The present invention provides a method for the estimation of a patient's drug-metabolizing capacity by CYP (cytochrome P450) -genotyping and CYP-phenotyping of said patient, comprising the following steps: obtaining CYP-genotyping data; obtaining CYP-phenotyping data; analyzing the CYP-phenotyping data and the CYP-genotyping data for the presence of one or more CYP enzyme variant(s); evaluating the mRNA expression level(s) of at least one CYP enzyme of which in the leukocytes show(s) a correlation with the enzyme activity of the same CYP enzyme(s) present in the liver; and thereby estimating the drug-metabolizing capacity of said patient based on CYP-genotype and the mRNA level of said at least one CYP enzyme.

Background

The lack of therapeutic effect of drugs or the appearance of undesired side -effects, resulting in adverse events for patients, is partly caused by differences or changes in drug metabolism. Of significant importance in interindividual differences is the genetic variability (polymorphism) of drug-metabolizing enzymes, causing reduced or even no enzyme activity. As an effect, drug-metabolizing capacity of a patient can be weaker ('poor metabolizer') compared with other members ('intermediate or extensive metabolizer') of population (Ingelman- Sundberg, 2001). An individual with poor drug metabolism capacity can live a normal life until he/she is treated with a drug metabolized by an enzyme with reduced or no activity. Patients with poor drug-metabolizing capacities produce significantly higher blood levels of certain drugs, causing more severe and frequent side- effects (Brockmoller et al, 2000; Wilke et al, 2005).

The principal organ of drug metabolism is the liver; however, every tissue has some ability to metabolize xenobiotics. Although drug-metabolizing activities of the gut wall, kidneys, lungs or even the brain can contribute to the overall biotransformation to some extent, a patient's drug-metabolizing capacity can be approximately estimated from the hepatic metabolism. Therapeutic failure or drug toxicity is strongly influenced by hepatic drug metabolism, primarily depending on the levels and activities of CYP enzymes. The enzymes belonging to the CYP 1-3 families play a central role in the biotransformation of various drugs to more polar compounds, which are readily excreted (Lewis, 2004; Monostory and Pascussi, 2008). One of the most important reasons for interindividual variations in drug metabolism is genetic polymorphism of CYP genes. Some CYP genes (CYP2C9, CYP2C19, CYP2D6, and CYP3A5) are highly polymorphic, resulting in enzyme variants with reduced or even no activity (Solus et al, 2004). The genetically determined variance in CYP enzyme activities is transiently modulated by environmental (nutrition, co-medication) or internal factors (age, hormonal status, liver function, and diseases), leading to different drug metabolism phenotypes (Monostory and Pascussi, 2008). Individuals with defective CYP alleles display permanent poor drug metabolism, whereas those who have wild-type CYP genes may become transient poor metabolizers. This means that the CYP-phenotype and drug-metabolizing capacity dynamically change in the course of external and internal influences, adapting to everyday chemical exposure. A transient decline in drug-metabolizing capacity may arise because of a decrease in physical and health conditions or the consumption of certain drugs or citruses. In contrast, an extensive metabolism can occur upon consumption of St. John's wort tea or during treatment with steroids or rifampicin (Monostory and Pascussi, 2008). By recognizing individual differences in drug-efficiency and toxicity, personalized drug therapy adjusted to a patient's drug-metabolizing capacity can help to avoid the potential side effects of drugs. Tailored medication as a part of modern medical practices requires establishing reliable diagnostic tools for the identification of inactivating mutations or the lack of functional CYP enzymes. Pharmacogenetic services for the estimation of drug-metabolizing capacity have expanded world-wide (Brockmoller et al., 2000; Wilke et al., 2005), however, the test services present in the market offer to determine non-functional CYP enzymes only by CYP-geno typing, and do not provide information about the drug-metabolizing capacity of patients who do not have CYP -mutations. These test services focus on CYP-genotyping analysis for clinically relevant mutations in CYP genes which determines the permanent poor metabolism, since the defective CYP alleles produce enzymes with reduced activity or even non- functional enzymes.

CYP -pheno typing analysis can provide information on the current hepatic CYP activities and can predict transient poor metabolism in those subjects who do not carry mutations in CYP genes. Since the liver is the main drug-metabolizing organ, the overall drug metabolism can be assessed by the hepatic activities. Drug metabolism in the liver can be approximately estimated by the activities of the most relevant drug-metabolizing CYP enzymes; however, the assays of CYP enzyme activities require a large amount of liver tissues. This can be considered as a significant drawback for human studies when testing the drug-metabolizing capacity from liver needle biopsies, where the available tissue is limited. It should be mentioned that substantially lower CYP enzyme activities can be measured in some extrahepatic tissues, such as kidneys; however, the availability of those tissues is as limited as for the liver. The levels of mRNAs as the prerequisite for enzyme proteins and enzyme activities can be determined in small amounts of biological material. Real-time PCR techniques for measuring CYP expressions may provide a useful method for an assessment of the hepatic and eventually of the overall drug-metabolizing capacity.

It can be seen from the above discussion of the state of the art that the currently available methodologies are in need of substantial improvements to provide better tools for the estimation of a patient's drug- metabolizing capacity to improve therapeutic strategies. The present inventors accomplish this goal, providing a global approach by combining CYP-genotyping and CYP -pheno typing tools for the estimation of patients' drug- metabolizing capacity. This approach can add novel elements to the available diagnostics, and a combination of CYP-genotyping and CYP -pheno typing enables a more accurate picture of a patient's drug metabolism. In contrast to the already existing assays, this multistep diagnostic system (sometimes referred herein by the descriptive name CYPtest) determines the patient's drug-metabolizing capacity, and suggests a more rational drug therapy that is adjusted to the CYPtest results. The complex diagnostic system provides an opportunity for predicting CYP gene deficiencies or an extremely reduced/increased CYP expression that identifies the limitations of drug therapy. With an estimation of the patient's drug-metabolizing capacity, a modification of the drug therapy in a rational, individually adjusted way can lower the incidences of adverse drug reactions. The quality of patients' life can eventually be improved, if the diminished drug-metabolizing capacity is recognized in time, and an individually adjusted therapy is applied.

During the development of the present diagnostic system, a further novel finding strengthened the robustness of the technology: a correlation between the activities of several drug-metabolizing CYP enzymes in the liver and the expressions of the respective CYPs in leukocytes (from peripheral blood) was established. The ability to utilize an easily accessible patient's sample provides significant additional advantages to the system according of the present invention. If CYP-status of leukocytes can inform us about the drug-metabolizing capacity of the liver, then the determination of a patient's drug-metabolizing capacity will have predictive power regarding future medication. A prospective investigation of the CYP-status allows a prediction of potential 'poor (or extensive) metabolizer' phenotypes and facilitates an improvement of individual therapy, leading to the optimization of drug choice and/or dosage for a more effective therapy, avoiding serious adverse effects, and decreasing medical costs.

Summary of the invention

Accordingly, the present invention provides a method for the estimation of a patient's drug-metabolizing capacity by CYP (cytochrome P450) -genotyping and CYP-phenotyping of said patient, comprising a) identification of specific allelic variants of one or more gene(s) encoding CYP enzyme(s) in a leukocyte sample obtained from said patient, thereby obtaining CYP-genotyping data,

b) quantitative determination of the mRNA expression level(s) of the one or more gene(s) encoding said CYP enzyme(s) in said leukocyte sample, thereby obtaining CYP-phenotyping data,

c) analysis of the CYP-phenotyping data and the CYP-genotyping data for the presence of one or more CYP enzyme variant(s),

d) identification, based on a pre-established correlation between leukocyte CYP mRNA expression level and liver CYP enzyme activity, of at least one CYP enzyme the mRNA expression level(s) of which in the leukocytes show(s) a correlation with the enzyme activity of the same CYP enzyme(s) present in the liver, e) estimation of the drug-metabolizing capacity of said patient based on the mRNA level of said at least one CYP enzyme, wherein

- said patient is qualified as a poor metabolizer for said CYP enzyme if the mRNA level of said at least one CYP enzyme is not higher than a low cut-off value,

- said patient is qualified as an extensive metabolizer for said CYP enzyme if the mRNA level of said at least one CYP enzyme is not lower than a high cut-off value, and

- said patient is qualified as an intermediate metabolizer for said CYP enzyme if the mRNA level of said at least one CYP enzyme is between a low cut-off value and a high cut-off value. For the invention as detailed above, WO2004/069189 may be considered as the closest prior art. In this application, a method is disclosed for assessing drug metabolism by data presented on CYP2D6. Genotyping and expression analysis is disclosed. The disclosure suggests the classification of patients as poor, intermediate, extensive or ultrarapid metabolizers based on the genotyping data (i.e. SNP analysis of CYP2D6). As a next step, for intermediate metabolizers, further mRNA expression determination is carried out to enhance the classification and therapy.

