing from increased plasma theophylline concentrations. Other CYP1A2-metabolized drugs which have adverse effect concerns, such as clozapine, tacrine, and cyclobenzaprine, may be influenced by taking echinacea concomitantly (Gorski et al., 2004[57]).

Echinacea dosing also significantly reduced (average by 11 %) the oral clearance and enhanced the systemic exposure of tolbutamide (Gorski et al., 2004[57]). This indicates that the in vivo hepatic CYP2C9 activity was slightly inhibited by echinacea. However, the maximum plasma tolbutamide concentration was not significantly modified by echinacea. These results showed a minor change in the pharmacokinetics of tolbutamide which is mainly metabolized by CYP2C9. The observed magnitude of changes in tolbutamide pharmacokinetic parameters, though statistically significant, is most likely lack clinical importance with this echinacea product. Thus it suggests that co-administration of echinacea has no considerable effect on the activity of CYP2C9 enzyme (Gorski et al., 2004[57]). However, the co-administration of echinacea with warfarin, a substrate known to be metabolized by multiple CYP enzymes including CYP2C9 and CYP3A4 (Abernethy et al., 1991[2]; Rettie et al., 1992[151]), may result in variable responses from a decrease in efficacy due to CYP3A4 induction to toxicity as a result of CYP2C9 inhibition to no effect (offsetting changes). Gorski and co-workers (Gorski et al., 2004[57]) commented that the co-administration of other echinacea products that differ in phytochemical content to the brand they studied (E. purpurea root, Nature's Bounty) with CYP2C9 substrates having a narrow therapeutic index such as phenytoin and warfarin may require to be cautiously monitored.

In the same study, it was also shown that echinacea co-administration did not significantly affect the pharmacokinetic parameters (i.e., AUC0-∞, oral clearance, t1/2) of the CYP2D6 probe dextromethorphan (Gorski et al., 2004[57]). This suggested that co-administration of echinacea does not alter the metabolism and pharmacokinetics of drugs metabolized by CYP2D6.

The systemic clearance of intravenously administered CYP3A4 probe midazolam was significantly enhanced by 42 %, and there was a corresponding significant decrease in mean AUC0-∞ 23 % (Gorski et al., 2004[57]). Collectively it indicates that hepatic CYP3A4 activity was increased. On the other hand, the oral clearance and the AUC0-∞ after oral midazolam dosing were not significantly changed by echinacea co-administration. Nevertheless, the mean absolute oral bioavailability of midazolam was increased from 24 % to 36 % (i.e., by 50 %). This is consistent with the inhibition of intestinal CYP3A4 by echinacea. The distinction effects of echinacea on intestinal and hepatic CYP3A4 activity may possibly be due to a diversity of mechanisms. These include, for example, locally acting CYP3A4 inhibiting constituents of echinacea that do not become systemically available, or fast absorption of CYP3A4 inducing constituents of echinacea, thus limiting intestinal exposure and intestinal CYP3A4 induction (Gorski et al., 2004[57]). Alternatively, it may be due to a systemically formed metabolite of an echinacea’s constituent that is capable of inducing hepatic CYP3A4 but not intestinal CYP3A4. In spite of this, these mechanistic considerations remain hypothetical and warrant further research.

A further study by Gurley and colleagues (Gurley et al., 2004[57]) employed single-time point phenotypic metabolic ratios to determine whether long-term supplementation of Echinacea purpurea affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. This clinical study was performed in 12 healthy volunteers. They were randomly assigned to receive E. purpurea (800 mg twice a day, or 1600 mg daily) for 28 days. Probe drug cocktails of midazolam (CYP3A4) and caffeine (CYP1A2), followed 24 hours later by chlorzoxazone (CYP2E1) and debrisoquine (CYP2D6) were administered before (baseline) and at the end of echinacea supplementation. Activities of CYP3A4, CYP1A2, CYP2E1, and CYP2D6 enzymes during pre-supplementation and post-supplementation were assessed by use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquine urinary recovery ratios (8-hour collection), respectively. In this study, high-dose E. purpurea given over 28 days did not significantly change the activities of CYP3A4, CYP2E1, and CYP2D6 as estimated by single timepoint metabolic ratios. The only appreciable alteration in mean phenotypic ratios appeared with 6-hour paraxanthine/caffeine values. Co-administration of Echinacea purpurea caused an approximately 13 % decrease in the ratio of paraxanthine/caffeine, this suggested that there was a possible inhibitory effect of E. purpurea on CYP1A2 enzyme. This minor difference, however, was not statistically significant (P = 0.07), nor was it thought clinically relevant. This finding suggests that prolonged use of E. purpurea poses a minimal risk of producing CYP1A2-, CYP2D6-, CYP2E1-, or CYP-3A4-mediated herbal drug interactions in humans. In respect to the effects of E. purpurea extract on CYP3A4 and CYP2D6, the findings of Gurley et al. (2004[65]) are consistent with those of Gorski et al. (2004[57]). This consistency happened regardless of differences in the type of E. purpurea products used (root extract versus whole plant extract), duration of supplementation (8 days versus 28 days), and CYP phenotype assessment methodologies (AUC versus single-time point phenotypic ratio). The lack of or little effect of E. purpurea supplementation on human CYP2D6 was supported by a clinical study by Gurley et al. (2008[72]). The evidence has shown that supplementation of a standardized E. purpurea extract (267 mg three times a day = 801 mg/day, for 14 days) caused no significant inhibition of CYP2D6 in 16 healthy subjects using debrisoquine as the CYP2D6 probe.

