Cytochrome P450 enzymes (CYP) play a pivot role in phase I of xenobiotic biotransformation. Many of CYP enzymes are known to be polymorphic, with many allelic variants. As a consequence of huge number of possible allele combinations, a wide range of metabolic activity can be observed among patients in population. Individual CYP activity may be also affected by various xenobiotics – either induction or inhibition of distinct CYP isoenzyme may cause failure or toxicity of pharmacotherapy. Due to this, some prediction of metabolic activity is worthwhile in clinical practice. This chapter is going to briefly summarize the possibilities of
2. In vivoenzyme activity assessment
2.1. Clinical need for CYP phenotyping
It is useful to assess CYP metabolic activity prior to or during the pharmacotherapy and to adjust the individual dosage according to the patient's phenotype at least in some cases.
The best studied CYP enzyme concerning polymorphisms, enzyme inhibition and dosage individualization according to phenotype is probably CYP2D6, followed by CYP2C9 and CYP2C19. These enzymes are highly polymorphic, contrary to CYP3A4, where the metabolic activity may vary due to differences in CYP3A4 gene expression and pharmacokinetic interactions [Ingelman-Sundberg 2004]. Nevertheless, any shift in metabolic capacity of individual CYP enzyme may result either in decreased or increased therapeutic response or intensity of adverse effects. For example, after paroxetine administration to the patients on tamoxifen, a decrease in plasma levels of active metabolite of tamoxifen was detected [Stearns et al. 2003]. This can be crucially important in breast cancer treatment. On the other hand, CYP2D6 ultrarapid metabolizer phenotype may cause failure of pharmacotherapy due to the very low and thus ineffective drug plasma levels [Corruble 2008]. In antipsychotic treatment, an association has been observed between extrapyramidal adverse effects and CYP2D6 genotype [Fleeman et al. 2011]. Significant clinical consequences of CYP2D6 genotype or enzyme inhibition were described also in beta-blockers, antianginal, antiarrythmic drugs, antihistamines and antiemetics. The clinical impact of enzyme polymorphism or changes due to inhibition or induction usually depends on the contribution of other CYP forms to the total drug's elimination. By this, the relative therapeutic potency of the parent drug or any of its metabolites may be altered [Zhou 2009].
Methodological approaches such as assessment of metabolic ratio of specific substrate to metabolite(s) in saliva, plasma / serum or urine are most widely used to assess metabolic activity
It is essentially important to differentiate between
One of the greatest advantages of genotype assessment is that it does not need to be repeated because it does not change with time or under the simultaneous influences of drugs and other factors. On the other side the disadvantage of the pharmacogenetic testing is, that genotype does not always correlate with observed metabolic activity recorded using probe drug(s). This discrepancy may be caused by various epigenetic factors as well as by inhibition or induction of enzyme metabolic activity caused by other xenobiotics coadministered.
2.2. Conventional probe substrates for
in vivometabolic activity assessment
Metabolic activity of various CYP enzymes is most often assessed using selective substrate of distinct CYP enzyme ("marker of metabolic activity"), i.e. a drug (or substance) which is ideally metabolized by the single CYP enzyme [Pelkonen et al. 1998, Pelkonen et al. 2008]. Ideal marker should be a non-toxic substance, with regard to its possible usage
The above mentioned principles are also utilized in drug development, where drugs are evaluated concerning their CYP-mediated interactions prior to the registration and launch on the basis of the enzyme-specific reaction [Zlokarnik et al. 2005].
Many substrates have been tried for CYP1A2 metabolic activity assessment, but caffeine is the most widely used one, although other enzymes are involved in biotransformation of caffeine and its metabolites (xanthinoxidase, N-acetyl transferase and with lesser extent also CYP2E1, CYP2A6, CYP3A4, CYP3A5) [Dorne et al. 2001, Tassaneeyakul et al. 1994]. Many metabolic ratios were used in phenotyping studies, with "caffeine metabolic ratio" (CMR) as the well determined marker of CYP1A2 metabolic activity [Hakooz 2009]. On the other hand, simple paraxanthine to caffeine molar concentration ratio assessed in serum/plasma or saliva is also used [van Troostwijk et al. 2003]. Other substrates are used much rarely in phenotyping studies. Probes for CYP1A2 metabolic activity assessment are summarized in Table 1. Moreover, various concentration ratios of caffeine and its metabolites are also used for determination of NAT2 (arylamine N-acetyltransferase), CYP2A6 and xanthinoxidase metabolic activities [Begas et al. 2007, Hakooz 2009, Nyeki et al. 2001].
CYP2C9 is known to be polymorphic, with more than 40 alleles identified [Ingelman-Sundberg et al. 2011]. Ten years ago it was thought that with CYP2C9 genotyping it will be possible to avoid adverse reactions in patients receiving warfarin [Ma et al. 2002]. These expectations were mostly calmed by reality, that CYP2C19 genotype is not the single major factor influencing warfarin toxicity.