It is clear that the above disclosure is sharply different from the present invention.

First, the person skilled in the art appreciates the following facts available in the state of the art:

- SNP analysis of CYP2D6 is well known, and the gene deficiencies determine the phenotype

- the regulation of CYP2D6 can be considered to be unique (i.e. according to our present knowledge, the CYP2D6 expression is not inducible and not influenced by the environmental conditions, in contrast to the regulation of the other drug metabolizing CYP enzymes) within the drug metabolizing enzymes, and definitely within the CYP 1-3 family, therefore its use as a demonstration of general principles for determining drug metabolizing status is not well supported

- when the genotyping of a blood sample shows no mutations in a gene (for example, for CYP2D6, it is about 90% of the population), the proposed method will give no data on the drug metabolizing status, since the level of CYP expression present in the blood has no predictive value for the current CYP enzyme activity in the liver, therefore classification based on the genotype is inherently flawed

- the expression analysis in itself gives no relevant data on the enzyme activity, since determining the expression level shows only the presence of the mRNS, but does not provide information on the presence of mutations, and thus the presence of an inactive enzyme (neither in the blood, nor in the liver)

Contrary the prior art, the present invention provides a method where the genotyping and phenotyping are carried out in parallel on the leukocyte sample in each case. Then the results obtained from the blood sample are evaluated on the basis of cut-off values derived from a study carried out on a large pool of patients' blood (leukocyte) and liver samples, establishing credible correlation between (i) the gene mutations and mRNS expression of the leukocytes and (ii) the enzyme activity of the liver. In fact, the studies presented in the present invention clearly show that no such generalization is possible that was suggested in WO2004/069189, i.e. no estimation of the CYP activity in the body (and especially the liver where those enzymes perform their main function) can be made based on the CYP expression in the blood. To the contrary, such correlation must be established for each and every drug metabolizing enzyme, and if the correlation is present, the appropriate cut- off values must be determined. Then, and only then the estimation of a patient's drug-metabolizing capacity may be made on the basis of the findings of the blood analysis.

In a preferred embodiment, the method according to the invention is used in connection with any of the CYP enzymes from the group consisting of CYP1A2, CYP2C9, CYP2C19 and/or CYP3A4/5.

In another embodiment, said CYP enzyme is not CYP2B6.

In another embodiment, said CYP enzyme is not CYP2D6.

In another preferred embodiment, said identification of specific allelic variants of the gene encoding said CYP enzyme is made by single nucleotide polymorphism analysis.

In a further preferred embodiment, said determination of the expression level of the gene encoding said CYP enzyme is made by real time-PCR analysis.

In a particularly preferred embodiment, the drug-metabolizing capacity of said patient is qualified using the cut-off values for CYP mRNA levels in leukocytes pre-established from the respective CYP enzyme activities in the liver. The cut-off values for the said CYP mRNA levels in leukocytes are applied that were proven to correlate with the respective CYP enzyme activity in the liver. For the CYP enzymes analyzed in the present study, the respective cut-off values shown in Table 6 can be used. The method of the present invention can be used for any drug-metabolizing CYP enzymes, if the correlation between the activity of the said CYP enzyme in the liver and the expression of the respective enzyme in leukocytes has been previously established.

In a more particular embodiment, the drug metabolizer status of the said patient consequently determines the therapeutic strategy (drug choice and dosage of a particular drug).

Furthermore, the invention provides tools for personalized medication adjusted to said patient's drug- metabolizing capacity. As an example, CYPtest characterizing said patient's CYP1, CYP2 and CYP3 enzymes provides the methods detecting the presence of single nucleotide polymorphisms of said CYP genes and quantitative assaying for the expression of said CYPs.

Accordingly, in a specific embodiment, the invention provides a method further comprising the step of determining and/or modifying the therapeutic strategy (drug choice and dosage of a particular drug) applied to said patient based on the estimated drug metabolizer status.

In another aspect, the present invention provides a method for determining and/or modifying the therapeutic strategy for a patient by the estimation of a patient's drug-metabolizing capacity by CYP (cytochrome P 450) -genotyping and CYP-phenotyping of said patient, comprising

a) identification of specific allelic variants of one or more gene(s) encoding CYP enzyme(s) in a leukocyte sample obtained from said patient, thereby obtaining CYP-genotyping data,

b) quantitative determination of the mRNA expression level(s) of the one or more gene(s) encoding said CYP enzyme(s) in said leukocyte sample, thereby obtaining CYP-phenotyping data,

c) analyzing the CYP-phenotyping data and the CYP-genotyping data for the presence of one or more CYP enzyme variant(s),

d) identifying, based on a pre-established correlation pattern between leukocyte CYP mRNA expression level and liver CYP enzyme activity, at least one CYP enzyme the mRNA expression level(s) of which in the leukocytes show(s) a correlation with the enzyme activity of the same CYP enzyme(s) present in the liver, e) estimating the drug-metabolizing capacity of said patient based on the mRNA level of said at least one CYP enzyme, wherein

- said patient is qualified as a poor metabolizer for said CYP enzyme if the mRNA level of said at least one CYP enzyme is not higher than a low cut-off value,

- said patient is qualified as an extensive metabolizer for said CYP enzyme if the mRNA level of said at least one CYP enzyme is not lower than a high cut-off value, and

- said patient is qualified as an intermediate metabolizer for said CYP enzyme if the mRNA level of said at least one CYP enzyme is between a low cut-off value and a high cut-off value, and f) applying the appropriate therapeutic strategy (drug choice and dosage of a particular drug) to said patient based on the estimated drug metabolizer status.

Detailed description

The personalized medication of modern therapy requires reliable diagnostic tools to estimate a patient's drug-metabolizing capacity. Although the assessment of overall drug metabolism is difficult to establish, much information can be obtained by using some simplification: 1) the enzymes involved in the biotransformation of drugs are located primarily in the liver; 2) the majority of drugs are metabolized by CYP enzymes; and 3) approximately 90% of drugs in clinical practice undergoing biotransformation involve one or more enzymes belonging to the CYPl-3 family (e.g. CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3As) (Chen et al, 2011). Thus, drug-metabolizing capacity can be approximately assessed through an integrative analysis of the current hepatic expression of CYP enzymes and the genomic identification of defective CYP alleles. The qualification of patient's drug-metabolizing status together with personalized medication can contribute to the improvement of drug therapy, resulting in increasing drug efficacy and a decreasing risk of adverse drug events. Liver biopsies are generally not available from patients, since it is risky and impractical to obtain specimens from the liver in patients. For CYP expression studies, the major advantage would be in easily accessible type of tissues, e.g., peripheral blood, presumably reflecting drug-metabolizing capacity of the liver. CYP enzyme activities are practically undetectable in the blood, thus, blood is not useful tissue for CYP activity assays. Nevertheless CYP mRNA levels in blood cells may reflect the hepatic CYP activities. The individual CYP enzymes vary in tissue distribution with the majority expressed in the liver, but there are various forms of CYPs expressed in other organs and tissues. There are various cellular factors controlling the expression of different CYPs in tissues which lead to an alteration in the levels of these enzymes in different tissues. Tissue specific variations in CYP expression result in differences in drug metabolism in various tissues. This means that the expression of a CYP gene in one tissue may not obviously reflect the liver expression of that CYP enzyme. Thus, CYP expression of each tissue including peripheral blood must be proven to be a surrogate for the respective CYP activities in the liver. Mature human erythrocytes, the main cellular components of blood are anucleate cells; thus, they are not capable of active RNA synthesis. However, Kabanova et al. (2009) provided strong evidence that red blood cells have substantial RNA content. This fact supports the assumption of nucleus- independent protein synthesis, and an obvious lack of the transcriptional regulation of gene expression in mature erythrocytes. Consequently, the mRNA levels of various genes indicate the current regulatory effects of environmental and internal factors before the moment of nucleus discarding, and do not display a prompt transcriptional response to transient modulation during the 120-day lifespan in circulation. Leukocytes are nucleated cells displaying active RNA synthesis; thus they can be chosen as the target cells of CYP expression studies. Although several efforts have been undertaken to establish CYP mRNA levels in liver tissues and in blood or peripheral blood mononuclear cells (Finnstrom et al., 2001 ; Nowakowski et al., 2002; Koch et al., 2002; Nowakowski-Gashaw et al., 2002; Furukawa et al, 2004; Lee et al, 2010), a comprehensive analysis of the relationship between hepatic CYP activities and CYP mRNA levels in leukocytes has not been reported.