Taken together, the study showed that an 8-day treatment with relatively high doses of E. purpurea root extract (i.e., 400 mg, four times daily = 1600 mg/day) displayed differential effects on the activity of various CYP enzymes. There were no alterations of CYP2D6 noted, negligible to modest inhibition of CYP2C9, modest inhibition of CYP1A2, and differential (i.e., inductive/inhibitory) effects on hepatic and intestinal CYP3A4.

Although the observed mean effects on CYP1A2 and CYP2C9 activities were generally moderate, they displayed some more pronounced effect sizes in individual subjects. Considering CYP-based Echinacea-drug interactions of potential clinical significance, the study suggests that the bioavailability of orally administered CYP3A4 substrates with low oral bioavailability may be significantly increased by Echinacea co-administration. Also the exposure to CYP1A2 and CYP2C9 substrates (e.g., theophylline, clozapine) may be modestly increased, at least in some individual subjects. The observed effects of high doses of E. purpurea root extract on CYP1A2 and CYP2C9, however, are overall small. Therefore, this is unlikely to be of clinical significance in clinical practice, unless drugs with a narrow therapeutic index are involved. These examples are theophylline in case of CYP1A2 and (S)-warfarin in case of CYP2C9.

A clinical study was conducted in 16 healthy volunteers received lopinavir-ritonavir (400/100 mg) twice daily for 30 days (Penzak et al., 2010[140]) to determine the effect of Echinacea purpurea on the pharmacokinetics of lopinavir-ritonavir, and on CYP3A4 and P-glycoprotein (P-gp) activity using the probe substrates midazolam, and fexofenadine, respectively. Echinacea purpurea extract was given in a dosage of 500 mg three times daily, which they continued for four weeks, the first two weeks in combination with lopinavir-ritonavir. The HIV protease inhibitor lopinavir is extensive metabolized by CYP3A4, whereas ritonavir is a potent inhibitor of CYP3A4 and P-gp. Ritonavir is combined with lopinavir to boost (i.e., increase) its systemic exposure. The results have shown that neither lopinavir nor ritonavir pharmacokinetics were significantly altered by 2 weeks of Echinacea co-administration (Penzak et al., 2010[140]). However, with respect to activity of CYP3A4, Echinacea co-administration considerably reduced AUC0–12h and increased oral clearance of midazolam, a CYP3A4 probe. These suggested that the Echinacea co-administration induced CYP3A4 enzyme activity and this in turn increased the clearance of midazolam (Penzak et al., 2010[140]). Thus co-administration of Echinacea purpurea may cause moderate decrease in plasma concentrations of other CYP3A4 substrates.

The above finding was also supported by a study conducted by Moltó et al. (2011[120]) in which they determined the potential of Echinacea purpurea to interact with the boosted protease inhibitor darunavir-ritonavir in 15 HIV-infected patients. Patients were receiving antiretroviral therapy including darunavir-ritonavir (600/100 mg twice a day) for at least 4 weeks. E. purpurea root extract capsules (500 mg every 6 h, for 14 days) were concomitantly given on top of their antiretroviral treatment. In general, supplementation of Echinacea purpurea did not affect the overall pharmacokinetics of darunavir and ritonavir, although individual patients did show a decrease in darunavir concentrations (Moltó et al., 2011[120]). While no dose adjustment is required, monitoring darunavir concentrations on an individual basis may give reassurance in this setting. Their findings with darunavir exposure was reduced in some individual patients by 30 to 40 % after E. purpurea supplementation, would be harmonious with the finding of a modest induction of hepatic CYP3A4 by echinacea administration previously reported by Gorski et al. (2004[57]). This evidence would be further consistent with the assumption that low-dose ritonavir (100 mg twice daily) might not efficiently inhibit hepatic CYP3A4 activity in individual patients (Hermann and von Richter, 2012[78]).

Overall, the outcomes from both studies (Moltó et al., 2011[120]; Penzak et al., 2010[140]) regarding the noticed changes in CYP3A4 activity is consistent for the effects of E. purpurea supplementation on a darunavir-ritonavir combination. These studies reliably suggest that E. purpurea co-administration is unlikely to considerably alter the pharmacokinetics of ritonavir-boosted protease inhibitors. This is most likely due to the presence of the potent CYP3A inhibitor ritonavir, but may be capable to moderately induce hepatic CYP3A activity.

Abdul and colleagues (Abdul et al., 2010[1]) studied the pharmacokinetic and pharmacodynamic interactions of Echinacea with warfarin in 12 healthy subjects who received a single oral 25 mg dose of warfarin alone and after 2 weeks of pre-treatment with high-dose Echinacea (1275 mg four times daily = 5100 mg/day). Pharmacodynamic parameters including the international normalized ratio (INR), platelet activity) and pharmacokinetic (warfarin enantiomer concentrations) end points were evaluated. The apparent oral clearance of (S)-warfarin was found to be significantly higher during concomitant treatment with echinacea but this did not lead to a clinically significant change in INR. Because (S)-warfarin is metabolized by CYP2C9 and CYP3A4, whereas (R)-warfarin is metabolized by CYP3A4 and CYP1A2 (Wittkowsky, 2003[188]), it was proposed that a 2-week treatment with high doses of Echinacea (i.e., 5100 mg/day) does not appreciably alter the activities of these CYP enzymes. Generally, the small extents of the effect observed are doubtful to be of clinical relevance.