Most often used CYP2C9-specific reactions are summarized in the Table 2. Diclofenac 4’-hydroxylation and tolbutamide methylhydroxylation [Zhou et al. 2009] seem to be the most frequently used, however, tolbutamide methylhydroxylation is also catalyzed by CYP2C19 [Wester et al. 2000]. Phenotyping with the use of substrates with low therapeutic index (phenytoin, oral anticoagulants) would be unsafe since the enzyme activity in CYP2C9*3 homozygotes is nearly absent, and these subjects could suffer from drug toxicities or adverse drug reactions [Zhou et al. 2009].
Folowing CYP2D6 and CYP2C9, CYP2C19 is the third well investigated polymorphic drug-metabolizing enzyme. CYP2C19 metabolizes many psychoactive drugs. Many of them are able to inhibit CYP2C19 metabolic activity. Probes for CYP2C19 metabolic activity assessment are summarized in Table 3.
|chloroguanine(proguanil)||conversion to cycloguanine|
Suitable substrates for
CYP3A4 is the predominant enzyme of CYP3A subfamily and plays the pivot role in drug metabolism [Guengrich 2005]. Hepatic CYP3A4 metabolizes about 50 % of clinically used drugs [Guengrich 1999] and CYP3A4 is the most abundant intestinal CYP enzyme [Guengerich 2005]. CYP3A4 is not supposed to be polymorphic as e.g. CYP2D6, but CYP3A4 activity may vary among individuals from 5 up to 50 fold [Ma et al. 2002]. Metabolic activity of CYP3A4 (expressed as log MR) has unimodal distribution; variation is probably a consequence of both genetic and non-genetic factors [Guengrich 1999, Ozdemir et al. 2000, Shimada et al. 1994, Wilkinson 1996]. Common probes for CYP3A4 metabolic ativity assessment are listed in the Table 5.
Since none of suggested probes for CYP3A4 phenotyping is metabolized uniquely by this enzyme, and the active site of CYP3A4 is thought to be large and able to bind multiple substrates simultaneously, it is recommended to use at least 2 structurally unrelated probe substrates for precise enzyme activity evaluation [Ekroos & Sjogren 2006, Foti et al. 2010, Liu et al. 2007]. The apparent metabolic activity of CYP3A4 (assessed using probe substrates) may be affected by P-glycoprotein mediated decrease of availability of probe substrates [Guengrich 2005].
By this, some authors suggest that there are no useful CYP3A4 substrates for accurate prediction of its metabolic activity [Benet 2005].
2.3. Differentiation between poor and extensive metabolizers: MR cut-off values
Metabolic ratio of AFMU/(AFMU +1U +1X) with an apparent antimode at 0.25 may serve for NAT2 phenotyping - subjects with metabolic ratios < 0.25 were then classified as slow acetylators and those with metabolic ratios > 0.25 as fast acetylators [Begas et al. 2007, Bendriss et al. 2000]. Some other authors suggest and use antimode of 0.34 of the same ratio [Nyeki et al. 2001, Tang et al. 1991].
Total salivary caffeine assessment (TOSCA) as a measure of general liver function was used for identification of patients with liver cirrhosis, with a cut-off value of 4.2 μg/ml, but TOSCA was not used for CYP1A2 phenotyping [Tarantino et al. 2006].
137X= 1,3,7 trimethylxanthine or caffeine
17X= 1,7-dimethylxanthine or paraxanthine
1U= 1-methyluric acid
17U= 1,7-dimethyluric acid
There is a lack of evidence for existence of a bimodal distribution of CYP3A4 activity and therefore there is no consensus for antimode or cut-off values in any of used probe substrates.
2.4. Breath tests and other approaches for CYP enzyme activity assessment
Erythromycine breath test (EBT) is probably one of the best proved models for CYP3A4 activity assessment. The hepatic CYP3A4 catalyzes
Similar breath tests detecting radiolabeled metabolites (or ratio of 14CO2 or 13CO2 to 12CO2) were designed for phynotyping CYP2C19 with use of [13C] pantoprazole [Furuta et al. 2009], and CYP1A2 with the use of [14C] caffeine [Kalow & Tang 1993]. Multiple CYP metabolic activities (CYP2C19, 1A2, 3A4, and 2C9) were evaluated with [13C]- aminophenazone ("aminopyrine breath test") [Kodaira et al. 2011]. CYP2E1 activity in rats was measured using [14C] nitrosodimethylamine as a probe substrate [Bastien & Villeneuve 1998].