Leukocytes would be of clinical interest if the CYP mRNA levels in leukocytes reflect the hepatic CYP activities. Nevertheless, a series of questions arises: 1) does the expression of drug-metabolizing CYP genes in leukocytes reflect hepatic CYP activities; 2) is the regulation of CYP expression in leukocytes similar to that of the liver; and 3) can we obtain information on hepatic drug-metabolizing capacity from leukocytes? To answer these questions, the present study was designed to investigate CYP mRNA levels in leukocytes collected simultaneously with liver tissues, and to study the correlation between CYP expression and hepatic CYP activities.

As outlined above, the present invention therefore provides a method for estimation of a patient's drug- metabolizing capacity by parallel CYP-genotyping and CYP-phenotyping. For obtaining meaningful data for correlation analysis, a baseline study was performed to establish the CYP enzyme activities in the liver. The basic methods for estimating drug-metabolizing capacity are determination of the catalytic activities of individual CYPs by CYP-selective activity probes (Yuan et al., 2002). The enzyme activities of the six most relevant drug-metabolizing CYPs (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were determined in liver tissues of cadaveric donors. The hepatic CYP activities of different donors showed a wide variation (Fig. 1) as was also reported by several authors (Transon et al, 1996; Blanco et al, 2000; Shu et al., 2001). The inter-individual variations of hepatic CYP activities toward selective marker substrates ranged from 80- to 750-fold variations for CYP2B6 and CYP3A4, respectively. In several cases the activities ranged from non-detectable to rather high values for CYP1A2, CYP2C9, CYP2C19 and CYP2D6. Although the techniques determining CYP activities by CYP-selective activity probes are reliable in hepatic microsomes, the catalytic analyses require a relatively large amount of liver tissues, which is a serious drawback for human studies. Additionally, CYP enzyme assays require relatively large amounts of microsomal proteins (0.2-0.25 mg), which in practice cannot be obtained from leukocytes, one of the easily accessible cells.

The method of the invention may use different types of biological samples. As detailed above, the liver sample, if available, could provide an obvious choice for the isolation of genomic DNA and total RNA, and for further analysis of CYP status. As also mentioned, the most widely available source is the blood sample, containing sufficient amount of genomic DNA and total RNA, can also be used for CYP -geno typing and CYP- fenotyping. Intact or analyzable nucleic acids are readily extracted from several various types of biological samples, such as saliva and other sources routinely used for genetic testing; however, CYP -pheno typing in these samples might face difficulties. In the method of the invention, the expression levels of the genes encoding the CYP enzymes are to be determined in liver and leukocyte samples. Methods for this determination are widely available, and are well known for the person skilled in the art. These include for example real time-PCR analysis. The measurements for different CYP expression levels from the same sample can be simultaneous (e.g. microarray arrangement), or the determinations may be done separately (manually or on automated equipment). The determination of mRNA expression level is usually done by using specific primer pairs and labelled probes. There is no specific constraint on the selection of the sequence of these primers and probes, other than that they should be capable to specifically identify the relevant nucleic acid molecules. Exemplary sequences are given in Table 2. Obviously the same principles apply to the sequences of the geno typing primers and probes.

Also, as the person skilled in the art appreciates, it is customary to normalize the expression level measurements. This is preferably done by calculating the quantity of target RNA relative to that of housekeeping genes well known and regularly used in the art for such purposes. Examples of said standard genes include the housekeeping gene glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) or beta-actin.

For establishing CYP mRNA expression, the liver samples used for CYP enzyme activity testing were also assayed for CYP expression by the real time PCR method. Variations in hepatic CYP mRNAs (ranging from 20- to 2000-fold) were more or less similar to the variations in CYP activities. The liver tissues of all donors contained detectable amounts of various CYP mRNAs in contrast to the hepatic activities which were non-detectable in some cases. All of the CYP mRNAs that were detected in liver tissues were also expressed in leukocytes, in contrast to those observed in other studies. Koch et al. (2002) could not detect mRNAs of the CYP3A subfamily, whereas Furukawa et al. (2004) could not display the expression of CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in peripheral blood cells at all. Artifactual causes due to inappropriate sample collection and storage, inefficient RNA isolation procedure, or inadequate primer/probe design might have led to their negative results. Koch et al. (2002) isolated the total RNA from lymphocytes by using the RNeasy kit (Qiagen, Hilden, Germany), whereas Furukawa et al. (2004) used the Qiagen miniprep kit for RNA extraction from leukocytes. Both kits offer an easy isolation procedure and produce high-purity RNA samples, and they are efficient in the isolation of transcripts expressed in relatively large amounts. According to our experience, the isolation of CYP mRNAs from leukocytes using these Qiagen kits seemed to be inefficient, whereas TRIzol reagent (Invitrogen) or TRI reagent (Molecular Research Center, Cincinnati, OH) was found to be appropriate for the extraction of the CYP mRNAs present in relatively small amounts. In our study, the relative expression of various CYP genes were generally lower in leukocytes than in liver tissues (about 20- to 3*10 ⁴ -fold lower), and the variations in CYP mR As in leukocyte were higher (ranging from 10 ² - to 10 ⁵ -fold variations) than in liver.

The relationship between the hepatic mR A levels and CYP activities were established to find evidence for CYP gene expression that could provide accurate information about CYP enzyme activities. A strong correlation (r _s >0.87) was displayed between CYP1A2 mRNA and phenacetin O-dealkylation, suggesting that the hepatic expression of the CYP1A2 gene reflects the CYP1A2 activities in the liver (Fig. 2). A similar conclusion was drawn from the results of Rodriguez-Antona et al. (2001) and George et al. (1995), obtaining a potential association between hepatic CYP1A2 mRNA levels and 7-methoxyresorufm O-demethylation activity or CYP1A2 protein content, respectively. In our study with 164 liver donors, we also found a close relationship between the hepatic mRNA levels and the mephenytoin N-demethylation of CYP2B6 (r _s =0.89) (Fig. 2). However, Rodriguez-Antona et al. (2001) reported a weaker correlation between the hepatic CYP2B6 mRNA levels and the benzoxyresorufm O-debenzylation activities of 12 human liver samples (r=0.52). The fact that benzoxyresorufm O-debenzylation is catalyzed by both CYP2B6 and CYP3A4 (Niwa et al., 2003) could account for the lower correlation coefficient found in CYP2B6 by Rodriguez-Antona et al. (2001).

The present study found that CYP2C9, CYP2C19, CYP2D6 and CYP3A4 a showed somewhat weaker correlation if the hepatic mRNA levels and activities were compared for all of the donors. Genetic polymorphisms, producing less active CYP enzymes or even null activities, are responsible for the fact that some liver tissues displayed relatively high mRNA levels but low activities of CYP2C9, CYP2C19, or CYP2D6. Accordingly, the method of the invention includes a step for the determination the presence and/or absence of specific mutation(s) within the genes encoding the CYP enzymes from a biological sample. The methods for this determination are widely available, and are well known for the person skilled in the art. These include for example single nucleotide polymorphism (SNP) analysis by high resolution melting curve analysis or by hydrolysis SNP analysis, and even complete gene sequencing. The analysis of the different CYP gene sequences from the same sample can be simultaneous (e.g. microarray arrangement), or the determinations may be done separately (manually or on automated equipment).

At present, more than 100 allelic variants of CYP families 1-3 are displayed in human populations, however, measuring all polymorphisms would not be cost effective. While there are many allelic variants for some CYP enzymes, some variants are rare in general and others vary substantially in prevalence by ethnic groups. Table 1 lists the frequencies of some CYP allelic variants in major ethnic groups, showing that one or few alleles with relatively high prevalence in one group may be rare in another.