In summary, the existing in vivo evidence reveals that echinacea products at recommended doses have little potential to produce clinically relevant or worrying metabolism-based pharmacokinetic interactions involving CYP1A2, CYP2C9, CYP2D6 and CYP2E1. Sequentially, there is reasonable clinical evidence suggesting that Echinacea products in fact have potential to moderately induce hepatic CYP3A4 activity. However, at the same time Echinacea products also may inhibit the pre-systemic (intestinal) metabolism of CYP3A4 drugs. As both mechanisms work against each other in terms of the net effects on the systemic exposure of CYP3A4 drugs, the onset of each mechanism probably occurs at different times (i.e., onset of inhibition occurs faster than onset of induction), and also will vary in their extent of effect on specific substrate characteristics (e.g., oral bioavailability of CYP3A4 drugs). General predictions on the clinical relevance of Echinacea and CYP3A4 drug interactions are complicated to generate. In spite of this, warning is advised when CYP3A4 drugs with low oral bioavailability due to pronounced intestinal CYP3A4-mediated metabolism (e.g. verapamil, cyclosporine A and tacrolimus) or CYP3A4 drugs with narrow therapeutic index, are co-administered with Echinacea supplementation. There are no clinical studies addressing the potential impact of Echinacea products on other important metabolic pathways such as CYP2C19 and phase II metabolism (e.g. glucuronidation).

Ginkgo biloba

Leaves from the tree Ginkgo (Ginkgo biloba; family Ginkgoaceae) have been used for 4000 years to improve mentation and respiratory function (Philp, 2004[142]). The Ginkgo tree, also known as maidenhair tree, is the surviving member of ancient family Ginkgoaceae, with no close living relatives. Ginkgo biloba is claimed to improve cerebral and peripheral blood flow. Currently available pharmaceutical products of Ginkgo biloba represent leave extracts. Ginkgo biloba extract contains two constituents: flavonoids and terpenoids, which have antioxidant properties (Kennedy et al., 2011[93]; Pierre et al., 2008[144]). Oral standardized dry extracts of Ginkgo biloba generally contain between 22-27 % flavones glycosides; 5-7 % terpene lactones, and should contain not more than 5 ppm of ginkgolic acids, constituents with known allergic potency (Kressmann et al., 2002[96]). Many studies have been conducted using EGb 761, a well-defined extract of Ginkgo biloba. Ginkgo biloba extract is widely used in some countries by patients with cognitive disorders such as memory decline with ageing and Alzheimer's disease. Early studies showed that the extract was superior to placebo in improving symptoms of dementia, and this has been confirmed by more recent research (Andrieu et al., 2003[4]; Ihl, 2012[82]). Standardized ginkgo biloba extract has a good safety profile, although some case reports have suggested an increased risk of bleeding.

Ginkgo biloba preparations may convene appreciable anti-platelet effects that are evidently caused by various ginkgolides. Consequently, information from case reports or controlled trials revealed that Ginkgo biloba extract potentiates the effects of anti-coagulant or anti-platelet drugs such as warfarin (Bone, 2008[17]; Vaes and Chyka, 2000[171]). However, these interactions are related to the pharmacodynamic interactions. While they are clinically important, the pharmacodynamic interactions are outside the scope of the current review on CYP mediated herbal drug interactions.

There were various studies showing the in vitro effects of Ginkgo biloba extracts or specific constituents to alter activities of CYP enzymes. However, these results were generally inconsistent. Overall, the obtained findings indicated no effect and either inhibition or induction of various human CYP enzymes. Some reports found the effects of Ginkgo biloba extracts on CYP are concentration-dependently (i.e. inhibition at low concentrations, induction at high concentrations). In some studies, a substrate-dependent fashion effects have been described. Of note, the results derived from in vitro studies have been obtained in part at very high concentrations of the herb (Ginkgo biloba). Such concentrations are unlikely to be achieved in humans (in vivo) after recommended doses of Ginkgo biloba extracts. This particular aspect is considered to be a major disadvantage of in vitro studies. Together, the buildup of evidence from in vitro studies offers little guidance in the reliable prediction of relevant metabolic-based Ginkgo biloba mediated drug interactions in vivo (Hermann and von Richter, 2012[78]).