2.5. Miotic response to opioids
Significant correlations were observed between alfentanil pharmacokinetic parameters and its induced miotic kinetic parameters either under ambient or dark conditions. This means that pupillary response after alfentanil administration may be used as noninvasive measure of CYP3A4 metabolic activity [Baririan et al. 2005, Kharasch et al. 2004, Klees et al. 2005].
2.6. Cocktail approaches
In early phase of drug development, there are utilized so called "high-throughput" methods increasing the efficiency and effectiveness of assay to assess metabolic activity of many CYP enzymes in short time [Smith et al. 2007, Testino & Patonay 2003, Zlokarnik et al. 2005]. Investigative methodologies for CYP enzymes often utilize so-called "cocktail" of markers to assess the metabolic activity of multiple CYP forms in one session [Asimus et al. 2007, Frye et al. 1997, Kumar et al. 2007, Smith et al. 2007, Tanaka et al. 2003, Yao, M. et al. 2007, Yao, Y.M. et al. 2007]. This approach is used either for
In vitroCYP activity assessment
The specific probe substrates for CYP activity assessment and inhibition studies are used in the preclinical drug development as well as for clinical purpose. Research in this field is carried out on various models (human liver microsomes, recombinant CYP enzymes, hepatocytes, precision-cut human liver slices, organ perfusions etc.) which enables use of wide range of techniques and model substrates (e.g. luminometric, fluorometric,
3.1. Conventional probe substrates used
Despite that probe substrates are in general supposed to be highly specific and selective towards individual CYP forms, it was reported that with use of (
Probe substrates for CYP2C19 activity assessment are summarized in the Table 9. As in the case of CYP2C9, also in CYP2C19 there were reported different sensitivities of probe substrates to inhibitors. (
Besides probe drugs used
Standard inhibitors are used in preclinical drug development for evaluation of the inhibitory potency of new chemical entity or to identify individual CYP enzymes responsible for a drug’s metabolism, and to determine the relative contribution of an individual CYP enzyme to biotransformation of evaluated chemical entity (Table 12).
|CYP enzyme||Inhibitor||Ki (μM)|
|furafylline||0.6 - 0.73|
|fluvoxamine||6.4 - 19|
|fluoxetine||18 - 41|
|CYP2D6||quinidine||0.027 - 0.4|
|ketoconazole||0.0037 - 0.18|
3.2. High throughput methods for CYP metabolic activity assays
In past two decades, the role of
Therefore, there is a need for new generation of assays with higher throughput capability, sensitivity and reproducibility. These assays are often automated and miniaturized. Plate-scanning readers and handling robots, multi-channel analyzers, high-density assay plates together with automated LC-MS systems are mostly mentioned as the major support for such high throughput methods [Zlokarnik et al. 2005]. Thanks to these techniques, these approaches are also cheaper than standard procedures, maintaining sufficient selectivity, sensitivity and precision, and enable testing of large sets of compounds.
3.2.1. Radiolabeled substrates
Detection of 13/14C or 3H metabolites (H13/14CHO and/or 13/14CO2) of radiolabeled substrates (erythromycin, caffeine, cyclosporine, aminophenazone, diazepam etc. ) is used in some
3.2.2. Fluorogenic substrates
Fluorogenic substrates are used
These difficulties are pronounced in many probe substrates - the most widely used fluorogenic substrate for assessment of CYP2C9 activity
|CYP enzyme||Probe substrate||Product / commercial name|
|CYP1A2||3-cyano-7-ethoxycoumarin (CEC)||3-cyano-7-hydroxycoumarin (CHC)|
|7-ethoxy-methyloxy-3-cyanocoumarin (EOMCC)||Vivid Blue|
|CYP2C9||7-methoxy-4-(trifluoromethyl)-coumarin (7-MFC)||7-hydroxy-4-(trifluoromethyl)-coumarin (HFC)|
|7-benzyloxy-methyloxy-3-cyanocoumarin (BOMCC)||Vivid Blue|
|N-octyloxymethyl-resorufin (OOMR)||Vivid Red|
|benzyloxy-methyl-fluorescein (BOMF)||Vivid Green|
|CYP2C19||3-cyano-7-ethyoxycoumarin (CEC)||3-cyano-7-hydroxycoumarin (CHC)|
|7-ethoxy-methyloxy-3-cyanocoumarin (EOMCC)||Vivid Blue|
|CYP2D6||7-methoxy-4-(aminomethyl)-coumarin (MAMC)||7-hydroxy-4-(aminomethyl)-coumarin (HAMC)|
|3-[2-(N,N diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC)||3-[2-(diethylamino)-ethyl]-7-hydroxy-4-methylcoumarin (AHMC)|
|7- p-methoxy-benzyloxy-4-trifluorocoumarin (MOBFC)||Vivid Cyan|
|7-ethoxy-methyloxy-3-cyanocoumarin (EOMCC)||Vivid Blue|
|CYP3A4||7-benzyloxy-trifluoromethylcoumarin (BFC)||7-hydroxy-4-(trifluoromethyl)-coumarin (HFC)|
|benzyloxy-methyl-resorufin (BOMR)||Vivid Red|