Table 1 : Frequencies of CYP allelic variants with clinically relevant consequences

Frequency (%)

CYP allele Consequence Caucasian Black African / Asian

(white) Afro-American

CYP2C9*2 Reduced enzyme 8-14 1 0

activity

CYP2C9*3 Reduced enzyme 4-16 1-2 2-3

activity

CYP2C19*2 Inactive 15 17 25-30

CYP2C19*3 Inactive 0.04 0.4 8

CYP2D6*3 Inactive 2 0 0 CYP2D6*4 Inactive 12-21 2-4 1

CYP2D6*5 No enzyme 2-7 4 6

CYP2D6*6 Inactive 1

CYP2D6*10 Reduced enzyme 1-2 6 51

activity

CYP2D6*17 Reduced enzyme 0 34 0

activity

CYP2D6duplication Ultra-rapid activity 1-5 2 0-2

CYP3A5*3 No enzyme 90-93 32 60-73

CYP3A5*6 0 12-13 0

CYP3A5*7 0 8-10 0

Thus, variants with serious clinical consequences and with relatively high frequency in the given population should be routinely measured. For instance, CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2D6*4 and CYP3A5*3 should be included in CYP-geno typing assays if Caucasian populations are studied, CYP2C19*2, CYP2D6*17, CYP3A5*3, CYP3A5*6 and CYP3A5*7 are necessary to be included if black African or Afro- American populations are studied, while CYP2C19*2, CYP2C19*3, CYP2D6*10 and CYP3A5*3 should be included in geno typing tests if Asians are studied. The person skilled in the art will be readily able to apply these modifications to the method of the invention according to the source of the samples to be analyzed.

In this step of our investigation, a preliminary CYP-genotyping study, detecting the frequent and clinically most relevant CYP polymorphisms in the population, was carried out before the estimation of the relationship between hepatic CYP mRNA levels and activities. The donors carrying mutant CYP alleles (heterozygous or homozygous for CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*3, CYP2D6*4, CYP2D6*6) were excluded from correlation analyses. The high correlation coefficients (r _s >0.88) indicated that the hepatic expression of CYP2C9, CYP2C19 and CYP2D6 genes is appropriate for an estimation of the respective CYP activities (Fig. 2). George et al. (1995) also reported a significant, but not close correlation between CYP2C9 mRNA and enzyme protein, which was caused by the poor selectivity of CYP2C antibody, recognizing not only CYP2C9 but several other CYP2Cs. Rodriguez- Antona et al. (2001) described the lack of correlation between the CYP2C9 mRNA levels and diclofenac 4'-hydroxylation; however, they did not take allelic variants producing nonfunctional enzymes into account. They found a lower correlation between the hepatic CYP2D6 mRNA levels and dextromethorphan O-demethylation than we observed, most likely because of neglecting the CYP2D6 polymorphic alleles.

For CYP3A, the liver tissues of some donors showed relatively high activities (nifedipine oxidation, midazolam Γ- and 4-hydroxylation) but low CYP3A4 expression. The weak correlation was assumed to be caused by CYP3A5 polymorphism, because the functional CYP3A5 enzyme as the product of CYP3A5*1 allele also catalyzes the metabolism of CYP3A substrates to some extent. The relatively rare CYP3A5*1 allele (5-10% of the Caucasian population) and eventually the functional CYP3A5 enzyme can contribute to the overall metabolism of CYP3A substrates, such as nifedipine, midazolam, cyclosporine, tacrolimus, erythromycin, carbamazepine, and lidocaine (Patki et al, 2003; Dai et al, 2006; Huang et al., 2004). Thus, we excluded the donors with CYP3A5*1 allele from the correlation analysis, which resulted in a much stronger correlation between the hepatic CYP3A4 mRNAs and CYP3A activities (r _s >0.8 for nifedipine oxidation, midazolam 1 '- and 4-hydroxylation) (Fig. 2). In previous studies by Sumida et al. (1999) and Rodriguez-Antona et al. (2001), a relatively high correlation between the CYP3A4 mRNA amounts and testosterone 6 -hydroxylation was observed. Hepatic CYP3A4 expression also seemed to be related to the normalized plasma concentrations (plasma level/dose*weight) of the CYP3A substrates, cyclosporine or tacrolimus in liver transplants (Thorn et al, 2004).

Hepatic CYP activities are considered to best characterize a patient's drug metabolism, although CYP mRNA levels in leukocytes may provide a tool for estimating the drug-metabolizing capacity of the liver. Information about intra-individual correlations between hepatic CYP activities and blood CYP mRNA levels is virtually non-existent. Finnstrom et al. (2001) demonstrated no correlation of CYP1A2 and CYP3A4 expression in blood with mRNA levels of CYP1A2 and CYP3A4 in the liver, which was caused by the RNA extraction from whole -blood samples. Leukocytes can be assumed, but red blood cells cannot be expected to reflect hepatic CYP expression, because peripheral red blood cells are in different maturation and turnover status, displaying different transcriptional responses to environmental and internal factors. No association was observed between the CYP1A2 mRNA levels in the liver and leukocytes by Furukawa et al. (2004); furthermore, CYP2C9, CYP2C19 or CYP3A4 mRNA was undetectable in the leukocytes. The yield rate of CYP mRNA from leukocytes using the Qiagen miniprep kit or a less sensitive analytical method could be the limitations for the detection of CYP expression in leukocytes. Lee et al. (2010) reported a poor correlation of CYP3A4 mRNA levels between the liver and leukocytes, although the limited samples (n=5) might not provide strong evidence for a correlation. We demonstrated that the mRNA levels of CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in leukocytes correlated strongly with the respective CYP activities in the liver, allowing a good estimation of the hepatic drug-metabolizing activities of these CYPs (Fig. 3). Preliminary CYP-genotyping for frequent and clinically relevant mutations in CYP genes was necessary to discard the subjects carrying polymorphic CYP alleles, which can be transcribed, but are not associated with enzyme activities. The leukocyte CYP expression of the donors with homozygous wild-type genotypes for CYP2C9 and CYP2C19, and with homozygous mutant genotypes for CYP3A5, strongly reflected the hepatic activities of CYP2C9, CYP2C19 and CYP3A. It should be noted that the mRNA levels of CYP2B6 and CYP2D6 in leukocytes did not display any relationship to the respective CYP activities in the liver (Fig. 3). Although polymorphic CYP2D6 alleles can provide information on CYP2D6 poor metabolism, the current CYP2D6 activities in the liver cannot be estimated from the CYP2D6 mRNA levels in leukocytes.

Accordingly, hepatic CYP activities can be estimated by combining CYP-genotyping and CYP- phenotyping analysis of liver biopsy samples or leukocytes. It is suggested that CYP-genotyping analysis for frequent and clinically relevant mutations in CYP genes (e.g. CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*3, CYP2D6*4, CYP2D6*6 and CYP3A5*3) should be carried out first. CYP-genotyping determines the permanent poor metabolism, because defective CYP alleles produce enzymes with reduced activity or even nonfunctional enzymes. CYP -phenotyping analysis of liver samples for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 provides information about the current hepatic CYP activities in those subjects who do not carry mutations in CYP2C9, CYP2C19 and CYP2D6 genes and have CYP3A5*3/*3 genotype. Peripheral blood is a more easily accessible biological sample; therefore, CYP -phenotyping analysis of leukocytes is preferred to that of liver tissues. CYP-phenotyping for CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in leukocytes provides information on the current hepatic CYP activities in those subjects who carry homozygous wild-type genotypes of CYP2C9 and CYP2C19 or homozygous mutant genotype of CYP3A5 (Fig. 4). In conclusion, a patient's drug-metabolizing capacity can be qualified by CYP-geno typing and CYP-phenotyping in liver samples and in leukocytes with some limitations, using the pre-established cut-off values of the expression level determined for the sample used, thereby qualifying said patient as poor, intermediate or extensive metabolizer. For this purpose, the present study provides said cut-off values based on a large sample size of 164 donors. The person skilled in the art will be able to readily apply these cut-off values when analyzing an actual sample for its drug-metabolizing capacity. The profile of a patient's genetic and non-genetic variations in drug metabolism can guide the selection of drugs and the optimal dose that can minimize the harmful side effects and ensure a more successful outcome. Tailored medication eventually will contribute to the improvement of the quality of patients' lives.

Brief description of the figures

Fig. 1. Frequency distribution of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities in

Hungarian liver donors (n=164).