Luckily, there are a number of clinical studies that have been carried out to determine the effects of Ginkgo biloba on many CYP isoforms and other drug metabolizing enzymes including phase II conjugations. These investigations mainly employed specific probe drugs as substrates for CYP enzymes in which it allows the identification and quantification of changes of specific CYP enzyme activity. Gurley et al. (2002[67]) employed single-time point phenotypic metabolic ratios to determine whether long-term supplementation of St. John’s wort, garlic oil, Panax ginseng, and Ginkgo biloba affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity in healthy young volunteers. Dosages of 60 mg Ginkgo biloba dietary supplements were given (four times daily) to 12 healthy subjects over 28 days. In this particular study, probe drug cocktails of caffeine, debrisoquine, chlorzoxazone, and midazolam were administered before and at the end of Ginkgo biloba supplementation. Activities of CYP1A2, 2D6, 2E1, and 3A4 were assessed by the use of paraxanthine/caffeine serum ratios (6-hour sample), debrisoquine urinary recovery ratios (8-hour collection), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), respectively. Comparisons of pre-treatment and post-treatment ratios revealed that Ginkgo biloba did not cause any appreciable change of CYP1A2, 2D6 and 3A4 activity. In contrast, Ginkgo biloba appeared to moderately increase the activity of CYP2E1 by 23 %, even though this effect was not statistically significant (Gurley et al., 2002[67]). This suggests a trend of CYP2E1 is being induced by Ginkgo biloba. A similar study was conducted by the same group of researchers (Gurley et al., 2005[66]) but in 12 elderly subjects, using the same Ginkgo biloba product, dose and treatment duration, as well as the same CYP probe drugs. Surprisingly, Ginkgo biloba had no significant effect on the activity of CYP1A2, 2D6, 2E1, and 3A4 in this elderly subjects. Thus these results do not confirm the earlier observed trend regarding a moderate CYP2E1 induction (Gurley et al., 2005[66]).

Lack of effect of Ginkgo biloba on the activity of CYP2D6 and CYP3A4 was supported by the evidence derived from a clinical study conducted by Markowitz and colleagues (Markowitz et al., 2003[115]). This study was similar in nature to those previously conducted by Gurley et al. (2005[66]). However, they assessed the influence of standardized Ginkgo biloba extract on the activity of CYP2D6 and CYP3A4 in healthy young adults, instead of elderly volunteers as used in Gurley and co-workers’ study (Gurley et al., 2005[66]). In the trial by Markowitz et al. (2003[115]), dextromethorphan (CYP2D6 activity) and alprazolam (CYP3A4 activity) were employed as specific substrates for the CYPs of interest. These probe drugs were co-administered orally at baseline, and following treatment with Ginkgo biloba extract (120 mg twice a day) for 14 days. There were no statistically significant differences between baseline and post-Ginkgo biloba treatment on dextromethorphan metabolic ratios. This indicated a lack of Ginkgo biloba effect on CYP2D6 activity. Regarding the CYP3A4 activity, a statistically significant decrease in the AUC of alprazolam was detected after treatment with Ginkgo biloba. The AUCs were decreased by 17 %, an amount that could be clinically significant for drugs with narrow therapeutic indices. However, when the effects of Ginkgo biloba are compared with synthetic medications known to induce alprazolam metabolism such as carbamazepine and rifampin, the magnitude of Ginkgo biloba effects were quite small (Markowitz et al., 2003[115]). Therefore, the authors concluded that standardized extracts of Ginkgo biloba, when taken in normally recommended doses, do not significantly alter the activity of human CYP2D6 and CYP3A4 enzymes. Thus, there appears to be little likelihood of significant herbal drug interactions between Ginkgo biloba and drugs predominantly metabolized by CYP2D6 or CYP3A4 isoforms (Markowitz et al., 2003[115]).

In addition, the effects of Ginkgo biloba supplementation (90 mg/day for 30 days) on the steady-state plasma concentration of donepezil were examined (Yasui-Furukori et al., 2004[194]) in elderly patients with Alzheimer's disease; the patients received donepezil 5 mg/day. Donepezil, the 'first-line' cholinesterase inhibitor, in the treatment of Alzheimer's disease, is metabolized by CYP2D6 and CYP3A4 (Jann et al., 2002[89]). The results from this study demonstrated that taking relatively low doses of Ginkgo biloba (90 mg/day) in elderly Alzheimer's Japanese patients did not alter the steady-state plasma concentrations of donepezil. This implies that daily doses of 90 mg of Ginkgo biloba do not have significant inhibitory or inducing effects on the CYP2D6- and CYP3A4-mediated metabolism of donepezil in the target population. This finding supports previous studies by Gurley et al. (2005[66]) and Markowitz et al. (2003[115]) in regard to the lack of interaction between Ginkgo biloba and drugs that are metabolized by CYP2D6 or CYP3A4.