|dibenzylmethylfluorescein (DBOMF)||Vivid Green|
|7-benzyloxy-methyloxy-3-cyanocoumarin (BOMCC)||Vivid Blue|
|7-benzyloxy-methyloxy-4-(trifluoromethyl)-coumarin (BOMFC)||Vivid Cyan|
Some of disadvantages (background fluorescence of unmetabolized substrate, low aqueous solubility, and low signal-to-noise ratio) which may limit the use of fluorescent substrates [Trubetskoy et al. 2005] were resolved by structurally related derivatives (e.g. oxyphenylmethyl-, oxymethyl-, octyloxymethyl-) of commonly available substrates such as resorufin (red), coumarin (blue and cyan) and fluorescein (green). Currently (2011), there are available 8 modified fluorescent substrates for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5 metabolic activities assessment [Makings & Zlokarnik 2011]. Similarly to other common fluorogenic substrates, these derivatives were synthetized as "blocked" fluorophores with negligible background fluorescence (lower than in common fluorogenic probes). Fluorescence signal is triggered after isoenzyme specific biotransformation (Fig. 1) [Marks et al. 2003]. Moreover, these modified (Vivid®) substrates exhibit higher aqueous solubility and their molecule contains 2 potential cleavage sites [Marks et al. 2003]. Fluorogenic assays may be performed in miniaturized form in microplates.
3.2.3. Luminometric substrates
Luminometric substrate probes are used for CYP activity assessment since 2003. These probes are derivatives of luciferin, substrate for the firefly luciferase which generates light. The derivatives have to be metabolized by CYP enzymes to form luciferin prior to the reaction with luciferase and light emission [Cali et al. 2003]. Then, recorded luminiscence is proportional to the amount of metabolite what is dependent on activity of the CYP enzyme. Illustrative example of reaction is given in Figure 2.
Various bioluminometric probe substrates (derivatives of luciferin) to test CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, MAO-A and MAO-B activity are available. Luciferin 6´ chloroethyl ether (Luciferin –CEE) is a substrate for CYP1A1 and CYP1B1, luciferin 6´ methyl ether (Luciferin-ME) for CYP1A2 and CYP2C8, 6´ deoxyluciferin (luciferin-H) for CYP2C9, ethylene glycol ester of luciferin 6'-methyl ether (Luciferin-ME EGE) for CYP2D6, 6-deoxyluciferin ethyleneglycol ester (Luciferin-H EGE) for CYP2C19, luciferin 6´ benzyl ether (Luciferin –BE) and luciferin 6´ pentafluorobenzyl ether (Luciferin-PFBE) for CYP3A4 and CYP3A7, luciferin phenylpiperazine xylene ether (Luciferin-PPXE) and the latest one developed, luciferin isopropylacetal (Luciferin-IPA) for CYP3A4.
These methods may be also automated, miniaturized and used in "high-throughput" mode, in 3 or 6 l 1536-well plates and low volume 384- well plates. The signal half-life of over 2 hours enables batch processing of plates [Cali et al. 2008].
The selection of probe drug for either assessment of CYP metabolic activity
In addition, in CYP3A4 it is recommended to use at least 2 structurally unrelated probe substrates because of presence of multiple substrate binding domains within CYP3A4 protein [Khan et al. 2002, Korzekwa et al. 1998, Schrag & Wienkers 2001, Tucker et al. 2001]. Published crystal structures of CYP enzymes confirmed the ability of individual CYP enzyme to metabolize a wide range of substrates or to bind multiple substrate/inhibitor molecules simultaneously [Ekroos & Sjogren 2006, Foti et al. 2010].
In some CYP forms, it seems that polymorphisms may have variable consequences in different substrates, as was shown in the case of CYP2D6 and CYP2C19. This phenomenon is also described as allele-dependent differences of substrates [Benet 2005]. In detail, this means that in the case of polymorphism CYP2D6*17, the rate of metabolism depends on the substrate used [Bogni et al. 2005, Zhou 2009].
Fluorogenic and bioluminometric probe substrates proved sufficiently their usability in early stages of drug development; these methods of CYP metabolic activity assessment are reproducible, robust and sensitive. On the other hand, since there is occasionally reported lack of correlation in specificity and sensitivity between fluorogenic and conventional probes (mostly in CYP3A4), follow-up studies with conventional probe substrates are strictly recommended to be performed in the clinical phase of new drug development [Cohen et al. 2003].
This chapter was supported by the project of Ministry of Health of Czech Republic: NS 9676-4/2008 and by the project “CEITEC - Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.