Fig. 2. Correlation of relative CYP gene expression and corresponding CYP activities in the liver. If Spearman's correlation coefficient (r _s ) was higher than 0.7, CYP expression levels in the liver and the corresponding hepatic CYP activities were defined as closely associated.

Fig. 3. Correlation of relative CYP gene expression in leukocytes and corresponding CYP activities in the liver.

If Spearman's correlation coefficient (r _s ) was higher than 0.7, CYP expression levels in leukocytes and the corresponding hepatic CYP activities were defined as closely associated.

Fig. 4. Estimation procedure of a patient's drug-metabolizing capacity in leukocytes isolated from peripheral blood.

The present invention is further illustrated by the experimental examples described below; however, the scope of the invention will by no means be limited to the specific embodiments described in the examples.

Example 1: Materials and methods

Liver and blood samples. The drug-metabolizing capacity of human livers not selected for transplantation (n=146) or liver tissues remaining after reduced-size transplantation (n=18) was determined by CYP-genotyping and CYP-phenotyping. In parallel, samples of peripheral blood were taken from all of the donors. The male/female ratio of the donors was 91/73, their age ranged between 3 and 74 years (43.4±14.46 years), and the cause of death included intracranial bleeding (65.2%) and cerebral contusion (34.8%). The livers were retrieved from hemodynamically stable brain death donors with normal liver function. The livers were perfused and stored in HTK (Fresenius AG, Bad Homburg v.d.H., Germany). The use of human tissues for scientific research was approved by the Hungarian Committee of Science and Research Ethics. All experimental activities were carried out under the regulation of Act CLIV of 1997 on Health and the decree 23/2002 of the Minister of Health of Hungary.

The microsomes and total RNA were isolated from the liver samples. The liver tissues were homogenized in Tris-HCl buffer (0.1 M, pH 7.4) containing 1 mM EDTA and 154 mM KC1. The hepatic microsomal fraction was prepared by differential centrifugation (van der Hoeven and Coon, 1974). All of the procedures of preparation were performed at 0-4°C. The protein content of the hepatic microsomes was determined by the method of Lowry et al. (1951), with bovine serum albumin as the standard. About 50 mg of liver tissues were homogenized in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA), and the total RNA was extracted according to the manufacturer's instructions. The RNA was precipitated by using ethanol and stored at -80 ^° C for further analyses.

Leukocytes were isolated from 0.5 ml of blood samples by red blood cell lysis buffer (Roche

Diagnostics, Mannheim, Germany) and suspended in either 0.2 ml of phosphate -buffered saline for DNA extraction or 1 ml of TRIzol reagent for isolation of the total RNA. The genomic DNA was extracted using high pure PCR template preparation kit (Roche Diagnostics). Total RNA was isolated from leukocytes in a similar way to the hepatic RNA extraction. The purity and the concentration of the DNA and RNA samples were determined with NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

To determine the effect of the period from liver sampling to RNA extraction, and from blood taking to leukocyte isolation and RNA extraction, CYP gene expression was compared among the RNA preparations extracted immediately after the liver sampling and blood taking from three donors, and after storage for 4h, 8h and 24h at 4°C. No effect of storage for 4h on the CYP mRNA levels was observed; however, some degradation of the CYP mRNAs occurred in samples stored for 8h and 24h. Thus, the time between liver tissue and blood delivery, and storage until leukocyte isolation and RNA extraction was limited up to 4h.

CYP enzyme assays. Published methods were followed to determine the CYP-selective enzyme activities: phenacetin O-dealkylation for CYP1A2 (Butler et al, 1989), mephenytoin N-demethylation for CYP2B6 (Heyn et al., 1996), tolbutamide 4-hydroxylation for CYP2C9 (Miners and Birkett, 1996), mephenytoin 4'-hydroxylation for CYP2C19 (Srivastava et al, 1991), dextromethorphan O-demethylation for CYP2D6 (Kronbach et al, 1987), and nifedipine oxidation (Guengerich et al., 1986), midazolam Γ- and 4-hydroxylation (Kronbach et al, 1989) for CYP3A4/5. The incubation mixture contained a NADPH-generating system (1 mM NADPH, 10 mM glucose 6-phosphate, 5 mM MgCl ₂ and 2 units/ml glucose 6-phosphate dehydrogenase), human liver microsomes and various substrates selective for CYP isoforms (phenacetin for CYP1A2, tolbutamide for CYP2C9, mephenytoin for CYP2B6 and CYP2C19, dextromethorphan for CYP2D6, or nifedipine and midazolam for CYP3A4/5). The amount of microsomal protein used in the enzymatic reactions was 0.8 mg/ml except for phenacetin O-dealkylation (1 mg/ml). The microsomal CYP enzyme reactions were linear in the 10- to 30-min incubation period. The enzyme reactions were terminated by the addition of ice-cold methanol. HPLC analyses were performed according to published methods (Guengerich et al, 1986; Kronbach et al, 1987 and 1989; Butler et al, 1989; Srivastava et al, 1991 ; Miners and Birkett, 1996; Heyn et al, 1996). All measurements were performed in duplicate with <5% inter- and intraday precision.

Quantitative real time-PCR. RNA (3 g) was reverse transcribed into single-stranded cDNA by using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules CA), and then real-time PCR with human cDNA was performed by using FastStart Taq DNA polymerase (LightCycler 480 Probes Master, Roche Diagnostics) and UPL probes for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (Roche Diagnostics). The sequences of primers and probes used for real-time PCR analyses are shown in Table 2. The quantity of target RNA relative to that of the housekeeping gene glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) was determined. The CYP mRNA levels were quantified by real-time PCR measurements in the liver tissues and leukocytes from all donors. Table 2: Sequences of PCR primers and probes for CYP-phenotyping and CYP-genotyping

Primer Sequence Probe Sequence

For CYP-phenotyping

CYP1A2 forward 5 ' - ACAACCCTGCCAATCTC AAG-3 ' CYP1A2 FAM-5'-CTGCCTCT-3 '-BHQ reverse 5 ' -GGGAAC AG ACTGGGAC AATG-3 '

CYP2B6 forward 5 ' -AAAGCGGAGTGTGGAGGA-3 ' CYP2B6 FAM-5'-AGGAGGAG-3 '-BHQ reverse 5 ' -AAGGTGGGGTCCATGAGG-3 '

CYP2C9 forward 5 ' -GTGCACGAGGTCCAGAGATAC-3 ' CYP2C9 FAM-5'-CTTCTCCC-3 '-BHQ reverse 5 ' -CAGGGAAATTAATATGGTTGTGC-3 '

CYP2C19 forward 5 ' -TGAAGGTGGAAATTTTAAGAAAAGTAA-3 ' CYP2C19 FAM-5'-CAGCAGGA-3 '-BHQ reverse 5 ' -CCCTCTCCCACACAAATCC-3 '

CYP2D6 forward 5 ' -TTCCTCAGGCTGCTGGAC-3 ' CYP2D6 FAM-5'-AGGAGGAG-3 '-BHQ reverse 5 ' -CGCTGGGATATGCAGGAG-3 '

CYP3A4 forward 5 ' -C ATGGACTTTTT AAGAAGCTTGG-3 ' CYP3A4 FAM-5'-CTCTGCCT-3 '-BHQ reverse 5 ' -TTCCATGTCAAACATACAAAAGC-3 '

GAPDH forward 5 ' -AGCCACATCGCTCAGACAC-3 ' GAPDH FAM-5'-TGGGGAAG-3 '-BHQ

5 ' -GCCCAAT ACGACCAAATCC-3 '