Robertson and coworkers (Robertson et al., 2008[153]) also used a similar methodology to determine whether Ginkgo biloba extract causes any alterations in the activity of human CYP3A4. Midazolam was employed as a probe drug for human CYP3A4. Besides midazolam, the investigators also used fexofenadine as a marker drug to monitor the influence of Ginkgo biloba on the activity of drug transporter protein, P-glycoprotein (P-gp). Effect of Ginkgo biloba extract on pharmacokinetics of midazolam and fexofenadine (after a single dose), and lopinavir’s pharmacokinetics at steady-state, in healthy subjects was assessed before and after the subjects received Ginkgo biloba at a dose of 120 mg, two times daily (for 4 weeks). Lopinavir, ritonavir, and fexofenadine exposures were not significantly affected by Ginkgo biloba co-administration, while total AUC and maximum plasma drug concentration (Cmax) of the CYP3A4 substrate midazolam were both significantly lower post Ginkgo biloba supplementation, i.e. by 34 % and 31 %, respectively, as compared to baseline. The apparent oral clearance of midazolam increased in 10 of 13 subjects studied post Ginkgo biloba supplementation. These results suggest that Ginkgo biloba moderately induces CYP3A4 metabolism. The authors commended that as the reductions in midazolam AUC and Cmax were similar, with no change in half-life of midazolam, it appears likely that the interaction occurred secondary to CYP3A4 induction at the intestinal level (Robertson et al., 2008[153]). However, it cannot be ruled out that hepatic induction of CYP3A4 may also have contributed. These findings obtained from the study of Robertson et al. (2008[153]) are in contrast with those previously reported by Gurley et al. (2005[66]) who found that 28 days of Ginkgo biloba extract (60 mg, four times daily) had no apparent effect on midazolam metabolism in 12 healthy subjects. Robertson and co-authors (Robertson et al., 2008[153]) thought that it is unlikely that the disparity in their findings is due to differences in Ginkgo biloba extract formulation, as the total daily dose of Ginkgo biloba (240 mg/day) was the same in both studies and both products were standardized to the same flavonol glycoside and terpene lactone contents. To our knowledge, as herbal dietary supplements generally have problems with quality control aspects, including Ginkgo biloba (Draves and Walker, 2003[34]; Foster et al., 2005[45]; Haller et al., 2004[76]), thus the differences in the content of active ingredients, dissolution and absorption characteristics of the formulation used cannot be excluded.

However, the two studies were different in their approach to assessing CYP3A4 phenotype. Robertson and colleagues (Robertson et al., 2008[153]) assessed CYP3A4 activity by determining midazolam AUC before and after 4 weeks of Ginkgo biloba extract administration. Whereas, in Gurley et al. (2005[66]) investigation, a 1-h post-dose plasma concentration ratio of 1-hydroxymidazolam/midazolam was used to assess human CYP3A4 activity. Use of this ratio to determine human CYP3A4 activity has produced inconsistent results in previous studies, perhaps caused by significant interpatient heterogeneity in the glucuronidation of 1-hydroxymidazolam. This can alter the 1-hydroxymidazolam/midazolam ratio independent of CYP3A4-mediated metabolism (Eap et al., 2004[36]; Rogers et al., 2002[156]; Streetman et al., 2000[164]). Therefore, it has been suggested that using midazolam concentrations alone, as opposed to 1-hydroxymidazolam ratios, offers a more accurate assessment of CYP3A4 activity (Nafziger and Bertino, 2007[129]; Penzak et al., 2008[139]).

Ginkgo biloba extract is also widely used as herbal dietary supplement in Japan. The effects of Ginkgo biloba extract on the pharmacokinetics and pharmacodynamics of nifedipine, a calcium-channel blocker, were studied using 8 healthy volunteers. Concurrent oral ingestion of Ginkgo biloba extract (240 mg/day) did not significantly affect any of the mean pharmacokinetic parameters of either nifedipine or its major metabolite dihydronifedipine (Yoshioka et al., 2004[197]). However, unexpected observation was found in which the maximal plasma nifedipine concentrations in 2 subjects were approximately doubled during these subjects were taking Ginkgo biloba extract concomitantly with nifedipine. The authors also observed that these 2 subjects had experienced more severe and longer-lasting headaches during Ginkgo biloba extract phase than the control phase (Yoshioka et al., 2004[197]). The mean heart rate after oral administration of nifedipine with Ginkgo biloba extract had a tendency to be faster than that without Ginkgo biloba extract at every time point. The adverse drug reactions observed were coincident with the abnormally high maximum plasma nifedipine concentrations in these particular two subjects. From this it was concluded that Ginkgo biloba extract and nifedipine should not be simultaneously administered, and careful monitoring is necessary when nifedipine and Ginkgo biloba extract need to be taken together in humans (Yoshioka et al., 2004[197]). Although the authors emphasized the finding of abnormally increased maximal plasma nifedipine concentrations in two subjects upon co-administration of Ginkgo biloba extract, they also discussed possible underlying mechanisms. Nifedipine has a well recognized pharmacokinetic variability this together with the overall study results implies the absence of any Ginkgo biloba effects in respect of the total nifedipine exposure (i.e., AUC). This supports the hypothesis that there is no systematic effect due to Ginkgo biloba on the bioavailability of nifedipine. As nifedipine is an antihypertensive drug known to be primarily metabolized by CYP3A4 (Bailey et al., 2013[8]; Nebert and Russell, 2002[132]), this interpretation would be in agreement with previous reports from other investigators showing lack of effect of Ginkgo biloba on metabolism and pharmacokinetics of CYP3A4 metabolized drugs.

Effects of a higher daily dose (360 mg/ day, for 28 days) of Ginkgo biloba extract on CYP3A4 and CYP2C9, were evaluated (Uchida et al., 2006[169]). CYP3A4 probe (midazolam) and CYP2C9 probe (tolbutamide) were orally administered as a single dose to 10 healthy volunteers before and after intake of Ginkgo biloba extract. AUC(0-infinity) for midazolam was significantly increased (25 %) by Ginkgo biloba extract intake and oral clearance was significantly decreased (26 %). These results suggested that Ginkgo biloba extract may inhibit the activity of CYP3A4, therefore it increased exposure to drugs cleared by CYP3A4 (Uchida et al., 2006[169]). Furthermore, these researchers examined the potential interaction between Ginkgo biloba extract and CYP2C9-metabolized drugs using tolbutamide as a CYP2C9 probe. Their results have shown that the AUC(0-infinity) for tolbutamide after Ginkgo biloba extract intake was slightly but significantly (16 %) lower than that before Ginkgo biloba extract intake. Co-administration of Ginkgo biloba extract with tolbutamide tended to reduce AUC(0-2h) of blood glucose-lowering effect of tolbutamide. Therefore, the authors suggested that the combination of Ginkgo biloba extract and drugs should be cautiously in terms of the potential interactions, especially in elderly patients or patients treated with drugs exerting relatively narrow therapeutic windows (Uchida et al., 2006[169]). When considering the magnitude of changes in pharmacokinetic parameters of CYP3A4 and CYP2C9-metabolized drugs caused by taking Ginkgo biloba extract concomitantly with the drugs, it revealed a minor alteration in the activities of these CYP enzymes of interest. This minor CYP2C9 induction and some inhibition of intestinal CYP3A4 were achieved with supra-therapeutic doses (360 mg/day) of Ginkgo biloba extract. The study results of drug interactions with Ginkgo biloba extract on CYP3A4- and CYP2C9 metabolically cleared drugs show minimal effect due to Ginkgo biloba extract (Hermann and von Richter, 2012[78]). Thus, this is in agreement with reports from other studies that suggested there was no effect by Ginkgo biloba extract on the activity of these enzymes at recommended doses.

Zuo et al. (2010[209]) employed diazepam as a probe substrate of CYP2C19 and CYP3A4 to determine the effects of Ginkgo biloba extract on the pharmacokinetics of diazepam. A single oral dose (10 mg) of diazepam was given either alone or concomitantly with oral Ginkgo biloba extract (120 mg twice daily) for 28 days to 12 healthy volunteers. They found that total AUC for diazepam and the main metabolite N-desmethyldiazepam were essentially unaltered. This data indicate that the disposition of CYP2C19 and CYP3A4 substrates is unlikely to be markedly modified by re¬commended doses of Ginkgo biloba products (Zuo et al., 2010[209]).

The effect of Ginkgo biloba extract on the pharmacokinetics and pharmacodynamics of warfarin was investigated in 12 healthy subjects (Jiang et al., 2005[90]). A single 25-mg dose of warfarin (Coumadin™) was given to each subject with and without pretreatment with multiple doses of Ginkgo biloba for 1 week (Tavonin™; 40 mg three times a day = 120 mg/day). Dosing of Ginkgo biloba was continued for a further week after warfarin administration. S-warfarin is mainly metabolized to S-7-hydroxywarfarin by CYP2C9, and R-warfarin is metabolized by CYP1A2 and CYP3A4. Using warfarin as a probe substrate, thereby allows for a separate mechanistic assessment of any potential change of these metabolic pathways by concomitant Ginkgo biloba treatment. Co-administration of recommended doses of a commonly used herbal supplement, Ginkgo biloba did not affect the pharmacokinetics and the pharmacodynamics of warfarin enantiomers after a single dose of warfarin in healthy male subjects (Jiang et al., 2005[90]). Also, Ginkgo biloba did not affect blood clotting status or platelet aggregation.

In addition, the effect of Ginkgo biloba on the activity of CYP2C9, the isoform responsible for S-warfarin clearance, was assessed in 11 healthy volunteers who received single 100-mg doses of the non-steroidal anti-inflammatory drug (NSAID) flurbiprofen, a probe substrate for CYP2C9 (Greenblatt et al., 2006[60]). Subjects also received either a standardized Ginkgo biloba leaf preparation (Ginkgold, 3 doses of 120 mg) or matching placebo in a randomized crossover study. The study showed that pretreatment of healthy subjects with usual clinical doses of Ginkgo biloba has no detectable effect on the pharmacokinetics of a single dose of flurbiprofen or on the apparent extent of formation of the principal hydroxylated metabolite. The findings suggest that short-term exposure to Ginkgo biloba does not inhibit CYP2C9 activity in vivo (Greenblatt et al., 2006[60]). As the study involved only short-term exposure to Ginkgo biloba, it was not intended to capture possible CYP2C9 induction that could occur with long-term treatment. The results confirm previous controlled clinical studies showing no effect of ginkgo on the pharmacokinetics or pharmacodynamics of warfarin which is metabolized by CYP2C9. This finding is also supported by an in vivo study conducted by Mohutsky et al. (2006[119]). They carried out two pharmacokinetic studies in healthy subjects using tolbutamide and diclofenac as probe substrates of CYP2C9. They found there were no interactions between Ginkgo biloba extract and these CYP2C9 probe substrates in vivo as demonstrated by the lack of effect on the steady-state pharmacokinetics of diclofenac and the urinary metabolic ratio of tolbutamide (Mohutsky et al., 2006[119]). This evidence seems to be contradictory to a number of case reports having documented possible interactions between Ginkgo biloba and warfarin (Fugh-Berman and Ernst, 2001[48]; Izzo and Ernst, 2001[86]). Such an interaction is mostly relevant to elderly patients on anticoagulant therapy. However, this interaction appears to be attributable to the inhibition of platelet activating factor by various ginkgolides (Zhu et al., 1999[207]). That is mediated by a pharmacodynamic mechanism. Explanation by a CYP-mediated metabolism for the Ginkgo biloba and warfarin interaction looks less possible based on the clinical data described above (Gurley et al., 2002[67]; Jiang et al., 2005[90]).