For CYP-genotyping

CYP2C9*2 forward 5 '-AGCAATGGAAAGAAATGGAAG-3 ' CYP2C9*2 wild FAM-CTCTTGAACACGGTCCTC-BHQ 1

reverse 5 '-TAAGGTCAGTGATATGGAGTAGG-3 ' mutant CalRed610-CTCTTGAACACAGTCCTC-BHQ2 CYP2C9*3 forward 5 '-GCAAGACAGGAGCCACATG-3 ' CYP2C9*3 wild CalFluorGold540-CGAGGTCCAGAGATACATTGAC-BHQl reverse 5 '-AGGAGAAACAAACTTACCTTGG-3 ' mutant Quasar670-CGAGGTCCAGAGATACCTTGAC-BHQ2 CYP2C19*2 forward 5 '-CTTAGATATGCAATAATTTTCCCAC-3 ' CYP2C19*2 wild CalGold540-TGATTATTTCCCGGGAACCCATAAC-BHQl reverse 5 '-GAAGCAATCAATAAAGTCCCGA-3 ' mutant Quasar670-TGATTATTTCCCAGGAACCCATAAC-BHQ2 CYP2C19*3 forward 5 '-AGATCAGCAATTTCTTAACTTGATG-3 ' CYP2C19*3 wild F AM-ACCCCCTGGATCC AGG-BHQ 1

reverse 5 '-TGTACTTCAGGGCTTGGTC-3 ' mutant CalRed610-ACCCCCTGAATCCAGG-BHQ2 CYP2D6*3 forward 5'-TGGCAAGGTCCTACGC-3 ' CYP2D6*3 wild FAM-CACAGGATGACCTGGGACC-BHQ 1

reverse 5 '-TCCATCTCTGCCAGGAAG-3 ' mutant CalRed610-CACGGATGACCTGGGACC-BHQ2 CYP2D6*4 forward 5 '-CTTCGCCAACCACTCC-3 ' CYP2D6*4 wild CalRed610-CCCCAGGACGCCC-BHQ2

reverse 5 '-GATCACGTTGCTCACG-3 ' mutant FAM-CCCCAAGACGCCC-BHQ1

CYP2D6*6 forward 5 '-TCTCCGTGTCCACCTTG-3 ' CYP2D6*6 wild FAM-GCTGGAGCAGTGGGTGAC-BHQ 1

reverse 5 ' -GCGAAGGCGGC AC A-3 ' mutant CalRed610-GCTGGAGCAGGGGTGAC-BHQ2 CYP3A5*3 forward 5 '-GAGAGTGGCATAGGAGATACC-3 ' CYP3A5*3 wild FAM-TTTGTCTTTCAATATCTCTTCCCTGT-BHQ1

5 '-TGTACGACACACAGCAACC-3 ' mutant CalRed610-TTTGTCTTTCAGTATCTCTTCCCTGT-BHQ2

FAM, CalRed610, CalFluorGold540, Quasar670: fluorescent labelling, BHQ: black hole quencher

CYP-genotyping with TaqMan probes. Hydrolysis SNP (single nucleotide polymorphism) analysis for CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*3, CYP2D6*4, CYP2D6*6 and CYP3A5*3 was performed by PCR with TaqMan probes (BioSearch Technologies, Novato, CA) using the CFX96 real-time PCR detection system (Bio-Rad Laboratories). Allelic discrimination was based on the design of two TaqMan probes, specific for the wild type-allele and the mutant allele labeled with different fluorescent tags (FAM, CalFlourGold540, CalRed610 or Quasar670; BioSearch Technologies). Primers and probes (Table 2) were designed based on the reference SNP sequences in the NCBI (National Center for Biotechnology Information) reference assembly. Real-time PCR was carried out with 80 ng genomic DNA by using FastStart Taq DNA polymerase (LightCycler 480 Probes Master, Roche Diagnostics, or iQ Supermix, Bio-Rad Laboratories). The CYP-genotypes were distinguished by post-PCR allelic discrimination plotting the relative fluorescence values for wild-type and mutant alleles. The allelic content of each sample was determined by a multicomponent algorithm yielding three allelic clusters representing the CYP-genotypic constituent: homozygous wild type, homozygous mutant type and heterozygous genotype. To confirm the results of the CYP-genotyping, a sequence analysis was also performed. 100 ng of DNA were amplified by using the primers designed for the hydrolysis SNP analysis and iQ Supermix. The PCR products were sequenced directly in an ABI PRISM 3100 Genetic Analyzer.

Data analysis. Hepatic CYP enzyme activities were determined individually in each donor, and the frequency distributions of the CYP activities were recorded for 164 donors. Three categories for each CYP activity (low, medium and high) were statistically distinguished by calculating the quartiles of the CYP activity distributions. The cut-off values between the three categories were set to the 1st and the 3rd quartiles of the donors. The quartiles were chosen over standard deviation values because the CYP activity distributions were skewed. The low, medium and high activity categories were used to describe the drug-metabolizer phenotypes, as poor, intermediate, and extensive metabolizers, respectively.

The gene expression of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 was also determined both in the liver and peripheral leukocytes. The correlation between the hepatic CYP activities and the relative CYP mRNA levels in the liver or leukocytes was estimated. The donors with homozygous mutant or heterozygous genotypes were excluded from the correlation analysis for all CYPs except for CYP3A4. For the evaluation of the CYP3A4 activity - mRNA correlation, the donors carrying the CYP3A5* 1 allele were excluded from the analysis. The correlation among the CYP activities and CYP gene expression levels was quantified, and correlation coefficients (r _s ) and 95% confidence intervals were calculated by using the Spearman approach (InStat version 3.05, GraphPad Software, San Diego, CA). A strong correlation between gene expression and hepatic CYP activities was considered, if the probability value (P) was under 0.0001.

Example 2: Correlation between CYP enzyme activities and CYP expression in liver tissues

Drug-metabolism in the liver can be approximately estimated by the activities of the most relevant drug- metabolizing CYP enzymes, thus we determined the catalytic activities of various CYPs in hepatic microsomal fractions of 164 Hungarian (Caucasian white) cadaveric donors. CYP -selective substrates that were metabolized by a single CYP isoenzyme to produce a given metabolite were used to measure CYP activities. Figure 1 shows the frequency distributions of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities in the Hungarian liver donors. The inter-individual variations of the CYP activities toward selective marker substrates were wide (ranged from 80- to 750-fold variations for CYP2B6 and CYP3A4, respectively); in several cases, the activities ranged from non-detectable to rather high values (Table 3). The CYP activity values did not show Gaussian distribution, rather they exhibited a skewed distribution. As a result, the median±QD values were calculated. The 1st and 3rd quartiles were considered to be the cut-off values between poor and intermediate metabolizers or between intermediate and extensive metabolizers, respectively.

Table 3: Characterization of human liver tissues (n=164) for selective substrates of CYP enzymes. The values are expressed as pmol product/mg microsomal protein*min.

CYP activity Enzyme activity Max. Cut-off values

median±QD PM-IM I M -EM

1st 3rd quartile quartile

CYP1A2

phenacetin O-dealkylation 155.9±110.75 0 1107.1 60.2 281.7

CYP2B6

mephenytoin 35.7±22.20 6.47 538.3 22.3 66.7

N-demethylation

CYP2C9

tolbutamide 4-hydroxylation 218.2±100.55 0 1056.0 104.4 305

CYP2C19

mephenytoin 25.2±19.15 0 342.9 11.9 50.2

4 ' -hydro xylation

CYP2D6

dextromethorphan 261.0±162.0 0 1461.0 140.6 464.6

O-demethylation

CYP3A4/5

nifedipine oxidation 414.2±272..25 3.79 2861.5 231.8 776.3 midazolam 4-hydroxylation 175.2±141.25 11.9 1180.0 101.1 383.6 midazolam Γ-hydroxylation 107.7±77.4 3.42 630.7 72.8 227.6

PM, poor metabolizer; IM, intermediate metabolizer; EM, extensive metabolizer

Assays of CYP enzyme activities require a large amount of liver tissues. This can be considered to be a significant drawback when testing the drug-metabolizing capacity from liver needle-biopsies, where the available tissue is limited. Real-time PCR techniques can measure CYP expressions in small liver samples. These techniques may provide a useful method for an assessment of the liver's drug-metabolizing capacity, if the hepatic CYP mRNA levels reflect the hepatic CYP activities. Total RNA was isolated, and the expression levels of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 genes were determined in the same liver samples in which the CYP activities were measured. The primers for each CYP mRNA assay were designed in two consecutive exons separated by an intron on the corresponding genomic DNA; thus the primer pairs amplified the cDNA generated from the CYP mRNA, and did not yield products on any possible contamination with genomic DNA. All of the CYP mRNA species investigated were detectable in liver tissues, but at varying levels (Table 4). The CYP3A4 mRNA displayed the highest inter -individual variation with the three-magnitude difference between the highest and lowest levels of hepatic expression; whereas the CYP2D6 expression exhibited the lowest variation (20-fold) between donors.