Yin and coworkers (Yin et al., 2004[195]) investigated the potential herbal drug interaction between Ginkgo biloba and omeprazole, a widely used CYP2C19 substrate, in subjects with different CYP2C19 genotypes. Eighteen healthy Chinese subjects previously genotyped for CYP2C19 were studied. All subjects received a single omeprazole 40 mg at baseline and then at the end of a 12-day treatment period with Ginkgo biloba (140 mg, twice a day). Multiple blood samples were collected over 12 h, and 24 h urine was collected post omeprazole dosing. Plasma concentrations of omeprazole and its metabolite omeprazole sulfone were significantly decreased, and 5-hydroxyomeprazole significantly increased following Ginkgo biloba administration in comparison to baseline. A significant decrease in the ratio of AUC of omeprazole to 5-hydroxyomeprazole was observed in the homozygous extensive metabolizers, heterozygous extensive metabolizers, and poor metabolizers, respectively. The decrease was greater in poor metabolizers than extensive metabolizers. No significant changes in the AUC ratios of omeprazole to omeprazole sulfone were observed. Renal clearance of 5-hydroxyomeprazole was significantly decreased after Ginkgo biloba, but the change was not significantly different among the three genotype groups. Their results show that Ginkgo biloba can induce omeprazole hydroxylation in a CYP2C19 genotype-dependent manner and concurrently reduce the renal clearance of 5-hydroxyomeprazole. Co-administration of Ginkgo biloba with omeprazole or other CYP2C19 substrates may significantly reduce their effects, but further studies are warranted (Yin et al., 2004[195]).

Possible effects of Ginkgo biloba as an inducer of CYP2C19 on single-dose pharmacokinetics of voriconazole were examined in 14 Chinese volunteers genotyped as either CYP2C19 extensive or poor metabolizers (Lei et al., 2009[101]). Pharmacokinetics of oral voriconazole 200 mg after administration of Ginkgo biloba 120 mg twice daily for 12 days were determined for up to 24 hours in a 2-phase randomized crossover study. For extensive metabolizers, the median value for voriconazole’s AUC(0 to infinity) after administration of voriconazole alone was not significantly different from that after voriconazole with Ginkgo biloba (P > 0.05). The other pharmacokinetic parameters of voriconazole such as time to reach maximum concentration, half-life, and apparent clearance also did not change significantly for extensive metabolizers in the presence of Ginkgo biloba. Pharmacokinetic parameters followed a similar pattern for poor metabolizers. The authors concluded that 12 days of treatment with Ginkgo biloba did not significantly alter the pharmacokinetics of voriconazole in either CYP2C19 extensive or poor metabolizers. Thus, the pharmacokinetic interactions between voriconazole and Ginkgo biloba may have limited clinical significance. Based on these results, it can be assumed that - even at the highest recommended doses - Ginkgo biloba-mediated CYP2C19-based pharmacokinetic drug interactions appear to be light and may have restricted clinical significance.

In recent study, cocktail phenotyping design was used to evaluate the metabolic drug interaction profile of Ginkgo biloba extract EGb 761® with respect to the activities of major CYP enzymes (Zadoyan et al., 2012[199]). These included CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP-3A4. In random order, the following pretreatments were administered to 18 healthy men and women for 8 days each: placebo twice daily, EGb 761® 120 mg twice daily, and EGb 761® 240 mg in the morning and placebo in the evening. The phenotyping cocktail was orally administered before and after the EGb 761®/placebo pretreatment periods. CYP probe drugs and metrics used were: tolbutamide (CYP2C9, plasma concentration 24-h postdose), omeprazole (CYP2C19, omeprazole/5-hydroxy omepra-zole plasma ratio 3-h postdose), dextromethorphan (CYP2D6, dextromethorphan/ dextrorphan plasma ratio 3-h postdose), and midazolam (CYP3A4, plasma concentration 6-h postdose). EGb 761®/placebo ratios for phenotyping metrics were close to unity for all CYP enzymes studied, except for CYP2C9, which may suggest a weak trend towards induction of these CYP2C family enzymes (Zadoyan et al., 2012[199]). Their data obviously show that even a relatively high dose of 240 mg daily doses of EGb 761® extract, it has no inhibitory effect towards CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, irrespective whether the product is given once or twice daily (Zadoyan et al., 2012[199]). The study also demonstrates that EGb 761® extract does not induce the activity of human CYP1A2, CYP2D6, and CYP3A4 enzymes. Supplementation of Ginkgo biloba (EGb 761® extract) may present a weak induction of CYP2C9 and CYP2C19 enzymes, which, however, seems very small to be of clinical relevance (Hermann and von Richter, 2012[78]; Zadoyan et al., 2012[199]).

Together, the existing evidence reveals that taking Ginkgo biloba extract at recommended doses up to daily doses of 240 mg do not have substantial or clinically meaningful effects on the activity of human CYP enzymes including CYP1A2, CYP-2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Apparently, CYP2C19 enzyme was shown to be moderately inducible by Ginkgo biloba extract products. This effect appears to occur at the highest recommended dose of level of 240 mg/day (Lei et al., 2009[100]). Also the induction effect on CYP2C19 may become somewhat more prominent with increasing doses of Ginkgo biloba extract (Yin et al., 2004[195]). Furthermore the extent of this induction effect might depend on the individual CYP2C19 genotype of subjects (Yin et al., 2004[195]) and possibly on characteristics of that particular CYP substrate. The existing evidence on this issue appears not to be consistent and further investigation is warranted.

Findings on pharmacokinetic alterations of CYP3A4 due to taking Ginkgo biloba concomitantly are not entirely consistent. Some studies suggested the possibility of presystemic induction of CYP3A4 with a high-dose treatment of Ginkgo biloba, whereas some data suggest CYP3A4 inhibition. For example, a study by Robertson et al. (2008[153]) showed a significant induction effect of Ginkgo biloba extract on CYP3A4 enzyme, but the extent of enzyme induction (approximately 30 % reduction in AUC of midazolam) was relatively small as compared to a large inter-individual variation in CYP3A4 activity observed in humans (Watkins, 1994[182]; Wrighton et al., 1993[189]; Zhang et al., 1997[200]). Thus the overall extent of CYP3A4-related effects produced by Ginkgo biloba are usually weak and doubtful to be of clinical relevance, unless for CYP3A4 drugs with a narrow therapeutic index are involved.

The evidence described previously also suggests that standard daily dose of Ginkgo biloba extract has no appreciable effect on the activity of two important drug metabolizing enzymes, human CYP3A4 and CYP2D6. Human CYP2D6 enzyme is involved in the metabolism of approximately 25 % of drugs on the market (Zhou, 2009[205]). It metabolizes several clinically used drugs such as antidepressants (imipramine, desipramine and fluoxetine), cardiovascular drugs especially antiarrhythmic drugs (encainide, flecainide), opioid analgesics (morphine, codeine and tramadol) in which some have a narrow therapeutic index. Also approximately 50 % of clinically used drugs are metabolized by human CYP3A4. Therefore, lack of drug interaction (via CYP metabolism) between majority of clinically used drugs metabolized by CYP2D6 and CYP3A4, and Ginkgo biloba provides a safety for patients who currently take this herbal supplement.

Ginkgo biloba happen to be at this time, the second best studied natural health product, in regards to the clinical investigation of its metabolic and transporter-based pharmacokinetic drug interaction potential (Hermann and von Richter, 2012[78]). The available array of published mechanistic herbal drug interaction studies permits reasonable interpretations on the particular drug metabolizing enzyme and transporter effects of Ginkgo biloba products in vivo. The information derived from these studies is largely consistent, with some minor disagreements that may be accounted for differences in study design, population studied, dose size, treatment durations, and perhaps differences in the composition of products investigated (Hermann and von Richter, 2012[78]).

St. John’s wort

St. John's wort (Hypericum perforatum L.; family Clusiaceae) is a popular medicinal herb used for the treatment of depression. Hypericum perforatum is a yellow-flowering, perennial herb native to Europe which has been introduced to many temperate areas of the world and grows wild in many meadows (Gurley et al., 2012[64]). The common name comes from its traditional flowering and harvesting on 24th June, the birthday of John the Baptist (St. John's Day). St. John's wort is the most studied botanical dietary supplement in the world. With more than 2000 peer-reviewed articles published on its safety and efficacy (Gurley et al., 2012[64]). Many clinical trials have shown antidepressive efficacy of St. John's wort superior to placebo and comparable to standard antidepressants but with fewer side effects than conventional antidepressive agents (Linde et al., 2008[104]). When used as a single agent, a risk/benefit ratio has made St. John's wort one of the most readily consumed dietary supplements in the world.

St. John's wort affects the pharmacokinetics of several drugs by inducing CYP isozymes, such as CYP3A4, CYP2C19, CYP2C9, and the drug transporter protein P-gp. This causes St. John's wort to be a highly problematic botanical with regard to CYP-mediated herbal drug interactions (Ang-Lee et al., 2001[5]; Brazier and Levine, 2003[21]; Gurley and Hagan, 2003[69]; Izzo and Ernst, 2001[86]). St. John’s wort contains numerous pharmacologically active compounds, including hyperforin, hypericin, pseudohypericin, and flavonoids (e.g., quercetin, quercitrin and I3,II8-biapigenin). Hyperforin, the principal mediator of St. John’s wort antidepressive action, and is the main reason for St. John’s wort herbal drug interaction potential. Hyperforin is a good ligand for the pregnane xenobiotic receptor (PXR) and thus acts as a potent inducer of CYP3A4 and P-gp, the gene product of MDR1 (Moore et al., 2000[122]; Watkins et al., 2003[183]). According to one estimate, hyperforin is the most potent PXR activator discovered to date. The clinical severity of St. John’s wort-drug interactions was first documented in 1999-2000 (Gurley et al., 2005[66]). At that time, numerous clinics around the world reported that concomitant use of St. John’s wort and cyclosporine A produced remarkable reductions in blood levels of the immunosuppressant among organ transplant recipients resulting in graft rejection. Since that time, an overabundance of clinical studies has investigated the effect of St. John’s wort on the pharmacokinetics of several medications (Gurley et al., 2005[66]).

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