Table 4: CYP mR A levels relative to GADPH mRNA in liver tissues and leukocytes of the donors. The values are the mRNA ratio *10 ^"3

CYP CYP mRNA levels Min. Max.

median±QD

CYP1A2 - liver 89.2±55.15 3.92 678.3

leukocytes 0.419±0.5026 0.0168 4.78

CYP2B6 - liver 57.91±42.80 10.9 607.1

leukocytes 3.26±4.643 0.00454 166.1

CYP2C9 - liver 453.8±237.32 30.4 1853.2

leukocytes 0.0178±0.01031 0.00022 0.0982

CYP2C19 - liver 179.9±130.14 10.17 1635.8

leukocytes 0.0057±0.0056 0.000048 0.0761

CYP2D6 - liver 219.7±132.86 67.92 1274.6

leukocytes 5.68±40.63 0.148 16.056

CYP3A4 - liver 484.7±406.8 2.31 4316.9

leukocytes 0.0362±0.0184 0.000009 0.218

A relationship between the hepatic CYP mRNA levels and enzyme activities has been reported by other investigators (Sumida et al., 1999; Rodriguez-Antona et al, 2001). A correlation between the CYP -selective activities and CYP gene expression, both determined in liver samples of Hungarian donors (n=l 17-124), was estimated. Although the expression of various CYPs would theoretically reflect the drug-metabolizing capacity of the liver, genetic polymorphism of CYPs can give rise to perpetually reduced or even extensive metabolism. Several SNPs frequently occuring in the Caucasian white population were determined in donors in parallel with CYP -pheno typing by CYP activities and CYP mRNA levels.

The hepatic CYP1A2 and CYP2B6 mRNA levels correlated well with the activities of phenacetin O- dealkylation and mephenytoin N-demethylation, respectively (Fig. 2 and Table 5).

Table 5: Relationship between CYP enzyme activities and CYP mRNA levels in liver and in leukocytes. Spearman's correlation coefficients (r _s ), 95% confidence intervals (95% CI) and probability values (P) were calculated.

CYP activity CYP mRNA

in liver in leukocytes

CYP1A2 CYP1A2 mRNA

phenacetin O-dealkylation 0.8780 0.8896

95% CI 0.8058 - 0.9245 0.7868 - 0.9443 P < 0.0001 < 0.0001

CYP2B6 CYP2B6 mRNA

mephenytoin r _s 0.8900 -0.1824

N-demethylation 95% CI 0.8342 - 0.9278 -0.4178 - 0.07595

P < 0.0001 0.1525

CYP2C9 CYP2C9 mRNA

tolbutamide 4-hydroxylation 0.9255 0.9179

95% CI 0.8858 - 0.9518 0.8703 - 0.9485 P < 0.0001 < 0.0001

CYP2C19 CYP2C19 mRNA

mephenytoin r _s 0.8808 0.7918

4 ' -hydroxylation 95% CI 0.8160 - 0.9237 0.6792 - 0.8679

P < 0.0001 < 0.0001

CYP2D6 CYP2D6 mRNA

dextromethorphan r _s 0.9130 0.07087

O-demethylation 95% CI 0.8532 - 0.9491 -0.1939 - 0.3260

P < 0.0001 0.5905

CYP 3 A CYP3A4 mRNA

nifedipine oxidation 0.8984 0.9475

95% CI 0.8436 - 0.9346 0.9145 - 0.9680 P < 0.0001 < 0.0001

CYP 3 A CYP3A4 mRNA

midazolam Γ -hydroxylation 0.8112 0.7718

95% CI 0.7165 - 0.8765 0.6484 - 0.8557 P < 0.0001 < 0.0001

CYP 3 A CYP3A4 mRNA

midazolam 4-hydroxylation r _s 0.8001 0.8078

95% CI 0.7005 - 0.8783 0.7005 - 0.8794 P < 0.0001 < 0.0001

A somewhat weaker correlation was found between the activities and mRNA expressions of CYP2C9, CYP2C19, CYP2D6 and CYP3A4, if all donors were included in the correlation analysis irrespective of their CYP-genotype (results of the analysis are not shown). The most common polymorphisms of CYP2C9 gene in white populations, CYP2C9*2 (430C>T) and CYP2C9*3 (1075A>C) alleles, produce enzymes with reduced function. The prevalence of the allelic variants for CYP2C9 was found to be 7.9% for CYP2C9*2 and 7.0% for CYP2C9*3. The donors with mutated CYP2C9 gene were excluded from the correlation analysis, and homozygous wild types were analyzed. A strong association between the hepatic CYP2C9 mRNA levels and tolbutamide 4-hydroxylation activity (r _s =0.9255) was displayed in donors with CYP2C9*1/*1 genotype (Fig. 2 and Table 5). Mutations in the CYP2C19 gene, resulting in non- functional CYP2C19 alleles, CYP2C19*2 (681G>A) and CYP2C19*3 (636G>A), were also determined in liver donors. The CYP2C19*2 allelic variant was detected with a frequency of 18.3% in the liver donors, whereas the CYP2C19*3 allele was not observed at all. Excluding the donors carrying the CYP2C19*2 allele from the correlation analysis, the hepatic mephenytoin 4-hydroxylation activity of the donors with the CYP2C19*1/*1 genotype significantly correlated with the CYP2C19 mRNA levels in the liver (r _s =0.8808). A deficiency of the CYP2D6 gene, resulting in CYP2D6*3 (2549delA) and CYP2D6*4 (1846G>A) alleles, is associated with the lack of enzyme activity, whereas CYP2D6*6 mutation (1795delT) leads to a lack of enzyme protein and consequently to the lack of CYP2D6 activity. The prevalence of CYP2D6*4 allelic variant was relatively high 17.4%, while the occurrence of both CYP2D6*3 and CYP2D6*6 was found to be 0.89%. A correlation analysis was carried out with the donors having exclusively the wild-type CYP2D6 gene, and a strong association was found between the hepatic CYP2D6 mRNA levels and dextromethorphan O-demethylase activities (r _s =0.9130).

CYP3A4 forms the bulk of the hepatic CYP3A protein and activity (approximately 95% of CYP3A pool); however, the other members, primarily CYP3A5 can also contribute to the metabolism of CYP3A substrates in an adult liver. The CYP3A5*3 mutation (6986A>G in intron 3) results in a splicing defect, leading to a lack of CYP3A5 enzyme. The estimated CYP3A5*3 allele frequency is more than 90% in Caucasian population. Those individuals who have a functional CYP3A5 enzyme (with CYP3A5*1/*1 and CYP3A5*l/*3 genotypes) metabolize some CYP3A substrates (e.g. tacrolimus, cyclosporine A, nifedipine, midazolam) more rapidly than CYP3A5 non-expressors. Some 89.5% of the Hungarian donors did not express functional CYP3A5, carrying CYP3A5*3/*3 genotype. We did not find homozygous wild-type genotype (CYP3A5*1/*1) among the donors investigated; but the functional CYP3A5*1 allele was detected in donors with heterozygous genotype. The frequency of wild-type (CYP3A5*1) allele was found to be 5.5% in the liver donors. Excluding the donors with the CYP3A5*l/*3 genotype from the correlation analysis, a strong correlation was displayed between the hepatic CYP3A4 expression and all three CYP3A activities (nifedipine oxidation, midazolam Γ- and 4- hydroxylation) (Fig. 2 and Table 5).

Example 3: Correlation between CYP enzyme activities in liver and CYP expression in leukocytes

Liver biopsies are generally not available from patients; however, information on drug-metabolizing capacity obtained from leukocytes would be of clinical interest, if CYP mRNA levels in leukocytes reflect the hepatic CYP activities. Leukocytes were isolated from peripheral blood samples, and the expression levels of CYP genes in leukocytes were determined in the same donors whose hepatic CYP activities and CYP mRNA levels were measured. The CYP expression profiles of the leukocytes showed some similarities to the liver; however, the expression levels displayed significant differences. All of the CYP mRNAs that were detected in liver tissues were also expressed in leukocytes. The relative expression of various CYPs was generally 10 ² - 10 ⁴ fold higher in liver tissues than in leukocytes, except for CYP2B6 (Table 4). The expression of the CYP2B6 genes in leukocytes was just 20-fold lower than in liver tissues. High inter-individual variations of CYP expression were also detected in leukocytes, similarly to the liver tissues. The largest variations (10 ⁴ - 10 ⁵ fold) between individuals were found in the expression of CYP2B6, CYP2D6 and CYP3A4 in leukocytes, whereas CYP1 A2 and CYP2C9 mRNA levels displayed only 285- and 445-fold variations, respectively.

Taking blood from patients and isolating leukocytes is a simple way to obtain biological material that may be assumed to provide sufficient information on hepatic drug-metabolizing capacity. Our aim was to study the relationship between CYP enzyme activities in the liver and the expression of CYP genes in leukocytes collected simultaneously. If Spearman's correlation coefficient was higher than 0.7, we defined CYP expression levels in leukocytes and the corresponding hepatic CYP activities as closely associated.

The CYP1A2 mRNA levels in leukocytes significantly correlated with the activity of phenacetin O- dealkylation in the liver (Fig. 3, Table 5). Thus we can conclude that the CYP1A2 expression in leukocytes reflects hepatic CYP1A2 activity. The donors carrying mutated CYP2C9 alleles (CYP2C9*2 or CYP2C9*3) were excluded from correlation analysis. The strong association between CYP2C9 mRNA levels in leukocytes and tolbutamide 4-hydroxylation activity in liver was also displayed in donors with the CYP2C9*1/*I genotype. The expression levels of CYP2C19 in leukocytes were closely associated with mephenytoin 4-hydroxylation in the liver of the donors carrying CYP2C19*1/*1 genotype. In conclusion, the leukocytes isolated from subjects carrying wild-type CYP2C9 or CYP2C19, reflect hepatic CYP2C9 and CYP2C19 activities. Furthermore, all three CYP3A activities in hepatic microsomes displayed close correlation with CYP3A4 mRNA levels in leukocytes, if we excluded the donors with CYP3A5*1 alleles from the correlation analysis. On the other hand, no association could be observed for the expression of CYP2B6 or CYP2D6 in leukocytes and hepatic enzyme activities, mephenytoin N-demethylation and dextromethorphan O-demethylation, respectively (r _s =-0.1824, P=0.1525 for CYP2B6 and r _s =0.07087, P=0.5905 for CYP2D6).

Example 4: Cut-off values for distinguishing poor, intermediate and extensive metabolizers

The drug-metabolizing capacity of the liver was qualified according to the frequency distributions of hepatic CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activities in 164 Hungarian donors. The 1st and 3rd quartiles determined the cut-off values between the categories of low, medium, and high CYP activities, characterizing poor, intermediate, and extensive metabolizer phenotypes, respectively (Table 3). The expression of these CYPs in the liver exhibited a strong correlation with hepatic CYP activities; therefore, an estimation of a patient's drug-metabolizing capacity could be carried out on the basis of the mRNA levels in a liver biopsy sample. The optimal cut-off values for mRNAs levels of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in the liver were set on the basis of the cut-off values for hepatic CYP activities, allowing a distinction between poor, intermediate and extensive metabolizers (Table 6). The mRNA levels of CYP2B6 and CYP2D6 in the leukocytes did not correlate with the respective CYP activities in the liver, and consequently the leukocytes could not serve as appropriate cells for the assessment of hepatic CYP2B6 and CYP2D6 activities. However, the expression of CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in leukocytes was proven to reflect the respective CYP activities in the liver, thus the hepatic activities of these CYP enzymes were suggested to be qualified by leukocyte mRNA levels of these CYP species. The cut-off values for the mRNAs levels of CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in leukocytes were also established on the basis of the cut-off values for the hepatic CYP activities, allowing a distinction between poor, intermediate and extensive metabolizers (Table 6). Table 6: Cut-off values for CYP mRNA levels relative to GADPH mR A in liver tissues and leukocytes distinguishing poor (PM), intermediate (IM) and extensive (EM) metabolizers.

CYP Cut-off values

PM-IM IM-EM

CYP1A2 - liver 4.5*1(T ² 1.5*10 ^_1

leukocytes lo- ⁴ 2*1(T ³

CYP2B6 - liver 3.5*10 ^-2 1.5*10 ^_1

leukocytes - -

CYP2C9 - liver 1.5*10 ^_1 ₇ * ₁₀ -i

leukocytes 5*10 ^-6 2.5*10 ^_i

CYP2C19 - liver 8*10 ^-2 3.5*10 ^"]

leukocytes 10 ^"6 lo- ⁵

CYP2D6 - liver 1.5*10 ^_1 5*10 ^_1

leukocytes - -

CYP3A4 - liver 2.5*10 ^_1 1.5

leukocytes 10 ^"6 10^

PM, poor metabolizer; IM, intermediate metabolizer; EM, extensive metabolizer

CYPtest investigations indicating reduced (or extensive) drug-metabolizing capacity of patients and rationalization of drug therapy resulted in successful outcomes.

Example 4: Case reports

1. An 11 -year old girl suffered from congenital liver fibrosis (liver disease leading to fibrotic degeneration) underwent liver transplantation. The postoperative course was uneventful and showed normal graft function; however, signs of rejection occurred two weeks after transplantation. Immunological reactions and infections were excluded as the reason of rejection. Testing drug-metabolizing capacity of the donor liver indicated extremely reduced CYP2C9 activity. This enzyme catalyzes the metabolism of the antifungal Mycosyst (fluconazole), the antibacterial Sumetrolim (sulfamethoxazole, trimethoprim) and the Losec (omeprazol) used in ulcus prevention therapy. The results of the histopathological examination also confirmed potential drug toxicity. Withdrawal of these drugs was suggested because of functional CYP2C9 deficiency of the graft. In consequence of alterations in medication, the liver graft recovered within one week, and the patient left the hospital. Currently, her days are similar to the lives of other healthy children.

2. The only option for a 17-year-old girl with primary sclerosing cholangitis, leading to cancerous processes, was the liver transplantation. One week after transplantation, significant increase in the levels of transaminases, alkaline phosphatase or bilirubin indicated the liver function failure. In the test of drug- metabolizing status, the liver graft showed strongly reduced activity of CYP2C19. Rationalization of medication, - withdrawal of Mycosyst (fluconazole) and replacement of Controloc (pantoprazole) to Quamatel (famotidine), - was proposed for the recipient. CYP2C19 is not involved in the famotidine metabolism. In consequence of alterations in medication, the patient recovered within one week, and left the clinic after two weeks.

3. A 1.5-month old new-born suffered from epilepsy was treated with valproic acid; however, some toxic symptoms were observed after two weeks (anemia, thrombocytopenia). CYPtest™ demonstrated genetic defects in CYP2C9 gene {CYP2C9*3/*3) which results in non- functional CYP2C9 enzyme. CYP2C9 is responsible for the metabolism of valproic acid. CYP-geno typing results were also confirmed by the high blood level of valproic acid which led to toxic symptoms. On the basis of CYPtest™ results, the drug was withdrawn and replaced with an alternative drug (carbamazepine). The patient's status is stable and her basic disease is successfully controlled by carbamazepine.

4. Gestation hypertension developed at the 21st gestation week of a 35-year old pregnant woman who was treated with methyl-dopa (Dopegyt). On the 23rd gestation week acute hepatitis developed, thus the antihypertensive therapy was changed from methyl-dopa to nifedipine. CYP3A4 enzyme plays a primary role in the metabolism of nifedipine. CYPtest™ indicated poor CYP3A4 metabolizing capacity, therefore reduced nifedipine dose (30 mg daily) was proposed. The patient's blood pressure was successfully controlled with reduced nifedipine therapy. Throughout the gestation period, CYP3A4 expression was followed and the nifedipine dosage was adjusted to the CYP3A4 status. Finally, a healthy baby was born on the 40th gestation week.

5. CYPtesting a 33 -year old woman with schizoid disorder, we demonstrated CYP3A5*l/*3 heterozygous genotype which produces functional CYP3A5 enzyme protein. The functional CYP3A5 enzyme is lacking in more than 90% of the Hungarian population which means that most of the population carries CYP3A5*3/*3 genotype. Production of functional CYP3A5 enzyme (CYP3A5*l/*3) leads to an increase in CYP3A activity, since the activities of CYP3A5 and CYP3A4 are summed resulting in faster metabolism of certain drugs. Dose increase of these drugs would be required for efficient therapy. The patient in our case was treated with carbamazepine in a daily dose of 400 mg. Since carbamazepine is primarily metabolized by CYP3 A enzymes, carbamazepine level in patient's blood was lower than the therapeutic concentration. Dose increase was proposed to 800 mg which resulted in less pronounced emotional fluctuation, stabilized mood and more balanced lifestyle.

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